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721. Dimethylnitrosamine and the newborn
636
CANCER RESEARCH
tumours in animals treated repeatedly with a carcinogenic polycyclic hydrocarbon given in sub-carcinogenic doses. Despite this new orientation, the effect of duration of promoting treatment on benign tumour induction is uncertain because of conflicting experimental results by different workers. No report could be found describing the effect of the length of promoting treatment on malignant tumour induction. In the present experiments the incidence of malignant skin tumours in mice, pretreated with dermally-applied doses of 300/lg 3,4-benzopyrene (BP), varied directly with the length of treatment with I which lasted between 10 and 50 weeks. Continuation of I from 50 to 77 weeks had no marked effect on the incidence of malignant tumours. In the control groups a few benign tumours developed in response to 300 ~g BP followed by acetone. Many benign and a few malignant tumours developed in mice treated for 35 or 77 weeks with I without pretreatment with. BP. The tumours in these groups were at no time as numerous as in the corresponding groups treated with BP. T1/ese results seem to support the general hypothesis that, in two-stage carcinogenesis the ultimate incidence of malignancy varies directly with the length of exposure to the "tumourpromoting" stimulus. There is some evidence to indicate that beyond a certain point in time of exposure or of the lifespan, cessation of exposure to the promoting stimulus has no effect. It has been shown that I is a complete carcinogen of mild potency for this strain of mice used and not merely a "pure promoter". The concept of "pure initiators" and "pure promoters" complementing each other's activities with resulting carcinogenesis is regarded as a myth in the light of present knowledge but it is felt that there still seems to be some value in classifying carcinogens as initiators or promoters.
720. A promising approach Roe, F. J. C., Mitchley, B. C. V. & Waiters, Margaret (1963). Tests for carcinogenesis using newborn mice: 1,2-benzanthracene, 2-naphthylamine, 2-naphthylhydroxylamine and ethyl methane sulphonate. Brit. J. Cancer 17, 255. Present-day procedures for carcinogenicity testing suffer from the handicap that a long interval time elapses between administration of the compound under test and the time of onset of tumours. The use of the newborn animal which is particularly sensitive to chemical carcinogens (Cited in F.C.T. 1964, 2, 33) shows promise of reducing this time lag. The present study involved subcutaneous injection of four compounds 1,2-benzanthracene (I), ethyl methane sulphonate (II), 2-naphthylamine (III) and 2-naphthylhydroxylamine (IV) into mice on the first day of life. Positive controls given the potent carcinogen 9,10-dimethylbenz[a]anthracene and solvent control groups were set up. Lung and other tumours developed in animals sacrificed during weeks 36-43. A single subcutaneous dose of 50 #g I or 100/ag II was considered to be definitely though weakly active while dosages of 50/~g of III or IV given by the same route were probably active but results require confirmation. In a second experiment in which 1, 2, 4 and 8-day-old mice were given single subcutaneous doses of 50/~g I the biggest crop of tumours in animals sacrificed during weeks 36-43 was obtained when administration was carried out on the first day of life. [It would be interesting to turn the clock back even further and see whether administration of carcinogens into pregnant animals would result in early induction of tumours in the progeny.] 721. Dimethylnitrosamine and the newborn Terracini, B. & Magee, P. N. (1964). Renal tumours in rats following injection of dimethylnitrosamine at birth. Nature, Lond. 202, 502. In a preliminary study the tumour incidence in rats receiving single subcutaneous doses of
TERATOGENE$1S
637
dimethylnitrosamine (I) on days 1 or 7 of life was compared (Cited in F.C.T. 1964, 2, 61). A dose of 250 pg I proved lethal to all animals within 2 weeks. By week 53 of the test, 14 animals had died after weaning; 10 had been given I when 1 day old and the other 4 animals received I at 7 days of age. Tumours were seen in 11/14 animals. Renal tumours developed in 8 animals; and the other 3 rats developed either renal tumour plus hepaloma, hepatoma alone or abdominal malignant lymphoma. The first renal tumour was first seen in a rat dying at 36 weeks of age. No controls were used in these preliminary experiments although the authors state that "no renal or liver tumours have been observed in untreated rats in their laboratory". The finding of Domsky et al. (Proc. Soc. exp. Biol., N. Y. 1963, 113, 110) that the potent carcinogen 9,10-dimethylbenz[a]anthracene is metabolized more slowly by the newborn than by the adult may be linked with the unusual susceptibility of the newborn to the carcinogen. With I, however, elimination from the body of adult and newborn rats ran parallel. The amount of unchanged I was estimated in animals at intervals of 0-48 hr after subcutaneous administration of 125 pg 1. In accordance with previous results in adult rats (Magee, Biochem. J. 1956, 64, 676) no I was detectable in the newborn animal 24 hr after dosing despite the fact that the rate of elimination of I during the first 12 hr was slower in the new-born than the adult. In preliminary metabolic studies similar doses of I labelled with carbon-14 were degraded to radioactive carbon dioxide detected in the expired air.
TERATOGENESIS 722. "Blinder E i f e r . . . " Bundesgesundheitsamt (1964). "Blinder E i f e r . . . " . Bundesgesundheitsblatt, 7, 122. The Bundesgesundheitsblatt has published a highly critical review of a paper appearing under this title by D6ring of Munich. The paper (Dtsch. Arztebl. 1964, 61, 733) is subtitled "Warnings of the Health Authorities about Meclizin". Readers are reminded that allegations of foetal malformations following the use of Meclizin (I) against pregnancy sickness appeared in the press in 1962. At that time, the German Federal Office of Health opposed the withdrawal of I from the market, but imposed a prescription obligation and compulsory labelling ("not to be taken during pregnancy"), pending animal testing. In 1963, the manufacturers, Union Chimique Beige, produced considerable evidence against any teratogenic action by I in man. The reviewer, however, consid:rs that d ~ctors prescribing I for use during pregnancy (dose range 12.5-50 rag) should realise that cleft palate has been produced in rats by oral administration of 25-250 mg/kg/day (King, Science 1963, 141, 353; Kendrick & Weaver, Proc. Soc. Exp. Biol., N.Y. 1963, 144, 747). There is therefore little justification for unqualified statements that preparations containing 1 are now deemed safe for use during pregnancy (Pharm. Ztg. 1964, No. 8, 277). An increase incidence of malformations is associated with both diabetes ar.d hyperemesis gravidarum. D6ring suggested that withdrawal of I might have an effect exactly opposite to that intended,, by depriving hyperemetic patients of treatment ar,d thus increasing the possibility of malformed offspring. The reviewer points out however, that there h,~tsnever been any suggestion of leaving this condition untreated. D6ring's statement, that I has never been limited to prescription sales in Switzerland, is also challenged. The Swiss regulations apparently exempt from prescription only preparations with a single d~se of up to 50 mg and a daily dose of up to 100 mg. In Austria, there are no exceptions to the prescription obligation for I, and formulations of I are not prescribed on Health Service accounts, except in specially approved cases. Apparently this limitation on the use of I has not led to the unfortunate results envisaged by the author of "Blind Z e a l . . . " .
CANCER RESEARCH
tumours in animals treated repeatedly with a carcinogenic polycyclic hydrocarbon given in sub-carcinogenic doses. Despite this new orientation, the effect of duration of promoting treatment on benign tumour induction is uncertain because of conflicting experimental results by different workers. No report could be found describing the effect of the length of promoting treatment on malignant tumour induction. In the present experiments the incidence of malignant skin tumours in mice, pretreated with dermally-applied doses of 300/lg 3,4-benzopyrene (BP), varied directly with the length of treatment with I which lasted between 10 and 50 weeks. Continuation of I from 50 to 77 weeks had no marked effect on the incidence of malignant tumours. In the control groups a few benign tumours developed in response to 300 ~g BP followed by acetone. Many benign and a few malignant tumours developed in mice treated for 35 or 77 weeks with I without pretreatment with. BP. The tumours in these groups were at no time as numerous as in the corresponding groups treated with BP. T1/ese results seem to support the general hypothesis that, in two-stage carcinogenesis the ultimate incidence of malignancy varies directly with the length of exposure to the "tumourpromoting" stimulus. There is some evidence to indicate that beyond a certain point in time of exposure or of the lifespan, cessation of exposure to the promoting stimulus has no effect. It has been shown that I is a complete carcinogen of mild potency for this strain of mice used and not merely a "pure promoter". The concept of "pure initiators" and "pure promoters" complementing each other's activities with resulting carcinogenesis is regarded as a myth in the light of present knowledge but it is felt that there still seems to be some value in classifying carcinogens as initiators or promoters.
720. A promising approach Roe, F. J. C., Mitchley, B. C. V. & Waiters, Margaret (1963). Tests for carcinogenesis using newborn mice: 1,2-benzanthracene, 2-naphthylamine, 2-naphthylhydroxylamine and ethyl methane sulphonate. Brit. J. Cancer 17, 255. Present-day procedures for carcinogenicity testing suffer from the handicap that a long interval time elapses between administration of the compound under test and the time of onset of tumours. The use of the newborn animal which is particularly sensitive to chemical carcinogens (Cited in F.C.T. 1964, 2, 33) shows promise of reducing this time lag. The present study involved subcutaneous injection of four compounds 1,2-benzanthracene (I), ethyl methane sulphonate (II), 2-naphthylamine (III) and 2-naphthylhydroxylamine (IV) into mice on the first day of life. Positive controls given the potent carcinogen 9,10-dimethylbenz[a]anthracene and solvent control groups were set up. Lung and other tumours developed in animals sacrificed during weeks 36-43. A single subcutaneous dose of 50 #g I or 100/ag II was considered to be definitely though weakly active while dosages of 50/~g of III or IV given by the same route were probably active but results require confirmation. In a second experiment in which 1, 2, 4 and 8-day-old mice were given single subcutaneous doses of 50/~g I the biggest crop of tumours in animals sacrificed during weeks 36-43 was obtained when administration was carried out on the first day of life. [It would be interesting to turn the clock back even further and see whether administration of carcinogens into pregnant animals would result in early induction of tumours in the progeny.] 721. Dimethylnitrosamine and the newborn Terracini, B. & Magee, P. N. (1964). Renal tumours in rats following injection of dimethylnitrosamine at birth. Nature, Lond. 202, 502. In a preliminary study the tumour incidence in rats receiving single subcutaneous doses of
TERATOGENE$1S
637
dimethylnitrosamine (I) on days 1 or 7 of life was compared (Cited in F.C.T. 1964, 2, 61). A dose of 250 pg I proved lethal to all animals within 2 weeks. By week 53 of the test, 14 animals had died after weaning; 10 had been given I when 1 day old and the other 4 animals received I at 7 days of age. Tumours were seen in 11/14 animals. Renal tumours developed in 8 animals; and the other 3 rats developed either renal tumour plus hepaloma, hepatoma alone or abdominal malignant lymphoma. The first renal tumour was first seen in a rat dying at 36 weeks of age. No controls were used in these preliminary experiments although the authors state that "no renal or liver tumours have been observed in untreated rats in their laboratory". The finding of Domsky et al. (Proc. Soc. exp. Biol., N. Y. 1963, 113, 110) that the potent carcinogen 9,10-dimethylbenz[a]anthracene is metabolized more slowly by the newborn than by the adult may be linked with the unusual susceptibility of the newborn to the carcinogen. With I, however, elimination from the body of adult and newborn rats ran parallel. The amount of unchanged I was estimated in animals at intervals of 0-48 hr after subcutaneous administration of 125 pg 1. In accordance with previous results in adult rats (Magee, Biochem. J. 1956, 64, 676) no I was detectable in the newborn animal 24 hr after dosing despite the fact that the rate of elimination of I during the first 12 hr was slower in the new-born than the adult. In preliminary metabolic studies similar doses of I labelled with carbon-14 were degraded to radioactive carbon dioxide detected in the expired air.
TERATOGENESIS 722. "Blinder E i f e r . . . " Bundesgesundheitsamt (1964). "Blinder E i f e r . . . " . Bundesgesundheitsblatt, 7, 122. The Bundesgesundheitsblatt has published a highly critical review of a paper appearing under this title by D6ring of Munich. The paper (Dtsch. Arztebl. 1964, 61, 733) is subtitled "Warnings of the Health Authorities about Meclizin". Readers are reminded that allegations of foetal malformations following the use of Meclizin (I) against pregnancy sickness appeared in the press in 1962. At that time, the German Federal Office of Health opposed the withdrawal of I from the market, but imposed a prescription obligation and compulsory labelling ("not to be taken during pregnancy"), pending animal testing. In 1963, the manufacturers, Union Chimique Beige, produced considerable evidence against any teratogenic action by I in man. The reviewer, however, consid:rs that d ~ctors prescribing I for use during pregnancy (dose range 12.5-50 rag) should realise that cleft palate has been produced in rats by oral administration of 25-250 mg/kg/day (King, Science 1963, 141, 353; Kendrick & Weaver, Proc. Soc. Exp. Biol., N.Y. 1963, 144, 747). There is therefore little justification for unqualified statements that preparations containing 1 are now deemed safe for use during pregnancy (Pharm. Ztg. 1964, No. 8, 277). An increase incidence of malformations is associated with both diabetes ar.d hyperemesis gravidarum. D6ring suggested that withdrawal of I might have an effect exactly opposite to that intended,, by depriving hyperemetic patients of treatment ar,d thus increasing the possibility of malformed offspring. The reviewer points out however, that there h,~tsnever been any suggestion of leaving this condition untreated. D6ring's statement, that I has never been limited to prescription sales in Switzerland, is also challenged. The Swiss regulations apparently exempt from prescription only preparations with a single d~se of up to 50 mg and a daily dose of up to 100 mg. In Austria, there are no exceptions to the prescription obligation for I, and formulations of I are not prescribed on Health Service accounts, except in specially approved cases. Apparently this limitation on the use of I has not led to the unfortunate results envisaged by the author of "Blind Z e a l . . . " .