Dieldrin ( 87% pure) was not found to be teratogenic in pregnant CD rats ( 9 to 25/ group) and CD 1 mice ( 6 to 16/ group) that were administered doses in peanut oil of 0, 1.5, 3.0, or 7 13 Aldrin/ Dieldrin   February 2003 6.0 mg/ kg bw/ day by gastric intubation on days 7 through 16 of gestation ( Chernoff et al., 1975). Fetal toxicity was reported in the mice, as indicated by a significant decrease in the numbers of caudal ossification centers at the 6.0 mg/ kg bw/ day dose level, and a significant increase in the number of supernumerary ribs in one study group at both the 3.0 and 6.0 mg/ kg bw/ day doses. In the second study group, the increase was significant only at the 3.0 mg/ kg bw/ day group. In contrast to these results in mice, exposed rat fetuses evidenced no differences from controls in body weight, mortality, or the occurrence of anomalies. Maternal toxicity in the high dose rats was indicated by a 4l% mortality and a significant decrease in weight gain; similarly, mice receiving 6.0 mg/ kg bw/ day showed a significant decrease in maternal weight gain. A significant increase in liver to body weight ratio in one group of maternal mice was reported at both the 3.0 and 6.0 mg/ kg bw/ day doses. Thus, any evidence of dieldrin's potential teratogenicity was accompanied by concomitant maternal toxicity.

CFW Swiss mice ( 100/ sex) fed 5 mg dieldrin/ kg diet ( purity not reported; approximately equal to 0.75 mg/ kg bw/ day based on Lehman, 1959) for 30 days prior to mating, and then for 90 days thereafter, experienced no adverse effects on fertility, fecundity, length of gestation period, size of first litters, or numbers of young produced per day ( Good and Ware, 1969). The only adverse reproductive effect observed in this study was a slight decrease ( 6%) in mean size of all litters combined.

Virgo and Bellward ( 1975) fed dieldrin ( purity not reported) to uniparous female Swiss Vancouver mice ( 18 to 19/ group) at dietary concentrations of 0, 2.5, 5, 10, 15, 20, or 25 mg/ kg ( 0 and approximately 0.375, 0.75, 1.5, 2.25, 3.0, or 3.75 mg/ kg bw/ day, respectively, based on Lehman, 1959) for a period extending from 4 weeks prior to their second mating through postpartum day 28. Males were exposed only during the 2 week mating period. Significant preparturition mortality was observed in 3.0 and 3.75 mg/ kg bw/ day females ( 89 and 56%, respectively), while fertility was decreased in the 1.5 and 2.25 mg/ kg bw/ day groups. Estrus and gestation period were unaffected by the treatment, but litter size was reduced at 3.75 mg/ kg bw/ day. Pre weaning pup mortality was increased from 31% in control animals to 47, 80, or 100% in the 0.375, 0.75, or 1.5 and higher mg/ kg bw/ day groups, respectively. Hyperactivity was exhibited by dams exposed to 1.5 or more mg/ kg bw/ day, which was a contributing factor to high pup mortality. Some higher dose dams violently shook their pups, ultimately killing them, and others neglected their litters. No gross abnormalities were observed in pups from any dose group.

In a subsequent cross fostering study, Virgo and Bellward ( 1977) fed primiparous female Swiss Vancouver mice ( number/ group not reported in citing references) diets containing dieldrin ( purity not reported) at concentrations of 0, 5, 10, or 15 mg/ kg ( 0 and approximately 0.75, 1.5, or 2.25 mg/ kg bw/ day based on Lehman, 1959) for 4 weeks prior to mating. Nursing was reduced in dams exposed to the two highest doses of dieldrin, although serum progesterone levels, milk production, and the dams' tendencies to build nests or retrieve pups were not adversely affected. When foster dams not exposed to dieldrin nursed pups from the 1.5 mg/ kg bw/ day group, all died within 4 days; the foster dams' own pups evidenced very low mortality and survived until weaning. Similar results were also reported for pups from the 0.75 mg/ kg bw/ day group. 7 14 Aldrin/ Dieldrin   February 2003 Aldrin/ Dieldrin Treon et al. ( 1954) reported increased mortality during the first 5 days of life in offspring from the first mating of a three generation reproduction study, in which rats were exposed to 0.275 mg/ kg bw/ day of either aldrin or dieldrin ( purity not reported). Reduced fertility during the parental generation's first mating was reported at doses of aldrin and dieldrin as low as 1.38 and 0.275 mg/ kg bw/ day, respectively. Subsequent parental matings did not demonstrate reproductive effects in the aldrin exposed groups, while fertility effects in the dieldrin exposed groups failed to exhibit consistent dose related responses. During matings of the offspring, reductions in fertility were not observed at the 0.275 mg/ kg bw/ day doses, but could not be adequately assessed at higher doses due to limited numbers of offspring surviving to be mated.

In a three generation study by Treon and Cleveland ( 1955), groups of Carworth rats ( number/ group not reported) were fed aldrin or dieldrin ( 95% purity) at concentrations of 0, 2.5, 12.5, or 25.0 ppm ( doses of 0 and approximately 0.125, 0.625, or 1.25 mg/ kg bw/ day, respectively, based on Lehman, 1959). Two litters/ generation were produced. No reductions in the numbers of live pups/ litter or pup weights were evident in dams fed any dose of either chemical. However, viability of the offspring during lactation was markedly decreased in the 0.625 and 1.25 mg/ kg bw/ day groups for both chemicals, and slightly to moderately decreased in the low dose groups. Pregnancy rates were reportedly initially reduced at the mid and high doses of aldrin, and at all three doses of dieldrin; this effect, however, gradually disappeared over successive generations.

In a study that examined two litters/ generation over six generations, Keplinger et al. ( 1970) fed Swiss white mice ( 4M to 14F/ group) diets containing aldrin ( purity not reported) at concentrations of 0, 3, 5, 10, or 25 mg/ kg ( 0 and approximately 0.45, 0.75, 1.5, or 3.75 mg/ kg bw/ day, respectively, according to Lehman, 1959). The 3.75 mg/ kg bw/ day group was discontinued due to excessive litter mortality in the few dams reaching gestation. Otherwise, the most pronounced effect reported was a reduction in suckling pup survival at 1.5 mg/ kg bw/ day, and to a lesser degree at 0.75 mg/ kg bw/ day. Similarly, groups of mice were fed diets containing dieldrin at concentrations of 0, 3, 10, or 25 mg/ kg ( 0 and approximately 0.45, 1.5, or 3.75 mg/ kg bw/ day). As with aldrin, the high dieldrin dose was soon discontinued for reasons of excessive litter toxicity, and the 1.5 mg/ kg bw/ day dose was discontinued after the first generation because of low pup survival. At the remaining 0.45 mg/ kg bw/ day dose, no effects on fertility, viability, or gestation were noted. Although a decrease in suckling pup survival was observed in the F 2b litters, a similar decrease also occurred in one of the six control groups.

Ottolenghi et al. ( 1974) exposed pregnant CD 1 mice ( 10/ group) and Syrian golden hamsters ( 41 to 43/ group) to high ( one half the oral LD 50), single oral doses of either aldrin or dieldrin (> 99% purity) in corn oil. Negative control groups consisted of untreated and corn oildosed animals. Mice were exposed on gestation day 9 to aldrin at 25 mg/ kg bw or dieldrin at 15 mg/ kg bw; hamsters on either gestation day 7, 8, or 9 to aldrin at 50 mg/ kg bw, or dieldrin at 30 mg/ kg bw. In mice, the aldrin treatment did not affect fetal survival or weight, but significantly increased the incidence of abnormalities such as webbed feet, cleft palate, and open eyes ( 33% of the live fetuses had malformations). In hamsters, aldrin treatment did cause a reduction in fetal 7 15 Aldrin/ Dieldrin   February 2003 survival and weight, as well as a significant increase in the incidence of the same types of abnormalities that were observed in mice; these effects were less pronounced when treatment was on gestation day 9, rather than on days 7 or 8. In mice, dieldrin produced the same types of abnormalities ( in 17% of the live fetuses) as seen with aldrin and the effects in hamsters were also similar to those described for aldrin with respect to fetal toxicity, malformation types, and degree of severity according to day of treatment.

No apparent effects on the fertility or pregnancy rates were evident in groups of mongrel dogs ( 2/ group, at least 1/ each sex) receiving 0, 0.2, 0.6, or 2.0 mg/ kg bw/ day of either aldrin or dieldrin ( purity 99%) in medicated meatballs for 1 year ( Kitselman, 1953). However, the majority of apparently healthy pups that were delivered from dams in all dose groups of aldrin, and from the mid and high dose groups of dieldrin, died within 3 days postpartum and evidenced degenerative liver and renal tubule changes upon histopathological examination. It should be noted that this study had several limitations with respect to size and design parameters.

In dominant lethal studies, Epstein et al. ( 1972) and Dean et al. ( 1975) reported no unequivocal adverse effects on reproduction subsequent to acute exposure of male mice to aldrin at doses up to 1 mg/ kg bw/ day for a period of 5 days, or to single oral doses of dieldrin ranging from 12.5 to 50 mg/ kg bw.

Inhalation Exposure No studies were obtained that investigated the developmental or reproductive effects of aldrin or dieldrin in animals following inhalation exposure.

Dermal Exposure No studies were obtained that investigated the developmental or reproductive effects of aldrin or dieldrin in animals following dermal exposure.

Other Routes of Exposure Castro et al. ( 1992) reported the effects of prenatal exposure to aldrin on the physical and behavioral developments of rats ( 1 to 21 day old and 90 day old groups). Pregnant female rats ( 10 to 20/ group) were subcutaneously injected with either aldrin ( 1.0 mg/ kg bw) or its vehicle ( 0.9% sodium chloride plus Tween 80), from day 1 of pregnancy until delivery. Pups from the aldrin group evidenced a decreased median effective time ( TE 50) for incisor teeth eruption, and an increased TE 50 for testes descent; other parameters indicative of physical development, such as pinna detachment, development of fur and ears, and eye opening, were not altered. No changes in body weight were observed between control and aldrin treated rats on the day of birth, at weaning, or at 90 days. Prenatal exposure to aldrin produced no changes in aldrin or dieldrin levels in serum, or in cellular and structural organization of cerebral cortex neurons, when tested at 90 days. 7 16 Aldrin/ Dieldrin   February 2003 Johns et al. ( 1998) reported no significant differences in birth weight, sex ratio, day of eye opening, or weight gain between the pups of control and dieldrin treated female rats, which had been intraperitoneally injected daily from E12 to E16 ( embryonic days 12 to 16) with 0, 5, or 10 mg/ kg bw of dieldrin.

7.2.6 Chronic Toxicity Oral Exposure Aldrin Treon and Cleveland ( 1955) administered aldrin in the diet to 40 Carworth rats/ sex at concentrations of 2.5, 12.5, or 25 ppm ( approximate doses of 0.125, 0.65, or 1.25 mg/ kg bw/ day, respectively, based on Lehman, 1959) for a period of 2 years. Forty animals/ sex served as controls. Mortality of the treated rats was greater than that of controls, with 50% surviving in the 2.5 and 12.5 ppm groups and 40% surviving in the 25 ppm group at the end of the experiment.

Fitzhugh et al. ( 1964) fed groups ( 12/ sex/ group) of Osborne Mendel rats aldrin ( purity 99%) in the diet at concentrations of 0.5, 2, 10, 50, 100, or 150 ppm ( approximated doses of 0.025, 0.1, 0.5, 2.5, 5.0, and 7.5 mg/ kg bw/ day, respectively, based on Lehman, 1959) for 2 years. A dose related increase in mortality was observed at dietary levels of 50 ppm or greater. In addition, significant ( p # 0.05) dose related increases in relative liver weights were observed. Histopathological changes observed in the livers of aldrin treated rats were referred to as primarily the characteristic " chlorinated insecticide" lesions that occur only in rodents. These lesions consist of enlarged centrilobular hepatic cells, with increased cytoplasmic oxyphilia, and peripheral migration of basophilic granules. The incidence and severity of these nonneoplastic histologic changes increased with increasing dietary aldrin level. In rats ingesting amounts of aldrin at 50 ppm or higher, distended and hemorrhagic urinary bladders, enlarged livers, and increased incidences of nephritis were reported. The apparent LOAEL for this study was 0.5 ppm ( 0.025 mg/ kg bw/ day), while a NOAEL was not established.

Deichmann et al. ( 1970) fed groups of Osborne Mendel rats ( 50/ sex/ dose) aldrin ( technical grade, 95% pure) for 31 months at concentrations of either 20, 30, or 50 ppm ( 1, 1.5, or 2.5 mg/ kg bw/ day, respectively, based on Lehman, 1959). Groups of 100 rats/ sex served as controls. Survival and body weight gains were comparable between the treated and the control groups, but treated animals exhibited tremors and clonic convulsions. Liver to body weight ratios were increased in males fed 30 or 50 ppm aldrin. Moderate increases ( not dose related) in the incidences of hepatic centrilobular cloudy swelling and necrosis were observed in all aldrintreated male and female rats, but not in the controls. A LOAEL of 20 ppm ( 1 mg/ kg bw/ day) was established by this study, but a NOAEL was not determined.

Groups of Osborne Mendel rats ( 50/ sex/ group) were exposed to 30 or 60 ppm of aldrin ( 95% purity) in the diet ( approximate doses of 1.5 or 3.0 mg/ kg bw/ day, based on Lehman, 1959) for 74/ 80 weeks ( M/ F), followed by 32 to 38 weeks of observation ( NCI, 1978). Pooled controls ( 58M/ 60F from similar bioassays, plus 10M/ 10F concurrent controls) were used for statistical 7 17 Aldrin/ Dieldrin   February 2003 evaluations. While no significant effects of aldrin exposure on mortality were observed, mean body weight gains during the second year were lower than control values. Signs typical of organochlorine intoxication ( hyperexcitability, tremors, convulsions), with frequency and severity increasing, especially during the second year. Routine gross and/ or microscopic evaluation revealed no adverse, non neoplastic respiratory, cardiovascular, gastrointestinal, musculoskeletal, hepatic, renal, endocrine, dermal, or ocular effects resulting from exposure to aldrin.

Aldrin ( technical grade, 95% pure) was administered in the diet for 80 weeks ( followed by 10 to 13 weeks of observation) at concentrations of 4 or 8 ppm ( approximate doses of 0.6 or 1.2 mg/ kg bw/ day, respectively, based on Lehman, 1959) to groups of 50 male mice, and at concentrations of 3 or 6 ppm ( approximate doses of 0.45 or 0.90 mg/ kg bw/ day, respectively, based on Lehman, 1959) to groups of 50 female mice ( NCI, 1978). Pooled controls ( 92M/ 79F from similar bioassays, plus 20M/ 10F concurrent controls) were used for statistical evaluations. In a trend test, a significant ( p = 0.037), dose dependent increase in mortality was observed in females; a similar effect was not observed in males. Hyperexcitability was observed in all exposed groups, with frequency and severity increasing during the second year. Mean body weight was unaffected during the first year, but somewhat lower than control values during the second year. Routine gross and/ or microscopic evaluation revealed no adverse, non neoplastic respiratory, cardiovascular, gastrointestinal, musculoskeletal, hepatic, renal, endocrine, dermal, or ocular effects resulting from exposure to aldrin. A NOAEL was not established because of toxicity at 3 ppm ( 0.45 mg/ kg bw/ day), the lowest dose tested.

Kitselman and Borgmann ( 1952) fed groups of 7 mongrel dogs of both sexes ( number/ sex not specified) either 0.2, 0.6, or 2 mg/ kg bw/ day of aldrin in medicated meatballs, for up to 228 days. The test material was reported to have been 99% pure. Dogs that were administered the 2 mg/ kg bw/ day dose exhibited marked body weight loss, and they all died between days 60 and 90. No treatment related effects were observed in dogs receiving the 0.2 mg/ kg bw/ day dose for 190 days, or in those administered the 0.6 mg/ kg bw/ day dose for 228 days. Based on body weight loss, 0.6 mg/ kg bw/ day and 2 mg/ kg bw/ day were considered to be the NOAEL and LOAEL, respectively, for this study.

In a long term feeding study by Treon and Cleveland ( 1955), beagles ( 2/ sex/ dose) fed diets containing aldrin ( purity 95%) at concentrations of 1 or 3 ppm ( approximate doses of 0.043 to 0.091 or 0.12 to 0.25 mg/ kg bw/ day, respectively, as reported by the authors) for 15.6 months, gained weight at rates similar to control dogs. However, at 3 ppm, significant ( p < 0.05) increases in absolute and relative liver weights were noted. Histopathologic changes, such as fatty degeneration of the liver and vacuolation of renal tubular cells, were also observed in both sexes at the 3 ppm level. At the 1 ppm level, females exhibited vacuolation of the distal renal tubules. The LOAEL for this study was 1 ppm ( 0.043 to 0.091 mg/ kg bw/ day), while a NOAEL was not established.

Fitzhugh et al. ( 1964) administered 0.2, 0.5, 1, 2, or 5 mg/ kg bw/ day aldrin ( purity 99%) to 12 mongrel dogs ( sexes combined), 6 days/ week, for periods of up to 25 months. Each group consisted of one dog/ sex except for the 0.5 mg/ kg bw/ day group, which had one male and three 7 18 Aldrin/ Dieldrin   February 2003 female dogs. All dogs receiving 1, 2, or 5 mg/ kg bw/ day died within 49 weeks; the first death occurred on day 22 in a female administered 5 mg/ kg bw/ day. Prior to death, the animals exhibited body weight loss, dehydration, and convulsions. Slight to moderate fatty degeneration was noted in hepatic and renal tubular cells and reduced numbers of mature erythroid cells were found in the bone marrow. In animals receiving 0.5 mg/ kg bw/ day, clinical signs of toxicity were limited to convulsions in one male dog during the 24th month. Dogs in the 0.2 mg/ kg bw/ day group exhibited no adverse effects. The NOAEL in this study thus appears to be 0.2 mg/ kg bw/ day, based on the absence of clinical signs of toxicity, body weight loss, and histopathological changes. However, the adequacy of this study for establishing a reliable NOAEL is limited by the small number of dogs used.

Dieldrin Groups of Osborne Mendel rats, 12/ sex/ dose, were fed 0, 0.5, 2, 10, 50, 100, or 150 ppm dieldrin ( recrystallized, 100% active ingredient) in their diet for 2 years ( Fitzhugh et al., 1964). These concentrations correspond to doses of 0 and approximately 0.025, 0.1, 0.5, 2.5, 5.0, or 7.5 mg/ kg bw/ day, respectively, based on Lehman ( 1959). Survival was markedly decreased at levels of 50 ppm and above. Liver to body weight ratios were significantly increased at all treatment levels, with females showing the effect beginning at 0.5 ppm, and males at $ 10 ppm. Microscopic lesions were described as being characteristic of chlorinated hydrocarbon exposure. These changes were minimal at the 0.5 ppm level. Male rats, at the two highest dose levels ( 100 and 150 ppm), developed hemorrhagic and/ or distended urinary bladders, usually associated with considerable nephritis. A LOAEL of 0.025 mg/ kg bw/ day, the lowest dose tested, was identified in this study.

Groups of Carworth Farm " E" strain rats ( 25/ sex/ dose level) were fed dieldrin (> 99% purity) in the diet at concentrations of 0, 0.1, 1.0, or 10.0 ppm for 2 years. These doses correspond to doses of 0 and approximately 0.005, 0.05, or 0.5 mg/ kg bw/ day, respectively, based on Lehman ( 1959). At 7 months, the 1 ppm intake level was equivalent to approximately 0.05 and 0.06 mg/ kg bw/ day for males and females, respectively. No effects on mortality, body weight, food intake, hematology or blood, and urine chemistries were reported. At the 10 ppm level, all animals became irritable after 8 to 13 weeks of treatment and developed tremors and occasional convulsions. Liver weights and liver to body weight ratios were significantly increased in females receiving both 1.0 and 10 ppm. Pathological findings, described as organochlorine insecticide changes of the liver, were found in one male and six females at the 10 ppm level. No evidence of tumorigenesis was found ( Walker et. al., 1969). Based on the significantly increased liver weight and relative liver weight reported for female rats, this study establishes a NOAEL and a LOAEL of 0.005 and 0.05 mg/ kg bw/ day, respectively.

Walker et al. ( 1972) administered dieldrin (> 99% pure) to groups of CF1 mice ( 30/ sex/ dose) in the diet for 128 weeks at concentrations of 1.25, 2.5, 5, 10, or 20 ppm ( approximate doses of 0.19, 0.38, 0.75, 1.5, or 3 mg/ kg bw/ day, respectively, based on Lehman, 1959). At the 20 ppm dose level, approximately 25% of the males and nearly 50% of the females died during the first 3 months of the experiment. Palpable intra abdominal masses were detected after 40, 75, or 100 weeks in the 10, 5, and 2.5 ppm treated groups, respectively. At 1.25 ppm, 7 19 Aldrin/ Dieldrin   February 2003 liver enlargement was not palpable and morbidity was similar to that of controls. A NOAEL cannot confidently be established from this study because clinical chemistry parameters were not determined.

Groups of Osborne Mendel rats ( 50/ sex/ group) were exposed to 29 or 65 ppm of dieldrin ( 95% purity) in the diet ( approximate doses of 1.45 or 3.25 mg/ kg bw/ day, respectively, based on Lehman, 1959) for 80 weeks followed by 30 to 31 weeks of observation for the low dose, or for 59 weeks followed by 51 to 52 weeks of observation for the high dose ( NCI, 1978). Pooled controls ( 58M/ 60F from similar bioassays, plus 10M/ 10F concurrent controls) were used for statistical evaluations. While no statistically significant end result effects of dieldrin exposure on mortality were observed, it was perhaps accelerated in treated animals, and mean body weight gains during the second year were lower than control values. Signs typical of organochlorine intoxication ( hyperexcitability, tremors, convulsions) were evident, with frequency and severity increasing, especially during the second year and in high dose animals. Routine gross and/ or microscopic evaluation revealed no adverse, non neoplastic respiratory, cardiovascular, gastrointestinal, musculoskeletal, hepatic, renal, endocrine, dermal, or ocular effects resulting from exposure to dieldrin.

In a related study, groups of Fischer 344 rats ( 24/ sex/ group) were exposed to 2, 10, or 50 ppm of dieldrin (" purified technical grade") in the diet ( approximate doses of 0.1, 0.5, or 2.5 mg/ kg bw/ day, respectively, based on Lehman, 1959) for 104 to 105 weeks ( NCI, 1978). Body weight and mortality were not significantly affected by dieldrin exposure, but signs typical of organochlorine intoxication ( hyperexcitability, tremors, convulsions) were noted in both sexes at the high dose after 80 weeks. As in the previously discussed study, no other significant adverse systemic effects were observed.

Dieldrin ( 95% pure) was administered in the diet for 80 weeks ( followed by 10 to 13 weeks of observation) at concentrations of 2.5 or 5 ppm ( approximate doses of 0.375 or 0.75 mg/ kg bw/ day, respectively, based on Lehman, 1959) to groups of B6C3F 1 mice ( 50/ sex/ group) ( NCI, 1978). Pooled controls ( 92M/ 79F from similar bioassays, plus 20M/ 10F concurrent controls) were used for statistical evaluations. Treatment had no appreciable effect on survival, while weight gains were non significantly lower than control values during the second year. Hyperexcitability, hyperactivity, fighting, and tremors were found to be treatment related, and were first observed in males, then later in females. Routine gross and/ or microscopic evaluation revealed no adverse, non neoplastic respiratory, cardiovascular, gastrointestinal, musculoskeletal, hepatic, renal, endocrine, dermal, or ocular effects resulting from exposure to aldrin.

Mongrel dogs, 1/ sex/ dose ( 2/ sex at 0.5 mg/ kg bw/ day), that were fed dieldrin ( recrystallized, 100% active ingredient) at dose levels of 0.2 to 10 mg/ kg bw/ day, 6 days/ week for up to 25 months, showed various toxic effects, including weight loss and convulsions at dosages of 0.5 mg/ kg bw/ day or more. Survival was inversely proportional to dose level. No toxic effects, gross or microscopic, were seen at a dose level of 0.2 mg/ kg bw/ day ( Fitzhugh et. al., 1964). A NOAEL of 0.2 mg/ kg bw/ day appears to have been established for this study, but its reliability is substantially limited because of the low number of animals studied. 7 20 Aldrin/ Dieldrin   February 2003 Groups of beagle dogs ( 5/ sex/ dose) were treated daily by capsule with dieldrin (> 99% purity) at 0.0, 0.005, or 0.05 mg/ kg in olive oil for 2 years. No treatment related effects were seen in general health, behavior, body weight, or urine chemistry. A significant increase in plasma alkaline phosphatase activity in both sexes and a significant decrease in serum protein concentration in males receiving the high dose were not associated with any clinical or pathological change. Liver weight and liver to body weight ratios were significantly increased in females receiving the high dose, 0.05 mg/ kg bw/ day, but no gross or microscopic lesions were found. There was no evidence of tumorigenic activity ( Walker et al., 1969).

Inhalation Exposure No studies were obtained that examined the chronic effects of aldrin or dieldrin in animals after chronic inhalation exposure.

Dermal Exposure There is one available study, which was conducted in rabbits, that examined the chronic effects of dermal exposure to dieldrin. Witherup et al. ( 1961) reported no effects on lung weight or pathology, heart weight or pathology, liver weight, serum proteins, thymol turbidity, serum alkaline phosphatase, or pathology in a chronic study in which rabbits were wrapped with material containing up to 0.04% dieldrin for up to 52 weeks. However, this study is limited in that some animals were treated with a variety of drugs to control " extraneous" diseases.

7.2.7 Carcinogenicity Oral Exposure Aldrin In a Food and Drug Administration ( FDA) long term carcinogenesis bioassay, Davis and Fitzhugh ( 1962) exposed a group of 215 C3HeB/ Fe mice ( numbers/ sex were not provided, but the group was reportedly divided approximately equally by sex) for up to 2 years to a diet containing aldrin ( purity not specified) at 10 ppm, constituting a dose of approximately 1.5 mg/ kg bw/ day using the conversion factor of Lehman ( 1959). The average long term survival rate of the treated group was approximately 2 months less than that of the controls, although these rates may have been affected by intercurrent diseases, pneumonia and intestinal parasitism. Results, reported for the combined sexes, indicated a significant ( p < 0.001) increase in the number of treated mice with hepatic cell adenomas ( 35/ 215 or 23%) when compared to that for the control group ( 9/ 217 or 7%). These hepatic cell adenomas were described as " expanding nodules of hepatic parenchymal tissue, usually with altered lobular architecture, and morphologically ranging from very benign lesions to borderline carcinomas." As reported by Epstein ( 1975a), an independent reevaluation of these lesions by other pathologists concluded that most were liver carcinomas. Despite the short comings of poor survival rate, lack of detailed pathology, loss of a large number of animals to the study, and failure to report the results 7 21 Aldrin/ Dieldrin   February 2003 separately by sex, the study provided evidence for aldrin's hepatocarcinogenicity to this strain of mouse.

In an FDA follow up to the previous study, aldrin of unspecified purity was fed to groups of C3H mice ( 100/ sex) at concentrations of 0 or 10 ppm ( approximately 1.5 mg/ kg bw/ day using the conversion factor of Lehman, 1959) for up to 2 years ( Davis, 1965). The incidences ( for both sexes combined) of hepatic hyperplasia and benign hepatomas in the treated group were reported to be approximately double those of the controls, whereas the incidence of hepatic carcinomas was judged to be about the same. This study suffered some of the deficiencies of its predecessor, and again an independent review concluded that most of the hepatomas were actually hepatocellular carcinomas ( Epstein, 1975a). This reevaluation provided incidences of hepatocellular carcinomas in the treated vs. control group of 82% vs. 30% for males, and 85% vs. 4% for females, both significant increases at p < 0.05.

Song and Harville ( 1964) fed a total of 55 C3H and CBA mice 15 ppm of aldrin ( unspecified purity) for an unspecified amount of time; 10 mice served as controls. In a companion study, mice were similarly fed dieldrin. Seven mice treated with aldrin or dieldrin were reported to have developed liver tumors by 330 to 375 days; however, as no further details were described, this report provides little useful information.

In a somewhat more recent carcinogenicity bioassay, technical grade aldrin ( 95% pure) was administered in the diet for 80 weeks to B6C3F 1 mice ( 50/ sex) at time weighted averages of 4 or 8 ppm for males, and at 3 or 6 ppm for females ( NCI, 1978). Based on Lehman ( 1959), these concentrations approximate doses of 0.6 and 1.2 mg/ kg bw/ day ( males), and 0.45 and 0.90 mg/ kg bw/ day ( females). The animals were observed for an additional 10 to 13 weeks. A significant ( p # 0.001) dose related increase in the incidence of hepatocellular carcinomas was observed in male, but not female, mice when compared to matched or pooled controls. Tumor incidences were 3/ 20, 17/ 92, 16/ 49, and 25/ 45 for the matched control, pooled control, low dose male, and high dose male groups, respectively.

When compared with control animals, NCI ( 1978) also reported increased incidences for combined follicular cell adenoma and carcinoma of the thyroid in both male and female Osborne Mendel rats. Treated animals ( 50/ sex) were fed technical grade aldrin ( 95% pure) at concentrations of 30 or 60 ppm ( 1.5 and 3 mg/ kg bw/ day, respectively, based on Lehman, 1959) for 74 or 80 weeks ( males or females, respectively), then observed for an additional 37 to 38 or 32 to 33 weeks ( males or females, respectively). The combined incidences from the pooled control, low dose, and high dose groups were respectively 4/ 48, 14/ 38, and 8/ 38 for males, and 3/ 52, 10/ 39, and 7/ 46 for females. Differences were significant ( p = 0.001) for the low dose groups, but not for the high dose groups. A significant ( p = 0.001) increase in the incidence of cortical adenomas of the adrenal gland were also observed in the low dose females, but this was not considered to be compound related by the authors. Aldrin produced no significant effect on the mortality of rats of either sex. Overall, the authors concluded that none of the observed tumors were associated with treatment, a view that has been echoed elsewhere ( USEPA, 1993a). However, other evaluations of the report have concluded that the occurrence of the thyroid and 7 22 Aldrin/ Dieldrin   February 2003 adrenal cortex tumors should be considered suggestive or equivocal evidence of aldrin's potential carcinogenicity in the rat ( Griesemer and Cueto, 1980; Haseman et al., 1987; USEPA, 1987).

A number of other carcinogenicity bioassays utilizing Carworth rats ( Treon and Cleveland, 1955), Holtzman rats ( Song and Harville, 1964), or Osborne Mendel rats ( Deichmann et al., 1967, 1970; Deichmann, 1974) failed to find evidence of aldrin induced tumors, but all suffered from substantial experimental and/ or reporting deficiencies that resulted in their being judged inadequate as tests of aldrin's possible carcinogenicity ( USEPA, 1987, 1993a).

Dieldrin In an FDA long term carcinogenicity bioassay, Davis and Fitzhugh ( 1962) exposed groups of approximately 218 C3HeB/ Fe mice ( numbers/ sex not specified, other than that they were approximately equal) for up to 2 years to dieldrin of unspecified purity at concentrations in the diet of either 0 or 10 ppm ( the latter corresponding to a dose of approximately 1.5 mg/ kg bw/ day, Lehman, 1959). Although compromised by poor survival rates, loss of a large percentage of the animals to the study and failure to treat the data separately by sex, the study did demonstrate a significantly increased incidence of hepatomas in the treated group when compared with the controls ( 36/ 148 or 24% vs. 9/ 134 or 7%). In a subsequent follow up study by FDA, groups of C3H mice ( 100/ sex) were fed either 0 or 10 ppm ( 0 or approximately 1.5 mg/ kg bw/ day) of dieldrin ( purity not specified) for up to 2 years ( Davis, 1965). This study suffered much the same limitations as its predecessor, but again demonstrated a significant increase in the incidence of benign hepatomas ( and in the combined incidence of benign hepatomas plus hepatocellular carcinomas) in the dieldrin group relative to controls. As for the companion aldrin studies discussed previously, a subsequent pathology reevaluation of both of these studies concluded that most of the hepatomas were in fact malignant hepatocellular carcinomas ( Epstein, 1975a, b).

As noted previously, Song and Harville ( 1964) fed a total of 55 C3H and CBA mice 15 ppm of dieldrin ( unspecified purity) for an unspecified amount of time; 10 mice served as controls. In a companion study, mice were similarly fed aldrin. Seven mice treated with aldrin or dieldrin were reported to have developed liver tumors by 330 to 375 days; however, as no further details were described, this report provides little useful information.

Epstein ( 1975a) reviewed and provided reevaluations of an unpublished study by MacDonald et al. ( 1972), in which " technical grade" dieldrin was fed for an uncertain period of time to groups of Swiss Webster mice ( 100/ sex/ group) at dietary concentrations of either 0, 3, or 10 ppm ( corresponding to approximate doses of 0, 0.45, or 1.5 mg/ kg bw/ day, respectively, Lehman, 1959). The authors concluded that dieldrin was not carcinogenic, but that it induced various nonneoplastic lesions of the liver, including a dose dependent increase in the incidence of hepatic nodules ( 0, 2.5, and 48% at 0, 3, and 10 ppm, respectively). However, a reevaluation of some of the histopathological data by independent pathologists ( as well as by one of the original authors) demonstrated that more than half of the reexamined livers from high dose mice contained hepatocellular carcinoma, thus confirming dieldrin's carcinogenicity to mice. 7 23 Aldrin/ Dieldrin   February 2003 Walker et al. ( 1972) conducted a number of studies in which they exposed groups of CF 1 mice ( 29 to 200/ sex/ dose; 29 to 300 controls/ sex/ study) for 2 to 132 weeks to dietary concentrations of dieldrin (> 99% pure) ranging from 0.1 to 20 ppm ( approximating doses of 0.015 to 3.0 mg/ kg bw/ day, Lehman, 1959). Significant dose related increases in the incidences of benign and total liver tumors were observed beginning at concentrations as low as 2.5 ppm, while the incidence of malignant liver tumors was significantly increased at concentrations of 5, 10, and 20 ppm. Liver tumors were also demonstrated to occur much earlier in treated than in control mice. In one of the studies, dieldrin also induced significant increases ( p < 0.05) in the incidences of lung, lymphoid, and " other" tumors in female mice.

In another study using CF 1 mice ( Thorpe and Walker, 1973), groups ( 30/ sex; 45 controls/ sex) were fed dieldrin (> 99% pure) in the diet for up to 110 weeks at concentrations of 0 or 10 ppm ( an approximate dose of 1.5 mg/ kg bw/ day, Lehman, 1959). Again, a statistically significant ( p < 0.01) increase in malignant liver tumors ( many of which metastasized to the lung) and a shortened latency period were induced by dieldrin.

In an NCI ( 1978) study, B6C3F 1 mice ( 50/ sex/ dose) were fed technical grade dieldrin (> 96% purity) for 80 weeks ( with an additional 10 to 13 weeks of observation) at time weighted average concentrations of 2.5 or 5 ppm ( equivalent to 0.375 or 0.75 mg/ kg bw/ day, respectively, based on Lehman, 1959). Matched ( 20 male, 10 female) and pooled ( 92 male, 91 female) controls received no dieldrin ( or other test chemical) in their feed. This assay was considered an acceptable test for carcinogenicity based on achieving a maximum tolerated dose without excess toxicity or mortality ( USEPA, 1987). When compared with pooled controls, male mice evidenced a significant ( p = 0.02) dose related increase in the incidence of hepatocellular carcinomas, as well as a significant ( p = 0.025) increase in such tumors at the high dose.

In a study by Tennekes et al. ( 1981, 1979), groups of 19 to 82 male CF 1 mice were fed dieldrin (> 99% pure) at concentrations of 0 or 10 ppm ( an approximate dose of 1.5 mg/ kg bw/ day, Lehman, 1959) for up to 110 weeks. Two types of diet and two types of bedding were examined as part of the study. Dieldrin treatment was reported to have shortened the liver tumor latency period, increased the incidence of combined liver tumors from 10 to 81%, and significantly ( p < 0.01) increased the incidences of hepatocellular carcinoma ( from 1 to 39%) and lung metastases ( from 0 to 14%).

In a large study intended to investigate dieldrin's enhancing affect on liver tumor formation ( Tennekes et al., 1982), a total of 1,800 CF 1 mice ( 17 to 297/ sex/ dose) were fed dieldrin (> 99.9% purity) over the course of their lifetimes at concentrations of 0, 0.1, 1, 2.5, 5, 10, or 20 ppm ( doses of 0 and approximately 0.015, 0.15, 0.375, 0.75, 1.5, or 3.0 mg/ kg bw/ day, respectively, based on Lehman, 1959). In both sexes, treatment appeared to result in dose related increases in the incidences of both combined ( benign plus malignant) and malignant liver tumors up to 10 ppm; somewhat lower incidences at 20 ppm were speculated to result from significant toxicity/ lethality at that concentration. Dieldrin also induced a dose dependent reduction in tumor latency periods; the lowest doses associated with a significant ( p < 0.05) reduction in median time to tumor formation were 0.1 and 1.0 ppm for females and males, respectively. The lack of a linear relationship between daily exposure level and median time to tumor formation or 7 24 Aldrin/ Dieldrin   February 2003 median total dose led the authors to speculate that dieldrin may affect tumor promotion rather than initiation.

Meierhenry et al. ( 1983) exposed groups of male C3H/ He, B6C3F 1, and C 57 BL/ 6J mice ( 50 to 71/ strain; 50 to 76 controls/ strain) for 85 weeks ( followed by 47 weeks of observation) to dieldrin (> 99% purity) at a dietary concentration of 10 ppm ( an approximate dose of 1.5 mg/ kg bw/ day, based on Lehman, 1959). Dieldrin induced significant ( p < 0.05) increases in the incidences of hepatocellular carcinomas relative to controls in all three strains of mice.

Osborne Mendel rats treated with dieldrin (> 96% purity) at time weighted average concentrations of 29 or 65 ppm in the diet ( approximate doses of 1.45 or 3.25 mg/ kg bw/ day, respectively, based on Lehman, 1959) for 80 weeks, and then observed for an additional 30 to 31 weeks, did not show any treatment related increase in tumors ( NCI, 1978). A second NCI ( 1978) study that exposed groups ( 24/ sex) of Fischer rats to dieldrin ( technical grade, purified) for 104 to 105 weeks at dietary concentrations of 0, 2, 10, or 50 ppm ( doses of 0 and approximately 0.1, 0.5, or 2.5 mg/ kg bw/ day, respectively, based on Lehman, 1959) produced similarly negative tumorigenic results. Both of these bioassays were judged to be adequate tests for carcinogenicity ( USEPA, 1987).

As evaluated by USEPA ( 1987), one other minimally acceptable study ( Deichmann et al., 1970) and four inadequate studies ( Treon and Cleveland, 1955; Fitzhugh et al., 1964; Song and Harville, 1964; Walker et al., 1969, which was reevaluated by Stevenson et al., 1976) collectively exposed several strains of rats ( Carworth, Osborne Mendel, or Holtzman) to dietary concentrations of dieldrin ( varying purities) ranging from 0.1 to 285 ppm ( approximate doses of 0.005 to 14.25 mg/ kg bw/ day, respectively, based on Lehman, 1959) for periods of 1 to 2 years. Although all of these studies failed to demonstrate any evidence for dieldrin's potential carcinogenicity, all suffered from one or more serious deficiencies ( e. g., too few animals, excessive mortality, inadequate duration, data missing or inadequately reported, etc.). Additionally, several dieldrin bioassays involving dogs or monkeys were evaluated by the USEPA ( 1987) as being inadequate or unacceptable tests of potential carcinogenicity due to serious limitations.

Inhalation Exposure No studies were obtained that examined the carcinogenicity of either aldrin or dieldrin in animals after inhalation exposure.

Dermal Exposure No studies were obtained that examined the carcinogenicity of either aldrin or dieldrin in animals after dermal exposure. 7 25 Aldrin/ Dieldrin   February 2003 7.3 Other Key Data 7.3.1 Mutagenicity/ Genotoxicity Effects Aldrin In bacterial reverse mutation assays that were conducted by several investigators, aldrin was not mutagenic to Salmonella typhimurium ( Simmon and Kauhanen, 1978; Cotruvo et al. 1977; Simmon et al., 1977; Probst et al., 1981; Nishimura et al., 1982) or E. coli ( Ashwood Smith et al., 1972; Probst et al., 1981), nor was it found to induce plasmid DNA breakage in E. coli, although it was tested only in the absence of S9 metabolic activation ( Griffin and Hill, 1978).

Simmon and Kauhanen ( 1978) reported that aldrin, at concentrations of 10 to 5,000 : g/ plate, did not cause gene conversion in Saccharomyces cerevisiae, either in the presence or absence of exogenous metabolic activation provided by Aroclor induced rat liver microsomes. It has, however, been reported to induce reverse mutation in the same organism ( Guerzoni et al., 1976).

Several doses of aldrin were tested in a mouse dominant lethal assay conducted by Epstein et al. ( 1972), and although some reductions in the level of implantation were demonstrated, they were judged to be statistically nonsignificant. Negative results have also been reported for its induction of sex linked recessive lethal mutation in Drosophila melanogaster ( Benes and Sram, 1969).

Georgian ( 1975) reported that aldrin induced chromosome aberrations in human lymphocytes in vitro and in rat and mouse bone marrow cells in vivo. However, the evidence for an in vivo clastogenic response is somewhat equivocal because the observed chromosomal aberration frequencies increased only at cytotoxic levels. Additionally, chromosome and chromatid gaps, which historically have been considered unreliable indicators of significant damage to genetic material, were included in the aberration totals. Therefore, the extent of the more meaningful, non gap, chromosomal damage cannot be ascertained. Negative results have also been reported for the in vivo induction of micronuclei in mice at an aldrin dose of 13 mg/ kg bw ( Rani et al., 1980).

Dulout et al. ( 1985) studied the incidences of sister chromatid exchanges ( SCEs) and chromosome aberrations in a population of floriculturists who were exposed to several pesticides, including aldrin. For those floriculturists who exhibited clinical symptoms of pesticide exposure, there were statistically significant increases in SCEs when compared with asymptomatic floriculturists, and in exchange type chromosome aberrations when compared with nonfloriculturists. However, interpretation of the role of aldrin in these findings is confounded by the concomitant exposure to other organophosphorous, carbamate, and organochlorine pesticides. Edwards and Priestly ( 1994) reported that occupational exposure to aldrin did not alter SCE frequencies in lymphocytes derived from workers ( n = 33) recruited from two south Australian suburban pesticide application companies. 7 26 Aldrin/ Dieldrin   February 2003 Unscheduled DNA synthesis ( UDS) was not induced when primary rat hepatocytes were exposed to aldrin at concentrations ranging from 0.5 to 1,000 nmol/ mL for 5 to 20 hours ( Probst et al., 1981), and most likely not when human lymphocytes were exposed to concentrations of up to 100 : g/ mL ( Rocchi et al., 1980). However, Ahmed et al. ( 1977a) reported the induction of UDS in transformed human cells at aldrin concentrations as low as 0.4 : g/ mL, and Sina et al. ( 1983) observed DNA strand breaks in the alkaline elution/ rat hepatocyte assay at an aldrin concentration of 110 : g/ mL.

Dieldrin Dieldrin was not mutagenic in the Salmonella/ microsome test ( Ames test), either with or without S 9 mix as a source of exogenous metabolic activation ( McCann et al., 1975). Similarly, nine other studies have collectively reported negative responses for dieldrin in at least eight different Ames tester strains of S. typhimurium, both with and without exogenous metabolic activation ( Anderson and Styles, 1978; Bidwell et al., 1975; Glatt et al., 1983; Haworth et al., 1983; Marshall et al., 1976; Nishimura et al., 1982; Probst et al., 1981; Shirasu et al., 1976; Wade et al., 1979). Negative responses have also been reported for dieldrin in E. coli using both a reverse mutation assay ( Ashwood Smith et al., 1972; Probst et al., 1981) and two forward mutation assay systems ( Gal Rz2 and streptomycin resistance) ( Fahrig, 1974). Additionally, Dean et al. ( 1975) reported negative findings in a host mediated assay ( microbial cells in animal hosts). However, in one contrary study, Majumdar et al. ( 1977) reported that dieldrin was mutagenic for S. typhimurium, both with and without exogenous metabolic activation.

Dieldrin produced negative responses in assays for forward mutation and aneuploidy induction in Aspergillus nidulans ( although it was not tested with exogenous metabolic activation) ( Crebelli et al., 1986), gene conversion in S. cerevisiae, and reverse mutation in S. marcesans ( Fahrig, 1974), in vitro DNA strand breaks in E. coli plasmids or in animal cell alkaline elution assays ( Swenberg et al., 1976; Swenberg, 1981), UDS in rat primary hepatocytes ( Probst et al., 1981), and most probably for UDS in human lymphocytes ( Rocchi et al., 1980). Dieldrin also failed to induce cell transformation in Syrian hamster embryo cells ( Mikalsen and Sanner, 1993). However, it was reported to induce forward mutation in Chinese hamster V79 cells ( Ahmed et al., 1977b), as well as UDS in transformed human cells ( Ahmed et al., 1977a).

In a dominant lethal study that orally exposed male CF 1 mice to dieldrin, mean implantation levels ( versus controls) were significantly reduced in females mated with males receiving 12.5 mg/ kg bw. However, in a subsequent experiment, mean implantations were not reduced, or even significantly increased, in females mated with males receiving 25 or 50 mg/ kg bw ( Dean et al., 1975). In another mouse dominant lethal assay, several doses of dieldrin up to 26 mg/ kg bw were found to be without mutagenic effect ( Epstein et al., 1972). Dieldrin also reportedly did not induce sex linked recessive mutation in D. melanogaster ( Benes and Sram, 1969).

Studies have demonstrated that dieldrin can cause chromosomal aberrations in mouse bone marrow cells following in vivo exposure ( Markaryan, 1966; Dean et al., 1975; Majumdar et al., 1976) in human lymphoblastoid cells ( Trepanier et al., 1977) and human WI 38 embryonic 7 27 Aldrin/ Dieldrin   February 2003 lung cells ( Majumdar et al., 1976) after in vitro exposure. In the latter case, the cytogenetic effects were accompanied by significant cytotoxicity, as there was evidence of cell degeneration. Dean et al. ( 1975) failed to find evidence of elevated frequencies of chromosomal aberrations in human lymphocytes after in vivo exposure to undetermined amounts of dieldrin. SCEs, but not chromosome aberrations, were induced by dieldrin in CHO cells, both in the presence and absence of S9 exogenous metabolic activation ( Galloway et al., 1987). Aneuploidy and/ or nuclear polyploidization were reportedly induced in the liver of CF 1 mice treated with dieldrin at 0.6 mg/ kg bw/ day ( van Ravenzwaay and Kunz, 1988). In a human occupational exposure study, Dean et al. ( 1975) compared the frequencies of both chromatid and chromosome type aberrations in lymphocytes that were isolated from workers exposed to dieldrin in a manufacturing facility with those from unexposed control subjects. No statistically significant differences in the frequencies were observed.

With respect to in vivo exposure, currently available data do not indicate unequivocally that either aldrin or dieldrin directly interacts with DNA to cause mutations in either the germ cells or the somatic cells of mammals.

7.3.2 Immunotoxicity No studies were obtained that examined the immunological effects of aldrin in either humans or animals, and only limited information was located regarding these types of effects in humans following exposure to dieldrin. A case report was located concerning a man who developed immunohemolytic anemia after eating fish that contained high levels of dieldrin ( Hamilton et al., 1978). Testing of the patient's serum revealed a positive response for antibodies to dieldrin coated red blood cells ( RBCs). Another case of immunohemolytic anemia was reported in a man who had had multiple exposures to dieldrin, heptachlor, and toxaphene while spraying cotton fields ( Muirhead et al., 1959); the individual's serum was found to contain antibodies to RBCs coated with either dieldrin or heptachlor. In contrast, volunteers who were re exposed to fabric that contained up to 0.5% dieldrin 2 weeks after an initial 4 day exposure did not reveal any evidence of sensitization ( Suskind, 1959).

Immunosuppression by dieldrin has been reported in a number of studies in mice. A decrease in the antigenic response to the mouse hepatitis virus 3 ( with a corresponding increase in its lethality) was observed in mice given a single oral dose of dieldrin ($ 18 mg/ kg) ( Krzystyniak et al., 1985). Similarly, an increase in the lethality of infections with the malaria parasite, Plasmodium berghei, or with Leishmania tropica was produced in mice by treatment with dieldrin in the diet at concentrations as low as 1 ppm ( approximately equivalent to 0.15 mg/ kg bw/ day based on Lehman, 1959 for 10 weeks [ Loose, 1982]). In addition, decreased tumor cell killing ability was observed in mice after dieldrin treatment with concentrations as low as 1 ppm ( approximately equivalent to 0.15 mg/ kg bw/ day based on Lehman, 1959) for 3, 6, or 18 weeks ( Loose et al., 1981).

Loose et al. ( 1981) also observed a decrease in antigen processing by alveolar macrophages in mice following administration of dieldrin at concentrations as low as 0.5 ppm ( approximately equivalent to 0.075 mg/ kg bw/ day based on Lehman, 1959) for 2 weeks. This 7 28 Aldrin/ Dieldrin   February 2003 occurred in the absence of observable effects on macrophage respiration, phagocytic activity or capacity, or microbial activity. In addition, macrophages from mice exposed for 10 weeks to dieldrin in the diet at 5 ppm ( approximately 0.75 mg/ kg bw/ day based on Lehman, 1959) were found to produce a soluble factor that induced T lymphocyte suppressor cells, suggesting suppressed immune system function ( Loose, 1982). In another limited study, lymphocyte proliferation appeared inhibited in a mixed lymphocyte reaction test in which splenic cells taken from mice treated twice with 16.6 mg/ kg bw of dieldrin were combined with stimulator cells taken from control animals ( Fournier et al., 1988).

7.3.3 Hormonal Disruption Wade et al. ( 1997) examined hormone levels in the serum and uterine tissues of young female Spargue Dawley rats after intraperitoneal exposure to 3 mg/ kg day dieldrin from days 18 to 21 after birth. As compared to the vehicle treated controls, this acute exposure to dieldrin produced no significant effects in serum thyroxine levels, or in uterine tissue levels of follicle stimulating hormone ( FSH), lutenizing hormone ( LH), thyroid stimulating hormone, prolactin, or growth hormone. Pituitary weight was also reported to be unaltered by dieldrin treatment.

In an in vitro study, Brown ( 1998) reported that very low dose of dieldrin decreased fetal testicular hormone output. Tissue samples from 6 human male fetuses, terminated after 12 to 19 weeks of gestation, were cultured and tested for the production of testosterone and inhibin after exposure to dieldrin, either in the presence or absence of a combination of FSH and LH ( 10 nM). Diledrin treatment alone did not reduce hormone secretions, but coadministration of FSH+ LH and dieldrin ( 10 12 M) significantly reduced ( p< 0.03) testosterone and inhibin B levels when compared to control levels.

7.3.4 Physiological or Mechanistic Studies One mechanism considered as a possible explanation for the aldrin/ dieldrin induced convulsions and tremors observed in animals and humans involves the effects of these insecticides on the GABA ( gamma aminobutyric acid) receptor. Several lines of evidence suggest that organochlorine insecticides, such as aldrin and dieldrin, can act as GABA A receptor antagonists, blocking the chloride ion channel in the central nervous system. Such inhibition of the chloride ion channel could be a significant contributing factor to convulsions and tremors ( Klaassen, 1996).

Using whole cell and single channel patch clamp techniques Nagata and Narahashi ( 1995) examined dorsal root ganglion ( DRG) neurons, isolated from the lumbo dorsal region of newborn rats, that had been treated with dieldrin ( 0.0001 to 10 : M). They reported that dieldrin exposure suppressed the GABA A receptor induced chloride currents ( both sensitive and lesssensitive types) in a time and dose dependent manner. The IC 50 values were estimated as being 3.7 nM and 98 nM for the sensitive and the less sensitive currents, respectively. Dieldrininduced suppression of chloride currents were directly dependent on GABA concentration ( up to 1000 : M) and appeared irreversible as the current did not recover after a 30 minute wash out 7 29 Aldrin/ Dieldrin   February 2003 with dieldrin free solution. This suppression of chloride currents in vitro may explain the in vivo hyperactivity that has been noted in animals exposed to dieldrin.

Nagata and Narahashi ( 1994) observed that dieldrin ( 1 : M) exhibited dual effects on chloride currents in primary cultures of DRG; i. e., initial transient enhancement followed by suppression after repeated co applications. The dieldrin caused desensitization and suppression of chloride current occurred at an EC 50 of 92 nM, but the enhancement of chloride current needed a higher EC 50 of 754 nM. These authors also reported that the dieldrin induced suppression of the GABA mediated chloride current was non competitive and irreversible, as recovery was not observed after prolonged washing of the neurons with dieldrin free solution.

Nagata et al. ( 1994) speculated that dieldrin may cause differential effects on the GABAinduced chloride currents in human embryonic kidney cells, depending upon the subunit combinations of the GABA A receptor chloride channel complex. The current molecular biological evidence indicates this complex to normally be a pentameric protein comprised of five subunits (", $, (, *, D) in various combinations. Using the whole cell variation of the patch clamp technique, the EC 50 values for GABA induction of chloride current were estimated as 9.8 : M for the " 1$ 2 ( 2s combination, 2.0 : M for the " 1$ 2 combination, and 3.0 : M for the " 6$ 2 ( 2s combination. When co applied with GABA, dieldrin ( 1 to 3 : M) produced mixed effects: initial transient enhancement, followed by suppression, was observed for the GABA induced chloride currents in the " 1$ 2 ( 2s and " 6$ 2 ( 2s combinations. However, suppression alone was observed in the " 1$ 2 combination, indicating that the ( subunit is necessary for dieldrin enhancing effects. The EC 50 values for dieldrin's suppression of GABA induced current were estimated as being 2.1 : M for the " 1$ 2 ( 2s combination, 2.8 : M for the " 1$ 2 combination, and 1.0 : M for the " 6$ 2 ( 2s combination. The authors concluded that dieldrin induced suppression of chloride current did not require specific subunit combinations, at least among the three combinations tested.

Using the radioligand, t 35S butyl bicyclophosphorothionate ( 35S TBPS), Brannen et al. ( 1998) demonstrated that dieldrin could interfere with GABA receptor binding. The authors observed that injection of dieldrin ( 10 mg/ kg bw/ day) to pregnant dams from days E12 through E17 ( embryonic days 12 to 17) caused a significant reduction in the amount of ( 35S TBPS binding to the GABA A receptor in E17 brainstem when compared to vehicle injected controls. However, dieldrin treatment caused no significant effect on radioligand binding in the " rest of the brain."

Johns et al. ( 1998) reported findings that suggest the prenatal binding of dieldrin to GABA receptor might alter the expression of GABA A subunit mRNAs in adult offspring, and thus might explain the postnatal behavioral changes observed in adult offspring. Pregnant rats were injected intraperitoneally daily during E12 through 16 with 0, 5, or 10 mg/ kg bw dieldrin. On postnatal day 56, testing of these adult offsprings in the elevated plus maze suggested that the dieldrin treated males spent less time in the closed arms of the elevated maze, indicating a lower level of anxiety. Consistent with these behavioral effects, 35STBPS binding and several GABA A subunit mRNA levels were elevated in regions of the brain from the adult offspring of dieldrintreated dams. 7 30 Aldrin/ Dieldrin   February 2003 In an in vitro study, Liu et al. ( 1997) studied the mechanisms involved in the neurotoxic effect of dieldrin on ( aminobutyric acid ( GABA A) receptors. Using embryonic day 14 ( E14) rat brain stem cell cultures, the authors examined the effects of dieldrin on the expression of five GABA receptor mRNA subunits (" 1, $ 3, ( 1, ( 2L, ( 2S). Following a 48 hour treatment of brain stem cells with 10 : M dieldrin, GABA A receptor subunit mRNA levels were found to be differentially regulated from those of control cultures. Levels of $ 3 subunits were significantly increased ( 300%; p< 0.05) by dieldrin, whereas expression of ( 2S and ( 2L transcripts were decreased by 50 and 40%, respectively ( p < 0.05). The levels of " 1 and ( 1 subunits, as well as the ratio of ( 2S to ( 2L, were not significantly affected by dieldrin treatment. As the evidence in general suggested a correlation between gene expression and receptor function, the altered expression of GABA receptor subunit mRNAs by prenatal dieldrin exposure may affect the functional properties of the GABA A receptor in the developing brain.

Liu et al. ( 1998) also studied the effect of prenatal in vivo exposure to dieldrin on the expression in the rat fetal brain stem of the five GABA A receptor subunit mRNAs (" 1, $ 3, ( 1, ( 2L, ( 2S). Pregnant rats, intraperitoneally administered dieldrin at 1 mg/ kg bw/ day from E12 to E17, evidenced a decrease in the mRNA levels of the " 1, $ 3, and ( 1 subunits, but not of those for ( 2S or ( 2L. Again, it was speculated that altered expression of these subunit mRNAs might impact the functional properties of the GABA A receptor, and thus GABA mediated behaviors.

In addition to its effects on GABA ergic neurons, dieldrin might also perturb or interact with dopaminergic neurons. In an in vitro study, Sanchez Ramos et al. ( 1998) examined the effects of a 24 or 48 hour treatment with dieldrin ( 0.01 to 100 : M) on primary cultures of mesencephalic neurons isolated from the fetal brains of Sprague Dawley rats or C 57/ BL mice. Toxicities toward dopaminergic and GABA ergic neurons were assessed by determining the survival of tyrosine hydroxylase immunoreactive ( TH ir) cells and glutamate decarboxylase ( GAD) ir neurons, respectively. Dieldrin exposure for 24 hours resulted in a dose dependent decrease in the survival of TH ir cells from rat mesencephalic cultures, with 50% relative cell survival occurring at 12 : M. The 24 hour dieldrin treatment also produced a dose dependent decrease in TH ir cell survival in mouse mesencephalic cultures, with 75% relative cell survival occurring at 10 : M. In general, the toxic effects of dieldrin were reported to be more severe for TH ir neurons than for GABA ergic neurons. Microscopic changes in neurons treated with dieldrin were observed in TH ir cells, such as diminished numbers and lengths of neurites, and rounded cell bodies, as opposed to the polygonal or spindle form found in control neurons. Consistent with the cell survival effects, dopamine uptake was impaired by lower concentration of dieldrin than was GABA uptake ( the EC 50 for DA uptake was 7.98 : M, compared to 43 : M for GABA uptake) suggesting that dieldrin had a greater functional effect on dopaminergic neurons than on GABA ergic neurons. Finally, the authors concluded that the greater toxicity of dieldrin on dopaminergic neurons might contribute to the Parkinsonism effects observed in workers exposed to pesticides, such as dieldrin.

Chatterjee et al. ( 1992) reported a number of estrogenic effects following subcutaneous administration of aldrin ( 1 mg/ kg bw/ day) for 3 days to groups ( 8/ group) of young ( 22 day old) or ovariectomized adult ( 90 day old) female Wistar rats. Aldrin exposure caused significant increases in both young and old rats, as compared to their respective control groups, for each of 7 31 Aldrin/ Dieldrin   February 2003 the following parameters indicative of positive estrogenic effects: uterine weight, endometrial gland thickness and proliferation, and the staining of periodic acid Schiff positive substance in the uterus. In ovariectomized adult rats, aldrin exposure also induced a persistent vaginal estrus, as compared to the constant diestrus seen in controls.

Several mechanistic studies have also been conducted to evaluate the estrogenic activity of dieldrin. Wade et al. ( 1997) examined the possible estrogenic effects of dieldrin in vivo by measuring the estrogen binding activity, peroxidase activity, and uterine weight in young female Spargue Dawley rats, and in vitro by measuring the cell proliferation activity in MCF 7 cells ( human breast cancer cells). Dieldrin treatment ( 2 to 10 : M) could competitively inhibit the binding of 3H 17$ estradiol ( E 2) to estrogen receptors in the rat uterus, indicating the similarities between the two compounds. The authors observed that the intraperitoneal administration of dieldrin at a dose of 3 mg/ kg bw to young female rats during the period of 18 to 21 days after birth, produced no changes in uterotrophic activity ( uterine weight, peroxidase activity, estrogen receptor number, and progesterone receptor number). In contrast to the lack of these specific in vivo estrogenic effects, dieldrin caused a positive response in the in vitro test; treatment of cultured MCF 7 cells with 50 : M dieldrin resulted in a 3.4 fold increase in cell proliferation, as compared to control cells. Wade et al. ( 1997) also reported that dieldrin lacked any synergistic effects in estrogenic activity when tested with endosulfan in both in vitro and in vivo assays. The weak in vivo estrogenic response of dieldrin is not likely attributable to study design limitations, as the positive control, diethylstilbesterol, produced significant estrogenic effects.

In another study, Soto et al. ( 1994) examined the estrogenic effects of dieldrin in vitro by measuring its proliferative effects on MCF 7 cells. Dieldrin treatment ( 1.0 pM to 10 : M) of MCF 7 cells produced a significant increase in the proliferation capacity only at the highest concentration. The relative proliferative efficiency for dieldrin at 10 : M was 54.89% that of estradiol, which induced its maximum level of proliferation ( i. e., 100% relative proliferation) at a concentration of only 10 pM ( 1 × 10 6 of the tested dieldrin concentration). This indicates the relatively very weak estrogenic effect of dieldrin.

Ramamoorthy et al. ( 1997) investigated the possible estrogenic activity of dieldrin using a series of molecular biology assays: estrogen binding activity and estrogen effects in 21 day old B6C3F 1 mouse uterus, estrogen mediated proliferation in MCF 7 cells, and reporter gene assays in yeast cells transformed with mouse or human estrogen receptor genes. They observed that dieldrin did not bind to the estrogen receptor in mouse uterus or MCF 7 cells in a competitive manner; produced no estrogen dependent effects, such as increase in uterine wet weight or progesterone binding in uterus excised from mice ( treated intraperitoneally with 2.5 to 60 : mol/ kg bw/ day for 3 days); did not induce MCF 7 cell proliferation at concentrations ranging from 10 8 to 10 5 M; and produced minimal induction of the reporter gene activity at concentrations of up to 2.5 × 10 5 M or 1 × 10 4 M in yeast cells transformed with either mouse estrogen receptor or human estrogen receptor, respectively. The negative findings reported in this study regarding dieldrin's estrogenic effects are not attributable to study design limitations, as the positive controls ( 17$ estradiol and diethylstilbesterol) had a strong estrogenic effects in these assays. Overall, the authors suggested that dieldrin produced only minimal estrogenic 7 32 Aldrin/ Dieldrin   February 2003 effects when tested alone, and moreover, when examined with toxaphene, exhibited no synergistic effects.

In contrast to the studies of Ramamoorthy and colleagues, Arnold et al. ( 1996) reported that dieldrin might have estrogenic effects when tested alone, or possess synergistic effects when combined with endosulfan. This was demonstrated by the induced expression of reporter gene activity in yeast or baculovirus ( insect) cells that had been transformed with human estrogen receptors.

Using an in vitro biomembrane assay, Demetrio et al. ( 1998) investigated the effects of incubating phosphatidylcholine and dimyristoylphosphatidylcholine ( DMPC) with aldrin concentrations of up to 100 : M for 18 to 20 hours, over the temperature range 12 to 40 ° C. They reported a decrease in the fluidity of the lipid bilayers, as measured by changes in fluorescence polarization of DPH ( 1,6 diphenyl 1,3,5 hexatriene) and of its propionic acid derivative DPHPA which indicated fluidity changes in the bilayer core and in the outer regions of the bilayer, respectively. Although these membrane fluidity changes may alter membrane functions, it is not known whether they occur in vivo.

Wright et al. ( 1972) reported that within 1 week of exposing rats or mice to 8 or 1.6 mg/ kg bw/ day of dieldrin, respectively, increases were observed in liver cell cytoplasmic vacuoles, smooth endoplasmic reticulum, microsomal protein, and mixed function oxidase activity. They also reported similar effects in dogs after 4 weeks of exposure to 2 mg/ kg bw/ day of dieldrin. Exposure of monkeys to concentrations as high as 0.1 mg/ kg bw/ day of dieldrin for up to 6 years also produced increased mixed function oxidase activity and cytochrome P 450 content in liver cells ( Wright et al., 1972, 1978).

Finally, in vitro studies have indicated that concentrations of aldrin as low as 5.0 to 6.0 : g/ ml can inhibit gap junctional intercellular communication among human teratocarcinoma cells ( Zhong Xiang et al., 1986) and metabolic cooperation among Chinese hamster cells ( Kurata et al., 1982). Similarly, dieldrin has also been reported to inhibit intercellular communication/ metabolic cooperation among human teratocarcinoma cells ( Wade et al., 1986; Zhong Xiang et al., 1986), Chinese hamster cells ( Kurata et al., 1982), and Syrian hamster embryo cells ( Mikalsen and Sanner, 1993).

7.3.5 Structure Activity Relationship Four compounds structurally related to aldrin and dieldrin chlordane, heptachlor, heptachlor epoxide, and chlorendic acid have induced malignant liver tumors in mice; chlorendic acid has also induced liver tumors in rats ( USEPA, 1993a, b). 7 33 Aldrin/ Dieldrin   February 2003 7.4 Hazard Characterization 7.4.1 Synthesis and Evaluation of Noncancer Effects Acute exposures to aldrin and/ or the metabolic product, dieldrin, could cause neurotoxic effects in humans characterized by hyperirritability, convulsions, and coma ( Jager, 1970; Spiotta, 1951; ACGIH, 1984). Cardiovascular effects, such as tachycardia and elevated blood pressure, may occur subsequent to convulsions ( Black, 1974).

Evidence suggesting that children would experience the neurotoxic toxic effects upon acute exposure to aldrin and/ or dieldrin is limited. In many instances, children may be more sensitive than adults as a result of their developing organ systems ( e. g., nervous system) and metabolic detoxication capacities ( Hayes, 1982; ATSDR, 2000). Long term effects of aldrin/ dieldrin in children have not been studied.

Occupational studies suggest that workers involved in the manufacture or application of aldrin/ dieldrin have increased dieldrin levels in plasma ( up to 250 ng/ mL). From the plasma, the aldrin and dieldrin could be distributed and stored in adipose tissue ( Nair et al., 1992).

Aldrin and dieldrin are quite toxic, as they have low acute toxicity values when tested in animals ( LD 50 values of generally # 100 mg/ kg) ( Borgmann et al., 1952; Gaines, 1960; Treon et al., 1952; Lu et al., 1965). Common acute or subchronic neurotoxic effects observed in animals are characterized by increased irritability, salivation, hyperexcitability, tremors followed by convulsions, loss of body weight, depression, prostration, and death ( Borgmann et al., 1952; Walker et al., 1969; Wagner and Greene, 1978; Wooley et al., 1985; NCI, 1978; Casteel, 1993). These symptoms are similar to those observed in humans exposed to aldrin or dieldrin ( Jager, 1970; Spiotta, 1951; ACGIH, 1984; ATSDR, 2000). In addition, cardiovascular effects, such as tachycardia and elevated blood pressure, may occur in humans subsequent to convulsions ( Black, 1974).

Evidence suggests that short term or subchronic oral exposure to aldrin at dietary concentrations of 300 ppm in rats and 80 ppm in mice could result in high mortality rates ( Treon and Cleveland, 1955; NCI, 1978).

Subchronic exposure of B6C3F 1 mice to dieldrin caused no significant effects in body weight gains, food consumption, or water consumption, but could increase relative liver weights ( liver weight to body weight ratios), and promote hepatic lesions induced by the hepatic carcinogen diethylnitrosamine. These dieldrin induced hepatic effects may be specific to mice, as they were not observed in rats ( Kolaja et al., 1996a, b).

Aldrin/ dieldrin exposure has been shown to produce developmental and reproductive toxic effects. In a 3 generation reproduction study conducted in rats, a reduction in the pregnancy rate was reported ( Treon and Cleveland, 1955). Prenatal exposure to aldrin also appears to have caused a reduction in pup survival in dogs ( Deichmann et al., 1971). An increase in fetal deaths, a decrease in live fetal weight, and increased incidences of webbed foot, cleft 7 34 Aldrin/ Dieldrin   February 2003 palate, and open eye were reported in Syrian golden hamsters and CD1 mice that were exposed to dieldrin prenatally ( Ottolenghi et al., 1974). Prenatal exposure to dieldrin may also cause maternal toxic effects, such as the increase in maternal mortality reported in mice ( Virgo and Bellward 1975).

However, evidence from several animal studies does not indicate that reproductive effects such as changes in fertility, fecundity, length of gestation, or perinatal mortality would be likely to result from exposure at environmental levels to either aldrin or dieldrin ( Kitselman, 1953; Good and Ware, 1969; Harr et al., 1970; Coulston et al., 1980).

Experimental evidence suggesting that dieldrin may be capable of producing estrogenic effects is not consistent ( Ramamoorthy et al., 1997; Arnold et al., 1996; Wade et al., 1997). Chatterjee et al. ( 1992) found that administration of dieldrin to rats increased uterine weight, endometrial gland thickness and proliferation, and the level of staining for periodic acid Schiff positive substances in the uterus in a fashion similar to that seen for estrogen. Soto et al. ( 1994) reported weak estrogenic like effects for dieldrin with respect to its enhancement of the proliferation of human breast cancer cells.

Chronic feeding of aldrin ( 1 to 10 mg/ kg bw/ day) to animals has in general produced high mortality effects ( Treon and Cleveland, 1955; Fitzhugh et al., 1964; NCI, 1978; Kitselman and Borgmann, 1952). Similar results were also reported for dieldrin exposure in mice ( Walker et al., 1972).

Increased liver weights and liver to body weight ratios were observed consistently in rats chronically exposed to aldrin ( Treon and Cleveland, 1955; Deichmann et al., 1970; Fitzhugh et al., 1964), and in rats, dogs, and mice exposed to dieldrin ( Fitzhugh et al., 1964; Walker et al., 1969; Walker et al., 1972).

Neurotoxic effects similar to those seen after acute exposure, such as tremors and convulsions, have also been reported in long term oral studies of animals exposed to aldrin ( Fitzhugh et al., 1964; Deichmann et al., 1970) or dieldrin ( Fitzhugh et al., 1964).

Chronic oral exposure of rats to aldrin has also caused some histopathological alterations in the liver, which were characterized by enlarged centrilobular hepatic cells having increased cytoplasmic oxyphilia and peripheral migration of basophilic granules ( Fitzhugh et al., 1964).

No studies were obtained that examined the toxic effects of aldrin or dieldrin in animals following inhalation exposure, and only very limited information was found regarding dermal exposure.

7.4.2 Synthesis and Evaluation of Carcinogenic Effects Long term follow up studies suggest that standardized mortality rates for all causes of death in workers employed in pesticide manufacturing plants are significantly lower than the 7 35 Aldrin/ Dieldrin   February 2003 corresponding national mortality rates ( de Jong, 1991; de Jong et al., 1997; Ditraglia et al., 1981; Brown, 1992; Amaoteng Adjepong et al., 1995). Slight increases in the incidence of rectal and liver cancers have been observed in the aldrin/ dieldrin exposed groups, but they are not robust or dose dependent ( de Jong et al., 1997; Ditraglia et al., 1981). Most of the results from the various occupational studies on the human health effects of aldrin/ dieldrin exposure are complicated to some degree by the simultaneous exposure to other pesticides; most plants were also involved in the manufacture of other pesticides, and the association of adverse human health effects with aldrin/ dieldrin contact is weakened by the lack of adequate exposure assessment.

Several lines of evidence suggest that chronic exposure to aldrin/ dieldrin selectively increases the incidence of liver cancer in several different strains of mice ( Meierhenry et al., 1983; Davis and Fitzhugh, 1962, Davis, 1965; Epstein, 1975a, b; NCI, 1978; Thorpe and Walker, 1973; Walker et al., 1972; Tennekes et al., 1981). These results were not observed in several strains of rats that have also been tested ( Treon and Cleveland, 1955; Fitzhugh et al., 1964; Song and Harville, 1964; Walker et al., 1969; Deichmann et al., 1970; NCI, 1978). Evidence from a single rat study ( NCI, 1978) suggesting possible increases in the incidences of follicular cell adenoma and carcinoma of the thyroid and of cortical adenoma of the adrenal gland after chronic aldrin exposure has not been supported by other studies. It must be kept in mind, however, that a number of these studies has been deemed inadequate tests for carcinogenicity due to a variety of significant study limitations.

Seven studies that collectively utilized 4 strains of rats, which were fed 0.1 to 285 ppm dieldrin for durations varying from 80 weeks to 31 months, did not produce positive results for carcinogenicity ( Treon and Cleveland, 1955; Fitzhugh et al., 1964; Song and Harville, 1964; Walker et al., 1969; Deichmann et al., 1970; NCI, 1978). Three of these studies used Osborne Mendel rats, two studies used Carworth rats, and one each used Fischer 344 and Holtzman strains. As noted above for aldrin, only three of the seven dieldrin studies were considered adequate in design and conduct ( USEPA, 1987, 1993b). The others used too few animals, had unacceptably high levels of mortality, were too short in duration, and/ or had inadequate pathology examination or reporting.

The status of aldrin and dieldrin as genotoxins is somewhat equivocal. Summarizing the studies reviewed in this document by certain genotoxicity endpoint categories, the assays performed on one or both of these chemicals have produced the following responses:

bacterial gene mutation: 21 (!) 1 (+) fungal gene mutation/ conversion: 4 (!) 1 (+) in vitro mammalian cell gene mutation: 1 (+) mammalian host mediated bacterial gene mutation: 1 (!) in vivo gene mutation  insects: 2 (!) in vitro chromosome damage/ aneuploidy: 2 (!) 3 (+) in vivo chromosome damage/ aneuploidy: 6 (!) 4 (+), 3 (?+) in vitro SCE: 1 (+) in vivo SCE: 1 (!) 1 (?+) bacterial/ plasmid DNA damage: 2 (!) in vitro mammalian cell DNA damage: 4 (!), 2 (?!) 3 (+) in vitro cell transformation: 1 (!) 7 36 Aldrin/ Dieldrin   February 2003 While the preponderance of these assay results are negative, some of the in vitro assays failed to employ some form of exogenous metabolic activation, such as S9 mix. Based on these data, it is currently difficult to reject at least the possibility that aldrin/ dieldrin can interact with chromosomes or induce DNA damage. However, as suggested in the following section, some or all of aldrin/ dieldrin's apparent genotoxicity may indirect or reflect epigenetic mechanisms.

7.4.3 Mode of Action and Implications in Cancer Assessment There have been several mechanistic studies conducted to explain the selective hepatocarcinogenic effects of aldrin and dieldrin in mice. Several studies suggest that these chemicals can induce at least hepatic tumors in mice, but are much less likely to do so, if at all, in rats. Although the mechanisms responsible for this species specificity are not fully understood, accumulating evidence indicates that increased hepatic DNA synthesis and oxidative stress may be involved.

Kolaja et al. ( 1996a) observed an increased DNA labeling index in the centrilobular region of liver in male B6C3F 1 mice, but not in male Fisher 344 rats, as early as 7 or 14 days after exposure to dieldrin at concentrations of 3.0 or 10.0 mg/ kg diet. Increases in the liver DNA labeling index in mice, but not in rats, were also reported by Kolaja et al. ( 1995) for the highdose groups when animals ( 5/ species/ dose) were fed with dieldrin in the diet at 0 ( control), 0.1, 1.0, or 10.0 mg dieldrin/ kg diet for 7 or 14 days. In a subsequent study, Kolaja et al. ( 1996b) described a selective promotion of hepatic focal lesions and an increase in DNA labeling at the highest dose in male B6C3F 1 mice, but not in male Fisher 344 rats. Groups of animals ( 5 animals/ species/ dose) were treated with the hepatic carcinogen, diethylnitrosamine ( 150 mg/ kg bw/ week, 2x for rats; 25 mg/ kg bw/ week, 8x for mice), prior to the administration of dieldrin at 0.1, 1.0, or 10.0 mg/ kg diet for 7 days.

In vivo experiments by Bachowski et al. ( 1997) demonstrated the following in B6C3F 1 mice, but not in F344 rats, upon feeding 10.0 mg dieldrin/ kg diet for up to 540 days: 1) increased production of 2,3 DHBA ( 2,3 dihydroxybenzoic acid, a marker used for measuring oxidative stress) in hepatocytes and their microsomes; 2) elevated production of MDA ( malondialdehyde, a marker for oxidative damage to lipids) in liver and urine; 3) increased OH8dG ( 8 hydroxy 2' deoxyguanosine, a marker for oxidative damage to DNA) levels in urine; and 4) decreased hepatic vitamin E (" tocopherol) content. The authors concluded that oxidative stress mechanisms may be involved in the mediation of dieldrin induced hepatic DNA synthesis that is observed in mice, but not in rats.

In a more recent study, Bachowski et al. ( 1998) also examined the in vivo association between dieldrin induced hepatic DNA synthesis and oxidative damage to lipids ( MDA), DNA ( OH8dG), or levels of nonenzymatic antioxidants ( ascorbic acid, glutathione, vitamin E) in male B6C3F 1 mice and F344 rats that were fed dieldrin ( 0.1, 1.0, or 10 mg/ kg diet) for up to 90 days. Consistent with the increase in hepatic DNA synthesis induced by dieldrin treatment, decreases in hepatic and serum vitamin E levels (" tocopherol), and increases in hepatic MDA and urinary MDA and OH8dG levels, were observed in mice. In contrast, these effects were less dramatic or 7 37 Aldrin/ Dieldrin   February 2003 not observed in rats, which may have been protected by higher basal levels of vitamin E ( and vitamin C) in their hepatic tissue.

Stevenson et al. ( 1995) found indirect evidence for an oxidative stress mechanism when they measured a partial protective effect of vitamin E in ameliorating the dieldrin induced hepatic DNA synthesis observed in B6C3F 1 mice ( fed dieldrin at 10 mg/ kg diet for 28 days). However, supplementation of the diet with vitamin C ( another antioxidant) at up to 400 mg/ kg diet resulted in only an inconsistent reduction in dieldrin induced hepatic DNA labeling.

Kolaja et al. ( 1998) also reported that supplementation of the diet with vitamin E ( 450 mg/ kg diet) for up to 60 days blocked the dieldrin ( 10 mg/ kg diet) treatment effects of increased hepatic focal lesion volume, focal lesion number, and focal lesion DNA labeling index that were observed in mice pre treated with the hepatic carcinogen, diethylnitrosamine.

Bauer Hofmann et al. ( 1992) analyzed the frequency and pattern of c Ha ras protooncogene mutations at codon 61 in polymerase chain reaction amplified DNA taken from glucose 6 phosphatase deficient ( G6P!) hepatic lesions in groups of male C3H/ He mice; groups had received either 10 ppm dieldrin, 500 ppm phenobarbital ( PB), or no treatment in the diet for 52 weeks. The incidence of G6P! hepatic lesions was reported to increase from 41% ( 15/ 37) in the control group to 67% ( 10/ 15) and 63% ( 10/ 16) in the dieldrin and PB groups, respectively. The corresponding average numbers of focal lesions/ mouse were 0.57, 1.5, and 1.0. Upon DNA analysis, c Ha ras mutations were observed in 57% ( 12/ 21) of the lesions from the control group, but in only 22% ( 5/ 23) and 25% ( 4/ 16) of those from the dieldrin and PB groups, respectively. As dieldrin ( like PB) increased the frequency of c Ha ras wild type, but not mutated, focal hepatic lesions, the authors concluded that the c Ha ras mutations had likely occurred spontaneously rather than as a result of dieldrin treatment. Further, no significant differences in the mutation spectra were noticed between control and dieldrin treated mice, the most prominent class of mutation being C6A transversion. These data suggest that the principal role for dieldrin in liver tumor formation may be one of promotion, rather than initiation.

Based on existing studies, Stevenson et al. ( 1999) have also suggested that aldrin/ dieldrin exposure induces hepatocarcinogenesis in mice through non genotoxic mechanisms such as increased production of reactive oxygen species ( ROS) in mouse hepatocytes ( possibly by futile cycling of P450 enzymes), increased hepatic DNA synthesis, and augmentation of tumorpromotional effects, rather than by causing point mutations or otherwise directly interacting with DNA. A possible mode of action for aldrin/ dieldrin in animals is depicted in Figure 7 l. Although the figure depicts aldrin/ dieldrin induction of hepatic DNA synthesis through modulation of proto oncogene expression ( via transcription factors such as Nf kB, AP 1, etc.), data directly relating the effects of aldrin/ dieldrin exposure to protooncogene expression remain to be established.

In addition to mechanisms that involve oxidative stress and the direct promotion of cellular proliferation, the previously discussed capacity of aldrin and dieldrin to inhibit various forms of in vitro intercellular communication in both human and animal cells may be significant 7 38 Aldrin/ Dieldrin   February 2003 Neoplasia Aldrin/ Dieldrin ROS Production Modulation of Gene Expression ( NF kB, AP 1, c Ha ras, second messengers)

Increased S phase DNA synthesis in hepatocytes Nongenotoxic ( Yes) Genotoxic ( No)

Genetic Instability Increase in focal lesion size Increased cell proliferation of spontaneously initiated hepatocytes Mitosis with respect to their in vivo effects on tumor production ( Kurata et al., 1982; Wade et al., 1986; Zhong Xiang et al., 1986; Mikalsen and Sanner, 1993).

7.4.4 Weight of Evidence Evaluation for Carcinogenicity Using current EPA ( 1986) cancer guidelines, aldrin and dieldrin are classified as B2 carcinogens, i. e. probable human carcinogens with little or no evidence of carcinogenicity in humans and sufficient evidence in animals ( different strains of mice). With inadequate data on carcinogenic effects in humans, under the USEPA's cancer risk assessment guidelines ( USEPA, 1996/ 1999), the weight of evidence indicates that aldrin and dieldrin could be classified as rodent carcinogens that are " likely to be carcinogenic to humans by the oral route of exposure, but whose carcinogenic potential by the inhalation and dermal routes of exposure cannot be determined because there are inadequate data to perform an assessment." This characterization is based on the tumor effects of aldrin and dieldrin observed in several strains of mice subsequent to oral exposures and must be tempered by the lack of evidence for significant human carcinogenicity from epidemiological studies. It should be noted that the USEPA has quantified the estimated carcinogenic risks from inhalation exposure to aldrin and dieldrin by extrapolating from available oral exposure route data ( USEPA, 1993a, b). Mechanistic studies performed in 7 39 Aldrin/ Dieldrin   February 2003 Figure 7 1. The Possible Mode of Action of Aldrin/ Dieldrin on Hepatocarcinogenesis Adapted from Stevenson et al. ( 1999)

vitro and in vivo suggest that one or more non genotoxic modes of action may underlie or contribute to the carcinogenic potential of aldrin and dieldrin, but these effects are not completely established, or can a role for genotoxic mechanisms confidently be eliminated based on the available data . In the absence of adequate data to fully support a non linear mechanism( s) of tumor formation, the quantitative cancer risk assessment of aldrin and dieldrin should conservatively be conducted using the linear default model.

7.4.5 Sensitive Populations No human studies were obtained that adequately address the effect of aldrin and dieldrin on sensitive populations, such as children. Several mechanistic studies, which describe the prenatal effects of aldrin/ dieldrin on GABA receptor malfunctions and on subsequent behavioral impairment, may suggest that children could be more sensitive to aldrin and dieldrin exposures than the general adult population ( Brannen et al., 1998; Liu et al., 1998; Johns et al., 1998; Castro et al., 1992). 7 40 Aldrin/ Dieldrin   February 2003 References ACGIH. 1984. American Conference of Governmental Industrial Hygienists. Documentation of the threshold limit values for substances in workroom air. 3rd ed. Cincinnati, OH: ACGIH, p. 139 ( as cited in USEPA, 1988).

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Zhong Xiang, L., T. Kavanagh, J. E. Trosko, and C. C. Chang. 1986. Inhibition of gap junctional intercellular communication in human teratocarcinoma cells by organochlorine pesticides. Toxicol. Appl. Pharmacol. 83: 10 19 ( as cited in GAP2000, 2000a, b). 8 1 Aldrin/ Dieldrin   February 2003 8.0 DOSE RESPONSE ASSESSMENTS 8.1 Dose Response for Non Cancer Effects 8.1.1 Reference Dose Determination The oral Reference Dose ( RfD), formerly termed the Acceptable Daily Intake ( ADI), is based on the assumption that thresholds exist for most, if not all, noncancer toxic effects. In general, the RfD is an estimate ( with uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human population ( including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a lifetime. The RfD is expressed in units of mg/ kg bw/ day, and has traditionally been derived from the NOAEL ( or LOAEL) identified from the data in a chronic ( or subchronic) study, divided by an uncertainty factor composed of one or more elements defined by EPA or NAS/ OW guidelines.

Aldrin Choice of Principal Study and Critical Effect The rat study by Fitzhugh et al. ( 1964), designed as a carcinogenesis bioassay, has been selected to serve as the basis for the Reference Dose principally because it displayed strength in histopathologic analysis, it examined a wider dose range ( 0.5 to 150 ppm in the diet) when compared with other available chronic studies, and in the absence of a reliable NOAEL, its data established the lowest available LOAEL. The database is fairly extensive and, generally, supportive of the principal study's findings, but is rated medium because of the lack of NOELs. Other chronic studies in rats ( using dietary exposures of 2.5 to 60 ppm) and dogs have also demonstrated aldrin's toxic effects on the liver ( Deichmann et al., 1970; Treon and Cleveland, 1955; NCI, 1978; the dog study in Fitzhugh et al., 1964).

In the principal study, groups of 24 rats ( 12/ sex) were fed aldrin in the diet at levels of 0, 0.5, 2, 10, 50, 100, or 150 ppm for 2 years. Liver lesions characteristic of chlorinated insecticide poisoning were observed at all exposure levels of aldrin. These lesions were characterized by enlarged centrilobular hepatic cells, with increased cytoplasmic oxyphilia and peripheral migration of basophilic granules. In addition, a statistically significant increase in liver to body weight ratio was observed at all dose levels. Kidney lesions at the highest dose levels were also reported and survival was markedly decreased at dose levels of 50 ppm and greater. The effect and no effect levels for liver toxicity are similar to those reported in the same study for dogs exposed to aldrin in the diet for 15 months ( Fitzhugh et al., 1964). While not permitting the determination of a NOAEL, the study does establish a LOAEL at the lowest aldrin concentration tested, 0.5 ppm.

RfD Derivation The RfD for aldrin was derived from the critical effect ( liver toxicity) that it induced in rats during a 2 year chronic feeding study ( Fitzhugh et al., 1964). This principal study reported 8 2 Aldrin/ Dieldrin   February 2003 that various toxic effects occurred in the liver at all aldrin concentrations tested ( 0.5 to 150 ppm). The resulting LOAEL dietary concentration of 0.5 ppm can be converted to a dose of 0.025 mg/ kg bw/ day by using an equivalency factor of 1 ppm in the diet = 0.05 mg/ kg bw/ day based on the food consumption rate in rats as described by Lehman ( 1959).

The RfD for aldrin is calculated as follows:

RfD = = 0.000025 mg/ kg bw/ day ( rounded to 3E 5 mg/ kg bw/ day) 0.025 mg / kg bw / day 1000 where:

0.025 mg/ kg bw/ day = LOAEL, based on liver toxicity in rats exposed to aldrin in the diet for 2 years 1000 = uncertainty factor; this composite uncertainty factor was chosen in accordance with EPA or NAS/ OW guidelines in which uncertainty factors of 10 each were applied to extrapolate from rats to humans, to account for uncertainty in the range of human sensitivity ( i. e., to protect sensitive human subpopulations), and to account for additional uncertainty because the study identified a LOAEL ( but not a NOAEL).

Dieldrin Choice of Principal Study and Critical Effect The study by Walker et al. ( 1969), also designed as a carcinogenesis bioassay, has been selected to serve as the basis for the Reference Dose principally because it was fairly extensively reported, the exposure period was of chronic duration, NOAELs were determined, and it is generally supported by other toxicity studies of dieldrin.