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Failure of hexachlorobenzene to induce dominant lethal mutations in the rat
MXICOLOGY
AND
APPLIED
PHARMACOLOOY
47, 415-419
(1979)
Failure of Hexachlorobenzene to Induce Dominant Lethal Mutations in the RaP” Failure of Hexachlorobenzene to Induce Dominant Lethal Mutations in the Rat. SIMON, R. G., AND BORZELLECA, J. F. (1979). Toxicol. Appl. Pharmacol. 47.415419. Three groups of 10 male rats each were gavaged with 0, 70, or 221 mg hexachlorobenzene (HCB)/kg daily for 5 consecutive days. Ten other male rats received a single oral dose of 0.5 mg triethylenemelamine (TEM)/kg. Both compounds were dissolved in corn oil. Following the last administration, each male rat was allowed to mate with two naive, nulliparous females per week for 14 consecutive weeks. Females were sacrificed on Day 14 of gestation. Their ovaries and uteri were exposed and examined for numbers of corpora lutea and total, living and dead implantations. Mating and fertility frequencies were calculated. TEM elicited typical dominant lethal events. HCB did not. Dose-dependent decreases in the numbers of females inseminated and impregnated were observed. G. S., TARDIFF,
The production of hexachlorobenzene (HCB) has increased markedly in recent years, from 0.8 metric tons in 1966 to 6.9 metric tons in 1971 (National Academy of Sciences, 1975). HCB is widely used as a seed dressing in wheat for prevention of fungal diseases and is a reported contaminant in organochlorine pesticides (Gilbertson and Reynolds, 1972). HCB has been found in samples of ocean and river water. Its persistence in the environment has also been reported (Seltzer, 1975; Yamato et al., 1975). Gilbertson and Reynolds (1972) have reported the presence of HCB in the eggs of common terns, presumably due to their consumption of contaminated fish. Brady and Siyali (1972) found HCB residues in human adipose tissue and milk. Experimentally administered HCB is stored in the adipose tissue of rats, dogs, and cows and persists for several months (Borzelleca et al., 1971). Over 3000 cases of human cutaneous porphyria in Turkey during 1955r Presented in part at the 17th Annual Meeting of the Society of Toxicology, San Francisco, California. March 13-16, 1978. 2 Support in part by EPA Grant R-804290010.
1959 have been attributed to the accidental ingestion of HCB-contaminated grain (Cam and Nigogosyan, 1963). HCB has also been shown to cause neuropathy (DeMatteis et al., 1961), liver damage (Medline et al., 1973), and cancer in Syrian golden hamsters (Cabral et al., 1977). The presence of HCB in the human food chain indicates the need for a thorough evaluation of the health effect of HCB, including potential mutagenicity. Khera (1974) reported the failure of HCB to induce dominant lethal mutations in male rats administered up to 60 mg/kg/day for 10 days. No adverse effects upon reproductive function or performance and no teratogenic effects due to HCB were reported. Grant et al. (1977) reported that the continual administration of HCB in the feed of male and female weanling rats at levels up to 640 ppm (approximately equal to Khera’s 60 mg/kg/ day) decreased lactation, fetal body weights, and fetal viability in a four-generation reproduction study. Our purpose was to determine whether the reported adverse reproductive effects of 415
0041-008x/79/020415~5802.0010 Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
416
SHORT
COMMUNICATIONS
Mating Index (MI) = (total number of females pregnant/total number of females allowed to mate) x 100. Fertility Index (FYI) = (total number of females pregnant/total number of females inseminated) x 100. Corpora Lutea Index (CLI) = total number of corporea lutes/total number of pregnant females. Implantation Index (II) = total number of implantation sites/total number of pregnant females. Preimplantation Loss Index (PILI) = (total number of corpora lutea- total number implantation sites)/total number of pregnant females. Fetal Index (FI) = total number of living fetuses/ total number of pregnant females. Resorption Index (RI) = total number of fetal deaths/total number of pregnant females. DF/LF = total number of dead fetuses/total number of living fetuses. DF>l = total number of females with one or more dead fetuses/total number of females with zero dead fetuses. DF>2 = total number of females with two or more dead fetuses/total number of females with one or zero dead fetuses.
continually fed low levels of HCB could be elicited by the subacute administration of high doses and to determine whether these high doses are mutagenic. METHODS Forty adults male rats (Flow Laboratories, Dublin, Va.) were divided equally into four groups. Each rat received either 0,70, or 221 mg HCB (po)/kg daily for 5 consecutive days (Bateman and Epstein, 1971; Green er al., 1976), or a single oral dose of 0.5 mg triethylemelamine (TEM)/kg. Both test compounds were dissolved in corn oil (Mazola). A preliminary study determined that the administration of 350 mg HCB/kg/day (one-tenth the acute, oral LD50) for 5 days resulted in 30% lethality within 10 days. Laboratory chow (Purina) and water were available ad libitum. Each male was allowed to mate with two naive, nulliparous females per week for 14 consecutive weeks. Insemination was determined by the microscopic examination of daily vaginal smears for sperm. Male rats were weighed daily during compound administration and weekly thereafter. Females were weighed before mating and immediately before sacrifice. Females were sacrificed on Day 14 of gestation, their uteri and ovaries exposed and examined for numbers of corpora lutea and total, living and dead implantations. Mating and fertility frequencies as described below were calculated. Data were subjected to the following statistical analyses: Green et al. (1976), Mantel and Haenszel (1959). Results were considered significant when ~~0.05. The following indexes were calculated:
RESULTS AND DISCUSSION No significant difference in body weights of male rats administered corn oil, HCB, or TEM were observed. TEM elicited the classical dominant lethal mutations during the first 7 weeks of the experimental period (Table I) as reported by Bateman and Epstein (1971).
TABLE REPRODUCTIVE
Number Week pregnant
DATA
MI
FOR FEMALE
FYI
Corn oil control (10 mg/kg/day) 1 14 70 93 2 16 80 100 3 17 85 100 4 13 65 93 5 17 85 100 6 15 75 100 7 18 90 100 8 17 85 100 9 15 75 100 10 16 80 89 11 16 80 94 12 13 65 100 13 15 75 94 14 13 65 100
RATS MATED
CL1
II
12.85 15.81 15.47 15.38 15.00 13.93 15.94 15.52 13.73 10.56 13.87 13.84 15.46 15.92
10.85 11.37 12.17 11.61 11.23 10.46 12.22 10.76 10.46 9.18 10.31 11.92 11.00 10.69
1
TO MALES
PILI 2.00 4.43 3.29 3.84 3.82 3.46 3.72 4.76 3.26 1.37 3.56 1.92 4.46 5.46
TREATED WITH HEXACHMROBENZENE?
FI
RI
DF/LF
10.28 10.81 11.17 11.07 10.88 10.13 11.66 10.35 9.86 8.87 9.68 11.46 10.53 9.53
0.57 0.62 1.00 0.53 0.35 0.33 0.55 0.41 0.66 0.31 0.66 0.46 0.46 1.15
71134 10/173 17/190 71144 6/185 5/152 10/210 71163 lo/148 51155 10/155 6149 6/158 151124
DFal 519 917 819 617 5112 3112 6112 5112 718 4/12 4/12 5/8
619 914
DF>2 2112 l/15 2115 l/12 l/16 l/14 3/15 2115 3112 3113 3113 l/12 l/14 8/9
SHORT
TABLE
Week TEM
Number pregnant
417
COMMUNICATIONS
l-Continued
MI
FYI
CL1
II
PILI
FI
RI
DF/LF
DFB
1
DFa2
50 65 15 a5 75 55 a5 65 80 65 80 55 85 80
100 93 94 94 94 100 100 86 94 81 94 79 100 94
13.10 16.84 15.80 15.88 15.40 13.81 15.47 15.61 13.50 11.23 16.37 17.27* 15.52 14.75
11.00 10.69 10.20* 11.47 9.66 10.81 10.358 10.92 a.43b 10.15 11.06 11.00 10.64 9.62
2.10 6.15 5.60 5.05 5.13 3.36 5.17 4.76 5.06 1.69 5.31 6.27b 4.88 5.12
6.5ob 6.92b 4.26” 8.52 a.93 9.21 9.17b 10.15 7.93 9.46 10.50 10.54 9.52 9.31
4.5ob 3.76b 5.93b 2.35 0.73 1.54 1.17 0.76 0.60 0.69 0.50 0.45 1.00 0.316
20168’ 49/9Oc 89/64c 40/45c 11/134 17/102c 20/156’ 101132 81127 91123 8/169 5/116 17/162 51149’
10/w 12/l 1s/oc 11/6 a/7 417 a/9 716 719 419 719 417 1017 5/l 1
10/o= 10/3’ 14/l’ 7110 2113 l/10 5112 2/l 1 l/15 2/l 1 l/15 l/10 2115 O/16
50 70 80 85 65 a5 65 15 75 85 15 75 70 65
83 93 94 93 87 100 100 100 94 100 79 100 93 93
15.60 14.71 13.68 17.46 14.23 16.11 16.76 15.40 14.26 10.94 16.60 15.20 12.42” 15.61
11.00 10.78 11.93 10.46 11.23 10.17 11.92 10.66 10.40 10.00 11.00 10.80 10.35 10.23
4.60 4.57 1.87 7.00 3.07 5.94 4.84 4.13 3.86 1.00 5.60 4.40 2.14b 5.38
10.80 10.00 11.31 9.92 10.84 9.58 11.38 9.66 9.86 9.52 10.53 960 9.78 8.84
0.20 0.85 0.62 0.53 0.38 0.58 0.53 1.00 0.66 0.47 0.46 0.53 0.57 0.69
21108 12/140 lo/181 71129 5/141 lo/163 71148 14/145 lo/148 811.62 7/158 81144 a/137 9/124
218 w
o/10 l/12 l/15 2/11 l/12 2115 o/13 3112 3/12 2115 l/14 l/14 2112 2/l 1
45 40 55 50 60 80 55 15 80 60 65 50 55 60
100 100 100 100 92 100 73 100 100 75 93 91 85 100
13.00 12.50 14.90 14.49 16.41 14.37 16.41 14.06 15.25 14.0ab 13.38 12.40 14.81 14.33
11.66 10.25 11.63 10.30 11.08 10.37 11.54 10.33 8.12 11.66* 9.69 9.2ob 10.81 10.66
1.44 1 .I5 3.21 4.10 5.33 4.00 4.90 3.13 4.62 2.41 3.69 3.20 4.09 3.66
10.88 9.75 10.45 10.10 10.66 10.00 11.00 9.60 10.06 10.91b 9.23 8.80b 9.72 10.58
0.77 0.62 1.18 0.20 0.41 0.31 0.54 0.73 0.56 0.75 0.46 0.40 1.09b O.OSb
7188 4178 13/115 2/101 51128 6/160 6/121 11/144 9/161 9/131 6/120 4188 12/107 l/127’
(0.5 mg/kg) 1 2 3 4 5 6 7 a 9 10 11 12 13 14
HCB
10 13 15 11 15 11 17 13 16 13 16 11 17 16 (70 mg/kg/day)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 HCB 1 2 3 4 5 6 7 8 9 10 11 12 13 14
10 14 16 17 13 17 13 15 15 17 15 15 14 13 (221 mg/kg/day) 9’ 8’ 11’ 10 12 16 11c 15 16 12 13 10 11 12
a HCB and corn oil, 5 days oral dosing: TEM, single oral dose. b Significantly different from control (Student’s t test, ~90.05). c Significantly different from control (x2 with Yates’ correction; psO.05).
818 5/a 419 a/9 716 817 4/l 1 5112 S/l0 718 519 716
316 315 a/3
m 4/a 6110 615 916 719
616 5/a 317 a/3 l/11’
217 l/7 219 o/10 l/11 O/16 O/l 1 2/13 2114 l/11 11/12 l/9 417 o/12
418
SHORT
COMMUNICATIONS
The female rats mated to males that received HCB showed the following statistically significant events. There was a decrease in corpora lutea and preimplantation losses during Week 13 at the low level. At the high level, there was an increase in corpora lutea, implantations, and living fetuses during Week 10; a decrease in implantations and living fetuses during Week 12; an increase in resorptions during Week 13 ; and a decrease in resorptions, the proportion of nonliving to living fetuses, and the proportion of females bearing one or more resorptions during Week 14. The events elicited by TEM follow the pattern of classical mutagens (Bateman, 1966). HCB did not induce any classical dominant lethal effects. Most chemicals that cause dominant lethal events manifest themselves during the early weeks (l-5) of mating (Bateman, 1966). Statistically significant effects among the HCB-treated groups were observed during Weeks 10-14. These effects were inconsistent in their extent and direction. Animals treated with HCB did not perform as well as controls during Weeks 12 and 13, but performed better than controls during Weeks 10 and 14. When compared with 14 weeks of pooled controls, no significant effects were seen among the HCBtreated groups. These inconsistent results may be attributed to weekly fluctuations in the test groups. HCB apparently had a dose-dependent effect upon male reproductive performance. At the low dose, the males impregnated fewer females (vs control males) 8 of the 14 weeks and, at the high dose, 12 of the 14 weeks (Mating Index). The effect at the high dose was significant (p < 0.001) using the sum of x2 procedure (Mantel and Haenszel, 1959). The percentage of inseminated females made pregnant (Fertility Index) was not adversely affected. HCB is well absorbed from the gastrointestinal tract (Mehendale et al., 1975) and persists in rat tissues for months following 33 weeks of administration via feed (Borzel-
leca et al., 1971). In the studies described herein, the administration of up to 221 mg/ kg HCB for 5 days failed to elicit dominant lethal effects, but did adversely affect male reproductive performance up to 14 weeks after HCB administration. Khera (1974) reported a lack of dominant lethal mutations in rats following administration of up to 60 mg/kg HCB for 10 days and reported no effects upon reproduction. Grant et al. (1977) reported decreased reproductive performance by males and females exposed to HCB at dietary levels of up to 640 ppm for life. Their experimental conditions were similar to those of Khera with three exceptions: (1) Both males and females were fed HCB; (2) Khera’s rats were exposed by gavage; and (3) exposure commenced during the weanling stage of life, and continued through the next three generations. These adverse effects reported to be due to HCB might be the result of increased sensitivity to HCB during the early stages of life. The work of Kimbrough and Linder (1974) and Grant et al. (1974) suggests that female rats may be more sensitive than males to some of the effects of HCB (lethality, respiratory, porphyria) but equally or less sensitive to others. The effects seen by Grant et al. (1977) might be explained by this differential sensitivity, though specific effects upon the female reproductive system were not reported by Kimbrough and Linder (1974). We conclude that HCB is not a dominant lethal mutant under the conditions tested, but can have adverse effects upon male reproductive performance.
REFERENCES BATEMAN, A. J. (1966). Testing chemicals for mutagenicity in a mammal. Nuture (London) 210, 205-206.
BATEMAN, A. J., and EPSTEIN, S. S. (1971). Dominant lethal mutations in mammals. In Chemical Mutagem. Principles and Methods for Their Detection (A. Hollander, ed.), Vol. 2, pp. 541-568. Plenum, New York.
SHORT
COMMUNICATIONS
BORZELLECA, J. F., LARSON, P. S., CRAWFORD,E. M., HENNIGAR, G. R., JR., KUCHAR, E. J., AND KLEIN, H. H. (1971). Toxicologic and metabolic studies on pentachloronitrobenzene. Toxicol. Appl. Pharmacol. 18, 522-534.
BRADY, M. N., AND SIYALI, D. S. (1972). Hexachlorobenzene in human body fat. Med. J. Aust. 1, 158. CABRAL, J. R. P., SHUBIK, P., MOLLNER, T., AND RAITANO, F. (1977). Carcinogenic activity of hexachlorobenzene in hamsters. Science 269, 510-511. CAM, C. AND NIGOGOSYAN,G. (1963). Acquired toxic porphyria cutanea tarda due to hexachlorobenzene. Report, of 348 cases caused by this fungicide. J. Amer. Med. Ass. 183, 88-91. DEMA?TEIS, F. PRIOR, B. E., AND RIMINGTON, C. (1961). Nervous and biochemical disturbances following hexachlorobenzene intoxication. Nafure (London) 191, 363-366. GILBERTSON, M., AND REYNOLDS,L. M. (1972). Hexachlorobenzene (HCB) in the eggs of common terns in Hamilton Harbor, Ontario. BUN. Environ. Contam. Toxicol. 7, 371-373. GRANT, D. L., IVERSON, F., HATINA, G. V., AND VILLENEUVE, D. C. (1974). Effects of hexachlorobenzene on liver porphyrin levels and microsomal enzymes in the rat, Environ. Physiol. Biochem. 4, 159-165.
419
KIMBROUGH, R. D., AND LINDER, R. E. (1974). The toxicity of technical hexachlorobenzene in the Sherman strain rat. A preliminary study. Res. Commun. Chem. Pathol. Pharmacol. 8(4), 653-664. KOEMAN, J. H., TEN NOEVER DE BRAUW, M. C., AND DE Vos, R. H. (1969). Chlorinated biphenyls in fish, mussels and birds from the river Rhine and the Netherlands coastal area. Nature (London) 221, 1125-1128. MANTEL, N., AND HAENSZEL, W. (1959). Statistical aspects of the analysis of data from retrospective studies of disease. J. Nat. Cancer Inst. 22, 719-748. MEDLINE, A., BAIN, E., MENON, A., AND HABERMAN, H. (1973). Hexachlorobenzene and the rat liver. Arch. Pathol. 96, 61-65. MEHENDALE, H. M., FIELDS, M., AND MATTHEWS, H. B. (1975). Metabolism and effects of hexachlorobenzene on hepatic microsomal enzymes in the rat. J. Agr. Food Chem. 23,261-265. NATIONAL ACADEMY OF SCIENCES (1975). Assessing Potential Ocean Pollutants, pp. 188-208. National Academy of Sciences, Washington, D.C. SELTZER, R. J. (1975). Ocean pollutants pose potential danger to man. Chem. Eng. News 53, 19-20. YAMAMOTO, Y. SUZUKI, M., AND AKIYAMA, T. (1975). Persistence of BHC in river water in the Kitakyushu District, Japan, 1970-1974. Bull. Environ. Contam. Toxicol. 14, 380-384. GLENN STUART SIMON~ ROBERT G. TARDIFF~ JOSEPH F. BORZELLECA Division of Toxicology Department of Pharmacology Medical College of Virginia Richmond, Virginia 23298 Received March 22, 1978; accepted September 24, 1978
GRANT, D. L., PHILLIPS, W. E., JR., AND HATINA, G. V. (1977). Effect of hexacfilorobenzene on reproduction in the rat. Arch. Environ. Contam. 5, 207-216. GREEN, S., ZEIGER, E., PALMER, K. A., SPRINGER, J. A., AND LEGATOR, M. S. (1976). Protocols for the dominant lethal test, host-medicated assay, and in vitro cytogenic test used in the Food and Drug Administration’s review of substances in the GRAS (Generally Recognized As Safe) list. J. ’ Recipient of NIH Predoctoral Training FellowToxicol. Environ. Health 1, 921-928. KHERA, K. S. (1974). Teratogenicity and dominant ship GM071 1 l-03. 4 Present address: National Academy of Sciences, lethal studies on hexachlorobenzene in rats. Food Cosmet. Toxicol. 12, 471-477. Washington, D.C.
AND
APPLIED
PHARMACOLOOY
47, 415-419
(1979)
Failure of Hexachlorobenzene to Induce Dominant Lethal Mutations in the RaP” Failure of Hexachlorobenzene to Induce Dominant Lethal Mutations in the Rat. SIMON, R. G., AND BORZELLECA, J. F. (1979). Toxicol. Appl. Pharmacol. 47.415419. Three groups of 10 male rats each were gavaged with 0, 70, or 221 mg hexachlorobenzene (HCB)/kg daily for 5 consecutive days. Ten other male rats received a single oral dose of 0.5 mg triethylenemelamine (TEM)/kg. Both compounds were dissolved in corn oil. Following the last administration, each male rat was allowed to mate with two naive, nulliparous females per week for 14 consecutive weeks. Females were sacrificed on Day 14 of gestation. Their ovaries and uteri were exposed and examined for numbers of corpora lutea and total, living and dead implantations. Mating and fertility frequencies were calculated. TEM elicited typical dominant lethal events. HCB did not. Dose-dependent decreases in the numbers of females inseminated and impregnated were observed. G. S., TARDIFF,
The production of hexachlorobenzene (HCB) has increased markedly in recent years, from 0.8 metric tons in 1966 to 6.9 metric tons in 1971 (National Academy of Sciences, 1975). HCB is widely used as a seed dressing in wheat for prevention of fungal diseases and is a reported contaminant in organochlorine pesticides (Gilbertson and Reynolds, 1972). HCB has been found in samples of ocean and river water. Its persistence in the environment has also been reported (Seltzer, 1975; Yamato et al., 1975). Gilbertson and Reynolds (1972) have reported the presence of HCB in the eggs of common terns, presumably due to their consumption of contaminated fish. Brady and Siyali (1972) found HCB residues in human adipose tissue and milk. Experimentally administered HCB is stored in the adipose tissue of rats, dogs, and cows and persists for several months (Borzelleca et al., 1971). Over 3000 cases of human cutaneous porphyria in Turkey during 1955r Presented in part at the 17th Annual Meeting of the Society of Toxicology, San Francisco, California. March 13-16, 1978. 2 Support in part by EPA Grant R-804290010.
1959 have been attributed to the accidental ingestion of HCB-contaminated grain (Cam and Nigogosyan, 1963). HCB has also been shown to cause neuropathy (DeMatteis et al., 1961), liver damage (Medline et al., 1973), and cancer in Syrian golden hamsters (Cabral et al., 1977). The presence of HCB in the human food chain indicates the need for a thorough evaluation of the health effect of HCB, including potential mutagenicity. Khera (1974) reported the failure of HCB to induce dominant lethal mutations in male rats administered up to 60 mg/kg/day for 10 days. No adverse effects upon reproductive function or performance and no teratogenic effects due to HCB were reported. Grant et al. (1977) reported that the continual administration of HCB in the feed of male and female weanling rats at levels up to 640 ppm (approximately equal to Khera’s 60 mg/kg/ day) decreased lactation, fetal body weights, and fetal viability in a four-generation reproduction study. Our purpose was to determine whether the reported adverse reproductive effects of 415
0041-008x/79/020415~5802.0010 Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
416
SHORT
COMMUNICATIONS
Mating Index (MI) = (total number of females pregnant/total number of females allowed to mate) x 100. Fertility Index (FYI) = (total number of females pregnant/total number of females inseminated) x 100. Corpora Lutea Index (CLI) = total number of corporea lutes/total number of pregnant females. Implantation Index (II) = total number of implantation sites/total number of pregnant females. Preimplantation Loss Index (PILI) = (total number of corpora lutea- total number implantation sites)/total number of pregnant females. Fetal Index (FI) = total number of living fetuses/ total number of pregnant females. Resorption Index (RI) = total number of fetal deaths/total number of pregnant females. DF/LF = total number of dead fetuses/total number of living fetuses. DF>l = total number of females with one or more dead fetuses/total number of females with zero dead fetuses. DF>2 = total number of females with two or more dead fetuses/total number of females with one or zero dead fetuses.
continually fed low levels of HCB could be elicited by the subacute administration of high doses and to determine whether these high doses are mutagenic. METHODS Forty adults male rats (Flow Laboratories, Dublin, Va.) were divided equally into four groups. Each rat received either 0,70, or 221 mg HCB (po)/kg daily for 5 consecutive days (Bateman and Epstein, 1971; Green er al., 1976), or a single oral dose of 0.5 mg triethylemelamine (TEM)/kg. Both test compounds were dissolved in corn oil (Mazola). A preliminary study determined that the administration of 350 mg HCB/kg/day (one-tenth the acute, oral LD50) for 5 days resulted in 30% lethality within 10 days. Laboratory chow (Purina) and water were available ad libitum. Each male was allowed to mate with two naive, nulliparous females per week for 14 consecutive weeks. Insemination was determined by the microscopic examination of daily vaginal smears for sperm. Male rats were weighed daily during compound administration and weekly thereafter. Females were weighed before mating and immediately before sacrifice. Females were sacrificed on Day 14 of gestation, their uteri and ovaries exposed and examined for numbers of corpora lutea and total, living and dead implantations. Mating and fertility frequencies as described below were calculated. Data were subjected to the following statistical analyses: Green et al. (1976), Mantel and Haenszel (1959). Results were considered significant when ~~0.05. The following indexes were calculated:
RESULTS AND DISCUSSION No significant difference in body weights of male rats administered corn oil, HCB, or TEM were observed. TEM elicited the classical dominant lethal mutations during the first 7 weeks of the experimental period (Table I) as reported by Bateman and Epstein (1971).
TABLE REPRODUCTIVE
Number Week pregnant
DATA
MI
FOR FEMALE
FYI
Corn oil control (10 mg/kg/day) 1 14 70 93 2 16 80 100 3 17 85 100 4 13 65 93 5 17 85 100 6 15 75 100 7 18 90 100 8 17 85 100 9 15 75 100 10 16 80 89 11 16 80 94 12 13 65 100 13 15 75 94 14 13 65 100
RATS MATED
CL1
II
12.85 15.81 15.47 15.38 15.00 13.93 15.94 15.52 13.73 10.56 13.87 13.84 15.46 15.92
10.85 11.37 12.17 11.61 11.23 10.46 12.22 10.76 10.46 9.18 10.31 11.92 11.00 10.69
1
TO MALES
PILI 2.00 4.43 3.29 3.84 3.82 3.46 3.72 4.76 3.26 1.37 3.56 1.92 4.46 5.46
TREATED WITH HEXACHMROBENZENE?
FI
RI
DF/LF
10.28 10.81 11.17 11.07 10.88 10.13 11.66 10.35 9.86 8.87 9.68 11.46 10.53 9.53
0.57 0.62 1.00 0.53 0.35 0.33 0.55 0.41 0.66 0.31 0.66 0.46 0.46 1.15
71134 10/173 17/190 71144 6/185 5/152 10/210 71163 lo/148 51155 10/155 6149 6/158 151124
DFal 519 917 819 617 5112 3112 6112 5112 718 4/12 4/12 5/8
619 914
DF>2 2112 l/15 2115 l/12 l/16 l/14 3/15 2115 3112 3113 3113 l/12 l/14 8/9
SHORT
TABLE
Week TEM
Number pregnant
417
COMMUNICATIONS
l-Continued
MI
FYI
CL1
II
PILI
FI
RI
DF/LF
DFB
1
DFa2
50 65 15 a5 75 55 a5 65 80 65 80 55 85 80
100 93 94 94 94 100 100 86 94 81 94 79 100 94
13.10 16.84 15.80 15.88 15.40 13.81 15.47 15.61 13.50 11.23 16.37 17.27* 15.52 14.75
11.00 10.69 10.20* 11.47 9.66 10.81 10.358 10.92 a.43b 10.15 11.06 11.00 10.64 9.62
2.10 6.15 5.60 5.05 5.13 3.36 5.17 4.76 5.06 1.69 5.31 6.27b 4.88 5.12
6.5ob 6.92b 4.26” 8.52 a.93 9.21 9.17b 10.15 7.93 9.46 10.50 10.54 9.52 9.31
4.5ob 3.76b 5.93b 2.35 0.73 1.54 1.17 0.76 0.60 0.69 0.50 0.45 1.00 0.316
20168’ 49/9Oc 89/64c 40/45c 11/134 17/102c 20/156’ 101132 81127 91123 8/169 5/116 17/162 51149’
10/w 12/l 1s/oc 11/6 a/7 417 a/9 716 719 419 719 417 1017 5/l 1
10/o= 10/3’ 14/l’ 7110 2113 l/10 5112 2/l 1 l/15 2/l 1 l/15 l/10 2115 O/16
50 70 80 85 65 a5 65 15 75 85 15 75 70 65
83 93 94 93 87 100 100 100 94 100 79 100 93 93
15.60 14.71 13.68 17.46 14.23 16.11 16.76 15.40 14.26 10.94 16.60 15.20 12.42” 15.61
11.00 10.78 11.93 10.46 11.23 10.17 11.92 10.66 10.40 10.00 11.00 10.80 10.35 10.23
4.60 4.57 1.87 7.00 3.07 5.94 4.84 4.13 3.86 1.00 5.60 4.40 2.14b 5.38
10.80 10.00 11.31 9.92 10.84 9.58 11.38 9.66 9.86 9.52 10.53 960 9.78 8.84
0.20 0.85 0.62 0.53 0.38 0.58 0.53 1.00 0.66 0.47 0.46 0.53 0.57 0.69
21108 12/140 lo/181 71129 5/141 lo/163 71148 14/145 lo/148 811.62 7/158 81144 a/137 9/124
218 w
o/10 l/12 l/15 2/11 l/12 2115 o/13 3112 3/12 2115 l/14 l/14 2112 2/l 1
45 40 55 50 60 80 55 15 80 60 65 50 55 60
100 100 100 100 92 100 73 100 100 75 93 91 85 100
13.00 12.50 14.90 14.49 16.41 14.37 16.41 14.06 15.25 14.0ab 13.38 12.40 14.81 14.33
11.66 10.25 11.63 10.30 11.08 10.37 11.54 10.33 8.12 11.66* 9.69 9.2ob 10.81 10.66
1.44 1 .I5 3.21 4.10 5.33 4.00 4.90 3.13 4.62 2.41 3.69 3.20 4.09 3.66
10.88 9.75 10.45 10.10 10.66 10.00 11.00 9.60 10.06 10.91b 9.23 8.80b 9.72 10.58
0.77 0.62 1.18 0.20 0.41 0.31 0.54 0.73 0.56 0.75 0.46 0.40 1.09b O.OSb
7188 4178 13/115 2/101 51128 6/160 6/121 11/144 9/161 9/131 6/120 4188 12/107 l/127’
(0.5 mg/kg) 1 2 3 4 5 6 7 a 9 10 11 12 13 14
HCB
10 13 15 11 15 11 17 13 16 13 16 11 17 16 (70 mg/kg/day)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 HCB 1 2 3 4 5 6 7 8 9 10 11 12 13 14
10 14 16 17 13 17 13 15 15 17 15 15 14 13 (221 mg/kg/day) 9’ 8’ 11’ 10 12 16 11c 15 16 12 13 10 11 12
a HCB and corn oil, 5 days oral dosing: TEM, single oral dose. b Significantly different from control (Student’s t test, ~90.05). c Significantly different from control (x2 with Yates’ correction; psO.05).
818 5/a 419 a/9 716 817 4/l 1 5112 S/l0 718 519 716
316 315 a/3
m 4/a 6110 615 916 719
616 5/a 317 a/3 l/11’
217 l/7 219 o/10 l/11 O/16 O/l 1 2/13 2114 l/11 11/12 l/9 417 o/12
418
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The female rats mated to males that received HCB showed the following statistically significant events. There was a decrease in corpora lutea and preimplantation losses during Week 13 at the low level. At the high level, there was an increase in corpora lutea, implantations, and living fetuses during Week 10; a decrease in implantations and living fetuses during Week 12; an increase in resorptions during Week 13 ; and a decrease in resorptions, the proportion of nonliving to living fetuses, and the proportion of females bearing one or more resorptions during Week 14. The events elicited by TEM follow the pattern of classical mutagens (Bateman, 1966). HCB did not induce any classical dominant lethal effects. Most chemicals that cause dominant lethal events manifest themselves during the early weeks (l-5) of mating (Bateman, 1966). Statistically significant effects among the HCB-treated groups were observed during Weeks 10-14. These effects were inconsistent in their extent and direction. Animals treated with HCB did not perform as well as controls during Weeks 12 and 13, but performed better than controls during Weeks 10 and 14. When compared with 14 weeks of pooled controls, no significant effects were seen among the HCBtreated groups. These inconsistent results may be attributed to weekly fluctuations in the test groups. HCB apparently had a dose-dependent effect upon male reproductive performance. At the low dose, the males impregnated fewer females (vs control males) 8 of the 14 weeks and, at the high dose, 12 of the 14 weeks (Mating Index). The effect at the high dose was significant (p < 0.001) using the sum of x2 procedure (Mantel and Haenszel, 1959). The percentage of inseminated females made pregnant (Fertility Index) was not adversely affected. HCB is well absorbed from the gastrointestinal tract (Mehendale et al., 1975) and persists in rat tissues for months following 33 weeks of administration via feed (Borzel-
leca et al., 1971). In the studies described herein, the administration of up to 221 mg/ kg HCB for 5 days failed to elicit dominant lethal effects, but did adversely affect male reproductive performance up to 14 weeks after HCB administration. Khera (1974) reported a lack of dominant lethal mutations in rats following administration of up to 60 mg/kg HCB for 10 days and reported no effects upon reproduction. Grant et al. (1977) reported decreased reproductive performance by males and females exposed to HCB at dietary levels of up to 640 ppm for life. Their experimental conditions were similar to those of Khera with three exceptions: (1) Both males and females were fed HCB; (2) Khera’s rats were exposed by gavage; and (3) exposure commenced during the weanling stage of life, and continued through the next three generations. These adverse effects reported to be due to HCB might be the result of increased sensitivity to HCB during the early stages of life. The work of Kimbrough and Linder (1974) and Grant et al. (1974) suggests that female rats may be more sensitive than males to some of the effects of HCB (lethality, respiratory, porphyria) but equally or less sensitive to others. The effects seen by Grant et al. (1977) might be explained by this differential sensitivity, though specific effects upon the female reproductive system were not reported by Kimbrough and Linder (1974). We conclude that HCB is not a dominant lethal mutant under the conditions tested, but can have adverse effects upon male reproductive performance.
REFERENCES BATEMAN, A. J. (1966). Testing chemicals for mutagenicity in a mammal. Nuture (London) 210, 205-206.
BATEMAN, A. J., and EPSTEIN, S. S. (1971). Dominant lethal mutations in mammals. In Chemical Mutagem. Principles and Methods for Their Detection (A. Hollander, ed.), Vol. 2, pp. 541-568. Plenum, New York.
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BORZELLECA, J. F., LARSON, P. S., CRAWFORD,E. M., HENNIGAR, G. R., JR., KUCHAR, E. J., AND KLEIN, H. H. (1971). Toxicologic and metabolic studies on pentachloronitrobenzene. Toxicol. Appl. Pharmacol. 18, 522-534.
BRADY, M. N., AND SIYALI, D. S. (1972). Hexachlorobenzene in human body fat. Med. J. Aust. 1, 158. CABRAL, J. R. P., SHUBIK, P., MOLLNER, T., AND RAITANO, F. (1977). Carcinogenic activity of hexachlorobenzene in hamsters. Science 269, 510-511. CAM, C. AND NIGOGOSYAN,G. (1963). Acquired toxic porphyria cutanea tarda due to hexachlorobenzene. Report, of 348 cases caused by this fungicide. J. Amer. Med. Ass. 183, 88-91. DEMA?TEIS, F. PRIOR, B. E., AND RIMINGTON, C. (1961). Nervous and biochemical disturbances following hexachlorobenzene intoxication. Nafure (London) 191, 363-366. GILBERTSON, M., AND REYNOLDS,L. M. (1972). Hexachlorobenzene (HCB) in the eggs of common terns in Hamilton Harbor, Ontario. BUN. Environ. Contam. Toxicol. 7, 371-373. GRANT, D. L., IVERSON, F., HATINA, G. V., AND VILLENEUVE, D. C. (1974). Effects of hexachlorobenzene on liver porphyrin levels and microsomal enzymes in the rat, Environ. Physiol. Biochem. 4, 159-165.
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KIMBROUGH, R. D., AND LINDER, R. E. (1974). The toxicity of technical hexachlorobenzene in the Sherman strain rat. A preliminary study. Res. Commun. Chem. Pathol. Pharmacol. 8(4), 653-664. KOEMAN, J. H., TEN NOEVER DE BRAUW, M. C., AND DE Vos, R. H. (1969). Chlorinated biphenyls in fish, mussels and birds from the river Rhine and the Netherlands coastal area. Nature (London) 221, 1125-1128. MANTEL, N., AND HAENSZEL, W. (1959). Statistical aspects of the analysis of data from retrospective studies of disease. J. Nat. Cancer Inst. 22, 719-748. MEDLINE, A., BAIN, E., MENON, A., AND HABERMAN, H. (1973). Hexachlorobenzene and the rat liver. Arch. Pathol. 96, 61-65. MEHENDALE, H. M., FIELDS, M., AND MATTHEWS, H. B. (1975). Metabolism and effects of hexachlorobenzene on hepatic microsomal enzymes in the rat. J. Agr. Food Chem. 23,261-265. NATIONAL ACADEMY OF SCIENCES (1975). Assessing Potential Ocean Pollutants, pp. 188-208. National Academy of Sciences, Washington, D.C. SELTZER, R. J. (1975). Ocean pollutants pose potential danger to man. Chem. Eng. News 53, 19-20. YAMAMOTO, Y. SUZUKI, M., AND AKIYAMA, T. (1975). Persistence of BHC in river water in the Kitakyushu District, Japan, 1970-1974. Bull. Environ. Contam. Toxicol. 14, 380-384. GLENN STUART SIMON~ ROBERT G. TARDIFF~ JOSEPH F. BORZELLECA Division of Toxicology Department of Pharmacology Medical College of Virginia Richmond, Virginia 23298 Received March 22, 1978; accepted September 24, 1978
GRANT, D. L., PHILLIPS, W. E., JR., AND HATINA, G. V. (1977). Effect of hexacfilorobenzene on reproduction in the rat. Arch. Environ. Contam. 5, 207-216. GREEN, S., ZEIGER, E., PALMER, K. A., SPRINGER, J. A., AND LEGATOR, M. S. (1976). Protocols for the dominant lethal test, host-medicated assay, and in vitro cytogenic test used in the Food and Drug Administration’s review of substances in the GRAS (Generally Recognized As Safe) list. J. ’ Recipient of NIH Predoctoral Training FellowToxicol. Environ. Health 1, 921-928. KHERA, K. S. (1974). Teratogenicity and dominant ship GM071 1 l-03. 4 Present address: National Academy of Sciences, lethal studies on hexachlorobenzene in rats. Food Cosmet. Toxicol. 12, 471-477. Washington, D.C.