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Pilot study of the mutagenicity of DDT in mice
PILOT STUDY OF THE MUTAGENICITY OF DDT IN MICE
MARGARET E. WALLACE 8/. P. KNIGHTS*
Department of Genetics, Milton Road, Cambridge CB4 1XH, Great Britain & A.
O. DYE
Department of Land Economy, University of Cambridge, Cambridge, Great Britain A BS TR A C T An inbred strain o f mice, half o f which had been given a diet containing 250 ppm of D D T over five generations, and half o f which were untreated, was examined to disclose whether there was a greater incidence o f mutants in the treated than in the control mice. The stock did not suffer unduly from litter competition, and the breeding programme used provided a fairly sensitive test. There was no evidence for a greater incidence o f recessive invisibles in the treated than in the control halves, but there were two recessive visibles in the treated and none in the control. It is concluded that there is no case for a grossly mutagenic effect o f DDT.
INTRODUCTION
To date, the mutagenicity of D D T has not been rigorously tested. This seems to be a serious omission in view of its known toxic effects, on account of which its use has become much more restricted than it was ten years ago. It is still the chemical of choice for the control of malaria; nonetheless the only tests of mutagenicity done as yet, using a mammal, are those of Epstein & Shafner (1968) and of Clark (1974), using mice. The tests were for dominant lethals, with treatment by injection in the former case and by oral dose in the latter. The recent discovery (Wallace, 1971) of a very high incidence of mutants in a colony of mice derived from wild ones trapped in a heavily DDT-treated area, calls for a new look at the possibility of mutagenicity. * Present address: Pembrokeshire Coast National Park, County Offices, Haverfordwest, Pembrokeshire, Great Britain. 217 Environ. Pollut. (11) (1976)--© Applied Science Publishers Ltd, England, 1976 Printed in Great Britain
218
MARGARET E. WALLACE, P. KNIGHTS, A. O. DYE
This paper presents new evidence. It reports on the incidence of recessive visible and invisible mutants in a colony of mice fed over five generations on a DDTtreated diet. It does not claim to be a rigorous test. It was, in fact, carried out because such a treated colony, and its untreated control, became available to the Genetics Department, and the intention was to make good use of this unusual material.
MATERIALS AND METHODS
Mice--History and treatment Through the International Agency for Research on Cancer, Lyons, France, a stock of DDT-treated and control mice was received. The strain, CF/I, ofgenotype aabbcc (nonagouti, brown, albino), had been sibmated to 20 generations in 1940, and was random bred until transfer to the Agency. There it was divided into a control stock and four treatment stocks, these receiving a diet containing D D T at dose levels of 2, 10, 50 and 250 parts per million (ppm) for their life span for five consecutive generations (Tomatis et al., 1972). The strain was maintained by minimal inbreeding (15-30 pairs per generation per stock), as part of a study of carcinogenesis (IARC, 1970). The sixth generation received treatment through their mothers (in utero and during lactation) until weaning; the seventh and eighth were minimally inbred without treatment. Late in 1970 the Genetics Department received 15 trios of the eighth generation of that treatment line which had received 250 ppm of D D T in their diet over five generations, and 21 trios of the same generation of the control stock. This concentration of DDT was chosen because it produced similar levels of DDT and its derivatives in the fatty tissues of mice fed 250 ppm DDT in their diet (214-600 ppm) as in men under occupational and high exposure (201-600 ppm) (Hayes et al., 1971 ; Tomatis et al., 1971 table 11 ; IARC, 1974). The two stocks were then maintained through one further generation by 48 matings minimally inbred, in order to clear them of a small outbreak of mastitis and infantile diarrhoea; this had developed as soon as they passed from an SPF environment to a conventional one--although no mice in the conventional environment showed overt symptoms. To check that there had been no great genetic divergence between the two stocks during the generations of physical separation, 20 males of the eighth generation of each were typed for biochemical polymorphisms at the following six loci: Gpi-1, Pgm-i, Pgm-2, ld-i, Hb-b and Dip-1 (glucose-6-phosphate dehydrogenase, phosphoglucomutase-I and -2, isocitrate dehydrogenase, haemoglobin beta chain and dipeptidase-l). A\ further five females of each stock were tested for resistance to DDT. The polymorphism test showed the two stocks to be sufficiently similar to preclude doubt about their common origin and genetic equivalence. The resistance test also showed them to be very similar; they were more resistant than most
MUTAGENIC1TY OF DDT IN M1CE
219
common laboratory strains, and the treated ones were insignificantly more resistant than the control. This agrees with the assumption that both were resistant before division and that the period of treatment was ineffectual as a selective force because there was no genetic diversity in this respect. A third check was made. In order to see whether the ninth generation was free of DDT, which is known to pass into milk and across the placental barrier (Research Committee on Toxic Chemicals, 1970), five mice of each sex of each stock were autopsied and assays made of the fatty tissue content of D D T and its derivatives. This showed both stocks to have an unusually low content (treated: 0.10-0.49 ppm, and control: 0.08-0.86 ppm). These later generations were not therefore exposed to D D T via any possible route.
Breeding programmes In the tenth generation, approximately 100 minimally inbred matings and 100 sibmatings were made up of each stock. In the eleventh generation, approximately 70 minimally inbred matings and 100 sibmatings were made up of each stock. These sets of matings and data from them are d e s i g n a t e d E M and PM when minimally inbred, and ES and PS when sibmated; E stands for control and P for treated. In the tenth generation, each mouse in each of the first litters of each stock was examined for visible abnormalities at birth, and at the phenocritical ages of 2, 7, 18 and 28 days; the litter sizes at each age were recorded. Every abnormality was investigated, by several types of mating, to see if it was inherited. In the eleventh generation, the females were sacrificed at 16 days gestation (as judged from the observation of vaginal plugs), and observations made on the numbers of corpora lutea, of implantation sites, of dead or partially absorbed foetuses, and of live young. The latter observations allow the inference of the numbers of fertilised eggs, of pre-imPlantation losses, of post-implantation losses to 16 days, and of live young at 16 days (McLaren & Michie, 1959). The incidence of recessive invisible mutants in the treated mice was compared with that of the control mice as follows. A lower litter size is expected in the sibmatings as compared, with the minimally inbred ones, due to homozygosity of lethals. If the amount by which litter size is reduced is the same in the control as in the treated stock, then there is probably the same level of invisible lethals segregating in both. If reduction in litter size is greater in the treated than in the control matings, then there are more invisibles in the treated stock than in the c o n t r o l - - o r there are a few invisibles more widely spread in the treated stock than in the control. In short, the comparison does not allow an assessment to be made of the quantity of invisibles in either stock; it simply shows whether or not the deathrate due to invisibles is or is not greater in the treated stock than in the control. If it is greater, then there is a case for an experiment specifically to test mutagenicity, such as the specific locus test. "Owing to the homozygosity of the albino gene in both stocks, the examination at
220
MARGARET E. WALLACE, P° KNIGHTS, A. O. DYE
birth and thereafter, while likely to show up such defects as behaviour defects and hair texture ones, would not show up coiour variation. An equal number (42) of females of the minimally inbred sections of each stock were therefore outcrossed to several laboratory stocks (each female to about .three different laboratory males); each of the latter carried a number of colour (and other) mutants. FI were also mated to each other and their progeny examined for coiour variation. Thus both stocks were scanned in general for all known dominant and recessive colour mutants, and specifically for mutants at the following loci: In, py (leaden, polydactyly, chromosome 1), a, un, we, pa (agouti, undulated tail, wellhaarig, pallid, chromosome 2), b (brown, chromosome 4), coh, p (chinchilla, pinkeyed dilution, chromosome 7), d, se (Maltese dilution, short-ear, chromosome 9), s (pied, chromosome 14), bt (belted, chromosome 15), dp and mr (dilute-Peru and maroon, location unknown).
RESULTS
Statistical analysis o f mortafity The analysis was carried out in three phases. First, stepwise linear regression was used on both the antenatal and the postnatal data to determine what proportion of the variation in litter losses was associated with competition due to litter size and what proportions with sibmating and the effect of the treatment. There were a few litters in each of the EM, PM, ES and PS stocks which showed an outstandingly high mortality due to disease; these were removed from the data. Next in the postnatal data, allowance for competition having been made, the total loss per litter up to 28 days, and the losses per litter in each of the four periods into which the 28 days were divided, were used in an analysis of variance to expose any significance in the differences between the relevant subgroupings of the data. Finally, the antenatal data were similarly analysed. The most useful variable for measuring competition was found to be litter size at 28 days. However, even with the high degree ofautocorrelation inevitable between variables, competition was shown to account for only a small proportion of the losses, and it did not mask the effect of the treatment or the breeding regime. For example, in the postnatal data, only 7 ~ of the losses could be accounted for on the basis of a loss rate of 0.25 per litter for every additional member of the litter. In neither the postnatal nor the antenatal data were the crucial differences significant, i.e. it could not be shown that litter loss due to sibmating was greater in the P than in the E data. The only significant comparisons in the postnatal data were within the minimally inbred groups as follows: The total loss per litter was greater in the PM data than in the EM, and losses per litter in the period 19-29 days were greater in the PM than in the EM data. In the antenatal study, similarly, differences were small by comparison with the variation within each group. The
MUTAGENICITY OF DDT IN MICE
221
largest differences were as follows: the corpora lutea count on a per litter basis for the PM group was larger than that in the PS group by 0.10, and the same count for the EM group was larger than for the ES group by 0.38; again the corpora iutea count in the stock (EM + ES) was greater than that in the stock (PM + PS) by 0.90, and in the ES group it was greater than that in the PS group by 0-81. These differences being small compared with the large amount of variation within each group, the effects of the treatment are not significant at the 5 % level--or indeed at the 30 % level. In short, since the effect of competition is so slight, any gross effect of the treatment would have shown itself, and it did not do so. Observations on visible mutants
There were 30 individuals, occurring at random between the sexes and over the four main groups of matings, which showed physical abnormalities. Two mice, one with gross limb malformation and one with no hind legs, occurred in the P stocks; the rest all had small defects, such as bifurcated left or right ear, tail kink or unusually small body size. No abnormality proved to be heritable. Of the 42 females of each stock outcrossed to test for colour and other mutants, three proved to be black-and-tan, ata, rather than nonagouti, aa; all three occurred in the treated (P) stock. From the 170 females in each stock autopsied at 16 days' gestation, a number of exencephalic young were seen. These also occurred only in the treated (P) stock, there being 12 matings segregating in this anomaly, distributed at random between the PM and PS groups. Careful scrutiny of young within an hour of birth, from females closely related to these, disclosed dead ones with the upper part of the head missing; since they occurred in about the same fraction within each litter, it could be concluded that these were also exencephalio and that the condition is inherited (Knights, 1972). Later work (Wallace & Knights, in preparation) has shown it to be due to a single recessive with imperfect penetrance.
DISCUSSION
The technique used here, for screening invisible mutants, has proved to be a reasonably sensitive one, especially where, as in the stocks under study, there is only a trivial amount of litter competition. It could probably be used again, therefore, with a similar stock or an inbred line. An unexpected result was the finding of a significant difference between the PM and EM groups both in total loss and in losses per litter in the 19-29 day period. Since the sibmatings PS and ES showed no corresponding significant difference, this result is probably best ascribed to chance. However, if there had been a significant difference between PS and ES, i.e. in the treated (P) group and the control
222
MARGARET E. WALLACE, P. KNIGHTS, A. O. DYE
(E) group as a whole, but no greater loss on sibmating as between the treated and control matings, an excess of segregating semi-lethals (or incompletely recessive genes), like exencephaly, would have been suspected of segregating in the treated compared with the control stock. The main interest in this study is the lack of evidence, despite the sensitivity of the method, for a higher incidence of invisibles in the treated than in the control data. There were, clearly, two visible mutants in the treated data and none in the control, but this number is too small in relation to the number of eighth generation ancestor females (60) from which they were descended, for the conclusion with any conviction that there is a significant difference between the stocks in this respect. In view of the overall lack of evidence for invisible mutants, it is reasonable to decide that, although the study cannot claim to have disproved completely a small but unacceptable level of mutagenicity of DDT, there is now less urgency for a more exhaustive (and expensive) test.
ACKNOWLEDGEMENTS
Our thanks are due to Dr U Tomatis, International Agency for Research on Cancer, Lyons, France, for sending us the treated and control CF/I mice; Dr J. Robinson, of Shell Research Ltd, for the fat and kidney assays; Dr J. M. Barnes and Dr R, Verschoyle, of the MRC Toxicology Unit, Carshalton, Surrey, for the tests of resistance to D D T ; Dr I. E. Lush, Royal Free Hospital School of Medicine, London, for the assay of biochemical mutants and Professor D. S. Falconer, ScD., FRS, for advice on the design of the main experiment. This work was funded by the International Agency for Research on Cancer, Lyons, France.
REFERENCES CLARK, J. M. (1974). Mutagenicity of DDT in mice, Drosophila melanogaster, and Neurospera crassa. Aust. J. bioL Sci., 27, 427--40. EPSTEIN, S. S. & SHAFNER,H. (1968). Chemical mutagens in the human environment. Nature,
Lond., 219, 385-7. HAYES,W. J., JR., DALE,W. E. & PmKLE,C. I. (1971). Evidence of safety of long-term, high, oral doses of DDT for man. Archs environ. Hlth, 22, 119-35. INTERNATIONAL AGENCYFOR RESEARCHON CANCER (1970). Annual Report for 1969, Lyons, France. INTERNATIONALAGENCYFOR RESEARCHON CANCER(1974). Monographs on the evaluation o f carcinogenic risk o f chemicals to man, 5, 90.
KNIGHTS,P. (1972). Hydrancephaly. Mouse News Letter, 47, 24. MCLAREN,A. & MICHXE,D. (1959). Sul~rpregnancy in the mouse. 1. Implantation and foetal mortality after induced SUl~rovulation in females of various ages. J. exp. Biol., 36, 281-300. RESEARCHCOMMITTEEON TOXIC CHEMICALS(1970). A.R.C. Third Report, London. TOMATIS, L., TURUSOv, V., TERRACINI, B., DAY, N., BARTHEL, W. L., CHARLES, R. T., COLLINS,
G. B. & BoloccHi, M. (1971). Storage levels of DDT metabolites in mouse tissues following long term exposure to technical DDT. Estr. Tumori, 57, 377-96. TOMATtS,L., TURUSOV,V., DAY,N. & CHARLES,R. T. (1972). The effect of long-term exposure to DDT on CF-I mice. Int. J. Cancer, 10, 489-506. WALLACE,M. E. (1971). An unprecedented number of mutants in a colony of wild mice. Environ. Pollut., 1, 175-84.
MARGARET E. WALLACE 8/. P. KNIGHTS*
Department of Genetics, Milton Road, Cambridge CB4 1XH, Great Britain & A.
O. DYE
Department of Land Economy, University of Cambridge, Cambridge, Great Britain A BS TR A C T An inbred strain o f mice, half o f which had been given a diet containing 250 ppm of D D T over five generations, and half o f which were untreated, was examined to disclose whether there was a greater incidence o f mutants in the treated than in the control mice. The stock did not suffer unduly from litter competition, and the breeding programme used provided a fairly sensitive test. There was no evidence for a greater incidence o f recessive invisibles in the treated than in the control halves, but there were two recessive visibles in the treated and none in the control. It is concluded that there is no case for a grossly mutagenic effect o f DDT.
INTRODUCTION
To date, the mutagenicity of D D T has not been rigorously tested. This seems to be a serious omission in view of its known toxic effects, on account of which its use has become much more restricted than it was ten years ago. It is still the chemical of choice for the control of malaria; nonetheless the only tests of mutagenicity done as yet, using a mammal, are those of Epstein & Shafner (1968) and of Clark (1974), using mice. The tests were for dominant lethals, with treatment by injection in the former case and by oral dose in the latter. The recent discovery (Wallace, 1971) of a very high incidence of mutants in a colony of mice derived from wild ones trapped in a heavily DDT-treated area, calls for a new look at the possibility of mutagenicity. * Present address: Pembrokeshire Coast National Park, County Offices, Haverfordwest, Pembrokeshire, Great Britain. 217 Environ. Pollut. (11) (1976)--© Applied Science Publishers Ltd, England, 1976 Printed in Great Britain
218
MARGARET E. WALLACE, P. KNIGHTS, A. O. DYE
This paper presents new evidence. It reports on the incidence of recessive visible and invisible mutants in a colony of mice fed over five generations on a DDTtreated diet. It does not claim to be a rigorous test. It was, in fact, carried out because such a treated colony, and its untreated control, became available to the Genetics Department, and the intention was to make good use of this unusual material.
MATERIALS AND METHODS
Mice--History and treatment Through the International Agency for Research on Cancer, Lyons, France, a stock of DDT-treated and control mice was received. The strain, CF/I, ofgenotype aabbcc (nonagouti, brown, albino), had been sibmated to 20 generations in 1940, and was random bred until transfer to the Agency. There it was divided into a control stock and four treatment stocks, these receiving a diet containing D D T at dose levels of 2, 10, 50 and 250 parts per million (ppm) for their life span for five consecutive generations (Tomatis et al., 1972). The strain was maintained by minimal inbreeding (15-30 pairs per generation per stock), as part of a study of carcinogenesis (IARC, 1970). The sixth generation received treatment through their mothers (in utero and during lactation) until weaning; the seventh and eighth were minimally inbred without treatment. Late in 1970 the Genetics Department received 15 trios of the eighth generation of that treatment line which had received 250 ppm of D D T in their diet over five generations, and 21 trios of the same generation of the control stock. This concentration of DDT was chosen because it produced similar levels of DDT and its derivatives in the fatty tissues of mice fed 250 ppm DDT in their diet (214-600 ppm) as in men under occupational and high exposure (201-600 ppm) (Hayes et al., 1971 ; Tomatis et al., 1971 table 11 ; IARC, 1974). The two stocks were then maintained through one further generation by 48 matings minimally inbred, in order to clear them of a small outbreak of mastitis and infantile diarrhoea; this had developed as soon as they passed from an SPF environment to a conventional one--although no mice in the conventional environment showed overt symptoms. To check that there had been no great genetic divergence between the two stocks during the generations of physical separation, 20 males of the eighth generation of each were typed for biochemical polymorphisms at the following six loci: Gpi-1, Pgm-i, Pgm-2, ld-i, Hb-b and Dip-1 (glucose-6-phosphate dehydrogenase, phosphoglucomutase-I and -2, isocitrate dehydrogenase, haemoglobin beta chain and dipeptidase-l). A\ further five females of each stock were tested for resistance to DDT. The polymorphism test showed the two stocks to be sufficiently similar to preclude doubt about their common origin and genetic equivalence. The resistance test also showed them to be very similar; they were more resistant than most
MUTAGENIC1TY OF DDT IN M1CE
219
common laboratory strains, and the treated ones were insignificantly more resistant than the control. This agrees with the assumption that both were resistant before division and that the period of treatment was ineffectual as a selective force because there was no genetic diversity in this respect. A third check was made. In order to see whether the ninth generation was free of DDT, which is known to pass into milk and across the placental barrier (Research Committee on Toxic Chemicals, 1970), five mice of each sex of each stock were autopsied and assays made of the fatty tissue content of D D T and its derivatives. This showed both stocks to have an unusually low content (treated: 0.10-0.49 ppm, and control: 0.08-0.86 ppm). These later generations were not therefore exposed to D D T via any possible route.
Breeding programmes In the tenth generation, approximately 100 minimally inbred matings and 100 sibmatings were made up of each stock. In the eleventh generation, approximately 70 minimally inbred matings and 100 sibmatings were made up of each stock. These sets of matings and data from them are d e s i g n a t e d E M and PM when minimally inbred, and ES and PS when sibmated; E stands for control and P for treated. In the tenth generation, each mouse in each of the first litters of each stock was examined for visible abnormalities at birth, and at the phenocritical ages of 2, 7, 18 and 28 days; the litter sizes at each age were recorded. Every abnormality was investigated, by several types of mating, to see if it was inherited. In the eleventh generation, the females were sacrificed at 16 days gestation (as judged from the observation of vaginal plugs), and observations made on the numbers of corpora lutea, of implantation sites, of dead or partially absorbed foetuses, and of live young. The latter observations allow the inference of the numbers of fertilised eggs, of pre-imPlantation losses, of post-implantation losses to 16 days, and of live young at 16 days (McLaren & Michie, 1959). The incidence of recessive invisible mutants in the treated mice was compared with that of the control mice as follows. A lower litter size is expected in the sibmatings as compared, with the minimally inbred ones, due to homozygosity of lethals. If the amount by which litter size is reduced is the same in the control as in the treated stock, then there is probably the same level of invisible lethals segregating in both. If reduction in litter size is greater in the treated than in the control matings, then there are more invisibles in the treated stock than in the c o n t r o l - - o r there are a few invisibles more widely spread in the treated stock than in the control. In short, the comparison does not allow an assessment to be made of the quantity of invisibles in either stock; it simply shows whether or not the deathrate due to invisibles is or is not greater in the treated stock than in the control. If it is greater, then there is a case for an experiment specifically to test mutagenicity, such as the specific locus test. "Owing to the homozygosity of the albino gene in both stocks, the examination at
220
MARGARET E. WALLACE, P° KNIGHTS, A. O. DYE
birth and thereafter, while likely to show up such defects as behaviour defects and hair texture ones, would not show up coiour variation. An equal number (42) of females of the minimally inbred sections of each stock were therefore outcrossed to several laboratory stocks (each female to about .three different laboratory males); each of the latter carried a number of colour (and other) mutants. FI were also mated to each other and their progeny examined for coiour variation. Thus both stocks were scanned in general for all known dominant and recessive colour mutants, and specifically for mutants at the following loci: In, py (leaden, polydactyly, chromosome 1), a, un, we, pa (agouti, undulated tail, wellhaarig, pallid, chromosome 2), b (brown, chromosome 4), coh, p (chinchilla, pinkeyed dilution, chromosome 7), d, se (Maltese dilution, short-ear, chromosome 9), s (pied, chromosome 14), bt (belted, chromosome 15), dp and mr (dilute-Peru and maroon, location unknown).
RESULTS
Statistical analysis o f mortafity The analysis was carried out in three phases. First, stepwise linear regression was used on both the antenatal and the postnatal data to determine what proportion of the variation in litter losses was associated with competition due to litter size and what proportions with sibmating and the effect of the treatment. There were a few litters in each of the EM, PM, ES and PS stocks which showed an outstandingly high mortality due to disease; these were removed from the data. Next in the postnatal data, allowance for competition having been made, the total loss per litter up to 28 days, and the losses per litter in each of the four periods into which the 28 days were divided, were used in an analysis of variance to expose any significance in the differences between the relevant subgroupings of the data. Finally, the antenatal data were similarly analysed. The most useful variable for measuring competition was found to be litter size at 28 days. However, even with the high degree ofautocorrelation inevitable between variables, competition was shown to account for only a small proportion of the losses, and it did not mask the effect of the treatment or the breeding regime. For example, in the postnatal data, only 7 ~ of the losses could be accounted for on the basis of a loss rate of 0.25 per litter for every additional member of the litter. In neither the postnatal nor the antenatal data were the crucial differences significant, i.e. it could not be shown that litter loss due to sibmating was greater in the P than in the E data. The only significant comparisons in the postnatal data were within the minimally inbred groups as follows: The total loss per litter was greater in the PM data than in the EM, and losses per litter in the period 19-29 days were greater in the PM than in the EM data. In the antenatal study, similarly, differences were small by comparison with the variation within each group. The
MUTAGENICITY OF DDT IN MICE
221
largest differences were as follows: the corpora lutea count on a per litter basis for the PM group was larger than that in the PS group by 0.10, and the same count for the EM group was larger than for the ES group by 0.38; again the corpora iutea count in the stock (EM + ES) was greater than that in the stock (PM + PS) by 0.90, and in the ES group it was greater than that in the PS group by 0-81. These differences being small compared with the large amount of variation within each group, the effects of the treatment are not significant at the 5 % level--or indeed at the 30 % level. In short, since the effect of competition is so slight, any gross effect of the treatment would have shown itself, and it did not do so. Observations on visible mutants
There were 30 individuals, occurring at random between the sexes and over the four main groups of matings, which showed physical abnormalities. Two mice, one with gross limb malformation and one with no hind legs, occurred in the P stocks; the rest all had small defects, such as bifurcated left or right ear, tail kink or unusually small body size. No abnormality proved to be heritable. Of the 42 females of each stock outcrossed to test for colour and other mutants, three proved to be black-and-tan, ata, rather than nonagouti, aa; all three occurred in the treated (P) stock. From the 170 females in each stock autopsied at 16 days' gestation, a number of exencephalic young were seen. These also occurred only in the treated (P) stock, there being 12 matings segregating in this anomaly, distributed at random between the PM and PS groups. Careful scrutiny of young within an hour of birth, from females closely related to these, disclosed dead ones with the upper part of the head missing; since they occurred in about the same fraction within each litter, it could be concluded that these were also exencephalio and that the condition is inherited (Knights, 1972). Later work (Wallace & Knights, in preparation) has shown it to be due to a single recessive with imperfect penetrance.
DISCUSSION
The technique used here, for screening invisible mutants, has proved to be a reasonably sensitive one, especially where, as in the stocks under study, there is only a trivial amount of litter competition. It could probably be used again, therefore, with a similar stock or an inbred line. An unexpected result was the finding of a significant difference between the PM and EM groups both in total loss and in losses per litter in the 19-29 day period. Since the sibmatings PS and ES showed no corresponding significant difference, this result is probably best ascribed to chance. However, if there had been a significant difference between PS and ES, i.e. in the treated (P) group and the control
222
MARGARET E. WALLACE, P. KNIGHTS, A. O. DYE
(E) group as a whole, but no greater loss on sibmating as between the treated and control matings, an excess of segregating semi-lethals (or incompletely recessive genes), like exencephaly, would have been suspected of segregating in the treated compared with the control stock. The main interest in this study is the lack of evidence, despite the sensitivity of the method, for a higher incidence of invisibles in the treated than in the control data. There were, clearly, two visible mutants in the treated data and none in the control, but this number is too small in relation to the number of eighth generation ancestor females (60) from which they were descended, for the conclusion with any conviction that there is a significant difference between the stocks in this respect. In view of the overall lack of evidence for invisible mutants, it is reasonable to decide that, although the study cannot claim to have disproved completely a small but unacceptable level of mutagenicity of DDT, there is now less urgency for a more exhaustive (and expensive) test.
ACKNOWLEDGEMENTS
Our thanks are due to Dr U Tomatis, International Agency for Research on Cancer, Lyons, France, for sending us the treated and control CF/I mice; Dr J. Robinson, of Shell Research Ltd, for the fat and kidney assays; Dr J. M. Barnes and Dr R, Verschoyle, of the MRC Toxicology Unit, Carshalton, Surrey, for the tests of resistance to D D T ; Dr I. E. Lush, Royal Free Hospital School of Medicine, London, for the assay of biochemical mutants and Professor D. S. Falconer, ScD., FRS, for advice on the design of the main experiment. This work was funded by the International Agency for Research on Cancer, Lyons, France.
REFERENCES CLARK, J. M. (1974). Mutagenicity of DDT in mice, Drosophila melanogaster, and Neurospera crassa. Aust. J. bioL Sci., 27, 427--40. EPSTEIN, S. S. & SHAFNER,H. (1968). Chemical mutagens in the human environment. Nature,
Lond., 219, 385-7. HAYES,W. J., JR., DALE,W. E. & PmKLE,C. I. (1971). Evidence of safety of long-term, high, oral doses of DDT for man. Archs environ. Hlth, 22, 119-35. INTERNATIONAL AGENCYFOR RESEARCHON CANCER (1970). Annual Report for 1969, Lyons, France. INTERNATIONALAGENCYFOR RESEARCHON CANCER(1974). Monographs on the evaluation o f carcinogenic risk o f chemicals to man, 5, 90.
KNIGHTS,P. (1972). Hydrancephaly. Mouse News Letter, 47, 24. MCLAREN,A. & MICHXE,D. (1959). Sul~rpregnancy in the mouse. 1. Implantation and foetal mortality after induced SUl~rovulation in females of various ages. J. exp. Biol., 36, 281-300. RESEARCHCOMMITTEEON TOXIC CHEMICALS(1970). A.R.C. Third Report, London. TOMATIS, L., TURUSOv, V., TERRACINI, B., DAY, N., BARTHEL, W. L., CHARLES, R. T., COLLINS,
G. B. & BoloccHi, M. (1971). Storage levels of DDT metabolites in mouse tissues following long term exposure to technical DDT. Estr. Tumori, 57, 377-96. TOMATtS,L., TURUSOV,V., DAY,N. & CHARLES,R. T. (1972). The effect of long-term exposure to DDT on CF-I mice. Int. J. Cancer, 10, 489-506. WALLACE,M. E. (1971). An unprecedented number of mutants in a colony of wild mice. Environ. Pollut., 1, 175-84.