Tables IV 8, IV 9 and IV 10 present data from the ICR database for the concentrations of MBA, BCA and DBA, respectively, as a function of influent TOC concentrations. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 24 Draft, do not cite or quote In contrast to the data presented by Arora ( 1997), a regression analysis of the ICR data indicates that there was a significant correlation (   = 0.05) between influent TOC concentration and the mean concentrations of MBA, BCA, and DBA in surface water ( Tabes IV 5, IV 6, and IV 7)/ This correlation with TOC levels is consistent with the formation of brominated acetic acids from the reaction of humic acid and hypobromous acid, a compound formed by the reaction of bromide ion with ozone and/ or chlorine in the disinfection process ( Chapter II). No such correlation was observed in groundwater, which had lower overall TOC levels.

Examination of the data using the Student's t test indicates that, with a few exceptions, at a given TOC concentration in surface water and groundwater, the mean concentrations of BCA were significantly higher ( p = 0.05) than the mean concentrations of DBA, which were significantly higher ( p = 0.05) than the mean MBA concentrations ( Tables IV 8, IV 9, and IV

10). ion. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 25 Draft, do not cite or quote Table IV 8. MBA by Influent Total Organic Carbon ( TOC) Concentration ( Quarterly Distribution System Average)

Source Influent TOC Conc. ( ppb) Plants N PctND % Mean µ g/ L Median µ g/ L STD µ g/ L Min µ g/ L Max µ g/ L p10 µ g/ L p90 µ g/ L SW < 1 12 61 98.36 0.00 0.00 0.04 ND 0.28 0.00 0.00 1  < 2 58 271 95.57 0.04 0.00 0.26 ND 3.20 0.00 0.00 2  < 3 100 479 84.13 0.22 0.00 0.72 ND 6.63 0.00 0.83 3  < 4 60 306 76.14 0.28 0.00 0.61 ND 3.33 0.00 1.33

4 69 324 74.69 0.50 0.00 1.75 ND 26.00 0.00 1.55 GW < 1 83 405 89.38 0.14 0.00 0.55 ND 6.68 0.00 0.28 1  < 2 13 52 65.38 0.38 0.00 0.65 ND 2.18 0.00 1.45 2  < 3 8 37 81.08 0.30 0.00 0.74 ND 3.08 0.00 1.23 3  < 4 3 7 85.71 0.10 0.00 0.26 ND 0.70 0.00 0.70

4 16 80 88.75 0.06 0.00 0.24 ND 1.95 0.00 0.28 1 Nondetects are treated as zero. Source: SW  Surface Water, GW  Groundwater Plants: Number of plants sampled N: Number of samples PctND: Percent samples nondetect Mean: Arithmetic mean of all samples Median: Median value of all samples STD: Standard deviation Min: Minimum Value Max: Maximum Value p10: 10th percentile p90: 90th percentile ND: Nondetect Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 26 Draft, do not cite or quote Table IV 9. BCA by Influent Total Organic Carbon ( TOC) Concentration ( Quarterly Distribution System Average)

Source Influent TOC Conc. ( ppb) Plants N PctND % Mean µ g/ L Median µ g/ L STD µ g/ L Min µ g/ L Max µ g/ L p10 µ g/ L p90 µ g/ L SW < 1 12 62 53.23 0.74 0.00 1.00 ND 4.10 0.00 2.25 1  < 2 58 270 15.19 2.52 1.98 2.41 ND 15.75 0.00 5.25 2  < 3 100 481 8.11 3.59 2.80 3.07 ND 23.80 0.35 7.73 3  < 4 60 309 4.21 4.17 3.38 3.14 ND 22.50 1.10 8.18

4 69 323 3.10 4.67 3.98 3.19 ND 24.18 1.48 8.53 GW < 1 83 405 63.46 0.65 0.00 1.40 ND 10.70 0.00 2.15 1  < 2 13 55 10.91 3.06 2.55 1.98 ND 7.35 0.00 5.93 2  < 3 8 36 38.89 2.20 1.39 2.43 ND 7.83 0.00 5.68 3  < 4 3 8 0.00 3.40 2.86 1.27 1.78 5.08 1.78 5.08

4 16 80 8.75 3.99 3.55 2.51 ND 11.28 0.43 7.64 1 Nondetects are treated as zero. Source: SW  Surface Water, GW  Groundwater Plants: Number of plants sampled N: Number of samples PctND: Percent samples nondetect Mean: Arithmetic mean of all samples Median: Median value of all samples STD: Standard deviation Min: Minimum Value Max: Maximum Value p10: 10th percentile p90: 90th percentile ND: Nondetect Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 27 Draft, do not cite or quote Table IV 10. DBA by Influent Total Organic Carbon ( TOC) Concentration ( Quarterly Distribution System Average)

Source Influent TOC Conc. ( ppb) Plants N PctND % Mean µ g/ L Median µ g/ L STD µ g/ L Min µ g/ L Max µ g/ L p10 µ g/ L p90 µ g/ L SW < 1 12 62 74.19 0.45 0.00 0.86 ND 2.68 0.00 2.08 1  < 2 58 272 72.79 0.46 0.00 1.10 ND 8.48 0.00 1.70 2  < 3 100 486 62.76 0.87 0.00 1.56 ND 7.65 0.00 3.25 3  < 4 60 309 55.02 1.69 0.00 2.81 ND 12.75 0.00 6.23

4 69 326 48.16 1.49 0.25 2.45 ND 14.25 0.00 4.33 GW < 1 83 406 60.59 0.69 0.00 1.36 ND 13.00 0.00 2.28 1  < 2 13 54 31.48 1.54 1.11 1.71 ND 6.58 0.00 3.75 2  < 3 8 36 61.11 1.71 0.00 2.68 ND 7.25 0.00 6.73 3  < 4 3 8 100.00 0.00 0.00 0.00 ND 0.00 0.00 0.00

4 16 80 43.75 0.67 0.26 0.85 ND 3.48 0.00 2.14 1 Nondetects are treated as zero. Source: SW  Surface Water, GW  Groundwater Plants: Number of plants sampled N: Number of samples PctND: Percent samples nondetect Mean: Arithmetic mean of all samples Median: Median value of all samples STD: Standard deviation Min: Minimum Value Max: Maximum Value p10: 10th percentile p90: 90th percentile ND: Nondetect Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 28 Draft, do not cite or quote A. 2.4 Seasonal Shifts Seasonal shifts in brominated acetic acids were investigated by Krasner et al. ( 1989). In September 1987, the USEPA's Office of Drinking Water entered into a cooperative agreement with the Association of Metropolitan Water Agencies ( AMWA) to perform a study of the occurrence and control of DBPs. The AMWA contracted with the Metropolitan Water District of Southern California ( MWD) to provide management services for the project and to perform the DBP analysis. In addition, the State of California Department of Health Services ( CDHS),

through the California Public Health Foundation ( CPHF), contracted with MWD to perform a similar study in California. Baseline data were gathered on 35 water treatment facilities, including 25 water utilities across the United States in the USEPA study and 10 California water utilities in the CDHS study. Levels of MBA and DBA were measured, but BCA was not evaluated.

During the first quarter ( spring 1988), a high correlation was found between DBA and the disinfectant byproduct dibromochloromethane. In addition, Krasner et al. ( 1989) reported that relatively high levels of the measured brominated DBPs were detected at some of the utilities.

These findings suggested that the influence of bromide in the raw water should be evaluated.

Therefore, chloride and bromide analyses was added to the protocol, beginning with the second quarter ( summer 1988) of sampling. Among the 35 facilities, bromide levels ranged from < 0.01 to 3.00 mg/ L. At the utility with the highest bromide levels there was a shift in the distribution of DBPs from the chlorinated DBPs to the brominated DBPs, resulting in DBA as the major haloacetic acid detected. While there were no clear trends of the concentrations of ions or Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 29 Draft, do not cite or quote brominated acetic acids with season in the composite analysis, DBA levels increased in the warmer months in the utility with the highest bromide levels. Some observed shifts in utilities were also seen as the result of drought conditions and saltwater intrusion.

A. 2.4.1 Seasonal Shifts in ICR Database The seasonal mean concentrations of MBA, BCA, and DBA are presented in Tables IV

11, IV 12, and IV 13, respectively. For simplicity of presentation, only the data required to conduct a Student's t test has been presented here. Additional data can be located in the ICR database ( U. S. EPA, 2000a). Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 30 Draft, do not cite or quote Table IV 11. MBA by Sample Quarter ( Quarterly Distribution System Average)

Sample Quarter MBA Surface Water Ground Water N Mean ( µ g/ L) STD ( µ g/ L) N Mean ( µ g/ L) STD ( µ g/ L)

Summer ` 97 239 0.29 0.73 92 0.21 0.79 Fall ` 97 250 0.23 0.55 86 0.21 0.60 Winter ` 98 240 0.25 1.74 101 0.18 0.53 Spring ` 98 262 0.17 0.53 105 0.14 0.46 Summer ` 98 250 0.28 0.83 104 0.09 0.30 Fall ` 98 229 0.32 1.03 93 0.13 0.51 N: Number of samples STD: Standard deviation Sample Quarter:

Summer ` 97: July, August, and September Fall ` 97: October, November, and December Winter ` 98: January, February, and March Spring ` 98: April, May, and June Summer ` 98: July, August, and September Fall ` 98: October, November, and December Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 31 Draft, do not cite or quote Table IV 12. BCA by Sample Quarter ( Quarterly Distribution System Average)

Sample Quarter BCA Surface Water Ground Water N Mean ( µ g/ L) STD ( µ g/ L) N Mean ( µ g/ L) STD ( µ g/ L)

Summer ` 97 236 4.12 3.83 92 1.60 2.54 Fall ` 97 252 3.52 2.82 88 1.40 2.09 Winter ` 98 243 3.11 2.54 103 1.48 2.02 Spring ` 98 260 3.54 2.90 104 1.33 1.99 Summer ` 98 251 3.90 3.42 103 1.55 2.11 Fall ` 98 232 3.46 2.76 94 1.46 2.17 N: Number of samples STD: Standard deviation Sample Quarter:

Summer ` 97: July, August, and September Fall ` 97: October, November, and December Winter ` 98: January, February, and March Spring ` 98: April, May, and June Summer ` 98: July, August, and September Fall ` 98: October, November, and December Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 32 Draft, do not cite or quote Table IV 13. DBA by Sample Quarter ( Quarterly Distribution System Average)

Sample Quarter DBA Surface Water Ground Water N Mean ( µ g/ L) STD ( µ g/ L) N Mean ( µ g/ L) STD ( µ g/ L)

Summer ` 97 241 1.30 2.19 92 0.89 1.92 Fall ` 97 252 1.12 2.08 88 0.88 1.39 Winter ` 98 244 0.94 2.04 103 0.81 1.33 Spring ` 98 262 0.88 1.86 104 0.67 1.30 Summer ` 98 253 1.17 2.16 104 0.80 1.53 Fall ` 98 232 1.14 2.16 93 0.88 1.41 N: Number of samples STD: Standard deviation Sample Quarter:

Summer ` 97: July, August, and September Fall ` 97: October, November, and December Winter ` 98: January, February, and March Spring ` 98: April, May, and June Summer ` 98: July, August, and September Fall ` 98: October, November, and December Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 33 Draft, do not cite or quote An examination of the data using the Student's t test showed that, based on only two seasons of analysis, the mean concentrtions of MBA in surface water were significantly higher ( p = 0.05) in the summer than in the spring ( Table IV 11). Also, based on only two seasons of analysis, the mean concentrations of BCA in surface water were higher in summer than in winter.

Aside from these exceptions, there were no consistently significant differences ( p = 0.05) in the mean concentrations of BCA or DBA between one season and other in surface water ( Table IV

13). This is in apparent contrast to the findings of Krasner et al. ( 1989), who found that DBA levels increased in the warmer months in the utility with the highest bromide levels. Seasonal variations in brominated acetic acids may be dependent on seasonal fluctuations in bromide ion concentration, which were not evaluated in this analysis. No seasonal differences in mean MBA,

BCA, or DBA concentrations in groundwater could be discerned.

B. Exposure to Sources Other Than Drinking Water MBA has been used in industry and in hospitals. Between 1981 to 1983, The National Institute of Occupational Safety ( NIOSH) conducted a survey of a sample of 4490 businesses employing nearly 1,800,000 workers ( NIOSH, 1990). Potential exposure estimates included surveyor observations of the use of MBA and trade name products known to contain MBA.

During the period from 1981 to 1983, 4874 workers were potentially exposed to MBA. The largest numbers of exposures ( 1999) occurred in the commercial printing letterpress business.

Used car dealers made up the next largest number of potential exposures ( 1723). The remainder Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 34 Draft, do not cite or quote of potential exposures, in decreasing numbers, included workers in the production of plastics ( 402), in hospitals ( 318), and in individuals working with electron tubes ( 192). Exposure levels were not reported in this survey, and more recent information on numbers of workers exposed was not available.

During the period from 1981 to 1983, there were no reported survey observations of the use of BCA, DBA, or trade name products known to contain BCA or DBA in the workplace ( NIOSH, 1990).

No data were located on exposure to MBA, BCA, or DBA in food, air, or via dermal exposure when showering or swimming.

C. Overall Exposure Only limited data on exposure to MBA, BCA, and DBA in sources other than drinking water exposure was located. Exposure to drinking water is discussed in Section IV. A.

D. Body Burden No data could be located on body burden. However, as discussed in Chapter 3, the brominated acetic acids are metabolized rapidly and are not lipophilic at physiological pH, and so would not be expected to bioaccumulate. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 35 Draft, do not cite or quote E. Summary The ICR database ( U. S. EPA, 2000a) contains extensive information on concentrations of MBA, BCA, and DBA in drinking water systems, and on how those concentrations vary with input water characteristics and treatment methods. The database contains information from six quarterly samples from 7/ 97 to 12/ 98, from approximately 300 large systems covering approximately 500 plants. The mean concentrations of BCA were 1.47 and 3.61 µ g/ L from groundwater and surface water respectively. The mean concentrations of DBA were 0.82 and 1.09 µ g/ L in groundwater and surface water, respectively. Examination of the ICR data using the Student's t test indicates that the mean concentrations of MBA, BCA, and DBA in surface water were significantly higher ( p = 0.05) than the mean concentrations of these chemicals in groundwater. In addition, the mean concentrations of BCA were significantly higher ( p = 0.05)

than the mean concentrations of DBA, which were significantly higher ( p = 0.05) than the mean MBA concentrations in both surface water and groundwater.

Examination of the ICR data using the Student's t test suggests that, although the concentrations of MBA in surface water treated with chlorine are similar to those treated with chlorine followed by chloramine. BCA and DBA concentrations were lower when free chlorine was used both in the treatment plant and the distribution system. Although ozonation appeared to significantly reduce the formation of BCA, there were no significant differences in MBA or DBA concentrations ( p = 0.05) with the use of ozone in treating surface water as compared to the common ( non ozonation) chemical disinfection processes. In addition there were no significant Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 36 Draft, do not cite or quote differences ( p = 0.05) between the two treatments using ozonation in treating surface water for MBA, BCA and DBA.

Consistent with the findings of other investigators, and the chemistry of the formation of bromoacetic acids, a regression analysis of the ICR data indicates that, with the exception of MBA in surface water, there was a significant correlation ( at   = 0.05) between influent bromide concentration and the mean concentrations of BCA and DBA in surface water and groundwater.

In addition, for a given influent bromide concentration range, the mean concentrations of BCA were generally higher ( p = 0.05) that the mean concentrations of DBA and MBA in both surface water and groundwater.

A regression analysis of the ICR data indicates that there was a significant correlation (   = 0.05) between influent TOC concentration and the mean concentrations of MBA, BCA, and DBA in surface water. This is consistent with the formation of brominated acetic acids from the reaction of humic acid and hypobromous acid, a compound formed by the reaction of bromide ion with ozone and/ or chlorine in the disinfection process. In addition, for a given influent TOC concentration range in surface water, the mean concentrations of BCA were significantly higher ( p = 0.05) than the mean concentrations of DBA, which were significantly higher ( p = 0.05) than the MBA mean concentrations.

Examination of the data using the Student's t test showed that, based on only two seasons of analysis, the mean concentrations of MBA in surface water were significantly higher ( p = 0.05)

in the summer than in the spring Also, based on only two seasons of analysis, mean concentrations of BCA in surface water were higher in summer than in winter. Aside from these Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD IV 37 Draft, do not cite or quote exceptions, there were no consistently significant differences ( p = 0.05) in the mean concentrations of MBA, BCA or DBA between one season and another in either surface water or groundwater. Seasonal variations in brominated acetic acids may be dependent on seasonal fluctuations in bromide ion concentration, which were not evaluated in this analysis.

The data on exposure to sources other than drinking water are limited, but MBA has been used in industry and in hospitals. Between 1981 to 1983, 4874 workers were potentially exposed to MBA. ( NIOSH, 1990). No data were located on exposure to MBA, BCA, or DBA in food, air,

or via dermal exposure.

No data could be located on body burden levels of MBA, BCA, or DBA.. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 1 Draft, do not cite or quote Chapter V. Health Effects in Animals The available database for the brominated acetic acids is limited and, therefore, many toxicity endpoints have not been fully explored. In recognition of this paucity of data, there is a large body of ongoing work, particularly for BCA and DBA. Preliminary results for many studies have been reported in published abstracts and are included here to provide a sense of the spectrum of effects induced by the brominated acetic acids. The full published studies would need to be evaluated for a complete understanding of the chemicals' effects and determination of the relevance of the data for quantitative risk assessment purposes.

A. Short Term Exposure Monobromoacetic acid Linder et al. ( 1994a) reported on the acute oral toxicity of MBA as part of a study on its spermatogenic effects. Five male Sprague Dawley rats per group were given single doses of 100 to 200 mg/ kg MBA ( specific dose levels not reported) by gavage in water. The LD 50 was reported as 177 mg/ kg, with a 95% ( fiducial) confidence limit range of 156 to 226 mg/ kg. Observed clinical symptoms included excess drinking, hypomobility, labored breathing, and diarrhea. No histopathologic changes were observed in either the epididymal sperm or testes of surviving animals. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 2 Draft, do not cite or quote MBA is irritating and corrosive to the human skin and mucous membranes ( NIOSH,

2000). The ability of a variety of carboxylic acids to cause skin corrosion was investigated in support of the development of a multivariate quantitative structure activity relationship ( QSAR)

analysis ( Eriksson et al., 1994). Forty five aliphatic carboxylic acids ( including MBA, DBA, and BCA) were evaluated in the QSAR analysis. Fifteen of the compounds, including MBA,

monochloroacetic acid, and dichloroacetic acid, were tested for cutaneous corrosion on adult rabbits. In these studies, the test chemical was applied to bare, shaved skin under an occlusive glass filter for one hour. The study description indicated that a 10 cm by 10 cm area on the trunk of the rabbit was shaved, but did not specify that this entire area was exposed. The observed and predicted lowest observed effect concentrations ( LOECs) for inducing corrosion for MBA were 0.2 M and 0.1 M, respectively.

No short term toxicity studies for exposure by the inhalation route were identified.

Bromochloroacetic acid Systemic toxicity was evaluated as part of a reproductive and developmental toxicityscreening assay for BCA ( NTP, 1998). As part of a range finding study, groups of male and female Sprague Dawley rats ( 6/ group) were exposed for 14 days to drinking water containing 0,

30, 100, 300, or 500 ppm BCA. The study authors reported that the estimated average doses resulting from these treatments were 0, 3, 10, 28, and 41 mg/ kg/ day. No mortality was observed and there were no treatment related differences in body weight, body weight gain, feed and water Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 3 Draft, do not cite or quote consumption, or clinical observations as compared with controls. The NOAEL for general toxicity was 41 mg/ kg/ day. A LOAEL could not be determined.

The results of the 14 day range finding study were used to select doses for a reproductive and developmental toxicity screening assay ( NTP, 1998). Sprague Dawley rats were administered BCA in their drinking water for various periods during a 35 day study period. Rats were divided into two groups of males and three groups of females. Group A males ( 10/ group) were exposed on study days 6  35 to doses of 0, 60, 200, or 600 ppm. Group B males were exposed on study days 6  31 ( 5/ group at 0, 60, or 200 ppm and 8/ group at 600 ppm ) and administered bromodeoxyuridine ( BrdU) via subcutaneously implanted pumps for 3 days prior to necropsy in order to measure cell proliferation. The female rats were grouped as follows: Group A rats (

periconception exposure group; 10/ dose at 0, 60, 200, or 600 ppm) were given BCA on study days 1  34 and were cohabitated with treated males on study days 13 18. Group B rats (

gestationalexposure group; 13/ dose) were cohabitated with treated males on study days 1  5 and exposed to BCA at doses of 0, 60, 200, or 600 ppm on gestation days ( GD) 6 to parturition. Group C (

periconception exposure group; 5/ dose group at 0, 60, 200 ppm and 8/ group at 600 ppm) were exposed to BCA on study days 1  30, cohabitated with treated males on study days 13 18, and administered BrdU via subcutaneously implanted pumps for 3 days prior to necropsy for cellproliferation assessment.

The average daily doses for male rats in both Group A and Group B were estimated by the study authors as equivalent to 0, 5, 15, and 39 mg/ kg/ day. No mortality, clinical signs of toxicity,

or body weight changes were observed. Water consumption was decreased by 21% to 34% at the Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 4 Draft, do not cite or quote high dose, presumably due to taste aversion. Clinical chemistry was evaluated in Group A males,

and the following statistically significant changes were observed: a 16% increase in albumin to globulin ratio in high dose animals; decreased alanine aminotransferase ( ALT) activity in both the mid dose ( 15% decrease) and high dose ( 20% decrease) groups; and increased albumin ( 5%

increase in the low dose group, no significant change in the mid dose group, and 9% increase in the high dose group). Clinical chemistry changes of these magnitudes are not generally considered to be toxicologically significant and the changes in the high dose group may have been secondary to dehydration. Although absolute and relative liver weights increased with increasing dose in both Group A and Group B, the relative liver weight was statistically different from controls ( 10% increase) only in the high dose group, and absolute liver weight was not significantly elevated. Gross necropsy did not reveal any major changes. No dose related increases in individual hepatocyte necrosis ( Group A) or labeling index ( Group B only) were observed.

Histopathologic examination showed an increase in cytoplasmic vacuolization of hepatocytes of treated animals in Group A. This effect was observed in all dose groups, was more prominent in the high dose group, and was absent in controls. However, cytoplasmic vacuolization was observed in both control and dosed animals of Group B, and was not increased with BCA treatment. Although the study authors suggested that the biological significance of these changes could not be determined without evaluation following longer term exposure, the highest dose was considered sufficient to induce general toxicity under the conditions of this study. In light of the questions concerning the biological significance of the clinical chemistry, liver weight and Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 5 Draft, do not cite or quote histopathology changes, 39 mg/ kg/ day ( the highest dose tested) was considered to be a marginal LOAEL in males, and the corresponding NOAEL was 15 mg/ kg/ day.

The estimated average daily doses for Group A and Group C females ( peri conception exposure groups) were 0, 6, 19, and 50 mg/ kg/ day. No mortality, clinical signs of toxicity, or body weight changes were observed in the female rats of these groups, but water consumption was decreased by 24 34% at the high dose; these findings were similar to those observed in treated male rats. Decreased water consumption was the only effect observed in Group A females.

However, Group C females exhibited a dose related increase in the incidence of renal tubular dilatation/ degeneration ( 0/ 5 in controls, 2/ 5 in the 19 mg/ kg/ day group, and 3/ 8 in the 50 mg/ kg/ day group). A statistical analysis of these data indicated that the incidences in treated groups were not statistically different from those in the controls ( p> 0.05 using the Fisher exact test); however, sample sizes were small and, thus, the power of the analysis was limited. No detailed pathology report was available to evaluate the biological significance of these histopathological changes in the kidneys. The study authors concluded that only the high dose resulted in renal toxicity. Cell proliferation analysis showed small differences between the labeling index of the treated and control groups for the kidney in Group B males and for the liver and urinary bladder in Group C females. However, these changes were not dose dependent and were not considered to be biologically significant.

Estimated average daily doses of BCA for Group B females ( gestational exposure group)

were 0, 10, 25, and 61 mg/ kg/ day. Similar to other groups, the only consistent treatment related effect in Group B females was decreased water consumption at the high dose. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 6 Draft, do not cite or quote Overall, some histopathology findings in this study suggest that the liver ( as evidenced by increased relative weight and increased cytoplasmic vacuolization in males) and kidney ( as indicated by increased renal tubular dilatation/ degeneration in females) may be target organs for BCA toxicity. Although liver histopathology was only observed in high dose Group A males,

these effects were considered to be treatment related by the study authors, yielding a marginal LOAEL of 39 mg/ kg/ day for equivocal liver effects. The study authors also concluded that the histopathological changes observed in the kidneys of Group C females, although not statistically different from controls, might be indicative of kidney toxicity. However, kidney histopathology was not corroborated by the kidney labeling index, suggesting that renal toxicity was not of sufficient severity to induce cellular proliferation and regeneration. Based on the study authors'

interpretation of the results, 39 mg/ kg/ day was selected as a LOAEL, and the corresponding NOAEL was 19 mg/ kg/ day. Effects on reproductive and developmental endpoints and determination of critical effect levels for these systems are described in Section V. C.

Parrish et al. ( 1996) tested whether the ability of brominated acetic acids to induce oxidative DNA damage was due to peroxisome proliferation. ( This study is described more fully in section V. D.) Male B6C3F1 mice ( 6/ dose group) were administered drinking water containing 0, 100, 500, or 2000 mg/ L BCA for 3 weeks. The authors did not provide information on average daily doses. Based on a default water intake value of 0.25 L/ kg/ day for male B6C3F1 mice ( U. S.

EPA, 1988), the corresponding doses were estimated to be 0, 25, 125, and 500 mg/ kg/ day.

General toxicity was assessed by measuring body weight and liver weight; these data for BCA are summarized in Table V 1. The effects of BCA on oxidative DNA damage and peroxisome Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 7 Draft, do not cite or quote proliferation were measured in the livers of male B6C3F1 mice, using the following measures: ( 1)

changes in the DNA adduct 8 hydroxy 2 deoxyguanosine ( 8 OhdG) as an indicator of oxidative stress, and( 2) changes in levels of cyanide insensitive Acyl CoA oxidase and 12 hydroxylation of lauric acid as indicators of peroxisome proliferation. An additional dose group exposed to 3000 mg/ L BCA ( 750 mg/ kg/ day) was evaluated for the Acyl CoA activity. Body weight was decreased by 8.5% at the highest dose tested. Absolute and relative liver weights were increased at the high dose by 20% and 33%, respectively. BCA had no effect on either measure of peroxisome proliferation after exposures up to 3000 mg/ L. BCA did induce oxidative DNA damage, with 8 OHdG levels in nuclear DNA of the liver significantly increased ( p< 0.05)

beginning at the lowest dose, 25 mg/ kg/ day. The level of 8 OHdG increased to a maximum of approximately 2 fold at the highest dose ( 500 mg/ kg/ day).

The absence of other measures of liver toxicity, such as histopathology or clinical chemistry results, clouds the classification of these liver weight changes as adverse. However, the accompanying increase in oxidative DNA damage suggests potentially adverse liver effects. Based on the data presented, the LOAEL for minimal liver effects was 500 mg/ kg/ day and the NOAEL was 125 mg/ kg/ day. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 8 Draft, do not cite or quote Table V 1. Body and Liver Weight Changes Induced by BCA and DBAa BCA Drinking water ( g/ L) Body weight ( g) Liver weight ( g) Relative liver weight (% body weight)

Control 27.2 ± 0.5c 1.5 ± 0.01 5.4 % ± 0.1 0.1 ( 25 mg/ kg/ day) b 26.1 ± 0.3 1.5 ± 0.1 5.8 % ± 0.4 0.5 ( 125 mg/ kg/ day) 28.3 ± 0.7 1.8 ± 0.1 6.2 % ± 0.6 2.0 ( 500 mg/ kg/ day) 24.9 ± 0.7** d 1.8 ± 0.1* 7.2% ± 0.4**

DBA Control 27.1 ± 0.5 1.5 ± 0.01 5.4 % ± 0.1 0.1 ( 25 mg/ kg/ day) 24.0 ± 0.7** 1.4 ± 0.1 5.8% ± 0.4 0.5 ( 125 mg/ kg/ day) 25.6 ± 0.8 2.1 ± 0.2** 8.0% ± 0.5**

2.0 ( 500 mg/ kg/ day) 26.1 ± 0.4 2.0 ± 0.1** 7.8% ± 0.6 Notes:

a. Adapted from Parrish et al., 1996 b. Estimated daily doses were calculated based on default drinking water values of 0.25 c. Mean ± standard error * Statistical significance: p< 0.05 ** Statistical significance: p< 0.01 The ability of a variety of carboxylic acids to cause skin corrosion was investigated using multivariate quantitative structure activity relationship QSAR ( Eriksson et al., 1994). The predicted lowest observed effect concentration ( LOEC) for corrosion for BCA was 0.7 M. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 9 Draft, do not cite or quote No short term toxicity studies for exposure to BCA by the inhalation route were identified.

Dibromoacetic acid The acute toxicity of DBA was examined as part of a study on the spermatogenic effects of this compound ( Linder et al., 1994a). Male Sprague Dawley rats ( 5/ group) were given single doses of 1000 to 2000 mg/ kg DBA ( specific dose levels not reported) by oral gavage in water.

Custom synthesized DBA of > 99% purity was used because the commercial chemical is generally contaminated with approximately 10% MBA. Surviving animals were killed 14  21 days after dosing. The oral LD 50 was 1737 mg/ kg, with a 95% ( fiducial) confidence limit range of 1411

1952 mg/ kg. Most of the animal deaths occurred within 48 hours of dosing. Observed symptoms included excess drinking, hypomobility, labored breathing, diarrhea, and ataxia. Histopathologic examination of the epididymal sperm in surviving animals showed the presence of mis shapen and degenerating sperm, as well as abnormal retention of Step 19 spermatids. Effects other than spermatotoxicity were not examined.

In another single dose spermatotoxicity study, Vetter et al. ( 1998) treated sexually mature male Crl: CD( SD) BR rats ( 4 5/ dose group) with 0, 600, or 1200 mg/ kg DBA in 10 mL/ kg deionized water. In the high dose group, signs of overt toxicity included lethargy, irregular gait,

decreased feces, ocular discharge, and dyspnea. Abnormal respiratory sounds were observed in some animals ( number of animals affected was not specified) and one animal in the high dose Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 10 Draft, do not cite or quote group died on study day 3. No overt toxicity was observed in the low dose group. No changes in measured sperm parameters ( motility, morphology, and cell membrane permeability) were reported at either dose; however, mi testes histopathology ( the presence of basophilic bodies) was observed in both dose groups. Based on the clinical findings, 1200 mg/ kg was considered to be an acute frank effects level ( FEL). The LOAEL was 600 mg/ kg for testes histopathology. and a A NOAEL could not be determined.

Parrish et al. ( 1996) tested whether the ability of brominated acetic acids to induce oxidative DNA damage was due to peroxisome proliferation. ( This study is described more fully in section V. D). Male B6C3F1 mice ( 6/ dose group) were given drinking water containing 0, 100,

500, or 2000 mg/ L DBA for 3 weeks. The authors did not provide information on average daily doses. However, based on a default water intake value of 0.25 L/ kg/ day for male B6C3F1 mice ( U. S. EPA, 1988), the corresponding daily doses were estimated to be 0, 25, 125, and 500 mg/ kg/ day, respectively. The effects of DBA on oxidative DNA damage and peroxisome proliferation were measured in the livers of male B6C3F1 mice, using the following measures: ( 1)

changes in the DNA adduct 8 hydroxy 2 deoxyguanosine ( 8 OhdG) as an indicator of oxidative stress, and ( 2) changes in levels of cyanide insensitive Acyl CoA oxidase and 12 hydroxylation of lauric acid as indicators of peroxisome proliferation. An additional dose group exposed to 3000 mg/ L DBA ( 750 mg/ kg/ day) was evaluated for the Acyl CoA activity . As part of this study,

general toxicity was assessed by measuring body weight and liver weight, as summarized in Table V 1. No dose related decrease in body weight was observed, but absolute and relative liver weights were increased at the mid and high doses. At the mid dose ( 125 mg/ kg/ day), absolute Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 11 Draft, do not cite or quote and relative liver weights were increased by 40% and 48%, respectively. At the high dose ( 500 mg/ kg/ day), absolute and relative liver weights were increased by 33% and 44%, respectively.

DBA induced Acyl CoA activity to a maximum of 3 fold following exposures up to 3000 mg/ L,

but did not induce the 12 hydroxylation of lauric acid. DBA induced oxidative DNA damage,

with 8 OHdG levels in hepatic nuclear DNA significantly increased ( p< 0.05) at the highest dose ( 500 mg/ kg/ day) to a maximum of approximately twice the control response. The absence of a clear dose response, and the lack of other measures of liver toxicity, such as histopathology or clinical chemistry, clouds the classification of the liver weight changes as adverse. However, the magnitude of the change and the accompanying increase in oxidative DNA damage suggests potentially adverse liver effects. Based on the data presented, the LOAEL for minimal liver effects was 125 mg/ kg/ day. The NOAEL was 25 mg/ kg/ day.

As part of a male reproductive study, Linder et al. ( 1995) administered daily gavage doses of 0 or 250 mg/ kg DBA to male Sprague Dawley rats ( 10/ dose group). Dosing was terminated after 42 days because severe toxic effects, including labored breathing, light tremor, difficulty moving the hind limbs, and significant weight loss, developed. In a subsequent study, male rats ( 10/ dose group) were given 0, 2, 10, or 50 mg/ kg/ day DBA by oral gavage for 31 or 79 days. The only observed effect was a slight decrease in body weight in the 50 mg/ kg/ day group on day 79 to approximately 95% of controls. Based on the findings of both studies, the FEL for general toxicity was 250 mg/ kg/ day and the NOAEL was 50 mg/ kg/ day. Adverse effect levels for reproductive endpoints are described in Section V. C. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 12 Draft, do not cite or quote NTP ( 1999) evaluated the immunotoxicity of DBA in female B6C3F1 mice ( 8/ dose group)

exposed to drinking water containing 0, 125, 250, 500, 1000, or 2000 mg/ L DBA for 28 days.

Four separate studies were conducted and different general and immunologic endpoints were examined in each study. Studies 1 3 investigated selected immunologic endpoints and bodyweight changes; body and organ weights, hematology, and gross pathology were examined in Study 4. Key immunotoxicity responses are presented in Table V 2, and body and organ weights are summarized in Table V 3. The study authors did not estimate DBA daily doses resulting from drinking water exposures; however, the average daily doses could be calculated based on waterconsumption and body weight data provided in the study report. DBA dose ranges were similar across the four studies and are presented below in conjunction with the experimental findings for each study. No significant differences ( p< 0.05) in drinking water consumption among dosed groups and no clinical signs of overt toxicity were observed in any of the studies. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 13 Draft, do not cite or quote Table V 2. Immunotoxicity of DBA in Female B6C3F1 mice a Study Number/ Endpoint Dose ( mg/ kg/ day) b Study # 1 0 19 39 73 150 285 Spleen cell number x 107 15.97 ± 0.50c 16.69 ± 0.51 17.55 ± 1.02 19.13 ± 0.58d** 18.49 ± 0.46* 18.45 ± 0.39*

Spleen Macrophages (% of cells) 2.8 ± 0.2 3.0 ± 0.2 2.9 ± 0.2 3.5 ± 0.3 4.2 ± 0.1** 4.5 ± 0.2**

Natural Killer cell lytic activity ( LU/ 10 7 cells) e 12 ± 1 14 ± 2 15 ± 1 16 ± 1* 20 ± 1** 24 ± 2**

Mixed leukocyte response ( CPM/ 105 spleen cells

responders) 633 ± 69 834 ± 160 863 ± 126 781 ± 52 693 ± 50 851 ± 69 Study # 2 0 20 38 70 143 280 IgM antibody forming colonies ( AFC/ 106 spleen cells) 1958 ± 145 1767 ± 62 1590 ± 149 1333 ± 89** 1251 ± 120** 985 ± 69**

Serum IgM titer ( OD) g 103 ± 7 100 ± 9 131 ± 19 102 ± 12 97 ± 5 85 ± 5 Study # 4 0 14 33 68 132 236 Macrophage Activating Factor (% suppression without stimulation)

(% suppression with stimulation) 29.08 ± 2.77 86.80 ± 4.26 19.61 ± 5.80 92.02 ± 2.04 16.45 ± 6.39 94.17 ± 2.16 10.22 ± 5.86*

92.01 ± 4.01 15.39 ± 2.50 93.62 ± 2.84 28.58 ± 3.62 89.05 ± 2.73 a. Adapted from NTP, 1999 b. Doses were estimated based on drinking water concentrations and water intake and body weight data provided c. Lytic unit ( LU) = the number of splenocytes required to kill 10% of the target cells.

d. CPM = counts per minute based on 3H thymidine incorporation in responder cells.

e. IgM titer based on enzyme linked immunosorbant assay.

* Statistical significance: p< 0.05 ** Statistical significance: p< 0.01 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 14 Draft, do not cite or quote Table V 3. General toxicity of DBA in Female B6C3F1 micea Estimated Dose ( mg/ kg/ day) b 0 14 33 68 132 236 Body weight ( g) 23.7 ± 0.4 24.0 ± 0.4 24.7 ± 0.6 24.8 ± 0.7 24.6 ± 0.4 23.0 ± 0.5 Liver weight ( mg) (% of body weight) 1071 ± 25 4.5 ± 0.1 1183 ± 32* 4.9 ± 0.1* 1293 ± 41** 5.2 ± 0.1** 1386 ± 62** 5.6 ± 0.1 1479 ± 14** 6.0 ± 0.1** 1567 ± 41** 6.8 ± 0.1**

Kidney weight ( mg) (% of body weight) 287 ± 15 1.21 ± 0.07 298 ± 6 1.24 ± 0.01 305 ± 5* 1.23 ± 0.02 312 ± 9* 1.26 ± 0.02 317 ± 6 * 1.29 ± 0.01* 335 ± 6* 1.46 ± 0.01a*

Spleen weight ( mg) (% body weight) 77 ± 3 0.328 ± 0.015 84 ± 3 0.349 ± 0.013 86 ± 4 0.350 ± 0.018 92 ± 4* 0.370 ± 0.015 88 ± 3 0.359 ± 0.011 96 ± 4** 0.418 ± 0.014**

Thymus weight ( mg) (% body weight) 67 ± 4 0.280 ± 0.014 60 ± 4 0.251 ± 0.017 65 ± 3 0.265 ± 0.010 63 ± 4 0.252 ± 0.011 58 ± 2 0.236 ± 0.007 43 ± 3** 0.189 ± 0.013**

Reticulocyte count (%) 4.01 ± 0.11 4.24 ± 0.27 4.70 ± 0.49 4.61 ± 0.16* 4.50 ± 0.16* 5.21 ± 0.25**

Notes:

a. Adapted from NTP, 1999 b. Doses were estimated based on drinking water concentrations and water intake and body weight data provided in the report.

c. Mean ± Standard Error.

* Statistical significance: p< 0.05 ** Statistical significance: p< 0.01 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 15 Draft, do not cite or quote In Study 1, doses estimated using group specific body weights and water consumption rates were 0, 19, 39, 73, 150, and 285 mg/ kg/ day. A statistically significant 8% decrease in terminal body weight was observed in the high dose group. Spleen cell number was significantly increased above controls ( p< 0.05) at 73 mg/ kg/ day and higher, but there was no dose response.

At 73 mg/ kg/ day, spleen cell number was elevated approximately 20% above controls, whereas at both 150 and 285 mg/ kg/ day, the increase was approximately 12%. With the notable exception of macrophages, the increase in the absolute number of most spleen cell types paralleled the increase in total number of spleen cells; thus the percentage of each cell type was generally unaltered. In contrast, spleen macrophages increased in a dose dependent manner to 50%, 77%, and 91%

above controls at 73, 150, and 285 mg/ kg/ day, respectively, ( 500, 1000, and 2000 mg/ L,

respectively) An increase in natural killer ( NK) cell lytic activity was also observed at the three highest DBA dose groups ( p< 0.05) when expressed as specific activity; a significant increase was observed in the four highest dose groups ( i. e., 39 mg/ kg/ day and above) when expressed as totalspleen activity. NK cell activity was maximal at the highest dose, showing an increase of 100%

when measured as specific activity and of 143% when measured as total spleen activity. However,

the significance of the treatment related changes in NK cell activity is unclear, because the positive control used in the experiment produced a significant decrease in NK cell activity. DBA treatment had no effect on mixed leukocyte response, which measures proliferative response of splenic leukocytes from treated animals to allogenic lymphocytes ( i. e., lymphocytic cells from a genetically distinct strain of the same species) of DBA/ 2 mice. Overall, the results of Study 1 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 16 Draft, do not cite or quote demonstrated an increase in several measures of cellular immunity, with statistically significant effects generally occurring at doses of 73 mg/ kg/ day and higher. The toxicological significance of the findings of Study 1 are unclear and are discussed in more detail later in this section, using a weight of evidence based on the results of all four studies.

In Study 2, estimated average daily doses were 0, 20, 38, 70, 143, and 280 mg/ kg/ day. No significant effects of DBA treatment on body weight were observed. In contrast to Study 1, no increase in spleen cell number was observed. A statistically significant ( p< 0.05) dose dependent decrease in spleen IgM antibody forming cell response to sheep erythrocytes was observed beginning

70 mg/ kg/ day. There was no change, however, in serum IgM titer to sheep erythrocytes. The study authors noted that the lack of concordance between these two measures of humoral response was not uncommon. They suggested that discordance might arise from the fact that the IgM antibody forming cell response is a specific measure of immune response in the spleen, whereas the IgM titer measures systemic humoral immunity and, thus, would reflect changes in both bone marrow and lymph node antibody production, in addition to antibody production in the spleen. Therefore, the IgM assay might not be a sensitive measure of toxicity for substances whose target organ of toxicity is only the spleen.

Study 3 evaluated macrophage activation. The estimated average daily doses were 0, 16,

35, 69, 134, and 229 mg/ kg/ day. No significant effects on body weight were observed. To assess macrophage activation, peritoneal macrophages were stimulated by treatment with a combination of gamma interferon and lipopolysaccharide, and their ability to kill or inhibit the growth of Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 17 Draft, do not cite or quote B16F10 tumor cells was measured. No clear dose dependent effects of DBA treatment on B16F10 tumor cell growth were observed with or without macrophage activation.

Body weight, organ weight, gross pathology, and hematology were evaluated in Study 4 ( Table V 3). The estimated average daily doses were 0, 14, 33, 68, 132, and 236 mg/ kg/ day.

Based on pooled body weight data for all studies, no significant change in terminal body weight was observed. However, body weight gain was decreased by 40% at the high dose. Statistically significant changes in organ weight were also reported. Thymus weight was significantly decreased only at the high dose. Absolute spleen weight was elevated at all the doses, but was statistically different from controls ( p< 0.05) only at 68 mg/ kg/ day and 236 mg/ kg/ day ( 19% and 24% increase, respectively, as compared with controls). Although absolute spleen weight was also elevated at 132 mg/ kg/ day ( 14% increase relative to controls), this difference was not statistically significant, indicating an ambiguous dose response. Relative spleen weight was statistically significantly increased only at the high dose, although there was an increasing trend with increasing dose. The absolute and relative liver weight increases were dose dependent and were significantly elevated at all doses tested (

14/ mg/ kg/ day). Relative liver weight increased with increasing dose to 9%, 16%, 24%, 33%, and 51% above control values for each of the dose groups. Kidney weight was also statistically increased in a dose dependent manner, beginning at 33 mg/ kg/ day for absolute weight ( 6% increase) and 132 mg/ kg/ day for relative kidney weight ( 7% increase). With the exception of a dose related increase in reticulocytes that achieved statistical significance at

68 mg/ kg/ day, no biologically significant hematology parameters were Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 18 Draft, do not cite or quote affected by treatment. It should be noted, however, that increases in reticulocytes are generally not considered adverse. No gross pathological lesions were identified. Although a histopathologic examination was conducted for Study 4, no results were provided. Therefore, treatment histopathology could not be assessed.

Overall, exposure to DBA in drinking water for 28 days resulted in body and organweight changes and alterations in several indicators of immunologic response. General toxicity indicators included decreased body weight gain in the highest dose group tested in all four studies, increased liver weights ( both absolute and relative) at all doses tested

14 mg/ kg/ day),

and increased absolute ( 33 mg/ kg/ day and above) and relative ( 132 mg/ kg/ day and above) kidney weights. However, the absence of supporting clinical chemistry and/ or histopathologic data precludes determination of liver and kidney effects as adverse. Therefore, the observed increase in liver weight was not selected as the critical effect. In Study 4, thymus weights were decreased and spleen weights ( absolute and relative) were increased at the highest dose tested ( 236 mg/ kg/ day).

Spleen weight was also increased in a dose dependent manner at lower doses; however, the doseresponse was not clear cut. Further, spleen weights were not increased in Study 2 at similar doses.

Therefore, the toxicologic significance of this finding remains unclear. A number of measures of cellular and humoral immunity were altered in a dose dependent manner by DBA treatment,

beginning in the animals treated with 500 mg/ L DBA in drinking water ( equivalent to 68 73 mg/ kg/ day, depending on the study). Spleen cell number was increased in Study 1 but was not elevated in Study 2, limiting interpretation of these findings. As previously discussed, NK cell Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 19 Draft, do not cite or quote lytic activity ( expressed as either specific activity or total spleen activity) was increased in DBAtreated animals in Study 2, but was decreased by exposure to a positive control in the same experiment. Therefore, these findings are inconclusive. The number of spleen macrophages,

however, increased statistically in a dose dependent manner a

500 mg/ L ( 73 mg/ kg/ day) DBA,

indicating an immunotoxic response in this target organ. In Study 3, exposure to 500 mg/ L ( 70 mg/ kg/ day) DBA and above also decreased spleen IgM antibody forming cell response, which represents a depression in humoral immunity. The decrease in spleen IgM antibody forming cell response and the increase in spleen macrophages are the clearest indicators of an immunotoxic effect. Based on these findings, the LOAEL for immunotoxicity under the conditions of this study was 70 mg/ kg/ day, and the NOAEL was 38 mg/ kg/ day.

No short term toxicity studies for exposure to DBA by the inhalation or dermal route were identified.

B. Long Term Exposure Monobromoacetic acid No long term toxicity studies for any exposure route were identified. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 20 Draft, do not cite or quote Bromochloroacetic acid In a published abstract, Stauber et al. ( 1995) reported on preliminary data suggesting that BCA induces liver tumors in B6C3F1 mice. Noncancer liver effects included glycogen accumulation and hepatocyte vacuolization. No long term toxicity studies for any exposure route were identified in the peer reviewed literature. However, BCA is currently undergoing 90 day subchronic and 2 year chronic bioassays ( NTP, 2000a).

Dibromoacetic acid In published abstracts, So and Bull ( 1995) reported that DBA increased the formation of aberrant crypt foci in the colon of treated rats, and Stauber et al. ( 1995) reported on preliminary data suggesting that DBA induces liver tumors in B6C3F1 mice. The effects of longer term DBA exposure on noncancer endpoints were not described.

Phillips et al. ( 2002, published abstract) examined the neurotoxicity of DBA in adolescent ( 28 day old) male and female F344 rats ( 12/ sex/ dose) given DBA in drinking water at concentrations of 0, 200, 600, or 1500 mg/ L ( mean doses calculated by the authors as 0, 20, 72,

and 161 mg/ kg/ day) for 6 months. In both sexes, body weight was significantly depressed in the high dose group but overall health status was unaltered. A neurobehavioral test battery was administered to all animals at 1, 2, 4, and 6 months. Dose dependent neuromuscular toxicity, Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 21 Draft, do not cite or quote characterized by mild gait abnormalities, hypotonia, and decreased forelimb and hindlimb grip strength, was observed in both sexes. Sensorimotor responsiveness, as measured by responses to a tail pinch and click, was reduced at all doses, but did not progress with continued exposure to DBA. Decreased motor activity was noted in both sexes in the high dose group, whereas a chest clasping response was only observed in high dose females. Neuropathologic examination revealed significant myelin sheath degeneration, axonal swelling, and axonal degeneration in the lateral and ventral areas of the spinal cord white matter in the high dose group. In the mid and high dose groups, small numbers of swollen, eosinophilic or faintly basophilic, and occasionally vacuolated neurons were observed in the spinal cord gray matter, and appeared to represent axonal degeneration. Neuropathologic examination has not yet been conducted in the low dose group.

No treatment related neuropathology was noted in the eyes, peripheral nerves, peripheral ganglia,

or brain. Based on neurobehavioral abnormalities, the LOAEL was 20 mg/ kg/ day, the lowest dose tested, and a NOAEL could not be determined.

No long term systemic toxicity studies for any exposure route were identified in the peerreviewed literature. However, DBA is currently undergoing 90 day subchronic and 2 year chronic bioassays ( NTP, 2000b). Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 22 Draft, do not cite or quote C. Reproductive and Developmental Effects Much of the emphasis on reproductive toxicity of brominated acetic acids has focused on the potential spermatotoxicity of these compounds. Therefore, to enhance the reader's evaluation of the following study descriptions, a short summary of spermatogenesis relevant to assessing male reproductive toxicity is provided here. For additional information the reader is referred to Zenick et al. ( 1994), from which the following summary text was largely developed.

The development of mature sperm ( spermatogenesis) is a multiple step process that begins within the seminiferous tubules in the testes and is completed with the movement of spermatids through the caput, corpus, and cauda epididymis for further functional development and transport to the vas deferens. The seminiferous tubule is comprised of spermatogenic cells and support cells such as Sertoli cells. The spermatogenic cells undergo a well defined step wise maturation process. As the cells mature, they move from the basal membrane of the seminiferous tubule until eventual release from the supporting Sertoli cells into the tubule lumen. The release of the spermatids from the Sertoli cells is termed spermiation. The resulting sperm cells are transported from the seminiferous tubule lumen to the epididymis where they undergo further functional development, including the acquisition of motility and reproductive viability.

The maturation of the spermatogenic cells in the seminiferous tubules occurs through a series of phases and the increasingly mature spermatogenic cells sequentially develop into spermatogonia, spermatocytes, and spermatids. Each of these three major developmental phases Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 23 Draft, do not cite or quote includes a series of smaller developmental steps. For example, for the rat there are 19 developmental phases or " steps" for spermatids. Within the seminiferous tubule, spermatogenic cells in various steps of development are found in distinct and repeatable associations. Each common set of associations is called a Stage of the seminiferous epithelium. For example, a cross section of a rat seminiferous tubule at Stage VIII would typically contain PI and P spermatocytes, and Step 8 and 19 spermatids. Thus, perturbations in normal cell associations can serve as an indication of spermatotoxicity. The time period between the appearance of the same Stage at a given point in the epithelium is called the cycle length of the seminiferous epithelium.

The stages and cycle length vary across species, but are nearly constant in the same species. The consistent length of time for spermatogenesis can be useful for identifying targets for spermatotoxicity, particularly for single dose studies. For example, information about potential targets of toxicity can often be gained by determining the amount of time from the time of exposure to a toxicant to the appearance of adverse effects by tracing back to the phase of development that the affected sperm was in at the time of exposure.

Spermatogenesis can also be perturbed through toxicity directed at cell populations that aid in the maturation of the spermatogenic cells. Sertoli cells provide support functions and developmental regulation of sperm cells, and thus can be an important target for toxicity. Sertoli cells play important roles in endocrine regulation of spermatogenesis, provide a protective semipermeable barrier for the seminiferous tubules, and provide direct support for development of Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 24 Draft, do not cite or quote spermatogenic cells through phagocytosis. For example, the Sertoli cells phagocytize a portion of the cytoplasm and overlying membrane of the spermatid to form a residual body at spermiation.

Monobromoacetic acid Linder et al. ( 1994a) reported the results of acute toxicity and acute spermatotoxicity studies of MBA. In the spermatotoxicity study, male Sprague Dawley rats ( 8/ group) were given a single dose of either 0 or 100 mg/ kg MBA in a volume of 5 mL/ kg in water, and were sacrificed 2 or 14 days after dosing. The selected single dose of 100 mg/ kg was an approximate LD 01, and was chosen to provide a relatively high dose with a minimal likelihood of mortality. Measures of male reproductive toxicity included reproductive organ weights, sperm counts, sperm morphology,

sperm motility, and histopathological examination of the seminiferous tubules. No adverse effects were observed in the single dose study; therefore, a repeated dosing protocol experiment was also conducted. Groups of eight rats were given daily doses of 0 or 25 mg/ kg/ day MBA in water for 14 days, and were sacrificed 24 hours after the last dose. MBA also failed to induce any spermatotoxicity in this repeated dosing study.

In a published abstract, Randall et al. ( 1991) reported on the reproductive and developmental toxicity of MBA. Pregnant Long Evans rats were given oral gavage doses of 0,

25, 50, or 100 mg/ kg/ day MBA in distilled water on gestation days 6 15. In the high dose group,

maternal weight gain was reduced and one dam died. No effects on reproduction were observed. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 25 Draft, do not cite or quote Several developmental effects were noted in the high dose group, including decreased size of live fetuses ( the affected measure of size was not provided in the study summary) and increased incidence of soft tissue malformations, most of which were cardiovascular and craniofacial. Based on the limited data provided in the abstract, the LOAEL for both maternal and developmental effects is 100 mg/ kg/ day and the NOAEL is 50 mg/ kg/ day.

No reproductive or developmental toxicity for MBA was identified following dosing by the inhalation or dermal routes.

Bromochloroacetic acid NTP ( 1998) reported the results of a short term reproductive and developmental toxicityscreening protocol for BCA. Details of the protocol for this study are provided in Section V. A.

Briefly, male and female Sprague Dawley rats were administered 0, 60, 200, or 600 ppm BCA in their drinking water for various periods during a 35 day study period. The rats were divided into two groups of males and three groups of females. Group A males ( 10/ group) were exposed on study days 6 35. Group B males were exposed on study days 6 31 to 0, 60, or 200 ppm ( 5/ group)

or to 600 ppm ( 8/ group), and were subsequently treated with BrdU for 3 days prior to necropsy to evaluate cell proliferation. The study authors reported that the estimated average doses for males were 0, 5, 15, and 39 mg/ kg/ day. Male rats were evaluated for clinical pathology, organ weights, sperm analysis ( group A only), and histopathology. No consistent treatment related Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 26 Draft, do not cite or quote effects on epididymal sperm measures, spermatid head counts, sperm morphology, or sperm motility were observed at necropsy.

Among females, Group A ( 10/ dose group) rats were treated with BCA on study days 1 34 and cohabitated with treated males on study days 13 18. Group B ( 13/ dose group) females were cohabitated with treated males on study days 1 5 and exposed on GD 6 through parturition.

Group C females ( 5/ group at 0, 60, 200 ppm, and 8 animals at 600 ppm) were exposed to a dosing regime similar to that of Group A, but were removed from treatment on study day 30 and subsequently administered BrdU to assess target tissue cell proliferation. Thus, the treatment protocol for Group A resulted in exposure for 12 days prior to mating and from GD 1 16 or 1 21,

depending on the number of days of cohabitation required for mating. The treatment protocol for Group C females resulted in 12 days of premating exposure and exposure beginning on GD1 and continuing through GD 12 16, depending on the number of days required for mating. Both groups were evaluated for indices of mating and fertility and number of corpora lutea, live and dead fetuses, and implantation sites. The study authors reported that the estimated average daily doses resulting for both groups were 0, 6, 19, and 50 mg/ kg/ day. No effects were observed on the mating index ( number of females with vaginal sperm / number of cohabitating pairs), pregnancy index ( number of fertile pairs / number of cohabitating pairs) or fertility index ( number of fertile pairs / number of females with vaginal sperm). Due to the limited number of pregnancies evaluated and the similar dosing protocols, reproductive outcome data were pooled for Groups A and C females. Analysis of the combined results revealed statistically significant decreases of up to Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 27 Draft, do not cite or quote 70% in the number of live fetuses per litter and up to 75% in total implants per litter, as compared with controls. Pre implantation losses increased up to 249% of controls in the combined highdose group, but this result was not statistically significant. A summary of selected endpoints for the combined Group A and C female data is provided in Table V 4.

Statistically significant treatment related effects by individual groups included a 16%

decrease in total implants per litter in the 600 ppm Group A females and a 50% decrease in number of live fetuses per litter in 600 ppm Group C females. A number of other outcomes for either Group A or C were reported to be adversely altered by BCA treatment but did not differ statistically from controls: ( 1) post implantation losses were increased in the 600 ppm Group C females; ( 2) pre implantation losses were increased in the 600 ppm Group A females and all dosed groups in Group C; ( 3) an increase in total resorptions was observed in the 600 ppm Group C females; and ( 4) decreased total implants per litter occurred in all dosed groups in Group C. The study author noted that the reason that many of these adverse outcomes lacked statistical significance may have been due to the small number of pregnancies ( N = 2 to 5) per treatment group evaluated in this screening protocol. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 28 Draft, do not cite or quote Table V 4. Reproductive and Developmental Toxicity of BCA following Peri conception Exposure ( Combined Data for Female Groups A and C) a Estimated Dose ( mg/ kg/ day)

Parameter 0 6 19 50 Live fetuses per litter (% of controls) 14.9 ± 1.05b ( 100%) 12.2 ± 1.36 ( 82%) 13.2 ± 0.63 ( 89%) 10.5 ± 1.14* ( 70%)

Total implants per litterc (% of controls) 16.4 ± 1.18 ( 100%) 13.7 ± 1.41 ( 84%) 14.6 ± 0.73 ( 89%) 12.3 ± 1.29* ( 75%)

% Pre implantation lossd (% of controls) 12.92 ± 6.24 ( 100%) 18.52 ± 6.81 ( 143%) 15.27 ± 4.18 ( 118%) 32.17 ± 7.48 ( 249%)

% Post implantation losse (% of controls) 8.67 ± 1.62 ( 100%) 11.50 ± 3.10 ( 133%) 8.65 ± 3.16 ( 100%) 12.68 ± 3.88 ( 146%)

Total resorptionsf (% of controls) 1.5 ± 0.27 ( 100%) 1.5 ± 0.41 ( 100%) 1.4 ± 0.54 ( 93%) 1.8 ± 0.62 ( 120%)

Dead fetuses per litter ( Number of pregnant females) 0.0 ± 0.00 ( 10) 0.0 ± 0.00 ( 11) 0.0 ± 0.00 ( 11) 0.0 ± 0.00 ( 12)

Notes:

a. Adapted from NTP, 1998.

b. Mean ± standard error.

c. Total implants = number of viable fetuses + early resorptions + late resorptions + dead fetuses d. % Pre implantation loss = [( corpora lutea  total implants) / corpora lutea] x 100 e. % Post implantation loss = [( resorptions + dead fetuses) / total implants] x 100 f. Total resorptions = early resorptions + late resorptions * Statistical significance: p< 0.05 Group B females ( cohabitation with males on study days 1 to 5 and exposure on GD 6 to parturition) were assessed for maternal body weight; feed and water consumption; number of uterine implantations; number, weight, and anogenital distance of pups; and evaluation of fetal heart and brain for soft tissue malformations. The study authors reported that the estimated Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 29 Draft, do not cite or quote average daily doses of BCA were 0, 10, 25, or 61 mg/ kg/ day for this group. The only observed effect was an increase in post implantation losses in all groups, which decreased with increasing dose, although all losses were elevated relative to controls ( 303%, 190%, and 184% of the control value in the 60, 200, and 600 ppm groups, respectively). In addition, total resorptions were increased to 200%, 137%, and 137% of controls in the 60, 200, and 600 ppm groups,

respectively. None of the effects were statistically different from controls, and the negative doseresponse makes it difficult to assess the biological significance of the findings. No treatmentrelated effects were observed in soft tissue examination ( heart and brain) of the fetuses.

Evaluation of the total data set of both significant and non significant effects suggested to the authors that BCA adversely affected the ability of females to conceive and carry a full litter to term. The effects of BCA appear to be particularly relevant for early gestation, as demonstrated by significantly increased pre implantation losses and decreased total implants per litter, and nonsignificant but elevated post implantation losses and increased number of resorptions.

Determination of a LOAEL and NOAEL for this study is undermined by the small sample sizes used in the screening protocol and the low number of pregnancies per dose group. Nonetheless, a number of reproductive and development effects of significant severity were reported in all dose groups. Based on biologically relevant changes that were statistically different from control values, the LOAEL for reproductive and developmental effects ( reduced implants per litter and live fetuses per litter) was 50 mg/ kg/ day ( high dose group) and the NOAEL was 19 mg/ kg/ day ( mid dose group). It should be noted, however, that the LOAEL and NOAEL might have been Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 30 Draft, do not cite or quote significantly lower if the statistical power of these experiments had been increased by the use of a larger sample size. As discussed in Section V. A., the high dose was a marginal LOAEL, and 19 mg/ kg/ day was a NOAEL for maternal toxicity.

The effects of BCA on reproduction in male mice have also been evaluated following oral gavage dosing. Luft et al. ( 2000) reported in an abstract on a study in which male C57BL/ 6 mice ( 12 mice/ group) were administered daily gavage doses of 0, 8, 24, 72, or 216 mg/ kg BCA for 14 days. After 14 days, 5 mice/ group were necropsied for histopathological examination of the testes, epididymis, and seminal vesicles. The remaining 7 males were used in a 40 day breeding assay to evaluate the effects of BCA treatment on fertility. Coital plug positive females ( presumably untreated) were replaced daily and uteri were dissected 14 days later; the numbers of implantations, resorptions, and fetuses were determined. No effects on body weight or reproductive organ weights were observed for any of the dose groups. The results of histopathologic examination of the male reproductive tissues were not reported in the abstract.

However, BCA treatment with 72 or 216 mg/ kg/ day resulted in adverse reproductive performance, but only during the first 10 days following treatment ( data not shown). Adverse measures of reproductive outcome included statistically significant decreases in both of the dose groups for ( 1) mean number of litters per male ( 1.1 for both dose groups compared to 3 for controls); ( 2) percentage of litters per mated female as measured by the percent of plug positive females that became pregnant ( 36% and 30% in the 72 and 216 mg/ kg/ day groups, respectively,

as compared to 68% for controls); and ( 3) total number of fetuses per male ( 10 and 9 in the 72 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 31 Draft, do not cite or quote and 215 mg/ kg/ day groups as compared with 27 for controls). There was no difference in the number of coital plugs, suggesting that treatment did not result in adverse behavioral effects on mating. The number of fetuses per litter, number of resorptions, and number of terata were also unaltered, indicating that, under the conditions of this study, adverse reproductive effects in male mice did not induce developmental toxicity. This study appears to have identified a LOAEL for decreased male fertility of 72 mg/ kg/ day and a NOAEL of 24 mg/ kg/ day, but a definitive conclusion would require a review of the full study.

Klinefelter et al. ( 2002a) administered BCA in a dose range finding study ( dissolved in deionized water and pH adjusted to 6.5) by gavage to adult male Sprague Dawley rats ( 12/ dose)

at doses of 0, 24, 72, or 216 mg/ kg/ day for 14 days. The doses were selected to represent the BCA molar equivalents of 0, 30, 90 and 270 mg/ kg/ day DBA, previously tested in the same laboratory ( Linder et al., 1994). Endpoints assessed included body weight; testes, epididymes, and seminal vesicle weights; ex vivo assessment of testosterone production; and serum levels of testosterone, luteinizing hormone ( LH), follicle stimulating hormone ( FSH), and prolactin. Sperm motility, sperm morphology ( cauda and caput), and sperm counts ( testicular sperm head count and epididymal sperm counts) were also evaluated. Testis sections were examined by light microscopy for delayed spermiation, formation of atypical residual bodies, and germ cell depletion.

Body weight was significantly decreased in the highest dose group. Testis, epididymis, and seminal vesicle weights were unaffected by BCA treatment. While spermatid numbers were not Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 32 Draft, do not cite or quote altered by BCA exposure, a significant dose related decline in epididymal sperm reserves was observed at 72 and 216 mg/ kg/ day, with the effect on cauda epididymal sperm being more severe than on caput epididymal sperm. Dose related decreases in serum LH, FSH, and prolactin were noted in all dosed groups, with statistical significance occurring in the two highest dose groups.

No effects on testis sperm production or serum testosterone were observed.

The percentage of motile and progressively motile cauda sperm decreased in a doserelated fashion, achieving significance in the two highest dose groups. Sperm motion parameters ( i. e., velocity and linearity) were similarly affected. A dose dependent reduction in the percentage of morphologically normal cauda and caput epididymal sperm was also observed. For cauda sperm, the percent normal sperm decreased to 33% in the 216 mg/ kg/ day group, as compared with 98.3% in controls. A similar decrease occurred in caput sperm, with the percent normal sperm being 31.2% in the 216 mg/ kg/ day dose group, as compared with 94.8% in controls. Caput epididymal sperm abnormalities were characterized by an increased number of sperm with misshapen heads or tail defects, whereas cauda sperm abnormalities consisted mainly of an increased number of isolated heads. Histological evaluation of the testis showed a dose related increase ( statistically significant in the two highest dose groups) in the number of Step 19 spermatids retained in Stage X and XI of the spermatogenic cycle. Other findings included a doserelated increase in the number and size of atypical residual bodies in Stages X and XI ( not quantified) and a shift in localization of these bodies, from basal migration to luminal release, with Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 33 Draft, do not cite or quote increasing BCA dose. According to the study authors, the LOAEL for altered spermiation in this study was 24 mg/ kg/ day, the lowest dose tested, and a NOAEL could not be determined.

In a subsequent definitive study by the same authors ( Klinefelter et al., 2002b), adult male Sprague Dawley rats ( 10/ dose) were administered 14 daily gavage doses of BCA ( dissolved in deionized water and pH adjusted) of 0, 8, 24, or 72 mg/ kg/ day. End points evaluated were the same as those assessed in the previous study. Additionally, sperm protein was extracted and analyzed, and a fertility assessment was conducted via in utero insemination of untreated females with sperm from treated males.

For the fertility assessment, the estrus cyclicity of a cohort of females was synchronized by administering a subcutaneous injection of an luteinizing hormone releasing hormone ( LHRH)

agonist at 115 hours prior to insemination. At the beginning of the dark cycle following proestrus,

each female was paired with a sexually experienced vasectomized male for 30 minutes. Receptive females ( as indicated by the presence of a copulatory plug) were subsequently anaesthetized, and epididymal sperm from treated males were injected into each uterine horn at an amount ( 5 x 106)

that results in approximately 75% fertility in control animals. The sperm from a single male was used to inseminate a single female. Inseminated females were sacrificed 9 days following treatment, and implanted embryos and corpora lutea of pregnancy were counted. Male fertility was expressed as a percentage equivalent to the number of implants/ corpora lutea x 100.

No treatment related changes in body weight, testes weight, and the weight of the seminal vesicles were observed. However, in contrast with the previous study conducted by the same Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 34 Draft, do not cite or quote authors, epididymal weights were reduced at 72 mg/ kg/ day, and there were no differences between treated and control groups in any of the hormonal measurements. Sperm motion parameters were consistently altered by BCA exposure. Although the percentage of motile sperm was only decreased in the high dose group ( 72 mg/ kg/ day), progressive sperm motility was decreased at all doses tested. Altered sperm morphology was only observed at 72 mg/ kg/ day;

abnormalities in both cauda and caput sperm were similar to those observed in the earlier study,

with the cauda sperm showing increased incidences of sperm with tail defects and the caput sperm showing increased incidences of sperm with isolated heads. In utero insemination of untreated females with the cauda epididymal sperm from treated males showed a significant reduction in fertility at all doses, but no dose response. Fertility rates in the 8, 24, and 72 mg/ kg/ day groups were 33%, 44%, and 37%, respectively, as compared with 75% in control animals. The LOAEL for this study was 8 mg/ kg/ day, the lowest dose tested, and a NOAEL could not be determined.

In the sperm protein extraction phase of the study, two dimensional evaluation of 120 proteins showed significant reductions in two proteins, SP22 and SP9. The shape of the doseresponse curve for SP22 paralleled the reduction in fertility, whereas that for SP9 did not. The Pearson correlation coefficient was 0.53 ( p < 0.001) for SP22 and fertility, and 0.23 for SP9 and fertility. When the data were fitted to a non linear threshold response model, the resulting correlation coefficient ( r2) for SP22 and fertility was 0.843. The study authors concluded that BCA, like DBA, is capable of perturbing spermatogenesis and fertility, and that SP22 appears to be useful as a sperm biomarker of fertility. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 35 Draft, do not cite or quote No reproductive or developmental toxicity studies for BCA were identified following dosing by the inhalation or dermal routes.

Dibromoacetic acid There has been considerable interest in the male reproductive effects of DBA, in part because its chlorinated analog, dichloroacetic acid ( DCA), is known to be a male reproductive toxicant. Linder et al. ( 1994a) reported the results of acute toxicity and acute spermatotoxicity studies of DBA. A single dose protocol was used to identify stages of spermatogenesis that might be impacted by DBA. In the spermatotoxicity study, male Sprague Dawley rats ( 8/ group) were administered a single gavage dose of 0 or 1250 mg/ kg DBA and sacrificed 2, 14, or 28 days after dosing. The approximate LD 01 dose of 1250 mg/ kg was selected to provide a relatively high dose with minimal likelihood of mortality. The study duration was extended to 28 days because of evidence from the acute toxicity study that effects on epididymal sperm could peak more than 14 days after dosing. Reproductive organ weights and sperm quality parameters were measured, and a histopathologic examination was performed. Only marginal reproductive organ weight changes were induced by DBA. Epididymis weights on Days 2 and 28 were decreased to 93% and 83% of control values ( p< 0.05), respectively, but were not different from controls on Day 14. Testes weights were decreased to 93% of controls on Day 28 ( p< 0.05). Prostate weights were significantly increased to 109% of controls on Day 2 ( p< 0.05). DBA treatment did not affect Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 36 Draft, do not cite or quote body weights, suggesting that reproductive organ weight changes were not secondary to general toxicity. Serum testosterone levels fell to 17% of control values 2 days after a single dose of 1250 mg/ kg, but returned to control levels by Day 14.

Several measures of spermatotoxicity were reported in this study. Caput sperm count was significantly reduced on Day 2 to 85% of controls, but was not affected on Days 14 or 28.

Caudasperm count was decreased to 54% and 44% of controls on study Days 14 and 28, respectively.

Testicular sperm head count was not affected, suggesting that DBA was not inhibiting overall sperm production. Sperm morphology was also seriously affected by exposure to DBA. The percent of sperm having flagellar defects and atypical heads was significantly increased in caput sperm on Day 28, with 16% showing abnormal morphology. In the control group, about 5% of sperm were estimated to be abnormal, based on direct inspection of the data presented in a figure in the paper. Cauda sperm showed a dramatic increase in flagellar defects on Day 14 ( p< 0.05), but not on Day 28. Significant increases in sperm with atypical heads and with both atypical head and flagellar defects were increased on Day 28 ( 64% of sperm displayed abnormal morphology).

According to the study authors, the appearance of different morphological changes ( flagellar versus acrosomal) on Days 14 and 28 indicated that the epididymal sperm underwent two sequential morphological changes as a result of DBA exposure. Several measures of sperm motility were significantly reduced at Day 14, including percent motile ( 38% of controls), percent progressive motility ( 32% of controls), straight line velocity ( 73% of controls), and curvilinear velocity ( 82% of controls). Only the first two measures were significantly reduced at Day 28 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 37 Draft, do not cite or quote ( 51% and 41% of controls, respectively). The study authors suggested that these decreases were related to the flagellar defects. Thus, treatment with a single dose of 1250 mg/ kg of DBA resulted in significantly adverse effects on sperm count, morphology, and motility.

Histopathology examination revealed altered spermiation at all three time points examined ( Days 2, 14, and 28). On all three days, Step 19 spermatids were retained beyond their normal release in Stage VIII of the seminiferous epithelium cycle. Other abnormal histological signs included the presence of remnants of residual bodies in Stages X and XI, and the presence of anucleate cytoplasmic debris in the lumen of the epididymal duct and in the caput epididymis on Day 2. On Day 14, debris from the testes was evident in the epididymis, much of which resembled residual bodies. On Days 14 and 28, abnormal late spermatids were observed in Stages I VIII.

Similar histological changes were observed on both days, although the changes were characterized by the authors as less severe on Day 28. Varying amounts of cytoplasmic debris were also observed in the epididymis on Day 28.

These results show that a single high dose of DBA is spermatotoxic in the rat. The target cells for adverse effects of DBA were not conclusively identified, although the authors described several aspects of sperm maturation that might be impacted, based on consideration of the normal transit times ( assuming that the kinetics of sperm development were not affected), and on consideration of the timing of the observed effects. The study authors noted that flagellar degeneration was observed in the cauda epididymis on Day 14, but not in the caput epididymis.

The flagellar changes might be due to a DBA effect during transit through the epididymis. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 38 Draft, do not cite or quote Alternatively, as the normal transit time of sperm through the caput epididymis is 3 days in the rat,

and accounting for the remaining 11 days since dosing, the authors suggested that late spermatids before or during spermiation might have been affected. On day 28, both altered sperm heads and flagellar degeneration were observed in both the caput and cauda sperm. As spermatids that were in Step 11 to 15 on Day 2 of exposure would have comprised the majority of caput sperm on Day 14 ( when only a minimal effect on head development was seen), the authors suggested that the abnormal head development may have resulted from an effect of DBA on Step 10 or earlier spermatids. Alternatively, they noted that the same effects would have been seen if the effect of DBA was on later steps but the action of DBA was delayed for several days following dosing. As the retention of Step 19 spermatids is an effect observed following treatments that alter hormone status, the observation that DBA reduced circulating testosterone levels is consistent with the effects on Step 19 spermatids noted in the study. According to the authors, another potential target for DBA might be Sertoli cells, since the presence of testicular debris might suggest disruption of the endocytic activity of these cells. While DBA treatment adversely affected sperm quality, it did not appear to inhibit sperm production, based on histological analysis of the testes and the absence of an effect on testicular sperm head counts.

Linder et al. ( 1994b) studied the spermatotoxicity of DBA following 14 daily exposures.

The effects on selected endpoints are presented in Table V 5. Male Sprague Dawley rats ( 8/ dose group), approximately four months old, were given daily gavage DBA doses of 0, 10, 30, 90, or Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 39 Draft, do not cite or quote 270 mg/ kg/ day, and sacrificed immediately after the last dose. The dose vehicle was distilled water and the dose volume was 5 mL/ kg, adjusted weekly for body weight. No effects on body weight or serum testosterone levels were noted at any dose level. Several parameters were affected ( p< 0.05), primarily at the highest dose level of 270 mg/ kg. These included mildly reduced testis ( 93% of controls) and epididymis weights ( 86% of controls). Absolute and relative ( to testis weight) testicular sperm head counts were also repressed to 81% and 88% of control values,

respectively. Dose dependent effects on various measures of sperm motility were also observed,

with statistically significant decreases observed at the highest dose. Caput sperm counts were reduced significantly in a dose dependent fashion beginning at the low dose of 10 mg/ kg/ day. The percent of morphologically normal sperm was statistically decreased ( 79% of controls) only in the high dose group, with atypical heads observed more frequently than degenerating flagella and a notable increase detected in fused sperm. Cauda sperm count was significantly reduced to 76%

and 30% of control values in the 90 and 270 mg/ kg/ day dose groups, respectively. The percent of morphologically normal sperm was decreased to 86% and 32% of controls in the same groups.

Morphological changes in cauda sperm were mainly related to degenerative changes of the flagella. Percent motile sperm and percent progressive motility were reduced to less than 10% of control values at 270 mg/ kg/ day. Straight line velocity and linearity were also reduced at the highest dose. Curvilinear velocity was significantly affected only at 90 mg/ kg/ day.

Histopathological evidence of altered spermiation was noted beginning at 10 mg/ kg/ day.

Histopathological findings included retention of Step 19 spermatids in Stages IX to XII and Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 40 Draft, do not cite or quote atypical acrosomal development of Step 15 spermatids at 10 mg/ kg/ day. The severity of these effects increased with increasing dose. The presence of atypical structures resembling residual bodies in the testis and caput epididymis was observed at the two highest doses. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 41 Draft, do not cite or quote Table V 5. Sperm Quality Parameters in Rats Given 14 Daily Doses of DBAa Dose ( mg/ kg/ day)

Parameter 0 10 30 90 270 Caput sperm 106 131 ± 14 b 112 ± 13* 118 ± 10* 108 ± 11* 100 ± 10*

Cauda sperm 106 264 ± 21 250 ± 70 243 ± 34 200 ± 59* 78 ± 15*

Caput sperm (% morphologically normal) 95 ± 4 90 ± 11 96 ± 1 93 ± 3 75 ± 12*

Cauda sperm (% morphologically normal) 98 ± 1 90 ± 15 96 ± 2 84 ± 10* 31 ± 17*

Percent motile 78 ± 7 74 ± 14 83 ± 6 66 ± 17 6 ± 7*

Progressive motility (%) 67 ± 4 61 ± 16 68 ± 12 56 ± 16 4 ± 5*

Straight line velocity ( µ m/ sec) 75 ± 12 68 ± 17 66 ± 16 63 ± 15 44 ± 22*

Curvilinear velocity ( µ m/ sec) 154 ± 7 145 ± 10 136 ± 16 128 ± 25* 141 ± 42 Linearity 52 ± 7 51 ± 9 49 ± 8 50 ± 5 30 ± 10**

Notes:

a. Adapted from Linder et al., 1994b b. Mean ± standard deviation.

* Statistical significance: p< 0.05 ** Statistical significance: p< 0.01 In summary, a variety of male reproductive tract toxicity parameters were affected by DBA in this study. Adverse spermatogenic effects were noted beginning at the lowest dose of 10 mg/ kg/ day and generally increased in severity with increasing dose. Mildly decreased caputsperm count was also observed at this dose, but the effect was not clearly dose dependent, with similar decrements ( ranging from 76% to 90% of control values) observed at all doses. Decreased Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 42 Draft, do not cite or quote cauda sperm count, frequency of abnormal cauda sperm morphology, and changes in sperm motility were statistically significant only at the two highest doses, 90 and 270 mg/ kg/ day.

Noticeable histopathological changes ( delayed release of Step 19 spermatids and atypical Step 15 spermatid acrosomal development) began at the low dose. The LOAEL for this study is the lowest dose tested, 10 mg/ kg/ day, based on histopathological changes in the male reproductive tract, and a NOAEL could not be determined.

Linder et al. ( 1995) studied the longer term ( up to 79 days) effects of DBA in male rats on both reproductive competence ( summarized in Table V 6 and Table V 7) and on sperm quality ( summarized in Table V 8 and Table V 9). The highest dose of 250 mg/ kg/ day was selected based on the expectation that it would produce substantial spermatotoxicity and permit the investigation of the time course of DBA effects on fertility and reproductive competence. Lower doses of 2,

10, or 50 mg/ kg/ day were selected to obtain dose response data. Selected doses were based on the results of previous short term studies ( Linder et al., 1994a; Linder et al., 1994b), which suggested that both no efect and significant effect dose levels would fall within this dose range.

Daily doses of custom synthesized, high purity DBA in a distilled water vehicle were given by gavage to 105 day old male Sprague Dawley rats whose reproductive competence had been proven. There were essentially two experimental protocols employed, each of which will be reviewed separately here. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 43 Draft, do not cite or quote Table V 6. Reproductive Outcomes in Rats Following Oral Dosing with DBAa Mated Days Dose mg/ kg Male Female Cop Pairsb Copulatory Plugsc Sperm positive females Fertile males No. litters Implants Fetuses 8 14 0 250 10 10 10 10 9 3* e 3.3 ± 1.3d 1.3 ± 1.5 9 3* 9 2* 9 2* 14.1 ± 1.6 5.5 ± 6.4* 13.0 ± 1.3 5.0 ± 5.7*

15 21 0 250 10 10 10 10 10 5* 3.6 ± 1.1 1.4 ± 0.9* 10 4* 10 0* 10 0* 13.7 ± 2.3  12.6 ± 2.9

30 37 0 2 10 50 250 10 10 10 10 10 10 10 10 10 10 9 9 7 7 7 3.6 ± 2.1 3.1 ± 1.3 2.6 ± 1.4 4.3 ± 2.2 1.4 ± 1.1* 9 9 7 7 1* 9 8 7 7 0* 9 8 7 7 0* 15.7 ± 2.5 15.3 ± 3.1 16.3 ± 1.7 13.0 ± 5.2  13.6 ± 3.2 14.3 ± 3.5 14.9 ± 2.0 11.9 ± 5.3

65 71 0 2 10 50 250 10 10 10 10 9 20 20 20 20 9 16 17 10* 13 6 3.4 ± 1.3 3.3 ± 1.3 2.6 ± 1.8 2.4 ± 1.8 1.3 ± 0.8* 14 16 10 13 2* 9 10 7 9 0* 15 ( 6) e 14 ( 4) 9 ( 2) 10 ( 1)* 0* 14.8 ± 1.4 15.9 ± 1.3 15.8 ± 2.9 14.9 ± 2.2  14.0 ± 1.4 15.1 ± 1.2 14.6 ± 2.4 14.1 ± 2.4

199 213 0 250 10 9 20 18 15 15 3.5 ± 2.0 2.9 ± 1.4 15 14 10 3* 15 ( 5) 5* ( 2) 14.9 ± 1.7 15.3 ± 1.8 14.4 ± 1.9 14.5 ± 2.2 a. Adapted from Linder et al., 1995.

b. Copulatory pairs as evidenced by the presence of copulatory plug or birth of a litter.

c. Per copulating pair d. Mean ± SD.

e. Numbers in parentheses are the number of males siring two litters.

* Statistical significance: p< 0.05 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 44 Draft, do not cite or quote Table V 7. Outcome of Artificial Insemination of Sperm from Rats Dosed with DBAa Day Dose mg/ kg Number of Inseminations Number of litters Implants Fetuses 9 0 250 6 6 5 5 5.40 ± 3.36b 6.00 ± 3.32 5.20 ± 3.63 6.00 ± 3.32 16 0 250 6 5 ( 1) c 6 1* 7.83 ± 4.79 4.00 7.67 ± 4.63 4.00 31 0 2 10 50 250 6 6 6 6 1 ( 5) 5 6 3 5 0 7.40 ± 4.77 9.50 ± 5.24 5.33 ± 2.08 5.80 ± 3.11  7.40 ± 4.77 9.17 ± 4.96 5.00 ± 1.73 5.80 ± 3.11

79 0 2 10 50 10 9 ( 1) 10 10 7 6 8 5 7.86 ± 2.91 7.17 ± 4.26 8.75 ± 2.43 9.00 ± 2.83 7.71 ± 2.69 6.83 ± 4.45 8.63 ± 2.67 9.00 ± 2.83 Notes:

a. Adapted from Linder et al., 1995.

b. Litter means ± SD.

c. Number in parentheses is the number of males with insufficient sperm for insemination.

* Statistical significance, p< 0.05 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 45 Draft, do not cite or quote Table V 8. Reproductive Organ Weights and Sperm Counts in Rats Given Daily Doses of DBAa Number of Doses mg/ kg Nb Body weight ( g) Testis weight ( g) Epididymis weight ( g) TSHCc ( million) TSHC per gram testis ( million) Caput sperm ( million) Cauda sperm ( million) Serum testosterone ( ng/ ml)

2 0 250 6 6 402 ± 17d 401 ± 14 1.97 ± 0.13 1.92 ± 0.09 0.64 ± 0.02 0.63 ± 0.02 271 ± 17 278 ± 15 149 ± 10 157 ± 8 124 ± 8 115 ± 9 254 ± 28 278 ± 41 8.0 ± 4.1 4.1 ± 2.1 5 0 250 6 6 400 ± 16 405 ± 14 1.89 ± 0.14 1.85 ± 0.29 0.62 ± 0.05 0.58 ± 0.08 261 ± 26 273 ± 56 151 ± 12 158 ± 10 115 ± 12 97 ± 24 239 ± 29 230 ± 72 10.7 ± 4.9 6.9 ± 6.1 9 0 250 6 6 411 ± 21 404 ± 28 1.94 ± 0.08 1.87 ± 0.14 0.62 ± 0.03 0.60 ± 0.05 295 ± 15 276 ± 34 162 ± 7 156 ± 8 124 ± 7 119 ± 14 250 ± 23 225 ± 42 3.4 ± 1.7 3.4 ± 3.2 16 0 250 5 6 407 ± 23 375 ± 58 1.96 ± 0.21 1.74 ± 0.18 0.64 ± 0.04 0.51 ± 0.07* 288 ± 26 263 ± 27 162 ± 19 162 ± 11 122 ± 27 92 ± 21 255 ± 23 75 ± 32* 4.1 ± 3.5 2.9 ± 1.5 31 0 2 10 50 250 6 5 6 6 6 432 ± 14 417 ± 16 424 ± 21 425 ± 22 368 ± 38* 1.93 ± 0.12 1.93 ± 0.08 1.94 ± 0.15 1.90 ± 0.14 1.81 ± 0.05 0.64 ± 0.03 0.65 ± 0.02 0.64 ± 0.06 0.60 ± 0.02 0.51 ± 0.03 265 ± 24 280 ± 26 264 ± 35 282 ± 28 283 ± 25 147 ± 8 155 ± 10 145 ± 16 160 ± 11 167 ± 13* 128 ± 9 125 ± 13 121 ± 9 111 ± 10* 47 ± 9* 240 ± 14 225 ± 44 225 ± 34 169 ± 34* 33 ± 5* 4.0 ± 3.3 11.8 ± 6.4 5.6 ± 5.7 3.9 ± 2.5 2.6 ± 1.6 79 0 2 10 50 10 10 10 10 458 ± 24 455 ± 16 446 ± 20 434 ± 24* 2.01 ± 0.15 1.96 ± 0.12 2.02 ± 0.14 1.97 ± 0.11 0.68 ± 0.03 0.67 ± 0.03 0.69 ± 0.04 0.64 ± 0.05 298 ± 32 270 ± 25 288 ± 21 289 ± 24 159 ± 11 148 ± 11* 155 ± 6 159 ± 11 126 ± 7 126 ± 10 122 ± 10 112 ± 11* 240 ± 40 247 ± 28 242 ± 34 196 ± 47* 10.5 ± 8.4 7.1 ± 3.8 6.2 ± 4.9 3.8 ± 1.6 0e 0 10 522 ± 22 2.02 ± 0.10 0.68 ± 0.06 260 ± 23 139 ± 6 118 ± 9 249 ± 45 2.7 ± 1.3 42f 250 9 483 ± 19* 0.99 ± 0.48* 0.49 ± 0.09* 50 ± 106* 34 ± 56* 20 ± 40* 37 ± 74* 2.7 ± 1.5 Notes:

a. Adapted from Linder et al., 1995.

b. N= number of rats.

c. TSHC = testicular sperm head count d. Group mean ± SD.

e. Nondosed controls.

f. Given 42 doses then allowed to recover for 186 days.

* Statistical significance: p< 0.05 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 46 Draft, do not cite or quote Table V 9. Sperm Quality Parameters in Rats Given Daily Doses of DBAa Number of Doses mg/ kg N b Motile sperm (%) Progressive motility (%) VSLc ( µ m/ sec) VCLc ( µ m/ sec) Linearity Caput sperm % normal Cauda sperm % normal 2 0 250 6 6 84 ± 5d 87 ± 7 75 ± 7 75 ± 5 80 ± 7 79 ± 7 105 ± 9 107 ± 7 48 ± 4 46 ± 5 96 ± 1 ( 0) d 96 ± 2 ( 0) 96 ± 2 ( 0) e 97 ± 1 ( 0)

5 0 250 6 6 84 ± 5 83 ± 8 72 ± 7 70 ± 7 80 ± 18 77 ± 9 108 ± 17 107 ± 11 46 ± 7 44 ± 3 97 ± 2 ( 0) 90 ± 4*( 3) 96 ± 2 ( 0) 95 ± 4 ( 0)

9 0 250 6 6 84 ± 8 84 ± 7 72 ± 8 73 ± 8 87 ± 17 79 ± 15 120 ± 21 107 ± 20 47 ± 4 49 ± 3 96 ± 2 ( 0) 55 ± 17* ( 8) 96 ± 1 ( 0) 95 ± 2 ( 0)

16 0 250 5 6 86 ± 5 13 ± 18* 70 ± 6 10 ± 15* 63 ± 24 31 ± 13* 92 ± 36 51 ± 24* 43 ± 3 32 ± 10* 98 ± 1 ( 0) 61 ± 37* ( 2) 97 ± 1 ( 0) 33 ± 20* ( 3)

31 0 2 10 50 250e 6 5 6 6 6 78 ± 3 81 ± 4 72 ± 20 47 ± 32* 3 ± 6 61 ± 7 65 ± 6 53 ± 16 36 ± 28 2 ± 4* 67 ± 15 74 ± 10 68 ± 16 55 ± 28  107 ± 22 113 ± 16 116 ± 31 91 ± 36  39 ± 5 42 ± 4 41 ± 5 35 ± 8  96 ± 2 ( 0) 97 ± 1 ( 0) 96 ± 6 ( 0) 88 ± 7* (. 3) 1 ± 2* ( 5) 96 ± 1 ( 0) 96 ± 2 ( 0) 96 ± 4 ( 0) 88 ± 10* ( 0) 2 ± 2* ( 3)

79 0 2 10 50 10 10 10 10 75 ± 8 77 ± 8 76 ± 11 66 ± 13 61 ± 8 63 ± 8 62 ± 9 53 ± 12 70 ± 9 72 ± 11 76 ± 10 64 ± 9 107 ± 11 106 ± 13 115 ± 17 96 ± 14 42 ± 3 42 ± 4 42 ± 2 41 ± 4 97 ± 1 ( 0) 94 ± 6 ( 0.02) 96 ± 2 ( 0) 68 ± 20* ( 1) 96 ± 2 ( 0) 93 ± 8 ( 0) 94 ± 4 ( 0) 75 ± 17* ( 0.06)

Notes:

a. Adapted from Linder et al., 1995.

b. N = number of rats.

b. VSL is straight line velocity and VCL is curvilinear velocity.

c. Group means ± SD.

d. Number in parentheses is the percent of fused sperm.

e. Motile sperm ( 15% and 1% motile) were present in only two rats.

* Statistical significance: p< 0.05 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 47 Draft, do not cite or quote In the first protocol, groups of 10 male rats were given daily gavage doses of either 0 or 250 mg/ kg/ day DBA. During study days 8 14, 15 21, and 30 37, the males were paired with females and allowed to mate by natural insemination. Dosing was terminated after 42 days because of the onset of overt toxicity, including labored breathing, light tremor, difficulty moving the hind limbs, and severe weight loss. The animals, however, were allowed to mate during recovery, on days 49 56, 65 71, and 199 213. During the mating period on study days 8 14, only 3/ 10 males copulated; only two males were fertile and only two litters were produced. The numbers of implants and fetuses in these litters were reduced by more than 50% as compared to controls. During the mating periods on days 15 21 and 30 37, there were no fertile males and no litters were produced, even though 5/ 10 and 7/ 10 males, respectively, copulated during these periods. To distinguish fertilization failure from pre implantation loss, females from the mating period on days 49 56 were sacrificed on GD 1 and examined for the presence of fertilized eggs.

There were no fertilized eggs. During the mating period on days 65 71, no males were fertile and no litters were produced. During the mating period on days 199 213 ( after 5 months of recovery),

only 3/ 9 males were fertile and 5 litters were produced, even though all males copulated. Thus,

although the reproductive performance of animals in the 250 mg/ kg/ day improved significantly during recovery, they never fully recovered.

Artificial insemination of luteinizing hormone releasing hormone ( LHRH) synchronized females, with sperm from treated males from an additional group of 6 animals, sacrificed on Days 9, 16, and 31 ( and also used for interim necropsy), was conducted to distinguish behavioral Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 48 Draft, do not cite or quote mating effects from effects due to physiologic reproductive competence. Five litters were sired with Day 9 sperm; no significant adverse effects were observed on the number of implants or fetuses. The absence of a significant effect on fertility by artificial insemination with Day 9 sperm suggested to the study authors that the reproductive effects observed from the Day 8 14 mating may have been due to a transient effect on libido. In contrast, only one litter was produced as a result of artificial insemination with Day 16 sperm, and no litters resulted from insemination with Day 31 sperm, indicating that reproductive incompetence was due to spermatotoxic effects.

Consistent with these reproductive outcomes, measures of sperm motility were not affected until Day 16, but were severely affected thereafter. The cauda sperm count was normal until Day 16, at which time it was reduced to 29% of controls. Caput sperm counts decreased progressively to as low as 37% of normal on Day 31. The percent of sperm with normal morphology was significantly decreased ( p< 0.05) beginning on Day 5 for caput sperm and on Day 16 for cauda sperm. Only minimal developmental toxicity data were provided in the paper. No effects on fetal weight were observed; minimal changes in the incidence of malformations were inconsistently observed and were not considered to be treatment related by the study authors.

In the second protocol, groups of 10 male rats were given daily gavage doses of 0, 2, 10,

or 50 mg/ kg/ day DBA for up to 79 days. The only systemic effect was a slight decrease in body weight ( to 95% of controls) that was apparent by Day 53 in the 50 mg/ kg/ day group. The rats were mated during study days 30 37 and 49 56 with one female per male, and during study days 65 71 with two females per male. No effects on the number of fertile males, litter size, fetal body Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 49 Draft, do not cite or quote weight, or number of implants per litter were observed. There was a dose dependent, but not always statistically significant, reduction in copulating pairs and copulatory plugs relative to controls during the 65 71 day mating period. During this final mating period, there was also a dose dependent decrease in the number of males siring two litters, which was statistically significant only at the highest dose tested, 50 mg/ kg/ day. The effects on mating behavior were similar at 10 mg/ kg/ day and 50 mg/ kg/ day, but there was no clear dose response. For example,

there were fewer copulatory plugs and multiple litters at 50 mg/ kg/ day than at 10 mg/ kg/ day, but the higher dose had more copulatory pairs and/ or inseminations ( depending on the mating period).

The mating behavior effects in the 10 mg/ kg/ day dose group included fewer copulating pairs,

fewer inseminations, fewer copulatory plugs, and fewer multiple litters, but the only statistically significant ( p< 0.05) effect at this dose was fewer copulating pairs in the Day 65 71 group.

Artificial insemination of LHRH synchronized females was performed with sperm from an ancillary group exposed to 0, 2, 10, or 50 mg/ kg/ day and sacrificed on Day 31 ( 6 males/ group) or sacrificed on Day 79 ( 10 males/ group). No significant effects on reproductive outcomes were observed. Necropsy results revealed that caput and cauda sperm counts were significantly reduced ( p< 0.05) at the high dose to 87% and 70% of control values, respectively, on Day 31, and to 89%

and 82% of control values, respectively, on Day 79. The percent motile sperm was affected on Day 31, but not on Day 79 at 50 mg/ kg/ day. Exposure to 50 mg/ kg resulted in moderate changes in sperm morphology, including head and tail defects and fused sperm, when examined at either Day 31 or Day 79. No gross effects on sperm morphology were seen at 2 or 10 mg/ kg/ day. A Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 50 Draft, do not cite or quote slight increase in dead fetuses of 5% ( compared to 0% in the controls) was reported for the Day 79 artificial insemination group dosed with 50 mg/ kg DBA. No other statistically significant,

developmentally toxic effects were reported.

Histopathologic results from the Linder et al. ( 1995) study were reported separately ( Linder et al., 1997a). Necropsies of rats dosed with 0 or 250 mg/ kg/ day DBA were performed 24 hours after the last of 2, 5, 9, 16, or 31 daily doses. A necropsy was also done at Day 228,

which included 42 days of exposure and a 6 month recovery period. At Days 2 and 5, there was moderate to extensive retention of Step 19 spermatids ( normally released in Stage VIII) in Stage IX of the cycle of the seminiferous epithelium. This retention was also seen in Stages IX and X at Days 9 and 16, and in virtually all Stage IX and X tubules at Day 31. Also at Day 31, there was retention of Step 19 spermatids and degenerating Step 19 spermatids in Stage XI to XIV tubules.

Basally located remnants of Step 19 nuclei were seen in Stages X to XII at Day 5, Stages X to XII at Day 9, Stages IX XII at Day 16, and in Stages XI XIV at Day 31. Fused Step 19 spermatid flagella in Stage IX were seen from Day 5 through Day 31. Atypical residual bodies were seen from Day 5 through Day 16 at numerous Stages, and appeared in the epididymis on Days 9, 16, and 31. Debris from these atypical residual bodies, along with other cytoplasmic debris from the testes, was observed throughout the epididymis on Day 31. Acrosomes and/ or heads of Step 12 and later spermatids were affected at Days 16 and 31, and maloriented late spermatids were also observed at these times. Vacuolization of the Sertoli cell cytoplasm was seen at Day 31. After a fairly long ( 6 month) recovery period, male rats dosed with 250 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 51 Draft, do not cite or quote mg/ kg/ day still displayed atrophic seminiferous tubules, disorganization of sperm producing tubules, and degeneration of mature and round spermatids, all likely resulting from effects on the structure and/ or function of Sertoli cells and indicative of permanent damage to the reproductive system.

In the same study, necropsies of rats given 0, 2, 10, or 50 mg/ kg/ day were performed 24 hours after the last of 31 or 79 daily doses. Retention of Step 19 spermatids near the tubule lumen in Stage IX was observed at Day 31 at doses of 10 mg/ kg/ day and higher. At Day 79 in the 10 mg/ kg/ day dose group, there was also retention of Step 19 spermatids in Stages IX XI at both the lumenal and basal surfaces. One animal at this dose also had disorganized and atrophied tubules,

but this effect was not considered to be treatment related because tubular disorganization was not seen at 50 mg/ kg/ day. The 50 mg/ kg/ day dose group ( after 31 doses) had increased retention of Step 19 spermatids, with moderate numbers observed at the lumenal surface at Stage IX and at the basement membrane at Stages X XII. Similar effects were seen when this dose group was treated for 79 days, with the retention of Step 19 spermatids also occurring at Stages IX and X.

Atypical residual bodies were present in Stage IX at Day 31 and occasionally at Stage IX and other stages at Day 79. Cytoplasmic debris was observed throughout the epididymis at Day 79 in the 50 mg/ kg/ day dose group. No histopathological changes were detected at 2 mg/ kg/ day on either Day 31 or Day 79.

Thus, the two papers for this study ( Linder et al., 1995; Linder et al., 1997a) describe increasingly severe effects with increasing dose and increasing exposure duration. No adverse Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 52 Draft, do not cite or quote effects were seen at the lowest dose tested, 2 mg/ kg/ day. Dosing with 250 mg/ kg/ day for as few as 8 14 days caused decreased mating and decreased fertility, and adverse reproductive effects were only partially reversible after 42 days of exposure and a 6 month recovery period. Overall,

this study identified an equivocal LOAEL of 10 mg/ kg/ day and a corresponding NOAEL of 2 mg/ kg/ day for male reproductive effects, based on histological evidence for changes in seminiferous tubule staging of altered spermatid development.

Collectively, the studies by Linder and colleagues have used a number of different experimental protocols to investigate the effects of DBA on spermatogenesis and the resulting effects on male fertility ( Linder et al., 1994a; Linder et al., 1994b; Linder et al., 1995; Linder et al.

1997a). Based on the results of these studies, DBA was clearly spermatotoxic in rats following high dose single exposures or repeated exposures for longer periods of time ( up to 79 days).

Effects on spermatogenesis were the most sensitive endpoint because they were observed in the absence of other toxicity indicators. Significant changes in sperm count, morphology, and motility were generally observed at doses higher than those associated with early histopathologic changes in spermatogenesis. However, the susceptibility of humans to DBA induced reproductive toxicity is not known and it is possible that rats are more sensitive than humans. In the absence of valid and reliable human data on the relationship between sperm quality and human fertility, and on the relative sensitivity of humans versus rats to DBA associated reproductive effects, adverse histopathologic or sperm quality changes in rodents are considered to be the more appropriate choice for the critical effect in the studies by Linder and his colleagues than fertility changes. Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 53 Draft, do not cite or quote In contrast to the results of Linder et al. ( 1994a), Vetter et al. ( 1998) did not observe a significant spermatotoxic effects of acute treatment with DBA. Vetter et al. ( 1998) evaluated spermatotoxic effects of DBA as a positive control to validate a computer assisted semen analysis and a flow cytometric assay for cell membrane integrity as alternatives to sperm motility assays for assessing male reproductive toxicity. Sexually mature male Crl: CD( SD) BR rats ( 4 5/ group)

were given single oral doses of 0, 600, or 1200 mg/ kg DBA in 10 mL/ kg deionized water. The high dose, but not the low dose, resulted in overt toxicity. The rats were sacrificed after 13 days,

at which time vas deferens sections were harvested for sperm analysis, and sections of the testes and epididymides were taken for histopathologic analysis. The average percent motile sperm was 74.4%, 74.8%, and 65.7% for the control, low, and high dose groups, respectively. The average percent of viable sperm was 90.9%, 91.4%, and 88.7%, for the control, low and high dose groups, respectively. Neither sperm motility nor membrane permeability following DBA treatment were statistically different from controls. The absence of an effect was not likely due to general failure of the assays since a second positive control,   chlorohydrin, did decrease sperm motility.

Following DBA treatment, no morphologic changes were observed in the sperm; however, large basophilic bodies were observed in the testes of low dose rats ( 3/ 5 males) and high dose rats ( 4/ 4 males), and in the epididymides of high dose rats ( 4/ 4 males). The study authors noted that differences in experiments, such as the source of the sperm from the vas deferens versus the cauda epididymis, the strain of rat, and the time of sacrifice ( 13 versus 14 days post treatment) are unlikely explanations for the absence of spermatotoxicity in this study as compared with the Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 54 Draft, do not cite or quote results reported by Linder et al. ( 1994a). In the absence of further data, the reasons for the differing results in the Linder et al. ( 1994a) and Vetter et al. ( 1998) experiments remain unresolved. The Vetter et al. ( 1998) study identified a LOAEL of 600 mg/ kg/ day, based on histopathologic changes in the testes. A NOAEL could not be determined.

Although the effects of DBA on male reproductive tract toxicity have been well studied,

fewer studies have evaluated the potential reproductive effects of DBA in females. Cummings and Hedge ( 1998) studied the effects of DBA exposure during early pregnancy in rats. Female Holtzman rats ( 8/ dose group) were administered gavage doses of 0, 62.5, 125, or 250 mg/ kg/ day DBA dissolved in water on GD 1 8. Administration of a higher dose, 500 mg/ kg/ day, induced moribund behavior and lethality; therefore, dosing at this level was discontinued and these animals were not further evaluated for reproductive endpoints. Treated animals from the other dose groups were sacrificed on GD 9, and body and reproductive organ weights, serum levels of progesterone, 17  estradiol, and luteinizing hormone, the number of implantation sites, the number of resorptions, the number of corpora lutea, and pre implantation losses were assessed.

The only affected response was a 170 % increase in serum 17  estradiol at 250 mg/ kg/ day. A second group of females was dosed similarly to the first group, sacrificed on GD 20, and evaluated for body weight, preimplantation losses, number of resorptions, number of pups per litter, pup weights, and placental weights. No differences in any of these measures were observed between treated and control animals. The authors concluded that DBA had little effect on female reproduction for the measures assessed in the study, but noted that effects on ovarian function and Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 55 Draft, do not cite or quote future fertility were not tested; such tests would be warranted by the observed increase in serum 17  estradiol. Based on the increase in serum 17  estradiol in this study, the LOAEL is the highest dose tested, 250 mg/ kg/ day, and the NOAEL is 125 mg/ kg/ day. An acute FEL of 500 mg/ kg/ day was also identified, based on moribund behavior and lethality in the pregnant dams.

This study, however, is limited by the small sample size of each of the groups.

Christian et al. ( 2001) evaluated the reproductive and developmental toxicity of DBA in Sprague Dawley rats. Male and female rats ( 10/ sex/ group) were given DBA in deionized drinking water at concentrations of 0, 125, 250, 500 or 1000 ppm, beginning 14 days prior to cohabitation and continuing through gestation and lactation ( 63 70 days of treatment). The average daily doses ( based on measured water consumption and body weights) varied, depending on the phase of reproduction. For males throughout the study ( SD 1 70), mean daily doses were 10.2, 20.4, 35.7,

and 66.1 mg/ kg/ day, respectively. For females on SD 1 15 ( pre mating), mean daily doses were 13.3, 26.2, 41.8 and 60.2 mg/ kg/ day, respectively; and 14.8, 30.3, 48.5 and 81.6, respectively, on gestation day ( GD) 0 21. During lactation ( LD 1 29), the estimated doses were 0, 43.5, 86.6,

150.7 and 211.7 for the 0, 125, 250, 500, and 1000 ppm groups, respectively; however, these doses included consumption of water by the pups and thus overestimated the mean daily intake for lactating females. Among the pups, two male and two female weanlings from each litter were selected for one additional week of observation ( postweanling days 1 8, commencing on LD 29);

daily food intake, drinking water consumption and body weights were recorded, and necropsy was conducted at sacrifice. The mean daily doses for the weanling pups were 0, 31.8, 58.5, 122.9 Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 56 Draft, do not cite or quote and 254.7 mg/ kg/ day for males, and 0, 33.3, 61.5, 123.8, and 241.2 for females in the 0, 125, 250,

500 and 1000 ppm groups, respectively.

Apparent taste aversion was associated with an exposure dependent reduction in water consumption, which was paralleled by a reduction in food intake at all concentrations. Decreased body weight gain was observed in parental animals and postweanling pups at the two highest exposure levels. Estrous cycling was unaffected in the female rats. The only observed adverse reproductive effect was a possible reduction in mating performance in the 1000 ppm group, as evidenced by a slight but nonsignificant increase in the number of days of cohabitation and a decrease in the number of mated pairs ( 6/ 10 in the 1000 ppm group versus 9 10/ 10 in all other groups). There were no effects on pre and postimplantation losses, live litter sizes, and gross external morphology or sex ratios in the pups. Although an exposure related decrease in pup body weights was noted, these findings were attributed to decreased water and food consumption resulting from the poor palatability of DBA treated drinking water. Based on a lack of statistically significant, treatment related findings, the parental and reproductive/ developmental NOAEL for this study is the highest dose tested, and a LOAEL could not be determined. For males, the paternal NOAEL is 66 mg/ kg/ day; for females, the corresponding NOAEL is not less than 60 mg/ kg/ day, and is likely to be higher, as water consumption and corresponding mean DBA daily doses were increased during gestation ( to 82 mg/ kg/ day) and lactation ( mean daily doses could not be determined due to the confounding effects of water consumption by the pups). Similarly,

the NOAEL for developmental effects is at least 82 mg/ kg/ day ( maternal dose during gestation). Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 57 Draft, do not cite or quote The Chemistry Council ( CCC, 2001; Christian et al., 2002) recently completed a twogeneration drinking water study of DBA in rats, conducted according to Good Laboratory Practice ( GLP) standards and U. S. EPA test guidelines. The report has recently been published and has also been independently reviewed and accepted by an EPA scientific advisory group.

Because this study addresses a key data gap, a fairly detailed summary of the reported findings is presented here. Male and female Crl: CD Sprague Dawley rats ( 30/ sex/ exposure group) were administered DBA in drinking water at concentrations of 0, 50, 250, or 650 ppm continuously from initiation of exposure of the parental ( P) generation male and female rats through weaning of the F2 offspring. The concentrations were chosen based on a range finding study that found that 650 ppm was the highest concentration expected to allow survival of the F1 offspring. For the P generation, DBA exposure was initiated at 43 days of age and continued from premating until study day ( SD) 92 for males; and from premating through gestation and a 29 day period of lactation ( LD 1 29) ( for approximately 120 days of exposure) for females. Parental generation offspring ( F1 males and females) were exposed in utero during gestation, and during lactation ( LD 1 29); selected F1 males and females ( 30/ sex/ exposure group) were further exposed during a postweaning period of at least 71 days, which continued through mating, gestation, and lactation.

All other F1 pups were sacrificed on LD 29. All F1 adult females and their offspring ( F2 generation) were sacrificed on LD 22. All females in the P and F1 generations were evaluated once daily for estrous cycling ( from 21 days before cohabitation through GD 0). All females were also assessed for duration of gestation, fertility index, gestation index, number and sex of Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 58 Draft, do not cite or quote offspring per litter, number of implantation sites, litter size and viability, viability index, lactation index, percent pup survival and litter sex ratio, general condition of the dam and litter during the postpartum period, and maternal behavior during lactation. Litters were examined to identify external abnormalities, physical signs of toxicity, pup weights, and litter viability. Necropsy of all P and F1 adults included gross evaluation of the cranial, thoracic, abdominal, and pelvic viscera.

Specialized measurements evaluating sperm parameters ( concentration, percent motility,

morphology, number of sperm, and testicular spermatid count). F1 generation pups were also evaluated for age at sexual maturation ( as determined by vaginal patency in females, preputial separation in males, and anogenital distance in both sexes) were performed. Individual organ weights were recorded for major organs, including testes and ovaries, as well as uterus with oviducts and cervix, epididymides, prostate gland, and seminal vesicles with coagulating glands.

All gross lesions were examined histologically. Histopathology was also conducted on adrenal and pituitary glands; testis, epididymides, prostate, seminal vesicles, coagulating glands in males;

ovaries, oviducts, uterus, cervix and vagina in females; and selected additional organs based on observed organ weight changes. Additionally, testicular histopathology examining the caput,

corpus, and cauda of the epidiymis was conducted in males, and the reproductive organs of all rats suspected of reduced fertility were subjected to histological examination.

The average daily doses ( based on measured water consumption and body weights) are presented in Table V 10. Daily doses varied both between exposure groups and among Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 59 Draft, do not cite or quote reproductive stages ( premating, gestation, lactation). Significant increases in pup mortality in F1 litters were considered to be unrelated to DBA exposure because the incidences were within the historical control of the testing facility. Other unscheduled deaths in the study were also unrelated to exposure to DBA. Clinical signs of toxicity were observed in various groups exposed to 250 and 650 ppm, and included soft or liquid feces, dehydration, and ungroomed coats. Water consumption were statistically significantly decreased in the P and F1 generation at all exposure levels, presumably due to taste aversion, and food intake was significantly reduced at the highest dose group in the P generation and the two high exposure groups in the F1 generation. Body weights and body weight gains for high dose P males and females were significantly reduced during the premating period and were significantly decreased for high dose P females during gestation and lactation. F1 male and female pups had significantly reduced body weights at all exposure levels during the lactation period, sufficient for the study authors to delay weaning until LD 29 to ensure pup survival.

Table V 10. Average Consumed Daily Doses ( mg/ kg/ day) for Male and Female Sprague Dawley Rats in the Two Generation Reproductive/ Developmental Toxicity Studya Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 60 Draft, do not cite or quote DBA Exposure Groups 0 ppm 50 ppm 250 ppm 650 ppm P Generation   Male Rats Premating to Termination ( SD 1 92) 0.0 4.4 22.4 52.4 P Generation   Female Rats Premating to Cohabitation ( SD 1 70) 0.0 6.0 28.1 69.1 Gestation ( GD 0 21) 0.0 6.4 30.1 76.1 Lactation ( LD 1 15) 0.0 11.6 55.6 132.0 F1 Generation   Male Rats Premating ( PD 1 71) 0.0 5.7 29.7 74.6 Weaning to Termination ( PD 1 134) 0.0 4.5 22.0 54.7 F1 Generation   Female Rats Weaning to Cohabitation ( PD 1 71) 0.0 6.6 32.1 83.4 Gestation ( GD 0 21) 0.0 6.2 28.5 67.1 Lactation ( LD 1 15) 0.0 10.0 49.6 114.7 a Chlorine Chemistry Council ( 2001), unpublished report; Christian et al., ( 2002) bSD = study day; GD = gestation day; LD = lactation day; PD = postweaning day By LD 29, the body weights of pups in the 50 ppm group were similar to control pup weights. Throughout the postweaning/ premating period, F1 males and females in the 250 and 650 ppm groups weighed significantly less than controls, and the females continued to exhibit significant reductions in body weight ( compared to controls) during gestation and lactation. The body weights of F2 pups in the two highest dose groups were also reduced by the end of lactation; however, these reductions were not reported to be statistically significant, relative to control group values. No treatment related effects were reported in either generation for estrous cycling, number of days in cohabitation, duration of gestation, mating indices, fertility indices, Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 61 Draft, do not cite or quote number and sex of offspring per litter, number of implantation sites, litter size, lactation index,

percent pup survival, pup sex ratio, and gross malformations. Total litter loss observed in the P generation for two dams in the 250 ppm exposure group and one dam in the 650 ppm group was not considered to be treatment related.

For the F1 generation 650 ppm exposure group, preputial separation was significantly delayed in the male rats ( 50.5 days versus 48.1 days in controls), and vaginal patency in female rats was also significantly retarded ( 36.3 days versus 33.4 days in controls); no significant difference was seen when the data were analyzed using body weight as a covariant. These effects were considered to be due to a general retardation of growth associated with the significant reduction in body weight in this exposure group at weaning. In F2 male and female pups,

anogenital distance did not differ from controls on LD 1 but was significantly reduced in male pups in the 250 and 650 ppm by LD 22; these findings were also considered to be associated with a general retardation of growth rather than being treatment related.

An increased incidence of malformations of the male reproductive tract, including small testes and small or absent epididymides, was observed in four males in the F1 group exposed to 650 ppm and was considered to be treatment related. Histomorphologic examination of these organs in these males revealed a minimal increase in abnormal residual bodies, retained Step 19 spermatids, hypospermia, atrophied epididymis and/ or atrophied testis. Histopathologic examination of reproductive organs of P and F1 male rats in the 250 and 650 ppm groups ( N =

30/ group/ generation) showed a consistent and significant exposure related increase in retained Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 62 Draft, do not cite or quote Step 19 spermatids in Stage IX and X tubules and in increased and abnormal residual bodies in affected seminiferous tubules ( Table V 11). Diffuse testicular atrophy and phagocytized Step 19 nuclei in the basilar area of affected seminiferous tubules were also observed, although at a lower incidence. Other testicular abnormalities in 250 and 650 ppm male rats of both generations included increased amounts of exfoliated spermatogenic cells/ residiual bodies in epididymal tubules, atrophy, and hypospermia. Percent motile sperm, sperm count, sperm density, and number and percent of morphologically abnormal sperm for exposed groups were within historical control values for the test laboratory and were unaffected by treatment. No effects were observed in the prostate gland, seminal vesicles, or coagulating glands of any of the male rats of either generation. All gross lesions in other organs in P and F1 parental rats and in F1 ands F2 pups were considered to be unrelated to DBA treatment.

Table V 11. Incidences of Exposure Related Histopathologic Findings in the Testes of Rats Consuming DBA in Drinking Water a P F1 DBA Concentrations ( ppm) 0 50 250 650 0 50 250 650 Number of Testes Examined 30 30 30 30 30 30 30 30 Retention of Step 19 Spermatids 4 3 13 23 0 1 12 20 Abnormal/ Increased Residual Bodies 3 5 15 25 1 2 10 14 a Chorine Chemistry Council ( 2001), unpublished report Histologic examination of the ovaries of ten P and F1 female rats in the control, 250 and 650 ppm exposure groups did not reveal any functional abnormalities; corpora lutea and growing Drinking Water Criteria Document for Brominated Acetic Acids EPA/ OW/ OST/ HECD V 63 Draft, do not cite or quote and antral follicles were present and apparently normal. There were no significant differences in the number of postlactational ovarian primordial follicles among any of these groups.