The carcinogenesis, mutagenesis and teratogenesis of insecticides. Review of studies in animals and man

Pharmac. Ther. Vo]. 6, pp. 147-166. ~) Pergamon Press Ltd. 1979 Printed in Great Britain.

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Specialist Subject Editor: FUMIO MATSUMURA

THE CARCINOGENESIS, MUTAGENESIS A N D TERATOGENESIS OF INSECTICIDES. REVIEW OF STUDIES IN ANIMALS A N D M A N STEPHEN S. STERNBERG Department of Pathology, Memorial Hospital and Laboratory of Pharmacology, Sloan-Kettering Institute New York, USA

1. INTRODUCTION To determine if any given chemical is carcinogenic for man is a formidable problem. Only a mere handful have thus far been uncovered, and the total number of cancers causally related to known carcinogens represents only a minute fraction of all cancers affecting man (to detect a mutagenic or teratogenic effect in man is even more difficult). One can suggest that 80 or 90 per cent of cancers are of 'environ° mental' origin but the fact is that the etiology of most cancers is unknown at the present time. Cancer was a major cause of morbidity and death long before food additives and chlorinated hydrocarbon insecticides were an intimate part of our daily life. The fact is that most carcinogens have been detected in the industrial environment, regardless of the availability of the data on animal experiments indicating carcinogenic activity. The information gained in uncovering industrial cancers could perhaps be used more profitably in determining risk of exposure to putative carcinogens. We know that in industrial cancers: (l) the population at risk is small; (2) the exposure is high and concentrated; (3) the incidence of cancer is high; and (4) the latent period is often short, at least in some of the affected individuals (Sternberg, 1976). However, in circumstances where there is a high and concentrated exposure, such as in a manufacturing plant, and where groups of workers have been followed for a reasonable number of years and no increased cancer incidence has developed, it is unlikely that a carcinogenic effect will ever be demonstrated in man. If there is exposure to the same agent in the general population but at a much lower dose and the incidence is such that only one person in, say, 100,000 develops a cancer from the chemical, the causal relationship becomes virtually impossible to detect. How do we assess the danger to man under the circumstances described? One approach (which has many advocates) is to ban everything which has been shown to have a tumorigenic effect in animals. While this may seem to be the safest approach, it is not necessarily the soundest. The decision should be based on benefit versus risk. A food dye generally represents a frivolous additive and can usually be eliminated without affecting our health. On the other hand, the potential danger of nitrites must be evaluated in terms of cancer risk versus the risk of botulism. And, perhaps more importantly, the risk of an insecticide such as DDT as a possible carcinogen vis-a-vis its ability to diminish or eliminate malaria, a disease no less malignant than cancer. Finally, what is considered a valid animal test for carcinogenesis is another thorny problem. Aflatoxin causes liver cancer in mice, rats, dogs, birds and other animal species. It does so with relative ease and with an extraordinarily high incidence in some tests. Such experiments leave little doubt as to the carcinogenicity of aflatoxin 147

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in wide ranges of animals. On the other hand, where the only susceptible test animal happens to be the mouse, and if it takes place only sporadically in some strains, or only in mice with a normally high incidence of tumors, the meaning of such a carcinogenic effect becomes doubtful. The reliability is further strained when it is realized that varying the diet, the caloric content, and so on, can alone affect tumor incidence. Obviously, a compound with such a history cannot be treated as seriously as aflatoxin (Newberne, 1975; Roe, 1966, 1975). There are certain aspects of carcinogenesis which are sometimes overlooked or insufficiently emphasized. What is not generally appreciated is that most carcinogens have an acute toxicity which results in cell damage. This is easily missed for the damage may be transient and the repair rapid. Full recovery may occur in as little as 24 hr with no histological evidence of altered cell function or death. To detect this type of acute cell damage requires: (1) the administration of appropriate doses which may be in the lethal range; and (2) examination of the tissues at proper intervals after dosing (hours or days). There are, of course, some compounds which produce cell death at relatively low doses and without death of the host. The acute toxicity may affect several organs but usually only one organ is susceptible in terms of a carcinogenic response. A minority of carcinogenic agents do not have an acute toxic effect, or at least one cannot demonstrate it. Such compounds, however, frequently show chronic toxicity. Many compounds show both acute and chronic toxicity. Finally, many, but not all, carcinogens produce premalignant lesions which portend the development of cancer. These three features of carcinogenesis, acute ~oxicity, chronic toxicity and premalignant change, are seen both in experimental animals and in man. Not all of these features are observed in every instance but, in the absence of such premonitory effects, one can consider the possibility that a carcinogenic action may not occur. In man, for instance, where the cancer has resulted from exposure to a known carcinogen, the tumor does not develop out of nowheremone or more of the premonitory changes, remote on recent, will presage the cancer. Such is the case for practically all of the known carcinogens affecting humans. Examples abound. The aromatic amines which produce urinary bladder cancer cause an acute hemorrhagic cystitis within 24 hr of an exposure to a high dose (Scott, 1962). A later effect is the development of a bladder papilloma which represents the precancerous phase. Long term ingestion of high doses of phenacetin is believed to cause renal cancer. This may be preceded by a benign but dangerous disorder, namely papillary necrosis of the kidney (Hultengren et al., 1965). Respiratory tract carcinogens have variable effects. It is unlikely that cigarette smoke (which can effect ciliary action) causes acute cell death. However, long-term inhalation frequently causes bronchitis and emphysema prior to or associated with the development of bronchial mucosa atypia, ultimately leading to carcinoma in situ and infiltrating cancer. Mustard gas workers, on the other hand, almost invariably develop acute or chronic bronchitis as an early effect (Wada et al., 1968). Arsenicals produce benign skin lesions (hyperkeratosis) which ultimately may go on to skin cancer (and visceral cancers). Pitch and tar workers develop acne prior to the skin cancer. Similarly, asbestosis foretells the pleural mesothelioma. Diethylstilbestrol causes a benign abnormality, vaginal adenosis, which ultimately may result in clear cell adenocarcinoma of the vagina. In view of what is known about the toxicity of carcinogens in terms of acute and chronic phases, and the appearance of premalignant and other changes, one can suggest that, in the absence of these findings in a group of susceptible individuals, it is unlikely that a carcinogenic action will take place. Our efforts might better be directed to the protection of individuals against known carcinogens rather than agents in which the carcinogenic effects is, at best, dubious. 1.1. MUTAGENESIS Interpretation of the result of mutagenesis testing appears to be more complicated than testing for carcinogenic activity although the two are closely related. In parti-

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cnlar, it is primarily the evaluation of results that presents the problem. These difficulties have been well summarized by Matter (1976). A major point to he determined is whether a compound reaches the genetic target. It is one thing to know what occurs in a bacterial system and quite another to know how the compound affects man. With whole body ionizing radiation, a model agent for mutagenesis, free-radicals aro produced and distributed throughout the body and are in contact literally with every cell. The reactivity is so consistent and clear-cut that genetic damage can be demonstrated in every test system used. Putative mutagens have to be absorbed and disseminated in the blood to possible target organs. However, the activity may reside in a metabolite rather than the original molecule, the dynamics of absorption and excretion of the metabolites may be quite different from the original compound, protein binding may vary the reactivity, and membrane permeability may be different and so on. Further, even if the target cell is reached, the toxicity may not be expressed as genetic damage. And, if the reactivity is a genetic one, it may not be detectable because of the rarity of the event. In particular, a factor to be considered in terms of mutagenesis of insecticides is the possible reactivity of treated plants to produce metabolic activation (Zimmerman, 1976). It has been pointed out that the test systems evaluate only the short-term effect of a mutagen and that little can be uncovered with these methods of the effects of chronic exposure (Fahrig, 1974). The relationship of mutagenesis and carcinogenesis has been greatly strengthened only during the last several years. The correlation is quite good for the majority of compounds tested in terms of carcinogens having mutagenic activity and mutagens being carcinogens. However, if the tests developed by Ames (1976) are used for screening purposes, the results should only be considered as preliminary until such time as a conclusive determination can be made. False negative tests, of course, are not acceptable. There are a number of Compounds which do not fit into the pattern of mutagens as carcinogens and vice versa. For instance, nitrous acid and hydroxylamine (base analogues with actions unrelated to electrophilic intermediates) are considered potent mutagens, yet have no carcinogenic activity (Miller and Miller, 1971). Similarly, certain steroids, sex hormones, are known carcinogens yet have shown no mutagenic activity. The steroid a,a'-dietbylstilbestrol (a human and animal carcinogen) also did not show mutagenic activity using the TA 100 strain of S. typhimurium (Bartsch, 1976). In the use of S. typhimurium and other mutagen test systems as a pre-screen, one should consider the points emphasized by Bartsch (1976) that: (1) positive results from mutagenicity tests do not automatically imply a carcinogenic effect on man; (2) positive or negative results from these tests are not a substitute for carcinogenicity tests in animals; (3) while the tissue=mediated mutagenicity assays can predict carcinogen potential in perhaps 80-90 per cent of the chemicals, the tests give no indication of target organs or specific specificity; and (4) the relative potency of a chemical mutagen cannot be correlated with its carcinogenic potency either in animals or man. 1.2. TERATOGENESIS

Toxicity from chemicals are generally manifested by acute and chronic toxicity'; some, as discussed, are mutagenic, and most of those which produce mutations are also found to be carcinogenic. A reflection of the toxicity of some chemicals is also observed in damage to embryos and fetuses when administered maternally during the period of gestation. One form of toxicity to the offspring is a teratogenic effect. It is most unusual for a chemical to be selectively toxic to the offspring--that is, to have no acute or chronic toxicity and to be neither mutagenic nor carcinogenic. Teratogenesis in nearly all instances represents only one of several aspects of toxicity. The fetus may react poorly to toxic substances absorbed in the maternal blood, in part due to the fetal liver being poorly developed in terms of its detoxffying ability. The so-called placental barrier, in reality, functions more as a sieve in terms of

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'filtering' out exogenous agents; the unbound chemicals pass to the embryo mostly by simple diffusion (Wilson, 1977). One would think that under these conditions the fetuses would be more susceptible to exogenous substances than they actually are. Remarkably, relatively few agents have actually been shown to be teratogenic, particularly those considered to be of environmental origin (DiPaolo, 1969). Wilson (1977) has estimated the part played by different agents in producing development defects in man. He indicates that all current known environmental factors together do not account for more than 10 per cent of the total teratogenic incidents. Among the factors considered 'environmental' are infections (rubella, herpes viruses, syphilis, etc.) 2-3 per cent, maternal metabolic imbalances (diabetes, virilizing tumors, etc.) 1-2 per cent, ionizing radiation 1 per cent, and drugs and environmental chemicals 4-6 per cent. A number of insecticides have been tested (as will be discussed later) and, on the whole, relatively few are teratogenic in laboratory animals and none have been shown to be so in man. However, it should be noted that there is evidence from animal studies that multiple environmental factors at low dosage may be additive or, indeed, be potentiating in terms of embryo-toxicity (Wilson, 1977; Fraser, 1977). 1.3. RELATIONSHIP OF TERATOGENESIS AND CARCINOGENESIS

That there is a link between congenital malformations and cancer has been known for some time. Down's syndrome and ataxia telangiectasia are examples of such a relationship. However, no such link has been discovered thus far in regard to environmental factors as causative agents (Miller, 1977). There are a number of chemicals and viruses which are known teratogens but thus far no carcinogenic activity has been demonstrated in man. These include busulfan, methotrexate, thalidomide and organic mercury (Shepard, 1973) and rubella, herpes virus hominis 1 and 2 and Venezuelan equine encephalitis (Sever, 1975). On the other hand, in a compilation by Miller (1977) in which there were thirty-one chemical carcinogens listed as active in humans, a significant number of compounds could not be, or were not, tested by the Ames test (McCann et al., 1975). Of the twelve tested, ten were found to be mutagenic, one 'weakly' mutagenic and one produced no effect. The correlation of the Ames test in animals is much closer than that in man, mainly for the reason that the methods are not adaptable in some instances to the known human carcinogen (i.e. asbestos and wood dust). 2. CHLORINATED HYDROCARBONS 2.1. DDT Fitzhugh and Nelson demonstrated in 1947 that DDT was associated with an increased incidence of hepatic cell tumors as well as what was then described as nodular adenomatoid hyperplasia. Their untreated controls had an incidence of liver tumors of 1 per cent. Very few present day studies using rats have that high an incidence in controls. Be that as it may, in the numerous carcinogenesis studies performed since, and mainly using mice, little has been added to our knowledge as to the possible carcinogenicity of DDT, either in rodents or in man. Many new questions have arisen in these studies, few of which have been answered, and a number not even addressed. Several experiments were performed with different mouse strains and also multigenerational studies. In general, strains of mice which normally develop liver tumors had an increased incidence with ingestion of DDT (Innes et al., 1969; Tomatis et al., 1972). The method of administration was unorthodox in one experiment; DDT was given by gavage for a 28 day period following which it was given in the diet (Innes et al., 1969). In nearly all the remaining studies, the route was by w a y of the diet. In one study, the total incidence of all tumors was increased by DDT (Tarj~in and Kem6ny, 1969). In another, lymphomas were increased in one strain of females but not in another, and not in the males of either strain (Innes et ai., 1969). Tomatis et al. (1972) showed in their studies that only liver tumors were increased but

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DDT had no effect in the incidence of a variety of other tumors that occur naturally in the strain used. In a multigenerational study (six consecutive generations given DDT), the tumor increase affected only those in the liver, and not in any of the other organs. However, the incidence of tumors in the liver, lungs and hematopoietic tissuesvaried considerably from one generation to the next. There was no progressive increase of liver ~umor incidence from generation to generation either in the controls or DDT treated animals. Typically, the average liver tumor incidence for six generations of male controls was 30 and 51 per cent, 50, 56 and 86 per cent in the mice treated with 2, 10, 50 and 250 ppm of DDT, respectively. Interestingly, among a number of tumors which occurred in treated animals that were not found in controls, were a few brain tumors. This may be of some significance in view of the fact that DDT affects the central nervous system as an acute (and chronic) toxic effect, producing dizziness and convulsions. Perhaps, if this finding was not lost in a multitude of other tumors these animals developed, it might have been viewed more seriously (Turusov et al., 1973). A carcinogenic study on hamsters (Cabral and Shubik, 1977) did not indicate any carcinogenic activity following a lifetime exposure to DDT. Syrian golden hamsters were given 125, 250 and 500 ppm in the diet and no differences were noted between the treated and control groups in terms of increasing the percentage of tumor bearing animals. Laws et al. (1967, 1973) studied a group of workers on two occasions some 6 years apart. In the first study (1967) thirty-five men had been occupationally exposed to DDT at levels that varied from 3.6 to 18 mg daily. This was 90 to 450 times the exposure of the general population. While no evidence of liver damage was found, specific tests for liver function were not performed. However, in 1973, thirty-one men of the original group were re-examined specifically in terms of liver function. At this time, the exposure time to DDT averaged 21 years. No evidence of liver disease was detected (Laws et al., 1973). Poland (1970) studied this same pool of workers to determine the effect of exposure on phenylbutazone and cortisol metabolism. Eighteen males had prolonged exposure (average duration of employment 14.4- 1.2 years); they had normal liver and renal function. Their levels of serum concentration and fat stores of DDT-related substances were 20-30 times those in the control population. The urinary excretion of 6/]-hydroxycortisol was increased 57 per cent, and the serum phenylbutazone half-life was reduced by 19 per cent. These effects were considered modest ones. It is not known if, in the general population, there is sufficient DDT storage to stimulate phenylbutazone metabolism or urinary excretion of 6~-hydroxycortisol. It was felt that if any induction at all occurred, it would be a minimal one. Other studies using volunteers likewise showed no evidence of liver damage. In one study, ten volunteers ingested 3.5 or 35 mg/man/day for 12 months; in another study twenty volunteers received the same dose levels for over 21 months and were followed for 5 years. Various liver function tests remained normal. It should be noted that the high dose of 35 mR represents a level of exposure which is about 555 times the general population. Other studies (factory workers) revealed similar negative findings (Ortelee, 1958). A Taiwanese family ingested varying amounts of DDT following contamination of pork dumplings. Eight of eleven members showed signs of toxication which appeared 2-6 hr after ingestion of 5.1-120.5 mg/kg of DDT. The symptoms consisted of excessive perspiration, nausea, vomiting, convulsions, headache, increased salivation, tremors, accelerated heartbeat and cyanosis of the lips. The highest dose (estimated to be 120.5 mg/kg) was in a 2 year old girl; she showed the toxic symptoms within 2 hr and had the more serious symptoms (including tremors and convulsions). All recovered within 2 days. Unfortunately, no biochemical or similar analyses could be performed (Hsieh, 1954). In none of the human studies were there ever any evidence of liver necrosis, although in early studies of the acute effects of DDT in animals it produced liver necrosis (Lillie and Smith, 1944; Cameron and Burgess, 1945). In terms of mutagenic activity, early studies with DDT showed chromosomal

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damage in mice (Johnson and Jalal, 1973) and in cultured rat kangaroo cell line (Palmer et al., 1970). However, in a system using SV 40 transformed human cells (VA-4), DDT failed to show unscheduled DNA synthesis with or without metabolic activation (Ahmed et al., 1977). Mahr ancl Miltenburger (1976) studied the relative toxicity of DDT and metabolites on Chinese hamster cell cultures. They found that the toxicity range from weak to strong was DDA, DDE, DDT and DDD. In one experiment, cultures were treated for 3 months at a dose of 8 ppm of DDT. The chromosome abberation rate was within the spontaneous range with this dose and it did not change the proliferation characteristics nor the sensitivity to acute higher dose. Of importance is the fact that the close used is a mean value reported for human fatty tissue accumulation (5-10 ppm). Larsen and Jalal (1974) examined the karyotypes of bone marrow cells of brown and Balb/c strain albino mice at 48 hr after exposure to 25, 50, 100 and 250ppm (mg/kg body weight). They found only deletions and gaps plus deletions to be significantly increased by DDT treatment at 50-100ppm. The dosages usecl are purported to be within the range found naturally in some secondary consumers (Larsen and Jalal, 1974). Higher frequencies of aberrations have been reported (Johnson and Jalal, 1973) but the duration of exposure was 3 weeks. Marshall et al. (1976), using the Ames system, tested the following insecticides: DDT, dieldrin, heptachlor, diazinon, carbaryl and limuron, as well as metabolites of DDT and heptachlor, DDE and heptachlor epoxide, respectively. These all proved to be negative in the system. Also tested at the same time were captan (a fungicide) ancl nitrosocarbaryl, a compound which potentially could form if carbaryl and nitrites, the latter being added in the form of food additives, were present simultaneously in the stomach. Nitrosocarbaryl was shown to be a potent base-pair substitution mutagen and also produced milcl frameshift activity. Captan also showed both types of mutagenic activity. These two compounds theoretically could be expected to interact with DNA. The negative compounds tested have no known 'groups' which could interact with DNA. A dominant lethal study of DDT in rats was performed by Palmer et al. (1973) and their conclusion was that the compound is only marginally positive to such a test. The particular test group was one in which the animals were mated at week 3 (post-meiotic stage of spermatogenesis) following oral administration with 100 mg/kg of DDT. This was the highest of three dose levels used, 25, 50 or 100 mg/kg. How this dose level was chosen is not indicated. Interestingly, other experiments included groups given 20, 40 or 80 mg/kg each day intraperitoneally for five consecutive days, and these showed no significant effects. Further, no dose-related responses to DDT were detected in any of the groups. Dominant lethal assays were performed in inbred Swiss Albino mice at two dosage levels: 2 x 150 mg DDT/kg over 2 days, and 2 x 100mg DDT/kg body weight for l0 weeks. Both doses produced dominant lethal mutations, the acute dose more so that the chronic. Clark (1974) suggests that the acute dose fails to induce the aryl hydrocarbon hydroxylase sufficiently to metabolize the DDT. The chronic dose, on the other hand, would continuously induce the system which, in turn, would metabolize some of the free DDT before it could induce dominant lethality. The results of these and other experiments led the author to conclude that DDT is a weak mutagen (Clark, 1974). DDT has been shown to prevent embryotoxic or teratogenic effects of several compounds (sodium salicylate, Benlate and chloridine) in rats when DDT was given during the first 10 or 12 days of pregnancy. The authors suggest that DDT by activation of liver microsomal enzymes accelerates the detoxification of teratogens (Shtenberg and Torchinskii, 1976). On the other hand, DDT has been shown to prolong the reproductive life in rats. Animals were given 200 ppm in the diet from the time of weaning and studied until eleven breedings had transpired. The average reproductive life-span was about 14 months in the treated rats as compared to 9 months in the controls. This occurred

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without effecting food consumption, body weight, litter size or viability (Ottoboni, 1972). 2.2. METHOXYCHLOR

Only a few carcinogenic studies have been performed and these have been either negative or u'ninterpretable because of inadequate reporting (see IARC Monograph Vol. 5, 1974, p. 193). Following the reports (Mazuch et al., 1975) that methoxychlor contained three impurities with polycyclic aromatic molecules, mutagenicity tests were performed by the method of Ames (1976). The impetus for the testing related to: (a) the known carcinogenicity of many polycyclic hydrocarbons; (b) the enhancing effect of methoxy substituents, the high reactivity of the ethylene; and (c) the widespread use of methoxychlor. Surprisingly, only one (3,6,11,14-tetramethoxydibenzo(g,p)chrysene) of the three compounds was found to be mutagenic. The others were negative; they were tetrakis (p-methoxyphenyl)ethylene, a reactive compound with marked change transfer activity, and 3,6-dimethoxy-9,10-bis (p-methoxyphenyl)-phenanthrene (Grant et al., 1976). 2.3. ALDRIN/DIELDRIN

?ddrin and dieldrin are organochloride insecticides formerly widely used mainly for soil insects on corn crops and, to a lesser extent, for termite control and mothproofing of woollen clothes and carpets. Aldrin is readily epoxidized to dieldrin, not only in plants and animals, but in the soil as well. However, the degradation of dieldrin is slow and, as a result, it has contaminated food, air and water in many areas of the world, but the amounts present are small. Because of the rapid metabolic transformation of aldrin to dieldrin, it is convenient to speak only of dieldrin in terms of toxicity, carcinogenicity and mutagenicity. Among a number of effects shared with other chlorinated hydrocarbon insecticides such as DDT is the acute effect on the liver. Both DDT and dieldrin cause an enlargements of hepatocytes, particularly in the central zones (Kimbrough et al., 1971; Ortega et al., 1957). In a series of carcinogenic studies following oral administration in mice, an increase in the number of liver tumors over controls was demonstrated by Thorpe and Walker (1973) and Walker et al. (1973). The tumors were both benign and malignant hepatomas and some metastasized to the lungs. In studies with primates (Zavon, 1970) and dogs (Walker et al., 1969), tumorogenicity was not demonstrated. In one study with rats (Walker et al., 1969), three animals given 10 ppm had microscopic nodules in the liver but similar nodules were present in a control rat. In a re-analysis of that study (Stevenson et al., 1976), the overall tumor incidence was studied (doses were 0.1, 1.0 and 10 ppm, over a 2 year period). The conclusions were that there was no treatment related increase in tumor incidence. The Carcinogenesis Program, Division of Cancer Cause and Prevention, National Cancer Institute, undertook a study of aldrin and dieldrin. Aldrin and dieldrin were tested in Osborne-Mendel rats and B6C3F1 mice and, in addition, dieldrin was tested in Fischer 344 rats. Alrin was given at a dose of 30 or 60 ppm to rats and among the tumors observed were those in the thyroid and adrenal. Both thyroid adenomas and carcinomas were seen in significant numbers in the low dose group when compared to the pooled controls. Matched control comparison, however, indicated no significant difference, Cortical adrenal adenoma was also higher in the low dose group but not in the high dose group. Again, control comparisons resulted in the conclusion that any association of adrenal tumors' with treatment was questionable. Only with mice who received 4 or 6 ppm were significant numbers of hepatocellular carcinomas seen. In male mice there was a significant dose-related increase in the incidence. These are mice who have a naturally high incidence of these tumors (seventeen of ninety-two animals in the pooled controls). It was concluded that this was the only experiment to

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show a dose-related increase in tumors. N o n e of the tumors in O s b o r n e - M e n d e l rats treated with aldrin or dieldrin could be associated with treatment. In the Fischer 344 rats (given, 2 10 or 50 ppm of dieldrin), many different tumors were found in control and treated animals. N o n e were related to treatment (Federal Register, 1978). Early studies with the cyclodiene pesticides revealed effects on animal reproduction. There were alterations in the estrus cycle in female rats with aldrin (Ball et al., 1953); in beagle dogs with aldrin (Deichmann et al., 1971); in mice with dieldrin (Guthrie et al., 1971); reduction in the number of pregnancies in rats given dieldrin (Treon and Cleveland, 1955) as well as in dogs (Deichmann et al., 1971). A review of other studies may be found in Ottolenghi et al. (1973). Teratogenic effects were first noted b y Ottolenghi et al. (1973) in studies with mice and hamsters. It should be noted, however, that doses used were one-half the LDs0 given as single oral doses in corn oil using aldrin, dieldrin and endrin. The anomalies included cleft palate, open eye and webbed feet. In addition, there were fetal deaths and growth retardation. Although the doses did not produce overt toxic effects (convulsions, respiratory difficulty, nosebleed and diarrhea), there is no indication how, if at all, the dams were affected, particularly in terms of weight. Later studies with dieldrin concerned the effects on reproduction in mice (Virgo and Bellward, 1975). Dams given 20 or 25 ppm in the diet had a mortality rate of up to 89 per cent. At 10 to 15 ppm, 18 per cent failed to b e c o m e pregnant. Other effects included a decrease in litter size and an increased loss of pups. At levels which caused hepatomegaly in the dam, there was an inability to rear the pups. Litter loss was often related to neglect by the mother. Bidwell et al. (1975) tested dieldrin at concentrations of 0.08, 0.8 and 8.0 mg/kg for dominant lethal effects as well as heritable translocation tests in mice; all were negative. Using salmonella tester strains, no increased mutants were found. Other mutagenic studies were performed b y Dean et al. (1975). In C F , male mice, oral doses of 12.5, 25 or 50 mg of H E O D / k g were used ( H E O D is 1,2,3,4,10, 10 - hexachloro - 6,7 - e p o x y - 1,4,4a,5,6,7,8,8a - octahydro - e x o - 1,4 - e n d o - 5,8 - dimethanonaphthalene, the major constituent of dieldrin). Chinese hamsters received single oral doses of 30 or 60 mg H E O D / k g . There was no evidence of dominant lethal effects in mice. In the hamsters, no evidence of c h r o m o s o m e breakage was found in femoral bone marrow. Other tests with mice showed no gene conversion. These same investigations performed short term lymphocyte cultures from workers in a dieldrin manufacturing plant. While c h r o m o s o m e damage was observed, it did not differ significantly from the control group. These studies confirmed the findings of Epstein et al. (1972), who found no dominant lethal mutations in male mice after single i.p. injections or repeated oral doses of dieldrin. It was concluded b y Dean et al. (1975) that dieldrin does not present a mutagenic hazard. In a survey of the toxicity of dieldrin in man, mainly in spray workers (Hayes, 1959), the acute toxicity related to central nervous system effects, including headache, weakness, loss of consciousness and convulsions. These changes are reversible. There was evidence of liver toxicity. Clastogenic effects with aldrin were observed in a narrow range of dosage (19.125 and 38.35 ~g/ml) using human peripheral blood cultures in vitro. These doses were near the limit for cell survival and the author felt that the observed c h r o m o s o m e lesions were probably not perpetuated in other abnormal ceils. On the other hand, the minimal dose which produced chromosomal aberrations was 9.56~g/g. The test system was bone marrow in mice and rats 24 hr following i.p. injection (Georgian, 1975). 2.4. KEPONE Chlordecone is the c o m m o n chemical name f o r a chlorinated insecticide better k n o w n under the trade name of K e p o n e ®. It became more familiar generally in 1975 when workers in a manufacturing plant became seriously ill necessitating closure of

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the factory. Shortly thereafter, sales were ordered stopped by the US Environmental Protection Agency as well as the use and the manufacture of Kepone. One of the earlier uses of Kepone was for the eradication of fire ants, Mirex, a structurally similar compound, subsequently replaced Kepone in the control of fire ants. Since then, it was found that Mirex can chemically break down into Kepone in amounts of 5-10 per cent (Carlson et al., 1976; Holden, 1976). Needless to say, Mirex is severely limited in its use. Among the early effects noted in animals (mice) was an adverse effect on reproduction (Good et al., 1965), and reduced hatchability in chickens at relatively high doses (Naber and Ware, 1965). McFarland and Lacy (1969) reported testicular atrophy in quail, later confirmed by Eroschenko (1978). The latter indicated, in addition, that the lesion is reversible in some animals. A bioassay sponsored by the Carcinogen Bioassay and Program Resources Branch of the National Cancer Institute revealed carcinogenic activity of Kepone. In this study, Osborne-Mendel rats and B6C3F1 mice were used. The time-weighted average dose for rats was 18, 24 and 26 ppm; mice received 20, 23 and 40 ppm. The compound was incorporated in the food and given for 80 weeks. Rats were killed 112 weeks after the start of the study and mice at 90 weeks. In the high dose group, three of forty-four (7 per cent) male and ten of forty-five (22 per cent) female rats developed hepatocelhilar carcinomas. Liver cancer did not develop in the controls. Some treated rats also had neoplastic nodules as did one control animal. In mice, the high dose group had an 88 and a 47 per cent incidence of hepatocellular carcinoma in male and female animals. The controls had a 16 and 0 per cent incidence, respectively. The low dose mice had an incidence which was quite similar to the high dose animals, 81 per cent males and 52 per cent females (National Cancer Institute, 1976, reported in Clinical Toxicology 1976). In a study of pregnant Sprague-Dawley rats, 1, 2 or 4 mg/kg/day of Kepone was given by gastric intubation beginning on day 2 until weaning. The control group and those receiving 1 mg/kg had normal pups. Stillbirths and abortions occurred at the other dose levels. Brain electrical activity and visual evoked responses were different in the treated animals as compared to the controls when examined at 24 days of age. This was considered to be a strong indication of central nervous system impairment. However, no pathological studies were mentioned (Rosenstein et al., 1977). A group of workers at a Kepone manufacturing plant were exposed for months to the insecticide and developed nervous system changes as well as liver and testicular damage. Although liver tests were normal, the livers were enlarged and showed non-specific changes by light and electron microscopy. Studies by the National Cancer Institute in 1976 showed that Kepone produced hepatocellular carcinoma in rats. It is likely that this was known or suspected long before those experiments. Thus far, there have been no instances of hepatocellular carcinoma reported in these workers, nor that chronic liver damage or dysfunction has occurred (Cohn et al., 1978; Huff and Gerstner, 1977; Taylor et al., J978. 2.5. CHLORDANE AND HEPTACHLOR

These two compounds have been severely restricted in usage in recent years• Chlordane is now used mainly for termite control. A number of carcinogenic studies have been performed with these two compounds and virtually none of them has been pubIished in the conventional manner. Epstein (1976) has selectively presented data accumulated from government documents, industry reports, review panels, and testimony from court proceedings. Chlordane apparently increases the incidence of, or produces hepatocellular carcinoma in rodents. In a dominant lethal study, groups of eight Charles River CD-1 random bred albino male mice that received 50 or 100 mg/kg of technical chlordane in single doses, either by gavage or i.p., were mated with three untreated females for 6 consecutive weeks. No increase in the frequency of dominant lethal changes were observed among the

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males; there was no increase in early deaths in utero among females. Other studies using HC-3260 (an experimental product containing a mixture of chlordane isomers) at the same dose levels, and heptachlor :heptachlor epoxide at 7.5 and 15 mg/kg also did not produce dominant lethal changes (Arnold et ai., 1977). 2.6. MIREX Mirex is an analog of Kepone and used principally in controlling fire ants. It is no longer permitted for that purpose. In a preliminary report in mice, an increased incidence of 'hepatomas' was noted (Innes et al., 1969). A long-term study in Charles River CD rats was performed by Ulland et al. (1977) using two dose levels, 50 and 100ppm in the diet. Hepatocellular carcinomas and neoplastic nodules were observed. No such tumors were seen in the control rats. Khera et al. (1976) studied the teratogenicity and dominant lethal effect of Mirex in rats. Doses of 1.5, 3.0, 6.0 and 12 mg/kg of Mirex were given orally on days 6 to 15 of gestation. Only the lowest dose was non-toxic to the mated rats. The other doses caused death and a reduction in the incidence of pregnancy, and both these effects were dose-related. The highest dose produced an increased incidence of deciduomas and fetal deaths, and a decrease in fetal weight. A significant increase of visceral anomalies occurred in the 6 and 12.5 mg/kg groups but no skeletal anomalies different from the controls occurred in any group. Since the teratogenic effects occurred only at doses which were toxic to the mothers, the significance of the finding is questionable. In the dominant lethal study, 6 mglkg was the highest dose used and this caused a consistent reduction in body weight gain during the 10 day dosing period. One male (of twenty in the group) died. The number of viable embryos, deciduomas and pregnancies in the three test groups (1.3, 3.0 or 6.0 mg/kg) were within the control range. A study of the oral toxicity and effects on reproduction was performed in rats (Gaines and Kimbrough, 1970). Particularly noteworthy were the liver changes; in addition to the cytomegaly of the hepatocytes and fat in the cytoplasm, cholestasis was noted. This last change is unusual and likely indicates significantly more damage than the cytomegaly. The enlargement of the hepatocytes are characteristics changes of the chlorinated hydrocarbons in rodents and have been reported with DDT and dieldrin. These compounds do not cause cholestasis. 2.7. LINDANE BHC is the common name for a mixture of isomers of 1,2,3,4,5,6,-hexachlorocyclohexane. BHC is an insecticide which has been in use for many years both on crops and biting insects, as well as for insects which attack buildings. In 1944, it was found that the gamma isomer was the only one which had insecticidal activity and this is known as lindane. Although it is an organochlorine insecticide, it is less of a problem than other organochlorine compounds because it is less persistent. There are various reasons for this, among the more important ones is that lesser amounts are needed for effective controls, it has a higher solubility in water, and it disappears more rapidly from soil (IARC, Monograph, Vol. 5, 1974). In common with many other organochlorine compounds, BHC produces liver tumors in mice. Nagasaki et al. (1972a,b) showed this in dd mice which normally have a very low spontaneous liver tumor incidence. In another study with CF1 mice, a strain in which the control mice had a liver tumor incidence of 24 per cent, those mice given 200 ppm of the beta isomer and 400 ppm of the alpha isomer had a liver tumor incidence of 73 and 93 per cent, respectively. Both treated groups also had lung metastases O'horpe and Walker, 1973). In earlier experiments with rats, there apparently were no differences between the control and treated animals (Fitzhugh et al., 1950; Truhaut, 1954). Several metabolites-(trichl0robenzene and trichlorophenol compounds) fed mice failed to show tumorigenic activity in concurrent experiments where several of the BHC isomers were found to be active.

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There was a report indicating that eight workers exposed to DDT or BHC or both developed cirrhosis and chronic hepatitis (Schfittmann, 1968). There have been no other reports in which this has been confirmed. Other reports indicated that BHC and lindane exposure, as well as exposure to BHC and DDT, resulted in the development of aplastic anemia (Loge, 1965; West, 1967; Woodliff et al., 1966). There were also two cases of leukemia reported in cousins exposed to lindane (Jedli6ka et al., 1958). These reports of hematopoietic toxicity in man do not necessarily indicate a causal relationship with the insecticides; the hematologic disorders may have been coincidental. CD strain rats were given 20, 50 and 100 ppm of lindane in the diet in a three generation study. There were no adverse effects upon reproductive function and no major malformations occurred. Selected rats given 50 or 100ppm had enlarged hepatocytes and vacuolated cytoplasm (Palmer et al., 1978b). New Zealand White rabbits and CFY rats were given 5, 10 and 15 mg/kg of lindane during the 6-18 day or the 6-16 day of pregnancy, respectively. There was no teratogenic effect or selective embryo toxicity. The dose was such that there was reduced weight gain in the treated mothers (Palmer et al., 1978a). In a 2 year feeding experiment, Beagle dogs of both sexes were given 25, 50 and 100ppm for 104 weeks and 200ppm for 32 weeks. The only noteworthy effect occurred in dogs given 100 and 200 ppm and this was enlargement of the liver. This. however, could not be correlated with any histopathological changes (Rivett et al. 1978). In a carcinogenicity study in mice, doses of 12.5, 25 or 50ppm (the last is about 8 mg/kg) did not have a tumorigenic effect. Further, electron microscopic studies did not show any changes in the liver. The mice used were Chbi NMR1 (SPF) and were kept for 80 weeks. Five of the control animals had liver 'adenomas'. Of note is the fact that only four of the treated mice in two of the three groups had 'adenomas' (Weisse and Herbst, 1977). Lindane (as BHC) was one of 193 pesticides tested for microbial mutagenicity by Shirasu et al. (1976). Only fifteen proved to be mutagenic and lindane was not among them. There were only two insecticides which were shown to be mutagenic in this series of tests, dichlorvos and vamidothion [dimethyl S-(2-[1-methylcarbamoylethylthio]ryhyl) phosphorothiolate]. The other fifty-eight insecticides were negative and included aldrin, carbaryl, DDT, diazinon, dieldrin, heptachlor and malathion (Shirasu et al., 1976, 1977; Kada et al., 1974). 2.8. TOXAPHENE Toxaphene is a chlorinated hydrocarbon containing a mixture of compounds and isomers. It is a long-lived agent and persists in soil and water as with other chlorinated hydrocarbons. Early toxicity studies revealed fatty liver changes in rats (Ortega et al., 1957). A three generation study on reproduction in rats given 25 and 100 ppm did not reveal effects on litter size, viability or both weight. Toxaphene given to mice and rats during gestation failed to produce effects at doses which were not toxic to the mothers. When maternal toxicity occurred, rat fetuses had skeletal abnormalities and in mice central nervous system changes were seen (Chernoff and Carver, 1976). 3. O R G A N O P H O S P H O R U S COMPOUNDS Lymphocyte cultures were examined in a series of patients exposed to a variety of organophosphate insecticides (malathion, trichlorfon, methyl parathion, mevinphos, dimethoate, diazinon and dichlorvos). These were people who attempted suicide with the agents or were overexposed accidentally during work. In these acutely intoxicated people, a temporary increase was noted in the frequency of chromatid breaks and chromosome aberrations. The authors correctly point out other factors must be ruled out and these include interactions by life-saving drugs and the possible effect of the solvents and vehicles used. From a practical point of view, the occurrence of acute intoxication is rare and a relatively minor problem. However, whether prolonged

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exposure at small doses produces a genetic effect is another matter, especially in view of the frequency of chromosome aberrations in instances of so-called mild intoxication. This, however, would concern, agricultural and factory workers; the organic phosphoric acid esters break down rapidly and residues in the general population'are insignificant (van Bao et al., 1974). 3.1. DICHLORVOS

Dichlorvos is one of the organophosphorus insecticides widely used in the form of plastic strips (Vapona) wherein advantage is taken of the volatility of the chemical to kill flying insects such as flies and mosquitoes. A 2 year inhalation study of dichlorvos was performed using rats exposed to nominal concentrations of 0, 0.05, 0.5 and 5.0 mg/m 3. (The FAO/WHO Joint Meetings on pesticide residues recommended an acceptable daily intake of 0.004mg/kg/day in man). In none of the groups were there any signs of 0rganophosphorus compound toxicity. This, despite the fact that additional intake of the compound occurred from other routes such as diet and drinking water. Further, percutaneous absorption occurred as well as absorption from grooming. Rats also have a high respiratory minute volume per kilogram of body weight as compared to man. Damage was not present in any tissue, including the respiratory tract where ultrastructural studies were also performed. Male rats had an increase in glutamic pyruvic transaminase and glutamic oxaloacetic transaminase at the high dose level (5.0 mg/m3), but apparently no pathological changes were present in the liver. There was no increased incidence of tumors (Blair et al., 1976). Other studies in rats and dogs also did not show carcinogenic activity (Witherup et al., 1971). The compound alkylates DNA in vitro but as yet in vivo alkylation has not been unequivocally shown. Long term studies have not revealed carcinogenic activity. However, there is considerable concern over the possibility that dichlorvos may be a mutagen. Its potential as a mutagen has been uncovered in a number of different microorganisms. On the other hand, all mammalian tests have been negative. Sax and Sax (1968) found that onion seeds germinated in air containing 'Vapona' strips had 13 per cent chromosomal aberrations in the germinated root tip cells. Gupta and Singh (1974) found 10 per cent of the cells studied at concentrations of 1 ppm had chromosomal aberrations. They studied the salivary gland chromosomes of fully grown, third instar, larvae of Drosophila. No abnormalities were noted in the controls. This report was of considerable concern because of the high incidence of chromosome aberrations at a dose which is quite low. Kramers and Knaap (1978) felt that dichlorvos should act as a very potent mutagen in Drosophila and produce sex-linked recessive lethals (compounds that induce chromosome breaks in Drosophila germ cells always induce recessive lethals (Vogel and Leigh, 1975)). They attempted to induce sex linked lethals in Drosophila using the same substance and the same route of administration as Gupta and Singh. The tests of Kramers and Knapp failed to demonstrate a transmissable genetic defect of the compound. An assay of dominant lethal mutations in female mice was undertaken by Dean and Blair (1976) using both oral administration and inhalation exposure to dichlorvos. In the oral studies, mice received either 25 or 50 mg/kg and the inhalation group 2 or 8 #g/l. One group (in addition to a control group) received 100mg/kg methyl methanesulphonate. Evaluation included the percentage of females pregnant, the number of fetal implants and the number of early fetal deaths. No significant effects were noted when compared with the corresponding negative control groups. Earlier studies using male mice in single or repeated exposure, by inhalation, intraperitoneal or oral routes, failed to induce dominant lethal mutation (Dean and Thorpe, 1972b; Epstein et al., 1972). Following mutation studies using other mammalian systems have also been negative: host-mediated assay (Buselmaier et ai., 1972; Dean et al., 1972; Voogd et al., 1972) and chromosome studies in rodents (Dean and Thorpe, 1972a,b). In regard to the host-mediated assay performed by Voogd, it was noted that only a small

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quantity (0.2 mg) could be applied to each animal; higher levels could not be tested because of its relatively high toxicity. In vitro studies of dichlorvos have revealed the alkylating properties (Bedford and Robinson, 1972; Lofroth, 1970). Mutagenic effects have been demonstrated in microorganisms: Escherichia coil (Ashwood-Smith et al., 1972; Bridges et al., 1973), Serratia m a r c e c e n s (Buselmaier et al., 1972; Dean, 1972) and S a c c h a r o m y c e s cerevisiae (Dean et al., 1972; Fahrig, 1973). 3.2. MALATHIONAND PARATHION

These compounds and similar related compounds are in part replacing the chlorinated hydrocarbons. Organophorus chemicals are degraded relatively quickly and do not present the problem of persistence as do the chlorinated hydrocarbons. They do, however, have an acute toxicity that may be fatal. Acute fatalities from DDT and other chlorinated hydrocarbons are extremely rare and some deaths may have been due in fact to the solvents. Malathion and other closely related compounds are teratogenic (for a review of toxicity and teratogenicity, see Fishbein, 1975). There have been no studies which indicate these compounds are carcinogenic. A more recent study of pregnant rats was made by Talens and Woolley (1973) wherein animals were given 1.5 and 2 mg/kg of parathion for 4 days, for 1, 7 or 13th day of gestation. Those treated in the last trimester had the most severe symptoms and several of the rats given the higher dose died. There were nine pup deaths in the treated groups and only two in the control. However, six of the nine deaths involved a litter of six. One would like a larger series to attach significance to this finding. Certain other effects on pups (body growth and righting reflexes) may have been a reflection of maternal toxicity. 3.3. TRICHLORFON(CHLoROPHOS, DIPTEREX), PHTHALOPHOS(IMIDAN), CRUFOMATE (RUELENE)

Wistar rats were given 80 mg/kg of chlorophos orally on day 9 or 13 of gestation. This dose was 1/10 of the LD50.Those treated at 9 days showed no significant increase in embryonic deaths. However, the rats treated at 13 days had fewer normal fetuses than the controls and the number of deaths increased. The dose of 90 mg/kg is calculated to be 2.5 x 103 times the average amount entering the human in a 24 hr period. Experiments with 8 mg/kg revealed no embryotoxic or teratogenic effects (250 times the estimated human dose) (Martson and Voronina, 1976). Experiments similar to those performed with chlorophos were done with phthalophos at dose that varied from 15 mg/kg (l]10kds0) to 0.06mg/kg. However, the only dose without embryotoxicity was the lowest one used (Martson and Voronina, 1976). Ruelene (4-tert-butyl-2-chlorophenyl methyl methyl-phosphoramidate) is a systemic organophosphorus pesticide used for grub control in cattle. A teratogenic study in pregnant cows (injected at 35 days of gestation) revealed no fetal abnormalities at doses of 8.8 g into the jugular vein plus 0.25 g in the amniotic sac. Other experiments indicated that Ruelene (or its metabolites or both) is transferred across the placental barrier. The fetus is believed to be protected by virtue of the rapid metabolism and excretion of the injected dose by the dam (Rumsey et al., 1974). 4. OTHER AGENTS 4.1. CARBARYL Carbaryl is a broad spectrum insecticide as well as an acaracide and molluscicide. Carbaryl can be nitrosated under conditions simulating those of the human stomach, but in relatively low yields. The product N-nitrosocarbaryl was given orally to thirty-one male Sprague-Dawley rats in high doses and nine developed squamous carcinomas of the forestomach. No such carcinomas were present in the controls. The

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dose (130 mg/kg) was given twice weekly by gavage in olive oil suspension (Eisenbrand et al., 1976). In experiments with nitroso-N-methylurethane, nitroso-N-ethylurethane, as well as nitrosocarbaryl, administration by gavage resulted in a high incidence of forestomach carcinoma in Sprague-Dawley rats with all compounds tested (Lijinsky and Taylor, 1976). The total dose given was 32 mg for nitroethylurethane, 29 mg for nitrosomethylurethane, and two dose levels of 50 and 300 mg for nitrosocarbaryl. Unfortunately, in these experiments, there is no indication that the controls were gavaged in a similar manner with olive oil alone and the findings in the control animals are not given. Carbaryl was administered by gavage to pregnant female rats (300 mg during a 10 day period) with and without a 4 per cent sodium nitrite solution. The authors conclude that the carbaryl alone did not induce malignant tumors, either in the dams or offspring, and that the carbaryl and sodium nitrite together resulted in no significant incidence of malignant tumors. However, there was no control group and the authors are probably relying upon historical controls (the report is listed as a preliminary communication). On the other hand, they do indicate that hepatocellular carcinomas were seen. Such cancers are ordinarily considered rare in control Sprague-Dawley rats (Lijinsky and Taylor, 1977). The difficulty in the interpretation of carcinogenic studies is exemplified by those experiments reported with carbaryl. Shimkin et al. (1969) noted six of fifteen mice to have lung tumors following administration of carbaryl in tricaprylin. Seven of twentyeight mice given tricaprylin alone developed lung tumors. Two of twenty-three untreated controls had lung tumors. This experiment, at most, should lead to the conclusion that further studies be performed. However, in an abstract summary of another article on the degradation of carbaryl, the abstractor refers to the 'tumorigenicity' of carbaryl based on this mouse experiment. The IARC, on the other hand, analyzing additional carcinogenic studies, concluded that 'the available data did not allow an evaluation of the carcinogenicity of carbaxyl to be made' (IARC Monograph, Vol. 12, p. 47, 1976). The IARC, unfortunately, considered that N-nitrosocarbaryl had been shown to be carcinogenic in rats in an experiment in which there also were no control animals (lbid, p. 47.) In experiments with carbaryl, beagle dogs (Smalley et al., 1968) and guinea pigs (Robens, 1969), no teratogenic effects were observed. In rats, Weil et al. (1972) observed no effects on reproduction but did find a diminution in fertility. Well et al. (1972) made a justifiable plea for uniformity in testing, pointing out that studies in rats which contradicted his, reflected another means of testing. Weft dosed his rats using carbaryl in the diet; other experimenters gave the entire dose at one time via gastric intubation unaccompanied by any food and producing a much higher and acute blood level of the compound. Needless to say, the administration via the diet would appear to be a more appropriate method. Nitroso carbamates (NO-carbamates) have been shown to irreversibly damage DNA (Regan et al., 1976). Five methyl carbamates (carbaryl, baygon, BUX-Ten, landrin and methomyl) were tested with a number of histidine auxotrophs of Salmonella typhyimurium LT 12 and they did not cause a significant increase in the number of revertant colonies. The nitroso derivatives, however, greatly increased the number of revertant colonies in two of the strains used. It is suggested that the NO-carbamates act as alkylating agents (as do other nitroso compounds). The Salmonella test failed to detect mutage/dcact~v~ty of carbaryl. However, using Drosophila melanogaster, weak mutagenic activity was demonstrated (Blevins et al., 1977). The addition of rat liver microsomal suspensions which enzymatically activates many mutagens did nothing to increase the mutagenicity of carbaryl using the Salmonella tester strains. 4.2. PIPERONYL BUTOXIDE Piperonyl butoxide is an insecticide synergist and is used in aerosols, wettable powders, etc. Its toxicity is very low, gram amounts being necessary to produce lethal

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effects in animals. However, liver damage is produced with long-term feeding of 5000 or 10,000ppm in rats. The changes are similar to those noted with chlorinated hydrocarbons and consist of liver enlargement and periportal hepatic cell enlargement (Lehman, 1948, 1952). In teratogenic studies with COBS Charles River Rats, no teratogenic effects occurred at the highest dose level fed which was not toxic to dams (Kennedy et al., 1977). 4.3. PYRETHRUM Pyrethrum is an insecticide prepared from flowers of the genus Chrysanthemum; the most important commercial species is cineruriaefolium (Trevirames) Boccone. Pyrethrum is unusual in that it is of 'natural' origin; indeed, the report of a symposium on this subject was titled Pyrethrum, The Natural Insecticide. The favorable connotation and the implication of non-toxicity of the word natural no doubt was a factor in this choice of a title. As a matter of fact, this insecticide probably is one of the safer ones, if not the most safe. However, some of the toxicity studies have uncovered some disquieting effects which will be discussed later. The material extracted from the pyrethrum flowers is 'pyrethrum extract'. The insecticidal components of this extract are 'the pyrethrins'. The six insecticidal constituents of pyrethrum extract are pyrethrins (! and II), cinerins (I and II) and the jasmolins (I and II) (for details and structural formulae, see Head, 1973). In 1949, a synergist piperonyl butoxide, was added to pyrethrum in part as a cost-reducing substance. Later, another compound with similar functions, N-octyl bicycloheptene dicarboximide, was added. Thus, in any study of pyrethrum these and any other compounds used in the commercial formula must also be examined. Although the pyrethrums literally represent insecticides used since antiquity, relatively few toxicological studies have been performed. Experiments by Bond and DeFeo (1969) revealed the LDs0in rats to be 7.2 gm/kg; (a pyrethrum extract containing 20 per cent pyrethrins was used). In another study, single doses of 450 mg/kg of 9 per cent pyrethrins in peanut oil were given to rats and several died, some with liver vacuolization (Kimbrough et al., 1968). Pyrethrum has been shown to cause liver enlargement similar to that caused by DDT as well as cytological changes. Oral administration of 200 mg/kg of pyrethrum to male rats increased their liver-to-body weight ratios by almost 25 per cent. This was not accounted for by an increase in total lipid nor by protein concentration of homogenates of microsomal fractions. Water levels were unaffected. There was decreased hepatic concentrations of DNA as compared to controls. It was suggested that the pyrethrum interfered with hepatic drug metabolism. After 6 days of pyrethrum administration, a marked decrease in hexabarbitone-induced narcosis was found but no change in the barbitone-induced sleeping time. Confirmation of this came from an increase of activity of several microsomal enzymes responsible for detoxification. Further, that this was not part of a general increase of cellular metabolism was shown by the oxidation of tryptophan which remained the same after 500 mg/kg of pyrethrum orally for 7 days (Springfield et al., 1973). In chronic toxicity studies by Bond and DeFeo (1969), rats were given 380 mg/kg/day of pyrethrins orally for 90 days (this is one-quarter of the acute LDs0 level). Two of twenty animals showed an 'increase in eosinophilia of the cytoplasm of liver, indicating the beginning of cell necrosis'. The same investigators found that when piperonyl butoxide was combined with pyrethrins, less toxicity resulted; this combination also produced the greatest liver enlargement of any of the compounds combined with the pyrethrins including ethyl alcohol, caffeine, sulfoxide and Tropitol. Other toxicity described with pyrethrins include diarrhea and central nervous system changes (tremors and convulsions) (see Barthel, 1973, for specific references). In man, the principal toxic effects relate to allergic and skin reactions. There apparently is no hazard of acute toxicity in humans. In 1973, Barthel (Casida, 1973) stated that 'although much work has been done on the toxicology of pyrethrum and pyrethrins, it is not complete enough to permit registration of the pyrethrins as a new insecticide if it were introduced to the market today'. JPT Vol. 6, No. I - - K

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More recently, a number of pyrethroids have been studied for acute and chronic t o x i c i t y as w e l l as f o r c a r c i n o g e n i c e f f e c t s . T h e c o m p o u n d s s t u d i e d w e r e a l l e t h r i n , fenothrin, furamethrin, permethrin and res'methren (details of the structural formulas a n d r e l a t e d inforn~ ~tion c a n b e f o u n d in M i y a m o t o , 1976). T h e t o x i c e f f e c t s a r e n o t unlike those seen with the natural substances. Hypersensitivity, tremors and motor a t a x i a w e r e o b s e r v e d in a c u t e o r a l t o x i c i t y s t u d i e s . I n c h r o n i c a n d l o n g - t e r m s t u d i e s , l i v e r a b n o r m a l i t i e s w e r e p r e s e n t in rats. T h e y w e r e bile d u c t p r o l i f e r a t i o n a n d o v a l cell infiltration at 12 w e e k s a n d 24 w e e k s ( a l l e t h r i n a n d f u r a m e t h r i n ) , bile d u c t p r o l i f e r a t i o n a t 80 w e e k s (allithrin) a n d h y p e r t r o p h y a n d f a t t y c h a n g e a t 24 w e e k s ( p e r m e t h r i n ) . U n f o r t u n a t e l y , d e t a i l s a r e l a c k i n g a s to t h e n u m b e r o f a n i m a l s a n d t h e findings w i t h s p e c i f i c d o s e s (the r a n g e s g i v e n v a r i e d f r o m 100 to 5000 p p m in s o m e e x p e r i m e n t s a n d 1000 to 1500 p p m in o t h e r s ) . M u t a g e n i c s t u d i e s w e r e a l s o r e p o r t e d b y M i y a m o t o (1976). A t d o s e s o f 10 m g / p l a t e o f p y r e t h r o i d s d i s s o l v e d in d i m e t h y l s u l f o x i d e , n o m u t a g e n l c e f f e c t s w e r e n o t e d (in t e r m s o f r e v e r t a n t s , c o m p a r e d to c o n t r o l s a n d n i t r o s o g u a n i d i n e ) . S i m i l a r l y , in t h e host-mediated assay, no significant number of revertants were detected (Miyamoto, 1976). Acknowledgement--Ms Martha Klapp gave invaluable assistance in the preparation of this manuscript.

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