<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>            Health Council of the Netherlands
          Benzene
            Health-based recommended occupational exposure limit
2014/03
</pre>

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<pre>Benzene
   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 Benzene
Uw kenmerk              : DGV/MBO/U-932342
Ons kenmerk             : U-8057/SV/fs/459-Q69
Bijlagen                :1
Datum                   : 21 februari 2014
Geachte minister,
Graag bied ik u hierbij aan het advies over de gevolgen van beroepsmatige blootstelling aan
benzeen.
Dit advies maakt deel uit van een uitgebreide reeks, waarin gezondheidskundige advies-
waarden worden afgeleid voor concentraties van stoffen op de werkplek. De conclusies van
het genoemde advies zijn opgesteld door de Commissie Gezondheid en beroepsmatige
blootstelling aan stoffen (GBBS) van de Gezondheidsraad en beoordeeld door de Beraads-
groep 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
Rijnstraat 50                                                   Postbus 16052
2515 XP          Den Haag                                       2500 BB         Den Haag
E - m a i l : s r. v i n k @ g r. n l                           w w w. g r. n l
Te l e f o o n ( 0 7 0 ) 3 4 0 5 5 0 8
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<pre></pre>

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<pre>Benzene
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. 2014/03, The Hague, February 21, 2014
</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.
This report can be downloaded from www.healthcouncil.nl.
Preferred citation:
Health Council of the Netherlands. Benzene - Health-based recommended
occupational exposure limit. The Hague: Health Council of the Netherlands,
2014; publication no. 2014/03.
all rights reserved
ISBN: 978-90-5549-988-5
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<pre>Contents
Samenvatting 9
Executive summary 15
Scope 21
Background 21
Committee and procedure 21
Data 22
Identity, properties and monitoring 23
Identity and physico-chemical properties 23
EU Classification and labeling 24
Validated analytical methods 25
Sources 27
Natural occurrence 27
Man-made sources 27
Exposure 29
General population 29
Working population 30
Kinetics 31
Absorption 31
Contents                                    7
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<pre>Distribution 32
Metabolism 33
Elimination 35
Possibilities for biological monitoring 35
Possibilities for biological effect monitoring 36
Summary 36
Mechanism of action 39
Effects 43
Observations in humans 43
Other effects 59
Observations in animals 60
Summary and evaluation 63
Existing guidelines, standards and evaluations 65
General population 65
Working population 65
Classification 66
Hazard assessment 69
Hazard identification 69
Quantitative assessment of the health risk 69
Groups at extra risk 74
Health-based recommended occupational exposure limit 74
References 77
Annexes 91
Request for advice 93
The Committee 95
The submission letter (in English) 99
Comments on the public review draft 101
Human data 103
Animal data 107
Genotoxicity data 113
Advice of the Subcommittee on Classification of Carcinogenic Substances 117
Classification of substances with respect to carcinogenicity 127
List of Abbreviations 129
Benzene
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<pre>Samenvatting
Vraagstelling
Op verzoek van de minister van Sociale Zaken en Werkgelegenheid leidt de
Commissie Gezondheid en beroepsmatige blootstelling aan stoffen (Commissie
GBBS) van de Gezondheidsraad gezondheidskundige advieswaarden af voor
stoffen in de lucht waaraan mensen blootgesteld worden tijdens de beroepsuitoe-
fening. De gezondheidskundige advieswaarden 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 ben-
zeen en stelt ze een gezondheidskundige advieswaarde vast. De conclusies van
de commissie berusten op de wetenschappelijke publicaties die vóór oktober
2013 zijn verschenen.
Fysische en chemische eigenschappen
Benzeen (CAS 71-43-2) is een kleurloze vloeistof met een zoete geur, die wordt
gewonnen uit koolstof- en petroleumbronnen. Benzeen heeft een moleculair
gewicht van 78,11 g/mol, een smeltpunt van 5,5°C en een kookpunt van 80,1°C.
Verder is benzeen vluchtig en zeer brandbaar.
Samenvatting                                                                    9
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<pre>  Gebruik
  Benzeen wordt voornamelijk gebruikt in de chemische en farmaceutische indus-
  trie als startmateriaal en tussenproduct voor de synthese van diverse chemische
  stoffen. Het wordt ook toegevoegd aan benzine, als anti-klopmiddel, voor een
  betere ontbranding.
  Monitoring
  De blootstelling aan benzeen kan goed gemeten worden door bepaling van
  benzeen in urine, bloed en uitgeademde lucht, en door de bepaling van S-fenyl-
  mercaptuurzuur (SPMA) in urine.
       De analyse van benzeen gebeurt meestal met gaschromatografie (GC). Mas-
  saspectrometrie (MS) kan gebruikt worden om benzeen te detecteren en te kwan-
  tificeren. De detectielimiet van benzeen in urine, bloed en uitgeademde lucht ligt
  in het gebied van enkele nanogrammen per liter.
       Er zijn verschillende GC-methoden om SPMA in urine te analyseren. In aan-
  vulling op de GC-methoden zijn verschillende high-performance liquid chroma-
  tography (HPLC) methoden beschikbaar. In het algemeen liggen de
  detectielimieten hiervan nabij de 1 microgram per liter.
  Huidige grenswaarden
  Er is voor benzeenblootstelling op de werkplek een Europese grenswaarde van
  3.25 mg per m3 lucht vastgesteld, voor een werkdag van 8 uur. Deze grenswaarde
  wordt ook in Nederland en enkele andere Europese landen toegepast. In de Ver-
  enigde Staten zijn grenswaarden van 3,2 mg/m3 (voor een 8-urige blootstelling)
  en 16 mg/m3 (voor een piekblootstelling van 15 minuten) vastgesteld door de
  Occupational Safety & Health Administration (OSHA).
  Kinetiek
  Benzeen wordt gemakkelijk opgenomen via alle blootstellingsroutes (de lucht-
  wegen, de huid en de mond), waarvan de luchtwegen de belangrijkste route vor-
  men). In de mens varieert de opname na inademing van ongeveer 50 tot 80
  procent, afhankelijk van de blootstellingsomstandigheden. Diergegevens wijzen
  erop dat opname van benzeen via de luchtwegen niet rechtevenredig afhankelijk
0 Benzene
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<pre>is van de concentratie in de lucht. De gegevens wijzen uit dat vloeibaar benzeen
door de menselijke huid kan worden geabsorbeerd. De geschatte opname via de
huid per tijdseenheid varieert van 200-400 µg per cm2 per uur. Benzeen wordt na
toediening via de mond efficiënt opgenomen in dieren; de mate hiervan varieert
van ongeveer 80 procent(in konijnen) tot meer dan 97 procent (in ratten en mui-
zen).
    Na opname verdeelt benzeen zich over het lichaam. Benzeen is bij de mens
gemeten in verschillende lichaamsvloeistoffen en weefsels, waarbij de hoogste
gehaltes zijn gemeten in weefsels met een hoog vetgehalte. Ook in dieren ver-
deelt benzeen zich na absorptie in vetrijke weefsels, vooral weefsels met een
hoge doorbloeding zoals de nier. In de rat werd in het bloed binnen 4 uur een
concentratie-evenwicht van benzeen bereikt, in vetweefsel binnen 6 uur en in
beenmerg in minder dan 2 uur, na blootstelling aan 1.600 mg/m3 (500 ppm)
benzeen.
    Hoewel nog niet alle stappen die leiden tot de toxiciteit van benzeen bekend
zijn, is het duidelijk dat het metabolisme in grote mate de toxiciteit van benzeen
bepaalt. De beschikbare gegevens wijzen erop dat de afbraakproducten van
benzeen voornamelijk in de lever gevormd worden. De eerste stap in de omzet-
ting van benzeen is de oxidatie van benzeen door het enzymsysteem cytochroom
P-450, voornamelijk het enzym CYP 2E1, en de vorming van benzeenoxide. Ver-
schillende mechanismen zijn betrokken bij de omzetting van benzeenoxide,
waarvan het mechanisme via niet-enzymatische omzetting tot fenol een promi-
nente rol speelt. Na blootstelling aan benzeen via de luchtwegen, is zowel in
dieren als mensen uitademing de belangrijkste route waarlangs benzeen het
lichaam verlaat. Het merendeel van het benzeen dat geabsorbeerd wordt, wordt
echter omgezet en uitgescheiden in de urine na binding met lichaamseigen
stoffen.
Effecten
Waarnemingen bij mensen
Benzeendamp irriteert de luchtwegen; benzeen is ook irriterend voor de huid.
    Kort na inademing van zeer hoge concentraties benzeen kunnen er bij men-
sen symptomen optreden die duiden op toxiciteit van het centraal zenuwstelsel,
zoals duizeligheid, stuiptrekkingen, trillingen en uiteindelijk een narcotisch
effect en overlijden door ademhalingsstilstand.
Samenvatting                                                                       11
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<pre>       Beroepsmatige blootstelling wordt sinds lange tijd in verband gebracht met
  nadelige effecten op het bloed en het beenmerg, waaronder een tekort aan rode
  én witte bloedcellen. Verschillende onderzoeken met werknemers die zijn bloot-
  gesteld aan benzeen tonen deze effecten aan bij hoge maar ook relatief lage
  blootstellingsconcentraties.
       Bij verschillende beroepsgroepen, in verschillende industrietakken, is een
  verhoogd risico gevonden op leukemie na blootstelling aan benzeen. Het ver-
  hoogde risico geldt in het bijzonder voor acute myeloide leukemie, maar recente-
  lijk is gebleken dat blootstelling aan benzeen ook kan leiden tot het myelodys-
  plastisch syndroom (MDS), een beenmergstoornis waarbij de productie van
  bloedcellen ernstig verstoord is.
       Er zijn onvoldoende en tegenstrijdige aanwijzingen om te kunnen conclude-
  ren dat benzeen nadelige effecten heeft op de vruchtbaarheid, of de ontwikkeling
  van het nageslacht van de mens.
  Waarnemingen bij dieren
  Onderzoek met ratten laat zien dat benzeen op korte termijn niet erg toxisch is,
  met een geschatte LD50-waarde hoger dan 2.000 mg/kg lichaamsgewicht en een
  LC50 waarde van 44.500 mg/m3 (13.700 ppm). Afhankelijk van de dosis, zijn de
  voornaamste acute effecten verdoving en narcose. Onderzoek met konijnen toont
  bij hen irritatie van de huid.
       Onafhankelijk van de blootstellingsroute, zijn de beenmergcellen en bloed-
  cellen het meest gevoelig voor herhaalde blootstellingen aan benzeen. Chroni-
  sche blootstelling aan benzeen kan leiden tot onderdrukking van het beenmerg,
  met als gevolg een tekort aan rode en witte bloedcellen.
       Studies waarbij dieren blootgesteld zijn via de luchtwegen of de mond laten
  zien dat benzeen tumoren veroorzaakt in meerdere organen. Doelorganen zijn,
  onafhankelijk van de blootstellingsroute, het hematopoetische systeem en een
  spectrum van epitheelweefsels. Bij muizen uit de carcinogeniteit van het hemato-
  poetische systeem zich voornamelijk in het ontstaan van lymfomen. Bij ratten
  daarentegen, wordt een verhoogd aantal dieren met leukemie gevonden na bloot-
  stelling aan benzeen. Daarnaast zijn verschillende typen epitheliale tumoren
  gevonden in muizen en ratten.
       Benzeen en de metabolieten van benzeen veroorzaakten geen mutaties in tes-
  ten met bacteriën, terwijl de resultaten van testen met zoogdiercellen dubbelzin-
  nig waren. Het merendeel van de testen voor chromosomale afwijkingen in
2 Benzene
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<pre>proefdieren was positief, zowel voor benzeen als voor de metabolieten van ben-
zeen.
    In vrouwtjesratten die voor het paren tien weken lang blootgesteld waren aan
975 mg/m3 benzeen (300 ppm), werden geen nadelige effecten gevonden op de
vruchtbaarheid, voortplanting, en lactatie. In muizen daarentegen, zijn er bij deze
concentratie aanwijzingen gevonden voor veranderingen in voortplantingsorga-
nen, met name in mannetjes. Deze effecten traden echter op bij concentraties die
duidelijk toxisch voor bloedcellen waren, bij zowel mannetjes als vrouwtjes. Er
zijn geen ontwikkelingsstoornissen gevonden na blootstelling aan benzeen, zelfs
niet bij concentraties die leidden tot toxiciteit bij de moederdieren.
Evaluatie en advies
De Subcommissie Classificatie carcinogene stoffen (een autonome subcommis-
sie van de Commissie GBBS) concludeert, overeenkomstig met de Europese
classificatie, dat benzeen kankerverwekkend is voor de mens (classificatie cate-
gorie 1A, zie Annex H en I). Ze beschouwt benzeen als een kankerverwekkende
stof met een niet-stochastisch genotoxisch werkingsmechanisme. Dit betekent
dat de subcommissie ervan uit gaat dat er een veilig blootstellingsniveau, een
drempelwaarde, voor benzeen bestaat. De commissie heeft het oordeel van de
subcommissie overgenomen en een gezondheidskundige advieswaarde afgeleid.
    Onafhankelijk van de blootstellingsroute, zijn het beenmerg en het bloedvor-
mend systeem het meest gevoelig voor de schadelijke effecten van benzeen. De
gegevens over het blootstellingsniveau waarbij effecten bij de mens kunnen
optreden zijn niet eenduidig. In meerdere studies zijn effecten gevonden bij een
benzeenconcentratie die lager was dan 3,3 mg/m3 (1 ppm), terwijl in andere stu-
dies geen effecten zijn beschreven bij deze concentratie. De commissie baseert
haar gezondheidskundige advieswaarde op het geheel van de gegevens en han-
teert, vanuit een pragmatisch oogpunt, 2 mg/m3 (0,6 ppm) als een reëel blootstel-
lingsniveau waarbij gezondheidseffecten kunnen optreden. De commissie past
voor het afleiden van een gezondheidskundige advieswaarde een onzekerheids-
factor toe, om rekening te houden met het feit dat er bij deze concentratie nog
effecten zijn te verwachten. Het toepassen van een standaard onzekerheidsfactor
van 3 levert een gezondheidskundige advieswaarde op van 0,7 mg/m3 (0,2 ppm).
Samenvatting                                                                        13
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<pre>  Gezondheidskundige advieswaarde
  De Commissie GBBS van de Gezondheidsraad beveelt een gezondheidskundige
  advieswaarde aan voor beroepsmatige blootstelling aan benzeen van 0,7 mg/m3
  (als een gemiddelde waarde over een achturige werkdag).
4 Benzene
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<pre>Executive summary
Scope
At request of the Minister of Social Affairs and Employment, the Dutch Expert
Committee on Occupational Safety (DECOS), a Committee of the Health
Council of the Netherlands, recommends health-based occupational exposure
limits for airborne substances to which people are exposed in the workplace.
These recommendations serve as a basis in setting legally binding occupational
exposure limits by the Minister. In this report, the Committee considers the
implications of exposure to benzene, and recommends a health-based
occupational exposure limit for this substance. The Committees’ conclusions
reflect the content of scientific publications that have appeared in the public
literature prior to October 2013.
Physical and chemical properties
Benzene (CAS 71-43-2) is a colourless liquid with a sweet odour, which is
commercially produced from coal and petroleum sources. Benzene has a
molecular weight of 78.11, a melting point of 5.5°C and a boiling point of
80.1°C. Benzene has a vapour pressure of 99.7 hPa at 20°C and is highly
flammable with flammability limits in air of 1.2% (lower limit) and 7.8%
(upper limit).
Executive summary                                                               15
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<pre>  Use
  Benzene is used primarily in the chemical and pharmaceutical industries, as a
  starting material and intermediate in the synthesis of numerous chemicals. It is
  also used as a gasoline additive, since benzene increases the octane rating and
  reduces knocking.
  Monitoring
  The determination of benzene in urine, blood and expired air, and the
  determination of S-phenylmercapturic acid (SPMA) in urine are suitable
  approaches for biomonitoring of benzene.
       The analysis of benzene generally involves dynamic headspace (purge and
  trap). Mass spectrometry can be used for the detection and quantification of
  benzene. The limit of detection for benzene in urine, blood and expired air falls
  within the low ng/L range.
       Commonly used methods for urinary SPMA analysis consist of extracting
  SPMA from the urine by liquid-liquid extraction, subsequent derivatisation, and
  detection by gas chromatography/mass spectromethry (GC/MS). In addition to
  the GC approach, several high-performance liquid chromatography methods are
  available. Generally, the limit of detection of urinary SPMA analysis are in the
  range of 1 µg/L.
  Exposure limits
  At the European level, there is currently a limit value of 3.25 mg/m3 (1 ppm) for
  occupational exposure to benzene. The legal time weighted average (TWA) (8h)
  occupational exposure limit for benzene in the Netherlands is 3.25 mg/m3 air.
  Also in Finland, France, and the UK, an occupational exposure limit of 3.25
  mg/m3 is being applied. In Germany, no MAK value (Maximum Concentration
  at the Workplace) has been derived. In the US, exposure limits of 3.2 mg/m3
  (8h-TWA) and 16 mg/m3 (15 min-TWA) have been set by OSHA. The American
  Conference of Governmental Industrial Hygienists (ACGIH) has specified a
  threshold limit value (TLV) of 1.6 mg/m3 (8h-TWA value).
6 Benzene
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<pre>Kinetics
Benzene is readily absorbed by all routes (inhalation, dermal and oral), of which
inhalation is considered to be the most important route of exposure. In humans,
extents of absorption have been reported ranging from approximately 50-80%,
depending on exposure conditions. Animal data suggest that the uptake of
benzene by the lungs is related to the concentration in a non-linear manner. The
amount of benzene absorbed and retained in the tissues and blood during a
6-hour exposure decreased from 33 to 15% in rats, and from 50 to 10% in mice,
when exposure was increased from 26 to 2,600 mg/m³ (8-812 ppm). Results from
in vivo experiments indicate that liquid benzene can be absorbed through human
skin, although not as substantial as the absorption following inhalation or oral
exposure.The estimated skin absorption rate ranges from 200-400 µg/cm2*h.
Benzene is efficiently absorbed following oral dosing in animals; absorption
levels have been reported of > 97% (in rats and mice) and 80% (in rabbits).
    Upon absorption, benzene is distributed throughout the body. Benzene has
been detected in various biological fluids and tissues of humans, the highest
levels amounting in lipid-rich tissues. Also in animals, benzene distributes in
tissues rich in lipids, particularly those with high perfusion rates, such as the
kidney. In rats, steady state concentrations of benzene were reached within 4
hours in blood, 6 hours in fat and less than 2 hours in bone marrow after
exposure to 1,600 mg/m³ (500 ppm).
    The metabolism of benzene is an important determinant for benzene-induced
toxicity, however the steps leading to benzene toxicity are not yet fully
understood. The available data indicate that metabolites are primarily generated
in the liver. Similar metabolic pathways exist in animals and human, although
remarkable species variability has been observed. The first step in the
metabolism of benzene is the oxidation of benzene to benzene oxide by the
cytochrome P-450, mainly CYP 2E1. Several pathways are involved in the
metabolism of benzene oxide, predominantly the pathway involving non-
enzymatic rearrangement to form phenol. In turn, phenol can be oxidised by
CYP2E1 to catechol or hydroquinone, which are subsequently oxidised to the
reactive metabolites 1,2- and 1,4-benzoquinone, respectively. The phenolic
metabolites of benzene can undergo conjugation. Other pathways of benzene
oxide metabolism include the reaction with glutathione to form SPMA and iron-
catalysed ring-opening to trans, trans-muconic acid.
    Following inhalation exposure to benzene, exhalation is the major route of
elimination of (unmetabolised) benzene in humans and animals. Most of the
Executive summary                                                                 17
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<pre>  absorbed benzene however, is metabolised and the metabolites are excreted after
  phase-II-conjugation, predominantly in the urine.
  Effects
  Observations in humans
  Benzene vapour is irritating to the respiratory tract; benzene is also irritating to
  skin.
      Following acute inhalation of high levels of benzene, humans exhibit
  symptoms of central nervous system toxicity, e.g., dizziness, convulsions,
  tremors and ultimately narcotic effects and death by respiratory arrest.
      Occupational exposure to benzene has long been associated with toxicity to
  the blood and bone marrow, including lymphocytopenia, pancytopenia, and
  aplastic anaemia. Several cross-sectional studies with workers who were exposed
  to benzene have shown haematological effects at a broad range of exposure
  levels.
      Increased risk of either leukaemia in general or acute myeloid leukaemia/
  acute non-lymphocytic leukaemia specifically, after exposure to benzene has
  been observed in several cohorts of workers, in various industries, with long-
  term exposure to benzene.
      There are insufficient, or inconsistent data on adverse effects of benzene
  exposure on fertility, or the development of offspring, in humans.
  Observations in animals
  Benzene has been shown to be irritating to the skin of rabbits, inducing moderate
  erythema, edema, and moderate necrosis following application.
      The acute toxicity of benzene is low, and suggest an oral LD50-value
  exceeding 2,000 mg/kg bw and a LC50 value of 44,500 mg/m³ (13,700 ppm) in
  rats. Depending on the dose, the main clinical signs are sedation and narcosis.
      Irrespective of the exposure route, the main and most sensitive targets of
  toxicity in animals after repeated dose application of benzene are the cells of the
  bone marrow and haematopoietic system. Chronic benzene exposure can result
  in bone marrow depression expressed as leucopenia, anaemia and/or
  thrombocytopenia, leading to pancytopenia, and aplastic anaemia.
      Inhalation and oral exposure studies provide evidence that benzene is a
  multipotential carcinogen in animals. Target organs of benzene, irrespective of
  exposure route, included the haematopoietic system and a spectrum of tissues of
8 Benzene
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<pre>epithelial origin. In mice, carcinogenicity of the haematopoietic system
predominantly involves the induction of lymphomas. In contrast, increased
frequencies of leukaemia were found in rats after exposure to benzene. In
addition, several epithelial tumours have been found in mice and rats.
    Bacterial mutagenicity assays conducted with benzene or its metabolites are
predominantly negative, whereas mixed results have been observed in
mammalian cell culture assays. In the majority of in vivo micronucleus tests and
in vivo chromosomal abberation assays, positive results have been observed for
benzene and its metabolites.
    In female rats exposed up to 975 mg/m3 (300 ppm) benzene for 10 weeks
during premating, no adverse effects on fertility, reproduction, and lactation were
observed. In mice, however, at this benzene concentration led to indications for
changes in reproductive organs. Most distinct for the males. These effects
however, were accompanied with clear-cut haematotoxicity (anaemia,
leucopenia and thrombocytopenia) in both sexes. None of the developmental
studies demonstrated a specific effect, even at levels that induced signs of
maternal toxicity.
Evaluation and recommendation
The Committee concludes that benzene, as recommended by the Subcommittee
on Clasification of Carcinogenic Substances, and in accordance with EU
classification, is known to be carcinogenic in humans (classification category
1A, see Annex H and I). The Subcommittee has further concluded that benzene
acts by a non-stochastic genotoxic mechanism. The Committee, therefore,
decided to apply a threshold approach.
    Irrespective of the exposure route, the main and most sensitive targets of
toxicity in animals and humans after repeated exposure to benzene are cells of
the bone marrow and haematopoietic system. Several studies address
haematological effects in humans at low benzene exposure levels, however, the
results are not consistent. Some studies report adverse effects below 3.25 mg/m3
(1 ppm), whereas other studies do not. The Committee applies a pragmatic
approach based on the aggregate of the accumulated evidence. It considers a
benzene effect level of 2 mg/m3 (0.6 ppm) an appropriate point of departure to
derive a health-based recommended occupational exposure limit (HBR-OEL).
The Committee applies an additional uncertainty factor to take into account the
use of an effect level instead of a no-effect level, as point of departure. By
applying a default uncertainty factor of 3, the Committee derives a HBR-OEL for
benzene of 0.7 mg/m3 (0.2 ppm).
Executive summary                                                                   19
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<pre>  Health-based recommended occupational exposure limit
  The Committee recommends a health-based occupational exposure limit for
  benzene of 0.7 mg/m3 (as an eight-hour weighted average concentration).
0 Benzene
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<pre> hapter 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 of the toxicity
        of substances that are used in the workplace. The purpose of the evaluation is to
        recommend a health-based occupational exposure limit, expressed as a
        concentration in the air, provided the database allows the derivation of such a
        value.
        This advisory report contains an evaluation of the health hazard and
        recommendation for a health-based occupational exposure limit for benzene.
1.2     Committee and procedure
        The present document contains the evaluation of the DECOS, hereafter called the
        Committee. The members of the Committee are mentioned in Annex B. The
        submission letter to the Minister can be found in Annex C.
             In 2013, 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
        Scope                                                                                21
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<pre>    deciding on the final version of the advisory report. The received comments, and
    the replies by the Committee, can be found on the website of the Health Council.
1.3 Data
    The Committee’s recommendation on the health-based occupational exposure
    limit of benzene has been based on scientific data, which are publicly available.
    Data were obtained from the online databases Toxline, Medline and Chemical
    Abstracts, In addition, in preparing this report several review documents were
    consulted.1-6 The last search was performed in October 2013.
        Finally, a list of abbreviations can be found at the end of this report in Annex J.
 2  Benzene
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<pre> hapter 2
        Identity, properties and monitoring
2.1     Identity and physico-chemical properties
        Benzene is a colourless liquid with a sweet odour. It is a ubiquitous
        environmental contaminant that is found in air, water and soil, and comes from
        both industrial and natural sources. It is commercially recovered from both coal
        and petroleum sources to be used primarily in the manufacture of organic
        chemicals.1,2,6 In Europe, benzene is mainly used to make styrene, phenol,
        cyclohexane, aniline, maleic anhydride, alkylbenzenes and chlorobenzenes. It is
        an intermediate in the production of anthraquinone, hydroquinone, benzene
        hexachloride, benzene sulfonic acid and other products used in drugs, dyes,
        insecticides and plastics. It is also added to gasoline for its octane-enhancing and
        anti-knock properties.6
        A summary of the identity and physical and chemical properties is given in
        Table 1.
        Identity, properties and monitoring                                                  23
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<pre>    Table 1 Identity, physical and chemical properties of Benzene.1,2
    IUPAC Name                         : Benzene
    Synonyms                           : Cyclohexatriene; Benzol
    CAS number                         : 71-43-2
    EINECS number                      : 200-753-7
    Chemical formula                   : C 6H 6
    Chemical structure                 :
    Molecular weight                   :   78.11 g/mol
    Colour                             :   Clear, colourless liquid
    Physical state                     :   Colourless to light yellow liquid
    Melting point                      :   5.5°C
    Boiling point                      :   80.1°C at 1,013 hPa
    Density at 20°C                    :   0.879 g/mL
    Odour                              :   Aromatic
    Solubility:
    •     Water at 25°C                    1,800 mg/L
    •     Organic solvents                 Miscible with acetone, chloroform, diethyl ether and ethanol;
                                           soluble in carbon tetrachloride
    Log Kow a                          :   2.13 (determined by HPLC)
    Vapour pressure                    :   99.7 hPa at 20°C
    Autoignition temperature           :   498°C
    Flashpoint                         :   -11°C (closed cup)
    Flammability limits in air         :   1.2% (lower limit); 7.8% (upper limit)
    Explosive limits                   :   1.4% (lower limit); 8% (upper limit)
    Conversion factors                 :   1 ppm = 3.26 mg/m3
    a    = octanol-water partitioning coefficient
2.2 EU Classification and labeling
    In the European Union, benzene is classified for carcinogenicity, category 1A
    (H350; May cause cancer) and for germ cell mutagenicity, category 1B (H340;
    May cause genetic defects)*.
    According to the Regulation on the classification, labelling and packaging of substances and mixtures
    (1272/2008/EC).
 4  Benzene
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<pre>2.3   Validated analytical methods
      In this section, the analytical methods which are available for detecting and/or
      measuring and monitoring benzene in air and in biological samples are
      described. The intention is not to provide an exhaustive list of analytical methods
      that could be used to detect and quantify benzene. Rather, the Committee aims to
      identify well-established methods that are used as the standard methods of
      analysis. For more details on analytical methods for benzene, the Committee
      refers to the review documents and subsequent references used.7,8
2.3.1 Biological monitoring
      The determination of benzene in urine, blood and expired air, and S-phenyl-
      mercapturic acid (SPMA) in urine are suitable approaches for biomonitoring
      of benzene (see also Sections 5.5 and 5.6).7 Despite the sensitive analytical
      methods available, no “standard” analytical method exists for benzene. For the
      determination of SPMA in urine, a standardised gas chromatography/mass
      spectrometry (GC/MS) approach has been published by the Deutsche Forshungs
      Gemeinschaft.9
          The analysis of blood and urine for benzene consists of an extraction step
      which is typically followed by GC/MS, hereby separating benzene from other
      volatile constituents. Extraction procedures include purge and trap, head space,
      solild phase extraction (SPE; i.e., using Tenax or charcoal) and solid phase
      microextraction (SPME). After extraction, benzene is transferred to a capillary
      column for GC separation. MS, or flame ionization or can subsequently be used
      for the detection and quantification of benzene. The limit of detection (LOD) for
      benzene in urine, blood and expired air falls within the low ng/L range.
          Several analytical methods for the determination of SPMA in urine exists.
      Commonly used methods consist of extracting SPMA from the urine by liquid-
      liquid extraction with ethyl acetate or by SPE. After subsequent derivatisation,
      SPMA can be detected by GC/MS with a LOD generally in the range of 1-5
      µg/L. Another approach is the detection of SPMA with high-performance liquid
      chromatography (HPLC) in combination with ultraviolet absorption detection,
      diode array detection, fluorescence detection, and MS. Generally, the LODs are
      below 1 µg/L.
      Identity, properties and monitoring                                                 25
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<pre>2.3.2 Environmental monitoring
      To determine benzene air levels, samples are collected from the air on either an
      adsorbent or by trapping whole air in a container.8 Passive vapour monitors or
      badges which collect volatile organic compounds based on diffusion are
      commonly used in occupational settings to measure ppm concentration levels
      present within the personal or breathing zone air. Environmental measurements,
      where the concentration is typically several orders of magnitude lower then
      occupational levels, are generally obtained by active sampling. Active sampling
      involves an air sampling pump and adsorbent held in an inert trap, and is the
      most sensitive sampling method. Recently, also passive badges have been used
      for environmental measurements.
           Benzene in air samples is then analysed by GC. The most common detector
      currently used with a GC is a MS detector. The LOD for personal air monitoring
      is typically around 0.1 mg/m3 (0.03 ppm) (for an 8-hour sampling period).
      Soil and water can be analysed in a similar fashion to air samples by purging the
      benzene from the water or soil and trapping the benzene on an adsorbent to
      facilitate its transference to GC. Alternate analytical methods for introducing a
      sample for GC analysis include headspace and SPE or SPME systems to
      concentrate the benzene from the sample followed by liquid concentration and
      injection.
 6    Benzene
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<pre> hapter 3
        Sources
3.1     Natural occurrence
        The natural sources of benzene include gas emissions from volcanoes and forest
        fires.1,2 Other sources of benzene are crude oil and, to a lesser extent, condensate
        from natural gas production.2
3.2     Man-made sources
        Benzene is produced by different petroleum conversion processes in petroleum
        refinery and chemical plant processes, primarily by catalytic reforming, steam
        cracking and dealkylation. Benzene is recovered during production of coal-
        derived chemicals, primarily from coke oven by-products. Benzene is extracted
        from these sources and purified for industrial use.2
            Benzene is released from a number of man-made sources. Approximately
        60% of the benzene emissions in The Netherlands is caused by road traffic. Other
        sources are combustion in wood-burning stoves and fire places, accounting for
        approximately 20% of the benzene emission, and storage and transshipment and
        chemical industry in the Rijnmond area.10
        Sources                                                                              27
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<pre>8 Benzene</pre>

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<pre> hapter 4
        Exposure
4.1     General population
        Exposure to low levels of environmental benzene is unavoidable due to the
        ubiquitous presence of benzene in the environment from a variety of natural and
        anthropogenic sources. Major sources of exposure to the general population
        include combustible fuel emissions and exhaust from motor vehicles,
        evaporation of gasoline and solvents (especially in attached garages), and
        industry or hazardous waste sites.2,7
             The highest air levels of benzene in The Netherlands are present in urban
        areas with a high industrial activity, such as locations of storage and
        transshipment, and surrounding highways (Figure 1a).10 Mean benzene levels
        have shown a decreasing trend, in particular since the 90’s, due to the
        introduction of the three-way catalytic converter, other technical improvements
        of motorised transport, and the reduction of the amount of benzene in petrol from
        5% to 1% (Figure 1b). The mean benzene concentration for the Netherlands in
        2011 was 0.50 µg/m³. Urban concentration levels were up to 4-fold higher than
        levels measured in rural areas.
             An additional source of exposure (for both workers and general population)
        is tobacco smoking (including second-hand smoking). It has been estimated that
        it accounts for 90% of the total benzene exposure of smokers.7
        Exposure                                                                          29
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<pre>  a
                                                                                                                         b
 igure 1 (a) Estimation of annual average benzene concentrations in air (µg/m3) in the Netherlands (2011), (b) Trend of the
 enzene concentration between 1990-2011. (Source: RIVM/DCMR, 2012; modified from10)
4.2          Working population
             Primary occupational exposure in Western countries is associated with
             employment in industries that use or make benzene or products containing
             benzene.1,7 In a number of other occupations, exposure to benzene can occur
             indirectly through the use of petroleum products (e.g., aviation workers,
             service station workers, bus drivers, cargo tank workers) or solvents. Long term
             air concentrations for these various exposures have been reported in the range
             of 0.01 to 2 mg/m3 (0.003 to 0.6 ppm) (arithmetic mean), measured during
             1991-2003.6
 0           Benzene
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<pre> hapter 5
        Kinetics
        The absorption, distribution and metabolism, and the possibilities of biological
        monitoring of benzene have been studied extensively. In this Chapter, the
        Committee provides a summary based on review documents.1,2,7,11
5.1     Absorption
        Benzene is readily absorbed by all routes (inhalation, dermal and oral), of which
        inhalation is considered to be the most important route of exposure.
        Inhalation
        Numerous studies on the absorption of benzene after inhalation exposure have
        been conducted. Mean absorption rates have been described, from approximately
        80% (during the first minutes of exposure to 150-350 mg/m3 (47-110 ppm) to
        approximately 50% after 4 hours of exposure in the 3.3-33 mg/m3 (1-10 ppm)
        range.12,13 Additional evidence of benzene absorption following inhalation
        exposure comes from data on cigarette smokers. Benzene levels were
        significantly higher in the venous blood of 14 smokers (median level of 547
        ng/L) than in a control group of 13 nonsmokers (median level of 190 ng/L).14
            Animal data suggest that the uptake of benzene by the lungs is related to the
        concentration in a non-linear manner.15 Mean percentage of inhaled 14C-benzene
        absorbed and retained in the tissues and blood during a 6h exposure decreased
        Kinetics                                                                          31
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<pre>    from 33-15% in rats, and from 50-10% in mice, as the exposure concentration
    was increased from approximately 26-2,600 mg/m³ (8-812 ppm). At similar
    vapour concentration exposures, mice take up 1.5 to 2.0-fold the dose per
    kilogram body weight compared to rats.
    Dermal
    Results from in vivo experiments indicate that liquid benzene can be absorbed
    through human skin, although not as substantial as absorption following
    inhalation or oral exposure. In vitro experiments with human skin indicate that
    benzene can be absorbed dermally.16
         In studies conducted in rhesus monkeys, miniature pigs, and hairless mice,
    dermal absorption was < 1% following a single direct (unoccluded) application
    of liquid benzene.16-19 In hairless mice, absorption was rapid and absorption rates
    increased linearly with dose and exposure time.20 Williams et al. analysed the
    experimental (both human and animal; in vitro and in vivo) skin absorption data
    of benzene, and concluded that the steady state absorption rate of benzene ranges
    from 200-400 µg/cm2*h.16
    Oral
    Data on the rate of absorption of benzene following oral ingestion in humans are
    not available. Case reports of accidental or intentional ingestion suggest that
    benzene is also readily absorbed after oral ingestion.2
         Benzene appears to be efficiently absorbed following oral dosing in animals.
    Approximately 80% of the administered radioactivity was eliminated in exhaled
    air and urine within 2-3 days after oral administration of 14C-labeled benzene to
    rabbits (340-500 mg/kg bw).21 Gastrointestinal absorption exceeded 97% in rats
    and in mice at oral doses between 0.5 and 150 mg benzene/kg bw.15
5.2 Distribution
    Information on the distribution of benzene in humans is primarily derived from
    case studies, and relates to exposure by inhalation.2 The available data suggest
    that benzene, upon absorption, is distributed throughout the body. Benzene has
    been detected in various biological fluids and tissues of humans. Benzene is
    lipophilic and the highest levels have been found in lipid-rich tissues. Benzene
    also has been shown to be able to cross the human placenta, and has been found
    in the cord blood in amounts equal to or greater than those in maternal blood.2
 2  Benzene
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<pre>        Animal data also show that benzene distributes in tissues rich in lipids and/or
    with high perfusion rates, such as the kidney, lung, liver, brain and spleen.
    Benzene can cross the placenta and distribute to developing offspring.2 The
    relative uptake in tissues appears to be dependent on the perfusion rate of tissues.
    Following inhalation exposure to 1,600 mg/m³ (500 ppm) benzene, steady state
    concentrations of benzene were reached in male F344 rats within 4 hours in
    blood (11.5 mg/mL), in 6 hours in fat (164.4 mg/g) and in less than 2 hours in
    bone marrow (37.0 mg/g).22 Levels in bone marrow exceeded the respective
    levels in blood. Benzene metabolites (phenol, catechol, and hydroquinone) were
    detected in blood and bone marrow of rats following 6 hours of inhalation
    exposure to benzene. For the more water soluble benzene metabolites, the
    distribution differs from that of benzene.23
5.3 Metabolism
    Although the metabolism of benzene has been studied extensively, the steps
    leading to benzene toxicity are not yet fully understood. The metabolism of
    benzene is an important determinant for benzene-induced toxicity; it is generally
    understood that both cancer and noncancer effects are caused by one or more
    (reactive) metabolites of benzene. Available data indicate that metabolites
    produced in the liver are carried to the bone marrow where benzene toxicity is
    expressed. Benzene metabolism may also occur, at least in part, in the bone
    marrow.
        Benzene metabolism appears to follow similar pathways in both animals and
    humans24; however remarkable species variability has been demonstrated.2 A
    general scheme of the metabolism of benzene is illustrated in Figure 2.
    The first step in the metabolism of benzene is the oxidation of benzene to
    benzene oxide by the cytochrome P-450 dependent mixed-function oxidase
    system. The P-450 enzyme CYP 2E1 appears to exhibit the greatest affinity for
    benzene and is the most active in benzene metabolism. CYP 2B1 is also capable
    of hydroxylating benzene, but contributes to the metabolism only at higher
    benzene concentrations.2 At low benzene exposures, involvement of an enzyme
    other than CYP 2E1 has been suggested.25
        Several pathways are involved in the metabolism of benzene oxide26, which
    exists in equilibrium with its oxepin.27 The predominant pathway involves non-
    enzymatic rearrangement to form phenol.28 In turn, phenol is oxidised in the
    presence of CYP2E1 to catechol or hydroquinone, which are subsequently
    oxidised via myeloperoxidase (MPO) to the reactive metabolites 1,2- and
    Kinetics                                                                             33
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<pre>Figure 2 Simplified benzene metabolism scheme. Benzene metabolism includes several metabolic pathways, involving various
 nzymatic and non-enzymatic steps. The framed compounds can be excreted as glucuronide or sulphate.
Abbreviations:CYP2E1 = cytochrome P-450 2E1; DHDH = dihydrodiol dehydrogenase; EH = epoxide hydrolase;
GSH = glutathione; GST = glutathione-S-transferase; MPO = myeloperoxidase; NQ01 = NAD(P)H: quinone oxidoreductase 1.
            1,4-benzoquinone, respectively.29 Benzoquinone formation via myeloperoxidase
            in the bone marrow is suggested as being a key step in the carcinogenicity of
            benzene.30 The sensitivity of bone marrow cells to benzene has been attributed to
            a relatively high level of peroxidases present in this tissue.2,31 The reverse
            reaction (reduction of 1,2- and 1,4-benzoquinone to catechol and hydroquinone,
            respectively) is catalysed by NAD(P)H: quinone oxidoreductase 1 (NQ01). Both
            catechol and hydroquinone may be converted to 1,2,4-benzenetriol via CYP2E1
            catalysis.
                 Alternatively, benzene oxide may undergo epoxide hydrolase-catalysed
            conversion to benzene dihydrodiol and subsequent dihydrodiol dehydrogenase-
            catalysed conversion to catechol.29,32,33 Each of the phenolic metabolites of
            benzene (phenol, catechol, hydroquinone, and 1,2,4-benzenetriol) can undergo
 4          Benzene
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<pre>    conjugation29,34 with sulfate or glucuronic acid; the conjugates of phenol and
    hydroquinone are the major urinary metabolites of benzene.35,36
         Other pathways of benzene oxide metabolism include: (1) reaction with
    glutathione to form SPMA29,37-41, and (2) iron-catalysed ring-opening to trans,
    trans-muconic acid (ttMA), presumably via the reactive trans, transmucon-
    aldehyde intermediate.29,42-46
5.4 Elimination
    Available human data indicate that following inhalation exposure to benzene,
    exhalation is the major route of elimination of unmetabolised benzene in humans
    and animals. Most of the absorbed benzene however, is metabolised and the
    metabolites are excreted after phase-II-conjugation predominantly in the urine
    (sulfates and glucuronides).24 Small amounts of the glucuronides may enter the
    bile and are found in the faeces. There is evidence that the elimination via some
    metabolic routes is saturable. No studies were available regarding excretion in
    humans after oral exposure to benzene.
         Experimental data in laboratory animals have shown an essentially similar
    pattern of benzene elimination and excretion as in humans. Also in animals,
    unmetabolised benzene is excreted mainly by exhalation and metabolised
    benzene is excreted primarily in urine. Only a small amount of an absorbed dose
    is eliminated in faeces. At higher concentrations, relatively high amounts of
    benzene are excreted by exhalation.
5.5 Possibilities for biological monitoring
    Biomarkers of exposure
    Several biomarkers of benzene exposure have been studied. These include
    benzene levels in blood, urine or expired air. In addition, benzene metabolites in
    urine and biological adducts of benzene have been used as biomarkers of
    exposure.8 The following approaches have been evaluated7:
    • benzene in blood, urine and expired air
    • SPMA in urine
    • ttMA in urine
    • phenol in urine
    • catechol and hydroquinone in urine, and
    • DNA and protein adducts in blood.
    Kinetics                                                                           35
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<pre>    Of the abovementioned parameters, only benzene in blood and urine, and SPMA
    in urine are considered reliable biomarkers. The use of benzene in expired air is
    hampered by practical issues during analysis (e.g., variability in breath sampling,
    transportation and storage; presence of contamination and losses, and absence of
    standardised methods), whereas the benzene metabolites ttMA, phenol, catechol
    and hydroquinone lack specificity. For the determination of DNA adducts,
    sensitive and specific analytical methods are not available.7
        Urinary SPMA, a benzene metabolite with a mean half life ranging from
    9-13 hours, has been shown to be a reliable biomarker for recent benzene
    exposure.7,41,47,48 For SPMA, there is no known endogenous or exogenous
    source, other than benzene exposure and SPMA levels can be detected below
    1 µg/L.
5.6 Possibilities for biological effect monitoring
    Biomarkers of effect (e.g., complete blood cell counts, red and white blood cell
    counts, chromosomal aberrations, sister chromatid exchanges, and examination
    of bone marrow) have been suggested for benzene. The Committee notes
    however, that these biomarkers can be informative on a population level but
    cannot easily be used for individual health surveillance, since they are not
    specific for exposure to benzene.
5.7 Summary
    Benzene is readily absorbed by all routes (inhalation, dermal and oral), of which
    inhalation is considered to be the most important route of occupational exposure.
    In humans, mean absorption has been reported ranging from approximately
    50-80%. In animals, the percentage of benzene absorbed and retained in the
    tissues and blood during a 6h exposure decreased from 33-15% of the dose in
    rats, and from 50-10% in mice, when exposure was increased from 26 to 2,600
    mg/m³ (8-812 ppm). The estimated skin absorption rate ranges from 200-400
    µg/cm2*h. Benzene is efficiently absorbed following oral dosing in animals;
    absorption levels have been reported of > 97% (in rats and mice) and 80% (in
    rabbits).
        Upon absorption, benzene is distributed throughout the body. Benzene has
    been detected in various biological fluids and tissues of humans, highest levels
    amounting in lipid-rich tissues. Also in animals, benzene distributes in tissues
    rich in lipids, particularly those with high perfusion rates, such as the kidney.
 6  Benzene
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<pre>    The metabolism of benzene is not yet fully understood. The available data
indicate that metabolites are primarily generated in the liver. Similar metabolic
pathways exist in animals and human, the first step is the oxidation of benzene to
benzene oxide by cytochrome P-450, mainly CYP 2E1. Several pathways are
involved in the metabolism of benzene oxide, predominantly the pathway
involving non-enzymatic rearrangement to form phenol. In turn, phenol is
oxidised in the presence of CYP2E1 to catechol or hydroquinone, which are
subsequently oxidised to the reactive metabolites 1,2- and 1,4-benzoquinone,
respectively. The phenolic metabolites of benzene undergo conjugation. Other
pathways of benzene oxide metabolism include the reaction with glutathione to
form SPMA and iron-catalysed ring-opening to ttMA.
    Following inhalation exposure to benzene, exhalation is the major route of
elimination of unmetabolized benzene in humans and animals. Most of the
absorbed benzene however, is metabolised and the metabolites are excreted after
phase-II-conjugation predominantly in the urine.
    The determination of benzene in urine, blood and expired air, and the
determination of SPMA in urine are suitable approaches for biological
biomonitoring of benzene.
Kinetics                                                                           37
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<pre>8 Benzene</pre>

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<pre>hapter 6
       Mechanism of action
       Benzene-induced carcinogenicity is caused by a complex mechanism involving
       the metabolism of benzene, subsequent toxicity to blood cells and blood-forming
       organs, genotoxicity and formation of initiated, mutated bone marrow target
       cells, altered oncogenic signalling and clonal proliferation.2,49 In this Chapter,
       the Committee will focus on haematotoxic and genotoxic effects.
       Haematotoxicity
       Benzene must be metabolised to induce haemototoxic and carcinogenic effects.43
       Several metabolising enzymes have been shown to be involved, in particular
       CYP2E1. Other enzymes associated with benzene toxicity include epoxide
       hydrolase and detoxifying NAD(P)H: quinone oxidoreductase 1 (NQ01).1
       Intermediates associated with hematotoxicity include hydroquinone, p-
       benzoquinone, catechol and muconaldehyde (see Figure 2).1,2 The contribution
       to benzene-induced toxicity of each intermediate is currently not known.
           Toxicity is thought to be a result of multiple reactive intermediates that
       interact with multiple targets within the bone marrow. The bone marrow is
       particularly sensitive to benzene, among others due to the presence of benzene-
       metabolising enzymes leading to the production of reactive oxygen species. In
       turn, reactive oxygen species can lead to a spectrum of cellular effects, including
       damage to tubulin, histone proteins, topoisomerase II, other DNA associated
       proteins and DNA itself (including structural and numerical aberrations).1
       Mechanism of action                                                                 39
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<pre>  Benzene-induced haematotoxicity manifests as pancytopenia (a decrease in
  various cellular elements of the circulating blood resulting in anaemia,
  leukopenia, or thrombocytopenia), and aplastic anaemia (along with
  myelofibrosis), i.e., when all cellular elements in the peripheral blood and bone
  marrow are reduced.
      Haematoxicity is considered to be an early indicator of developing acute
  myeloid leukaemia (AML)/myelodysplastic syndrome (MDS) after benzene
  exposure.50 Persistent cytopenias and other blood disorders frequently precede
  the onset of leukaemia in patients developing AML secondary to exposure to
  benzene or alkylating agents.3 Also, workers suffering from benzene poisoning
  are at increased risk of developing leukaemia.51 Currently however, it is not
  proven that benzene-induced haematotoxicity forms an initial (required) step to
  neoplastic disease, or simply represents bone marrow damage.52
  Genotoxicity
  Leukaemia develops from genotoxic effects in the CD34 progenitor cells in the
  bone marrow, a primary target in benzene-toxicity.53,54 Overwhelming evidence
  exists that benzene causes chromosomal aberrations in haematopoetic cells in
  humans and experimental animals.1,2,55 The Committee considers this induction
  of chromosomal aberrations the most plausible explanation for benzene
  carcinogenicity.
      Multiple pathways leading to MDS/AML have been identified. These
  involve different oncogenes and tumour suppressor genes and can be
  distinguished by their specific chromosomal aberration. Several typical
  cytogenetic or mutagenic profiles are commonly observed in AML:56,57
  • unbalanced aberrations (primarily 5q-/-5 or 7q-/-7 and +8)
  • balanced rearrangements (e.g., t(11q23), t(8;21) and t(15;17)) or inversions
      (e.g., inv(16))
  • karyotypically normal but with mutations (e.g., mutations of NPM1 or
      C/EBPα, duplications of FLT3).
  These profiles are quite similar for therapy-related MDS/AML (i.e., MDS/AML
  caused by treatment with alkylating agents, radiation, or topoisomerase II
  inhibitors) and spontaneous MDS/AML, although the frequencies at which these
  typical chromosomal aberrations occur may differ.57 MDS/AML associated with
  benzene exposure has been reported to share a similar genetic profile with
  therapy-related MDS/AML, i.e., a high frequency of loss of all or part of
  chromosomes 5/7.30,58,59 AML/MDS related to therapy and AML/MDS related to
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<pre>benzene exposure have therefore been considered biologically similar
diseases.50,57,60 Recent data however, suggest that the pattern of clonal
cytogenetic abnormalities in benzene-exposed cases more closely resemble that
of spontaneous AML than therapy-related AML.61
    Several underlying mechanisms of benzene-induced AML/MDS have been
suggested in literature (reviewed by McHale et al.62), i.e.,:
1 Inhibition of topoisomerase II. The inhibition of topoisomerase II is the most
    well established explanation for the genotoxic mode of action of benzene,
    and the most likely mechanism through which benzene induces chromosomal
    translocations.62 Several studies have shown that benzene and its metabolites
    hydroquinone and 1,4-benzoquinone act as inhibitors of topoisomerase II
    (“topoisomerase II poisons”), potentially leading to DNA strand breaks,
    aberrant mitotic recombination and subsequent chromosomal
    aberrations.30,55,63-66 Topoisomerase II inhibitors are indeed also known to
    produce leukaemia in humans and some share structural and biological
    similarities with benzene.64,67 Furthermore, several genetic pathways that
    have been implicated in benzene-induced MDS/AML are associated with the
    inhibition of topoisomerase II.56 Whysner et al. compared the genotoxic
    profiles of benzene and its metabolites with those of other genotoxic agents,
    and concluded that it was most similar to genotoxicity induced by
    topoisomerase II inhibitors.55
2 Adduct formation of reactive metabolites. Adduct formation has been
    observed for benzene metabolites in multiple organs in animals, and in blood
    of benzene exposed workers.68 This mainly involved binding to proteins, for
    which benzene oxide and p-benzochinon have been considered as the most
    important metabolites involved. Based on the very low level of DNA adducts
    found, in particular in target tissues, it has been suggested that covalent
    binding does not play a significant role in benzene-induced carcinogenicity.55
3 Oxidative DNA damage. Several benzene metabolites have been associated
    with the generation of reactive oxygen species (ROS). Subsequently, reactive
    oxygen species and oxidative damage after exposure to benzene have been
    linked with the induction of DNA strand breaks and point mutations.
4 Error prone DNA repair. It has been suggested that induction and activation
    of DNA-PKcs may contribute to benzene carcinogenesis by increasing the
    error-prone, non-homologous end joining (NHEJ) DNA repair pathway..
    This has also been suggested to explain the high susceptibility of
    haematopoetic stem cells to benzene, as these cells preferentially initiate
    DNA repair instead of undergoing apoptosis.
Mechanism of action                                                                41
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<pre>  5   Epigenetic alterations. Benzene has been shown to alter the expression of
      many genes in the peripheral blood of exposed workers. Epigenetic changes
      are major mechanisms by which gene expression is regulated, and epigenetic
      marks including histone modification, DNA methylation and microRNA
      expression, activate or repress expression of individual genes (e.g.,
      oncogenes and tumor suppressor genes).
  Consequently, these events in the stem or progenitor cell most likely result in
  genomic instability, and subsequent activation of key protooncogenes, loss of
  heterozygosity, and inactivation of tumour suppressor genes. Dysregulation of
  the p53 pathway resulting in alterations in cell cycle checkpoints, apoptosis, or
  the DNA repair system may be an event leading to haematopoietic
  malignancies.52
  Conclusion
  Benzene-induced haematotoxicity and genotoxicity result from a complex
  cascade of events. Multiple mechanisms have been suggested to be involved
  (cytogenetic alterations, aberrant mitotic recombination, gene mutations and/or
  epigenetic alterations). Most evidence has been provided for a role of inhibition
  of topoisomerase II in the induction of chromosomal damage; however, other
  modes of action, such as oxidative stress and inhibition of DNA repair, may add
  to the effect.
      Based on the weight of evidence. the Subcommittee on Classification of
  Carcinogen Substances considers that the above mentioned mechanisms, which
  ultimately may lead to genotoxicity and altered gene expression, are most likely
  to be explained by mechanisms for which a threshold exists. Therefore, the
  Subcommittee concludes that benzene acts by an indirect (non-stochastic)
  genotoxic mode of action (see Annex H).
2 Benzene
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<pre> hapter 7
        Effects
        Numerous human studies and animal studies regarding the carcinogenic and non-
        carcinogenic effects of benzene have been published. As the Committee prefers
        the use of human data, and considers the available human data on benzene
        adequate, it will focus its current evaluation of benzene to human studies.
        Furthermore, the Committee limits its evaluation to the human studies most
        relevant for the quantitative risk assessment of benzene.
7.1     Observations in humans
7.1.1   Carcinogenicity
        Classification of haematological malignancies
        There is extensive literature available on benzene carcinogenicity in humans.
        These carcinogenic effects mainly relate to the haematopoietic and lymphoid
        system. The classification of tumours of the haematopoietic and lymphoid tissues
        has been revised by the World Health Organisation in 2008, due to the
        availability of new scientific and clinical information.69 This revised classifi-
        cation is based on the integration of clinical, morphologic, immunopheno-typic,
        genetic and other biological features.70 In the revised classification,
        haematological disorders are no longer classified according to their localisation,
        but according to their cells of origin. The traditional classification and the 2008
        Effects                                                                             43
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<pre>Figure 3 Former and current classification of the main haematopoietic and lymphoid malignancies.
            revision of the main haematopoietic and lymphoid tumour types are illustrated in
            Figure 3.
            Due to the revision, the classification of several haematological disorders that
            have been associated with exposure to benzene have changed in time, hereby
            complicating an historical analysis of these disorders.71 For instance, acute
            lymphatic leukaemia (ALL) has been reported as a type of leukaemia in past
            studies, whereas in more recent studies, ALL is classified as a lymphoid
            neoplasm. In addition, in the new classification additional haematological
            disorders are distinguished. Particularly relevant for benzene is the current
            discrimination between myelodysplastic syndrome (MDS) and acute myeloid
            leukaemia (AML; also referred to as acute non-lymphatic leukaemia (ANLL)).
            These malignancies are both associated with benzene exposure and have
            historically been difficult to discern.
            Critical carcinogenic endpoints
            Despite the use of different classifications in time, the Committee notes that
            clear evidence has been published for a relationship of benzene exposure and
            development of leukaemia and AML/MDS. For other haematological
            malignancies, this relationship is less clear. Several positive associations have
            been reported, including two recent meta-analyses in which an association was
            suggested between benzene exposure and multiple myeloma (MM), chronic
            lymphocytic leukaemia (CLL), acute lymphocytic leukaemia (ALL), and chronic
            myeloid leukaemia (CML).72,73
 4          Benzene
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<pre>    Overall, the Committee finds the data available on subtypes of
haematological malignancy other than leukaemia AML/MDS, most notably non-
Hodgkin lymphoma (NHL), currently inconsistent (see e.g., references72,74-79).
Therefore, the Committee will focus on the key cohorts for exposure-response
analysis – including recent updates, and meta-analyses – on leukaemia in general
and AML.80 These studies are summarised below and represented in Annex E.
The malignancies that are less consistently linked to benzene exposure are not
specifically discussed by the Committee, as these do not influence the
carcinogenic classification of benzene nor do these provide a basis for a
quantitative risk analysis.
Cohort studies
Pliofilm cohort
The Pliofilm cohort is an extensively studied cohort consisting of workers
exposed to benzene in three rubber hydrochloride manufacturing plants at two
locations in Ohio.81 This cohort has historically been considered to be a carefully
performed study, with a low probability of co-exposures which served as a basis
for earlier quantitative risk analyses of several authorities or organisations.4 This
cohort has been updated several times.
    The Pliofilm cohort initially consisted of a total of 748 workers who were
exposed to benzene for at least 1 day between 1940 and 1950.81 Mean exposure
duration was short but high exposures (> 325 mg/m3 (> 100 ppm)) occurred
frequently; 58% of the workers were exposed to benzene less than 1 year. Death
rates for age-matched U.S. white males during the same calendar period were
used for comparison with death rates observed in the cohort. An increased risk of
dying from leukaemia (all myelocytic or monocytic cell types) was found
(standardised mortality ratio (SMR) of 5.6, p < 0.001). Workers exposed for
more than 5 years had a SMR of 21.00.
    An update of this cohort, providing more detailed exposure estimates,
included 1,165 white males employed between 1940 and 1965 who were
followed through 1981.82 Cumulative exposure for each cohort member was
estimated from historical air-sampling data, or alternatively, from interpolation
based on existing data. A statistically significant increased mortality from all
leukaemias (9 observed versus 2.7 expected; SMR = 3.37; 95% CI 1.54-6.41)
and multiple myeloma was noted (4 observed versus 1 expected; SMR = 4.09;
95% CI 1.10-10.47). Stratification of exposure identified a relationship between
cumulative exposure and an increasing risk of leukaemia.
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<pre>      Assessment of the Pliofilm cohort after an additional follow-up period up to
  1996 (i.e., 15 years after the original report from Rinsky (1981)) also showed an
  increased leukaemia mortality risk at cumulative exposures exceeding 650
  mg/m3-years (200 ppm-years).83 For MM, a non-significant increased relative
  risk was found whereas for NHL, no association was found. Although in this
  update 5 new leukaemia cases were observed, the relative risk had declined
  compared to the previous analysis, suggesting that the excess risks diminished
  with prolonged time since exposure. Exposures in the most recent 10 years were
  most strongly associated with leukaemia risk; there was no significant
  relationship observed between leukaemia deaths and benzene exposures of more
  than 20 years ago.
      Although several exposure estimates have been derived in time84-86, the
  temporal trend observed in this cohort has been undisputed and has been
  confirmed by others.87-89
  NCI/CAPM*
  The largest cohort study conducted to date is the NCI/CAPM study.90-93 In this
  study incidence rates (occurrence of disease and cause of death) for lympho-
  haematopoietic malignancies and other haematological disorders were evaluated
  in a cohort of 74,828 benzene exposed and 35,805 non-exposed workers
  employed in 672 factories at 12 cities in China. The workers were employed
  from 1972 to 1987, and were followed for an average of nearly 12 years.
  Estimates of benzene exposure were derived from work histories and available
  historic benzene measurements. Mean exposure was estimated to be 73.4 mg/m3
  (22.5 ppm) (time-weighted average), with a mean exposure duration of 9.3 years.
  A total of 82 exposed cases were diagnosed with leukaemia.
      For all haematological neoplasms (leukaemia, ANLL, ANLL/MDS and
  NHL), increased RRs and statistically significant trends were found when
  benzene exposure was expressed as either average or cumulative exposure level.
  Analysis by duration of exposure (< 5, 5-9, or ≥ 10 years) did not show increased
  risk with increasing exposure duration.
  UK Petrol cohort
  In a case-control study in the United Kingdom, 91 cases of leukaemia were
  compared with matched controls (four per case) in a cohort of workers in the
  petroleum distribution industry who were exposed to low levels of benzene.94,95
  Study by the U.S. National Cancer Institute (NCI) and the Chinese Acadamy of Preventive Medicine
  (CAMP).
6 Benzene
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<pre>Exposure to benzene was estimated based on work histories obtained, and
specific information on the exposure conditions at the terminals. Analyses were
done for all leukaemia and separately for acute lymphoblastic leukaemia, CLL,
AML and acute monocytic leukaemia, and CLL. Potential confounding or effect
modifying variables considered were smoking, employment status at end date,
socioeconomic status based on the job title of longest duration, age, and the
starting date of work, ever had a previous job, and ever had a previous job as a
driver.
    No significantly increased risk was observed in the risk of overall leukaemia
or subtypes. The authors note the suggestion of a relationship between myeloid
leukaemia (in particular acute myeloid and monocytic leukaemia) and exposure
to benzene.
    The UK Petrol cohort was included in a pooled analysis, recently published
by Schnatter et al.96 (see below).
Dow cohort
Ott et al. described a retrospective cohort study in 594 individuals exposed to
benzene at a chemical plant in Michigan between 1938 and 1970, with follow up
from 1940 to 1973.97 Workers were exposed to an estimated 6.5-29.5 mg/m3 (2-9
ppm) (TWA), based on data derived from work histories and industrial hygiene
records. No significant increases in total leukaemia were found in chemical
industry workers. A statistically significant increased number of cases of
myelogenous leukaemia was noted (4 observed compared to 0.9 expected;
p < 0.011). The study, however, suffers from limitations in design (e.g., a small
sample size).
    In a subsequent update of this cohort, with an additional follow up of 9 years
included 956 employees, the mortaility risk of myelogenous leukaemia was
increased but did not reach statistical significance (SMR = 1.94; 95% CI
0.52-4.88).98
    In 2004, a follow-up study was published on this cohort study in which
cause-specific mortality was determined in a prospective study of 2,266 chemical
workers.99 The risk for leukaemia was slightly above background (SMR = 1.14).
No significantly increased risk was found for any of the lymphohaematopoietic
cancers. Only a weak trend of increasing SMRs for leukaemia, and possibly
ANLL, with increasing cumulative exposure to benzene was noted.
Chemical Manufacturers Association (CMA) cohort
This cohort study consisted of 4,602 male workers in US chemical industry
between 1946 and 1975.100,101 The control group consisted of 3,074 unexposed
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<pre>  males from the same company in the same period. Benzene exposure was
  estimated based on work histories and benzene measurements. Half of the
  exposed workers were exposed to average benzene concentrations of less than
  3.3 to over 160 mg/m3 (1-50 ppm) for six months to over 35 years, on average 8
  hours per day. The control group consisted of 3,074 unexposed workers from the
  same chemical industry. Death rates were also compared to the general
  population.
      No increased risk of leukaemia or other types of lympho-haematopoietic
  cancer were noted among the exposed and unexposed group when compared
  with the general population. When compared to the unexposed group, the
  exposed group showed a statistically significant positive trend in the risk of
  leukaemia and other types of lympho-haematopoietic cancer.
  Australian Health Watch study
  A large nested case-control study within a cohort of 17,525 employees in the
  Australian petroleum industry working for more than 5 years found an increased
  risk of leukaemia.102-104 Workers were employed between 1935 and 1985, the
  majority after 1965. Exposure to benzene was retrospectively estimated based on
  individual occupational history and benzene exposure measurements supplied by
  Australian petroleum companies.102 79 cases of lympho-haematopoietic cancers
  were identified between 1981 and 1999, including 33 cases of leukaemia – 11 of
  these cases were diagnosed as ANLL. A strong association was found between
  leukaemia risk and exposure to benzene. Increasing risk was reported from
  2.6-5.2 mg/m3-years (0.8-1.6 ppm-years) onwards, to significantly increased
  ORs of 5.9 and 98.2, for exposure estimates of > 26-32 mg/m3-years (> 8-16
  ppm-years) and 52 mg/m3-years (>16 ppm-years), respectively. Risk for the
  subtype ANNL was increased at exposures greater than 26 mg/m3-years (8 ppm-
  years) (OR = 7.17; 95% CI 1.27-40.4).103 NHL and MM were not associated
  with benzene exposure.
      In a re-analysis of the exposure-response relationship, the 7 leukaemia cases
  with the highest cumulative exposure (52 mg/m3-years (>16 ppm-years)) were
  compared with a different reference exposure category (i.e., the two lowest
  exposed categories).104 This new reference category contained 9 cases of
  leukaemia, as opposed to 3 cases in the previous analysis. An increase in
  leukaemia risk with increasing cumulative benzene exposure was observed,
  hereby confirming the earlier analysis of this cohort by Glass in 2003.
      This cohort was included in a pooled analysis, recently published by
  Schnatter et al.96 (see pooled analysis below).
8 Benzene
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<pre>Canadian cohort
In 1996, Schnatter conducted a nested case-control study on
lymphohaematopoetic cancers in a previously defined cohort105,106, consisting of
a total of 6,672 petrochemical workers (average age 38 years) exposed to
benzene for a median 18 years 63 and followed up for an average of
approximately 15 years.107 A total of 31 cases (16 leukaemia cases, 7 cases of
MM and 8 cases of NHL) were matched with 4 controls for each case. Benzene
exposure was estimated based on work histories and historical industrial hygiene
surveys, and ranged from 0.04-20.2 mg/m3 (0.01-6.2 ppm). Additional potential
confounders including smoking, dermal contact, hobbies, previous exposures
and occupations, diagnostic radiation exposure and familial cancer were
accounted for.
    No significant association was found between the cumulative benzene
exposure and mortality due to leukaemia, NHL or multiple myeloma. However,
the power of the study was limited and the authors report incomplete correction
for confounders.
    This cohort was included in a pooled analysis, recently published by
Schnatter et al.96 (see pooled analysis below).
Pooled analysis of the AHW, Canadian and UK Petrol cohorts
Recently, Schnatter et al. presented an update of three nested case–control
studies among petroleum workers from Australia, Canada, and the United
Kingdom.96 Cases were re-assessed based on the new classification for
hematopoetic malignancies and exposure to benzene was standardised across the
three studies. Cumulative benzene exposure showed a monotonic dose-response
relationship with MDS (highest vs lowest tertile, > 2.93 vs ≤ 0.35 ppm-years,
OR = 4.3; 95% CI 1.3 to 14.3). In contrast to previous findings in these cohorts,
there was little evidence of a dose–response relationships for AML.
Shoe factory worker cohort
A cohort consisting of 891 men and 796 women employed in the shoe factory
industry was followed between 1939 and 1984.108 Workers were followed from
1950 to 1999. Exposures were estimated based on work histories and limited air
sampling data. Estimated benzene concentrations ranged from 0-300 mg/m3
(0-92 ppm), and mean cumulative exposures of 190.4 ± 304.2 mg/m3-years
(58.4 ± 93.9 ppm-years), respectively. Duration of exposure and duration of
employment were not reported. The general population was used as control
reference.
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<pre>      Leukaemia risk was significantly increased at >652 mg/m3-years (> 200
  ppm-years) for men only (SMR = 7.0; 95% CI 1.9-18.0) and for men and women
  combined (SMR = 5.1; 95% CI 1.4-13.0). Leukaemia subtypes were not
  evaluated.
  Offshore worker cohort study
  A recent historical cohort study included 27,919 offshore workers and 366,114
  controls from the general working population.109 Workers were employed
  between 1981 and 2003. Benzene exposure and variability therein, was however
  not quantified in this study. Based on previous studies assessing benzene
  exposure for this type of industry, the authors estimated that exposure of the
  workers ranged from below 0.003 to 2.3 mg/m3 (0.001 to 0.7 ppm).
      An increased risk of AML (RR = 2.89; 95% CI 1.25-6.67) and multiple
  myeloma (RR = 2.49; 95% CI 1.21-5.13) was observed. An increased risk of
  AML was only present among workers who had their first registered engagement
  in this industry in the period 1981-1985 (RR = 3.26). In contrast, for the period
  1986-2003 there was no statistically significant increased risk for AML found.
  Meta-analyses
  A meta-analysis by Raabe and Wong consisted of 19 cohorts with a total of
  208,741 workers in the petrochemical industry employed between 1937 and
  1989 in the United States and the United Kingdom.110 Average exposure levels
  were estimated to be 0.70 mg/m3 (0.22 ppm) based on mean benzene exposure
  levels reported for general plant operations in petroleum refineries. No increased
  risk of AML or other types of leukaemia were found. Analyses that were limited
  to studies of refinery workers or studies with at least 15 years of follow-up
  yielded similar results.
      In a more recent analysis, Vlaanderen et al. fitted meta-regression models to
  30 aggregated risk estimates, extracted from 9 observational studies to assess the
  benzene-leukaemia exposure-response relationship.111 In addition, relative risks
  (RRs) were calculated for several cumulative exposure levels, based on either all
  studies, or cohort studies only, using four different modelling scenarios (i.e.,
  natural spline or linear model with and without intercept). All scenarios predicted
  a significantly increased, exposure-dependent RR. The highest RRs were
  predicted by applying a natural spline on all studies, whereas these RRs dropped
  considerably after correction for the predicted intercept, or when only cohort
  studies were used.
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<pre>    Khalade et al. conducted a systematic review and meta-analysis on the
relationship between occupational benzene exposure and the risk of leukaemia,
including all types combined and the four main subgroups separately (AML,
ALL, CLL and CML).112 This meta-analysis included 15 studies providing 16
risk estimates in total. A statistically significant increase in summary effect size
for all leukaemias combined was found. Dose-response analysis for the 9 studies
in which exposures were expressed in ppm-years resulted in significant increased
risks of leukaemia for the different exposure categories and a significant trend.
The risk for AML was also increased, but the trend was not statistically
significant. Some evidence of an increased risk was also noted for CLL, no
association was found for CML.
    A meta-analysis of benzene exposure and leukaemia subtypes, including nine
cohorts and 13 case-control studies from several industries, found a high and
significant risk of AML.113 Furthermore, a positive dose-response relationship
across study designs was noted. No clear evidence for a dose-response
relationship was obtained for other leukaemia subtypes (CLL, CML and ALL).
This meta-analysis however, did not attempt to couple exposure concentrations
to AML incidence but rather described patterns among industrial sectors and
different study designs in relation to relative risk.
    Vlaanderen et al. conducted a meta-analysis on a total of 44 publications on 5
different haematological malignancies (HL, NHL, MM, ALL and CLL).72 The
authors aimed to identify the most informative subgroups of cohort studies by the
stratification of three different study quality dimensions. These study quality
dimensions involved (a) the year of start of follow-up, (b) the strength of the
reported association benzene-AML, and (c) the quality of the exposure
assessment. For MM, ALL and CLL the relative risk increased with increasing
study quality for all three stratification approaches, thereby suggesting an
association with the exposure to benzene.
    A similar approach by these authors was applied for CML. The overall meta-
relative risk (mRR) was non-significantly elevated (1.23; 95% CI 0.93-1.63).73
An increasing meta-RRs with increasing study quality was reported for all
dimensions. For studies with start of follow-up after 1970 this increase was
statistically significant (1.67; 95% CI 1.02-2.74. For AML, the highest study
quality stratum significance and exposure quality showed an elevated but non-
significant increased mRR.
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<pre>      Conclusion
      The Committee concludes that benzene is carcinogenic in humans by causing
      leukaemia and more specifically, AML/ANNL.
7.1.2 Genotoxicity
      The Committee limits its evaluation of the genotoxicity data (mainly on
      chromosomal aberrations in peripheral white blood cells) to studies with low
      exposure levels (i.e., below 33 mg/m3 (10 ppm) of benzene (Table 2). For a
      complete overview of the genotoxicity data, the Committee refers to extensive
      review documents available (e.g., references1,2,6,55,59).
      Chromosomal aberrations
      A large biomonitoring study (n=171) in Chinese factory workers exposed to
      benzene also included the assessment of chromosomal aberrations.114 A broad
      range of benzene exposures was studied, on the day of biological sample
      collection, exposures ranged from 0.20-400 mg/m3 (0.06-123 ppm) with a
      median exposure of 10.4 mg/m3 (3.2 ppm). The median of the 4-week mean
      benzene exposures was 12.4 mg/m3 (3.8 ppm), and the median lifetime
      cumulative exposure was 166.1 mg/m3 (51.1 ppm-years). The results analysed
      using 4-week mean exposures showed a significant trend in the increase of
      chromatid breaks, total chromatid-type aberrations, total chromosomal-type
      aberrations, and total aberrations. When the low portion of the benzene exposure
      spectrum (< 1.6 mg/m3; mean 0.5 mg/m3 (< 0.5 ppm; mean 0.14 ppm); n=16)
      was examined, there were positive associations for total chromatid aberrations,
      total chromosomal aberrations, total aberrations, chromatid breaks, and acentric
      fragments.
          The specificity of benzene-induced aneuploidy and the influence of genetic
      polymorphisms on chromosomal aberrations were studied in workers at a coke
      oven plant by Kim et al.115 The benzene concentration ranged from 0.03-2.4
      mg/m3 (0.01-0.7 ppm) (geometric mean 1.8 mg/m3 (0.6 ppm)). Multiple
      regression analysis indicated that the frequencies of chromosome aberrations
      were significantly associated with benzene exposure and polymorphisms in the
      metabolic enzyme genes.
          In a cytogenetic monitoring study by De Jong et al., 32 workers exposed
      for periods between 1 and 13 years to low levels of benzene (0.1-2.5 mg/m3;
      < 0.03-0.8 ppm)) in a petrochemical complex in the Netherlands were monitored
 2    Benzene
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<pre>for the induction of structural chromosomal aberrations.116 Control samples were
obtained from 42 employees working in an remote administrative office, and
matched for sex, age and smoking habits. No increase in frequencies of
chromosome aberrations was observed.
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<pre> able 2 Selection of genotoxicity studies with human peripheral lymphocytes at low benzene concentrations.
ndustry                         Number of Mean exposure (range)                     Result           Reference
                                workers      mg/m3               ppm                (result at < 3.3
                                exposed                                             mg/m3 (1 ppm))
  hromosomal aberration tests in pheripheral lymphocytes
 hoe and glue manufacturing     130          10.4 (0.2-400)      3.2 (0.1-122)      + (+)            Qu et al. (2003)114
                                16           0.5 (0-1.6)         0.14 (0-0.5)       + (+)
                                (subgroup)
  oke oven plant                82           1.8 (0.0-2.4)       0.6 (0.0-0.7)      + (+)            Kim et al. (2004)115
 etrochemicals                  32           < 0.3 (< 0.1-2.5)   < 0.1 (< 0.0-0.8)  - (-)            De Jong et al. (1988)116
Oil-refinery                    42           7.2 (2.9-20.5)      2.2 (0.9-6.3)      + (ND)           Major et al. (1994)117
Oil-refinery                    49           (3.0-68.7)          (0.9-21.5)         + (ND)           Tompa et al. (1994)118
                                subgroup (1.0-18.4)              (0.3-5.6)          + (ND)
  enzene production             22           (0.7-40.3)          (0.2-12.4)         + (ND)           Sarto et al. (1984)119
 hoe manufacturing              49           < 16.2              <5                 + (ND)           Bogadi Sare et al.
                                                                                                     (1997)120
 hoe manufacturing              38           25.5 (5.2-48.6)     7.8 (1.6-15.0)     + (ND)           Karacic et al. (1995)121
                                45           15.9 (6.2-40.8)     4.9 (1.9-12.6)
 ingle strand breaks
 hoe manufacturing              20           4.2 (0.8-16.1)      1.3 (0.2-4.8)      + (ND)           Popp et al. (1992)123
 everal, with exposure to       33           0.4 (0.0-2.0)       0.13 (0.0-0.6)     + (+)            Nilsson et al. (1996)124
 asoline
Micronucleus assay
 hoe manufacturing              35           2.6                 0.8                + (+)            Liu et al. (1996)125
 ister chromatid exchange
 hoe manufacturing              38           25.5 (5.2-48.6)     7.8 (1.6-15.0)     + (ND)           Karacic et al. (1995)121
                                45           15.9 (6.2-40.8)     4.9 (1.9-12.6)     - (ND)
Oil-refinery                    42           7.2 (2.9-20.5)      2.2 (0.9-6.3)      + (ND)           Major et al. (1994)117
Oil-refinery                    49           (3.0-68.7)          (0.9-21.5),        + (ND)           Tompa et al. (1994)118
                                subgroup (1.0-18.4)              (0.3-5.6)          + (ND)
  enzene production             22           (0.7-40.3)          (0.2-12.4)         - (ND)           Sarto et al. (1984)119
Other: chromosomal aberrations in sperm
 arious, including shoe and     33           9.4 (< 78)          2.9 (< 24)         + (+)            Xing et al. (2010)126
 lue manufacturing                9          < 3.3               <1                 + (+)            Ji et al. (2012)127
ND: Not determined; positive results were observed but exposure below 3.3 mg/m3 (1 ppm) was not specifically addressed.
              Chromosomal aberrations were measured in peripheral blood lymphocytes of
              42 oil-refinery workers exposed to benzene, and 42 controls.117 The benzene
              concentrations in the ambient air samples varied from 3-20 mg/m3 (mean:
              7 mg/m3) (0.9-6.3 ppm; mean 2.2 ppm). The continuous low-dose benzene
              exposure statistically significantly increased the numbers of chromosomal
              aberrations.
                  Tompa et al. assessed the induction of chromosomal aberrations, SCEs and
              UV-induced DNA synthesis as indicators of genotoxic effects in peripheral blood
              lymphocytes of 49 workers occupationally exposed to benzene (3-68.7 mg/m3;
 4            Benzene
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<pre>0.9-21.1 ppm).118 Most of the workers were followed up in a period in which the
benzene concentrations were reduced to 1-18.4 mg/m3 (0.3-5.6 ppm). Overall,
the frequencies of chromosomal aberrations were significantly higher in the
exposed groups than in controls.
    A cytogenetic study was performed with 22 healthy workers engaged in
benzene production and exposed to low concentrations of benzene and 22
matched controls. Exposure concentrations ranged from 0.7-40.4 mg/m3
(0.2-12.4 ppm).119 Benzene exposure was confirmed by biological monitoring.
A statistically significant increase of structural chromosomal aberrations was
observed in the exposed workers.
    The incidence of structural chromosome aberrations and SCE was studied by
Bogadi Sare et al. in the peripheral blood lymphocytes cell genome of 49 female
shoe-makers.120 Workers were exposed to concentrations of benzene up to 49
mg/m3 (15 ppm). Chromosomal aberration analysis revealed a significant
increase in dicentric incidence in the exposed group compared to the controls,
however the authors noted a presence of potential confounders.
    Structural chromosome aberrations in peripheral blood lymphocytes were
studied in female workers employed in the shoe-making industry, the first group
in 1987 and the second group in 1992.121 Mean benzene exposures were 25.5 and
15.9 mg/m3 (7.8 and 4.9 ppm) for the first and the second group, respectively.
The results were compared with those obtained from 35 controls. This
cytogenetic study showed a significant increase in dicentric chromosomes in
exposed groups I and II when compared to the control group.
Single strand breaks
The induction of single strand breaks was studied in workers exposed to benzene
in one of five occupational work places, including six industrial process types,
namely, printing, shoe-making, methylene di-aniline (MDA), nitrobenzene,
carbomer, and benzene production.122 Benzene concentration in breath was less
than 9.8 mg/m3 (3 ppm). The mean value of DNA damage was 1.73 ± 0.81.
Dose-dependent DNA damage occurred at higher levels of exposure, and DNA
damage exhibited a strong correlation with benzene breath levels.
    Peripheral lymphocyte DNA damage was investigated in a group of 20
female workers of a shoemaking plant who were exposed to benzene (mean
concentration of 4.16 mg/m3; range 0.80-16.10 mg/m3 (1.3; 0.2-5.0 ppm)) and
toluene.123 The relative DNA elution rate was statistically significantly higher in
workers compared to controls.
Effects                                                                             55
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<pre>      Nilsson et al. determined the induction of single-strand breaks in DNA of
  leukocytes, and urinary levels of the oxidative DNA adduct 8-hydroxydeoxy-
  guanosine (8OHdG), in 33 men occupationally exposed to benzene from
  gasoline and in 33 controls.124 The average exposure to benzene over a shift was
  determined by personal air sampling. The 8-hr TWA exposure to benzene was
  0.4 mg/m3 (range: 0.01-2.0 mg/m3) (0.13; 0.003-0.6 ppm). Exposed workers had
  a significant increase of single strand breaks (p = 0.04) over the shift compared
  with controls. Urinary 8OHdG increased over the shift in exposed workers but
  not in controls.
  Induction of micronuclei
  The induction of MN in blood lymphocytes was measured in 87 shoe makers
  exposed to different concentrations of benzene, and 30 controls.125 In the 35
  individuals in the lowest exposure category, exposed to a mean concentration of
  2.6 mg/m3 (0.8 ppm), a statistically significant increase in micronucleated
  lymphocytes was reported.
  Sister chromatid exchange (SCE)
  In several studies with peripheral lymphocytes, the induction SCE was also
  assessed, mostly with positive results.117-119,121,123
  Other
  Xing et al. used multicolour fluorescence in situ hybridization to measure the
  incidence of sperm with numerical abnormalities of chromosomes X, Y, and 21
  among 33 benzene-exposed men and 33 unexposed men from Chinese
  factories.126 Exposure levels ranged from below the detection limit to 78 mg/m3
  (24 ppm) (median, 9.4 mg/m3 (2.9 ppm)). From the exposed men, 27% (n = 9)
  were exposed to concentrations of ≤ 3.3 mg/m3 (1 ppm). Increased sperm
  aneuploidy was observed within low- and high-exposed groups, including the
  ≤ 3.3 mg/m3 (1 ppm) exposure group.
      These authors subsequently compared aneuploidies in blood lymphocytes
  and sperm within the same individuals.127 The results showed that benzene
  exposure was positively associated with the gain of chromosome 21 but not sex
  chromosomes in blood lymphocytes. This was in contrast to analysis of sperm,
  where the gain of sex chromosomes, but not chromosome 21, was significantly
  increased in the exposed workers. Furthermore, a significant correlation in the
6 Benzene
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<pre>      gain of sex chromosomes between blood lymphocytes and sperm was observed
      among the unexposed subjects, but not among the exposed workers.
      Conclusion on genotoxicity
      The Committee notes that multiple studies are available that indicate that
      benzene is genotoxic in humans by inducing cytogenic effects. Nearly all studies
      were performed with isolated peripheral lymphocytes. Several studies however,
      suffer from poor exposure information and methodological insufficiencies, in
      particular lack of a proper control groups and insufficient information on
      benzene exposure (i.e., the occurrence of peak concentrations co-exposures to
      other chemicals).2
7.1.3 Haemototoxicity
      Occupational exposure to benzene has long been associated with toxicity to the
      blood and bone marrow, including lymphocytopenia, pancytopenia, and aplastic
      anaemia. Several cross-sectional studies with workers who were exposed to
      benzene have shown haematological effects at a broad range of exposure levels.
      The Committee limits its assessment to haematological effects observed at low
      concentrations, i.e., at or below 1 ppm: these are relevant for setting a health-
      based recommended occupational exposure level (HBR-OEL).
      Lan et al. assessed the white blood cell and platelet count in 250 shoe workers
      exposed to benzene and 140 controls.128 For each subject, individual benzene
      and toluene exposure was monitored repeatedly up to 16 months before
      phlebotomy, and postshift urine samples were collected from each subject.
      Subjects were categorised into four groups (control; < 3.3 mg/m3 (< 1 ppm);
      3.3-32.5 mg/m3 (1-10 ppm); and > 32.5 mg/m3 (> 10 ppm)) by mean benzene
      levels measured during the month before phlebotomy. All types of white blood
      cells measured, but also platelets were significantly decreased in workers from
      the lowest exposure group (mean exposure of 1.9 mg/m3 (0.6 ppm)) to the
      highest exposure group (93.3 mg/m3 (28.7 ppm)). Also in a subpopulation that
      included workers exposed to a mean exposure level of 0.9 mg/m3 (0.3 ppm)
      benzene, the decrease in the number of peripheral blood cells was statistically
      significant. In addition, highly significant dose-dependent decreases in colony
      forming capacity of progenitor cells were observed. The effect on colony
      formation appeared greater than the effect on differentiated white blood cells and
      Effects                                                                            57
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<pre>  granulocytes, suggesting a relatively higher sensitivity towards benzene of
  progenitor cells as compared to mature cells.
  In a study by Qu et al., personal benzene exposure was monitored and peripheral
  blood cells were counted in a total of 181 Chinese factory workers.114,129,130 The
  population studied had a broad range of benzene exposures: on the day of
  biological sample collection, exposures ranged from 0.2-396 mg/m3 (0.1-122
  ppm) with a median exposure of 10.4 mg/m3 (3.2 ppm). The median of the
  4-week mean benzene exposures was 12.4 mg/m3 (3.8 ppm), and the median
  lifetime cumulative exposure was 166.1 mg/m3-years (51.1 ppm-years). In this
  study, a decrease in neutrophil number and red blood cells was observed at
  benzene exposures of 1.6 mg/m3 (0.5 ppm) or lower (4-week mean of 0.5 mg/m3
  (0.1 ppm)). The Committee notes that 24/51 control subjects were males,
  whereas no males were included in the exposure group.
       In a study on the haematotoxic effects of benzene, 928 workers in 5 factories
  in China were monitored weekly and 12 peripheral blood indices were
  examed.131 According to the authors, the most sensitive parameters to benzene
  appeared to be neutrophils and the mean platelet volume, where effects were
  seen, with a linear exposure-response relationship, for benzene air concentrations
  of 25.3-26.7 mg/m3 (7.8-8.2 ppm). Logistic regression analysis revealed
  statistically significant occurrence of anemia in the < 3.3 mg/m3 (< 1 ppm)
  category.
  In studies conducted in the Western chemical industry, in general, no
  haematological effects have been observed at benzene exposure levels at or
  below 3.3 mg/m3 (1 ppm).
       Collins et al. found no adverse effects on routinely collected haematological
  parameters in chemical workers exposed to either 0.0-4.6 mg/m3 (0.0-1.4 ppm)
  or 1.8 mg/m3 (0.6 ppm) (8h TWA mean).132,133
  Swaen et al. compared 8,532 blood samples collected during routine health
  surveillance of workers exposed to low levels of benzene at a chemical plant in
  The Netherlands, with 12,173 samples of employees with no occupational
  benzene exposure.134 A Job Exposure Matrix was constructed to estimate
  benzene exposure. Depending on the job and operational status, the mean 8h
  TWA benzene air concentration ranged from 0.5-3.0 mg/m3 (0.1-0.9 ppm). No
  adverse effect on any of the haematological parameters was observed.
8 Benzene
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<pre> able 3 Overview of studies of adverse effects on haematological parameters at low benzene concentrations.
ndustry                Number exposed        Mean exposure (range)                      Effect        Reference
                                             mg/m3                 ppm                  observed <
                                                                                        3.3 mg/m3
                                                                                        (1 ppm)
 hoe production         250                  0.9                   0.3                  +             Lan et al. (2004)128
Glu and shoe            131                  0.5 (0-1.6)           0.14 (0.0-0.5)       +             Qu et al. (2002)129
 roduction
 ubber/shoe /           928                  7.4 (0.1-872)         2.3 (0.0-268)        +             Schnatter et al.
 harmaceutical                                                                                        (2010)131
 hemical                200                  0.0-4.6               0.0-1.4              -             Collins et al. (1991)132
 hemical                387                  1.8                   0.6                  -             Collins et al. (1997)133
 hemical                701                  (0.5-3.0)             (0.1-0.9)            -             Swaen et al. (2010)134
 etroleum production 1,200                   < 2.0                 < 0.6                -             Tsai et al. (2004)135
             A large study included 1,200 petrochemical workers with routinely collected
             haematological parameters, with mean benzene exposure (TWA-8h) of 2.0
             mg/m3 (0.6 ppm) from 1977 to 1988 and 0.5 mg/m3 (0.1 ppm) since 1988 who
             were compared to 3,227 unexposed controls. No increased abnormality in any of
             the included haematological parameters was found.135
             An overview of findings in blood cell counts in the low exposure range is
             provided in Table 3.
7.2          Other effects
             Several other effects have been associated with exposure to benzene. As these
             effects are generally observed at exposure levels far exceeding exposure levels at
             which haematological effects occur, the Committee considers them as non-
             critical for deriving a health-based recommend occupational exposure level.
             Therefore, only a short summary for these other effects is provided here, based
             on review documents.1,2,11
             Irritation and sensitisation
             High concentrations of benzene vapours are irritating to the mucous membranes
             of the eyes, nose, and respiratory tract. In humans, benzene is a skin irritant.136
             By defattening the keratin layer, it may cause erythema, vesiculation, and dry
             and scaly dermatitis.137 The ingestion of liquid benzene causes local irritation of
             the mucous membranes of the mouth, throat, esophagus and stomach.2
             Effects                                                                                                        59
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<pre>    Acute and short-term toxicity
    Following acute inhalation of benzene, humans exhibit symptoms indicative of
    central nervous system effects at levels ranging from 975-9,750 mg/m3.1 Very
    high concentrations of benzene vapours produce narcotic effects and can lead to
    death by respiratory arrest. Case reports have been described that report an
    acceleration (of the respiratory rate) followed by drowsiness, fatigue, dizziness,
    headache and nausea after inhalation of a high concentration of benzene vapour.
    At high exposure levels, pulse rate increases, there may be a sensation of
    tightness in the chest accompanied by breathlessness, and ultimately people
    exposed may lose consciousness. Convulsions and tremors have occurred , from
    which it can be concluded that death may follow in a few minutes or several
    hours following severe exposure. Cyanosis, hemolysis, and congestion or
    hemorrhage of organs were reported in the cases for which there were autopsy
    reports.138-141
    Long-term toxicity
    With the exception of haematological toxicity, genotoxicity and carcinogenicity
    (described in Section 7.1.1 to 7.1.3), limited data are available regarding toxicity
    in humans following long-term inhalation exposure to benzene.1 Most findings
    are inconclusive due to uncertainties in exposure assessment and limitations in
    reporting. Both humoral and cellular immunological effects have been described
    in humans exposed to benzene. In 2008, the Dutch Health council evaluated the
    effects of organic solvents, including benzene, on reproduction.142 The evaluated
    data did not indicate that effects on development or fertility could be attributed to
    exposure to benzene.
7.3 Observations in animals
    An enormous amount of animal data on benzene toxicity is available, and
    involves relatively high benzene exposure levels. In view of the available human
    data, the Committee considers the animal data not relevant for establishing a
    HBR-OEL. Only a short summary is provide here, for more details the
    Committee refers to extensive reviews available.1,2,11,55
 0  Benzene
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<pre>Irritation and sensitisation
Benzene has been shown to be irritating to the skin of rabbits, inducing moderate
erythema, edema, and moderate necrosis following application.143 Benzene can
also cause irritation of the mucous membranes (eye, respiratory tract and mouth,
esophagus and stomach).2
Acute and short-term toxicity
Acute inhalation toxicity is low with a LC50 value of 44,500 mg/m³ (13,700 ppm)
after a 4-hour exposure for rats.144 Depression of the central nervous system
appeared to be related to death. The main pathological findings were congestion
of the lungs and liver. A dermal LD50 value of > 8,260 mg/kg bw for rabbits and
guinea pigs has been reported.145 Acute oral toxicity data for rats suggest that
the oral LD50 is above 2,000 mg/kg bw, ranging from 810 mg/kg bw to 10,000
mg/kg bw. 146-148 Depending on the dose, the main clinical signs are sedation and
narcosis. Pathological findings include among others hyperemic and
haemorragic lungs, adrenals and spine.
Long-term toxicity
Irrespective of the exposure route, the main and most sensitive targets of toxicity
in animals after repeated dose application of benzene are the cells of the bone
marrow and haematopoietic system.1,2,11 The rapidly proliferating stem cells,
myeloid progenitor cells and stromal cells are sensitive targets. Chronic benzene
exposure has been reported to result in bone marrow depression expressed as
leucopenia, anaemia and/or thrombocytopenia, leading to pancytopenia, and
aplastic anaemia at concentrations > 33 mg/m3 (10 ppm).
Carcinogenicity
Several studies with inhalation and oral exposure provide evidence that benzene
is carcinogenic in animals.149-162 Target organs of benzene, irrespective of
exposure route, included the haematopoietic system and a spectrum of tissues of
epithelial origin.
     In mice, carcinogenicity of the haematopoietic system predominantly
involves the induction of lymphomas. In contrast, increased frequencies of
leukaemia in comparison to controls were found in rats after exposure to
benzene.
Effects                                                                             61
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<pre>      In addition, several epithelial tumours have been found in mice (e.g., Zymbal
  gland, lung, Harderian gland, preputial gland, forestomach, mammary gland and
  liver) and rats (e.g., Zymbal gland, oral cavity, forestomach, nasal cavity, and
  skin).
  An overview of carcinogenic effects observed in animals is provided in Annex F.
  Genotoxicity
  Bacterial mutagenicity assays conducted with benzene or its metabolites are
  predominantly negative, whereas mixed results have been observed in
  mammalian cell culture assays.1,2,55
      In the vast majority of in vivo micronucleus tests and in vivo chromosomal
  abberation assays, positive results have been observed for benzene and its
  metabolites (i.e., phenol, hydroquinone, catechol, and benzenetriol). Only one
  micronucleus test was conducted with a target organ, the Zymbal gland in the rat,
  which was also positive.163 Benzene and some metabolites tested caused
  structural chromosomal aberrations and sister chromatid exchange (SCE),
  although some of the SCE results were considered only weakly positive.
  Furthermore, benzene has been tested positive in a majority of DNA damage
  assays.55 Two in vivo mutagenicity assays in transgenic mice have been
  described in which marginal responses were noted.164,165 In contrast to the
  conclusion of the authors, the Subcommittee on Classification of Carcinogenic
  Substances considered these as negative results (for details on the view of the
  Subcommittee, see Annex H).
      Finally, most of the genotoxicity studies conducted in Drosophila
  melanogaster (heritable translocations, sex-linked recessive lethal mutations,
  somatic mutation and recombinations) gave negative results.
  An overview of the outcome of the in vivo genotoxicity assays is provided in
  Annex G.
  Reproduction toxicity
  Fertility
  Aspects related to male and female fertility have been investigated in laboratory
  animals in studies of different quality and validity and with the inhalatory route
  of administration only. In a fertility study with female rats exposed up to 300
2 Benzene
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<pre>    ppm benzene for 10 weeks during premating, mating, gestation, and lactation
    showed no effect on indices of fertility, reproduction, and lactation.166
        Available data from subchronic toxicity studies indicate that mice are more
    sensitive to benzene exposure than rats. With respect to possible effects on the
    organs of the reproductive system, no effects for either sex have been observed in
    rats with concentration levels of up to and including 300 ppm (960 mg/m3)
    benzene. In mice, however, this benzene concentration level led to some
    indications for changes in reproductive organs. These appeared to be more
    distinct for the males (testes weight and histopathology affected) than for the
    females (occasional ovarian cysts), but were accompanied with clear-cut
    haematotoxicity (anaemia, leucopenia and thrombocytopenia) in both sexes.2
    Developmental effects
    There are numerous inhalation studies available in which rats or mice have been
    exposed to benzene during pregnancy.166-172 None of these studies demonstrated
    a specific embryotoxic or teratogenic potential even at levels that induced signs
    of maternal toxicity. However, impairment of fetal development as evidenced by
    decreased body weights of the offspring and increased skeletal variants as well as
    delayed ossification were observed at levels > 162.5 mg/m3 (> 50 ppm) often
    associated with maternal toxicity.
7.4 Summary and evaluation
    Epidemiologic studies and case studies provide clear evidence of a causal
    association between exposure to benzene and leukaemia, especially AML/
    ANLL. More recently, an increased risk of MDS is being linked with the
    exposure to benzene. Also for MM, CLL, CML and ALL, although to a lesser
    extent, associations with benzene exposure have been reported. The association
    with other B-cell lymphomas such as follicular lymphoma and diffuse large
    B-cell lymphoma remains unclear.
        Benzene induces tumours in several target organs, including the
    haematopoietic system and several organs of epithelial origin, in rats as well as in
    mice.
        Convincing evidence exists that exposure to benzene causes the induction of
    micronuclei, chromosomal aberrations, sister chromatid exchanges and DNA
    strand breaks, both in humans and in animals. Gene mutation assays are overall
    negative, whereas no DNA adducts have been measured in target tissues in vivo
    after exposure to either benzene or its metabolites.
    Effects                                                                              63
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<pre>      Impairment of the haemopoietic system is the primary (non-carcinogenic)
  adverse health effect after long-term exposure to benzene. This includes the
  manifestation of bone marrow depression, leading to aplastic anaemia,
  leukopenia, agranulocytosis, and pancytopenia.
      In humans, high concentrations of benzene vapour are irritant to mucous
  membranes of the eyes, nose and respiratory tract. Liquid benzene on direct
  contact may cause erythema dryness and cracking of the skin. In animals,
  benzene is irritant to the skin and may cause serious damage to eyes.
      Following acute inhalation of benzene, humans exhibit symptoms indicative
  of central nervous system effects at high exposure levels. Convulsions and
  tremors occur frequently, and death may follow in a few minutes or several hours
  following severe exposure.
      The acute toxicity of benzene in animals is low. The oral LD50 is estimated to
  be > 2,000 mg/kg bw and depending on the dose, the main clinical signs are
  sedation and narcosis. An LC50 value of 44,500 mg/m³ (13,700 ppm) is reported
  in rats; depression of the central nervous system appeared to be related to death.
      With the exemption of carcinogenic, haematotoxic and genotoxic effects, no
  clear evidence for critical effects after long-term exposure to benzene is
  available. Information on the reproductive toxicity of benzene in humans is
  limited. There are no indications that benzene is teratogenic in humans. In
  animals, some effects associated with reproduction were observed, however only
  at haematotoxic exposure levels of benzene.
  Based on the available carcinogenicity data, the Subcommittee on Classification
  of Carcinogenic Substances confirms the classification of benzene in category
  1A (the compound is known to be carcinogenic to humans). Based on the data
  available on the mode of action, the Subcommittee furthermore concluded that
  benzene acts by a non-stochastic genotoxic mode of action (see Annex H for
  further details on the Subcommittee’s opinion).
4 Benzene
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<pre> hapter 8
        Existing guidelines, standards and
        evaluations
8.1     General population
        As of 2010, the European Union has an environmental exposure limit value for
        benzene of 5 µg/m3 (year-average).
8.2     Working population
        The currently applicable occupational exposure limits are summarised in
        Table 4.
            In addition, biological limit values* have been set several international
        authorities. For example, ACGIH established biological exposure limits
        (biological exposure index (BEI)) of 25 µg/g creatinine for SPMA, and 500 µg/g
        creatinine for ttMA.173 DFG (Deutsche Forschungsgemeinschaft) applies
        biological exposure limits (Expositions equivalents für krebserzeugende
        Arbeitsstoffe (EKA)**; exposure-equivalents for carcinogenic substances) of 5 µg
        benzene/L in blood, and 45 µg SPMA/g creatinine and 2 mg/L ttMA in urine.174
        Time of sampling: end of shift.
 *      EKA value corresponding to external benzene exposure of 3.3 mg/m3.
        Existing guidelines, standards and evaluations                                   65
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<pre>    Table 4 Existing Occupational Exposure Limits (OELs) for benzene.
    Country                            OEL (mg/m3)            Time-weighted         Type of OELa
    - Organisation                                            average
    The Netherlandsb                   3.25                   8h                    ns
    - Ministry of Social Affairs
      and Employment
    European Unionc                    3.25                   8h                    BLV
    Denmarkb                           1.6                    8h                    ns
    Finlandb                           3.25                   8h                    BLV
    Franceb                            3.25                   8h                    BEL
    Germany
    - AGSd                             1.9 (1)/ 0.2 (2)
    - DFG175                           none                   -                     -
    Norwayb                            3                      8h                    ns
    Spainb                             3.25                   8h                    ns
    Sweden                             1.5                    8h                    ns
                                       9                      15 min                ns
    United Kingdomb                    3.25                   8h                    WEL
    USA1
    - ACGIH                            1.6                                          TLV
                                       8.0                                          STEL
    - OSHA                             3.2                    8h                    PEL
                                       16.0                   15 min                STEL
    - NIOSH                            0.3                    10h                   REL
                                       3.2                    15 min                STEL
    a    Abbreviations: ns = not specified; TLV = threshold limit value; STEL = short-term exposure
         limit; PEL = permissible exposure limit; REL = recommended exposure limit; WEL =
         workplace exposure limit; BLV = binding limit value; BEL = binding exposure limit
    b    Source: Social Economic Council (http://www.ser.nl/en/grenswaarden/benzeen.aspx)
    c    Directive 2004/37/EC of the European Parliament and of the Council of 29 April 2004 on the
         protection of workers from the risks related to exposure to carcinogens or mutagens at work,
         Annex III (https://osha.europa.eu/nl/legislation/directives/exposure-to-chemical-agents-and-
         chemical-safety/osh-directives/directive-2004-37-ec-indicative-occupational-exposure-limit-
         values).
    d    IFA Gestis database (http://limitvalue.ifa.dguv.de/Webform_gw.aspx) ((1) Workplace exposure
         concentration corresponding to the proposed tolerable cancer risk. (2) Workplace exposure
         concentration corresponding to the proposed preliminary acceptable cancer risk.)
8.3 Classification
    In the European Union, benzene is classified for carcinogenicity (category 1A/
    H350; may cause cancer); and for mutagenicity (category 1B/H340, may cause
    genetic defects).
    In 1982, the International Agency for Research on Cancer (IARC) concluded that
    there is sufficient evidence that benzene is carcinogenic to man and that there is
 6  Benzene
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<pre>limited evidence that benzene is carcinogenic in experimental animals.5
Therefore, IARC classified benzene as a Group 1 carcinogen (carcinogenic to
humans).
    In the subsequent updates of the monograph, the body of evidence for
carcinogenicity in experimental animals was considered to be sufficient. The
conclusion to classify benzene as a Group 1 carcinogen was confirmed in an
update of the monograph in 1987.176
    In the latest update, IARC concluded that there is sufficient evidence in
humans for the carcinogenicity of benzene (Group 1).6 IARC further concluded
that benzene causes acute myeloid leukaemia/acute non-lymphocytic leukaemia,
whereas a positive association has been observed between exposure to benzene
and acute lymphocytic leukaemia, chronic lymphocytic leukaemia, multiple
myeloma, and non-Hodgkin lymphoma. There is sufficient evidence for the
carcinogenicity of benzene in experimental animals.
Existing guidelines, standards and evaluations                                67
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<pre>8 Benzene</pre>

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<pre> hapter 9
        Hazard assessment
9.1     Hazard identification
        Irrespective of the exposure route, the main and most sensitive targets of toxicity
        in animals and humans after repeated exposure to benzene are the rapidly
        proliferating stem cells, myeloid progenitor cells and stromal cells of the bone
        marrow and haematopoietic system.1-6 As has been summarised in Chapter 7, the
        available data on the critical toxic effects of benzene in humans mainly involve
        the development of leukaemia (in particular leukaemia from myeloid lineage),
        the induction of chromosomal aberrations, and the reduction of the number of
        peripheral blood cells.
9.2     Quantitative assessment of the health risk
        The Subcommittee on Classification of Carcinogenic Substances of DECOS
        has concluded that benzene acts by a non-stochastic genotoxic mechanism
        (Annex H). DECOS has decided to adopt this conclusion of the Subcommittee,
        and therefore applies a threshold approach by deriving a HBR-OEL for benzene.
        Several exposure-response analyses of the benzene-leukaemia association have
        been reported. However, as the power at low levels of benzene exposure is low,
        these studies do not allow the determination of a reliable point of departure for
        derivation of a HBR-OEL. Studies on the induction of chromosomal aberrations
        Hazard assessment                                                                   69
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<pre>  show limitations in design and reporting and as a consequence, also cannot serve
  as a basis to derive a reliable point of departure. Data on haematological effects
  after benzene exposure include quantitative data on low benzene exposures, from
  properly studies in several occupationally exposed populations. The Committee
  therefore considers the data on haematotoxicity most suitable for derivation of a
  HBR-OEL for benzene.
  The Committee notes that a large amount of data is available concerning the
  haemototoxicity of benzene at low exposure levels. At benzene concentrations
  below 3.25 mg/m3 (1 ppm), several haematological studies have shown adverse
  effects whereas several others have not. For the purpose of deriving a HBR-OEL,
  all of these studies have their strengths and weaknesses. The Committee has
  therefore decided to apply a weight of evidence approach to derive a HBR-OEL,
  using the aggregate of evidence of the available studies.
  Assessment of relevant studies
  The Committee considers the cross-sectional study published by Lan et al.128
  important for its weight of evidence approach, as it provides information on the
  exposure-response relationship in the exposure range below 3.25 mg/m3 (1 ppm).
       This study on shoe factory workers in China (two-third females) was
  designed particularly for studying effects of low benzene exposures. Exposure
  was assessed using personal monitoring for 16 months prior to biological sample
  collection. Exposure in the last month before blood collection was used in the
  analyses due to the relatively short half-life of most peripheral blood cells. Lan et
  al. reported significantly reduced white blood cells (of all types) in workers
  exposed to 1.9 mg/m3 (0.57 ppm) benzene. In a subgroup of workers (gender not
  specified), exposed to low benzene levels, effects were observed at a mean
  benzene concentration of 0.9 mg/m3 (0.29 ppm). Details on the exposure
  assessment in general (Vermeulen et al.177) and the individual measurements in
  the low exposure subgroup (online supplemental to the publication*) were
  published separately.
       The Committee considers the exposure assessment reported by Lan et al. a
  realistic estimate of the exposure of the population that was studied. Although
  subjects were categorised by mean benzene exposure levels measured the month
  before blood sampling, long-term benzene exposure of these workers is
  anticipated to have been similar, as it was reported that little or no task rotation
   www.sciencemag.org/cgi/content/full/306/5702/1774/DC1.
0 Benzene
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<pre>occurred. Also, the benzene levels in the factories during the 16 months
preceeding blood sampling showed only seasonal fluctuations in exposure,
mainly caused by differences in ventilation pattern.177 Further, the Committee
considers the likelihood of bias due to the involvement of peak exposures low.
First, the low exposure subgroup was not involved in gluing and exposed only
to background levels of benzene. The excursions above 3.3 mg/m3 (1ppm),
ranging from 3.3-5.7 mg/m3 (1.01-1.74 ppm), were reported to occur
infrequently. Also a contribution of dermal exposure in these shoe workers is
considered unlikely. The overall probability of dermal exposure has been
reported to be low, and importantly, the low exposure group had no direct contact
with benzene-containing glue.178
    The Committee notes that some comments can be made on the effects that
were observed by Lan et al. at low exposure concentrations. Noteworthy, a more
pronounced reduction in various blood cell types was found in the < 3.3 mg/m3
(< 1 ppm) category than in the 3.3 to < 33 mg/m3 (1 to < 10 ppm) category. These
cut off effects have been discussed by the authors in a response to a letter to the
editor.179,180 In their response, Lan et al. presented spline regression analyses for
white blood cells based on all individual measurements, showing a monotonic
decrease along the whole exposure range (0.7 to < 49 mg/m3 (0.2-15 ppm)). With
respect to the differences in blood cells counts between the low exposed and the
controls, it can be argued that these are due to demographic and lifestyle
differences between the unexposed and exposed population. However, the
Committee considers the selection of controls from workers involved in clothes-
manufacturing from the same geographical region, in this case, appropriate.
A second study that provides information on haematological effects at exposure
levels below 3.3 mg/m3 (1 ppm) is that of Qu et al., who conducted a
biomonitoring study in workers from three different factories in China.129 An
analysis based on all 130 workers exposed to a broad range of exposures and 51
controls revealed a concentration-dependent decrease in red blood cells, white
blood cells, and neutrophils (with and without adjustment for sex, age, smoking,
and toluene exposure). Furthermore, a statistically significant reduction of red
blood cells, white blood cells, and neutrophils was observed in a subpopulation
workers exposed to low levels of benzene (4-wk mean exposure of 0.46 mg/m3
(0.14 ppm)). Qu et al. also measured chromosomal aberrations and observed
statistically significant effects in the low exposure group.
    The Committee notes several limitations of this study, in particular in relation
to the effects reported in the low exposure group. First, a possible contribution of
dermal exposure to the effects observed has not been addressed. Also, the control
Hazard assessment                                                                     71
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<pre>  group and exposure groups are not properly matched. The low exposure group
  consisted only of 16 individuals, all women (all non-smoking), whereas about
  half of the control subjects were female (of which 31% smoked). Since there has
  not been corrected for both gender (since women have relatively lower levels of
  haemoglobine, haematocrite, and red blood cells) and smoking (as smoking is a
  known cause of increased neutrophils), the effect levels reported need to be
  interpreted with caution as these can lead to an overestimation of the true
  benzene hazard. The Committee considers the study of Qu et al. supporting, but
  not critical, to its weight of evidence approach.
  The findings of Lan et al. and Qu et al. at benzene concentrations below 1 ppm
  are supported by several other studies. In a study involving 5 factories in China,
  with workers exposed to a broad range of exposures, a statistically significant
  increase in risk was found for a clinically relevant reduction in red blood cells.131
  Several other studies addressed endpoints other than blood cell counts.91,96 In
  two studies on leukaemia, a statistically significantly increased risk was observed
  at cumulative exposures of benzene that correspond to mean exposure levels of
  < 3.3 mg/m3 (< 1 ppm)*. Hayes et al. observed an increased risk of haematologic
  neoplasms < 130 mg/m3-years (< 40 ppm-years) (equivalent to a time-weighed
  average exposure of 3.3 mg/m3 (1 ppm)), while in a pooled analysis, Schnatter et
  al. reported an increased risk of MDS at 9.5 mg/m3-years (> 2.9 ppm-year)
  (equivalent to a time-weighed average exposure of 0.1 ppm).96 However, as the
  exposure period in the study by Schnatter et al. was far less than 40 years, the
  corresponding mean exposure level is anticipated to have been higher. As no
  clear indications of an adverse effect were seen below an average exposure of 0.7
  mg/m3 (0.2 ppm) in the same study, the Committee considers the risk of MDS at
  this mean exposure level in practice to be negligible.
  In addition to the studies mentioned above, the Committee points to several
  studies that have been conducted on workers in the (petro)chemical industry
  (mainly males), which have not shown effects of benzene below an exposure
  concentration of 3.3 mg/m3 (1 ppm).132-135 These routine health surveillance
  studies involved large numbers of workers and blood samples, and have been
  conducted using standard clinical methods. Importantly, the working conditions
  in these studies represent the occupational exposure conditions in The
  Netherlands and Europe. The estimation of benzene exposures is based on
  sampling strategies targeted at representative jobs and workplace combinations.
  When assuming 40 years of occupational exposure.
2 Benzene
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<pre>Therefore, the subsequent air measurements do not provide information on the
workers exposed but rather on the job or task for which it was taken. The
Committee notes that such sampling strategy is the method applied in routine
monitoring industry, rather than using individual monitoring which is more
suitable for the detection of subtle changes in blood cell numbers.
Recommendation of the health-based occupational exposure limit
The Committee has decided to apply a weight of evidence approach to derive a
HBR-OEL for benzene, taking into account the aggregate of the accumulated
evidence, including the apparent discrepancies between some of the reported
results. The Committee therefore does not derive a point of departure by
rounding off one (or more) of the reported effect levels, but pragmatically sets a
point of departure. Based on the studies discussed above and summarised in
Table 5, in which both NOAELs and LOAELs in the range of 0.5 to 3.3 mg/m3
are reported, the Committee considers a benzene effect level of 2 mg/m3
(0.6 ppm) a realistic starting point for deriving a HBR-OEL. The Committee
applies a default uncertainty factor of 3, because of the use of a LOAEL instead
of a NOAEL. In view of the use of an aggregate of evidence based on multiple
studies, the Committee does not apply any additional uncertainty factors (e.g.,
for intra-individual differences or the size of the study population), and sets a
HBR-OEL for benzene at 0.7 mg/m3 (0.2 ppm), 8h time-weighted average
(8h-TWA).
Skin notation
To decide whether a skin notation should be recommended to the substance, the
Committee uses the ECETOC criteria for assigning a skin notation.181 According
to the ECETOC methodology, a skin notation is needed for a substance, in
absence of relevant circumstantial evidence on human skin exposure, when the
Critical Absorption Value (CAV; the rate of absorption above which dermal
exposure is considered to be an important contributor to the total exposure)
exceeds:
    (10 [m3] x OEL [mg/m3] x f x 0.1)/2,000 [cm2]
in which 10 m3 is the human inhalation volume per 8h working day, f is the
absorption factor for inhalation (here assumed to be 1), 0.1 denotes the 10%
criterion, 2,000 cm2 is the surface area of the hands and forearms, and OEL is the
Hazard assessment                                                                  73
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<pre>    Occupational Exposure Limit, in this case the HBR-OEL. Thus the CAV will be:
        (10 [m3] x 0.7 [mg/m3] x 1 x 0.1)/2,000 [cm2] = 0.35 µg/cm2*h
    Williams et al. analysed the experimental skin absorption data of benzene, and
    concluded that the steady state absorption rate ranges from 200-400 µg/cm2*h.16
    This rate exceeds the CAV of 0.35 µg/cm2*h by far. The Committee therefore
    recommended to apply a skin notation.182
9.3 Groups at extra risk
    A high variation of the level of toxicity has been observed among workers
    exposed to comparable levels of benzene, but no specific group at risk has yet
    been identified. This variation may be partly explained by biological factors such
    as gender, age, and extrinsic factors such as physical activity, coexposures
    smoking and dietary habits.1,2
        In addition, genetic factors play a role such as single-nucleotide
    polymorphisms (SNPs) in genes related to metabolism of benzene and genes
    otherwise involved in benzene-induced toxicity (e.g., cytokine and chemokine
    coding genes).1,2,183
9.4 Health-based recommended occupational exposure limit
    DECOS recommends a health-based occupational exposure limit for benzene of
    0.7 mg/m3 (0.2 ppm), as an eight-hour weighed average concentration.
 4  Benzene
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<pre> able 5 Epidemiological studies addressing effects at low (<3.3 mg/m3; 1 ppm) benzene concentrations.
 opulation           Experimental design            Effect studied      Mean (No) Effect level Critical effect   Reference
Haematotoxicity (key studies)
Workers in shoe      Individual measurements of     Blood cell counts 1.9 mg/m3                Reduction in      Lan et al.
manufacturing        250 exposed workers and 140                        (0.6 ppm)              blood cell counts (2004)128
actories             controls                                           (LOAEL)
                                                                        Subgroup:
                                                                        0.9 mg/m3
                                                                        (0.3 ppm)
                                                                        (LOAEL)
Workers in glue      Biomarker study in 105         Blood cell counts 1.6 mg/m3                Reduction in      Qu et al.
actory or a small benzene-exposed workers and                           (0.5 ppm)              blood cell counts (2002)129
 hoe factory         26 unexposed workers                               (LOAEL)
Workers in rubber, Individual monitoring of 928 Blood cell counts 3.3 mg/m3                    Anaemia (based    Schnatter
 hoe and             workers                                            (1 ppm)                on logistic       et al.
 harmaceutical                                                          (LOAEL)                regression        (2010)131
ndustry                                                                                        analysis)
Workers in           Routine biomonitoring study Blood cell counts 1.8 mg/m3                   Not observed      Collins
 hemical industry with 387 workers and 553                              (0.6 ppm)                                (1991)132;
                     unexposed workers                                  (NOAEL)                                  Collin
                                                                                                                 et al.
                                                                                                                 (1997)133
Workers in           Routine biomonitoring study in Blood cell counts   3.0 mg/m3              Not observed      Swaen et al.
 hemical industry    volving 701 exposed workers                        (0.9 ppm)                                (2010)134
                     (8,532 blood samples) and                          (NOAEL)
                     1,059 non-exposed workers
                     (12,173 blood samples)
Workers in petro- Routine haematology               Blood cell counts   2.0 mg/m3              Not observed      Tsai et al.
 hemical industry surveillance study in 1200                            (0.6 ppm)                                (2004)135
                     exposed workers and 3,227                          (NOAEL)
                     controls
Genotoxicity (supporting study)
Workers in glue      Biomarker study in 105         Chromosomal         1.6 mg/m3              Chromatid and     Qu et al.
actory or a small benzene-exposed workers and       aberrations         (0.5 ppm)              chromosomal       (2002)129
 hoe factory         26 unexposed workers                               (LOAEL)                aberrations
  arcinogenicity (supporting studies)
Workers in a         Cohort study with 74,828       NHL, leukaemia,     Cumulative exposure:   Increased risk of Hayes et al.
 ariety of           exposed workers and 35,805     ANLL, ANLL/         < 130 mg/m3-y          haematologic      (1997)85
ndustries            controls                       MDS, other          (< 40 ppm-y)           neoplasms
                                                    hematologic         Equivalent to < 3.3    combined
                                                    neoplasms;          mg/m3 TWA
                                                    separately and      (< 1 ppm ppm TWA)a
                                                    combined
 etroleum workers Pooled analysis of 3 nested       AML, CML, and       Cumulative exposure: Increased risk of Schnatter
                     case-control studies from      CLL and two         > 9.5 mg/m3-y          MDS               et al.
                     Australia, Canada, and the UK myeloid neoplasms     (> 2.9 ppm-y)                           (2012)96
                                                    (MDS and MPD)       Equivalent to > 0.2
                                                                        mg/m3 TWA
                                                                        (> 0.1 ppm
                                                                        ppm TWA)a
      Assuming 40 years of occupational exposure.
             Hazard assessment                                                                                             75
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<pre>6 Benzene</pre>

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8  Benzene
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<pre>84 Wong O. Risk of acute myeloid leukaemia and multiple myeloma in workers exposed to benzene.
   Occup Environ Med 1995; 52(6): 380-384.
85 Yin SN, Li GL, Tain FD, Fu ZI, Jin C, Chen YJ et al. Leukaemia in benzene workers: a retrospective
   cohort study. Br J Ind Med 1987; 44(2): 124-128.
86 Yin SN, Li GL, Tain FD, Fu ZI, Jin C, Chen YJ et al. A retrospective cohort study of leukemia and
   other cancers in benzene workers. Environ Health Perspect 1989; 82: 207-213.
87 Guideline to the classification of carcinogenic compounds. The Hague: Health Council of the
   Netherlands; 2010: publication no. A10/07E.
   References                                                                                         89
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<pre>0 Benzene</pre>

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<pre>A Request for advice
B The Committee
C The submission letter (in English)
D Comments on the public review draft
E Human data
F Animal data
G Genotoxicity data
H Advice of the Subcommittee on Classification of Carcinogenic
  Substances
  Classification of substances with respect to carcinogenicity
  List of Abbreviations
  Annexes
                                                               91
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<pre>2 Benzene</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 Standards (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                                                                                        93
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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, a ‘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.
4 Benzene
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<pre>nnex B
     The Committee
     •  R.A. Woutersen, chairman
        Toxicologic Pathologist, TNO Quality of Life, Zeist, and Professor of
        Translational Toxicology, Wageningen University and Research Centre,
        Wageningen
     •  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
     •  G.J. Mulder
        Emeritus Professor of Toxicology, Leiden University, Leiden
     •  T.M. Pal
        Occupational Physician, Netherlands Centre for Occupational Diseases,
        University of Amsterdam, Amsterdam
     The Committee                                                               95
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<pre>  •   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 Antoni van
      Leeuwenhoek, Amsterdam
  •   I.M.C.M. Rietjens
      Professor of Toxicology, Wageningen University and Research Centre,
      Wageningen
  •   G.M.H. Swaen
      Epidemiologist, Dow Benelux NV, Terneuzen (until April 1, 2013);
      Exponent, Menlo Park, United States (from August 15, 2013, until
      February 1, 2014)
  •   R.C.H. Vermeulen
      Epidemiologist, Institute for Risk Assessment Sciences, Utrecht University,
      Utrecht
  •   P.B. Wulp
      Occupational physician, Labour Inspectorate, Groningen
  •   B.P.F.D. Hendrikx, advisor
      Social and Economic Council, The Hague
  •   A.S.A.M. Van der Burght, scientific secretary
      Health Council of the Netherlands, The Hague
  •   S.R. Vink, 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
6 Benzene
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<pre>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.
The Committee                                                               97
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<pre>8 Benzene</pre>

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<pre>nnex C
     The submission letter (in English)
     Subject         : Submission of the advisory report Benzene
     Your Reference: DGV/MBO/U-932342
     Our reference : U-8057/SV/fs/459-Q69
     Enclosed        :1
     Date            : February 21, 2014
     Dear Minister,
     I hereby submit the advisory report on the effects of occupational exposure to
     benzene.
     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.
     The submission letter (in English)                                               99
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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
00 Benzene
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<pre>nnex D
     Comments on the public review draft
     A draft of the present report was released in 2013 for public review. The
     following organisations and persons have commented on the draft document:
     • V. Gálvez Pérez, Instituto Nacional de Seguridad e Higiene en el Trabajo,
         Madrid, Spain.
     • D. Gombert, Agence nationale de sécurité sanitaire de l’alimentation, de
         l’environnement et du travail, Maisons-Alfort Cedex, France
     • T.J. Lentz, National Institute for Occupational Safety and Health, Cincinnati
         (OH), USA
     • A. M. Rohde and G. Wallace, CONCAWE/European Chemical Industry
         Council (CEFIC), Aromatics Producers Association, Brussels, Belgium
     • T. Scheffers, Theo Scheffers Arbo Consultancy, Maastricht, The Netherlands
     Comments on the public review draft                                             101
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<pre>02 Benzene</pre>

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<pre> nnex      E
           Human data
 ohort       Cohort description    Exposure          Haemato-   Exposure (ppm)  No of  Relative risk
Reference)                         assessment        logical    TWAa            cases/ (95% CI)
                                                     malignancy                 deaths Numbers in bold =
                                                                                       statistically
                                                                                       significant)
 liofilm     748 rubber workers    Estimation based  Leukaemia  Estimated range        SMR
Rinsky                             on work histories mortality  35-100 ppm      7      5.6
981)81                             and air sampling
                                   data
 liofilm     1,165 rubber workers Estimation based   Leukaemia  ppm-years              SMR
update)      (white males)         on work histories mortality  0-40            2      1.1 (0.1-3.9)
Rinsky       Control: 10 subjects and air sampling              40-199          2      3.2 (0.4-11.75)
987)82       for each case,        data                         200-399         2      11.9 (1.3-42.9)
             matched for age and                                ≥ 400           3      66.4 (13.3-193.9
             year of first                                      Total           9      3.4 (1.5-6.4)
             employment
 liofilm     1,165 rubber workers Estimation based   AML        ppm-years              SMR
update)      (white males)         on work histories            < 40            1      1.2 (0.0-6.6)
Wong                               and air sampling             40-200          0      0 (0-14.8)
995)184                            data                         200-400         2      27.2 (3.3-98.2)
                                                                > 400           3      98.4 (20.3-287.75)
 liofilm     1,845 rubber workers  Estimation based  Leukaemia  ppm-years              SMR
update)      (males and females,   on work histories mortality  1 ppm-day-40    6      1.5 (0.5-3.3)
Rinsky       all races) (including and air sampling             40-200          4      3.2 (0.9-8.9)
002)83       1,291 workers         data                         200-400         2      5.6 (0.1-24.1)
             exposed to at least 1                              ≥ 400           3      24.0 (4.8-78.5)
             ppm-day
             (control=554
             unexposed workers)
           Human data                                                                                  103
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<pre> liofilm        See Rinsky (2002)     See Rinsky (2002) Leukaemia      See Rinsky (2002)           SMR
update)         Follow-up until 1996                     mortality                       1 in 1950 From 32.5 (0.8-
Silver 2002)89                                                         Average 174.5 ±   to 15 in 180.6) in 1950 to
                                                                       214.4 ppm-years)  1996      2.5 (1.4-4.1) in
                                                                                                   1996
 liofilm        1,845 rubber workers  Estimation based   Leukaemia     ppm-years
update)         (males and females,   on work histories  mortality     <1                  5       1
Richardson      all races)            and air sampling                 1-< 50              3       0.8 (0.2-3.2)
 008)88         (control: see Rinsky  data                             50-250              4       2.5 (0.6-10-2)
                1987) Follow-up until                                  250-500             4       10.5 (2.3-46.6)
                1996                                                   ≥ 500               1       13.9 (0.7-116.1)
 CI-CAPM        74,828 workers        Estimation based                                             RR
Hayes 1997,     (males and females)   on work histories  All           < 10              24        2.2 (1.1-4.2)
Dosemeci        in 672 factories in   and benzene        haematologic  10-24             16        3.1 (1.5-6.5)
 994, Travis,   China (control =      measurements       neoplasms     ≥ 25              18        2.8 (1.4-5.7)
 994, Yin,      35,805 unexposed
 987, 1989,     workers)                                 Leukaemia     < 10              15        2.0 (0.9-4.5)
 994)90-                                                               10-24             13        3.7 (1.6-8.7)
3,185,186                                                              ≥ 25              10        2.3 (0.9-5.7)
                                                         ANNL          < 10                7       2.0 (0.6-7.0)
                                                                       10-24               9       5.8 (1.8-18.9)
                                                                       ≥ 25                5       2.6 (0.7-9.9)
                                                         ANNL/MDS      < 10              11        3.2 (1.0-10.1)
                                                                       10-24               9       5.8 (1.8-18.8)
                                                                       ≥ 25                8       4.1 (1.2-13.2)
Dow Chemical 594 workers in           Estimated based on Leukaemia     2-9                 1       1 case of leukaemia
Ott 1978)97     chlorobenzol,         industrial hygiene                                           (vs. 0.9 expected;
                alkylbenzene and      measurements and                                             no SMR calculated)
                ethylcelulose         work histories.
                production
Dow Chemical 956 workers in           See Ott 1978                     ppm-months                  SMR (0-year lag)
update)         chlorobenzol,                            Leukaemia     0-500               2       1.7 (CI not reported)
Bond 1986)98 alkylbenzene and                                          500-1,000           0       2.5     ,,
                ethylcelulose                                          ≥ 1,000             1       1.6     ,,
                production
Dow Chemical 2,266 workers in         See Ott 1978                                                 SMR (0-year lag)
update)         chlorobenzol,                            Leukaemia and < 5                 3       0.8 (0.2-2.2)
Bloemen         alkylbenzene and                         aleukaemia    5-14                5       1.6 (0.5-3.7)
 004)99         ethylcelulose                                          15+                 4       1.2 (0.3-3.1)
                production
                                                         AML           <5                  0       0.0 (0.0-2.6)
                                                                       5-14                3       2.7 (0.6-7.8)
                                                                       15+                 1       0.9 (0.02-5.1)
 04           Benzene
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<pre>  hemical       4,602 exposed          Estimation work                                             RR (Mantel-
Manufacturers   workers (males) in     histories and                        ppm-months             Haenszel; no CI)
  ssociation    US chemical industry   benzene               Lymphatic and non-exposed          3  1.0
CMA)            (control=3,074         measurements          haematopoietic < 180               5  2.1
Wong            unexposed males                                             180-719             5  3.0
 987)100        from same company                                           ≥ 720               5  3.9
                in same period)                                                                    (χ2=5.4 (p=0.02))
                                                             Leukaemia and non-exposed          0  undefined
                                                             aleukaemia     < 180               2  χ2=6.5 (p=0.01)
                                                                            180-719             1
                                                                            ≥720                3
UK Petrol       23,306 distribution    Based on work         All leukaemia, Exposure               No statistically sign.
Nested case     workers in petroleum history records         acute          categories up to       increased risk
 ontrol study   distribution industry                        lymphoblastic, 45 ppm-y
Lewis 1997,     (cohort, control =                           chronic
  ushton,       men from same oil                            lymphocytic,
 997)94,95      company of                                   acute myeloid
                equivalent age)                              and monocytic,
                                                             and chronic
                                                             myeloid
                                                             leukaemia
                                                                                                   OR
  ustralian     17,525 employees       Estimation work       Leukaemia      ≤ 0.1               5  1.0
Health Watch    (males and females)    histories and                        > 0.1-0.2           9  3.8 (1.2-12.7)
AHW)            in Australian          benzene                              > 0.2-0.4           4  2.2 (0.5-9.4)
Glass 2000,     petroleum industry     measurements                         > 0.4-0.8           4  6.3 (1.5-26.2)
 003, 2005)102- (cohort, control =                                          > 0.8-1.6           6  1.5 (0.3-6.7)
 04             from same company                                           > 1.6-3.2           3  5.6 (1.0-31.3)
                of equivalent age)                                          > 3.2               2  19.6 (1.4-270.8)
Canadian        6,672 male petroleum   Estimated based on Myeloid/          Within the range of NA No increase risk of
 ohort          marketing and          work histories and lymphatic         0.01 to 6.2 ppm        leukaemia observed
 Schnatter      distribution workers,  historical industrial leukaemia
 993)106        226 locations (control hygiene surveys
                = unexposed workers
                (number not
                specified))
  anadian       Nested case-control    Estimated based on Leukaemia         Within the range of NA No increase risk of
 ohort          study involving 31     work histories and                   0.01 to 6.2 ppm        leukaemia observed
update)         cases of               historical industrial
Schnatter       lymphohaematopoieti    hygiene surveys
 996) 107       c cancer (control=124
                unexposed subjects
                from the same cohort
                matched for age)
 hoe Factory    1,687 workers (males   Estimation based      Leukaemia      ppm-years              SMR
Costantini      and females)           on work histories     mortality      < 40                3  1.3 (0.3-3.7)
 003)108        (control=general       and limited air                      40-99               2  4.1 (0.5-14.7)
                population death       sampling data                        100-199             2  2.5 (0.3-9.1)
                rates)                                                      ≥ 200               4  5.1 (1.4-13.0)
              Human data                                                                                            105
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<pre>Offshore         27,919 Norway           Estimation        All blood/bone 0.001 to 0.69
 ohort           petroleum industry                        marrow
Kirkeleit        workers                                   neoplasms                          20        1.90 (1.19-3.02)
 008)109         (control=366,114
                 persons from general                      AML                                6         2.89 (1.25-6.67)
                 working population)
Schnatter        UK Petrol, AHW and Estimated using                       ppm-years
 012) (pooled    Canadian cohorts        historical        AML, CLL,      0 -> 2.93 ppm                 No increased risk
 nalysis)96                              monitoring data   CML, MPD
                                                                                              6
                                                           MDS            ≤ 0.348             8         1.00
                                                                          0.348-2.93          15        1.73 (0.55-5.47)
                                                                          > 2.93                        4.33 (1.31 -14.3)
                                                                          ≤ 0.016             7         1.00
                                                                          0.016-0.081         5         1.99 (0.51-7.76)
                                                                          0.081-0.259         7         1.85 (0.51-6.75)
                                                                          > 2.59              10        3.12 (0.9-10.8)
Meta analysis
Raabe and        208,741 mainly                                                                         Meta-SMR
Wong 1996)110 petroleum refinery                           AML            Generally less than           0.9
                 workers in the US and                                    1 ppm for most
                 UK                                                       petroleum refinery
                                                                          jobs (0.22 ppm
                                                                          based on an
                                                                          industry-wide
                                                                          survey including
                                                                          14,824 samples)
Vlaanderen       Meta regression         0.32-554.3 ppm-   Leukaemia                                    Highest RR (natural
 010)111         analysis of 6 cohort    years                            ppm-years                     spline; all studies)
                 studies and 3 nested                                     10                            1.5 (1.1-2.2)
                 case-control studies)                                    20                            1.7 (1.3-2.3)
                                                                          40                            2.1 (1.5-3.0)
Khalade          Various cohorts                           Leukaemia      Summary effect
 010)112         derived from a total of                                  size                          1.4 (1.2-1.6)
                 15 studies
                                                                          < 40 ppm-years                1.6 (1.1-2.4)
                                                                          40-99.9 ppm-years             1.9 (1.3-2.9)
                                                                          > 100 ppm-years               2.6 (1.6-4.4)
                                                           AML            < 40 ppm-years                1.9 (1.0-4.0)
                                                                          40-99.9 ppm-years             2.3 (0.9-5.9)
                                                                          > 100 ppm-years               3.2 (1.1-9.5)
Schnatter        9 cohorts and 13        Qualitatively     AML            NS                            Increased risk
 005)113         case-control studies                                                                   across study designs
     In those references where exposure is expressed in both ppm-years and ppm, only exposure in ppm is reported.
 06          Benzene
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<pre> nnex       F
            Animal data
 arcinogenicity studies reported in the EU RAR.2
 oute      Species Study             Mortality    Hyperplasia      Tumor response                                Ref. from
                      design         rate         related to tumor                                               EU RAR
                                                  response
nhalation Mouse/C 300 ppm,           Reduced      Granulopoietic/  8/40 animals with hematopoietic lymphoma Snyder et al.
           57Bl/6J 6h/d, 5d/w median              myeloid bone      (6 lymphocytic lymphoma, 1 plasmocytoma, (1980)161
           (males) lifetime;         survival (41 marrow           1 hemocytoblastic leukaemia)
           AKR/J                     vs 75 weeks hyperplasia in    2/40 control animals with lymphocytic
           (males)                   in control   C57Bl mice       lymphoma
                                                  (13/32 animals
                      100 ppm,       Ø            without tumors)  No increase of tumour rate of lymphomas
                      6hr/d,5d/w,                                  (29/49 in treated animals, 24/50 in controls)
                      lifetime
           Mouse/ Intermitt.: Ø                   -                C57Bl/intermittend:                           Snyder et al.
           C57Bl/6J 300 ppm,                                       tumor bearing animals (TBA): total TBA 25/ (1988)162
           and CD- 6h/d, 5d/w,                                     54 vs 8/46 controls; malignant TBA 24/54 vs
           1 (males) 1 w                                           2/46 controls; Zymbal gland carcinoma 19/54
                      interrupted                                  vs 0/46 controls
                      by 2 weeks                                   CD-1/interrmittend:
                      unexposed,                                   total TBA 25/54 vs 4/46 controls; malignant
                      until death                                  TBA 2/54 vs 1/46 controls; lung adenoma 4/54
                                                                   vs 3/46 controls
            Animal data                                                                                                  107
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<pre>            1,200 ppm                                   C57Bl/10 weeks:
            6hr/d,5d/w,                                 no significant tumor response
            10 weeks,                                   CD-1/10 weeks:
            untreated                                   total TBA 45/71 vs 36/71controls; malignant
            until death                                 TBA 24/71 vs 22/71 controls; benign TBA.35/
                                                        71 vs 21/71 controls; lung adenoma 33/71 vs
                                                        17/71 controls; Zymbal gland carcinoma 4/71
                                                        vs 0/71 controls
   Mouse/   300 ppm,       Increased    -               Lymphomas/ leukaemia all types:              Cronkite
   C57Bl/6  6h/d, 5d/w,                                 20/89 vs 8/88 controls;                      et al. (1984,
   BNL      16 weeks,                                   thymic lymphoma: 10/89 vs 1/88 controls;     1985)149,150
   (female) observation                                 nonthymic lymphoma: 6/89 vs 2/88 controls;
            until death                                 myelogenous leukaemia 0/89 vs 3/88 controls;
                                                        leukaemia NOS: 4/89 vs 2/88 controls;
                                                        Zymbal tumors: 16/89 vs 1/88 controls;
                                                        ovarian tumors: 8/89 vs 0/88 controls
   Mouse/C 100, 300        Increased at -               300 ppm: Myelogenous neoplasms, males:       Cronkite
   BA/Ca ppm, 6h/d,        100 and 300                  19.3% vs 0% control; females: 11% vs 1.7%    et al.
   BNL      5d/w, 16       ppm                          control                                      (1989)151
            weeks,                                      100 ppm: Myelogenous neoplasms, males:
            observation                                 2.4% vs 0% control
            until death
   Mouse/ 6 hr/d, 5 d/     No data      CD-1 mice:      CD-1 mice:                                   Goldstein
   AKR,     w, Lifetime                 Granulocytic    chronic myelogenous leukaemia 1/40, acute    et al.
   C57Bl, AKR: 100                      hyperplasia     myeloblastic leukaemia 1/40 vs none in       (1982)154
   CD-1 (no and 300                     1/40            control
   data on ppm, C57Bl
   sex)     and CD-1:
            300 ppm
   Mouse 300 ppm,          Increased    Increased       Malignant lymphoma 14/118 vs 2/119 controls Farris et al.
   CBA/Ca 6h/d, 5d/w,                   granulocytic    lung adenoma 42/118 vs 17/119 controls       (1993)152
   (males) 16 weeks,                    hyperplasia in  preputial gland squamous cell carcinoma 71/
            observation                 bone marrow     118 vs 0/118 controls Zymbal gland carcinoma
            until month                 (42/116 vs      14/125 vs 1/125 controls forestomach
            22 after start              9/117 controls) squamous cell carcinoma 9/125 vs 6/125
                                        and in spleen   controls
                                        (7/114 vs 0/116
                                        controls)
   Rat      100, 300       No data      -               100 ppm: chronic myelogenous leukaemia 1/    Goldstein
   Sprague- ppm                                         40 vs none in control                        et al.
   Dawley   6 h/d, 5 d/w,                                                                            (1982)154
   (no data lifetime
   on sex)
08  Benzene
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<pre>Rat/     Pregnant    Increased in At week 118:     Increase of tumor incidences at week 150: No. Maltoni,
Sprague- females +   offspring    liver: nodular   of animals with tumors were related to the No. (1982; 1983;
Dawley   male/       after 104    hyperplasias in  of animals at study begin:                      1985)156-158
adult    female      weeks of     5/48 adult       Zymbal gland carcinoma: adult females
females  offspring:  treatment    females          (parent) 3/54 vs 1/60 controls; offspring males
and male 200 ppm,                 (parent), 1/74   6/75 vs 2/158 controls; offspring females 8/65
and      4h/d, 5d/w,              offspring male,  vs 0/149 controls
female   7 week,                  5/59 offspring   oral cavity carcinomas:
12-day   then 200                 females nodular  adult females (parent) 2/54 vs 0/60 controls;
embryos  ppm 7h/d,                dysplasia 2/59   offspring males 1/75 vs 0/158 controls;
         5d/w, 12                 offspring        offspring females 10/65 vs 0/149 controls
         week, then               females versus   nasal cavity carcinomas:
         300 ppm,                 none of each     adult females (parent) 1/54 vs0/60 controls;
         7h/d, 5d/w,              lesions was seen offspring males 1/75 vs0/158 controls;
         85 week                  in controls      offspring females 2/65 vs 0/149 controls
                                                   skin carcinomas:
                                                   adult females (parent) 0/54 vs 0/60 controls;
                                                   offspring males 1/75 vs 0/158 controls;
                                                   offspring females 1/65 vs 0/149 controls;
                                                   forestomach carcinomas:
                                                   adult females (parent) 0/65 vs 0/149 controls;
                                                   offspring males 0/75 vs 0/158 controls;
                                                   offspring females 3/65 vs 1/149 controls
                                                   hepatomas:
                                                   adult females (parent) 1/54 vs 0/60 controls;
                                                   offspring males 2/75 vs 1/158 controls;
                                                   offspring females 7/65 vs 0/149 controls;
                                                   hemolymphoreticular neoplasia:
                                                   adult females (parent): 0/54 vs 2/158 controls;
                                                   offspring males 6/75 vs 12/158 controls;
                                                   offspring females 0/65 vs 1/149 controls
         Male+                    At week 118:     No. of animals with tumors were related to the
         female                   liver: nodular   No. of animals at study begin:
         offspring:               hyperplasia      Zymbal gland carcinoma:
         200 ppm,                 2/64 offspring   offspring males 4/70 vs 2/158 controls;
         4h/d, 5d/w,              males and 7/59   offspring females 1/59 vs 0/149 controls; oral
         7week, then              offspring        cavity carcinomas: offspring males 2/70 vs
         200 ppm,                 females versus   0/158 controls; offspring females 6/59 vs
         7h/d, 5h/d,              none in the      0/149 controls
         12 week,                 controls         nasal cavity carcinomas:
         observation                               offspring males 1/70 vs0/158 controls;
         until death                               offspring females 1/59 vs 0/149 controls
                                                   hepatomas:
                                                   offspring males 2/70 vs 1/158 controls;
                                                   offspring females 5/59 vs 0/149 controls
 Animal data                                                                                              109
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<pre>Oral Mouse 0, 25, 50,    Decreased  Hematopoietic    Malignant lymphomas:                         NTP,
     B6C3F1 100 mg/kg    with       marrow           males: 9/48,9/50,15/49 vs 4/49 controls;     (1986)160;
            bw/d         increasing hyperplasia      females: 24/45,24/50,20/49 vs 15/49 controls Huff et al.
            (gavage)     doses      males: 11/48,    Zymbal gland carcinomas:                     (1989)155
                                    0/50, 25/49 vs   males: 1/34,4/40,21/39 vs 0/43 controls;
                                    0/49 control;    females: 0/32,1/37,3/31 vs 0/43 controls
                                    females:         squamous cell papilloma or carcinoma
                                    14/45,8/50,      (combined) of the forestomach:
                                    13/49 vs 3/49    males: 2/42,3/44,5/38 vs 2/45 controls;
                                    control;         females: 3/40,6/45,5/42 vs 1/42 controls
                                    hyperplasia      alveolar/bronchiolar carcinomas:
                                    Zymbal gland     males: 11/48,12/50,14/49 vs 5/49 controls;
                                    males: 4/34,     females: 3/42,6/50,6/49 vs 0/49 controls
                                    12/40,10/39 vs   alveolar/bronchiolar adenomas:
                                    0/42 control;    females: 2/42,5/50,9/49 vs 4/49 controls;
                                    females:1/32,    alveolar/bronchiolar adenomas or carcinomas
                                    2/37, 6/31 vs 1/ (combined):
                                    43 control;      males: 16/48,19/50,21/49 vs 10/49 controls;
                                    alveolar/bronch. females: 5/42,10/50,13/49 vs 4/49 controls
                                    hyperplasia:     Harderian gland adenomas:
                                    males: 3/48,     males: 9/46,13/49,11/48 vs 0/49 controls
                                    7/50, 10/49 vs   Harderian gland adenomas or carcinomas
                                    2/49 control;    (combined):
                                    females:         females: 6/44,10/50,10/47 vs 5/48 controls
                                    11/48, 12/50,    squamous cell carcinomas of preputial gland
                                    14/49 vs 1/49    males: 3/28,18/29,28/35 vs 0/21 controls
                                    control          mammary gland carcinomas:
                                    hyperplasia of   females: 2/45,5/50,10/49 vs 0/49 controls
                                    preputial gland: mammary gland carcinosarcomas
                                    males: 18/28,    females: 0/45,1/50,4/49 vs 0/40 controls
                                    9/29, 1/35 vs    ovarian granulosa cell tumors
                                    1/21 control     females: 1/44,6/49,7/48 vs 1/47 controls
                                                     overian benign mixed tumors
                                                     females: 1/44,12/49,7/48 vs 0/47 controls
                                                     hepatocellular adenomas:
                                                     females:8/44,5/50,4/49 vs 1/49 controls
                                                     hepatocellular adenomas or carcinomas
                                                     (combined)
                                                     12/44,13/50,7/49 vs 4/49 controls
     Rat/F- Males: 0,    Increased  -                Zymbal gland carcinomas:                     NTP,
     344    50, 100, 200 with dose                   males: 6/46,10/42,17/42 vs 2/32 controls     (1986)160;
            mg/kg bw/d                               females: 5/40,5/44,14/46 vs 0/45 controls    Huff et al.
            females 0,                               squamous cell papilloma of the skin:         (1989)155
            25, 50,                                  males: 2/50,1/50,5/50 vs 0/50 controls
            100 mg/kg                                squamous cell carcinoma of the skin:
            bw/d                                     males: 5/50,3/50,8/50 vs 0/50 controls
                                                     squamous cell papilloma or carcinomas
                                                     (combined) of
                                                     the oral cavity:
                                                     males: 9/50,16/50,19/50 vs 1/50 controls
                                                     females: 5/50,12/50,9/50 vs 1/50 controls
 10   Benzene
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<pre>            Rat/       50, 250 mg/ No data       SD: liver        52 weeks: Zymbal gland carcinomas: 50/      Maltoni
            Sprague- kg                          acanthomas and   males: 0%; 50/females: 6.5%; 250/males: 0%; et al.
            Dawley bw/d, 4-5 d/                  dysplasias:      250/females: 22.9%;control/males: 0%        (1989)159
                       w, 52 weeks               males: 25%,      control/females: 0%;
                                                 females: 17.5%   leukaemia: 50/males: 0%; 50/females 6.7%;
                                                 control males    250/males: 11.4% 250/females:2.9%; control/
                                                 and control      males: 0%; control/females 3.3%;oral cavity
                                                 females: 0%      carcinomas; 250/males: 0% 250/females:
                                                                  5.7%; control/males: 0% control/females: 0%
                       500 mg/kg                                  104 week: Zymbal gland carcinomas:
                       bw/d, 4-5                                  SD-males: 45%; SD-females 40%; SD-control
                       d/w, 104                                   males: 2% SD-control females:0%; W-males:
                       weeks in                                   17.5% W-females: 15%; W-control males:0%
                       Sprague-                                   W-control females 0%
                       Dawley                                     leukaemia: SD-males: 2.5%; SD-females:
                       (SD) and                                   7.5%;SD-control males: 6%; SD-control
                       Wistar (W)                                 females: 2%; W-males: 5%; W-females: 10%;
                       rats                                       W-control males: 2.5%; W-control
                                                                  females:7.5%
                                                                  oral cavity carcinomas: SD-males 52.5%;
                                                                  SD-females: 50%; SD-control males: 0%;
                                                                  SD-control females:0%; W-males: 5%;
                                                                  W-females: 10%; W-control males: 2.5%;
                                                                  W-control females: 0%
                                                                  forestomach carcinomas in situ:
                                                                  SD-males:0%; SD-females: 15%; SD control
                                                                  males: 0%; SD-control females: 0%
                                                                  forestomach invasive carcinomas:
                                                                  SD-males: 2.5% SD-females: 0%; SD-control
                                                                  males: 0% SD-control females:0%
                                                                  skin carcinomas:
                                                                  SD-males: 22.5% SD-females: 0%;
                                                                  SD-control males:0% SD-control females 2%
                                                                  liver angiosarcomas:
                                                                  SD-males:5% SD-females 7.5%; SD-control
                                                                  males: 0% SD-control females. 0%
                                                                  liver hepatomas:
                                                                  SD-males: 7.5% SD-females: 2.5%;
                                                                  SD-control males: 6% SD-control females:
                                                                  0% nasal cavity carcinomas:
                                                                  SD-males: 7.5% SD-females: 2.5%;
                                                                  SD-control males: 0% SD-control
                                                                  females:0%; W-males: 5% W-females: 2.5%;
                                                                  W-control males:0% W-control females: 0%
Ø = no treatment-related effect; NOS = not other specified; TBA = tumor bearing animals.
             Animal data                                                                                              111
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<pre>12 Benzene</pre>

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<pre>nnex G
     Genotoxicity data
     The genotoxicity results are derived from the review by Whysner et al.55
     Results of benzene genotoxicity studies in humans.
                                       Results    Number of
                                                  studies
     Micronucleus                      +           2
                                       −           5
     Chromosomal aberrations           +          15a
                                       (+)         2
                                       −           5
     Aneuploidy                        +           5
                                       −           2
     Sister chromatid exchange         +           5
                                       -           9
     DNA damage/SS. DS breaks          +           3
                                       −           1
     a    Seperate exposure group within the same study.
     Genotoxicity data                                                        113
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<pre>   Results obtained in rodents for benzene and its metabolites.
                                                                          Resultsa     Number of
                                                                                       studiesb
   Micronucleus                                  Benzene                  +            59
                                                                          (+)           1
                                                                          −             5
                                                 Phenol                   +             4
                                                                          (+)           2
                                                                          −             6
                                                 Hydroquinone             +            21
                                                                          −             1
                                                 Benzoquinone             +             2
                                                                          (+)           1
                                                 Catechol                 +             3
                                                                          (+)           1
                                                                          −             3
                                                 Benzenetriol             +             1
                                                                          −             1
                                                 Muconaldehyde            −             1
   Chromosomal aberrations                       Benzene                  +            18
                                                                          −             2
                                                 Phenol                   +             2
                                                 Hydroqinone              +             5
   Aneuploidy                                    Benzene                  +             2
                                                 Hydroquinone             +             3
   Sister chromatid exchange                     Benzene                  +             3
                                                                          (+)           4
                                                 Phenol                   +             1
                                                 Muconaldehyde            +             1
   DNA damage/SS, DS breaks                      Benzene                  +             5
                                                                          −             4
                                                 Phenol                   −             1
   Transgenic mouse mutation                     Benzene                  +c            2
   a    +                               +
           indicates positive results; ( ) indicates a weakly positive result or positive but in a
                                       −
        study of limited quality; and indicates negative results.
   b    Investigations of different strains of animals and different tissues within one publication
        are counted as separate studies.
   c    Result reported by the authors. The Subcommittee on Classification Carcinogenic
        Substances considers the marginal responses reported in the transgenic mouse mutation
        assay as negative results (see Annex H).
   Note: only categories for which at least one result is present are included.
14 Benzene
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<pre>Results of in vitro genotoxicity tests for benzene and its metabolites in cells derived
from rodents and humans.
                                                           Resultsa                   Number of studies
Micronucleus
Benzene without activation                                 -                            1
Benzene with activation                                    -                            1
Phenol                                                     +                            3
                                                           -                            1
Hydroquinone                                               +                            9
                                                           (+)                          4
                                                           -                            7
Benzoquinone                                               +                            5
Catechol                                                   +                            2
                                                           -                            1
Benzenetriol                                               +                            3
Muconaldehyde                                              +                            2
Chromosomal aberrations
Benzene without activation                                 +                            6
                                                           -                            7
Benzene with activation                                    +                            3
                                                           -                            2
Hydroquinone                                               +                            2
                                                           -                            2
Catechol                                                   +                            2
Aneuploidy
Benzene without activation                                 +                            2
                                                           -                            2
Benzene with activation                                    -                            1
Phenol                                                     -                            1
Hydroquinone                                               +                            4
                                                           -                            2
Catechol                                                   +                            1
Benzotriol                                                 +                            1
Sister chromatid exchange
Benzene without activation                                 +                            1
                                                           -                          11
Benzene with activation                                    +                            1
                                                           -                            6
Phenol                                                     +                            3
                                                           (+)                          1
                                                           -                            2
Hydroquinone                                               +                            8
Benzoquinone                                               +                            1
                                                           -                            1
Catechol                                                   +                            7
Genotoxicity data                                                                                       115
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<pre>   Benzenetriol                                           +                         2
   Muconaldehyde                                          -                         1
   DNA damage/SS breaks
   Benzene without activation                             +                         2
                                                          -                         8
   Benzene with activation                                +                         2
                                                          -                         3
   Phenol                                                 -                         3
   Hydroquinone                                           +                         4
                                                          (+)                       2
   Benzoquinone                                           +                         4
                                                          (+)                       1
                                                          -                         2
   Catechol                                               +                         2
                                                          -                         4
   Benzenetriol                                           +                         6
                                                          -                         2
   Muconaldehyde                                          -                         3
   Unscheduled DNA synthesis
   Benzene without activation                             +                         1
                                                          -                         6
   Benzene with activation                                -                         2
   Phenol                                                 +                         1
   Hydroquinone                                           +                         1
   Catechol                                               +                         1
   Muconaldehyde                                          -                         1
   Mammalian gene mutation
   Benzene without activation                             +                         3
                                                          (+)                       1
                                                          -                        12
   Benzene with activation                                +                         2
                                                          (+)                       2
                                                          -                        13
   Phenol                                                 +                         1
                                                          -                         2
   Hydroquinone                                           +                         3
   Benzoquinone                                           +                         2
   Catechol                                               +                         3
   Benzenetriol                                           +                         1
   Muconaldehyde                                          +                         2
   Muconic acid                                           -                         3
   a   +                               +
          indicates positive results; ( ) indicates a weakly positive result or positive but in a study of
       limited quality; and - indicates negative results.
16 Benzene
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<pre> nnex H
      Advice of the Subcommittee on
      Classification of Carcinogenic
      Substances
H.1   Scope
      For carcinogens, the Dutch Expert Committee on Occupational Safety (DECOS)
      of the Health Council derives either a health-based recommended occupational
      exposure limit (HBR-OEL) or a health-based calculated occupational cancer risk
      value (HBC-OCRV), dependent on their mechanism of action. For non-
      genotoxic carcinogens and non-stochastic genotoxic carcinogens, it is assumed
      that the carcinogenic effects only occur when exposure levels exceed a certain
      threshold. For such substances, the Committee derives a HBR-OEL. For
      stochastic genotoxic carcinogens, and genotoxic carcinogens for which the
      mechanism of action is unknown but for which a stochastic mechanism is not
      unlikely, it is assumed that any level of exposure is associated with a certain risk
      for developing cancer. For these substances, a HBC-OCRV is derived.
      In order to establish the appropriate approach, the Subcommittee on the
      Classification of carcinogenic substances was requested by DECOS to evaluate
      the carcinogenic properties of benzene and in particular, its genotoxic mode of
      action. The members of the Subcommittee are listed at the end of this Annex.
      Advice of the Subcommittee on Classification of Carcinogenic Substances              117
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<pre>H.2 Carcinogenicity of benzene
    Human data
    Increased risk of either leukaemia in general or acute myeloid leukaemia/acute
    non-lymphocytic leukaemia specifically, after exposure to benzene has been
    observed in several cohorts of workers exposed to benzene in various industries
    (in particular the Pliofilm cohort of rubber workers in the US, the Chinese
    Academy of Preventive Medicine (CAMP) cohort of benzene-exposed workers
    in China and the Australian Healthwatch cohort of petroleum workers)(i.e.
    Rinsky et al. 1981, 1987, 2002; Wong et al. 1995; Silver et al. 2002; Richardson
    et al. 2008; Hayes et al. 1997; Travis et al. 1994; Yin et al. 1994; Glass et al.
    2003). In addition, several meta-analyses underline the positive relationship
    between benzene exposure and leukaemia, or acute myeloid leukaemia (AML)
    specifically (Schnatter et al. 2005; Vlaanderen et al. 2010; Khalade et al. 2010).
    In one pooled analysis, an increased risk of MDS was observed (Schnatter et al.
    2012).
        Several positive associations have been reported between benzene exposure
    and other haematological malignancies, including (based on meta-analyses)
    multiple myeloma (MM), chronic lymphocytic leukaemia (CLL), acute
    lymphocytic leukaemia (ALL), and chronic myeloid leukaemia (CML)
    (Vlaanderen et al. 2011; Vlaanderen et al. 2012).
    The Subcommittee concludes that there is sufficient evidence for a causal
    relationship between benzene exposure and haematological malignancies in
    humans.
    Animal data
    In rats as well as in mice, benzene induces tumours in several target organs,
    involving the haematopoietic system and several organs of epithelial origin.
        Tumours that have been reported in rats exposed by inhalation included
    carcinomas of the Zymbal gland and oral cavity. Tumours found at other sites
    involved carcinoma of the nasal cavity, mammary gland tumours and hepatomas
    (Maltoni et al. 1982a, 1982b, 1983, 1985, 1989; Snyder et al. 1984). Mice
    developed a variety of tumours, including lymphomas, myelogenous leukaemias,
    Zymbal gland tumours, ovarian tumours and lung tumours (Cronkite et al. 1986,
    1984, 1985, 1989; Farris et al. 1993; Snyder et al. 1980).
 18 Benzene
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<pre>        Oral administration induced a similar spectrum of tumours (Huff et al. 1989;
    Maltoni et al. 1983, 1985, 1989; NTP 1986). Exposure of rats to benzene induced
    Zymbal gland carcinomas, squamous cell papillomas and carcinomas of the oral
    cavity and skin. In mice, benzene caused tumours that included lymphomas,
    Zymbal gland carcinomas, lung alveolar/bronchiolar adenomas and carcinomas,
    Harderian gland adenomas, preputial gland squamous cell carcinomas, and
    mammary gland carcinomas.
    The Subcommittee concludes that benzene is carcinogenic to man, based on
    epidemiological evidence for AML and total leukaemia, and supporting evidence
    in experimental animals. The Subcommittee recommends, in accordance with
    the EU-classification, to classify benzene in category 1A (‘the compound is
    known to be carcinogenic to humans’).*
H.3 Genotoxicity of benzene
    Cytogenicity
    An important basis for the Subcommittee’s evaluation of the genotoxicity of
    benzene are the results obtained in standardised, guideline-compliant assays
    (reviewed in Whysner et al. 2004; JRC-IHCP 2008). These assays are designed
    to identify the potential to induce (numerical and structural) chromosomal
    aberrations and gene mutations. From these results, the Subcommittee concludes
    that a large amount of convincing evidence shows that exposure to benzene
    causes the induction of micronuclei, chromosomal aberrations, sister chromatid
    exchanges and DNA strand breaks, both in humans and in animals.
        Typical genetic profiles of MDS/AML are found in patients associated with
    benzene exposure, i.e. higher levels of chromosomal changes commonly
    observed in AML, including 5q-/-5 or 7q-/-7, +8, and t(8;21) (Smith et al. 2010).
    The Subcommittee concludes that benzene is genotoxic by the induction of
    structural and numerical chromosomal aberrations.
    Mutagenicity
    Standardised mutagenicity assays have indicated a low mutagenic potential. The
    majority of the gene mutation assays in bacteria did not reveal any mutagenic
    See Annex I for the classification system of the Health Council.
    Advice of the Subcommittee on Classification of Carcinogenic Substances           119
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<pre>   responses after exposure to benzene and its metabolites. In vitro mammalian
   gene mutation assays conducted with benzene are predominantly negative
   (Whysner et al. 2004; JRC-IHCP 2008).
       Billet et al. (2010) studied the mutational pattern induced by benzene on the
   TP53 gene in human type II-like alveolar epithelial A549 cells by using the
   Functional Analysis of Separated Alleles in Yeast (FASAY). Seventeen
   mutations linked to benzene exposure were found, including one – or two – base
   deletions and single nucleotide substitutions, of which A>G and G>A transitions
   were the most prevalent.
       Two transgenic mouse mutation assays are available that both have shown
   marginal responses at relatively high benzene exposure levels (Mullin et al.
   1995; Provost et al. 1996), in particular an increase in the frequency and length
   of deletion mutations (Mullin et al. 1998). The Subcommittee notes that one
   study only applied one dose whereas in the other, a dose-response relationship is
   only reported in the spleen. Furthermore, a relatively high variation is observed
   in the controls, within each study as well as between both studies.
       Mutations have been found in the glycophorin A (GPA) gene mutation assay
   (Rothman et al. 1995). The GPA assay measures somatic cell mutation frequency
   in MN heterozygous peripheral erythrocytes; mutations that are considered to
   have occurred exclusively in precursor erythroid cells or stem cells in the bone
   marrow. Both NN variants (which are associated with chromosomal damage and
   mitotic recombination) and NØ variants (which are associated with gene
   inactivation through point mutations and deletions) were scored. In 24 workers
   heavily exposed to benzene (mean exposure was approximately 234 mg/m3; 72
   ppm), an increase in NN but not in NØ mutant variants was observed compared
   to 23 matched controls.
   The Subcommittee concludes that it cannot be excluded that benzene has a low
   mutagenic potential.
   Adducts
   Several DNA-adduct formation (with 32P-postlabeling) studies and DNA-
   binding studies (with 14C-labeled benzene) have been published (reviewed
   Whysner et al. (2004)). The Subcommittee considers 32P-postlabeling analysis
   most specific to measure covalent binding. In most in vivo studies with either
   benzene or its metabolites, DNA adducts were below the limit of detection. Only
   relatively low levels of DNA binding have been reported, with an apparent lack
20 Benzene
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<pre>of concordance of target organs between DNA-binding studies and the results
obtained in bioassays.
In line with the considerations made by Whysner et al. (2004), the Subcommittee
concludes that DNA adducts do not play a significant role in the carcinogenicity
of benzene.
Mechanisms of action
Several mechanisms of actions have been implicated with the carcinogenic
properties of benzene, including:
• Inhibition of topoisomerase II
• Adduct formation of reactive metabolites
• Oxidative stress
• Error-prone DNA repair
• Epigenetic alterations.
For more details on these mechanisms of action and benzene carcinogenicity, the
Subcommittee refers to reviews published recently by McHale et al. (2012) and
Wang et al. (2012).
     The Subcommittee notes that all mechanisms of action that have been
proposed, with the exception of the formation of adducts, are currently
considered to be thresholded phenomena. The Subcommittee considers covalent
binding by benzene, in view of the low electrophilic nature of the prominent
metabolites of benzene, the absence of positive findings in standardised gene
mutation assays and the lack of substantial adduct formation, of no concern for
the risk assessment of benzene.
     Whereas there is a lack of evidence for a direct mechanism of genotoxicity,
there is a large amount of evidence suggesting that benzene acts by thresholded
mechanisms of action. (McHale et al. (2012); Wang et al. (2012); Whysner et al.
(2004)) The Subcommittee acknowledges that currently, not all findings can
undeniably be attributed to a particular mode of action (either direct or indirect).
In particular, the induction of gene mutations and unbalanced chromosomal
aberrations have been noted in this context. The Subcommittee concludes
however, that also these findings can be the result of indirect genotoxicity and do
therefore not provide evidence for a direct genotoxic mode of action per se.
     The Subcommittee further acknowledges that the contribution to the toxicity
and carcinogenicity of benzene of each of the proposed mechanisms of actions,
cannot be quantified. In this context, a systems biology approach has been
Advice of the Subcommittee on Classification of Carcinogenic Substances              121
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<pre>    proposed for benzene to identify potential biomarkers of exposure, early effect
    and susceptibility (Zhang et al. 2010), which may lead to more refined risk
    assessment approaches.
    Overall, the weight of evidence points to an indirect genotoxic mode of action
    (e.g., inhibition of topoisomerase II, generation of oxidative stress, etc.), whereas
    there is no evidence to substantiate a direct genotoxic mode of action. Therefore,
    the Subcommittee considers an indirect genotoxic mode of action most likely for
    benzene.
H.4 Recommendation for classification
    Based on the available data, the Subcommittee recommends, in accordance with
    EU classification, to classify benzene in category 1A (the compound is known to
    be carcinogenic to humans). The Subcommittee concludes that benzene acts by a
    non-stochastic genotoxic mode of action.
H.5 References
    Billet S, Paget V, Garçon G, Heutte N, André V, Shirali P, Sichel F. (2010) Benzene-induced
    mutational pattern in the tumour suppressor gene TP53 analysed by use of a functional assay, the
    functional analysis of separated alleles in yeast, in human lung cells. Arch Toxicol. 84(2):99-107.
    Cronkite, EP, Bullis, J., Inoue, T, Drew, RT (1984): Benzene inhalation produces leukaemia in mice.
    Toxicol. Appl. Pharmacol. 75: 358-361.
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Advice of the Subcommittee on Classification of Carcinogenic Substances                              123
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24 Benzene
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<pre>    Yin SN, Hayes RB, Linet MS, Li GL, Dosemeci M, Travis LB, Li CY, Zhang ZN, Li DG, Chow WH,
    Wacholder S, Wang YZ, Jiang ZL, Dai TR, Zhang WY, Chao XJ, Ye PZ, Kou QR, Zhang XC, Lin
    XF, Meng JF, Ding CY, Zho JS, Blot WJ. (1996) A cohort study of cancer among benzene-exposed
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    Systems biology of human benzene exposure. Chem Biol Interact. 19;184(1-2):86-93.
H.6 The Subcommittee on Classification of Carcinogenic Substances
    •   R.A. Woutersen, chairman
        Toxicologic Pathologist, TNO Quality of Life, Zeist, and Professor of
        Translational Toxicology, Wageningen University and Research Centre,
        Wageningen
    •   J. van Benthem
        Genetic Toxicologist, National Institute for Public Health and the
        Environment, Bilthoven
    •   P.J. Boogaard
        Toxicologist, SHELL International BV, The Hague
    •   G.J. Mulder
        Emeritus Professor of Toxicology, Leiden University, Leiden
    •   Ms. M.J.M. Nivard
        Molecular Biologist and Genetic Toxicologist, Leiden University Medical
        Center, Leiden
    •   G.M.H. Swaen
        Epidemiologist, Dow Chemicals NV, Terneuzen (until April 1, 2013);
        Exponent, Menlo Park, United States (from August 15, 2013 until
        February 1, 2014)
    •   E.J.J. van Zoelen
        Professor of Cell Biology, Radboud University Nijmegen, Nijmegen
    •   A.S.A.M. Van der Burght, scientific secretary
        Health Council of the Netherlands, The Hague
    •   S.R. Vink, scientific secretary
        Health Council of the Netherlands, The Hague
    Date last meeting: October 25th, 2013.
    Advice of the Subcommittee on Classification of Carcinogenic Substances                      125
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<pre> nnex      I
           Classification of substances with
           respect to carcinogenicity
           The Committee expresses its conclusions in the form of standard phrases:
 ategory   Judgement of the Committee (GRGHS)                                 Comparable with EU Category
                                                                              67/548/EEC          EC No 1272/2008
                                                                              before              as from
                                                                              12/16/2008          12/16/2008
A          The compound is known to be carcinogenic to humans.                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, it is unclear whether the compound is genotoxic.
B          The compound is presumed to be as carcinogenic to humans.          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, it is unclear whether the compound is genotoxic.
           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. A10/07E.187
           Classification of substances with respect to carcinogenicity                                           127
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<pre>nnex J
     List of Abbreviations
     AML                  acute myeloid leukaemia
     ALL                  acute lymphatic leukaemia
     ANNL                 acute non-lymphatic leukaemia
     CLL                  chronic lymphatic leukaemia
     CML                  chronic myeloid leukaemia
     GC                   gas chromatography
     HBC-OCRV             health-based calculated occupational cancer risk value
     HBR-OEL              health-based recommended occupational exposure limit
     HPLC                 high-performance liquid chromatography
     LOD                  limit of detection
     MDS                  myelodysplastic syndrome
     MM                   multiple myeloma
     MPO                  myeloperoxidase
     MS                   mass spectometry
     NHL                  non-Hodgkin lymphoma
     NQ01                 NAD(P)H: quinone oxidoreductase 1
     SMR                  standardised mortality ratio
     SPE                  solid phase extraction
     SPMA                 S-phenylmercapturic acid
     SPME                 solid phase microextraction
     ttMA                 trans, trans-muconic acid
     List of Abbreviations                                                       129
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<pre>Health Council of the Netherlands
Advisory Reports
The Health Council’s task is to       In addition, the Health Council
advise ministers and parliament on    issues unsolicited advice that
issues in the field of public health. has an ‘alerting’ function. In some
Most of the advisory reports that     cases, such an alerting report
the Council produces every year       leads to a minister requesting
are prepared at the request of one    further advice on the subject.
of the ministers.
Areas of activity
Optimum healthcare                    Prevention                          Healthy nutrition
What is the optimum                   Which forms of                      Which foods promote
result of cure and care               prevention can help                 good health and
in view of the risks and              realise significant                 which carry certain
opportunities?                        health benefits?                    health risks?
Environmental health                  Healthy working                     Innovation and
Which environmental                   conditions                          the knowledge
influences could have                 How can employees                   infrastructure
a positive or negative                be protected against                Before we can harvest
effect on health?                     working conditions                  knowledge in the
                                      that could harm their               field of healthcare,
                                      health?                             we first need to
                                                                          ensure that the right
                                                                          seeds are sown.
www.healthcouncil.nl
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