<b>Bijsluiter</b>. De hyperlink naar het originele document werkt niet meer. Daarom laat Woogle de tekst zien die in dat document stond. Deze tekst kan vreemde foutieve woorden of zinnen bevatten en de opmaak kan verdwenen of veranderd zijn. Dit komt door het zwartlakken van vertrouwelijke informatie of doordat de tekst niet digitaal beschikbaar was en dus ingescand en vervolgens via OCR weer ingelezen is. Voor het originele document, neem contact op met de Woo-contactpersoon van het bestuursorgaan.<br><br>====================================================================== Pagina 1 ======================================================================

<pre>Hydroquinone and benzoquinone
    Health-based recommended occupational exposure limit
</pre>

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<pre></pre>

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<pre>Aan de minister van Sociale Zaken en Werkgelegenheid
Onderwerp              : aanbieding advies over Hydroquinone and benzoquinone
Uw kenmerk             : DGV/MBO/U-932342
Ons kenmerk            : U-7449/BJB/fs/459-S67
Bijlagen               :1
Datum                  : 3 december 2012
Geachte minister,
Graag bied ik u hierbij aan het advies over de gevolgen van beroepsmatige blootstelling
aan hydrochinon en benzochinon.
Dit advies maakt deel uit van een uitgebreide reeks, waarin gezondheidskundige advies-
waarden worden afgeleid voor concentraties van stoffen op de werkplek. Het genoemde
advies is opgesteld door de Commissie Gezondheid en beroepsmatige blootstelling aan
stoffen (GBBS) van de Gezondheidsraad en beoordeeld door de Beraadsgroep Gezondheid
en omgeving.
Ik heb dit advies vandaag ter kennisname toegezonden aan de staatssecretaris van Infra-
structuur en Milieu en aan de minister van Volksgezondheid, Welzijn en Sport.
Met vriendelijke groet,
prof. dr. W.A. van Gool,
voorzitter
Bezoekadres                                                           Postadres
Parnassusplein 5                                                      Postbus 16052
2 5 11 V X D e n         Haag                                         2500 BB Den     Haag
E - m a il : a . j. b a a r s @ g r. n l                              w w w. g r. n l
Te l e f o o n ( 0 7 0 ) 3 4 0 7 0 5 8
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<pre></pre>

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<pre>Hydroquinone and benzoquinone
Health-based recommended occupational exposure limit
Dutch Expert Committee on Occupational Safety
a committee of the Health Council of the Netherlands
to:
the Minister of Social Affairs and Employment
No. 2012/26, The Hague, December 3, 2012
</pre>

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<pre>The Health Council of the Netherlands, established in 1902, is an independent
scientific advisory body. Its remit is “to advise the government and Parliament on
the current level of knowledge with respect to public health issues and health
(services) research...” (Section 22, Health Act).
     The Health Council receives most requests for advice from the Ministers of
Health, Welfare & Sport, Infrastructure & the Environment, Social Affairs &
Employment, Economic Affairs, and Education, Culture & Science. The Council
can publish advisory reports on its own initiative. It usually does this in order to
ask attention for developments or trends that are thought to be relevant to
government policy.
     Most Health Council reports are prepared by multidisciplinary committees of
Dutch or, sometimes, foreign experts, appointed in a personal capacity. The
reports are available to the public.
                 The Health Council of the Netherlands is a member of the European
                 Science Advisory Network for Health (EuSANH), a network of science
                 advisory bodies in Europe.
                 The Health Council of the Netherlands is a member of the International Network
                 of Agencies for Health Technology Assessment (INAHTA), an international
                 collaboration of organisations engaged with health technology assessment.
 I NA HTA
This report can be downloaded from www.healthcouncil.nl.
Preferred citation:
Health Council of the Netherlands. Hydroquinone and benzoquinone. Health-
based recommended occupational exposure limit. The Hague: Health Council of
the Netherlands, 2012; publication no. 2012/26.
all rights reserved
ISBN: 978-90-5549-942-7
</pre>

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<pre>   Contents
   Samenvatting en advieswaarde 11
   Executive summary 21
   Scope 29
.1 Background 29
.2 Committee and procedure 29
.3 Data 30
   Identity, properties and monitoring 31
.1 Chemical identity 31
.2 Physical and chemical properties 32
.3 EU Classification and labelling 34
.4 Validated analytical methods 34
   Sources 39
.1 Natural occurrence 39
.2 Man-made sources 40
   Exposure 43
.1 General population 43
.2 Working population 46
   Contents                               7
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<pre>   Kinetics 51
.1 Absorption and distribution 51
.2 Biotransformation 53
.3 Elimination 54
.4 Physiologically based pharmacokinetic models 55
.5 Summary 56
   Mechanisms of action 59
.1 Bioactivation 59
.2 Detoxification 62
.3 Summary 63
   Effects 65
.1 Observations in humans 65
.2 Animal experiments 70
.3 Summary and evaluation 101
   Existing guidelines, standards and evaluations 105
.1 General population 105
.2 Working population 106
   Hazard assessment 113
.1 Assessment of the health risk 113
.2 Recommendation of the health-based occupational exposure limit 117
.3 Groups at extra risk 120
.4 Additional considerations - benzoquinone 120
.5 Health-based recommended occupational exposure limits 122
0  Recommendation for research 123
   References 125
   Hydroquinone and benzoquinone
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<pre>  Annexes 137
A Request for advice 139
B The Committee 141
C Letter of submission 143
D Comments on the public review draft 145
E Human data 147
F Animal data (in vivo) 151
G In vitro data 169
H Mutagenicity studies 171
  Evaluation of the Subcommittee on the Classification of carcinogenic substances 179
  Carcinogenic classification of substances by the Committee 185
K BMD analysis of thyroid hyperplasia in mice exposed to hydroquinone 187
  Contents                                                                            9
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<pre>0 Hydroquinone and benzoquinone</pre>

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<pre>Samenvatting en advieswaarde
Vraagstelling
Op verzoek van de minister van Sociale Zaken en Werkgelegenheid leidt de
Commissie Gezondheid en Beroepsmatige Blootstelling aan Stoffen (GBBS) van
de Gezondheidsraad gezondheidskundige advieswaarden af voor stoffen in lucht
waaraan mensen beroepsmatig blootgesteld kunnen worden. Deze advies-
waarden vormen vervolgens de basis voor grenswaarden – vast te stellen door de
minister – waarmee de gezondheid van werknemers beschermd kan worden.
    In dit advies bespreekt de commissie de gevolgen van blootstelling aan
hydrochinon en benzochinon en stelt zij een gezondheidskundige advieswaarde
vast. De conclusies van de commissie zijn gebaseerd op wetenschappelijke
publicaties die vóór oktober 2012 zijn verschenen.
Fysische en chemische eigenschappen
Hydrochinon (CAS nr. 123-31-9) is een wateroplosbare kristallijne stof die in
grote hoeveelheden wordt geproduceerd, maar in kleine hoeveelheden ook van
nature in sommige planten aanwezig is. De stof heeft een molecuulmassa van
110,11 Dalton; het smeltpunt is 172 ºC en het kookpunt is 286 ºC.
    In waterige oplossing is hydrochinon gevoelig voor zowel zuur-base
omzettingen als redox-cycling, wat leidt tot de vorming van benzochinon en
Samenvatting en advieswaarde                                                   11
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<pre>  semichinon, alsmede verschillende reactieve vormen van zuurstof (ROS:
  reactive oxygen species).
      Hydrochinon wordt vrijwel uitsluitend gebruikt als industriële stof: als
  antioxidant in de productie van rubbers, als remmer in de stabilisatie van
  monomeren, in fotografische en lithografische bedrijven, als stabilisator voor
  onder andere verven, lakken en vernisssen, en als antioxidant voor industriële
  vetten en oliën.
      Benzochinon (CAS nr. 106-51-4) is een slecht wateroplosbare kristallijne
  stof, die in grote hoeveelheden wordt geproduceerd. Het is in kleine hoeveel-
  heden echter ook in de natuur aanwezig: het wordt bijvoorbeeld door vele
  insecten gesynthetiseerd en uitgescheiden. De stof heeft een molecuulmassa van
  108,09 Dalton en een smeltpunt van 116 ºC; bij deze temperatuur sublimeert de
  stof.
      In waterige oplossing is benzochinon gevoelig voor zowel redox-cycling als
  zuur-base transformaties, leidend tot de vorming van hydrochinon en semi-
  chinon, alsmede verschillende reactieve vormen van zuurstof.
      Benzochinon wordt voornamelijk gebruikt voor de productie van hydro-
  chinon, maar wordt ook gebruikt als polymerisatieremmer in de productie van
  plastics, en als intermediair in de productie van verschillende chemicaliën, zoals
  rubber-acceleratoren en oxidatiemiddelen. Daarnaast vindt het toepassing in de
  fotografie.
  Monitoring
  Voor monitoring van hydrochinon in lucht zijn methoden beschikbaar voor
  concentraties tussen 0,7 en 25 mg hydrochinon per m3. Naast deze milieu-
  monitoring is ook biologische monitoring van aan meer dan 2 mg/m3 hydro-
  chinon blootgestelde werknemers mogelijk, namelijk door analyse van hydro-
  chinon in de urine en door analyse van adducten van hydrochinon en albumine in
  het bloed.
      Voor benzochinon zijn methoden voor milieu- en biologische monitoring
  beschikbaar die gebaseerd zijn op albumine-adducten. In urine is benzochinon
  niet aantoonbaar.
  Grenswaarden
  Nederland heeft momenteel geen grenswaarden voor beroepsmatige blootstelling
  aan hydrochinon en benzochinon. De American Conference of Governmental
  Industrial Hygienists (ACGIH) heeft in 2008 voor hydrochinon een TLV
2 Hydroquinone and benzoquinone
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<pre>(threshold limit value) van 1 mg/m3 vastgesteld als 8-uurs tijdgewogen
gemiddelde (TWA), en in 2001 voor benzochinon een TLV-TWA van 0,44 mg/
m3. Het Verenigd Koninkrijk heeft voor hydrochinon een beroepsmatige
blootstellingslimiet van 0,5 mg/m3 vastgesteld; zij hebben benzochinon niet
geëvalueerd. Duitsland heeft geen beroepsmatige blootstellingslimieten voor
hydrochinon en benzochinon.
Kinetiek
In proefdieronderzoek met ratten zijn de absorptie, distributie, metabolisme en
excretie van hydrochinon onderzocht, daarnaast is er een huidabsorptie-studie
met humane huid in vitro beschikbaar. Er is geen informatie beschikbaar over de
kinetiek na inademing van hydrochinon (inhalatoire blootstelling). De absorptie
via de huid werd geclassificeerd als “langzaam”. Na toediening via het maag-
darmkanaal (orale toediening) wordt hydrochinon snel en goed geabsorbeerd. De
distributie na orale toediening, intraveneuze injectie of inbrengen in de luchtpijp
is nagenoeg identiek.
    Hydrochinon wordt enzymatisch via semichinon geoxideerd tot benzo-
chinon; deze reactie kan ook spontaan verlopen. De reductie van benzochinon tot
hydrochinon wordt eveneens enzymatisch gekatalyseerd, maar ook deze kan
spontaan verlopen. Hydrochinon kan conjugeren met glucuronide en sulfaat; de
gevormde conjugaten worden via de urine uitgescheiden. Benzochinon (en
semichinon) kunnen conjugeren met glutathion, resulterend in mono-, di- en tri-
glutathionconjugaten, die in de gal worden teruggevonden. De glutathionc-
onjugaten kunnen verder worden omgezet tot cysteine-conjugaten en
mercaptuurzuren.
    De belangrijkste uitscheidingsroute is via de urine (> 85%), als waterop-
losbare metabolieten, waarvan glucuronides (45-56%) en sulfaten (19-43%) de
belangrijkste zijn. Mercaptuurzuren zijn in kleinere hoeveelheden aanwezig
(< 5%). Slechts een klein deel (1-3%) wordt onveranderd in de urine
uitgescheiden.
    Voor benzochinon is geen informatie over de kinetiek na inademing
beschikbaar. De stof wordt vanuit het maag-darmkanaal en via de huid snel
geabsorbeerd. Het wordt gedeeltelijk onveranderd en gedeeltelijk als
hydrochinon uitgescheiden; van de laatste het grootste deel als conjugaten.
Samenvatting en advieswaarde                                                        13
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<pre>  Effecten
  Hydrochinon
  Mens
  Uit de beschikbare humaan-epidemiologische studies met hydrochinon konden
  geen conclusies met betrekking tot de kankerverwekkende eigenschappen
  worden getrokken.
  Bij beroepsmatige blootstelling induceert hydrochinon depigmentatie van de
  huid, pigmentatie van het hoornvlies, en sensibilisering.
  Dier
  In proefdieronderzoek werden oog- en huidirritatie, depigmentatie en
  sensibilisering waargenomen. In de acute toxiciteitsstudies werden neuro-
  logische effecten zoals overmatige opwinding, rillingen, toevallen en coma
  gerapporteerd.
      Er zijn subacute studies met toedieningen via de huid (dermaal) en via het
  maag-darmkanaal (oraal) uitgevoerd. Na dermale toediening gedurende 14 dagen
  aan ratten werd verminderd lichaamsgewicht vastgesteld. Na orale toediening
  gedurende 13 weken aan ratten en muizen werden veranderde nier- en lever-
  gewichten gevonden, bij ratten werden ook milde, tijdelijke rillingen en een
  verminderde kooi-activiteit waargenomen.
      Er zijn orale carcinogeniteitsstudies uitgevoerd (toediening via maagsonde
  en via het voer); studies via de dermale route of via de ademhaling zijn niet
  beschikbaar.
      Bij 50 mg/kg lichaamsgewicht/dag oraal toegediend aan ratten gedurende
  104 weken werden verminderde lichaamsgewichten gevonden, alsmede renale
  tubulaire adenomen (goedaardige gezwellen in de niertubulus) in mannetjes en
  mononucleaire celleukemieën (een bepaalde vorm van kanker van de witte
  bloedcellen) in vrouwtjes. In de dieetstudie werden eveneens renale renale
  tubulaire adenomen geïnduceerd. In de rattenstudies werd tevens chronische
  progressieve nefropathie (chronische en voortschrijdende nierschade)
4 Hydroquinone and benzoquinone
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<pre>waargenomen. Alleen uit de sonde-studie kon een NOAEL* van 25 mg/kg
lichaamsgewicht/dag worden afgeleid, uit de dieetstudie kon dat niet.
     In muizen induceerde 50 mg/kg lichaamsgewicht/dag gedurende 104 weken
toegenomen relatieve levergewichten in mannetjes en hepatocellulaire adenomen
(goedaardige gezwellen in de lever) in vrouwtjes. In de dieet-studie werden
eveneens hepatocellulaire adenomen gevonden, maar alleen in mannetjes. Ook in
deze studies konden geen NOAEL’s worden afgeleid.
     Hydrochinon kan het DNA en de chromosomen beschadigen.
     Op basis van de reproductietoxiciteitstudies met hydrochinon concludeert de
commissie dat de stof niet toxisch is voor de vruchtbaarheid en het nageslacht.
De NOAEL voor ontwikkelingstoxiciteit was 75 mg/kg lichaamsgewicht/dag,
voor maternale toxiciteit was de NOAEL 25 mg/kg lichaamsgewicht/dag.
     De beschikbare gegevens over neurotoxiciteit tonen milde en tijdelijke
tremoren en een verminderde kooi-activiteit; de NOAEL voor neurotoxiciteit
was 20 mg/kg lichaamsgewicht/dag.
Benzochinon
Mens
Ten aanzien van de carcinogeniteit van benzochinon zijn geen relevante
epidemiologische studies beschikbaar.
     Blootstelling van mensen aan hoge luchtconcentraties benzochinon kan
leiden tot oogirritatie, terwijl dermale blootstelling kan resulteren in ernstige
huidirritatie en dermatitis. In oudere onderzoekingen werden na beroepsmatige
blootstellingen tot 5 jaar aan 0,44 mg/m3 benzochinon geen systemische effecten
gezien.
Dier
Op basis van diverse dierstudies lijkt benzochinon huid-sensibiliserend te zijn.
De stof induceerde schade aan de huid en irritatie na onderhuidse injectie in
cavia’s.
     In acute toxiciteitsstudies in proefdieren werden verschillende neurologische
effecten waargenomen, waaronder verlies van reflexen, tremoren, en verlamming
van de achterpoten. Er zijn geen acute dermale of inhalatoire studies beschikbaar.
No observed adverse effect level (NOAEL): het hoogste niveau waarbij nog juist geen toxische
effecten optreden.
Samenvatting en advieswaarde                                                                 15
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<pre>      Met benzochinon zijn subacute studies uitgevoerd na inhalatoire blootstelling
  en na intraperitoneale en subcutane injecties. De kritische effecten waren
  hematologisch van aard: vermindering van aantal bloedcellen, methemoglo-
  binevorming (een vergrote hoeveelheid van een geoxideerde vorm van
  hemoglobine in het bloed, leidend tot een verminderde zuurstofcapaciteit) en
  thrombopenie (een verminderde hoeveelheid bloedplaatjes). Een inhalatoire
  NOAEL kon niet worden vastgesteld.
      Afgezien van twee zeer beperkte carcinogeniteitsstudies (een orale muizen-
  en een dermale rattenstudie) zijn er geen chronische studies met benzochinon
  beschikbaar. In de dermale studie werden na subcutane injectie enkele locale
  fibrosarcomen (kwaadaardige bindweefselgezwellen) waargenomen; in de orale
  studie werden tumoren in de lever en de milt gevonden. Getuige de hoge sterfte
  nog vóór de inductie van tumoren was er in beide studies sprake van een
  substantiële toxiciteit. Daardoor en door de lage kwaliteit van de studies kunnen
  geen conclusies worden getrokken met betrekking tot de carcinogene potentie
  van benzochinon.
      Benzochinon kan het DNA en de chromosomen beschadigen.
      Er zijn geen studies betreffende reproductietoxiciteit beschikbaar.
  Evaluatie en advies
  Benzochinon en hydrochinon zijn metabolieten van elkaar. De toxiciteit van de
  een is daarom ook relevant voor de ander, hoewel er een verschil in toxische
  potentie kan bestaan.
      Er zijn geen bruikbare humane gegevens op basis waarvan gezondheids-
  kundige advieswaarden voor hydrochinon en benzochinon afgeleid kunnen
  worden.
      Als de beschikbare proefdierstudies in ogenschouw worden genomen,
  hebben de toxiciteitsprofielen van hydrochinon en benzochinon veel gemeen:
  beiden zijn irriterend voor huid en ogen, werken sensibiliserend op de huid en
  hebben een vergelijkbaar genotoxiciteitsprofiel. De meer complete gegevens
  voor hydrochinon kunnen mogelijk als indicatie voor de potentiële effecten van
  benzochinon dienen.
  Hydrochinon
  Subchronische en chronische blootstelling van proefdieren aan hydrochinon leidt
  tot veranderingen in lichaams-, lever- en niergewichten, en tot tijdelijke tremoren
  en veranderingen in kooi-activiteit. In dieet- en sondestudies met muizen en
6 Hydroquinone and benzoquinone
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<pre>ratten werd tumorinductie in levers en nieren geconstateerd, voorafgegaan door
toenemende lever- en niergewichten en histopathologische veranderingen, in de
nier met hyperplasie (vergroting van een bepaald orgaan of weefsel als gevolg
van een abnormaal hoge celdeling), in de lever met hypertrofie (toenemende
grootte van weefsels of organen door vergroting van het volume van de
afzonderlijke cellen). In de rat werd tevens nefropathie waargenomen; terwijl in
de muis schildklier-effecten (hyperplasie, en adenomen en carcinomen) werden
gezien. De waargenomen nefropathie bij ratten is soortspecifiek en niet relevant
voor de mens. De Subcommissie Classificatie van carcinogene stoffen is van
oordeel dat de resultaten betreffende de waargenomen tumoren inconsistent
waren, en tumoren betroffen die niet relevant voor de mens worden geacht. Op
basis van de beschikbare gegevens is de Subcommissie Classificatie van
carcinogene stoffen van mening dat de gegevens over hydrochinon onvoldoende
zijn om de kankerverwekkende eigenschappen te beoordelen (categorie 3).
Verder concludeerde de subcommissie dat hydrochinon mutaties kan induceren,
doch uitsluitend bij hoge blootstellingen, waarschijnlijk via reactieve vormen
van zuurstof. Zolang blootstellingen aan hydrochinon onder de concentraties
blijven die locale cytotoxiciteit (en derhalve hyperplasie) veroorzaken, worden
de risico’s op carcinogene effecten verwaarloosbaar geacht.
    Op basis van deze conclusies en de overweging van de Subcommissie
Classificatie van carcinogene stoffen dat het mechanisme van de carcinogeniteit
van hydrochinon in proefdieren naar alle waarschijnlijkheid berust op een niet-
stochastisch genotoxisch werkingsmechanisme (zoals het genereren van ROS,
maar mogelijk speelt ook remming van topo-isomerase II een rol) kiest de
commissie dan ook voor de drempelwaarde-benadering om een gezondheids-
kundige advieswaarde voor hydrochinon af te leiden.
    Als startpunt voor de afleiding van de gezondheidskundige advieswaarde
voor hydrochinon neemt de commissie de chronische muizenstudie met sonde-
toediening, met schildklierhyperplasie als kritisch effect. In deze studie vertonen
de vrouwtjes een ongeveer drie maal hogere gevoeligheid dan de mannetjes,
zodat de vrouwtjes als uitgangspunt zijn genomen. De commissie prefereert de
BMD* methode, echter, een analyse van de onderzoeksgegevens toonde aan dat
die van de vrouwtjes niet geschikt zijn voor een BMD analyse; de data van de
mannetjes zijn dat wel, maar de daaruit afgeleide BMDL moet dan gecorrigeerd
worden voor de grotere gevoeligheid van de vrouwtjes. Uit de gegevens van de
mannetjes wordt een BMDL voor 10% extra risico afgeleid van 15,7 mg/kg
lichaamsgewicht/dag. Aangezien vrouwtjes ongeveer drie maal gevoeliger zijn
BMD: benchmark dosis; BMDL: benchmark dosis bij de onderste 95% betrouwbaarheidsgrens.
Samenvatting en advieswaarde                                                           17
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<pre>  dan mannetjes voor dit effect wordt een correctiefactor van 3 op deze waarde
  toegepast, hetgeen resulteert in een gecorrigeerde BMDL van 5,2 mg per kg
  lichaamsgewicht per dag. Om te extrapoleren van de muis naar de mens wordt
  een factor van 3 toegepast, wat leidt tot een waarde van 1,7 mg per kg lichaams-
  gewicht per dag. Route-to-route extrapolatie (van orale naar inhalatoire bloot-
  stelling; 70 kg lichaamsgewicht, 10 m3 ademvolume per 8-urige werkdag) leidt
  vervolgens tot een waarde voor dagelijkse blootstelling van 12 mg/m3. Om te
  corrigeren voor de verschillen in gevoeligheid tussen mensen onderling wordt
  een factor van 3 toegepast. Daaruit volgt dan een gezondheidskundige advies-
  waarde van 4 mg/m3.
      Hoewel er slechts beperkte gegevens beschikbaar zijn, verwacht de
  commissie dat bij deze concentratie de risico’s voor oog- en huidirritatie en
  sensibilisering te verwaarlozen zijn.
  Benzochinon
  De commissie volgt dezelfde lijn van argumentatie voor benzochinon, met
  inachtneming van het vermoeden dat deze stof de hoogste intrinsieke toxiciteit
  van de twee heeft. Omdat geschikte gegevens voor benzochinon zelf ontbreken
  heeft de commissie geprobeerd om de gezondheidskundige advieswaarde voor
  hydrochinon te gebruiken voor de afleiding van een gezondheidskundige advies-
  waarde voor benzochinon. De commissie concludeert echter dat de beschikbare
  gegevens over de relatieve toxiciteit van benzochinon ten opzichte van hydro-
  chinon van onvoldoende kwaliteit zijn om op basis daarvan een betrouwbare
  gezondheidskundige advieswaarde voor benzochinon af te leiden. Op basis van
  de beschikbare gegevens is de Subcommissie Classificatie van carcinogene
  stoffen van mening dat de gegevens over benzochinon onvoldoende zijn om de
  kankerverwekkende eigenschappen te beoordelen (categorie 3). In hoofdstuk 9.4
  geeft de commissie enkele nadere overwegingen met betrekking tot een gezond-
  heidskundige advieswaarde voor beroepsmatige blootstelling aan benzochinon.
      Om de verschillen in toxische potentie van hydrochinon en benzochinon te
  kunnen vergelijken beveelt de commissie voorts aan om de inhalatoire toxiciteit
  van beide stoffen te onderzoeken, bij voorkeur na chronische blootstelling,
  Gezondheidskundige advieswaarden
  De commissie Gezondheid en Beroepsmatige Blootstelling aan Stoffen van de
  Gezondheidsraad stelt bij beroepsmatige blootstelling aan hydrochinon een
8 Hydroquinone and benzoquinone
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<pre>gezondheidskundige advieswaarde voor van 4 mg/m3, als 8 uurs tijdgewogen
gemiddelde.
    De commissie is van mening dat de beschikbare gegevens over de toxiciteit
van benzochinon van onvoldoende kwaliteit zijn om op basis daarvan een
betrouwbare gezondheidskundige advieswaarde voor benzochinon af te leiden.
Samenvatting en advieswaarde                                                  19
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<pre>0 Hydroquinone and benzoquinone</pre>

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<pre>Executive summary
Scope
At request of the Minister of Social Affairs and Employment (Annex A), the
Dutch Expert Committee on Occupational Safety (DECOS), a committee of the
Health Council of the Netherlands, performs scientific evaluations on the toxicity
of existing substances that are used in the workplace. The purpose of the evalua-
tions is to recommend a health-based recommended occupational exposure limit
(HBROEL) for concentrations in the air at the workplace, provided the database
allows derivation of such a value. In the Netherlands, these recommendations
serve as a basis in setting public occupational exposure limits by the minister.
    In this report, the Committee discusses the consequences of occupational
exposure to hydroquinone and benzoquinone, and recommends a HBROEL. The
Committee’s conclusions are based on scientific papers published prior to
October 2012.
Physical and chemical properties
Hydroquinone (CAS no. 123-31-9) is a water soluble crystalline substance, that
is produced in large amounts, but also occurs in small amounts naturally in some
plants. Hydroquinone has a molecular weight of 110.11, a melting point of
172 °C, and a boiling point of 286 °C.
Executive summary                                                                  21
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<pre>       When present in aqueous solution, hydroquinone is susceptible to both redox
  cycling and acid-base transformations, leading to the formation of benzoquinone
  and semiquinone, and also various reactive oxygen species (ROS).
       Hydroquinone is used almost exclusively as an industrial chemical, i.e., as
  antioxidant in rubber manufacturing, as inhibitor in stabilization of monomers, in
  photographic industry and lithography, as stabilizer for paints and varnishes, and
  as antioxidant for industrially used fats and oils.
       Benzoquinone (CAS no. 106-51-4) is a poorly water soluble crystalline
  substance, that is produced in large amounts, but also occurs in small amounts
  naturally, i.e., in a variety of arthropods: the substance is synthesized and
  excreted by many insects. Benzoquinone has a molecular weight of 108.09, and a
  melting point of 116 °C (sublimation).
       When present in aqueous solution, benzoquinone is susceptible to both redox
  and acid-base transformations, leading to the formation of hydroquinone,
  semiquinone, and also ROS.
       Benzoquinone’s major use is in hydroquinone production, but it is also used
  as a polymerization inhibitor, photographic chemical, tanning agent, and as an
  intermediate in the production of a variety of substances, including rubber
  accelerators and oxidizing agents.
  Monitoring
  Methods for air monitoring of hydroquinone have a working range of 0.7 to 25
  mg per m3.
       Biological monitoring of workers exposed to hydroquinone is possible by
  the analysis of hydroquinone in urine, at or above an airborne concentration of
  2 mg/m3. Where urinary levels of hydroquinone give exposure on a daily basis,
  adducts of hydroquinone to albumin might reflect exposures over weeks to
  months prior to collection of a blood sample: serum albumin typically has a half
  life of 21 days in humans.
       For benzoquinone methods for environmental monitoring and biomonitoring
  based on albumin adducts are available. Benzoquinone is not detectable in urine.
  Exposure limits
  Currently The Netherlands do not have limit values for occupational exposure to
  hydroquinone and benzoquinone. The American Conference of Governmental
  Industrial Hygienists (ACGIH) established in 2008 a threshold limit value (TLV)
  for exposure to hydroquinone of 1.0 mg/m3 as 8 hr time weighted average
2 Hydroquinone and benzoquinone
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<pre>(TWA). For benzoquinone ACGIH established a TLV-TWA of 0.44 mg/m3 in
2001. The United Kingdom has set an occupational exposure limit (8 hr TWA)
for hydroquinone of 0.5 mg/m3, based on its (suspected) genotoxicity. No short
term exposure level (STEL) was proposed. In Germany no Maximal Acceptable
Concentration (MAK) value was derived; hydroquinone was classified in
category 2 for carcinogenicity and category 3A for germ cell mutagenicity. The
United Kingdom has not evaluated benzoquinone. In Germany no MAK value
was derived, and benzoquinone was classified in category 3B for carcinogenicity
and category 3B for germ cell mutagenicity.
Kinetics
Various ADME (absorption, distribution, metabolisme and excretion) studies
with hydroquinone in rats are available, as well as an in vitro percutaneous
absorption study using human skin. No information is available on kinetics of
hydroquinone after inhalation. Hydroquinone absorption via the skin was
determined to be “slow” (0.5-1 µg/cm2/hr), but may be more rapid with vehicles
such as alcohols. After oral administration or intratracheal instillation,
hydroquinone was rapidly and extensively absorbed. Distribution was similar for
administration via gavage, intravenous injection and intratracheal instillation.
    Hydroquinone can be oxidised via semiquinone to benzoquinone by various
enzymes, and also spontaneously (auto-oxidation). The reverse reaction can also
occur both spontaneously and enzyme mediated. Hydroquinone can be
conjugated with sulphate and glucuronide resulting in the respective conjugates
which are excreted via the urine. Benzoquinone can be conjugated with
glutathione resulting in mono-, di-, and tri-glutathione conjugates which are
detectable in the bile. The glutathione conjugates can be further metabolised to
cysteine conjugates and mercapturic acids.
    The primary route of elimination is via the urine (> 85%) in the form of water
soluble metabolites. The major urinary metabolites are glucuronide conjugates
(45-56%) and sulphate conjugates (19-43%). Mercapturic acids are present at
lower levels (< 5%). Only a small fraction (about 1-3%) is excreted unchanged in
the urine.
    For benzoquinone no information is available on kinetics after inhalation.
Benzoquinone is reported to be readily absorbed from the gastrointestinal tract
and subcutaneous tissue. It is excreted partly unchanged and partly as
hydroquinone, the major proportion of which is eliminated as conjugates. No
quantitative information was available.
Executive summary                                                                  23
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<pre>  Effects
  Hydroquinone
  Hydroquinone was reported to induce skin depigmentation, cornea pigmentation
  and sensitization in humans after occupational exposure. Eye irritation, skin
  irritation, depigmentation and sensitization were also observed in animal studies.
  The results from the studies on sensitization show “weak” to “extreme”
  sensitization, depending on the methods or vehicle used.
       In the acute toxicity studies in animals, various neurological symptoms were
  observed, including hyperexcitability, tremors, convulsions and coma.
       Sub-acute toxicity studies have been performed via dermal and oral (gavage)
  exposure. Reduced body weight was observed after topical application to rats for
  14 days. After oral repeated dose administration, hydroquinone induced changes
  in kidney and liver weights of rats and mice for 13 weeks; with rats also mild and
  transient tremors and reduced home-cage activity were observed.
       Only oral carcinogenicity studies (diet studies and gavage application) were
  performed; carcinogenicity studies via dermal or inhalation exposure were not
  available. In F344 rats administered 50 mg hydroquinone per kg bw per day by
  gavage for 104 weeks, reduced body weights were observed, and renal tubular
  adenomas in males and mononuclear cell leukaemia in females were found. In
  the diet study also renal tubular adenomas were induced. In both studies chronic
  progressive nephropathy was observed. Only for the gavage study a NOAEL* of
  25 mg/kg bw/day was derived; the diet study did not allow the derivation of a
  NOAEL.
       In B6C3F1 mice, 50 mg/kg bw/day hydroquinone for 104 weeks induced
  increased relative liver weights in males and hepatocellular adenomas in females.
  In the diet study induction of hepatocellular adenomas was also observed, but in
  this study only in males. Also in these studies no NOAELs were derived.
       No conclusion regarding the carcinogenic properties of hydroquinone could
  be drawn from the human epidemiological studies with the substance.
       Hydroquinone was negative in Salmonella strains TA 97, 98, 100, 1535, and
  1537, with or without metabolic activation, but positive in strains TA102 and
  104, both sensitive for oxidative mutagens. Hydroquinone was able to induce
  DNA strandbreaks, SCEs (sister chromatid exchanges), gene mutations,
  chromosomal aberrations and micronuclei in mammalian cells and cell lines in
  no observed adverse effect level.
4 Hydroquinone and benzoquinone
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<pre>vitro. Numerous in vivo studies were performed with hydroquinone,
investigating the induction of micronuclei, chromosomal aberrations or
polyploidy in bone marrow, and chromosomal aberrations or hyperploidy in
germ cells. All but one of these studies used intraperitoneal administration and
most of them were positive. One oral study gave a weak positive result. The
positive responses may well be explained by the postulated underlying
mechanisms, demonstrated to occur under in vitro conditions: redox-cycling with
ROS generation, inhibition of topoisomerase II, and direct covalent binding to
macromolecules (either directly or following conjugation with glutathione).
Although this latter capability may underlie the in vitro observed genotoxicity,
several attempts to demonstrate in vivo formation of hydroquinone-derived
DNA-adducts failed so far.
     Based on the studies on reproduction, it is concluded that hydroquinone is
not a reproductive toxicant. The NOAEL for developmental toxicity was 75 mg/
kg bw/day, for maternal toxicity it was 25 mg/kg bw/day.
     The available data on neurotoxicity showed mild and transient tremors and
reduced home-cage activity at 50 and 64 mg/kg bw/day. The NOAEL for
neurotoxicity was 20 mg/kg bw/day.
Benzoquinone
In humans, exposure to high air levels of benzoquinone may result in irritation of
the eyes. Dermal exposure of humans may result in dermatitis and severe
irritation. Older studies reported that no systemic effects were observed
following prolonged occupational exposure to benzoquinone vapour at a level of
0.44 mg/m3 for periods up to 5 years.
     Benzoquinone appeared to be a skin sensitizer based on results of various
animal studies. It induced skin lesions and irritation when injected subcutane-
ously in guinea pigs.
     In acute toxicity studies in animals, various neurological symptoms were
observed, including loss of reflexes, writhing and paralysis of the hind limbs. No
acute dermal or inhalation studies are available.
     Subacute toxicity studies with benzoquinone have been performed via
inhalation exposure, and intraperitonal and subcutanous injections. The critical
effects were all of a haematological character and included decreases in blood
cells, methaemoglobin formation and thrombopenia. A NOAEL by inhalation
could not be established.
     There are no chronic toxicity studies on benzoquinone except two very
limited carcinogenicity studies: one dermal study (via subcutanous injections)
Executive summary                                                                  25
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<pre>  with rats and one oral study with mice. After dermal exposure a few local
  fibrosarcomas were found, whereas after oral exposure liver and spleen tumours
  were observed. In both studies toxicity by benzoquinone was quite substantial, as
  evidenced by a high mortality before tumour occurrence. Due to this and because
  of the poor quality of the studies no conclusions can be drawn about the potential
  of benzoquinone to induce carcinogenic effects.
      No epidemiological data relevant to the carcinogenicity of benzoquinone
  were available.
      Benzoquinone was negative in Salmonella strains TA 98, 1535, and 1537,
  with or without metabolic activation, and positive in strain TA 100 without
  metabolic activation. Salmonella strains TA102 and 104, both sensitive for
  oxidative mutagens and responding positive towards hydroquinone, were not
  tested. Benzoquinone was able to induce DNA strandbreaks, SCEs, gene
  mutations, and micronuclei in mammalian cells, and cell lines in vitro. In vivo
  micronuclei induction studies – via the oral route – were weakly positive, while
  one dominant lethal test with C3H mice was negative.
      These positive responses may well be explained by the postulated underlying
  mechanisms, demonstrated to occur under in vitro conditions: redox-cycling with
  ROS generation, inhibition of topoisomerase II, and covalent binding to
  macromolecules (either directly or after conjugation with glutathione). Although
  this latter capability may underlie the in vitro observed genotoxicity, several
  attempts to demonstrate in vivo formation of benzoquinone-derived DNA-
  adducts failed so far.
      No studies on reproduction and developmental toxicity of benzoquinone
  were available.
  Evaluation and recommendation
  Since benzoquinone and hydroquinone are metabolites of each other
  (interconverted at reaction rates dependent on prevailing local conditions), the
  toxicity observed with one of the two is also relevant for the other, although there
  may be a difference in potency.
      There are no suitable data from human studies that can be used for deriving a
  HBROEL for either hydroquinone or benzoquinone.
      As far as available animal data permit, toxicity profiles of hydroquinone and
  benzoquinone have indeed a lot in common: both substances are irritating to eyes
  and skin, act as skin sensitizers, and have a comparable genotoxicity profile. The
  database on hydroquinone, however, is more complete: i.e., for benzoquinone
  adequate data on (sub)chronic toxicity, carcinogenicity, and reproductive toxicity
6 Hydroquinone and benzoquinone
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<pre>are not available. Thus, for these data gaps for benzoquinone, available data on
hydroquinone could be used to indicate its potential effects.
Hydroquinone
Subchronic or chronic exposure of experimental animals to hydroquinone leads
to changes in body weight, changes in liver and kidney weight, and in transient
tremors and changes in home-cage activity. Hydroquinone is not a reproductive
toxicant.
     In diet and gavage carcinogenicity studies with mice and rats, tumour
induction was observed in kidney and liver, preceded by increased kidney and
liver weights and histopathological changes: in the kidney with hyperplasia, and
in liver with hypertrophy. In the rat also nephropathy was seen, while in the
mouse the thyroid was affected (hyperplasie and adenomas and carcinomas). The
nephropathy observed in rats is species-specific and not relevant for humans. The
Subcommittee on the Classification of carcinogenic substances is of the opinion
that the results regarding tumour induction were inconsistent and concerned
tumour types that are considered not relevant for humans. Based on the available
information, the subcommittee is of the opinion that the data are as yet
insufficient to evaluate the carcinogenic properties of hydroquinone (category 3).
     Further, the Subcommittee concluded that hydroquinone may induce
mutations under high exposure conditions, probably due to the formation of
ROS. But as long as exposure levels to hydroquinone are below levels inducing
local cytotoxicity (and consequently hyperplasia), the risk for carcinogenic
effects were considered to be negligible.
     Based on these conclusions and the consideration of the Subcommittee on
Classification of carcinogenic substances that the carcinogenic mechanism of
hydroquinone in animal studies is in all probability a non-stochastic genotoxic
mechanism (due to ROS-generation, but inhibition of topoisomerase II may also
play a role) the Committee adopts a threshold approach for deriving a HBROEL
for hydroquinone.
     As starting point for deriving a HBROEL for hydroquinone, the Committee
selects the chronic mouse gavage study as the pivotal one, with thyroid
hyperplasia as the critical effect. In this study the females were approximately
three times as sensitive compared to the males, and thus the effect in the females
should be taken as the starting point. However, analysis of the study data of the
females showed that these are not suited for a BMD* analysis. Only with the data
BMD: benchmark dose; BMDL: benchmark dose at lower 95% confidence limit.
Executive summary                                                                  27
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<pre>  of the males a BMD analysis is possible, but then the resulting BMDL needs to
  be corrected for the higher susceptibility of the females. From the data of the
  males a BMDL for 10% extra risk of 15.7 mg/kg bw/day is derived. In view of
  the higher susceptibility of the females a correction factor of 3 is applied,
  resulting in an adjusted BMDL of 5.2 mg/kg bw/day. To extrapolate from mice to
  men a factor of 3 is applied, leading to a value of 1.7 mg/kg bw/day. Extra-
  polation to man, including route-to-route extrapolation (from oral to inhalation
  exposure; 70 kg bw, breathing volume 10 mg/m3 during a 8-h working day),
  results in a value for daily exposure of 12 mg/m3. To correct for potential
  differences in interindividual susceptibility, a factor of 3 is applied. Applying this
  factor results in a HBROEL of 4 mg/m3. Although only limited data are
  available, the Committee expects that at this level the risk for eye and skin
  irritation and sensitization is negligible.
  Benzoquinone
  The Committee adopts a similar line of reasoning for benzoquinone, recognizing
  that this chemical intrinsically may be the more potent of the two toxicants. As
  suitable data for benzoquinone itself are lacking, the Committee tried to use the
  established HBROEL for hydroquinone for deriving a HBROEL for benzo-
  quinone. However, the Committee concludes that the available data on the
  relative toxicity of hydroquinone and benzoquinone are of insufficient quality for
  deriving a solid HBROEL for benzoquinone. The Subcommittee on the Clas-
  sification of carcinogenic substances is of the opinion that the data are as yet
  insufficient to evaluate the carcinogenic properties of benzoquinone (category 3).
  In Section 9.4 the Committee presents some further considerations with respect
  to a health-based limit value for occupational exposure to benzoquinone.
  Health-based recommended occupational exposure limits
  The Dutch Expert Committee on Occupational Safety recommends a health-
  based recommended occupational exposure limit for hydroquinone of 4 mg/m3,
  as 8 hr time weighted average.
  The Committee concludes that the available data on the toxicity of benzoquinone
  are of insufficient quality to derive a health-based recommended occupational
  exposure limit for benzoquinone.
8 Hydroquinone and benzoquinone
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<pre>Chapter 1
        Scope
1.1     Background
        At request of the Minister of Social Affairs and Employment (Annex A), the
        Dutch Expert Committee on Occupational Safety (DECOS), a committee of the
        Health Council of the Netherlands, performs scientific evaluations on the toxicity
        of existing substances that are used in the workplace. The purpose of the
        evaluations is to recommend a health-based occupational exposure limit
        (HBROEL) for concentrations in the air at the workplace, provided the database
        allows derivation of such a value. In the Netherlands, these recommendations
        serve as a basis in setting public occupational exposure limits by the minister.
1.2     Committee and procedure
        This document contains the assessment by DECOS (hereafter called the
        Committee) of the health hazard of hydroquinone and benzoquinone. The
        members of the Committee are listed in Annex B. The submission letter (in
        English) to the Minister can be found in Annex C.
             In 2012, the President of the Health Council released a draft of the report for
        public review. The individuals and organisations that commented on the draft are
        listed in Annex D. The Committee has taken these comments into account in
        deciding on the final version of the report.
        Scope                                                                                29
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<pre>1.3 Data
    The Committee’s recommendations on the health-based occupational exposure
    limit of hydroquinone and benzoquinone have been based on scientific data,
    which are publicly available. Data were obtained from on-line databases,
    Toxline, Medline, Current Contents, and Chemical Abstracts and TSCATS. The
    names and CAS numbers of hydroquinone (123-31-9) and benzoquinone
    (106-51-4) were used in combination with the following key words: adverse
    effects, occupation exposure, kinetics, human, animal and in vitro. Literature
    references containing the term therapeutic were excluded. The literature from
    this search was selected based on titles and abstract. The final search was
    performed in October, 2012.
 0  Hydroquinone and benzoquinone
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<pre> hapter    2
           Identity, properties and monitoring
2.1        Chemical identity
 able 1 Chemical identity of hydroquinone and benzoquinone
 hemical name             Hydroquinone                                                Benzoquinone
UPAC name                 benzene-1,4-diol                                            cyclohexa-2,5-diene-1,4-dione
 ynonyms                  1,4-benzenediol; p-dihydroxybenzene; p-hydroxyphenol; 1,4-  1,4-benzoquinone, p-benzoquinone;
                          dihydroxybenzene; p-benzenediol; benzohydroquinone;         p-quinone; quinone
                          benzoquinol; p-dioxobenzene; p-dioxybenzene; hydroquinol;
                          hydroquinole; alpha-hydroquinone; p-hydroquinone; ß-quinol;
                          quinol
 AS-number                123-31-9                                                    106-51-4
 INEC-number              204-617-8                                                   203-405-2
 EC-number                604-005-00-4                                                606-013-00-3
 TECS-number              MX 3500 000                                                 DK2625000
           Identity, properties and monitoring                                                                       31
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<pre>2.2           Physical and chemical properties
 able 2 Physical and chemical properties of hydroquinone and benzoquinone1-13
                                             Hydroquinone                          Benzoquinone
 hysical description                         White crystalline                     Yellow crystalline
Molecular formula                            C6H4(OH)2                             C6 H 4 O 2
 tructural formula
Molecular weight                             110.11                                108.09
Melting point (oC)                           172.3                                 115.7
 oiling point (oC)                           286                                   sublimes
 apour pressure (Pa)                         2.4 x 10-3 (at 25 oC)                 12 (at 20 oC)
 pecific gravity (g/cm3)                     1.332 (at 15 oC)                      1.318 (at 20 oC)
Auto ignition temperature (oC)               515 oC                                560 oC
 og Poctanol/water                           0.59                                  0.2
 olubility                                   Highly soluble in water (70 g/L at 25 Slightly soluble in water; soluble in
                                             °C); highly soluble in alcohol and    alcohol, ether, and alkaline solutions
                                             ether; slightly soluble in benzene
Odour threshold (mg/m3)                      Odourless                             0.084 ppm
 onversion factors (at 25 oC and 760 torr)   1 ppm = 4.50 mg/m3;                   1 ppm = 4.42 mg/m3;
                                             1 mg/m3 = 0.222 ppm                   1 mg/m3 = 0.226 ppm
 elative vapour density (air=1)              3.81                                  3.7
              Oxidation-reduction equilibrium
              Hydroquinone is a water-soluble, crystalline solid. When present in aqueous
              solution, hydroquinone is susceptible to both redox cycling and acid-base
              transformations (Figure 1). The products of these reactions include benzo-
              quinone and semiquinone, and various reactive oxygen species (ROS). Hydro-
              quinone, semiquinone and benzoquinone are the most stable species under
              aqueous physiological conditions. While all possible conversions are indicated in
              Figure 1, in vivo interconversions between hydroquinone, semiquinone and
              benzoquinone likely proceed via simultaneous electron-proton (i.e. hydrogen
              atom) transfers (diagonal arrows).14
 2            Hydroquinone and benzoquinone
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<pre> igure 1 Acid-base and oxidation-reduction scheme for hydroquinone, semiquinone and benzoquinone.14 Electron and proton
ransfer reactions are shown on vertical and horizontal axes, respectively. For the biotransformations of hydroquinone and
 enzoquinone, see Section 5.2.
             Identity, properties and monitoring                                                                          33
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<pre>                  The rate of hydroquinone autooxidation in an aqueous medium is pH
            dependent, occurring rapidly under alkaline, but slowly under acidic
            conditions.13 At pH above neutrality, hydroquinone can oxidize spontaneously to
            yield semiquinone and benzoquinone. This may give rise to the formation of
            superoxide anion (O2ֿ˙)15 and subsequently to H2O2.16 Both H2O2 and O2ֿ˙ can
            be converted to OH˙ which (in contrast to H2O2, and O2ֿ˙) can directly induce
            DNA breaks.17 These ROS are also capable of inducing cytotoxicity and cell
            damage.
                  The differences in physical and chemical properties between hydroquinone
            and benzoquinone clearly have their impact on both exposure conditions and
            toxicokinetics. But considering the possible interconversion, observations made
            for one of the compounds may also be relevant for the other. The likelihood that
            toxicity observed after exposure to one of the compounds will also occur after
            exposure to the other will depend on both exposure levels and local physiological
            conditions.
2.3         EU Classification and labelling
 able 3 Classification of hydro- and benzoquinone.
 ompound                            Classification                            Reference
Hydroquinone                        Carc. Cat. 3; R40                         http://www.inchem.org/documents/icsc/icsc/
CAS no.123-31-9)                    Muta. Cat. 3; R68                         eics0166.htm (December 13, 2011)
                                    Xn; R22
                                    Xi; R41-43
                                    N; R50
Hazard and precaution phrases       H302, H317, H318, H341, H351, H400        EG 1271/2008
                                    P273, P280. P305, P351, P338
 enzoquinone                        T; R23/25                                 http://www.inchem.org/documents/icsc/icsc/
CAS no.106-51-4)                    Xi; R36/37/38                             eics0799.htm (December 13, 2011)
                                    N; R50
Hazard and precaution phrases       H301, H315, H319, H331, H335, H400        EG 1271/2008
                                    P261, P273, P301, P310, P305, P351, P338,
                                    P311
2.4         Validated analytical methods
2.4.1       Environmental monitoring
            hydroquinone
            Hydroquinone in air is present both as vapour and in particulate form. The
            proportion of total hydroquinone captured by particle filters is strongly
 4          Hydroquinone and benzoquinone
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<pre>temperature-dependent in the range of 10-30ºC, so measurement methods must
take the vapour fraction into account.18
     NIOSH (1994) developed a fully validated method (method 5004) for air
monitoring of hydroquinone with a working range of 0.7 to 8 mg/m3 for a 90-L
air sample or 2 to 25 mg/m3 for a 30-L air sample. Samples are collected by
drawing a known volume of air through a 0.8 µm cellulose ester membrane.
Samples are immediately desorbed with 1% acetic acid and analyzed by HPLC-
UV (290 nm) using an ID Partisil 10-ODS m-Bondapack C18 column.19
     OSHA described a partially evaluated method for air monitoring of
hydroquinone with a target concentration of 2 mg/m³ (TWA) (method no.
PV2094). Samples are collected by drawing a known volume of air through an
XAD-7 tube coated with 10% phosphoric acid (maximum air volume: 20 litres
and sampling rate: 0.2 L/min). Samples are desorbed with methanol and
analyzed by gas chromatography with a flame ionization detector (GC-FID)
using capillary column. Liquid chromatography with an ultraviolet detector (LC-
UV) can be used for better sensitivity. The status of method is “Stopgap method”
and is presented for information and trial use.20
     The UK Health and Safety Executive (HSE)21 describes a method that has
been validated to demonstrate that it complies with BS EN 482. The method
covers the concentration range 0.05 mg/m3 to 3.0 mg/m3 for 15 minute or 8 hour
sampling. Samples are taken using a glass fibre filter containing in a personal
inhalable dust sampler which is backed up with a Tenax sorbent tube. After
sampling, the samplers are desorbed into acetonitrile and analysed by HPLC.
Separation is achieved using a Zorbax CN column or equivalent and separated
species are detected using a photodiode array detector.21
     In 1999, a method for measuring hydroquinone in air, both in the laboratory
and at the workplace, was evaluated. The method involved sampling the
inhalable fraction onto a filter contained in a multi-holed sampler with a back-up
of Tenax TA, followed by desorption into acetonitrile and analysis by High
Performance Liquid Chromatography. It was shown to be effective at measuring
hydroquinone over the range 0.1 to 2 times a concentration of 0.5 mg/m3 for 8
h.22
benzoquinone
For the monitoring of benzoquinone, Clayton and Clayton (1981) referred to a
method described in 1947. Samples are collected in a midget impinger
containing isopropanol. After reaction with phloroglucinol the absorption is
measured spectrophotometrically at 520 nm.23
Identity, properties and monitoring                                                35
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<pre>          OSHA describes a method which is based on a modified method of NIOSH,
      method S18.1.24,25 The method is fully validated and has a working range of
      0.17-0.75 mg/m³. Samples are collected by drawing a known volume of air
      through a XAD-2 tube #SKC ST226-30-04 (100/50 mg sections, 20/50 mesh;
      maximum air volume: 24 litres and sampling rate: 0.2 L/min). Samples are
      desorbed with isopropanol/hexane (20/80) and analyzed by HPLC-UV (HPLC/
      UL68).24,25
2.4.2 Biological monitoring
      hydroquinone in urine
      Biological monitoring of workers exposed to hydroquinone is possible by the
      analysis of hydroquinone in urine. Biological monitoring may be able to detect
      occupational exposure to hydroquinone at or above an airborne concentration of
      2 mg/m3. However, the background levels of hydroquinone in urine produced
      from dietary and environmental sources may make detection and interpretation
      of low levels of exposure difficult.18 The mean amount of hydroquinone detected
      in human urine was reported to be 53 ± 37 µg/day (n=7; Niwa et al., 1981). Inoue
      et al. (1988) found a mean concentration of 4.2 ± 4.7 mg/L (n=131). In a study of
      Waidyanatha et al. (2004) hydroquinone levels in urine of control workers (i.e.
      not (occupationally) exposed to benzene or hydroquinone) were between 0.066
      and 2.1 mg/L (mean 0.45 mg/L).26 It has also been determined that urinary
      hydroquinone excretion is positively correlated with cigarette smoking.27 There
      are no data in the literature on which to base a biological monitoring guidance
      value.18
          Analytical methods for the determination of hydroquinone in urine are based
      on the acid-hydrolysis of glucuronide and sulphate conjugates followed by
      solvent extraction. Chromatography can be via HPLC-UV, HPLC with
      fluorescence detection, GC of the trimethylsilyl derivates with flame ionization
      detection, GCMS detection of GC of the heptofluorobutyryl derivatives with
      electron capture detection. The most sensitive is the HPLC-fluorescence method,
      with a detection limit of 0.03 mg/L and a coefficient of variation of 3% within
      day and 6% day to day. There are no international quality assurance schemes for
      urinary hydroquinone at present.18
          Nikolic et al. (2004) described a salting out extraction of hydroquinone from
      aqueous solutions (such as urine). The extraction was performed with
      diisopropyl ether and NaCl-saturated samples. The extracts were analysed by
      UV-VIS spectrophotometry.28
 6    Hydroquinone and benzoquinone
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<pre>benzoquinone in urine
No biomonitoring method of benzoquinone in urine exists. Probably benzo-
quinone is not detectable in urine, as the acidity of urine will result in the
conversion of benzoquinone to hydroquinone.
hydroquinone and benzoquinone adducts
Since hydroquinone and benzoquinone are metabolites of benzene, monitoring
tools used for benzene might also be suitable for hydroquinone and benzo-
quinone.
    Urinary metabolites of benzene are eliminated rapidly and as a result can
only serve as biomarkers for approximately 24 hours following exposure. On the
contrary, serum albumin typically has a half life of 21 days in humans. Therefore,
measurement of its adducts might reflect exposures over weeks to months prior
to collection of a blood sample.29
    In studies of Rappaport et al. (2002 and 2005), benzene exposure over longer
periods of time was monitored by measuring of adducts of benzoquinone with
serum albumin. To determine levels of benzoquinone-albumin (BQ-Alb) adducts
in blood serum, cysteinyl groups in the protein were selectively cleaved resulting
in volatile derivatives, which are suitable for GC-MS analysis.30,31 The authors
reported a half life of 13.5 days for these adducts.30,31
    Identical to the situation of urinary monitoring, high background levels of
benzoquinone adducts to albumin and haemoglobin were determined in un-
exposed mice, rats and humans27 making detection and interpretation of low
levels of exposure difficult.
    In the study of Rappaport, mean BQ-Alb levels in non-smokers and smokers
were 2.1 ± 1.1 nmol/g and 3.5 ± 2.0 nmol/g, respectively. The lowest level of
BQ-Alb detected in unexposed workers was 0.9 nmol/g.27
Identity, properties and monitoring                                                37
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<pre>8 Hydroquinone and benzoquinone</pre>

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<pre> hapter 3
        Sources
3.1     Natural occurrence
        hydroquinone and benzoquinone
        There are no natural sources of benzoquinone or hydroquinone at large scale.
           No data on benzo- and hydroquinone concentrations in air, soil or water have
        been found.
           Due to its physicochemical properties, hydroquinone will be distributed
        mainly to the water compartment when released into the environment. It
        degrades both as a result of photochemical and biological processes;
        consequently, it does not persist in the environment. No bioaccumulation is
        observed.13
           At small scale, benzoquinone occurs in a variety of arthropods. It is excreted
        and synthesized by many insects.32 Hydroquinone occurs in various plant
        products both as free, unbound hydroquinone and as a β-D-glucopyranoside
        conjugate, called arbutin (also see Section 4.1).
        Sources                                                                           39
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<pre>3.2   Man-made sources
3.2.1  Production
      hydroquinone
      In 1996, the world capacity for the production of hydroquinone was estimated to
      be 40,000 - 45,000 tonnes per year.33 More recent information is not available.34
           There are three current manufacturing processes for hydroquinone: oxidative
      cleavage of diisopropylbenzene, hydroxylation of phenol, and oxidation of
      aniline. For oxidative cleavage of diisopropylbenzene, the para-isomer is isolated
      and oxidized with oxygen to produce the corresponding dihydroperoxide, which
      is treated with sulphuric acid to produce acetone and hydroquinone. In the phenol
      hydroxylation process, hydrogen peroxide is used as a hydroxylation agent.
      Strong mineral acids or ferrous or cobaltous salts are used as catalysts. In the
      aniline oxidation process, aniline is oxidized with manganese dioxide and
      sulphuric acid to benzoquinone, which is subsequently reduced to hydroquinone
      with iron dust and water, or by catalytic hydrogenation.13,33
      benzoquinone
      Benzoquinone was first produced commercially in 1919, and has since been
      manufactured in several European countries, Japan and the United States.9 Large
      scale preparations involve oxidation of aniline or phenol. Benzoquinone is
      subsequently steam-distilled, chilled and obtained in high purity and yield.35
3.2.2 Use
      hydroquinone
      Virtually all the uses of hydroquinone are industrial. In the USA, approximately
      25% of the hydroquinone manufactured is used as an intermediate for chemical
      conversion to hydroquinone-based rubber antioxidants and antiozonants.
      Another 25% is used as an intermediate for chemical conversion to inhibitors
      used to stabilize monomers. An additional 33% is used in the photographic
      industry including black and white photographic film, lithography, and hospital
      x-ray film. Other uses (11-12%) include chemical conversion to stabilizers for
      paints, varnishes, motor oils, and fuels, and for antioxidants in the industrial use
 0    Hydroquinone and benzoquinone
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<pre>of fats and oils.33 A small, but not quantified, amount of the hydroquinone is
used by the photo-hobbyist, and about 0.05% is used in non-prescription drugs
such as skin bleaching creams. Both are considered consumer use.33 In recent
years the demand in photographic processing has stabilized because conversion
to digital photography is largely complete and film usage has leveled off.36
benzoquinone
The major use of benzoquinone is in hydroquinone production9, but it is also
used as a polymerization inhibitor, photographic chemical, tanning agent, and as
an intermediate in the production of a variety of substances, including rubber
accelerators and oxidizing agents.6,9
Sources                                                                          41
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<pre>2 Hydroquinone and benzoquinone</pre>

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<pre> hapter 4
        Exposure
4.1     General population
        Tobacco smoke
        hydroquinone and benzoquinone
        Inhalation exposure to benzoquinone and hydroquinone may occur from tobacco
        smoke.37 Hydroquinone was measured in mainstream smoke from non-filter
        cigarettes at amounts ranging from 110 to 300 µg per cigarette.38,39 Levels of
        benzoquinone have not been measured, but semiquinone has been found in
        cigarette smoke.40
        Diet
        hydroquinone
        Significant exposure to hydroquinone can occur through dietary sources.
        Hydroquinone occurs in nature as the β-D-glucopyranoside conjugate (arbutin)
        and as free, unbound hydroquinone. The concentrations of free and total hydro-
        quinone have been measured in a variety of foods and beverages by Hill et al.
        (1993)41, summarized in Table 4. Also in the study of Deisinger et al. (1996),
        significant amounts of arbutin were detected in wheat products (1-10 mg/kg),
        Exposure                                                                       43
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<pre>             pears (4-15 mg/kg), and coffee and tea (0.1 mg/kg). Free hydroquinone was
             found in coffee (0.2 mg/kg), red wine (0.5 mg/kg), wheat cereals (0.2-0.4
             mg/kg), and broccoli (0.1 mg/kg).42
                 In most of the samples derived from plant sources, the levels of arbutin are
             considerably higher than those of free hydroquinone. However, arbutin is
             hydrolysed readily by dilute acids yielding hydroquinone and glucose.
             Therefore, both free hydroquinone and arbutin may contribute to hydroquinone
             exposure from natural sources.13
 able 4 Concentrations of free and total hydroquinone in various foods and beverages (Hill et al. 1993).41
 ood sample                                                  Concentrations hydroquinone (mg/kg ± SD)
                                                             Free (unbound)                   Total (bound+unbound)
Wheat germ, toasted                                          0.59                             8.4
Whole wheat bread (100% whole wheat)                         0.6 ± 0.2                        0.9 ± 0.5
Whole wheat cereal (commercially available)                  0.21 ± 0.02                      0.99 ± 0.16
Dip-brewed coffee (pre-ground)                               0.29 ± 0.003                     0.39 ± 0.02
Diet cola                                                    0.036                            0.029
 ear skin (D’Anjou, fresh)                                   -a                               38
 ear flesh (D’Anjou, fresh)                                  -a                               1.3
     Background levels comparable to that observed in control blanks
             benzoquinone
             Exposure to benzoquinone via the diet can occur due to the excretion of benzo-
             quinone by insects. Benzoquinone occurs in a variety of arthropods, and many
             insects synthesize and excrete a mixture of quinones including benzoquinone.32
                 Deisinger et al. (1996) investigated dietary and other potential sources of
             hydroquinone and their contribution to hydroquinone concentrations in the
             plasma and urine of human volunteers. Low concentrations of hydroquinone
             were detected in the urine and plasma of humans with no occupational or other
             known exposure to hydroquinone. After consuming a meal including arbutin-
             and hydroquinone-containing foods, volunteers showed significant increases in
             plasma and urinary levels of hydroquinone and its conjugated metabolites (total
             hydroquinone). Mean plasma concentrations of total hydroquinone peaked at 5
             times background levels at 2 h after the completion of the meal, and mean
             urinary excretion rates of total hydroquinone peaked at 12 times background at
             2-3 h after the meal. Immediately after smoking four cigarettes in approximately
             30 min, mean plasma concentrations of total hydroquinone were maximally 1.5
             times background levels; mean urinary excretion rates of total hydroquinone
             peaked at 2.5 times background at 1-3 h after smoking. These data indicate that
 4           Hydroquinone and benzoquinone
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<pre>considerable human exposure to hydroquinone can result from plant-derived
dietary sources and, to a lesser extent, from cigarette smoke.42
    With a meal of 200 grams of bread and a pear of 300 grams, a person might
consume at least 0.6 mg hydroquinone per meal. A moderate smoker (10
cigarettes per day) may inhale 3 mg hydroquinone/day due to smoking alone.
Other routes of exposure
hydroquinone and benzoquinone
Since benzene and p-phenylenediamine can be metabolized enzymatically and
via air oxidation, respectively, to benzoquinone, exposure to these substances
may result in exposure to benzoquinone (and hydroquinone).43
hydroquinone
Photo-hobbyists can be exposed to hydroquinone dermally or by inhalation. In
1980, the number of photo-hobbyists was estimated to be about 2.2 million in the
USA; more recent data, or data on exposure levels are not available.13,34
    Dermal exposure may also result from the use of cosmetic and medical
products containing hydroquinone, such as skin lighteners. In the USA, hydro-
quinone has been used in cosmetics. Both over-the-counter and prescription
drugs are used to lighten areas of hyper-pigmented skin. Over-the-counter skin
lighteners may contain up to 2% hydroquinone. Concentrations up to 4% may be
found in prescription drugs. In some countries even higher concentrations may
be found in skin lighteners.13
    In the Cosmetic Ingredient Review (CIR), the Environmental Working Group
(EWG) determined that hydroquinone should not be used in non-drug cosmetic
products that are left on the skin and not immediately rinsed off.44 The panel
drew this conclusion based on studies linking (orally administered) hydro-
quinone to cancer and immune system damage that targets bone marrow. EWG
identified hydroquinone on the ingredients list of 18 products left on the skin,
most designed as depigmenters or skin lighteners.
    No data on hydroquinone concentrations in air, soil or water have been found
(see Section 3.1).13
    Based on various calculations, in 1995 the US Environmental Protection
Agency (EPA) considered hydroquinone exposure through drinking water and
fish consumption negligible for processor/user sites.
Exposure                                                                         45
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<pre>    benzoquinone
    No specific data available.
4.2 Working population
    hydroquinone
    Hydroquinone can be encountered in a solid form or in solution during its
    production and use.10 It has a very low vapour pressure, but can be oxidized in
    the presence of moisture to form benzoquinone, which is more volatile. The
    saturated concentration in air for hydroquinone vapour under standard conditions
    is estimated to be 0.108 mg/m3 (approximately 0.024 ppm at 25°C).10
        Occupations in which hydroquinone exposure may occur are: hydroquinone
    manufacturing workers, antioxidant makers, drug makers, hair dressers and
    cosmetologists, paint makers, photographic developer makers, photo processors,
    organic chemical synthesizers, plastic stabilizer workers, and rubber coating
    workers.10,13
        Hydroquinone manufacture is a heavily automated procedure, consequently
    only 81 employees in the USA are potentially exposed during manufacture,
    materials handling, maintenance, quality control sampling and analysis.
    Employee protective clothing and engineering controls, such as local exhaust
    ventilation systems, are used to further prevent exposure at points in the process
    where exposure can occur.33 No information on the number of employees
    exposed during manufacturing in Europe was found.
        The average airborne hydroquinone concentration (~8,000 breathing zone
    and area samples) in one hydroquinone manufacturing plant in the USA has
    decreased from 6.0 mg/m3 in the 1950s to 0.1 mg/m3 in 1990.45
        Inhalation exposure levels in two US manufacturing plants were estimated
    and calculated dose rates are shown in Table 5. These estimated exposure levels
    assume that no personal protective devices were used. However, producers in the
    USA have reported the use of engineering controls (local exhaust and dust
    collectors) and personal protective devices (e.g. gloves, uniforms, goggles,
    respirators, boots) to control exposure. Therefore, the calculated dose rates may
    well exceed actual expected dose rates.33
        Very rough worst-case estimates of total dermal contact with hydroquinone
    (650-18,200 mg/day) were calculated using models which assume that emplo-
    yees regularly immerse their hands in hydroquinone for the entire work day
    without protective equipment. All US manufacturers of hydroquinone report that
 6  Hydroquinone and benzoquinone
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<pre>            engineering controls and personal protective equipment are used to minimize
            exposure, and thus the bounding estimates are likely to overestimate the potential
            contact that employees can have with hydroquinone dust.33
                 The number of employees working in US industries which use hydroquinone
            has been estimated at 350,000 to 560,000 at 16,000 to 66,000 facilities. The
            amount of exposure to hydroquinone at these work sites was not available and it
            was assumed that the exposures were equivalent to that of hydroquinone
            manufacturing employees (Table 5).33 There are, however, several reasons to
            believe that this assumption will overestimate occupational exposures at
            processor/user sites. The first is that many operations which use or process
            hydroquinone are batch operations in which hydroquinone is added to hoppers,
            reactors, or mixing vessels which are then closed. In these types of operations,
            bags or drums are opened and their contents emptied out during periods of 10-60
            minutes. Automation also reduces exposure to hydroquinone. For example,
            commercial photo processors are heavily automated and emissions from
            machines are typically below detectable levels.33
                 A study of the US National Institute of Occupational Safety and Health
            (NIOSH) did not detect hydroquinone in the atmosphere of rooms were films
            were being developed. Monitoring data were also available from the US
            Occupational and Safety Administration (OSHA) Compliance Information
            System on airborne concentrations of hydroquinone in processor/user facilities.
            The data base included 78 air samples from facilities in 16 different standard
            industrial code categories. All samples, except of one, were less than the
            analytical limit of detection or less than 1 mg/m3. The majority of the samples
            were below detectable levels.33
 able 5 Estimated occupational exposure associated with the manufacture of hydroquinone in the USA33
 ype of worker              No. of workers; h/day; Airborne concentrations (n=29) Potential inhalation dose ratea (mg/kg
                            days/year               (mg/m3)                         bw/day)
                                                    Average         Maximum         Average            Maximum
Operator/sampler            34; 1-8; 100-250        0.116           0.300           0.0166             0.0429
 oader/packager             34; 1-8; 100-250        0.254           1.870           0.0363             0.2670
Maintenance personnel/      13; 1-8; 1-250          0.135           0.295           0.0193             0.0421
 ousekeeper
    Dose rates assume: the medium work inhalation rate of 1.25 m3 air/h; the maximum number of hours in a range; no use of
    personal protective equipment; the chemical is 100% concentrated, and 70 kg bw.
            Exposure                                                                                                    47
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<pre>                   In the manufacturing of photographic developers, monitoring data are
             commonly collected as total dust in the breathing zone. Exposure levels recorded
             by manufacturers indicated the 8-hr TWA values were below 1 mg/m3 with
             average exposures at ~0.15 mg/m3.33 Measurements for hydroquinone among
             users of photographic developers indicate hydroquinone levels in air below the
             analytical limit of detection (0.01 mg/m3).33 However, spills of photo developers
             in the workplace could result in exposure to hydroquinone.33
                   It has been estimated by the UK Health and Safety Executive (HSE) that in
             the UK 3,000-5,000 people are exposed to hydroquinone at work. The main route
             for work-related potential exposure is by inhalation (UK-HSE 1993, cited in
             OECD 1996).33 Estimated occupational exposure associated with the processing
             of hydroquinone in the UK in different industries is presented in Table 6.
                   The potential dose rates shown in Table 6 assume that no personal protective
             equipment is worn. Available information indicated that hydroquinone users in
             the UK rely on personal protective equipment for control of exposure. Current
             control measures in the UK maintain exposure levels below the TLV of 2 mg/m3
             and in many cases below 1 mg/m3. A combination of local ventilation and
             appropriate handling procedures should be adequate to control exposure below
             0.5 mg/m3 8-hr TWA.33
                   Biological monitoring of darkroom workers did not indicate an increase in
             work-related hydroquinone exposure as urinary levels were less than control
             worker values.33 However, as is already mentioned in Section 2.4.2, the
             background levels of hydroquinone in urine produced form dietary and
             environmental sources may make detection and interpretation of low levels of
             exposure difficult.18
 able 6 Estimated occupational exposure associated with the processing of hydroquinone in the UK.33
ndustry category         Average airborne concentrationa Potential inhalation dose rate for typical exposure periods (mg/kg)b
                         (mg/m3)
                         average (maximum)                10 min average (maximum)            60 min average (maximum)
 ood                     0.42                             1.2 x 10-3                          7.5 x 10-3
 ubber                   4.3 (10)                         1.2 x 10-2 (2.9 x 10-2)             7.7 x 10-2 (1.8 x 10-1)
Agrochemical             0.03 (0.1)                       8.6 x 10-5 (2.9 x 10-4)             5.4 x 10-4 (1.8 x 10-3)
    Sampling periods were 5-120 min.
    Assumes 1.25 m3 of air inhaled in 1 h, 0.2 m3 of air inhaled in 10 min, 70 kg bw, and that chemical hoppers or reaction
    vessels are filled once per 8-h work shift.
 8           Hydroquinone and benzoquinone
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<pre>benzoquinone
Occupational exposure to benzoquinone may occur in the dye -, textile -,
chemical -, tanning -, and cosmetic industries.6 No detailed information on
occupational exposure to benzoquinone was available.
Exposure                                                                    49
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<pre>0 Hydroquinone and benzoquinone</pre>

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<pre> hapter 5
        Kinetics
5.1     Absorption and distribution
        hydroquinone
        For an overview of studies performed on absorption see Annex F-1.
             In the study of Barber et al. (1995)46, the rate of percutaneous absorption of a
        5% aqueous hydroquinone solution through human stratum corneum and whole
        rat skin was measured in vitro. The absorption rate through human skin was
        determined to be 0.522 µg/cm2/h; the absorption rate through rat skin was 1.09
        µg/cm2/h. These absorption rates were considered to be “slow” based on
        published definitions of absorption (OECD 1996).33
             In the study of Stenius (1989)47, a solution of 4% hydroquinone in oil was
        applied to human and rat skin in vitro (40 mg/cm2). After 24 h 1.68% of the dose
        had been absorbed by the rat skin and 0.28% by the human skin.
             The actual amount of hydroquinone absorbed following dermal exposure
        depends on the exposure concentration, the time of exposure, and the vehicle, as
        well as other factors. Bucks et al. (1988) applied a solution of 14C-hydroquinone
        to human foreskin at a concentration of 125 µg/cm2. Percutaneous absorption
        was estimated by measurement of radioactivity in the urine. Peak elimination
        was observed within the first 12 h, and elimination was completed within 5 days.
        Average absorption per hour was 1.6, 2.3 and 2.5% of the dose in the first,
        Kinetics                                                                              51
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<pre>  second and third 4 h period after application, respectively (Bucks et al. in WHO
  1994).13
      In the study of DiVincenzo et al. (1984), groups of male Sprague-Dawley rats
  were orally administered 14C-labelled hydroquinone at single doses of 3, 30, or
  200 mg/kg bw by gavage. In addition, one group of rats was given 4 daily doses
  of 200 mg/kg bw unlabelled hydroquinone followed by a single dose of 200
  mg/kg bw 14C-labelled hydroquinone by gavage. Radioactivity was widely
  distributed throughout the tissues with higher concentrations in the liver and
  kidneys. Less than 2% remained in the carcass after 96 h. The distribution did not
  change with repeated dosing.48
      Fox et al. (1986, cited in OECD 199633) administered 14C-labelled
  hydroquinone to groups of F344 rats (males) at a single dose of 50 mg/kg bw by
  oral gavage, or by intravenous injection, or by intratracheal instillation. Of some
  groups, blood was collected for up to 8 h and other groups were euthanized at 10,
  20, 40, 60, 120, and 480 h. Absorption was rapid (t1/2 after gavage administration
  was approximately 1 min). Distribution was similar for the three routes. Initially,
  radioactivity in the blood was associated with the plasma, but these levels
  declined rapidly in the first hour. After 4 h, 64% of the radioactivity was
  associated with the erythrocytes.31
      English et al. (1988, cited in OECD 199633; English and Deisinger 200549),
  administered 14C-labelled hydroquinone to groups of F344 rats (males and
  females) at single oral doses of 25 or 350 mg/kg bw by gavage, or after 14
  repeated oral doses of unlabelled hydroquinone. In addition, some groups of
  animals were dermally treated (occlusive) with 14C-labelled hydroquinone at
  levels of 25 or 150 mg/kg bw for 24 h. After oral administration, hydroquinone
  was rapidly absorbed. Less than 1% remained in the tissues. The liver and
  kidneys contained the highest concentrations, with females generally having
  higher concentrations than males. After dermal application 61-71% of the dose
  was recovered from the application site immediately after dosing. Blood
  concentrations after topical application were generally below the limit of
  detection. After 168 h, the skin at the application site contained 0.1-2% of the
  total radioactivity. Approximately 2-13% of the total radioactivity was associated
  with the tissues and carcass.33
  benzoquinone
  Few data on absorption and distribution of benzoquinone are available. Benzo-
  quinone is reported to be readily absorbed from the gastrointestinal tract and
2 Hydroquinone and benzoquinone
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<pre>    after subcutaneous injection (species not specified).9,50 However, no quantitative
    information was available.
5.2 Biotransformation
    Since hydroquinone and benzoquinone can be converted into each other, the
    description of biotransformation of these compounds is combined.
    hydroquinone and benzoquinone
    In Figure 2, a comprehensive metabolic scheme for hydroquinone and
    benzoquinone is given. The Figure shows the enzyme-mediated conversions
    between hydroquinone and benzoquinone and the intermediate semiquinone.
    Furthermore, the conjugation reactions, the process of redox cycling resulting in
    formation of ROS, and the covalent binding to macromolecules such as proteins
    (haemoglobin) and DNA are depicted.
    Oxidation and reduction reactions
    The oxidation of hydroquinone into semiquinone and subsequently into
    benzoquinone can be exerted by cytochrome P450 (CYP) and various
    peroxidases (e.g., prostaglandin H synthase and myeloperoxidase).51,52
        In turn, benzoquinone can be reduced to hydroquinone via a one-electron
    reduction by enzymes such as CYP, cytochrome P450 reductase, ubiquinone
    oxidoreductase, xanthine oxidoreductase, and cytochrome b5 reductase.
    Additionally, reduction of benzoquinone to hydroquinone via a two-electron
    reduction can be catalysed by the flavoproteins NAD(P)H-quinone
    oxidoreductases (NQO1 and NQO2). NQO1 and NQO2 are cytosolic proteins
    that catalyze metabolic reduction of quinones and their derivatives to protect
    cells against redox-cycling and oxidative stress53, as demonstrated by the
    observation that over-expression of NQO1 in CHO cells protects cells from the
    toxicity of benzoquinone.54
    Conjugation reactions
    Hydroquinone can be conjugated with sulphate or glucuronic acid. Quantit-
    atively these conjugations are the most important reactions: after single oral
    administration of hydroquinone to rats, approximately 65-85% was excreted in
    the urine as glucuronide- and sulfate conjugates within 8 h (see Section 5.3).
    Kinetics                                                                           53
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<pre>         Semiquinone and benzoquinone can be conjugated with glutathione (GSH),
    both spontaneously as well as mediated by glutathione S-transferases (GSTs) to
    form mono-, di-, or tri-glutathione-conjugates (depicted as Q-SG1,2,3 in Figure 2,
    in which Q is the quinone moiety, SG is the conjugated glutathione, and 1,2,3
    indicates the number of conjugated glutathione moieties). The glutathione
    conjugates can be further metabolized by γ-glutamyl transpeptidase (γ-GT) and
    subsequently by dipeptidase to cysteine (cys-) conjugates; finally, N-acetylation
    yields the corresponding mercapturic acids.
5.3 Elimination
    hydroquinone
    For an overview of studies performed on elimination see Annex F-1.
         In the study of Fox et al. (1986, cited in OECD 199633) with F344 rats,
    elimination of hydroquinone was rapid. Analysis of plasma indicated rapid
    metabolism of hydroquinone; only 1% of the total radioactivity in plasma
    ultrafiltrate was unaltered hydroquinone. Glucuronide and glutathione
    conjugates of hydroquinone were detected 40 min after oral gavage
    administration.33
         In the study of DiVincenzo et al. (1984; see Section 5.1) with male rats
    administered 14C-labelled hydroquinone, the primary route of elimination was
    the urine (92-99%) with 87% of the total radioactivity recovered excreted in the
    first 24 h. The faeces contained 2-4% of the total radioactivity recovered. Less
    than 1% of the dose was expired as CO2. Only 1% of the urinary metabolites was
    unchanged hydroquinone; the majority was identified as glucuronide conjugates
    with a smaller portion as sulphate conjugates. After repeated oral dosing 72%
    was glucuronide and 23% sulphate conjugate, while after a single oral dose this
    was 56% and 43%, respectively.48
         In the study of English et al. (1988, cited in OECD 199633; English and
    Deisinger 200549; see Section 5.1), elimination followed a biphasic character and
    occurred within the first 8 hrs after oral administration to F344 rats. Dose-related
    differences were observed, suggesting that elimination processes are saturated at
    high-dose levels. Hydroquinone was excreted mainly (> 86%) in the form of
    water soluble metabolites via the urine; 1-2% was excreted in the faeces.
    Approximately 45-53% of the urinary metabolites consisted of glucuronide
    conjugates and 19-33% of sulphate conjugates. A mercapturic acid conjugate
    was also identified at levels of 4.7% and 2.3% of the dose in females and in
    males, respectively. Less than 3% of the dose was excreted as the parent
 4  Hydroquinone and benzoquinone
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<pre>    compound. After dermal application only 15-18% of the total radioactivity was
    recovered in the urine with 2-4% in the feces, 168 h after dosing.33
        In the study of Hill et al. (1993) hydroquinone (1.8 mmol/kg, ip) was
    administered to acivicin*-pretreated male Sprague-Dawley rats. Five S-
    conjugates of hydroquinone were identified in bile, and one S-conjugate was
    identified in urine. The major biliary S-conjugate identified was 2-(GS-S-
    yl)HQ** (18 µmol/4 h). Additional biliary metabolites were, 2,5-bis(GS-S-yl)HQ
    (2 µmol/4 h), 2,6-bis(GS-S-yl)HQ (0.7 µmol/4 h), and 2,3,5-tris(GS-S-yl)HQ
    (1.2 µmol/4 h). 2-(N-acetylcystein-S-yl)HQ was the only urinary thioether
    metabolite identified (11.4 µmol/4 h). The quantity of S-conjugates excreted in
    urine and bile within 4 h of hydroquinone administration was 34.3 ± 4.5 µmol/4
    hr (4.3 ± 1.1% of dose).41
        In the study of Lau et al. (1996), male F344 rats were administered 14C-
    labelled hydroquinone either 1.8 mmol/kg bw by gavage alone or after 14 daily
    doses of unlabelled hydroquinone. The metabolites detected in urine were
    hydroquinone-glucuronide, hydroquinone-sulphate, hydroquinone-mercapturate,
    2-(GS-S-yl)HQ, 2,5-bis(GS-S-yl)HQ, 2,6-bis(GS-S-yl)HQ, and 2,3,5-tris(GS-S-
    yl)HQ. Subchronic administration of hydroquinone appeared to increase the rate
    and extent by which hydroquinone was metabolized to its thioethers.55
    benzoquinone
    Few data on elimination of benzoquinone are available. Benzoquinone is
    reported to be excreted partly unchanged and partly as hydroquinone, the major
    proportion of which is eliminated as conjugates.9 However, no quantitative
    information was available.
5.4 Physiologically based pharmacokinetic models
    hydroquinone
    A physiologically based pharmacokinetic (PBPK) model for hydroquinone was
    described by Corley et al. (2000)56 and refined by Poet et al. (2010)57 to include a
    description of dermal exposure.
    Acivicin is an irreverible inhibitor of γ-glutamyltranspeptidase.
 *  GS: conjugated glutathione; HQ: hydroquinone.
    Kinetics                                                                             55
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<pre>    benzoquinone
    PBPK models for benzoquinone are not available.
5.5 Summary
    hydroquinone
    Various absorption, distribution, metabolism and excretion studies on
    hydroquinone were performed in rats. No information is available on kinetics of
    hydroquinone after inhalation. Hydroquinone absorption via the skin was
    determined and classified as “slow” (0.5-1 µg/cm2/h) but may be more rapid with
    vehicles such as alcohols. After oral administration or intratracheal instillation,
    hydroquinone was rapidly and extensively absorbed. Distribution was similar for
    administration via gavage, intravenous injection and intratracheal instillation.
        Hydroquinone can be oxidized via semiquinone to benzoquinone by various
    enzymes. The reverse reaction can also occur both spontaneously and
    enzymatically mediated.
        Hydroquinone can be conjugated with sulphate and glucuronic acid resulting
    in the respective conjugates, which are excreted via the urine. Benzoquinone and
    semiquinone can also be conjugated with glutathione, resulting in mono-, di-,
    and tri-glutathione conjugates which are detectable in the bile. The glutathione
    conjugates can be further metabolised to cysteine conjugates and mercapturic
    acids.
        The primary route of elimination is via the urine (> 85%) in the form of water
    soluble metabolites. The major urinary metabolites are glucuronide conjugates
    (45-56%) and sulphate conjugates (19-43%). Mercapturic acids are present at
    lower levels (< 5%). Only a small fraction (about 1-3%) of the urinary
    metabolites consists of the parent compound.
    benzoquinone
    No information is available on kinetics of benzoquinone after inhalation.
    Benzoquinone is reported to be readily absorbed from the gastrointestinal tract
    and subcutaneous tissue. It is excreted partly unchanged and partly as hydro-
    quinone, the major proportion of which is eliminated as conjugates. However, no
    quantitative information was available.
 6  Hydroquinone and benzoquinone
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<pre>igure 2 Biotransformation of hydroquinone and benzoquinone (modified from DeCaprio 199914 and English et al. 1994b58).
           Kinetics                                                                                                 57
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<pre>8 Hydroquinone and benzoquinone</pre>

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<pre> hapter 6
        Mechanisms of action
        The molecular mechanisms of quinone toxicity are well described by O’Brien
        (1991).59 Specific mechanistic studies are summarized in Annex F-8.
6.1     Bioactivation
        Potentially several pathways may be involved in toxicity induced by
        occupational exposure to hydroquinone and benzoquinone:
        • Production of reactive oxygen species (ROS)
        • Inhibition of topoisomerase II
        • Macromolecular binding of benzoquinone and semiquinone.
        ROS production
        As described in Section 5.2, the conversions between benzoquinone, hydro-
        quinone and the semiquinone (SQ) can occur both spontaneously and enzym-
        atically (see Figure 2). Once the semiquinone is formed, redox cycling can occur
        in the presence of molecular oxygen, resulting in ROS, including superoxide
        anion (O2ֿ˙) and H2O2.60
            ROS potentially may cause lipid peroxidation and membrane damage,
        cytotoxicity, DNA damage, mutagenicity, and carcinogenicity.60 Benzoquinone
        and hydroquinone have been demonstrated to induce double strand breaks when
        incubated with naked DNA in a cell free system. The absence or presence of
        Mechanisms of action                                                             59
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<pre>  various protective compounds (glutathione, trolox*, etc.) and enzymes (catalase,
  superoxide dismutase) in these studies resulted in clear differences in effects
  which indicated that free radicals may play a role.17,61
       Also the glutathione conjugates of benzoquinone, particularly 2,6-bis
  (GS-S-yl)HQ, and 2,3,5-tris(GS-S-yl)HQ, are capable of redox cycling and have
  been demonstrated to be efficient generators of ROS.62 The latter has been
  shown to induce a mutation spectrum in isolated DNA that was consistent with a
  OH˙-induced mutation spectrum.14,60
  Inhibition of topoisomerase II
  The enzyme topoisomerasse II is essential for the maintenance of proper
  chromosome structure and segregation. Several studies have shown that
  hydroquinone and benzoquinone inhibit in vitro the funtionality of
  topoisomerase II and enhance DNA cleavage (Lindsey et al. 200563; Smith
  201064). In vitro, the presence of glutathione protected topoisomerase II from
  inhibition (Chen and Eastmond 199565). Bioactivation of hydroquinone by
  peroxidase to benzoquinone enhanced topoisomerase II inhibion (Eastmond et al.
  2005).66 In a cell-free system benzoquinone is a more potent inhibitor of
  topoisomerase II than hydroquinone is (Hutt and Kalf 199667; Baker et al.
  200168).
  Macromolecular binding
  Unlike hydroquinone, the reactive benzoquinone and the unstable semiquinone
  are capable of direct covalent binding to macromolecules to form DNA- and
  protein adducts in vitro.16,60 It was observed that hydroquinone in the presence of
  prostaglandin H synthase was converted to reactive metabolite(s) that irrever-
  sibly bind to DNA and sulphydryl groups. Additionally, benzoquinone appeared
  to have been formed61, implying that it might have been one of the reactive
  metabolites.
       Benzoquinone has been demonstrated to alkylate isolated, naked thymus
  DNA to form exocyclic DNA adducts.69 The adducts formed have been iden-
  tified as (3’-hydroxy)-3,N4-benzetheno-2’-deoxycytidine-3’-phosphate, (3’-
  hydroxy)-1,N6-benzetheno-2’-deoxyadenosine-3’-phosphate, and (3’-hydroxy)-
  ‘Trolox’ is the trade name for 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-
  soluble derivative of vitamin E. Like vitamin E it is an antioxidant, and is used in biological or
  biochemical applications to reduce oxidative stress or damage.
0 Hydroquinone and benzoquinone
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<pre>1,N2-benzetheno-2’-deoxyguanosine-3’-phosphate.70 This type of adduct can be
excised by DNA repair mechanisms, as was proven in in vitro studies by using
HeLa cell extracts.69 However, the DNA adduct formed in HL-60 cells and
human bone marrow in vitro did not correspond to any of the principal adducts
formed in naked DNA reacted with benzoquinone. This suggests that cellular
environment modifies the DNA adduct formation by benzoquinone. The same
DNA adducts were detected if HL-60 cells and human bone marrow in vitro
were exposed to hydroquinone.70,71
    After oxidation to the corresponding quinones the glutathione conjugates are
also capable of direct covalent binding to macromolecules.16,60
    Potentially, macromolecular binding may also cause membrane damage,
lipid peroxidation, cytotoxicity, DNA damage, mutagenicity, and carcino-
genicity.60,72 Benzoquinone can bind to critical thiol groups of tubulin resulting
in inhibition of microtubule formation. As a consequence, the formation of a
functional spindle apparatus in the mitotic cell may be disturbed, leading to
abnormal chromosome segregation and aneuploidy.73
    DNA alkylation by benzoquinone (and hydroquinone upon its oxidation to
benzoquinone) has been demonstrated in vitro only. The existence of such DNA-
adducts in vivo has not yet been proven74,75, although several attempts have been
undertaken to demonstrate these.
Mechanistic studies on hydroquinone-induced kidney toxicity
Prostaglandin H synthase is selectively located in the kidney medulla, and γ-GT
activity is found in the brush border membrane of proximal tubular cells.76 In
Section 5.2 (and also above) it has already been mentioned that prostaglandin H
synthase is capable of activating hydroquinone to form reactive metabolites that
might be benzoquinone and/or semiquinone.
    γ-GT is involved in the further metabolism of the glutathione conjugates of
hydro- and benzoquinone.
    The role of γ-GT in the hydroquinone-induced kidney toxicity was investiged
in the study of Peters et al. (1997) (see Annex F-8). The study showed that
hydroquinone was selectively toxic to the proximal tubular cells of the male rat
kidney, and causes injury at the junction of the medullary rays and outer strips of
the outer medulla.77 In these regions, prostaglandin H synthase and γ-GT
activities are high. They also showed that inhibition of γ-GT resulted in a
significant reduction of hydroquinone-induced nephrotoxicity77, indicating that
γ-GT plays a role in the hydroquinone-induced nephrotoxicity.
Mechanisms of action                                                                61
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<pre>        Administration of 2,3,5-tris(GS-S-yl)HQ to rats caused hyperplasia in the
    necrotic proximal tubular cells in kidneys of F344 rats (Peters et al. 1997)77 and
    of Eker rats (Lau et al. 2001)78, corresponding to the γ-GT region specificity in
    the kidney. The conjugate was about 600 times more potent in inducing
    nephrotoxicity than hydroquinone77, indicating that this might be a major part of
    the mechanism of the hydroquinone-mediated nephrotoxicity.
        In the study of Hill et al. (1994) the potency of inducing nephrotoxicity of
    2,3,5-tris(GS-S-yl)HQ and 2-(GS-S-yl)HQ were compared in the in situ perfused
    rat kidney. The compound 2,3,5-tris(GS-S-yl)HQ was considerably more potent
    than 2-(GS-S-yl)HQ, probably due to the fact that the metabolites derived from
    the former are more reactive.79 Apparently the damage to the kidneys is
    selectively induced in the areas where enzymes are located involved in the
    activation of hydroquinone. Key enzymes in the hydroquinone-induced
    nephrotoxicity are likely to be γ-GT and prostaglandin H synthase.
        In order to study the mechanism of induction of kidney neoplasms by
    hydroquinone, English et al. (1994a,b) studied both hydroquinone-induced
    covalent DNA binding and cell proliferation in kidneys of F344 rats
    administered hydroquinone by gavage at levels of 2.5, 25, 50 mg/kg bw, 5
    days/week, for 6 weeks. No DNA-adducts were observed80, but a dose level of
    50 mg/kg bw hydroquinone resulted in cell proliferation in the kidney, and
    excretion of urinary enzymes indicative of proximal tubule damage, though only
    in males.58 They also observed that this latter effect did not occur in F344
    females and in Sprague-Dawley rats. Therefore, a F344 rat-specific nongeno-
    toxic etiology of tumours in the kidney was suggested, with hydroquinone-
    induced cell proliferation secondary to cytotoxicity.
        In the study of Peters et al. (1997)77, hydroquinone was found to induce
    hyperplasia in the necrotic proximal tubular cells in kidneys of F344 rat (see
    above). The hyperplasic regions were at the same location at which tumours
    ultimately developed (Jeong et al. 1999).60 Thus the hydroquinone-induced
    nephrocarcinogenesis appears to be linked to the increased cell proliferation as a
    response to the hydroquinone-induced nephrotoxicity and cell necrosis.
6.2 Detoxification
    Detoxicification of ROS formed due to redox cycling between hydro- and
    benzoquinone and their glutathione conjugates can be accomplished by the cell’s
    cytoprotective mechanisms consisting of antioxidants, scavenging enzymes and
    repair processes.
 2  Hydroquinone and benzoquinone
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<pre>    Reduction of the reactive benzoquinone to the less reactive hydroquinone via
    two-electron reduction is catalysed by NQO1 and NQO2 (see Section 5.2).54
    Further detoxification of hydroquinone may proceed via conjugation with
    sulphate or glucuronide (see Figure 2).
6.3 Summary
    Hydroquinone can be detoxified by sulphation and glucuronidation. Benzo-
    quinone can be converted to hydroquinone via two-electron reduction by NQO1
    and NQO2.
        Redox cycling between hydro- and benzoquinone as well as of their
    glutathione conjugates can lead to the generation of ROS, which potentially
    causes lipid peroxidation, membrane damage, cytotoxicity, DNA damage,
    mutagenicity, and carcinogenicity. The cytoprotective mechanisms of the cell
    (antioxidants, scavenging enzymes, repair processes) counteract the effects of
    ROS production. The net effect of free radicals on cellular function thus depends
    on the balance between radical production and the capacity of these cyto-
    protective systems.
        Benzoquinone, and to a lesser extent hydroquinone, are in vitro inhibitors of
    topoisomerase II, an enzyme responsible for proper chromosome structure and
    segregation.
        The reactive benzoquinone, the unstable semiquinone and the quinones of
    their glutathione conjugates are capable of direct covalent binding to macro-
    molecules such as proteins and DNA. This results in protein- and DNA-adducts,
    and may consequently result in cytotoxicity and/or genotoxicity. The binding of
    benzoquinone to tubulin may result in the inhibition of microtubule formation.
        DNA-alkylation by benzoquinone (and hydroquinone upon its oxidation to
    benzoquinone) has been demonstrated in vitro only; despite several attempts, the
    existence of such DNA-adducts in vivo has not yet been proven, probably
    because of extensive biotransformation.
        Mechanistic studies regarding the hydroquinone-induced kidney toxicity
    showed that the induction of kidney adenomas by hydroquinone is not
    accompanied by any covalent binding to DNA, but is apparently associated with
    local cytotoxicity and necrosis in proximal tubular cells, indicating that hydro-
    quinone induced nephrocarcinogenesis is likely linked to increased cell
    proliferation.
        Overall, the balance of the various metabolic pathways involved, as well as
    other local physiological conditions (e.g., pH) determine the toxicological
    outcome of exposure to either hydroquinone or benzoquinone.
    Mechanisms of action                                                              63
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<pre>4 Hydroquinone and benzoquinone</pre>

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<pre> hapter 7
        Effects
        The mechanism of action of hydroquinone and benzoquinone, with the
        semiquinone as intermediate, is rather complex (see Chapter 6), resulting in a
        number of target sites/organs. Various enzymes are influencing the ultimate
        toxicity of hydro- and benzoquinone, both by increasing and decreasing the
        reactivity/toxic potential. Consequently, the vulnerability of a particular tissue
        for hydro- and benzoquinone depends on the levels of these enzymes.
            In this Chapter, hydroquinone and benzoquinone are described separately.
        However, due to the possible interconversion between them, observations made
        for one compound might also be relevant for the other. The toxicological
        properties of both compounds may be similar in a qualitative sense (i.e. inducing
        the same toxicological endpoint via the same mechanism of action). Whether
        toxicity induced by one of both will also be induced by the other, will be
        dependent on aspects as exposure and local physiological conditions, such as pH
        and enzyme levels.
7.1     Observations in humans
        The studies on effects of hydro- and benzoquinone in humans are summarized in
        Annex E.
        Effects                                                                            65
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<pre>7.1.1 Irritation and sensitization
      hydroquinone
      Sterner et al. (1947; cited in ACGIH 2008)1 reported that occupational exposure
      to hydroquinone dust and benzoquinone vapour caused eye irritation, photo-
      phobia, lacrimation, and corneal ulceration, with no serious cases appearing from
      exposures of duration shorter than five years. Benzoquinone was believed to be
      the chief causative agent, although hydroquinone dust was suspected as a
      contributory cause. Hydroquinone dust concentrations and benzoquinone vapour
      concentrations were reported to vary between 1 and 55 mg/m3, and 0.04 and 14
      mg/m3, respectively.
           Anderson and Oglesby (1958, cited in ACGIH 2008)1 found corneal changes
      consisting of changes in the curvature of the lens, long after (occupational)
      exposure had ceased and after the staining and pigmentation of the cornea had
      disappeared. They concluded that that the corneal changes observed were caused
      by benzoquinone vapour or hydroquinone dust. However, exposure levels were
      not reported.
           Hydroquinone can be considered as a common contact allergen, and sources
      of non-occupational exposure are for example rubber products in which hydro-
      quinone is present as preservative.81 Allergic contact dermatitis represents a
      delayed (type IV) hypersensitivity reaction (which is distinguished from irritant
      contact dermatitis).
           Cross-reactions between chemicals may occur if they share similar functional
      groups critical to the formation of complete allergens (hapten + carrier protein).
      A cross-reactor of hydroquinone is resorcinol and hydroquinone is a cross-
      reactor of methyl hydroxybenzoate and phenol.81
      benzoquinone
      Local cutaneous effects of exposure to benzoquinone include discolouration,
      severe irritation, erythema, swelling, and the formation of papules and vesicles.6
      Prolonged contact can lead to necrosis. Vapour condensing on the eyes can
      produce serious disturbance in vision (Anderson and Oglesby 1958, Fasset 1960,
      cited in ACGIH 2001a).2 Sterner et al. (1947) reported that the vapour of
      benzoquinone and the dust of hydroquinone, arising in the manufacture of
      hydroquinone, produced characteristic ocular injuries in workers. The injuries
      developed gradually over a period of years with no serious cases appearing from
 6    Hydroquinone and benzoquinone
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<pre>      exposures of durations shorter than 5 years. Benzoquinone was believed to be the
      chief causative agent, although hydroquinone dust was suspected as a
      contributory cause. Hydroquinone dust concentrations and benzoquinone vapour
      concentrations were reported to vary between 1 and 55 mg/m3, and 0.04 and 14
      mg/m3, respectively (Sterner et al. 1947, cited in ACGIH 2001a, 2008).1,2
7.1.2 Acute and short-term toxicity
      hydroquinone
      No information on effects on humans after acute or short-term exposure to
      hydroquinone was found, other than the effects mentioned in Section 7.1.1.
      benzoquinone
      No information on effects on humans after acute or short-term exposure to
      benzoquinone was found, other than the effects mentioned in Section 7.1.1.
7.1.3 Long-term toxicity
      hydroquinone
      Friedlander et al. (1982)82 reported a cohort mortality and cancer incidence study
      of 478 workers (7162 person-years of follow-up) engaged in colour printing and
      processing at nine laboratories in the continental USA during the period 1964-
      1976. Six job activities were combined within the processing definition:
      chemical mix, analytical laboratory, film processing, film take-off, print
      processing and print take-off, but in only one of these (film processing) was
      hydroquinone identified as an occupational exposure. A single industrial hygiene
      measurement of hydroquinone indicated a concentration range and annual mean
      time-weighed average of < 0.01 mg/m3 air. The control populations were (1) two
      separate groups of employees with the same company not defined as processors
      for mortality, and (2) the up-state New York population for cancer incidence.
      There were 36 deaths (12 from malignancies) observed, giving standardized
      mortality ratios (SMRs) well below 1.0 for all mortalities and about 1.0 for most
      malignancies. Overall, there were 7 cases of cancer and usually the standardized
      incidence ratios (SIRs) were either below or about 1.0. The only exception was
      central nervous system tumours, but this was based on just two cases. Given the
      mixed population observed, only some of whom appear to have been exposed to
      Effects                                                                            67
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<pre>  hydroquinone, and the level of characterisation of exposure to this chemical, it is
  considered that this study is not informative with regard to the carcinogenicity of
  hydroquinone.67
      Pifer et al. (1995)45 reported a cohort mortality study of 879 workers (22,895
  person-years of follow-up) at a Tennessee (USA) plant in which hydroquinone
  was manufactured and used over several decades. Job history records were
  linked to extensive industrial hygiene data and expertise to estimate cumulative
  exposure to hydroquinone. Average hydroquinone dust levels ranged from 0.1 to
  6.0 mg/m3, with levels over 2 mg/m3 for most of the period of operation of the
  plant. Mean employment duration was 13.7 years and mean follow-up from first
  exposure was 26.8 years. Relative risk estimates (SMRs) for this cohort were
  derived by comparison with the general population of Tennessee as well as with
  an occupational cohort not exposed to hydroquinone (a plant of the same
  company, located in New York State). The SMR for all causes of death combined
  (n=168) was significantly below 1.0, as was the SMR for all cancers combined
  (n=33). Only two sites, colon (n=5) and lung (n=14) had more than three
  observed cases. Most site-specific SMRs were well below 1.0. The results were
  similar for both comparison populations. The dose-response analyses of selected
  cancer sites did not reveal any meaningful trend of heterogeneity.45 The
  International Agency for Research on Cancer (IARC) noted in 1999 that the
  numbers of individual cancer sites were small and the power to detect effects was
  weak, and that this cohort had systematically lower SMRs than the comparison
  industrial cohort (IARC 1999a).8
      Nielsen et al. (1996)83 carried out a cohort incidence study among 837
  Danish lithographers born between 1933 and 1942 and registered with the
  Danish Union of Lithographers in 1947 or later. Questionnaires were sent to
  cohort members in 1989 to obtain information on job exposures; usable
  responses were received from 620 workers. About one-quarter of the cohort
  members reported working regularly with hydroquinone for photographic
  development. The entire cohort was traced in the Danish Cancer Registry from
  1947 to 1989. Relative risk estimates (SIRs) for this cohort were derived by
  comparison with the general population of Denmark. There were a total of 24
  cancers registered, giving an SIR of 0.9. For no site except skin were there more
  than three cases. Five cases of malignant melanoma occurred, with 1.5 expected
  (SIR 3.4, 95% confidence interval (CI) 1.2-7.5). Among these five, two had
  reportedly been exposed to photochemicals (such as hydroquinone). In the study
  it was not possible to distinguish between the carcinogenic effects of the
  exposure to pigments, dyes, and organic solvents.83 It must be noted that the
  power to detect effects was weak considering the co-exposure with other
8 Hydroquinone and benzoquinone
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<pre>photochemicals and the unestablished exposure to hydroquinone of lithographers
suffering from malignant melanomas.
     Fryzek et al. (2005)84 reported a retrospective cohort mortality study of 2,624
workers engaged in motion picture film processing at a California (USA)
laboratory that is the oldest continuously operating laboratory of this type in the
world. All workers were employed for at least 3 months between January 1960
and December 2000. There were 54,462 person-years of follow up with 666
observed deaths (hourly workers, 44,019 person-years with 561 deaths;
administrative workers, 10,444 person-years with 105 deaths. Thirty three
percent of hourly workers and 19% of administrative workers had worked 10
years or more. The SMR ± 95% CI for all causes of death combined among
hourly and administrative workers, respectively, were 1.1 (1.0-1.2) and 1.1 (0.9-
1.3), and for all malignancies combined, 1.0 (0.9-1.2) based on 135 deaths, and
1.2 (0.8-1.7) based on 30 deaths. In most instances the SMRs for individual
malignancies were < 1.0. Borderline significant excesses of non-Hodgkin
lymphoma were observed among hourly workers, SMR = 2.2 (1.0-4.0) based on
10 cases and borderline significant excesses of malignancies of the respiratory
system were observed among all workers combined, SMR = 1.3 (1.0-1.6) based
on 62 cases. However, there was no exposure-relationship for these malignancies
if duration of employment was used as an indicator of exposure. Also no
information on cigarette smoking habits of personal was available, so no
adjustment for this potential confounder was possible. In the case of non-
Hodgkin lymphoma, there was no simple relationship with either duration of
employment or year of first employment for hourly workers as a group. As a
minimum, 75 chemicals were identified as being used on film production since
1960, including hydroquinone which was used in film development. Only three
air sampling data were collected for hydroquinone. One sample was taken before
1981 and was below the detection limit. Two samples taken after 1981 contained
0.014 and 0.052 mg/m3, respectively.84
benzoquinone
Chronic (long-term) inhalation exposure to benzoquinone (exposure levels not
reported) of humans resulted in visual disturbances; chronic dermal contact
caused skin ulceration.6
     No systemic effects were observed in the study of Sterner et al. (1947, cited
in ACGIH 2001a, 2008).1,2 Since this publication, a large number of clinical and
environmental studies were performed on workers in plants where benzoquinone
(and hydroquinone) were produced. These studies confirmed the findings of
Effects                                                                              69
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<pre>      Sterner et al. that no systemic effects arose at a level of 0.44 mg/m3 (0.1 ppm)
      benzoquinone vapour during a period of 5 years (Fasset 1960, cited in ACGIH
      2001a).2
           No information is available on the reproductive, developmental, or
      carcinogenic effects of benzoquinone in humans. No epidemiological data of
      benzoquinone were available.
7.2   Animal experiments
7.2.1 Irritation and sensitization
      The studies on irritation and sensitization of hydroquinone and benzoquinone in
      animals are summarized in Annex F-2. Relevant studies are described in more
      detail below.
      hydroquinone
      Eye irritation
      There are several data on eye irritation of hydroquinone. Hydroquinone in
      aqueous solution, e.g., in tears, is oxidized by air, forming a brown coloured
      substance partly due to conversion to benzoquinone.13
           In a study performed by the Eastman Kodak Company (1971) several
      crystals of hydroquinone powder were placed into the tight eye of two rabbits.
      The treated eye of one animal was washed, while the treated eye of the other
      animal was unwashed. Irritation was scored at 1, 24, 48 h and 14 days after
      treatment. Slight erythrema of the palpebra developed after 1 h in both washed
      and unwashed eyes. Eythrema of the nictitating membrane was also evident in
      the unwashed eye at 1 h. By 24 h, the washed eye appeared normal, but the
      unwashed eye continued to demonstrate slight erythrema of the palpebra, orbital,
      and nictitating membranes. Erythrema of the nictitating membranes persisted to
      48 h after instillation but was not observed 14 days after treatment (Eastman
      Kodak Company 1971, cited in OECD 1996).33
           In the study of Ferraris de Gaspare (1949), the effect of light on
      hydroquinone induced eye irritation was studied in rabbits. Hydroquinone (pure
      substance) was applied daily, for 2-4 months, to the eyes of rabbits which were,
      respectively, kept in the dark, in sunlight, in normal light, irradiated with UV
      light, or pre-sensitized with haematoporphyrin and then kept under either
      reduced light or sunlight. Most rabbits developed pigmentation, first in the
 0    Hydroquinone and benzoquinone
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<pre>conjunctiva and then in the cornea. Degenerative alterations of the corneal
parenchyma were also observed. Pigment formation appeared earlier in animals
exposed to light. Older animals seemed more prone to develop pigment than
younger ones. Pigment was deposited in albino rabbit eyes as well as in those of
rabbits with normal pigmentation (Ferraris de Gaspare 1949, cited in WHO
1994).13
     Following an injection of 0.1 ml of a solution of hydroquinone (0.012-0.05
mol/L) into the cornea of rabbits, the resultant reaction was graded 5 out of the
possible maximum of 100 (Hughes 1948, cited in WHO 1994).13
     In guinea pigs, hydroquinone (1-3 mg pure substance instilled into the eyes
twice daily for 9 weeks) caused immediate but transient irritation. During the
second day of application a slight corneal opacity was observed in some animals,
and on the third day opacity to varying degrees occurred in most of the animals.
Ulcers appeared in two animals. The eyes had fully recovered 3 days after
cessation of treatment.13
     In dogs, hydroquinone (2-5 mg pure substance) instilled twice daily (5 days
per week for 9 weeks) into the eyes caused immediate but transient irritation and
lacrimation. Opacity of the cornea, lacrimation and redness of the conjunctiva
were produced within 4 days, but ulcers were not observed. The eyes returned to
normal within two days after cessation of treatment (Dreyer 1940, cited in WHO
1994).13
Skin irritation
Hydroquinone in concentrations up to 0.1% in water was not found to be irritant
when administered intracutaneously to female guinea pigs.85
     Bleehen et al. (1968)86 observed skin irritation in black guinea pigs after non-
occlusive, topical applications of creams containing 5% or 10% hydroquinone, 5
days/week for one month (surface treated not indicated). No irritation was seen at
levels of 3% hydroquinone.86
     In the study of Maibach and Patrick (1989)13 male and female black guinea
pigs were administered hydroquinone in a hydrophilic ointment at concentrations
of 0.1, 1.0, and 5.0% via non-occlusive, topical application (surface treated not
indicated) for 5 days/week for 13 weeks. The lowest concentration caused
marginal irritation, and the medium concentration resulted in a slight to marginal
irritation in 30% of the animals (mainly females). Moderate to severe irritation
and severe ulcerated inflammatory responses occurred at the highest
concentration (Maibach and Patrick 1989, cited in WHO 1994).13
Effects                                                                               71
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<pre>  Depigmentation
  In consistence with its application in skin bleaching creams (see Section 3.2.2)
  and its mechanism of action of hypo-pigmentation (see Section 6.1),
  depigmentation of the skin was observed in different skin irritation studies.
  Bleehen et al. (1968)86 reported weak to moderate depigmentation of the skin of
  black guinea pigs after topical, dermal application of creams containing 1-10%
  hydroquinone, once daily, five times per week for one month.86 In the study of
  Jimbow et al. (1974)87, depigmentation in the epilated skin of 24 black guinea
  pigs (males and females) after topical applications of hydroquinone was
  observed. Creams containing 2 or 5% hydroquinone in an oil-water emulsion
  were applied daily, 6 days per week, for 3 weeks. The depigmentation was first
  seen within 8-10 days and was greatest between 14 and 20 days. It was more
  marked at the higher concentration. Inflammatory changes and thickening of the
  epidermis were also reported. When hydroquinone was applied topically for
  three weeks, biopsy specimens showed that it had caused a marked reduction
  both in the numbers of melanised melanosomes in the cells and the number of
  actively functioning melanocytes.73
      Depigmentation of the skin was also observed in the study of Maibach and
  Patrick (198913; study details are outlined in the Section on skin irritation above).
  At the highest concentration, approximately 40% of the animals dosed showed
  moderate depigmentation of the skin (females only). At the medium
  concentration, weak depigmentation was observed in the females (not in males),
  and at the lowest concentration no depigmentation effects were seen. At the
  highest concentration, hypopigmentation was noticed in 80-100% of the animals
  in all dose groups (Maibach and Patrick 1989, cited in WHO 1994).13
  Sensitization
  Several sensitization studies on guinea pigs with hydroquinone have been
  reported. The methods and results are summarized in Annex F-2.
      In the sensitization study of Rajka and Blohm (1970)85, the induction and
  challenge were performed by injecting 0.1 ml 0.001% hydroquinone solution. At
  challenge 4/18 animals had a positive sensitization reaction, which was
  considered a “weak” sensitivity reaction. A challenge with 0.001% p-phenyl-
  enediamine or benzoquinone after a hydroquinone induction resulted in a
  positive reaction in 6/18 and 9/18 animals, respectively. After an induction with
  0.001% p-phenylenediamine and a challenge with hydroquinone, 4/20 animals
  were sensitized.85
2 Hydroquinone and benzoquinone
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<pre>     Goodwin et al. (1981)88, compared three guinea pig sensitization procedures.
Sensitization induced by hydroquinone was “strong” when assayed by the
Magnusson and Kligman maximization test, “moderate” by the single injection
adjuvant test and “weak” by the modified Draize procedure.88
     Hydroquinone was found to be a “moderate” sensitizer in female guinea pigs
in both the guinea pig maximization test and Freund's complete adjuvant test as
performed by Van der Walle et al. (1982a,b).89,90 Hydroquinone produced
identical sensitization potentials in the Freund's complete adjuvant test using
induction concentrations of 0.5 mol/litre and 0.45 µmol/litre.
     In 1988, Basketter and Goodwin91 used three sensitization test methods
representing both topical and intradermal routes of application. Groups of 10
guinea pigs were sensitized by using the guinea pig maximization test, a
modified single injection adjuvant test, and a cumulative contact enhancement
test. The sensitization potential of hydroquinone was assessed as “strong”,
“weak”, and “moderate”, respectively, in these three tests. Subsequent cross-
challenges with p-phenylenediamine, sulfanilic acid, and benzoquinone gave
only “restricted evidence” of cross-reactions.90
     In the maximization test of Basketter and Scholes (1992)92, the induction
injections were performed with 2.0% hydroquinone by injection and 1.0% by
patch, followed by a challenge with 0.5% by patch. All guinea pigs (number not
indicated) were sensitized and therefore the compound was classified as
“extreme” sensitizer. In the local lymph node assay (LLNA) hydroquinone was
positive for sensitisation.92 Roberts et al. (2007)93 reported a murine LLNA
threshold (EC3*) of 0.11% for hydroquinone, classifying it as a strong sensitizer.
     The data in the above cited studies clearly indicate that hydroquinone is a
strong dermal sensitizer.
benzoquinone
Eye irritation
No data on eye irritation were available.
EC3 value: the effective concentration for stimulation of a three-fold increase in lymph node cell
proliferation.
Effects                                                                                            73
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<pre>  Skin irritation
  In the study of Rajka and Blohm (1970)85, intracutaneous injections of 0.1%
  benzoquinone solutions in female guinea pigs gave necrotic reactions. At a
  concentration of 0.01%, redness and slight infiltration was observed. Injecting a
  0.001% solution did not result in effects of irritancy.85
  Sensitization
  In the study of Rajka and Blohm (1970)85, the induction and challenge were
  performed by injecting 0.1 ml 0.001% benzoquinone solution. At challenge,
  19/20 animals had a positive sensitization reaction. A challenge with 0.001% p-
  phenylenediamine or hydroquinone after a benzoquinone induction resulted in a
  positive reaction in 5/20 and 1/20 animals, respectively. After an induction with
  0.001% p-phenylenediamine and a challenge with benzoquinone, 16/20 animals
  were sensitised.85
      In a guinea pig maximization test performed by Möllgaard et al. (1990)94, the
  cross-reactivity between p-phenylenediamine and benzoquinone has also been
  established. However, no data are available on concentrations used and the time
  schedule for challenge and induction.94
      In the study of Basketter and Scholes (1992)92, the local lymph node assay
  was compared with the guinea pig maximization test using various compounds
  among which benzoquinone and hydroquinone. In the maximization test the
  induction injections were performed with benzoquinone dissolved in 0.09%
  NaCl aided by acetone if required. For the induction and challenge patch
  benzoquinone was dissolved in acetone with 30% polyethyleneglycol. The
  concentrations were 0.005 (injection induction), 10 (induction patch) and 2.5%
  (challenge patch). All guinea pigs (number not indicated) were sensitized and
  therefore benzoquinone was classified as “extreme” sensitizer.92 In the local
  lymph node assay, groups of 4 mice were treated with benzoquinone by a daily
  topical application of 25 µL of a series of concentrations, from 0.5 to 2.5%
  dissolved in acetone with 20% olive oil on the dorsal surface of each ear for 3
  consecutive days. Four to five days after the first topical application, all mice
  were injected intraveneously with phosphate buffered saline containing 3H-
  methylthymidine. After 5 h the mice were killed and the amount of 3H-methyl-
  thymidine incorporation in the auricular lymph nodes was assayed. Benzo-
  quinone was positive in this assay.92 Roberts et al. (2007)93 reported a murine
  LLNA threshold (EC3) of 0.01% for benzoquinone, classifying it as an extreme
  dermal sensitizer.
4 Hydroquinone and benzoquinone
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<pre>          The data in the above cited studies clearly indicate that benzoquinone is an
      extreme dermal sensitizer.
7.2.2 Acute toxicity
      The acute toxicity studies on hydroquinone and benzoquinone are summarized in
      Annex F-3.
      hydroquinone
      Inhalation LC50 values are not available.
          The dermal LD50 value was determined to exceed 1,000 mg/kg bw in guinea
      pigs.33 The HSG (Health and Safety Guide; IPCS INCHEM) estimated that the
      dermal LD50 value for hydroquinone may exceed 3,800 mg/kg bw in rodents.95
          Oral LD50 values for several animal species ranged between 300 and 1,300
      mg/kg bw33,94 (see also Annex F-3). Acute high-level exposure to hydroquinone
      caused severe effects on the central nervous system (CNS) including hyper-
      excitability, tremor, convulsions, coma, and death. At sublethal doses, these
      effects were reversible. According to the OECD hydroquinone is moderately
      acute toxic via the oral route.33
      benzoquinone
      Inhalation LC50 and dermal LD50 values were not available.
          In the study of Omaye et al. (1980)32 in rats, the oral toxicity of benzo-
      quinone was primarily caused by respiratory impairment. The secondary
      symptoms appeared within 6 hours after dosing. These include ataxia, some loss
      of righting reflex, loss of corneal reflex, depressed respiration and hypothermia.
      Mild to extreme skin blanching was frequently observed and cyanosis was seen
      occasionally. At autopsy, petechial haemorrhages on the lungs, reddened lungs,
      dark livers, darkened spleens and green to black coloured intestines were
      observed.32 In the experiment of Serif and Seymour (1963)97 with mice intra-
      peritoneally administered hydroquinone, the animals experienced writhing,
      paralysis of the hind limbs and cyanosis due to benzoquinone administration.
          In conclusion, oral LD50 values for benzoquinone for rats ranged between
      130 and 165 mg/kg bw (see also Annex F-3). Acute high-level exposure to
      benzoquinone caused severe effects on the central nervous system (CNS)
      including loss of reflexes, depressed respiration and paralysis. Intraperitoneal
      LD50s were 8.5 mg/kg bw for mice97, and 25 mg/kg bw for rats.2
      Effects                                                                            75
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<pre>7.2.3        Sub-acute/sub-chronic toxicity
             The short-term toxicity studies on hydroquinone and benzoquinone are
             summarized in Annex F-4.
             hydroquinone
             No inhalation repeated dose toxicity studies are available. Values of other
             repeated dose studies are presented in Table 7.
 able 7 Subacute/subchronic toxicity of hydroquinone.
 pecies                Exposure         NOAEL              LOAEL                               Reference
                       duration                            Critical effects
Dermal
 at (F344)             14 days          1,920              3,840 mg/kg bw/day                  NTP 198998
                                        mg/kg bw/day       Reduced body weight (m).
 at (F344)             13 weeks         ~74a               > 74 mg/kg bw/day                   David et al. 199899
                                        mg/kg bw/day       No (overt) toxicity.
Mouse (B6C3F1)         14 days          4,800              > 4,800 mg/kg bw/day                NTP 198998
                                        mg/kg bw/day
Oral (all gavage)
 at (Sprague-Dawley) 13 weeks           20                 64 mg/kg bw/day                     Bernard 1988, cited in OECD
                                        mg/kg bw/day       Mild, transient tremors and         199633
                                                           reduced home-cage activity.
 at (F344)             14 days          250                500 mg/kg bw/day                    NTP 198998; Kari et al.
                                        mg/kg bw/day       Mortality, tremors, reduced final 1992100
                                                           mean body weight.
 at (F344)             13 weeks         < 25               25 mg/kg bw/day                     NTP 198998; Kari et al.
                                        mg/kg bw/day       Decreased absolute and relative 1992100
                                                           liver weights (m). Increased
                                                           absolute and relative liver weights
                                                           (f) at doses ≥ 100 mg/kg bw/day.
Mouse (B6C3F1)         14 days          125                250 mg/kg bw/day                    NTP 198998; Kari et al.
                                        mg/kg bw/day       Mortality, tremors, decreased final 1992100
                                                           mean body weight.
Mouse (B6C3F1)         13 weeks         < 25               25 mg/kg bw/day                     NTP 198998; Kari et al.
                                        mg/kg bw/day       Increased absolute and relative     1992100
                                                           liver weights (m).
     Actual dosing was 5.0% oil-in-water emulsion (equivalent to ~74 mg/kg bw/day).
 6           Hydroquinone and benzoquinone
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<pre>Dermal exposure
A fourteen-day dermal study in F344 rats and B6C3F1 mice was conducted in
the National Toxicology Program (NTP 1989)98. During 14 days, 12 doses were
dermally administered, ranging between 240 and 3,840 mg/kg bw/day to rat and
between 300 and 4,800 mg/kg bw/day to mice. The final mean body weight of
male rats that received 3,840 mg/kg bw/day was 6% lower than that of the
vehicle controls. Body weights of female rats at this dose level and of mice at
4,800 mg/kg bw/day were comparable to controls. The final mean body weights
of mice at all dose groups did not deviate from the vehicle controls. There were
no compound-related clinical signs of toxicity in either species.98
    In the study of David (1998)99, prolonged dermal dosing of F344 rats for
5 days/week during 13 weeks with 2.0, 3.5 or 5.0% hydroquinone in an oil-in-
water emulsion cream resulted in minimal to minor dermal irritation, but no
overt toxicity. No adverse effects or compound related effects occurred in organ
weight, clinical pathology, or histopathology.99
Oral exposure
In the programme of NTP (1989)98, 14-days gavage studies were conducted by
administering hydroquinone in corn oil to groups of 5 F344/N rats of each sexe
in doses ranging from 63 to 1,000 mg/kg bw/day, and to groups of 5 B6C3F1
mice of each sexe in doses ranging from 31 to 500 mg/kg bw/day. All rats
receiving 1,000 mg/kg bw/day and 1/5 male and 4/5 female rats receiving 500
mg/kg bw/day died before the end of the 14 days. The final mean body weight of
rats receiving 500 mg/kg bw/day were 9% lower than that of the vehicle controls
for male and 18% lower for females. Compound-related clinical signs in rats
included tremors lasting up to 30 minutes after each dosing at 500 and 1,000
mg/kg bw/day. In the 14-day study with mice, 4/5 male mice and 5/5 female
mice receiving 500 mg/kg bw/day and 3/5 males receiving 250 mg/kg bw/day
died before the end of the study. The final mean body weights of male mice that
received 250 or 125 mg/kg bw/day were 8% or 4% lower than those of the
vehicle controls. Tremors followed by convulsions were seen at 250 and 500
mg/kg bw/day.98
    In 13-weeks gavage studies conducted by NTP (1989)98, doses for F344 rats
and B6C3F1 mice (groups of 10 males and 10 females each) ranged from 25 to
400 mg/kg bw/daty. All rats receiving 400 mg/kg bw and 3/10 female rats
receiving 200 mg/kg bw/day died before the end of the study. The mean body
Effects                                                                          77
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<pre>  weight at necropsy of male rats administered 100 or 200 mg/kg bw was about
  8-9% lower than that of vehicle controls. Mean body weights of vehicle control
  and dosed female rats at necropsy were similar. The liver weight to body weight
  ratios for the dosed male rats were lower than those for vehicle controls. These
  ratios for the three highest dose groups of female rats were significantly greater
  than those for the vehicle controls. Tremors and convulsions were observed after
  dosing in most rats receiving 400 mg/kg bw/day and in several female rats
  receiving 200 mg/kg bw/day. Inflammation and/or epithelial hyperplasia
  (acanthosis) of the forestomach were seen in 4/10 male rats and 1/10 female rats
  receiving 200 mg/kg bw/day. Toxic nephropathy, characterized by tubular cell
  degeneration in the renal cortex, was seen in 7/10 male and 6/10 female rats
  receiving 200 mg/kg bw/day and in 1/10 females receiving 100 mg/kg bw/day.98
  In the studies with mice, 8/10 males and 8/10 females receiving 400 mg/kg
  bw/day and 2/10 male mice receiving 200 mg/kg bw/day died early. Mean body
  weights of dosed and vehicle control mice at necropsy were similar. Liver weight
  to body weight ratios for dosed male mice were significantly greater than those
  for vehicle controls. Ulceration and inflammation, or epithelial hyperplasia of the
  forestomach was found in 3/10 male and 2/10 female mice receiving 400 mg/kg
  bw/day and 1/10 females receiving 200 mg/kg bw/day.98
      No adverse effects on the kidney were reported in Sprague-Dawley rats
  treated by gavage with 0, 20, 64 or 200 mg/kg bw/day hydroquinone in distilled
  water, 5 days per week for 13 weeks (Bernard 1988, cited in OECD 1996;
  Topping et al. 2007).33,101 Neurohistopathology was conducted on various brain
  areas. The kidneys of the high-dose and control male rats were also processed for
  histopathology. No mortality occurred. Mild, transient tremors and reduced
  home-cage activity were observed in the mid- and high-dose groups immediately
  after dosing, with the incidence increased in a dose-dependent manner
  (unfortunately no more detailed quantitative data were reported). No differences
  in body weight, feed consumption, and brain or kidney weight were noted.
  Morphologic lesions associated with the treatment were not observed.
  benzoquinone
  No oral and dermal repeated dose toxicity studies are available. Other repeated
  dose studies are presented in Table 8.
      The studies performed before 1970 were evaluated by the German MAK
  Committee in 1970. Two evaluated studies were summarized in the MAK
  document of 2000.102 No references to the original studies or publications were
  given.
8 Hydroquinone and benzoquinone
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<pre> able 8 Subacute/subchronic toxicity of benzoquinone.
 pecies              Exposure duration NOAEL               LOAEL                        Reference
                                                           Critical effects
nhalation
 at                  4 months,           < 0.27-0.36 mg/m3 0.27-0.36 mg/m3              Anonymous 2, no date (cited in
                     4 h/day                               Thrombopenia                 MAK 2000)100
 ubcutaneous injection
 at                  2.5-5 months,       <7a               7 mg/kg bw/day               Anonymous 1, no date (cited in
                     2 times/week        mg/kg bw/day      Anaemia,                     MAK 2000)100
                                                           methaemoglobinemia,
                                                           decreases in serum albumin
                                                           and serum cholinesterase
                                                           activity
ntraperitoneal injection
Mice (Swiss)         6 weeks,            <2                2 mg/kg bw/day               Rao et al. 1988101
                     6 days/week         mg/kg bw/day      Decreases in red blood cells
                                                           and bone marrow cell counts,
                                                           and haemoglobin content
     Actual dosing scheme was 25 mg/kg bw, twice weekly (equivalent to 7 mg/kg bw/day).
             In the first study, rats were exposed to benzoquinone at levels of 0.27-0.36 or
             2.7-3.6 mg/m3 for 4 h per day, during 4 months by inhalation. All animals
             survived. However, in the high concentration group, weight loss, easy tiredness,
             transient anaemia and thrombopenia was observed. In the low concentration
             group, 2 out of 8 rats suffered from trombopenia.81 Since the original document
             can not be retrieved, the relevance and accuracy of these data can not be
             assessed.
                 In the second study described, 25 mg/kg bw benzoquinone was administered
             twice weekly to rats by subcutaneous injection during 2.5-5 months. This
             resulted in anaemia, methaemoglobinemia, decrease in serum albumin and serum
             cholinesterase activity. Furthermore, changes in heart and liver were observed.102
                 The most recent study on short-term toxicity of benzoquinone was performed
             in 1988 by Rao et al.103 In this study benzoquinone (2 mg/kg bw/day) was
             administered intraperitoneally to Swiss mice 6 days per week for 6 weeks. The
             animals were killed after the last exposure. Benzoquinone produced significant
             decreases in red blood cells and bone marrow cell counts, and haemoglobin
             content, together with relative changes in organ weights. In addition, benzo-
             quinone elicited histopathological injuries in liver, thymus, spleen, kidneys and
             peripheral lymph nodes.103
             Effects                                                                                                79
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<pre>7.2.4         Chronic toxicity and carcinogenicity
              The long-term toxicity and carcinogenicity studies on benzoquinone and
              hydroquinone are summarized in Annex F-5.
              hydroquinone
              Dermal and inhalation carcinogenicity studies are not available. The neoplastic
              and non-neoplastic effects observed in the oral carcinogenicity studies are
              presented in Table 9. In the following, firstly the rat studies, and secondly the
              mouse studies are described.
 able 9 Long-term toxicity of hydroquinone.
 pecies                  Exposure duration    NOAEL                 LOAEL                     Reference
                                                                    Critical effects
 at (F344)               104 weeks            < 350 a               350 mg/kg bw/day          Shibata et al. 1991104
 ral (with diet)                              mg/kg bw/day          Reduced body weight
                                                                    gain (m, f).
                                                                    Higher absolute and
                                                                    relative liver and kidney
                                                                    weights (m).
                                                                    Higher relative kidney
                                                                    weight (f).
                                                                    Chronic nephropathy (m,
                                                                    f).
                                                                    Renal tubular adenomas
                                                                    (m)
 at (F344)               104 weeks,           25                    50 mg/kg bw/day           NTP 198998; Kari et al.
 ral (gavage)            5 days/week          mg/kg bw/day          Lower mean body weight 1992100
                                                                    (m).
                                                                    Renal tubular adenomas
                                                                    (m).
Mouse (B6C3F1)           96 weeks             < 1,050 a             1,050 mg/kg bw/day        Shibata et al. 1991104
 ral (with diet)                              mg/kg bw/day          Reduced body weight
                                                                    gain (m).
                                                                    Higher relative liver and
                                                                    kidney weight (f).
                                                                    Hepatocellular adenomas
                                                                    (m).
Mouse (B6C3F1)           104 weeks,           < 50                  50 mg/kg bw/day           NTP 198998; Kari et al.
 ral (gavage)            5 days/week          mg/kg bw/day          Increased relative liver 1992100
                                                                    weights (m).
                                                                    Hepatocellular adenomas
                                                                    (f).
     rounded figures from 0.8% w/w in diet.
 0            Hydroquinone and benzoquinone
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<pre>Rat studies
In the study of Shibata et al. (1991)104, groups of 28-30 male and 28-30 female
Fischer 344/N rats were given hydroquinone in the diet at concentrations of 0 or
0.8% for 104 weeks (equivalent to 351 and 368 mg/kg bw/day for males and
females, respectively). Body weight gain was decreased in both exposed males
and females. Absolute and relative liver and kidney weight were increased in
exposed males. Relative kidney weights were increased in females. The
neoplastic lesions observed are presented in Table 10. Chronic nephropathy was
more severe in males than in female. In the kidneys of exposed males, the
incidence of tubule hyperplasia was 30/30 and that of adenomas was 14/30 (p =
0.01), compared with 1/30 and 0/30, respectively, in unexposed controls. No
other tumour type was increased by exposure.8,104
Table 10 Histopathological lesions in the 2-year rat diet study with hydroquinone by Shibata et al.
(1991).104
Rat (F344)                      No. of animals with lesions
                                males                              females
Dose (mg/kg bw/day)             Control (30)a   350 b (30)         Control (30)    370 b (30)
Kidneys
Chronic nephropathy+ c          17              10                1               8
++                               0                9**             0               0**
+++                              0                5               0               0
Hyperplasia, papilla             2              11*                0               0
Hyperplasia, tubular             1              30**               0               2
Adenoma                          0              14**               0               0
a    in parentheses no. of animals studied.
b    rounded figures from 0.8% w/w in diet.
c    +, ++ and +++ denote severity of effect.
* and ** denote significant differences from control values (*p<0.05, **p<0.01); for severity for
chronic nephropathy by the two-sided Fischer’s exact test, for severity of non-neoplastic lesions by
the Mann-Whitney test.
In the study conducted by NTP98, groups of 65 F344 rats of each sex were
administered 0, 25, or 50 mg/kg bw hydroquinone in deionised water by gavage,
5 days per week for 104 weeks. Ten rats from each group were killed after 15
months for an interim evaluation.84 In the rats killed at 15 months, the relative
kidney weight of high dose male rats was greater than that for vehicle controls.
The haematocrit value, haemoglobin concentration, and erythrocyte count for
high dose female rats were decreased. Compound-related increased severity of
nephropathy was observed in male rats.98
     In the 2-year study, mean body weight of high dose males was 5-13% lower
than that of vehicle controls after week 73, and that of low dose males was 5-9%
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<pre>  lower than that of vehicle controls after week 89. Mean body weight of dosed
  females was similar to that of vehicle controls throughout the study. The relative
  kidney and liver weights for high dose males were higher than those for vehicle
  controls. No significant differences in survival were observed between any groups
  of rats of either sex after 2 years (male rats: vehicle control, 27/55; low dose,
  18/55; high dose, 18/55; female rats: 40/55; 27/55; 32/55, respect-ively).98,103 The
  neoplastic lesions observed are presented in Table 11. Renal tubular cell adenomas
  developed in 4/55 low-dose (p=0.069) and 8/55 high-dose (p=0.003) males,
  compared with 0/55 controls.83 In exposed females, mono-nuclear cell leukaemia
  developed in 15/55 of the low-dose group (p=0.048) and 22/55 of the high-dose
  group (p=0.003), compared with 9/55 controls. The historical incidence of
  leukaemia for water/vehicle control females was 25±15%.98
  Table 11 Histopathological lesions in the 2-year rat gavage study with hydroquinone by NTP
  (1989).98
  Rat (F344)                                  No. of animals with lesions
                                              males (55)a                       females (55)
  Dose (mg/kg bw/day)                         Control      25         50        Control 25        50
  Kidneys
  • Chronic nephropathy none                   2            3           0
                                minimaal       3            1           3      No treatment related
                                mild          12           12           5*     effects
                                noderate      26           31         15
                                marked        12            8         32
  • Hyperplasia                                0            0           2
  • Adenoma                                    0            4           8*
  Lymphoid system
  • Mononuclear cell leukaemia                No treatment related effects      9         15      22*
  a    in parentheses no. of animals studied.
  *    denotes significant differences (p<0.01); for severity of chronic nephropathy by the two-sided
       Fischer’s exact test, for severity of non-neoplastic lesions by the Mann-Whitney test.
  Hard et al. (1997)105 re-evaluated the renal histopathology of the NTP study in
  rats. They reviewed the grade of chronic progressive nephropathy (CPN) and
  presence of atypical tubular hyperplasia and adenomas; see Table 12. Hydro-
  quinone exposure in males at 50 mg/kg bw/day produced a statistically
  significant increase in the grade of CPN. There was no statistically significant
  difference between low-dose males and controls regarding the CPN grade
  (p= 0.63). At 0, 25 and 50 mg/kg bw/day, 0/44, 4/49 and 15/51 male rats had
  either atypical tubular hyperplasias or adenomas. All were within areas of severe
  or end-stage CPN and were statistically significantly associated with CPN grade.
  Additionally, there was a dose-related increase in profiles believed to represent
  new tubule proliferation within areas of advanced CPN, as well as an apparent
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<pre>expansion of these into unusual complex tubule profiles in end-stage kidneys of
the high-dose male group. Based on their re-evaluation the authors suggested a
mechanism for hydroquinone-related adenoma formation that includes enhance-
ment of the severity of CPN coupled with stimulation of tubular proliferation.
     Hard and Khan reviewed CPN in 2004.106 They argued that CPN is a
spontaneous age-related disease that occurs in high incidence in F344 and
Sprague-Dawley rats, exhibiting a male predisposition. The disease generally
starts at about 2 months of age, when some rats develop basophilic renal tubules
with a thickened basement membrane. Progression involves an increase in
number of tubules affected, tubular degeneration and atrophy, and an ongoing
tubule-cell proliferation. By the time that end-stage is reached, there are virtually
no normal tubules remaining and death from renal failure is highly probable. The
authors stated that this degenerative and regenerative disease is not the result of
any chemical treatment, and it is necessary to distinguish its regenerative aspects
from preneoplasia (atypical hyperplasia) from which adenomas develop.
     Furthermore they expressed the opinion that, although the precise etiology of
CPN and the mechanisms underlying its pathogenesis remain unknown, evidence
is emerging that advanced CPN is a risk factor for a marginal increase in the
background incidence of renal tubular tumours. Reviewing the pathological
entities associated with chronic renal failure in man, the authors finally
concluded that this rodent CPN has no strict human counterpart.106
     McGregor (2007)107 reviewed the human risks of hydroquinone from its
carcinogenic and mutagenic properties including the pathology re-evaluation by
Hard et al (1997).105 His evaluation showed that all renal tubular adenomas and
all cases of renal tubular atypical hyperplasia occurred in areas of severe or end-
stage CPN and that the neoplasms were not otherwise confined to any particular
part of the kidney. The author proposed and evaluated a non-genotoxic mode of
action involving exacerbation of CPN, considering CPN to be a spontaneously
occurring rodent renal disease process.
Mouse studies
In the study of Shibata et al. (1991)104, groups of 28-30 male and 28-30 female
B6C3F1 mice were given hydroquinone in the diet at concentrations of 0 or 0.8%
for 96 weeks (equivalent to 1,046 and 1,486 mg/kg bw/day for males and
females, respectively). Statistically significant reduction in body weight gain was
noted in males. The neoplastic lesions observed are presented in Table 13. No
increase in tumour incidence was found in females. In males the combined
incidence of hepatocellular adenomas and carcinomas was increased to 20/30 in
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<pre>  exposed animals (p<0.05) compared with 13/28 in controls. This increase was
  solely due to an increased number of adenomas: from 6/28 in controls to 14/30 in
  exposed animals.
  Table 12 Re-evaluation (Hard et al. 1997)105 of the microscopic slides for chronic progressive
  nephropathy of the 2-year rat gavage study (NTP 1989).98
  Rat (F344)                                No. of animals with lesions
                                            males                             females
  Dose (mg/kg bw/day)                       Control 25           50           Control 25         50
                                            (44)a     (49)       (51)         (53)     -         (46)
  Kidneys
  Chronic progressive nephropathy
            minimal                          0         2          0           4       -          2
            mild                             0         2          2           6       -          7
            low moderate                     2         2          2 *        15       -         18
            high moderate                   20        20         11          27       -         15
            severe                          20        21         16           1       -          1
            end-stage                        2         2         20           0       -          3
  Atypical.tubule hyperplasia/adenoma        0         4         15*           -       -          -
  a    in parentheses no. of animals graded for chronic progressive nephropathy (CPN).
  * denotes significant differences (for CPN regarding grade of severity): p<0.001 by Χ2 test.
  Table 13 Histopathological lesions in the chronic mouse diet study with hydroquinone by
  Shibata et al. (1991).104
  Mouse (B6C3F1)                         No. of animals with lesions
                                         males                              females
  Dose (mg/kg bw/day)a                   Control (28) b 1,050 (30)          Control (29)    1,490 (30)
  Kidneys
     Hyperplasia, tubular                0                 9**              0                0
     Adenoma                             0                 3                0                0
  Liver
     Hypertrophy                         0               26**               0                3
     Foci of cellular alteration         4               14*                0                2
     Hepatocellular adenoma              6               14*                0                1
     Hepatocellular carcinoma            7                 6                1                0
  Forestomach
     Hyperplasia                         1               11**               3               14**
     Squamous cell carcinoma             0                 1                0                1
  a    rounded figures from 0.8% w/w in diet.
  b    in parentheses no. of animals studied.
  * and ** denote significant differences from control values (*p<0.05, **p<0.01); for severity of
  neoplastic lesions by the two-sided Fischer’s exact test, for severity of non-neoplastic lesions by the
  Mann-Whitney test.
  While no other tumour type was significantly increased, 9/28 and 3/28 males
  showed tubular hyperplasia, and adenomas in the kidney, respectively, against no
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<pre>such lesions in controls; this incidence of hyperplasia was significant (p<0.01).
In addition, significantly increased incidences of hyperplasia in the forestomach
were observed in both sexes.8,105
      In the study conducted by the NTP98, groups of 65 B6C3F1 mice of each sex
were administered 0, 50, or 100 mg/kg by gavage, 5 days per week for 104
weeks. Ten mice from each group were killed after 15 months for an interim
evaluation.98
      In mice killed at 15 months, the relative liver weights for high dose male and
female mice were significantly greater than those for vehicle controls. Lesions
seen in the liver of male mice included increased syncytial cells and diffuse
cytomegaly.98
      In the 2-years study, mean body weights of high dose male mice were 5-8%
lower than those of vehicle controls after week 93, and those of high dose female
mice were 5-14% lower after week 20. Relative liver weights were increased for
all dosed male and high dose female mice. No significant differences in survival
were observed between any groups of mice of either sex after 2 years (male
mice: vehicle control 33/55; low dose 37/54; high dose 36/55; female mice:
37/55; 39/55; 36/55). The neoplastic lesions observed are presented in Table 14.
No increase in tumours was found in exposed males. In females,
Table 14 Histopathological lesions in the 2-year mouse gavage study with hydroquinone by NTP
(1989).98
Mouse (B6C3F1)                         No. of animals with lesions
                                       males (55)a                      females (55)
Dose (mg/kg bw/day)                    Control 50 b         100 c       Control 50           100
Liver
    Basophilic focus                    2          5        11           2          6          3
    Hepatocellular adenoma              9         21        20           2         15*       12*
    Hepatocellular carcinoma           13         11         7           1          2          2
    Adenoma or carcinoma               20         29        25           3         16*       13*
Thyroid
    Hyperplasia                         5         15*       19*         13         47*       45*
    Adenoma                             2          1         2           3          5          6
    Carcinoma                           0          0         0           0          0          1
    Adenoma or carcinoma                2          1         2           3          5          7
a     in parentheses no. of animals studied.
b     53 animals in this group.
c     54 animals in this group.
* denotes significant differences from control values (p <0.01); for severity of neoplastic lesions by
the by two-sided Fischer’s exact test, for severity of non-neoplastic lesions by the Mann-Whitney
test.
combined incidences of hepatocellular adenomas and carcinomas found were
3/55 in controls, 17/55 in the low-dose group (p=0.001) and 14/55 in the high-
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<pre>  dose group (p=0.005), i.e. entirely due to an increase in the incidence of
  adenomas (2/55 controls, 15/55 low-dose group (p=0.001) and 12/55 high-dose
  group (p=0.005)), and without a clear dose-response relationship.8,98 In addition,
  significantly increased incidences of hyperplasia in the thyroid were observed in
  both sexes.98,100
       NTP concluded from their observations in the carcinogenicity studies in rats
  and mice that there was hydroquinone-related carcinogenicity in male rats, as
  indicated by increased incidences of tubular adenomas of the kidney, in female
  rats as shown by increases in mononuclear cell leukaemia, and in female mice
  based on increases in hepatocellular neoplasms, mainly adenomas. There was no
  evidence of carcinogenicity in male mice.98
       IARC (1999a) noted that the incidences of leukaemia in the exposed female
  rats were within the historical control range.8 Additionally, it is noted that the
  increase in kidney tumours entirely concerns adenomas, i.e., in none of these
  studies adenocarcinomas were observed. Also remarkable is the finding that with
  the same B6C3F1 mouse strain NTP (1989)98 found an increased incidence of
  liver tumours in females without any clear effects in males, while Shibata et al.
  (1991)104 found an increased effect in males without any effect in females.
       IARC (1999a) considered these studies and concluded that they provided
  limited evidence for experimental carcinogenicity by hydroquinone.8 EPA has
  not classified hydroquinone for carcinogenicity (EPA 1999).5
  Initiation-promotion studies
  Whysner et al. (1995)108 gave a good overview of initiation-promotion studies
  investigating the potential of hydroquinone to promote carcinogenesis in various
  rat organs (i.e. after administration of organ-specific initiators), such as bladder
  (Miyata et al. 1985, cited in IARC 1999a8; Kurata et al. 1990109), stomach
  (Hirose et al. 1989, cited in IARC 1999a8), liver (Stenius et al. 198947, Okazaki
  et al. 1993110), upper digestive tract (Yamaguchi et al. 1989111, Hasegawa et al.
  1990112), kidney (Okazaki et al. 1993110), and pancreas in hamsters (Maruyama
  et al. 1991113) (see Annex F-8). In some of the studies, the final body weights of
  hydroquinone-treated rats were reduced as compared to controls. Under the
  applied exposure regimens, hydroquinone was unable to increase the incidence
  or multiplicity of tumours, when either given alone or after organ-specific
  initiators.
       Stenius et al. (1989) performed a similar kind of initiation-promotion study
  with lesions considered as preneoplastic, i.e., enzyme-altered (γ-GT) foci (GGT-
  foci) in male Sprague-Dawley rats. Groups of 7-10 rats were given hydroquinone
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<pre>in dietary concentrations of 0, 100 or 200 mg/kg (equivalent with 0 or
approximately 5 or 10 mg/kg bw/day) for six weeks, beginning one week after
partial hepatectomy (PH) and i.p. injection of 300 mg/kg bw N- nitrosodi-
ethylamine (DEN) to initiate liver carcinogenesis. In the group submitted to PH
and fed the high dose of hydroquinone no GGT-foci were induced. Hydro-
quinone after initiation (PH and DEN) increased the multiplicity of foci in the
low-dose group and, though less marked, in the high-dose group. In a second
experiment in which groups of 10 rats received 0 or 1 mg/kg bw hydroquinone
(by oral gavage, five days per week for seven weeks) after initiation of liver
carcinogenesis, no increase in multiplicity of enzyme-altered foci was found, but
an increase of their area (p<0.05), and volume (p<0.001). According to the
authors the results indicate that GSH depletion may act to develop enzyme-
altered foci.47
    In a study of Okazaki et al. (1993), groups of 15 or 20 male Wistar/Crj rats
were given hydroquinone at concentrations of 0 or 0.8% in the diet (equivalent to
350 mg/kg bw/day) for 36 weeks with or without a preceding exposure to 0.1%
N-ethyl-N-hydroxyethylnitrosamine (EHEN) via the dinking water for three
weeks to initiate liver and kidney carcinogenesis. The final body weights of rats
given hydroquinone were lower than the concurrent controls. The relative liver
and kidney weights of rats receiving hydroquinone were higher than those of the
basal diet group. Hydroquinone alone did not induce preneoplastic or neoplastic
liver or kidney lesions. In the kidney, hydroquinone exposure after initiation
increased the multiplicity of renal cell tumours (from 2.58 after initiation to 5.22
per rat; p<0.01) and of microadenomas (from 0.94 after initiation to 2.77 per rat;
p<0.05). The authors concluded that hydroquinone potentially enhances the
second stage of EHEN-induced renal carcinogenesis.110
Summary
Long term oral exposure to hydroquinone resulted exclusively in renal tubular
adenomas in male F344 rats (and in males of one of the mouse studies), and an
increased incidence of hepatocellular tumours in B6C3F1 mice, i.e., in one study
in males, in the other in females: in both cases, however, the increase consisted
entirely of an increase of adenomas only. In addition, significantly increased
incidences of hyperplasia in the thyroids of both sexes were observed in the 2-
years mouse gavage study.
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<pre>  benzoquinone
  No long term inhalation toxicity and carcinogenicity studies are available. Some
  information on long-term non-neoplastic toxicity effects of benzoquinone could
  be derived from the dermal and oral carcinogenicity studies described below.
  Dermal exposure
  In a study of Umeda (1957), 15 Wistar and 9 hybrid strain rats were administered
  benzoquinone dissolved in propylene glycol and injected subcutaneous on the
  back once weekly. The first 53 days, the rats received 0.5 ml of a 10 g/L solution,
  from day 53 to 173 0.5 ml of 2 g/L and from day 173 to 394 0.5 ml of 4 g/L.
  Seventeen rats (about 70%) survived the injection period of 394 days. Three
  fibrosarcomas developed in two rats at the site of injection. No other tumours
  were found. In the control group (18 animals of different strains receiving only
  the vehicle) no tumours were found (Umeda 1957).114 Due to the low number of
  treated animals and the use of different strains, limited conclusions can be drawn.
  Oral exposure
  El-Mofty et al. (1992) investigated the carcinogenic effect of benzoquinone
  using Swiss albino mice. Male (69) and female (65) animals were administered 2
  mg benzoquinone by gavage, twice weekly for 13 months. Within 7 months,
  36% of the males and 43% of the females died. The tumour incidence was
  33.6%; the tumours were characterised as lympholeukaemias* and were located
  in the liver and spleen.115 At the time the first tumours appeared (7-9 months
  after start of exposure), mortality was 36% in males, and 43% in females.
  Because of this high mortality and the absence of any histopathological
  information no conclusion can be drawn whether benzoquinone induced tumours
  via a secondary effect, such as chronic cell and/or organ damage, or via a direct
  genotoxic mechanism.115
  The authors described these lympholeukaemias as follows: `The liver tumors appeared as white
  nodules on the outer surface. Microscopically, the liver tissue was infiltrated with typically
  lymphocytic cells and was diagnosed as lympholeukemia. The nuclei of the neoplastic cells were
  hyperchromatic, and mitotic figures were usually not numerous. The spleen in most cases was much
  larger than normal, and histologically, spleen tumors were diagnosed as lympholeukemia.`
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<pre>           Information on other endpoints than mortality and carcinogenicity was not
      given in the oral and dermal carcinogenicity studies on benzoquinone described
      above.
      Initiation-promotion studies
      In the study of Gwynn and Salaman (1953), groups of 19 albino “S” mice
      received a topical, dermal application of a 0.15% 7,12-dimethylbenzanthracene
      solution in acetone as an initiation treatment. Three weeks later benzoquinone at
      a concentration of 3.6 g/L in acetone was applied weekly for 27 weeks. At the
      end of the study no tumours or hyperplasia of the epidermis were observed.116
           In the study of Monks et al. (1990), groups of 25 female Sencar mice
      received a dermal application of 25 nmol 7,12-dimethylbenzanthracene. Two
      weeks later benzoquinone was applied weekly on a shaved skin area at doses of
      0, 440, 880 or 1,760 nmol/mouse. After 31 weeks of application of
      benzoquinone, no signs of possible promoter capacity had been observed.117
           IARC considered that the available data did not allow a conclusion on the
      carcinogenicity of benzoquinone in animals (IARC 1999b).9 EPA considered the
      results of available animal studies insufficient to evaluate the carcinogenicity of
      benzoquinone (EPA 1992, revised in 2000).6
      Summary
      Benzoquinone was reported to induce lympholeukaemias in liver and spleen in
      mice after gavage administration for 13 months. Due to the high mortality and
      the absence of any histopathological information, this study is considered of too
      limited value to draw conclusions on the carcinogenic potential of benzoquinone.
      The initiation-promotion studies available did not indicate any sign of promoter
      capacity of benzoquinone.
7.2.5 Mutagenicity and genotoxicity
      Mutagenicity and genotoxicity of hydroquinone and benzoquinone were
      extensively and well described by IARC in 1999a,b.8,9 The reviews generated by
      IARC are presented in Annexes H-1 (hydroquinone) and H-2 (benzoquinone).
      Below, IARC data and additional studies found in the literature are summarised.
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<pre>  hydroquinone
  DNA strand breaks / SCEs
  Hydroquinone induced SCEs in vitro in V79 Chinese hamster cells118, in CHO
  cells either with or without exogenous metabolic activation98, and in human
  lymphocytes in the absence of metabolic activation. Hydroquinone tested
  negative in an in vivo SCE test using bone marrow cells.8
  DNA adduct formation
  Covalent binding of hydroquinone to DNA was observed in vitro in various cell
  types.8 However, covalent DNA binding of hydroquinone could not be demon-
  strated in vivo.
  Clastogenic effects
  Hydroquinone tested positive in the in vitro micronuclei test in the absence of
  metabolic activation using embryonic human liver cells, human lymphocytes and
  V79, IEC-17 and 18 cells.8,118 Chromosome aberrations were induced by hydro-
  quinone in vitro in human lymphocytes in the absence of metabolic activation.8,98
      The effect of polymorphism of the glutathione S-transferases GST-M1, GST-
  T1 and GST-P1 on induction of micronuclei (MN) and SCEs was studied by in
  vitro hydroquinone treatment of human lymphocytes isolated from healthy
  volunteers. Hydroquinone induced a significant higher frequency of MN in
  lymphocytes with the GST-M1 null genotype than with GST-M1 present, while
  this effect was not seen for SCEs. Additionally, the other polymorphisms did not
  significantly affect the frequency of MN or SCEs. This suggests that GST-M1 is
  involved in the metabolic fate of hydroquinone and that polymorphisms in GST-
  M1 could be related to inter-individual differences in DNA damage arising from
  the exposure to this compound.74
      Hydroquinone was in vivo weakly positive in the micronucleus test in mouse
  bone-marrow cells and in mouse liver cells in utero.8 Chromosome aberrations
  were induced by hydroquinone in vivo in mouse bone marrow and spermato-
  cytes/spermatogonia.8
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<pre>DNA mutation
Hydroquinone was negative in Salmonella strains TA 97, 98, 100, 1535, and
1537, with or without metabolic activation.8,98 Hydroquinone was mutagenic in
Salmonella strains TA102 and TA104 (strains sensitive for oxidative mutagens).8
Hydroquinone tested positive for genotoxicity in S. cerevisiae without exo-
genous metabolic activation, and induced gene mutations in mouse lymphoma
and Syrian hamster embryo cells in the absence of metabolic activation system.8
Hydroquinone induced gene mutations in mouse L5178Y/TK lymphoma cells in
the presence or absence of metabolic activation.98 Hydroquinone was a potent
inducer of gene mutations (6-thioguanine resistance) in V79 cells.118
    No information on in vivo induction of gene mutations by hydroquinone was
available.
Aneuploidy induction
Aneuploidy was induced in vitro by hydroquinone in Chinese hamster cells,
Syrian hamster embryo cells and human lymphocytes, in the absence of
metabolic activation.8
    Hydroquinone clearly increased the frequencies of hyperploid secondary
spermatocytes, which indicated non-disjunction induction during the first
meiotic division. Concomitantly, hydroquinone induced meiotic delay in primary
and/or secondary spermatocytes.119
    Aneuploidy was also induced in vivo in mouse bone marrow and
spermatocytes.8
Overall, the observations reported above may indicate the potency of hydro-
quinone to (1) induce DNA strand breaks and SCEs, (2) bind covalently to DNA,
(3) induce clastogenic effects both in vitro and in vivo, (4) induce DNA muta-
tions in vitro, and (5) induce aneuploidy both in vitro and in vivo. It should be
noted, however, that generally effects in the in vitro tests were seen only at
relatively high doses.
benzoquinone
DNA strand breaks and SCEs
Benzoquinone was demonstrated to induce double strand breaks in an in vitro
cell-free system, incubated with λ-DNA in the absence and presence of various
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<pre>  anti-oxidants (GSH, trolox, etc.) and anti-oxidant enzymes (catalase, superoxide
  dismutase).17 Benzoquinone induced DNA strand breaks in mouse lymphoma
  cells in vitro in the absence of metabolic activation, and in human lymphocytes
  in the presence of metabolic activation.9 No in vivo data on DNA strand break
  induction by benzoquinone are available. Benzoquinone tested positive in an in
  vitro SCE test using human lymphocytes.9
  DNA adduct formation
  In the study of Gut et al. (1996) it was demonstrated that benzoquinone reacted
  spontaneously with DNA in vitro to form DNA-adducts.16 No additional studies
  were found that show the potency of benzoquinone to form DNA-adducts.
  Clastogenic effects
  Benzoquinone increased the frequency of micronuclei formation in V79 cells.118
  Benzoquinone also tested positive in various micronucleus tests in vitro in the
  absence of metabolic activation, and weakly positive in vivo.9
  DNA mutation
  Benzoquinone tested negative for genotoxicity in S. typhimurium.118 The IARC
  data on DNA mutations induced in S. typhimurium by benzoquinone were
  inconclusive.9 Benzoquinone was a potent inducer of gene mutations in V79
  cells9, as it produced an approximately 100-fold increase in the frequency of
  6-thioguanine-resistant cells at a concentration of 1 µmol/L.118 Benzoquinone
  was positive in vivo in the TK-assay.9,120
  Aneuploidy induction
  No information on aneuploidy induction by benzoquinone was available.
  Overall, it can be concluded that benzoquinone has the potency of inducing DNA
  strand breaks in vitro, may induce clastogenic effects both in vitro and in vivo,
  and has the potency to induce DNA mutations. In one study it was demonstrated
  that benzoquinone was able to form DNA-adducts in vitro. It should be noted,
  however, that generally effects in the in vitro tests were seen only at relatively
  high doses.
2 Hydroquinone and benzoquinone
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<pre>7.2.6 Evaluation of the carcinogenicity and genotoxicity data
      In this Chapter, the evaluation of the Subcommittee on the Classification of
      carcinogenic substances is summarized. The full evaluation of the Subcommittee
      is reproduced in Annex I.
      hydroquinone
      Only oral carcinogenicity studies with hydroquinone were performed (NTP
      198998, Kari et al. 1992100, Shibata et al. 1991104); no carcinogenicity studies via
      dermal or inhalation exposure were located. Carcinogenicity studies (via diet and
      gavage) with B6C3F1 mice and F344 rats on hydroquinone are available for
      evaluation of this endpoint. The following tumour responses were observed:
      In gavage studies with two test doses:
      • Kidney adenomas in male rats
      • Mononuclear cell leukaemia in female rats
      • Hepatocellular adenomas in female mice.
      In diet studies with a single test dose:
      • Kidney adenomas in male rats
      • Hepatocellular adenomas in male mice.
      As to the hepatocellular adenomas, the Subcommittee noted that they were not
      consistently seen, and did not progress to malignant carcinomas. Because of this
      inconsistent picture and the susceptibility of the B6C3F1 mouse to develop liver
      tumours, the Subcommittee considered that the induction of these hepatocellular
      adenomas is not relevant for humans.
           The renal tubular adenomas were found in both the diet and the gavage study,
      but in male rats only. These benign tumours did not progress to malignant ones.
      Severe kidney damage appeared to be a prerequisite for the development of the
      adenomas. This chronic progressive nephropathy is a spontaneous disease in,
      particularly, ageing male rats. The Subcommittee was of the opinion that
      hydroquinone plays an indirect role in the development of the adenomas by
      exacerbating already existing chronic progressive nephropathy and enhancing
      proliferative aspects of this disease process. Because of this indirect role
      involving exacerbation of an already existing spontaneous disease which is
      further common in rats but without a human counterpart, the Subcommittee was
      Effects                                                                              93
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<pre>  of the opinion that the induction of these renal tubular adenomas in male rats is
  not relevant for humans.
      The mononuclear cell leukaemias were found at statistically significantly
  increased incidences in the high-dose female rats in only one study. The
  Subcommittee noted that especially F344 rats show high background incidences
  of this tumour type while the rate in controls is very variable and depends upon
  several factors such as e.g., sex, vehicle and diet. In view of the susceptibility of
  these rats to develop monuclear cell leukaemias, the very variable background
  incidences, and the fact these leukaemias were seen in one sex in one study only,
  the Subcommittee considered that the induction of these mononuclear cell
  leukaemias in female F344 rats is not relevant for humans.
      The Subcommittee concluded that hydroquinone is a genotoxic compound.
  In vitro, it induced amongst others, gene mutations, micronuclei, chromosomal
  aberrations, aneuploidy, sister chromatid exchanges, DNA single strand breaks
  and oxidative DNA damage in mammalian cell systems. In in vivo tests, mainly
  performed in mice using intraperitoneal injection, hydroquinone produced
  increases in the incidences of micronuclei and chromosomal aberrations in bone
  marrow and of chromosomal aberrations in spermatogonia. In vitro, hydro-
  quinone is an inhibitor of topoisomerase II. Redox-cycling of hydroquinone/
  benzoquinone generates ROS. Based on the literature it is quite likely that the
  mode of action of hydroquinone’s genotoxicity is by a non-stochastic
  mechanism.
  benzoquinone
  One dermal (Umeda1957) and one oral study (El-Mofty et al. 1992) addressed
  the (potential) carcinogenicity of benzoquinone. The Subcommittee was of the
  opinion that because of several serious flaws in design and reporting, these
  studies cannot be used to evaluate the carcinogenicity of benzoquinone.
  Recommendation for classification
  hydroquinone
  The Subcommittee concluded that there are two valid carcinogenicity studies in
  which hydroquinone was orally administered to rats and mice. The findings were
  inconsistent and concerned tumour types that were considered not relevant for
  humans. Based on the available information, the Subcommittee was of the
4 Hydroquinone and benzoquinone
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<pre>            opinion that the data are as yet insufficient to evaluate the carcinogenic
            properties of hydroquinone (Health Council category 3; see Annex J).
            benzoquinone
            The Subcommittee concluded that there are no valid carcinogenicity studies,
            Based on the available information, the Subcommittee was of the opinion that the
            data are as yet insufficient to evaluate the carcinogenic properties of
            benzoquinone (Health Council category 3; see Annex J).
            Approach for deriving health-based occupational exposure limits
            Overall, the Committee adopts the conclusions and recommendation of the
            Subcommittee on the Classification of carcinogenic substances. Since a non-
            stochastic genotoxic mechanism is considered responsible for the
            carcinogenicity of hydroquinone in animals (probably ROS-generation, while
            also inhibition of topoisomerase II may play a role), the Committee concludes
            that with respect to hydroquinone a threshold approach is appropriate for
            deriving a health-based occupational exposure limit for hydroquinone.
                Furthermore the Committee is of the opinion that the line of reasoning
            described above for hydroquinone is in principle also valid for benzoquinone.
7.2.7       Reproduction toxicity (fertility and development)
            hydroquinone
            Two 2-generation studies were conducted in rats. The results are summarized in
            Annex F-7 and in Table 15.
 able 15 Reproductive toxicity studies with hydroquinone.
 pecies                   NOEL                             LOEL (mg/kg bw/day) /                    Reference
                          (mg/kg bw/day)                   Critical effects
 at (Sprague-Dawley)      15        General toxicity       50          General toxicity F0 and F1:  Blacker et al.
                          150       Parental reprotoxicity             mild, transient tremors and  1993121
                          150       Toxicity F1                        decreased body weight
                                                           >150        Reproduction and fertility:
                                                                       no treatment related effects
 at (Sprague-Dawley)      15        General toxicity       50          General toxicity F0 and F1:  Schroeder 1989
                          150       Parental reprotoxicity             transient tremors            (unpublished), cited
                          150       Reprotoxicity F1                                                in OECD 199633
            Effects                                                                                                   95
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<pre>  In the study of Blacker et al. (1993), performed according to guideline OECD
  416, hydroquinone was administered in an aqueous solution by gavage at doses
  of 0, 15, 50, or 150 mg/kg bw/day. F0 and F1 parental animals were dosed daily
  for at least 10 weeks prior to cohabitation, during cohabitation, and until
  scheduled termination. At all dose levels tested, no adverse effects were
  observed on feed consumption, survival, or reproductive parameters for the F0 or
  F1 parental animals. Mild, transient tremors were observed shortly after dosing
  at 150 mg/kg bw/day in several F0 and F1 parental animals and in a single F0
  male at 50 mg/kg bw/day. These tremors occurred infrequently and were consi-
  dered to be due to an acute stimulatory effect of hydroquinone on the nervous
  system. Body weights for F0 and F1 parental females were similar between all
  dose groups throughout the study. Body weights for F0 parental males were also
  comparable to those of control throughout the study. Statistically significant
  differences in body weights were noted for the F1 parental males in the 50 and
  150 mg/kg bw/day dose groups at several intervals during the pre-mating,
  mating, and post-mating periods. No treatment-related effects on pup weight, sex
  distribution, or survival were noted for pups of either generation. Upon post-
  mortem examination, no treatment-related gross lesions were observed in either
  the F0 or F1 parental animals or their weanlings. Histopathologic examination of
  reproductive tissues and pituitary glands from high-dose F0 and F1 parental
  animals did not reveal any changes related to treatment with hydroquinone. Thus
  no adverse effects on reproduction or fertility were observed in either generation
  at any dose level, and the results of the present study indicate that hydroquinone
  is not a selective reproductive toxicant. The NOAELs for general and repro-
  ductive toxicity were 15 and 150 mg/kg bw/day, respectively.121
      In the study of Schroeder (1989; guideline OECD 416), Sprague-Dawley rats
  were treated with 0, 15, 50 or 150 mg/kg bw/day hydroquinone in distilled water
  by gavage. In the maternal and paternal generation, no adverse effects of treat-
  ment were evident from mortality, body weight, or feed consumption at the 15
  and 50 mg/kg bw/day dose levels. Mild and transient tremors were observed in
  one male at 50 mg/kg bw/day, and in several males and females in the 150 mg/kg
  bw dose groups. Concerning reproductive toxicity evaluated both in parental
  animals (fertility gestation, reproductive organ toxicity, etc) and in offspring
  (weights of litter, post-natal growth, viability, etc.), no adverse effects were seen
  in body weight, sex distribution, survival, or gross pathology of pups delivered
  (Schroeder 1989, cited in OECD 1996).33
      Teratogenicity/developmental studies were conducted in rats and rabbits. The
  results are summarized in Annex F-7 and Table 16.
6 Hydroquinone and benzoquinone
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<pre>    In the study of Krasavage et al. (1992; guideline OECD 414), pregnant rats
(COBS-CD-BR) were given 0, 30, 100, or 300 mg/kg bw/day hydroquinone by
gavage on gestation days (GD) 6 to 16. Maternal effects included a slight, but
significant (p ≤ 0.05) reduction in body weight gain and feed consumption for the
300 mg/kg bw/day dams. Reproductive indices, i.e., pregnancy rate, numbers of
corpora lutea, implantation sites, viable foetuses, and early and late resorptions,
foetal sex ratio, pre- and post-implantation losses, and gravid uterine weights,
were not affected by treatment. A slightly reduced (p ≤ 0.05) mean foetal body
weight seen at the 300 mg/kg bw/day dose level was associated with the slightly
reduced body weight gain seen for the dams at this dose level. Gross external,
internal soft tissue, and skeletal examinations of the foetuses revealed no
treatment-related malformations. The incidences of gross external variations
(small hematomas) and internal soft tissue variations (dilated renal pelvis,
hydronephrosis, and hydrourether) in the treated litters were not statistically
different from the control incidences. Skeletal variations (delayed ossification of
membranous skull bones, hyoid bone, thoracic centre 1-3, sacral arches 3 and 4,
and bilobed thoracic centre 9-13) were seen with similar frequency in the control
and treated groups. A statistically significant increase in the incidence of total
common vertebral variations seen at the 300 mg/kg bw/day dose level was not
considered toxicologically significant (according to the authors, analyses of
individual skeletal veriations, including individual vertebral variations, and
statistical analysis of the the total number of fetuses with a skeletal alteration
indicated no significant effects on skeletal development in the treated groups
compared with the control group). The authors concluded that hydroquinone was
not selectively toxic to the developing rat conceptus and, thus, appears not to
have the properties of a developmental toxicant. The NOAEL for both maternal
and developmental toxicity was 100 mg/kg bw/day, whereas 300 mg/kg bw/day
was the LOAEL (maternal and developmental).122
    In the study of Murphy et al. (1992; guideline OECD 414), hydroquinone
was administered to pregnant New Zealand White rabbits (18 mated per dose
group) in aqueous solution (0, 25, 75, or 150 mg/kg bw/day) by gavage on GDs 6
to 18. Caesarean sections were performed on GD 30. Doses of 75 and 150 mg/kg
bw/day adversely affected feed consumption and/or body weight of dams during
the treatment period. At these doses, however, treatment-related effects were not
evident from physical observations, liver and kidney weights, premature delivery
incidence, and caesarean sectioning data. The NOAEL for maternal toxicity was
25 mg/kg bw/day. In the 150 mg/kg bw/day dose group, total incidences of
external, visceral, and skeletal findings for fetuses did not differ statistically from
controls. Slight but statistically insignificant increases were found, however, in
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<pre>           the incidences of ocular and minor skeletal malformations (micro-ophthalmia,
           vertebral/rib defects, angulated hyoid arch) on both a per foetus basis and a per
           litter basis. Under the conditions of this study, the authors concluded that
           hydroquinone at 150 mg/kg bw/day produced minimal developmental alterations
           in the presence of maternal toxicity. The NOAEL for developmental toxicity was
           75 mg/kg bw/day.123
 able 16 Developmental toxicity studies with hydroquinone.
 pecies                 NOAEL                          LOAEL (mg/kg bw/day) /                          Reference
                        (mg/kg bw/day)                 Critical effects
Rat (COBS-CD-BR)        100        General toxicity    300        Maternal: decreased food consumption Krasavage et al.
                        300        Pregnancy/litter               and body weight gain                 1992122
                        100        Fetal data          300        Fetuses: decreased body weight
 abbit (NZW)             25        General toxicity     75        Maternal: decreased food consumption Murphy et al.
                        150        Pregnancy/litter                                                    1992123
                         75        Fetal data          150        Developmental: minimal developmental
                                                                  alterations
           benzoquinone
           No data were available.
7.2.8      Immunotoxicological and haematological effects
           hydroquinone
           The cytotoxic as well as immunosuppressive potential of hydroquinone was
           demonstrated in in vivo (ip injection in mice) and in vitro studies that showed a
           reduced cellularity of bone marrow and spleen, and an inhibition of maturation of
           B-lymphocytes.
                Wierda and Irons (1982) investigated the in vivo toxicity of hydroquinone
           toward the development of polyclonal, plaque-forming cells (PC-PFC) from
           progenitor B lymphocytes. Dextran sulfate (DxS), lipopolysaccharide (LPS), or
           the two mitogens combined (DxS + LPS) were used to induce proliferation and
           maturation of these progenitors to PC-PFC. Groups of 4 C57BL/6 mice were
           exposed to 2 daily doses of hydroquinone (100 mg/kg bw), administered either iv
           or ip for 3 consecutive days. Spleen and marrow cells were harvested for culture
           one day later. Hydroquinone (100 mg/kg bw) was cytotoxic to spleen cells and
           reduced bone marrow cellularity. It reduced the frequency of PC-PFC developed
           from the spleens and bone marrows of treated mice. These experiments
           demonstrate the immunotoxic potential of hydroquinone in vivo through the
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<pre>reduction of progenitor B lymphocytes124, though via routes irrelevant for the
occupational situation.
    Several studies have been performed on the effects of hydroquinone on
various haemopoietic cells aiming at unravelling the mechanism of myelo-
toxicity induced by benzene, of which hydroquinone and benzoquinone are
metabolites. Most of the studies performed were on haemopoietic cells in vitro.
However, the relevance for the in vivo situation is difficult to estimate.
Observations were:
• Macrophage peroxidase catalyzed the metabolic oxidation of hydroquinone
    to benzoquinone, and benzoquinone and/or its semiquinone intermediate did
    bind to protein and cysteine (Schlosser 1989).125
• Hydroquinone may exert many of its haematopoietic effects via synergism
    with granulocyte-macrophage colony stimulating factor (GM-CSF) in TF-1
    erythroleukaemia cells, in human CD34+ bone marrow cells, and in
    granulocyte-macrophage colonies from mouse lineage restricted marrow
    cells117 (Irons et al. 1992, cited in Klaassen 1996).79
• Hydroquinone inhibited mitogen-stimulated activation of both T and B
    lymphocytes (Guy et al. 1991).128
• Hydroquinone inhibited the production of many cytokines including IL-1,
    IL-2, IFN, and TNF (Kin et al. 1989; Post et al. 1985; Cheung et al., cited in
    Pyatt et al. 2000).126
• Hydroquinone directly inhibited T cells by blocking the activity of the
    transcription factor nuclear factor kB (NFkB) (Pyatt et al. 2000).126
• Hydroquinone disrupted normal NFkB activity in B cells, which accounted
    for the observed loss in B cell function and maturation, and thus may play a
    role in mediating the immunosuppressive effects of hydroquinone on B cells
    (Patt et al. 1988, cited in Pyatt et al. 2000).126
• Hydroquinone induced an increase in total granulocyte-macrophage colony
    forming cells (GM-CFC) in the bone marrow of mice in vivo (and in vitro;
    see for the in vitro part Annex G-3) (Henschler et al. 1996).127
• Hydroquinone was demonstrated to decrease 59Fe uptake in erythrocytes
    after administration to Swiss albino mice. Hydroquinone was 25-100 times
    less potent than benzoquinone (Guy et al. 1991).128
• Hydroquinone appeared to activate circulating neutrophils of mice in vivo,
    thereby impairing further stimulatory responses (Ribeiro et al. 2011).129
• Hydroquinone induced apoptosis of neutrophils and eosinophils isolated
    from the blood of healthy humans, through the caspase 9/3-dependent
    pathway and the increased ROS production (Yang et al. 2011).130
Effects                                                                            99
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<pre>      Myelotoxic effects were not observed in a long-term bioassay on rodents and are,
      therefore, not considered to be critical effects of hydroquinone-induced toxicity.
      benzoquinone
      Reactions of benzoquinone with whole blood of both F344 rats and humans
      resulted in linear formation of adducts with haemoglobin and albumin131 (see
      also Section 7.2.3).
          Benzoquinone has been reported to decrease 59Fe uptake in erythrocytes after
      administration to Swiss albino mice128, demonstrating its erythrotoxic potential.
          Benzoquinone has been shown to inhibit Fc receptor-mediated phagocytosis
      in cultured murine peritoneal macrophages. This is likely to be due to disruption
      of filamentous actin via an effect other than the direct alkylation of actin by
      benzoquinone.132
7.2.9 Neurological effects
      hydroquinone
      Acute high-level exposure to hydroquinone causes hyper-excitability tremor,
      convulsions and coma (see Sections 7.2.2 and 7.2.3).
          A 90-day study including neurological examinations was performed by
      Bernard 1988, cited in OECD 199633) and Topping et al. 2007.101 Sprague-
      Dawley rats were administered hydroquinone at dose levels of 0, 20, 64, or 200
      mg/kg bw/day by gavage, 5 days per week for 13 weeks. A functional-
      observational battery was performed. Mild and transient tremors and reduced
      home-cage activity were observed in mid- and high-dose groups immediately
      after dosing, with the incidence increasing in a dose dependent manner, but
      unfortunately no more detailed quantitative data were reported. The results of
      neuropathological examinations were negative. The NOAEL in this study was 20
      mg/kg bw/day. Note that the NOAEL for parental toxicity in the 2-generation
      reproductive study with rats (Blacker et al. 1993121) was 15 mg/kg bw/day, based
      on mild, transient tremors, and decreased body weight; see Section 7.2.7).
      benzoquinone
      Neurological effects were observed in the acute toxicity test described in Section
      7.2.2. Rats treated with benzoquinone experienced ataxia, loss of righting reflex
      and corneal reflex. Mice treated with benzoquinone suffered from writhing and
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<pre>    paralysis of the hind limbs. No long-term neurological studies with
    benzoquinone have been reported.
7.3 Summary and evaluation
    hydroquinone
    No conclusion regarding the carcinogenic properties of hydroquinone could be
    drawn from the epidemiological studies with the substance.
         Hydroquinone was reported to induce skin depigmentation, cornea
    pigmentation and sensitization in humans after occupational exposure. Eye
    irritation, skin irritation, depigmentation and sensitization was also observed in
    animal studies. According to the authors, the results from the studies on
    sensitization showed “weak” to “extreme” sensitization, depending on the
    methods or vehicle used. Cross-reactivity for allergic contact dermatitis exists
    between p-phenylenediamine, hydroquinone and benzoquinone.
         In the acute toxicity studies in animals, various neurological symptoms were
    observed, including hyperexcitability, tremors, convulsions, and coma. The
    lowest oral LD50 was about 300 mg/kg bw in rat. The dermal LD50 may be well
    above 1,000 mg/kg bw. No acute inhalation studies were available.
         Sub-acute toxicity studies with hydroquinone have been performed via
    dermal and oral (gavage) exposure. Reduced body weight was observed after
    dermal application of 3,840 mg/kg bw/day for 14 days. No adverse effects were
    observed at dermal application of 74 mg/kg bw/day for 13 weeks. After oral
    repeated dose administration, hydroquinone induced changes in kidney and liver
    weights of F344 rats and B6C3F1 mice at a level of 25 mg/kg bw/day for 13
    weeks. Toxic nephropathy, characterised by tubular cell degeneration in the renal
    cortex, was seen in 7/10 male and 6/10 female rats receiving of 200 mg/kg bw/
    day and 1 female receiving 100 mg/kg bw/day. However, because the Committee
    does not consider changes in liver and kidney weights to be a adverse effect, a
    NOAEL for toxic nephropathy of 25 mg/kg bw/day can be derived. In Sprague
    Dawley rats, no effects were observed at 20 mg/kg bw/day for 13 weeks. At 64
    mg/kg bw/day, mild, transient tremors and reduced home-cage activity were
    observed.
         With hydroquinone only oral carcinogenicity studies were performed;
    carcinogenicity studies via dermal or inhalation exposure are not available. At
    levels of 50 mg/kg bw/day administered to F344 rats for 104 weeks, reduced
    body weights were observed, and renal tubular adenomas in males and
    mononuclear cell leukaemias in females were found. The Committee does not
    Effects                                                                            101
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<pre>   consider these tumours as relevant (see Section 7.2.6). The NOAEL was set at
   25 mg/kg bw/day.
       In B6C3F1 mice, 50 mg/kg bw/day for 104 weeks induced increased relative
   liver weights in males and hepatocellular adenomas in males and females. Based
   on the considerations of the Subcommittee on the Classification of carcinogenic
   substances, the Committee does not consider these tumours as relevant (see
   Section 7.2.6). No NOAEL was derived in this study.
       In vitro, hydroquinone is an inhibitor of topoisomerase II, an enzyme
   responsible for the maintenance of proper chromosome structure and
   segregation.
       Hydroquinone was negative in Salmonella strains TA 97, 98, 100, 1535, and
   1537, with or without metabolic activation, but positive in strains TA102 and
   TA104, both sensitive for oxidative mutagens. Hydroquinone was able to induce
   DNA strandbreaks, SCEs, gene mutations, chromosomal aberrations and
   micronuclei in mammalian cells, and cell lines in vitro. Numerous in vivo studies
   were performed with hydroquinone, investigating induction of micronuclei,
   chromosomal aberrations or polyploidy in the bone marrow, and chromosomal
   aberrations or hyperploidy in germ cells. All but one of these studies used ip
   administration and most of them were positive. One oral study gave a weakly
   positive result.
       These positive responses may well be explained by the postulated underlying
   mechanisms, demonstrated to occur under in vitro conditions: redox-cycling with
   ROS generation, inhibition of topoisomerase II, and direct covalent binding to
   macromolecules (either directly or after conjugation with glutathione). Although
   this latter capability may underlie the in vitro observed genotoxicity, several
   attempts to demonstrate the in vivo formation of hydroquinone-derived DNA-
   adducts failed.
       Based on the studies on reproduction, the Committee concludes that
   hydroquinone is not a reproductive toxicant. In one of the developmental studies,
   slight (statistically insignificant) malformations were observed. The NOEL for
   developmental toxicity was set at 75 mg/kg bw/day.
       The available data on neurotoxicity showed mild, transient tremors and
   reduced home-cage activity at 64 mg/kg bw/day; the NOAEL for neurotoxicity
   was 20 mg/kg bw/day. The tremors observed in the 2-generation rat reproductive
   study with a NOAEL of 15 and a LOAEL of 50 mg/kg bw/day were also only
   mild and transient.
       Following the Subcommittee on the Classification of carcinogenic
   substances the Committee concludes that there are two valid carcinogenicity
   studies in which hydroquinone was orally administered to rats and mice. The
02 Hydroquinone and benzoquinone
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<pre>findings were inconsistent and concerned tumour types that were considered not
relevant for humans. Based on the available information, the Committee is of the
opinion that the data are as yet insufficient to evaluate the carcinogenic
properties of hydroquinone (category 3; see Annex J).
     The Committee concludes that a threshold approach is suitable for deriving a
health-based occupational exposure limit for hydroquinone.
benzoquinone
Human epidemiological studies with benzoquinone are not available.
     In humans, exposure to high air levels of benzoquinone may result in
irritation of the eyes. Dermal exposure of humans may result in dermatitis and
severe irritation. No systemic effects arose upon occupational exposure of
humans at a level of 0.44 mg/m3 benzoquinone vapour during a period of 5 years.
Based on the results of various animal studies, benzoquinone appeared to be a
skin sensitizer. It induced skin lesion and irritation when injected subcutaneously
in guinea pigs. Cross-reactivity for allergic contact dermatitis exists between p-
phenylenediamine, hydroquinone and benzoquinone.
     In the acute toxicity studies in animals, various neurological symptoms were
observed, including loss of reflexes, writhing and paralysis of the hind limbs.
The lowest oral LD50 in rats was 130-160 mg/kg bw. No acute dermal or
inhalation studies are available.
     Sub-acute toxicity studies with benzoquinone have been performed via
inhalation exposure, and ip and sc injection. The critical effects were all of a
haematological character and included decreases in blood cells,
methaemoglobinaemia and trombopenia. A NOAEL by inhalation in rats was not
established, since effects were already observed at the lowest dose of 0.27-0.36
mg/m3. Since the original document of this 4 months study could not be
retrieved, the relevance and accuracy of these data can not be assessed.
     There are only two very limited carcinogenicity studies. Because of the poor
quality of the studies and the high mortality, no conclusions can be drawn
regarding the potential of benzoquinone to induce carcinogenic effects at
maximum tolerated levels. The initiation-promotion studies indicated no signs of
promoter capacity of benzoquinone. Epidemiological data relevant to the
carcinogenicity of benzoquinone were not available.
     In vitro, benzoquinone is an inhibitor of topoisomerase II, an enzyme
responsible for the maintenance of proper chromosome structure and
segregation.
Effects                                                                             103
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<pre>       Benzoquinone was negative in Salmonella strains TA 98, 1535, and 1537,
   with or without metabolic activation. Benzoquinone was able to induce DNA
   strand breaks, SCEs, gene mutations, and micronuclei in mammalian cells, and
   cell lines in vitro. In vivo micronuclei induction studies – via the oral route –
   were weakly positive, while one dominant lethal test with C3H mice was
   negative.
       Studies on reproduction and developmental toxicity with benzoquinone were
   not available.
       Overall (based on the information available) a NOAEL for benzoquinone
   could not be derived.
       In line with the Subcommittee on the Classification of carcinogenic
   substances the Committee concludes that too little information on carcino-
   genicity of benzoquinone is available, and thus it is as yet not possible to
   evaluate its carcinogenic properties (category 3; see Annex J).
       As is clarified in Sections 2.2 and 6, the mechanism of action of
   benzoquinone and hydroquinone may be similar due to the fact that they are
   metabolites of each other and because conversions between them are part of the
   mechanism of action. Therefore, the Committee adopts the opinion that the line
   of reasoning described above for hydroquinone is also valid for benzoquinone.
04 Hydroquinone and benzoquinone
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<pre> hapter 8
        Existing guidelines, standards and
        evaluations
8.1     General population
        Over-the-counter skin lighteners may contain up to 2% hydroquinone, and
        prescription drugs may contain higher concentrations. A 2% upper limit on
        hydroquinone concentration was set by the South African government in 1980
        and followed by the United Kingdom and USA.13
            In 2002, the European Scientific Committee on Cosmetic Products and Non-
        Food Products intended for Consumers (SCCNFP) concluded that due to very
        low exposure to the consumer hydroquinone is safe for use, within certain
        restrictions and conditions, as technical aids in artificial nail systems.
        Hydroquinone is listed in the “restrictive list”, Annex III - Part 1 (substances
        which cosmetic products must not contain except subject to restrictions and
        conditions) of the European Cosmetics Directive (76/768 as amended). This
        Directive has also been amended in order to allow the use of this substance as a
        technical aid in artificial nail systems. Hydroquinone is allowed at a maximum
        level of 0.02% (after mixing for use). The following warnings must be printed on
        the label: “For professional use only”, “Avoid skin contact”, “Read directions for
        use carefully”. Details are laid down in Directive 2003/83/EC of 24 September
        2003, which was published in issue no. L238/23 of the Official Journal of the
        European Union.
            In 2003 the Cosmetic Ingredient Review (CIR) Expert Panel of the EU
        determined that hydroquinone should not be used in non-drug cosmetic products
        Existing guidelines, standards and evaluations                                     105
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<pre>    that are left on the skin and not immediately rinsed off. The panel drew this
    conclusion based on studies linking hydroquinone to cancer and immune system
    damage that targets bone marrow (EWG 200344).
         No international guideline for hydroquinone in drinking water has been
    established (WHO 1993, cited in IARC 1999a8).
8.2 Working population
    hydroquinone
    Occupational exposure limits
    See Table 17 for occupational exposure limits for hydroquinone previously
    derived by national and international organisations.
         The American Conference of Governmental Industrial Hygienists (ACGIH)
    recommended in 2008 a 8-h TWA (time weighted average) TLV (threshold limit
    value) of 1 mg/m3 stating that this exposure limit will minimize the potential risk to
    workers from eye irritation and eye damage associated with occupational exposure
    to hydroquinone (Sterner et al. 1947; Oglesby et al. 1947, Anderson et al. 1958133,
    cited in ACGIH 20081). Insufficient data were considered available to elaborate a
    TLV-STEL (short-term exposure limit, generally for a period of 15 min).1
         EPA concluded in 1992 (revised in 2000) that no information is available on
    the reproductive or developmental effects of hydroquinone in humans. A slight
    reduction in maternal body weight gain, decreased foetal weight, increased
    resorption rate, and reduced fertility in males have been observed in rats orally
    exposed to hydroquinone via gavage or in the diet. Exposure of rabbits to
    hydroquinone via gavage produced negligible developmental alterations. EPA
    has not established a Reference Concentration (RfC) for hydroquinone. EPA has
    calculated a provisional Reference Dose (RfD) of 0.04 mg/kg bw/day for
    hydroquinone based on haematological effects in humans.5 The provisional RfD
    is a value that has had some form of Agency review but is not in the Integrated
    Risk Information System (IRIS). The provisional RfD is an estimate (with
    uncertainty spanning perhaps an order of magnitude) of a daily oral exposure to
    the human population (including sensitive subgroups) that is likely to be without
    appreciable risk of deleterious noncancer effects during a lifetime. It is not a
    direct estimator of risk but rather a reference point to gauge the potential
    effects. At exposures increasingly greater than the RfD, the potential for adverse
    health effects increases. Lifetime exposure above the RfD does not imply that an
    adverse health effect would necessarily occur.
 06 Hydroquinone and benzoquinone
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<pre>Table 17 Established limits for occupational exposure to hydroquinon in various countries.
Country                         Occupational        Time-weighted     Note b      Reference
Organisation                    exposure limit      average a
                                (mg/m3)
Denmark                         2.0                 8h                OEL         2007
Finland                         0.5                 8h                -           2005
                                2.0                 15 min            STEL
Germany
   AGS c                        -                   -                 -           2007
   DFG d                        -                   -                 -
Norway                          0.5                 8h                -           2007
Sweden                          0.5                 8h                -           2005
                                1.5                 15 min            STEL
UK
   HSE e                        0.5                 8h                MEL         2007
USA
   ACGIH f                      1.0                 8h                TLV         2008
   NIOSH g                      2.0                 15 min            REL         2004
   OSHA h                       2.0                 8h                PEL         2004
Iceland                         0.5                 8h                -           1999
European Union
   SCOEL i                      -                   -                 -           -
a    Time-weighted average (TWA): a maximum mean exposure limit based generally over the
     period of a working day.
b    Abbreviations - OEL: occupational exposure limit; STEL: short-term exposure limit; MEL:
     maximum exposure limit; TLV: threshold limit value; REL: recommended exposure limit; PEL:
     permissible exposure limit.
c    Ausschuss für Gefahrstoffe.
d    Deutsche Forschungsgemeinschaft.
e    Health and Safety Executive.
f    American Conference of Governmental Industrial Hygienists.
g    National Institute for Occupational Safety and Health.
h    Occupational Safety and Health Administration.
i    Scientific Committee on Occupational Exposure Limit Values.
In the UK, the current 8-h TWA MEL (maximum exposure limit) at 0.5 mg/m3 is
based on a HSE review (2001)18 concluding that the key health effect of concern
is genotoxicity. A level of exposure below which there would be no concerns for
this endpoint would not be identified, and therefore the criteria for establishing
an OES (occupational exposure standard) were not met. Hence, in view of the
serious health concerns associated with genotoxicity, a MEL was established. A
STEL was not proposed.
     In Germany, no MAK (Maximale Arbeitsplatz-Konzentration) value has
been derived (2005).102
Existing guidelines, standards and evaluations                                                 107
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<pre>       Up to January 1, 2007, the limit value for occupational exposure to
   hydroquinone in The Netherlands was 2.0 mg/m3; this value has been withdrawn
   due to the introduction of a new system of legal limit values. The value had been
   adopted from the ACGIH 8-h TWA TLV of 2001.134 ACGIH stated that this
   exposure limit will minimize the potential risk to workers from eye injury,
   dermatitis, central nervous system effects, and other systemic effects potentially
   associated with occupational exposure to hydroquinone (ACGIH 2001b134).
   Insufficient data were considered available to recommend skin, sensitization, or
   carcinogenicity notations.
   Classification and labelling
   See Table 18 for classification and labelling of hydroquinone derived by national
   and international organisations.
       In Germany, hydroquinone was labelled for sensitization; carcinogen
   category 2; germ cell mutagen category 3A (last German update in 2003136;
   Greim 1998135). The argumentation was the following: Hydroquinone is
   genotoxic. In mammalian cells in vitro and in vivo it induces micronuclei,
   chromosomal aberrations, DNA single strand breaks, oxidative damage to DNA
   and in vitro also gene mutations and SCEs. In addition it has C-mitotic effects.
   DNA-adducts were detected in vitro, adducts in cellular macromolecules also in
   vivo. In two well-documented carcinogenicity studies with oral administration of
   hydroquinone an increase was found in the incidence of hyperplasia and
   adenomas of the kidneys in male rats and mice; liver adenomas were also
   detected in the mice. In one study thyroid gland hyperplasia and adenomas, and
   forestomach hyperplasia occurred in mice, and in another study mononuclear
   leukaemia was found in female rats. Pathological changes in the blood count and
   damage to the bone marrow were also variously reported in studies with repeated
   administration. A cohort study, which, however, is only of limited meaning due
   Table 18 Classification and labelling of hydroquinone in various countries
   Country            Classification/labelling                  Reference
   Germany            H (skin absorption)                       Greim 1998135;
                      Sh (skin sensitization)                   MAK-Werten 1994, 2000136
                      Group 2 (carcinogenicity)
                      Group 3A (germ cell mutagen)
   USA                Group A3 (carcinogenicity)                ACGIH 20081
   USA                Not classified for carcinogenicity        EPA 1992 (revised 1999)5
   UK                 Not classified for carcinogenicity        HSE 200018
   IARC               Group 3 (carcinogenicity)                 IARC 1999a8
08 Hydroquinone and benzoquinone
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<pre>to the small number of participants, yielded no evidence of hydroquinone-
induced tumours in man. Because of its genotoxicity and the results of
carcinogenicity studies in animals hydroquinone is classified in Category IIIA2
in the “List of MAK and BAT Values”. Hydroquinone induces chromosomal
aberration and hyperploidy in germ cells of male mice and is therefore classified
in Germ cell mutagens group 3. Allergological investigations in man and animals
have shown hydroquinone to have sensitizing effects. However, despite the
numerous opportunities for exposure, sensitization is rarely observed at the usual
maximum concentrations used of 2 %. For this reason the substance has
provisionally not been designated with an “S”.135
    ACGIH concluded that the incidence of bladder carcinomas in mice bladder-
implanted with cholesterol pellets containing 20% hydroquinone was
significantly greater than in mice receiving a cholesterol-only pellet implant
(Boyland et al. 1964, cited in ACGIH 2008).1 Equivocal evidence of
carcinogenicity expressed as hepatocellular neoplasm in rats treated with
hydroquinone via water-gavage has also been reported.1,98 Accordingly, an A3
(Confirmed Animal Carcinogen with Unknown Relevance to Humans) notation
was assigned to hydroquinone. Insufficient data were considered available to
recommend labelling for skin irritation or sensitization.1
    EPA has not classified hydroquinone for carcinogenicity.5
    HSE considered the available data on the mechanism(s) of tumour formation
in experimental animals not clearly identified and consequently found it difficult
to judge the relevance for humans. But in view of some positive genotoxicity
findings in vitro, HSE had also difficulties in deciding to a “non-genotoxic”
mechanism involved in the tumour production.18
    IARC concluded in 1999 that there is inadequate evidence in humans for the
carcinogenicity of hydroquinone. There is limited evidence in experimental
animals for the carcinogenicity of hydroquinone. The conclusion was that
hydroquinone is not classifiable as to its carcinogenicity to humans (Group 3).
(IARC 1999a8; see also the section on benzoquinone in IARC 1999b).9
benzoquinone
Occupational exposure limits
See Table 19 for occupational exposure limits for benzoquinone previously
derived by national and international organisations.
Existing guidelines, standards and evaluations                                     109
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<pre>   Table 19 Established limits for occupational exposure to benzoquinone in various countries.
   Country                            Occupational      Time-weighted     Note b     Reference
   Organisation                       exposure limit    average a
                                      (mg/m3)
   Denmark                            0.4               8h                OEL        2007
   Finland                            0.45              8h                -          2005
                                      1.3               15 min            STEL
   Germany
      AGS c                           -                 -                 -          2007
      DFG d                           -                 -                 -
   Norway                             0.4               8h                -          2007
   Sweden                             0.4               8h                -          2005
                                      1.3               15 min            STEL
   UK                                                                     -
      HSE e                           -                 8h                MEL        2007
   USA
      ACGIH f                         0.44              8h                TLV        2001
      NIOSH g                         0.4               8h                REL        2004
      OSHA h                          0.4               8h                PEL        2004
   Iceland                            0.4               8h                -          1999
   European Union
      SCOEL i                         -                 -                 -          -
   a    Time-weighted average (TWA): a maximum mean exposure limit based generally over the
        period of a working day.
   b    Abbreviations - OEL: occupational exposure limit; STEL: short-term exposure limit; MEL:
        maximum exposure limit; TLV: threshold limit value; REL: recommended exposure limit; PEL:
        permissible exposure limit.
   c    Ausschuss für Gefahrstoffe.
   d    Deutsche Forschungsgemeinschaft.
   e    Health and Safety Executive.
   f    American Conference of Governmental Industrial Hygienists.
   g    National Institute for Occupational Safety and Health.
   h    Occupational Safety and Health Administration.
   i    Scientific Committee on Occupational Exposure Limit Values.
   Up to January 1, 2007, the limit value for occupational exposure to benzoqui-
   none in The Netherlands was 0.4 mg/m3; this value has been withdrawn due to
   the introduction of a new system of legal limit values. The value had been
   adopted from the ACGIH 8-h TWA TLV, which is based on industrial experience
   that showed mild, transient ocular irritation at air concentrations of 0.44 mg/m3
   (0.1 ppm) or greater, whereas no systemic effects have been observed at this
   concentration (0.44 mg/m3). Insufficient data were considered available to
   recommend skin, sensitization, or carcinogenicity notations (ACGIH 2001a).2
10 Hydroquinone and benzoquinone
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<pre>Classification and labelling
In Germany, benzoquinone was labelled for skin sensitization; carcinogen
category 3B (substances for which in vitro tests or animal studies have yielded
evidence for carcinogenic effect that is not sufficient for classification in one
other categories); germ cell mutagen category 3B136 (last German update in
2000).102
    ACGIH was of the opinion that insufficient data were available to
recommend skin, sensitization, or carcinogenicity notations, or a TLV-STEL.2
    EPA concluded in 1992 (revised in 2000) that the available data were
insufficient to evaluate the carcinogenicity of the compound. EPA has not
classified benzoquinone for carcinogenicity.6
    IARC concluded in 1999 that the available data on benzoquinone did not
allow an evaluation of its carcinogenicity. No epidemiological data relevant to
the carcinogenicity of benzoquinone were available. There is inadequate
evidence in experimental animals for the carcinogenicity of benzoquinone. The
overall evaluation was that benzoquinone is not classifiable as to its carcino-
genicity to humans (Group 3) (IARC 1999b9; see also the section on hydro-
quinone in IARC 1999a).8
Existing guidelines, standards and evaluations                                    111
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<pre>12 Hydroquinone and benzoquinone</pre>

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<pre> hapter 9
        Hazard assessment
9.1     Assessment of the health risk
        hydroquinone
        There are no human data on hydroquinone that might justify their use for
        deriving a health-based occupational exposure limit: studies lack exposure data,
        exposure is not exclusively to hydroquinone, or the group size is too small.
             Hydroquinone was reported to induce skin depigmentation, cornea
        pigmentation and sensitization in humans after occupational exposure. Eye
        irritation, skin irritation, depigmentation and sensitization were also observed in
        animal studies. In a local lymph node assay hydroquinone was classified as a
        strong sensitizer with an EC3 of 0.11%. The EC3 can be used as a point of
        departure for deriving a limit value for dermal sensitization (IPCS 2012), but
        there is as yet no scientifically reliable method to extrapolate dermal
        sensitization to respiratory sensitization (IPCS 2012).137 Hence the derivation of
        a health-based limit value for inhalation on the basis of dermal sensitization is
        not applicable.
             Although ACGIH derived in 20081 a TLV of 1 mg/m3 for hydroquinone
        based upon its eye irritating properties, the Committee is of the opinion that
        ACGIH appears to have evaluated it in a rather worst-case approach. Firstly the
        human occupational studies which formed the basis of its evaluation all date
        from over four decades ago, with exposure data that are not very reliable in view
        Hazard assessment                                                                   113
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<pre>   of the then available chemical analytical power, and secondly there were co-
   exposures to benzoquinone and aniline which are likely to have influenced
   negatively the eye and skin irritations observed.
        A number of animals studies on hydroquinone toxicity is available. The
   effects observed in the relevant studies are summarised in Table 20, arranged in
   order of increasing NOAELs.
        The lowest NOAELs were observed in the 13- and 104-week oral exposure
   studies with F344 rats and B6C3F1 mice, and in the two generation reproduction
   studies.
        In the 13-week oral exposure study of hydroquinone with F344 rats and
   B6C3F1 mice,100,102 gavage administration at a level of 25 mg/kg bw/day
   induced changes in liver weights. Toxic nephropathy, characterised by tubular
   cell degeneration in the renal cortex, was seen in 7/10 male and 6/10 female rat at
   200 mg/kg bw/day and in 1/10 females receiving 100 mg/kg bw/day. The
   Committee does not consider the changes in liver and kidney weights as relevant
   effects, and set the NOAEL for toxic nephropathy at 25 mg/kg bw/day.
        In the 104-week oral carcinogenicity rat study, reduced body weights and
   renal tubular adenomas in males were seen at 50 mg/kg bw/day. The NOAEL in
   this study was 25 mg/kg bw/day. In the mouse study, increased relative liver
   weights in males, hepatocellular adenomas in females, and thyroid hyperplasia in
   males and females were observed at 50 mg/kg bw/day for 104 weeks. No
   NOAEL was derived in this study. The Committee considers these liver and
   kidney adenomas not to be relevant for humans.
        The available data on reproduction showed that hydroquinone is not a
   reproductive toxicant. In one of the developmental studies, slight but statistically
   insignificant malformations were observed, when expressed per foetus or on a
   litter basis, resulting in a NOAEL for developmental toxicity of 75 mg/kg bw/
   day.
        The available data on neurotoxicity showed mild, transient tremors and
   reduced home-cage activity at 64 mg/kg bw/day in a 13-week rat study; the
   NOAEL for neurotoxicity was 20 mg/kg bw/day. Also in a 2-generation rat study
   mild and transient tremors were seen at 50 mg/kg bw/day, with a NOAEL of 15
   mg/kg bw/day.
14 Hydroquinone and benzoquinone
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<pre> able 20 Effects induced by hydroquinone at different exposure levels and time frames
including NOAELs/LOAELs as reported by the authors)
NOAEL             LOAEL          Duration         Species             Effects                               Reference
mg/kg bw/day) (mg/kg bw/
                  day)
 ye irritation
                  -              Eye instillation Rabbit, dog         Eye irritation (no or only semi-      WHO 199413,
                                                                      quantitative data)                    OECD 199633
Dermal (skin itrritation and sensitization)
                  -              Up to 13 weeks Guinea pig            Skin irritation and depigmentation    WHO 199413,
                                                                      (no or only semi-quantitative data)   OECD 199633
                  -              Induction and    Guinea pig, mouse Skin sensitization (no or only semi-    WHO 199413,
                                 challenge                            quantitative data)                    OECD 199633
                  EC3: 0.11% -                    Local lymph node Strong sensitizer                        Roberts 200793
                                                  assay
Dermal (acute and short term toxicity)
                  > 1,000        single exposure Guinea pig           None                                  OECD 199633
 ,900             3,840          14 days          Rat                 Reduced body weight                   NTP 198998
 ,800             > 4,800        14 days          Mouse               None                                  NTP 198998
Oral toxicity
 5                50             2 generations    Rat                 Mild, transient tremors and           Blacker 1993121
                                                  (gavage)            decreased body weight (F0 and F1)
 0                64             13 weeks         Rat                 Mild, transient tremors and reduced   Bernard 1988, in
                                                  (gavage)            home-cage activity                    OECD 199633;
                                                                                                            Topping 2007101
  25              25             13 weeks         Rat                 Decreased absolute and relative liver NTP 198998;
                                                  (gavage)            weights (m). Increased absolute and Kari 1992100
                                                                      relative liver weights (f) at doses ≥
                                                                      100 mg/kg bw/day. No toxic
                                                                      nephropathy.
  25              25             13 weeks         Mouse               Increased absolute and relative liver NTP 198998;
                                                  (gavage)            weights (m).                          Kari 1992100
 5                50             104 weeks,       Rat                 Lower mean body weights (m).          NTP 198998;
                                 5 days/week      (gavage)            Renal tubular adenomas (m).           Kari 1992100
 5                75             2 generations    Rabbit              Decreased food consumption            Murphy 1992123
                                                  (gavage)            (maternal)
 5                150            2 generations    Rabbit              Minimal developmental alterations Murphy 1992123
                                                  (gavage)            (developmental)
  50              50             104 weeks,       Mouse               Increased relative liver weights (m). NTP 198998;
                                 5 days/week      (gavage)            Hepatocellular adenomas (f).          Kari 1992100
 00               300            2 generations    Rat                 Decreased food consumption and Krasavage 1992122
                                                  (gavage)            body weight (maternal and
                                                                      development).
 25               250            14 days          Mouse               Mortality, tremors, decreased final NTP 198998;
                                                  (gavage)            mean body weight.                     Kari 1992100
              Hazard assessment                                                                                          115
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<pre> 350       350          104 weeks       Rat              Reduced body weight gain (m, f). Shibata 1991104
                                        (via diet)       Higher absolute and relative liver
                                                         and kidney weights (m). Higher
                                                         relative kidney weight (f).
                                                         Chronic nephropathy (m, f). Renal
                                                         tubular adenomas (m)
           298 - 390    single exposure Rat              LD50                               OECD 199633;
                                        (gavage)                                            Carlson 195396
           390 - 680    single exposure Mouse            LD50                               OECD 199633
                                        (gavage)
50         500          14 days         Rat              Mortality, tremors, reduced final  NTP 198998; Kari
                                        (gavage)         mean body weight.                  1992100
 1,050     1,050        96 weeks        Mouse            Reduced body weight gain (m).      Shibata 1991104
                                        (via diet)       Higher relative liver and kidney
                                                         weights (f). Hepatocellular
                                                         adenomas (m).
       Furthermore the Subcommittee on the Classification of carcinogenic substances
       concluded that hydroquinone may induce mutations under high exposure
       conditions, probably due to the formation of ROS. But as long as exposure levels
       to hydroquinone are below levels inducing local cytotoxicity (and consequently
       hyperplasia), the risk for carcinogenic effects was considered to be negligible.
       Based on the available information, the subcommittee was of the opinion that the
       data are as yet insufficient to evaluate the carcinogenic properties of
       hydroquinone.
            Based on these conclusions, and the consideration of the Subcommittee on
       Classification of carcinogenic substances that the carcinogenic mechanism of
       hydroquinone in animal studies is in all probability a non-stochastic genotoxic
       mechanism (due to ROS-generation, while inhibition of topoisomerase II may
       also play a role), the Committee adopts a threshold approach for deriving a
       HBROEL for hydroquinone.
       benzoquinone
       In humans, exposure to high air levels of benzoquinone may result in irritation of
       the eyes. Dermal exposure of humans may result in dermatitis and severe
       irritation. Older studies reported that no systemic effects were observed
       following prolonged occupational exposure to benzoquinone vapour at a level of
       0.44 mg/m3 for periods up to 5 years.
            Only a limited number of animal studies is available. In a local lymph node
       assay benzoquinone was classified as an extreme sensitizer with an EC3 of
       0.01%. The EC3 can be used as a point of departure for deriving a limit value for
16     Hydroquinone and benzoquinone
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<pre>    dermal sensitization (IPCS 2012), but there is as yet no scientifically reliable
    method to extrapolate dermal sensitization to respiratory sensitization (IPCS
    2012).137 Hence the derivation of a health-based limit value for inhalation on the
    basis of dermal sensitization is not applicable.
        Sub-acute toxicity studies on benzoquinone have been performed via
    inhalation exposure and intraperitoneal and subcutaneous injections. The critical
    effects were all of a haematological character and included decreases in blood
    cells, methaemoglobinemia and trombopenia. A LOAEL of 0.27-0.36 mg/m3
    was derived in 4 months inhalation study in rats. However, as the original
    document could not be retrieved, the relevance and accuracy of these data can
    not be assessed.
        There are only two very limited carcinogenicity studies on benzoquinone.
    Due to their poor quality and high mortality rates, no NO(A)ELs or LO(A)ELs
    could be derived from these.
        With regard to the current standard on scientific reliability there are no
    human data on benzoquinone that might justify their use for deriving a
    HBROEL. Also the data from experimental animal studies provide too little
    information to derive a HBROEL.
        In Section 9.4 some additional considerations regarding the toxic potency of
    benzoquinone are presented.
9.2 Recommendation of the health-based occupational exposure limit
    hydroquinone
    With regard to the current standard on scientific reliability there are no human
    data on hydroquinone that might justify their use for deriving a HBROEL, and
    hence the Committee used experimental animal data as the starting point in
    deriving a HBROEL. The Committee selected the 2-year mouse carcinogenicity
    study by NTP as the pivotal one (NTP 198998; Kari et al. 1992100). In this study a
    statistically significant increase of the incidence of thyroid hyperplasia was
    observed in both sexes at 50 and 100 mg/kg bw/day, albeit with a serious
    difference in susceptibility for this effect between males and females. The
    Committee also took into account the neurotoxic effects with NOAELs of 20 and
    15 mg/kg bw/day (tremors and reduced home-cage activity at 64 mg/kg bw/day
    in the 13-week rat study of Bernard 1988 (cited in OECD 199633) and Topping
    2007101, and tremors and decreased body weight at 50 mg/kg bw/day in the
    2-generation study of Blacker 1993121, respectively). However, these neurotoxic
    Hazard assessment                                                                  117
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<pre>   effects were mild and of a transient character. Moreover, a BMD* analysis of the
   study by Blacker (the only one with quantitative data) showed the lowest BMDL
   for 10% extra risk to be 48 mg/kg bw/day, which is much higher than the BMDL
   derived from the thyroid effects (see below).
       The 2-years NTP mouse gavage study (NTP 198998, Kari et al. 1992100) with
   the data on thyroid hyperplasia (Table 21; see also Table 14) was thus selected as
   the pivotal study for deriving the HBROEL. With these data BMD analyses were
   done.
       However, analysis of the study data using the software programme PROAST
   (a software package developed for analysing dose-response data of toxicity
   studies and calculating BMDs; Slob 2002138, 2009139; EFSA 2011140) showed
   that the data of the males and the females can not be combined. The incidences
   of the lesions in the males differ too much from the incidences in the females,
   both in the controls and in the dosed groups. In addition, a BMD analysis of the
   results with the females is not possible: the incidences flattens off already at the
   low dose, and since there are only two dose groups it is not possible to estimate a
   BMD for e.g. 10% extra risk (Slob, personal communication). Only for the
   results with the males a BMD analysis is possible, and consequently an
   adjustment factor will be needed to correct for the difference in susceptibility
   between males and females. The BMD analysis of the data of the males, using
   US-EPA’s BMD software141, is outlined in Annex K. Only a BMD/BMDL for
   10% extra risk could be estimated, the data were of insufficient quality to derive
   a reliable BMD/BMDL for 5% extra risk.
       The lowest BMD and BMDL for 10% extra risk are produced by the log-
   logistic model, with a BMD of 25.1 and BMDL of 15.7 mg/kg bw/day. The other
   models resulted in BMDs varying from 28.5 - 43.5 mg/kg bw/day and BMDLs
   varying from 19.1 - 34.0 mg/kg bw/day. The Committee considers the BMDL of
   15.7 mg/kg bw/day the appropriate starting point for the derivation of the
   HBROEL.
       Since the exposure levels inducing effects in the acute and short term
   exposure studies are significantly higher than those at in the sub-acute and
   Table 21 Thyroid hyperplasia in mice (2-year gavage study; NTP 198998, Kari et al. 1992100).
   Thyroid hyperplasia in   Number of animals with lesions / total number of animals per dose group
   mice (B6C3F1)            males                                females
   Dose (mg/kg bw/day)      Control      50          100         Control      50         100
   Thyroid hyperplasia      5/55         15/53       19/54       13/55        47/55      45/55
   BMD: benchmark dose; BMDL: benchmark dose at lower 95% confidence limit.
18 Hydroquinone and benzoquinone
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<pre>chronic studies, no elaboration of a Short Term Exposure Limit (STEL) or
ceiling value for hydroquinone is considered necessary.
In order to convert this animal BMDL into a HBROEL the following corrections
are made:
• Correction for the difference in susceptibility for thyroid hyperplasia
     between male and female mice;
• Differences in sensitivity between mice and men;
• Route-to-route extrapolation, i.e., from the oral to the inhalation route of
     exposure.
From the data shown in Table 21 it appears that females are approximately three
times as sensitive as males for the thyroid hyperplasia resulting from exposure to
hydroquinone. Hence an adjustment factor of 3 is considered appropriate to
correct for this difference in susceptibility (there are no data that would justify
another factor). The observed BMDL of 15.7 mg/kg bw/day is thus adjusted to
5.2 mg/kg bw/day.
     With regard to the possible difference in sensitivity between mice and man
(i.e., both kinetic and dynamic differences) an uncertainty factor of 3 will be
used. Applying this results in a value of 1.7 mg/kg bw/day.
     Based on the available data it is concluded that oral absorption is nearly
complete, and similar for mice and man. It is also assumed, from a precautionary
principle view, that absorption by inhalation is 100%. Consequently, an external
burden of 1.7 mg/kg bw/day will result in an equal internal daily human body
burden of (1.7 mg/kg bw x 70 kg =) 119 mg. When this internal dose is divided
by the total volume of air inhaled by a human during a working day of 8 hours
(10 m3) the adjusted mouse BMDL is converted to an inhalation BMDL in
humans of 12 mg/m3.
     For intraspecies differences, i.e., to compensate for possible differences in
sensitivity among workers, an uncertainty factor of 3 will be used. Taking this
last adjustment into account results in a HBROEL for hydroquinone of 12/3
mg/m3 = 4 mg/m3.
     Most occupational exposure limits for hydroquinone of other countries are
2 mg/m3 or lower. In general these are based on its eye and skin irritation
properties. But the quantitative data of these properties date from over four
decades ago, with exposure data that are not very reliable in view of the then
available chemical analytical power. Moreover, there were co-exposures to
benzoquinone and aniline which are likely to have influenced negatively the eye
and skin irritations observed. Although only limited data are available, the
Hazard assessment                                                                   119
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<pre>    Committee expects that at the level of 4 mg/m3 the risk for eye and skin irritation
    and sensitization is negligible (see also Sections 7.1.1 and 8.2).
    Skin notation
    Absorption of hydroquinone through the human skin averaged 1.6, 2.3 and 2.5%
    per hour of the dose in the first, second and third 4 hours after application of
    125 µg/cm2 (see Section 5.1). When 2,000 cm2 human skin would be exposed for
    1 hr, the quantity absorbed would be at least 2,000 x 125 x 0.016 µg = 4,000 µg.
    In view of the exposure level advised, dermal absorption can add considerably
    (more than 10%) of the amount taken up via the inhalatory route. For benzo-
    quinone the same way of reasoning is assumed, in absence of experimental data.
    Therefore the Committee recommends a skin notation for hydroquinone and
    benzoquinone.
    benzoquinone
    The available data do not allow the derivation of a HBROEL for benzoquinone
    (see also additional considerations in Section 9.4).
9.3 Groups at extra risk
    No groups at extra risk have been identified.
9.4 Additional considerations - benzoquinone
    Since benzoquinone and hydroquinone are metabolites of each other (converted
    at reaction rates dependent on prevailing local conditions), it is clear that toxicity
    observed with one of the two will be also relevant for the other, though there may
    be a difference in potency.
         Indeed, as far as available data permit, their toxicity profiles have a lot in
    common: both substances are irritating to eyes and skin, act as skin sensitizers,
    and have a comparable genotoxicity profile. The database on hydroquinone,
    however, is more complete: i.e., for benzoquinone there are no adequate data on
    (sub)chronic toxicity, carcinogenicity, and reproductive toxicity. Thus, for these
    data gaps for benzoquinone, available data on hydroquinone could be used to
    indicate its potential effects.
         As suitable data for benzoquinone itself are lacking, the Committee tries to
    use the HBROEL established for hydroquinone to derive a HBROEL for
 20 Hydroquinone and benzoquinone
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<pre>             benzoquinone, addressing the difference in potency between the two toxicants.
             The Committee is of the opinion that a threshold approach is suitable for
             benzoquinone because the reasoning followed for hydroquinone (see Section
             7.2.6) is also considered valid for benzoquinone.
                  The data that allow a comparison on differences in toxic potency between
             hydroquinone and benzoquinone are summarized in Table 22. In addition to a
             few quantitative data on skin irritation and dermal sensitization, only some
             comparable data for acute oral toxicity are available. Already from these limited
             data it appears that the relative toxic potency of benzoquinone as compared to
             that of hydroquinone varies with the endpoint considered. Therefore the
             Committee concludes that this is not a sufficient basis for deriving a HBROEL
             for benzoquinone.
                  The value of 0.4 mg/m3 (8 hr TWA) that was valid in the Netherlands until
             January 1, 2007 (due to the introduction of the new system of legal limit values;
             see Table 19) was adopted from the ACGIH (2001a).2 It was based on industrial
             experience that showed ocular irritation at air concentrations of 0.44 mg/m3,
             whereas no systemic effects have been observed during approximately 5 years of
             occupational exposure at this concentration. Likely this limit value protects
             against adverse systemic effects of exposure to benzoquinone. ACGIH (2001)2
             was of the opinion that insufficient data were available to recommend skin,
             sensitization, or carcinogenicity notations.
 able 22 Comparative toxicity of hydroquinone and benzoquinone.
 oxicity parameter                                       Hydroquinone       Benzoquinone        Reference
Oral LD50 rat (mg/kg bw)                                 300-390            130-165             Tables E-3A and E-3B
NOAEL for irritation in female guinea pig                0.1%               0.001%              Rajka and Blohm 197086
% solution in water; sc injection)
 C3 for sensitization in a murine local lymph node assay 0.11%              0.01%               Roberts et al. 200793
nternal daily dose inducing systemic toxicity
mg/kg bw/day) in the rat:
 rat (oral, gavage, 3 months)                            < 25a                                  NTP 198998;
                                                                                                Kari et al. 1992100
 rat (inhalation, 4 h/day, 4 months)                                        < 0.6a              Anonymous, cited in MAK
                                                                                                2000102
    derived assuming 100% absorption, default body weight 300 g, and default respiration rate 200 ml/min.
             Hazard assessment                                                                                        121
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<pre>9.5 Health-based recommended occupational exposure limits
    The Dutch Expert Committee on Occupational Safety recommends a health-
    based recommended occupational exposure limit for hydroquinone of 4 mg/m3,
    as 8 hour time weighted average.
    The Committee concludes that the available data on the toxicity of benzoquinone
    are of insufficient quality to derive a health-based recommended occupational
    exposure limit for benzoquinone.
 22 Hydroquinone and benzoquinone
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<pre>hapter 10
       Recommendation for research
       It is recommended to investigate the toxicity of hydroquinone and benzoquinone
       after inhalation, preferably in a chronic exposure experiment, to compare the
       difference in toxic potential between benzoquinone and hydroquinone.
            In addition, it is recommended to investigate the irritating properties of
       hydroquinone and benzoquinone in studies according to OECD guidelines, in
       order to get an accurate insight at which concentrations occupationally exposed
       people might start to develop local toxic effects. If possible, human studies
       should be used to support any animal data.
       Recommendation for research                                                     123
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<pre>24 Hydroquinone and benzoquinone</pre>

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8  NTP. Toxicology and carcinogenesis studies of hydroquinone (CAS No. 123-31-9) in F344/N rats
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9  David RM, English JC, Totman LC, Moyer C, O'Donoghue JL. Lack of nephrotoxicity and renal cell
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00 Kari FW, Bucher J, Eustis SL, Haseman JK, Huff JE. Toxicity and carcinogenicity of hydroquinone
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01 Topping DC, Bernard LG, O'Donoghue JL, English JC. Hydroquinone: acute and subchronic toxicity
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   70-78.
02 Deutsche Forschungsgemeinschaft (DFG). 1,4-Benzochinon. Gesundheitsschadliche
   Arbeitsstoffe.Toxikologische-arbeitsmedizinische Begrundungen von MAK-Werten. [31], 1-26.
   2000. Wiley,VCH Verlag GmbH and Co., KGaA, Weinheim, Germany.
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   Vet Hum Toxicol 1988; 30(6): 517-520.
   References                                                                                            131
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<pre>04 Shibata MA, Hirose M, Tanaka H, Asakawa E, Shirai T, Ito N. Induction of renal cell tumors in rats
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05 Hard GC, Whysner J, English JC, Zang E, Williams GM. Relationship of hydroquinone-associated
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06 Hard GC, Khan KN. A contemporary overview of chronic progressive nephropathy in the laboratory
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07 McGregor D. Hydroquinone: an evaluation of the human risks from its carcinogenic and mutagenic
   properties. Crit Rev Toxicol 2007; 37(10): 887-914.
08 Whysner J, Verna L, English JC, Williams GM. Analysis of studies related to tumorigenicity induced
   by hydroquinone. Regul Toxicol Pharmacol 1995; 21(1): 158-176.
09 Kurata Y, Fukushima S, Hasegawa R, Hirose M, Shibata M, Shirai T et al. Structure-activity relations
   in promotion of rat urinary bladder carcinogenesis by phenolic anti-oxidants. Jpn J Cancer Res 1990;
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10 Okazaki S, Hoshiya T, Takahashi S, Futakuchi M, Saito K, Hirose M. Modification of hepato- and
   renal carcinogenesis by catechol and its isomers in rats pretreated with N-ethyl-N-
   hydroxyethylnitrosamine. Teratog Carcinog Mutagen 1993; 13(3): 127-137.
11 Yamaguchi S, Hirose M, Fukushima S, Hasegawa R, Ito N. Modification by catechol and resorcinol
   of upper digestive tract carcinogenesis in rats treated with methyl-N-amyl-nitrosamine. Cancer Res
   1989; 49(21): 6015-6018.
12 Hasegawa R, Furukawa F, Toyoda K, Takahashi M, Hayashi Y, Hirose M et al. Inhibitory effects of
   antioxidants on N-bis(2-hydroxypropyl)nitrosamine-induced lung carcinogenesis in rats. Jpn J
   Cancer Res 1990; 81(9): 871-877.
13 Maruyama H, Amanuma T, Nakae D, Tsutsumi M, Kondo S, Tsujiuchi T et al. Effects of catechol and
   its analogs on pancreatic carcinogenesis initiated by N-nitrosobis(2-oxo-propyl)amine in Syrian
   hamsters. Carcinogenesis 1991; 12(7): 1331-1334.
14 Umeda M. Production of rat sarcoma by injections of propylene glycol solution of p-quinone. Gan
   1957; 48(2): 139-144.
15 El-Mofty MM, Khudoley VV, Sakr SA, Fathala NG. Flour infested with Tribolium castaneum,
   biscuits made of this flour, and 1,4-benzoquinone induce neoplastic lesions in Swiss albino mice.
   Nutr Cancer 1992; 17(1): 97-104.
16 Gwynn RH, Salaman MH. Studies on co-carcinogenesis. SH-reactors and other sub-stances tested for
   co-carcinogenic action in mouse skin. Br J Cancer 1953; 7(4): 482-489.
17 Monks TJ, Walker SE, Flynn LM, Conti CJ, DiGiovanni J. Epidermal ornithine de-carboxylase
   induction and mouse skin tumor promotion by quinones. Carcinogenesis 1990; 11(10): 1795-1801.
18 Glatt HR, Padykula R, Berchtold GA, Ludewig G, Platt KL, Klein J et al. Multiple activation
   pathways of benzene leading to products with varying genotoxic characteristics. Environ Health
   Perspect 1989; 82: 81-89.
32 Hydroquinone and benzoquinone
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<pre>19 Miller BM, Adler I-D. Aneuploidy in mouse spermatocytes. Mutagenesis 1992; 7(1): 69-76.
20 Wangenheim J, Bolcsfoldi G. Mouse lymphoma L5178Y thymidine kinase locus assay of 50
   compounds. Mutagenesis 1988; 3(3): 193-205.
21 Blacker AM, Schroeder RE, English JC, Murphy SJ, Krasavage WJ, Simon GS. A two-generation
   reproduction study with hydroquinone in rats. Fundam Appl Toxicol 1993; 21(4): 420-424.
22 Krasavage WJ, Blacker AM, English JC, Murphy SJ. Hydroquinone: a developmental toxicity study
   in rats. Fundam Appl Toxicol 1992; 18(3): 370-375.
23 Murphy SJ, Schroeder RE, Blacker AM, Krasavage WJ, English JC. A study of developmental
   toxicity of hydroquinone in the rabbit. Fundam Appl Toxicol 1992; 19(2): 214-221.
24 Wierda D, Irons RD. Hydroquinone and catechol reduce the frequency of progenitor B lymphocytes
   in mouse spleen and bone marrow. Immunopharmacology 1982; 4(1): 41-54.
25 Schlosser MJ, Kalf GF. Metabolic activation of hydroquinone by macrophage peroxidase. Chem-Biol
   Interact 1989; 72(1-2): 191-207.
26 Pyatt DW, Yang Y, Stillman WS, Cano LL, Irons RD. Hydroquinone inhibits PMA-induced
   activation of NFkappaB in primary human CD19+ B lymphocytes. Cell-Biol Toxicol 2000; 16(1):
   41-51.
27 Henschler R, Glatt HR, Heyworth CM. Hydroquinone stimulates granulocyte-macrophage progenitor
   cells in vitro and in vivo. Environ Health Perspect 1996; 104(suppl 6): 1271-1274.
28 Guy RL, Hu P, Witz G, Goldstein BD, Snyder R. Depression of iron uptake into erythro-cytes in mice
   by treatment with the combined benzene metabolites p-benzoquinone, muconaldehyde and
   hydroquinone. J Appl Toxicol 1991; 11(6): 443-446.
29 Ribeiro AL, Shimada AL, Hebeda CB, de Oliveira TF, de Melo Loureiro AP, Filho Wdos R, Santos
   AM, de Lima WT, Farsky SH. In vivo hydroquinone exposure alters circulating neutrophil activities
   and impairs LPS-induced lung inflammation in mice. Toxicol 2011; 288(1-3): 1-7.
30 Yang EJ, Lee JS, Yun CY, Kim IS. The pro-apoptotic effect of hydroquinone in human neutrophils
   and eosinophils. Toxicol in Vitro 2011; 25(1): 131-137.
31 McDonald TA, Waidyanatha S, Rappaport SM. Measurement of adducts of benzoquinone with
   hemoglobin and albumin. Carcinogenesis 1993; 14(9): 1927-1932.
32 Manning BW, Adams DO, Lewis JG. Effects of benzene metabolites on receptor-mediated
   phagocytosis and cytoskeletal integrity in mouse peritoneal macrophages. Toxicol Appl Pharmacol
   1994; 126(2): 214-223.
33 Anderson B, Durham NC, Oglesby F. Corneal changes from quinone-hydroquinone exposure. AMA
   Arch Ophthalmol 1958; 59(4): 495-501.
34 ACGIH. Hydroquinone. In: Documentation of the Threshold Limit Values and Biological Exposure
   Indices. American Conference of Governmental Industrial Hygienists, Cincinnati, Ohio, USA;
   2001b.
35 Greim H. Hydroquinone. In: Occupational Toxicants: Critical Data Evaluation for MAK Values and
   Classification of Carcinogens, Volume 10. Wiley-VCH; 1998: 113-145.
   References                                                                                         133
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<pre>36 Occupational toxicants and MAK values. Wiley Interscience, Weinheim, Germany; 2000. http://
   www3.interscience.wiley.com/cgi-bin/mrwhome/104554790/home consulted: June 4, 2008.
37 IPCS. Guidance for immunotoxicity risk assessment for chemicals. International Programme on
   Chemical Safety, Harmonization Project Document no. 10. World Health Organization, Geneva,
   Switzerland; 2012.
38 Slob W. Dose-response modeling of continous endpoints. Toxicol. Sci. 2002; 66: 298-312.
39 Slob W. PROAST: software for dose-response modeling and benchmark dose analysis. National
   Institute for Public Health and the Environment, Bilthoven, The Netherlands; 2009.
40 EFSA. Use of BMDS and PROAST software packages by EFSA Scientific Panels and Units for
   applying the Benchmark Dose (BMD) approach in risk assessment. Technical report, European Food
   Safety Authority (EFSA), Parma, Italy; 2011.
41 US-EPA benchmark dose software. US Environmental Protection Agency, Washington DC, USA.
   www.epa.gov/ncea/bmds/ (November 2011).
42 Adebajo SB. An epidemiological survey of the use of cosmetic skin lightening cosmetics among
   traders in Lagos, Nigeria. West Afr J Med 2002; 21(1): 51-55.
43 Raynaud E, Cellier C, Perret JL. Depigmentation for cosmetic purposes: prevalence and side-effects
   in a female population in Senegal (original title: Depigmentation cutanee a visee cosmetique'. Ann
   Dermatol Venereol 2001; 128(6-7): 720-724.
44 Hoshaw RA, Zimmerman KG, Menter A. Ochronosislike pigmentation from hydroquinone bleaching
   creams in American blacks. Arch Dermatol 1985; 121(1): 105-108.
45 Naumann G. Corneal damage in hydroquinone workers. Arch Ophthalmol 1966; 76(2): 189-194.
46 Lidén C. Occupational dermatoses at a film laboratory. Follow-up after modernization. Contact
   Dermatitis 1989; 20(3): 191-200.
47 Makropoulos V, Alexopoulos EC. Case report: hydroquinone and/or glutaraldehyde induced acute
   myeloid leukaemia? J Occup Med Toxicol 2006; 1: 19.
48 Pifer JW, Hearne FT, Friedlander BR, McDonough JR. Mortality study of men employed at a large
   chemical plant, 1972 through 1982. J Occup Med 1986; 28(6): 438-444.
49 Zhang L, Rothman N, Wang Y, Hayes RB, Bechtold W, Venkatesh P et al. Interphase cytogenetics of
   workers exposed to benzene. Environ Health Perspect 1996; 104 (suppl 6): 1325-1329.
50 Choudat D, Neukirch F, Brochard P, Barrat G, Marsac J, Conso F et al. Allergy and occupational
   exposure to hydroquinone and to methionine. Br J Ind Med 1988; 45(6): 376-380.
51 Boatman RJ, English JC, Perry LG, Bialecki VE. Differences in the nephrotoxicity of hydroquinone
   among Fischer 344 and Sprague-Dawley rats and B6C3F1 mice. J Toxicol Environ Health 1996;
   47(2): 159-172.
52 Ciranni R, Adler I-D. Clastogenic effects of hydroquinone: induction of chromosomal aberrations in
   mouse germ cells. Mutat Res 1991; 263(4): 223-229.
53 Leopardi P, Zijno A, Bassani B, Pacchierotti F. In vivo studies on chemically induced aneuploidy in
   mouse somatic and germinal cells. Mutat Res 1993; 287(1): 119-130.
34 Hydroquinone and benzoquinone
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<pre>54 Ludewig G, Dogra S, Glatt HR. Genotoxicity of 1,4-benzoquinone and 1,4-naphthaquinone in
   relation to effects on glutathione and NAD(P)H levels in V79 cells. Environ Health Perspect 1989;
   82: 223-228.
   References                                                                                        135
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<pre>36 Hydroquinone and benzoquinone</pre>

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<pre>A Request for advice
B The Committee
C Letter of submission
D Comments on the public review draft
E Human data
F Animal data (in vivo)
G In vitro data
H Mutagenicity studies
  Evaluation of the Subcommittee on the classification of carcinogenic
  substances
  Carcinogenic classification of substances by the Committee
K BMD analysis of thyroid hyperplasia in mice following oral exposure to
  hydroquinone
  Annexes
                                                                         137
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<pre>38 Hydroquinone and benzoquinone</pre>

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<pre>nnex A
     Request for advice
     In a letter dated October 11, 1993, ref DGA/G/TOS/93/07732A, to, the State
     Secretary of Welfare, Health and Cultural Affairs, the Minister of Social Affairs
     and Employment wrote:
     Some time ago a policy proposal has been formulated, as part of the simplification of the
     governmental advisory structure, to improve the integration of the development of recommendations
     for health based occupation standards and the development of comparable standards for the general
     population. A consequence of this policy proposal is the initiative to transfer the activities of the
     Dutch Expert Committee on Occupational Safety (DECOS) to the Health Council. DECOS has been
     established by ministerial decree of 2 June 1976. Its primary task is to recommend health based
     occupational exposure limits as the first step in the process of establishing Maximal Accepted
     Concentrations (MAC-values) for substances at the work place.
     In an addendum, the Minister detailed his request to the Health Council as
     follows:
     The Health Council should advice the Minister of Social Affairs and Employment on the hygienic
     aspects of his policy to protect workers against exposure to chemicals. Primarily, the Council should
     report on health based recommended exposure limits as a basis for (regulatory) exposure limits for air
     quality at the work place. This implies:
     •    A scientific evaluation of all relevant data on the health effects of exposure to substances using a
          criteria-document that will be made available to the Health Council as part of a specific request
     Request for advice                                                                                        139
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<pre>       for advice. If possible this evaluation should lead to a health based recommended exposure limit,
       or, in the case of genotoxic carcinogens, an ‘exposure versus tumour incidence range’ and a
       calculated concentration in air corresponding with reference tumour incidences of 10-4 and 10-6
       per year.
   •   The evaluation of documents review the basis of occupational exposure limits that have been
       recently established in other countries.
   •   Recommending classifications for substances as part of the occupational hygiene policy of the
       government. In any case this regards the list of carcinogenic substances, for which the
       classification criteria of the Directive of the European Communities of 27 June 1967 (67/548/
       EEG) are used.
   •   Reporting on other subjects that will be specified at a later date.
   In his letter of 14 December 1993, ref U 6102/WP/MK/459, to the Minister of
   Social Affairs and Employment the President of the Health Council agreed to
   establish DECOS as a Committee of the Health Council. The membership of the
   Committee is given in Annex B.
40 Hydroquinone and benzoquinone
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<pre>nnex B
     The Committee
     •  G.J. Mulder, chairman
        Emeritus Professor of Toxicology, Leiden University, Leiden
     •  P.J. Boogaard
        Toxicologist, Shell International BV, The Hague
     •  D.J.J. Heederik
        Professor of Risk Assessment in Occupational Epidemiology, Institute for
        Risk Assessment Sciences, Utrecht University, Utrecht
     •  R. Houba
        Occupational Hygienist, Netherlands Expertise Centre for Occupational
        Respiratory Disorders, Utrecht
     •  H. van Loveren
        Professor of Immunotoxicology, Maastricht University, Maastricht, and
        National Institute for Public Health and the Environment, Bilthoven
     •  T.M. Pal
        Occupational Physician, Netherlands Centre for Occupational Diseases,
        University of Amsterdam, Amsterdam
     •  A.H. Piersma
        Professor of Reproductive Toxicology, Utrecht University, Utrecht, and
        National Institute for Public Health and the Environment, Bilthoven
     •  H.P.J. te Riele
        Professor of Molecular Biology, VU University Amsterdam, and Netherlands
        Cancer Institute, Amsterdam
     The Committee                                                               141
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<pre>   •   I.M.C.M. Rietjens
       Professor of Toxicology, Wageningen University and Research Centre,
       Wageningen
   •   G.M.H. Swaen
       Epidemiologist, Dow Benelux NV, Terneuzen
   •   R.C.H. Vermeulen
       Epidemiologist, Institute for Risk Assessment Sciences, Utrecht University,
       Utrecht
   •   R.A. Woutersen
       Toxicologic Pathologist, TNO Quality of Life, Zeist, and Professor of
       Translational Toxicology, Wageningen University and Research Centre,
       Wageningen
   •   P.B. Wulp
       Occupational Physician, Labour Inspectorate, Groningen
   •   B.P.F.D. Hendrikx, advisor
       Social and Economic Council, The Hague
   •   A.J. Baars, scientific secretary
       Health Council of the Netherlands, The Hague
   The Health Council and interests
   Members of Health Council Committees are appointed in a personal capacity
   because of their special expertise in the matters to be addressed. Nonetheless, it
   is precisely because of this expertise that they may also have interests. This in
   itself does not necessarily present an obstacle for membership of a Health
   Council Committee. Transparency regarding possible conflicts of interest is
   nonetheless important, both for the chairperson and members of a Committee
   and for the President of the Health Council. On being invited to join a
   Committee, members are asked to submit a form detailing the functions they
   hold and any other material and immaterial interests which could be relevant for
   the Committee’s work. It is the responsibility of the President of the Health
   Council to assess whether the interests indicated constitute grounds for non-
   appointment. An advisorship will then sometimes make it possible to exploit the
   expertise of the specialist involved. During the inaugural meeting the
   declarations issued are discussed, so that all members of the Committee are
   aware of each other’s possible interests.
42 Hydroquinone and benzoquinone
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<pre>nnex C
     Letter of submission
     Subject                : Submission of the advisory report Hydroquinone
                              and benzoquinone
     Your Reference         : DGV/MBO/U-932342
     Our reference          : U-7449/BJB/fs/459-S67
     Enclosed               :1
     Date                   : December 3, 2012
     Dear Minister,
     I hereby submit the advisory report on the effects of occupational exposure to
     hydroquinone and benzoquinone.
     This advisory report is part of an extensive series in which health-based
     recommended exposure limits are derived for the concentrations of various
     substances in the workplace. The advisory report in question was prepared by the
     Health Council’s Dutch Expert Committee on Occupational Safety (DECOS)
     and assessed by the Standing Committee on Health and the Environment.
     Letter of submission                                                             143
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<pre>   I have today sent copies of this advisory report to the State Secretary of
   Infrastructure and the Environment and to the Minister of Health, Welfare and
   Sport, for their consideration.
   Yours sincerely,
   (signed)
   Professor W.A. van Gool,
   President
44 Hydroquinone and benzoquinone
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<pre>nnex D
     Comments on the public review draft
     A draft of the present report was released in 2012 for public review. The
     following organisation has commented on the draft document:
     • Th.J. Lentz, National Institute for Occupational Safety and Health,
         Cincinnati (OH), USA.
     Comments on the public review draft                                       145
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<pre>46 Hydroquinone and benzoquinone</pre>

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<pre>nnex E
     Human data
     Human data 147
</pre>

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<pre>Human studies, hydroquinone.
                 Compound      Exposure        Subjects,        Exposure       Exposure       Effects         Reference
                               through         sample size      duration       concentration
Oral
Acute            Hydroquinone Accidental       -                -              80-200 mg/kg Death             WHO 199413
 oisoning                      ingestion                                       bw
  ontrolled      Hydroquinone Ingestion        2 males,         5 months       500 mg/day     No effects in EC 20007,
 tudy                                          volunteers                                     blood and urine WHO 199413
                                               17 males +       3-5 months     300-500 mg/
                                               females,                        day
                                               volunteers                      (~6 mg/kg bw/
                                                                               day)
Dermal
Cohort           Hydroquinone Occupational     Danish           About one-     No information The SIRa for Nielsen et al.
                               exposure        lithographers quarter of the                   cancers was     200383
                               Controls:       registered with cohort                         0.9.
                               general         the Danish       members                       For no site
                               population of   Union of         reported                      except skin
                               Denmark         Lithographers working                          there were
                                               in 1947 or later regularly with                more than three
                                               (n=837)          hydroquinone                  cases. Five
                                                                for                           cases of
                                                                photographic                  malignant
                                                                development.                  melanoma
                                                                                              occurred, of
                                                                                              which two had
                                                                                              reportedly been
                                                                                              exposed to
                                                                                              hydroquinone.
  pidemio-       Hydroquino- Skin lightening Traders from Modal: 1-3           Crèmes contain Ochronosisb Adebajo
ogical           lone, among cosmetics,        the Lagos        years          maximal 2% (stated to be 2002142
                 other active  bleaching       metropolis,      8.3% < 6       hydroquino- associated with
                 compounds     creams          Selected (ad months             lone           hydroquinolone
                                               random): 130 12.6%: 5 years                    exposure)c
                                               males, 320
                                               females
                                               Exposed: 96
                                               males,
                                               252 females
  ross sectional Hydroquinone, Skin lightening Hospitalized -                  -              Ochronosisc     Raynaud et al.
                 among other cosmetics         women                                                          2001143
                 active                        Exposed: 47
                 compounds                     Not exposed
                                               100
  ase study      Hydroquinone Skin lightening Two patients -                   Crèmes         Ochronosis-     Hoshaw et al.
                               cosmetics       experiencing                    containing 2% like             1995144
                                               effects                         hydroquinone pigmentation
                                               Exposed 2/2
  48          Hydroquinone and benzoquinone
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<pre> ase study      Hydroquinone Occupational     Three workers Prolonged         No information Corneal           Naumann
                             exposure         in a             period (about                  damage;          1966145
                                              hydroquinone 10 years)                          conjunctival
                                              producing                                       pigmentation
                                              factory                                         and
                                              experiencing                                    degenerative
                                              effects                                         changes
                                              Exposed 3/3
 ohort study Hydroquinone Occupational 105 people              No information No information Altered           Anderson et al.
                             exposure         experiencing                                    curvature of the 1958133
                                              evidence of                                     cornea, which
                                              exposure (out                                   may become
                                              of 201 persons                                  apparent long
                                              that might have                                 after exposure
                                              received
                                              significant
                                              exposure) were
                                              followed for 10
                                              years
 ollow up after Hydroquinone Working in a 78 employees Various                No information 34/54 exposed Lidén 1989146
modernization,               film laboratory, of which 54                                     subjects had
nterviews,                   study period chemically                                          dermatoses;
 ermatological               1983-1986        exposed.                                        12/54 were
 xaminations,                                                                                 contact allergic
 atch testing                                                                                 to film
                                                                                              chemicals.
                                                                                              Reduced
                                                                                              chemical
                                                                                              exposure
                                                                                              reduced the
                                                                                              occurrence and
                                                                                              severity of skin
                                                                                              problems
nhalation
 ase study      Hydroquinone Occupational Two female           16 yr          No information Acute             Makropoulos &
                             exposure         medical                                         monocytic        Alexopoulos
                                              radiation                                       leukaemia;       2006147
                                              technologists in                                promyelo-
                                              same primary                                    genous
                                              case center                                     leukaemia
 ohort          Hydroquinone Occupational Workers in           At least 3     Three           SMRd most        Fryzek et al.
                             exposure         motion picture months           measurements: malignancies 200584
                                              film processing between         before 1981: below 1.0; Non
                                              in California 1960-2000         below detection Hodgkin
                                              n=2646                          level; after    lymphoma
                                                                              1981: < 0.014 SMR 2.2
                                                                              mg/m3 and       (1.0-4.0).
                                                                              0.052 mg/m3 Respiratory
                                                                                              mailignancies
                                                                                              SMR 1.9 (1.1-
                                                                                              3.0) n=18
              Human data                                                                                                   149
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<br><br>====================================================================== Pagina 150 ======================================================================

<pre>  ohort, death Hydroquinone Occupational         Workers at a No information No information Mortality was Pifer et al.
 auses                           exposure        Tennessee                                       lower than that 1986148
                                 Controls:       chemical plant                                  in the
                                 general         (USA), study                                    population at
                                 population and  period                                          large and
                                 occupational    1972-1982,                                      consistent with
                                 controls        n= 9,000                                        the
                                                                                                 occupational
                                                                                                 controls
  ohort         Hydroquinone     Occupational Workers at a Mean                  Average dust The SMR for Pifer et al.
                                 exposure        Tennessee        employment levels from         all causes of 199545
                                 Controls:       chemical plant duration 13.7 0.1-6.0 mg/m3, death as well as
                                 general         (USA), study years; mean        with levels     for cancers
                                 population and period            follow-up from > 2 mg/m3 for combined was
                                 occupational 1942-1990           exposure year most of the      significantly
                                 controls        n=879            26.8 year      period of       below 1.0 for
                                                                                 operation       both
                                                                                                 comparison
                                                                                                 populations.
  pidemiologic  Hydroquinone,    Occupational Workers in nine -                  Hydroquinone No significant Friedlander
nvestigations,  quinines and     exposure; study Eastman Kodak                   < 0.01 mg/cm3 excess of          et al. 198282
mortality,      other            period          Color Print and                 (1 sample)      mortality,
 ancer          chemicals        1964-1979       Processing                      Quinone         sickness-
ncidence,                                        laboratories in                 vapours         absence or
 ickness-                                        the US                          0.2-0.9 ppm     cancer
 bsence                                           n=478                          (8 samples)     incidence
                                                 Plant controls
                Benzene          Occupational Workers in          Medium 31      # 31 ppm        Trisomy of       Zhang et al.
                                 exposure,       Shanghai,        ppm 8-hr time (n= 21)          chromosome 1996149
                                 quantified by China              weighted       > 31 ppm        9 statistically
                                 using batches Controls 44        average        (n= 22)         significant
                                 and measuring Exposed 43                                        increased in the
                                 phenol in urine                                                 > 31 ppm group
                Hydroquinone,    Occupational Workers in a -                     -               Increased        Choudat et al.
                trimethyl-       exposure        large chemical                                  prevalence of 1988150
                hydroquinone,                    plant engaged                                   allergic
                retinene-                        in the synthesis                                symptoms
                hydroquinone                     of methionine                                   (dyspnoca and
                                                 and vitamins                                    reversible
                                                 Exposed 33                                      obstruction)
                                                 Not exposed 55
     SIR:standardised incidence ratio.
     Ochronosis: a brown, blue, or black pigmentation that develops in the skin, cartilage, and various organs of patients; the
     condition is often associated with alkaptonuria.143
     Skin lightening cosmetics based on different active ingredients have been studied. The side effects experienced are
     presented without splitting up. Therefore no conclusions with respect to hydroquinolone can be drawn.
     SMR:standardised mortality ratio.
  50          Hydroquinone and benzoquinone
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<pre>nnex F
     Animal data (in vivo)
     Animal data (in vivo) 151
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<pre> able F-1 Absorption, distribution, metabolism, and excretion.
Animal species               Test conditions                     Results                                   Reference
Dermal absorption
 at (F344) skin in vitro     Hydroquinone 5% aqueous solution Absorption rate through rat skin is 1.09    Barber et al.
                                                                 µg/cm2/h                                  199546
Human abdominal skin         Hydroquinone 5% aqueous solution    Absorption rate through human skin is     Barber et al.
n vitro                                                          0.522 µg/cm2/h                            199546
 oxicokinetics
 at (Sprague-Dawley), males [14C-]Hydroquinone 3, 30, or 200     The primary route of elimination was the  DiVincenzo et al.
                             mg/kg bw, single dose, by gavage    urine. Of the tissues examined highest    198448
                             (3 groups of animals)               concentrations in the liver and kidneys.
                             Or                                  The majority of the urinary metabolites
                             Hydroquinone (unlabelled) 200       was identified as glucuronide conjugates
                             mg/kg bw, 4 daily doses, by gavage, with a smaller portion as sulphate
                             followed by                         conjugates; only 1% was unchanged
                             [14C-]hydroquinone, 200 mg/kg bw,   hydroquinone.
                             single dose, by gavage (1 group of  After repeated dose there was a shift to
                             animals)                            glucuronide conjugates at the expense of
                                                                 sulphate conjugates.
 at (F344), males            [U-14C-]Hydroquinone 50 mg/kg       Absorption and elimination were rapid;    Fox et al. 1986
                             bw, single dose, gavage, iv, or it  distribution was similar for the three    (unpublished),
                             instillation (4 animals per         routes.                                   cited in OECD
                             treatment).                         Radioactivity in the blood was            199633
                             Of some groups, blood was collected associated initially with the plasma, but
                             for up to 8 h and other groups were by 4 h most of the radioactivity is
                             euthanized at 10, 20, 40, 60, 120,  associated with the red blood cells. Only
                             and 480 h.                          1% of the total radioactivity in plasma
                                                                 ultrafiltrate was unaltered hydroquinone.
                                                                 Glucuronide and GSH conjugates of
                                                                 hydroquinone were detected 40 min
                                                                 after gavage administration.
 at (F344), males and        [14C-]Hydroquinone 25 or 350        After oral administration, absorption     English et al.
emales                       mg/kg bw, po, single dose, or after was rapid. Blood concentrations after     1988
                             14 repeated doses of unlabelled     dermal application were generally below   (unpublished),
                             hydroquinone                        the limit of detection.                   cited in OECD
                             or [14C-]hydroquinone, 25 or 150    Elimination followed a biphasic           199633; English
                             mg/kg bw, dermal application, 24 h character and occurred within the first    & Deisinger
                             occlusive.                          8 h after oral administration. The        200549
                             (OECD 417)                          primary route of elimination was the
                                                                 urine. The liver and kidneys contained
                                                                 the highest concentrations.
                                                                 Approximately 45-53% of the urinary
                                                                 metabolites after an oral dose was the
                                                                 glucuronide conjugate and 19-33% was
                                                                 the sulphate conjugate. The parent
                                                                 compound was less than 3% of the dose.
 52          Hydroquinone and benzoquinone
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<pre>  at (F344), male                [14C]-Hydroquinone 1.8 mmol/kg,    Metabolites found in urine:               Lau et al. 199655
                                 po,                                hydroquinone-glucuronide,
                                 or 1.8 mmol/kg po, at day 15 after hydroquinone-sulphate, hydroquinone-
                                 14 daily doses of unlabelled       mercapturates [2-(GS-S-yl)HQ, 2,5-
                                 hydroquinone.                      bis(GS-S-yl)HQ, 2,6-bis(GS-S-yl)HQ,
                                                                    2,3,5-tris(GS-S-yl)HQ].
                                                                    Subchronic administration of
                                                                    hydroquinone increases the rate and
                                                                    extant by which hydroquinone is
                                                                    metabolized to its nephrotoxic thioethers
  at (Sprague-Dawley), male [14C]-Hydroquinone, 1.8 mmol/kg,        Metabolites detected in bile: 2-(GS-S-    Hill et al. 199341
                                 ip, after acivicin* pre-treatment  yl)HQ (18.9 ± 2.7 µmol), 2,5-bis(GS-S-
                                                                    yl)HQ (2.2 ± 0.6 µmol), 2,6-bis(GS-S-
                                                                    yl)HQ (0.7 ± 0.3 µmol), 2,3,5-tris(GS-S-
                                                                    yl)HQ (1.2 ± 0.1 µmol), and 2-(cystein-
                                                                    S-ylglycyl)hydroquinone.
                                                                    2-(N-Acetylcystein-S-yl)HQ was the
                                                                    only urinary thioether metabolite (11.4 ±
                                                                    3.6 µmol) identified. The quantity of S-
                                                                    conjugates excreted in urine and bile
                                                                    within 4 h of hydroquinone
                                                                    administration was 34.3 ± 4.5 µmol
                                                                    (4.3 ± 1.1% of dose).
Dog (Beagle), male               [14C-]Hydroquinone, 25 mL of a     No measurable radioactivity was found     Hamilton 1985
                                 4.5 g/L solution in water, dermal  in blood. Urinary excretion of            (unpublished),
                                 application, 60 min.               radioactivity was low with the peak       cited in OECD
                                 The solution was held in contact   between 24 and 48 h. The dermal           199633
                                 with the shaved skin for 60 min    absorption rate was calculated to be
                                 using absorption cells.            1.1/µg/cm2/h
HQ: hydroquinone
GS: glutathione moiety
p: intraperitoneal
v: intraveneous
t: intratracheal
 o: per os
  acivicin is an irreversible inhibitor of γ-glutamyltransferase
               Animal data (in vivo)                                                                                        153
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<pre> able F-2 Irritation and sensitisation studies.
Animal species                   Test conditions                    Result                                       Reference
 kin irritation / Depigmentation
Guinea pig (strain ns), female Hydroquinone; intracutaneous         Not primarily irritating                     Rajka & Blohm
                                 injections, 0.1 ml, 0.001%,                                                     197085
                                 0.01%, and 0.1% in water, 10
                                 days
Guinea-pig (black)               Hydroquinone; dermal               Skin irritation occurred with creams         Bleehen et al.
                                 application, creams containing     containing 5% or 10% hydroquinone. No        196886
                                 0%, 1%, 3%, 5%, 7% or 10%,         irritation was seen at levels of 3%
                                 5 days/week for one month.         hydroquinone. Weak to moderate
                                                                    depigmentation of the skin at all dose
                                                                    levels.
Guinea-pig (black), male and     Hydroquinone; dermal               The 0.1% dose caused marginal irritation;    Maibach & Patrick
emale                            administration in a hydrophilic    the 1.0% dose resulted in a slight to        1989, cited in
                                 ointment at concentrations of      marginal irritation in 30% of the animals    WHO, 199413
                                 0.1%, 1.0%, and 5.0%, 5 days/      (mainly females); the 5.0% dose induced
                                 week for 13 weeks.                 moderate to severe irritation and severe
                                                                    ulcerated inflammatory responses. At the
                                                                    5% dose, approximately 40% of the
                                                                    animals dosed showed moderate
                                                                    depigmentation of the skin (females only)
Guinea-pig (black), male and     Hydroquinone; dermal               Depigmentation, inflammatory changes          Jimbow et al.
emale                            application, creams containing     and thickening of the epidermis. A           197487
                                 2% or 5%, in an oil-water          marked reduction both in the numbers of
                                 emulsion, daily, 6 days/week       melanized melanosomes in the cells and
                                 for 3 weeks (n=24).                the number of actively functioning
                                                                    melanocytes
Guinea pig (strain ns), female   Benzoquinone; intracutaneous       0.1%: necrotic reactions                     Rajka & Blohm
                                 injections, 0.1 ml, 0.001%,        0.01%: redness and slight infiltration       197085
                                 0.01%, and 0.1% in water, 10       0.001%: not primarily irritating
                                 days
 ye irritation
 abbit (strain ns), sex ns       Hydroquinone crystals were         Eyrthrema of the nictitating membrane of     Toxicity report
                                 placed into the right eye (n=2). the unwashed eye persisted to 48 h after       Kodak 1971, cited
                                                                    instillation but was not observed 14 days    in OECD 199633
                                                                    after treatment
 abbit (strain ns), sex ns       Hydroquinone powder was            Most rabbits developed pigmentation, first   Ferraris de Gaspare
                                 instilled into the eyes daily, for in the conjunctiva and then in the cornea.   1949, cited in
                                 2-4 months, to the eyes of         Pigment formation appeared earlier in        WHO 199413
                                 rabbits which were kept in the animals exposed to light. Older animals
                                 dark, in sunlight, in normal       seemed more prone to develop pigment
                                 light, irradiated with UV light, than younger ones. Pigment was deposited
                                 or pre-sensitized with             in albino rabbit eyes as well as in those of
                                 haematoporphyrin and then          rabbits with normal pigmentation.
                                 kept under either reduced light
                                 or sunlight, respectively.
 abbit (strain ns), sex ns       Hydroquinone injections into The resultant reaction was graded 5 out of         Hughes 1948, cited
                                 the cornea, 0.1 ml, 0.012-0.05 the possible maximum of 100                      in WHO 199413
                                 mol/L
 54           Hydroquinone and benzoquinone
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<pre>Guinea-pig (strain ns)       Hydroquinone; 1-3 mg powder Immediate but transient irritation. During WHO 199413
                             was instilled into the eyes,    the second day of application a slight
                             twice daily for 9 weeks         corneal opacity was observed in some
                                                             animals, and on the third day opacity of
                                                             varying degrees occurred in most of the
                                                             animals. Ulcers appeared in two animals.
                                                             The eyes had fully recovered 3 days after
                                                             cessation of treatment
Dog (strain ns)              Hydroquinone; 2-5 mg powder Immediate but transient irritation and         Dreyer, 1940, cited
                             was instilled into the eyes,    lacrimation. Opacity of the cornea,        in WHO 199413
                             twice daily, 5 days/week for 9 lacrimation and redness of the conjunctiva
                             weeks                           were produced within 4 days, but no
                                                             ulcers were seen. The eye returned to
                                                             normal within 2 days after cessation of
                                                             treatment
 kin sensitisation
Guinea pig (strain ns)       Hydroquinone; induction with    Number of animals with skin reaction at    Rajka & Blohm
                             0.1 ml 0.001% by injection;     challenge: 4/18. Cross-reactivity with p-  197085
                             challenge with 0.001% by        phenylenediamine and benzoquinone.
                             injection (n=18)
                             (Intracutaneous sensitization)
Guinea pig (strain ns)       Hydroquinone; induction with    Number of animals with skin reaction at    Goodwin (1981)88
                             0.1 ml 2.0% by injection, or    challenge: 7/10; “strong” sensitizer
                             10% by patch; challenge with
                             5.0% by patch
                             (Maximization test in
                             compliance with OECD 406)
Guinea pig (strain ns)       Hydroquinone; induction with    Number of animals with skin reaction at    Goodwin 198188
                             2.5%, by injection; challenge   challenge: 3/10; “weak” sensitizer
                             with 1.0% by injection or 30%
                             by topical application.
                             (Draize test in compliance with
                             OECD 406)
Guinea pig (strain ns)       Hydroquinone; induction with    Number of animals with skin reaction at van der Walle et al.
                             0.5 mol/L (day 0) and 1 mol/L   challenge: 5/10 (day 21) and 5/10 (day 35) 198290
                             (day 7) by patch; challenge
                             with 0.125 mol/L (day 21) and
                             0.250 mol/L (day 35).
                             (Maximization test)
Guinea pig (strain ns)       Hydroquinone; induction with    Number of animals with skin reaction at    van der Walle et al.
                             5 x 0.1 ml 0.45 µmol/L and 5 x  challenge:                                 198290
                             0.5 mol/L by injection;         3/8 (day 21) and 4/8 (day 35)
                             challenge with 0.115 µmol/L     4/8 (day 21) and 4/8 (day 35)
                             and 0.125 mol/L by patch.
                             (Freund’s complete adjuvant
                             test)
Guinea pig (strain ns)       Hydroquinone; induction with    “Strong” sensitizer                        Basketter &
                             0.1 ml 2.0% by injection, or                                               Goodwin 198891
                             1% by patch; challenge with
                             5.0% by patch. N=10.
                             (Maximization test)
              Animal data (in vivo)                                                                                   155
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<pre>Guinea pig (strain ns)     Hydroquinone; induction with     “Weak” sensitizer                          Basketter &
                           0.1 ml 2.0% by injection;                                                   Goodwin 198891
                           challenge with 10% by patch.
                           N=10. (Modified single
                           injection adjuvant test)
Guinea pig (strain ns)     Hydroquinone; induction with     “Moderate” sensitizer                      Basketter &
                           1.0% by patch; challenge with                                               Goodwin 198891
                           20% by patch. N=10.
                           (Cumulative contact
                           enhancement test)
Guinea pig (strain ns)     Hydroquinone; induction with     “Extreme” sensitizer                       Basketter &
                           2.0% by injection, or 1.0% by                                               Scholes 199292
                           patch; challenge with 0.5% by
                           patch. N=ns. (Maximization
                           test),
Mouse (strain ns)          Hydroquinone; daily topical      Positive                                   Basketter &
                           applications on the dorsal                                                  Scholes 199292
                           surface of each ear, 0.5 to 2.5%
                           during 3 days. At day 4-5, i.v.
                           injection of [3H] methyl-
                           thymidine. Determination of
                           3H-incorporation in lymph
                           nodes 5 h later. N=4. (Local
                           lymph node assay)
Guinea pig (strain ns)     Benzoquinone; induction with     Number of animals with skin reaction at Rajka & Blohm
                           0.1 ml, 0.001% by injection;     challenge: 19/20. Cross-reactivity with p- 197085
                           challenge with 0.001% by         phenylenediamine.
                           injection (n=20)
                           (Intracutaneous sensitization)
Guinea pig (strain ns)     Benzoquinone; Guinea Pig         Cross-reactivity between p-                Möllgaard et al.
                           Maximization test, no data on    phenylenediamine and benzoquinone.         199094
                           concentrations used and the
                           time schedule for challenge
                           and induction
Guinea pig (strain ns)     Benzoquinone; induction with     “Extreme” sensitizer                       Basketter &
                           0.005% by injection, or 10%                                                 Scholes 199292
                           by patch; challenge with 2.5%
                           by patch. (Maximization test)
Mouse (strain ns)          Benzoquinone; daily topical      Positive                                   Basketter &
                           applications on the dorsal                                                  Scholes 199292
                           surface of each ear, 0.5 to 2.5%
                           during 3 days. At day 4-5, iv
                           injection of [3H] methyl-
                           thymidine. Determination of
                           3H-incorporation in lymph
                           nodes 5 h later (n=4).
                           (Local lymph node assay)
 ocal lymph node assay     -                                Hydroquinone EC3: 0.11%                    Roberts et al.
murine)                                                     Benzoquinone EC3: 0.01%                    200793
 s: not specified
 56           Hydroquinone and benzoquinone
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<pre> able F-3A Acute toxicity studies, hydroquinone.
Animal species                      Test conditions                        LD50             Reference
                                                                           (mg/kg bw)
Oral
  at (strain ns)                    Fasted / water *                       390              Hodge et al. 1949
Mouse (strain ns)                   (similar to OECD 401)                  680              (unpublished report), cited in
                                                                                            OECD 199633
  at (Priestly), sex ns             Unfasted / glycerin                    1,295            Carlson & Brewer 195396
                                    (similar to OECD 401)
Rat (Prague-Dawley), sex ns         Unfasted / propylene glycol            1,090            Carlson & Brewer 195396
                                    Unfasted / water                       1,182
                                    Unfasted / glycerine                   1,081
                                    Fasted / propylene glycol              323
                                    (similar to OECD 401)
  at (Wistar), sex ns               Unfasted / propylene glycol            731
                                    Fasted / propylene glycol              298
                                    (similar to OECD 401)
  at (Osborne-Mendel), male and     Fasted / water *                       302              Woodard 1951, cited in
emale                               (similar to OECD 401)                                   OECD 199633
Mouse (Swiss), male and female      Unfasted / water *                     390              Woodard 1951, cited in
                                                                                            OECD 199633
Dermal
Guinea pig (strain ns)              24 h, occlusive                        > 1,000          Toxicity report Kodak 1971,
                                    (similar to OECD 402)                                   cited in OECD 199633
  : carrier used
 s: not specified
 able F-3B Acute toxicity studies, benzoquinone.
Animal species                         Test conditions          LD50                  Reference
                                                                (mg/kg bw)
Oral
  at (strain ns)                       ns                       130                   Woodward et al. 1949, cited in
                                                                                      ACGIH 2001a2
Gastric intubation
  at (Sprague-Dawley), male            ns                       165                   Omaye et al. 198032
njection iv
  at (strain ns)                       ns                       25                    Woodward et al. 1949, cited in
                                                                                      ACGIH 2001a2
njection ip
Mouse, white (Beyers)                  ns                       8.5                   Serif & Seymour 196397
 s: not specified
p: intraperitoneal
v: intraveneous
               Animal data (in vivo)                                                                                  157
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<pre> able F-4 Short-term and subchronic toxicity studies.
Animal species       Test conditions                    NOAEL          LOAEL (mg/kg bw/day) /          Reference
                                                        (mg/kg bw/day) Critical effects
Oral
 at                  Hydroquinone                       20             64                              Bernard 1988
Sprague-Dawley)      0, 20, 64, 200 mg/kg bw/day, by                   Tremors and reduced             (unpublished),
                     gavage, 13 weeks, 5 days/week                     home-cage activity              cited in OECD
                                                                                                       199633
 at (F344)           Hydroquinone                       250            500                             NTP 198998, Kari
                     0, 63, 125, 250, 500, 1,000 mg/kg                 Tremors and decreased final     et al. 1992100
                     bw/day, by gavage (n=5/sex/dose),                 mean body weight
                     14 days, 5 days/week
Mouse (B6C3F1)       Hydroquinone                       125            250                             NTP 198998, Kari
                     0, 31, 63, 125, 250, 500 mg/kg                    Mortality, tremors, and         et al. 1992100
                     bw/day, by gavage (n=5/sex/dose),                 decreased final mean body
                     14 days, 5 days/week                              weight
Rat (F344)           Hydroquinone                       < 25           25                              NTP 198998, Kari
                     0, 25, 50, 100, 200, 400 mg/kg                    Decreased absolute and          et al. 1992100
                     bw/day, by gavage (n=10/sex/                      relative liver weights in males
                     dose), 13 weeks, 5 days/week                      (at all dose levels). Increased
                     (similar to OECD 408)                             absolute and relative liver
                                                                       weights in females at ≥ 100
                                                                       mg/kg bw/day.
Mouse (B6C3F1)       Hydroquinone                       < 25           25                              NTP 198998, Kari
                     0, 25, 50, 100, 200, 400 mg/kg                    Increased absolute and relative et al. 1992100
                     bw/day, by gavage (n=10/sex/                      liver weights in males (at all
                     dose), 13 weeks, 5 days/week                      dose levels).
                     (similar toe OECD 408)
Dermal
 at (F344)           Hydroquinone                       1,920          3,840                           NTP 198998, Kari
                     0, 240, 480, 960, 1,920, 3,840 mg/                Reduced body weight             et al. 1992100
                     kg bw/day in ethanol, 14 days, 12
                     doses
Mouse (B6C3F1)       Hydroquinone                       4,800          > 4,800                         NTP 198998, Kari
                     0, 300, 600, 1,200, 2,400, 4,800                                                  et al. 1992100
                     mg/kg bw/day in ethanol, 14 days,
                     12 doses
Rat (F344),          Hydroquinone                       5.0%           > 5.0%                          David et al. 199899
male and female      0, 2.0, 3.5, 5.0% in oil-in-water  (~74 mg        No overt toxicity (based on
                     emulsion,                          per kg bw/day) standard tox parameters,
                     13 weeks, 5 days/week                             BrdU-incorporation and
                     (similar to OECD 411)                             histopathology of the kidney)
 58         Hydroquinone and benzoquinone
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<pre>njection ip
Mouse (Swiss)       Benzoquinone                      <2          2                               Rao et al. 1988103
                    2 mg/kg bw/day, ip, 6 weeks, 6                Relative changes in organ
                    days/week                                     weights. Significant decreases
                                                                  in red blood cells and bone
                                                                  marrow cell counts and
                                                                  haemoglobin content.
                                                                  Histological injuries in liver,
                                                                  thymus, spleen, kidney and
                                                                  peripheral lymph nodes.
njection sc
 at                 Benzoquinone                      <7          7                               Rao et al. 1988103
                    25 mg/kg bw twice weekly, sc,                 Anaemia,
                    2.5-5 months (equivalent to 7 mg/             methaemoglobinemia,
                    kg bw/day)                                    decrease in serum albumin and
                                                                  serum cholinesterase activity
nhalation
 at                 Benzoquinone                      < 0.27-0.36 0.27-0.36 mg/m3                 Anonymous 2 (not
                    0.27-0.36 mg/m3 or 2.7-3.6        mg/m3       Thrombopenia. At 2.7-3.6        traceable, cited in
                    mg/m3, 4 months, 4 h/day                      mg/m3 weight lost, easy         MAK 2000102)
                                                                  tiredness, transient anaemia,
                                                                  thrombopenia
p: intraperitoneal
c: subcutaneous
 rdU: bromodeoxyuridine
              Animal data (in vivo)                                                                              159
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<pre> able F-5 Long-term toxicity and carcinogenicity studies.
Animal species Test conditions                  NOAEL          LOAEL (mg/kg bw/day) / Critical         Reference
                                                (mg/kg bw/day) effects
Oral
 at (F344)       Hydroquinone                   < 351          351 (0.8% in diet)                      Shibata et al.
                 0.8% in the diet [351 and 368                 Reduced body weight gain (males and     1991104
                 mg/kg bw/day, m and f]                        females). Higher absolute and relative
                 (n=30/sex/dose), 104 weeks                    liver and kidney weight in males.
                                                               Higher relative kidney weight in
                                                               females.
                                                               Chronic nephropathy was more severe
                                                               in males. In the kidneys of exposed
                                                               male rats, the incidence of tubule
                                                               hyperplasia was 30/30 and that of
                                                               adenomas was 14/30 (p< 0.01),
                                                               compared with 1/30 and 0/30,
                                                               respectively, in controls. Incidence of
                                                               other tumour types was not increased
                                                               by exposure
Mouse (B6C3F1) Hydroquinone                     < 1,046        1,046 (0.8% in diet)                    Shibata et al.
                 0.8% in the diet [1,046 and                   Reduced body weight gain (males).       1991104
                 1,486 mg/kg bw/day, m and f]                  The incidence of hepatocellular
                 (n=30/sex/dose), 94 weeks                     adenoma was increased to 14/30 in
                                                               exposed male (p<0.05) compared with
                                                               6/28 in controls. Incidence of other
                                                               tumour type was not significantly
                                                               increased by exposure of males,
                                                               although three renal adenomas
                                                               occurred. No increase in tumour
                                                               incidence was found in females.
 at (F344)       Hydroquinone                   < 25           25                                      NTP 198998,
                 0, 25, 50 mg/kg bw/day, by                    Mean body weights of both low and       Kari et al.
                 gavage (n=65/sex/dose),                       high dosed male rats were decreased.    1992100
                 2 year, 5 days/week                           The relative kidney and liver weights
                 (similar to OECD 451)                         of high dosed male rats were higher
                                                               than those of controls. Relative liver
                                                               weights were increased for dosed males
                                                               and high dosed females.
                                                               In exposed males, renal tubule cell
                                                               adenomas developed in 4/55 low-dose
                                                               group (p = 0.069) and 8/55 high-dose
                                                               group (p = 0.003) compared with 0/55
                                                               controls. In exposed females,
                                                               mononuclear cell leukaemia developed
                                                               in 15/55 low-dose group (p = 0.048)
                                                               and 22/55 high-dose group (p = 0.003)
                                                               compared with 9/55 controls.
 60         Hydroquinone and benzoquinone
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<pre>Mouse (B6C3F1) Hydroquinone                        < 50           50                                      NTP 198998,
                  0, 50, 100 mg/kg bw/day, by                     Mean body weights of high dosed         Kari et al.
                  gavage (n=65/sex/dose),                         males and females were decreased.       1992100
                  2 year, 5 days/week                             Relative liver weights were increased
                  (similar to OECD 451)                           for dosed males and high dosed
                                                                  females.
                                                                  In males and females, increased thyroid
                                                                  hyperplasia was found in 5/55 male and
                                                                  13/55 female controls, 15/53 male and
                                                                  47/55 female low-dose group (p<0.01),
                                                                  and 19/54 male and 45/55 female high-
                                                                  dose groups (p<0.01).
                                                                  In females, hepatocellular adenomas
                                                                  were found in 2/55 controls, 15/55 low-
                                                                  dose group (p<0.01) and 12/55 high-
                                                                  dose group (p<0.01). No increase in
                                                                  tumours was found in exposed males.
  at (F344)       Histopathological                < 25           25                                      Hard et al.
                  examination of kidney                           Hydroquinone-related adenoma            1997105
                  material from the study of                      formation may include enhancement of
                  NTP 198998                                      the severity of chronic progressive
                                                                  nephropathy coupled with stimulation
                                                                  of tubule proliferation
Mouse             Benzoquinone, gavage 2 mg; <2                   2                                       El-Mofty et al.
Swiss albino)     13 weeks, 2 days/week.                          Lympholeukaemias in liver and spleen    1992115
njection sc
  at (Wistar)     Benzoquinone, injection sc       -              Seventeen rats survived the injection   Umeda 1957114
                  0.5 ml of a 10 g/L solution                     period of 394 days. Three
                  (first 53 days), 0.5 ml of 2 g/L                fibrosarcomas developed in two rats at
                  (days 53-173) and 0.5 ml of 4                   the site of injection. No other tumours
                  g/L (days 173-294) (n=15+9)                     were found.
 c: subcutaneous.
 able F-6 Neurotoxicity.
Animal species Test conditions                     NOAEL             LOAEL (mg/kg bw/day) / Critical      Reference
                                                   (mg/kg bw/day)    effects
Oral
  at (Sprague-    Hydroquinone                     20                64                                   Bernard 1988
Dawley)           0, 20, 64, 200 mg/kg bw/day                        Tremors and reduced home-cage        (unpublished),
                  by gavage, 13 weeks, 5 days/                       activity were observed in mid-and    cited in OECD
                  week. Full battery of                              high-dose groups immediately after   199633
                  neurological tests                                 dosing with the incidence increased
                                                                     in a dose dependent manner.
              Animal data (in vivo)                                                                                   161
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<pre> able F-7 Reproduction toxicity (fertility and development).
Animal species      Test conditions                 NOAEL            LOAEL (mg/kg bw/day)                     Ref Reference
                                                    (mg/kg bw /day)  / Critical effects
  generation repro
  at                Hydroquinone                    General: 15      General F0 and F1: 50                    Blacker et al.
CD Sprague-         0, 15, 50, 150 mg/kg            Reproductive:    Mild, transient tremors (F0: 0/58, 0/58, 1993121
Dawley)             bw/day by gavage, until         150              1/58, 24/58; F1: 0/58, 0/58, 0/58, 21/
                    scheduled termination                            58) and decreased body weight.
                    (OECD 416)                                       Reproduction and fertility: > 150
                                                                     No treatment related effects
  at                Hydroquinone                    P generation: 15 General F0 and F1: 50                    Schroeder 1989
CD Sprague-         0, 15, 50, 150 mg/kg            F1 generation:   Transient tremors (no quantitative       (unpublished),
Dawley)             bw/day by gavage, until         150              data)                                    cited in OECD
                    scheduled termination           F2 generation:   Reproduction and fertility: > 150        199633
                                                    150              No treatment related effects
Developmental
  abbit             Hydroquinone                    Maternal: 25     Maternal: 75                             Murphy et al.
New Zealand         0, 25, 75, 150 mg/kg            Development: 75 Decreased food consumption                1992123
White)              bw/day by gavage (18                             Development: 150
                    mated/dose), GD 6-18,                            Minimal developmental alterations
                    section on GD 30 (OECD
                    414)
Rat                 Hydroquinone                    Maternal: 100    Maternal: 300                            Krasavage et al.
 COBS-CD-BR)        0, 30, 100, 300 mg/kg           Development:     Decreased food consumption and body 1992122
                    bw/day by gavage, GD 6-15       100              weight gain
                    (OECD 414)                                       Development: 300
                                                                     Decreased body weight
 62          Hydroquinone and benzoquinone
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<pre> able F-8 Mechanistic studies in vivo.
Animal species           Test conditions                               Results / Conclusions                 Reference
 arcinogenesis, benzoquinone after initiation
Mouse (albino “S”),      Benzoquinone                                  No tumours or hyperplasia of the      Gwynn &
emale                    0, 3.6 g/L, dermal application, weekly for 27 epidermis                             Salaman 1953116
                         weeks, three weeks after initiation with
                         0.15% 7,12-dimethylbenzanthracene (n=19)
Mouse (Sencar), female Benzoquinone,                                   No signs of possible promoter         Monks et al.
                         0, 444, 880 or 1,760 nmol/mouse, dermal       capacity                              1990117
                         application, weekly for 31 weeks, two weeks
                         after initiation with 25 nmol 7,12-
                         dimethylbenzanthracene (n=25)
 arcinogenesis, hydroquinone after initiation
 at (Fischer 344), male Hydroquinone,                                  Hydroquinone alone induced no         Miyata et al.
                         0 or 0.2% in the diet for 22 weeks, after two bladder lesions. When                 1985, cited in
                         weeks 0 or 0.05% N-nitrosobutyl-N-(4-         hydroquinone was given after          IARC 1999a8
                         hydroxybutyl)amine in the drinking-water      initiation, no increase in bladder
                         followed by uteric ligation one week later.   lesions was observed
                         Animals were killed at week 24 (n=15)
 at (Fischer 344), male Hydroquinone,                                  The body weights of rates given       Hirose et al.
                         0 or 0.8% in the diet for 51 weeks, one week  hydroquinone after initiation were    1989, cited in
                         after exposure to 0 or 150 mg/kg bw           lower than those given initiator      IARC 1999a8
                         N-methyl-N’-nitro-N-nitrosoguanidine by       only. Hydroquinone alone did not
                         oral gavage (n=10-16)                         induce forestomach lesions, nor did
                                                                       it enhance the incidence of
                                                                       forestomach or glandular stomach
                                                                       lesions induced by the initiator
 at (Sprague-Dawley),    Hydroquinone,                                 In the hepatectomized group           Stenius et al.
male                     0, 100 or 200 mg/kg in the diet for six weeks exposed only to hydroquinone, no      198947
 irst experiment         one week after partial hepatectomy and ip     liver enzyme-altered (γ-GT) foci
                         injection of 300 mg/kg N-                     were induced. Hydroquinone after
                         nitrosodiethylamine One group underwent       initiation increased the multiplicity
                         only partial hepatectomy and was fed the      of foci from 0.08 per cm2 to 0.68 in
                         high dose of hydroquinone (n=7-10)            the low-dose group and to 0.34 in
                                                                       the high-dose group
 at (Sprague-Dawley),    Hydroquinone,                                 Hydroquinone did not increase the     Stenius et al.
male                     0 or 1 mg/kg bw by oral gavage, five days     multiplicity of enzyme-altered foci,  198947
 econd experiment        per week for seven weeks after the regimen but their area was increased from a
                         to initiate liver carcinogenesis (n=10)       mean of 1.00x10-4 cm2 to 1.30x10-4
                                                                       cm2 (p<0.05) and their volume from
                                                                       1.49x10-4 cm3 to 3.12x10-4 cm3
                                                                       (p<0.001)
             Animal data (in vivo)                                                                                         163
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<pre>at (Fischer 344), male Hydroquinone,                               Hydroquinone alone reduced              Yamaguchi et al.
                       0 or 0.8% in the diet for 49 weeks either   weight gain. In animals given           1989111
                       alone or starting one week after six        hydroquinone after carcinogen, the
                       intraperitoneal injections of 25 mg/kg bw   incidence of oesophageal carcinoma
                       N-nitrosomethyl-n-amylamine (n=12-15)       was 4/12 rats (not significant)
                                                                   compared with 0/11 in the group
                                                                   given initiator only, and the
                                                                   multiplicity was increased to 0.33
                                                                   tumours per rats (p<0.05) compared
                                                                   with 0 in the controls
at (Fischer 344/Du     Hydroquinone,                               Body weight was reduced by              Hasegawa et al.
rj), male              0 or 0.8% in the diet for 30 weeks either   hydroquinone given after the            1990112
                       alone or after exposure to 0.1% N-nitroso-  initiator and liver weight was
                       bis(2-hydroxypropyl)amine in the drinking   increased compared with the group
                       water for two weeks (n=10 or 20). No        given initiator only. Hydroquinone
                       unexposed controls were included.           alone induced no lung or thyroid
                                                                   tumours. Rats given initiator
                                                                   developed low incidences of
                                                                   tumours in the thyroid, lung, urinary
                                                                   bladder and kidney. None of these
                                                                   incidences was increased by
                                                                   hydroquinone
at (Fischer 344), male Hydroquinone,                               Hydroquinone alone did not affect       Kurate et al.
                       0 or 0.8% in the diet for 36 weeks either   body weight or bladder weight.          1990, cited in
                       alone or after exposure to 0.05% N-         Hydroquinone exposure alone did         IARC 1999a8
                       nitrosobutyl-N-(4-hydroxybutyl)amine in the not induce bladder tumours and
                       drinking water for four weeks (n=10 or 20) feeding of hydroquinone after
                                                                   initiator did not increase the
                                                                   incidence or multiplicity of bladder
                                                                   neoplasms induced by the initiator
                                                                   alone
at (Wistar/Crj), male  Hydroquinone,                               Hydroquinone alone resulted in          Okazaki et al.
                       0 or 0.8% in the diet for 36 weeks alone or decreased final body weights            1993, cited in
                       one week after exposure to 0.1% N-          compared to controls (basal diet and    IARC 1999a8
                       nitrosoethyl-N-hydroxyethylamine in the     initiator) and increased relative liver
                       dinking water for three weeks (n=15 or 20) and kidney weights compared to the
                                                                   basal diet group. Hydroquinone
                                                                   alone did not induce preneoplastic
                                                                   or neoplastic liver or kidney lesions.
                                                                   In the kidney, hydroquinone
                                                                   exposure after initiation increased
                                                                   the multiplicity of renal cell
                                                                   tumours to 5.22 per rat (p<0.01)
                                                                   compared with 2.58 after initiation
                                                                   only, and increased the multiplicity
                                                                   of microadenomas to 2.77 (p<0.05)
                                                                   compared with 0.94 after initiation
                                                                   only
64          Hydroquinone and benzoquinone
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<pre>Hamster (Syrian golden), Hydroquinone,                                 Hydroquinone alone did not affect Maruyama et al.
emale                    0 or 1.5% in the diet for 16 weeks either     body weights or liver or pancreas     1991113
                         alone or after two subcutaneous injections of weights compared with untreated
                         50 mg/kg bw N-nitrosobis                      controls. Given after initiation,
                         (2-oxopropyl)amine (n=10-20)                  hydroquinone did not affect body
                                                                       weight or liver weight, but reduced
                                                                       pancreas weight compared with
                                                                       animals given initiator only.
                                                                       Hydroquinone alone did not induce
                                                                       neoplastic lesions in the pancreas or
                                                                       liver. In animals given
                                                                       hydroquinone after initiation, the
                                                                       multiplicity of pancreatic lesions
                                                                       was reduced
 ephrotoxicity and carcinogenicity
 at (F344, Sprague-      Hydroquinone,                                 Nephrotoxicity and renal tubule cell Boatman
Dawley)                  200 or 400 mg/kg bw, single dose by gavage    proliferation in F344 rats at ≥ 200 1996151
                         (n=4-6)                                       mg/kg bw. Females were more
Mouse (B6C3F1)           Hydroquinone,                                 sensitive than males. Sprague-
                         350 mg/kg bw, single dose by gavage           Dawley rats and mice were not/
                         (n=4-6)                                       hardly susceptible
 at (F344), male         Hydroquinone,                                 Effects were seen at ≥ 1.8 mmol/kg    Peters et al.
                         single dose, po in corn oil                   Tremors after dosing.                 199777
                         1.8 mmol/kg (n=3)                             Nephrotoxicity in some rats
                         4.5 mmol/kg (n=3)                             accompanied with elevation of γ-
                         4.5 mmol/kg, 1 h after 10 mg/kg acivicin ip   GT, ALP, GST, and glucose.
                         (n=3)                                         Selectively toxic to the proximal
                         Controls (corn oil; n=3)                      tubular cells of the kidney and
                                                                       injury at the junction of the
                                                                       medullary rays and the OSOM.
                                                                       Increased cell proliferation. Steep
                                                                       dose response curve. γ-GT
                                                                       inhibition by acivicin strongly
                                                                       inhibited the hydroquinone-
                                                                       mediated kidney toxicity
 at (F344), male         2,3,5-tris(GS-S-yl)HQ,                        Nephrotoxicity evidenced by the       Peters et al.
                         7.5 µmol/kg, single dose, iv (n=3)            urinary excretion of γ-GT and ALP     199777
                                                                       (brush-border enzymes), GST
                                                                       (indicator of the loss of cell
                                                                       membrane integrity) and decreased
                                                                       glucose excretion (indicating loss of
                                                                       renal function). The primary site
                                                                       was the OSOM. Single cell and
                                                                       tubular necrosis and necrosis at the
                                                                       junctions of the medullary rays and
                                                                       the OSOM. Increased cell
                                                                       proliferation
             Animal data (in vivo)                                                                                         165
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<pre>  ker rat,             2,3,5-tris(GS-S-yl)HQ,                   Effects after 4 months: increased    Lau et al. 200178
mutant Tsc-2Ek/+       2.5 µmol/kg bw/day ip for 4 months       cell proliferation (determined by
                       followed by 3.5 µmol/kg bw/day ip for 6  BrdU-incorporation) in the kidney.
                       month, 5 days/week.                      Numerous toxic tubular dysplasias.
                       Treated n=10, controls n=20              Increased number of basophilic
                                                                dysplasias.
                                                                After 12 months: significant
                                                                increase in the number of renal
                                                                tumours. Loss of the normal Tsc-2
                                                                allele in 12/12 tumours investigated
  at (F344), male and  Hydroquinone,                            Increased NAG excretion in males     English et al.
emale                  0, 2.5, 25, 50 mg/kg bw/day by gavage, 5 of the 50 mg/kg bw/day dosed         1994b80
                       days per week, 6 weeks                   group indicating mild kidney
                                                                toxicity.
                                                                Hydroquinone does not produce
                                                                covalent DNA adducts in the
                                                                kidneys, suggesting a nongenotoxic
                                                                aetiology of tumours in the kidney.
Rat (F344), male and   Hydroquinone,                            Hydroquinone induces cell            English et al.
 emale                 0, 2.5, 25, 50 mg/kg bw/day by gavage, 5 proliferation (determined by BrdU-   1994a58
                       days per week, 1, 3 or 6 weeks (n=5/sex/ incorporation) and nephrotoxicity
                       dose),                                   (determined by urinalysis and
  at (Sprague-Dawley), Hydroquinone,                            histopathology) in male F344 rats
male                   0, 50 mg/kg bw, by gavage,5 days/week, 6 (50 mg/kg), but not in female F344
                       weeks (n=5/dose)                         or male SD rats. Hydroquinone
                                                                induced cell proliferation might be
                                                                secondary to toxicity.
Heamatotoxicity
  at (Sprague-Dawley), Hydroquinone,                            2-(GS-S-yl)HQ, 2,3,5-tris(G-S-       Bratton et al.
male                   0.9 mmol/kg bw + phenol, 1.1 mmol/kg bw, yl)HQ, 2,5-bis(G-S-yl)HQ and 2,6-    199762
Mouse (DBA/2), male    ip, single dose (co-administration)      bis(GS-S-yl)HQ were detected in
                                                                bone marrow, reaching maximum
                                                                values at 15 minutes. The γ-GT
                                                                metabolite 2-(Cys-Gly)HQ, the
                                                                dipeptidase metabolite 2-(Cys)HQ,
                                                                and the mercapturic acid metabolite
                                                                2-(NAC)HQ reached maximum
                                                                levels at 30 min
  at (Sprague-Dawley), 2,3,5-tris(GS-S-yl)HQ,                   Effects 2,3,5-tris(GS-S-yl)HQ at     Bratton et al.
male                   17µmol/kg bw, single dose iv             17µmol/kg bw and of 2,6-bis(GS-S-    199762
                       2,6-bis(GS-S-yl)HQ,                      yl)HQ at 50 µmol/kg bw:
                       50 µmol/kg bw, single dose iv            Significant (45 and 28 %,
                                                                respectively) reducing of 59Fe
                                                                incorporation into immature
                                                                erythrocytes
  66          Hydroquinone and benzoquinone
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<pre>Mouse (Swiss, albino)       Hydroquinone,                                    59Fe  uptake is significantly       Guy et al.
emale                       25, 50, 75, 100 mg/kg bw ip, 3 doses, at t = 0,  decreased (25-100 times less potent 1991128
                            16, 24 h59                                       than benzoquinone)
                            Fe was administered at t = 64 h
Mouse (Swiss, albino)       Benzoquinone,                                    59Fe uptake is 46% decreased at
emale                       0.5, 1.0, 2.0, 3.0, 4.0 mg/kg bw ip, 3 doses, at 3.0 mg/kg
                            t = 0, 16, 24 h.59
                            Fe was administered at t = 64 h
 roliferation/differentiation disturbing of heamapoetic cells
Mouse (C57BL6)              Hydroquinone,                                    Doubling of GM-CFC per femur,       Henschler et al.
                            75 mg/kg bw, twice daily for 11 days (n=6)       hydroquinone induce an increase in 1996127
                                                                             total GM-CFC in the bone marrow
                                                                             of mice in vivo (and in vitro;
                                                                             see Table F-3)
 ,3,5-tris(GS-S-yl)HQ:           2,3,5-tris(glutathione-S-yl)hydroquinone
 ,5-bis(GS-S-yl)HQ:              2,5-bis(glutathione-S-yl)hydroquinone
 ,6-bis(GS-S-yl)HQ:              2,6-bis(glutathione-S-yl)hydroquinone
 -(GS-S-yl)HQ:                   2-(glutathione-S-yl)hydroquinone
Acivicin:                        γ–GT inhibitor
ALP:                             alkaline phosphatase
ALT:                             alanine transaminase
AST:                             aspartate transaminase
  rdU:                           bromodeoxyuridine
  UN:                            blood urea nitrogen
GM-CFC:                          granulocyte-macrophage colony forming cells
GST:                             glutathione S-transferase
 –GT:                            γ-glutamyl transpeptidase
p:                               intraperitoneal
v:                               intravenous
NAG:                             N-acetyl-β-D-glucosaminidase
 o:                              per os
OSOM:                            outer stripe of the outer medulla
 sc-2:                           tuberous sclerosis-2
             Animal data (in vivo)                                                                                           167
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<pre>68 Hydroquinone and benzoquinone</pre>

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<pre>  nnex        G
              In vitro data
 roliferation/differentiation disturbing of heamapoetic cells.
 ystem / cell Test conditions                           Results / conclusions                                        Reference
ine
 DCP-mix        Hydroquinone, 10-12 – 10-5 M            Almost a doubling of GM-CFC between 10-9 -10-6 M,            Henschler
 ells                                                   hydroquinone induced an increase in the number of GM-        et al. 1996127
                                                        colonies in vitro
n situ perfused 2,3,5-(TriGSyl)HQ, 2-(GSyl)HQ,          5 and 10 µmol 2,3,5-(triGSyl)HQ and 40 µmol                  Hill et al.
at kidney       infusion into the right renal artery    2-(GSyl)HQ caused a time-dependent elevation in γ-GT.        199479
                                                        Metabolites of 2,3,5-(triGSyl)HQ (10 µmol) were observed
                                                        in urine and bile only within the first 30 min of perfusion.
                                                        At the lower dose (5 µmol) neither parent compound nor
                                                        metabolites were found.
                                                        After 2-(GSyl)HQ perfusion, 2-(N-acetyl-cystein-S-yl)HQ
                                                        (9.2 ± 0.5 µmol). 2-(cystein-S-ylglycine)HQ
                                                        (0.8 ± 0.3µmol), and 2-(cystein-S-yl)HQ (1.3 ± 0.3 µmol)
                                                        were detected in urine and bile. Unchanged 2-(GSyl)HQ
                                                        was also detected (0.8 ± 0.1 µmol). A greater fraction of
                                                        the dose was retained by the kidney following treatment
                                                        with 10 µmol 2,3,5-(triGSyl)[14C]HQ than following
                                                        treatment with 40µmol 2-(GSyl)[14C]HQ (36 and 11%,
                                                        respectively).
 ,3,5-(triGSyl)HQ:               2,3,5-(triglutathione-S-yl)hydroquinone
 -(GSyl)HQ:                      2-(glutathione-S-yl)hydroquinone
 DCP:                            factor-dependent cells Paterson
GM-CFC:                          granolocyte-macrophage colony forming cells
 -GT:                            γ-glutamyl transpeptidase
              In vitro data                                                                                                     169
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<pre>70 Hydroquinone and benzoquinone</pre>

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<pre>nnex H
     Mutagenicity studies
     Mutagenicity studies 171
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<pre> able H-1 Genetic and related effects of hydroquinone (adapted from IARC, 1999a8).
 est system                                          Results                    Dose         Reference
                                                     Exogenous metabolic system (LED or HID)
                                                     without       with
DNA strand breaks / SCEs
Mammalian cells, in vitro
DIA, DNA strand breaks, cross-links or related       -             n.t.         11           Pellack-Walker & Blumer
 amage, LYS mouse lymphoma cells, alkaline elution                                           1986
DIA, DNA strand breaks, isolated rat hepatocytes     +             n.t.         33           Walles 1992
 ells, alkaline elution
DIH, DNA strand breaks, cross-links or related       ?             +            11           Anderson et al. 1995
 amage, human lymphocytes (comet assay)
DIH, DNA strand breaks, human promyelocytic HL60 +                 n.t.         1.1          Hiraku & Kawanishi 1996
 ells, pulse field electrophoresis
DNA single-strand breaks on supercoiled Bluescript -               +            11           Schlosser et al. 1990
 lasmid DNA
 IC, SCE, CHO cells                                  +             +            0.5          Galloway et al. 1987
 IS, SCE, Syrian hamster embryo cells                +             n.t.         0.11         Tsutsui et al. 1997
 HL, SCE, human lymphocytes                          +             n.t.         4.4          Morimoto & Wolff 1980
 HL, SCE, human lymphocytes                          +             +            110          Morimoto et al. 1983
 HL, SCE, human lymphocytes                          +             n.t.         6            Erexson et al. 1985
 HL, SCE, human lymphocytes                          ?a            n.t.         4.4          Knadle 1985
Mammalian cells, in vivo
 VA, SCE, (C57BL/Cnc x C3H/Cn3)F1 mouse bone -                                  120 ip x 1   Pacchierotti et al. 1991
marrow
DNA adduct formation
Mammalian cells, in vitro
BID, binding (covalent) to DNA, mouse P388D1 cells +               n.t.         5.5          Kalf et al. 1990
  ID, binding (covalent) to calf thymus DNA          +             n.t.         5.5          Leanderson & Tagesson
                                                                                             1990
  ID, binding (covalent) to DNA, cultured rat Zymbal +             n.t.         750          Reddy et al. 1990
 lands
  ID, binding (covalent) to calf thymus DNA          -             +b           11           Schlosser et al. 1990
BID, binding (covalent) to DNA, human                +             n.t.         5.5          Lévay et al. 1991
 romyelocytic HL-60 cells
BID, binding (covalent) to DNA, male B6C3F1 mouse +                n.t.         11           Lévay et al. 1993
 one-marrow cells
BID, binding (covalent) to DNA, human bone-          +             n.t.         11           Lévay et al. 1993
marrow macrophages
BID, binding (covalent) to DNA, human                +             n.t.         27.5         Pathak et al. 1995
 romyelocytic HL-60 cells
BID, binding (covalent) to DNA, B6CeF1 mouse bone +                n.t.         27.5         Pathak et al. 1995
marrow
  ID, binding (covalent) to DNA, human               +             n.t.         5.5          Lévay & Bodell 1996
 romyelocytic HL-60 cells
 72            Hydroquinone and benzoquinone
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<pre>  inding (covalent) to porcine brain tubulin [porcine -     n.t. 2,750          Brunner et al. 1991
 rain tubulin assembly assay]
Mammalian cells, in vivo
  VD, binding (covalent) to DNA, Sprague-Dawley       -          150 po x 4     Reddy et al. 1990
at Zymbal gland, liver or spleen
  VD, binding (covalent) to DNA, Fischer 344 rat      -          50 po, 5 d/wk, English et al. 1994b
 idneys                                                          6 wk
Clastogenic effects
Mammalian cells, in vitro
MIA, micronucleus test, CL-1 cells                    +c    n.t. 1              Antoccia et al. 1991
MIA, micronucleus test, V79 cells                     (+)   n.t. 31.6           Seelbach et al. 1993
MIA, micronucleus test, V79 cells                     +     n.t. 2.8            Ellard & Parry 1993
MIA, micronucleus test, XEM2 cells                    +     n.t. 2.8            Ellard & Parry 1993
MIA, micronucleus test, SD1 cells                     +     n.t. 2.8            Ellard & Parry 1993
MIA, micronucleus test, V79 cells                     NT    +d   11.5           Dobo & Eastmond 1994
CIC, chrom. ab., CHO cells                            -     +    450            Galloway et al. 1987
CIS, chrom. ab., Syrian hamster embryo cells          +     n.t. 3.3            Tsutsui et al. 1997
MIH, micronucleus test, human lymphocytes             +     n.t. 2.8            Yager et al. 1990
 kinetochore-positive)
MIH, micronucleus test, human lymphocytes             +     n.t. 8.2            Robertson et al. 1991
 kinetochore-positive)
MIH, micronucleus test, human lymphocytes             ?     ?    1              Van Hummelen & Kirsch-
                                                                                Volders 1992
MIH, micronucleus test, human lymphocytes             (+) e n.t. 20             Ferguson et al. 1993
MIH, micronucleus test, human lymphocytes             -     +    50             Vian et al. 1995
CHL, chrom. abb. human lymphocytes (FISH)             +     n.t. 11             Eastmond et al. 1994
Mammalian cells, in vivo
MVM, micronucleus test, NMRI mouse bone marrow +                 50 sc x 6      Tunek et al. 1982
MVM, micronucleus test, S mouse bone marrow           +          80 ip x 1      Ciranni et al. 1988
MVM, micronucleus test, S mouse bone marrow           (+)        80 po x 1      Ciranni et al. 1988
MVM, micronucleus test, (101/E1 x C3H/E1)F1           +          50 ip x 1      Adler & Kliesch 1990
mouse bone marrow
MVM, micronucleus test, (101/E1 x C3H/E1)F1,          +          15 ip x 3      Adler & Kliesch 1990
mouse bone marrow
MVM, micronucleus test, Swiss CD-1 mouse bone (+)                60 ip x 1      Barale et al. 1990
marrow
MVM, micronucleus test, (102/E1 x C3H/E1)F1,          +          50 ip x 1      Adler et al. 1991
mouse bone marrow
MVM, micronucleus test, (102/E1 x C3H/E1)F1,          +c         100 ip x 1     Miller et al. 1991
mouse bone marrow
MVM, micronucleus test, (C57BL/Cnc x C3H/Cne)F1 +                40 ip x 1      Pacchierotti et al. (1991)
mouse bone marrow
MVM, micronucleus test, Swiss CD-1 mouse bone +                  20 ip x 1      Marrazinni et al. 1994a
marrow
MVM, micronucleus test, Swiss CD-1 mouse bone +                  80 ip x 1      Marrazinni et al. 1994b
marrow
              Mutagenicity studies                                                                     173
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<pre>MVM, micronucleus test, CD-1 mouse bone marrow    +      60 ip x 3     Chen & Eastmond 1995a
  BA, chrom. ab., (102/E1 x C3H/E1)F1 mouse bone  +      75 ip x 1     Xu & Adler 1990
marrow
  BA, chrom. ab., Swiss CD-1 mouse bone marrow    +      80 ip x 1     Marrazinni et al. 1994b
  CC, chrom. ab. (102/E1 x C3H/E1)F1 mouse        +      40 ip x 1     Ciranni & Adler 1991152
 permatocytes treated
  GG, chrom. ab. (102/E1 x C3H/E1)F1 mouse        +      40 ip x 1     Ciranni & Adler 1991152
 permatogonia treated
DNA mutation
 acteria
 RB, SOS repair activity, S. typhimurium TA1535/  - -    3,300         Nakamura et al. 1987
 SK1002, umu test
 A0, S. typhimurium TA100, rev. mut.              - -    333           Haword et al. 1983
 A0, S. typhimurium TA100, rev. mut.              - -    125           Sakai et al. 1985
 A5, S. typhimurium TA1535, rev. mut.             - -    333           Haword et al. 1983
 A7, S. typhimurium TA1537, rev. mut.             - -    333           Haword et al. 1983
 A9, S. typhimurium TA98, rev. mut.               - -    333           Haword et al. 1983
 A9, S. typhimurium TA98, rev. mut.               - -    125           Sakai et al. 1985
 AS, S. typhimurium TA97, rev. mut.               - -    125           Sakai et al. 1985
 A2, S. typhimurium TA102, rev. mut.              + n.t. NG            Hakura et al. 1996
 A4, S. typhimurium TA104, rev. mut.              + n.t. 25            Hakura et al. 1996
 CG, S. cerevisiae MP1 gene conversion            + n.t. 1,320         Fahrig 1984
 CH, S. cerevisiae MP1 homozygosis by mitotic     - n.t. 1,320         Fahrig 1984
ecombination or gene conversion
 CF, S. cerevisiae MP1, for. mut.                 + n.t.               Fahrig 1984
nsect cells
DMX, D. melanogaster, sex-linked rec. lethal mut. ?      28,000 ppm    Foureman et al. 1994
                                                         feed
DMX, D. melanogaster, sex-linked rec. lethal mut. -      1,500 ppm inj Foureman et al. 1994
                                                         x1
Mammalian cells, in vitro
G5T, Gene mutation, mouse lymphoma L5178Y cells, +  n.t. 2.5           McGregor et al. 1988a,b
k locus
GIA, Gene mutation, Syrian hamster embryo cells, +  n.t. 1.1           Tsutsui et al. 1997
 prt locus
GIA, Gene mutation, Syrian hamster embryo cells, +  n.t. 1.1           Tsutsui et al. 1997
 uabain resistance
Aneuploidy induction
Mammalian cells, in vitro
AIA, aneupoidy, DON:Wg3h Chinese hamster cells, +   n.t. 10            Warr et al. 1993
 islocating metaphase chromosomes
AIA, aneupoidy, LUC2 Chinese hamster cells        - n.t. 5             Warr et al. 1993
AIA, aneupoidy, Syrian hamster embryo cells       - n.t. 3.3           Tsutsui et al. 1997
 CS, cell transformation, Syrian hamster embryo   + n.t. 0.33          Tsutsui et al. 1997
 ells, clonal assay
AIA, aneupoidy, human lymphocytes MN multicolour +  n.t. 8.3           Eastmond et al. 1994
 hrom. staining (FISH)
 74            Hydroquinone and benzoquinone
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<pre>CR, Inhibition of intercellular communication,          +              n.t.             0.055           Vang et al. 1993
V79MZ Chinese hamster cells
nhibition of assembly of bovine microtubules            (+)            n.t.             110             Wallin & Hartley-Hasp
                                                                                                        1993
Mammalian cells, in vivo
 VA, aneuploidy, (102/E1xC3H/Ea)F1 mouse bone           -                               100 ip x 1      Xu & Adler 1990
marrow polyploidy
 VA, aneuploidy, (C57BL/Cnc x C3H/Cne)F1 mouse          +                               80 ip x 1       Pacchierotti et al. 1991
 one marrow hyperploidy
 VA, aneuploidy, (C57BL/Cnc x C3H/Cne)F1 mouse          -                               120 ip x 1      Pacchierotti et al. 1991
 one marrow polyploidy
 VA, aneuploidy, (C57BL/Cnc x C3H/Cne)F1 mouse          +                               80 ip x 1       Leopardi et al. 1993152
 permatocytes hyperploidy
 VA, aneuploidy, Swiss CD-1 mouse bone marrow           -                               80 ip x 1       Marrazinni et al. 1994b
 olyploidy
 VA, aneuploidy, Swiss CD-1 mouse bone marrow           +                               80 ip x 1       Marrazinni et al. 1994b
 yperploidy
 VA, aneuploidy, CD-1 mouse bone marrow, MN             +                               60 ip x 3       Chen & Eastmond 1995a
multicolour chromosome staining (FISH)
 ; (+); -; ?:            positive; weakly positive; negative; inconclusive
 ED; HID:                lowest effective dose; highest ineffective dose (in vitro tests: µg/mL; in vivo tests: mg/kg bw/day)
 .t.; n.g.:              not tested; not given
nj.; ip; sc; po:         injection; intraperitoneral; subcutaneous; oral
ev. mut.; for. mut.:     reverse mutation; forward mutation
 CE:                     sister chromatid exhange
V79 cells:               Chinese hamster lung V79 cells
  HO cells:              Chinese hamster ovary cells
  L-1 cells :            Chinese hamster embryonic lung CL-1 cells
XEM2 cells :             Chinese hamster V79 exp CYP1A1 cells
 D1:                     Chinese hamster V79 exp CYP2B1 cells
 Positive if glutathione was depleted with diethyl maleate
 With prostaglandin H synthetase for oxidation
 No increase in % kinetochore-positive micronuclei compared with controls.
 Supplemented with arachidonic acid; increase in both kinetochore-positive and
  -negative micronucleated cells compared with controls (CREST-labelling procedure).
 Size ratio of micronuclei to nucleus was not significantly different from controls.
               Mutagenicity studies                                                                                            175
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<pre> able H-2 Genetic and related effects of benzoquinone (adapted from IARC, 1999b9).
 est system                                               Results                 Dose            Reference
                                                          Exogenous metabolic     (LED or HID)
                                                          system
                                                          without     with
DNA strand breaks / SCEs
Mammalian cells, in vitro
DIA, DNA strand breaks, mouse lymphoma L5178YS cells +                n.t.        0.11            Pellack-Walker &
                                                                                                  Blumer 1986
DIH, DNA strand breaks, cross-links or related damage,    -           +           11              Anderson et al. 1995
 uman lymphocytes (comet assay)
 IC, SCE, V79 cells                                       -           n.t.        11              Ludewig et al.
                                                                                                  1989154
 HL, SCE, human lymphocytes                               +           n.t.        0.55            Erexson et al. 1985
DNA adduct formation
  o IARC data available
Clastogenic effects
Mammalian cells, in vitro
MIA, micronucleus test, V79 cells                         +           n.t.        5.4             Ludewig et al.
                                                                                                  1989154
MIA, micronucleus test, V79, IEC-17 and 18 cells          +           n.t.        0.01            Glatt et al. 1990
MIH, micronucleus test, HuFoe-15 embryonal human liver    +           n.t.        0.01            Glatt et al. 1990
 ells
MIH, micronucleus test, human lymphocytes                 +           n.t.        0.275           Yager et al. 1990
Mammalian cells, in vivo
MVM, micronucleus test, pregnant CD-1 mouse bone-         (+)                     20 po x 1       Ciranni et al. 1988a
marrow cells
MVM, micronucleus test, foetal CD-1 mouse liver cells in  (+)                     20 (to dam) x 1 Ciranni et al. 1988a
 tero
MVM, micronucleus test, CD-1 mouse                        (+)                     20 po x 1       Ciranni et al. 1988b
DLM, dominant lethal test, male C3H and (C3Hx101)F1       -                       6.25 ip x 1     Rohrborn & Vogel
mice                                                                                              1967
DNA mutation
 acteria
 A0, S. typhimurium TA100, rev. mut.                      +           n.t.        5.0             Nazar et al. 1981
 A0, S. typhimurium TA100, rev. mut.                      -           -           16.5            Mortelmans et al.
                                                                                                  1986
 A5, S. typhimurium TA1535, rev. mut.                     -           -           16.5            Mortelmans et al.
                                                                                                  1986
 A7, S. typhimurium TA1537, rev. mut.                     -           -           16.5            Mortelmans et al.
                                                                                                  1986
 A9, S. typhimurium TA98, rev. mut.                       -           -           16.5            Mortelmans et al.
                                                                                                  1986
NCR, N. crassa, rev. mut. to arg+                         -           n.t.        n.g.            Reissig 1963
NCF, N. crassa, for. mut. to pyrimidine dependence        -           n.t.        n.g.            Reissig 1963
 76          Hydroquinone and benzoquinone
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<pre>Mammalian cells, in vitro
G9H, gene mut., V79 cells, hprt locus                        +            n.t.           0.54                Ludewig et al.
                                                                                                             1989154
Aneuploidy induction
  o IARC data available
 ; (+); -:           positive; weakly positive; negative
 ED; HID:            lowest effective dose; highest ineffective dose (in vitro tests: µg/mL; in vivo tests: mg/kg bw/day)
 .t.; n.g.:          not tested; not given
ev. mut.; for. mut.: reverse mutation; forwards mutation
 o; ip:              oral; intraperitoneal
 CE:                 sister chromatid exchange
V79 cells:           Chinese hamster lung V79 cells
              Mutagenicity studies                                                                                          177
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<pre>78 Hydroquinone and benzoquinone</pre>

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<pre> nnex I
      Evaluation of the Subcommittee on
      the Classification of carcinogenic
      substances
.1    Scope
      On request of the Dutch Expert Committee on Occupational Safety of the Health
      Council, the Subcommittee on the Classification of Carcinogenic Substances
      evaluated the carcinogenic properties of hydroquinone and benzoquinone.
.2    Carcinogenicity of hydroquinone
      The International Agency for Research on Cancer (IARC) classified
      hydroquinone as not classifiable as to its carcinogenicity to humans (Group 3),
      based on inadequate evidence in humans for the carcinogenicity of
      hydroquinone, and limited evidence in experimental animals for the
      carcinogenicity of hydroquinone (IARC 1999).
          For the evaluation of the carcinogenicity of hydroquinone, two oral studies
      were available. In the first study (study I), performed and comprehensively
      reported by the US National Toxicology Program (NTP), F344 rats and B6C3F1
      mice (n=55/species/sex/group) were exposed by gavage to concentrations of
      hydroquinone (in deionised water) of 25 and 50 mg/kg bw/day and of 50 and 100
      mg/kg bw/day, respectively, 5 days/week, for 104 weeks (Kari et al. 1992, NTP
      1989). In the second study (study II), performed and limitedly reported by
      Shibata et al. 1991, hydroquinone was administered in the diet to F344 rats
      (n=30/sex/group) at doses of ca. 350 and 370 mg/kg bw/day for males and
      Evaluation of the Subcommittee on the Classification of carcinogenic substances 179
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<pre>               females, respectively, and to B6C3F1 mice (n=30/sex/group) at doses of ca. 1,050
               and 1,490 mg/kg bw/day for males and females, respectively.
                    In these studies, administration of hydroquinone induced increased tumour
               incidences (control, low dose, high dose) as summarized in Table I.1.
                    As to the hepatocellular adenomas, the Subcommittee notes that they were
               not consistently seen, viz., in males in (diet) study II and in females in (gavage)
               study I; that they were benign and did not progress to malignant carcinomas; and
               that there was a relatively small increase in (diet) study I and no dose response in
               (gavage) study II. Because of this inconsistent picture and the susceptibility of
               the B6C3F1 mouse to develop liver tumours, the Subcommittee considers that
               the induction of hepatocellular adenomas is not relevant for humans.
                    The renal tubular adenomas were found in both studies, but in male rats only.
               These benign tumours did not progress to malignant ones. Severe kidney damage
               appeared to be a prerequisite for the development of the adenomas. This chronic
               progressive nephropathy is a spontaneous disease in, particularly, ageing male
               rats. The Subcommittee is of the opinion that hydroquinone plays an indirect role
               in the development of the adenomas by exacerbating already existing chronic
               progressive nephropathy and enhancing proliferative aspects of this disease
               process. This view of an indirect role is supported by the findings that increased
               cell proliferation but no DNA adducts were detected in the F344 male rat kidney
               following repeated oral administration of nephrotoxic doses.
                    Because of this indirect role involving exacerbation of an already existing
               spontaneous disease which is further common in rats but without a human
               counterpart, the Subcommittee is of the opinion that the induction of renal
               tubular adenomas in male rats is not relevant for humans.
 able I.1 Increased tumour incidences in chronic rat and mouse studies (study I: NTP 1989, Kari et al. 1992; study II: Shibata
 t al. 1991).
 pecies; study                         Dose                Control               Low dose               High dose
  at, male; study I                    0, 25, 50           0 / 55                4 / 55                 8 / 55
enal tubular adenomas                  mg/kg bw/day                              (7%, p=0.059)          (14%, p=0.003)
  at, male; study I re-evaluated       0, 25, 50           0 / 44                4 / 49                 15 / 51
Hard et al. 1997)                      mg/kg bw/day                              (8%)                   (29%, p<0.01
enal tubular adenomas
  at, male; study II                   0, 350              0 / 30                -                      14 / 30
enal tubular adenomas                  mg/kg bw/day                                                     (47%, p<0.01)
  at, female; study I                  0, 25, 50           9 / 55                15 / 55                22 / 55
mononucleaer cell leukaemia            mg/kg bw/day                              (27%.p=0.124)          (40%, p=0.005)
Mouse, male; study II                  0, 1050             6 / 28                -                      14 / 30
 epatocellular adenomas                mg/kg bw/day        (22%)                                        (47%, p<0.05)
Mouse, female; study I                 0, 50, 100          2 / 55                15 / 55                12 / 55
 epatocellular adenomas                mg/kg bw/day        (4%)                  (27%, p=0.001)         (22%, p=0.004)
  80           Hydroquinone and benzoquinone
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<pre>   The mononuclear cell leukaemias were found at statistically significantly
   increased incidences in the high-dose female rats in study I only. The
   Subcommittee notes that especially F344 and Wistar-Furth rats show high
   background incidences of this tumour type while they are low (i.e., ≤ 6%) in
   other rat strains and species. The rate in control F344 rats is very variable and
   depends upon several factors such as e.g., sex, vehicle and diet. In addition, the
   rates recorded in the laboratories conducting NTP studies increased in the course
   of time. In the period 1980-1984, in which the hydroquinone study was
   performed, the average incidences of mononuclear cell leukaemia in water-
   treated (gavage) control F344 rats were 43.8±12.6 and 26.2±10.6% in males and
   females, respectively.
        In view of the unique susceptibility of F344 rats to develop monuclear cell
   leukaemias, the very variable background incidences, and the fact these
   leukaemias were seen in one sex in one study only, the Subcommittee considers
   that the induction of mononuclear cell leukaemias in female F344 rats is not
   relevant for humans.
   Mode of action
   The Subcommittee concludes that hydroquinone is a genotoxic compound. In
   vitro, amongst others, it induced gene mutations, micronuclei, chromosomal
   aberrations, aneuploidy, sister chromatid exchanges, DNA single strand breaks,
   and oxidative DNA damage in mammalian cell systems. In in vivo tests, mainly
   performed in mice using intraperitoneal injection, hydroquinone produced
   increases in the incidences of micronuclei and chromosomal aberrations in bone
   marrow and of chromosomal aberrations in spermatogonia. Based on the
   literature it is quite likely that the mode of action of hydroquinone’s genotoxicity
   is by a non-stochastic mechanism.
.3 Carcinogenicity of benzoquinone
   One dermal (Umeda 1957) and one oral study (El-Mofty et al. 1992) addressed
   the (potential) carcinogenicity of benzoquinone. The Subcommittee is of the
   opinion that because of several serious flaws in design and reporting these
   studies cannot be used to evaluate the carcinogenicity of benzoquinone.
   Evaluation of the Subcommittee on the Classification of carcinogenic substances      181
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<pre>.4  Recommendation for classification
    Hydroquinone
    The Subcommittee concludes that there are two valid carcinogenicity studies in
    which hydroquinone was orally administered to rats and mice. The findings are
    inconsistent and concern tumour types that are considered not relevant for
    humans.
          The Subcommittee is of the opinion that the available data are insufficient to
    evaluate the carcinogenic properties of hydroquinone (category 3).
    Benzoquinone
    The Subcommittee concludes that there are no valid carcinogenicity studies.
          The Subcommittee is of the opinion that the available data are insufficient to
    evaluate the carcinogenic properties of benzoquinone (category 3).
.5  References
    El-Mofty MM, Khudoley VV, Sakr SA, Fathala NG. Flour infested with Tribolium castaneum,
    biscuits made of this flour, and 1,4-benzoquinone induce neoplastic lesions in Swiss albino mice.
    Nutr Cancer 1992; 17(1): 97-104.
    Hard GC, Whysner J, English JC, Zang E, Williams GM. Relationship of hydroquinone-associated
    rat renal tumors with spontaneous chronic progressive nephropathy. Toxicol Pathol 1997; 25(2): 132-
    143.
    IARC. Hydroquinone. In: Re-evaluation of some organic chemicals, hydrazine and hydrogen
    peroxide. Proceedings of the IARC Working Group on the Evaluation of Carcinogenic Risks to
    Humans. 1999: 691-720.
    Kari FW, Bucher J, Eustis SL, Haseman JK, Huff JE. Toxicity and carcinogenicity of hydroquinone
    in F344/N rats and B6C3F1 mice. Food Chem Toxicol 1992; 30(9): 737-747.
    NTP (National Toxicology Program USA). Toxicology and carcinogenesis studies of hydroquinone
    (CAS No. 123-31-9) in F344/N rats and B6C3F1 mice (gavages studies). Research Triangle Park,
    NC: U.S. Department of Health and Human Studies, National Institute of Health; 1989: NTP
    Technical Report No. 366.
    Shibata MA, Hirose M, Tanaka H, Asakawa E, Shirai T, Ito N. Induction of renal cell tumors in rats
    and mice, and enhancement of hepatocellular tumor development in mice after long-term
    hydroquinone treatment. Jpn J Cancer Res 1991; 82(11): 1211-1219.
 82 Hydroquinone and benzoquinone
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<pre>Umeda M. Production of rat sarcoma by injections of propylene glycol solution of p-quinone. Gan
1957; 48(2): 139-144.
Evaluation of the Subcommittee on the Classification of carcinogenic substances                 183
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<pre>84 Hydroquinone and benzoquinone</pre>

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<pre> nnex        J
             Carcinogenic classification of
             substances by the Committee
             The Committee expresses its conclusions in the form of standard phrases:
 ategory    Judgement of the committee (GRGHS)                                  Comparable with EU Category
                                                                                67/584/EEC           EC No 1272/2008
                                                                                before 12/16/2008    as from 12/16/2008
A           The compound is known to be carcinogenic to man.                    1                    1A
            • It acts by a stochastic genotoxic mechanism.
            • It acts by a non-stochastic genotoxic mechanism.
            • It acts by a non-genotoxic mechanism.
            • Its potential genotoxicity has been insufficiently investigated.
               Therefore, the mechanism of action is not known.
B           The compound is presumed to be carcinogenic to man.                 2                    1B
            • It acts by a stochastic genotoxic mechanism.
            • It acts by a non-stochastic genotoxic mechanism.
            • It acts by a non-genotoxic mechanism.
            • Its potential genotoxicity has been insufficiently investigated.
               Therefore, the mechanism of action is not known.
            The compound is suspected to be carcinogenic to man.                3                    2
3)          The available data are insufficient to evaluate the carcinogenic    Not applicable       Not applicable
            properties of the compound.
4)          The compound is probably not carcinogenic to man.                   Not applicable       Not applicable
ource: Health Council of The Netherlands. Guideline to the classification of carcinogenic compounds. The Hague: Health
 ouncil of the Netherlands, 2010; publication no. A10/07E.
             Carcinogenic classification of substances by the Committee                                               185
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<pre>86 Hydroquinone and benzoquinone</pre>

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<pre> nnex       K
            BMD analysis of thyroid hyperplasia in
            mice exposed to hydroquinone
            The results of the NTP study with hydroquinone administered orally to mice
            (NTP 1989; Kari et al. 1992), resulting (among others, see Section 7.2.4) in
            hyperplasia of the thyroid, are shown in Table K-1.
                 Analysis of these study data using the software programme PROAST (a
            software package developed for analysing dose-response data of toxicity studies
            and calculating BMDs [Slob 2002, 2009; EFSA 2011]) showed that the data of
            the males and the females can not be combined. The incidences of the lesions in
            the males differ too much from the incidences in the females, both in the controls
            and in the dosed groups.
                 In addition, a BMD analysis of the results with the females is also not
            possible: there are only two dose groups and the incidences flattens off already at
            the low dose, resulting in the impossibility to estimate a BMD for e.g. 10% extra
            risk (Slob, personal communication).
                 Only for the results with the males a BMD analysis is possible. This analysis,
            using the BMD software program of US-EPA (US-EPA 2011) is shown in Table
            K-2 and Figure K-1.
 able K-1 Thyroid hyperplasia in mice (2-year gavage study; NTP 1989, Kari et al. 1992).
 hyroid hyperplasia in mice    Number of animals with lesions / total number of animals per dose group
B6C3F1)                        males                                          females
Dose (mg/kg bw/day)            Control         50            100              Control          50      100
 hyroid hyperplasia            5/55            15/53         19/54            13/55            47/55   45/55
            BMD analysis of thyroid hyperplasia in mice exposed to hydroquinone                              187
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<pre>   The lowest BMD and BMDL for 10% extra risk (25.1 and 15.7 mg/kg bw/day,
   respectively) are produced by the log-logistic model. Other BMDs and BMDLs
   vary from 28.5 - 43.5 and 19.1 - 34.0 mg/kg bw/day, respectively.
   References
   EFSA, 2011. Use of BMDS and PROAST software packages by EFSA Scientific Panels and Units
   for applying the Benchmark Dose (BMD) approach in risk assessment. Technical report, European
   Food Safety Authority (EFSA), Parma, Italy; 2011.
   Kari FW, Bucher J, Eustis SL, Haseman JK, Huff JE, 1992. Toxicity and carcinogenicity of
   hydroquinone in F344/N rats and B6C3F1 mice. Food Chem Toxicol 1992; 30(9): 737-747.
   NTP, 1989. Toxicology and carcinogenesis studies of hydroquinone (CAS No. 123-31-9) in F344/N
   rats and B6C3F1 mice (gavages studies). Research Triangle Park, NC: U.S. Department of Health
   and Human Studies, National Institute of Health; 1989: NTP Technical Report No. 366.
   Slob W, 2002. Dose-response modeling of continous endpoints. Toxicol. Sci. 66: 298-312.
   Slob W, 2009. PROAST: software for dose-response modeling and benchmark dose analysis.
   National Institute for Public Health and the Environment, Bilthoven, The Netherlands; 2004.
   US-EPA, 2011. US Environmental Protection Ageny, benchmark dose software (BMDS) v. 2.2;
   www.epa.gov/ncea/bmds/ (November 2011).
   Table K-2 BMD analysis for 10% extra risk of thyroid hyperplasia in males using US-EPA’s BMD
   software.
   Model                      BMD           BMDL       Log-likelihood fitted Degrees of    Model
                                                       vs. full model        freedom       accepted
   Gamma                      28.5          19.1       0.236                 1             yes
   Logistic                    43.5         34.0       0.827                 1             yes
   LogLogistic                 25.1         15.7       0.134                 1             yes
   Multistage                 28.5          19.1       0.236                 1             yes
   Multistage Cancer          28.5          19.1       0.236                 1             yes
   Probit                      41.3         32.1       0.730                 1             yes
   Weibull                     28.5         19.1       0.236                 1             yes
   Quantal Linear             28.5          19.1       0.236                 1             yes
88 Hydroquinone and benzoquinone
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<pre>                                                                               L og -L o gis tic M o de l with 0 .9 5 C on fid en ce Le v e l
                                                                        L o g- Lo g is tic
                                               0 .5
                                               0 .4
   F r a c ti o n A ff e c t e d
                                               0 .3
                                               0 .2
                                               0 .1
                                                 0              BM DL           BM D
                                                            0           20                      40                      60                      80   100
                                                                                                          d os e
                                   2 0: 16 0 2 /2 3 20 12
igure K-1 Log-logistic model.
                                            BMD analysis of thyroid hyperplasia in mice exposed to hydroquinone                                            189
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<pre>90 Hydroquinone and benzoquinone</pre>

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