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- Toxicology and Hazard IdentiB~cation In this chapter, the National Research Council's subcommittee on methyl bro- mide reviews the toxicokinetic and toxicological information on methyl bro- mide as presented in the CaTifomia Department of Pesticide Regulation's (DPR's) October 1999 draft report Methyl Bromide: Risk Characterization Document for Inhalation Exposure (DPR 1999~. The information reviewed by the subcommittee is presented in Section ITT, "Toxicology Profile," and Appendices B and D, of the DPR draft report. In the sections below, the sub- committee comments on DPR's selection of the critical study and toxicologi- cal endpoints for acute, subchronic, and chronic exposures. In Table 2-l be- Tow, taken from the DPR risk characterization document (DPR 1999, p. 10), the critical no-observed-adverse-effect levels (NOAELs),~ toxicity endpoints, and reference concentrations2 (RfCs) are summarized. The subcommittee has used the term no-observed-adverse-effect level (NOAEL) rather than DPR's term no-observed-effect level (NOEL). NOAEL is defined as an exposure level at which there are no statistically or biologically significant increases in the frequency or severity of adverse effects between the exposed population and its appropriate control. Many organizations use the terms interchangeably. 2A reference concentration (RfC) is an estimate of the concentration of a substance that is unlikely to cause noncancer health effects in humans during a lifetime. It is used by DPR as a regulatory value for establishing buffer zones to protect residents from adverse effects of methyl bromide exposure. 12

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TOXICOLOGYA ND Hall ~NDENTIFICATION 13 TABLE 2-l Summary of Critical NOAELs Used by DPR Experimental NOAEL (ppm) 40 Human Equivalent NOAEL (ppm)a Adult Child RfC(ppb)b 21 na 210 Effect in Animal Studies Developmental toxicity (pregnant rabbit) Neurotoxicity (dog) 120 (adult) Neurotoxicity 70 (child) (pregnant rabbit) 2 (adult) 1 (child) 2 (adult) 1 (child) Reference 103 45 25 Sub- 20 chronic: 1 wk 6wk 0.5 (estimated) Chronic 0.3 (estimated) 12 ~ 0.2 0.1 0.2 0.1 Breslin et al. 1990b Newton 1994b Sikov et al. 1981 Neurotoxicity (dog) Newton 1994b Nasal epithelial Reuzel et hyperplasia (rat) al. 1987, 1991 aThe human equivalent NOAEL in parts per million (ppm) is derived from the experimental NOAEL, taking into account the relative breading rates and exposure durations of animals and humans. bThe inhalation reference concentration (RfC), In parts per billion (ppb) is the ratio of the human equivalent NOAEL and an uncertainty factor of 100. NOTE: na = not applicable. Source: Adapted from DPR 1999, p. 10. PHARMACOKINETICS The absorption, distribution, and, to some extent, the biotransformation and excretion of methyl bromide are reviewed in the DPR report. Following inha- lation or ingestion, methyl bromide was absorbed rapidly and distributed to most tissues in the body, based on a 14C label, which can reflect either parent or metabolite (Bond et al. 1985~. After 6 hr of nose-only inhalation, rats ab- sorbed 38/O to 48/O of the methyl bromide concentrations at 1.6 to 170 ppm, but only 27% at 310 ppm (Medinsky et al. 1985~; dogs absorbed approxi- mately 40/O after 3 hr of exposure to concentrations at 174 to 361 ppb (Raabe 1986, as cited in DPR 1999~; and humans absorbed 52% to 55% of concentra- tions of ~ ~ ppb after 2 hr of exposure (Raabe 198S, as cited in DPR 1999~. In

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~ 4 METHYL BROMIDE RISK CHARA CTERIZA TION IN CALIFORNIA rats, a radioactive label (radiolabel) was found in the nasal turbinates, lungs, testes, brain, thymus, and adrenal glands of the rat (Medinsky et al. 1984; Bond et al. 1985; Jaskot et al. 1988~. The tissues with the highest radioactiv- ity were lung, liver, and nasal turbinates. Following oral administration in rats, more than 90/O of the dose was absorbed (Medinsky et al. 1984~. Fol- lowing a high-dose accidental exposure of a man who died 4 hr later, methyl bromide was detected in all tissues, except the spleen, at an autopsy ~ hr after death (Michalodimitrakis et al. 1997 as cited in DPR 1999~. Methyl bromide is rapidly biotransformed following inhalation (Bond et al. 1985~. In rats, more than 75/O of the inhaled dose was excreted within 65 hr (Bond et al. ~ 985~. Over 90/O of the inhaled radioactivity in rats was associ- atecI with metabolites with an elimination half-life from tissues of I.5 to ~ hr (Bond et al. 1985~. About half of the inhaled dose in rats is excreted as ex- haled CO2 with a biphasic half-life of approximately 4 hr and 1 1 to ~ 7 hr.; less than 5% is exhaled as methyl bromide (Medinsky et al. 1985~. About 20% of the absorbed radioactivity is excreted in the urine and only about ID via the feces. The estimated clearance half times of the radiolabels in dogs and hu- mans are about 41 hr and 72 hr. respectively (Raabe 1986, as cited in DPR 1999~. Although the discussion on the absorption, distribution, and excretion of '4C-labeled methyl bromide (based on following the radiolabel) in the DPR reports appears complete, there is only limited discussion of its metabolism. The studies on the excretion of 14C following the inhalation of radiolabeled methyl bromide are consistent with the hypothesis that the methyl group of methyl bromide joins the 1-carbon pool after metabolism. Thus, it is impor- tant that the literature on the metabolism of methyl bromide be reviewed in the DPR document. The metabolism of methyl bromide could have implica- tions for its toxicity and subsequent risk assessment, especially for some seg- ments of the population, as noted below. Following absorption, conjugation with glutathionea common detoxifica- tion mechanism appears to be the primary metabolic pathway for monohalo- methanes, including methyl bromide (Hallier et al. 1990; Peter et al. 1989; Bonnefoi et al. 1991~. Methy~glutathione is then metabolized to S-methyI- cysteine by transpeptidases. In turn, S-methy~cysteine is metabolized to methanethiol through methy~thioacetic acid. Methanethiol is oxidized to for- maldehyde and hydrogen sulfide and then to formate and sulfate (Kornburst end Bus 1983~. The toxicity of methyl bromide might result from the direct methylation of cellular macromolecules or the toxic metabolites methanethiol, formaldehyde, or hydrogen sulfide. Exposure to methyl bromide has induced glutathione

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TOXICOLOGYA ND HAZARD INDENTIFICATION 15 depletion in some in vivo and in vitro studies. It is possible that in cases ot cumulative exposure to chemicals involving glutathione conjugation, less glutathione would be available to detoxify methyl bromide, resulting in more acute effects. Reports on the effect of glutathione depletion or administration of the glutathione precursor (N-acety~cysteine) on the toxicity of methyl bro- mide are not consistent and vary with the species and endpoint examined (Gamier et al. 1996~. The reasons for these inconsistencies are not clear. However, it is evident that the interactions of methyl bromide with glutathione or the metabolites of methyl bromide play a role in its toxicity (Gamier et al. 1996). A genetically determined polymorphism in glutathione transferase activity has been reported in humans (Hallier et al. 1993~. Humans can have at least 14 classes of this enzyme with 11 subunits. Approximately 75% of humans have erythrocytes with a form ofthis enzyme, which is selective for the conju- gation of methyl bromide with glutathione (fast conjugators), but about 25% of the population does not have this enzyme phenotype (slow conjugators). The specific class of glutathione transferase responsible for conjugating methyl bromide and its metabolites is thought to be glutathione transferase theta (GST Tl-l) (Gamier et al. 1996~. Ethnic differences in the prevalence of the genotype have been reported (Nelson et al. ~ 995~. In the case of sister chromatic exchanges in blood cells exposed in vitro to methyl bromide, cells from fast conjugators appeared to be protected from the production of sister chromatic exchanges, whereas cells from slow conjuga- tors were not. On the other hand, in a single report involving one fast conju- gator and one slow conjugator, the slow conjugator appeared to have consid- erably fewer and less severe neurological symptoms than did the fast conjuga- tor, indicating that the proximate toxin might have been one ofthe metabolites of methyl bromide conjugated with glutathione as discussed above (Gamier et al. ~ 996~. These very limited data suggest that the ability to quickly conjugate methyl bromide to glutathione might have profound ramifications on whether or not an exposed individual is likely to experience neurotoxic effects or be more susceptible to genotoxic effects. it is possible that the ability to conju- gate methyl bromide with glutathione could influence the dose response for the carcinogenicity, mutagenicity, neurotoxicity, or other toxic effects for cer- tain segments of the population who either possess or lack this polymorphism. Such differences in metabolism and toxic outcomes, if better substantiated, might be a factor in identifying susceptible subpopulations that should re- ceive special consideration in the risk assessment process (Hallier et al. ~ 993~. in addition, animals used in toxicity studies might not mimic the polymorph- isms seen in the human population.

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6 METHYL BROMIDE RISK CHARACTERIZATIONIN CALIFORNIA GENOTOXICITY Methyl bromide is a methylating agent and reacts with cellular macromole- cules. As reviewed in Section IIl.E of the DPR report, methyl bromide is genotoxic in a number of in vitro and in vivo assays. In in vitro studies, it is a direct-acting mutagen in Salmonella typhimurium strains TAl00 and TA1535, Escherichia cold strains Sd-4 and WP2hcr, and in Saccharomyces cerevisiae (Simmon et al. 1977, as cited in DPR 1999; Kramers et al. 1985, as cited in DPR 1999; Moriya et al. 1983; NTP 1992; DjalaTi-Behzad et al. 1982; Mortelmans and Sheperd 1980, as cited in DPR 1999~. It produces a dose- dependent induction of sex-linked recessive lethality in Drosophila melano- gaster (Kramers et al. 1985, as cited in DPR 1999; McGregor 1981, as cited in DPR 1999) and forward mutations in a mouse lymphoma assay (Kramers et al. 1985, as cited in DPR 1999~. The DPR report states that, ofthe gene muta- tion studies summarized above and specifically cited in the document, only the study that used Saccharomyces cerevisiae was considered acceptable by DPR. DPR does not provide an explanation for this assessment, although the subcommittee finds that such an evaluation should be justified. The subcom- mittee is particularly concerned that DPR has implied that the gene mutation studies performed by National Toxicology Program (NTP) are not satisfac- tory. If this is the case, DPR should provide a detailed explanation as to why these tests are not acceptable. DPR evaluated several in vivo assays. In female mice exposed to methyl bromide by inhalation at ~ 00 and 200 ppm for 10 days, an increase of micro- nuclei was observed (NTP 1992~. No increase of micronuclei was seen fol- Towing intraperitoneal injection of methyl bromide at up to 123 mg/kg (Put- nam and Morris ~ 99 i, as cited in DPR ~ 999~. Dominant lethal mutations were not observed in rats exposed to methyl bromide by inhalation. A dose-related increase in the frequency of sister chromatic exchanges in bone marrow of female mice exposed to concentrations of methyl bromide at 100 or 200 ppm for 10 days was reported (NTP 1992~. However, this was not seen in another study in which mice were exposed to a concentration of methyl bromide at 120 ppm for 12 weeks (NTP 1992~. DNA abducts were detected in liver, Jung, stomach, and forestomach of rats exposed to high concentrations of methyl bromide at 130 to 260 ppm by inhalation (Gansewendt et al. 1991~. Bentley (1994, as cited by DPR 1999) exposed rats for 5 days to concentrations of methyl bromide up to 250 ppm and examined testicular damage. DPR deter- mined that at 250 ppm, methyl bromide was considered positive for genotoxic potential to the DNA of testicular cells, whereas, at Tower exposure levels, results were inconclusive. Because of the lack of raw data concerning this study, both DPR and the subcommittee consider this study to be unacceptable.

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TOXICOLOGYAND HAZARD INDENTIFICATION ~ 7 Although the available literature on the genotoxicity of methyl bromide appears to have been adequately reviewed in the DPR report, there is no dis- cussion of the human erythrocyte polymorphism (Hallier et al. ~ 993~. In addi- tion, there is no mention of the significance of the genotoxicity of methyl bro- mide in relation to the potential carcinogenicity of methyl bromide. Because methyl bromide is a direct-acting mutagen, especially in in vitro systems, some discussion for its lack of correlation with carcinogenicity should be pre- sented in the DPR report. ACUTE TOXICITY In Section ITT.B of the DPR report, four rat, one mouse, two dog and one guinea pig inhalation toxicity studies were examined for acute effects, as were one rat and one rabbit inhalation developmental studies, and one dog oral study. In addition, human effects from dermal exposures were analyzed. In general, dogs appear to be the most sensitive species of laboratory animals studied. The study by Newton (1994b) in which dogs were exposed via inha- lation for 7 hr/day for 34 days over 6 weeks, was considered by DPR as a crit- ical study for neurotoxicity endpoints. The NOAEL and Towest-observed- adverse-effect level (LOAEL) were 103 and 158 ppm, respectively, based on no recorded adverse effects until 9 days of exposure at 103 ppm, and de- creased activity on the second day of exposure at 158 ppm, and brain lesions following 6 days of exposure at 158 ppm. This study appears to have been appropriate for selecting a NOAEL because the neurotoxicity endpoint is highly relevant to humans, and the critical signs of neurotoxicity (decreased activity) seen at the LOAEL were noted on the second day of exposure, and not following multiple days of exposure. In addition, most of the other stud- ies would have identified higher NOAELs. It should be noted that the DPR document is quite confusing with respect to its discussion of the acute critical studies. There is substantial discussion of acute effects under the section on acute toxicity, but the critical study (New- ton 1994b) is discussed under the section on subchronic toxicity, probably because this was also the critical study for the subchronic NOAEL. The sub- committee recommends that DPR revise its acute toxicity section to include a discussion of these observations so that there is greater clarity on the end- points selected for the critical study for the acute NOAEL. In addition, the observation of toxicity after the single dose was a functional one, that is, de- creased activity, which is likely to be a more sensitive manifestation of toxic- ity than pathological lesions. A similar NOAEL was noted in a rat inhalation study with a single 6-fur exposure (Driscoll and Huricy 1993~; however, the

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15 METHYLBROMIDE RISK CHARACTERIZATIONIN CALIFORNIA LOAEL was considerably higher (350 ppm, based on deficits in a functional observational battery, which is also a functional endpoint similar to that of the critical dog study). Further discussion of the critical dog study (Newton 994b) is found in the neurotoxicity section of this chapter. The greater sensitivity of the dog compared with the rat makes the dog study the appropriate critical study for regulatory purposes. Also, inhalation exposure is more likely for acute effects than is oral exposure, and therefore, an inhalation study is more appropriate to designate as the critical study than an oral study. Human dermal exposure data were too limited to consider for regulatory purposes. Lower NOAELs and LOAELs were observed in some in vivo and in vitro studies (Honma et al. 1987 and 1991~; however, a critical analysis of these data by DPR revealed a number of inconsistencies in the data sets, rendering them unsuitable as critical studies. The subcommittee concurs with DPR that the Honma et al. studies (1987, 1991) are not appropriate for regulatory purposes because these studies examined neurochemical endpoints that are not necessarily indicative of toxicity. In addition to neurotoxicity, DPR also considered developmental toxicity for its acute exposure risk assessment, based on the assumption that only a single exposure at a critical time is necessary for the induction of a develop- mental effect. This is a well-accepted principle in developmental toxicology that has been incorporated into the U.S. Environmental Protection Agency's (EPA's) Guidelines for Developmental Toxicity Risk Assessment (1991~. A developmental toxicity study in rabbits was considered by DPR as the critical study for developmental endpoints (Breslin et al. 199Ob). The LOAEL and NOAEL were 80 and 40 ppm, respectively, with gallbladder agenesis, fused sternebrae, and reduced fetal weights observed at 80 ppm. This study was considered appropriate by the subcommittee for selection of the NOAEL for acute toxicity for women of childbearing age in the workforce and in the gen- eral population. This study is discussed in more detail in the developmental toxicity section. SUBCIlRONIC TOXICITY A large number of studies were available for analysis by DPR for sub- chronic toxicity involving several species and dosing protocols. Eight rat, three mouse, seven rabbit, two dog, one guinea pig, and two monkey inhala- tion studies, and four rat oral studies were considered by DPR. As was true for acute toxicity, it is most appropriate to consider inhalation exposure be- cause this route most accurately reflects the primary route of repeated expo- sures in humans. Because there is a convincing database on neurotoxic effects

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TOXICOLOGY AND HAZARD INDENTIFICATION 19

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20 METHYL BROMIDE RISK CHAR 4CTERIZATIONIN CALIFORNIA Biogenics Corporation 1986~. Thus, the LOAEL seen in rats exposed to 30 ppm for 132 to 145 days suggests that this species is less sensitive to methyl bromide than rabbits, which had a NOAEL of 20 ppm for a 7-day exposure. Another rat study also identified a lower NOAEL based on biochemical mea- sures (i.e., decreased brain monoamine levels) (Honma et al. ~ 982~; however, because it is unclear whether these biochemical changes genuinely reflect or correspond with any functional changes, and because of the concerns of de- sign and consistency in the results from the series of Honma et al. studies, this is not a suitable study to select as a critical study. Therefore, the subcommit- tee concurs with the selection of studies by DPR as the critical studies for sub- chronic toxicity. ClIRONIC INHALATION AND ONCOGENICITY Two chronic inhalation studies were reviewed by DPR for the assessment of the chronic toxicity and oncogenicity of methyl bromide (Section TTT.D). The first study (Reuzel et al. 1987, 1991) involved exposure of male and fe- maTe Wistar rats to methyl bromide concentrations at 0, 3, 30, or 90 ppm for 6 hr/day, 5 days/wk, for 29 months. Each exposure group comprised 90 males and 90 females with interim sacrifices of 10 rats/sex/group at 13, 52, and 104 weeks. Body weights, clinical signs, hematology, biochemistry, and gross and microscopic effects were examined at these times. Exposure to 90 ppm was clearly toxic with early mortalities reported (not statistically significant at the terminal sacrifice); body weights of both sexes in this exposure group were significantly lower than those of the respective control groups throughout most of the study. At terminal sacrifice, effects on the heart were apparent in the 90-ppm exposure group. Statistically significant higher incidences of heart lesions in this group included cartilaginous metaplasia (males only), moderate to severe myocardial degeneration (females only), and thrombi (males and females). Myocardial degeneration also occurred in aged control rats. Therefore, when total incidences of myocardial degeneration were con- sidered, incidences in the control and 90-ppm groups were similar for both sexes. At the 29-month sacrifice, the 3-ppm concentration was a NOAEL for endpoints of body weight and absolute and relative brain weight. The sub- committee noted that the absolute brain weights were significantly reduced for both sexes in the 3- and 30-ppm groups, but did not consider the reductions biologically significant (98% of control values for both sexes in the 3-ppm group, and 94/O and 96% of control values for mates and females, respec- tively, in the 30-ppm group), especially in the absence of histological corre- lates. DPR reevaluated the absolute brain-weight data by combining inci-

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TOXICOLOGYAND HAZARD INDENTIFICATION 21 dences at 29 months with data from animals that died during the study. With the reevaluation they found that the NOAEL remained 3 ppm for the reduc- tion in absolute brain weight. Basal cell hyperplasia of the olfactory epithelium was present in both males and females in a dose-related manner at the 29-month terminal sacrifice, but not at the other time points. Incidences were statistically significant in the 3- ppm group at the 29-month terminal sacrifice when total incidences were con- sidered (13 of 48 and 19 of 58 in males and female subgroups, respectively, compared with 4 of 46 and 9 of 58 in the respective control subgroups). In the 3-ppm group, the majority of lesions were characterized as "very slight"; the severity of these lesions became greater ("slight" to "moderate") in the higher exposure groups. These lesions were not present in either males or females in the 3-ppm group at the 52-week interim sacrifice and were not sig- nificantly elevated over those ofthe respective control groups at the 04-week interim sacrifice. But these lesions were present in the female control group at the 104-week interim sacrifice at an incidence (4 of 10, 40/O) similar to that in the female 3-ppm group at terminal sacrifice (19 of 4S, 40/O). At terminal sacrifice, the incidence of total olfactory lesions in males in the 3-ppm group was 13 of 48 (27%) compared with 4 of 46 (9%) in the male control group. The subcommittee made the following observations regarding the nasal lesions: (~) they increased in control rats in an age-dependent manner from 12 to 29 months; (2) all but one of the lesions were classified as slight or very slight in the 3-ppm group; and (3) one moderate lesion of the nasal mucosa was observed in controls at the 24-month observation (accompanied by a 40% incidence of total lesions in control females). The incidence in the control males at 24 months was 3 of 10 (30%~. Therefore, the effect in the 3-ppm group at the 29-month te~inal sacrifice, although dose-related and statisti- cally significant, must be considered slight or equivocal. This study was well conducted, used a relevant route of administration, used adequate numbers of rats of both sexes, and examined all relevant endpoints of methyl bromide toxicity. In addition, the same critical endpoint of more pronounced lesions, was observed in rats exposed for shorter periods of time at higher concentra- tions (Eustis et al. 1988; Hurtt et al. 1988~. Therefore, the subcommittee con- curs that this study should be the critical study for the chronic risk assessment. A separate chronic RfC for children based on this study is not necessary be- cause the endpoint is not applicable or relevant to children. The study shows that the endpoint occurs in aged rats; children exposed throughout their child- hood will not show this particular endpoint. The second chronic inhalation study reviewed by DPR was conducted by the National Toxicology Program (NTP 1992~. This chronic study with BSC3F1 mice included neurobehavioral evaluations at 3-month intervals and

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22 METHYL BROMIDE RISK CHARD CTERIZA TION IN CA LIFORNIA was an equally suitable study for derivation of the RfC. Tn this study, each group of 70 mate and 70 female mice was exposed to concentrations of methyl bromide at 0, 10, 33, or 100 ppm for two years the typical duration of chronic studies. Sixteen mice (eight males and eight females) per group were used for neurotoxicity testing only. Interim sacrifices of 10 mice/sex/ group took place at 6 and ~5 months. The exposure to 100 ppm was discon- tinued after 20 weeks because of neurotoxicity and early mortalities. This study identified the same organ and tissue endpoints as the Reuzel et al. (1987, 1991) study, the nose, heart, and brain, and also included an endpoint for affected bone. Aside from increased mortality in the lOO-ppm dose group, statistically significant LOAELs and NOAELs for target organ effects were cerebellar and cerebral degeneration, 100 and 33 ppm; myocardial degener- ation and chronic cardiopathy,lOO ppm and 33 ppm; sternal dysplasia, 100 ppm and 33 ppm (increased but not statistically significant for either mates or females over controls in the 33-ppm group); and olfactory metaplasia or ne- crosis, 100 ppm and 33 ppm. it was noted that, similar to results observed in rats at the 3-ppm exposure group in the Reuzel et al. (1987, 1991) study, no olfactory lesions were present in mice at the end of 24 months. DPR identified a LOAEL of 10 ppm for neurotoxicity in the NTP (1992) study based on statistically decreased locomotor activity at 6 and 12 months. The subcommittee disagrees with DPR that the LOAEL for neurotoxicity is 10 ppm. A statistically significant decrease occurred at only ~ of ~ time peri- ods for each sex (6 months for males and 12 months for females) and these decreases were offset by random nonstatistically significant increases over control values at other test times. The authors of the NTP study found "no consistent neurobehavioral differences in animals from the two lower dose groups" (10 and 33 ppm). Therefore, the LOAEL for neurotoxicity is 100 ppm. The NTP Peer Review Panel concurred with the findings of the authors of the NTP study with one member advising caution in the interpretation of the behavioral and functional neurotoxicity results. The Peer Review Panel comments are incorporated into the NTP report. DPR also reviewed the chronic inhalation study of Gotoh et al. (1994, as cited in DPR 1999~. This study was not considered acceptable by either DPR or the subcommittee because the study was reported in summary form and individual data were not available for evaluation. The t~vo-generation repro- duction study by American Biogenics Corporation (1986) can also be consid- ered when evaluating chronic toxicity. However, the NOAEL and LOAEL of 3 ppm and 30 ppm, respectively, for reduced growth of neonatal rats were higher than in the Reuzel et al. (1987, 1991) study. In addition to the two chronic inhalation studies, DPR reviewed four dietary studies. One was a study in which rats were administered encapsu-

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24 METHYL BROMIDE RISK CHARACTERIZATIONIN CALIFORNIA

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TOXICOLOGYA ND HAZARD INDENTIFICATION 25 In addition to the effects on fertility, effects on offspring body weights, or- gan weights, and brain weight and dimensions are discussed in Section ITT.F. ~ of the DPR report. These should also be mentioned in Section ITT.G under developmental effects. The significant, dose-dependent reductions in the body weights during lactation of the offspring of all four mating trials might be due to gestational or lactational exposure to methyl bromide. The latter is suggested by the fact that pup weights were not decreased consistently on post-natal day O or day 4, but were decreased on postnatal days 14, 2l, and 28 in the 30- and 90-ppm groups. In the F2a and F2b progeny, pup weights were also significantly reduced on postnatal days 0, 4, and 7, suggesting that at least some of the effect on pup weight was due to gestational exposure. Methyl bromide exposure of the dams was temporarily halted from gestational day 21 until postnatal day 4, and exposure of the pups was not begun until weaning at postnatal day 28 in all four trials. Therefore, from post-natal days 4 through 28 the pups would have been exposed to methyl bromide only via the breast milk. A literature search revealed no data on the excretion of methyl bromide in breast milk; however, as a lipophilic molecule, it might well be excreted in breast milk. (Data are available that suggest that bromide, that is, sodium bromide, will be excreted in milk tDisse et al. 19963.) The body weight differences did not remain significant in the FIb animals during adulthood. None of the Fla, F2a, or F2b animals was followed into adult- hood. The body weights of the F0 females were not affected by methyl bro- mide exposure beginning in early adulthood (62 days old), but the 90-ppm F0 mates showed decreased body weights compared with controls from exposure week 3 (around 80 days old) until the final sacrifice (about 250 days old). The offspring of the Fib and F2b mating trials and the F0 adult mates showed dose-related reductions in brain weights, which were significant for the 90-ppm F0 males, the 90-ppm FIb males and females that were sacnficed as adults, and the 90-ppm F2b females sacrificed at 28 days. Unfortunately, brain weights and other organ weights were not measured in the Fla or F2a offspring. Brain weights were not significantly reduced in the subset of FIb offspring that were sacrificed at 28 days ~ = 0.17, analysis of variance fANOVA] performed by the subcommittee; see Table 2-2~. Cerebral cortex widths were measured in the 0- and 90-ppm groups of the adult F0 and FIb animals, and were significantly reduced only in the 90-ppm FIb animals. Ab- solute organ weights (heart, kidney, liver), but not organ weights adjusted for body weight, were significantly reduced in the 90-ppm F2b female progeny and nonsignificantly reduced in the male F2b progeny (kidney, liver, testis). Taken together, the cortex width data, the body and organ weight data, and the fertility data suggest that the developing rat is more sensitive to methyl bro- mide toxicity than adults are. The subcommittee concurs with DPR that the

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26 METHYL BROMIDE RISK CHAR 4CTERIZATIONIN CALIFORNIA TABLE 2-2 Brain-Weight Summary for Fib WeanTings Sacrificed at 28 Days, by Exposure Level Flb weanlings O ppm 3 ppm 30 ppm 90 ppm Malesa 1.51 +0.14g 1.5210.11 g 1.47~0.10g l.50~0.09g Femalesa 1.48 ~ 0.09 g 1.42 ~ 0.12 g 1.41 ~ 0.12 g 1.45 ~ 0.06 g aNo statistically significant differences by ANOVA. developmental NOAEL for this study is 3 ppm, based on significant body- weight reductions in the offspring of the 30- and 90-ppm groups. The second reproductive study (Kaneda et al. 1993) discussed in Section ITI.F.2 of the DPR report was designed to test the effects of methyI-bromide- fumigated feeds. Because the feeds were allowed to aerate for 21 days after fumigation, the doses of methyl bromide were quite Tow (maximally 200 ng/kg/day, actual doses not determined) compared with the bromine doses (about 1 10 to 730 ,ug/kg/day). No effects on mating index or fertility index were observed. The concluding sentence of this paragraph, is confusing "since the actual methyl bromide concentration in each dose is not known, it is not possible to determine whether the effects were due to bromine or methyl bromide" (DPR 1999, p. 72~. It would be more accurate to say that the study cannot be used to establish NOAELs for reproductive effects for methyl bromide because the actual doses of methyl bromide were not determined. Moreover, the inhalation route of exposure is more relevant to humans. l:n addition to the two reproductive studies reviewed by DPR, there is an- other study (Sikov et al. 1981), reviewed by DPR as a developmental toxicity study, which provides some additional information about the reproductive effects of methyl bromide. The report describes a study of pregestational and gestational inhalation exposure of female Wistar rats to nominal concentra- tions of methyl bromide at 0, 20, and 70 ppm for 7 Friday, 5 days/wk. The pregestational exposures occurred for 3 weeks before mating with untreated males. No significant effects were observed on fertility rates (92% to 100/O in the exposed groups compared with 98/O in the controls), on corpora lutea per dam, implants per litter, or implants per corpus luteum. These data are con- sistent with the results of the inhalation study discussed above (American Biogenics Corporation 1986; Hardisty 1992, as cited in DPR 1999; Busey 1993, as cited in DPR 1999) that found no effects on fertility indices of the F0 generation exposed for up to 105 days before mating. The subcommittee found DPR's discussion of the reproductive studies of methyl bromide to be somewhat contradictory. In Section II of the DPR re- .

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ToXICOLOGYA ND HAZARD INDENTIFIC~TION 27 port, DPR found that methyl bromide is a direct reproductive toxicant; how- ever, in Section rv, in its discussion of the risk assessment of methyl bromide, DPR suggested that the reduced fertility seen in the American Biogenics Cor- poration (1986) study might be the result of developmental effects on the re- productive system, resulting in altered reproductive function in adult life. The subcommittee concurs with DPR's latter conclusion that, taken together, the results ofthe Sikov et al. (1981) and American Biogenics Corporation (1986) studies suggest that methyl bromide might affect the development of the re- productive system, but the subcommittee does not agree that the studies sup- port DPR's conclusion that methyl bromide is a direct reproductive toxicant in adult animals. DEVELOPMENTAL TOXICITY Four inhalation and two oral exposure studies in two species were reviewed by DPR for the assessment of the developmental toxicity of methyl bromide (Section TIT.G). The developmental aspects of the rat inhalation study (Amer- ican Biogenics Corporation 1986; Hardisty ~ 992, as cited in DPR I 999; Busey 1993, as cited in DPR 1999) discussed in Section TII.F. ~ of the DPR report should also have been discussed in Section lII.G. The inhalation study described above (Sikov et al. 1981) also examined developmental endpoints in rats. This study examined the effects of exposure to methyl bromide at 0, 20, and 70 ppm before or during gestation. Minimal maternal toxicity, in the form of reduced gestational body weights on some gestational days in the dams exposed to 70 ppm during gestation, was observed. The investigators also observed higher, although not statistically significant, rates of various ossification defects in the rat fetuses whose dams were exposed to methyl bromide, compared with those who were not. For the most part, these did not show a clear dose-related pattern. However, for the supraoccipital, interparietal, and parietal bones of the skull, the percent of lit- ters and the percent of fetuses that displayed ossification defects were higher in the 20- and 70-ppm gestationally exposed groups than in the controls or in the groups with only pregestational exposure (see Table 2-3~. Although such ossification defects are often considered to represent variants when they are consistently elevated in the experimental groups in a manner that suggests dose-dependency, there should be concern about subtle developmental effects (EPA 1991; Sikov et al. 1981~. The skull ossification defects, as well as the total skeletal anomalies and total ossification defects listed in Table 2-3, fulfill the consistency criterion (all gestationally exposed groups have higher rates

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28 METHYL BROMIDE RISK CHARA CTERIZA TION IN CALIFORNIA than the controls), but the dose-dependency criterion is only fulfilled for supraoccipital ossification and total ossification defects. lEn summary, these data suggest that methyl bromide might be a developmental toxicant at doses as Tow as 20 ppm; however, they do not unequivocally establish it as a devel- opmental toxicant. Therefore, this study should not be used to set a `develop- mental NOAEL. The same paper (Sikov et al. 1981) also described an inhalation study in rabbits using similar exposure regimens that were planned to continue for ges- tation days ~ to 24, without pregestational exposure. All of the does in the 70- ppm group manifested severe toxicity, and all but one of them died, even though the exposures were terminated early, on gestation day ~ 5. The NOAEL for maternal toxicity was 20 ppm. No adverse effects were observed in the offspring of the 20-ppm group or of the one survivor in the 70-ppm group. However, because the exposures were terminated early in all groups, this study was not considered to be a valid study of developmental toxicity by DPR. Nonetheless, it provides evidence that gestational exposure to 20 ppm methyl bromide from gestation days ~ to 15 does not cause developmental toxicity in rabbits. TABLE 2-3 Summary of Skull Ossification Defects in Sikov et al. (1981) Rat Substudy Exposure levels in ppm (pre-mating/gestation) Ossification defect 010 20/0 70/0 0/20 0/70 20/20 19.4 4.3 19.4 3.9 20/0 70/0 11.8 0 1.9 <0.4 13.9 2.1 0/20 70/70 Supraoccipital Interparietal 13.5 14.7 2 gb 2 3 6.5 1.1 29.0 7.0 5.3 0.8 21.1 3.7 19.4 8.7 38.9 8.7 Parietal 8.1a 5.9 11.1 12.9 13.9 28.9 19.4 1.9 1.4 4.1 4.3 2.6 8.2 4.8 Total skeletal anomalies 0.18C 0.30 0.19 0.22 0.26 1.16 1.4 1.2 1.3 1.7 Total ossification defects 0.03e 0.06 0.07 0.07 0.11 0.24f 0.48 0.61 0.68 0.81 aPercent litters with defect bPercent pups with defect CNumber of skeletal anomalies/fetus Number of skeletal anomalies/litter Number of ossification defects/fetus Number of ossification defects/litter

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TOXICOLOGYAND HAZARD INDENTIFICATION 29 A preliminary study by Breslin et al. (1990a) examined the effects of inha- lation exposure in rabbits to methyl bromide at 0, 10, 30, or 50 ppm (Part 1) and at 0, 50, 70, and 140 ppm (Part 2) for 6 hr/day on gestation days 7 to 19. The exposure regimens used in both Parts ~ and 2 of the study were designed to assess the levels at which maternal toxicity and embryo lethality might oc- cur. No toxicity was observed in the dams or in the offspring in Part I. In Part 2, the dams exposed to 140 ppm showed severe neurotoxicity with meningeal inflammation and midbrain necrosis, and were sacrificed early on gestation day 17. The dams exposed to 70 ppm exhibited statistically significant de- creased body weight at gestation day ~ 6 and decreased weight gain on gesta- tion days 13 to 16 only. Information on maternal weight gain corrected for gravid uterine weight was not given. Therefore, the subcommittee cannot judge whether the effects on maternal weight gain represented maternal or fetal toxicity or both. This study was appropriately considered supplemental by DPR. A definitive study by Breslin et al. (199Ob) exposed rabbits to methyl bro- mide at 0, 20, 40, or 80 ppm (Part 1) and at 0 or 80 ppm (Part 2) for 6 hr/day from gestation days 7 to 19. Part 2 of the study was designed to determine whether observations made in Part ~ could be replicated. In Part I, at the 80- ppm dose, 3 of 26 does exhibited clinical signs of neurotoxicity beginning on the last day of exposure. One of these rabbits delivered its litter early, on ges- tation day 27. Unfortunately, the brains of these animals were not examined. No maternal neurotoxicity was observed in Part 2, but one doe died of unde- termined causes in the 80 ppm group. Statistically significant decreases in ma- ternal weights in the 80-ppm group were observed on gestation days 13 and 16 in Part 1, but not at all in Part 2. The two animals with the largest weight Tosses in Part 1 were also two of the three that displayed neurotoxicity. Statis- tically significant decreases in maternal weight gains were observed for the interval gestation days 13 to 16 in Part ~ and for the interval gestation days 10 to ~ 3, 7 to 20, and 0 to 28 in Part 2. However, fetal and gravid uterine weights were also significantly decreased in Part 2 and gravid uterine weights were nonsignificantly reduced in the 40- and 80-ppm groups compared with con- trols in Part I, suggesting that the reduced maternal weight gain might repre- sent a developmental effect rather than or in addition to a maternal effect. In Section T.C of the DPR report (1999, p. 6), maternal weight gain corrected for gravid uterine weighta better indicator of strictly maternal toxicity was described as being unaffected by treatment; however, it does not appear in the results sections of the Breslin et al. (199Ob) report. If DPR calculated this parameter from data in the study, the results of the calculation should be in- cluded in an appendix. Fetal malformations were observed primarily in the 80-ppm groups in both

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30 METHYL BROMIDE RISK CHARA CTERIZA TION IN CALIFORNIA Parts ~ and 2 (Breslin et al. ~ 990b). The total rate of malformations in Part ~ was 14.5% in the 80-ppm fetuses, compared with 2.~% in the sham-treated control fetuses. In Part 2, a similar rate was observed in the 80-ppm fetuses (14.1%), but a much higher rate was observed in the sham-treated controls (12.3%) compared with Part ~ and compared with the naive controls in Part 2 (5.9%~. Malformations included statistically significant increases in galIblad- der agenesis and fused sternebrae in Part 1. These defects were observed in fetuses from dams with and without neurotoxicity. A similar incidence of gallbladder agenesis was observed in Part 2, although it did not reach statisti- cal significance with a smaller number (N) of animals. Although it is not considered to be a major malformation by some experts (Ty} 1991; OEHHA 1993), several arguments favor considering the increased incidence of gallbladder agenesis in the Breslin et al. (199Ob) study to be evi- dence of developmental toxicity of methyl bromide. First, it represents the failure of an entire organ to form. Second, the 8.2% incidence at 80 ppm (af- fected fetuses/total fetuses) of galIbladder agenesis in Part ~ was statistically significantly elevated over the control incidence of 1.~%. The incidence of 4.3% at 80 ppm in Part 2, although not statistically significant, was nearly 5 times higher than the 0.9/O incidence observed in the sham-treated control group. Moreover, the incidence of galIbladder agenesis in the methyI- bromide-treated fetuses is much higher than the 0.09/O to 0. ~ 9/O observed in historically untreated control New Zealand White rabbits from Dow Chemical Company (where the Breslin study was performed), WTE Research Laborato- ries, the Middle Atiantic Reproduction and Teratology Association, and StadIer et al. (1983) (summarized in Tables 5-8, Appendix B of the DPR re- port). Third, the same laboratory which performed the Breslin et al. (199Ob) study (Dow Chemical Company) has reported gallbladder agenesis as a possi- ble treatment-induced effect of other test compounds (DPR 1999, Appendix B,p. 179~. Kaneda et al. (1998) studied the developmental toxicity of oral methyl bro- mide exposure in rats (at 0, 3, 10, or 30 mg/kg/day, gestation days 6 to 15) and rabbits (at 0, 1, 3, or 10 mg/kg/day, gestation days 6 to 18~. Maternal tox- icity in the form of erosion and thickening of the stomach wall and adhesions between stomach and other organs was observed in the rats given 30 mg/l~g. Fetuses from that group exhibited statistically nonsignificant increases in microphthaimia and skeletal variations. No maternal effects were noted in the rabbits. In the fetuses, skeletal malformations were observed more frequently in the methyI-bromide-treated groups than in controls, but the increases were not dose-dependent or statistically significant. Because the oral route of expo- sure to methyl bromide is less significant than the inhalation route for humans, these studies were appropriately considered supplemental informa-

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TOXICOLOGY AND HARM ]NDENTIFICA TION 31 lion by DPR. To better be able to compare the inhalation with the oral devel- opmental studies, it would be useful to calculate the estimated absorbed doses for these studies. In conclusion, DPR found that the currently available evidence suggests that methyl bromide might be a developmental toxicant by the inhalation route in two species. In rats, this evidence includes significant, dose-depend- ent reductions in pup body and brain weights and cerebral cortex widths and a nonsignificant reduction in fertility in gestationally exposed offspring (Arner- ican Biogenics Corporation 1986; Hardisty 1992, as cited in DPR ~ 999, Busey 1993, as cited in DPR 1999~. As this was a two-generation study, it is not possible to determine the critical period or periods for exposure for these ef- fects. However, the patterns of occurrence are consistent with developmental effects. In a separate rat developmental study, a nonsignificant, but consis- tent, increase in the incidence of skull ossification defects was observed in gestationally exposed animals (Sikov et al. 1981~. In rabbits, the evidence for developmental toxicity includes gallbladder agenesis, reduced fetal weights, and increased frequency of fused sternebrae (Breslin et al. 199Ob). Taken alone any of these studies could be considered equivocal; however, taken to- gether, the subcommittee agrees with DPR that they suggest that methyl bro- mide might be a developmental toxicant. NEUROTOXICITY There is ample evidence that neurotoxicity is the most prominent type of toxicity elicited by methyl bromide in humans. Therefore, selection of neuro- toxic endpoints for the critical studies in the risk characterization is most con- sistent with protection of humans from the adverse effects of methyl bromide. Many of the studies did not include neurobehavioral analysis as their end- points. The critical studies selected for acute and subchronic exposures were based on functional endpoints, and thus appear to reflect the most sensitive indicators measured. The subcommittee finds that a change in behavior (of the dogs in the Newton tI994b] study) is a functional one, which might be more sensitive than a pathological endpoint, because the functional change might be due to a biochemical deficit (or change) even though a histological change has not occurred. As mentioned above, some of the observations in the subchronic study (Newton 1994b) were equivocal, such as the two non- responsive dogs at the lowest concentration tested; there were low animal numbers in this experiment, not a good dose-response relationship, and the observations were outside the standardized protocol. Nevertheless, the obser- vation of low levels of responsiveness is very consistent with the nervous sys-

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32 METHYL BROMIDE RISK CHARACTERIZATIONIN CALIFORNIA tem depression observed in a number of the animal studies, and therefore is believed to reflect a true neurotoxic response. One possible exception to DPR's use of the most sensitive functional data available as the critical study would be DPR's rejection of the Honma et al. studies (1987, 1991) on mechanisms for use in the risk characterization. These studies investigated neurochemical endpoints in an attempt to elucidate the mechanisms by which methyl bromide exerts its neurotoxic effects, and implicated primarily the monoaminergic and catecholaminergic systems as potential targets. However, changes in neurochemistry alone do not necessar- iTy indicate toxicity (Overstreet et al. ~ 974), and these studies did not appear to correlate these neurochemical changes with functional deficits; therefore, the significance ofthese neurochemical changes cannot be discerned. In addi- tion, there were serious concerns raised by the DPR staff about the design of these experiments because the descriptions of design and methods were overly brief, and it was not clear to the staff whether confounders that could have caused the observed effects had been ruled out. Such issues as contradictions in the data sets obtained or lack of clear dose-response relationships leave these data sets suspect with respect to their suitability for use in a risk charac- terization. Therefore, the subcommittee concurs with DPR's conclusions that these Honma et al. studies, although suggesting highly sensitive endpoints, are not suitable for use in a risk characterization. Therefore, neurotoxicity appears to be a prominent foe oftoxicity elicited by methyl bromide in a variety of laboratory animal studies and is consistent with evidence of neurotoxicity from human studies. Of particular note are studies indicating neurological deficits occurring in those occupationally ex- posed to methyl bromide at apparently relatively Tow exposure levels (Anger et al. 1986~; these human observations suggest toxic endpoints that need to be considered for a health-protective risk characterization. SELECTION OF CRITICAL EFFECTS FOR ACUTE TOXICITY The use of a developmental toxicity study for the assessment of the risks of acute exposure to methyl bromide is a reasonable one, given the principle that a single gestational exposure is sufficient to produce an adverse developmen- tal effect, and in light of the large numbers of women of childbearing age in the workforce. In fact, the EPA Guidelines for Developmental Toxicity Risk Assessment (EPA 1991) state that data from reproductive and developmental toxicity studies should be used in the overall assessment of risk of a com- pound. DPR's rationale for using the developmental toxicity studies by Breslin et

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TOXICOLOGYAND Hall ~NDENTIFICATION 33 al. (1 990a,b) for assessment of the risk of acute exposure in women of child- bearing age is outlined in the document and will be briefly summarized here. First, gallbladder development in the rabbit occurs over ~ to 2 days beginning on gestation day ~1.5. Therefore, the assumption that only a single exposure is necessary for the induction of adverse developmental effects is very likely to hold true for this particular effect. Second, the finding of gallbladder agenesis was confirmed when the experiment was repeated. Third, the devel- opmental effects of gallbladder agenesis and fused sternebrae should not be discounted because maternal toxicity occurred in some does. Both defects occurred in offspring of does who did not exhibit neurotoxicity as well as in the offspring of the minority of does who did. In Part 2 of the study, none of the does exhibited neurotoxicity, yet a similar incidence of gallbladder agenesis was observed as in Part I. In addition, signs of neurotoxicity appeared on gestation day ~9, the day 12 ofthe 13-day exposure. Given that gallbladder development would have been completed 5.5 to 6.5 days before maternal toxicity occurred, it is unlikely that this defect was the result of the maternal toxicity. Moreover, it is not clear that the inconsistent maternal weight changes, which were observed in the SO-ppm groups (significantly lower maternal weight gain compared with controls on some, but not all, ges- tational days, primarily in Part 2), represented maternal rather than fetal toxic- ity, because the fetal weights and gravid uterine weights were also signifi- cantly Tower in the 80-ppm group in Part 2. In addition to DPR's arguments, it should be noted that maternal toxicity has been associated with some devel- opmental abnormalities, including fused sternebrae, but not gallbladder agenesis (Khera 1984; Khera et al. 1989~. Moreover, a recent study found no correlation between maternal body-weight change as an indicator of maternal toxicity and various embryo or fetal parameters, including number of anoma- lies per litter and reduced fetal weight (Chahoud et al. ~ 999~. Finally, another interpretation of the occurrence of maternal and fetal toxicity at the same dose level is that the threshold for toxicity of the test compound is similar in mother and fetus. The subcommittee considers there to be several counterarguments to using the Breslin et al. (199Ob) study to determine the critical NOAEL for acute ex- posure in women of childbearing age. One, alluded to above, is that the find- ings of this study, taken on their own, might be considered equivocal evidence for developmental toxicity. The three significant effects fetal weight de- cline, fused sternebrae, and gallbladder agenesis were not statistically con- sistent between Parts 1 and 2 of the stu(ly. Fetal weight was statistically sig- nificantly Tower in Part 2 in the 80-ppm group, but not in Part 1; gallbladder agenesis was statistically significantly elevated only in Part 1; and skeletal examinations were only performed in Part 1. In addition, fused sternebrae are

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34 METHYL BROMIDE RISK CHARACTERIZATIONIN CALIFORNIA considered a morphological variation that occurs in 0.27% to 0.92% of un- treated control New Zealand White rabbit fetuses (DPR ~ 999, Tables 5-7, Ap- pendix B). GalIbladder agenesis is a less common developmental abnormal- ity, but it is also considered a variation by some experts. These arguments against the Breslin et al. (199Ob) study are weakened by the finding of proba- ble developmental toxicity associated with methyl bromide exposure in an- other species, the rat, at nonmaternally toxic doses (American Biogenics Cor- poration 1986; Hardisty 1992, as cited in DPR 1999; Busey 1993, as cited in DPR 1999~. Another counterargument is that developmental studies should not be used for acute exposure risk assessment if appropriately performed acute exposure studies exist for the agent in question. This argument ignores the possibility that fetuses might be more sensitive than adults to a given agent and that developmental effects caused by multiday gestational exposures would theoretically be caused by single exposures as well. Based on the above considerations, DPR's use ofthe Breslin et al. (199Ob) developmental toxicity study to determine the critical NOAEL for acute toxic- ity for workers and residents appears to be a conservative approach, but one that is justified in the absence of additional data that show that a single expo- sure at the time of galIbladder development does not cause galIbladder agenesis.