Data from Epidemiological Studies There is evidence from epidemiological studies which suggests that exposure to bromodichloromethane is possibly associated with reproductive effects. An epidemiological study by Waller et al. ( 1998) found an association between consumption of trihalomethanes in drinking water and increased risk of spontaneous abortion. Analysis of personal exposure to individual trihalomethanes demonstrated that bromodichloromethane had the strongest association to with spontaneous abortion. When all four individual trihalomethanes ( including chloroform) were simultaneously included as variables in a logistic regression model, high personal bromodichloromethane exposure had an odds ratio of 3.0 for spontaneous abortion ( 95% C. I. 1.4, 6.6). King et al. ( 2000) examined the relationship between stillbirth of fetuses weighing more than 500 g and chlorination byproducts in drinking water. Increased risk of stillbirth was associated with total trihalomethane concentration, chloroform concentration, and bromodichloromethane concentration. The strongest association was observed for bromodichloromethane, where risk doubled in women exposed to concentrations of 20 µ g/ L or more when compared to women consuming water containing concentrations less than 5 µ g/ L.

Both population based studies had reasonable cohorts, recruitment procedures, and screening protocols. The number of subjects was adequate, and the information regarding fetal losses was credible. Therefore, the conclusion that increased fetal loss in humans occurs in association with the exposure measurements that were made is quite convincing. A weakness of both population based studies, however, is that the measurement of the trihalomethanes appeared to be at the source or at intermediate sources rather than at the tap. Thus, the Draft  Do Not Cite or Quote February 20, 2003 VII  8 bromodichloromethane concentrations that were measured could be different than the actual exposures ( levels at the tap) and/ or the bromodichloromethane could be a surrogate for the actual toxicant in both population based studies. Although these human studies raise significant concern, the occurrence of multiple disinfection byproducts in drinking water prevents the conclusion that bromodichloromethane was the specific causative agent for the observed effects.

Full Litter Resorption in F344 Rats As indicated above, two oral exposure studies of bromodichloromethane have observed increased incidences of full litter resorption ( FLR) in the F344 strain of rat. Narotsky et al. ( 1997) observed a significantly increased incidence of full litter resorption ( FLR) in F344 rats treated with 75 mg/ kg day bromodichloromethane by aqueous gavage throughout the period of organogenesis. These studies are of particular interest because of epidemiological association between bromodichloromethane ingestion and increased risk of spontaneous abortion.

In light of human epidemiological studies possibly implicating bromodichloromethane in pregnancy loss, Bielmeier et al. ( 2001) conducted a series of experiments to further elucidate the mode of action of this compound on full litter resorption in F344 rats. Their experiments investigated the effects of bromodichloromethane on serum levels of progesterone ( necessary for the maintenance of pregnancy) and luteinizing hormone ( LH), which helps maintain the corpus luteum and luteal function ( the secretion of progesterone). In the rat, secretion of progesterone by the corpus luteum is required up to GD 17 for maintenance of pregnancy. Luteal function is established and maintained in the rat through a series of mechanisms that require cervical stimulation by coitus, a pituitary response to this cervical stimulation, and a secretion of hormones by the products of conception. In the newly formed corpus luteum, progesterone secretion is autonomous. However, continued function requires luteotropic hormones secreted by the pituitary. In many species LH is considered to be the most important luteotropic factor. However, in rats prolactin has been identified as the primary luteotropic factor.

The hormonal requirements of the corpus luteum change with the physiological stage of the pregnancy. Early in the rat pregnancy, prolactin secreted by the pituitary is essential for luteal function. Starting at about GD 6, LH also is required for maintenance of luteal function in addition to prolactin. The corpora lutea are dependent on LH until GD 11, at which time luteal maintenance becomes dependent on one or more placental prolactin like hormones ( prolactogens) ( Gibori et al., 1988).

Bielmeier et al. ( 2001) investigated the effects of bromodichloromethane in rats during two physiologically different stages of pregnancy: GD 6 to 10, which encompasses the LHdependent stage of pregnancy and GD 11 to 15, which is an LH independent stage. These authors observed increased incidences of FLR in F344 rats treated with 75 or 100 mg/ kg day BDCM on one or more days during the LH dependent interval from gestation day 6 to 10, but not in rats treated during the LH independent period. Thus, the critical period for induction of FLR was limited to the luteinizing hormone ( LH) dependent phase of pregnancy, suggesting that bromodichloromethane may disrupt pregnancy via a LH mediated mode of action. The treatment Draft  Do Not Cite or Quote February 20, 2003 VII  9 period also overlapped the period when maintenance of pregnancy is dependent on progesterone produced by the ovary. The response to bromodichloromethane was strain specific and FLR was not observed in Sprague Dawley rats treated with up to 100 mg/ kg day on GD 6 to10. Measurement of serum LH and progesterone levels indicated that FLR was accompanied by a marked reduction in progesterone concentration without a corresponding drop in LH levels. The failure of bromodichloromethane to exert adverse effects after the LH dependent window, the reduction in serum progesterone level, and the unchanged serum luteinizing hormone levels led this group to conclude that the target of toxicity was the ovary and that the mode of action was a reduced sensitivity of the corpus luteum to luteinizing hormone.

Bielmeier et al. ( 2001) have emphasized that there are significant differences between rats and humans in the hormonal maintenance of pregnancy. However, these authors have noted that rats and humans are similar in that either LH or human chorionic gonadotropin ( hCG), which act via the same receptor, are required during specific gestational periods to maintain pregnancy. In the opinion of these authors, the findings in rats may be relevant to observations of adverse pregnancy outcomes in human epidemiological studies if bromodichloromethane disrupts pregnancy by diminishing luteal responsiveness to LH via an effect on the LH/ hCG receptor,. However, additional research is required to determine whether the findings in rats are potentially relevant to humans.

As noted by Narotsky et al. ( 1997), it is important to recognize that bromodichloromethane induced FLR occurs in F344 rats at doses several orders of magnitude greater than would be experienced by humans consuming highly contaminated water. For example, a dose of 75 mg/ kg day is approximately 15,000 fold higher than human intake assuming a body weight of 70 kg, a bromodichloromethane concentration of 180 µ g/ L, and water consumption of 2L/ day. However, the possibility that humans are more sensitive to bromodichloromethane than rats cannot be dismissed. At this time, data are insufficient to evaluate this possibility.

b. Childhood Cancer and Noncancer Effects Bioactivation to reactive metabolites is an apparent prerequisite for toxicity and carcinogenicity of the brominated trihalomethanes. Therefore, an important issue in the assessment of childhood risk of cancer and other adverse effects is whether the enzymes responsible for metabolism are more active in fetuses, neonates, and or children than in adults. This section evaluates the available data for developmental expression and/ or activity of three key metabolizing enzymes that are known or anticipated to bioactivate the brominated trihalomethanes: CYP2E1, CYP2B1/ 2 ( in rodents only), and glutathione S transferase theta.

CYP2E1 Carcinogenicity of brominated trihalomethanes has been shown to be at least partly related to bioactivation by the cytochrome P450 isoform CYP2E1 ( U. S. EPA, 1994b). Thus, a higher level of CYP2E1 activity in children relative to adults might predispose children to greater Draft  Do Not Cite or Quote February 20, 2003 VII  10 toxicity. Studies of human fetal liver have produced contradictory results, but suggest that CYP2E1 protein is either not expressed or is expressed at levels lower than in adults ( Hakkola et al., 1998). Carpenter et al. ( 1996) detected immunoreactive CYP2E1 protein in liver samples from fetuses ranging from 16 to 24 weeks in gestational age. The samples obtained were from fetuses whose mothers did not have a history of alcohol use. The immunoreactive protein exhibited a slightly lower molecular weight than observed for CYP2E1 from adult liver samples. Expression of the corresponding mRNA was confirmed in a fetal liver sample of 19 weeks gestational age by reverse transcriptase polymerase chain reaction ( RT PCR). However, CYP2E1 mRNA was not detectable in a fetal liver sample of 10 weeks gestational age, suggesting ( in the opinion of the study authors) that CYP2E1 expression may be related to specific stages of fetal development. The catalytic capability of CYP2E1 protein in human fetal microsomes was demonstrated by measuring the rate of ethanol oxidation to acetaldehyde. The rate of conversion varied from 12 to 27% of that measured in adult microsomes. Treatment of fetal hepatocytes in primary culture with ethanol or clofibrate indicated that fetal CYP2E1 protein is inducible ( approximately two fold compared to untreated cells).

Viera et al. ( 1996) detected small amounts of CYP2E1 mRNA in fetal liver samples ( approximately 5 to 10% of the levels in adult liver) collected from fetuses aged 14 to 40 weeks. However, these authors could not detect immunoreactive CYP2E1 protein in any of 27 fetal liver samples Other studies have failed to detect either CYP2E1 protein or mRNA in fetal liver samples. Cresteil et al. ( 1985) and Komori et al. ( 1989) did not detect immunoreactive protein or mRNA in fetal liver samples of less than 16 weeks gestational age. Jones et al. ( 1992) did not detect CYP2E1 mRNA or protein in liver samples that were of similar gestational age ( 16 to 18 weeks) to the samples examined by Carpenter et al. ( 1996). Juchau and Yang ( 1996) did not detect CYP2E1 mRNA by RT PCR in human embryonic tissues between days 45 and 60 of gestation. The factors contributing to the different results are unknown, but may include interindividual variability, gestational age of the tissue examined ( for the samples less than 16 weeks gestational age), or the existence of factors other than developmental stage that control expression.

Information on the presence of CYP2E1 in human fetal tissues other than the liver is limited. Viera et al. ( 1998) examined the mRNA content of human fetal lung and kidney. CYP2E1 mRNA was expressed at a very low level in both tissues and the levels remained stable after birth. Studies of human fetal brain tissue indicate that CYP2E1 is expressed in human embryonic brain tissue ( see Juchau et al., 1994) and that relatively low levels of CYP2E1 mRNA, immunoreactive protein, and catalytically active protein are present during the early fetal period of development ( Brzezinski et al., 1999). In one study, a dramatic increase in CYP2E1 was observed at approximately gestation day 50, and a fairly constant level was maintained until at least day 113 ( Brzezinski et al., 1999). The relevance of the data for lung and brain is uncertain, since these organs are not known to be targets for brominated trihalomethane toxicity.

Viera et al. ( 1996) investigated age related variations in human CYP2E1 protein levels and catalytic activity from birth through adulthood. These authors observed a rapid increase in the immunoreactive CYP2E1 microsomal content within 24 hours after birth that was independent Draft  Do Not Cite or Quote February 20, 2003 VII  11 of the gestational age of the newborn. This activation was accompanied by a demethylation of cytosine residues in the 5' regulatory region of the gene, suggesting that methylation of specific residues prevents transcription in the fetal liver. The CYP2E1 protein level gradually increased during the first year and reached the adult level in children aged 1 to 10 years. CYP2E1 catalytic activity was assessed by determination of in vitro hydroxylation of chlorzoxazone in 89 microsomal preparations. Chlorzoxazone hydroxylation activity increased within 24 hours after birth and steadily increased during the first year. Catalytic activity reached adult levels at age 1 to 10 years.

Animal studies of CYP2E1 expression during development have given variable results. One study indicated that CYP2E1 is expressed in the fetal rat liver and placenta and that levels are increased in rat pups exposed to ethanol in utero or via lactation ( Carpenter et al., 1997). Liver samples from rat fetuses exposed to ethanol in utero showed a 2.4 fold increase in protein levels and 1.5 fold increase in catalytic activity ( Carpenter et al., 1997). Other authors have reported that hepatic CYP2E1 gene transcription in rats is activated at birth and that the amount of CYP2E1 reaches a peak prior to weaning ( see Ronis et al., 1996). The protein level then falls to approximately 25% of the peak level and remains stable into adulthood ( Ronis et al., 1996).

The regulation of CYP2E1 is complex when examined at both the molecular ( Lieber 1997) and physiological ( Ronis et al. 1996) levels. The factors and processes responsible for the increase in CYP2E1 protein levels and activity at birth have not been clearly identified. At the physiological level, there is some evidence from rodent studies to suggest that growth hormone regulates the constitutive expression of CYP2E1 ( Ronis et al., 1996). The reduction of CYP2E1 from peak levels before weaning is reported to coincide with the increased levels of growth hormone and with development of adult levels of growth hormone receptors ( Ronis et al., 1996). The occurrence of peak expression after birth has been attributed to a role of CYP2E1 in gluconeogenesis, since there is a very high demand for energy production from glucose at this developmental stage ( see Ronis et al. 1996; Viera et al. 1996).

CYP2B1/ 2 ( Rodents)

Research conducted by Pankow et al. ( 1997) suggests that the closely related CYP isoforms 2B1 and 2B2 participate in the catabolism of dibromochloromethane in rats. These isoforms show greater than 97% homology of amino acid sequence and have highly similar genomic organization. To date, these isoforms have not been reported in adult or fetal human tissues ( Nelson et al., 1996; Juchau et al., 1998). Omiecinski et al. ( 1990) detected low levels of CYP2B isoform mRNA in fetal rat liver on gestation day 15 ( the earliest day in development when the authors were able to macroscopically recognize and dissect the fetal liver) using the polymerase chain reaction ( PCR). Although the levels of mRNA expression were " substantially lower" lower at day 15 than observed later in development, expression was clearly inducible by pretreatment of pregnant rats with phenobarbital. Both constitutive and phenobarbital induced levels of mRNA increased with developmental age, reaching maximal levels at approximately three weeks postpartum. No measurements of CYP2B activity were made in this study, so it is not known whether changes in mRNA levels were paralleled by changes in catalytic activity. Draft  Do Not Cite or Quote February 20, 2003 VII  12 Juchau et al. ( 1998) reviewed a series of experiments that employed the selective substrate probe pentoxyresorufin to test for CYP2B1/ 2 catalytic activity in fetal rat tissues. The overall conclusion upon examination of all results was that if CYP2B isoforms are expressed in fetal rats, they occur at biologically insignificant levels. Asoh et al. ( 1999) examined the catalytic activity of CYP2B isoforms in fetal rat liver and found very low activity, a finding consistent with the conclusion of Juchau et al. ( 1998).

Gebremichael et al. ( 1995) investigated the postnatal developmental profile of CYP2B1 in Sprague Dawley rats. CYP2B1 activity was detectable as early as seven days postnatally and exhibited a variable pattern of expression ( no clear trend evident) when assayed at Days 14, 21, 50, and 100. Asoh et al. ( 1999) examined the induction of CYP2B isoforms in neonatal rats. The level of CYP2B catalytic activity was markedly higher at five days after birth relative to levels observed in fetal hepatic tissue. Oral or intraperitoneal administration of phenobarbital to pregnant rats increased the level of CYP2B expression and activity in neonates. Overall, these findings suggest that CYP2B isoform activity is likely to be lower in fetuses than in neonates or adults and that increased levels of activity may be observed in fetuses and neonates exposed to inducing xenobiotics. The significance of this information for risk of cancer in human fetuses, neonates, and children is uncertain since, as noted above, the CYP2B1/ 2 isoforms have not been identified in humans.

Glutathione S Transferase Theta Genotoxicity studies in genetically engineered bacteria indicate that brominated trihalomethanes can also be activated to mutagens by the product of the glutathione S transferase ( GST) theta gene GSTT1 1 ( DeMarini et al., 1997; Landi et al., 1999). Children and the fetus could potentially experience increased risk of adverse effects if the activity of this enzyme was higher at these life stages than in adults. Information on the developmental expression of GST genes is currently limited. Although other classes of GSTs ( alpha, mu, and pi) are expressed in fetal liver, Mera et al. ( 1994) reported that theta class GSTs were expressed in only adult liver. This finding suggests that the fetus does not experience increased risk as a result of GST thetamediated mutagenicity. The occurrence of increased risk in children cannot be evaluated, since at present the age at which synthesis of the GST theta is induced is unknown.

c. Childhood Cancers: Other Considerations Examination of childhood cancer data compiled by the National Cancer Institute ( Ries et al. 1999) indicates that the incidence of hepatic, renal, and intestinal cancer ( the types of cancer observed in animal studies of brominated trihalomethane cancer potential) from causes other than genetic predisposition are low. Primary neoplasms of the liver are rare in children younger than 15 years of age. There has been little change in the liver cancer incidence ( total hepatic tumors) in this age group over the last 21 years, with rates between 1.4 and 1.7 cases per million throughout the time period. The incidence of hepatocellular carcinoma ( the type of neoplasm observed in mice treated with bromodichloromethane or dibromochloromethane) decreased in children younger than 15 years of age during the period 1975 to 1995. The incidence rate of renal Draft  Do Not Cite or Quote February 20, 2003 VII  13 carcinoma remained very low ( well under one case per million) in children younger than 15 years during the period 1975 to 1995. Trend data were not available in Ries et al. ( 1999) for intestinal cancer. These observations are consistent with ( but do not prove) a conclusion that children are not more susceptible than adults.

d. Conclusion The available evidence for developmental expression of enzymes known to metabolize brominated trihalomethanes supports the conclusion that children do not experience greater risk from exposure to these compounds than do adults. At present there are no cancer incidence data from humans to suggest that brominated trihalomethanes contribute to increased risk of cancer in children.

3. Other Potentially Susceptible Populations a. Subpopulations with altered levels of CYP2E1 CYP2E1 catalyzes the metabolism of brominated trihalomethanes to reactive intermediates that mediate toxicity. Individuals with higher levels of CYP2E1 activity may therefore be at greater risk for adverse health effects. This section describes factors associated with increased levels of CYP2E1 activity and subpopulations who may be at increased risk as a result of these factors.

Genetic Polymorphisms Significant inter ethnic differences exist in CYP2E1 polymorphism ( Ronis et al., 1996; Lieber 1997)) and it is possible that these differences could influence susceptibility to toxic effects. The CYP2E1 polymorphisms currently reported in the literature are located in the 5' flanking ( noncoding) regions of the gene, while the coding regions of the gene which specify sequence appear to be well conserved among various ethnic groups ( Ronis et al. 1996). Mutations in the 5' region of a gene can affect the regulation gene expression. The rare mutant c2 polymorphism of CYP2E1 is reported to be associated with higher transcriptional activity, protein levels, and catalytic activity than the more common wild type allele ( Lieber et al. 1997). As reported by Lieber et al. ( 1997), the highest frequency of the c2 allele occurs in the Taiwanese ( 0.28) and Japanese ( 0.19 to 0.27) populations. The frequencies in African Americans, European Americans, and Scandinavians are much lower, generally in the range 0.01 and 0.05. Efforts to link the occurrence of the c2 allele to higher rates of CYP2E1 mediated liver disease have yielded inconsistent results. Thus, the functional significance of CYP2E1 polymorphism is presently uncertain, and no conclusion can as yet be drawn about the relative risk for different ethnic populations exposed to brominated trihalomethanes.

Altered Physiological or Health States Draft  Do Not Cite or Quote February 20, 2003 VII  14 The physiological functions of CYP2E1 include lipid metabolism and ketone utilization ( Lieber, 1997). Induction of CYP2E1 is observed in many conditions that elevate circulating levels of lipids, including consumption of a high fat or low carbohydrate diet, starvation, obesity, and insulin dependent diabetes. Among the individuals likely to be affected by such conditions, diabetics constitute the most clearly defined susceptible population. Induction of CYP2E1 in uncontrolled insulin dependent diabetes is well studied. In animals, this induction results in elevated levels of CYP2E1 in the liver, kidney, and lung ( Ioannides et al., 1996). Acetone ( a substrate of CYP2E1) is thought to be the inducing compound ( Ronis et al., 1996). As a result of induction, diabetic animals are more susceptible to the toxicity of some chemicals metabolized by CYP2E1. While there are no specific data for the brominated trihalomethanes, this phenomenon has been demonstrated for other halogenated compounds including chloroform, carbon tetrachloride, trichloroethylene, and bromobenzene ( Ioannides et al., 1996). Because the animal and human orthologues of CYP2E1 show similar substrate specificity and bioactivation potential, it is possible that diabetic humans may also be more susceptible to CYP2E1 mediated toxicity. As CYP2E1 levels are reduced by insulin treatment, increased toxicity would be anticipated only in poorly controlled or uncontrolled diabetics ( Ioannides et al., 1996).

Alcohol consumption CYP2E1 contributes to the metabolism of ethanol in humans and animals. Consumption of ethanol induces CYP2E1 and chronic alcohol consumption is reported to result in as much as a 10 fold induction ( Lieber, 1997). Hence, concurrent exposure to ethanol and brominated trihalomethanes may increase susceptibility to adverse health effects. This interaction is of concern because concurrent exposure to brominated trihalomethanes and ethanol is likely to occur in a significant number of people. At present, there are no human or animal studies which examine this interaction for brominated trihalomethanes. However, Wang et al. ( 1994) reported that a single 100 mg/ kg oral dose of ethanol administered to rats significantly increased the toxicity of the structurally related trihalomethane chloroform ( also metabolized by CYP2E1). Lieber ( 1997) noted that the hepatotoxicity of commonly used industrial solvents ( e. g. carbon tetrachloride, bromobenzene, and vinylidene chloride) and anesthetics ( enflurane and halothane) was increased in heavy drinkers, with a pattern of damage that was consistent with the selective expression and induction of CYP2E1 in certain regions of the liver.

Concurrent exposure to other CYP2E1 inducers including pharmaceuticals Because CYP2E1 is highly inducible by a wide range of xenobiotic compounds, prior exposure to such inducers may potentially play a significant role in brominated trihalomethane toxicity. Known inducers of CYP2E1 include certain therapeutic agents ( acetaminophen, isoniazid), volatile anaesthetics ( halothane, isoflurane), and solvents ( acetone, benzene, carbon tetrachloride, trichloroethylene) ( Raucy, 1995). Individuals exposed to or consuming these inducers on a regular basis may therefore be at greater risk for brominated trihalomethane toxicity.

b. Subpopulations with altered levels of glutathione S transferase theta Draft  Do Not Cite or Quote February 20, 2003 VII  15 Individuals with Genetic Polymorphisms Genotoxicity studies in bacteria ( discussed in section V. F. 1) indicate that brominated trihalomethanes can be activated to mutagens by the product of the glutathione S transferase theta gene GSTT1 1 ( DeMarini et al., 1997; Landi et al., 1999). If similar pathways for bioactivation exist in humans, GSTT1 1 polymorphism may influence susceptibility to brominated trihalomethane mediated toxicity. GSTT1 1 is characterized by a deletion polymorphism which results in total loss of glutathione S transferase   activity in individuals ( 10 to 60% of the population depending upon ethnicity and race) homozygous for the null genotype ( GSTT1 1/). Individuals who are heterozygous for GSTT 1 ( GSTT1 1+/) have intermediate levels of enzyme activity, while individuals homozygous for GSTT 1 ( GSTT1 1+/+) have the highest levels. Landi et al. ( 1999) have suggested that GSTT1 1+/+ individuals may experience excess genotoxic risk when exposed to brominated trihalomethanes, particularly in organs which express glutathione Stransferase theta and come in direct contact with brominated trihalomethanes. Potential target sites would include the gastrointestinal tract and the bladder.

Concurrent Exposure to Inducers If GSTT 1 mediated pathways for bioactivation of brominated trihalomethanes exist in humans, factors which induce this enzyme may increase the risk of adverse health effects from exposure. Although GSTT 1 is constitutively expressed, the level of its expression can be altered by exposure to exogenous chemicals. Landi ( 2000) has summarized information on factors which increase expression of the enzyme. In rats, aspirin increased GSTT 1 levels in the colon. Alphatocopherol coumarin; and other anticarcinogenic drugs increased gastric and esophageal levels; and indole 3 carbinol and coumarin increased GSTT 1 levels in the liver. In mice, phenobarbital induced hepatic GSTT 1 levels. Data for humans are limited, but there are indications that the dietary intake of cruciferous vegetables enhances the expression of GSTT 1. It is possible that consumption of these substances by GSTT 1 positive individuals could result in increased risk of adverse effects. However, there are presently no data available for evaluation of this hypothesis.

c. Subpopulations with altered levels of putative protective compounds Glutathione depletion has been observed to increase the hepatotoxicity of bromodichloromethane ( Gao et al., 1996). On the basis of these data, Gao et al. ( 1996) proposed that populations with low baseline levels of glutathione ( e. g., due to dietary deficiencies of glutathione precursors such as cysteine and selenium) may be more sensitive to bromodichloromethane induced toxicity.

d. Possible gender differences Apparent gender related differences in the toxicity of brominated trihalomethanes have been noted in studies where male and female animals were exposed concurrently ( e. g. Aida, 1992a; Daniel, 1990; NTP, 1987, 1989a; Tobe et al., 1982a). In general, male rats and mice Draft  Do Not Cite or Quote February 20, 2003 VII  16 appear to be somewhat more sensitive to the hepatic and renal toxicity induced by brominated trihalomethanes than are females, although there are exceptions to this pattern ( eg. the chronic oral exposure study of bromoform conducted by NTP, 1989a in mice and the short term study of bromoform conducted by Aida et al., 1992a). While the basis for the apparent greater sensitivity of males is unknown, the difference may be related to gender specific differences in the level of enzymes responsible for bioactivation of brominated trihalomethanes to toxic metabolites, or to gender specific differences in cellular protective mechanisms. It is important to note that at present there is no evidence for gender related differences in the activity levels of CYP2E1 or GSTT 1 in humans.

E. Summary It is generally believed that the toxicity of the brominated trihalomethanes is related to their metabolism. This conclusion is based largely on the observation that liver and kidney, the chief target tissues for these compounds, are also the primary sites of their metabolism. In addition, treatments which increase or decrease metabolism also tend to increase or decrease trihalomethane induced toxicity in parallel.

Metabolism of brominated trihalomethanes is believed to occur via oxidative and reductive pathways. Limited structure activity data for brominated trihalomethanes and the structurallyrelated trihalomethane chloroform suggest that bromination may influence the proportion of compound metabolized via the oxidative and reductive pathways, with brominated compounds being more extensively metabolized by the reductive pathway. Additional evidence suggests that a GSH mediated pathway may play an important role in metabolism of brominated trihalomethanes. These data raise the possibility that brominated trihalomethanes may induce adverse effects ( toxicity and carcinogenicity) via several different pathways.

The precise biochemical mechanisms which link brominated trihalomethane metabolism to toxicity have not been characterized, but many researchers have proposed that toxicity results from the production of reactive intermediates. Reactive intermediates may arise from either the oxidative ( dihalocarbonyls) or the reductive ( free radicals) pathways of metabolism. Such reactive intermediates are known to form covalent adducts with various cellular molecules, and may impair the function of those molecules and cause cell injury. Free radical production may also lead to cell injury by inducing lipid peroxidation in cellular membranes. Direct evidence showing a relationship between the level of covalent binding intermediates generated by either pathway and the extent of toxicity is not available for the brominated trihalomethanes. Manipulation of cellular glutathione levels suggests that this compound may play a protective role in brominated trihalomethane induced toxicity.

Individual brominated trihalomethanes have been shown to induce tumors in laboratory animals. The mechanism by which brominated trihalomethanes induce tumors in target tissues has not been fully characterized. DNA adducts can be formed by interaction of reactive metabolites ( resulting from oxidative and reductive metabolism) with DNA. In addition, preliminary evidence suggested that DNA adducts can be formed through conjugation with glutathione and Draft  Do Not Cite or Quote February 20, 2003 VII  17 bioactivation of the resulting conjugates. Comparison of dose response data for liver toxicity ( including cell proliferation) and tumorigenicity in mice suggests that tumor formation occurs at concentrations lower than those which stimulate cell proliferation. No evidence for increased cell proliferation in kidney was obtained in studies using doses up to 246 mg/ kg day for bromodichloromethane, 312 mg/ kg day for dibromochloromethane, or 379 mg/ kg day for bromoform.

Interaction with agents which increase or decrease the activity of enzymes responsible for metabolism of brominated trihalomethanes may modify carcinogenicity/ toxicity. Pretreatment with inducers of CYP2E1 has been observed to increase the hepatotoxicity of bromodichloromethane and dibromochloromethane in male rats. Pretreatment with m xylene, an inducer of the CYP2B1/ CYP2B2 isoforms, increased the hepatotoxicity of dibromochloromethane in male rats. Conversely, administration of the cytochrome P450 inhibitor 1 aminobenzotriazole prevented bromodichloromethane induced hepatotoxicity in rats. Recent findings indicating possible glutathione mediated metabolism of brominated trihalomethanes suggest that treatments or agents which alter glutathione S transferase activity could potentially modify the toxicity of brominated trihalomethanes.

The severity of brominated trihalomethane toxicity is potentially affected by the vehicle of administration. In a study of vehicle effects on the acute toxicity of bromodichloromethane, a high dose ( 400 mg/ kg) of the chemical was more hepato and nephrotoxic when given in corn oil compared to aqueous administration, but this difference was not evident at a lower dose ( 200 mg/ kg).

A number of potentially sensitive subpopulations have been identified for health effects of brominated trihalomethanes. A growing body of scientific evidence indicates that children may suffer disproportionately from some environmental health risks. These risks may arise because the neurological, immunological, and digestive systems of children are still developing. In addition, children may incur greater exposure because they eat more food, consume more fluids, and breathe more air in proportion to their body weight when compared to adults. U. S. EPA has identified three key questions to consider when evaluating health risks to children from drinking water disinfection byproducts ( DBP), including the brominated trihalomethanes: 1) Is there information which shows that the DBP causes effects in the developing fetus or impairs ability to conceive and bear children? 2) If the DBP causes cancer, are children more likely to be affected by it than are adults? and 3) If the DBP causes a noncancer toxic effect, are children more likely to be affected by it than are adults? The available data for dibromochloromethane and bromoform suggest that developmental effects in animals occur only at doses which cause maternal toxicity and at doses lower than those which induce histopathological effects in the liver and kidney. There is no evidence that these compounds impair the ability to conceive or have children. In animal studies, exposure to bromodichloromethane resulted in reduced sperm motility; this effect was not accompanied by histopathologic changes in the male reproductive system. Exposure of pregnant F344 rats during gestation days 6 9 caused full litter resorption. This response was not observed in similarly exposed pregnant Sprague Dawley rats. Epidemiological studies have found an association between exposure to bromodichloromethane in drinking water and increased Draft  Do Not Cite or Quote February 20, 2003 VII  18 spontaneous abortion and increased stillbirth. Although these studies raise concern for human health effects, the occurrence of multiple disinfection byproducts in drinking water prevents the conclusion that bromodichloromethane is the causative agent.

At present, there are no cancer data which indicate that brominated trihalomethanes contribute to increased risk of cancer in children. No studies were located which examined preor post pubertal cancer rates in humans in relation to brominated trihalomethane exposure. Cancer bioassays of brominated trihalomethanes conducted in mice and rats have not used study designs that included perinatal exposure.

The available evidence suggests that the toxic effects of brominated trihalomethanes are mediated by the enzymes CYP2E1, CYP2B1/ 2 ( in rodents), and glutathione S transferase theta ( GSTT 1). The weight of evidence from studies of the developmental expression of these enzymes supports the conclusion that children do not experience greater risk from brominated trihalomethane exposure as a result of higher metabolic activity.

In addition to children, other potentially sensitive populations include those with altered levels or activity of CYP2E1 or GSTT 1 and those with altered levels of glutathione. Factors contributing to increases in CYP2E1 activity potentially include genetic polymorphisms; altered physiological or health states; alcohol consumption; and concurrent exposure to other inducers, including some pharmaceuticals and solvents. Factors contributing to increased GSTT 1 activity include genetic polymorphisms and concurrent exposure to inducers. Based on observations in animals, human populations with reduced levels of glutathione as a result of dietary deficiency or other factors may experience increased sensitivity to the toxic effects of bromodichloromethane.

Apparent gender related differences in the toxicity of brominated trihalomethanes have been noted in studies where male and female animals were exposed concurrently. In general, male rats and mice appeared to be more sensitive than females to liver and renal toxicity, although some exceptions to this pattern have been noted. There is no evidence for a similar pattern of gender response in humans. Draft  Do Not Cite or Quote February 20, 2003 VIII  1 VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS This section quantifies the toxicological effects of brominated trihalomethanes based on health effects information presented in Sections V and VI. At present, there are two basic approaches to quantification of toxicological effects: the conventional NOAEL/ LOAEL approach and benchmark dose modeling. Benchmark dose ( BMD) modeling ( U. S. EPA, 1995; 2000b) was chosen as the preferred approach for quantifying toxicological effects of the brominated trihalomethanes. BMD modeling avoids several limitations of the NOAEL/ LOAEL approach, including: 1) the slope of the dose response plays little role in determining the NOAEL; 2) the NOAEL ( or LOAEL) is limited to the doses tested experimentally; 3) the determination of the NOAEL is based on scientific judgement, and is subject to inconsistency; and 4) experiments using fewer animals tend to produce larger NOAELs, and as a result may produce larger health advisories ( HAs) or reference doses ( RfDs) ( U. S. EPA, 1995) that may not be sufficiently protective of human health. In contrast, benchmark doses ( BMDs) are not limited to the experimental doses, appropriately reflect the sample size, and can be defined in a statistically consistent manner. The BMD approach was therefore selected for quantification of toxicological effects of the brominated trihalomethanes. Values for HAs and RfDs derived using the conventional NOAEL/ LOAEL approach are presented in the text for comparison with those obtained using the BMD approach.

The methods employed for BMD modeling are described in Appendix A. The modeling was performed using the BMDS software ( Version 1.2) developed by the U. S. EPA National Center for Environmental Assessment. The BMDs and BMDLs were calculated based on a BMR of 10% extra risk for all quantal endpoints analyzed. For continuous data, the BMR was defined as 1.1 standard deviations, which corresponds to an additional risk of approximately 10% when the background response rate is assumed to be 1% with normal variation around the mean and constant standard deviation ( Crump, 1995). The BMDL 10 was defined as the 95% lower bound on the corresponding BMD estimate. Confidence bounds were automatically calculated by the BMDS software using a likelihood profile method.

A. Bromodichloromethane 1. Noncarcinogenic effects a. One day Health Advisory Studies of the acute toxicity of bromodichloromethane are summarized in Table VIII 1. Lilly et al. ( 1994) administered single doses of bromodichloromethane by either oil or aqueous gavage to male F344 rats at dose levels of 200 or 400 mg/ kg. This study identified a LOAEL of 200 mg/ kg day based on histologic lesions in the kidney and changes in urinary parameters. A NOAEL value was not identified for either vehicle. Data for hepatic vacuolar degeneration and renal tubular degeneration obtained using the aqueous vehicle were modeled using the BMDS software. BMD and BMDL 10 values of 263 and 182 mg/ kg day, respectively, were calculated using the hepatic data. BMD and BMDL 10 values of 131 and 8.9 mg/ kg day, respectively, were Draft  Do Not Cite or Quote February 20, 2003 VIII  2 obtained using the renal data. The BMDL 10 for renal tubular degeneration is the lowest calculated across studies, but is not considered a reliable estimate because there is insufficient information to accurately characterize the shape of the dose response curve in the region of interest.

Thornton Manning et al. ( 1994) administered bromodichloromethane to female F344 rats by aqueous gavage for five consecutive days at dose levels ranging from 75 to 300 mg/ kg day. This study identified a NOAEL of 75 mg/ kg day and a LOAEL of 150 mg/ kg day based on increased liver and kidney weights and histologic lesions in the liver ( mild centrilobular hepatocellular vacuolar degeneration) and in the kidney ( mild renal tubule vacuolar degeneration). An analogous study ( Thornton Manning et al., 1994) conducted in female C57BL/ 6J mice indicated that the mice were less sensitive to bromodichloromethane than the rats, as no treatment related histologic lesions were observed in the liver or kidney. However, similar NOAEL and LOAEL values were identified based on increased liver weight and changes in serum chemistry parameters. Data for renal tubular degeneration in rats were analyzed using the BMD approach. BMD and BMDL 10 values of 133 and 65 mg/ kg day, respectively, were calculated for this endpoint. The BMD is in close agreement with the BMD value calculated for the same endpoint using the data of Lilly et al. ( 1994).

Two reproductive studies which examined full litter resorption were also considered for derivation of the One day HA. Bielmeier et al. ( 2001) examined the occurrence of full litter resorption in F344 rats treated with 0, 75 or 100 mg/ kg day bromodichloromethane by aqueous gavage on gestation day 9. The LOAEL for this effect was 75 mg/ kg day. Narotsky et al. ( 1997) evaluated the same endpoint in F344 rats administered 0, 25, 50, or 75 mg/ kg day on gestation days 6 through 15. Full litter resorption was observed at 50 and 75 mg/ kg day. The NOAEL and LOAEL in this study were thus identified as 25 and 50 mg/ kg day, respectively. When data from these studies were analyzed using the BMD approach, BMD values of 48 and 23 mg/ kg day were obtained for the Narotsky et al. ( 1997) and Bielmeier et al. ( 2001) studies, respectively. The higher value from the Narotsky et al. ( 1997) study was considered the more reliable estimate of the BMD because it was based on response data that included lower doses, one of which was an apparent NOAEL. The BMDL 10 calculated from the Narotsky et al. ( 1997) data was 30 mg/ kgday

Three additional studies were considered as candidates for derivation of the One day HA. Lilly et al. ( 1997) administered single doses of bromodichloromethane by aqueous gavage to male F344 rats at dose levels ranging from 123 to 492 mg/ kg. Based on changes in urinary parameters, this study identified a NOAEL of 164 mg/ kg day and a LOAEL of 246 mg/ kg day. No histopathological examination was conducted in this study. The study by French et al. ( 1999), which investigated immune system response, identified a similar NOAEL value. However, the database for immune response to bromodichloromethane is limited when compared to information on hepatic and renal toxicity. Adverse effects were noted only at the highest dose and frank effect level, and evidence for vehicle effects on immunotoxicity endpoints was observed. Keegan et al. ( 1998) administered single doses of bromodichloromethane to male F344 rats by gavage at dose levels ranging from 21 to 246 mg/ kg. The study authors identified a NOAEL of 41.0 mg/ kg day and a LOAEL of 81.9 mg/ kg day based on elevations in serum markers of hepatotoxicity ( ALT, Draft  Do Not Cite or Quote February 20, 2003 VIII  3 AST, and SDH). Histopathological examination was not conducted and this was considered to be a limitation of the investigation. These three studies were not considered further for derivation of the One day Health Advisory ( HA), and thus data reported in them were not analyzed using the BMD approach.

The study conducted by Narotsky et al. ( 1997) was selected for derivation of the One day HA. The critical effect in this study was full litter resorption observed in pregnant F344 rats treated with bromodichloromethane. The BMDL 10 value calculated for this endpoint was 30 mg/ kg day, which is roughly half of the most reliable BMDL 10 value calculated for histopathological changes in kidney ( Thornton Manning et al., 1994). Although dosing in the Narotsky et al. ( 1997) study lasted from gestation days 6 through 15, a subsequent study by Bielmeier et al. ( 2001) indicated that a single dose ( 75 mg/ kg) of bromodichloromethane on gestation day 9 was sufficient to elicit full litter resorption in the same strain of rats. Since there is presently insufficient information available to fully assess the occurrence of reproductive effects in humans exposed to bromodichloromethane, use of data for full litter resorption was adopted as a conservative approach to derivation of the One day HA. The One day HA for a 10 kg child is calculated using the following equation:

One day HA = ( 30 mg/ kg day) ( 10 kg) = 1.0 mg/ L ( 300) ( 1 L/ day)

where:

30 mg/ kg day = BMDL 10 based on incidence of full litter resorption in F344 rats treated with bromodichloromethane on gestation days 6 to 15.

10 kg = Assumed body weight of a child 300 = Uncertainty factor based on NAS/ OW guidelines. This value includes a factor of 10 to protect sensitive human populations; a factor of 10 for extrapolation from animals to humans; and a factor of 3 to account for database limitations and uncertainty regarding possible reproductive effects of bromodichloro methane in humans 1 L/ day = Assumed water consumption of a 10 kg child For comparative purposes, the One day HA derived using the conventional NOAEL/ LOAEL approach would also be based on data from the Narotsky et al. ( 1997) study. This study identified a NOAEL of 25 mg/ kg day based on FLR, which was the lowest value among the candidate studies. Using this NOAEL and an uncertainty factor of 300 as described Draft  Do Not Cite or Quote February 20, 2003 VIII  4 Table VIII 1 Summary of Candidate Studies for Derivation of the One day HA for Bromodichloromethane Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL mg/ kg day LOAEL mg/ kg day BMD mg/ kg day BMDL 10 mg/ kg day Lilly et al.

( 1994) Rat F344 M 6 0 200 400 Gavage ( oil) Single Dose Body, liver, and kidney weights,

serum and urine chemistry, liver and kidney histology  200 ( minimal renal tubule degeneration and necrosis,

changes in urinary parameters) Not modeled

Lilly et al.

( 1994) Rat F344 M 6 0 200 400 Gavage ( aqueous) Single Dose Body, liver, and kidney weights,

serum and urine chemistry, liver and kidney histology  200 ( minimal renal tubule degeneration,

changes in urinary parameters) 263 182 ( Hepatic vacuolar degeneration in males)

131 8.9 ( Renal tubule degeneration in males)

Lilly et al.

( 1997) Rat F344 M 5 0 123 164 246 328 492 Gavage ( aqueous) Single Dose Body, liver, and kidney weights,

serum and urine chemistry 164 246 ( changes in urinary parameters) Not modeled

Keegan et al.

( 1998) Rat F344 M 6 0 21 31 41 82 123 164 246 Gavage ( aqueous) Single Dose Body, liver, and kidney weights,

serum chemistry 41 82 ( elevated ALT,

AST, and SDH activities) Not modeled  Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL mg/ kg day LOAEL mg/ kg day BMD mg/ kg day BMDL 10 mg/ kg day Draft  Do Not Cite or Quote February 20, 2003 VIII  5 French et al.

( 1999) Rat C57BL/ 6 F 6 0 75 150 300 Gavage ( aqueous) 5 days Body, spleen, and thymus weights,

immune function 150 300 ( FEL)

( mortality,

decreased body weight, altered immune response) Not modeled

Thornton

Manning et al. ( 1994) Rat F344 F 6 0 75 150 300 Gavage ( aqueous) 5 days Body, liver, and kidney weights,

serum chemistry,

liver and kidney histology 75 150 ( increased liver and kidney weights, mild centrilobular hepatocellular vacuolar degeneration,

mild renal tubule vacuolar degeneration) 133 65 ( renal tubular degeneration)

Thornton

Manning et al. ( 1994) Mouse C57BL/

6J F 6 0 75 100 Gavage ( aqueous) 5 days Body, liver, and kidney weights,

serum chemistry,

liver and kidney histology 75 150 ( increased liver weight, elevated ALT and SDH activities) Not modeled   Narotsky et al.

( 1997) * Rat F344 F 12

14 0 25 50 75 Gavage ( oil)

( water)** Gestation days 6 15 Body weight,

clinical signs, developmental parameters 25 50 ( full litter resorption) 48 30 ( full litter resorption)

Bielmeier et al. ( 2001)* Rat F344 F 8 11 0 75 100 Gavage ( aq) Gestation day 9 Full litter resorption;

hormone profiles  75 ( full litter resorption) 23 4.2 ( full litter resorption)

* The NOAEL and LOAEL values listed are for reproductive or developmental effects.

** The NOAEL and LOAEL values were the same in either vehicle. BMD modeling was performed on aqueous vehicle data only. Draft  Do Not Cite or Quote February 20, 2003 VIII  6 above, the One day HA for a 10 kg child calculated using the conventional approach would be 0.8 mg/ L ( rounded from 0.83 mg/ L).

b. Ten day Health Advisory Sixteen studies were considered for derivation of the Ten day HA for bromodichloromethane. These studies are summarized in Table VIII 2 below. Aida et al. ( 1992a) administered microencapsulated bromodichloromethane in the diet to Wistar rats for one month at dose levels ranging from 20.6 to 203.8 mg/ kg day. This study identified a NOAEL of 61.7 mg/ kg day and a LOAEL of 189.0 mg/ kg day in male rats based on histologic changes in the liver ( swelling of hepatocytes, single cell necrosis, hepatic cord irregularity, and bile duct proliferation). Analysis using the BMD approach calculated BMD and BMDL 10 values of 34 and 17 mg/ kg day, respectively, based on data for liver cell vacuolization in females.

Data from four of the other candidate studies are consistent with the histopathological results obtained by Aida et al. ( 1992a). Melnick et al. ( 1998) administered bromodichloromethane by gavage to female B6C3F 1 mice for 5 days/ week for 3 weeks and identified a NOAEL of 75 mg/ kg day ( duration adjusted NOAEL of 54 mg/ kg day) and a LOAEL of 150 mg/ kg day ( duration adjusted LOAEL of 107 mg/ kg day) based on histologic changes in the liver ( hepatocyte hydropic degeneration). Analysis using the BMD approach calculated duration adjusted BMD and BMDL 10 values of 31 and 8.4 mg/ kg day, respectively, for this endpoint. Condie et al. ( 1983) administered bromodichloromethane by gavage to male CD 1 mice for 14 days and identified a NOAEL of 74 mg/ kg day and a LOAEL of 148 mg/ kg day based on minimal to moderate liver and kidney lesions. Analysis using the BMD approach calculated BMD and BMDL 10 values of 24 and 7.5 mg/ kg day, respectively, based on data for histopathological changes in the liver. NTP ( 1998) conducted histopathologic examinations in conjunction with a study of reproductive and developmental toxicity in Sprague Dawley rats. Although no reproductive or developmental toxicity was observed at the dose levels investigated, histopathological changes were noted in the liver of males rats treated with the compound for 35 days. The NOAEL and LOAEL for this effect were 9 and 38 mg/ kg day, respectively. Analysis of data for single cell hepatic necrosis using the BMD approach calculated BMD and BMDL 10 values of 35 and 18 mg/ kg day, respectively, which are virtually identical to the values calculated using the liver cell vacuolization data for females from the Aida et al. ( 1992b) study. Coffin et al. ( 2000) observed hydropic degeneration in female mice treated with 150 mg/ kg day bromodichloromethane in corn oil for 11 days. These data were not modeled because other studies utilized doses lower than 150 mg/ kg day, which allowed better characterization of response in the low dose region of the dose response curve.

In contrast to the studies described above, Chu et al. ( 1982a) did not observe any microscopic lesions in the liver when Sprague Dawley rats were administered bromodichloromethane at doses up to 68 mg/ kg day in the drinking water for 28 days. The studies of Munson et al. ( 1982) and NTP ( 1987) did not conduct histopathological examinations. These studies identified NOAELs ranging from 50 to 150 mg/ kg day and LOAELs ranging from 125 to 300 mg/ kg day for other endpoints, including depressed humoral immunity ( Munson et al., Draft  Do Not Cite or Quote February 20, 2003 VIII  7 1982), decreased weight gain ( NTP, 1987; rats), and increased mortality and gross renal pathology ( NTP, 1987; mice). These data were not analyzed by the BMD approach.

Seven studies that examined developmental and/ or reproductive endpoints were evaluated. Two studies reported the incidence of full litter resorption in F344 rats following treatment with bromodichloromethane. Bielmeier et al. ( 2001) examined the occurrence of full litter resorption in F344 rats treated with 0, 75 or 100 mg/ kg day bromodichloromethane by aqueous gavage on gestation day 9. The LOAEL for this effect was 75 mg/ kg day. Narotsky et al. ( 1997) evaluated the same endpoint in F344 rats administered 0, 25, 50, or 75 mg/ kg day on gestation days 6 through 15. Full litter resorption was observed at 50 and 75 mg/ kg day. The NOAEL and LOAEL in this study were thus identified as 25 and 50 mg/ kg day, respectively. When data from these studies were analyzed using the BMD approach, BMD values of 48 and 23 mg/ kg day were obtained for the Narotsky et al. ( 1997) and Bielmeier et al. ( 2001) studies, respectively. The higher value from the Narotsky et al. ( 1997) study was considered the more reliable estimate of the BMD because it was based on response data that included lower doses, one of which was an apparent NOAEL. The BMDL 10 calculated from the Narotsky et al. ( 1997) data was 30 mg/ kgday Ruddick et al. ( 1983) observed an increased incidence of sternebral aberrations in the pups of Sprague Dawley rats administered bromodichloromethane in corn oil by gavage. Statistical analysis of the published data indicated that the NOAEL and LOAEL for this effect were 100 mg/ kg day and 200 mg/ kg day, respectively. The BMD and BMDL 10 obtained for this study were 27 and 15 mg/ kg day, respectively.

The remaining reproductive/ developmental studies sponsored by the Chlorine Chemistry Council ( CCC) were also evaluated. CCC ( 2000a, b) examined developmental toxicity in New Zealand rabbits and identified developmental NOAELs of 76 and 55 mg/ kg day ( the highest doses tested in each study). The CCC ( CCC, 2000c, d) also examined reproductive and developmental toxicity in Sprague Dawley rats. In a range finding study ( CCC, 2000c), F 1 generation pups exposed to bromodichloromethane via lactation and possibly by consumption of water supplied to the dams exhibited reduced body weights and body weight gains. These effects occurred at exposure levels which also resulted in maternal toxicity. Biologically meaningful average daily doses could not be established in this experiment; therefore, the concentration based NOAEL and LOAEL values for developmental effects were 50 and 150 ppm. based on changes in F 1 pup body weight and body weight gain. In a subsequent developmental study, CCC ( 2000d) identified NOAEL and LOAEL values of 45 and 82 mg/ kg day, respectively, based on decreased number of ossification sites per fetus for the forelimb phalanges and hindlimb metatarsals and phalanges. This effect was observed at doses associated with maternal toxicity. Endpoints from these studies were not modeled because other studies identified adverse effects at lower doses.

Data for maternal toxicity from three reproductive/ developmental studies were also considered for derivation of the 10 day HA for bromodichloromethane. Narotsky et al. ( 1997) observed decreased maternal body weight gain on gestation days 6 to 8 in female rats administered 25 mg/ kg day ( the lowest dose tested) by aqueous gavage in 10% Emulphor. BMD modeling identified BMD and BMDL 10 values of 18 and 10 mg/ kg day, respectively, for this endpoint. The data reported in this study did not permit evaluation of body weight or body Draft  Do Not Cite or Quote February 20, 2003 VIII  8 weight gain at other time points during the treatment period. CCC ( 2000d) reported decreased maternal body weight gain at several time points in pregnant rats administered bromodichloromethane in drinking water, with the most severe effect observed immediately after initiation of treatment on gestation days 6 to 7. The NOAEL and LOAEL for decreased body weight on gestation days 6 to 7 were 18.4 and 45 mg/ kg day, respectively. When body weight gain for this interval was modeled, the resulting BMD and BMDL 10 values were approximately 18 and 15 mg/ kg day, respectively. However, the modeled fits to the data were poor, and the results were not considered sufficiently reliable for derivation of a health advisory. To address this problem, body weight gain data for gestation days 6 to 9 were also modeled. Reliable values of 23 and 18 mg/ kg day were obtained for the BMD and BMDL 10, respectively. The CCC ( 2000b) study observed decreased maternal body weight gain at several time points in pregnant rabbits administered bromodichloromethane in the drinking water on gestation days 6 to 29. The NOAEL and LOAEL for decreased maternal body weight gain ( corrected for gravid uterine weight) on gestation days 6 to 21 were 13.4 and 35.3 mg/ kg day, respectively. A BMD value of 50 mg/ kg day was obtained for this data set, but the BMDS software failed to identify the corresponding BMDL 10. No further modeling was attempted since this value was well above the lowest BMDs obtained in some other candidate studies.

As evident from the data in Table VIII 2, the four studies that examined histopathological changes in the liver are in close agreement, having identified BMD values ranging from 24 to 35 mg/ kg day. The corresponding BMDL 10 values ranged from 7.5 to 18 mg/ kg day. Maternal toxicity occurred in rats at similar levels in two developmental studies. These studies identified BMD values of 18 and 23 mg/ kg day, with corresponding BMDL 10 values of 10 and 18 mg/ kgday The NTP ( 1998) and CCC ( 2000d) drinking water studies were selected to derive the Tenday HA. Selection of these studies was based on the administration of bromodichloromethane in drinking water, the most relevant route of exposure. In addition, these studies utilized a lower range of doses, which provided information on the shape of the dose response curve in the region of interest. The Ten day HA is calculated according to the following equation:

Ten day HA = ( 18 mg/ kg day) ( 10 kg) = 0.60 mg/ L ( rounded to 0.6 mg/ L) ( 300) ( 1 L/ day)

where:

18 mg/ kg day = BMDL 10 based on single cell hepatic necrosis in rats administered bromodichloromethane in the drinking water for 35 days or decreased maternal body weight gain on gestation days 6 9 in pregnant female rats administered bromodichloromethane in the drinking water.

10 kg = Assumed body weight of a child Draft  Do Not Cite or Quote February 20, 2003 VIII  9 300 = Uncertainty factor based on NAS/ OW guidelines. This value includes a factor of 10 to protect sensitive human populations and a factor of 10 for extrapolation from animals to humans, and a factor of 3 to account for database limitations and uncertainty regarding possible reproductive effects of bromodichloromethane in humans 1 L/ day = Assumed water consumption of a 10 kg child For comparative purposes, the Ten day HA derived using the conventional NOAEL/ LOAEL approach would be based on data from the CCC ( 2000d) study. This study identified a NOAEL of 18 mg/ kg day based on reduced maternal body weight gain in pregnant rabbits. Using this NOAEL and an uncertainty factor of 300 as described above, the Ten day HA for a 10 kg child calculated using the conventional approach would be 0.60 mg/ L ( rounded to 0.6 mg/ L). Draft  Do Not Cite or Quote February 20, 2003 VIII  10 Table VIII 2 Summary of Candidate Studies for Derivation of the Ten day HA for Bromodichloromethane Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) BMD ( mg/ kg day) BMDL 10 ( mg/ kg day)

Aida et al.

( 1992a) Rat Wistar M, F 7 Male 0 21 62 189 Females 0 21 66 204 Feed 1 month Clinical signs, body weight, serum chemistry, hematology, histology 62 189 ( liver histopathology in males) 34 17 ( liver cell vacuolation in females)

Chu et al.

( 1982a) Rat SD M 10 0 0.8 8 68 Drinking water 28 days Clinical signs, serum chemistry, histology 68  No data to model

Condie et al.

( 1983) Mouse CD 1 M 8 16 0 37 74 148 Gavage ( oil) 14 days Serum enzymes, PAH uptake in vitro,

histology 74 148 ( elevated ALT,

decreased PAH uptake, liver and kidney histopathology) 24 7.5 ( hepatic centrilobular pallor)

125 53 ( Renal epithelial hyperplasia)

Melnick et al.

( 1998) Mouse B6C3F 1 F 10 0 75 150 326 Gavage ( oil) 3 weeks ( 5 d/ wk) Body and liver weights, serum chemistry, liver histology 75 150 ( liver histopathology) 31 * 8.4 ( Hepatocyte hydropic degeneration)

Munson et al.

( 1982) Mouse CD 1 M, F 8 12 Males 0 50 125 250 Gavage ( aq.) 14 days Body and organ weights, serum chemistry, hematology, and immune function 50 125 ( depressed humoral immunity) Not modeled  Table VIII 2 ( cont.)

Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) BMD ( mg/ kg day) BMDL 10 ( mg/ kg day)

Draft  Do Not Cite or Quote February 20, 2003 VIII  11 NTP ( 1987) Rat F344/ N M, F 5 0 38 75 150 300 600 Gavage ( oil) 14 days Body weight, clinical signs, gross necropsy 150 300 ( decreased weight gain) Not modeled

NTP ( 1987) Mouse B6C3F 1 M, F 5 0 19 38 75 150 300 Gavage ( oil) 14 days Body weight, clinical signs, gross necropsy 75 150 ( FEL) *

( mortality,

lethargy, gross renal pathology) Not modeled

Coffin et al.

( 2000) Mouse B6C3F 1 F 10 0 150 300 Gavage ( oil) 11 days Relative liver wt.,

liver histopathology;

labeling index  150 Not modeled

NTP ( 1998) Rat SD M ( group A) 5 13 0 9 38 67 Drinking water 35 days Body and organ weights, serum chemistry, hematology, gross necropsy, histology,

sperm evaluation 9 38 ( liver histopathology) 35 18 ( liver cell necrosis)

Ruddick et al. ( 1983)* Rat SD F 9 14 0 50 100 200 Gavage ( oil) Gestation days 6 15 Body and organ weights; maternal serum chemistry;

hematology, and histopathology;

developmental parameters 100 200 ( sternebral aberrations) 27 15 ( sternebral aberrations) Table VIII 2 ( cont.)

Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) BMD ( mg/ kg day) BMDL 10 ( mg/ kg day)

Draft  Do Not Cite or Quote February 20, 2003 VIII  12 Narotsky et al. ( 1997)* Rat F344 F 12

14 0 25 50 75 Gavage ( oil)

( Emulphor Gestation days 6 15 Body weight, clinical signs, developmental parameters 25 ( developmental ) 50 ( full litter resorption) 48 30 ( full litter resorption)

( maternal) 25 ( reduced matermal body weight gain gestation days 6 8, aqueous vehicle only) 18 10 ( reduced matermal body weight gain gestation days 6

8, aqueous vehicle only)

Bielmeier et al. ( 2001)* Rat F344 F 8 11 0 75 100 Gavage ( aq) Gestation day 9 Full litter resorption;

hormone profiles  75 ( full litter resorption) 23 4.2 ( full litter resorption)

CCC ( 2000c)* Rat SD M, F 10 0 ppm 50 ppm 150 ppm 450 ppm 1350 ppm Drinking water Males 64 days Females 74 days Reproductive and developmental parameters 50 ppm 150 ppm ( reduced F 1 pup weight and weight gain) Not modeled

CCC ( 2000d)* Rat SD F 25 0.0 2.2 18.4 45.0 82.0 Drinking water Gestation days 6 21 Reproductive and developmental parameters 45.3 ( developmental) 82.0 ( reduced number of ossification sites in phalanges or metatarsals occurring with maternal toxicity) Not modeled

18.4 ( maternal) 45 ( reduced maternal body weight gain gestation days 6 7) 23 18 ( reduced maternal body weight gain gestation days 6

9; see text for comment) Table VIII 2 ( cont.)

Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) BMD ( mg/ kg day) BMDL 10 ( mg/ kg day)

Draft  Do Not Cite or Quote February 20, 2003 VIII  13 CCC ( 2000a)* Rabbit NZW F 5 0.0 13.9 32.3 76.3 Drinking water Gestation days 6 29 Reproductive and developmental parameters 76.3 ( developmental)  Not modeled

CCC ( 2000b)* Rabbit NZW F 25 0 1.4 13.4 35.6 55.3 Drinking Water Gestation days 6 29 Clinical sign, gross lesions, reproductive and developmental endpoints 55 ( developmental)  Not modeled

13.4 ( maternal) 35.3 ( reduced corrected maternal body weight gain gestation days 6 29) 50 BMDS software failed * The NOAEL and LOAEL values listed are for reproductive or developmental effects.

** The NOAEL and LOAEL values were the same for developmental effects in either vehicle. The LOAEL for maternal toxicity was 25 mg/ kg day for the aqueous vehicle ( 10% Emulphor). The NOAEL and LOAEL for maternal toxicity using the corn oil vehicle were 25 mg/ kg day and 50 mg/ kg day,

respectively. BMD modeling was performed on aqueous vehicle data only.

* BMD and BMDL10 calculated using duration adjusted doses Abbreviations: FEL, Frank effect level; SD, Sprague Dawley; NZW, New Zealand White Draft  Do Not Cite or Quote February 20, 2003 VIII  14 c. Longer term Health Advisory Two rodent oral exposure studies conducted by NTP ( 1987) were considered for derivation of the Longer term HA for bromodichloromethane. In addition, eight reproductive studies were considered. These studies are summarized in Table VIII 3 below.

NTP ( 1987) administered bromodichloromethane by gavage to F344/ N rats for 5 days/ week for 13 weeks at dose levels ranging from 19 to 300 mg/ kg day. Based on decreased weight gain, this study identified a NOAEL of 75 mg/ kg day and a LOAEL of 150 mg/ kg day. Treatment related hepatic and renal lesions were observed only at the high dose. In a similar study, NTP ( 1987) administered bromodichloromethane by gavage to B6C3F 1 mice for 5 days/ week for 13 weeks at dose levels ranging from 6.25 to 100 mg/ kg day for males and from 25 to 400 mg/ kg day for females. This study identified a NOAEL of 50 mg/ kg day and a LOAEL of 100 mg/ kg day based on histologic alterations in the kidney ( focal necrosis of the proximal renal tubular epithelium and nephrosis) of male mice. BMD analysis using the BMDS program identified duration adjusted BMD values of 63 and 75 mg/ kg day for focal necrosis of renal tubular epithelium in males and vacuolated cytoplasm in the liver of females, respectively. The corresponding BMDL 10 values for these renal and hepatic effects were 35 and 47 mg/ kg day, respectively.

Eight reproductive or developmental studies ( Ruddick et al., 1983; Narotsky et al., 1997; Bielmeier et al. 2001; CCC, 2000a, b, c, d; CCC, 2002) were also considered for derivation of the Longer term HA. CCC ( 2002) identified a LOAEL of 150 ppm ( approximately 11.6 to 40.2 mg/ kg day) for delayed sexual maturation in F 1 male rats in a two generation study of bromodichloromethane administered in drinking water. The LOAEL for parental effects in the study was also 150 ppm, based on decreased body weight and body weight gain in F 1 females and F 1 males and females. Bielmeier et al. ( 2001) examined the occurrence of full litter resorption in F344 rats treated with 0, 75 or 100 mg/ kg day bromodichloromethane by aqueous gavage on gestation day 9. The LOAEL for this effect was 75 mg/ kg day. Narotsky et al. ( 1997) evaluated the same endpoint in F344 rats administered 0, 25, 50, or 75 mg/ kg day on gestation days 6 through 15. Full litter resorption was observed at 50 and 75 mg/ kg day. The NOAEL and LOAEL in this study were thus identified as 25 and 50 mg/ kg day, respectively. When data from these studies were analyzed using the BMD approach, BMD values of 48 and 23 mg/ kg day were obtained for the Narotsky et al. ( 1997) and Bielmeier et al. ( 2001) studies, respectively. The higher value from the Narotsky et al. ( 1997) study was considered the more reliable estimate of the BMD because it was based on response data that included lower doses, one of which was an apparent NOAEL. The BMDL 10 calculated from the Narotsky et al. ( 1997) data was 30 mg/ kgday Studies conducted by NTP ( 1998) did not detect reproductive or developmental toxicity at doses up to 116 mg/ kg day. Two studies conducted in New Zealand White rabbits did not detect developmental effects at doses up to 55 and 76 mg/ kg day, respectively ( CCC, 2000a, b).

Three additional studies in rats identified developmental effects that occurred at dose levels that also resulted in maternal toxicity. In a range finding study ( CCC, 2000c), F 1 generation pups exposed to bromodichloromethane via lactation and possibly by consumption of Draft  Do Not Cite or Quote February 20, 2003 VIII  15 water supplied to the dams exhibited reduced body weights and body weight gains. Biologically meaningful daily doses could not be established in this experiment; therefore, the concentrationbased NOAEL and LOAEL values are 50 ppm and 150 ppm. based on reduced body weight and body weight gain in the F 1 pups. In a subsequent developmental study, CCC ( 2000d) identified NOAEL and LOAEL values of 45 and 82 mg/ kg day, respectively, based on decreased number of ossification sites per fetus for the forelimb phalanges and hindlimb metatarsals and phalanges. These reversible developmental delays occurred at doses which also resulted in maternal. Endpoints from these studies were not modeled because other effects were observed at lower doses. Ruddick et al. ( 1983) observed an increased incidence of sternebral aberrations in the pups of Sprague Dawley rats administered bromodichloromethane in corn oil by gavage. Statistical analysis of the published data indicated that the NOAEL and LOAEL for this effect were 100 mg/ kg day and 200 mg/ kg day, respectively. The lowest dose tested was 50 mg/ kg day. The BMD and BMDL 10 obtained for this study were 27 and 15 mg/ kg day, respectively. However, examination of the modeling output indicated that none of the available models fit the data well in the low dose region of the curve. Therefore, the reliability of these values is questionable.

The Narotsky et al. ( 1997) and CCC ( 2000d) studies identified BMDL 10 values of 10 and 18 mg/ kg day based on reduced maternal body weight gain. The CCC data were considered the most relevant since they were obtained from a drinking water study which utilized concentrations that resulted in daily doses well below those used in the Narotsky study. The NTP ( 1987) and the Narotsky et al. ( 1997) studies provided similar, but higher, BMDL 10 values, based on reproductive and histopathological endpoints. The NTP ( 1987) study utilized bromodichloromethane doses as low as 6.3 mg/ kg day, in contrast to the reproductive study conducted by Narotsky et al. ( 1997) in which the lowest dose was 25 mg/ kg day. The NTP ( 1987) data thus provide more information about the shape of the dose response curve in the region of interest. The BMD data for focal necrosis of renal tubular epithelium and reduced body weight gain in pregnant female rats were thus selected as the most reliable basis for determining the Longer term HA. Using the lower of the two values, the duration adjusted BMDL 10 of 18 mg/ kg day for reduced maternal body weight gain, the Longer term HA for a 10 kg child is calculated according to the following equation:

Longer term HA = ( 18 mg/ kg day) ( 10 kg) = 0.60 mg/ L ( rounded to 0.6 mg/ L) ( 300) ( 1 L/ day)

where:

18 mg/ kg day = BMDL 10 based on decreased maternal body weight gain on gestation days 6 9 in pregnant female rats administered bromodichloromethane in the drinking water.

10 kg = Assumed body weight of a child Draft  Do Not Cite or Quote February 20, 2003 VIII  16 300 = Uncertainty factor based on NAS/ OW guidelines. This value includes a factor of 10 to protect sensitive human populations and a factor of 10 for extrapolation from animals to humans, and a factor of 3 to account for uncertainty regarding possible reproductive effects of bromodichloromethane in humans 1 L/ day = Assumed water consumption of a 10 kg child The Longer term HA for adults would be calculated as follows:

Longer term HA = ( 18 mg/ kg day) ( 70 kg) = 2.1 mg/ L ( rounded to 2 mg/ L) ( 300) ( 2 L/ day)

where:

18 mg/ kg day = BMDL 10 based on reduced body weight gain in female rats administered bromodichloromethane in the drinking water 70 kg = Assumed body weight of an adult 300 = Uncertainty factor based on NAS/ OW guidelines. This value includes a factor of 10 to protect sensitive human populations and a factor of 10 for extrapolation from animals to humans, and a factor of 3 to account for database limitations and uncertainty related to potential reproductive effects of bromodichloromethane in humans 2 L/ day = Assumed water consumption of a 70 kg adult For purposes of comparison , a Longer term HA derived using the conventional NOAEL/ LOAEL approach would be based on the study conducted by CCC ( 2000d). This study identified a NOAEL of 18.4 mg/ kg day and a LOAEL of 45 mg/ kg day based on reduced body weight gain on gestation days 6 to 7. Using the NOAEL of 18.4 mg/ kg day, and assuming drinking water ingestion of 1 L/ day and an uncertainty factor of 300 ( including factors of 10 for interspecies extrapolation and protection of susceptible populations, and a factor of 3 for database limitations and uncertainty regarding potential reproductive effects in humans), the Longer term HA for a 10 kg child would be 0.6 mg/ L. The Longer term HA for a 70 kg adult consuming 2 L/ day would be 2 mg/ kg day. Draft  Do Not Cite or Quote February 20, 2003 VIII  17 Table VIII 3 Summary of Candidate Studies for Derivation of the Longer term HA for Bromodichloromethane Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) BMD ( mg/ kg day) BMDL 10 ( mg/ kg day) *

NTP ( 1987) Rat F344/ N M, F 10 0 19 38 75 150 300 Gavage ( oil) 13 weeks ( 5 d/ wk) Body weight,

clinical signs,

histology 75 150 ( decreased weight gain)

( hepatic and renal lesions at 300) Not modeled

NTP ( 1987) Mouse B6C3F 1 M, F 10 Male 0 6.3 13 25 50 100 Female 0 25 50 100 200 400 Gavage ( oil) 13 weeks ( 5 d/ wk) Body weight,

clinical signs,

histology 50 100 ( renal lesions) 63 35 ( focal necrosis of renal tubular epithelium in males)

75 47 ( Hepatic vacuolated cytoplasm in females)

Ruddick et al. ( 1983)* Rat SD F 9 14 0 50 100 200 Gavage ( oil) Gestation days 6 15 Body and organ weights; maternal serum chemistry; hematology, and histopathology;

developmental parameters 100 200 ( increased incidence of sternebral variations) 27 15 ( increased incidence of sternebral variations)

Narotsky et al.

( 1997) * Rat F344 F 12

14 0 25 50 75 Gavage ( oil)

( aq)** Gestation days 6 15 Body weight,

clinical signs, developmental parameters 25 ( developmental) 50 ( full litter resorption) 48 30 ( full litter resorption)

( maternal) 25 ( reduced maternal body weight gain gestation days 6

8, aq. vehicle) 18 10 ( reduced maternal body weight gain,

gestation days 6

8, aq. vehicle) Table VIII 3 ( cont.)

Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) BMD ( mg/ kg day) BMDL 10 ( mg/ kg day) *

Draft  Do Not Cite or Quote February 20, 2003 VIII  18 Bielmeier et al. ( 2001)* Rat F344 F 8 11 0 75 100 Gavage ( aq) Gestation day 9 Full litter resorption;

hormone profiles  75 ( full litter resorption) 23 4.2 ( full litter resorption)

CCC ( 2000c)* Rat SD M, F 10 0 ppm 50 ppm 150 ppm 450 ppm 1350 ppm Drinking water Males 64 days Females 74 days Reproductive/ developmental parameters Developmental 50 ppm Parental 50 ppm Developmental 150 ppm

Parental 50 ppm Not modeled

CCC ( 2000d)* Rat SD F 25 0.0 2.2 18.4 45.0 82.0 Drinking water Gestation days 6 21 Reproductive/ developmental parameters 45.3 ( developmental) 82.0 ( reduced number of ossification sites in phalanges or metatarsals occurring with maternal toxicity) Not modeled

18.4 ( maternal) 45 ( reduced maternal body weight gain gestation days 6

7) 23 18 ( reduced maternal body weight gain gestation days 6

9; see comments in text)

CCC ( 2002)* Rat SD M, F 30 0 ppm 50 ppm 150 ppm 450 ppm Drinking water Two generations Reproductive parameters 50 ppm ( offspring) 150 ppm ( delayed sexual maturation in F 1 males) Not modeled   50 ppm ( parental) 150 ppm ( Reduced body wt and body wt gain in F 0 females and F 1 males and females) Table VIII 3 ( cont.)

Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) BMD ( mg/ kg day) BMDL 10 ( mg/ kg day) *

Draft  Do Not Cite or Quote February 20, 2003 VIII  19 CCC ( 2000a)* Rabbit NZW F 5 0 4.9 13.9 32.3 76.3 Drinking Water Gestation day 6 to 29 Body weight,

clinical signs,

reproductive and developmental parameters 76.3 ( developmental)  Not modeled

CCC ( 2000b)* Rabbit NZW F 25 0 1.4 13.4 35.6 55.3 Drinking Water Gestation days 6 29 Clinical sign,

gross lesions,

reproductive and developmental endpoints 55.3 ( developmental)  Not modeled

13.4 ( maternal) 35.3 ( reduced maternal body weight gain 50 ( developmental) BMD software failed * BMDL10 value was derived using duration adjusted doses.

* Modeled using Crump Benchmark Dose Software * The NOAEL and LOAEL values listed are for reproductive/ developmental effects.

** The developmental NOAEL and LOAEL values were the same in either vehicle. The LOAELs for maternal toxicity were 25 mg/ kg day and 50 mg/

kgday for the aqueous and corn oil vehicles respectively. BMD modeling was performed on aqueous vehicle data only.

Abbreviations: NA, Not available; SD, Sprague Dawley; NZW, New Zealand White Draft  Do Not Cite or Quote February 20, 2003 VIII  20 d. Reference dose, Drinking Water Equivalent Level and Lifetime Health Advisory This section reports the existing RfD value for bromodichloromethane and describes the derivation of the RfD for this compound. This section also describes the calculation of Drinking Water Equivalent Level and Lifetime Health Advisory values which require the RfD as input. For this document, new and existing studies were reviewed and appropriate candidate data were selected for benchmark dose ( BMD) modeling. The results of BMD modeling were used in conjunction with appropriate uncertainty factors to calculate the RfD. A comparison of the RfD derived using the BMD approach to the results obtained using the conventional NOAEL/ LOAEL approach is also provided.

Description of the Existing RfD The existing RfD for bromodichloromethane is 0.02 mg/ kg day ( IRIS, 1993a). This value was derived using a duration adjusted LOAEL of 17.9 mg/ kg day identified for renal cytomegaly in B6C3F 1 mice administered bromodichloromethane by corn oil gavage for 5 days/ week for 102 weeks ( NTP, 1987). An uncertainty factor of 100 was used to account for extrapolation from animal data and for protection of sensitive human subpopulations. An additional factor of 10 was used because the RfD was based on a LOAEL ( although it was considered minimally adverse) and to account for lack of reproductive data.

Identification of Candidate Studies for Derivation of the RfD Several studies of chronic duration were considered for derivation of the RfD for bromodichloromethane. These studies are summarized in Table VIII 4 below. NTP ( 1987) administered bromodichloromethane to F344/ N rats by gavage in corn oil at doses of 50 or 100 mg/ kg day for 5 day/ week for 102 weeks. This study identified a LOAEL of 50 mg/ kg day based on histologic alterations in the liver and kidney. In a similar study, NTP ( 1987) administered bromodichloromethane by gavage in corn oil to B6C3F 1 mice for 5 days/ week for 102 weeks at dose levels of 25 or 50 mg/ kg day for males and 75 or 150 mg/ kg day for females. Based on histologic alterations in the liver, kidney, and thyroid of male mice, this study identified a LOAEL of 25 mg/ kg day, which is consistent with the value identified in the rat study. In a third study, Tobe et al. ( 1982) administered microencapsulated bromodichloromethane to Wistar rats in the diet at dose levels ranging from 6 to 168 mg/ kg day. Histologic data for the animals exposed to bromodichloromethane were reported by Aida et al. ( 1992b). This study identified a LOAEL for male rats of 6 mg/ kg day on the basis of histopathologic changes in the liver.

Ten reproductive and/ or developmental toxicity studies ( Ruddick et al., 1983; Klinefelter et al., 1995; Narotsky et al., 1997; NTP, 1998; Bielmeier et al. 2001; CCC, 2000a, b, c, d; CCC, 2002) were considered for derivation of the RfD in addition to the chronic studies. The investigations of Ruddick et al. ( 1983), Klinefelter et al. ( 1995), and Narotsky et al. ( 1997) identified NOAELs or LOAELs in rats that were substantially higher than the LOAEL identified by Aida et al. ( 1992b) ( Table VIII 4 below). The studies conducted by NTP ( 1998) did not observe developmental or reproductive effects at doses up to 116 mg/ kg day. The study by Bielmeier et al. ( 2001) identified a free standing LOAEL of 75 mg/ kg day in F344 rats. The studies conducted by CCC ( 2000a, b) identified NOAELs of 55 and 76 mg/ kg day, respectively, Draft  Do Not Cite or Quote February 20, 2003 VIII  21 for developmental effects in New Zealand White rabbits. The study conducted in rats by CCC ( 2000c) identified concentration based NOAEL and LOAEL values of 50 ppm and 150 ppm for reduced body weight and body weight gain in F 1 pups. The study conducted in rats by CCC ( 2000d) identified NOAEL and LOAEL values of 45 mg/ kg day and 82 mg/ kg day, respectively on the basis of decreased ossification sites per fetus per litter in the forelimb and hindlimb. Lowrange LOAEL values for maternal toxicity ranged from 13 to 25 mg/ kg day ( CCC, 2000b; CCC 2000d; Narotsky et al., 1997). CCC ( 2002) identified a LOAEL of 150 ppm ( approximately 11.6 to 40.2 mg/ kg day) for delayed sexual maturation in F 1 male rats in a two generation study of bromodichloromethane administered in drinking water. The LOAEL for parental effects in the study was also 150 ppm, based on decreased body weight and body weight gain in F 1 females and F 1 males and females. Since these studies identified NOAEL and/ or LOAEL values substantially higher than that identified by Aida et al. ( 1992b), they were not further considered for derivation of the RfD.

Method of Analysis Selected data from the candidate studies were analyzed using the benchmark dose ( BMD) modeling approach. Initially, data sets for potentially sensitive endpoints were selected as described in U. S. EPA ( 1998b) and analyzed using the Crump Benchmark Dose Modeling Software ( K. S. Crump, Inc.). Results of this analysis are summarized in Table VIII 5. Following the release of Version 1.2 of the BMDS program ( U. S. EPA, 2000a), a subset of the most sensitive endpoints identified using the Crump software was reanalyzed in accordance with proposed U. S. EPA ( 2000b) recommendations. An advantage of analysis with the BMDS software is that several additional models are available to fit the data. The results of the analysis with the BMDS software are included in Table VIII 4.

Choice of Principal Study and Critical Effect for the RfD Three data sets for histopathological effects in liver were analyzed using the BMDS software ( Table VIII 4). BMD modeling identified several endpoints with BMD values lower than the conventionally determined LOAEL of 6 mg/ kg day ( Aida, 1992b). The lowest BMD values were obtained for fatty degeneration ( 1.9 mg/ kg day) in male rats ( Aida et al., 1992b) and for kidney cytomegaly ( 2.0 mg/ kg day) in male mice ( NTP, 1987). Comparably low BMD values were also obtained for granulomas observed in the liver of male rats ( 2.1 mg/ kg day) and for fatty degeneration in the liver of female rats ( 3.1 mg/ kg day) in the study conducted by Aida et al. ( 1992b). In contrast, the BMD values calculated for endpoints examined in other studies were approximately 10 to 20 fold higher. Draft  Do Not Cite or Quote February 20, 2003 VIII  22 Table VIII 4 Summary of Candidate Studies for Derivation of the RfD for Bromodichloromethane Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) BMD ( mg/ kg day) BMDL 10 ( mg/ kg day) a NTP ( 1987) Rat F344/ N M, F 50 0 50 100 Gavage ( oil) 102 weeks ( 5 d/ wk) Body weight,

clinical signs,

gross necropsy,

histology  50 ( lesions of kidney and liver)  36.5c ( liver necrosis in male rats)

NTP ( 1987) Mouse B6C3F 1 M, F 50 0 25 50 Gavage ( oil) 102 weeks ( 5 d/ wk) Body weight,

clinical signs,

gross necropsy,

histology  25 ( lesions of liver,

kidney, and thyroid) 2.0 1.5 ( kidney cytomegaly in male mice)

Aida et al.

( 1992b) Rat Wistar M, F 40 Male 0 6 26 138 Female 0 8 32 168 Diet 24 months Body weight,

clinical signs,

serum biochemistry,

gross necropsy,

histology  6 ( liver fatty degeneration and granuloma) 3.1 2.1 ( fatty degeneration in liver of females )

1.9 0.8 ( fatty degeneration in liver of males)

2.1 1.4 ( Granulomas in livers of males)

Ruddick et al. ( 1983) b Rat SD F 9 14 0 ppm 50 ppm 150 ppm 450 ppm 1350 ppm Gavage ( oil) Gestation days 6 15 Body and organ weights; maternal serum chemistry; hematology, and histopathology;

developmental parameters 100 200 ( sternebral variations) 27 15 ( sternebral variations)

Narotsky et al. ( 1997) b Rat F344 13

14 0 75 100 Gavage ( oil)

( water) Gestation days 6 15 Body weight,

clinical signs, developmental parameters 25 ( developmental) 50 ( full litter resorption) 48 30 ( full litter resorption) Table VIII 4 ( cont.)

Draft  Do Not Cite or Quote February 20, 2003 VIII  23 Klinefelter et al. ( 1995) b Rat F344 M 7 0 22 39 Drinking water 52 weeks Body and organ weights, gross necropsy, histology, sperm motion parameters 22 39 ( decreased sperm velocities)   d Bielmeier et al. ( 2001) b Rat F344 F 8 11 0 75 100 Gavage ( aq) Gestation day 9 Full litter resorption,

hormone profiles, body weight  75 ( full litter resorption) 23 4.2 ( full litter resorption)

CCC ( 2000a) b Rabbit NZW F 5 0 4.9 13.9 32.3 76.3 Drinking Water Gestation days 6 29 Body wt.,

clinical signs,

reproductive developmental parameters 76    e CCC ( 2000b) b Rabbit NZW F 25 0 1.4 13 36 55 Drinking water Gestation days 6 29 Maternal feed and water intake, body wt.; gross lesions; uterine weight, no. implantation sites, uterine contents, and no.

corpora; Fetal wt., gross ext.

alterations, skel.

alterations, sex,

visceral alterations 55 ( developmental)    e 13.4 ( maternal) 35.3 ( reduced maternal body weight gain) 50 ( maternal) BMDS software failed CCC ( 2000c) b Rat SD M, F 10 0 ppm 50 ppm 150 ppm 450 ppm 1350 ppm Drinking water Males 64 days Females 74 days Reproductive/ developmental parameters 50 ppm 150 ppm   Table VIII 4 ( cont.)

Draft  Do Not Cite or Quote February 20, 2003 VIII  24 CCC ( 2000d) b Rat SD F 25 0.0 2.2 18.4 45.0 82.0 Drinking water Gestation days 6 21 Reproductive/ developmental parameters 45 ( developmental) 82 ( reduced no. of ossification sites in phalanges or metatarsals occurring with maternal toxicity)   ( developmental)   e 18.4 ( maternal) 45 ( reduced maternal body weight gain) 23 ( maternal) 18 ( reduced maternal body weight gain)

CCC ( 2002) Rat SD M, F 30 0 ppm 50 ppm 150 ppm 450 ppm Drinking water Two generations Reproductive/ developmental parameters 50 ( offspring) 150 ( delayed sexual maturation in F 1 males) Not modeled

50 ( parental) 150 ( decrecreased body weight and body weight gain in F 0 females and F 1 males and females) Not modeled

a BMDL10 values were derived using duration adjusted doses.

b Ruddick et al. ( 1983); Klinefelter et al. ( 1995), Narotsky et al. ( 1997), Bielmeier et al. ( 2001), and CCC ( 2000a d) are included in this table because they are reproductive and/ or developmental studies. The NOAEL, LOAEL, BMD, and BMDL10 values listed are for reproductive and/ or developmental endpoints.

c Data modeled using Crump BMD software d No histopathological abnormalities were noted in this study, and similar effects on sperm velocity were not observed in NTP ( 1998); therefore, data for sperm velocity were not modeled e Data were not modeled since effects occurred at higher doses than other candidate endpoints   Indicates that data were not modeled Abbreviations: NZW, New Zealand White; SD, Sprague Dawley NOTE: The short term reproductive and developmental toxicity study conducted by NTP ( 1998) was not included in this table because no developmental or reproductive effects were noted at dose levels ranging from 67 to 126 mg/ kg day. Draft  Do Not Cite or Quote February 20, 2003 VIII  25 Table VIII 5 Summary of Preliminary BMD Modeling Results for the Bromodichloromethane RfD Study Endpoint Modeled BMDL10 ( mg/ kg day) *

Subchronic NTP ( 1987) mouse study Focal necrosis of renal tubular epithelium in males 34 Vacuolated hepatocytes in females 64 Chronic NTP ( 1987) rat study Kidney cytomegaly in males No acceptable fit Liver necrosis in males 36.5 Liver fatty metamorphosis in males No acceptable fit Clear cell changes in liver of females No acceptable fit Chronic NTP ( 1987) mouse study Kidney cytomegaly in males 0.96 Liver fatty metamorphosis in males 7.5 Thyroid follicular cell hyperplasia in females 15 Chronic Aida et al. ( 1992b) rat study Fatty degeneration in liver of males 2.38 Granulomas in liver of males 4.5 Fatty degeneration in liver of females 1.20 Granulomas in liver of females No acceptable fit * BMD modeling conducted on duration adjusted doses using the Crump BMD Software ( K. S. Crump, Inc.). Draft  Do Not Cite or Quote February 20, 2003 VIII  26 The chronic study conducted by Aida et al. ( 1992b) was selected for derivation of the RfD. The lowest dose utilized in this study was 6 mg/ kg day ( in contrast to low doses of 22 to 75 mg/ kg day utilized in other candidate studies), which provides some information on the shape of the dose response curve in the region of interest. The lowest BMD ( 1.9 mg/ kg day) was obtained for fatty degeneration in the liver of male mice. The corresponding BMDL 10 for this endpoint was 0.8 mg/ kg day. The incidence of this lesion was strongly dose dependent, with incidences of 0/ 24, 5/ 14, 12/ 13, and 19/ 19 observed at the doses of 0, 6, 25, and 138 mg/ kg day, respectively. The occurrence of this lesion in rats treated with bromodichloromethane is consistent with current understanding of the mode of action of brominated trihalomethanes. This endpoint was therefore selected to derive the RfD for bromodichloromethane.

Derivation of the RfD The BMDL 10 calculated for fatty degeneration in the liver of male rats in the chronic rat study conducted by Aida et al. ( 1992b) was selected as the most appropriate basis for derivation of the RfD for bromodichloromethane. The RfD is calculated according to the following equation:

RfD = ( 0.8 mg/ kg day) = 0.003 mg/ kg day ( 3 µ g/ L) ( 300)

where:

0.8 mg/ kg day = Duration adjusted BMDL 10 based on fatty degeneration of the liver in male rats 300 = Uncertainty factor based on NAS/ OW guidelines. This value includes a factor of 10 to account for intrahuman variability, and a factor of 10 for interspecies variability, and a factor of 3 to account for database deficiencies, including lack of a multigeneration reproductive toxicity study and database limitations and uncertainty related to possible human reproductive effects suggested ( causality can not be established from available data) by epidemiological studies.

A composite UF of 300 was used. The standard factors of 10 were used for interspecies extrapolation and for protection of sensitive subpopulations. An additional factor of 3 was used to account for database deficiencies related to possible reproductive or developmental effects in humans. Use of an additional uncertainty factor of 3 is supported by findings in epidemiological studies ( Waller et al., 1998; King et al., 2000) which suggest potential associations between bromodichloromethane exposure via drinking water and adverse pregnancy outcomes. Although the results of the epidemiological studies can not establish that bromodichloromethane caused the observed effects, they do raise significant concern for potential reproductive effects in exposed Draft  Do Not Cite or Quote February 20, 2003 VIII  27 human populations, and the inclusion of an additional uncertainty factor is thus considered appropriate for protection of human health.

The DWEL for bromodichloromethane is calculated as follows:

DWEL = ( 0.003 mg/ kg day) ( 70 kg) = 0.100 mg/ L ( 100 µ g/ L) 2 L/ day where:

0.003 mg/ kg day = RfD for bromodichloromethane 70 kg = Assumed weight of an adult 2 L/ day = Assumed water consumption by a 70 kg adult Lifetime Health Advisory The Lifetime Health Advisory ( HA) represents that portion of an individual's total exposure that is attributed to drinking water and is considered protective of noncarcinogenic health effects over a lifetime of exposure. Bromodichloromethane has been categorized with respect to carcinogenic potential as Group B2: Probable human carcinogen ( IRIS, 1993a). Therefore, in accordance with U. S. EPA Policy, a Lifetime HA is not recommended.

Alternative Approach for Derivation of the RfD Use of the conventional NOAEL/ LOAEL approach represents an alternative means for deriving the RfD and DWEL. Aida et al. ( 1992b) identified a LOAEL of 6 mg/ kg day in male rats on the basis of histopathological changes in the liver. Using this value and a composite uncertainty factor of 3,000 ( including factors of 10 for interspecies extrapolation, protection of sensitive subpopulations, and use of a LOAEL, and a factor of 3 for database limitations and uncertainty regarding potential reproductive effects in humans), the RfD derived using the conventional approach is 0.002 mg/ kg day. Assuming a body weight of 70 kg and drinking water ingestion of 2 L/ day, the corresponding DWEL is 0.07 mg/ L ( 70 µ g/ L). Draft  Do Not Cite or Quote February 20, 2003 VIII  28 2. Carcinogenic Effects a. Categorization of Carcinogenic Potential Previous Evaluations The Carcinogenic Risk Assessment Verification Endeavor ( CRAVE) group of the U. S. EPA reviewed the available evidence on the carcinogenicity of bromodichloromethane and assigned it to Group B2: probable human carcinogen ( IRIS, 1993a). Assignment to this category is appropriate for chemicals where there are no or inadequate human data, but which have sufficient animal data to indicate carcinogenic potential.

IARC has recently re evaluated the carcinogenic potential of bromodichloromethane ( IARC 1999a). IARC concluded that there is sufficient evidence of carcinogenicity for bromodichloromethane in experimental animals, but inadequate evidence in humans. Thus, IARC classified bromodichloromethane as a Group 2B carcinogen: possibly carcinogenic to humans.

Categorization of Carcinogenic Potential Under the Proposed 1999 Cancer Guidelines Cancer Hazard Summary Under the proposed guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1999) bromodichloromethane is likely to be carcinogenic to humans by all routes. This descriptor is appropriate when the available tumor data and other key data are adequate to demonstrate carcinogenic potential to humans. This finding is based on the weight of experimental evidence in animal models which shows carcinogenicity by modes of action that are relevant to humans.

Supporting Information for Cancer Hazard Assessment Human Data The information on the carcinogenicity of bromodichloromethane from human studies is inadequate. There are no epidemiological data specifically relating increased incidence of cancer to exposure to bromodichloromethane. There are equivocal epidemiological data describing a weak association of chlorinated drinking water exposures with increased incidences of bladder, rectal, and colon cancer. U. S. EPA has determined that these studies cannot attribute the observed effects to a single compound, as chlorinated water contains numerous other disinfection byproducts that are potentially carcinogenic.

Animal Data The carcinogenicity of bromodichloromethane in male and female animals has been investigated in a well designed and conducted corn oil gavage study conducted in rats and mice, a dietary exposure study in rats, and two drinking water studies in rats. Additional data are available from a study in which male Strain A mice were exposed to bromodichloromethane by Draft  Do Not Cite or Quote February 20, 2003 VIII  29 intraperitoneal injection. No data are available on the carcinogenic potential of bromodichloromethane administered via the inhalation or dermal routes.

In the corn oil gavage study ( NTP, 1987), statistically significant increases were observed in the incidences of neoplasms of the large intestine and kidney in male and female rats, the kidney in male mice, and liver in female mice. The neoplasms observed in the large intestine and kidney are considered rare neoplasms based on historical control data for the tested strains. In the feeding study ( Aida et al., 1992b), exposure to microencapsulated bromodichloromethane did not result in statistically significant increases in any tumor type. Observed neoplastic lesions included three cholangiocarcinomas and two hepatocellular adenomas in high dose females, one hepatocellular adenoma in a control female, one cholangiosarcoma in a high dose male, and one hepatocellular carcinoma each in a low and a high dose male. In the drinking water study conducted by Tumasonis et al. ( 1985), hepatic neoplastic nodules, hepatic adenofibrosis, and lymphosarcoma were significantly increased in female rats. No significant increase in the occurrence of any tumor type was observed in male rats. Renal adenoma or adenocarcinoma were noted in two males and one female treated with bromodichloromethane, while neither tumor type was reported in the control group. In the drinking water study conducted by George et al. ( 2002), the prevalence of neoplastic lesions in the liver was significantly increased only at the lowest administered dose. Intraperitoneal injection of Strain A mice with bromodichloromethane resulted in an apparent increase in the number of pulmonary adenomas per animals, although the response did not reach statistical significance in any dose group.

Structural Analogue Data Trihalomethanes structurally related to bromodichloromethane have shown varying degrees of carcinogenic potential in rodents. Chloroform, the most extensively characterized trihalomethane, is reported to be carcinogenic at high doses in several chronic animal bioassays, with significant increases in the incidence of liver tumors in male and female mice and significant increases in the incidence of kidney tumors in male rats and mice ( U. S. EPA, 2001). The occurrence of tumors in animals exposed to chloroform is demonstrably species, strain, and gender specific, and has only been observed under dose conditions that caused cytotoxicity and regenerative cell proliferation in the target organ. The cancer database for structurally related brominated trihalomethanes is more limited, but includes well conducted studies performed by the National Toxicology Program. In a two year corn oil gavage study of bromoform, NTP ( 1989a) found clear evidence for carcinogenicity in female rats and some evidence of carcinogenicity in male rats based on occurrence of tumors of the large intestine ( adenomatous polyps or adenocarcinoma). In a two year corn oil gavage study of dibromochloromethane, NTP ( 1985) determined that there was some evidence of carcinogenicity in female mice and equivocal evidence of carcinogenicity in male mice, based on the occurrence of hepatocellular adenomas and carcinomas. Other, less well documented, oral exposure studies ( Tobe et al., 1982; Kurokawa, 1987; Voronin et al., 1987) found no evidence for carcinogenicity of bromoform or dibromochloromethane.

Other Key Data Draft  Do Not Cite or Quote February 20, 2003 VIII  30 Bromodichloromethane is formed as a byproduct of drinking water disinfection with chlorine. Exposure to bromodichloromethane may occur via ingestion of tap water, via dermal contact during showering or bathing, or by inhalation of bromodichloromethane volatilized during household activities. Absorption of single oral doses appears to be extensive. Bromodichloromethane is rapidly metabolized and eliminated predominately as expired volatiles, CO 2, or CO. Only a small amount ( less than 10%) is eliminated in urine or in feces. No comprehensive tissue data are available regarding the bioaccumulation or retention of bromodichloromethane following repeated exposure. However, because of the rapid metabolism and excretion of bromodichloromethane, marked accumulation and retention is not expected.

Bromodichloromethane itself is not directly reactive with DNA. Metabolism to reactive species is a prerequisite for toxicity, as inferred from metabolic induction and inhibition studies. In vitro and in vivo studies of the mutagenic and genotoxic potential of bromodichloromethane have yielded both positive and negative results. Synthesis of the overall weight of evidence from these studies is complicated by the use of a variety of testing protocols, different strains of test organisms, different activating systems, different dose levels, different exposure methods ( gas versus liquid) and, in some cases, problems due to evaporation of the test chemical. However, because a majority of studies yielded positive results, bromodichloromethane is considered to be at least weakly mutagenic and genotoxic. Recent studies conducted with strains of Salmonella engineered to express rat theta class glutathione S transferase suggest that mutagenicity of the brominated trihalomethanes may be mediated by glutathione conjugation.

Mode of Action The mode of action for tumor induction by bromodichloromethane has not been clearly elucidated and may involve contributions from multiple bioactivation pathways. In each case, toxicity is believed to result from interaction of reactive metabolites with cellular macromolecules. Proposed bioactivation pathways for bromodichloromethane include: 1) production of reactive dihalocarbonyls by oxidative metabolism; 2) production of reactive dihalomethyl radicals by oxidative metabolism; and 3) formation of DNA reactive species via a glutathione dependent pathway. The relative contribution of each pathway to tumor induction by bromodichloromethane has not been characterized. It is possible that only the latter two processes lead to DNA damage in vivo, because the highly reactive dihalocarbonyl intermediate may not survive long enough to enter the nucleus and react with DNA. For this reason, cytotoxicity may be the primary consequence of the oxidative pathway. Cytotoxicity coupled with regenerative hyperplasia is considered the primary mode of action for tumor formation following exposure to high concentrations of chloroform, a structurally related trihalomethane which has low genotoxic potential. Data to support a similar primary mode of action for tumor development in liver, kidney, and large intestine are currently lacking for bromodichloromethane. In the absence of such information, combined with a positive weight of evidence evaluation for genotoxicity, the mode of action for tumor development is assumed to be a linear process. The processes leading to tumor formation in animals are expected to be relevant to humans.

Conclusion Draft  Do Not Cite or Quote February 20, 2003 VIII  31 Under the proposed guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1999) bromodichloromethane is likely to be carcinogenic to humans by the oral route. This weight ofevidence evaluation is based on 1) observations of tumors in animals treated by oral pathways; 2) lack of epidemiological data specific to bromodichloromethane and equivocal data for drinking water drinking water exposures that cannot reliably be attributed to bromodichloromethane among multiple other disinfection byproducts; 3) positive results for a majority of the available genotoxicity and mutagenicity tests; and 4) metabolism and mode of action that are reasonably expected to be comparable across species. Although no cancer data exist for exposures via the dermal or inhalation pathways, the weight of evidence conclusion is considered to be applicable to these pathways as well. The finding for inhalation is based on the observation that patterns of metabolizing enzyme activity in male rats are similar for exposure via the inhalation and gavage routes. Bromodichloromethane absorbed through the skin is expected to be metabolized and cause toxicity in much the same way as bromodichloromethane absorbed by the oral and inhalation routes.

b. Choice of Study for Quantification of Carcinogenic Risk In accordance with the Proposed 1999 Cancer Guidelines ( U. S. EPA, 1999), quantification of cancer risk is appropriate for compounds categorized as likely to be carcinogenic to humans. Five oral exposure studies were available for quantification of the carcinogenic risk associated with exposure to bromodichloromethane. Detailed summaries of these studies are provided in Section V. G. 1. The two year study conducted by NTP ( 1987) in rats and mice was selected for quantification of carcinogenic effects associated with exposure to bromodichloromethane. Selection of this study was based on significantly increased incidence of several tumor types, monotonic dose response curves, and comprehensive documentation of study design and results.

In the NTP ( 1987) study, groups of male and female F344/ N rats ( 50/ sex/ dose) received 0, 50, or 100 mg/ kg day gavage doses of bromodichloromethane in corn oil. The doses were administered 5 days/ week for 102 weeks. In a similar experiment, groups of male and female B6C3F 1 mice ( 50/ sex/ dose) were administered doses of 0, 25, or 50 mg/ kg day ( males) or 0, 75, or 150 mg/ kg day ( females) for 5 days/ week for 102 weeks. All animals were subjected to gross and microscopic examinations for neoplastic lesions. Survival of all dosed animals was comparable to or greater than the corresponding control group. Statistically significant increases were observed in the incidences of neoplasms of the large intestine and kidney in male and female rats, the kidney in male mice, and the liver in female mice ( Table VIII 6). The authors noted that neoplasms of the large intestine and kidney are uncommon tumors in F344/ N rats and B6C3F 1 mice. They concluded that under the conditions of these 2 year gavage studies, clear evidence of carcinogenic activity existed for male and female rats and mice. Draft  Do Not Cite or Quote February 20, 2003 VIII  32 Table VIII 6 Tumor Frequencies in Rats and Mice Exposed to Bromodichloromethane in Corn Oil for 2 Years  Adapted from NTP ( 1987)

Animal Tissue/ Tumor Tumor Frequency Male rat Control 50 mg/ kg 100 mg/ kg Large intestinea Adenomatous polyp 0/ 50 3/ 49 33/ 50b Adenocarcinoma 0/ 50 11/ 49b 38/ 50b Combined 0/ 50 13/ 49b 45/ 50b Kidney a Tubular cell adenoma 0/ 50 1/ 49 3/ 50 Tubular cell adenocarcinoma 0/ 50 0/ 49 10/ 50b Combined 0/ 50 1/ 49 13/ 50b Female rat Control 50 mg/ kg 100 mg/ kg Large intestine c Adenomatous polyp 0/ 46 0/ 50 7/ 47b Adenocarcinoma 0/ 46 0/ 50 6/ 47b Combined 0/ 46 0/ 50 12/ 47b Kidney Tubular cell adenoma 0/ 50 1/ 50 6/ 50b Tubular cell adenocarcinoma 0/ 50 0/ 50 9/ 50b Combined 0/ 50 1/ 50 15/ 50b Male mouse Control 25 mg/ kg 50 mg/ kg Kidney d Tubular cell adenoma 1/ 46 2/ 49 6/ 50 Tubular cell adenocarcinoma 0/ 46 0/ 49 4/ 50 Combined 1/ 46 2/ 49 9/ 50b Female mouse Control 75 mg/ kg 150 mg/ kg Liver Hepatocellular adenoma 1/ 50 13/ 48b 23/ 50b Hepatocellular carcinoma 2/ 50 5/ 48 10/ 50b Combined 3/ 50 18/ 48b 29/ 50b a One rat died at week 33 in the low dose group and was eliminated from the cancer risk calculation. b Statistically significant at p< 0.05, compared to controls. c Intestine not examined in four rats from control group and three rats from high dose group. d In the control group, two mice died during the first week, one mouse died during week, nine and one escaped in week 79. One mouse in the low dose group died in the first week. All of these mice were eliminated from the cancer risk calculations. Draft  Do Not Cite or Quote February 20, 2003 VIII  33 Use of the NTP rodent studies ( NTP, 1987) for derivation of cancer risk estimates for bromodichloromethane is complicated by the use of a corn oil as a dosing vehicle. Although a vehicle effect has not been reported for brominated trihalomethanes, it can be inferred from studies of chloroform carcinogenicity that such an effect might exist, at least for hepatic tumors in mice. Therefore, in the case of bromodichloromethane, the U. S. EPA believes that the most appropriate basis of the cancer risk estimate is the incidence of renal tumors in male mice. Renal tumors are considered to be appropriate because these tumors were increased in a dose dependent manner in both mice ( male) and rats ( both sexes).

c. Choice of Approach and Rationale The LMS model ( U. S. EPA, 1986) and the default linear approach described by the Proposed Guidelines for Carcinogen Risk Assessment ( U. S. EPA, 1996; 1999) were used to quantify the cancer risk associated with exposure to bromodichloromethane. Although data are mixed, the weight of evidence indicates that bromodichloromethane is mutagenic ( see Section V. F. 1). Furthermore, recent studies ( Melnick, et al. 1998; George et al., 2002) suggest that induction of hepatic tumors occurs at doses of bromodichloromethane that have marginal or no effect on hepatocyte labeling index, indicating that regenerative hyperplasia is not required for tumor induction. Thus, use of a linear approach was considered appropriate for quantification of cancer risk associated with exposure to bromodichloromethane.

d. Cancer Potency and Risk Estimates The available estimates for cancer risk associated with bromodichloromethane are summarized in Table VIII 7. U. S. EPA ( 1994b) recommended use of a cancer potency estimate of 6.2 x 10 2 ( mg/ kg day) 1 as reported in IRIS ( 1993a). This value was derived in accordance with the 1986 Guidelines for Carcinogenic Risk Assessment ( U. S. EPA, 1986), based on the occurrence of renal tumors in male mice. A unit risk of 1.8 x 10 6 was estimated using an assumed body weight of 70 kg and a drinking water ingestion rate of 2 L. This estimate was used to calculate a drinking water concentration of approximately 6 µ g/ L associated with a 10 5 risk ( 0.6 µ g/ L for 10 6 risk).

A cancer potency value of 3.5 x 10 2 ( mg/ kg day) 1 ( U. S. EPA, 1998b) was derived using the LMS method and an animal to human conversion factor of body weight3/ 4 ( Table VIII 7). The use of body weight3/ 4 is consistent with recommendations in U. S. EPA ( 1992b). This potency factor is also based on the occurrence of renal tumors in male mice. A unit risk of 1 x 10 6 ( µ g/ L) 1 was estimated for bromodichloromethane using an assumed body weight of 70 kg and a drinking water ingestion rate of 2 L. This estimate was used to calculate a drinking water concentration of approximately 10 µ g/ L associated with a 10 5 risk ( 1 µ g/ L for 10 6 risk).

Cancer risk estimates were also obtained using the LED 10 ( the lower 95% confidence limit on a dose associated with 10% extra risk) for renal tumors in mice and assuming a linear mode of action for the carcinogenicity of bromodichloromethane ( Table VIII 7). A cancer potency value of 3.4 x 10 2 ( mg/ kg day) 1 was derived using this approach. A unit risk of 9.6 x 10 7 ( µ g/ L) 1 was estimated for bromodichloromethane using an assumed body weight of 70 kg and a drinking water ingestion rate of 2 L. This estimate was used to calculate a drinking water concentration of Draft  Do Not Cite or Quote February 20, 2003 VIII  34 approximately 10 µ g/ L associated with a 10 5 risk ( 1 µ g/ L for 10 6 risk). These values are closely similar to corresponding values derived using the LMS approach with body weight scaling to the 3/ 4 power.

Table VIII 7 Summary of Cancer Risk Estimates for Bromodichloromethane Method of Estimation Tumor Site Species Sex Slope Factor ( mg/ kg day) 1 Unit Risk ( µ g/ L) 1 LED 10 ( µ g/ kgday 10 5 Risk Concentration ( µ g/ L)

LMS Method Using BW3/ 4Conversion ( U. S. EPA, 1998b) Liver Mouse F 6.9 × 10 2 2.0 × 10 6  5 Kidney Rat M F 5.5 × 10 3 6.1 × 10 3 1.6 × 10 7 1.7 × 10 7  64 57 Mouse M 3.5 × 10 2 1.0 × 10 6  10 Large intestine Rat M F 1.7 × 10 2 6.1 × 10 3 4.9 × 10 7 1.7 × 10 7  20 57 LMS Method Using BW2/ 3Conversion U. S. EPA ( 1994b)* Liver Mouse F 1.3 × 10 1 3.7 × 10 6  3 Kidney Rat M F 8.7 × 10 3 9.5 × 10 3 2.5 × 10 7 2.7 × 10 7  40 37 Mouse M 6.2 × 10 2 1.8 × 10 6  6 Large intestine Rat M F 2.5 × 10 2 4.9 × 10 3 7.1 × 10 7 1.4 × 10 7  14 72 LED 10/ Linear Method ( U. S. EPA, 1998b) Liver Mouse F 6.5 × 10 2 1.9x10 6 1.5 × 103 5 Kidney Rat M F 8.1 × 10 3 8.8 × 10 3 2.3x10 7 2.5x10 7 1.2 × 104 1.1 × 104 43 40 Mouse M 3.4 x 10 2 9.6 x10 7 3.0 x103 10 Large intestine Rat M F 2.8 × 10 2 1.0 × 10 2 8x10 7 3x10 7 3.5 × 103 9.6 × 103 12 34 * Based on information adapted from IRIS ( 1993a) Draft  Do Not Cite or Quote February 20, 2003 VIII  35 B. Dibromochloromethane 1. Noncarcinogenic effects a. One day Health Advisory Four candidate studies that investigated the acute oral toxicity of dibromochloromethane were available. These studies are summarized in Table VIII 8 ( below). Bowman et al. ( 1978) administered dibromochloromethane by gavage to ICR Swiss mice at doses ranging from 500 to 4,000 mg/ kg and found that sedation and anesthesia resulted at doses of 500 mg/ kg or higher. NTP ( 1985) conducted a preliminary range finding study in which F344/ N rats and B6C3F 1 mice were administered dibromochloromethane by gavage at doses ranging from 160 to 2,500 mg/ kg and found that death may result from doses at 310 mg/ kg or higher in mice or rats. More recently, Müller et al. ( 1997) investigated the cardiotoxic effects of acute oral dibromochloromethane exposure in male Wistar rats. In this study, rats administered doses of 83 or 167 mg/ kg exhibited transient changes in cardiovascular parameters, while rats administered doses of 333 or 667 mg/ kg exhibited persistent alterations in at least one of the cardiovascular parameters that lasted throughout the postexposure observation period. Finally, Korz and Gatterman ( 1997) investigated the behavioral toxicity of acute oral dibromochloromethane exposure in male golden hamsters and observed only transient effects on the behavioral parameters investigated.

These studies were not considered adequate for deriving the One day HA, since evaluated more sensitive endpoints such as histopathology. Therefore, the Ten day HA for dibromochloromethane ( 0.6 mg/ L) calculated below is recommended for use as the One day HA.

b. Ten day Health Advisory Studies considered for derivation of the Ten day HA for dibromochloromethane are summarized in Table VIII 9 below. The key studies in this group are those of Aida et al. ( 1992a), Condie et al. ( 1983), and Melnick et al. ( 1998). These studies reported effects on sensitive endpoints and had data suitable for BMD analysis. Use of the remaining studies was limited by a variety of considerations, including lack of data suitable for BMD analysis ( Chu et al., 1982a; NTP, 1996; Coffin et al., 2000), toxicological relevance or difficulty in interpretation of the most sensitive endpoint ( NTP, 1985; Munson et al. 1982), and occurrence of effects only at the frank toxicity level ( NTP, 1985).

Melnick et al. ( 1998) administered dibromochloromethane to female B6C3F 1 mice by gavage for 5 days/ week for 3 weeks and identified a NOAEL of 100 mg/ kg day ( durationadjusted NOAEL of 71 mg/ kg day) and a LOAEL of 192 mg/ kg day ( duration adjusted LOAEL of 137 mg/ kg day) based on histologic changes in the liver ( hepatocyte hydropic degeneration). BMD analysis calculated BMD and BMDL 10 values of 112 and 68 mg/ kg day, respectively. Draft  Do Not Cite or Quote February 20, 2003 VIII  36 Table VIII 8 Summary of Candidate Studies for Derivation of the One day HA for Dibromochloromethane Reference Species Route Exposure Duration Dose ( mg/ kg day) Result Bowman et al.

( 1978) Mouse ICR Swiss Gavage ( aqueous) Single dose 500  4000 Sedation; anesthesia; increased mortality NTP ( 1985) Rat F344/ N Mouse B6C3F1 Gavage ( corn oil) Single dose 160  2500 Lethargy; death Müller et al.

( 1997) Rat Wistar Gavage ( olive oil) Single dose 83  667 Transient changes in blood pressure; effects on cardiac muscle contractility Korz and Gatterman ( 1997) Hamster Gavage ( olive oil) Single dose 50 Transient changes in posttreatment behavior Draft  Do Not Cite or Quote February 20, 2003 VIII  37 Table VIII 9 Summary of Candidate Studies for Derivation of the Ten day HA for Dibromochloromethane Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) BMD ( mg/ kg day) BMDL 10 ( mg/ kg day)

Aida et al.

( 1992a) Rat Wistar M, F 7 Males 0 18 56 173 Females 0 34 101 332 Feed 1 month Body weight, clinical signs, serum biochemistry,

hematology, histology 18.3 56 ( liver histopathology) 29 14 6.7 ( Liver cell vacuolation in females)

5.5 ( Liver cell vacuolation in males)

Chu et al.

( 1982a) Rat SD M 10 0 0.7 8.5 68 Drinking water 28 days Clinical signs, serum biochemistry,

histology 68  Not modeled

Condie et al.

( 1983) Mouse CD 1 M 8 16 0 37 74 147 Gavage ( oil) 14 days Serum enzymes, PAH uptake in vitro,

histology 74 147 ( elevated ALT,

decreased PAH,

liver and kidney histopathology) 3.5 11 1.6 ( Renal mesangial hypertrophy)

6.9 ( hepatic cytoplasmic vacuolation)

Melnick et al.

( 1998) Mouse B6C3F 1 F 10 0 50/ 37 100/ 71 192/ 137 417/ 298 Gavage ( oil) 3 weeks ( 5 d/ wk) Body and liver weights, serum chemistry, liver histology 100 ( marginal) 192 ( liver histopathology) 112* 68 ( hepatic hydropic degeneration)

Munson et al.

( 1982) Mouse CD 1 M, F 8 12 0 50 125 250 Gavage ( aqueous) 14 days Body and organ weights, serum chemistry, hematology, immune function 50 125 ( decreased humoral immunity) Not modeled  Table VIII 9 ( cont.)

Reference Species Sex n Dose Route Exposure Duration Endpoints NOAEL ( mg/ kg day) LOAEL ( mg/ kg day) BMD ( mg/ kg day) BMDL 10 ( mg/ kg day)

Draft  Do Not Cite or Quote February 20, 2003 VIII  38 NTP ( 1985) Rat F344/ N M, F 5 0 60 125 250 500 1000 Gavage ( oil) 14 days Body weight, clinical signs, gross necropsy 250 500 ( FEL)

( mortality,

lethargy, gross pathology) Not modeled

NTP ( 1985) Mouse B6C3F 1 M, F 5 0 30 60 125 250 500 Gavage ( oil) 14 days Body weight, clinical signs, gross necropsy 60 125 ( stomach lesions) 143 218 54 ( stomach nodules  males)

77 ( stomach nodules  females)

NTP ( 1996) Rat F344/ N M, F 10 Males 0 4 12 28 Females 0 6 7 17 20 48 48 Drinking water 29 days Body weight, serum chemistry, hematology, gross necropsy, histology,

sperm evaluation 28  Not modeled   Coffin et al.

( 2000) Mouse B6C3F 1 F 10 0 100 300 Gavage ( oil) 11 days Relative liver wt.;

liver histopathology;

labeling index  100 Not modeled

* BMD and BMDL10 calculated using duration adjusted doses Abbreviations : FEL, Frank Effect Level; SD, Sprague Dawley   No data Draft  Do Not Cite or Quote February 20, 2003 VIII  39 These values are considerably higher ( approximately 5 to 10 fold) than BMD and BMDL 10 values calculated for hepatic effects using data from Condie et al. ( 1983) or Aida et al. ( 1992a).

Condie et al. ( 1983) administered dibromochloromethane by gavage to male CD 1 mice for 14 days and identified a NOAEL of 74 mg/ kg day and a LOAEL of 148 mg/ kg day based on minimal to moderate liver and kidney injury. Histologic changes in the liver included focal inflammation and cytoplasmic vacuolization similar to that observed in the study by Aida et al. ( 1992a). Effects in the kidney included minimal to slight epithelial hyperplasia at the high dose and minimal to slight mesangial hypertrophy at all ( non control) doses. Data for cytoplasmic vacuolization and renal mesangial hypertrophy were analyzed by BMD modeling. The lowest BMD and BMDL 10 ( 3.5 and 1.6 mg/ kg day, respectively) among all candidate studies were identified for mesangial hypertrophy. However, the pattern of dose response for this endpoint ( 0/ 16, 4/ 5, 7/ 10, 7/ 10 at doses of 0, 37, 74, and 147 mg/ kg day, respectively) resulted in generally poor curve fits ( 0.1< goodness of fit p value> 0.49) and a high degree of model dependence ( See summary of modeling results in Appendix A). Thus, confidence in the reliability of the BMD for renal effects was low. The BMD and BMDL 10 values for hepatic cytoplasmic vacuolization were higher ( 11 and 6.9 mg/ kg day, respectively). These results were based on incidences of 1/ 16, 3/ 5, 4/ 10, and 8/ 10 at doses of 0, 37, 74, and 147 mg/ kg day, respectively.

The study by Aida et al. ( 1992a) was selected as the basis for derivation of the Ten day HA. In this study, Wistar rats of both sexes were administered microencapsulated dibromochloromethane in the diet for one month. The dose levels ranged from 18.3 to 173.3 mg/ kg day for males and from 34.0 to 332.5 mg/ kg day for females. A NOAEL of 18.3 mg/ kgday and a LOAEL of 56.2 mg/ kg day were identified based on histologic changes ( cell vacuolization, swelling, and single cell necrosis) in the livers of male rats. BMD analysis of data for hepatic cell vacuolization calculated BMD and BMDL 10 values of 29 and 6.7 mg/ kg day in females and 14 and 5.5 mg/ kg day in males, respectively. The BDML 10 for hepatic cell vacuolization in male rats was selected for calculation of the 10 day HA because it was considered the lowest reliable value based on examination of the raw data and modeling results. The incidence of this endpoint was 0/ 7, 1/ 7, 3/ 7, and 7/ 7 at doses of 0, 18, 56, and 173 mg/ kgday respectively.

Based on the BMDL 10 calculated from the data of Aida et al. ( 1992a), the Ten day HA is derived as follows:

Ten day HA = ( 5.5 mg/ kg day) ( 10 kg) = 0.55 mg/ L ( rounded to 0.6 mg/ L) ( 100) ( 1 L/ day)

5.5 mg/ kg day = BMDL 10 based on hepatic cell vacuolization in rats fed dibromochloromethane for one month 10 kg = Assumed body weight of a child Draft  Do Not Cite or Quote February 20, 2003 VIII  40 100 = Composite uncertainty factor based on NAS/ OW guidelines; includes a factor of 10 for interspecies variation and a factor of 10 for protection of sensitive human populations 1 L/ day = Assumed water consumption of a 10 kg child The Ten day HA was calculated using the conventional NOAEL/ LOAEL approach for comparison with the value obtained using the BMD approach. The Aida et al. ( 1992a) study identified a NOAEL of 18.3 mg/ kg day based on the absence of hepatic effects in rats. Using this value and and the assumptions described above, the Ten day HA would be 1.8 mg/ L ( rounded to 2 mg/ L).

c. Longer term Health Advisory Four candidate studies were considered for derivation of the Longer term HA for dibromochloromethane. These studies are summarized in Table VIII 10 ( below). Selected data from three of these studies were modeled using the BMD approach. The results of BMD analysis are included in Table VIII 11.

Chu et al. ( 1982b) administered dibromochloromethane to Sprague Dawley rats in the drinking water at doses ranging from 0.57 to 236 mg/ kg day. This study identified a NOAEL of 49 mg/ kg day, and a LOAEL of 224 mg/ kg day based on mild hepatic lesions ( increased cytoplasmic volume and vacuolation due to fatty infiltration) observed in males. BMD and BMDL 10 values of 18 and 5.3 mg/ kg day, respectively, were calculated for hepatic vacuolization using the BMDS software. Daniel et al. ( 1990) identified a LOAEL of 50 mg/ kg day based on hepatic lesions ( centrilobular lipidosis) observed in male Sprague Dawley rats and on kidney lesions ( tubular degeneration) observed in female Sprague Dawley rats administered dibromochloromethane by gavage for 90 consecutive days. BMD and BMDL 10 values of 20 and 4.2 mg/ kg day, respectively, were calculated for renal tubular degeneration using the Crump BMD software ( K. S. Crump, Inc.). NTP ( 1985) administered doses of dibromochloromethane ranging from 15 to 250 mg/ kg day to male and female mice. The compound was administered by gavage in corn oil, five days per week for 13 weeks. NOAEL and LOAEL values of 125 and 250 mg/ kg day were identified on the basis of renal and hepatic lesions. BMD and BMDL 10 values were not calculated since lesions occurred only at the high dose of 250 mg/ kg day.