<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>www.healthcouncil.nl Innovation and the knowledge infrastructure Before we can harvest knowledge in the field of healthcare, we first need to ensure that the right seeds are sown. Healthy working conditions How can employees be protected against working conditions that could harm their health? Environmental health Which environmental influences could have a positive or negative effect on health? Healthy nutrition Which foods promote good health and which carry certain health risks? Prevention Which forms of prevention can help realise significant health benefits? Optimum healthcare What is the optimum result of cure and care in view of the risks and opportunities? Areas of activity Advisory Reports The Health Council’s task is to advise ministers and parliament on issues in the field of public health. Most of the advisory opinions that the Council produces every year are prepared at the request of one of the ministers. In addition, the Health Council issues unsolicited advice that has an ‘alerting’ function. In some cases, such an alerting report leads to a minister requesting further advice on the subject. Health Council of the Netherlands Chromium VI compounds 2016/04 2016/04 Evaluation of the effects on reproduction, recommendation for classification Chromium VI compounds Health Council of the Netherlands</pre>

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<pre>Chromium VI compounds
    Evaluation of the effects on reproduction,
    recommendation for classification
</pre>

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<pre></pre>

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<pre>Aan de minister van Sociale Zaken en Werkgelegenheid
Onderwerp               : aanbieding advies Chromium VI compounds
Uw kenmerk              : DGV/BMO/U-932542
Ons kenmerk             : U-963412/SV/jh/543-G16
Bijlagen                :1
Datum                   : 18 mei 2016
Geachte minister,
Graag bied ik u hierbij het advies aan over de effecten van een groep chroom VI-verbin-
dingen op de vruchtbaarheid en de ontwikkeling van het nageslacht; het betreft ook effecten
op de lactatie en via de moedermelk op de zuigeling.
      Dit advies maakt deel uit van een uitgebreide reeks waarin voor de voortplanting giftige
stoffen worden geclassificeerd volgens richtlijnen van de Europese Unie. Het gaat om stof-
fen waaraan mensen tijdens de beroepsuitoefening kunnen worden blootgesteld. Dit advies
is een actualisatie van een advies dat in 2001 door de Gezondheidsraad is uitgebracht. De
raad is gevraagd om deze actualisatie omdat de voorgestelde classificatie uit het eerdere
advies afwijkt van de classificatie die op dit moment in de Europese Unie wordt gehanteerd.
Dit advies is opgesteld door een vaste commissie van de Gezondheidsraad, de Subcommis-
sie Classificatie reproductietoxische stoffen. Het is vervolgens getoetst door de Beraads-
groep Volksgezondheid.
Ik heb dit advies vandaag ter kennisname toegezonden aan de minister van VWS en aan de
staatssecretaris van IenM.
Met vriendelijke groet,
prof. dr. J.L. Severens,
vicevoorzitter
Bezoekadres                                                       Postadres
Parnassusplein 5                                                  Postbus 16052
2 5 11 V X D e n H a a g                                          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 6 5 2 7 8 1 5 8 4
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<pre></pre>

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<pre>Chromium VI compounds
Evaluation of the effects on reproduction,
recommendation for classification
Subcommittee on the Classification of Reproduction Toxic Substances,
a Committee of the Health Council of the Netherlands
to:
the Minister of Social Affairs and Employment
No. 2016/04, The Hague, May 18, 2016
</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 and Sport, Infrastructure and the Environment, Social Affairs
and Employment, and Economic Affairs. 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. Chromium VI compounds. Evaluation of the
effects on reproduction, recommendation for classification. The Hague: Health
Council of the Netherlands, 2016; publication no. 2016/04.
all rights reserved
ISBN: 978-94-6281-075-4
</pre>

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<pre>   Contents
   Samenvatting 9
   Executive summary 11
   Scope 13
.1 Background 13
.2 Committee and procedure 14
.3 Classification for lactation 15
.4 Data 15
   Identity of the substance 17
.1 Name and other identifiers of the substance 17
.2 Composition of the substance 18
.3 Physico-chemical properties 18
.4 International classifications 19
   Manufacture and uses 21
.1 Manufacture 21
.2 Identified uses 21
   Contents                                       7
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<pre>    Read-across and toxicokinetics (absorption, metabolism, distribution and elimination) 23
 .1 Read-across/grouping 23
 .2 Non-human studies 25
 .3 Human studies 30
 .4 Summary and discussion on toxicokinetics 34
    Toxicity for reproduction 37
 .1 Effects on fertility 37
 .2 Developmental toxicity 55
 .3 Other relevant information 70
 .4 Summary and discussion of reproductive toxicity 73
 .5 Comparison with criteria 79
 .6 Conclusions on classification and labelling 80
    References 83
    Annexes 87
A   The Committee 89
B   The submission letter (in English) 91
C   Comments on the public draft 93
D   Regulation (EC) 1272/2008 of the European Community 95
E   Additional considerations to Regulation (EC) 1272/2008 107
    Chromium VI compounds
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<pre>Samenvatting
In het voorliggende advies heeft de Gezondheidsraad chroomtrioxide, natrium-
chromaat, natriumdichromaat, kaliumdichromaat, chroomzuur, ammoniumchro-
maat, ammoniumdichromaat, calciumchromaat, kaliumchromaat, en dichroom-
trischromaat onder de loep genomen. Deze chroom VI-verbindingen worden
gebruikt in metaalafwerking, synthese van andere chroomverbindingen, houtver-
duurzamingsmiddelen, katalysatoren en pigmenten/kleurstoffen. Dit advies past
in een reeks adviezen waarin de Gezondheidsraad op verzoek van de minister
van Sociale Zaken en Werkgelegenheid de effecten van stoffen op de voortplan-
ting beoordeelt. Het gaat vooral om stoffen waaraan mensen tijdens de beroep-
suitoefening kunnen worden blootgesteld. De Subcommissie Classificatie
reproductietoxische stoffen van de Commissie Gezondheid en beroepsmatige
blootstelling aan stoffen (GBBS) van de raad, hierna aangeduid als de commis-
sie, kijkt zowel naar effecten op de vruchtbaarheid van mannen en vrouwen als
naar effecten op de ontwikkeling van het nageslacht. Daarnaast worden effecten
op de lactatie en via de moedermelk op de zuigeling beoordeeld.
Op basis van Verordening (EG) 1272/2008 van de Europese Unie doet de
commissie een voorstel voor classificatie. Voor de bovengenoemde chroom VI-
verbindingen komt de commissie tot de volgende aanbevelingen:
• voor effecten op de fertiliteit adviseert de commissie ze te classificeren in
     categorie 1B (stoffen waarvan verondersteld wordt dat zij toxisch zijn voor
Samenvatting                                                                     9
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<pre>     de menselijke voortplanting) en te kenmerken met H360F (kan de
     vruchtbaarheid schaden)
  •  voor effecten op de ontwikkeling adviseert de commissie ze te classificeren
     in categorie 1B (stoffen waarvan verondersteld wordt dat zij toxisch zijn voor
     de menselijke voortplanting) en te kenmerken met H360D (kan het
     ongeboren kind schaden)
  •  voor effecten tijdens de lactatie adviseert de commissie om ze te kenmerken
     met H362 (kan schadelijk zijn via de borstvoeding).
0 Chromium VI compounds
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<pre>Executive summary
In the present report the Health Council of the Netherlands reviewed chromium
trioxide, sodium chromate, sodium dichromate, potassium dichromate, chromic
acid, ammonium chromate, ammonium dichromate, calcium chromate,
potassium chromate, and dichromium tris(chromate). These chromium VI
compounds are used in metal finishing, manufacture of other chromium
compounds, wood preservation products, catalysts, and pigments/dyes. This
report is part of a series, in which the Health Council evaluates the effects of
substances on reproduction, at the request of the Minister of Social Affairs and
Employment. It mainly concerns substances to which man can be occupationally
exposed. The Subcommittee on the Classification of Reproduction Toxic
Substances of the Dutch Expert Committee on Occupational Safety (DECOS) of
the Health Council, hereafter called the Committee, evaluates the effects on male
and female fertility and on the development of the progeny. Moreover, the
Committee considers the effects of a substance on lactation and on the progeny
via lactation.
The Committee recommends classification according to Regulation (EC) 1272/
2008 of the European Union. For the abovementioned chromium VI compounds,
the Committee recommends:
• for effects on fertility, to classify these compounds in category 1B (presumed
    human reproductive toxicant), and to label them with H360F (may damage
    fertility)
Executive summary                                                                 11
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<pre>  •  for effects on development, to classify these compounds in category 1B
     (presumed human reproductive toxicant) and to label them with H360D (may
     damage the unborn child)
  •  for effects during lactation, to label these compounds with H362 (may cause
     harm to breastfed babies).
2 Chromium VI compounds
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<pre> hapter 1
        Scope
1.1     Background
        As a result of the Dutch regulation on registration of compounds toxic to
        reproduction that came into force on 1 April 1995, the Minister of Social Affairs
        and Employment requested the Health Council of the Netherlands to classify
        compounds toxic to reproduction. This classification is performed by the Health
        Council’s Subcommittee on the Classification of reproduction toxic substances
        of the Dutch Expert Committee on Occupational Safety (DECOS). The
        classification is performed according to European Union Regulation (EC) 1272/
        2008 on classification, labelling and packaging (CLP) of substances and
        mixtures. The CLP guideline is based on the Globally Harmonised System of
        Classification and Labelling of Chemicals (GHS). The Subcommittee’s advice
        on the classification will be applied by the Ministry of Social Affairs and
        Employment to extend the existing list of compounds classified as reproductive
        toxicant (category 1A and 1B and 2) or compound with effects on or via
        lactation.
            The ministry of Social Affairs and Employment asked the Health Council to
        update the evaluation and classification on reproduction toxicity of a series of
        substances. In this report, such an update was performed for a group of
        chromium VI compounds. For those specified in Chapter 2.1, this document
        replaces the recommendation regarding the classification for reproductive
        toxicity of chromium VI compounds that was published in 2001.1
        Scope                                                                             13
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<pre>1.2 Committee and procedure
    This document contains the classification of a group of chromium VI compounds
    by the Health Council’s Subcommittee on the Classification of Reproduction
    Toxic Substances, hereafter called the Committee. The members of the
    Committee are listed in Annex A. A submission letter (in English) to the
    Minister can be found in Annex B.
        In 2015, the President of the Health Council released a draft of the report for
    public review. The individuals and organisations that commented on the draft
    report are listed in Annex C. The Committee has taken these comments into
    account in deciding on the final version of the report. The received comments,
    and the replies by the Committee, can be found on the website of the Health
    Council.
    The classification is based on the evaluation of published human and animal
    studies concerning adverse effects with respect to fertility and development as
    well as lactation of the above mentioned compound.
    Classification for reproduction (fertility (F) and development (D):
    Category 1                  Known or presumed human reproductive toxicant (H360(F/D))
       Category 1A              Known human reproductive toxicant
       Category 1B              Presumed human reproductive toxicant
    Category 2                  Suspected human reproductive toxicant (H361(f/d))
    No classification for effects on fertility or development
    Classification for lactation:
                                Effects on or via lactation (H362)
                                No labelling for lactation
        The classification and labelling of substances is performed according to the
    criteria of the European Union (Regulation (EC) 1272/2008) presented in
    Annex D. The classification of substances is ultimately dependent on an
    integrated assessment of the nature of all parental and developmental effects
    observed, their specificity and adversity, and the dosages at which the various
    effects occur. The criteria necessarily leave room for interpretation, dependent on
    the specific data set under consideration. In the process of using the regulation,
    the Committee has agreed upon a number of additional considerations (see
    Annex E).
 4  Chromium VI compounds
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<pre>1.3 Classification for lactation
    The recommendation for classifying substances for effects on or via lactation is
    also based on Regulation (EC) 1272/2008. The criteria define that substances
    which are absorbed by women and have been shown to interfere with lactation or
    which may be present (including metabolites) in breast milk in amounts
    sufficient to cause concern for the health of a breastfed child, shall be classified
    and labelled. Unlike the classification of substances for fertility and
    developmental effects, which is based on hazard identification only (largely
    independent of dosage), the labelling for effects during lactation is based on a
    risk characterization and therefore, it also includes the consideration of the level
    of potential exposure of the breastfed child.
        Consequently, a substance should be labelled for effects during lactation
    when it is likely that the substance would be present in breast milk at potentially
    toxic levels. The Committee considers a concentration of a substance as
    potentially toxic to the breastfed child when this concentration leads to
    exceeding the exposure limit for the general population, e.g. the acceptable daily
    intake (ADI).
1.4 Data
    Literature searches were conducted in the on-line databases TOXLINE,
    MEDLINE and CAPLUS. A final search was performed in November 2015.
    Publications cited in the selected articles, but not selected during the primary
    search, were reviewed if considered appropriate. In addition, handbooks and
    most recent reviews were consulted.
    Scope                                                                                15
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<pre>6 Chromium VI compounds</pre>

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<pre> hapter       2
              Identity of the substance
2.1           Name and other identifiers of the substance
              Data are derived from ECHA and HSDB.2,3
 C number             215-607-8         231-889-5       234-190-3         231-906-6        231-801-5
 C name               Chromium trioxide Sodium chromate Sodium dichromate Potassium        Chromic acid
                                                                          dichromate
 AS number            1333-82-0         7775-11-3       10588-01-9        7778-50-9        7738-94-5
 AS name              Chromium oxide,   Chromic acid,   Chromic acid,     Chromic acid,    Chromic acid,
                      (CrO3)            (H2CrO4),       (H2Cr2O7),        (H2Cr2O7),       (H2CrO4)
                                        disodium salt   disodium salt     dipotassium salt
UPAC name             Trioxochromium    Disodium        Sodium dichromate Dipotassium      dihydroxy(dioxo)
                                        dioxido(dioxo)                    dichromate       chromium
                                        chromium
 LP Annex VI          024-001-00-0      024-018-00-3    024-004-00-7      024-002-00-6     -
ndex number
Molecular formula     CrO3              Na2CrO4(•4H2O)  Na2Cr2O7(•2H2O)   K2Cr2O7          CrH2O4
Molecular weight      99.99             161.99          261.96            294.22           118.0
g/mol)
 tructural formula
              Identity of the substance                                                                   17
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<pre>  C number           232-138-4           232-143-1       237-366-8            232-140-5          246-356-2
  C name             Ammonium            Ammonium        Calcium chromate     Potassium chromate Dichromium
                     chromate            dichromate                                              tris(chromate)
CAS number           7788-98-9           7789-09-5       13765-19-0           7789-00-6          24613-89-6
CAS name                                 Chromic acid,                        Chromic acid,      Chromic acid
                                         (H2Cr2O7),                           (H2CrO4),          (H2CrO4),
                                         diammonium salt                      dipotassium salt   chromium(3+)
                                                                                                 salt (3:2)
 UPAC name           Ammonium            Diammonium      Calcium dioxido-     Potassium          Chromium (3+)
                     chromate            dichromate      (dioxo) chromium     chromate           dioxido (dioxo)
                                                                                                 chromium
CLP Annex VI                             024-003-00-1    024-008-00-9         024-006-00-8       024-010-00-X
 ndex number
Molecular formula    (NH4)2CrO4          (NH4)2Cr2O7     CaCrO4               K2CrO4             CrH2O4.2/3Cr
Molecular weight     152.07              252.06          156.09               194.20             451.97
g/mol)
 tructural formula
2.2           Composition of the substance
              Not applicable.
2.3           Physico-chemical properties
              Data are derived from EU RAR and ECHA.2-4
 roperty             Chromium trioxide Sodium chromate   Sodium dichromate Potassium             Chromic acid
                                                                              dichromate
 tate of the         Dark red            Yellow          Reddish to bright    Bright orange-red  Dark purplish-red
 ubstance at normal  deliquescent        orthorhombic    orange deliquescent crystals - not      crystals
emperature and       crystals, flakes or crystals        crystals in hydrated hygroscopic or
 ressure and 101,3   powder                              form usually         deliquescent
 Pa                                                      dihydrated
Melting/freezing     196 °C              792 °C          Becomes anhydrous ~398 °C               196 °C
 oint                                                    at 100 °C and melts
                                                         at ~357 °C
Relative density     ~2.7                ~2.4-2.7        ~2.5                 ~2.7               1.67-2.82
Water solubility (at ~1 667 g/L          ~530 g/L        ~2 355 g/L           ~115 g/L           1 854 g/L
 oom temperature)
  8           Chromium VI compounds
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<pre>Autoignition and       Decomposes at        -                     Decomposes above Decomposes above Decomposes at
lammability            250 °C to Cr2O3                            400 °C               500 °C               about 250 °C
                       and O2
Oxidizing properties Violent oxidizing      Mildly oxidizing;     Strong oxidizing     Strong oxidizing     Strong oxidizing
                       agent                strong oxidizer in    agent                agent                agent
                                            acidic conditions
 roperty               Ammonium             Ammonium              Calcium chromate     Potassium chromate Dichromium
                       chromate             dichromate                                                      tris(chromate)
 tate of the           Yellow crystals      Orange crystals       Yellow crystals      Yellow crystals      Dark purple/black
 ubstance at normal                                                                                         granular solid, with
emperature and                                                                                              an amorphous non-
 ressure and                                                                                                crystalline structure
 01,3 kPa
Melting/freezing       185 °C               180 °C                2,710 °C             968.3 °C             >300 °C
 oint                  (decomposes)         (decomposes)
Relative density       ~1.9 g/cm3           ~2.2 g/cm3            ~2.9 g/cm3           ~2.7 g/cm3           ~2.3 g/cm3
Water solubility (at   ~405 g/L             ~360 g/L              163 g/L              394 g/L              96.6 g/L
oom temperature)
Autoignition and       -                    -                     -                    -
lammability
Oxidizing properties   Oxidizing            Oxidizing             -                    -                    Oxidizing
                       substance            substance                                                       substance
2.4          International classifications
             EU harmonised classification
 hromium      Sodium       Sodium      Potassium     Chromic acid Ammonium    Ammonium     Calcium      Potassium   Dichromium
rioxide       chromate     dichromate  dichromate                 chromate    dichromate   chromate     chromate    tris(chromate)
 epr 2        Repr 1B      Repr 1B     Repr 1B       -            -           Repr 1B      -            -           -
H361f)        (H360FD)     (H360FD)    (H360FD)                               (H360FD)
             In addition to classification for reproductive toxicity, chromium VI compounds
             have been classified for several other endpoints, including carcinogenicity and
             mutagenicity:
 hromium      Sodium       Sodium      Potassium     Chromic acid Ammonium    Ammonium     Calcium      Potassium   Dichromium
rioxide       chromate     dichromate  dichromate                 chromate    dichromate   chromate     chromate    tris(chromate)
 arc. 1A      Carc. 1B     Carc. 1B    Carc. 1B      Carc. 1B     Carc. 1B    Carc. 1B     Carc. 1B     Carc. 1B    Carc. 1B
H350)         (H350)       (H350)      (H350)        (H350)       (H350)      (H350)       (H350)       (H350)      (H350)
Muta. 1B      Muta. 1B     Muta. 1B    Muta. 1B                               Muta. 1B                  Muta. 1B
H340)         (H340)       (H340)      (H340)                                 (H340)                    (H340)
             The classification of chromium VI compounds was last updated in the 29th ATP
             (2004), for which the proposal was discussed in 2002.
             Identity of the substance                                                                                        19
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<pre>0 Chromium VI compounds</pre>

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<pre> hapter 3
        Manufacture and uses
3.1     Manufacture
        Not relevant for classification.
3.2     Identified uses
        Main uses2-4
        •  Chromium trioxide: Used in metal finishing; for manufacturing of wood
           preservation products, catalysts, chromium dioxide and pigments
        •  Sodium chromate: Used for manufacturing of other chromium compounds
        •  Sodium dichromate: Used for manufacturing of other chromium compounds,
           wood preservative products, vitamin K and wax; as mordant in dyeing; in
           metal finishing
        •  Potassium dichromate: Used for manufacturing of pigments, wood
           preservation products, dyes, catalysts and chromium metal; as colouring
           agent in ceramics
        •  Chromic acid: Used in production of various chemicals (chromates,
           oxidizing agents, catalysts); as intermediate in chromium-plating, in ceramic
           glazes and colored glass. Ammonium chromate: Used as sensitiser for gelatin
           coatings used in photography; in textile printing pastes, and fixing chromate
           dyes on wool; as an analytical reagent, catalyst, and corrosion inhibitor
        Manufacture and uses                                                             21
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<pre>  •  Ammonium dichromate: Used as an intermediate and laboratory reagent
  •  Calcium chromate: Used as a pigment, a corrosion inhibitor; in
     electroplating, photochemical processing, and industrial waste treatment
  •  Potassium chromate: Used as a mordant in dyeing fabrics; as tanning agent in
     the leather industry, in bleach oils and waxes; as an oxidizing agent in
     organic synthesis
  •  Dichromium tris(chromate): Used as corrosion inhibitor; as catalyst in the
     mordanting of yarns.
2 Chromium VI compounds
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<pre> hapter 4
        Read-across and toxicokinetics
        (absorption, metabolism, distribution
        and elimination)
4.1     Read-across/grouping
        Absorption of chromium VI compounds has been reported after inhalation, oral
        and dermal exposure, in both humans and animals, albeit at different degrees (see
        next sections). The absorption data have all been considered relevant for humans,
        including the oral absorption data. Importantly, in a species comparison in rats,
        mice and guinea pigs, it was concluded that the presence of a forestomach did
        not influence the toxicokinetics of sodium dichromate dehydrate.5
            Toxicity data on chromium VI compounds mainly involve sodium chromate
        and potassium chromate. These data suggest that in addition to local toxicity,
        exposure can lead to systemic effects.
            A number of epidemiological studies have considered the association of
        exposure to chromium VI and systemic effects, as some evidence of kidney
        damage has been found among chromate production and chromium plating
        workers.4,6
            In rats, adverse effects on liver function and the immune/lymphatic system
        have been reported after repeated inhalation exposure. After repeated oral
        exposure, haematoxicity, hepatotoxicity, and renal toxicity was observed in rats
        and mice.4,6 In addition, reproductive toxicity has been reported for several
        species (see this report).
            Although the exact mode of action is not known, chromium VI-induced
        toxicity has been attributed to the formation of reactive intermediates.
        Read-across and toxicokinetics (absorption, metabolism, distribution and elimination) 23
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<pre>  Information available indicates that most if not all chromium compounds
  specified in this document are either corrosive or irritating to skin. Under
  physiological conditions, chromium VI can be reduced intracellularly by e.g.
  hydrogen peroxide, glutathione reductase, ascorbic acid, to produce reactive
  intermediates, including chromium V, chromium IV, hydroxyl radicals, and
  ultimately, chromium III. These reactive intermediates can affect DNA, proteins,
  and membrane lipids, thereby disrupting cellular integrity and functions.7 The
  reduction of chromium VI is therefore considered to serve as a detoxification
  process when it occurs at a distance from the target site. Reduction of chromium
  VI in or near the cell nucleus of target organs however, may be the cause of the
  toxicity that is observed.8 If chromium VI is reduced to chromium III
  extracellularly, it is not readily transported into cells thereby limiting toxicity.
       The Committee concludes that the available toxicokinetic, mechanistic and
  toxicity data suggest that the amount of systemically available chromium VI is
  responsible for the systemic effects observed after exposure to sodium chromate
  and potassium chromate. All the soluble chromium VI compounds specified in
  this report are, dependent on chromium ion concentration and pH, present as
  either chromium acid (HCrO4-), or chromate/dichromate anions (Figure 1).
  Overall, the data indicate that soluble chromium VI compounds can be present in
  different species, dependent on pH and irrespective of the form from which
  chromium VI enters solution.
   Figure 1 A predominance diagram showing the influence of concentration of chromium and pH on
   the predominance of chromium species in solution, in which pCr stands for minus the logarithm of
   the chromium concentration and pH stands for minus the logarithm of the hydrogen ion
   concentration. The lines on a predominance diagram indicate where adjacent species have the same
   concentration. As chromium VI is a strong oxidising agent, it only exists as oxygenated species in the
   environment. The actual species present in solution depends on the pH. At very low pH (e.g. near 0)
   the dominant species in solution will be the fully protonated form (H2CrO4). At pHs between 0 and
   6-6.5, the dominant chromate species in solution would be HCrO4-, and at pHs above 6-6.5 the main
   chromate species in solution would be CrO42-.4
4 Chromium VI compounds
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<pre>    Therefore, the Committee assumes that a similar toxicity profile is likely for all
    soluble chromium VI compounds, although differences in potency may exist.
    Conform REACH Annex XI section 1.5, read-across may be applied for
    substances with ‘common precursors and/or the likelihood of common
    breakdown products via physical and biological processes, which result in
    structurally similar chemicals’. Consequently, for the purpose of classification
    and labelling, the Committee applies read-across for all chromium VI
    compounds specified in this document.
    The information provided in the next sections is summarised from the ATSDR6
    and EU RAR4. Additional sources are referenced separately.
4.2 Non-human studies
    Absorption
    Oral
    Studies with 51chromium in animals indicate that chromium and its compounds
    are poorly absorbed from the gastrointestinal tract after oral exposure. The
    absorption fraction of soluble chromium VI is higher than that of soluble
    chromium III. Absorption amounts of ≤1.4% of the administered oral dose have
    been reported for chromium VI in rats. When food was given ad libitum, only
    1-3% of orally administered chromium VI was absorbed in rats and mice; the
    amount increased if food had been withdrawn for 16-48 hours. In contrast, one
    study reported at least 18% absorption in unstarved guinea pigs receiving
    potassium chromate orally. The question whether chromium VI absorption
    mainly occurs when the reducing capacity of the gastrointestinal tract is
    exhausted, has been noted as an issue to consider when evaluating and
    interpreting oral dosing bioassays.6
    Inhalation
    Following inhalation or intratracheal instillation of highly water-soluble
    chromium VI compounds, approximately 20-30% of the chromium dose has
    been reported to be rapidly absorbed into the bloodstream. Rats exposed for a
    single inhalation of chromium trioxide (3.18 mg chromiumVI/m3) for 30 minutes
    rapidly absorbed chromium from the lungs. The kinetics of three chromium VI
    compounds, sodium chromate, zinc chromate, and lead chromate, have been
    Read-across and toxicokinetics (absorption, metabolism, distribution and elimination) 25
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<pre>  compared in rats in relation to their solubility. The results indicated that zinc
  chromate, which is ~1,000 times less soluble than sodium chromate, is more
  slowly absorbed from the lungs, but peak blood levels are higher than for sodium
  chromate. Lead chromate was more poorly and slowly absorbed, as indicated by
  very low levels in blood and other tissues, and greater retention in the lungs.
  Dermal
  Dermal absorption of highly water-soluble chromium VI compounds in guinea
  pigs varied between <1% and 4% of the applied aqueous dose, depending on the
  chromium concentration. The dermal absorption of sodium chromate (chromium
  VI) by guinea pigs was somewhat higher, though not statistically significantly,
  than that of chromium III trichloride.
  Distribution
  Oral
  Several tissue distribution studies of chromium VI compounds have been
  described. In one experiment, 0-5.8 mg chromium VI/kg/day (administrated as
  sodium chromate in water by gavage) for 7 days. At low doses, chromium levels
  were comparable to controls whereas at 5.8 mg/kg/day, the largest amount of
  chromium (expressed as µg chromium/whole organ) was found in the liver
  (≈22 µg), followed by the kidney (≈7.5 µg), lung (≈4.5 µg), blood (≈2 µg), and
  spleen (≈1 µg).
      In another experiment, rats were exposed by gavage to 7 mg chromium/
  kg/day for 7 days from various sources, including sodium chromate; calcium
  chromate; soil containing chromium (30% chromium VI). The highest levels of
  chromium were found in liver, spleen, kidney, lung, blood, brain, and testes after
  dosing with sodium chromate, but the relative levels in these tissues after the
  other treatments followed no consistent pattern.
      The chromium content in major organs (heart, lung, kidney, liver, spleen,
  testes) of mice receiving drinking water that provided doses of 4.4, 5.0, or
  14.2 mg chromium VI/kg/day as potassium dichromate was determined after 1
  year of exposure. Accumulation was reported in all of the above organs, with the
  highest levels reported in the liver and spleen.
      In rats exposed to 100 and 200 mg/L potassium chromate in the drinking
  water for 3 or 6 weeks, a general trend of increasing chromium concentration
  with time of exposure was apparent in the liver and kidneys, but only the kidneys
6 Chromium VI compounds
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<pre>showed a difference in the concentration after exposure to 100 and 200 mg/L.
Blood concentrations were almost saturated by 3 weeks with little further
accumulation by 6 weeks. No chromium was detected in the lungs after drinking
water exposure.
    The distribution of potassium chromate was compared in male Fisher rats
and C57BL/6J mice exposed either by drinking water (8 mg chromium VI/
kg/day for 4 and 8 weeks). The concentrations of chromium (µg/g wet tissue)
after drinking water exposures for 8 weeks in mice were: liver 13.83, kidney
4.72, spleen 10.09, femur 12.55, lung 1.08, heart 1.02, muscle 0.60, and blood
0.42. These concentrations were not markedly different than for 4-week
exposures. For rats, the concentrations were: liver 3.59, kidney 9.49, spleen 4.38,
femur 1.78, lung 0.67, heart 1.05, muscle 0.17, and blood 0.58. These results
indicate that, although blood levels are comparable, considerable species
differences exist between mice and rats.
    A comparative absorption study of sodium dichromate dihydrate was
performed by the National Toxicity Program (NTP) in F344/N rats, B6C3F1
mice and Hartley guinea pigs. prior to its repeated dose study.5 Dose
concentrations reported were 0, 2.87, 8.62, 28.7, 86.2, 287, and 862 mg
chromium VI/L (equivalent to 0, 1, 3, 10, 30, 100, and 300 mg chromium/L,
respectively. Chromium in blood and kidney increased with exposure
concentration in all three species and although differences were seen in the
absolute amounts of chromium in kidney and blood, uptake as a function of
exposure concentration did not appear to differ qualitatively in guinea pigs when
compared to rats and mice.
    Twelve pregnant female albino rats (Druckrey strain) and 13 Swiss albino
mice were exposed to 500 mg/L potassium dichromate in their drinking water
(calculated to correspond to 11.9 and 3.6 mg chromium VI/day, respectively)
during pregnancy up to 1 day before delivery. In untreated rats, concentrations of
chromium were 0.067, 0.219, and 0.142 µg/g fresh weight in maternal blood,
placenta, and foetuses, respectively, and 0.064, 0.304, and 0.366 µg/g fresh
weight in mice, respectively. In treated rats, chromium levels were 3.2-fold
higher in maternal blood, 3-fold higher in placenta, and 3.1-fold higher in foetal
tissue when compared to control values. In treated mice, chromium levels were
2.5-fold higher in maternal blood, 3.2-fold higher in placenta, and 9.6-fold higher
in foetuses when compared to control values. In treated mice, there was a
significant elevation in chromium levels in placental and foetal tissues over
maternal blood levels, and a significant increase in chromium levels in foetal
tissue over placental concentrations when compared to controls.
Read-across and toxicokinetics (absorption, metabolism, distribution and elimination) 27
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<pre>  Inhalation
  Steady-state concentrations in the blood have been reported after ~4 days in rats
  exposed to 2.1 mg chromium VI/m3 as zinc chromate for 6 hours/day.
       Rats exposed for a single inhalation of chromium trioxide mist from
  electroplating (18 mg chromium VI/m3) for 30 minutes rapidly absorbed
  chromium from the lungs. The content of chromium in the lungs declined from
  13.0 mg immediately after exposure to 1.1 mg at 4 weeks.
  Dermal
  Chromium compounds are absorbed after dermal administration by guinea pigs.
  Measurement of 51chromium in the organs and body fluids revealed distribution,
  due to dermal absorption of chromium VI compounds, to the blood, spleen, bone
  marrow, lymph glands, urine, and kidneys. Absorption was greater for chromium
  VI than for chromium III.
  Metabolism
  Chromium VI is unstable inside the body and is ultimately reduced to chromium
  III, which is essential to physiological processes, including glucose, protein, and
  fat metabolism and intracellular reduction and oxidation reactions.
       In vivo and in vitro experiments in rats indicated that, in the lungs, chromium
  VI can be reduced to chromium III by ascorbate. When ascorbate is depleted
  from the lungs, chromium VI can also be reduced by glutathione. The reduction
  of chromium VI by glutathione is slower and results in greater residence time of
  chromium in the lungs, compared to reduction by ascorbate. Because chromium
  III does not readily enter cells, these data suggest that reduction of chromium VI
  by the epithelial lining fluid may constitute the first line of defense against
  toxicity of inhaled chromium compounds. Furthermore, uptake and reduction of
  chromium compounds by the pulmonary alveolar macrophages may constitute a
  second line of defense against pulmonary toxicity of chromium VI compounds.
       The first defense against chromium VI after oral exposure is the reduction of
  chromium VI to chromium III in the gastric environment where gastric juice and
  ascorbate play important roles.
       In addition to the reduction of chromium VI by ascorbate or glutathione, in
  vitro studies have demonstrated reduction of chromium VI by microsomal
  enzymes. Microsomal reduction of chromium VI occurs in the lungs mainly as it
  does in the liver, involving cytochrome P450, NADPH-dependent-cytochrome
8 Chromium VI compounds
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<pre>P450 reductase, and to a lesser extent cytochrome b5 and NADH-dependent-
cytochrome b5 reductase.
Elimination
Inhalation
It is generally believed that that absorbed chromium VI compounds may be
excreted more slowly than absorbed chromium III compounds due to a higher
ability to enter cells. Inhaled or intratracheal instilled chromium VI is excreted in
urine and faeces in similar amounts (in the range 20-70% of administered dose).
Peak urinary chromium concentrations were observed at 6 hours (the first time
point examined) in rats exposed intratracheally to chromium VI as sodium
dichromate. Chromium urinary concentrations decreased rapidly, falling from
2,947 µg chromium/g creatinine at 6 hours to 339 µg chromium/g at 72 hours.
     Elimination of chromium was very slow in rats exposed to 2.1 mg chromium
VI/m3 as zinc chromate 6 hours/day for 4 days. Urinary levels of chromium
remained almost constant for 4 days after exposure and then decreased, from
which it has been concluded that chromium bound inside the erythrocyte is
released slowly.
Oral
Given the low absorption of chromium compounds by the oral route, the major
pathway of excretion after oral exposure is through the faeces. Chromium in
urine and faeces is in the form of chromium III complexes, with glutathione for
example.
     Rats given 18 mg chromium VI/kg as potassium dichromate by gavage
excreted about 25 µg chromium in the first 24 hours after dosing and appr. 10 µg
chromium in each of the next 24-hour periods.
     In rats and hamsters fed chromium compounds, faecal excretion of chromium
varied slightly from 97 to 99% of the administered dose. Urinary excretion of
chromium varied from 0.6 to 1.4% of the dose administered as either chromium
VI or chromium III compounds.
Read-across and toxicokinetics (absorption, metabolism, distribution and elimination) 29
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<pre>    Dermal
    51Chromium     was detected in the urine of guinea pigs after a radiolabeled sodium
    chromate solution was placed over skin depots that were monitored by
    scintillation counting to determine the dermal absorption.
4.3 Human studies
    Absorption
    Oral
    Gastrointestinal absorption of chromium in humans is estimated to be <10% of
    the ingested dose, with the absorption fraction of soluble chromium compounds
    being higher than that of insoluble forms. The absorption of chromium VI was
    measured in four male and two female volunteers (ages ranging from 25 to 39
    years) treated orally with potassium chromate (chromium VI) in capsules at
    doses of 0.005 mg/kg/day. Subjects were exposed for 3 days. Based on urinary
    excretion data, the mean absorption of potassium chromate was 3.4% (range
    0.69-11.9%).
         In a follow-up study by the same group, five male volunteers ingested in total
    a litre of deionised water containing chromium VI concentrations ranging from
    0.1 to 10.0 mg/L (approximately 0.001-0.1 mg chromium VI/kg/day) for 3 days.
    A dose-related increase in chromium levels was seen in urine, plasma, and
    erythrocytes. The percentage of the dose excreted in urine ranged from <2 to 8%.
         In a repeated dose study, three healthy adults ingested chromium VI (as
    K2Cr2O7) in water at 5 mg chromium/day for 3 consecutive days. Three divided
    doses were taken at approximately 6-hour intervals over a 5-15-minute period.
    After at least 2 days without dosing, the 3-day exposure regimen was repeated at
    10 mg chromium/day. Estimated doses based on body weight were 0.05 and 0.1
    mg/kg/day, respectively. Bioavailability based on 4-day urinary excretion was
    1.7% (range 0.5-2.7%) at 0.05 mg chromium VI/kg/day and 3.4% (range 0.8-
    8.0%) at 0.1 mg chromium VI/kg/day. Absorption of 0.05 mg chromium VI/kg
    appeared to be somewhat lower when given as three divided doses rather than
    when given as a single bolus dose (1.7 versus 5.7%).
         Uptake of potassium dichromate was determined in a man who was given 0.8
    mg of chromium VI in drinking water 5 times each day for 17 days. Steady-state
    concentrations of chromium in blood were attained after 7 days. Red blood cell
    and plasma levels returned to background levels within a few days after exposure
 0  Chromium VI compounds
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<pre>was stopped. The data are consistent with a bioavailability of 2% and a plasma
elimination half-life of 36 hours.
    The average absorption fractions that were determined from cumulative
urinary excretion in 8 healthy adults, who ingested 5 mg chromium (in 10 mg
chromium/L drinking water as K2Cr2O7), amounted to 6.9% (± 3.7).
    Experimental studies in humans did not find evidence for an effect of
conditions that limit the reduction of chromium on the absorption of chromium.
The range of doses of chromium administered to humans in these different
studies was considerable and demonstrated oral bioavailability at all doses.
Inhalation
The absorption of inhaled chromium compounds depends on a number of factors,
including physical and chemical properties of the particles (oxidation state, size,
solubility) and the activity of alveolar macrophages. The identification of
chromium in urine, serum and tissues of humans occupationally exposed to
soluble chromium VI compounds in air indicates that chromium can be absorbed
from the lungs. In most cases, chromium VI compounds were found to be more
readily absorbed from the lungs than chromium III compounds, in part due to
differences in the capacity to penetrate biological membranes.
Dermal
Chromium VI can penetrate human skin to some extent, especially if the skin is
damaged. Fourteen days after a salve containing potassium chromate was applied
to the skin of an individual to treat scabies, appreciable amounts of chromium
were found in the blood, urine, feces, and stomach contents. Potassium
dichromate has also been reported to penetrate the excised intact epidermis of
humans.
    An average rate of systemic uptake of chromium in four volunteers
submersed up to the shoulders in a tub of chlorinated water containing a 22 mg
chromium VI/L solution of potassium dichromate for 3 hours was measured to
be 3.3x10-5-4.1x10-4 µg Cr/cm2-hour (based on urinary excretion of total
chromium).
    The influence of solvent on the cutaneous penetration of potassium
dichromate by humans has been studied. Results with dichromate in water and in
petrolatum revealed that about 10 times more chromium penetrated when
potassium dichromate was applied in petrolatum than when applied in water.
Read-across and toxicokinetics (absorption, metabolism, distribution and elimination) 31
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<pre>  About 5 times more chromium penetrated when potassium dichromate was
  applied than when a chromium trichloride glycine complex was applied.
  Distribution
  Oral
  The distribution of chromium in human body tissue after acute oral exposure was
  determined in the case of a 14-year-old boy who ingested 7.5 mg chromium VI/
  kg as potassium dichromate. Despite extensive treatment by dialysis and
  chelation, the boy died 8 days after admission to the hospital. Upon autopsy, the
  chromium concentrations were as follows: liver, 2.94 mg/100 cc (normal, 0.016
  mg/100 cc); kidneys, 0.64 and 0.82 mg/100 cc (normal, 0.06 mg/100 cc); and
  brain, 0.06 mg/100 cc (normal, 0.002 mg/100 cc).
  Inhalation
  Examination of tissues from Japanese chrome platers and chromate refining
  workers at autopsy revealed higher chromium levels in the hilar lymph node,
  lung, spleen, liver, kidney, and heart, compared to normal healthy males.
      Chromium concentrations have been measured in organs and tissues at
  autopsy of a man who died of lung cancer, 10 years after his retirement from
  working in a chromate producing plant for 30 years and exposed mainly to
  chromium VI. This analysis revealed measurable levels of chromium in the
  brain, pharyngeal wall, lung, liver, aorta, kidney, abdominal rectal muscle,
  suprarenal gland, sternal bone marrow, and abdominal skin. These levels were
  higher than in five controls without occupational exposure to chromium.
      The levels of chromium were higher in the lungs, but not in the liver or
  kidneys, of autopsy specimens from 21 smeltery and refinery workers in North
  Sweden compared with that for a control group of 8 individuals.
      In tissues from three individuals having lung cancer who were industrially
  exposed to chromium, cumulative chromium exposures amounted to 3.45, 4.59,
  and 11.38 mg/m3 years. All tissues from the three workers had elevated levels of
  chromium with the possible exception of neural tissues. Levels were orders of
  magnitude higher in lungs than in other tissues. The highest lung level reported
  was 456 mg/10 g tissue in the first worker, 178 in the second worker, and 1,920
  for the third worker. Significant chromium levels were found in the tissue of the
  second worker even though he had not been exposed to chromium for 18 years.
2 Chromium VI compounds
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<pre>Dermal
The findings of toxic effects in the heart, stomach, blood, muscles, and kidneys
of humans who were dermally exposed to chromium compounds are suggestive
of distribution to these organs. Fourteen days after a salve containing potassium
chromate was applied to the skin of an individual to treat scabies, appreciable
amounts of chromium were found in the blood (2-5 mg/100 mL), urine
(8 mg/L), feces (0.61 mg/100 g), and stomach contents (0.63 mg/100 mL).
    A transient increase in the levels of total chromium in erythrocytes and
plasma was observed in subjects immersed in a tank of chlorinated water
containing potassium dichromate.
Metabolism
No data are available.
Elimination
Oral
Given the low absorption of chromium compounds by the oral route, the major
pathway of excretion after oral exposure is through the faeces. Urinary excretion
of inorganic chromium VI compounds has been reported to be higher than that of
inorganic chromium III compounds, indicating a higher absorption.
    An amount of 89.4% of an acute, oral dose of chromium VI as sodium
chromate, was retrieved in a 6-day fecal collection, whereas 2.1% of the dose
was found in a 24-hour urine collection. In subjects drinking increasing doses of
chromium (0.001-0.1 mg chromium VI/kg/day as potassium chromate in water
for 3 days), increasing doses ranging from < 2 to 8% of the dose were excreted in
the urine.
    After ingestion of 0.05 mg chromium VI/kg, approximately 76-82% of the
14-day total amount of chromium in the urine was excreted within the first 4
days (mean peak concentration 209 µg chromium/g creatinine; range 29-585 µg
chromium/g creatinine). The average urinary excretion half-life for four of the
volunteers was 39 hours at this dose. All subjects had returned to background
concentrations (0.5-2.0 µg chromium/g creatinine) by 14 days post-dosing.
    Uptake of potassium dichromate was determined in a man who was given 0.8
mg of chromium VI in drinking water 5 times each day for 17 days. Steady-state
Read-across and toxicokinetics (absorption, metabolism, distribution and elimination) 33
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<pre>    concentrations of chromium in blood were attained after 7 days and a plasma
    elimination half-life of 36 hours was estimated.
    Inhalation
    In workers exposed to chromium VI as chromium trioxide in the chrome plating
    industry, an association was observed between exposure concentrations and post-
    shift urinary chromium levels. Workers mainly exposed to chromium VI
    compounds have been reported to have higher urinary chromium levels than
    workers primarily exposed to chromium III compounds.
    Dermal
    Fourteen days after application of a salve containing potassium chromate VI,
    which resulted in skin necrosis and sloughing at the application site, chromium
    was found at 8 mg/L in the urine and 0.61 mg/100 g in the faeces of one
    individual.
        A slight increase (over background levels) in urinary chromium levels was
    observed in four subjects submersed in a tub of chlorinated water containing 22
    mg chromium VI/L as potassium dichromate for 3 hours. For three of the four
    subjects, the increase in urinary chromium excretion was <1 µg/day over the
    5-day collection period.
4.4 Summary and discussion on toxicokinetics
    There is a reasonably good database available on the toxicokinetics of the
    chromium VI compounds, although there are relatively few human data. For all
    routes, it is generally assumed that chromium VI is better absorbed than
    chromium III. It is further assumed that absorption is higher for soluble
    chromium compounds than for insoluble.
        Chromium VI is poorly absorbed after oral administration, with absorption
    fractions reported from 1-3% (in rats and mice) to 18% in guinea pigs. For
    humans, gastrointestinal absorption is estimated to be <10% of the administered
    dose. Amounts of 20-30% absorption have been reported in animals after
    inhalation or intratracheal instillation. Systemic exposure after inhalation
    exposure has been reported in humans, but no quantitative studies are available.
    Dermal absorption of highly water-soluble chromium VI compounds varied
    between 1-4% in guinea pigs. Chromium VI can penetrate human skin to some
    extent, especially if the skin is damaged.
 4  Chromium VI compounds
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<pre>    Results of animal testing indicate that chromium can remain in the lungs for
several weeks after inhalation exposure and also becomes bound to haemoglobin
in erythrocytes for the lifespan of the cells. In the body, chromium VI is reduced
to chromium III, for example by glutathione. The observed distribution appears
widespread, even after a single dose, and includes transfer of across the placenta
and accumulation in foetal tissue.
    Ingested chromium is excreted primarily in the faeces, whereas absorbed
chromium appears to be primarily excreted in the urine. Repeated exposure leads
to accumulation of chromium in several tissues, particularly the spleen because
of uptake of senescent erythrocytes.
    The available data indicate that generally the chromium VI compounds are
likely to behave in a similar manner in respect of toxicokinetics, and that the
kinetic behaviour of these substances would be similar in those species studied,
including humans.
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<pre>6 Chromium VI compounds</pre>

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<pre>  hapter        5
                Toxicity for reproduction
5.1             Effects on fertility
5.1.1           Non-human information
                Animal fertility studies are summarised in the next table and described in the
                following text. Changes and differences mentioned are statistically significant
                unless stated otherwise.
 able 1 Summary table of fertility studies in animals.
 uthors          species        experimental        dose/route                general toxicity         effects on reproduction
                                period/design
  laser et al.,   Rat, males     130 days per        0 or 200 µg Cr/m3 as No clinical signs; no effect  No effect on reproduction
 984              and female, generation for 3 sodium dichromate (0 on food- and water                  NOAEL= 0.3 mg Cr/kg bw/d
                  Wistar; n =    generations         or 0.2 mg Cr/kg bw/d consumption
                  8-11           (time/d not         (assuming a breathing NOAEL=0.3 mg Cr/kg
                                 indicated)          volume of 200 ml/         bw/d
                                                     min))/inhalation
 ubramanian et Monkey;           During 6            0, 50, 100, 200 or 400 Not reported                6 mg/kg bw and higher: decreased sperm
 l., 2006         Macaca         months; highest mg Cr as potassium            NOAEL=23 mg Cr/kg        count, decreased sperm forward motility,
                  radiata; n =   dose 6 m-           dichromate/L drinking bw/d                         decreased activity of superoxide
                  not specified; recovery;           water (0, 3, 6, 11 or 23                           dismutase and catalase, and decreased
                  males; 7-9 kg monthly semen mg Cr/kg bw/d)/oral                                       concentration of reduced glutathione in
                                 sampling                                                               both seminal plasma and sperm
                                                                                                        NOAEL=3 mg Cr/kg bw/d
  ruldhas         Monkey;        During 6            0, 100, 200 or 400 mg Not reported                 All doses: decreased testis weight;
 t al., 2005      Macaca         months; 3/dose      Cr as potassium           NOAEL= 23 mg Cr/kg       disrupted spermatogenesis leading to
                  radiata; n =   6 m-recovery;       dichromate/L drinking bw/d                         accumulation of prematurely released
                  3/dose;        ultrastructrural    water (0, 6, 11 or 23                              spermatocytes, spermatids and uni- and
                  males; 7-8 kg  and biochemical     mg Cr/kg bw/d)/oral                                multinucleate giant cells in the lumen of
                                 analysis of testis                                                     seminiferous tubules
                                                                                                        LOAEL=6 mg Cr/kg bw/d
                Toxicity for reproduction                                                                                                      37
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<pre> i et al., 2001  Rat; Wistar;   For 6 days;        0, 10 or 20 mg         Not reported                 Both levels: decreased sperm count
                 males;         sacrificed after 6 chromium trioxide/kg   NOAEL=10 mg Cr/kg            increased sperm abnormality; decreased
                 n = 8-11       weeks              bw/d by gavage (0, 5   bw/d                         diameter of seminiferous tubules and germ
                                                   or 10 mg Cr/kg bw/d)/                               cell rearrangement within tubules
                                                   oral                                                LOAEL=5 mg Cr/kg bw/d
 anojia et al.,  Rat; Swiss     During 20 days 0, 250, 500 or 750 mg      No mortality or change in    All doses: reduced mating index and
 996             albino;        (one               Cr/L as potassium      clinical signs; decreased    fertility index; persistent diestrus phase
                 females;       folliculogenesis dichromate in            water consumption;           At the two highest doses: increase in
                 n = 10         cycle) prior to    drinking water (0, 31, decreased body weight gain   estrus cycle, decrease in the number of
                                gestation          59 and 76 mg/kg        in all doses                 corpora lutea and implantations
                                                   bw/d)/oral             LOAEL=31 mg Cr/kg bw/d       LOAEL=31 mg Cr/kg bw/d
 anojia et al.,  Rat;           during 3 months 0, 250, 500 or 750 mg     89 and 124 mg/kg bw: 15%     At the end of treatment, all exposed
 998             Druckrey;      prior to           Cr/L as potassium      and 10% of animals, resp.,   animals showed persistent dioestrous
                 females;       gestation          dichromate in          died within 14 days during   phase; oestrous cycle became regular
                 n = 10                            drinking water (0, 45, treatment                    again within 15-20 days and they began to
                                                   89 and 124 mg/kg       NOAEL=45 mg Cr/kg            mate
                                                   bw/d); drinking water  bw/d                         LOAEL=45 mg Cr/kg bw/d
 howdhury and Rat; Charles during 90 days rats 0, 20, 40 or 60            16 and 24 mg: decreased      All doses: decreased testicular protein and
Mitra, 1995      Foster; males;                    mg/kg bw/d of sodium   body weight gain             serum testosterone, decreased activity of
                 n = 10                            dichromate by gavage   NOAEL=8 mg Cr/kg bw/d        3β-∆5-hydroxysteroid dehydrogenase
                                                   (0, 8, 16 or 24 mg                                  16 and 24 mg/kg: decreased testis weight,
                                                   Cr/kg bw/d)/oral                                    number of resting spermatocytes (high
                                                                                                       dose), number of pachytene spermatocytes
                                                                                                       and stage-7 spermatids, population of
                                                                                                       Leydig cells, seminiferous tubular
                                                                                                       diameter, and DNA and RNA and succinic
                                                                                                       dehydrogenase; increased testicular
                                                                                                       cholesterol
                                                                                                       Testicular ascorbic acid: increased at 8 and
                                                                                                       16 mg/kg and decreased at 24 mg/kg
                                                                                                       LOAEL=8 mg Cr/kg bw/d
 TP, 1996a       Rat; Sprague-  Daily for 9        0, 15, 50, 100, and    Mean corpuscular volume      No effect observed on testicular cell
                 Dawley;        weeks; 8 weeks     400 mg potassium       and haemoglobin values in    counts for Sertoli nuclei and preleptotene
                 n = 24 males   recovery;          dichromate/kg diet (0, high-dose group were         spermatocyte in Stage X and XI, or with
                 and 48         necropsy after 3,  0.4, 1, 2 and 9 mg Cr/ decreased for both males     microscopy of ovaries
                 females        6, 9 and 17        kg bw/d)(mean doses    and females (reversible), no NOAEL=9 mg Cr/kg bw/d
                                weeks              of males and females   other treatment related
                                                   combined)/oral         findings were observed
                                                                          NOAEL=2 mg Cr/kg bw/d
 TP, 1996b       Mouse,         Daily for 9        0, 15, 50, 100, and    Decrease in mean             Testicular cell counts for Sertoli nuclei
                 BALB/c,        weeks; 8 weeks     400 mg potassium       corpuscular volume (MCV)     and preleptotene spermatocyte counts in
                 n = 24 males   recovery;          dichromate/kg diet (0, and mean corpuscular         Stage X and XI did not reveal any
                 and 48         necropsy after 3,  1, 5, 10 and 41 mg Cr/ hemoglobin (MCH) in          differences compared to controls
                 females        6, 9 and 17        kg bw/d) (mean doses   males and females in the     NOAEL=41 mg Cr/kg bw/d
                                weeks              of males and females   highest dose group.
                                                   combined)/oral         Cytoplasmic vacuolisation
                                                                          in hepatocytes was noted in
                                                                          the 5, 10, and 41 mg/kg
                                                                          bw/d in males and females.
                                                                          High dose group: loss of
                                                                          body weight (10%) for
                                                                          females, decrease of
                                                                          absolute liver weight for
                                                                          males and females (10%)
                                                                          NOAEL=1 mg Cr/kg bw/d
 8              Chromium VI compounds
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<pre> TP, 1997        Mouse,        F0: 1 week prior  0, 100, 200, and 400    Slight decrease in body   F0: no effect on pregnancy index and
                 BALB/c,       to mating and 12  mg/potassium/kg diet    weights, increased feed   number of litters per pair; no effect on
                 n = 10-20/sex week mating       dichromate (F0: 0, 7,   consumption, and small    epididymal sperm density, percent
                               period            14 and 30 mg Cr/kg      decreases in MCV, MCH,    abnormal sperm and testicular sperm
                               (continuous       bw/d; F1: 0, 8, 16 and  and haemoglobin (F1       count; no effect on gonadal organ weights
                               breeding phase);  37 mg Cr/kg bw/d)/      animals only).            NOAEL F0=30 mg Cr/kg bw/d
                               F1: after         oral                    NOAEL F0=30 mg Cr/kg      F1: no effect on mating index, pregnancy
                               weaning until 74                          bw/d                      index, fertility index or gestation length,
                               +/- 10 days of                            LOAEL F1=8 mg Cr/kg       oestrous cycle and sperm parameters
                               age continued                             bw/d                      NOAEL F1=37 mg Cr/kg bw/d
                               with 1 week
                               mating period
 TP, 2007        Mouse;        For 3 m;          0, 62.5, 125, or 250    Decreased body weight in: No effects on sperm parameters reported.
                 B6C3F1,       sacrificed 2w     mg sodium               -5 and 9 mg/kg bw/d       NOAEL= 9 mg/kg bw/d
                 BALB/c, and later               dichromate dihydrate/   (B6C3F1 and BALB/C)
                 am3-C57BL/                      L in drinking water (3, -all treatment groups
                 6 males;                        5 or 9 mg Cr/kg bw, as  (C5BL/6); conc. dep.
                 n = 5-10                        reported by authors)/   decrease in mean red cell
                                                 oral                    volume and haemoglobin
                                                                         (all strains);
                                                                         Increase in erythrocyte
                                                                         counts (B6C3F1 and
                                                                         BALB/C)
                                                                         LOAEL=3 mg/kg bw/d
 rivedi et al.,  Mouse,        entire gestation 0, 250, 500 or 1000      Reduction in body weight  Pre-implantation loss at 120 and 234
 989             females,      period, sacrifice mg potassium            gain                      mg/kg
                 Swiss;        GD 19             dichromate/L in         NOAEL=59 mg Cr/kg         NOAEL=59 mg Cr/kg bw/d
                 n = 10-13                       drinking water (0, 59, bw/d
                                                 120 and 234 mg Cr/kg
                                                 bw/d)
 ahid et al.,    Mouse;        from weaning      0, 100, 200 or 400 mg Body weight gain and food   All doses: slight, but significantly
 990             males, Balb- during 7 weeks; potassium                  consumption not affected  increased number of seminiferous tubules
                 C Swiss, n =7 testis and        dichromate/kg diet      NOAEL=46 mg Cr/kg         degenerated; increased number of intact
                               epididymis        (0, 11, 21 or 46 mg     bw/d                      tubules without spermatogonia; decreased
                                                 Cr/kg bw/d)/oral                                  number of spermatogonia/tubules and
                                                                                                   increased number of resting and pachytene
                                                                                                   spermatocytes
                                                                                                   21 and 46 mg Cr/kg bw/d: decreased
                                                                                                   sperm count and increased percentage of
                                                                                                   abnormal sperms
                                                                                                   LOAEL=11 mg Cr/kg bw/d
Murthy et al.,   Mouse,        During 20 days;   0, 250, 500 or 750 mg Not reported                Decrease in number of follicles at
 996             Swiss,        subgroups:        Cr/L as potassium       NOAEL=163 mg Cr/kg        different stages of maturation at all doses;
                 females, n =  1. follicle       dichromate in           bw/d                      109 and 163 mg/kg: decreased number of
                 10/subgroup   counting;         drinking water (0, 54,                            ova; proliferated, dilated, and congested
                               2. ova counting;  109 or 163 mg Cr/kg                               blood vessels, pyknotic nuclei in follicular
                               3. measuring      bw/d)/oral                                        cells, and atretic follicles; 163 mg/kg:
                               estrous cycle                                                       increased length of oestrous cycle
                                                                                                   LOAEL=54 mg Cr/kg bw/d
Murthy et al.,   Mouse,        During 90 days;   0, 0.05, 0.5 or 5 mg    Not reported              1 mg/kg: disintegrated cell membranes of
 996             Swiss,        electron          Cr/L as potassium       NOAEL=1 mg Cr/kg          two layered follicular cells and altered
                 females, n =  microscopic       dichromate in           bw/d                      villiform mitochondria in thecal cells
                 10/subgroup   study of ovaries  drinking water                                    NOAEL=0.1 mg Cr/kg bw/d
                                                 (0, 0.01, 0.1 or 1
                                                 mg/kg bw/d)/oral
                Toxicity for reproduction                                                                                                   39
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<pre> lbetieha and   Mouse,        12 weeks prior     0, 1000, 2000, 4000 or  148 and 370 mg: decreased    148 mgc: increased relative testis weight;
 l-Hamood,      males, Swiss; to mating; body    5000 mg potassium       body weight;                 reduced number of implantations and
 997            n = 9-20      weight and rel.    dichromate/L drinking   NOAEL=74 mg Cr/kg            viable foetuses
                              organ weight       water (0, 74, 148, 296, bw/d                         296 mg/kg: decreased number of
                              only studied at    370 mg Cr/kg bw/d)/                                  implantations and viable foetuses
                              2000 and 5000      oral                                                 370 mg/kg: increased relative testis
                              mg/L                                                                    weight; decreased weight of seminal
                                                                                                      vesicles and preputial gland; increased
                                                                                                      total number of resorptions and dead
                                                                                                      foetuses
                                                                                                      NOAEL=74 mg Cr/kg bw/d
                Mouse,        12 weeks prior     0, 2000 or 5000 mg      NOAEL=383 mg Cr/kg           383 mg/kg: increased ovary weight; in
                females,      to mating          potassium               bw/d                         both groups no effect on fertility or on
                Swiss,                           dichromate/L drinking                                uterine weight; decreased number of
                n = 11-18                        water (0, 153 or 383                                 implantations and live foetuses
                                                 mg Cr/kg bw/d)                                       NOAEL=153 mg Cr/kg bw/d
 ousef et al.,  Rabbit; New Daily during 10 0 or 4 mg Cr/kg bw/d         No adverse clinical signs;   Decrease in relative weight of testes and
 006            Zealand       w; semen           as potassium            no effect on food            epididymis and plasma testosterone
                White; males; collected          dichromate by           consumption; decrease        concentration; decrease in various sperm
                n=6           weekly; blood gavage/oral                  body weight (p<0.05);        parameters, initial fructose and libido (by
                              every other                                NOAEL=4 mg Cr/kg bw/d        decreasing reaction time); increase in
                              week; sacrificed                                                        percentage of dead sperm and initial pH;
                              after 10 w                                                              no effect on semen ejaculate volume
                                                                                                      LOAEL=4 mg Cr/kg bw/d
 rnst, 1990     Rat, males,   5 consecutive      0, 1, 2 or 4 mg Cr/kg   Decreased body weight        1, 2 and 4 mg/kg: no effect 7 days after
                Wistar, n = 8 days, sacrifice at bw/d as sodium          gain at all doses; no        last administration
                              7 or 60 days       chromate/ip             mortality or clinical signs; 60 d: dose dependent reduction in relative
                              after the last                             no effect on food and water  testis weight; increased number of
                              dose                                       intake                       atrophic seminiferous tubules with loss of
                                                                         LOAEL=1 mg Cr/kg bw/d        spermiogenic epithelium; decreased
                                                                                                      number of epididymal spermatozoa;
                                                                                                      additionally at 4 mg/kg: testis, loss of
                                                                                                      cellular organisation; complete
                                                                                                      degeneration of seminiferous tubules and
                                                                                                      atrophy of Leydig cells
                                                                                                      LOAEL=1 mg Cr/kg bw/d
 rnst and       Rat, Wistar, 5 d/w for 8 w;      0 or 0.5 mg Cr/kg       No clinical signs; no effect Decreased sperm motility and serum
 onde, 1992     males, n = 10 recovery group     bw/d as sodium          on body weight, food- and    testosterone (T), increase in follicle
                              8w                 chromate/ip             water consumption            stimulating hormone (FSH) and
                                                                         NOAEL=0.5 mg Cr/kg           luteinizing hormone (LH); after recovery
                                                                         bw/d                         period sperm motility, T and FSH returned
                                                                                                      to normal levels, LH level remained
                                                                                                      decreased
                                                                                                      LOAEL=0.5 mg/kg bw/d
 ehari et al.,  Rabbit, male, daily injection    0 or 2 mg/kg bw/d       No mortality or morbidity    0.7 mg/kg (3w): testis, decreased level of
 978            ITRC colony, for 3 or 6 w,       potassium dichromate NOAEL=0.7 mg Cr/kg              in succinic dehydrogenase and mild
                n = 8-10      sacrifice 72 h     (0 or 0.7 mg Cr/kg      bw/d                         oedema of interstitial tissue
                              after last         bw/d)/ ip                                            0.7 mg/kg (6w): testis, decreased level of
                              injection                                                               succinic dehydrogenase and adenosine
                                                                                                      triphosphate, marked oedema of
                                                                                                      interstitial tissue and no spermatocytes in
                                                                                                      seminiferous tubules
                                                                                                      LOAEL=0.7 mg Cr/kg bw/d
Abbrevations used: GD = gestation day; iv = intravenous; ip = intraperitoneal; PND = post-natal day; P1/2/3 = first/second/third
 arental generation. If applicable, doses were converted to amount of chromium based on molecular weight. Conversion of
 oses to mg/kg bw/d, if applicable, was performed using the general conversion factor for body weight to food or water
 onsumption (assuming dry diet), and the default values for body weights (if not specified by the authors) as specified in the
 echnical Guidance on Risk Assessment, Part 1, Appendix 6. The Committee notes that this conversion does not specifically
ake into account pregnant animals.
 0             Chromium VI compounds
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<pre>Inhalation
Glaser et al. studied the effects of continuous inhalation of 200 µg Cr/m3 as
sodium dichromate on the reproduction and development of offspring in 3
generations of Wistar rats (n = 8-11).9 Pregnant F0 females were exposed directly
after giving birth together with their offspring till day 25 and then sacrificed.
Their offspring was further continuously exposed. No effects on reproductive
parameters were observed, including number of pregnancies, implantations and
foetuses. From generation to generation, a decrease in serum-immunoglobulin
level and increased relative lung weight was noted.
    The Committee notes that the exposure concentrations of 200 µg chromium
VI/m3 applied in this study correspond to an equivalent oral dose of 0.3 mg Cr/kg
bw, respectively (assuming a breathing volume of 200 mL/min).
Oral
Subramanian et al. collected monthly semen samples from adult monkeys
(Macaca radiata; number not specified) exposed to 0, 50, 100, 200 or 400 mg
chromium VI/L (equivalent to 0, 3, 6, 11 or 23 mg Cr/kg bw/d) as potassium
dichromate for 6 months via drinking water.10 No effects were observed at 50
mg/L. A reduction of sperm concentration and motility was observed at 100, 200
and 400 mg/L. Activities of superoxide dismutase and catalase, and glutathione
concentration were decreased and hydrogen peroxide concentration increased in
seminal plasma and sperm. All the effects were dependent on dose and duration:
the higher the dose the earlier the effect was observed and the increase/decrease
was larger with time exposed. All observed effects were restored with 3-6
months of a chromium-free exposure period. Vitamin C co-administration of 0.5,
1 or 2 g/L prevented the above effects.
In an earlier histological investigation, Aruldhas et al. had shown that exposure
of Macaca monkeys (n = 3/group) to 100, 200 or 400 mg chromiumVI/L
(equivalent to 0, 6, 11 or 23 mg Cr/kg bw/d) for 6 months via the drinking water
resulted in decreased testis weight, disrupted spermatogenesis, leading to
accumulation of prematurely released spermatocytes, spermatids and uni- and
multinucleate giant cells in the lumen of seminiferous tubules.11 Withdrawal of
chromium treatment for 6 months normalized plasma chromium concentrations,
spermatogenesis and the status of pro- and antioxidants in the testis.
Toxicity for reproduction                                                         41
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<pre>              Li et al.12 treated male Wistar rats (n = 8-11/group) with 0, 10 or 20 mg
              chromium trioxide/kg ‘by oral feeding’ (it is not clear whether this dose was
              administered by gavage or via the diet; equivalent to 5 or 10 mg Cr/kg bw/d by
              gavage) for 6 days. Rats were sacrificed after 6 weeks. Sperm count was
              decreased and sperm abnormality increased in both exposed groups.
              Histopathological evaluation of the testes showed a decreased diameter of the
              seminiferous tubules and germ cell rearrangement within the tubules.
              Table 2 Summary of sperm analysis data of Li et al., 2001.
                                                       Control              5 mg/kg bw/d       10 mg/kg bw/d
              Sperm count × 106/g epididymis           87.40 ± 3.85         21.4 ± 1.2*        17.48 ± 1.04*
              % abnormal sperm                         2.75 ± 0.06          6.68 ± 0.32**      7.6 ± 0.15**
              * p<0.05; ** p<0.01
              Kanojia et al. exposed Swiss albino rats (n = 10/group) with levels of 0, 250, 500
              or 750 mg CrVI/L in the drinking water (equivalent to 31, 59 or 76 mg Cr/kg
              bw/d) for 20 days prior to gestation. Dams were sacrificed on gestation day 19.
              Kanojia et al. also investigated these exposure levels in drinking water in female
              Druckrey rats (corresponding to 45, 89 or 124 mg Cr/kg bw/d), for 3 months
              prior to gestation.13,14 A reduced body weight gain was noted for both strains. All
              CrVI rats from both strains showed a reduced mating index, a reduced fertility
              index, an increased oestrous cycle length and a reduction in the number of
              corpora lutea and number of implantations. The 3-month exposure of Druckrey
 able 3 Summary of fertility effects reported by Kanojia et al., 1996, 1998.
 wiss albino rats (Kanojia et al. 1996)            Control             31 mg/kg bw/d      59 mg/kg bw/d     76 mg/kg bw/d
Maternal weight gain (g)                           70.50 ± 5.19        65.02 ± 3.17a      60.92 ± 2.13ab    55.5 ± 3.01abc
Mating index (%)                                   100                 80                 70                40
 ertility index (%)                                96                  75                 57                31
Number of corpora lutea                            10.02 ± 0.91        9.81 ± 0.95        7.13 ± 0.61ab     4.43 ± 0.50abc
Number of implantations                            9.51 ± 0.96         9.61 ± 0.83        5.91 ± 0.39ab     2.27 ± 0.36 abc
 re-implantation loss                              5.08 ± 0.65         2.03 ± 0.31        17.11 ± 2.13ab    48.75 ± 5.81abc
Druckrey rats (Kanojia et al. 1998)                Control             54 mg/kg bw/d      89 mg/kg bw/d     124 mg/kg bw/d
Maternal weight gain (g)                           69.16 ± 3.66        61.66 ± 4.23       57.50 ± 2.82a     54.16 ± 3.11 a
Mating index (%)                                   100                 70                 60                40
 ertility index (%)                                98                  67                 58                50
Number of corpora lutea                            10.50 ± 0.56        9.56 ± 0.73        8.33 ± 0.49a      7.13 ± 0.60ab
Number of implantations                            9.83 ± 0.92         8.32 ± 0.82        6.86 ± 0.42a      5.70 ± 0.72ab
 re-implantation loss (%)                          6.38 ± 0.63         12.97 ± 0.75a      17.64 ± 0.45ab    20.05 ± 0.45abc
 alues represent mean ± S.E. of 10 rats in each group. The significance of the difference among various groups was evaluated
 y applying one-way ANOVA (p<0.05). Comparison between two groups: avs control, bvs 31 mg/kg bw, cvs 59 mg kg/bw.
 2            Chromium VI compounds
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<pre>rats resulted in a persistent diestrous phase. The oestrous cycle became regular
again within 15-20 days when animals began to mate. This recovery period was
not investigated after 20-day exposure where they were allowed to mate for only
one night.
Chowdhury and Mitra15 administered male Charles Foster rats (n = 10/group) 0,
20, 40 or 60 mg/kg bw/d sodium dichromate (equivalent to 0, 8, 16 or 24 mg
Cr/kg bw/d) for 90 days by gavage. Body weight gain was lower at 16 and 24
mg/kg bw. Testis weight was decreased at these doses compared to controls.
Spermatogenesis was inhibited as shown by a decreased count of resting
spermatocytes (high dose), pachytene spermatocytes and stage-7 spermatids at
40 and 60 mg/kg bw/d. At these dose levels also the population of Leydig cells,
seminiferous tubular diameter, DNA and RNA were reduced compared to
control. Testicular protein and serum testosterone were reduced at all dose levels,
and testicular cholesterol was increased at 40 and 60 mg/kg bw/d indicating
steroidogenic impairment, which was confirmed by inhibition of
3β-∆5-hydroxysteroid dehydrogenase at all dose levels.
In a study of the National Toxicology Program (NTP) 0, 15, 50, 100 or 400 mg
potassium dichromate/kg diet was administered to Sprague Dawley rats (24
males and 48 females/group) (equivalent to mean doses of 0, 0.4, 1, 2, or 9 mg
Cr/kg bw/d for males and females combined) for 9 weeks with a recovery period
of 8 weeks.16 Interim necropsies were performed after 3, 6 and 9 weeks of
administration and a terminal necropsy after week 17. No treatment-related
findings were observed on body weight, water and food consumption, organ
weights, macroscopy, and microscopy of the liver and kidneys, and
haematological parameters, except for a decrease in mean corpuscular volume
and mean corpuscular haemoglobin at the highest dose in both sexes, which had
disappeared after the recovery period. Testicular cell counts for Sertoli nuclei and
preleptotene spermatocyte counts investigated in Stage X and XI did not reveal
any differences between the treated groups and control, nor were any treatment
related changes in microscopical lesions found in ovaries.
Table 4 Summary of testicular cell counts in rats reported by NTP, 1996.
Weeks of interim kill           Control                            9 mg Cr/kg bw/d
3                               2.401 ± 0.274 (6)                  2.800 ± 0.235 (6)
6                               3.053 ± 0.190 (6)                  2.710 ± 0.101 (6)
9                               2.825 ± 0.486 (6)                  2.891 ± 0.380 (6)
Note: Ratio = number of germ cells / number of Sertoli cells.
Toxicity for reproduction                                                            43
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<pre>  The same study design was used to expose Balb/C mice to potassium (equivalent
  doses of 0, 1, 5, 10 and 41 mg Cr/kg bw/d, for males and females combined).17
  No treatment-related findings were observed on clinical signs, and macroscopic
  evaluation. Body weight of males in the highest dose group was decreased during
  the dosing period. Food consumption was generally increased in both sexes,
  especially at 41 mg/kg bw/d. Water consumption was decreased (8-9%) in the
  highest dose group in both sexes during the first half of the dosing period. Mean
  corpuscular volume and mean corpuscular haemoglobin at 41 mg/kg bw/d were
  decreased in both sexes, and returned to normal in both sexes during recovery.
  Cytoplasmic vacuolization in hepatocytes was noted at 1 mg/kg bw/d in females
  and in both sexes at 5 and 41 mg/kg bw/d. Testicular cell counts for Sertoli nuclei
  and preleptotene spermatocyte counts in Stage X and XI did not reveal any
  difference compared to controls.
  Table 5 Summary of testicular cell counts in mice reported by NTP, 1996.
  Weeks of interim kill           Control                          41 mg Cr/kg bw/d
  3                               5.79 ± 0.62 (5)                  5.71 ± 0.46 (6)
  6                               6.00 ± 1.06 (6)                  5.73 ± 0.49 (6)
  9                               5.44 ± 0.68 (6)                  5.37 ± 0.79 (6)
  Note: Ratio = number of germ cells / number of Sertoli cells.
  Subsequently, the NTP investigated reproduction and sperm parameters in a
  continuous breeding study with BALB/c mice dosed via the diet with 0, 100, 200
  and 400 mg/kg diet potassium dichromate.18 F0 animals (20 mice/sex/group)
  received 0, 7, 14, or 30 mg Cr/kg bw/d for 7 days pre-mating and 12 weeks
  during cohabitation. Litters produced during cohabitation were counted and
  weighed by sex and sacrificed on postnatal day 1. F0 pairs were separated and
  any litter born after the continuous breeding phase were reared and weaned on
  postnatal day 21. F0 animals were sacrificed and examined. In each dose group
  20 weanlings/sex/group were selected, received diets containing 0, 100, 200 or
  400 mg/kg diet potassium dichromate (equivalent to 8, 16 and 37 mg/kg bw/d)
  and were cohabitated on postnatal day 74 +/-10 for 7 days and separated.
  Offspring was counted and weighed by sex on postnatal day 1. At necropsy,
  F1 terminal body weight and organ weight were obtained, sperm analysis and
  macroscopy performed and histopathology of liver and kidney.
      F0 fertility was not affected. No treatment-related findings on pregnancy
  index and number of litters per pair were observed. No effect on epididymal
  sperm density, percent abnormal sperm and testicular sperm count was noted. No
  effect on gonadal organ weights was seen. In week 14 and during lactation and at
  necropsy, body weight of F0 dams at the two highest doses was decreased at
4 Chromium VI compounds
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<pre>              delivery of one or more litters. No treatment-related effect was observed on
              mortality, body weight of sires, clinical signs, food consumption, macroscopy or
              microscopy. Liver weight was decreased at the highest dose level in both sexes.
                    Also in the F1-generation no effect was found on mating index, pregnancy
              index, fertility index or gestation length. The oestrous cycle and sperm
              parameters were not affected. Again no treatment-related mortality, clinical
              signs, gross or microscopic lesions were observed in F1 animals. Adult body
              weights were decreased at the highest dose level (body weights were already
              decreased at weaning; see developmental toxicity), whereas food consumption
              was increased. At necropsy, mean corpuscular volume was decreased at the two
              highest dose groups in males and all dose groups in females. Mean corpuscular
              haemoglobin was decreased in males and mean haemoglobin was decreased in
              females in the highest dose group. To further evaluate the decreased mean
              corpuscular volume, erythrocyte morphology was investigated and found to be
              not affected. Developmental details are described under ‘developmental
              toxicity’.
 able 6 Summary of pregnancy indexa of breeding pairs reported by NTP, 1997.
 itter            Control                  7 mg Cr/kg bw/d      14 mg Cr/kg bw/d        30 mg Cr/kg bw/d        Trendb
                  20/20 (100)              20/20 (100)          20/20 (100)             20/20 (100)             p=0.500
                  20/20 (100)              20/20 (100)          20/20 (100)             20/20 (100)             p=0.500
                  19/20 (95)               20/20 (100)          17/20 (85)              19/20 (95)              p=0.241
                  15/20 (75)               18/20 (90)           13/20 (65)              14/20 (70)              p=0.147
 c                2/20 (10)                3/20 (15)            4/20 (20)               2/20 (10)               p=0.392
     Number of females delivering / number of cohabiting pairs (percent pregnant).
     P-value, from Cochran-Armitage trend test for decrease in pregnancies. Each dose group is compared to the control group
     with a chi-square test (*=p<0.05).
     The cohabitation period for this study was reduced from the standard 4 weeks to 12 weeks so only four litters would be
     produced during this study. However, eleven animals produced five litters.
 able 7 Summary of number of live pupsa reported by NTP, 1997.
 itter             Control                 7 mg Cr/kg bw/d      14 mg Cr/kg bw/d        30 mg Cr/kg bw/d        Trendb
                   5.2 ± 0.5 (20)          6.6 ± 0.5 (20)       5.3 ± 0.5 (20)          4.5 ± 0.5 (20)          p=0.151
                   6.0 ± 0.5 (20)          6.1 ± 0.6 (20)       6.4 ± 0.7 (20)          5.9 ± 0.7 (20)          p=0.795
                   7.7 ± 0.5 (19)          7.6 ± 0.5 (20)       7.1 ± 0.9 (17)          6.5 ± 0.4 (10)          p=0.127
                   7.6 ± 0.9 (15)          6.4 ± 0.5 (18)       7.4 ± 0.7 (13)          6.7 ± 0.9 (14)          p=0.680
                   9.0 ± 1.0 (2)           6.3 ± 2.0 (3)        9.0 ± 0.9 (4)           5.5 ± 1.5 (2)           p=0.562
Combinedc          6.6 ± 0.3 (20)          6.7 ± 0.2 (20)       6.5 ± 0.3 (20)          5.9 ± 0.3 (20)          p=0.081
     Mean ± standard error (number of pairs producing live pups).
     Each dose group is compared to the control group with Shirley’s test when a trend is present (p<0.01 from Jonckheere’s
     trend test), otherwise Dunn’s test is applied (*=p<0.05).
     Mean of the average number of live pups produced by each fertile pair ± standard error (number of fertile pairs).
              Toxicity for reproduction                                                                                     45
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<pre> able 8 Summary of sperm analysis dataa reported by NTP, 1997.
 ndpoint                              Control                 7 mg Cr/kg    14 mg Cr/kg       30 mg Cr/kg bw/d       Trendb
                                                              bw/d          bw/d
 omputed assisted sperm analysis
   Total cells analyzed               204.00 ± 5.26 (20)      -             -                 201 ± 5.59 (10)        p=0.201
   Sperm motility, %                  75.89 ± 1.09 (20)       -             -                 73.00 ± 1.45 (10)      p=0.065
   motile
   Velocity (µm/s)                    121.45 ± 1.95 (20)                                      119.48 ± 2.34 (10)     p=0.538
   Linearity                          6.07 ± 0.11 (20)        -             -                 6.04 ± 0.15 (10)       p=0.628
   ALH max (µm)                       6.40 ± 0.17 (20)        -             -                 6.36 ± 0.26 (10)       p=0.725
   ALH mean (µm)                      5.44 ± 0.15 (20)        -             -                 5.40 ± 0.20 (10)       p=0.775
   Beat/cross frequency (hz/sec)      13.98 ± 0.27 (20)       -             -                 13.92 ± 0.29 (10)      p=0.660
   Average radius (µm)                33.72 ± 1.18 (20)       -             -                 34.56 ± 1.92 (10)      p=0.895
   Circular cells                     22.70 ± 1.21 (20)       -             -                 22.70 ± 1.98 (10)      p=0.895
   Circular over all cells (%)        11.16 ± 0.54 (54)       -             -                 11.36 ± 0.98 (10)      p=0.860
   Circular over motile cells (%)     14.74 ± 0.72 (20)       -             -                 15.57 ± 1.32 (10)      p=0.524
 pididymal sperm densityc             582.66 ± 50.31 (19) 530.68 ±                            540.94 ± 44.29 (10)    p=0.343
                                                              79.36 (10)
 pid. Sperm morphology,               26.93 ± 0.69 (20)       -                               24.65 ± 1.55 (10)      p=0.098
% abnormald
 permatids/mg testise                 174.46 ± 7.06 (20)      193.55 ±      212.36 ±          184.79 ± 6.39 (10) p=0.122
                                                              713.79 (10)   12.06 (10)
 otal spermatids/testisf              15.30 ± 0.84 (20)       18.11 ±       17.55 ±           15.47 ± 1.16 (10)      p=0.516
                                                              1.30 (10)     1.04 (10)
    Endpoint mean ± standard error (number of animals).
    When only two dose groups are present, the dosed group is compared to the control group using wilcoxon’s test. However,
    when more than two groups are present, each dose group is compared with the control group by Shirley’s test if p<0.01
    from Jonckheere’s trend test; otherwise Dunn’s test is applied (*=p<0.05).
    Sperm density, expressed as 1,000 sperm per mg right caudal tissue.
    Dose group means and standard errors are computed only from samples with at least 200 sperm.
    Spermatid heads per mg testis (x 1,000) = (total number of spermatid heads per right testis) / (right testis weight in mg).
    Total number of spermatid heads (x 1,000,000) = (average number of spermatid heads x 2.5 x 100) / 0.0001.
Note: Computer assisted sperm analysis and sperm morphology were not required for the low and mid-dose groups.
              The NTP also conducted two more recent studies in rats and mice.5 In the first
              study, groups of male and female F344/N rats and B6C3F1 mice (n = 10) were
              given drinking water containing 0, 62.5, 125, 250, 500, or 1,000 mg sodium
              dichromate dihydrate/L for 3 months (equivalent to approximately 2, 4, 6, 11,
              and 21 mg hexavalent chromium/kg body weight per day to rats and 3, 5, 9, 16,
              and 28 mg/kg per day to mice). No histological findings on reproductive organs
              were reported.
                   In the second study, sodium dichromate dihydrate was administered in
              drinking water to male B6C3F1 (n = 10), BALB/c (n = 10), and am3-C57BL/6 (n
              = 5) mice for 3 months at exposure concentrations of 0, 62.5, 125, or 250 mg/L
 6            Chromium VI compounds
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<pre>             (equivalent to 3, 5, or 9 mg Cr/kg bw/d) to B6C3F1, BALB/c, and am3-C57BL/6
             mice. No effects on sperm count and motility were observed.
 able 9 Summary of sperm analysis dataa reported by NTP, 2007.
                                     Control               3 mg Cr/kg bw/d 5 mg Cr/kg bw/d 9 mg Cr/kg bw/d
 6C3F1
Weights (g)
   Necropsy bw                       46.8 ± 0.8            46.7 ± 0.6      42.7 ± 1.0**    36.4 ± 0.5**
   L Cauda epididymis                0.0142 ± 0.0005       0.0140 ± 0.0005 0.0150 ± 0.0004 0.0146 ± 0.0005
   L. Epididymis                     0.0457 ± 0.0008       0.0446 ± 0.0010 0.0439 ± 0.0006 0.0430 ± 0.0009
   L. Testis                         0.1192 ± 0.0020       0.1230 ± 0.0016 0.1176 ± 0.0010 0.1183 ± 0.0016
 permatid measurements
   Spermatid heads (107/g testis)    19.52 ± 0.42          20.31 ± 0.68    20.08 ± 0.62    20.72 ± 0.33
   Spermatid heads (107/testis)      2.19 ± 0.07           2.33 ± 0.07     2.19 ± 0.07     2.32 ± 0.05
 pididymal spermatozoal measurements
   Sperm heads (107/g cauda testis)  87.02 ± 8.41          103.26 ± 6.69   87.04 ± 7.05    102.93 ± 11.18
   Sperm heads (107/cauda testis)    1.21 ± 0.09           1.45 ± 0.11     1.26 ± 0.11     1.48 ± 0.14
   Sperm motility (%)                87.67 ± 0.89          88.05 ± 0.65    88.61 ± 0.83    87.63 ± 1.19
BALB/c
Weights (g)
   Necropsy bw                       28.8 ± 0.5            28.2 ± 0.6      27.0 ± 0.3**    25.7 ± 0.4**
   L Cauda epididymis                0.0101 ± 0.0006       0.0109 ± 0.0003 0.0097 ± 0.0004 0.0101 ± 0.0005
   L. Epididymis                     0.0351 ± 0.0006       0.0355 ± 0.0009 0.0349 ± 0.0006 0.0333 ± 0.0007
   L. Testis                         0.0973 ± 0.0029       0.0962 ± 0.0021 0.0928 ± 0.0017 0.0929 ± 0.0022
 permatid measurements
   Spermatid heads (107/g testis)    14.07 ± 1.05          14.90 ± 0.45    14.72 ± 0.29    15.87 ± 0.57
   Spermatid heads (107/testis)      1.29 ± 0.12           1.35 ± 0.06     1.31 ± 0.04     1.35 ± 0.06
 pididymal spermatozoal measurements
   Sperm heads (107/g cauda testis)  111.07 ± 7.40         110.40 ± 5.50   120.70 ± 5.70   114.90 ± 11.60
   Sperm heads (107/cauda testis)    1.10 ± 0.03           1.20 ± 0.05     1.17 ± 0.07     1.15 ± 0.11
   Sperm motility (%)                91.42 ± 0.40          89.75 ± 0.37    90.54 ± 0.43    91.28 ± 0.46
Am3-C57BL/6
Weights (g)
   Necropsy bw                       42.6 ± 1.5            36.0 ± 2.6*     35.4 ± 1.3**    27.4 ± 0.2**
   L Cauda epididymis                0.0131 ± 0.0003       0.0114 ± 0.0007 0.0175 ± 0.0040 0.0119 ± 0.0009
   L. Epididymis                     0.0426 ± 0.0021       0.0387 ± 0.0016 0.0471 ± 0.0062 0.0429 ± 0.0053
   L. Testis                         0.1124 ± 0.0019       0.1061 ± 0.0038 0.1098 ± 0.0024 0.0996 ± 0.0015**
 permatid measurements
   Spermatid heads (107/g testis)    20.91 ± 1.41          19.55 ± 0.33    19.60 ± 0.65    20.04 ± 0.72
   Spermatid heads (107/testis)      2.24 ± 0.16           2.01 ± 0.09     2.06 ± 0.03     1.93 ± 0.08
             Toxicity for reproduction                                                                    47
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<pre> pididymal spermatozoal measurements
   Sperm heads (107/g cauda testis)      123.00 ± 11.20         114.20 ± 9.80           108.70 ± 18.10        123.30 ± 8.40
   Sperm heads (107/cauda testis)        1.61 ± 0.15            1.30 ± 0.14             1.66 ± 0.15           1.47 ± 0.16
   Sperm motility (%)                    89.90 ± 0.53           90.28 ± 0.17            90.22 ± 0.92          90.10 ± 0.59
  Significantly different (P≤0.05) from the control group by Dunn’s test.
 * Significantly different (P≤0.01) from the control group by Williams’ test.
 Data are presented as mean ± standard error. Differences from the control group are not significant by Dunnett’s test (tissue
weights) or Dunn’s test (spermatid and epididymal spermatozoal measurements).
              In the developmental toxicity study (more details provided in section
              developmental toxicity) with mice (n = 10-13) from Trivedi et al., an increased
              pre-implantation loss was noted at 500 mg/L drinking water (120 mg Cr/kg
              bw/d) and no implantations at all were seen at 1,000 mg/L (234 mg Cr/kg
              bw/d).19
 able 10 Developmental effects reported by Trivedi et al., 1989.
 xposure group                              Control                59 mg/kg bw/d          120 mg/kg bw/d       234 mg/kg bw/d
  orpora lutea                              6.6 ± 0.72             5.0 ± 0.71             8.0 ± 0.54           No implantation
 reimplantation loss (%) (number of         3.60 ± 2.60 (2)        7.88 ± 3.36 (3)        26.19 ± 1.54***(6)   No implantation
ncidences)
 ** p<0.001
              Zahid et al. fed male Balb/C mice (5-7 animals/group) 0, 100, 200 or 400 mg
              potassium dichromate/kg diet (equivalent to 0, 11, 21 or 46 mg Cr/kg bw/d) in
              the diet for 7 weeks.20 Body weight and food consumption were not affected as
              were testis and epididymis weight. A slight, but significant increase of the
              number of degenerated seminiferous tubules was observed at all dose levels
              (dose-related). The number of spermatogonia per tubule was decreased related to
              the dose with an increase of the number of resting and pachyteen spermatocytes
              at all dose levels. At 200 and 400 mg/kg diet, sperm count was decreased without
              a dose relation, whereas the number of abnormal sperm cells increased dose-
              dependently.
 able 11 Results of sperm analysis data reported by Zahid et al., 1990.
                                 Control                 11 mg Cr/kg bw/d         21 mg Cr/kg bw/d       46 mg Cr/kg bw/d
Degenerated tubules              0/1400 (0%)             21/1774* (1.2%)          26/1129 *(2.3%)        33/1372 *(2.4%)
Ungenerated tubules without 18/90 (20.0%)                45/90 (50%)**            54/90 (60%)*           81/90 (90%)*
 permatogonia
 permatogonia                    11.1 ± 6.4              2.2 ± 3.8*               1.2 ± 1.7*             0.6 ± 0.3*
Resting spermatocytes            7.8 ± 11.7              20.2 ± 16.9***           19.6 ± 16.3***         22.8 ± 19.2*
 perm count (x105/mm3)           10.2 ± 1.3              5.7 ± 1.1                4.5 ± 1.1*             4.4 ± 0.9*
Abnormal sperm                   385/3193 (12.1%)        165/1242 (13.3%)         304/1607 (18.9%)*      318/1296 (24.5)*
  p<0.001; ** p<0.01; *** p<0.05
 8            Chromium VI compounds
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<pre>              Murthy et al. investigated the ovarian effects in Swiss mice (n = 10/group)
              exposed to 0, 250, 500 or 750 mg CrVI/L drinking water (equivalent to 0, 54,
              109 or 163 mg Cr/kg bw/d) for 20 days.21 A reduction in the number of follicles
              at different stages of maturation was seen at all dose levels in a dose-dependent
              manner. The number of ova was decreased at 109 and 163 mg/kg bw and
              histopathological examination revealed proliferated, dilated, and congested
              blood vessels, pyknotic nuclei in follicular cells, and atretic follicles to be
              present. The oestrous cycle was extended at 163 mg/kg bw.
 able 12 Results of female reproductive toxicity data reported by Murthy et al., 1996.
                                 Control                54 mg/kg bw/d          109 mg/kg bw/d         163 mg/kg bw/d
No. small follicles              39.4 ± 0.5             36.2 ± 0.4             34.0 ± 0.3a            25.2 ± 0.4abc
No. med follicles                9.8 ± 0.4              7.6 ± 0.2a             6.2 ± 0.3ab            4.6 ± 0.2abc
No. large follicles              7.6 ± 0.2              6.6 ± 0.2a             5.2 ± 0.2ab            2.4 ± 0.24abc
 strous cycle length (days)      4.4 ± 0.6              4.7 ± 0.6              5.8 ± 0.4              7.7 ± 0.8ab
No. ova/mouse                    26.2 ± 1.1             25.4 ± 0.5             18.4 ± 1.2ab           2.4 ± 0.9abc
 he values represent mean +/- S.E. of 10 mice per group. The significance of the differences was evaluated using one-way
ANOVA (abcp<0.05; avs control, bvs54 mg/kg bw/d, cvs109 mg/kg bw/d).
              They also administered another group of Swiss mice (n = 10/group) 0, 0.05, 0.5
              or 5 mg CrVI/L for 90 days (equivalent to 0, 0.01, 0.1 and 1 mg Cr/kg bw/d) and
              examined the ovaries with electron microscopy. At the highest dose level
              disintegrated cell membranes of two layered follicular cells and altered villiform
              mitochondria in thecal cells were observed.
              Elbetieha and Al-Hamood studied the effects on fertility in male Swiss (n = 9-20/
              group) mice after exposure to 1,000, 2,000, 4,000 and 5,000 mg potassium
              dichromate/L in the drinking water (equivalent to 74, 148, 296 and 370 mg Cr/kg
              bw/d) during 12 weeks.22 In the male mice exposed to the two highest doses,
              body weight, testis, seminal vesicles and preputial gland of the parental animals
              were weighed at necropsy after 12 weeks of exposure. In both exposure groups,
              body weight was decreased and relative testis weight was increased.
              Furthermore, in the highest dose group, relative seminal vesicle and preputial
              gland weights were decreased. In female mice mated with male mice exposed to
              296 and 370 mg Cr/kg bw/d, the number of implantations and the number of
              viable foetuses were reduced. In the female mice mated with male mice from the
              highest dose group, these reductions were not statistically significant.
              Toxicity for reproduction                                                                                  49
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<pre> able 13 Results of male reproductive toxicity data reported by Elbetieha and Al-Hamood, 1997.
                               0 mg/L              74 mg Cr/kg bw/d 148 Cr/kg bw/d        296 Cr/kg bw/d     370 Cr/kg bw/d
Number of pregnant             33/40 (82.5)        33/38 (86.8)       20/22 (90.9)        16/18 (88.8)       19/26 (73)
emales (%)
Mean number of                 8.18 ± 1.59 (33) 7.84 ± 1.56 (33) 6.33 ± 2.79 (20)** 6.86 ± 1.88 (16)*        7.84 ± 2.73 (16)
mplantations
Mean number of                 8.18 ± 1.59 (33) 7.75 ± 1.80 (33) 6.33 ± 2.79 (20)** 6.86 ± 1.88 (16)*        7.15 ± 2.98 (19)
 iable foetuses
 otal number of resorptions 0                      3 resorptions      0                   0                  6 resorptions,
 nd dead foetuses                                                                                            6 dead foetuses
  p<0.05, ** p<0.01; Significance when compared to control value (Student’s t test).
             In addition, Elbetieha and Al-Hamood studied the effects of chromium VI in
             female mice (n = 15 and 11) after exposure to 2,000 and 5,000 mg potassium
             dichromate/L in the drinking water (equivalent to 153 and 384 mg Cr/kg bw/d)
             during 12 weeks, after which the treated females were mated with untreated
             males for 10 days. No effect on body weight was observed, but the relative ovary
             weight was increased at 384 mg Cr/kg bw/d. No effect on the number of pregnant
             females was noted. However, the number of implantations and the number of
             viable foetuses were reduced in both groups of chromium VI treated female mice
             mated with untreated male mice.
 able 14 Results of female reproductive toxicity data reported by Elbetieha and Al-Hamood, 1997.
                                              Control                    153 Cr/kg bw/d             384 Cr/kg bw/d
Number of pregnant females                    17/18                      14/15                      9/11
Mean number of implantations                  9.00 ± 1.36 (17)           7.35 ± 1.54 (14)**         7.44 ± 1.50 (9)*
Mean number of viable foetuses                8.76 ± 1.39 (17)           6.55 ± 2.18 (9)*           5.88 ± 2.47 (9)**
Number of mice with resorptions               2/18 (11)                  8/15 (53)***               7/11 (63)****
 otal number of resorptions                   4                          37                         14
  p<0.05, ** p<0.01; Student’s t-test; *** p<0.01, **** p<0.005; Chi-square test; Significance when compared to control value.
             Yousef et al. administered 0 or 4 mg Cr/kg bw as potassium dichromate to male
             rabbits (n = 6/group) by gavage daily during ten weeks. Semen was collected
             weekly, blood every other week and rabbits were sacrificed at the end of the
             exposure period.23 Rabbits showed no adverse clinical signs. Body weight,
             relative weight of testes and epididymis and plasma testosterone concentration
             were decreased in chromium VI-exposed rabbits. Packed sperm volume, sperm
             concentration, total sperm output, sperm motility, total motile sperm per
             ejaculate, total functional sperm fraction, normal sperm, initial fructose and
             libido (by decreasing reaction time) were decreased compared to controls, while
             dead sperm and initial pH were increased. No effect on semen ejaculate volume
  0          Chromium VI compounds
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<pre>was noted. In seminal plasma of chromium VI-treated rabbits, the concentration
of thiobarbituric acid-reactive substances was elevated and activities of
glutathione S-transferase, aspartate aminotransferase and acid phosphatase were
decreased. Seminal plasma lipids, glucose and urea were increased in chromium
VI-treated rabbits, while total cholesterol, low-density-lipoprotein and high-
density-lipoprotein cholesterol were decreased. No effect on triglycerides, total
protein and albumin in seminal plasma was noted.
Table 15 Results of male reproductive toxicity data reported by Yousef, 2006.
                                                Control                   4 mg Cr/kg bw/d
Ejaculate volume (mL)                           0.69 ± 0.01               0.66 ± 0.01
pH                                              7.4 ± 0.04                7.7 ± 0.05*
Reaction time (s)                               10.5 ± 0.3*               22.0 ± 0.9*
Packed sperm volume (%)                         16.2 ± 0.47               14.6 ± 0.33*
Sperm concentration (x106/mL)                   266 ± 6.08                218 ± 7.60*
Total sperm output (x106)                       185 ± 4.17                137 ± 3.96
Sperm motility (%)                              66.9 ± 0.90               63.8 ± 1.30*
Total motile sperm (x106)                       137 ±1.8                  90 ± 2.9*
Dead sperm (%)                                  24.7 ± 0.78               30.6 ± 0.79*
Normal sperm (%)                                85 ± 0.4                  82 ± 0.6*
Total functional sperm fraction (x106)          116 ± 1.66                73 ± 2.53*
Initial fructose (mg/dL)                        140 ± 0.95                134 ± 0.89*
* p<0.05
Intraperitoneal
As the Committee considers the intraperitoneal route of administration of limited
relevance for classification, no summarising tables are provided for these studies.
Ernst treated male Wistar rats intraperitoneally for 5 consecutive days with 0, 1,
2 or 4 mg Cr/kg bw/d (as sodium chromate) (n = 8/group). At all dose levels no
testicular toxicity was observed 7 days after the last administration.24 Sixty days
after the last administration, a dose-dependent reduction in relative testis weight,
an increase in the number of atrophic seminiferous tubules with a loss of
spermiogenic epithelium and a reduced number of epididymal spermatozoa were
observed in all chromium treated groups. In the highest dose group, the cellular
organisation of the testis was lost; complete degeneration was observed in almost
every seminiferous tubule and the Leydig cells appeared to be atrophic. In all
CrVI dose groups a dose-related decreased weight gain was observed, not
accompanied by decreased food or water consumption.
Toxicity for reproduction                                                                 51
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<pre>      Ernst and Bonde studied in male Wistar rats (n = 10) the effects of subchronic
      intraperitoneal treatment (5 days/week during 8 weeks) to 0.5 mg Cr/kg bw/d (as
      sodium chromate).25 No clinical signs or effect on body weight, food or water
      consumption were noted. At the end of the exposure period, a reduction in
      epididymal sperm motility, a decrease in serum testosterone (T) and an increase
      in follicle stimulating hormone (FSH) and in luteinizing hormone (LH) were
      observed. After a recovery period of 8 weeks sperm motility and T and FSH
      levels were normal again, whereas LH level was decreased.
      Behari et al. studied male rabbits (n = 8-10/group) after a daily intraperitoneal
      injection of 0 or 2 mg potassium dichromate/kg bw for 3 or 6 weeks(equivalent
      to 0 or 0.7 mg Cr/kg bw/d).26 The animals were sacrificed 72 hours after the last
      injection. After 3 weeks of exposure, a decrease in succinic dehydrogenase
      activity and mild oedema of interstitial tissue in the testis were observed in the
      CrVI treated group. After 6 weeks of dosing, a decrease in succinic
      dehydrogenase and adenosine triphosphatase activities, and marked oedema of
      the interstitial tissue were observed. Furthermore, no spermatocytes were
      observed in the seminiferous tubules of the testis after 6 weeks.
5.1.2 Human studies
      Most studies pertain to chromium and other metals present in welding fumes, but
      exposure levels were not determined.
      In a case-control study, Rachootin and Olson studied among others the
      associations between chromium VI during welding and male sperm
      abnormalities, female hormonal disturbances and infertility.27 All cases (n = 927)
      and controls (n = 3,728) were identified at Odense University Hospital in
      Denmark during the period 1977-1980. No statistically significant associations
      were observed between welding of stainless steel (containing chromium VI) and
      male sperm abnormalities, female hormonal disturbances, delayed conception or
      idiopathic infertility.
      Jelnes and Knudsen found that the semen quality of stainless steel welders
      (n = 75-77) was similar to that of non-welders within the same plant in the period
      January to July 1987, when matched with respect to age and smoking habits.28
      The semen quality was also comparable to that of referents not working in the
      metal industry.
 2    Chromium VI compounds
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<pre>Mortensen studied the semen quality of workers visiting fertility clinics in
Denmark.29 Based on information from questionnaires, workers could be placed
in one of the following groups: welders, metal workers not exposed to welding,
other industrial workers and unexposed workers (not exposed to chemical or
physical agents suspected of influencing sperm quality). Cases were defined as
having at least one of the following: sperm concentration less than 20 million/
mL, less than 50% of sperm cells motile or less than 50% of sperm cells with a
normal morphological appearance. Men with sperm that did not fulfill any of the
criteria were considered to have normal sperm and were classified as controls.
The odds ratio of reduced sperm quality among welders (27 cases; 28 controls)
was 2.00 (95% CI 1.16-3.45). The odds ratio was even higher when stainless
steel welders were separated from nonstainless steel welders: 2.34 (95% CI 0.95-
5.73). Potential confounders, such as living quarters, age, smoking habits,
alcohol intake, medication use, and earlier diagnosis of mumps with or without
orchitis were not associated with the occupation of welding.
Bonde and Ernst examined the associations of chromium VI with sex hormones
and semen quality in welders in Denmark.30 The study included 30 tungsten inert
gas stainless steel welders, 30 mild steel welders and 47 non-welding workers
from six different plants. Participants were classified into three groups according
to the concentration of chromium in their urine defined by the median and the 75
percentile of the distribution of urine chromium concentration: <1.07, 1.07-1.78
and >1.78 nmol/mmol creatinine. All groups had subjects in all three classes, but
the number of stainless steel welders increased with increasing concentration,
while for mild steel workers and non-welding metal workers most subjects were
in the lowest class. For electricians, all subjects were in the lowest class. None of
the sperm parameters and serum concentrations of sexual hormones showed an
inverse association with chromium concentration in urine.
In a review of 1993, Bonde reported on several studies on male metal workers,
including results from the above-mentioned field study, but with an increased
group of mild steel welders (46) and non-welding referents (54).31 All workers
were employed on 1 December 1986 at one of six plants in Aalborg, Denmark.
While the sperm concentration in seminal plasma was not reduced among
welders and no increased rate of subjects with oligospermia was found, sperm
count and degree of sperm motility were decreased in stainless steel and mild
steel workers. The linear penetration rate in egg white and the proportion of
normal sperm forms were decreased in mild steel welders only. Serum
testosterone was lower in stainless steel workers compared to referents, whereas
Toxicity for reproduction                                                             53
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<pre>  no difference in the level of follicle stimulating hormone was observed. Among
  538 questionnaire respondents working at the six plants, an increased risk of
  infertility (inability to conceive within 2 years) was noted with an odds ratio of
  2.2 (95% CI 1.1-4.6) when compared to age-matched non-welding referents. In a
  nationwide cohort of metalworkers (n = 3,507), no association was found
  between fertility and welding exposure.
  Li et al. investigated the semen status of 21 workers exposed to chromium VI in
  an electroplating factory in Henan, China for 1-15 years.12 A control group of
  workers not exposed to any harmful chemicals was recruited from the same
  factory. The average age was similar in both groups. In exposed workers, semen
  samples were collected preferably 5 days after sexual abstinence. Sperm counts
  and motility were reduced to 53 and 85% of those in the control group,
  respectively. Seminal volume and liquefaction time were not affected. Serum
  follicle stimulating hormone was increased compared to controls (7.34 and 2.41
  IU/mL in exposed and control groups, respectively; p<0.01), while serum
  luteinizing hormone was not affected. The chromium concentration was 1.40 and
  1.26 µmol/mL in serum and 7.55 and 6.38 µmol/mL in seminal fluid in exposed
  and control group, respectively; these differences were not statistically
  significant. The zinc concentration (role in oxidant defense system) and activity
  of lactate dehydrogenase and lactate dehydrogenase C4 isoenzyme (specific for
  seminal fluid of mature testicles) in seminal fluid were decreased in exposed
  workers to 26, 60 and 70% of those in the control group.
  Kumar et al. reported a cross-sectional study on 61 workers exposed to
  chromium during chromium sulphate manufacturing and 15 controls from a
  nearby industry not involved in the manufacturing of chromium compounds.
  Lifestyle differences between groups included differences in smoking, drinking,
  and chewing habits. Chromium blood levels were higher in exposed workers
  compared to control subjects (63.7 ± 50.4 µg/L versus 22.8 ± 17.7 µg/L).
  A higher number of morphologically abnormal sperm cells were reported in the
  exposure group, and a statistically significant correlation (r=301) was found for
  this parameter and blood chromium levels. However, no significant differences
  were found in semen volume, liquefaction time, mean pH value, and sperm
  viability, concentration or motility.
4 Chromium VI compounds
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<pre>5.2            Developmental toxicity
5.2.1          Non-human information
               Animal developmental toxicity studies are summarised in Table 16 and described
               below. Changes and differences mentioned are statistically significant unless
               stated otherwise.
 able 16 Summary table of developmental toxicity studies in animals.
 uthors species      experimental period/ dose/route            general toxicity                      developmental toxicity
                     design
  laser   Rat;       Daily during gestation 0, 50, 100 or 200 No mortality or clinical signs;         No developmental effects observed
 t al.,   Wistar;    (time/d not indicated); µg Cr/m3 as        increased relative organ weight,      (macroscopy, number of foetuses, foetal
 984      n = 10-12 sacrificed on GD 21 sodium                  particularly lung. Hyperplasia at the weight and placental weight)
                                             dichromate (0,     highest concentration                 NOAEL=0.3 mg Cr/kg bw/d
                                             0.1, 0.2 and 0.3 LOAEL=0.3 mg Cr/kg bw/d
                                             mg Cr/kg bw/d
                                             (assuming a
                                             breathing volume
                                             of 200 ml/min))/
                                             inhalation
  anojia Rat; Swiss During 20 days (one 0, 250, 500 or 750 No mortality or change in clinical         All doses: decreased number of live
 t al.,   albino;    folliculogenesis cycle) mg Cr/L as         signs; decreased water                foetuses, number of resorptions, and post-
 996      n = 10     prior to gestation;     potassium          consumption; decreased weight gain    implantation loss;
                     sacrificed on GD 19; dichromate in         in all doses                          59 and 76 mg/kg: decreased placental
                     1/3 foetuses visceral drinking water       LOAEL=31 mg Cr/kg bw/d                weight; non-significant decrease foetal
                     abnormalities,          (31, 59 and 76 mg                                        weight and crown-rump length; subdermal
                     remaining external      Cr/kg bw/d)/oral                                         haemorrhagic patches, short tail, and
                     and skeletal                                                                     reduced caudal ossification
                                                                                                      76 mg/kg: kinky tail, reduced parietal and
                                                                                                      interparietal ossification
                                                                                                      LOAEL=31 mg Cr/kg bw/d
  anojia  Rat;       During 3 months prior   0, 250, 500 or 750 Gestational weight gain decrease in   All dose levels: decreased number of live
 t al.,   Druckrey; to gestation; sacrificed mg Cr/L as         all dose groups; 89 and 124 mg/kg:    foetuses/litter, foetal weight and crown-
 998      n = 10     on GD 19; 1/3           potassium          15% and 10% of animals, resp.,        rump length; increased number of
                     foetuses visceral       dichromate in      died within 14 days; liver and        resorptions; increased pre- and post
                     abnormalities,          drinking water (0, kidney; reduced water consumption     implantation loss; decreased placental
                     remaining external      45, 89 or 124 mg/  LOAEL=45 mg Cr/kg bw/d                weight at 89 and 124 mg/kg; drooping wrist
                     and skeletal            kg bw/d)/oral                                            and subdermal haemorrhagic patches on
                                                                                                      thoracic and abdominal region at all doses;
                                                                                                      kinky and/or short tail at 89 and 124 mg/kg;
                                                                                                      no visceral abnormalities; reduced caudal
                                                                                                      ossification at all doses, and reduced
                                                                                                      parietal and interparietal ossification at 89
                                                                                                      and 124 mg/kg; increased Cr level in
                                                                                                      placenta and foetus
                                                                                                      LOAEL=45 mg Cr/kg bw/d
  lsaieed Rat;       Gestation day 6-15;     0 or 50 mg/L Cr    No adverse clinical signs; no effect  Increase of pre- and post-implantation
 nd       Wistar;    sacrifice GD 20; 1/3    as potassium       on food intake and water              losses, number of resorbed and dead
  ada,    n = 10     visceral, remainder     dichromate in      consumption; decrease gestational     foetuses per litter; decreased foetal weight,
 002                 skeletal examination    drinking water (7  weight (40%)                          number of visceral (renal pelvis dilatation)
                                             mg Cr/kg bw/d)/    LOAEL=7 mg Cr/kg bw/d                 and skeletal (incomplete ossification of
                                             oral                                                     skull bone) anomalies per litter
                                                                                                      LOAEL=7 mg Cr/kg bw/d
               Toxicity for reproduction                                                                                                         55
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<pre>oudani Rat;         on GD 14 to postnatal    0 or 700 mg /L     No mortality or clinical signs; slight Changes of biochemical parameters in
t al.,   Wistar;    day 14; biochemical      potassium          decrease in food and water             treated dams and histopathological
011a,b,c n = 6      parameters, lipid        dichromate in      consumption; decrease in body          examination indicated damage to kidney of
                    peroxidation,            drinking water (0  weight. Changes of biochemical         pups.
                    oxidative stress and     or 24 mg Cr/kg     parameters in treated dams and         Also reduced femur weight and length were
                    histopathology in        bw/d)/oral         histopathological examination          reported in pups, and changes in bone
                    kidney of dams and                          indicated damage to kidney             mineralization, observed after biochemical
                    offspring (culled to 4/                     Haemorrhage, leukocyte infiltration    and histological analysis
                    sex); 24h-urine                             and necrosis in liver (more            Haemorrhage, leukocyte infiltration and
                    sample before                               pronounced than in pups)               necrosis in liver (less pronounced than in
                    sacrifice; blood                            LOAEL=24 mg Cr/kg bw/d                 dams)
                    sample after sacrifice                                                             LOAEL=24 mg Cr/kg bw/d
 TP,     Mouse;     F0: 1 week prior to      0, 100, 200 or 400 F0 : decreased body weight dams at F1: no influence on number of live pups per
997      BALB/c; mating and 12-week          mg potassium       mid and high dose at delivery of one litter, proportion of pups born alive, sex
         n = 20/sex mating period            dichromate/kg      or more litters, in week 14, during ratio and live pup weight; decreased body
                    (continuous breeding     diet (F0: 0, 7, 14 lactation and at necropsy; no          weight at PND 21 (M) at 37 mg/kg;
                    phase; intermediate      or 30 mg Cr/kg     treatment-related effect on            NOAEL F0=30 mg Cr/kg bw/d
                    litters evaluated on     bw/d; F1: 0, 8, 16 mortality, body weight of sires,       F2: no influence on number of live pups per
                    PND 1); F1: after        or 37 mg Cr/kg     clinical signs, food consumption,      litter, proportion of pups born alive, sex
                    weaning until 74 +/-     bw/d)/oral         macroscopy or microscopy;              ratio and adjusted live pup weight
                    10 days of age                              decreased absolute liver weight at NOAEL F1=37 mg Cr/kg bw/d
                    continued with 1 week                       30 mg Cr/kg bw/d (M/F)
                    mating period                               NOAEL F0=7 mg Cr/kg bw/d
                                                                F1: no treatment-related mortality,
                                                                clinical signs, gross or microscopic
                                                                lesions; decreased body weight and
                                                                increased food consumption at 37
                                                                mg/kg; at necropsy, decreased MCV
                                                                at 16 and 37 (M/F) and at 8 mg/kg
                                                                (F); decreased MCH (M) and mean
                                                                haemoglobin (F) at 37 mg/kg; no
                                                                effect on erythrocyte morphology
                                                                LOAEL F1=8 mg Cr/kg bw/d
rivedi   Mouse;     During gestation;        0, 250, 500 or     No effect on clinical signs or         59 mg/kg: increased number of resorptions
t al.,   ITCR;fema  sacrifice on GD 19; 1/   1,000 mg           placental weight; 234 mg/kg: severe and postimplantation loss; decreased litter
989      les; n =   3 of foetuses visceral   potassium          effect on body weight                  size; decrease foetal weight and length;
         10-13      abnormalities;           dichromate/L in    NOAEL=120 mg Cr/kg bw/d                retarded ossification of skull bones;
                    remainder skeletal       drinking water (0,                                        additionally, at 120 mg/kg: increased in
                    abnormalities            59, 120 and 234                                           external abnormalities (kinking of tail,
                                             mg Cr/kg bw/d)/                                           subdermal haemorrhagic patches; retarded
                                             oral                                                      ossification of fore- and hindlimb
                                                                                                       phalanges, vertebrae and sternebrae;
                                                                                                       accumulation of Cr in foetus at 120 mg/kg
                                                                                                       LOAEL=59 mg Cr/kg bw/d
 e Flora Mouse;     During gestation;        0, 5 or 10 mg Cr/  No effect on frequency of              No effect on number of foetuses/litter,
t al..   Swiss;     sacrifice GD 18;         L (0, 1, or 2 mg   micronuclei in polychromatic           foetal weight, frequency of micronuclei in
006      n=5        number of foetuses/      Cr/kg bw/d); or    erythrocytes and PCE/NCE ratio in polychromatic erythrocytes and PCE/NCE
                    litter and foetal weight 10 mg Cr/L         bone marrow                            ratio in foetal liver and peripheral blood
                    and micronuclei in       drinking water as  NOAEL=2 mg Cr/kg bw/d                  NOAEL=2 mg Cr/kg bw/d (repeated dose)
                    foetal liver and blood   potassium          (repeated dose)
                                             dichromate (2 mg
                                             Cr/kg bw/d)/oral.
                                             50 mg Cr/kg bw     NOAEL=50 mg Cr/kg bw (single           NOAEL=50 mg Cr/kg bw (single dose)
                                             once, as sodium    dose)
                                             dichromate
                                             dehydrate or
                                             potassium
                                             dichromate/ip
 6            Chromium VI compounds
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<pre>unaid      Mouse;      GD 6-14; sacrificed     0, 250, 500 or 750 No clinical signs; 125 and 182        67 mg/kg: increased number of resorptions
 t al.,    Swiss; 30 g on GD 19; 1/3 of        mg Cr/L as         mg/kg: reduced body weight gain       and post-implantation loss; 125 and 182
 996       bw; n = 10 foetuses visceral        potassium          (8 and 30% reduction, respectively)   mg/kg: decreased number of foetuses,
                       abnormalities;          dichromate in      NOAEL=67 mg Cr/kg bw/d                increased number of dead foetuses,
                       remainder first         drinking water (0,                                       resorptions and post-implantation loss;
                       external then skeletal  67, 125 and 182                                          decreased foetal weight; no effect on
                       deformities             mg Cr/kg bw/d)/                                          crown-rump length;
                                               oral                                                     Gross and skeletal defects: 125 mg/kg:
                                                                                                        increased incidence of reduced caudal
                                                                                                        ossification;182 mg/kg: increased incidence
                                                                                                        of drooping wrist and subdermal
                                                                                                        haemorrhagic patches; increased incidence
                                                                                                        of reduced ossification (nasal, frontal,
                                                                                                        parietal, interparietal, caudal, tarsal); Cr
                                                                                                        foetal levels increased at all doses
                                                                                                        LOAEL=67 mg Cr/kg bw/d
  lbetieha Mouse,      12 weeks prior to       0, 2,000, or 5,000 153 and 384 mg/kg: decrease body 153 and 384 mg/kg: reduced number of
 nd Al-    females,    mating; body weight     mg potassium       weight;                               viable foetuses; increase in total number of
  amood,   Swiss;      and relative organ      dichromate/L       LOAEL=153 mg Cr/kg bw/d               pregnant mice with resorptions
 997       n = 9-20    weight only studied at  drinking water                                           LOAEL=153 mg Cr/kg bw/d
                       2000 and 5,000 mg/L     (153 or 384 mg
                                               Cr/kg bw/d)/oral
  l-       Mouse;      From GD 12 to PND       0 or 1000 mg Cr no effect on water consumption; no No effect on pup weight, weight of ovaries,
  amood    Swiss;      20; mating of treated   potassium          further effects reported              uterus, testes, seminal vesicles and
 t al.,    n =25/      M/F with untreated      dichromate/L as NOAEL=71 mg Cr/kg bw/d                   preputial glands; no effect on male fertility
 998       group       pups at PND 60 for 10   in drinking water                                        (as measured by the number of pregnant
                       d; sacrificed 1w after  (0 or 71 mg Cr/kg                                        females, number of implantations and
                       removal of male         bw/d)/oral                                               number of viable foetuses); delayed vaginal
                       (fertility parameters);                                                          opening (27.1 d compared to 24.6 d in
                       other group sacrificed                                                           controls); female fertility was affected:
                       at PND 50 (body and                                                              decreased number of pregnant females,
                       organ weight)                                                                    implantations and viable foetuses, and
                                                                                                        increased number of resorptions
                                                                                                        LOAEL=71 mg Cr/kg bw/d
 ivakum Rat; n = 5 From GD 9.5-14.5. At        0 and 25 mg/L      Not reported                          Increased germ cell apoptosis; up-
 r et al.,            PND 60, F1 female        potassium                                                regulation p53, p27, Bax, Caspase-3;
 014                  pups (n-15) were         dichromate in                                            increase p53 and SOD-2 co-localisation;
                      allowed to mate with     drinking water (0                                        accelerated germ cell cyst breakdown;
                      non-exposed males.       and 1 mg Cr/kg                                           advanced primordial follicle assembly and
                                               bw/d)/oral                                               primary follicle transition; down regulation
                                                                                                        of p-AKT,p-ERK and XIAP; induction of
                                                                                                        early reproductive senescence and decrease
                                                                                                        in litter size in F1 female progeny
                                                                                                        LOAEL=1 mg Cr/kg bw/d
Marouani Rat;          GD 6-15; killed on      0, 1 or 2 mg Cr/kg No mortality or changes in clinical At 1 and 2 mg/kg: Reduced foetal bw,
 t al.,    Wistar;     GD 19                   bw/d as potassium signs; reduction in body weight gain retarded foetal development, reduced
 010       n=8                                 dichromate/ip      (to 71 and 40% of control at 1 and 2 number of foetuses per mother and high
                                                                  mg/kg, resp.) and relative weight of incidences in dead foetuses and resorptions;
                                                                  the uterus (to 79 and 70% of control gross morphological abnormalities;
                                                                  at 1 and 2 mg/kg, resp.)              incomplete ossification in nasal, cranium,
                                                                  NOAEL=2 mg Cr/kg bw/d                 abdominal or caudal bones. At 2 mg/kg:
                                                                                                        absence of sacral vertebrae; histological
                                                                                                        changes with atrophy of vital organs
                                                                                                        LOAEL=1 mg Cr/kg bw/d
  ndo and Mouse;       Single injection on     0 or 15 mg         Stated to be observed daily for signs No effect on live, dead or resorbed foetuses,
Watanabe Jcl:ICR;      GD 9; sacrifice GD      chromium           of toxicity (no results reported); no foetal weight (male/female) or number of
 988       n = 15-17   17; foetuses sexed,     trioxide/kg bw     effect on body weight                 malformations
                       examined externally     (8 mg Cr/kg bw)/ NOAEL=8 mg Cr/kg bw/d                   NOAEL=8 mg Cr/kg bw/d
                       and 1/3 for skeletal    ip
                       abnormalities
               Toxicity for reproduction                                                                                                             57
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<pre> ale      Hamster, Single injection GD 8;    0, 5, 7.5, 10 or 15 3 mg/kg: no effects;                 3, 4 and 5 mg/kg: increased number of
 978      Golden      sacrifice GD 12, 14 or mg chromium         4 and 5 mg/kg: most lost weight;     resorptions; increased number of
          (LAK:       15                     trioxide/kg bw (0, urinary bladders distended with       malformations (cleft palate,
          LVG(SYR,                           3, 4, 5 or 8 mg Cr/ clear urine (control milky urine);   hydrocephalus), retarded ossification
          n = 4-21                           kg bw/d)/iv         mottled kidneys, gall bladders       (vertebral column and sternum (skull at 4
                                                                 abnormally distended with bile       and 5), hyoid bone, hind limb, fore limb (5
                                                                 (histologically normal); clear       and 8)
                                                                 vacuoles in periportal hepatocytes;  LOAEL=3 mg Cr/kg bw/d
                                                                 moderate to extensive tubular
                                                                 necrosis with hyaline casts in
                                                                 kidney;
                                                                 8 mg/kg: 3 out of 4 died shortly
                                                                 after treatment
                                                                 NOAEL=3 mg Cr/kg bw/d
 ale and Hamster;     Single injection GD 7, 0 or 8 mg           Weight loss; mottled kidneys with    GD 7, 8, 9, 10: decreased crown-rump
 unch     Golden      8, 9, 10 or 11;        chromium            varying degrees of tubular necrosis; length
 979      (LAK:       sacrifice GD 12, 14 or trioxide/kg bw (0 some gall bladders (GD 8 and 9)        GD 7,8,9: increased number of resorptions,
          LVG         15                     or 4 mg Cr/kg       were distended with bile             increased number of live foetuses with
          (SYR));                            bw)/iv              (histologically normal)              external defects and cleft palate; some
          n=6                                                    LOAEL=4 mg Cr/kg bw/d                kidneys small or absent at GD 7 and 8;
          (control 3)                                                                                 GD 11: no effects
                                                                                                      LOAEL=4 mg Cr/kg bw/d
Abbrevations used: GD = gestation day; iv = intravenous; ip = intraperitoneal; PND = post-natal day; P1/2/3 = first/second/third
 arental generation. If applicable, doses were converted to amount of chromium based on molecular weight. Conversion of
 oses to mg/kg bw/d, if applicable, was performed using the general conversion factor for body weight to food or water
 onsumption (assuming dry diet), and the default values for body weights (if not specified by the authors) as specified in the
 echnical Guidance on Risk Assessment, Part 1, Appendix 6. The Committee notes that this conversion does not specifically
ake into account pregnant animals.
              Inhalation
              Glaser et al. treated pregnant Wistar rats with 0, 50, 100 or 200 µg chromium
              VI/m3 as sodium dichromate by inhalation for 21 days after which animals were
              sacrificed and foetuses were weighed.9 The only effects observed on dams was
              an increased relative lung weight at all dose levels and increased relative liver
              weight at 100 and 200 µg/m3. The number of foetuses, foetal weight or placental
              weight were not affected. In foetuses at 200 µg/m3 the lymphocyte count was
              decreased, plasma levels of total bilirubin were increased, whereas triglycerides
              were decreased. The latter was also decreased at 100 µg/m3.
                    Glaser et al. also studied the effects of 200 µg chromium VI/m3 on the
              reproduction and development of offspring in 3 generations of rats (see details
              under fertility). Effects on parents included haematological, clinical biochemistry
              effects, decreased serum-immunoglobulin levels and increased lung weights. No
              effects on number of foetuses, live foetuses at postnatal day 25, foetal weight or
              skeletal development were noted.
                    The Committee notes that the equivalent oral doses corresponding to the
              exposure concentrations of 50, 100, and 200 µg chromium VI/m3 applied in this
              study are rather low, i.e. 0.1, 0.2 and 0.3 mg Cr/kg bw/d, respectively (assuming
              a breathing volume of 200 mL/min).
 8            Chromium VI compounds
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<pre>             Oral
             In the first study by Kanojia et al.13 with rats (for details see section ‘effects on
             fertility’), dams showed a decreased weight gain at all doses (i.e. 250, 500 and
             750 mg Cr/L, equivalent to 31, 59 and 76 mg Cr/kg bw/d). The number of live
             foetuses was dose-related decreased and the number of resorptions (not dose-
             related) and post-implantation loss (dose-related) was increased at all doses. At
             the two highest dose levels decreases were seen in the foetal and placental
             weights, and increased incidences of subdermal haemorrhagic patches, short tail,
             and reduced caudal ossification. A kinky tail and reduced parietal and
             interparietal ossification was observed in foetuses at 76 mg/kg bw/d only. No
             visceral abnormalities were noted.
 able 17 Results as reported by Kanojia et al., 1996.
                                              Control               31 mg/kg bw/d       59 mg/kg bw/d         76 mg/kg bw/d
Weight gain in mothers (g)                    70.50 ± 5.19          65 ± 3.17a          60.92 ± 2.13ab        55.5 ± 3.01abc
Number of live foetuses                       9.11 ± 0.87           8.29 ± 0.93 a       4.12 ± 0.51 ab        1.21 ± 0.13abc
Number of resorptions                         0.40 ± 0.24           1.09 ± 0.34a        1.72 ± 0.23a          1.03 ± 0.29a
 ost-implantation loss                        4.20 ± 0.41           13.73 ± 1.57a       30.28 ± 4.19ab        46.69 ± 5.21abc
 oetal weight (g)                             3.54 ± 41             3.46 ± 0.29         3.08 ± 0.37           2.53 ± 0.31
 lacental weight (g)                          0.67 ± 0.08           0.71 ± 0.09a        0.79 ± 0.19a          0.86 ± 0.12a
 rown-rump length (cm)                        3.18 ± 0.19           3.01 ± 0.27         2.78 ± 0.31           2.61 ± 0.23
 alues represent mean ± S.E. of 10 rats in each group. The significance of the difference among various groups was evaluated
 y applying one-way ANOVA (p<0.05). Comparison between two groups: avs control, bvs 31 mg/kg bw/d, cvs 59 mg/kg bw/d.
 able 18 Results as reported by Kanojia et al., 1996.
                                              Control               31 mg/kg bw/d       59 mg/kg bw/d         76 mg/kg bw/d
Gross abnormalities
Number of pups/litter observed                72/10                 70/10               51/10                 19/10
Drooping wrist                                0                     0                   0                     6/4 (32)
 ub-dermal hemorrhagic patches                0                     0                   8/6 (16)              8/4 (42)a
Kinky tail                                    0                     0                   0                     8/6 (42)a
 hort tail                                    0                     0                   4/4                   10/4 (53)a
 keletal abnormalities
Number of foetuses/litter observed            48/10                 45/10               34/10                 19/10
 educed parietal ossification                 0                     0                   0                     12/10 (63)a
 educed inter-parietal ossification           0                     0                   0                     10/10 (53)a
 educed caudal ossfication                    6/4 (12)              8/5 (18)            18/8 (53)a            18/10 (95)a
Gross and skeletal abnormalities are represented as number of abnormal pups/litter observed; percentage in parentheses
 alculated by the total number of pups observed. Statistical significance evaluated by Fisher’s Exact test; comparison between
wo groups: avs. Control (p<0.05).
             Toxicity for reproduction                                                                                        59
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<pre>             In another study using the same exposure levels of chromium in drinking water,
             Kanojia et al.14 (for details see section ‘effects on fertility’) reported that at 500
             and 750 mg/L (equivalent to 89 and 124 mg/kg bw/d) 15% and 10% of animals,
             respectively, died within 14 days of start of exposure. Gestational weight gain of
             dams was decreased in all dose groups. The number of live foetuses/litter, foetal
             weight and crown-rump length were decreased in all treated groups in a dose-
             related manner. The number of resorptions, and pre- and post-implantation loss
             were increased at all chromium VI dose levels with a dose-response relationship.
             Placental weight was decreased at 500 and 750 mg/L. In foetuses subdermal
             haemorrhagic patches on the thoracic and abdominal region were noted in all
             dose groups with a dose-relationship compared to none in controls. No visceral
             abnormalities were seen. A dose-dependent reduced ossification of caudal bones
 able 19 Results as reported by Kanojia et al., 1998.
                                              Control               45 mg/kg bw/d       89 mg/kg bw/d        124 mg/kg bw/d
 ody weight (g)                               182 ± 10              168 ± 14            150 ± 16             138 ± 15a
Weight gain in mothers (g)                    69.16 ± 3.66          61.66 ± 4.23        57.50 ± 2.82a        54.16 ± 3.11a
Number of live foetuses per litter            9.30 ± 0.92           7.30 ± 0.53         5.45 ± 0.42ab        4.16 ± 0.31abc
Number of resorptions per litter              0.53 ± 0.25           1.02 ± 0.30         1.41 ± 0.37a         1.67 ± 0.34a
 re-implantation loss (%)                     6.38 ± 0.63           12.97 ± 0.75a       17.64 ± 0.45ab       20.05 ± 0.45abc
 ost-implantation loss (%)                    5.39 ± 0.35           12.25 ± 0.63a       20.25 ± 0.54ab       22.80 ± 0.91abc
 oetal weight (g)                             4.36 ± 0.28           3.44 ± 0.22a        3.07 ± 0.16a         2.75 ± 0.22a
 lacental weight (g)                          0.72 ± 0.06           0.67 ± 0.07         0.52 ± 0.05a         0.37 ± 0.08ab
 rown-rump length (cm)                        3.68 ± 0.16           3.15 ± 0.13         2.76 ± 0.09 a        2.65 ± 0.16ab
 alues represent mean ± S.E. of 10 rats in each group. The significance of the difference among various groups was evaluated
 y applying one-way ANOVA (p<0.05). Comparison between two groups: avs control, bvs 89 mg/kg bw/d, cvs 124 mg/kg bw/d.
 able 20 Results as reported by Kanojia et al., 1998.
                                              Control               45 mg/kg bw/d       89 mg/kg bw/d        124 mg/kg bw/d
Gross abnormalities
Number of pups/litter observed                77/10                 63/10               46/10                25/10
Drooping wrist                                0                     3/3 (5)a            7/5 (15)ab           7/5 (25)ab
 ub-dermal hemorrhagic patches                0                     5/4 (8)a            8/6 (17)ab           6/6 (24)ab
Kinky tail                                    0                     0                   5/3 (11) ab          5/3 (20)ab
 hort tail                                    0                     0                   6/4 (13)ab           12/7 (48)abc
 keletal abnormalities
Number of foetuses/litter observed            42/10                 35/10               29/10                22/10
 educed parietal ossification                 0                     0                   7/6 (24)ab           22/9 (50)abc
 educed inter-parietal ossification           0                     0                   8/6 (28)ab           10/8 (45)abc
 educed caudal ossfication                    5/3 (12)              9/6 (25)a           17/7 (59)ab          20/9 (91)abc
Gross and skeletal abnormalities are represented as number of abnormal pups/litter observed; percentage in parentheses
 alculated by the total number of pups observed. Statistical significance evaluated by Fisher’s Exact test (p<0.05); comparison
 etween two groups: avs. Control, bvs 250 mg/L, cvs 500 mg/L.
 0           Chromium VI compounds
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<pre>was observed in all dose groups, while reduced ossification of parietal and
interparietal bones was seen at 500 and 750 mg/L only.
Elsaieed and Nada administered 0 or 50 mg chromium VI/L as potassium
dichromate in their drinking water (equivalent to 7 mg/kg bw/d) to pregnant
Wistar rats during gestation day 6-15.32 Dams were sacrificed on gestation day
20. Gestational weight gain was decreased 40% relative to control in chromium
VI treated dams, which may be related to the foetal toxicity observed. Pre- and
post-implantation losses and the number of resorbed and dead foetuses per litter
were increased compared to controls. Foetal weight was decreased by 33% and
the number of visceral (renal pelvis dilatation) and skeletal (incomplete
ossification of skull bone) anomalies per litter were increased. Exposed dams
showed an increase of chromium levels in blood, placental and foetal tissues.
Table 21 Results as reported by Elsaieed and Nada, 2002.
                                                   Control      7 mg kg/bw/d
Gestation weight gain (g)                          23.6 ± 1.3   14.2 ± 1.7a
Number of corpora lutea/litter                     7 ± 0.49     7.1 ± 0.48a
Number of pre-implantation loss/litter             0            2.1 ± 0.36a
Number of post-implantation loss/litter            0            1.5 ± 0.34a
Number of resorbed foetuses/litter                 0            1.2 ± 0.13a
Number of dead foetuses/litter                     0.1 ± 0.099  1.2 ± 0.24a
Number of live foetuses/litter                     6.8 ± 0.44   1.5 ± 0.29a
Foetal weight (g)                                  3.9 ± 0.42   2.6 ± 0.23a
Number of visceral anomalies/litter                0            2.1 ± 0.39a
Number of skeletal anomalies/litter                0            1.0 ± 0.34a
a   significantly different at p<0.05
Soudani et al. studied the effects of potassium dichromate on liver and kidney
function of female rats and their progeny and on the bones of offspring.33-35
Female Wistar rats (6 animals/group) received 700 mg/L potassium dichromate
in their drinking water (equivalent to 24 mg Cr/kg bw/d) from gestation day 14 to
postnatal day 14. Pups were culled to 4/sex after parturition. No mortality or
clinical signs were observed in the treated group. A reduction in body weight was
observed in both mothers and offspring. An increase of relative liver and kidney
weight was observed in the pups of exposed dams. Renal and hepatic toxicity
was measured in both mothers and pups by biochemical, urinary and histological
analysis.
    In pups, the femur weight and length was decreased compared to controls.
Bone calcium and phosphorus levels of pups were decreased. Calcium was
Toxicity for reproduction                                                         61
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<pre>              increased in plasma and decreased in urine, while phosphorus was decreased in
              plasma and increased in urine. Impairment of bone function was confirmed
              histologically.
              Potassium dichromate administered at 0, 100, 200 and 400 mg/kg diet
              (equivalent to 0, 7, 14 and 30 mg Cr/kg bw/d) to BALB/c mice in a NTP RACB
              (Reproductive Assessment by Continuous Breeding)-study did not influence the
              number of live pups per litter, proportion of pups born alive, sex ratio and live
              pup weight (for additional data see under fertility).18 For the last litter produced
              in the continuous breeding phase – treated with potassium dichromate from birth
              (via lactation) and after weaning via the diet – litter parameters were
              investigated. Mean average pup weights of the parents (F1) were slightly less
              (9-16%; not statistically significant) than controls on postpartum day 14 and 21
              for both sexes. At 21 days postpartum after culling, males of the high dose group
              had lower body weights. At 74 days postpartum at the start of mating, males of
              the high dose group and females of the mid and high dose group had lower body
 able 22 Results as reported by the NTP, 1997.
                                        Control           7 mg Cr/kg          14 mg Cr/kg       30 mg Cr/kg         Trend
                                                          bw/d                bw/d              bw/d
 verage litters per pairabd             3.8 ± 0.2 (20)    4.1 ± 0.1 (20)      3.7 ± 0.2 (20)    3.8 ± 0.2 (20)      p=0.554
 ive pups per littercd
   Male                                 3.0 ± 0.2 (20)    3.0 ± 0.2 (20)      2.8 ± 0.2 (20)    2.7 ± 0.2 (20)      p=0.191
   Female                               3.6 ± 0.2 (20)    3.8 ± 0.1 (20)      3.7 ± 0.2 (20)    3.2 ± 0.2 (20)      p=0.150
   Combined                             6.6 ± 0.3 (20)    6.7 ± 0.2 (20)      6.5 ± 0.3 (20)    5.9 ± 0.3 (20)      p=0.081
 roportion of pups born alivecd         0.99 ± 0.00 (20) 0.96 ± 0.01 (20)* 0.96 ± 0.02 (20) 0.96 ± 0.01 (20) p=0.393
 ex of pups born aliveed                0.45 ± 0.02 (20) 0.44 ± 0.02 (20) 0.43 ± 0.02 (20) 0.46 ± 0.03 (20) p=0.966
 ive pups weight (g)cd
   Male                                 1.52 ± 0.03 (20) 1.50 ± 0.02 (20) 1.45 ± 0.02 (20) 1.53 ± 0.03 (20) p=0.928
   Female                               1.56 ± 0.08 (20) 1.50 ± 0.03 (20) 1.44 ± 0.02 (20) 1.49 ± 0.03 (20) p=0.305
   Combined                             1.51 ± 0.02 (20) 1.49 ± 0.02 (20) 1.44 ± 0.02 (20)) 1.52 ± 0.03 (20) p=0.630
Adjusted live pup weight (g)fg                                                                                      Overall
   Male                                 1.52 ± 0.02 (20) 1.51 ± 0.02 (20) 1.46 ± 0.02 (20) 1.50 ± 0.02 (20) p=0.214
   Female                               1.56 ± 0.04 (20) 1.52 ± 0.05 (20) 1.44 ± 0.04 (20) 1.46 ± 0.05 (20) p=0.246
   Combined                             1.51 ± 0.02 (20) 1.51 ± 0.02 (20) 1.45 ± 0.02 (20) 1.49 ± 0.02 (20) p=0.090
 p<0.05
    Only pairs surviving to the end of task 2 were included for statistical analysis of data.
    Mean ± standard error (number of cohabited pairs).
    Mean ± standard error (number of fertile pairs).
    Each dose group is compared to the control group with Shirley’s test when a trend is present (p<0.01 from Jonckeere’s
    trend test), otherwise Dunn’s test is used.
    Mean ± standard error (number of fertile pairs producing live pups).
    Least squares estimate of the mean of the average pup weight from each fertile pair, adjusted for average litter size ±
    standard error (number of fertile pairs producing live pups).
    Overall p-values from an F-test for equality of group means. Dunnett’s test was used to compared each dosed group to the
    control group.
 2            Chromium VI compounds
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<pre>             weights, whereas feed consumption was increased for all dose-groups. Body
             weights of males stayed lower than controls for the final 4 weeks of the test.
             Three blood parameters (mean corpuscular volume, mean corpuscular
             haemoglobin and mean haemoglobin) were decreased in all dose groups at
             necropsy. For the F2 generation no differences were observed in number of live
             pups per litter, proportion of pups born alive, sex ratio and adjusted live pup
             weight.
             Trivedi et al. administered 0, 250, 500 and 1,000 mg/L in drinking water
             (probably specified as doses chromium and administered as potassium
             dichromate, equivalent to 0, 59, 120 and 234 mg Cr/kg bw/d) to ITRC mice
             (n = 10-13) during the entire gestation period.19 Dams were sacrificed on
             gestation day 19 and foetuses were examined. Dams at 234 mg/kg bw showed a
             body weight loss, while dams at 120 mg/kg bw showed a reduced body weight
             gain. No effects on clinical signs were noted. At the highest dose, no
             implantations were observed. In the 59 and 120 mg/kg bw group an increased
             number of resorptions and post-implantation loss, a decreased litter size, foetal
             weight and length were observed with a dose dependence. Furthermore, a
             retarded ossification of skull bones was observed. Additionally, in the 120 mg/kg
             bw group an increase in external abnormalities (kinking of tail, subdermal
             haemorrhagic patches), retarded ossification of fore- and hindlimb phalanges,
             vertebrae and sternebrae was noted.
 able 23 Results reported by Trivedi et al.,1989.
                                              Control          59 mg/kg bw/d        120 mg/kg bw/d       234 mg/kg bw/d
Number of litters (number of females)         10 (10)          10 (13)              10 (12)              0 (10)
 ody weight gain (g)                          16.43 ± 2.10     13.5 ± 1.94          13.0 ± 0.65a*        -1.19 ± 2.8abc***
 itter size                                   6.3 ± 0.61       3.8 ± 0.73           3.5 ± 0.57a*         No implantation
 lacental weight (mg)                         134.0 ± 5.00     123.0 ± 4.37         126.0 ± 4.30         No implantation
 ex ratio (M/F)                               2.40/4.00        2.25/1.50            1.50/2.00            No implantation
 oetal weight (g)                             1.95 ± 0.08      1.32 ± 0.05 ***      1.09 ± 0.03a*** b** No implantation
 rown-rump length (CRL) (cm)                  2.87 ± 0.12      2.38 ± 0.08a***      2.05 ± 0.12 a*** b** No implantation
ncidences of resorptions (%)                  1 (10.0)         9 (32.69)a***        7 (51.70)a**         No implantation
 ostimplantation loss (%) (number of          1.66 ± 1.25 (1)  26.48 ± 1.46 *** (3) 88.09 ± 2.12ab*** No implantation
                                                                           a
ncidences)                                                                          (7)
 he values represent means +/- SEM of 10 mice/group. The significance of the difference among various groups was evaulated
 y applying one-way ANOVA, except for incidence of resorptions, where Fisher Exact Test was used for comparison.
 omparison between two groups: avs control; bvs 250 mg/L, cvs 500 mg/L.*p<0.05; **p<0.01, ***p<0.001.
             Toxicity for reproduction                                                                                     63
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<pre>             As part of a genotoxicity study, timed-pregnant Swiss albino mice (n = 5) were
             given sodium dichromate dihydrate or potassium dichromate up to a
             concentration of 10 mg CrVI/L drinking water throughout gestation.36 All dams
             were sacrificed on gestation day 18. Neither litter size nor foetal weights were
             affected by treatment (no further details were provided).
             Junaid et al. administered potassium dichromate at levels of 0, 250, 500 and 750
             mg Cr/L in drinking water (equivalent to 67, 125 and 182 mg Cr/kg bw/d) to
             Swiss mice (n = 10/group) during gestation days 6-14.37 No effect on clinical
             signs or behaviour of dams was noted. At the two highest dose levels, a reduced
             body weight gain was observed; no effect on placental weight was seen. In the 67
             mg/kg bw group an increased number of resorptions (p<0.05) and post-
             implantation loss (not significant) was noted. In the 125 mg/kg bw group a
             reduced number of foetuses, a reduced foetal weight, an increased number of
             resorptions, an increased post-implantation loss and an increased number of
             foetuses with reduced caudal ossification were observed. In addition, at 182
             mg/kg bw an increased number of foetuses with reduced ossification of several
             bones (nasal, frontal, parietal, interparietal, caudal, tarsal) and an increased
             number of foetuses with external abnormalities (drooping wrist and subdermal
             haemorrhagic patches) were observed. No effect on crown-rump length was
             noted.
 able 24 Results reported by Junaid et al.,1996.
                                              Control             67 mg/kg bw/d        125 mg/kg bw/d     182 mg/kg bw/d
Weight gain in mothers (g)                    15.57 ± 0.20        15.21 ± 0.31         14.29 ± 0.54a,b    11.79 ± 0.49a,b,c
Number of corpora lutea                       9.5 ± 0.42          9.80 ± 0.24          9.20 ± 0.40        9.80 ± 0.24
Number of foetuses (dead and live)/litter     8.8 ± 0.29          8.20 ± 0.20          7.00 ± 0.36ab      7.20 ± 0.24ab
Number of dead foetuses (number of litters) All alive             All alive            3 (2)              12 (7)
 oetal weight (g)                             1.31 ± 0.41         1.27 ± 0.03          1.14 ± 0.03ab      1.06 ± 0.029ab
 lacental weight (g)                          0.15 ± 0.007        0.15 ± 0.005         0.16 ± 0.006       0.15 ± 0.010
 rown-rump length (cm)                        2.82 ± 0.38         2.75 ± 0.03          2.67 ± 0.04        2.60 ± 0.044
Number of resorption sites                    0.30 ± 0.21         1.00 ± 0.21a         1.70 ± 0.3a,b      2.30 ± 0.273abc
 ost-implantation loss (%)                    4.32 ± 2.34         10.60 ± 2.11         21.93 ± 3.96a,b    34.60 ± 2.54abc
 alues represent mean ± S.E. of 10 rats in each group. The significance of the difference among various groups was evaluated
 y applying one-way ANOVA (p<0.05). Comparison between two groups: avs control, bvs 67mg/kg bw/d, cvs 125 mg/kg bw/d.
 4           Chromium VI compounds
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<pre> able 25 Results reported by Junaid et al.,1996.
                                             Control                67 mg/kg bw/d        125 mg/kg bw/d      182 mg/kg bw/d
  umber of pups/litter observed              40/10                  40/10                30/10               25/10
Drooping wrist                               0                      3/2 (7.5)            3/2 (10)            4/3 (16)a
 ub-dermal hemorrhagic patches               0                      0                    3/2 (10)            4/3 (16)a
Kinky tail                                   0                      0                    2/1 (6.66)          3/3 (12)
 hort tail                                   0                      0                    0                   2/1 (8)
 keletal abnormalities
Number of pups/litter observed               40/10                  40/10                30/10               25/10
  educed nasal ossfication                   0                      0                    0                   16/8 (32)a
  educed frontal ossification                0                      0                    0                   10/6 (40)a
  educed parietal ossification               0                      0                    0                   8/5 (32)a
  educed inter-parietal ossification         0                      0                    0                   10/7 (40)a
  educed caudal ossification                 1/1 (2.5)              3/1 (7.5)            14/5 (46.6)a        21/7 (84)a
  educed carpals ossification                0                      0                    0                   0
  educed metacarpals ossification            0                      0                    0                   0
  educed tarsals ossification                0                      0                    0                   19/8 (76)a
  educed claws ossification                  0                      0                    0                   0
Gross and skeletal abnormalities are represented as number of abnormal pups/litter observed; percentage in parentheses
 alculated by the total number of pups observed. Statistical significance evaluated by Fisher’s Exact test (p<0.05); comparison
 etween two groups: avs. Control.
             Elbetieha and Al-Hamood studied the effects of CrVI in female mice (n = 11-15)
             after exposure to 2000 and 5000 mg potassium dichromate/L in the drinking
             water (equivalent to 153 and 384 mg Cr/kg bw/d) during 12 weeks (see also
             under fertility).22 A reduced water consumption was noted for the highest dose
             group. The relative ovary weight was significantly increased at the highest dose
             group. No effect on the number of pregnant females was noted. However, the
             number of implantations and the number of viable foetuses were significantly
             reduced in the chromium VI treated female mice mated with untreated male
             mice. The number of mice with resorptions was significantly increased in both
             dose groups compared to the control group.
 able 26 Developmental effects reported by Elbetieha and Al-Hamood, 1997.
                                             Control                       153 mg/kg bw/d             384 mg/kg bw/d
Number of females                            18                            15                         11
Number of pregnant females                   17/18                         14/15                      9/11
Number of implantations                      9.00 ± 1.36 (17)              7.35 ± 1.54 (14)**         7.44 ± 1.50 (9)
Number of viable foetusesa                   8.76 ± 1.39 (17)              6.55 ± 2.18 (9)*           5.88 ± 2.47 (9)**
Number of mice with resorptions              2/18 (11%)                    8/15 (53%)***              7/11 (63%)****
 otal number of resorptions                  4                             37                         14
  p<0.05, ** p<0.01 (compared to control value, Student’s t-test); *** p<0.01, **** p<0.005 (compared to control value, Chi-
 quare test)
             Toxicity for reproduction                                                                                       65
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<pre>  Al-Hamood et al. exposed female Swiss mice from day 12 of gestation till day 20
  of lactation to 0 or 1000 mg/L of potassium dichromate in their drinking water
  (equivalent to 71 mg Cr/kg bw/d).38 Pups were culled to 8/litter at birth and
  weaned on postnatal day 21. At postnatal day 60 treated male offspring was
  mated with untreated females (1:2) and vice versa. Male fertility was not affected
  as measured by the number of pregnant females; the number of viable foetuses in
  females impregnated by exposed males was not affected. Female fertility was
  affected as shown by a reduced number of pregnant females, implantations and
  viable foetuses and increased number of resorptions. Vaginal opening was
  delayed. Another group of pups sacrificed at postnatal day 50 showed no effect
  on body weight, and weight of ovaries, uterus, testes, seminal vesicles and
  preputial glands.
  Table 27 Developmental effects reported by Al-Hamood et al., 1998.
                                  Control                         71 mg/kg bw/d
  Number of animals               12                              22
  Number of pregnant animals      12/12 (100%)                    14/22 (63.6%)
  Number of implantations         10.25 ± 1.48                    9.07 ± 1.14*
  Number of viable foetuses       10.25 ± 1.48                    8.85 ± 1.56*
  Total number of resorptions     0                               3
  * p<0.05 Student’s t-test
  In a recent study, Sivakumar et al. exposed pregnant rats (n = 5) to 25 mg/L
  potassium dichromate in drinking water (equivalent to 1 mg Cr/kg bw/d) from
  gestational day 9.5 to 14.5.51 Ovaries were removed from the F1 offspring on
  post natal day 1 and analyses were performed. Increased germ cell apoptosis was
  observed and was correlated with up-regulating of proteins associated with
  apoptosis. Also accelerated germ cell cyst breakdown was shown, together with
  advanced primordial follicle assembly and primary follicle transition and
  downregulation of survival pathway proteins. According to the authors, these
  event led to early reproductive senescence and a decrease in litters size in F1
  female progeny.
  Intraperitoneal or intravenous administration
  As the Committee considers the intraperitoneal and intravenous route of
  administration of limited relevance for classification, no summarising tables are
  provided for these studies.
6 Chromium VI compounds
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<pre>Marouani et al. have investigated the embryo- and foetotoxicity of potassium
dichromate in rats.39 Female Wistar rats (n = 8) were administered 0, 1 or 2 mg
Cr/kg bw/d as potassium dichromate in saline intraperitoneally from day 6 to15
of gestation. Treated dams showed no mortality or changes in clinical signs, but a
significant reduction in body weight gain (to 71 and 40% of control at 1 and 2
mg/kg bw/d, respectively) and relative weight of the uterus (79 and 70% of
control at 1 and 2 mg/kg bw/d, respectively) was seen. A significantly reduced
number of foetuses (dead and alive)/litter, increased number of dead foetuses and
litters with dead foetuses, reduced foetal weight and crown rump length were
noted at both dose levels. The number of resorptions was increased at both doses.
Consequently, post implantation loss had significantly increased in a dose-
dependent manner. Placenta weight was also significantly reduced at both dose
levels. External examination of the foetuses revealed oedema, and subdermal
haemorrhagic patches on the thoracic and abdominal regions at both doses. At
2 mg/kg bw/d effects were more severe and also facial defect, lack of tail and
hypotrophy were observed. Skeletal abnormalities observed by radiography at
1 mg/kg bw/d included incomplete ossification in nasal, cranium, abdominal or
caudal bones, while at 2 mg/kg bw/d also absence of ossification of the sacral
vertebrae was noted. Microscopic inspection revealed atrophy of organs such as
liver, lung, heart and sacral vertebrae at 2 mg/kg bw/d. Histopathological
examination of the placenta showed dose-dependent atrophy of decidual cells,
degeneration of chorionic villi and hypertrophy of blood lacuna.
Endo and Watanabe dosed Jcl:ICR mice intraperitoneally on gestation day 9 with
0 or 15 mg chromium trioxide/kg bw (equivalent to 0 or 8 mg Cr/kg bw).40 Dams
were killed on gestation day 17 and foetuses were examined for external
malformations, sexed and about one third of the foetuses were examined for
skeletal malformations and variations. No effect was observed on number of live,
dead or resorbed foetuses, male and female foetal weight or number of
malformations.
Gale treated golden hamsters (LAK:LVG(SYR) intravenously with 0, 5, 7.5, 10
and 15 mg chromium trioxide/kg bw (equivalent to 0, 3, 4, 5 or 8 mg Cr/kg bw)
on day 8 of gestation.41 Dams were killed on gestation day 12, 14 or 15 and all
foetuses were examined for external malformations. Of the 15-day-old foetuses
approximately half were examined for visceral abnormalities and half for
skeletal abnormalities. Some of the 14- and 15-day-old foetuses were examined
by dissection of the abdominal, thoracic and cephalic region. The high dose
induced severe maternal mortality shortly after treatment; 3 out of 4 females
Toxicity for reproduction                                                          67
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<pre>      died. At 4 and 5 mg/kg bw weight loss, mottled kidneys with extensive tubular
      necrosis and hyaline casts was described; some livers exhibited clear vacuoles in
      some of the periportal hepatocytes. A dose-related increased number of
      resorptions and foetal abnormalities, including cleft palates and skeletal defects,
      and hydrocephalus was observed. At all dose levels retarded ossification of the
      vertebral column and sternum, skull (4 and 5 mg/kg bw), hyoid bone, hind limb,
      fore limb (4 and 5 mg/kg bw) was noted.
      Gale and Bunch dosed hamsters intravenously with a single injection of 0 and
      8 mg chromium trioxide/kg bw (equivalent to 4 mg Cr/kg bw) on gestation days
      7, 8, 9, 10 or 11.42 Dams were killed on gestation day 15 and foetuses were
      examined for external and internal malformations. All chromium-treated dams
      lost weight with the most severe effect in dams dosed on gestation day 7. The
      kidneys of most treated dams were mottled with varying degrees of tubular
      necrosis. Examination of pups showed after dosing on gestation days 7, 8, 9 and
      10 a decreased average crown-rump length compared to controls. From dams
      treated on gestation day 7, 8 and 9, an increased number of live foetuses with
      external defects and cleft palates were observed. Based on the average crown-
      rump length, the chromium-exposed foetuses with cleft palates are smaller than
      the chromium-treated foetuses with normal palates. Internal defects observed
      were small or absent kidney in one and four foetuses after dosing on gestation
      day 7 and 8, respectively, compared to none in the control groups. No effects
      were observed after dosing on gestation days 11.
5.2.2 Human studies
      In the human studies on exposure to chromium and other metals present in
      welding fumes, exposure levels were not determined.
      Bonde investigated the effects on offspring among male welders in a nationwide
      Danish cohort consisting of male workers employed for at least 1 year at 79
      Danish stainless steel or mild steel manufacturing companies from April 1964
      through December 1984 (n = 10,059), who fathered 3569 children in 1973
      through 1986.31 Occurrences of low birth weight, pre-term delivery, infant
      mortality and congenital malformations were not increased among children at
      risk from either paternal stainless steel or mild steel welding. Increased
      occurrences of any organ-specific congenital malformation were not observed
      either.
 8    Chromium VI compounds
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<pre>Hjollund et al. recruited a cohort of first-pregnancy planners from members of
the union of metal workers and three other trade unions in Denmark.43 The
cohort (n = 406) was followed for 6 menstrual cycles from cessation of
contraceptive use. In total, 280 pregnancies were conceived, of which 203 had a
paternal background without welding exposure, 54 with mild steel welding (but
not stainless steel) and 23 with stainless steel welding. Of the spontaneous
abortions, 36 were clinically diagnosed and 35 were detected by human
chorionic gonadotrophic hormone analysis but did not survive to a clinically
recognized pregnancy. The relative risk for spontaneous abortion from paternal
exposure to stainless steel welding (10 cases) was increased in comparison with
pregnancies without paternal welding exposure (RR 3.5, 95% CI 1.3-9.1),
adjusted for the confounders centre, female age, female body mass index,
menstrual cycle length, male and female smoking, caffeine and alcohol
consumption, and reproductive disease. Separate analysis of early pregnancy loss
and loss of clinically recognized pregnancies also showed increased risks (RR
3.0, 95% CI 1.1-8.0, and RR 3.2, 95% CI 1.1-9.8, respectively). All spontaneous
abortions in spouses of stainless steel workers happened before the 10th
gestational week. The risk of pregnancy loss increased with the number of years
of stainless-steel welding, being 2.6 (95% CI 1.1-6.1) after 5 years. Mild steel
welding, which does not involve exposure to hexavalent chromium, was not
associated with an increased risk of pregnancy loss.
Hjollund et al. also investigated the association between welding exposure and
survival of pregnancies after in vitro fertilization (IVF).44 From the Danish IVF
register, couples with the first treatment after 1 January 1996 were selected after
completing a questionnaire (n = 4,007). Excluded were treatment with donated or
thawed eggs, fertilization with donor sperm and treatment cycles ending with
ectopic pregnancies, mola or induced abortion. A subgroup of male metal
workers received a second questionnaire on exposure to welding (n = 319) and
was classified as stainless steel welder (n = 91), mild steel welder (n = 128),
other welder or non-welder (n = 100). The proportion of miscarriages before the
28th gestational week was 17.6% (16 out of 91) in pregnancies where the father
welded stainless steel compared to 25.0% (32 out of 128) for mild steel welding
and 28.4% in the group of unexposed reference pregnancies (830 out of 2,925).
The risk ratios for miscarriages from pregnancies with paternal exposure to
stainless steel and mild steel welding were 0.6 (95% CI 0.4-1.0) and 0.95 (95%
CI 0.7-1.4), respectively, after adjustment for IVF center, male and female
smoking, coffee and alcohol consumption, age and number of transferred
embryos. Number of hours with stainless steel welding or number of years with
Toxicity for reproduction                                                           69
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<pre>            stainless steel welding had no effect on the estimates. Risk estimates were similar
            when based on analyses with spontaneous abortion defined as foetal death before
            gestational week 20. According to the authors, this result should not be
            extrapolated to natural pregnancies as the cohort was preselected by their
            reproductive problems and spontaneous abortion in IVF is only registered in case
            of death of all implanted embryos (usually the two most viable ones) resulting in
            an overestimation of foetal survival.
            Aschengrau et al., investigated potential associations between levels of drinking
            water contaminants, including chromium, and the occurrence of late adverse
            pregnancy outcomes in a case-control study.45 Trace element levels were
            gathered from routine analyses of public water supplies from the residing
            communities and compared between 1039 congenital anomaly cases, 77 stillbirth
            cases, 55 neonatal death cases, and 1177 controls. No differences were found
            between women residing in areas with detectable chromium levels, and those
            who resided in areas with undetectable chromium levels. The Committee notes
            that this study provides no information on actual chromium VI exposure.
5.3         Other relevant information
            Lactation
            Animal studies
 able 28 Developmental toxicity studies in animals after lactational exposure.
 uthors species experimental period/ dose/route              general toxicity    developmental toxicity
                   design
  anu    Rat;      Postnatal day 1-20; 0, 50, 100, 200 or 50 mg/kg: weight       25 and 50 mg/kg: decreased pup weight;
 t al.,  Wistar; pilot study             400 mg Cr/L as      loss; weakness;     follicle number, increased follicular atresia
 008     n not                           potassium           decrease intake of  NOAEL=12 mg Cr/kg bw/d
         specified                       dichromate in       food and water;
                                         drinking water      decrease uterus and
                                         (6, 12, 25 or 50    ovary weights;
                                         mg Cr/kg bw/d)/ 25 mg/kg: similar
                                         oral                symptoms but less
                                                             severe
                                                             NOAEL=12 mg
                                                             Cr/kg bw/d
  0         Chromium VI compounds
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<pre> anu   Rat;     Litters culled to 4   0 or 200 mg Cr/L No maternal          Delay in vaginal opening, extended dioestrous
t al., Wistar;  female pups; dams     as potassium       toxicity for main  phase of the oestrous cycle, At different days
008    n = 18   exposed on postnatal  dichromate in      study described    after partus: impaired follicle development,
                day 1-21; groups of   drinking water                        decreased plasma levels of of oestradiol,
                6 dams (= 24 pups)    (0 or 25 mg Cr/kg                     testosterone and progesterone; decreased
                sacrifice on PND 21,  bw/d)/oral                            growth hormone and prolactin plasma levels
                45 and 65; blood and                                        and increased follicle stimulating hormone
                ovary parameters;                                           LOAEL= 25 mg Cr/kg bw/d
                onset of puberty;
                follicle number
amuel  Rat;     During PND 1-21;      0, 50 or 200 mg    Decreased body     Decreased uterus weight at 200 mg; delayed
t al., Wistar;  at birth litters were Cr/L as potassium  weight at both     puberty at both doses (dose-related); extended
011    n = 12   culled to 4 female    dichromate in      doses, severely at oestrous cycle (dioestrous stage) at both
                pups/dam; pups        drinking water     25 mg/kg           doses; also extended metoestrous stage at 200
                sacrificed on PND     (6 or 25 mg Cr/kg  NOAEL=6 mg         mg; decreased uterine activity of antioxidants
                42 and 65             bw/d)/oral         Cr/kg bw/d         and serum testosterone and progesterone at
                                                                            both doses (dose-related); increased H2O2
                                                                            and lipid peroxidation, and FSH at both doses
                                                                            (dose-related)
                                                                            LOAEL=6 mg Cr/kg bw/d
tanley Rat; SD; During PND 1-21;      0, 50 100, or 200 No maternal         Increased follicular atresia and decreased
t al., n=5      at birth litters were mg Cr/L as         toxicity for main  steroidogenesis in PND 25, 45 and 65,
013             culled to 4 female    potassium          study described    Vitamin C (partly) inhibited these effects; ic
                pups/dam; pups        dichromate in                         hydrogen peroxide and lipid hydroperoxide in
                sacrificed on PND     drinking water                        plasma and ovary; decreased antioxidant
                25, 45 and 65         (0, 6, 12 or 25 mg                    enzymes (AOXs)GPx1, GR, SOD, and
                                      Cr/kg bw/d)/oral                      catalase; ic glutathione S-transferase in
                                                                            plasma and ovary
                                                                            LOAEL=6 mg Cr/kg bw/d
          Banu et al. investigated lactational exposure to chromium VI on the puberty of
          female offspring from Wistar rats.46 Litters from pregnant rats (n = 18) were
          culled to four female pups on the day of birth. Dams were given 0 or 200 mg/L
          potassium dichromate in the drinking water (equivalent to 25 mg Cr/kg bw/d)
          from the day of parturition to postnatal day 21. On postnatal day 21, 45 and 65
          each, female pups from 6 dams/group (n = 24) were sacrificed and blood and
          ovaries collected. Chromium levels in plasma and ovarian tissue were higher in
          exposed female offspring than in controls and decreased with aging of the pups.
          Vaginal opening was delayed (55 days in treated rats compared to 33 days in
          control rats). While the dioestrous phase of the estrous cycle was extended, the
          length of the pro-oestrous, oestrous and metoestrous phases was not affected. On
          postnatal day 21 with chromium CI treatment decreased primordial, primary and
          secondary follicle numbers and resulted in no antral follicle development. On
          postnatal day 45 all follicle numbers were decreased. On postnatal day 65 only
          primordial and primary follicle numbers were decreased. Plasma levels of
          oestradiol, testosterone and progesterone were decreased in all three age groups
          Toxicity for reproduction                                                                                     71
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<pre>  compared to controls. Chromium VI treatment did not change plasma luteinizing
  hormone, but increased follicle stimulating hormone on postnatal day 21 and 45.
  Both hormones were not affected on postnatal day 65. Growth hormone and
  prolactin plasma levels were decreased compared to controls in all three age
  groups. In the main study no maternal effects were described, but in the pilot
  study where dams were exposed to 50, 100, 200 or 400 mg/L potassium
  dichromate (6, 12, 25 or 50 mg Cr/kg bw/d) for postnatal day 1-20, severe toxic
  symptoms such as weight loss, weakness, and reduced intake of food and water,
  reduced uterus and ovary weights were observed at 50 mg Cr/kg bw, while these
  symptoms were low to moderate at 25 mg Cr/kg bw. Simultaneous
  administration of vitamin C (500 mg/L) protected against the CrVI
  developmental effects.
  The same group hypothesized that lactational exposure to chromium VI would
  induce oxidative stress and disrupt uterine function in female offspring from
  Wistar rats.47 Litters from pregnant rats (n = 12) were culled to four female pups
  on the day of birth. Dams were given 0, 50 or 200 mg/L potassium dichromate in
  the drinking water (equivalent to 6 and 25 mg Cr/kg bw/d) from the day of
  parturition to postnatal day 21. On postnatal day 42 and 65 each, female pups
  from 6 dams/group (n = 24) were sacrificed and blood and uterus collected.
  Chromium levels in plasma and uterine tissue were higher in exposed female
  offspring than in controls and decreased with aging of the pups. Pup weight was
  decreased in exposed groups in a dose-related manner at postnatal day 42 and 65.
  Uterus weight was decreased at 200 mg/L only at both times. As in the former
  study the dioestrous phase of the oestrous cycle was extended at both dose levels,
  while the metoestrous stage was extended at 200 mg/L. Uterine activities of
  superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase
  and glutathione S-transferase were decreased at both dose levels with only partial
  recovery at postnatal day 65. Increased concentrations of lipid peroxidation and
  hydrogen peroxide were observed in chromium VI-treated pups with higher
  levels at postnatal day 65. The steroid hormones (testosterone, oestradiol and
  progesterone) showed a dose-related decrease in both age groups. Follicle
  stimulating hormone was elevated at both dose levels and age groups.
  Luteinizing hormone was only markedly increased at 200 mg/L at postnatal day
  45.
  In a subsequent study with a similar design, this research group tested the
  hypothesis that lactational exposure to potassium dichromate accelerates follicle
  atresia in F1 offspring by increasing reactive oxygen species and decreasing
2 Chromium VI compounds
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<pre>    cellular antioxidants.52 The results showed that lactational exposure to potassium
    chromate increased follicular atresia and decreased steroidogenesis at postnatal
    day 25, 45, and 65 in a dose dependent manner. In addition, potassium chromate
    increased hydrogen peroxide and lipid hydroperoxide in plasma and ovary and
    decreased several antioxidant enzymes while glutathione S-transferase was
    increased.
    In a study of Al-Hamood et al.38 mice were exposed from day 12 of gestation till
    day 20 of lactation and in the study of Soudani et al.33-35 rats were exposed from
    gestation day 14 to postnatal day 14. The effects observed are described under
    developmental toxicity. Since it is not clear whether effects observed are due to
    exposure in utero or via lactation, these studies were not considered for drawing
    conclusions on labelling for lactation.
    Human studies
    Chromium concentrations in human breast milk within Europe range from 0.09
    to 19.8 µg/L as presented in the review of University of East Anglia.48 Two
    studies from this review measured maternal chromium intake. In the first study, a
    mean maternal chromium intake of 41.08 ± 0.416 mg/day (n = 17) was found
    with a concomitant chromium level of 0.184 ± 0.021 µg/L in breast milk
    (number of samples not specified). In the second study, the mean maternal
    chromium intake was 256 ± 187 mg/day (n = 19) with a chromium level of 10.8
    µg/L (range 3.1-19.4 µg/L; 536 samples) in breast milk. Both studies showed no
    direct correlation between chromium intake and breast milk concentration. This
    was confirmed by an isotope study in which mothers were given 53Cr orally,
    which was, subsequently, not found in their breast milk. No reliable data on
    occupationally exposed women are available.
5.4 Summary and discussion of reproductive toxicity
    Fertility and developmental effects of chromium VI have been studied in humans
    and animals.
    Human studies investigated male and female fertility parameters in relation to
    stainless steel welding, which involves chromium VI exposure in the
    fumes.12,27-31,49 Inconsistent results were described and in all cases co-exposure
    to other substances was present in the fumes.
    Toxicity for reproduction                                                          73
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<pre>                   In multiple species, effects on the gonads were observed after exposure to
               chromium VI; the chromium VI was administered as sodium dichromate,
               potassium dichromate or chromium trioxide (one study only). In two studies in
               monkeys, and two studies in rabbits, various sperm parameters were affected
               (involving a decrease in testis weight, disruption of spermatogenesis, and a
               decrease in sperm number and motility).10,11,23,26,27 Sperm quality and quantity
               were also affected in several studies with rats and mice.12,15,20,22,24 In contrast,
               two NTP-studies in rats and three in mice did not show any adverse effects on
               sperm.3,5,16-18,50 The Committee notes that in rats the NTP applied relatively low
               doses of chromium compared to other oral studies, and only mild signs of
               general toxicity were observed and only at the highest dose (i.e. effects on blood
               parameters, small decreases of body weight and absolute liver weight,
               cytoplasmic vacuolisation in hepatocytes). In mice, the oral results were
               inconsistent as one study20 showed sperm effects at dose levels of 11 mg Cr/kg
               bw/day and above, which did not induce any effects in the NTP studies.
               However, effects on testes and fertility were observed in the only study with 148
               mg Cr/kg bw/day and above.22 In this study, a correlation was observed of
               decreased seminal vesicle and preputial gland weight with a decreased number of
               implantations and number of viable foetuses when non-exposed female mice
               were mated with exposed males. Overall, all four tested species show clear
               effects on testes, sperm counts and/or fertility. The absence of effects in some
               studies can at least partly be explained by the low dose levels tested.
 able 29 Summary of effects on fertility in male animals.
Males                  Species    Substance           Exposure route General toxicity       Effects on fertility
 ousef et al., 2006 Rabbit        Potassium           Oral (gavage)  NOAEL=4 mg/kg bw/d     LOAEL=4 mg/kg bw/d
                                  dichromate                         (no effects observed)  (various sperm parameters)
 ehari et al., 1978 Rabbit        Potassium           i.p.           NOAEL=0.7 mg/kg bw/d   LOAEL=0.7 mg/kg bw/d
                                  dichromate                         (no effects observed)  (number of spermatocytes)
 ubramanian et al., Monkey Potasium                   Oral (drinking NOAEL=23 mg/kg bw/d    NOAEL<6 mg/kg bw/d
 006; Aruldas et al.              dichromate          water)         (no effects observed)  (several sperm parameters)
 005
Glaser et al., 1984    Rat        Sodium dichromate Inhalation       NOAEL=0.2 mg/kg bw/d   NOAEL=0.2 mg/kg bw/d
                                                                     (no effects observed)  (no effects observed)
 i et al., 2001        Rat        Chromium trioxide Oral (diet)      NOAEL=10 mg/kg bw/d    LOAEL=5 mg/kg bw/d
                                                                     (no effects observed)  (several sperm parameters)
 howdhur and           Rat        Sodium dichromate Oral (gavage)    NOAEL=8 mg/kg bw/d     LOAEL=8 mg/kg bw/d
Mitra, 1995                                                          (decreased body weight (reduction testicular
                                                                     gain)                  protein and serum,
                                                                                            testosterone)
NTP, 1996; 2007        Rat        Potassium           Oral (diet)    NOAEL=2 mg/kg bw/d     NOAEL=9 mg/kg bw/d
                                  dichromate                         (haematotoxicity)      (no effects observed)
 4             Chromium VI compounds
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<pre> rnst, 1990             Rat       Sodium chromate     i.p.           LOAEL= 1 mg/kg bw/d        LOAEL=1 mg/kg bw/d
                                                                     (decreased body weight     (number of testicular
                                                                     gain)                      parameters (cellular and
                                                                                                morphology))
 rnst and Bond,         Rat       Sodium chromate     i.p.           NOAEL= 0.5 mg/kg bw/d LOAEL=0.5 mg/kg bw/d
 992                                                                 (no effects observed)      (hormones; sperm
                                                                                                motility)
 ahid et al., 1990      Mouse     Potassium           Oral (diet)    NOAEL=46 mg/kg bw/d LOAEL=11 mg/kg bw/d
                                  dichromate                         (no effects observed)      (several sperm parameters)
NTP, 1996               Mouse     Potassium           Oral (diet)    NOAEL=1 mg/kg bw/d         NOAEL=41 mg/kg bw/d
                                  dichromate                         (haematotoxicity)          (no effects observed)
NTP, 1997               Mouse     Potassium           Oral (diet)    NOAEL F0=30 mg/kg          NOAEL F0=30 mg/kg bw
                                  dichromate                         bw/d (no effects observed) (no effects observed)
                                                                     NOAEL F1=8 mg/kg bw/d NOAEL F1=37 mg/kg bw/
                                                                     (haematotoxicity)          d (no effects observed)
NTP, 2007               Mouse     Potassium           Oral (diet)    LOAEL=3 mg/kg bw/d         NOAEL=9 mg/kg bw/d
                                  dichromate                         (haematotoxicity)          (no effects observed)
 lbetieha and Al-       Mouse     Potassium           Oral (drinking NOAEL=74 mg/kg bw/d NOAEL=74 mg/kg bw/d
Hamood, 1997                      dichromate          water)         (reduced body weight)      (testis weight; number of
                                                                                                implantations)
                   For female reproduction toxicity, several studies with rats and mice are
               available with different results. Effects on the oestrous cycle were observed in
               rats and mice.13,14,21 Further, a reduced mating index was observed in female
               rats.13,14 In studies with female mice, also effects on ovary were observed, and a
               reduced number of implantations. In contrast, no histological lesions were
               observed in the ovaries of rats and mice in studies by the NTP.5,16,17 Also no
               effect was observed on fertility of the F0- and F1-generation in an NTP
               continuous breeding study with mice and no effect on the oestrous cycle or
               gonadal organ weights were noted either.18 However, the highest dose levels
               (30 and 41 mg Cr/kg bw/d) applied in the NTP studies were below the LOAEL
               (60 mg Cr/kg bw/d and above) in the oral studies that showed reductions in
               implantations and decreased number of foetuses in mice.
 able 30 Summary of effects on fertility in female animals.
 emales                 Species   Substance           Exposure route     General toxicity         Effects on fertility
Kanojia et al., 1996 Rat          Potassium           Oral (drinking     LOAEL=31 mg/kg bw/d      LOAEL=31 mg/kg bw/d
                                  dichromate          water)             (body weight)            (reduced mating index)
Kanojia et al., 1998 Rat          Potassium           Oral (drinking     NOAEL=45 mg/kg bw/d      LOAEL=45 mg/kg bw/d
                                  dichromate          water)             (mortality)              (persistent dioestrous
                                                                                                  phase)
NTP, 1996; 2007         Rat       Potassium           Oral (drinking     LOAEL=4 mg/kg bw/d       NOAEL = 21 mg/kg
                                  dichromate          water)             (haematotoxicity)        bw/d (no abnormalities
                                                                                                  ovaries)
               Toxicity for reproduction                                                                                 75
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<pre>NTP, 1996; 2007         Mouse    Potassium         Oral (drinking   NOAEL=1 mg/kg bw/d NOAEL = 41 mg/kg
                                 dichromate        water)           (haematotoxicity)      bw/d (no abnormalities
                                                                                           ovaries)
 rivedi et al., 1989    Mouse    Potassium         Oral (drinking   NOAEL=59 mg/kg bw/d NOAEL=59 mg/kg
                                 dichromate        water)           (reduced body weight) bw/d (pre-implantation
                                                                                           loss)
Murthy et al., 1996     Mouse    Potassium         Oral (drinking   Sub-acute: NOAEL=163 Sub-acute: LOAEL=54
                                 dichromate        water)           mg/kg bw/d (no effects mg/kg bw/d (decreased
                                                                    observed)              number follicles, ova,
                                                                    90-d: NOAEL=1.1        etc.)
                                                                    mg/kg bw/d (no effects 90-d: NOAEL=0.1 mg/
                                                                    observed)              kg bw/d (abnormailities
                                                                                           follicular and fecal cells)
 lbetieha and           Mouse    Potassium         Oral (drinking   NOAEL=74 mg/kg bw/d NOAEL=74 mg/kg bw/d
Al-Hamood, 1997                  dichromate        water)           (reduced body weight) (ovary weight; number
                                                                                           of implantations)
                   In conclusion, animal studies show effects on structural and functional
               fertility parameters in males and females of several species after exposure to
               chromium VI compounds, although a relation of the effects on structural
               parameters with functional fertility is not clear. Therefore, classification of
               chromium VI compounds for effects on fertility is warranted.
               With regard to developmental toxicity, one of the three human studies available
               indicates an increased risk after exposure to stainless steel welding.31,43,44
               However, no conclusions can be drawn as the stainless steel welding fumes have
               not been analysed for chromium VI and co-exposures to other toxic substances
               occurred.
                   Developmental toxicity studies with chromium VI in rats, mice and hamsters
               are available, exposed via various routes. Developmental toxicity studies with
               chromium VI in rats and mice have shown effects of exposure via drinking water
               pre-mating (mice) and during gestation (both species). Male or female mice
               exposed before mating resulted in an increased number of resorptions, reduced
               number of implantations and viable foetuses.13,14,22 After oral gestational
               exposure in rats and mice, clear adverse effects on offspring were seen in
               absence of maternal toxicity (i.e. increased number of resorptions, pre- and post-
               implantation loss, reduced litter size, foetal weight and length, and retarded
               ossification).19,32,37-39 Furthermore, an increased number of cleft palate, skeletal
               defects and hydrocephalus were noted after intravenous administration of
               chromium trioxide hamster in the absence and presence of maternal toxicity.41,42
 6             Chromium VI compounds
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<pre> able 31 Summary of effects on development in animals.
 emales               Species   Substance          Exposure route General toxicity         Effects on development
Kanojia et al., 1996 Rat        Potassium          Oral (drinking LOAEL = 31 mg/kg         LOAEL = 31 mg/kg
                                dichromate         water)         bw/d (decreased body     bw/d (decreased number
                                                                  weight gain)             of live foetuses,
                                                                                           increased number of
                                                                                           resorptions, post-
                                                                                           implantation loss)
Kanojia et al., 1998 Rat        Potassium          Oral (drinking LOAEL = 45 mg/kg         LOAEL = 45 mg/kg
                                dichromate         water)         bw/d (decreased          bw/d (decreased number
                                                                  gestational body weight  of live foetuses/litter,
                                                                  gain)                    foetal weight and crown-
                                                                                           rump length, increased
                                                                                           number of resorptions
                                                                                           and implantation loss
 lsaieed and Nada,    Rat       Potassium          Oral (drinking LOAEL = 7 mg/kg bw/d     LOAEL = 7 mg/kg bw/d
 002                            dichromate         water)         (decreased gestational   (increased post-
                                                                  weight)                  implantation loss,
                                                                                           resorptions, dead
                                                                                           foetuses, visceral and
                                                                                           skeletal anomalies,
                                                                                           decrease foetal weight)
 oudani et al.,       Rat       Potassium          Oral (drinking LOAEL = 24 mg/kg         LOAEL = 24 mg/kg
 011a,b,c                       dichromate         water)         bw/d (decreased body     bw/d (decreased body
                                                                  weight, changes in       weight, changes in
                                                                  biochemical parameters   biochemical parameters
                                                                  and histopathology in    and histopathology in
                                                                  dams)                    pups)
 ivakum et al., 2014 Rat        Potassium          Oral (drinking (not reported)           LOAEL = 1 mg/kg bw/d
                                dichromate         water)                                  (decreased litter size,
                                                                                           cellular abnormalities)
Glaser et al., 1984   Rat       Sodium dichromate Inhalation      LOAEL= 0.2 mg/kg         NOAEL= 0.2 mg/kg
                                                                  bw/d (local hyperplasia, bw/d (no effects
                                                                  increased relative organ observed)
                                                                  weight)
Maroua et al., 2010   Rat       Potassium          ip             LOAEL = 1 mg/kg bw/d     LOAEL = 1 mg/kg bw/d
                                dichromate                        (decreased body weight   (decreased foetal body
                                                                  gain and relative uterus weight gain, reduced
                                                                  weight)                  number of foetuses/
                                                                                           mother, increased
                                                                                           incidences of dead
                                                                                           foetuses and resorption,
                                                                                           morphological
                                                                                           abnormalities and
                                                                                           incomplete ossification)
               Toxicity for reproduction                                                                            77
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<pre> rivedi et al., 1989  Mouse   Potassium         Oral (drinking NOAEL = 120 mg/kg       LOAEL = 60 mg/kg
                              dichromate        water)         bw/d (decreased body    bw/d (increased number
                                                               weight)                 of resorptions,
                                                                                       postimplantation loss,
                                                                                       decreased litter size,
                                                                                       retarded ossification)
 unaid et al., 1996   Mouse   Potassium         Oral (drinking NOAEL = 125 mg/kg       LOAEL = 67 mg/kg
                              dichromate        water)         bw/d (decreased body    bw/d (increased number
                                                               weight gain)            of resorptions, post-
                                                                                       implantation loss,
                                                                                       decreased number of
                                                                                       foetuses)
 lbetieha and Al-     Mouse   Potassium         Oral (drinking LOAEL = 148 mg/kg       LOAEL = 148 mg/kg/
Hamood 1996                   dichromate        water)         bw/d (decreased body    bw/d (decreased number
                                                               weight)                 of viable foetuses,
                                                                                       increase number
                                                                                       resorptions)
NTP, 1997             Mouse   Potassium         Oral (diet)    NOAEL( F0) = 7 mg/kg NOAEL( F0) = 30 mg/kg
                              dichromate                       bw/d (decreased body    bw/d (no effects
                                                               weight)                 observed)
                                                               LOAEL (F1) = 8 mg/kg NOAEL (F1) = 37 mg/kg
                                                               bw/d (decreased MCH bw/d (no effects
                                                               and haemoglobin)        observed)
Al-Hamood et al.,     Mouse   Potassium         Oral (drinking NOAEL = 71 mg/kg        LOAEL = 71 mg/kg
 998                          dichromate        water)         bw/d (no effects        bw/d (increased number
                                                               observed)               of resorptions,
                                                                                       implantations and viable
                                                                                       foetuses)
De Flora et al., 2006 Mouse   Sodium dichromate Oral (drinking NOAEL = 2 mg/kg bw/d NOAEL 2 mg/kg bw/d
genotoxicity study)           dihydrate and     water)         (no effects observed)   (no effects observed)
                              potassium
                              dichromate
 ndo and Watana,      Mouse   Chromium trioxide ip             NOAEL = 8 mg/kg bw/d    NOAEL = 8 mg/kg bw/d
 988                                                           (no effects observed)   (no effects observed)
De Flora et al., 2006 Mouse   Potassium         ip             NOAEL = 50 mg/kg        NOAEL 50 mg/kg bw/d
genotoxicity study)           dichromate                       bw/d (no effects        (no effects observed)
                                                               observed)
Gale (1978)           Hamster Chromium trioxide iv             NOAEL= 2 mg/kg bw/d     LOAEL= 2 mg/kg bw/d
                                                               (decreased body weight, (increased number of
                                                               nephrotoxicity and      resorptions and
                                                               hepatotoxicity)         malformations, retarded
                                                                                       ossifcation)
Gale and Bunch        Hamster Chromium trioxide iv             LOAEL= 4 mg/kg bw/d LOAEL= 4 mg/kg bw/d
1979)                                                          (decreased body weight, (increased number of
                                                               nephrotoxicity and      resorptions and
                                                               hepatotoxicity)         malformations, decrease
                                                                                       crown-rump length)
 8             Chromium VI compounds
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<pre>    In conclusion, despite the lack of clear evidence from human studies, animal
    studies with several species demonstrate chromium VI-induced developmental
    toxicity in the absence of maternal toxicity. Therefore, classification for
    developmental toxicity is warranted.
        Two studies are available in which rats were exposed during lactation only. In
    the first study dams were exposed to 25 mg Cr/kg bw/d via drinking water from
    parturition until postnatal day 21 and puberty of female pups was studied.
    Various adverse effects were observed, including delayed vaginal opening,
    extended diestrus phase, decreased follicle numbers, and lack of antral follicle
    development. In an additional study from the same group in which dams were
    exposed to 6 or 25 mg Cr/kg bw/d, female pups showed an extended metestrus
    phase and increased level of luteinizing hormone at the high dose, and at both
    dose levels decreased pup weight, and increased oxidative stress. In both studies,
    chromium levels in plasma and uterine/ovarian tissue of female pups were higher
    than in those of controls and decreased with aging of the pups. Since no levels in
    breast milk were measured, toxicity cannot be directly attributed to the
    chromium VI level in breast milk.
5.5 Comparison with criteria
    For effects on fertility and development, substances can be classified in category
    1 (further divided in 1A and 1B) and category 2. The classification of substances
    in category 1A (known human reproductive toxicants) is largely based on
    evidence from humans, whereas the classification in category 1B (presumed
    human reproductive toxicant) is largely based on specific reproduction toxic
    effects observed in animals, which are considered relevant for humans. When
    evidence is found (either in humans or animals) which is not sufficient for
    classification in category 1, a substance is classified in category 2.
    From human data, no clear association between chromium VI exposure and
    fertility parameters was observed.
        The Committee notes that in animals, effects on fertility parameters have
    been observed in multiple species. These studies show consistent adverse effects
    on sperm parameters. In addition, reduced fertility (i.e. reduced number of
    implantations, reduced number of viable fetuses) has been observed after
    exposure of males and females. The Committee notes that it is not completely
    clear to what extent general toxicity was induced at the conditions under which
    these studies were performed. Several studies however, suggest that toxicity to
    fertility is induced in absence of general toxicity. Thus, the fertility effects should
    Toxicity for reproduction                                                               79
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<pre>    be regarded as direct effects on sex organ function. Given the convincing
    findings in the studies showing adverse effects on fertility, which are considered
    relevant for humans, the Committee proposes to classify chromium VI
    compounds for effects on fertility in category 1B (presumed human reproductive
    toxicant) and to label with H360F (may damage fertility) according to
    Regulation (EC) 1272/2008.
    From human data, no clear association between chromium VI exposure and
    developmental parameters was observed.
        Various adverse effects on development have been observed in animals
    exposed to chromium VI. For the developmental toxicity studies, the Committee
    also notes that it is not completely clear to what extent general toxicity was
    induced at the conditions under which these studies were performed. Several
    studies however, suggest that severe developmental toxicity (i.e. prenatal deaths
    and malformations) is induced in absence of maternal toxicity. Thus, the
    developmental toxicity observed should be considered as a direct effect on the
    developing offspring and is unlikely to have been mediated by maternal toxicity.
    The Committee therefore considers this relevant for humans, and recommends to
    classify chromium VI compounds in category 1B (presumed human reproductive
    toxicant) and label with H360D (May damage the unborn child) according to
    Regulation (EC) 1272/2008.
    Several studies have shown developmental effects and oxidative stress in pups
    after chromium VI exposure to lactating rats. The Committee further notes that
    most chromium VI compounds are classified for carcinogenic and genotoxic
    properties (see Section 2.4). No threshold can be established for (stochastic)
    genotoxic carcinogens and the presence of chromium VI in breast milk cannot be
    excluded. Therefore, the Committee proposes to label chromium VI compounds
    for effects on or via lactation.
    Although the vast amount of data is derived from sodium dichromate and
    potassium dichromate, the Committee considers the conclusions applicable to all
    chromium VI compounds specified in this report (see Chapter 4 on read-across
    and toxicokinetics).
5.6 Conclusions on classification and labelling
    The Committee recommends classification according to Regulation (EC) 1272/
    2008 of the European Union. For chromium trioxide, sodium chromate, sodium
 0  Chromium VI compounds
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<pre>dichromate, potassium dichromate, chromic acid, ammonium chromate,
ammonium dichromate, calcium chromate, potassium chromate, dichromium
tris(chromate), the Committee recommends:
• for effects on fertility, to classify these compounds in category 1B (presumed
     human reproductive toxicant), and to label them with H360F (may damage
     fertility)
• for effects on development, to classify these compounds in category 1B
     (presumed human reproductive toxicant) and to label them with H360D (may
     damage the unborn child)
• for effects during lactation, to label these compounds with H362 (may cause
     harm to breastfed babies).
Proposed classification for fertility
Category 1B, H360F.
Proposed classification for developmental toxicity
Category 1B, H360D.
Proposed labelling for effect during lactation
H362.
Toxicity for reproduction                                                        81
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<pre>2 Chromium VI compounds</pre>

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<pre>References
Health Council of the Netherlands. Chromium VI and its compounds; Evaluation of the effects on
reproduction, recommendation for classification. The Hague: Health Council of the Netherlands;
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ECHA (website). Registered substances. Chemical substance search. European Chemicals Agency
(ECHA). Internet: http://echa.europa.eu/web/guest/information-on-chemicals/registered-substances.
Consulted November, 2015.
National Institutes of Health (NIH), U.S. National Library of Medicine. Hazardous Substance
Database. Internet: http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB. Consulted November 2015.
European Union Risk Assessment Report: chromium trioxide, sodium chromate, sodium dichromate,
ammonium dichromate and potassium dichromate risk assessment. European Chemical Bureau,
editor. Italy: European Communities; 2005.
National Toxicology Program (NTP). NTP Technical Report on the toxicity studies of sodium
dichromate dihydrate administrated in drinking water to male and female F344/N rats and B6C3F1
mice and male BALB/c and am3-C57BL/6 mice. National Institutes of Health, Public Health Service,
U.S. Department of Health and Human Services; 2007: Toxicity Report Series Number 72.
Toxicological profile for chromium. US Department of Health and Human Services; ATSDR; 2012.
De Mattia G, Bravi MC, Laurenti O, De LO, Palmeri A, Sabatucci A et al. Impairment of cell and
plasma redox state in subjects professionally exposed to chromium. Am J Ind Med 2004; 46(2): 120-
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Dayan AD, Paine AJ. Mechanisms of chromium toxicity, carcinogenicity and allergenicity: review of
the literature from 1985 to 2000. Hum Exp Toxicol 2001; 20(9): 439-451.
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<pre>  Glaser U, Hochrainen D, Kloppel H, Kordel W, Kuhnen H. Inhalation studies with Wistar rats and
  pathophysiological effects of chromium. Berlin: 1984: D-1 UFOPLAN F+E 10606007/2.
0 Subramanian S, Rajendiran G, Sekhar P, Gowri C, Govindarajulu P, Aruldhas MM. Reproductive
  toxicity of chromium in adult bonnet monkeys (Macaca radiata Geoffrey). Reversible oxidative stress
  in the semen. Toxicol Appl Pharmacol 2006; 215(3): 237-249.
1 Aruldhas M, Subramanian S, Sekar P, Vengatesh G et al. Chronic chromium exposure-induced
  changes in testicular histoarchitecture are associated with oxidative stress: study in a non-human
  primate (Macaca radiata Geoffroy). Human Reproduction 2005; 20(10): 2801-2813.
2 Li H, Chen Q, Li S, Yao W, Li L, Shi X et al. Effect of Cr(VI) exposure on sperm quality: human and
  animal studies. Ann Occup Hyg 2001; 45: 505-511.
3 Kanojia R, Junaid M, Murthy R. Chromium induced teratogenicity in female rat. Toxicol Lett 1996;
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4 Kanojia R, Junaid M, Murthy R. Embryo and fetotoxicity of hexavalent chromium: a long-term
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5 Chowdhury A, Mitra C. Spermatogenic and steroidogenic impairment after chromium treatment in
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6 National Toxicology Program (NTP). Final report on the reproductive toxicity of potassium
  dichromate (hexavalent) (CAS No. 7778-50-9) administered in diet to SD rats. US Department of
  Health and Human Services; 1996: RACB95001.
7 National Toxicology Program (NTP). Final report on the reproductive toxicity of potassium
  dichromate (hexavalent) (CAS No. 7778-50-9) administered in diet to BALB/c mice. US Department
  of Health and Human Services; 1996: RACB95002.
8 National Toxicology Program (NTP). Final report on the reproductive toxicity of potassium
  dichromate (CAS No. 7778-50-9) administered in diet to BALB/c mice. US Department of Health
  and Human Services; 1997: RACB94014.
9 Trivedi B, Saxena D, Murthy R, Chandra S. Embryotoxicity and fetotoxicity of orally administered
  hexavalent chromium in mice. Reprod Toxicol 1989; 3: 275-278.
0 Zahid Z, Al-Hakkak Z, Kadhim A, Elias E, Al-Jumaily I. Comparative effects of trivalent and
  hexavalent chromium on spermatogenesis of the mouse. Toxicol Environ Chem 1990; 25: 131-136.
1 Murthy RC, Junaid M, Saxena DK. Ovarian dysfunction in mice following chromium (VI) exposure.
  Toxicol Lett 1996; 89(2): 147-154.
2 Elbetieha A, Al-Hamood M. Long-term exposure of male and female mice to trivalent and
  hexavalent chromium compounds: effect on fertility. Toxicology 1997; 116: 39-47.
3 Yousef MI, El-Demerdash FM, Kamil KI, Elaswad FAM. Ameliorating effect of folic acid on
  chromium(VI)-induced changes in reproductive performance and seminal plasma biochemistry in
  male rabbits. Reprod Toxicol 2006; 21(3): 322-328.
4 Ernst E. Testicular toxicity following short-term exposure to tri- and hexavalent chromium: an
  experimental study in the rat. Toxicol Lett 1990; 51: 269-275.
4 Chromium VI compounds
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<pre>5 Ernst E, Bonde J. Sex hormones and epididymal sperm parameters in rats following subchronic
  treatment with hexavalent chromium. Human Exp Toxicol 1992; 11: 255-258.
6 Behari J, Chandra S, Tandon S. Comparative toxicity of trivalent and hexavalent chromium to
  rabbits. Acta Biol Med Germ 1978; 37: 463-468.
7 Rachootin P, Olsen J. The risk of infertility and delayed conception associated with exposures in the
  Danish workplace. J Occup Med 1983; 25: 394-402.
8 Jelnes J, Knudsen L. Stainless steel welding and semen quality. Reprod Toxicol 1988; 2: 213-215.
9 Mortensen J. Risk for reduced sperm quality among metal workers, with special reference to welders.
  Scand J Work Environ Health 1988; 14: 27-30.
0 Bonde J, Ernst E. Sex hormones and semen quality in welders exposed to hexavalent chromium.
  Human Exp Toxicol 1992; 11: 259-263.
1 Bonde J. The risk of male subfecundity attributable to welding of metals. Studies of semen quality,
  infertility, fertility, adverse pregnancy outcome and childhood malignancy. Int J Androl 1993; 16,
  suppl 1: 1-29.
2 Elsaieed EM, Nada SA. Teratogenicity of hexavalent chromium in rats and the beneficial role of
  ginseng. Bull Environ Contamin Toxicol 2002; 68(3): 361-368.
3 Soudani N, Ibtissem BA, Troudi A, Bouaziz H, Boudawara T, Zeghal N. Oxidative stress induced by
  chromium (VI) in bone of suckling rats. Toxicol Ind Health 2011; 27(8): 724-734.
4 Soudani N, Sefi M, Bouaziz H, Chtourou Y, Boudawara T, Zeghal N. Nephrotoxicity induced by
  chromium (VI) in adult rats and their progeny. Hum Exp Toxicol 2011; 30(9): 1233-1245.
5 Soudani N, Bouaziz H, Sefi M, Chtourou Y, Boudawara T, Zeghal N. Toxic effects of chromium (VI)
  by maternal ingestion on liver function of female rats and their suckling pups. Environ Toxicol 2011;
  28(1): 11-20.
6 De Flora S, Iltcheva M, Balansky RM. Oral chromium(VI) does not affect the frequency of
  micronuclei in hematopoietic cells of adult mice and of transplacentally exposed fetuses. Mutat Res,
  Genet Toxicol Environ Mutagen 2006; 610(1-2): 38-47.
7 Junaid M, Murthy R, Saxena D. Embryotoxicity of orally administered chromium in mice: exposure
  during the period of organogenesis. Toxicol Lett 1996; 84: 143-148.
8 Al-Hamood M, Elbetieha A, Bataineh H. Sexual maturation and fertility of male and female mice
  exposed prenatally and postnatally to trivalent and hexavalent chromium compounds. Reprod Fertil
  Dev 1998; 10: 179-183.
9 Marouani N, Tebourbi O, Mokni M, Yacoubi MT, Sakly M, Benkhalifa M et al. Embryotoxicity and
  fetotoxicity following intraperitoneal administrations of hexavalent chromium to pregnant rats.
  Zygote 2011; 19(3): 229-235.
0 Endo A, Watanabe T. Analysis of protective activity of N-acetyl cysteine against teratogenicity of
  heavy metals. Reprod Toxicol 1988; 2: 141-144.
1 Gale T. Embryotoxic effects of chromium trioxide in hamsters. Environ Res 1978; 16: 101-109.
2 Gale T, Bunch J. The effect of the time of administration of chromium trioxide on the embryotoxic
  response in hamsters. Teratology 1979; 19: 81-86.
  References                                                                                            85
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<pre>3 Hjollund NH, Bonde JP, Jensen TK, Henriksen TB, Andersson AM, Kolstad HA et al. Male-
  mediated spontaneous abortion among spouses of stainless steel welders. Scand J Work Environ
  Health 2000; 26(3): 187-192.
4 Hjollund NH, Bonde JP, Ernst E, Lindenberg S, Andersen AN, Olsen J. Spontaneous abortion in IVF
  couples--a role of male welding exposure. Human reproduction (Oxford, England) 2005; 20(7):
  1793-1797.
5 Aschengrau A, Zierler S, Cohen A. Quality of community drinking water and the occurrence of late
  adverse pregnancy outcomes. Arch Environ Health 1993; 48(2): 105-113.
6 Banu SK, Samuel JB, Arosh JA, Burghardt RC, Aruldhas MM. Lactational exposure to hexavalent
  chromium delays puberty by impairing ovarian development, steroidogenesis and pituitary hormone
  synthesis in developing Wistar rats. Toxicol Appl Pharmacol 2008; 232(2): 180-189.
7 Samuel JB, Stanley JA, Roopha DP, Vengatesh G, Anbalagan J, Banu SK et al. Lactational hexavalent
  chromium exposure-induced oxidative stress in rat uterus is associated with delayed puberty and
  impaired gonadotropin levels. Human Exp Toxicol 2011; 30(2): 91-101.
8 Mullee A, Brown T, Collings R, Harvey L, Hooper L, Fairweather-Tait S. Literature search and
  review related to specific preparatory work in the establishment of Dietary Reference Values;
  preparation of an evidence report identifying health outcomes upon which Dietary Reference Values
  could potentially be based for chromium, manganese and molybdenum. Scientific report submitted to
  EFSA. 2012.
9 Kumar S, Sathwara NG, Gautam AK, Agarwal K, Shah B, Kulkarni PK et al. Semen quality of
  industrial workers occupationally exposed to chromium. J Occup Health 2005; 47(5): 424-430.
0 National Toxicology Program (NTP). NTP Technical Report on the toxicology and carcinogenesis
  studies of sodium dichromate dihydrate in F344/N rats and B6C3F1 mice (drinking water studies).
  National Institutes of Health, Public Health Service, U.S. Department of Health and Human Services;
  2008: NTP Technical Report number 546.
1 Sivakumar KK, Stanley JA, Arosh JA, Pepling ME, Burghardt RC, Banu SK. Prenatal exposure to
  chromium induces early reproductive senescence by increasing germ cell apoptosis and advancing
  germ cell cyst breakdown in the F1 offspring. Dev Biol. 2014; 388(1): 22-34.
2 Stanley JA, Sivakumar KK, Nithy TK, Arosh JA, Hoyer PB, Burghardt RC, et al. Postnatal exposure
  to chromium through mother's milk accelerates follicular atresia in F1 offspring through increased
  oxidative stress and depletion of antioxidant enzymes. Free Radic Biol Med. 2013; 61: 179-96.
6 Chromium VI compounds
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<pre>A The Committee
B The submission letter (in English)
C Comments on the public review draft
D Regulation (EC) 1272/2008 of the European Community
E Additional considerations to Regulation (EC) 1272/2008
  Annexes
                                                         87
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<pre>8 Chromium VI compounds</pre>

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<pre>nnex A
     The Committee
     •   D. Lindhout, chairman
         Professor of Medical Genetics, Paediatrician (not practising), clinical
         geneticist, University Medical Centre, Utrecht
     •   N. Roeleveld
         Reproductive Epidemiologist, Radboud university medical center, Nijmegen
     •   J.G. Theuns-van Vliet
         Reproductive Toxicologist, Triskelion BV, Zeist
     •   T.G.M. Vrijkotte (since February 1, 2016)
         Epidemiologist, Academic Medical Center, Amsterdam
     •   D.H. Waalkens-Berendsen
         Reproductive Toxicologist, Zeist
     •   P.J.J.M. Weterings
         Toxicologist, Weterings Consultancy BV, Rosmalen
     •   A.H. Piersma, structurally consulted expert
         Professor of Reproductive and Developmental Toxicology, National Institute
         of Public Health and the Environment, Bilthoven
     •   S.R. Vink, scientific secretary
         Health Council of the Netherlands, Den Haag
     With respect to the data presentation and interpretation, the Committee consulted
     Ing. J.J.A. Muller and dr. G. Tiesjema as additional experts from Bureau
     REACH, National Institute of Public Health and the Environment, Bilthoven.
     The Committee                                                                     89
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<pre>  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, persons 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 Health Council to assess
  whether or not someone can become a member. An expert who has no financial
  but another clearly definable interest, can become a member under the restriction
  that he will not be involved in the debate on the subject to which his interest
  relates. If a person’s interest is not clearly definable, he can sometimes be
  consulted as an expert. Experts working for a ministry or governmental
  organisation can be structurally consulted. During the inaugural meeting the
  declarations issued are discussed, so that all members of the Committee are
  aware of each other’s possible interests.
0 Chromium VI compounds
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<pre>nnex B
     The submission letter (in English)
     Subject          : Submission of the advisory report Chromium VI compounds
     Your reference   : DGV/BMO/U-932542
     Our reference    : U-963412/SV/jh/543-G16
     Enclosure(s)     :1
     Date             : May 18, 2016
     Dear Minister,
     I hereby submit the advisory report on the effects of a group of chromium VI
     compounds on fertility and on the development of the progeny; it also concerns
     effects on lactation and on the progeny via lactation.
         This advisory report is part of an extensive series in which reproduction toxic
     substances are classified in accordance with European guidelines. It concerns
     substances to which people may be exposed occupationally. This advisory report
     is an update of an advisory report that was published by the Health Council in
     2001. This update was requested since the previously recommended
     classification deviates from the current classification in the EU.
     The advisory report was prepared by a permanent Committee of the Health
     Council of the Netherlands, the Subcommittee on the Classification of
     Reproduction Toxic Substances. The advisory report was consequently reviewed
     by the Health Council’s Standing Committee on Public Health.
     The submission letter (in English)                                                  91
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<pre>  Today I sent copies of this advisory report to the Minister of Health, Welfare and
  Sport and to the State Secretary of Infrastructure and the Environment, for their
  information.
  Yours sincerely,
  (signed)
  Prof. dr. J.L. Severens,
  Vice President
2 Chromium VI compounds
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<pre>nnex C
     Comments on the public draft
     A draft of the present report was released in 2015 for public review. The
     following organisations and persons have commented on the draft document:
     • T.J. Lentz, L. Greenawald, S.S. Leonard, National Institute for Occupational
         Safety and Health (NIOSH), Cincinnati, OH, USA
     • J. Arts, AkzoNobel NV.
     The comments received, and the reply by the Committee can be found on the
     website of the Health Council.
     Comments on the public draft                                                   93
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<pre>4 Chromium VI compounds</pre>

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<pre>nnex D
     Regulation (EC) 1272/2008 of the
     European Community
     3.7            Reproductive toxicity
     3.7.1          Definitions and general considerations
     3.7.1.1        Reproductive toxicity includes adverse effects on sexual function and fertility in adult
     males and females, as well as developmental toxicity in the offspring. The definitions presented
     below are adapted from those agreed as working definitions in IPCS/EHC Document No 225, Princi-
     ples for Evaluating Health Risks to Reproduction Associated with Exposure to Chemicals. For classi-
     fication purposes, the known induction of genetically based heritable effects in the offspring is
     addressed in Germ Cell Mutagenicity (section 3.5), since in the present classification system it is con-
     sidered more appropriate to address such effects under the separate hazard class of germ cell muta-
     genicity.
     In this classification system, reproductive toxicity is subdivided under two main headings:
     (a) adverse effects on sexual function and fertility;
     (b) adverse effects on development of the offspring.
     Some reproductive toxic effects cannot be clearly assigned to either impairment of sexual function
     and fertility or to developmental toxicity. Nonetheless, substances with these effects, or mixtures con-
     taining them, shall be classified as reproductive toxicants.
     Regulation (EC) 1272/2008 of the European Community                                                      95
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<pre>  3.7.1.2         For the purpose of classification the hazard class Reproductive Toxicity is differentiated
                  into:
  •     adverse effects
        •    on sexual function and fertility, or
        •    on development;
  •     effects on or via lactation.
  3.7.1.3         Adverse effects on sexual function and fertility
  Any effect of substances that has the potential to interfere with sexual function and fertility. This
  includes, but is not limited to, alterations to the female and male reproductive system, adverse effects
  on onset of puberty, gamete production and transport, reproductive cycle normality, sexual behaviour,
  fertility, parturition, pregnancy outcomes, premature reproductive senescence, or modifications in
  other functions that are dependent on the integrity of the reproductive systems.
  3.7.1.4         Adverse effects on development of the offspring
  Developmental toxicity includes, in its widest sense, any effect which interferes with normal devel-
  opment of the conceptus, either before or after birth, and resulting from exposure of either parent
  prior to conception, or exposure of the developing offspring during prenatal development, or postna-
  tally, to the time of sexual maturation. However, it is considered that classification under the heading
  of developmental toxicity is primarily intended to provide a hazard warning for pregnant women, and
  for men and women of reproductive capacity. Therefore, for pragmatic purposes of classification,
  developmental toxicity essentially means adverse effects induced during pregnancy, or as a result of
  parental exposure. These effects can be manifested at any point in the life span of the organism. The
  major manifestations of developmental toxicity include (1) death of the developing organism, (2)
  structural abnormality, (3) altered growth, and (4) functional deficiency.
  3.7.1.5         Adverse effects on or via lactation are also included in reproductive toxicity, but for
  classification purposes, such effects are treated separately (see Table 3.7.1 (b)). This is because it is
  desirable to be able to classify substances specifically for an adverse effect on lactation so that a spe-
  cific hazard warning about this effect can be provided for lactating mothers.
6 Chromium VI compounds
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<pre>3.7.2        Classification criteria for substances
3.7.2.1      Hazard categories
3.7.2.1.1    For the purpose of classification for reproductive toxicity, substances are allocated to
one of two categories. Within each category, effects on sexual function and fertility, and on develop-
ment, are considered separately. In addition, effects on lactation are allocated to a separate hazard cat-
egory.
Table 3.7.1(a) Hazard categories for reproductive toxicants.
Categories                      Criteria
CATEGORY 1                      Known or presumed human reproductive toxicant
                                Substances are classified in Category 1 for reproductive toxicity when
                                they are known to have produced an adverse effect on sexual function
                                and fertility, or on development in humans or when there is evidence
                                from animal studies, possibly supplemented with other information, to
                                provide a strong presumption that the substance has the capacity to
                                interfere with reproduction in humans. The classification of a sub-
                                stance is further distinguished on the basis of whether the evidence for
                                classification is primarily from human data (Category 1A) or from
                                animal data (Category 1B).
                Category 1A Known human reproductive toxicant
                                The classification of a substance in Category 1A is largely based on
                                evidence from humans.
                Category 1B Presumed human reproductive toxicant
                                The classification of a substance in Category 1B is largely based on
                                data from animal studies. Such data shall provide clear evidence of an
                                adverse effect on sexual function and fertility or on development in
                                the absence of other toxic effects, or if occurring together with other
                                toxic effects the adverse effect on reproduction is considered not to be
                                a secondary non-specific consequence of other toxic effects. However,
                                when there is mechanistic information that raises doubt about the rele-
                                vance of the effect for humans, classification in Category 2 may be
                                more appropriate.
CATEGORY 2                      Suspected human reproductive toxicant
                                Substances are classified in Category 2 for reproductive toxicity when
                                there is some evidence from humans or experimental animals, possi-
                                bly supplemented with other information, of an adverse effect on sex-
                                ual function and fertility, or on development, and where the evidence
                                is not sufficiently convincing to place the substance in Category 1. If
                                deficiencies in the study make the quality of evidence less convincing,
                                Category 2 could be the more appropriate classification.
                                Such effects shall have been observed in the absence of other toxic
                                effects, or if occurring together with other toxic effects the adverse
                                effect on reproduction is considered not to be a secondary non-specific
                                consequence of the other toxic effects.
Regulation (EC) 1272/2008 of the European Community                                                        97
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<pre>  Table 3.7.1(b) Hazard category for lactation effects.
  EFFECTS ON OR VIA LACTATION
  Effects on or via lactation are allocated to a separate single category. It is recognised that for many
  substances there is no information on the potential to cause adverse effects on the offspring via lacta-
  tion. However, substances which are absorbed by women and have been shown to interfere with lac-
  tation, or which may be present (including metabolites) in breast milk in amounts sufficient to cause
  concern for the health of a breastfed child, shall be classified and labelled to indicate this property
  hazardous to breastfed babies. This classification can be assigned on the:
  (a) human evidence indicating a hazard to babies during the lactation period; and/or
  (b) results of one or two generation studies in animals which provide clear evidence of adverse effect
  in the offspring due to transfer in the milk or adverse effect on the quality of the milk; and/or
  (c) absorption, metabolism, distribution and excretion studies that indicate the likelihood that the sub-
  stance is present in potentially toxic levels in breast milk.
  3.7.2.2        Basis of classification
  3.7.2.2.1      Classification is made on the basis of the appropriate criteria, outlined above, and an
  assessment of the total weight of evidence (see 1.1.1). Classification as a reproductive toxicant is
  intended to be used for substances which have an intrinsic, specific property to produce an adverse
  effect on reproduction and substances shall not be so classified if such an effect is produced solely as
  a non-specific secondary consequence of other toxic effects.
  The classification of a substance is derived from the hazard categories in the following order of pre-
  cedence: Category 1A, Category 1B, Category 2 and the additional Category for effects on or via lac-
  tation. If a substance meets the criteria for classification into both of the main categories (for example
  Category 1B for effects on sexual function and fertility and also Category 2 for development) then
  both hazard differentiations shall be communicated by the respective hazard statements. Classifica-
  tion in the additional category for effects on or via lactation will be considered irrespective of a clas-
  sification into Category 1A, Category 1B or Category 2.
  3.7.2.2.2      In the evaluation of toxic effects on the developing offspring, it is important to consider
  the possible influence of maternal toxicity (see section 3.7.2.4).
  3.7.2.2.3      For human evidence to provide the primary basis for a Category 1A classification there
  must be reliable evidence of an adverse effect on reproduction in humans. Evidence used for classifi-
  cation shall ideally be from well conducted epidemiological studies which include the use of appro-
  priate controls, balanced assessment, and due consideration of bias or confounding factors. Less
  rigorous data from studies in humans shall be supplemented with adequate data from studies in
  experimental animals and classification in Category 1B shall be considered.
8 Chromium VI compounds
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<pre>3.7.2.3         Weight of evidence
3.7.2.3.1       Classification as a reproductive toxicant is made on the basis of an assessment of the
total weight of evidence, see section 1.1.1. This means that all available information that bears on the
determination of reproductive toxicity is considered together, such as epidemiological studies and
case reports in humans and specific reproduction studies along with sub-chronic, chronic and special
study results in animals that provide relevant information regarding toxicity to reproductive and
related endocrine organs. Evaluation of substances chemically related to the substance under study
may also be included, particularly when information on the substance is scarce. The weight given to
the available evidence will be influenced by factors such as the quality of the studies, consistency of
results, nature and severity of effects, the presence of maternal toxicity in experimental animal stud-
ies, level of statistical significance for inter-group differences, number of endpoints affected, rele-
vance of route of administration to humans and freedom from bias. Both positive and negative results
are assembled together into a weight of evidence determination. A single, positive study performed
according to good scientific principles and with statistically or biologically significant positive results
may justify classification (see also 3.7.2.2.3).
3.7.2.3.2       Toxicokinetic studies in animals and humans, site of action and mechanism or mode of
action study results may provide relevant information which reduces or increases concerns about the
hazard to human health. If it is conclusively demonstrated that the clearly identified mechanism or
mode of action has no relevance for humans or when the toxicokinetic differences are so marked that
it is certain that the hazardous property will not be expressed in humans then a substance which pro-
duces an adverse effect on reproduction in experimental animals should not be classified.
3.7.2.3.3       If, in some reproductive toxicity studies in experimental animals the only effects
recorded are considered to be of low or minimal toxicological significance, classification may not
necessarily be the outcome. These effects include small changes in semen parameters or in the inci-
dence of spontaneous defects in the foetus, small changes in the proportions of common foetal vari-
ants such as are observed in skeletal examinations, or in foetal weights, or small differences in
postnatal developmental assessments.
3.7.2.3.4       Data from animal studies ideally shall provide clear evidence of specific reproductive
toxicity in the absence of other systemic toxic effects. However, if developmental toxicity occurs
together with other toxic effects in the dam, the potential influence of the generalised adverse effects
shall be assessed to the extent possible. The preferred approach is to consider adverse effects in the
embryo/foetus first, and then evaluate maternal toxicity, along with any other factors which are likely
to have influenced these effects, as part of the weight of evidence. In general, developmental effects
that are observed at maternally toxic doses shall not be automatically discounted. Discounting devel-
Regulation (EC) 1272/2008 of the European Community                                                         99
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<pre>   opmental effects that are observed at maternally toxic doses can only be done on a case-by-case basis
   when a causal relationship is established or refuted.
   3.7.2.3.5      If appropriate information is available it is important to try to determine whether devel-
   opmental toxicity is due to a specific maternally mediated mechanism or to a non-specific secondary
   mechanism, like maternal stress and the disruption of homeostasis. Generally, the presence of mater-
   nal toxicity shall not be used to negate findings of embryo/foetal effects, unless it can be clearly dem-
   onstrated that the effects are secondary non-specific effects. This is especially the case when the
   effects in the offspring are significant, e.g. irreversible effects such as structural malformations. In
   some situations it can be assumed that reproductive toxicity is due to a secondary consequence of
   maternal toxicity and discount the effects, if the substance is so toxic that dams fail to thrive and there
   is severe inanition, they are incapable of nursing pups; or they are prostrate or dying.
   3.7.2.4        Maternal toxicity
   3.7.2.4.1      Development of the offspring throughout gestation and during the early postnatal stages
   can be influenced by toxic effects in the mother either through non-specific mechanisms related to
   stress and the disruption of maternal homeostasis, or by specific maternally-mediated mechanisms. In
   the interpretation of the developmental outcome to decide classification for developmental effects it
   is important to consider the possible influence of maternal toxicity. This is a complex issue because
   of uncertainties surrounding the relationship between maternal toxicity and developmental outcome.
   Expert judgement and a weight of evidence approach, using all available studies, shall be used to
   determine the degree of influence that shall be attributed to maternal toxicity when interpreting the
   criteria for classification for developmental effects. The adverse effects in the embryo/foetus shall be
   first considered, and then maternal toxicity, along with any other factors which are likely to have
   influenced these effects, as weight of evidence, to help reach a conclusion about classification.
   3.7.2.4.2      Based on pragmatic observation, maternal toxicity may, depending on severity, influ-
   ence development via non-specific secondary mechanisms, producing effects such as depressed foe-
   tal weight, retarded ossification, and possibly resorptions and certain malformations in some strains
   of certain species. However, the limited number of studies which have investigated the relationship
   between developmental effects and general maternal toxicity have failed to demonstrate a consistent,
   reproducible relationship across species. Developmental effects which occur even in the presence of
   maternal toxicity are considered to be evidence of developmental toxicity, unless it can be unequivo-
   cally demonstrated on a case-by-case basis that the developmental effects are secondary to maternal
   toxicity. Moreover, classification shall be considered where there is a significant toxic effect in the
   offspring, e.g. irreversible effects such as structural malformations, embryo/foetal lethality, signifi-
   cant post-natal functional deficiencies.
00 Chromium VI compounds
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<pre>3.7.2.4.3      Classification shall not automatically be discounted for substances that produce devel-
opmental toxicity only in association with maternal toxicity, even if a specific maternally-mediated
mechanism has been demonstrated. In such a case, classification in Category 2 may be considered
more appropriate than Category 1. However, when a substance is so toxic that maternal death or
severe inanition results, or the dams are prostrate and incapable of nursing the pups, it is reasonable
to assume that developmental toxicity is produced solely as a secondary consequence of maternal
toxicity and discount the developmental effects. Classification is not necessarily the outcome in the
case of minor developmental changes, when there is only a small reduction in foetal/pup body weight
or retardation of ossification when seen in association with maternal toxicity.
3.7.2.4.4      Some of the end points used to assess maternal effects are provided below. Data on
these end points, if available, need to be evaluated in light of their statistical or biological signifi-
cance and dose response relationship.
Maternal mortality:
an increased incidence of mortality among the treated dams over the controls shall be considered evi-
dence of maternal toxicity if the increase occurs in a dose-related manner and can be attributed to the
systemic toxicity of the test material. Maternal mortality greater than 10 % is considered excessive
and the data for that dose level shall not normally be considered for further evaluation.
Mating index
(no. animals with seminal plugs or sperm/no. mated × 100) (*)
Fertility index
(no. animals with implants/no. of matings × 100)
Gestation length
(if allowed to deliver)
Body weight and body weight change:
Consideration of the maternal body weight change and/or adjusted (corrected) maternal body weight
shall be included in the evaluation of maternal toxicity whenever such data are available. The calcula-
() It is recognised that the Mating index and the Fertility index can also be affected by the male.
Regulation (EC) 1272/2008 of the European Community                                                       101
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<pre>   tion of an adjusted (corrected) mean maternal body weight change, which is the difference between
   the initial and terminal body weight minus the gravid uterine weight (or alternatively, the sum of the
   weights of the foetuses), may indicate whether the effect is maternal or intrauterine. In rabbits, the
   body weight gain may not be useful indicators of maternal toxicity because of normal fluctuations in
   body weight during pregnancy.
   Food and water consumption (if relevant):
   The observation of a significant decrease in the average food or water consumption in treated dams
   compared to the control group is useful in evaluating maternal toxicity, particularly when the test
   material is administered in the diet or drinking water. Changes in food or water consumption need to
   be evaluated in conjunction with maternal body weights when determining if the effects noted are
   reflective of maternal toxicity or more simply, unpalatability of the test material in feed or water.
   Clinical evaluations (including clinical signs, markers, haematology and clinical chemistry studies):
   The observation of increased incidence of significant clinical signs of toxicity in treated dams relative
   to the control group is useful in evaluating maternal toxicity. If this is to be used as the basis for the
   assessment of maternal toxicity, the types, incidence, degree and duration of clinical signs shall be
   reported in the study. Clinical signs of maternal intoxication include: coma, prostration, hyperactivity,
   loss of righting reflex, ataxia, or laboured breathing.
   Post-mortem data:
   Increased incidence and/or severity of post-mortem findings may be indicative of maternal toxicity.
   This can include gross or microscopic pathological findings or organ weight data, including absolute
   organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio. When supported by find-
   ings of adverse histopathological effects in the affected organ(s), the observation of a significant
   change in the average weight of suspected target organ(s) of treated dams, compared to those in the
   control group, may be considered evidence of maternal toxicity.
   3.7.2.5        Animal and experimental data
   3.7.2.5.1      A number of internationally accepted test methods are available; these include methods
   for developmental toxicity testing (e.g. OECD Test Guideline 414), and methods for one or two-gen-
   eration toxicity testing (e.g. OECD Test Guidelines 415, 416).
   3.7.2.5.2      Results obtained from Screening Tests (e.g. OECD Guidelines 421 — Reproduction/
   Developmental Toxicity Screening Test, and 422 — Combined Repeated Dose Toxicity Study with
02 Chromium VI compounds
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<pre>Reproduction/Development Toxicity Screening Test) can also be used to justify classification,
although it is recognised that the quality of this evidence is less reliable than that obtained through
full studies.
3.7.2.5.3      Adverse effects or changes, seen in short- or long-term repeated dose toxicity studies,
which are judged likely to impair reproductive function and which occur in the absence of significant
generalised toxicity, may be used as a basis for classification, e.g. histopathological changes in the
gonads.
3.7.2.5.4      Evidence from in vitro assays, or non-mammalian tests, and from analogous substances
using structure-activity relationship (SAR), can contribute to the procedure for classification. In all
cases of this nature, expert judgement must be used to assess the adequacy of the data. Inadequate
data shall not be used as a primary support for classification.
3.7.2.5.5      It is preferable that animal studies are conducted using appropriate routes of administra-
tion which relate to the potential route of human exposure. However, in practice, reproductive toxic-
ity studies are commonly conducted using the oral route, and such studies will normally be suitable
for evaluating the hazardous properties of the substance with respect to reproductive toxicity. How-
ever, if it can be conclusively demonstrated that the clearly identified mechanism or mode of action
has no relevance for humans or when the toxicokinetic differences are so marked that it is certain that
the hazardous property will not be expressed in humans then a substance which produces an adverse
effect on reproduction in experimental animals shall not be classified.
3.7.2.5.6      Studies involving routes of administration such as intravenous or intraperitoneal injec-
tion, which result in exposure of the reproductive organs to unrealistically high levels of the test sub-
stance, or elicit local damage to the reproductive organs, including irritation, must be interpreted with
extreme caution and on their own are not normally the basis for classification.
3.7.2.5.7      There is general agreement about the concept of a limit dose, above which the produc-
tion of an adverse effect is considered to be outside the criteria which lead to classification, but not
regarding the inclusion within the criteria of a specific dose as a limit dose. However, some guide-
lines for test methods, specify a limit dose, others qualify the limit dose with a statement that higher
doses may be necessary if anticipated human exposure is sufficiently high that an adequate margin of
exposure is not achieved. Also, due to species differences in toxicokinetics, establishing a specific
limit dose may not be adequate for situations where humans are more sensitive than the animal
model.
3.7.2.5.8      In principle, adverse effects on reproduction seen only at very high dose levels in animal
studies (for example doses that induce prostration, severe inappetence, excessive mortality) would
Regulation (EC) 1272/2008 of the European Community                                                       103
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<pre>              not normally lead to classification, unless other information is available, e.g. toxicokinetics informa-
              tion indicating that humans may be more susceptible than animals, to suggest that classification is
              appropriate. Please also refer to the section on maternal toxicity (3.7.2.4) for further guidance in this
              area.
              3.7.2.5.9      However, specification of the actual ‘limit dose’ will depend upon the test method that
              has been employed to provide the test results, e.g. in the OECD Test Guideline for repeated dose tox-
              icity studies by the oral route, an upper dose of 1 000 mg/kg has been recommended as a limit dose,
              unless expected human response indicates the need for a higher dose level.
              3.7.3          Classification criteria for mixtures
              3.7.3.1        Classification of mixtures when data are available for all ingredients or only for some
              ingredients of the mixture
              3.7.3.1.1      The mixture shall be classified as a reproductive toxicant when at least one ingredient
              has been classified as a Category 1A, Category 1B or Category 2 reproductive toxicant and is present
              at or above the appropriate generic concentration limit as shown in Table 3.7.2 for Category 1A, Cat-
              egory 1B and Category 2 respectively.
              3.7.3.1.2      The mixture shall be classified for effects on or via lactation when at least one ingredi-
              ent has been classified for effects on or via lactation and is present at or above the appropriate generic
              concentration limit as shown in Table 3.7.2 for the additional category for effects on or via lactation.
 able 3.7.2 Generic concentration limits of ingredients of a mixture classified as reproduction toxicants or foreffects on or via
actation that trigger classification of the mixture.
 ngredient classified as:     Generic concentration limits triggering classification of a mixture as:
                              Category 1A                Category 1B               Category 2                Additional category
                              reproductive toxicant reproductive toxicant reproductive toxicant for effects on or via l
                                                                                                             actation
Category 1A                   ≥ 0,3 %
 eproductive toxicant         [Note 1]
  ategory 1B                                             ≥ 0,3 %
eproductive toxicant                                     [Note 1]
Category 2                                                                         ≥ 3,0 %
 eproductive toxicant                                                              [Note 1]
Additional category                                                                                          ≥ 0,3 %
or effects on or via                                                                                         [Note 1]
actation
  ote The concentration limits in the table above apply to solids and liquids (w/w units) as well as gases (v/v units).
  ote 1 If a Category 1 or Category 2 reproductive toxicant or a substance classified for effects on or via lactation is present in
he mixture as an ingredient at a concentration above 0,1 %, a SDS shall be available for the mixture upon request.
  04          Chromium VI compounds
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<pre>3.7.3.2        Classification of mixtures when data are available for the complete mixture
3.7.3.2.1      Classification of mixtures will be based on the available test data for the individual
ingredients of the mixture using concentration limits for the ingredients of the mixture. On a case-by-
case basis, test data on mixtures may be used for classification when demonstrating effects that have
not been established from the evaluation based on the individual components. In such cases, the test
results for the mixture as a whole must be shown to be conclusive taking into account dose and other
factors such as duration, observations, sensitivity and statistical analysis of reproduction test systems.
Adequate documentation supporting the classification shall be retained and made available for review
upon request.
3.7.3.3        Classification of mixtures when data are not available for the complete mixture:
               bridging principles
3.7.3.3.1      Subject to paragraph 3.7.3.2.1, where the mixture itself has not been tested to determine
its reproductive toxicity, but there are sufficient data on the individual ingredients and similar tested
mixtures to adequately characterise the hazards of the mixture, these data shall be used in accordance
with the applicable bridging rules set out in section 1.1.3.
3.7.4          Hazard Communication
3.7.4.1        Label elements shall be used for substances or mixtures meeting the criteria for
               classification in this hazard class in accordance with Table 3.7.3
Regulation (EC) 1272/2008 of the European Community                                                        105
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<pre> able 3.7.3 Label elements for reproductive toxicity.
 lassification              Category 1A or Category 1B              Category 2                              Additional category
                                                                                                            for effects on or via
                                                                                                            lactation
GHS Pictograms                                                                                              No pictogram
 ignal Word                 Danger                                  Warning                                 No signal word
Hazard Statement            H360: May damage fertility or the       H361: Suspected of damaging fertil-     H362: May cause
                            unborn child (state specific effect if  ity or the unborn child (state specific harm to breast-fed
                            known)(state route of exposure if it is effect if known) (state route of expo-  children.
                            conclusively proven that no other       sure if it is conclusively proven that
                            routes of exposure cause the hazard)    no other routes of exposure cause the
                                                                    hazard)
 recautionary Statement     P201                                    P201                                    P201
 revention                  P202                                    P202                                    P260
                            P281                                    P281                                    P263
                                                                                                            P264
                                                                                                            P270
 recautionary Statement     P308 + P313                             P308 + P313                             P308 + P313
 esponse
 recautionary Statement     P405                                    P405
 torage
 recautionary Statement     P501                                    P501
Disposal
 06           Chromium VI compounds
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<pre>nnex E
     Additional considerations to
     Regulation (EC) 1272/2008
     The classification and labelling of substances is performed according to the
     guidelines of the European Union (Regulation (EC)1272/2008) presented in
     Annex D. The classification of compounds is ultimately dependent on an
     integrated assessment of the nature of all parental and developmental effects
     observed, their specificity and adversity, and the dosages at which the various
     effects occur. The guideline necessarily leaves room for interpretation, dependent
     on the specific data set under consideration. In the process of using the
     regulation, the committee has agreed upon a number of additional
     considerations:
     • If there is sufficient evidence to establish a causal relationship between
         human exposure to the substance and impaired fertility or subsequent
         developmental toxic effects in the offspring, the compound will be classified
         in category 1A, irrespective of the general toxic effects (see Annex D,
         3.7.2.2.1.).
     • Adverse effects in a reproductive study, occurring without reporting the
         parental or maternal toxicity, may lead to a classification other than category
         1B, when the effects occur at dose levels which cause severe toxicity in
         general toxicity studies.
     • Clear adverse reproductive effects will not be disregarded on the basis of
         reversibility per se.
     Additional considerations to Regulation (EC) 1272/2008                              107
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<pre>   •   The committee does not only use guideline studies (studies performed
       according to OECD* standard protocols) for the classification of compounds,
       but non-guideline studies are taken into consideration as well.
    Organisation for Economic Cooperation and Development.
08 Chromium VI compounds
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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 opinions 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                  realise significant                 which carry certain
and opportunities?                    health benefits?                    health risks?
Environmental                         Healthy working                     Innovation and
health                                conditions                          the knowledge
Which environmental                   How can employees                   infrastructure
influences could have                 be protected against                Before we can harvest
a positive or negative                working conditions                  knowledge in the
effect on health?                     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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<br><br>