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Embryotoxicity of chemical contaminants of foods
Fd Cosmet. ToxicoL Vol. 9, pp. 195-205. Pergamon Press 1971. Printed in Great Britain
Embryotoxicity of Chemical Contaminants of Foods* D. J. CLEGG Food Advisory Bureau, Food and Drug Directorate, Carlingwood Plaza, Curling Avenue, Ottawa, Ontario, Canada (Received 20 August 1970)
SUIVIIVIARY A brief review of a variety of contaminants that may occur in foodstuffs indicates that in the majority of cases embryotoxicity or teratogenicity are toxic hazards unlikely to arise in practice as a result of such contamination. The major possible exception is mercury, especially methylmercury, on which further data are being obtained. Meanwhile mercury intake should be restricted, and this is being done where possible, at least as far as the Canadian diet is concerned. Finally, further data are required on the effect of polychlorinated biphenyls on mammalian foetal development. The probability of hazard is at present regarded as being low, but insufficient information is available to make an accurate assessment. Introduction
Following the discovery of the embryotoxic and teratogenic effects of thalidomide in the early 1960s, considerable interest has been generated in the possible embryotoxie and/or teratogenic potential of a variety of compounds. This review is concerned with the data that have accumulated on chemicals that may occur as contaminants in the diet of man, either as a result of environmental contamination or from sources such as agricultural practice or food processing. Mycotoxins The best known of the mycotoxins that may appear in the human diet are the aflatoxins, comprising five major closely-related toxins produced by fungi of the genus Aspergillus. These compounds are extremely toxic to the developing chick embryo (Platt, Stewart & Gupta, 1962), and in the developing 5-day embryo as little as 0-3 ~g of a crude groundnut extract causes mortality within 2 days (D. J. Clegg, unpublished data, 1962). This extreme sensitivity of the hen egg is the basis of a bioassay technique for aflatoxins proposed by Verrett, Marliac & McLaughlin (1964). This technique, in which the fertile hen egg is injected prior to incubation, is still widely used. *Presented at a Symposium on "Chemical Contaminants in Foods--H~Tard or Not ?", sponsored by the Food and Drug Directorate, Department of National Health and Welfare, Ottawa, Ontario, and held in Ottawa on 18-19 June 1970. 195
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In the rat, intraperitoneal injection of aflatoxin B~ has been shown to cause haemorrhagic lesions at the utero-placental junction, with subsequent embryonic death (Le Breton, Frayssinet, Lafarge & de Recondo, 1964) while repeated injection of small doses retarded foetal growth. Butler & Wigglesworth (1966), feeding aflatoxin Bz to rats on day 16 of pregnancy, failed to induce embryonic death but noted growth retardation of the foetus. In neither instance was a teratogenic effect reported. In the hamster, Elis & DiPaulo "(1967) demonstrated teratogenic effects in the central nervous system following intraperitoneal injection of 4 mg aflatoxin B1/kg on day 8 of pregnancy. In farm animals, 2 ppm aflatoxin Bx fed for 3-12 months in the diet of cows had no apparent effect on conception, gestation or parturition (Allcroft & Lewis, 1963), and very high doses of aflatoxin Bz (0-9-1.75 mg/day) had no effect on gestation or parturition in sheep although fertility was reduced (Lewis, Markson & Allcroft, 1967). From the available data on aflatoxins, it is apparent that teratogenicity and embryotoxicity are relatively insignificant effects compared with the known carcinogenic effects of this group of compounds. Metals Zinc. Although frank zinc deficiency is known to result in congenital malformations in rats (Hurley & Swenerton, 1966), such a deficiency has never been observed in man. Zinc intoxication has occurred, however, following preparation of food in galvanized vessels (Brown, Thom, Orth, Cova & Juarez, 1964). Fern & Carpenter (1968) have investigated the effects of excess zinc on the developing hamster. At 30 mg zinc sulphate/kg, injected intravenously, maternal animals succumbed within 24 hr. Lower doses (10-25 mg/kg) induced about 12% foetal resorptions, and with 2 mg/kg given intravenously, teratogenic effects (fused ribs and exencephaly) occurred in about 4 % of cases. In multigeneration studies in rats (Heller & Burke, 1927), levels of 2500 ppm in the diet as zinc chloride caused no ill-effect, although 5000 ppm increased mortality in the offspring, a finding that has been tentatively attributed to the destruction of the fl-cells of the Islets of Langerhans (Underwood, 1962). However, 5000 ppm zinc as the oxide caused very little increase in the mortality of the offspring. Although intravenous injection of soluble zinc salts causes effects in the hamster, probably because of the high blood levels resulting from the method of administration, zinc taken orally is relatively poorly absorbed and sufficiently high blood levels are unlikely to be attained. It seems improbable, therefore, that zinc contamination of food will increase the incidence of teratogenic or embryotoxic effects in man. Cadmium. Plated cooking utensils have been known to cause cadmium poisoning (Monier-Williams, 1949). In the hamster, Ferm & Carpenter (1967a) have shown that intravenous administration of cadmium sulphate, at a dose of 2 mg/kg on day 8 of gestation, results in a 66% incidence of malformations in the embryos. These malformations are mainly craniofacial. Cadmium is known to cross the placenta of the hamster (Ferm, Hanlon and Urban, 1969) and since it is found in foetal and newborn human liver and kidney (Underwood, 1962), it seems reasonable to infer transplacental passage in man. However, the levels of cadmium found in man are low, and cannot be related to the high levels administered to the hamsters. Furthermore, in the experiment cited, the use of intravenous injections to produce effects makes extrapolation to man impossible, and it seems likely that cadmium, like zinc, is not a significant factor in human teratogenicity or embryotoxicity.
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Calcium. Although calcium is an essential element for development, a bizarre phenomenon has been observed following injection of 0.1 mg calcium pantothenate into the yolk sac of hen eggs incubated for 2 days (Grabowski, 1966). Of the resultant embryos, 17~ were grossly abnormal. Follow-up experiments, using 10--40/~g calcium chloride injected into the yolk sac (of 2-day-incubated eggs) or allantois (3- and 5-day-incubated eggs), resulted in a slightly increased mortality rate, with 12-17 ~ abnormalities among the offspring. Thus, trace quantities of calcium appeared to be the causal agent. The author suggests that membrane permeability may be affected and that this results in abnormalities. These data illustrate the dangers of extrapolating teratogenic data from hen-egg experiments; there are no data implicating calcium as a teratogen in mammals. Selenium. Robertson (1970) has suggested that selenium may be a possible teratogen in man. Out of one possible and four certain pregnancies among women exposed to selenite, only one pregnancy went to term, and the infant showed bilateral clubfoot. Of the other pregnancies, two could have terminated because of other clinical factors. Widespread enquiries in other laboratories where exposure to selenite could occur have not revealed any similar pattern, except for one miscarriage in the only pregnancy in 5 years in one small laboratory. A further letter (Hadjimarkos, 1970) pointed out the potential danger of high concentrations of selenium in foods grown on soils with a high selenium content. Selenium has been shown to cross the placenta in rats and cats (Westfall, Stohlman & Smith, 1938) and its passage can be inferred in other species from the symptoms of selenium poisoning observed in the offspring of animals grazing on seleniferous rangeland. Foals and calves in affected areas are sometimes born with deformed hoofs. The offspring of rats (Rosenfeld & Beath, 1954), pigs (Wahlstrom & Olson, 1959) and sheep (Rosenfeld & Beath, 1947) have all shown abnormalities following selenium administration during pregnancy. On the other hand, 2 mg sodium selenite/kg (an almost lethal dose) administered intravenously had no teratogenic or embryotoxic effects in the hamster (Holmberg & Ferm, 1969). Evidence is, therefore, controversial and difficult to relate to the possible effects of selenium on human embryonic development. It seems unlikely that food contamination with selenium would be a major problem in cities because of the wide variations in the source of foodstuffs. However, a close watch should be kept on rural populations, especially in areas where the soil has a high selenium content. Lead. Severe exposure of the pregnant human female to high levels of lead has apparently resulted in abortion (Wilson, 1966; Taussig, 1936). Since lead is known to cross the human placenta in cases of high-level exposure (Barltrop, 1968), the occurrence of teratogenic effects at a sublethal level is not surprising, and following excessive industrial exposure offspring with neurological symptoms have been encountered (Angle & Mclntire, 1964). Growth, both intrauterine and postnatal, may be retarded (Palmisano, Sneed & Cassady, 1969). In hamsters, lead nitrate, lead chloride or lead acetate injected intravenously in doses of 50 mg/kg on day 8 of pregnancy resulted in a high incidence of teratogenic malformations in the sacral and tail regions of the offspring (Ferm & Carpenter, 1967b). Foetal mortality was not significantly increased. No evidence of teratogenic activity has been observed in the cow (Shupe, Binns, James & Keeler, 1967) or the sheep (James, Lazar & Binns, 1966). The dose levels involved could not be calculated in the cases of human exposure, but in all cases were obviously considerably in excess of dietary exposure. Similarly, the route of administration and extremely high dosage involved in the hamster teratogenicity experiment render extrapolation to man almost impossible.
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Mercury. Several studies of the embryotoxic or teratogenic activity of mercurial salts have been performed in the laboratory. In avian species, mercuric chloride has been injected into fresh, fertile, unincubated hen-egg yolk sacs at doses of 0-5 or 0.25 mg/egg. At the higher dose all the embryos died, and at the lower dose hatchability was reduced by 80~o (McLaughlin, Marliac, Verrett, Mutchler & Fitzhugh, 1963). Pheasants fed wheat containing 15-20 ppm methylmercury dicyandiamide for 9 days laid eggs with reduced hatchability and containing levels of 1.3-2.0 ppm mercury (Borg, Wanntorp, Erne & Hanko, 1969). Eggs from abandoned pheasants' nests frequently contained more than 1 ppm mercury and addled eggs from eagle and owl nests contained 3.5-11 ppm. Thus, it would appear that mercury entering the egg, either by injection or via the maternal organism, can produce embryotoxic effects. Autoradiographic studies in mice showed that ethylmercuric chloride passed through the placenta more easily than either mercuric chloride or phenylmercuric chloride (Ukita, Takeda, Sato & Takakashi, 1967). Once through the placenta, mercury appeared to have a greater affinity for the central nervous system than was the case in the adult. Later studies (Suzuki, Matsumoto, Myama & Katsunuma, 1967) have confirmed that alkylmercury does cross the placenta more readily than aryl or inorganic compounds, as indicated by the ratios of maternal: placental: foetal blood levels of mercury (1 : 19: 0-4 for mercuric chloride; 1 : 1.9:2.1 for methylmercuric acetate; 1:4-5:0.3 for phenylmercuric acetate). The placenta appears to act as a barrier to mercuric chloride and phenylmercuric acetate--possibly in the region of the yolk-sac membrane. Similarly, methylmercury has been shown to cross the rat placenta (Fujita, 1969). In mouse experiments, ethylmercuric phosphate injected subcutaneously at a dose level of 40 mg/kg on day 10 of pregnancy did not affect litter size (Oharazawa, 1968). However, foetal body weight was reduced and a 31.6 ~o incidence of cleft palate was observed. Since no mice were permitted to litter and no histopathology was reported, the incidence of central nervous system damage was not established. Intraperitoneal administration of 0.1 mg methylmercury dicyandiamide to CBA mice prior to mating had no effect on the size of subsequent litters (H. Fr616n and C. Ramel, cited by L6froth, 1969), but the incidence of pregnancies was reduced either as a result of reduced libido or of damage to gametes. Injection on day I0 of pregnancy resulted in a significant increase in embryonic mortality (17 ~o compared with 7.2 ~o in controls). Methylmercury is known to cause chromosomal aberrations in the lymphocytes of persons consuming fish containing high mercury levels (J. Lindsten, personal communication cited by Lffroth, 1969). Such damage, if it occurs in the mouse, could be responsible either for the reduced incidence of pregnancies or for the teratogenic effects reported in the above experiments. In the rat, daily administration of 0.1 mg mercury/kg (as methylmercury) has been shown to reduce birth weight and cause neurological disorders during postnatal development (Fujita, 1969). Embryotoxicity in the rat has also been demonstrated by feeding 1, 2, 4 or 8 ppm mercury as phenylmercuric acetate in the diet (Piechoka, 1968). At 8 ppm, litter size was reduced by 44-3 Yo. No data on neurological involvement were given. Wistar rats given a single oral dose of 2 or 20 mg methylmercury chloride/kg on day 9, 10 or 11 of pregnancy showed cerebellar changes (Matsumoto, Suzuki, Monta, Nakamura & Saeki, 1967). At the higher dose, the whole shape of the cerebellum was distorted and, in addition, the midbraln showed enlarged ventricular cavities and degeneration of the nerve cells. At the lower dose, histological changes were detectable in the cerebellum. The histological changes observed in this experiment contrasted with the degeneration and loss of
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nerve cells in the cerebral cortex observed in the adult rat, in that the changes were confined to the cerebellum. In a similar experiment, rats were dosed orally with 0.2 mg methylmercuric chloride/rat during the first 20 days of gestation; there were no signs of maternal toxicity up to sacrifice, and litter size and embryo weight were normal. Histology of the embryonic brain revealed a disappearance of nucleoli, a reduction of chromatin granules and a decrease of ribosomes in the granular cells of the cerebral cortex. Purkinje cells were also observed to be vacuolated or to be electron-dense. These changes (which were also observed in 100-day-old offspring of a second identically-treated group) were similar to the changes observed in the adult rat following experimentally-induced Minamata disease (Nonaka, 1969). The offspring of one cat exposed during pregnancy to between 150 and 220 mg bisethylmercuric sulphide showed changes in the few remaining cerebellar granular cells and a delayed migration of the Purkinje cells (Morikawa, 1961). Thus, animal experimentation indicates that mercury may have both embryotoxic and teratogenic potential in rats, mice and cats. The greater ease with which alkylmercury crosses the placenta leads to the speculation that methyl- or ethylmercury may possibly be more important in foetal toxicity than aryl or inorganic mercury. There is also some evidence of preferential accumulation of methylmercury, especially in foetal nervous tissue. In man, 19 of 111 cases of Minamata disease were encountered in newborn children (Irukayama, 1969). Since a population is unlikely to contain 17-3 ~ of pregnant women, these figures indicate that the unborn infant is at greater risk from methylmercury poisoning than the adult. The mothers of the 19 congenital cases of Minamata disease had all ingested fish or shellfish contaminated with methylmercury. However, no symptoms had been observed in the majority of mothers, and in the few cases where symptoms did occur, they were confined to slight numbness of the finger (Kakita, 1961). This lack of symptoms in the mothers may well be due to the relative ease of placental transfer of methylmercury, like that observed in laboratory animals. The passage of methylmercury across the human placenta, and its possible preferential concentration in the foetus, has been demonstrated in normal pregnant women, human foetal erythrocyte levels showing 28 ~ more mercury than the maternal erythroeytes (Tejning, 1968). (Plasma levels of mercury are 19 ~ higher in the mother than in the foetus, but plasma levels are relatively insignificant when compared with those in the erythrocytes.) In congenital cases of Minamata disease, cerebral palsy is the major effect. In the two cases reported in detail, examination of the brain tissue revealed degenerative and regressive changes in the cerebral cortical nerve cells, stated to be similar to those observed in adult autopsy material (Matsumato, Koya & Takeuchi, 1965). Thus, there seems to be very little doubt that methylmercury can exert an embryotoxic effect in man. Since effects have been reported in the rat following the oral ingestion of 0.1 mg methylmercury/kg, it seems probable that the safe ingestion level for the pregnant human female is extremely low. Strontium-90. Although the effects of embryonic irradiation have been extensively studied, relatively little is known about the effects of internally-deposited radioactive isotopes on embryonic development. Hopkins, Casarett, Tuttle & Baxter (1967) injected doses of 0, 191 or 382 t~c strontium-90 intravenously into 2- or 10-day postconception rats. In 2-day postconception rats, mortality rates were increased from 7 ~ in the control group to 22 ~ in the group given 191 t~c and 64 ~ in that given 382 tzc strontium-90. Foetal weight was decreased to 95 and 89 ~, respectively, of the control weight, and the incidence of runts was increased from 5 ~ to 13 and 23 ~ respectively. In 10-day postconception embryos,
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mortality was virtually unaffected by either dose, but birth weight was decreased to 96 and 89 ~ of the control value and the incidence of abnormalities increased from 3.1 ~o to 17.6 and 51~o at the two dose levels. The intrauterine radiation dose after the 10-day postconception injection was 12 rads/day after 6 hr, decreasing to 0.3 rads/day by day 2 after injection. The dose rate remained low until late in foetal life, when deposition of strontium90 in foetal bone caused a second peak in activity at 20 rads/day. In Canada, the dietary intake of strontium-90 is considerably below the levels used in these studies (Belkhode, Mar & Quastel, 1969). Nitrosamines
A recent paper by DiPaulo (1969) reviewed the embryotoxicity and teratogenicity of the alkylating agents, including nitroso compounds. Briefly, dimethylnitrosamine, although non-teratogenic, is embryotoxic both orally and by intraperitoneal injection in the rat, hamster and chick. Pesticides
Since Khera & Clegg (1969) presented a paper on the perinatal toxicity of pesticides, the Report of the Secretary's Commission on Pesticides and their Relationship to Environmental Health (the 'Mrak Report') has been published (Commission on Pesticides, 1969). In this report, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), various esters of 2,4-dichlorophenoxyacetic acid (2,4-D), PCNB, captan and folpet are all listed as causing a significant increase in the incidence of anomalies in C57BL/6 mice. 2,4-D and 2,4,5-T are also stated to have increased the incidence of anomalies in A K R mice. Both of these pesticides have been the subject of further investigation since the report was issued. 2,4,5-T. On 29 October 1969, Dr. L. A. DuBridge, Scientific Adviser to the President, announced the partial curtailment of the use of the herbicide 2,4,5-T in the USA. According to Nelson (1969), a reporter on the staff of the Los Angeles Times, this decision was based on "scientific studies which would soon become widely known" and on South Vietnamese newspaper stories (so far unsubstantiated in any scientific journal) attributing an increase in congenital defects to the American defoliation programme, which uses 2,4,5-T. Although various comments on the scientific studies appeared in the lay press in November and December, the first scientific account did not appear until the Mrak report was published in December 1969. The data reported by the Commission on Pesticides (1969) was derived from a large-scale teratogenic screening study involving several commonlyused pesticides. These pesticides were administered to mice daily from day 6 to day 14 (strain C57BL/6) or to day 15 (AKR) of pregnancy, either subcutaneously in dimethylsulphoxide (DMSO) or orally in 50~o honey solution. In the case of 2,4,5-T, repeated administration of 113 mg/kg subcutaneously caused a significant increase in the incidence of cleft palate and cystic kidneys in embryos removed at day 18 (C57BL/6) or 19 (AKR) of pregnancy. Foetal mortality was increased in the C57BL/6 strain only. Oral administration of 113 mg/kg]day increased foetal mortality in both strains, and induced cleft palate and cystic kidney in C57BL/6 mice and cleft palate in A K R mice. Repeated doses of 46.4 mg/kg given orally to C57BL/6 mice resulted in an increased incidence of abnormal litters, and an increased incidence of abnormal foetuses per litter, but the types of abnormalities were not specified. Foetal mortality was apparently unaffected at 46-4 mg/kg/day in C57BL/6
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mice. Although no data are provided, the text of the report of the Commission on Pesticides (1969) indicates that subcutaneous administration of repeated doses of 113 mg/kg to a hybrid C57BL/6 × AKR mouse strain significantly increased the incidence of cystic kidney in particular. A study on Sprague-Dawley rats given 4.6, 10 or 46.4 mg/kg/day indicated that teratogenic (kidney anomalies) and embryotoxic effects were observed at all levels. These experiments have since been reported in more detail in the conventional literature (Courtney, Gaylor, Hogan, Falk, Bates & Mitchell, 1970a), and basically confirm the information originally published (Commission on Pesticides, 1969). However, the abnormalities in the C57BL/6 mice treated with 46.4 mg/kg/day were confined to cystic kidneys (which have still not been described). The increased embryonic mortality in the rat experiment was significant at 10 mg/kg/day and above, but not at 4.6 mg/kg/day. In addition, a further possible embryotoxic effect in rats was demonstrated, comprising a dose-related incidence of haemorrhage in the gastro-intestinal tract. Additional data indicate that in C57BL/6 mice, 21.5 mg/kg/day administered subcutaneously in DMSO on days 6-14 of pregnancy caused no significant increase in any teratogenic or embryotoxic effects. Finally, in an additional experiment on C57BL/6 mice, subcutaneous treatment with 113 mg/kg/day in DMSO on days 9-17 of pregnancy caused cleft palate and cystic kidney, but embryonic mortality was unaffected. The 2,4,5-T used in these studies was analysed by Dow Chemical Co. and shown to contain approximately 30 ppm 2,3,7,8-tetrachlorodibenzo-p-dioxin (referred to henceforth as dioxin). Emerson, Thompson, Gerbig & Robinson (1970) administered 2,4,5-T containing less than 1 ppm dioxin orally to groups of 25 female Sprague-Dawley rats in doses of 1, 3, 6, 12 or 24 mg/kg/day on days 6-15 of pregnancy (day 0 being the day on which positive vaginal smears were observed). A further 50 female rats were dosed orally with the vehicle, 0-25 ~ hydroxypropylmethyl cellulose. Foetuses were removed and examined for soft-tissue and skeletal abnormalities on day 20 of gestation, and ten high-dose foetuses were examined histologically. There were no clinical symptoms in the dams, and no indications of increased mortality, intestinal haemorrhage or teratogenic effects in the foetuses. A study was performed using 91 ~ pure dioxin, the impurities comprising 7 Yotrichloro-, 2 Yopentachloro- and < 0.02 ~o dichlorodibenzo-p-dioxin (Sparschu, Dunn & Rowe, 1970). The test material was administered by gavage in doses of 0-03, 0.125, 0.5, 2.0 and 8.0 /zg/kg/day to groups of 8-12 rats on days 6-15 of gestation, and 24 control rats were given the corn oil-acetone (9 : 1, v/v) vehicle. Doses were based throughout on pre-breeding body weights. In the dams, 3/11 given 2/~g/kg showed vaginal haemorrhage from days 15-20 of gestation, and 6/8 given 8/~g/kg showed vaginal haemorrhage at times between days 13 and 20 of gestation. Maternal body-weight gain was depressed at 0.5 /zg/kg and above. With regard to the foetuses, the numbers of corpora lutea and of implantation sites were comparable in all groups. Resorption was significantly increased at 0.5 ~g/kg and above, all foetuses being resorbed at 9/~g/kg. The sex ratio of the foetuses was normal, but foetal weight was depressed at 0-125 /zg/kg and above. Gross abnormalities comprised foetal oedema at 2.0/~g/kg, a rudimentary tail at 0.125/zg/kg, and two misshapen limbs and a kinked tail at 2.0 tzg/kg. Skeletal examination revealed no compound or dose-related effects. Soft-tissue examination showed a dose-related incidence of gastro-intestinal haemorrhage at 0.125/zg/kg and above. Dilation of the renal pelvis was observed in all groups, with a doubtful increase in incidence at 0.5 and 2.0/zg/kg. Microscopically, a dose-related incidence of subcutaneous oedema occurred at 0.125 /zg/kg and above. The evidence from the l).C.r. 912---C
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experiments to date, therefore, suggests that the dioxin may be the agent inducing the embryotoxic and teratogenic effects in rats. Courtney, Moore, Gaylor, Hogan & Falk (1970b) presented data from further teratogenic studies on 2,4,5-T in April 1970. Three strains of mice were studied, the test materials being given on days 6-15 of gestation by subcutaneous injection in DMSO. Charles River mice showed an increased incidence of cleft palate and kidney involvement following treatment with 2,4,5-T at 100 mg/kg/day (providing a dose of less than 0.01/~g dioxin/kg). No effects were observed at a dose of 50 mg]kg/day with a 2,4,5-T sample providing 0.075 tzg/kg dioxin. Foetal mortality was unaffected at either dose level. Dioxin alone increased the incidence of kidney involvement at 1 /zg/kg and of cleft palate at 3/zg]kg. In DBA mice, 2,4,5-T caused an increased incidence of cleft palate at 100 mg]kg]day ( <0.05/zg dioxin/kg), but foetal mortality and the incidence of kidney involvement were unaffected. Dioxin alone, at 3 t~g/kg, caused an increase only in the incidence of kidney involvement. In C57BL]6 mice, 100 mg 2,4,5-T]kg]day ( <0.05 tzg/kg dioxin) caused a significant increase in mortality only. Dioxin alone, at 3/~g]g, significantly increased the incidence of cleft palate and kidney involvement, but did not increase foetal mortality. In summary, mice are susceptible to the teratogenic effects of dioxin and possibly, at high levels, to those induced by 2,4,5-T. However, until the susceptibility of the various mouse strains to dioxin-induced effects is more accurately known, the teratogenicity of 2,4,5-T must remain in some doubt. Further rat studies (Courtney et al. 1970b) have confirmed that 2,4,5-T per se is not teratogenic in the rat, although dioxin causes kidney abnormalities. One further mammalian study has been completed. J. L. Emerson, D. J. Thompson, C. G. Gerbig & V. B. Robinson, (unpublished report of Dow Chemical Co., 1970) have studied the teratogenic activity of 2,4,5-T containing less than 1 ppm dioxin. Groups of 20 rabbits were treated orally on day 6-18 of pregnancy with doses of 0, 10, 20 or 40 mg/kg/day. There were no indications of embryotoxic or teratogenic effects. Dioxin, therefore, appears to be the main agent responsible for the teratogenic effects associated with 2,4,5-T in mammals. If 2,4,5-T is teratogenic in mice, it appears that the dose levels involved are in excess of 50 mg/kg, a dose level that provides an adequate safety margin for man when one considers that 2,4,5-T residues have rarely been detected in food. Even in those eases where 2,4,5-T residues have been detected, the levels in food have been extremely low. Similarly, it must be remembered that the mouse studies involved administration of 2,4,5-T by subcutaneous injection, a route which would not be directly comparable to oral ingestion of contaminated foods. Finally the solvent, DMSO, has itself been implicated as a teratogen in the hamster (Ferm, 1966) and in the rat, mouse and chick embryo (Caujolle, Caujolle, Cros & Calvet, 1967), and as an embryotoxin in the rat (Juma & Staples, 1967) at very high dose levels. The doses used by Courtney et al. 1970a,b) were far below those inducing teratogenic or embryotoxic effects, but the possibility of potentiation of the teratogenic and embryotoxic activity of DMSO by 2,4,5-T and/or dioxin has not been investigated and cannot, therefore, be excluded. In the case of dioxin, a no-effect level of 0.03 t~g/kg has been demonstrated in rats. The amount of dioxin applied with 2,4,5,-T is small, but until more information is available on environmental contamination with dioxin, its rate of degradation and the residue situation in food, the use of 2,4,5-T has been restricted as a precautionary measure to prevent possible exposure of pregnant women to dioxin. 2,4-D. The Mrak report also indicated that a number of esters of 2,4,-D could be teratogenic, according to the teratogenic screening test results (Commission on Pesticides,
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1969). K. K h e r a (personal c o m m u n i c a t i o n , 1970) has investigated this p r o b l e m a n d reports as follows: "2,4-D and its formulations (isooctyl, butyl, d i m e t h y l a m i n e a n d b u t o x y ethanol) were a d m i n i s t e r e d in corn oil singly by i n t u b a t i o n as one daily dose from 6-15 days o f gestation in rats. All the c o m p o u n d s at doses a p p a r e n t l y non-toxic for the d a m s (100-150 m g / k g / d a y ) induced r e d u c t i o n in average litter weight a n d significantly increased incidence o f s p o n t a n e o u s l y occurring skeleton m a l f o r m a t i o n s . These effects were n o t a p p a r e n t at doses o f 25 mg/kg. Similar studies with 2,4,5-T are in progress." Since 25 m g / k g seems to be a no-effect level for 2,4-D, a n d its residues (if any) are m i n i m a l (and d o not c o n t a i n dioxin), no p r o b l e m s are anticipated with this herbicide as far as c a n be predicted f r o m d a t a at present available.
Polyehlorinated biphenyls Recent d a t a suggest t h a t p o l y c h l o r i n a t e d biphenyls are as u b i q u i t o u s as D D T . A t present, very little is k n o w n a b o u t their possible effects on the h u m a n foetus. I n the a d u l t rabbit, the acute oral LDso for the various p o l y c h l o r i n a t e d biphenyls t h a t have been e x a m i n e d lies between 8 and 11 g/kg. In a very p r e l i m i n a r y study (which is being repeated) 50 m g / k g was e m b r y o t o x i c in the r a b b i t (W. J. Philips, D. C. Villeneuve & D. G r a n t , personal c o m m u n i c a t i o n , 1970). N o effects were observed at 1 or 10 mg/kg. T h e e m b r y o n i c r a b b i t thus a p p e a r s to be much m o r e susceptible t h a n the adult, b u t this remains t o be confirmed.
REFERENCES Allcroft, Ruth & Lewis, Gwyneth (1963). Groundnut toxicity in cattle: Experimental poisoning of calves and a report on clinical effects in older cattle. Vet. Rec. 75, 487. Angle, Carol R. & McIntire, Matilda S. (1964). Lead poisoning during pregnancy. Am. d. Dis. Child. 108 436. Barltrop, D. (1968). The transfer of lead to the human foetus. In Mineral Metabolism in Paediatrics. Blackwell Scientific Publications, Oxford. Belkhode, M. L., Mar, P. G. & Quastel, M. R. (1969). Strontium 90 and Cesium 137 in Canadian Diet and Milk, 1966-1968. Radiation Protection Division, Publication No. 75. Borg, K., Warmtorp, H., Erne, K. & Hanko, E. (1969). Alkyl mercury poisoning in terrestrial Swedish wildlife. Viltrevy 6, 301. Brown, M. Alice, Them, J. V., Orth, G. L., Cova, Patricia & Juarez, J. (1964). Food poisoning involving zinc contamination. Archs envir. Hlth 8, 657. Butler, W. H. & Wigglesworth, J. S. (1966). The effects of aflatoxin BI on the pregnant rat. Br. J. exp. Path. 47, 242. Caujolle, F. M. E., Caujolle, D. H., Cros, S. B. & Calvet, M.-M. J. (1967). Limits of toxic and teratogenic tolerance of dimethyl sulphoxide. Ann. N. Y. Acad. Sci. 141, 110. Commission on Pesticides (1969). Report o f the Secretary's Commission on Pesticides and Their Relationship to Environmental Health. Parts I and II. U.S. Department of Health, Education, and Welfare, Washington, D.C. Courtney, K. Diane, Gaylor, D. W., Hogan, M. D., Falk, H. L., Bates, R. R. & Mitchell, I. (1970a). Teratogenic evaluation of 2,4,5-T. Science, N. Y. 168, 864. Courtney, K. D., Moore, J. A., Gaylor, D. W., Hogan, M. D. & Falk, H. L. (1970b). Record of the Hearing on 2,4,5-T, before the Subcommittee on Energy, Natural Resources and the Environment of the Senate Committee on Commerce. Chairman, P. A. Hart, 15 April 1970. p. 225. DiPaulo, J. A. (1969). Teratogenic agents: Mammalian test systems and chemicals. Ann. N.Y. Acad. Sci. 163, 801. Elis, J. & DiPaulo, J. A. (1967). Aflatoxin B,. Induction of malformations. Archs Path. 83, 53.
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Emerson, J. L., Thompson, D. J., Gerbig, C. G. & Robinson, V. B. (1970). Teratogenic study of 2,4,5trichlorophenoxyacetic acid in the rat. Toxic. appl. Pharmac. 17, 317. Ferm, V. H. (1966). Congenital malformations induced by dimethyl sulphoxide in the golden hamster. J. Embryol. exp. Morph. 16, 49. Ferm, V. H. & Carpenter, S. J. (1967a). Teratogenic effect of cadmiuna and its inhibition by zinc. Nature, Lond. 216, 1123. Ferm, V. H. & Carpenter, S. J. (1967b). Developmental malformations resulting from the administration of lead salts. Expl moL Path. 7, 208. •Ferm, V. H. & Carpenter, S. J, (1968). The relationship of cadmium and zinc in experimental mammalian teratogenesis. Lab. Invest. 18, 429. Ferm, V. H., I-Ianlon, D. P. & Urban, J. (1969). The permeability of the hamster placenta to radioactive cadmium. J. Embryol. exp. Morph. 22, 107. Fujita, E. (1969). Experimental studies of organic mercury poisoning; the behaviour of the Minamata disease causal agent in maternal bodies, and its transfer to their infants via either placenta, or breast milk. J. Kumamoto med. Soc. 43, 47. Grabowski, C. T. (1966). Teratogenic effects of calcium salts on chick embryos. J. EmbryoL exp. Morph. 15, 113. Hadjimarkos, D. M. (1970). Selenium. Lancet i, 721. Heller, V. G. & Burke, A. D. (1927). Toxicity of zinc. J. biol. Chem. 74, 85. Holmberg, R. E., Jr. & Ferm, V. H. (1969). Interrelationships of selenium, cadmium and arsenic in mammalian teratogenesis. Archs envir. Hlth 18, 873. Hopkins, B. J., Casarett, G. W., Tuttle, L. W. & Baxter, R. C. (1967). Strontium-90 and intrauterine development in the rat. J. EmbryoL exp. Morph. 17, 583. Hurley, Lucille S. & Swinerton, Helene (1966). Congenitial malformations resulting from zinc deficiency in rats. Proc. Soc. exp. BioL Med. 123, 692. Irukayama, K. (1969). The pollution of Minamata bay and Minamata disease. Adv. pollut. Res. 3, 153. James, L. F., Lazar, V. A. & Binns, W. (1966). Effects of sublethal doses of certain minerals on pregnant ewes and foetal development. 4n:..t. vet. Res. 27, 132. Juma, M. B. & Staples, R. E. (I'S .',~. Effect of maternal administration of dimethyl sulphoxide on the development of rat fetuses. Proc. ~ ,'. exp. BioL Med. 125, 567. Kakita, T. (1961). Kumamoto lgakukai Zasshi 35, 289. Khera, K. & Clegg, D. J. (1969). Perinatal toxicity of pesticides. Can. med. Ass. J. 100, 167. Le Breton, Eliane, Frayssinet, C., Lafarge, C. et de Recondo, A. M. (1964). Aflatoxine---M6canisme de l'action. Fd Cosmet. ToxicoL 2, 675. Lewis, G., Markson, L. M. & Allcroft, R. (1967). The effect of feeding toxic groundnut meal to sheep over a period of five years. Vet. Rec. 80, 312. L6froth, G. (1969). A review of health h~7~rds and side effects associated with the emission of mercury compounds into natural systems. Ecological Research Committee Bulletin no. 4. Swedish Natural Science Research Council, Stockholm, Sweden. Matsumoto, H., Koya, G. & Takeuchi, T. (1965). Fetal Minamata disase. A neuropathological study of two cases of intrauterine intoxication by a methyl mercury compound. J. Neuropath. exp. NeuroL 24, 563. Matsumoto, H., Suzuki, A., Monta, C., Nakamura, K. & Saeki, S. (1967). Preventative effect of penicillamine on the brain defect of fetal rat poisoned transplacentally with methyl mercury. Life Sci. 6, 2321. McLaugldin, J., Jr., Marliac, J.-P., Verrett, M. Jacqueline, Mutchler, Mary K. & Fitzhugh, O. G. (1963). The injection of chemicals into the yolk sac of fertile eggs prior to incubation as a toxicity test. Toxic. appl. Pharmac. 5, 760. Monier-Williams, G. W. (1949). Trace Elements in Food. Chapman & Hall, London. Morikawa, N. (1961). Pathological studies in organic mercury poisoning. Kumamoto Med. J. 14, 71. Nelson, B. (1969). Herbicides: Order on 2,4,5-T issued at unusually high level. Science, N.Y. 166, 977. Nonaka, I. (1969). An electron microscopic study of the experimental congenital Minamata disease in rat. Kumamoto Med. J. 22, 27. Oharazawa, H. (1968). Chromosomal abnormalities and teratogenesis induced by ethyl mercuric phosphate in pregnant mice. Nippon Sanka-Fujinka Gakkai Zasshi 20, 1479. Palmisano, P. A., Sneed, R. C. & Cassady, G. (1969). Untaxed whiskey and fetal lead exposure. J'. Pediat. 75, 869. Piechoka, J. (1968). Chemical and toxicological studies of the fungicide phenyl mercuric acetate. 1. Studies on the effect of food contaminated with Fungitox OR, on rats. Roczn. palest. Zakl. Hig. 19, 385. Platt, B. S., Stewart, 1L J. C. & Gupta, S. R. (1962). The chick embryo as a test organism for toxic substances in food. Proe. Nutr. Soc. 21, xxx. Robertson, D. S. F. (1970). Selenium--A possible teratogen ? Lancet i, 518. Rosenfeld, Irene & Beath, O. A. (1947). Congenital malformations of eyes of sheep. J. agric. Res. 75, 93.
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Rosenfeld, Irene & Beath, O. A. (1954). Effect of selenium on reproduction in rats. Proc. Soe. exp. Biol. Med. 87, 295. Shupe, J. L., Binns, W., James, L. F. & Keeler, R. F. (1967). Lupine, a cause of crooked calf disease. J. Am. vet. med. Ass. 151, 198. Sparschu, G. L., Dunn, F. L. & Rowe, V. K. (1970). Teratogenic study of 2,3,7,8-tetraehlorodibenzop-dioxin in the rat. Toxic. appl. Pharmac. 17, 317, Suzuki, T., Matsumoto, N., Myama, T. & Katsunurna, H. (1967). Placental transfer of mercuric chloride, phenyl mercuric acetate, and methyl mercuric acetate in mice. Ind. Health (Jap.) S, 149. Taussig, F. J. (1936). Abortion, Spontaneous and lnduced, pp. 111 & 354. Kimpton, London. Tejning, S. (1968). Report 68.02.20 from the Department of Occupational Medicine, University Hospital, Lund, Sweden. Ukita, T., Takeda, Y., Sato, Y. & Takakashl,T. (1967). Distribution of 2°3Hg labelled mercury compounds in adult and pregnant mice determined by whole-body autoradiography. Radioisotopes 16, 31. Underwood, E. J. (1962). Trace Elements in Human and Animal Nutrition. p. 336. Academic Press, New York. Verrett, M. Jacqueline, Marliac, J.-P. & McLaughlin, J., Jr. (1964). Use of the chicken embryo in the assay of aflatoxin toxicity. J. Ass. off. agric. Chem. 47, 1003. Wahlstrom, R. C. & Olson, O.E. (1959). The effects of selenium reproduction in swine. J. Anita. Sci. 18, 141. Westfall, B. B., Stohlman, E. F. & Smith, M. I. (1938). The placental transmission of selenium./. Pharmae. exp. Ther. 64, 55. Wilson, A. T. (1966). Effects of abnormal lead content of water supplies on maternity patients. The use of a simple industrial screening test in ante-natal care in general practice. Scott. med. J. 11, 73.
Embryotoxicity of Chemical Contaminants of Foods* D. J. CLEGG Food Advisory Bureau, Food and Drug Directorate, Carlingwood Plaza, Curling Avenue, Ottawa, Ontario, Canada (Received 20 August 1970)
SUIVIIVIARY A brief review of a variety of contaminants that may occur in foodstuffs indicates that in the majority of cases embryotoxicity or teratogenicity are toxic hazards unlikely to arise in practice as a result of such contamination. The major possible exception is mercury, especially methylmercury, on which further data are being obtained. Meanwhile mercury intake should be restricted, and this is being done where possible, at least as far as the Canadian diet is concerned. Finally, further data are required on the effect of polychlorinated biphenyls on mammalian foetal development. The probability of hazard is at present regarded as being low, but insufficient information is available to make an accurate assessment. Introduction
Following the discovery of the embryotoxic and teratogenic effects of thalidomide in the early 1960s, considerable interest has been generated in the possible embryotoxie and/or teratogenic potential of a variety of compounds. This review is concerned with the data that have accumulated on chemicals that may occur as contaminants in the diet of man, either as a result of environmental contamination or from sources such as agricultural practice or food processing. Mycotoxins The best known of the mycotoxins that may appear in the human diet are the aflatoxins, comprising five major closely-related toxins produced by fungi of the genus Aspergillus. These compounds are extremely toxic to the developing chick embryo (Platt, Stewart & Gupta, 1962), and in the developing 5-day embryo as little as 0-3 ~g of a crude groundnut extract causes mortality within 2 days (D. J. Clegg, unpublished data, 1962). This extreme sensitivity of the hen egg is the basis of a bioassay technique for aflatoxins proposed by Verrett, Marliac & McLaughlin (1964). This technique, in which the fertile hen egg is injected prior to incubation, is still widely used. *Presented at a Symposium on "Chemical Contaminants in Foods--H~Tard or Not ?", sponsored by the Food and Drug Directorate, Department of National Health and Welfare, Ottawa, Ontario, and held in Ottawa on 18-19 June 1970. 195
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In the rat, intraperitoneal injection of aflatoxin B~ has been shown to cause haemorrhagic lesions at the utero-placental junction, with subsequent embryonic death (Le Breton, Frayssinet, Lafarge & de Recondo, 1964) while repeated injection of small doses retarded foetal growth. Butler & Wigglesworth (1966), feeding aflatoxin Bz to rats on day 16 of pregnancy, failed to induce embryonic death but noted growth retardation of the foetus. In neither instance was a teratogenic effect reported. In the hamster, Elis & DiPaulo "(1967) demonstrated teratogenic effects in the central nervous system following intraperitoneal injection of 4 mg aflatoxin B1/kg on day 8 of pregnancy. In farm animals, 2 ppm aflatoxin Bx fed for 3-12 months in the diet of cows had no apparent effect on conception, gestation or parturition (Allcroft & Lewis, 1963), and very high doses of aflatoxin Bz (0-9-1.75 mg/day) had no effect on gestation or parturition in sheep although fertility was reduced (Lewis, Markson & Allcroft, 1967). From the available data on aflatoxins, it is apparent that teratogenicity and embryotoxicity are relatively insignificant effects compared with the known carcinogenic effects of this group of compounds. Metals Zinc. Although frank zinc deficiency is known to result in congenital malformations in rats (Hurley & Swenerton, 1966), such a deficiency has never been observed in man. Zinc intoxication has occurred, however, following preparation of food in galvanized vessels (Brown, Thom, Orth, Cova & Juarez, 1964). Fern & Carpenter (1968) have investigated the effects of excess zinc on the developing hamster. At 30 mg zinc sulphate/kg, injected intravenously, maternal animals succumbed within 24 hr. Lower doses (10-25 mg/kg) induced about 12% foetal resorptions, and with 2 mg/kg given intravenously, teratogenic effects (fused ribs and exencephaly) occurred in about 4 % of cases. In multigeneration studies in rats (Heller & Burke, 1927), levels of 2500 ppm in the diet as zinc chloride caused no ill-effect, although 5000 ppm increased mortality in the offspring, a finding that has been tentatively attributed to the destruction of the fl-cells of the Islets of Langerhans (Underwood, 1962). However, 5000 ppm zinc as the oxide caused very little increase in the mortality of the offspring. Although intravenous injection of soluble zinc salts causes effects in the hamster, probably because of the high blood levels resulting from the method of administration, zinc taken orally is relatively poorly absorbed and sufficiently high blood levels are unlikely to be attained. It seems improbable, therefore, that zinc contamination of food will increase the incidence of teratogenic or embryotoxic effects in man. Cadmium. Plated cooking utensils have been known to cause cadmium poisoning (Monier-Williams, 1949). In the hamster, Ferm & Carpenter (1967a) have shown that intravenous administration of cadmium sulphate, at a dose of 2 mg/kg on day 8 of gestation, results in a 66% incidence of malformations in the embryos. These malformations are mainly craniofacial. Cadmium is known to cross the placenta of the hamster (Ferm, Hanlon and Urban, 1969) and since it is found in foetal and newborn human liver and kidney (Underwood, 1962), it seems reasonable to infer transplacental passage in man. However, the levels of cadmium found in man are low, and cannot be related to the high levels administered to the hamsters. Furthermore, in the experiment cited, the use of intravenous injections to produce effects makes extrapolation to man impossible, and it seems likely that cadmium, like zinc, is not a significant factor in human teratogenicity or embryotoxicity.
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Calcium. Although calcium is an essential element for development, a bizarre phenomenon has been observed following injection of 0.1 mg calcium pantothenate into the yolk sac of hen eggs incubated for 2 days (Grabowski, 1966). Of the resultant embryos, 17~ were grossly abnormal. Follow-up experiments, using 10--40/~g calcium chloride injected into the yolk sac (of 2-day-incubated eggs) or allantois (3- and 5-day-incubated eggs), resulted in a slightly increased mortality rate, with 12-17 ~ abnormalities among the offspring. Thus, trace quantities of calcium appeared to be the causal agent. The author suggests that membrane permeability may be affected and that this results in abnormalities. These data illustrate the dangers of extrapolating teratogenic data from hen-egg experiments; there are no data implicating calcium as a teratogen in mammals. Selenium. Robertson (1970) has suggested that selenium may be a possible teratogen in man. Out of one possible and four certain pregnancies among women exposed to selenite, only one pregnancy went to term, and the infant showed bilateral clubfoot. Of the other pregnancies, two could have terminated because of other clinical factors. Widespread enquiries in other laboratories where exposure to selenite could occur have not revealed any similar pattern, except for one miscarriage in the only pregnancy in 5 years in one small laboratory. A further letter (Hadjimarkos, 1970) pointed out the potential danger of high concentrations of selenium in foods grown on soils with a high selenium content. Selenium has been shown to cross the placenta in rats and cats (Westfall, Stohlman & Smith, 1938) and its passage can be inferred in other species from the symptoms of selenium poisoning observed in the offspring of animals grazing on seleniferous rangeland. Foals and calves in affected areas are sometimes born with deformed hoofs. The offspring of rats (Rosenfeld & Beath, 1954), pigs (Wahlstrom & Olson, 1959) and sheep (Rosenfeld & Beath, 1947) have all shown abnormalities following selenium administration during pregnancy. On the other hand, 2 mg sodium selenite/kg (an almost lethal dose) administered intravenously had no teratogenic or embryotoxic effects in the hamster (Holmberg & Ferm, 1969). Evidence is, therefore, controversial and difficult to relate to the possible effects of selenium on human embryonic development. It seems unlikely that food contamination with selenium would be a major problem in cities because of the wide variations in the source of foodstuffs. However, a close watch should be kept on rural populations, especially in areas where the soil has a high selenium content. Lead. Severe exposure of the pregnant human female to high levels of lead has apparently resulted in abortion (Wilson, 1966; Taussig, 1936). Since lead is known to cross the human placenta in cases of high-level exposure (Barltrop, 1968), the occurrence of teratogenic effects at a sublethal level is not surprising, and following excessive industrial exposure offspring with neurological symptoms have been encountered (Angle & Mclntire, 1964). Growth, both intrauterine and postnatal, may be retarded (Palmisano, Sneed & Cassady, 1969). In hamsters, lead nitrate, lead chloride or lead acetate injected intravenously in doses of 50 mg/kg on day 8 of pregnancy resulted in a high incidence of teratogenic malformations in the sacral and tail regions of the offspring (Ferm & Carpenter, 1967b). Foetal mortality was not significantly increased. No evidence of teratogenic activity has been observed in the cow (Shupe, Binns, James & Keeler, 1967) or the sheep (James, Lazar & Binns, 1966). The dose levels involved could not be calculated in the cases of human exposure, but in all cases were obviously considerably in excess of dietary exposure. Similarly, the route of administration and extremely high dosage involved in the hamster teratogenicity experiment render extrapolation to man almost impossible.
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Mercury. Several studies of the embryotoxic or teratogenic activity of mercurial salts have been performed in the laboratory. In avian species, mercuric chloride has been injected into fresh, fertile, unincubated hen-egg yolk sacs at doses of 0-5 or 0.25 mg/egg. At the higher dose all the embryos died, and at the lower dose hatchability was reduced by 80~o (McLaughlin, Marliac, Verrett, Mutchler & Fitzhugh, 1963). Pheasants fed wheat containing 15-20 ppm methylmercury dicyandiamide for 9 days laid eggs with reduced hatchability and containing levels of 1.3-2.0 ppm mercury (Borg, Wanntorp, Erne & Hanko, 1969). Eggs from abandoned pheasants' nests frequently contained more than 1 ppm mercury and addled eggs from eagle and owl nests contained 3.5-11 ppm. Thus, it would appear that mercury entering the egg, either by injection or via the maternal organism, can produce embryotoxic effects. Autoradiographic studies in mice showed that ethylmercuric chloride passed through the placenta more easily than either mercuric chloride or phenylmercuric chloride (Ukita, Takeda, Sato & Takakashi, 1967). Once through the placenta, mercury appeared to have a greater affinity for the central nervous system than was the case in the adult. Later studies (Suzuki, Matsumoto, Myama & Katsunuma, 1967) have confirmed that alkylmercury does cross the placenta more readily than aryl or inorganic compounds, as indicated by the ratios of maternal: placental: foetal blood levels of mercury (1 : 19: 0-4 for mercuric chloride; 1 : 1.9:2.1 for methylmercuric acetate; 1:4-5:0.3 for phenylmercuric acetate). The placenta appears to act as a barrier to mercuric chloride and phenylmercuric acetate--possibly in the region of the yolk-sac membrane. Similarly, methylmercury has been shown to cross the rat placenta (Fujita, 1969). In mouse experiments, ethylmercuric phosphate injected subcutaneously at a dose level of 40 mg/kg on day 10 of pregnancy did not affect litter size (Oharazawa, 1968). However, foetal body weight was reduced and a 31.6 ~o incidence of cleft palate was observed. Since no mice were permitted to litter and no histopathology was reported, the incidence of central nervous system damage was not established. Intraperitoneal administration of 0.1 mg methylmercury dicyandiamide to CBA mice prior to mating had no effect on the size of subsequent litters (H. Fr616n and C. Ramel, cited by L6froth, 1969), but the incidence of pregnancies was reduced either as a result of reduced libido or of damage to gametes. Injection on day I0 of pregnancy resulted in a significant increase in embryonic mortality (17 ~o compared with 7.2 ~o in controls). Methylmercury is known to cause chromosomal aberrations in the lymphocytes of persons consuming fish containing high mercury levels (J. Lindsten, personal communication cited by Lffroth, 1969). Such damage, if it occurs in the mouse, could be responsible either for the reduced incidence of pregnancies or for the teratogenic effects reported in the above experiments. In the rat, daily administration of 0.1 mg mercury/kg (as methylmercury) has been shown to reduce birth weight and cause neurological disorders during postnatal development (Fujita, 1969). Embryotoxicity in the rat has also been demonstrated by feeding 1, 2, 4 or 8 ppm mercury as phenylmercuric acetate in the diet (Piechoka, 1968). At 8 ppm, litter size was reduced by 44-3 Yo. No data on neurological involvement were given. Wistar rats given a single oral dose of 2 or 20 mg methylmercury chloride/kg on day 9, 10 or 11 of pregnancy showed cerebellar changes (Matsumoto, Suzuki, Monta, Nakamura & Saeki, 1967). At the higher dose, the whole shape of the cerebellum was distorted and, in addition, the midbraln showed enlarged ventricular cavities and degeneration of the nerve cells. At the lower dose, histological changes were detectable in the cerebellum. The histological changes observed in this experiment contrasted with the degeneration and loss of
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nerve cells in the cerebral cortex observed in the adult rat, in that the changes were confined to the cerebellum. In a similar experiment, rats were dosed orally with 0.2 mg methylmercuric chloride/rat during the first 20 days of gestation; there were no signs of maternal toxicity up to sacrifice, and litter size and embryo weight were normal. Histology of the embryonic brain revealed a disappearance of nucleoli, a reduction of chromatin granules and a decrease of ribosomes in the granular cells of the cerebral cortex. Purkinje cells were also observed to be vacuolated or to be electron-dense. These changes (which were also observed in 100-day-old offspring of a second identically-treated group) were similar to the changes observed in the adult rat following experimentally-induced Minamata disease (Nonaka, 1969). The offspring of one cat exposed during pregnancy to between 150 and 220 mg bisethylmercuric sulphide showed changes in the few remaining cerebellar granular cells and a delayed migration of the Purkinje cells (Morikawa, 1961). Thus, animal experimentation indicates that mercury may have both embryotoxic and teratogenic potential in rats, mice and cats. The greater ease with which alkylmercury crosses the placenta leads to the speculation that methyl- or ethylmercury may possibly be more important in foetal toxicity than aryl or inorganic mercury. There is also some evidence of preferential accumulation of methylmercury, especially in foetal nervous tissue. In man, 19 of 111 cases of Minamata disease were encountered in newborn children (Irukayama, 1969). Since a population is unlikely to contain 17-3 ~ of pregnant women, these figures indicate that the unborn infant is at greater risk from methylmercury poisoning than the adult. The mothers of the 19 congenital cases of Minamata disease had all ingested fish or shellfish contaminated with methylmercury. However, no symptoms had been observed in the majority of mothers, and in the few cases where symptoms did occur, they were confined to slight numbness of the finger (Kakita, 1961). This lack of symptoms in the mothers may well be due to the relative ease of placental transfer of methylmercury, like that observed in laboratory animals. The passage of methylmercury across the human placenta, and its possible preferential concentration in the foetus, has been demonstrated in normal pregnant women, human foetal erythrocyte levels showing 28 ~ more mercury than the maternal erythroeytes (Tejning, 1968). (Plasma levels of mercury are 19 ~ higher in the mother than in the foetus, but plasma levels are relatively insignificant when compared with those in the erythrocytes.) In congenital cases of Minamata disease, cerebral palsy is the major effect. In the two cases reported in detail, examination of the brain tissue revealed degenerative and regressive changes in the cerebral cortical nerve cells, stated to be similar to those observed in adult autopsy material (Matsumato, Koya & Takeuchi, 1965). Thus, there seems to be very little doubt that methylmercury can exert an embryotoxic effect in man. Since effects have been reported in the rat following the oral ingestion of 0.1 mg methylmercury/kg, it seems probable that the safe ingestion level for the pregnant human female is extremely low. Strontium-90. Although the effects of embryonic irradiation have been extensively studied, relatively little is known about the effects of internally-deposited radioactive isotopes on embryonic development. Hopkins, Casarett, Tuttle & Baxter (1967) injected doses of 0, 191 or 382 t~c strontium-90 intravenously into 2- or 10-day postconception rats. In 2-day postconception rats, mortality rates were increased from 7 ~ in the control group to 22 ~ in the group given 191 t~c and 64 ~ in that given 382 tzc strontium-90. Foetal weight was decreased to 95 and 89 ~, respectively, of the control weight, and the incidence of runts was increased from 5 ~ to 13 and 23 ~ respectively. In 10-day postconception embryos,
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mortality was virtually unaffected by either dose, but birth weight was decreased to 96 and 89 ~ of the control value and the incidence of abnormalities increased from 3.1 ~o to 17.6 and 51~o at the two dose levels. The intrauterine radiation dose after the 10-day postconception injection was 12 rads/day after 6 hr, decreasing to 0.3 rads/day by day 2 after injection. The dose rate remained low until late in foetal life, when deposition of strontium90 in foetal bone caused a second peak in activity at 20 rads/day. In Canada, the dietary intake of strontium-90 is considerably below the levels used in these studies (Belkhode, Mar & Quastel, 1969). Nitrosamines
A recent paper by DiPaulo (1969) reviewed the embryotoxicity and teratogenicity of the alkylating agents, including nitroso compounds. Briefly, dimethylnitrosamine, although non-teratogenic, is embryotoxic both orally and by intraperitoneal injection in the rat, hamster and chick. Pesticides
Since Khera & Clegg (1969) presented a paper on the perinatal toxicity of pesticides, the Report of the Secretary's Commission on Pesticides and their Relationship to Environmental Health (the 'Mrak Report') has been published (Commission on Pesticides, 1969). In this report, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), various esters of 2,4-dichlorophenoxyacetic acid (2,4-D), PCNB, captan and folpet are all listed as causing a significant increase in the incidence of anomalies in C57BL/6 mice. 2,4-D and 2,4,5-T are also stated to have increased the incidence of anomalies in A K R mice. Both of these pesticides have been the subject of further investigation since the report was issued. 2,4,5-T. On 29 October 1969, Dr. L. A. DuBridge, Scientific Adviser to the President, announced the partial curtailment of the use of the herbicide 2,4,5-T in the USA. According to Nelson (1969), a reporter on the staff of the Los Angeles Times, this decision was based on "scientific studies which would soon become widely known" and on South Vietnamese newspaper stories (so far unsubstantiated in any scientific journal) attributing an increase in congenital defects to the American defoliation programme, which uses 2,4,5-T. Although various comments on the scientific studies appeared in the lay press in November and December, the first scientific account did not appear until the Mrak report was published in December 1969. The data reported by the Commission on Pesticides (1969) was derived from a large-scale teratogenic screening study involving several commonlyused pesticides. These pesticides were administered to mice daily from day 6 to day 14 (strain C57BL/6) or to day 15 (AKR) of pregnancy, either subcutaneously in dimethylsulphoxide (DMSO) or orally in 50~o honey solution. In the case of 2,4,5-T, repeated administration of 113 mg/kg subcutaneously caused a significant increase in the incidence of cleft palate and cystic kidneys in embryos removed at day 18 (C57BL/6) or 19 (AKR) of pregnancy. Foetal mortality was increased in the C57BL/6 strain only. Oral administration of 113 mg/kg]day increased foetal mortality in both strains, and induced cleft palate and cystic kidney in C57BL/6 mice and cleft palate in A K R mice. Repeated doses of 46.4 mg/kg given orally to C57BL/6 mice resulted in an increased incidence of abnormal litters, and an increased incidence of abnormal foetuses per litter, but the types of abnormalities were not specified. Foetal mortality was apparently unaffected at 46-4 mg/kg/day in C57BL/6
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mice. Although no data are provided, the text of the report of the Commission on Pesticides (1969) indicates that subcutaneous administration of repeated doses of 113 mg/kg to a hybrid C57BL/6 × AKR mouse strain significantly increased the incidence of cystic kidney in particular. A study on Sprague-Dawley rats given 4.6, 10 or 46.4 mg/kg/day indicated that teratogenic (kidney anomalies) and embryotoxic effects were observed at all levels. These experiments have since been reported in more detail in the conventional literature (Courtney, Gaylor, Hogan, Falk, Bates & Mitchell, 1970a), and basically confirm the information originally published (Commission on Pesticides, 1969). However, the abnormalities in the C57BL/6 mice treated with 46.4 mg/kg/day were confined to cystic kidneys (which have still not been described). The increased embryonic mortality in the rat experiment was significant at 10 mg/kg/day and above, but not at 4.6 mg/kg/day. In addition, a further possible embryotoxic effect in rats was demonstrated, comprising a dose-related incidence of haemorrhage in the gastro-intestinal tract. Additional data indicate that in C57BL/6 mice, 21.5 mg/kg/day administered subcutaneously in DMSO on days 6-14 of pregnancy caused no significant increase in any teratogenic or embryotoxic effects. Finally, in an additional experiment on C57BL/6 mice, subcutaneous treatment with 113 mg/kg/day in DMSO on days 9-17 of pregnancy caused cleft palate and cystic kidney, but embryonic mortality was unaffected. The 2,4,5-T used in these studies was analysed by Dow Chemical Co. and shown to contain approximately 30 ppm 2,3,7,8-tetrachlorodibenzo-p-dioxin (referred to henceforth as dioxin). Emerson, Thompson, Gerbig & Robinson (1970) administered 2,4,5-T containing less than 1 ppm dioxin orally to groups of 25 female Sprague-Dawley rats in doses of 1, 3, 6, 12 or 24 mg/kg/day on days 6-15 of pregnancy (day 0 being the day on which positive vaginal smears were observed). A further 50 female rats were dosed orally with the vehicle, 0-25 ~ hydroxypropylmethyl cellulose. Foetuses were removed and examined for soft-tissue and skeletal abnormalities on day 20 of gestation, and ten high-dose foetuses were examined histologically. There were no clinical symptoms in the dams, and no indications of increased mortality, intestinal haemorrhage or teratogenic effects in the foetuses. A study was performed using 91 ~ pure dioxin, the impurities comprising 7 Yotrichloro-, 2 Yopentachloro- and < 0.02 ~o dichlorodibenzo-p-dioxin (Sparschu, Dunn & Rowe, 1970). The test material was administered by gavage in doses of 0-03, 0.125, 0.5, 2.0 and 8.0 /zg/kg/day to groups of 8-12 rats on days 6-15 of gestation, and 24 control rats were given the corn oil-acetone (9 : 1, v/v) vehicle. Doses were based throughout on pre-breeding body weights. In the dams, 3/11 given 2/~g/kg showed vaginal haemorrhage from days 15-20 of gestation, and 6/8 given 8/~g/kg showed vaginal haemorrhage at times between days 13 and 20 of gestation. Maternal body-weight gain was depressed at 0.5 /zg/kg and above. With regard to the foetuses, the numbers of corpora lutea and of implantation sites were comparable in all groups. Resorption was significantly increased at 0.5 ~g/kg and above, all foetuses being resorbed at 9/~g/kg. The sex ratio of the foetuses was normal, but foetal weight was depressed at 0-125 /zg/kg and above. Gross abnormalities comprised foetal oedema at 2.0/~g/kg, a rudimentary tail at 0.125/zg/kg, and two misshapen limbs and a kinked tail at 2.0 tzg/kg. Skeletal examination revealed no compound or dose-related effects. Soft-tissue examination showed a dose-related incidence of gastro-intestinal haemorrhage at 0.125/zg/kg and above. Dilation of the renal pelvis was observed in all groups, with a doubtful increase in incidence at 0.5 and 2.0/zg/kg. Microscopically, a dose-related incidence of subcutaneous oedema occurred at 0.125 /zg/kg and above. The evidence from the l).C.r. 912---C
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experiments to date, therefore, suggests that the dioxin may be the agent inducing the embryotoxic and teratogenic effects in rats. Courtney, Moore, Gaylor, Hogan & Falk (1970b) presented data from further teratogenic studies on 2,4,5-T in April 1970. Three strains of mice were studied, the test materials being given on days 6-15 of gestation by subcutaneous injection in DMSO. Charles River mice showed an increased incidence of cleft palate and kidney involvement following treatment with 2,4,5-T at 100 mg/kg/day (providing a dose of less than 0.01/~g dioxin/kg). No effects were observed at a dose of 50 mg]kg/day with a 2,4,5-T sample providing 0.075 tzg/kg dioxin. Foetal mortality was unaffected at either dose level. Dioxin alone increased the incidence of kidney involvement at 1 /zg/kg and of cleft palate at 3/zg]kg. In DBA mice, 2,4,5-T caused an increased incidence of cleft palate at 100 mg]kg]day ( <0.05/zg dioxin/kg), but foetal mortality and the incidence of kidney involvement were unaffected. Dioxin alone, at 3 t~g/kg, caused an increase only in the incidence of kidney involvement. In C57BL]6 mice, 100 mg 2,4,5-T]kg]day ( <0.05 tzg/kg dioxin) caused a significant increase in mortality only. Dioxin alone, at 3/~g]g, significantly increased the incidence of cleft palate and kidney involvement, but did not increase foetal mortality. In summary, mice are susceptible to the teratogenic effects of dioxin and possibly, at high levels, to those induced by 2,4,5-T. However, until the susceptibility of the various mouse strains to dioxin-induced effects is more accurately known, the teratogenicity of 2,4,5-T must remain in some doubt. Further rat studies (Courtney et al. 1970b) have confirmed that 2,4,5-T per se is not teratogenic in the rat, although dioxin causes kidney abnormalities. One further mammalian study has been completed. J. L. Emerson, D. J. Thompson, C. G. Gerbig & V. B. Robinson, (unpublished report of Dow Chemical Co., 1970) have studied the teratogenic activity of 2,4,5-T containing less than 1 ppm dioxin. Groups of 20 rabbits were treated orally on day 6-18 of pregnancy with doses of 0, 10, 20 or 40 mg/kg/day. There were no indications of embryotoxic or teratogenic effects. Dioxin, therefore, appears to be the main agent responsible for the teratogenic effects associated with 2,4,5-T in mammals. If 2,4,5-T is teratogenic in mice, it appears that the dose levels involved are in excess of 50 mg/kg, a dose level that provides an adequate safety margin for man when one considers that 2,4,5-T residues have rarely been detected in food. Even in those eases where 2,4,5-T residues have been detected, the levels in food have been extremely low. Similarly, it must be remembered that the mouse studies involved administration of 2,4,5-T by subcutaneous injection, a route which would not be directly comparable to oral ingestion of contaminated foods. Finally the solvent, DMSO, has itself been implicated as a teratogen in the hamster (Ferm, 1966) and in the rat, mouse and chick embryo (Caujolle, Caujolle, Cros & Calvet, 1967), and as an embryotoxin in the rat (Juma & Staples, 1967) at very high dose levels. The doses used by Courtney et al. 1970a,b) were far below those inducing teratogenic or embryotoxic effects, but the possibility of potentiation of the teratogenic and embryotoxic activity of DMSO by 2,4,5-T and/or dioxin has not been investigated and cannot, therefore, be excluded. In the case of dioxin, a no-effect level of 0.03 t~g/kg has been demonstrated in rats. The amount of dioxin applied with 2,4,5,-T is small, but until more information is available on environmental contamination with dioxin, its rate of degradation and the residue situation in food, the use of 2,4,5-T has been restricted as a precautionary measure to prevent possible exposure of pregnant women to dioxin. 2,4-D. The Mrak report also indicated that a number of esters of 2,4,-D could be teratogenic, according to the teratogenic screening test results (Commission on Pesticides,
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1969). K. K h e r a (personal c o m m u n i c a t i o n , 1970) has investigated this p r o b l e m a n d reports as follows: "2,4-D and its formulations (isooctyl, butyl, d i m e t h y l a m i n e a n d b u t o x y ethanol) were a d m i n i s t e r e d in corn oil singly by i n t u b a t i o n as one daily dose from 6-15 days o f gestation in rats. All the c o m p o u n d s at doses a p p a r e n t l y non-toxic for the d a m s (100-150 m g / k g / d a y ) induced r e d u c t i o n in average litter weight a n d significantly increased incidence o f s p o n t a n e o u s l y occurring skeleton m a l f o r m a t i o n s . These effects were n o t a p p a r e n t at doses o f 25 mg/kg. Similar studies with 2,4,5-T are in progress." Since 25 m g / k g seems to be a no-effect level for 2,4-D, a n d its residues (if any) are m i n i m a l (and d o not c o n t a i n dioxin), no p r o b l e m s are anticipated with this herbicide as far as c a n be predicted f r o m d a t a at present available.
Polyehlorinated biphenyls Recent d a t a suggest t h a t p o l y c h l o r i n a t e d biphenyls are as u b i q u i t o u s as D D T . A t present, very little is k n o w n a b o u t their possible effects on the h u m a n foetus. I n the a d u l t rabbit, the acute oral LDso for the various p o l y c h l o r i n a t e d biphenyls t h a t have been e x a m i n e d lies between 8 and 11 g/kg. In a very p r e l i m i n a r y study (which is being repeated) 50 m g / k g was e m b r y o t o x i c in the r a b b i t (W. J. Philips, D. C. Villeneuve & D. G r a n t , personal c o m m u n i c a t i o n , 1970). N o effects were observed at 1 or 10 mg/kg. T h e e m b r y o n i c r a b b i t thus a p p e a r s to be much m o r e susceptible t h a n the adult, b u t this remains t o be confirmed.
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