HEPTACHLOR First draft prepared by Dr. W. Phang, US Environmental Protection Agency, Washington, D.C., United States EXPLANATION The JMPR evaluated heptachlor in 1970 (Annex I, 14) and concluded that an adequate carcinogenicity study in a second species of animal other than the rat was needed. An estimated ADI of 0.0005 mg/kg/bw was allocated. Since the last Meeting, several toxicology, metabolism, and epidemiology studies, along with many case reports, have become available, which are summarized in this monograph. Some of the previously published reports reviewed in FAO/WHO monographs of 1967 and 1970 (Annex I, 7 and 15) are also included. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA Biochemical Aspects Absorption, distribution, and excretion Rat Although quantitative data on the absorption of heptachlor are not available, heptachlor is readily absorbed from the gastrointestinal tract as demonstrated by the presence of the parent compound and/or its metabolites in the urine, faeces, and tissues of rats which had been fed 14C-labelled heptachlor and in the serum of humans who had consumed undiluted raw milk products known to be contamin-ated with residues of heptachlor. Two male rats were given a single oral dose of 14C-heptachlor (0.464 µCi) in corn oil. During a 10 day monitoring period, 6% of the administered radioactivity was found in the urine while 60% was found in the faeces. Approximately 26% of the total radioactivity recovered from the faeces was unchanged heptachlor and the rest was in the form of metabolites (Tashiro & Matsumura, 1978). Heptachlor has been shown to be readily metabolized to heptachlor epoxide which was found to be distributed in adipose tissue of dogs and rats (Radomski & Davidow, 1953; Davidow & Radomski, 1953). When rats were fed a diet containing 35 ppm heptachlor for 2 months or more, the highest concentration of heptachlor epoxide was found in fat with substantially lower levels in liver, kidneys, and muscles. However, no heptachlor or heptachlor epoxide was found in the brain (Radomski & Davidow, 1953). The rate of heptachlor epoxide accumulation and elimination from the body fat was examined by placing male and female rats on a diet containing heptachlor for 12 weeks and then on the untreated diet for 12 more weeks. In males, at approximately 2 to 8 weeks of dosing the heptachlor epoxide level in fat reached a plateau; by 6 weeks after dosing the level was below the detection limit. In females, the heptachlor epoxide level in fat was much higher than that in males by 2 weeks and throughout the remaining duration of the study; by the 8th week after dosing the level was below the limit of detection (Radomski & Davidow, 1953). Biotransformation Rat In a metabolism study on heptachlor reported by Tashiro and Matsumura (1978), two male rats were administered a single oral dose of 14C-heptachlor (0.464 µCi) in 250 µl of corn oil; most of the radioactivity (approximately 60% of the administered dose) was recovered in the faeces. The major metabolites in the faeces were heptachlor epoxide, 1-hydroxychlordene, 1-hydroxychlordene epoxide, and 1,2-dihydroxydihydrochlordane, as well as two unindentified products. Similar metabolites were found in an in vitro comparative and qualitative study using human or rat liver microsomes. Based upon these findings, the metabolic scheme for heptachlor shown in Figure 1 was proposed. Heptachlor epoxide was also isolated from fat of dogs which had been administered heptachlor at dose levels of 1 to 3 mg/kg and rats fed 30 mg/kg heptachlor for 3 months (Davidow & Radomski, 1953; Radomski & Davidow, 1953). Effect on enzymes and other biochemical parameters From the information published in the 1971 FAO/WHO Monograph, heptachlor and aldrin appear to be substrates for the same enzyme, which is inhibited by the epoxide. In vitro microsomal metabolism of heptachlor does not proceed beyond formation of the epoxide. The treatment of rats with heptachlor increases the metabolism of fenitrothion (Annex 1, 15). Acute toxicity Some of the acute toxicity data of heptachlor and heptachlor epoxide were considered by the 1966 Joint Meeting and published in the 1967 FAO/WHO Monograph. Additional data have become available in the open literature; these data have been evaluated and are summarized with previously published data in Table 1. Clinical signs of acute toxicity of heptachlor include hypoactivity, tremor, convulsion, ataxia, and changes in EEG patterns. Histopathologically, severe liver damage was reported. Acute toxicity of metabolites The oral LD50 for four other metabolites of heptachlor (chlordene, 3-chlorochlordene, 1-hydroxychlordene, and chlordene epoxide) was reported to be greater than 4600 mg/kg body weight in male and female rats (Annex 1, 15).Table 1. Acute toxicity data of heptachlor and heptachlor epoxide Species Sex Route LD50 Reference (mg/kg bw) A. Heptachlor Rat M oral 71 Podowski et al. (1979) M&F oral 60-142a Velsicol Corp. (1959)b Chick d oral 63 Sherman & Ross (1961)b Mouse d oral 70 Gak et al. (1976) Hamster d oral 100 Gak et al. (1976) Rat M dermal 195c Gaines (1969) F dermal 250c Gaines (1969) B. Heptachlor epoxide Rat M&F oral 34-48a Velsicol Corp. (1959)b Mouse M oral 32-48 Velsicol Corp. (1959)b a Sex differences b Data excerpted from the 1966 JMPR monograph (Annex 1, 7) c Xylene was the solvent. d Not stated Short-term studies Mouse Groups of 10 Charles River CD-1 mice/sex/dose were fed a mixture of heptachlor-heptachlor epoxide (25:75) at dietary concentrations of 1, 5, 10, 25, and 50 mg/kg for 30 days. Following 30 days of treatment, the animals were sacrificed and necropsied. Microscopic examinations were conducted on liver tissue. No deaths occurred in the 1, 5, nor 10 mg/kg groups. One female died in the 25 mg/kg group, and 9 males and 8 females died in the 50 mg/kg group. No marked differences were seen between the treated animals and the controls with respect to body weights and food consumption. Mice which were sacrificed at the end of the study from the 10, 25 and 50 mg/kg groups showed liver enlargement associated with accentuated lobulation. The liver weight of the only surviving male mouse from the 50 mg/kg group was markedly increased (control mean: 1.69 g; 50 mg/kg; 4.00 gm), and increased mean liver weight was also found in the 25 ppm male mice (control, 1.69 g; 25 ppm, 4.35 gm). Histopathology findings indicated that livers of male mice in the 5, 10, 25, and 50 ppm and of female mice in the 10, 25, and 50 ppm groups had enlargement of centrilobular and midzonal hepatocytes with replacement of the normal coarse cytoplasmic granularity by finely granular and homogeneous cytoplasm. The severity of this lesion was dose-related. The incidences of the microscopic findings were not reported (Wazeter et al., 1971a). Based upon the histopathology findings, the NOAEL was 1 mg/kg. Rat Two short-term studies on heptachlor in rats were summarized in the 1971 monograph. The dietary levels tested in these two studies ranged from 5 to 45 ppm. At 10 ppm and above, rat liver cells showed an increase in smooth endoplasmic reticulum and mitochondria. The liver effects regressed after the treated rats were placed on a control diet for 120 days (Annex 1, 15). Long-term studies Some of the unpublished long-term studies on heptachlor were evaluated by the 1966 Joint Meeting and published in the 1967 Monograph. To facilitate consideration of the toxicity of this chemical the older toxicity studies are also summarized here. Rat Groups of Carworth Farms (CF) rats (20/sex/dose) were fed diets containing heptachlor (purity not specified) at a concentration of 0, 1.5, 3.0, 5.0, 7.0, or 10 ppm for 110 weeks. The treatment diet was prepared by spraying standard rat diet with an ethanol solution of heptachlor. After 7 weeks on the test diet, 5 females and 5 males from each dose group were mated to determine whether or not heptachlor would affect the reproductive capacity of the treated animals or the survival and subsequent growth of the offspring. Following similar procedures, another mating was conducted using different animals from each dose group after 22 weeks of treatment. The offspring were kept with their mothers until weaning at 3 weeks of age. After weaning, the offspring were placed on the control diet for 8 weeks for further observations. After 110 weeks of treatment, the surviving animals were sacrificed, certain haematological parameters were examined. Histopathology was conducted on the heart, liver, lungs, brain, spleen, kidneys, thyroids, and the adrenal glands. Heptachlor treatment did not affect mortality, body weight, or food consumption. The limited reproductive parameters measured did not indicate any effect on the body weights at birth or at weaning. Heptachlor administration did not appear to significantly affect the mortality of the offspring. Organ weights of the heptachlor-treated rats were not markedly different from those of controls. Haematology parameters (erythrocyte counts, haemoglobin, leukocyte counts, and differential white cell counts) did not show any compound-related effect. The histopathology data indicated 6/16 males and 3/18 females at 7.0 ppm and 2/12 males and 9/16 females at 10 ppm had slight hepatic cellular alterations characterized by swelling, homogeneity of the cytoplasm and peripheral arrangement of the cytoplasmic granules in the hepatic cell in the central zone of the lobules. Under the conditions of this study, heptachlor at doses up to 10 ppm did not produce an increase in the tumour incidence in CF rats (Witherup et al., 1955). Groups of CFN (Carworth Farms, Nelson) rats (25/sex/dose) were fed heptachlor epoxide at dietary concentrations of 0, 0.5, 2.5, 5.0, 7.5, and 10.0 ppm for 108 weeks. The test diet was prepared by spraying an ethanol solution of heptachlor epoxide on Purina Laboratory Chow. The survival rate was greater than 47% in the treated and control groups. There was an increase in the liver weights of treated females and a slight increase in treated males. The test chemical was not reported to cause any additional effects. This study has methodological deficiencies which include uncertain concentrations in the feed and insufficient number of rats in each dose level. Also the report does not contain histopathological data and is therefore not adequate for evaluating the long-term toxicity of heptachlor (Witherup et al., 1959). However, a panel of independent pathologists convened by the US National Academy of Sciences (1977) reviewed the histopathology and concluded that there was no increase in liver tumours in treated rats. Four groups of Charles River CD female rats (25/dose) were fed a 3:1 mixture of heptachlor:heptachlor epoxide at dietary concentrations of 5, 7.5, 10, and 12.5 ppm. A control group of 54 females was fed a diet containing the vehicle, corn oil. Interim sacrifices were conducted at 5 and 19 months to determine the nature and degree of the histological changes in the liver. At 5 months, 3 controls and 5 rats from the 7.5 ppm group were sacrificed. At 19 months, 5 controls, 2 rats from 5 ppm, 2 rats from 10 ppm, and 1 rat from 12.5 ppm were sacrificed. After 24 months of treatment, the surviving rats were sacrificed, and gross and histological examinations were conducted. There was an increase in mortality at 12.5 ppm relative to that of controls (control, 21%; 12.5 ppm, 50%). The test article did not affect body weight gain. Liver changes which were described as fatty liver and enlarged cells in the central zone of the hepatic lobules were reported. However, no histopathology data were included in the report (Jolley, 1966). Groups of Osborne-Mendel rats (50/sex/dose) were fed technical grade heptachlor (73% heptachlor, 18% trans-chlordane, and 2% cis-chlordane) in the diet at time-weighted doses of 38.9 and 77.9 ppm for males and 25.7 and 51.3 ppm for females. The matched controls consisted of 10 males and 10 females, and pooled controls were 60 rats/sex. The treated animals received the chemical for 80 weeks and were observed for 30 weeks. During the 80 weeks of treatment, the dietary concentrations changed three times for each treatment group based on changes in body weight. There was a decrease in the mean body weight of high dose males which almost returned to the control level after the treatment was terminated. There was also a slight increase in mortality in high dose male and female rats. Histopathology data revealed numerous inflammatory, degenerative, and proliferative lesions with approximately equal frequencies in treated and control animals. There was an increased incidence of thyroid follicular cell neoplasms, including combined adenomas and carcinomas in the high dose female rats (high dose, 14/38; low dose, 3/43; matched controls, 1/9; p<0.001) while, in male rats, a non-significant increase was observed only at the low dose (high dose, 3/38; low dose, 9/38; matched control, 1/8). At the same time, the incidences of the thyroid C-cell neoplasms decreased in high dose female rats (high dose, 3/38; low dose, 7/43; matched controls, 3/9; p<0.05). The results of analyses of the thyroid lesions were ambiguous and further complicated by variability in different experiments conducted at about the same time with this strain of rats (NCI, 1977). Dog Groups of Beagle dogs (4/sex/dose) were fed heptachlor epoxide (purity not specified) at concentrations of 0, 1, 3, 5, 7, and 10 ppm for two years. After 2 years on test, 2 dogs/sex/dose were sacrificed and necropsied while the other two dogs/sex/dose were maintained on the control diet for an additional 6 months (recovery period). In addition, the test animals in this study were also mated during the study and employed as the P1 parental animals for a 2-generation reproduction and teratology study. No death nor compound-related behavioural change was seen during the study. The test article did not affect the body weights or food consumptions of the treated animals. The parameters for haematology and urinalysis were comparable between treated and control dogs. There were increases in alkaline phosphatase activities in males and females at 3 ppm and above; these increases, in some dogs, were more marked towards the end of the treatment period and tended to persist throughout the recovery period. The serum albumin and total protein levels were slightly decreased in 10 ppm male and female dogs during the treatment and the recovery periods. An increase in the SGPT level was also seen in 10 ppm animals during the treatment, extending into the recovery period. After one year of treatment, the animals in 7 ppm group also showed an increase in the SGPT level which lasted into the recovery period. There was an increase in liver weights in 10 ppm male and female dogs relative to those of the controls, and this increase persisted with a slight attenuation during the recovery period. Histopathologic examination on dogs (2 dogs/sex/dose) sacrificed at the end of the treatment period showed an increase in the incidence of liver changes in animals at 3.0 ppm or above. The changes were described as enlargement and vacuolation of groups of centrilobular hepatocytes or scattered individual hepatocytes, presence of finely granular, and a "ground glass" appearance of the cytoplasm of large numbers of hepatocytes. These histopathological changes were also seen in dogs at 3 ppm and above after 6 months of recovery period. No compound-related histopathology changes were seen in 1 ppm dogs. Based upon the changes in biochemical parameters and the histological changes in the liver, the NOAEL was 1 ppm (Wazeter et al., 1971b). Although this study has certain deficiencies, including an insufficient number of experimental animals, the results provide useful information on the toxicity of the test compound in dogs. Groups of 23 to 27 week old beagle dogs (2 males or 3 females/dose) were fed heptachlor epoxide at dietary concentrations of 0, 0.5, 2.5, 5.5, and 7.5 ppm for sixty weeks. The test diet was prepared by spraying an ethyl alcohol solution of heptachlor epoxide on the food. The experimental results were limited to clinical observation, mortality, body weight changes, food consumption, and organ weights. The limited results indicated that there was an increase in liver weights of all the heptachlor treated male and female dogs relative to that of the controls. For 7.5 ppm males, the increase was approximately twice that of the controls. However, the pathology data were not included in the report (Witherup et al., 1958). Mouse Groups of Charles River CD-1 mice (100/sex/dose) received a mixture of heptachlor/heptachlor epoxide (25%/75%) at dietary concentrations of 0, 1.0, 5.0, and 10.0 ppm for 18 months. A positive control group (100 mice/sex) received 2-acetaminofluorene at a dietary concentration of 250 ppm. An interim sacrifice was performed on 10 mice/sex/dose including the positive control group. Gross pathology and histology examinations were conducted on animals which died during the study or were sacrificed on schedule. There was a decrease in the number of survivors in 10 ppm male and female mice, in 5 ppm females, and in positive control males relative to those of the negative controls as shown in Table 2. Mean body weights and food consumptions of the treated mice were not affected. There were increases in the mean liver weights of males and females in 5 and 10 ppm groups. An increase in the incidence of hepatocytomegaly was seen in all heptachlor-heptachlor epoxide treated mice relative to that of the controls. The incidence of hepatic nodular hyperplasia was markedly increased in 5 and 10 ppm males and females relative to that of the controls. For heptachlor-treated animals, hepatoma was observed in only one male mouse at 1 ppm (Wazeter, 1973). Based on the incidence of hepatocytomegaly, a NOAEL was not established (LOAEL <1ppm). The histological slides of this study were later evaluated by a panel of pathologists convened by the Pesticide Information Review and Evaluation Committee of the National Academy of Sciences, USA. The results of the evaluation are presented in Table 2. Levels of 1, 5, and 10 ppm of heptachlor-heptachlor epoxide did not produce a statistically significant increase in the incidence of hepatocellular carcinoma. However, at 10 ppm there was a statistically significant increase in the incidence of combined hepatocellular carcinomas and nodular changes (NAS, 1977). Epstein (1976) reported a previously unpublished study conducted by the US-FDA. Groups of C3H mice (100/sex/dose) were fed 0 or 10 ppm of either heptachlor or heptachlor epoxide (purity unspecified) for 24 months. The liver histopathology was re-evaluated by a panel of pathologists convened by the Pesticide Information Review and Evaluation Committee of the US National Academy of Sciences. Their results indicated a significant increase in hepatocellular carcinomas in females given heptachlor and in both males and females given heptachlor expoxide. Table 2. The incidence of hepatocellular carcinomas and nodular changes in CD-1 mice fed heptachlor-epoxide Male Female Group HC HC+N HC HC+N Control 1/59 2/59 1/74 1/74 1 ppm H-HE 2/66 4/66 1/65 3/65 5 ppm H-HE 2/66 4/66 1/65 3/65 10 ppm H-HE 1/73 27/73* 4/52 16/52* AAF 5/58 9/58 5/75 13/75 HC hepatocellular carcinomas HC+N hepatocellular carcinomas and nodular changes * (p<.001) Groups of B6C3F1 mice (50/sex/dose) received technical-grade heptachlor (72% heptachlor, 18% trans-chlordane, and 2% cis-chlordane) in the diet at initial concentrations of 10 and 20 ppm for males and 20 and 40 ppm for females. The initial concentrations were reduced due to adverse toxic effects, with time-weighted average concentrations of 6 and 14 ppm for males and 9 and 18 ppm for females. The mice were treated for 80 weeks and placed on the control diet for another 10 weeks. Matched-controls consisted of 20 males or 10 females, and the pooled controls were 100 males and 80 females. Under the conditions of the study, heptachlor had no significant effects on the body weights. Survival at 90 weeks was high: 70% of treated and control males and 60% of treated and control females. Survival of female mice showed a significant trend towards lower survival in treated groups compared to that in the control group. The incidence of hepatocellular carcinomas was significantly increased in the high-dose males and females as shown in Table 3 (NCI,1977). A review of liver histopathology from this study by a panel of pathologists convened by the Pesticide Information Review and Evaluation Committee of the US National Academy of Sciences concluded that there was a low incidence of induced hepatocellular carcinomas (Table 3). According to this review, the incidence of hepatocellular carcinomas was statistically increased (p<0.001) in groups of males and females receiving heptachlor at the high dose only when combined with the diagnosis of nodular changes (NAS, 1977). Table 3. Incidence of proliferative lesions of the liver in B6C3F1 mice fed technical grade heptachlor Lesion Pooled Matched Low Dose High Dose Control Control Males Hepatocellular carcinomas 17/92 5/19 11/46 34/47* Nodular hyperplasia 3/92 1/19 9/46 6/47 Diffuse hyperplasia 3/92 0/19 1/46 3/47 Hepatocytomegaly 0/92 0/19 1/46 0/47 Females Hepatocellular carcinomas 3/78 1/10 3/47 30/42* Nodular hyperplasia 2/78 0/10 3/47 0/42 Diffuse hyperplasia 1/78 0/10 0/47 0/42 Hepatocytomegaly 1/78 0/10 0/47 1/42 The denominator represents the number of tissues examined microscopically. * p<0.001 (Fisher exact test) Reproduction studies Dogs Groups of 6-9 month old Beagle dogs (4/sex/dose) were fed heptachlor epoxide at dietary concentrations of 0, 1, 3, 5, 7, and 10 ppm. When the female dogs attained an age of 14 months, they were mated twice with male dogs from the same dose group. The females were allowed to deliver and to nurse their pups. For the F2 generation, 4 female and 2 male pups of the F1 generation were selected from each dose level to be the parental animals (P2) of the F2 generation. At an approximate age of 14 months, the animals were mated, and the pregnant females were allowed to deliver and to nurse their pups to 6 weeks of age. At this time, the females and their pups were sacrificed. No clinical signs or behaviour changes were seen in treated P1, P2, or their offspring. There were no indications that body weight or food consumption were affected by the test chemical. Whether or not the test chemical produced any reproductive effects could not be determined with the very limited data presented in this report. There was a significant increase in the mortality rate of F1 pups in the 10 ppm group (control, 9/20; 10 ppm, 17/18). Only 1 male pup survived to the scheduled sacrifice, and no female was available to serve as a P2 parental animal. There were slight increases in death rates of F2 pups at 3 and 7 ppm relative to the controls (Control, 0/4; 3 ppm, 3/8; 7 ppm, 3/8). The 5 ppm group had no pups. Haematology and urinalysis results did not indicate any compound-related effects. Very limited clinical biochemistry data showed that alkaline phosphatase and/or SGPT activities in certain dogs were increased. However, the changes in the clinical chemistry parameters could not be correlated with pathology findings because the necropsy data on animals showing clinical biochemistry changes were not presented in the report. The limited necropsy data showed that, in F1 pups, 1/4 females at 7 ppm and 3/10 males and 3/7 females at 10 ppm had pale or greasy liver. This observation was compound-related. Based on the increase in the mortality rate, the NOAEL was 1 ppm (Wazeter et al., 1971c). Rat Three reproduction studies in rats were evaluated by the JMPR in 1966 and 1970. In a 3-generation reproduction study, a group of 80 rats were given 6.9 mg/kg of heptachlor daily for 3 months prior to mating. Cataracts, which became obvious between the 19th and 26th days, were found in 6.8% of the young. A similar lesion was present in 15.2% of the parental animals after 9 months. The only effect on reproduction was a decrease in the litter size (Annex 1, 7). In two other reproduction studies in rats, dietary levels of heptachlor ranging from 0.3 to 10 ppm were tested; no cataracts nor adverse effects on fertility and reproduction were found. A slight increase in post-natal mortality of pups was seen at 10 ppm (Annex 1, 15). Special studies for teratology and embryotoxicity Rabbit A rabbit teratology study was previously evaluated by the 1970 Joint Meeting, and it was concluded that no teratogenic effects could be attributed to the compound (Annex 1, 15). Special studies on genotoxicity In a large number of genotoxicity assays (Table 4), heptachlor did not induce genetic damage. Exceptions were the induction of small numbers of mutations in bacteria in the presence of plant extracts, mutations and a chromosomal aberrations in plants and in a single study, mutations at the highly sensitive tk locus in mouse lymphoma cells. Gap-junctional intercellular communication was inhibited in several in vitro assays. In both a sister chromatid exchange assay and a chromosome aberration assay in Chinese hamster ovary cells, there were "positive" results in the presence of rat S9 metabolic activation. In the absence of metabolic activation the chromosome aberration assay was negative (NTP, 1985). However, the tests were not repeated to confirm the positive results. The mutagenic potential of heptachlor epoxide was also tested. The results are presented in the following table. Table 4. Results of genotoxicity assays on heptachlor Test system Test object Concentration Resultsa Reference Ames test S. typhimurium 1000 µg/plate -/- Marshall et al. (1976) TA1535, TA1536 TA1537, TA1538 Ames test S. typhimurium 2500 µg/ml -/0 Simmon et al. (1977) TA98, TA100, TA1535, TA1537, TA1538 Ames test S. typhimurium not stated -/- Probst et al. (1981) TA98, TA100, TA1535, TA1537, TA1538, G46, C3076, D3052 E. coli WP2 & WP2 uvr A- Ames test S. typhimurium 0.1 µg/plate -/- NTP (1983) TA98, TA100, TA1535, to 10 mg/plate TA1537 Ames test S. typhimurium 5-10 µg/ml +/-b Gentile et al. (1982) TA98, TA100, TA1535 Ames test S. typhimurium 2500 µg/ml -/- Moriya et al. (1983) TA98, TA100, TA1535, TA1537, TA1538 E. coli WP2 hcr Table 4 (contd). Test system Test object Concentration Resultsa Reference Rec assay B. subtilis 356 µg/ml -/- Matsui et al. (1989) Differential E. coli WP 2 2000 µg/ml 0/- Rashid & Mumma (1986) toxicity S. typhimurium TA1538 Ames test S. typhimurium 1 mg/ml -/- Mersch-Sundermann (1988) TA97, TA98, TA100, TA102 Ames test S. typhimurium 167 µg/ml -/- Zeiger et al. (1987) TA98, TA100, TA1535, TA1537 Gene conversion S. cerevisia f -/- Gentile et al. (1982) Mutation Zea mays 1.12 kg/ha 0/+ Gentile et al. (1982) Chromosomal Lens sp. 0.1-0.3% 0/+ Jain (1988) aberrations Pisum sp. Micronuclei Tradescantia 1.88 ppm 0/+ Sandhu et al. (1989) SLRSc mutation D. melanogaster 5 µg/ml (feeding 0/- Benes & Sram solution) (1969) tk locus Mouse lymphoma 25 µg/ml 0/- McGregor et al. L5178Y mutation cells (1988 Table 4 (contd). Test system Test object Concentration Resultsa Reference ARL-HGPRT Adult rat liver 10-6 to 10-4 M -/NAd Telang et al. epithelial cell line (1982) Unscheduled DNA Mouse, rat and hamster 10-5 - Maslansky & Williams (1981) synthesis assay primary hepatocytes Primary liver cell explants from rats 10 nmole/ml - Probst et al. (1981) SV-40 transformed human fibroblasts 100 & 1000 µM ?/- Ahmed et al. (1977) (VA-4) Dominant Swiss mice 4.8 & 24 mg/kg (IP) - Epstein et al. (1972) lethal 5 & 10 mg/kg (PO) CD-1 mce 7.5 & 15 mg/kg - Arnold et al. (1977) 25:75e Inhibition of Mouse testicular cells 40 mg/kg 0/- Seiler (1977) DNA synthesis in vivo Inhibition of Chinese hamster V79 10 µg/ml 0/+ Kurata et al. (1982) metabolic cells cooperation Table 4 (contd). Test system Test object Concentration Resultsa Reference Inhibition of Rat hepatocytes 20 µg/ml 0/+ Ruch et al. (1990) metabolic Mouse hepatocytes cooperation a with S9 activation/without S9 activation; phi indicates that the test was not performed b with plant S9 c sex-linked recessive lethal d NA = not applicable e 25:75 blend w/w of heptachlor:heptachlor-epoxide f concentration in original paper cited as "an appropriate amount". Table 5. Results of genotoxicity assays on heptachlor epoxide Test system Test object Concentration Resultsa Reference Ames assay S. typhimurium 1000 µg/plate -/- Marshall et al. (1976) Unscheduled DNA SV-40 transformed 100 & 1000 µM +/- Ahmed et al. (1977) synthesis assay human fibroblasts (VA-4) Dominant lethal Swiss mice 6 & 30 mg/kg (IP) - Epstein et al. (1972) 8 mg/kg (PO) a with S9 activation/without S9 activation Observations in humans In 1986, a study was conducted in the Midwest United States on a group of 45 dairy farm family members who had consumed undiluted raw milk products known to have been contaminated with heptachlor at concentrations as high as 89.2 ppm (fat basis). The serum levels of heptachlor or its metabolites in the exposed individuals were compared to those from an unexposed group of 94 persons from the same geographical area and to results from the Second National Health and Nutrition Examination Survey (2nd NHANES). The exposed group had significantly higher mean levels of primary metabolites such as heptachlor expoxide (0.84±1.0 vs 0.50±0.9 ppb) and oxychlordane (0.71±0.8 vs 0.49±1.1 ppb) than the unexposed group. In addition, the percentage of individuals in exposed group with elevated serum concentrations of these same metabolites (21.2%) was higher than that for the unexposed group (heptachlor expoxide, 3.8%; oxychlordane, 6.3%) and the second National Health and Nutrition Survey (2.5% for both metabolites) (Stehr-Green et al., 1988). There is evidence of transplacental transfer of heptachlor epoxide. The following heptachlor epoxide levels were detected in various tissues of mother and newborn infants (Polishuk, 1977a): adipose tissue (maternal): 0.2856 ± 0.3950 ppm maternal blood: 0.2798 ± 0.4626 ppm uterine muscle: 0.4895 ± 0.5086 ppm fetal blood: 0.9959 ± 0.9458 ppm placenta: 0.5000 ± 0.3950 ppm amniotic fluid: 0.6730 ± 1.1645 ppm Several investigators detected heptachlor epoxide levels ranging from non-detectable to 0.46 ppm in human milk (Kroger, 1972; Polishuk et al., 1977b; Strassman & Kutz, 1977; Savage et al., 1981; Takahashi et al., 1981; Takei et al., 1983). No adverse effects on human fetal development were reported following ingestion of milk containing heptachlor for 27 to 29 months among women of child bearing age in Oahu, Hawaii, USA. The levels of heptachlor in milk fat ranged from 0.12 to 5.00 ppm (Le Marchand et al., 1986). A retrospective mortality study was conducted on workers employed in the manufacture of heptachlor and chlordane between 1946 and 1976. The study group consisted of 1403 white males who were employed for more than 3 months at either of the two plants which produced heptachlor and chlordane in the United States. The mortality information was obtained from the Social Security Administration files and supplemented by information collected by another investigator through follow-up. The results indicated an excess of death from cerebrovascular disease (17 observed, 9.3 expected) (Wang & MacMahon, 1979a). The mortality of 16 126 males employed as pesticide applicators was evaluated. The cohort was employed between 1967 and 1976 by any of the nationwide (US) pest control companies for 3 months or more. The information on mortality was traced in Social Security Administration files. The results did not demonstrate a significant difference in mortality from the norm in the cohort (Wang & MacMahon, 1979b). An update of the above study group was conducted by following the same cohort to the end of 1984. The analysis showed an excess of lung cancer among the pesticide applicators. However, these applicators were exposed to a broad spectrum of pesticides, and the increased lung cancer risk among the pesticide applicators might not relate to their exposure to heptachlor alone (MacMahon et al., 1987). A retrospective mortality study of 2141 workers from 4 manufacturing plants of chlorinated hydrocarbon pesticides, which included chlordane, heptachlor, DDT, aldrin, dieldrin, and endrin was reported by Ditraglia et al., 1981. The workers in each plant were considered as a separate cohort, each had worked at least 6 months prior to 31 December 1964. The results showed too few deaths to allow any meaningful conclusions (Ditraglia et al., 1981). Another retrospective study was conducted by Shindell and Associates (1981) on a cohort of 1115 employees who worked 3 months or more at the Memphis, Tennessee, USA, plant of Velsicol Chemical Corp., from 1 January 1952 to 31 December 1979. The plant began to manufacture heptachlor in 1952. The data on morbidity and mortality were obtained for 93.1% of the male and 90% of the female cohort. The workers were classified according to their job or product exposure. Mortality data on the cohorts were compared to the overall mortality experience of like segments (by age and sex) of the United States population at large to determine whether Memphis plant exposure caused any discernible variations from the expected mortality from all causes and from selected significant causes. No significant difference was found. COMMENTS Heptachlor is structurally similar to chlordane. An ADI of 0.0005 mg/kg bw was estimated for heptachlor by JMPR in 1970. Heptachlor is well absorbed through the GI tract and metabolized to a large extent to heptachlor epoxide and minor metabolites. Heptachlor epoxide has been shown to accumulate in adipose tissue, and to cross the placenta, but it was not found in the brain. In rats, the major route of elimination is via the faeces. In both short- and long-term studies in dogs, rats, and mice, the liver was found to be the target organ. In a 30-day feeding study in mice at dietary concentrations of 0, 1, 5, 10, 25 or 50 ppm, histopathology findings indicated that males at 5 ppm or above and females at 10 ppm or above had enlargement of centrilobular and midzonal hepatocytes. The severity was dose-related. Based upon these results, the NOAEL was 1 ppm, equivalent to 0.15 mg/kg bw/day. Several long-term/carcinogenicity studies in rats were reviewed, but the majority of them had severe methodological limitations. In one study, rats received time-weighted dietary concentrations of 39 or 78 ppm (males) and 26 or 51 ppm (females) of heptachlor for 80 weeks. On the basis of decreased body-weight in high-dose males and increases in mortality in high-dose males and females, the NOAEL was 26 ppm, equivalent to 1.3 mg/kg bw/day. Deficiencies in this study precluded proper evaluation of the carcinogenic potential of heptachlor in rats. In a 2-year feeding study, dogs fed heptachlor epoxide at dietary concentrations of 0, 1, 3, 5, 7 or 10 ppm, exhibited an increase in liver weight at 10 ppm and an increase in the incidence of liver histopathological changes at 3 ppm and above. The histopathological changes were enlargement and vacuolation of centrilobular or scattered hepatocytes. Similar histological changes persisted through six months of the recovery period. The NOAEL based upon these findings was 1 ppm, equivalent to 0.025 mg/kg bw/day. Heptachlor/heptachlor epoxide is carcinogenic in mice; two studies demonstrated an increase in liver tumour incidence in male and female Charles River CD-1 and B6C3F1 mice. Heptachlor/heptachlor epoxide also caused a marginal increase in the incidence of hepatocytomegaly in all treated CD-1 mice; a NOAEL was not established in this study. In a 2-generation reproduction study in dogs at dietary concentrations of 1, 3, 5, 7, or 10 ppm of heptachlor epoxide, there was an increase in mortality of F2 pups at 3 ppm and above. The NOAEL based on this finding was 1 ppm. The available epidemiology studies have not shown a clear relationship between any effects in humans and exposure to heptachlor. After reviewing the available in vitro and in vivo short-term test data, it was concluded that, although it can interfere with intercellular communication, heptachlor is not genotoxic. Many studies available for evaluation were conducted more than 20 years ago and have severe deficiencies. Because of these deficiencies, its carcinogenicity in mice and its ability to bioaccumulate, the Meeting recommended that heptachlor should not be used directly on food crops and its use in the production of food commodities should be phased out. Because of its environmental persistence it is found as a contaminant in food commodities. 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See Also: Toxicological Abbreviations Heptachlor (EHC 38, 1984) Heptachlor (HSG 14, 1988) Heptachlor (ICSC) Heptachlor (PIM 578) Heptachlor (FAO Meeting Report PL/1965/10/1) Heptachlor (FAO/PL:CP/15) Heptachlor (FAO/PL:1967/M/11/1) Heptachlor (FAO/PL:1968/M/9/1) Heptachlor (FAO/PL:1969/M/17/1) Heptachlor (AGP:1970/M/12/1) Heptachlor (WHO Pesticide Residues Series 4) Heptachlor (WHO Pesticide Residues Series 5) Heptachlor (CICADS 70, 2006)