<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>Bromodichloromethane
Evaluation of the carcinogenicity and genotoxicity
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<pre></pre>

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<pre>Gezondheidsraad                                          Vo o r z i t t e r
Health Council of the Netherlands
Aan de minister van Sociale Zaken en Werkgelegenheid
Onderwerp               : Aanbieding advies Bromodichloromethane
Uw kenmerk              : DGV/MBO/U-932542
Ons kenmerk             : U-1477/JR/pg/246-R11
Bijlagen                :1
Datum                   : 12 december 2007
Geachte minister,
Graag bied ik u hierbij het advies aan over de kankerverwekkendheid van broomdichloor-
methaan. Het maakt deel uit van een uitgebreide reeks waarin kankerverwekkende stoffen
worden geclassificeerd volgens richtlijnen van de Europese Unie. Het gaat om stoffen waar-
aan mensen tijdens de beroepsmatige uitoefening kunnen worden blootgesteld.
     Het advies is opgesteld door een vaste subcommissie van de Commissie Gezondheid en
beroepsmatige blootstelling aan stoffen (GBBS), de Subcommissie Classificatie van carci-
nogene stoffen. Het advies is voorgelegd aan de Commissie GBBS en vervolgens getoetst
door de Beraadsgroep Gezondheid en omgeving van de Gezondheidsraad.
Ik heb dit advies vandaag ter kennisname toegezonden aan de minister van Volksgezond-
heid, Welzijn en Sport en de minister van Volkshuisvesting, Ruimtelijke Ordening en
Milieubeheer.
Hoogachtend,
prof. dr. J.A. Knottnerus
Bezoekadres                                                                 Postadres
Parnassusplein 5                                                            Postbus 16052
2 5 11 V X D e n          Haag                                              2500 BB Den            Haag
Te l e f o o n ( 0 7 0 ) 3 4 0 6 6 3 1                                      Te l e f a x ( 0 7 0 ) 3 4 0 7 5 2 3
E - m a i l : j o l a n d a . r i j n k e l s @ g r. n l                    w w w. g r. n l
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<pre></pre>

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<pre>Bromodichloromethane
Evaluation of the carcinogenicity and genotoxicity
Subcommittee on the classification of carcinogenic substances of the
Dutch Expert Committee on Occupational Standards,
a committee of the Health Council of the Netherlands
to:
the Minister of Social Affairs and Employment
No. 2007/05OSH, The Hague, December 12, 2007
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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...” (Section
22, Health Act).
     The Health Council receives most requests for advice from the Ministers of
Health, Welfare & Sport, Housing, Spatial Planning & the Environment, Social
Affairs & Employment, and Agriculture, Nature & Food Quality. 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 gov-
ernment policy.
     Most Health Council reports are prepared by multidisciplinary committees of
Dutch or, sometimes, foreign experts, appointed in a personal capacity. The
reports are available to the public.
                 The Health Council of the Netherlands is a member of the European
                 Science Advisory Network for Health (EuSANH), a network of science
                 advisory bodies in Europe.
                 The Health Council of the Netherlands is a member of the International Network
                 of Agencies for Health Technology Assessment (INAHTA), an international
                 collaboration of organisations engaged with health technology assessment.
 I NA HTA
This report can be downloaded from www.healthcouncil.nl.
Preferred citation:
Health Council of the Netherlands. Bromodichloromethane; Evaluation of the
carcinogenicity and genotoxicity. The Hague: Health Council of the Netherlands,
2007; publication no. 2007/05OSH.
all rights reserved
ISBN: 978-90-5549-668-6
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<pre>    Contents
    Samenvatting 9
    Executive summary 11
1   Scope 13
1.1 Background 13
1.2 Committee and procedure 13
1.3 Data 14
2   General information 15
2.1 Identity, and physical and chemical properties 15
2.2 IARC classification 16
3   Carcinogenicity studies 17
3.1 Observations in humans 17
3.2 Carcinogenicity studies in animals 17
4   Mutagenicity and genotoxicity 23
4.1 In vitro assays 23
4.2 In vivo assays 24
4.3 Carcinogenic mode of action 26
    Contents                                          7
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<pre>5   Classification 29
5.1 Evaluation of data on carcinogenicity and genotoxicity 29
5.2 Recommendation for classification 30
    References 31
    Annexes 35
A   Request for advice 37
B   The committee 39
C   Comments on the public review draft 41
D   IARC Monograph 43
E   Carcinogenic classification of substances by the committee 47
F   Guideline 93/21/EEG of the European Union 49
8   Bromodichloromethane
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<pre>Samenvatting
Op verzoek van de minister van Sociale Zaken en Werkgelegenheid evalueert en
beoordeelt de Gezondheidsraad de kankerverwekkende eigenschappen van stof-
fen waaraan mensen tijdens de beroepsmatige uitoefening kunnen worden bloot-
gesteld. De evaluatie en beoordeling worden verricht door de subcommissie
Classificatie van Carcinogene Stoffen van de Commissie Gezondheid en
Beroepsmatige Blootstelling aan Stoffen van de Raad, hierna kortweg aangeduid
als de commissie. In het voorliggende advies neemt de commissie broomdi-
chloormethaan onder de loep. Broomdichloormethaan is een stof dat onder
andere wordt gebruikt bij de synthese van organische stoffen.
Op basis van de beschikbare gegevens leidt de commissie af dat broomdichloor-
methaan beschouwd moet worden als kankerverwekkend voor de mens. Dit komt
overeen met een classificatie in categorie 2 volgens de richtlijnen van de Euro-
pese Unie. De commissie concludeert verder dat broomdichloormethaan een sto-
chastisch genotoxisch werkingsmechanisme heeft.
Samenvatting                                                                     9
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<pre>10 Bromodichloromethane</pre>

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<pre>Executive summary
At request of the Minister of Social Affairs and Employment, the Health Council
of the Netherlands evaluates and judges the carcinogenic properties of sub-
stances to which workers are occupationally exposed. The evaluation is per-
formed by the subcommittee on Classifying Carcinogenic Substances of the
Dutch Expert Committee on Occupational Standards of the Health Council, here-
after called the committee. In this report, the committee evaluated bromodichlo-
romethane. Bromodichloromethane is an agent that is used in the synthesis of
organic chemicals.
Based on the available information, the committee is of the opinion that bromod-
ichloromethane should be considered as carcinogenic to humans. This recom-
mendation corresponds to the EU classification in category 2. The committee
concludes furthermore that bromodichloromethane acts by a stochastic genotoxic
mechanism.
Executive summary                                                                11
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<pre>12 Bromodichloromethane</pre>

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<pre>Chapter 1
        Scope
1.1     Background
        In the Netherlands a special policy is in force with respect to occupational use
        and exposure to carcinogenic substances. Regarding this policy, the Minister of
        Social Affairs and Employment has asked the Health Council of the Netherlands
        to evaluate the carcinogenic properties of substances, and to propose a classifica-
        tion with reference to an EU-directive (see annex A and F). In addition to classi-
        fying substances, the Health Council also assesses the genotoxic properties of the
        substance in question. The assessment and the proposal for a classification are
        expressed in the form of standard sentences (see annex E). This report contains
        the evaluation of the carcinogenicity of bromodichloromethane.
1.2     Committee and procedure
        The evaluation is performed by the subcommittee on Classifying Carcinogenic
        Substances of the Dutch Expert Committee on Occupational Standards of the
        Health Council, hereafter called the committee. The members of the committee
        are listed in annex B. The first draft was prepared by MI Willems, from the
        Department of Occupational Toxicology of the TNO Nutrition and Food
        Research, by contract with the Ministry of Social Affairs and Employment.
            In 2007 the President of the Health Council released a draft of the report for
        public review. The individuals and organisations that commented on the draft are
        Scope                                                                               13
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<pre>    listed in annex C. The committee has taken these comments into account in
    deciding on the final version of the report.
1.3 Data
    The evaluation and recommendation of the committee is standardly based on
    scientific data, which are publicly available. The starting points of the commit-
    tees’ reports are, if possible, the monographs of the International Agency for
    Research on Cancer (IARC). This means that the original sources of the studies,
    which are mentioned in the IARC-monograph, are reviewed only by the commit-
    tee when these are considered most relevant in assessing the carcinogenicity and
    genotoxicity of the substance in question. In the case of bromodichloromethane,
    such an IARC-monograph is available, of which the summary and conclusion of
    IARC is inserted in annex D.
         More recently published data were retrieved from the online databases Med-
    line, Toxline, Chemical Abstracts, and RTECS. The last updated online search
    was in March 2007. The new relevant data were included in this report.
14  Bromodichloromethane
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<pre>Chapter 2
        General information
2.1     Identity, and physical and chemical properties
        Bromodichloromethane (BDCM) is used in the synthesis of organic chemicals
        and as a reagent in laboratory research.1-3 It has also been used to: separate miner-
        als and salts; as a flame retardant; in fire extinguishers; and, as a solvent for
        waxes, fats and resins. Furthermore, it is found as a by-product of chlorinated
        drinking- and swimming pool water.
            The major source of occupational exposure is when the compound is used in
        laboratory research, or when the agent is inhaled in drinking water facilities and
        swimming pool facilities. The general population is mainly exposed through con-
        sumption of contaminated drinking water, or through ingestion of chlorinated
        swimming pool water.1-3
            Below is given the identity and some of its physical and chemical properties.
        Chemical name                  : bromodichloromethane
        CAS registry number            : 75-27-4
        EC/EINECS number               : 200-856-7
        Synonyms                       : dichlorobromomethane; dichloromonobromomethane;
                                         monobromodichloromethane; BDCM
        Description                    : colourless liquid
        Molecular formula              : CHBrCI
                                                  2
        General information                                                                   15
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<pre>    Structure                    :     Cl
                                        l
                                   H – C – Br
                                        l
                                       Cl
    Molecular weight             : 163.83
    Boiling point                : 90.1 °C
    Melting point                : -57.1 °C
    Relative density of vapour/  : 1.3
    air mixture at 20 °C
    Vapour pressure              : 6.7 kPa at 20 °C
    Solubility                   : Soluble in water (4.5 g/L at 20 °C); acetone; ethanol; ben-
                                   zene; chloroform; and, diethyl ether
    N-octanol/water partition    : 2.10
    coefficient as log Pow
    Conversion factors (25°C,    : 1 ppm = 0.15 mg/m3
    760 mm Hg)                     1 mg/m3 = 6.70 ppm
2.2 IARC classification
    In 1999, IARC concluded that there is sufficient evidence for the carcinogenicity
    of BDCM in experimental animals, but that there were no carcinogenicity data
    available from studies in humans.2 Therefore, according to the IARC guidelines,
    it classified BDCM in Group 2B, which means that the agent is possibly carcino-
    genic to humans.
16  Bromodichloromethane
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<pre>Chapter 3
        Carcinogenicity studies
3.1     Observations in humans
        Several population-based case control studies suggested that exposure to chlori-
        nated water, that predominantly contained BDCM, is associated with increased
        risk of colorectal and bladder cancer in humans.1,2,4-6 However, a problem in these
        studies is that bromodichloromethane exposure is almost always accompanied by
        exposure to other trihalomethane agents, such as chloroform, dibromochlo-
        romethane, bromoform, and many other by-products that can be present in chlo-
        rinated water. Therefore, it is far from certain whether BDCM was the
        responsible agent.
            No epidemiological data or case reports are available on the carcinogenicity
        of BDCM alone.
3.2     Carcinogenicity studies in animals
        The American National Toxicology Program (NTP) carried out three long-term
        animal carcinogenicity studies. 6-8 In the first, groups of 50 male and female Fis-
        cher F344 rats and B6C3F1 mice, were given BDCM in corn oil by gavage, five
        days per week for 102 weeks (Dunnick et al. 1993).7 Control groups of 50 male
        and female animals received normal diet containing an empty capsule alone. The
        results are shown in Tables 3.1 (rats) and 3.2 (mice).
        Carcinogenicity studies                                                             17
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<pre>Table 3.1 Tumour development in Fischer rats, given BDCM in corn oil by gavage for two years (Dunnick et al. 1993).
                                           Males                                     Females
Dose in mg BDCM/kg bw                      Control     50            100             Control     50             100
Survival at 104 weeks                      28/50       36/50        28/50            34/50       27/50          41/50
No. of tumour bearing animals
Large Intestines:
•    Adenomatous polyps                    0/50        3/50         33/50            0/46        0/50           7/47
•    Adenocarcinomas                       0/50        11/50        38/50            0/46        0/50           6/47
The kidneys
•    Tubular cell adenomas                 0/50        1/50          3/50            0/50        1/50           6/50
•    Tubular cell adenocarcinomas          0/50        0/50          10/50           0/50        0/50           9/50
•    Adrenal medullary                     18/50       14/50        5/50             -           -              -
     phaeochromocytomas
Anterior pituitary neoplasms               -           -            -                31/49       20/49          14/49
Mammary gland fibroadenomas                -           -            -                20/50       15/50          1/50
-, no evidence for compound-related neoplasms.
Table 3.2 Tumour development in B6C3F1 mice, given BDCM in corn oil by gavage for two years (Dunnick et al. 1993).
                                           Males                                     Females
Dose in mg BDCM/kg bw                      Control     25            50              Control     75             150
Survival at 104 weeks                      34/50       32/50        42/50            26/50       13/50          15/50
No. of tumour bearing animals
The liver
•    Hepatocellular adenomas               -           -            -                1/50        13/48          23/50
•    Hepatocellular carcinomas             -           -            -                2/50        5/48           10/50
The kidneys
•    Tubular cell adenomas                 1/49        2/50          6/50            -           -              -
•    Tubular cell adenocarcinomas          0/50        0/50          4/50            -           -              -
Anterior pituitary neoplasms               -           -            -                17/44       8/43           3/38
The thyroid
•    Follicular cell hyperplasia           0/48        3/44          5/49            6/50        18/45          21/48
-, no evidence for compound-related neoplasms.
              Regarding male and female rats, the numbers of tumour-bearing animals having
              neoplasms in the large intestines and in the kidneys were significantly greater in
              the high dose group than in controls (p<0.001). On the other hand, the number of
              female animals with anterior pituitary neoplasms and mammary gland fibroade-
              nomas was significantly lower in the high dose group than in controls (p<0.001).
              This was also the case for adrenal medullary phaeochromocytomas (benign and
              malignant) in males (p=0.003).
                   In mice, the numbers of tumour-bearing animals having neoplasms in the
              liver (males) and in the kidneys (females) were significantly greater in the high
              dose groups than in controls (p<0.05). Also follicular cell hyperplasia in the thy-
18            Bromodichloromethane
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<pre>             roid was more common in the high dose groups than in controls. But again, the
             number of female animals with anterior pituitary neoplasms was significantly
             lower in the high dose group than in controls (p=0.006).
                 In the second study, groups of 50 male F344/N rats and B6C3F1 mice were
             given BDCM in drinking water for up to 100 or 104 weeks (George et al. 2002).8
             Control groups of 50 male animals received normal drinking water with 0.25%
             Emulphor, a nontoxic dissolvent. In rats and mice, no changes in feed consump-
             tion, final body weight and survival were observed. In both animal species,
             BDCM did not increase cancer in the large bowel, kidney, spleen, bladder, or in
             any other organ tissues examined, except in the liver of rats. As shown in Table
             3.3, in rats receiving the lowest dose, the prevalence of hepatocellular adenomas
             was significantly increased compared to controls. However, in the mid dose
             group the increase was marginal when hepatocellular adenomas were combined
             with carcinomas, while in the high dose group no clear increase in prevalence
             was observed. In the kidneys of the rats, an increase in the prevalence of renal
             tubular hyperplasia was observed in the highest dose group compared to controls
             (15.8% versus 8.7%). Overall, the authors concluded that under the study condi-
             tions, BDCM in drinking water was not carcinogenic in male B6C3F1 mice, but
             that it was carcinogenic in male F344/N rats.
             To compare exposure doses to reality, concentrations of BDCM in chlorinated
             drinking water are reported to be between 1 and 50 μg/L, with a maximum
             detected over 200 g/L.9
Table 3.3 Tumour prevalence of male F344/N rats and B6C3F1 mice, given BDCM in drinking water for two years (George et
al. 2002).
                                     F344/N rats                            B6C3F1 mice
BDCM concentration in                Control    70    350        700        Control 50             250       500
drinking water (mg/L)
Mean daily dose                      0         3.9    20.6       36.3       0          8.1         27.2      43.4
(mg BDCM/kg bw/day)
Survival at 104 weeks                32/50     31/50  33/50      33/50      36/50      32/50       36/50     40/50
Prevalence (percent of animals with a tumour)
Hepatocellular adenomas              2.2       15.5*  6.2        4.1        20.5       22.2        14.3      15.9
Hepatocellular carcinomas            2.2       2.2    8.3        4.1        23.1       25.0        19.0      15.9
Hepatocellular adenomas and          4.4       17.8*  14.6**     8.2        n          n           n         n
carcinomas
* p< 0.05; ** p<0.10; n, not reported.
             Carcinogenicity studies                                                                                19
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<pre>        In the third study, which had the same design as the second study, no
   increased incidences of neoplasms to BDCM concentrations of 0, 175, 350, and
   700 mg/L was observed in male F344/N rats and B6C3F1 mice (NTP 2006).6 In
   fact, in mice a negative trend was observed of hepatocellular adenomas and car-
   cinomas (combined). Furthermore, in the mid-dose group, the incidence of
   hemangiosarcomas in all organs was significantly decreased compared to control
   mice. Survival rates did not differ among the exposed and control groups.
        The American National Toxicology Program analysed the different outcomes
   of its studies. Using a pharmacokinetic model, NTP concluded that “The differ-
   ent responses observed in these studies were attributed to differences in organ
   dosimetry by these routes of exposure and possible influences of dietary factors
   and differences in body weight on neoplasm development”.6
        In the IARC Monograph of 1991, the Working Group evaluated two other
   animal carcinogenicity studies, but it noted the incomplete reporting and diagno-
   sis of these studies.9 One study concerned life-long exposure of BDCM in drink-
   ing water of male and female Wistar rats (Tumasonis et al. 1985).10 In female
   animals increased number of tumour-bearing animals were observed for neoplas-
   tic nodules and adenofibrosis in the liver compared to controls, while the propor-
   tion of lymphosarcomas was increased in males, but decreased in females.
   Furthermore, in exposed female animals, the prevalence of pituitary gland or
   mammary gland tumours was decreased compared to controls. The other study
   concerned CBA x C57B1/6 hybrid mice, which were given BDCM in drinking
   water for a life-long period (Voronin et al. 1987)11 No treatment-related tumours
   were observed in any of the mice.
        Aida et al. (1992) carried out a carcinogenicity study on male and female
   Wistar rats, which were given microencapsuled BDCM in the diet for up to two
   years.12 The mean daily intake was calculated to be 6-8, 25-32, and 138-168 mg/
   kg bw for males and females, respectively. Control groups were given the diet
   containing an empty capsule alone. At several time points (6, 12 and 18 months)
   interim kills were scheduled, leaving a maximum of 20 animals per group alive
   for the total exposure period. Data on neoplasms were only presented for the ini-
   tial number of animals at the start of the experiment. No significant treatment-
   related increases in the number of neoplastic lesions were observed in any of the
   groups and in any of the organs examined, including the liver and kidneys.
   Regarding non-neoplastic lesions in the liver, dose-related changes included:
   fatty degeneration (mid dose, males); fatty degeneration and granulomas (two
   highest doses, females); and, bile duct proliferation and cholangiofibrosis (high-
   est dose, males and females). No non-neoplastic lesions were observed in the
   kidneys of either sex.
20 Bromodichloromethane
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<pre>In 2007 the National Toxicology Program published a report on the toxicity and
carcinogenicity of BDCM in genetically modified mice, which were susceptible
for developing cancer.13 Tg.AC mice (hemizygous for mutant v-Ha-ras trans-
gene; Ha-ras is an oncogene), and p53 haploinsufficient mice were given BDCM
in drinking water, by gavage, or dermally. In the drinking water studies, groups
of 15 male and 15 female mice (study duration, 26 weeks), or 10 male and 10
female mice (study duration, 42 weeks) received daily 0, 175, 350 or 750 mg
BDCM per liter drinking water; in the gavage studies, mice received 0, 25, 50 or
100 mg BDCM/kg bw for 5 days per week; and, in the dermal study (study dura-
tion, 26 and 39 weeks; Tg.AC strain only), doses of 0, 64, 128, or 256 mg
BDCM/kg bw were administered for 5 days per week. Survival of all the exposed
animals were similar to that of there respective vehicle controls. Overall, no
treatment-related neoplasms were observed in any of the exposed animals. In
both drinking water and gavage studies in Tg.AC and p53 haploinsufficient
mice, BDCM-exposed males had increased renal tubule degeneration, and
BDCM-exposed females had fatty changes of the hepatocytes. These non-neo-
plastic changes were not observed in vehicle controls. Since BDCM caused can-
cer in other animal studies, the authors suggested that the genetically modified
mice, which were used in their studies, may not be as sensitive for detecting can-
cer-causing agents.
     In 2002 and 2003, a few animal studies have been published by Hooth et al.
and McDorman et al. of the same research group.14-16 In these studies, male and
female Tsc2 mutant Long-Evans (Eker) rats received BDCM in their drinking
water at 70 and 700 mg/L for 4 or 10 months. The concentrations corresponded
to a mean daily intake of 3,5 and 35 mg BDCM/kg bw, and to 6.5 and 56 mg
BDCM/kg bw, for male and females, respectively. Rats carrying a mutation in
the Tsc2 tumour suppressor gene readily develop renal preneoplastic and neo-
plastic lesions, and are highly susceptible to the effects of renal carcinogens. In
all studies, no more than eight animals per group were used. The number of
tumour-bearing animals, and the total number of renal adenomas and carcinomas
per animal, did not differ from the data obtained from control animals. Further-
more, no lesions were observed in the urinary bladder, while the incidence of
aberrant crypt foci in the colon was non-significantly increased compared to con-
trol group.
     DeAngelo et al. (2002) administered BDCM in the drinking water of male
F344/N rats, and B6C3F1 mice at a dose of 700 mg/L for 13 weeks.17 They also
used A/J mice, which were given 500 mg BDCM/L for 13 and 30 weeks. The
study was performed to investigate the development of aberrant crypt foci, which
are considered early putative preneoplastic lesions in the colon. No aberrant
Carcinogenicity studies                                                             21
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<pre>   crypt foci were observed in the two mouse strains at any time. In rats, BDCM
   induced a low but significant increase in aberrant crypt foci compared to con-
   trols. In an additional study, male F344/N rats were given the same concentration
   in drinking water, but also a group was added, in which the animals received
   BDCM in corn oil by gavage (50 mg/kg bw) for 26 weeks.18 In all BDCM-treated
   groups, the incidence of aberrant crypt foci was significantly increased compared
   to negative controls. Corn oil did not promote BDCM-induced aberrant crypt
   foci.
       Toussaint et al. (2001) allowed three groups of Japanese medaka (Oryzias
   latipes) fish plus a control group to swim in BDCM containing water for up to
   nine months.19 The three groups were exposed to 18, 143, and 1424 μg/L, respec-
   tively. At the end of the exposure period, in none of the exposed fish treatment-
   related neoplastic lesions in the liver or any other organs were observed. The
   main non-neoplastic lesions observed, were a statistically significant increase in
   gall bladder hyperplasia and bile duct abnormalities in fish exposed to the high-
   est dose at six and nine months.
22 Bromodichloromethane
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<pre>Chapter 4
        Mutagenicity and genotoxicity
4.1     In vitro assays
        Conflicting findings have been reported concerning the mutagenic potential of
        BDCM in Salmonella typhimurium assays. For instance, it did induce reverse
        mutations in tester strains TA100, TA98, and TA97, all in the presence of a meta-
        bolic activation system, but no mutations were observed in TA102, TA1535,
        TA1537, and in some tests using TA100, and TA98.2,20 Le Curieux et al. (1995)
        used a normal TA100 strain in an Ames-fluctuation assay.21 This is a modified
        Salmonella assay, in which bacteria are exposed in a liquid medium instead of an
        agar plate. However, the test results were negative. Also no treatment-related
        reverse mutations in strain TA1535 was observed by Pegram et al. (1997).22 But
        in the same study, in a TA1535 strain, which was transfected with a glutathione
        transferase gene (TA1535+GST), a clear dose-dependent increase in mutation
        frequencies was observed.22,23
            In 2006, the American National Toxicology Program repeated mutagenicity
        testing using the standard Salmonella assay and various tester strains, in the pres-
        ence and absence of a metabolic activation system.6 Again conflicting results
        were found. For instance, equivocal results were obtained for strains TA100,
        TA97, and TA98, without a metabolic activation system (one of the two trials
        only). No reverse point mutations were found for strain TA1535 and TA1537.
        Also, no treatment-related mutations were found when a metabolic activation
        system was used in any of the strains, except a weak positive outcome in strain
        Mutagenicity and genotoxicity                                                        23
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<pre>    TA1535 (hamster S9; one of the two trials only). Based on its data and from data
    presented by others, The National Toxicology Program suggested that the con-
    flicting results may, in some cases, be related to improper control for the volatil-
    ity of the agent.
         In cultured animal cells, BDCM induced gene mutations in the tk locus of
    L5178Y mouse lymphoma cells.2 Using the same lymphoma assay, inconclusive
    outcomes were obtained in an international collaborative study, because a mar-
    ginal response was found in one laboratory and a negative response in the other
    (Sofuni et al. 1996).24 BDCM induced trifluorothymidine resistance in L5178Y
    mouse lymphoma cells, in the presence of a metabolic activation system, but not
    without such a metabolic activation (NTP 2006).6
    Regarding DNA damage, BDCM induced primary DNA damage in Escherichia
    coli PQ37, when the SOS chromotest was used (Le Curieux et al. 1995).21 Fur-
    thermore, exposure of primary rat and human kidney cells to 0.5 – 4.0 mM
    BDCM resulted in a statistically significant increase in DNA damage (Robbiano
    et al. 2004).25
    Bromodichloromethane provoked a slight increase in chromosomal aberrations
    in Chinese hamster lung fibroblasts, in the presence and absence of a metabolic
    activation system (Matsuoka et al. 1996).26 However, in the same study, it did not
    induce polyploidy. In another study, no increased frequencies in chromosomal
    aberrations due to BDCM exposure could be detected in Chinese hamster ovary
    cells.6
         Conflicting outcomes were reported of sister chromatid exchanges (SCEs). In
    its Monograph, IARC reported on two studies using Chinese hamster ovary and
    FAF cells with negative outcomes.2 In the absence of a metabolic activation sys-
    tem, BDCM significantly induced SCEs in a clear dose-dependent manner in rat
    erythroblastic leukemia cells (Fujie et al. 1993).27 Also, a small increase in SCEs
    was observed in one of the four trials, using Chinese hamster ovary cells, but
    only in the presence of a metabolic activation system (NTP 2006).6
         In a micronucleus assay, using primary rat and human kidney cells, a signifi-
    cant increase in micronucleated cells occurred after BDCM exposure.25
4.2 In vivo assays
    No data were available on the induction of gene mutations by bromodichlo-
    romethane.
24  Bromodichloromethane
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<pre>No unscheduled DNA synthesis occurred in the liver cells of male Sprague-Daw-
ley rats, which were given a single dose of 135 and 450 mg BDCM/kg bw by
gastric intubation (Stocker et al. 1997).28
    In another study, using the same animal species and the Comet assay, a single
oral administration of 458 mg BDCM/kg bw (half of the LD50 dose) did induce
DNA damage in the kidney cells of the rats (Robbiano et al. 2004).25
    Potter et al. (1996) did not find increased frequencies of DNA strand breaks
in the kidneys of male F344 rats, which were given daily oral doses of 0.75 and
1.5 mmol BDCM/kg bw by gavage for 7 days.29
Regarding its clastogenic potential, in the bone marrow cells of male and female
Long-Evans rats, statistically significantly increased frequencies of chromo-
somal aberrations were observed after a single intraperitoneal injection of 164
mg BDCM/kg bw, but not at lower doses (Fujie et al. 1990).30 However, when the
agent was given orally by gastric intubation at the same dose levels, five times on
five successive days, no treatment-related chromosomal aberrations in the bone
marrow cells could be detected.
    Morimoto and Koizumi (1983) reported on the induction of sister chromatid
exchanges in bone marrow cells of mice, which were given orally 50 mg BDCM/
kg bw, four times on four different days.2,31
    Conflicting results were obtained in showing chromosomal fragmentations
by micronucleus tests. For instance, no BDCM-induce increases in micronucle-
ated cells were observed in: the bone marrow cells and peripheral blood lym-
phocytes of mice, given intraperitoneal injections of 200 or 500 mg BDCM/kg
bw (Hayashi et al. 1988; IARC 1999)2,32; in bone marrow polychromatic erythro-
cytes of male B6C3F1 mice, which were given intraperitoneal injections of 200 to
500 mg BDCM/kg bw, three times at 24-hour intervals (killing 24 hours after last
injection; NTP 2006)6; and, in peripheral blood erythrocytes of female B6C3F1
mice, given 44 – 700 mg BDCM per liter drinking water for 3 weeks (NTP
2006)6. However, significant positive outcomes were found in: the kidney cells
of Sprague-Dawley rats, which were given a single oral administration of 458 mg
BDCM/kg bw (Robbiano et al. 2004)25; and, in peripheral blood lymphocytes of
the amphibian Pleurodeles waltl larvae, which were exposed to 50 μg BDCM/
mL for 12 days (Le Curieux et al. 1995).21
    Torti et al. (2002) investigated the induction of micronuclei in peripheral
blood cells of wild type and heterozygous p53 C57BL/6 and FVB/N mice by
inhalation exposure.33 In short, groups of five to six animals were daily exposed
to up to 30 or 300 ppm BDCM (≈ 4.5 or 45 mg/m3 at 25°C) for 6 hours per day
Mutagenicity and genotoxicity                                                       25
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<pre>    for one, three or thirteen weeks. The outcome indicated a weak induction of
    micronuclei by BDCM at concentrations higher than 30 ppm.
4.3 Carcinogenic mode of action
    The mechanisms through which bromodichloromethane may exert its genotoxic
    and carcinogenic potential are not clarified yet. Nevertheless, some ideas are
    given in the literature, as stated below.
        Firstly, the biotransformation of BDCM. There are three potential (compet-
    ing) metabolic pathways for BDCM suggested, which give rise to the formation
    of reactive intermediates (NTP 2006).6 The first is oxidative metabolism by a
    cytochrome P450 mixed function oxidase system (predominantly the cyto-
    chrome P450 2E1 isoform), resulting in highly reactive dihalocarbonyl interme-
    diates that are further detoxified into CO2 (hydrolysis) or CO (reduction by
    glutathione binding). The second is reductive metabolism mediated by cyto-
    chrome P450, which results in free radical intermediates. Thirdly, BDCM may
    undergo glutathione-S-transferase-catalyzed conjunction with glutathione,
    resulting in DNA reactive S-dihalomethyl metabolites. Reactive intermediates
    may bind to proteins and even DNA. That BDCM is able to covalently modify
    DNA and to form DNA (2’-deoxyguanosine) adducts, after conjunction with glu-
    tathione, is shown by Ross et al. (2004).23 Based on their results and those from
    others, these authors suggested that the carcinogenicity of BDCM could depend
    on the bioactivation capacity, in particular the activity of P450 2E1 and glu-
    tathione-S-transferase (GSTT1-1) isoenzymes.
        The rate of activity of these enzymes are genetically determined and species
    dependent. Such differences between species may explain the different outcomes
    between rats and mice in carcinogenicity studies. Furthermore, the central role of
    biotransformation in the toxicity of BDCM may explain why toxic effects are
    restricted to a few organs, such as the liver and kidneys, since biotransformation
    of xenobiotics is mainly localized in these (and some other) organs.
        Secondly, in a few animal studies using B6C3F1 mice or F344 rats, it was
    shown that BDCM induced DNA hypomethylation, when the compound was
    given in the drinking water or by gavage (Coffin et al. 2000; Pereira et al. 2004;
    Tao et al. 2005).35-37 In B6C3F1 mice, it was furthermore shown that BDCM
    caused DNA hypomethylation of the c-myc tumour promotor gene. DNA methy-
    lation is a key epigenetic mechanism that results in the silencing or down regula-
    tion of genes, without changing their coding sequence. As such, DNA
    methylation plays an important role in DNA repair and genome instability.38,39 It
26  Bromodichloromethane
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<pre>is hypothesized that hypomethylation may contribute to oncogenesis, for
instance by activating oncogenes or stimulating chromosome instability.38
Mutagenicity and genotoxicity                                             27
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<pre>28 Bromodichloromethane</pre>

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<pre>Chapter 5
        Classification
5.1     Evaluation of data on carcinogenicity and genotoxicity
        No data on the genotoxicity and carcinogenicity in humans were available.
            The main routes of BDCM-exposure in animal carcinogenicity studies were
        by gavage and drinking water. This plus the use of different animal species
        resulted in mixed results. For instance, in mice, liver and kidney tumours were
        found when BDCM was given by gavage, but not in drinking water. Also tumour
        development in rats depended on the route of exposure. For instance, liver
        tumours were found after exposure by gavage diet and drinking water, but kidney
        tumours and tumours in the large intestines were only found when BDCM was
        given by gavage. Furthermore, under comparable study and exposure conditions,
        conflicting results were obtained in that in one study tumours were observed, but
        not in another study. However, despite the somewhat contradictory findings, the
        study was well designed, and no reasons could be found that the tumours found
        in the animals were not relevant. All in all, the animal studies give sufficient evi-
        dence that exposure to bromodichloromethane can result in cancer development.
            Also mutagenicity and genotoxicity testing revealed mixed results. For
        instance, gene mutations were observed in certain bacterial Salmonella strains
        and mammalian cells, but not in all cases. DNA damage was observed in vitro
        and in vivo, but in at least one in vivo study no damage (strand breaks) was
        found. Also testing for its clastogenic potential (i.e., chromosomal aberrations,
        sister chromatid exchanges, micronuclei) resulted in mixed outcomes in both in
        Classification                                                                        29
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<pre>    vitro and in vivo assays. It seems that the conflicting results could partly be
    explained by differences in experimental conditions, such as the route of expo-
    sure, and the absence or presence of certain biotransformation enzymes, such as
    glutathione-S transferase (GSTT1-1). At the moment it is unclear how bromo-
    dichloromethane exerts its genotoxic and carcinogenic effects, although it is
    hypothesized that through biotransformation, reactive intermediates are formed,
    which can bind to DNA and thus cause DNA damage. Another possible (non-
    genotoxic) route is DNA hypomethylation. Based on the available mutagenicity
    and genotoxicity data, the committee is of the opinion that there are sufficient
    indications that BDCM could exert its carcinogenic effect by a stochastic geno-
    toxic mechanism.
        The committee did not find indications that the observations in animals, and
    the proposed carcinogenic mechanism would not occur in humans.
5.2 Recommendation for classification
    The committee is of the opinion that bromodichloromethane should be consid-
    ered as carcinogenic to humans. This recommendation corresponds to the EU
    classification in category 2. The committee concludes furthermore that bromo-
    dichloromethane acts by a stochastic genotoxic mechanism.
30  Bromodichloromethane
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<pre>   References
1  Agency for Toxic Substances and Disease Registry (ATSDR). Toxicological profile for
   bromodichloromethane. ATSDR; 1989.
2  IARC. Bromodichloromethane. IARC Monogr Eval Carcinog Risks Hum 1999; 71 Pt 3: 1295-1304.
3  National Toxicology Program. Substance Profiles: Bromodichloromethane. In: Report on
   carcinogens, 11th edition. U.S. Department of Health and Human Services; 2005: 35-36.
4  Doyle TJ, Zheng W, Cerhan JR, Hong CP, Sellers TA, Kushi LH e.a. The association of drinking
   water source and chlorination by-products with cancer incidence among postmenopausal women in
   Iowa: a prospective cohort study. Am J Public Health 1997; 87(7): 1168-1176.
5  Morris RD, Audet AM, Angelillo IF, Chalmers TC, Mosteller F. Chlorination, chlorination by-
   products, and cancer: a meta-analysis. Am J Public Health 1992; 82(7): 955-963.
6  National Toxicology Program (NTP). Toxicology and carcinogenesis studies of
   bromodichloromethane (CAS No. 75-27-4) in male F344/N rats and female B6C3F1 mice (Drinking
   Water Studies). Natl Toxicol Program Tech Rep Ser 2006;(532): 1-248.
7  Dunnick JK, Melnick RL. Assessment of the carcinogenic potential of chlorinated water:
   experimental studies of chlorine, chloramine, and trihalomethanes. J Natl Cancer Inst 1993; 85(10):
   817-822.
8  George MH, Olson GR, Doerfler D, Moore T, Kilburn S, DeAngelo AB. Carcinogenicity of
   bromodichloromethane administered in drinking water to Male F344/N Rats and B6C3F1 mice. Int J
   Toxicol 2002; 21(3): 219-230.
9  IARC. Bromodichloromethane. IARC Monogr Eval Carcinog Risks Hum 1991; 52: 179-212.
10 Tumasonis CF, McMartin DN, Bush B. Lifetime toxicity of chloroform and bromodichloromethane
   when administered over a lifetime in rats. Ecotoxicol Environ Saf 1985; 9(2): 233-240.
   References                                                                                          31
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<pre>11 Voronin VM, Donchenko AI, Korolev AA. [Experimental study of the carcinogenicity of
   dichlorobromomethane and dibromochloromethane formed during the chlorination of water]. Gig
   Sanit 1987;(1): 19-21.
12 Aida Y, Yasuhara K, Takada K, Kurokawa Y, Tobe M. Chronic toxicity of microencapsulated
   bromodichloromethane administered in the diet to Wistar rats. J Toxicol Sci 1992; 17(2): 51-68.
13 National Toxicology Program. Toxicology studies of bromodichloromethane (Cas no. 75-27-4) in
   genetically modified (FVB Tg.AC hemizygous) mice (dermal, drinking water, and gavage studies),
   and carcinogenicity studies of bromodichloromethane in genetically modified [B6.129-Trp53tm1Brd (N5)
   haploinsufficient] mice (drinking water and gavage studies. National Institute of Health Public
   Service, NIH publication No. 07-4422, NC, USA; 2007.
14 Hooth MJ, McDorman KS, Hester SD, George MH, Brooks LR, Swank AE e.a. The carcinogenic
   response of Tsc2 mutant Long-Evans (Eker) rats to a mixture of drinking water disinfection by-
   products was less than additive. Toxicol Sci 2002; 69(2): 322-331.
15 McDorman KS, Hooth MJ, Starr TB, Wolf DC. Analysis of preneoplastic and neoplastic renal lesions
   in Tsc2 mutant Long-Evans (Eker) rats following exposure to a mixture of drinking water
   disinfection by-products. Toxicology 2003; 187(1): 1-12.
16 McDorman KS, Chandra S, Hooth MJ, Hester SD, Schoonhoven R, Wolf DC. Induction of
   transitional cell hyperplasia in the urinary bladder and aberrant crypt foci in the colon of rats treated
   with individual and a mixture of drinking water disinfection by-products. Toxicol Pathol 2003; 31(2):
   235-242.
17 DeAngelo AB, Geter DR, Rosenberg DW, Crary CK, George MH. The induction of aberrant crypt
   foci (ACF) in the colons of rats by trihalomethanes administered in the drinking water. Cancer Lett
   2002; 187(1-2): 25-31.
18 Geter DR, George MH, Moore TM, Kilburn S, Huggins-Clark G, DeAngelo AB. Vehicle and mode of
   administration effects on the induction of aberrant crypt foci in the colons of male F344/N rats
   exposed to bromodichloromethane. J Toxicol Environ Health A 2004; 67(1): 23-29.
19 Toussaint MW, Rosencrance AB, Brennan LM, Dennis WE, Beaman JR, Wolfe MJ e.a. Chronic
   toxicity of bromodichloromethane to the Japanese medaka (Oryzias latipes). Toxicol Pathol 2001;
   29(6): 662-669.
20 Kundu B, Richardson SD, Granville CA, Shaughnessy DT, Hanley NM, Swartz PD e.a. Comparative
   mutagenicity of halomethanes and halonitromethanes in Salmonella TA100: structure-activity
   analysis and mutation spectra. Mutat Res 2004; 554(1-2): 335-350.
21 Le Curieux F, Gauthier L, Erb F, Marzin D. Use of the SOS chromotest, the Ames-fluctuation test and
   the newt micronucleus test to study the genotoxicity of four trihalomethanes. Mutagenesis 1995;
   10(4): 333-341.
22 Pegram RA, Andersen ME, Warren SH, Ross TM, Claxton LD. Glutathione S-transferase-mediated
   mutagenicity of trihalomethanes in Salmonella typhimurium: contrasting results with
   bromodichloromethane off chloroform. Toxicol Appl Pharmacol 1997; 144(1): 183-188.
32 Bromodichloromethane
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<pre>23 Ross MK, Pegram RA. In vitro biotransformation and genotoxicity of the drinking water disinfection
   byproduct bromodichloromethane: DNA binding mediated by glutathione transferase theta 1-1.
   Toxicol Appl Pharmacol 2004; 195(2): 166-181.
24 Sofuni T, Honma M, Hayashi M, Shimada H, Tanaka N, Wakuri S e.a. Detection of in vitro
   clastogens and spindle poisons by the mouse lymphoma assay using the microwell method: interim
   report of an international collaborative study. Mutagenesis 1996; 11(4): 349-355.
25 Robbiano L, Baroni D, Carrozzino R, Mereto E, Brambilla G. DNA damage and micronuclei induced
   in rat and human kidney cells by six chemicals carcinogenic to the rat kidney. Toxicology 2004;
   204(2-3): 187-195.
26 Matsuoka A, Yamakage K, Kusakabe H, Wakuri S, Asakura M, Noguchi T e.a. Re-evaluation of
   chromosomal aberration induction on nine mouse lymphoma assay "unique positive' NTP
   carcinogens. Mutat Res 1996; 369(3-4): 243-252.
27 Fujie K, Aoki T, Ito Y, Maeda S. Sister-chromatid exchanges induced by trihalomethanes in rat
   erythroblastic cells and their suppression by crude catechin extracted from green tea. Mutat Res
   1993; 300(3-4): 241-246.
28 Stocker KJ, Statham J, Howard WR, Proudlock RJ. Assessment of the potential in vivo genotoxicity
   of three trihalomethanes: chlorodibromomethane, bromodichloromethane and bromoform.
   Mutagenesis 1997; 12(3): 169-173.
29 Potter CL, Chang LW, DeAngelo AB, Daniel FB. Effects of four trihalomethanes on DNA strand
   breaks, renal hyaline droplet formation and serum testosterone in male F-344 rats. Cancer Lett 1996;
   106(2): 235-242.
30 Fujie K, Aoki T, Wada M. Acute and subacute cytogenetic effects of the trihalomethanes on rat bone
   marrow cells in vivo. Mutat Res 1990; 242(2): 111-119.
31 Morimoto K, Koizumi A. Trihalomethanes induce sister chromatid exchanges in human lymphocytes
   in vitro and mouse bone marrow cells in vivo. Environ Res 1983; 32(1): 72-79.
32 Hayashi M, Norppa H, Sofuni T, Ishidate MJ. Flow cytometric micronucleus test woth mouse bone
   marrow and peripheral bloos erythrocytes. Environ Mol Mutagen 1989; 14: 85.
33 Torti VR, Cobb AJ, Wong VA, Butterworth BE. Induction of micronuclei in wild-type and p53(+/-)
   transgenic mice by inhaled bromodichloromethane. Mutat Res 2002; 520(1-2): 171-178.
34 Kundu B, Richardson SD, Swartz PD, Matthews PP, Richard AM, DeMarini DM. Mutagenicity in
   Salmonella of halonitromethanes: a recently recognized class of disinfection by-products in drinking
   water. Mutat Res 2004; 562(1-2): 39-65.
35 Coffin JC, Ge R, Yang S, Kramer PM, Tao L, Pereira MA. Effect of trihalomethanes on cell
   proliferation and DNA methylation in female B6C3F1 mouse liver. Toxicol Sci 2000; 58(2): 243-
   252.
36 Pereira MA, Wang W, Kramer PM, Tao L. DNA hypomethylation induced by non-genotoxic
   carcinogens in mouse and rat colon. Cancer Lett 2004; 212(2): 145-151.
37 Tao L, Wang W, Li L, Kramer PK, Pereira MA. DNA hypomethylation induced by drinking water
   disinfection by-products in mouse and rat kidney. Toxicol Sci 2005; 87(2): 344-352.
   References                                                                                           33
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<pre>38 Das PM, Singal R. DNA methylation and cancer. J Clin Oncol 2004; 22(22): 4632-4642.
39 Robertson KD, Jones PA. DNA methylation: past, present and future directions. Carcinogenesis
   2000; 21(3): 461-467.
34 Bromodichloromethane
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<pre>A Request for advice
B The committee
C Comments on the public review draft
D IARC Monograph
E Carcinogenic classification of substances by the committee
F Guideline 93/21/EEG of the European Union
  Annexes
                                                             35
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<pre>36 Bromodichloromethane</pre>

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<pre>Annex A
      Request for advice
      In a letter dated October 11, 1993, ref DGA/G/TOS/93/07732A, to, the State
      Secretary of Welfare, Health and Cultural Affairs, the Minister of Social Affairs
      and Employment wrote:
      Some time ago a policy proposal has been formulated, as part of the simplification of the governmen-
      tal advisory structure, to improve the integration of the development of recommendations for health
      based occupation standards and the development of comparable standards for the general population.
      A consequence of this policy proposal is the initiative to transfer the activities of the Dutch Expert
      Committee on Occupational Standards (DECOS) to the Health Council. DECOS has been established
      by ministerial decree of 2 June 1976. Its primary task is to recommend health based occupational
      exposure limits as the first step in the process of establishing Maximal Accepted Concentrations
      (MAC-values) for substances at the work place.
      In an addendum, the Minister detailed his request to the Health Council as fol-
      lows:
      The Health Council should advice the Minister of Social Affairs and Employment on the hygienic
      aspects of his policy to protect workers against exposure to chemicals. Primarily, the Council should
      report on health based recommended exposure limits as a basis for (regulatory) exposure limits for air
      quality at the work place. This implies:
      •    A scientific evaluation of all relevant data on the health effects of exposure to substances using a
           criteria-document that will be made available to the Health Council as part of a specific request
      Request for advice                                                                                        37
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<pre>       for advice. If possible this evaluation should lead to a health based recommended exposure limit,
       or, in the case of genotoxic carcinogens, a ‘exposure versus tumour incidence range’ and a calcu-
       lated concentration in air corresponding with reference tumour incidences of 10-4 and 10-6 per
       year.
   •   The evaluation of documents review the basis of occupational exposure limits that have been
       recently established in other countries.
   •   Recommending classifications for substances as part of the occupational hygiene policy of the
       government. In any case this regards the list of carcinogenic substances, for which the classifica-
       tion criteria of the Directive of the European Communities of 27 June 1967 (67/548/EEG) are
       used.
   •   Reporting on other subjects that will be specified at a later date.
   In his letter of 14 December 1993, ref U 6102/WP/MK/459, to the Minister of
   Social Affairs and Employment the President of the Health Council agreed to
   establish DECOS as a Committee of the Health Council.
38 Bromodichloromethane
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<pre>Annex B
      The committee
      •   G..J. Mulder, chairman
          emeritus professor of toxicology, Leiden University, Leiden
      •   P.J. Boogaard
          toxicologist, SHELL International BV, The Hague
      •   Ms. M.J.M. Nivard
          Molecular biologist and genetic toxicologist, Leiden University Medical
          Center, Leiden
      •   G.M.H. Swaen
          epidemiologist, Dow Chemicals NV, Terneuzen
      •   R.A. Woutersen
          toxicologic pathologist, TNO Nutrition and Food Research, Zeist
      •   A.A. van Zeeland
          professor of molecular radiation dosimetry and radiation mutagenesis,
          University Medical Center, Leiden
      •   E.J.J. van Zoelen
          professor of cell biology, Radboud University Nijmegen, Nijmegen
      •   J.M. Rijnkels, scientific secretary
          Health Council of the Netherlands, The Hague
      The committee consulted an additional expert, Prof dr G Mohn, working at
      Department of Radiation Genetics and Chemical Mutagenesis of the University
      of Leiden, with respect to the genotoxic data.
      The committee                                                               39
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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 Coun-
   cil Committee. Transparency regarding possible conflicts of interest is nonethe-
   less important, both for the President and members of a Committee and for the
   President of the Health Council. On being invited to join a Committee, members
   are asked to submit a form detailing the functions they hold and any other mate-
   rial and immaterial interests which could be relevant for the Committee’s work.
   It is the responsibility of the President of the Health Council to assess whether
   the interests indicated constitute grounds for non-appointment. An advisorship
   will then sometimes make it possible to exploit the expertise of the specialist
   involved. During the establishment meeting the declarations issued are dis-
   cussed, so that all members of the Committee are aware of each other’s possible
   interests.
40 Bromodichloromethane
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<pre>Annex C
      Comments on the public review draft
      A draft of the present report was released in 2007 for public review. The follow-
      ing organisations and persons have commented on the draft document:
      • G. Jonkers, Vereniging van Verf en Drukinktfabrikanten, the Netherlands;
      • E. González-Fernández, Ministerio de Trabajo y Asuntos Sociales, Spain;
      • R.D. Zumwalde, National Institute for Occupational Safety and Health, the
          USA.
      Comments on the public review draft                                               41
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<pre>42 Bromodichloromethane</pre>

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<pre>Annex D
      IARC Monograph
D.1   VOL: 52 (1991) (p. 179)9
      CAS No.: 75-27-4
      Summary of Data Reported and Evaluation
      Exposure data
      Bromodichloromethane is found in chlorinated drinking-water as a consequence
      of the reaction between chlorine, added during water treatment, and natural
      organic substances in the presence of bromide. The major route of human expo-
      sure to bromodichloromethane is via drinking-water. It has been detected in chlo-
      rinated drinking-water in many parts of the world; it has also been detected in
      some untreated waters, but at much lower levels. Bromodichloromethane is a
      major component of the organohalides produced by marine algae.
      Experimental carcinogenicity data
      Bromodichloromethane was tested for carcinogenicity in two-year studies in
      male and female Fischer 344 rats and B6C3F1 mice by oral gavage, in life-span
      studies in male and female Wistar rats and in CBA x C57Bl/6 hybrid mice by
      administration in drinking-water. In the gavage studies, bromodichloromethane
      IARC Monograph                                                                    43
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<pre>   increased the incidences of adenomatous polyps and adenocarcinomas of the
   large intestine and of tubular-cell adenomas and adenocarcinomas of the kidney
   in male and female rats, of tubular-cell adenomas and adenocarcinomas of the
   kidney in male mice and of hepatocellular adenomas and carcinomas in female
   mice. In the study by administration in drinking-water, it induced neoplastic nod-
   ules and adenofibrosis of the liver in rats; no increase in tumour incidence was
   seen in mice. In a screening test for lung adenomas by intraperitoneal injection,
   bromodichloromethane did not increase the incidence of lung tumours in strain A
   mice.
   Human carcinogenicity data
   No relevant data were available to the Working Group.
   Other relevant data
   Repeated exposure of rats and mice to bromodichloromethane resulted in toxic
   effects in several organs, including the liver and kidney.
   A study of developmental toxicity in rats given bromodichloromethane through-
   out the period of major organogenesis showed skeletal variations in the presence
   of maternal toxicity but no teratogenic effect.
   Bromodichloromethane induced mutations in some studies with bacteria and, in
   a single study, in cultured mammalian cells. Chromosomal aberrations but not
   sister chromatid exchange were observed in cultured mammalian cells. In single
   studies, sister chromatid exchange was observed in cultured human cells and in
   mouse bone marrow in vivo. In one study, bromodichloromethane did not induce
   micronuclei in bone-marrow cells of mice treated in vivo.
   Evaluation
   There is inadequate evidence for the carcinogenicity of bromodichloromethane
   in humans.
   There is sufficient evidence for the carcinogenicity of bromodichloromethane in
   experimental animals.
44 Bromodichloromethane
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<pre>    Overall evaluation
    Bromodichloromethane is possibly carcinogenic to humans (Group 2B).
D.2 VOL: 71 (1999) (p. 1295)2
    Evaluation
    No epidemiological data relevant to the carcinogenicity of bromodichlo-
    romethane were available.
    There is sufficient evidence in experimental animals for the carcinogenicity of
    bromodichloromethane.
    Overall evaluation
    Bromodichloromethane is possibly carcinogenic to humans (Group 2B).
    IARC Monograph                                                                  45
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<pre>46 Bromodichloromethane</pre>

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<pre>Annex         E
              Carcinogenic classification of
              substances by the committee
The committee expresses its conclusions in the form of standard phrases:
Judgment of the committee                                                                       Comparable with EU class
This compound is known to be carcinogenic to humans                                             1
•   It is stochastic or non-stochastic genotoxic
•   It is non-genotoxic
•   Its potential genotoxicity has been insufficiently investigated. Therefore, it is unclear
    whether it is genotoxic
This compound should be regarded as carcinogenic to humans                                      2
•   It is stochastic or non-stochastic genotoxic
•   It is non-genotoxic
•   Its potential genotoxicity has been insufficiently investigated. Therefore, it is unclear
    whether it is genotoxic
This compound is a suspected human carcinogen.                                                  3
•   This compound has been extensively investigated. Although there is insufficient evidence    (A)
    for a carcinogenic effect to warrant a classification as ‘known to be carcinogenic to
    humans’ or as ‘should be regarded as carcinogenic to humans’, they indicate that there is
    cause for concern.
•   This compound has been insufficiently investigated. While the available data do not war-    (B)
    rant a classification as ‘known to be carcinogenic to humans’ or as ‘should be regarded as
    carcinogenic to humans’, they indicate that there is a cause for concern.
This compound cannot be classified                                                              not classifiable
•   There is a lack of carcinogenicity and genotoxicity data.
•   Its carcinogenicity is extensively investigated. The data indicate sufficient evidence sug-
    gesting lack of carcinogenicity.
              Carcinogenic classification of substances by the committee                                             47
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<pre>48 Bromodichloromethane</pre>

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<pre>Annex F
      Guideline 93/21/EEG of the European
      Union
      4.2            Criteria for classification, indication of danger, choice of risk phrases
      4.2.1          Carcinogenic substances
      For the purpose of classification and labelling, and having regard to the current state of knowledge,
      such substances are divided into three categories:
      Category 1:
      Substances known to be carcinogenic to man.
      There is sufficient evidence to establish a causal association between human exposure to a substance
      and the development of cancer.
      Category 2:
      Substances which should be regarded as if they are carcinogenic to man.
      There is sufficient evidence to provide a strong presumption that human exposure to a substance may
      result in the development of cancer, generally on the basis of:
      •    appropriate long-term animal studies
      •    other relevant information.
      Guideline 93/21/EEG of the European Union                                                             49
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<pre>   Category 3:
   Substances which cause concern for man owing to possible carcinogenic effects but in
   respect of which the available information is not adequate for making a satisfactory assess-
   ment.
   There is some evidence from appropriate animal studies, but this is insufficient to place the substance
   in Category 2.
   4.2.1.1       The following symbols and specific risk phrases apply:
   Category 1 and 2:
   T; R45 May cause cancer
   However for substances and preparations which present a carcinogenic risk only when inhaled, for
   example, as dust, vapour or fumes, (other routes of exposure e.g. by swallowing or in contact with
   skin do not present any carcinogenic risk), the following symbol and specific risk phrase should be
   used:
   T; R49 May cause cancer by inhalation
   Category 3:
   Xn; R40 Possible risk of irreversible effects
   4.2.1.2       Comments regarding the categorisation of carcinogenic substances
   The placing of a substance into Category 1 is done on the basis of epidemiological data; placing into
   Categories 2 and 3 is based primarily on animal experiments.
   For classification as a Category 2 carcinogen either positive results in two animal species should be
   available or clear positive evidence in one species; together with supporting evidence such as geno-
   toxicity data, metabolic or biochemical studies, induction of benign tumours, structural relationship
   with other known carcinogens, or data from epidemiological studies suggesting an association.
50 Bromodichloromethane
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<pre>Category 3 actually comprises 2 sub-categories:
a    substances which are well investigated but for which the evidence of a tumour-inducing effect is
     insufficient for classification in Category 2. Additional experiments would not be expected to
     yield further relevant information with respect to classification.
b    substances which are insufficiently investigated. The available data are inadequate, but they
     raise concern for man. This classification is provisional; further experiments are necessary
     before a final decision can be made.
For a distinction between Categories 2 and 3 the arguments listed below are relevant which reduce
the significance of experimental tumour induction in view of possible human exposure. These argu-
ments, especially in combination, would lead in most cases to classification in Category 3, even
though tumours have been induced in animals:
•    carcinogenic effects only at very high levels exceeding the 'maximal tolerated dose'. The maxi-
     mal tolerated dose is characterized by toxic effects which, although not yet reducing lifespan, go
     along with physical changes such as about 10% retardation in weight gain;
•    appearance of tumours, especially at high dose levels, only in particular organs of certain species
     is known to be susceptible to a high spontaneous tumour formation;
•    appearance of tumours, only at the site of application, in very sensitive test systems (e.g. i.p. or
     s.c. application of certain locally active compounds);
•    if the particular target is not relevant to man;
•    lack of genotoxicity in short-term tests in vivo and in vitro;
•    existence of a secondary mechanism of action with the implication of a practical threshold above
     a certain dose level (e.g. hormonal effects on target organs or on mechanisms of physiological
     regulation, chronic stimulation of cell proliferation;
•    existence of a species - specific mechanism of tumour formation (e.g. by specific metabolic
     pathways) irrelevant for man.
For a distinction between Category 3 and no classification arguments are relevant which exclude a
concern for man:
•    a substance should not be classified in any of the categories if the mechanism of experimental
     tumour formation is clearly identified, with good evidence that this process cannot be extrapo-
     lated to man;
•    if the only available tumour data are liver tumours in certain sensitive strains of mice, without
     any other supplementary evidence, the substance may not be classified in any of the categories;
•    particular attention should be paid to cases where the only available tumour data are the occur-
     rence of neoplasms at sites and in strains where they are well known to occur spontaneously with
     a high incidence.
Guideline 93/21/EEG of the European Union                                                                 51
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<pre>52 Bromodichloromethane</pre>

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<br><br>