Fd Cosmrr. Toxicol. Vol. IS. pp. 229-232. Pergamon Press 1977. Printed in Great Britain
PRELIMINARY TRANSPLACENTAL CARBARYL
COMMUNICATION
CHRONIC TOXICITY TEST .OF WITH NITRITE IN RATS*
W. LrrrNsrcvt and H. W. TAYLOR~ Carcinogenesis Program. Biology Division Oak Ridge National Oak Ridge, (Received
Tennessee, 26 October
Laboratory,
USA 1976)
Summary-Pregnant female rats were given by gavage a total of 300mg carbaryl during a lo-day period. No malignant tumours were induced in the dams or their offspring during their natural lifespan. Pregnant and non-pregnant female rats were given a suspension of carbaryl in sodium nitrite solution on several successive days, a total of 90 mg carbaryl and 120 mg sodium nitrite being administered to each adult rat. There was no significant incidence of malignant tumours in the dams or their offspring as a result of the treatment.
Introduction Carbaryl, the l-naphthyl ester of N-methylcarbamic acid, is a widely used insecticide which is permitted as a residue on crops, a tolerance level having been set for it by the Food and Drug Administration. Carbaryl itself is of interest as a possible environmental contaminant, and as a member of the large group of N-methylcarbamate esters which are used as insecticides. Several of them have been tested for toxicity and appear to be relatively non-toxic in mammals; neither do they show mutagenic activity in bacterial systems. However, because they can react with nitrous acid to form N-nitroso derivatives (Elespuru & Lijinsky, 1973), another aspect of their presence in the environment must be considered, namely the possible adverse biological effects of the nitroso compounds so formed. It has been demonstrated that nitrosocarbaryl, which can interact with human DNA in oitro (Regan, Setlow, Francis & Lijinsky, 1976), is a potent bacterial mutagen (Elespuru, Lijinsky & Setlow, 1974), as are the nitroso derivatives of several other methylcarbamate insecticides (Lijinsky & Elespuru, 1976). Nitrosocarbaryl is also a fairly potent carcinogen when administered orally to rats (Eisenbrand, Schmlhl & Preussman, 1976; Lijinsky & Taylor, 1976) and, because formation of nitrosocarbaryl from ingested carbaryl and nitrite is the most likely mode of human exposure, it was decided to investigate its formation in this way in rats. Since the amount formed in a rat’s stomach was likely to be small, the transplacental route of exposure for the rats was chosen. This *Research supported by the National Cancer Institute under contract with the Energy Research and Development Administration and Union Carbide Corporation. fPresent address: Chemical Carcinogenesis Prog&, Frederick Cancer Research Center, Frederick, Maryland 21701, USA. IPresent address: Department of Pathology and Parasitology, School of Veterinary Medicine, Auburn University, Auburn, Alabama 36830, USA. 229
is considered the most sensitive mammalian test system, and has responded to very small doses of nitroso compounds formed in this way in uiuo (Ivankovic & Preussmann, 1970).
Experimental Technical-grade carbaryl (‘Sevin’, kindly supplied by Union Carbide Corp., Bound Brook, NJ) was crystallized from acetone, after insoluble dark material had been filtered off, colourless crystals, m.p. 143-144°C being obtained. These were finely powdered in a mortar and mixed with water to give a fairly uniform suspension which would pass through the needle of a small syringe. The suspensions given to the animals by gavage contained carbaryl (30mg/ml) in water or in sodium nitrite solution, (40 mg NaN02/ml). Twenty-four female Sprague-Dawley rats, 1214 wk old, were mated by leaving them with males for a week-end, in which time the majority would normally be expected to become pregnant. They were divided into three groups of eight animals. Those in group 1 were given 1 ml of a suspension of 30 mg carbaryl in water on each of 10 days between day 4 and 18 of gestation (weekends omitted). One animal died during the treatment and three of the remainder gave birth to a total of nine female and 13 male rats. Group II rats were given 1 ml of a suspension of 30 mg carbaryl in 4% sodium nitrite solution on days 4, 5 and 6 of gestation (first trimester). At that time six of the eight animals died of nitrite poisoning and the treatment was discontinued. Only one of the remaining animals gave birth, to a litter of seven females and three males. Group III rats were given only 0.6 ml of the suspension given to group II, to avoid nitrite poisoning. Treatment was given on each of days 14-18 of gestation (last trimester). No animals died and six gave birth to litters totalling 32 females and 32 males. To provide additional numbers for the significance of tumour incidences in the animals given carbaryl with nitrite to be assessed, an additional
.
Carbaryl + NaN02 Carbaryl + NaN02
Carbaryl + NaN02
II, III, IV II
III
Transpiacental
Intragastric Transplacental
Route Intragastric Transplacental
14-18
14-18t 4-6
Time of exposure* 4-18 4-18
of rats treated
*Days of gestation on which test solution was administered to dams. Non-pregnant tOr days 4-6 (group II).
Treatment Carbaryl Carbaryl
Group no. I I
Table 1. Survival
60 I 9 12 22 7 3 30 32
or carbaryl
IO 7 9 12 20 I 3 28 32
and
9 11 20 I 3 28 31
1
80
nitrite
90 5 9 10 18 6 3 26 21
13 5 3 23 24
100 3 l
110 2 6 5 9 4 2 15 16
120 2 3 2 7 2 1 10 5
t!l 0 2 0 5 2
130 1
No. of animals surviving at week
females of group IV were treated on an equivalent no. of days.
No. of rats/group 7F 9F 13M 22F 7F 3M 32F 32M
with carbaryl
3 0
1
0
1
1
140 - 150 1 0
0
0
160
e F 2 9 !i ii r el 5 i
Transplacental
Carbaryl + NaNO*
III
1418
4-6
1418t
4-18
4-18
treated
1 22 4
3M 32F 32M
13
ii
4
Breast
rats
5
in
7F
22F
9F 13M
6F
rats/ w-w
No. of
Tumours
*Days of gestation on which test solution was administered to dams. Non-pregnant tOr days 46 (group II).
Transplacental
Carbaryl + NaNO,
II
Intragastric
Carbaryl + NaNO,
Transplacental
Carbaryl
I
II, III, IV
Intragastric
Carbaryl
I
Route
Treatment
Group no.
Time of exposure*
Table 2.
carbaryl
nitrite
6
1 1
1 4 6
0
Pancreas
1
Adrenal
and
0
2
0
0
Liver
0
1
0
0
Uterus
No. of animals with turnours of
or carbaryl
Stomach papilloma (1) Lymphosarcoma (1) Mesothelioma (1) Myeloid leukaemia (2) Osteoadenocarcinoma of gut (1) Adenocarcinoma of gut (1) Lymphosarcoma (1) Myeloid leukaemia (1) Mesothelioma (1) Zymbal gland carcinoma (1) Skin trichoepithelioma (1)
Lymphosarcoma (1) Oesophageal papilloma (1) Lymphangioma (1) Lymphosarcoma (1) Myeloid leukaemia (1) Thyroid carcinoma (1)
(1)
(1)
Other tissues Lymphosarcoma Leukemia (1) Adenocarcinoma
females of group IV were treated on an equivalent no. of days.
4
1 17
2
11
7 2
3
Pituitary
with
232
W.
LUINSKY
and
twelve rats (group IV) were given the same treatment as group III on five successive days. ‘All the adults and offspring, were observed until natural death except a few that were killed when moribund. Complete autopsy was performed and all tumours and other lesions were fixed for histological examination, together with most grossly normal major organs.
Results and Discussion As seen in Table 1, survival of all of the rats was good (with the exception of those killed by nitrite poisoning) and there was little difference between the groups when account was taken of the longer period of experimental observation of the offspring which were treated in utero. In each group at least half of the animals were alive after 2 years and a number survived beyond 2.5 years. Therefore, the dose of carbaryl given, with or without nitrite, had no apparent life-shortening effect. The distribution of tumours in the rats of the various groups is shown in Table 2. The common tumours in all groups were those normally seen in Sprague-Dawley rats, notably mammary fibroadenomas and tumours of the pituitary (predominantly in females) and adrenal tumours (mostly cortical adenomas). There were a few islet-cell adenomas of the pancreas and some hepatocellular carcinomas. The distribution of these tumours did not suggest any treatment-related effect on their incidence. In the females treated with carbaryl alone, and in their offspring, there were very few tumours other than those mentioned, and it appears that the dose of carbaryl administered, 300 mg or 1.5 mmol was without tumorigenic effect. Although this is by no means an exhaustive test of the carcinogenicity of carbaryl, the lack of response of the transplacentally exposed rats suggests that the tumorigenic effect of carbaryl, if any, is exceedingly weak. There is a small possibility that the compound does not cross the placenta, but it has been shown to be readily absorbed from the stomach (C&per, Pekas & Dinusson, 1973). The offspring of the females receiving carbaryl plus nitrite in the latter part of pregnancy gave no indication of a transplacental carcinogenic effect of the nitrosocarbaryl formed in the stomach. Nitrosocarbaryl is a fairly potent carcinogen when administered to rats, producing squamous carcinomas of the stomach when given intragastrically (Eisenbrand et al., 1976; Lijinsky & Taylor, 1976) and sarcomas when given SC (Eisenbrand, Ungerer & Preussmann, 1975), and can be formed to some extent in vitro even at
H. W. TAYLOR
low concentrations of carbaryl and nitrite (Eisenbrand et al. 1975). We conclude that either insufficient nitrosocarbaryl was formed in the rats in uiuo during the 5 days of treatment with 18mg carbaryl plus 24mg sodium nitrite, or that unlike methylnitrosourea and ethylnitrosurea a significant proportion of the nitrosocarbaryl formed does not cross the placenta of rats. While transplacental treatment of rats with larger quantities of nitrosocarbaryl might establish this point, the results of these experiments demonstrate no overt transplacental carcinogenic hazard from ingestion of carbaryl and nitrite together. Neither do they establish conclusively the safety of a combination of carbaryl and nitrite when ingested by humans, who might be a more sensitive species to nitrosocarbaryl (received directly or transplacentally). However, an experiment of this kind with its negative results does give a little more comfort than results that are clearly positive. REFERENCES
Casper, H. H., Pekas, J. C. & Dinusson, W. E. (1973). Gastric absorption of a pesticide (I-napthyl-N-methylcarbamate) in the fasted rat. Pesric. Biochem. Pkysiol. 2, 391. Eisenbrand, G., Schmlhl, D. & Preussman, R. (1976). Carcinogenicity in rats of high oral doses of N-nitrosocarbaryl, a nitrosated pesticide. Cancer Letters 1, 281. Eisenbrand, G., Ungerer, 0. & Preussmann, R. (1975). The reaction of nitrite with pesticides. II. Formation, chemical properties and carcinogenic activity of the N-nitroso derivative of N-methyl-1-naphthyl carbamate (carbaryl). Fd Cosmet. Toxicol. 13, 365. Elespuru, R. K. & Lijinsky, W. (1973). The formation of carcinogenic nitroso compounds from nitrite and some types of agricultural chemicals. Fd Cosmet. Toxicol. II, 807. Elespuru, R., Lijinsky, W. & Setlow, J. K. (1974). Nitrosocarbaryl as a potent mutagen of environmental significance. Nature, Lond. 241, 386. Ivankovic, S. u. Preussmann, R. (1970). Transplazantare Erzeugung maligner Tumoren nach oraler Gabe von Athylharnstoff und Nitrit an Ratten. Naturwissenschafterl 57, 460. Lijinsky, W. & Elespuru, R. K. (1976). Mutagenicity and carcinogenicity of N-nitroso derivatives of carbamate insecticides. 1; Enuironmental N-Nitroso Compounds: Analvsis and Formation. Edited bv E. A. Walker, P. Bog&ski and L. Griciute. IARC Scientific Publns.‘no. 14, p. 425. International Agency for Research on Cancer Lyon. Lijinsky, W. & Taylor, H. W. (1976). Carcinogenesis in Sprague-Dawley rats of N-nitroso-N-alkylcarbamate esters. Cancer Letters 1, 275. Regan, J. D., Setlow, R. B., Francis. A. A. & Lijinsky, W. (1976). Nitrosocarbaryl: its effect on human DNA. Mutation
Res. 38, 293.
PRELIMINARY TRANSPLACENTAL CARBARYL
COMMUNICATION
CHRONIC TOXICITY TEST .OF WITH NITRITE IN RATS*
W. LrrrNsrcvt and H. W. TAYLOR~ Carcinogenesis Program. Biology Division Oak Ridge National Oak Ridge, (Received
Tennessee, 26 October
Laboratory,
USA 1976)
Summary-Pregnant female rats were given by gavage a total of 300mg carbaryl during a lo-day period. No malignant tumours were induced in the dams or their offspring during their natural lifespan. Pregnant and non-pregnant female rats were given a suspension of carbaryl in sodium nitrite solution on several successive days, a total of 90 mg carbaryl and 120 mg sodium nitrite being administered to each adult rat. There was no significant incidence of malignant tumours in the dams or their offspring as a result of the treatment.
Introduction Carbaryl, the l-naphthyl ester of N-methylcarbamic acid, is a widely used insecticide which is permitted as a residue on crops, a tolerance level having been set for it by the Food and Drug Administration. Carbaryl itself is of interest as a possible environmental contaminant, and as a member of the large group of N-methylcarbamate esters which are used as insecticides. Several of them have been tested for toxicity and appear to be relatively non-toxic in mammals; neither do they show mutagenic activity in bacterial systems. However, because they can react with nitrous acid to form N-nitroso derivatives (Elespuru & Lijinsky, 1973), another aspect of their presence in the environment must be considered, namely the possible adverse biological effects of the nitroso compounds so formed. It has been demonstrated that nitrosocarbaryl, which can interact with human DNA in oitro (Regan, Setlow, Francis & Lijinsky, 1976), is a potent bacterial mutagen (Elespuru, Lijinsky & Setlow, 1974), as are the nitroso derivatives of several other methylcarbamate insecticides (Lijinsky & Elespuru, 1976). Nitrosocarbaryl is also a fairly potent carcinogen when administered orally to rats (Eisenbrand, Schmlhl & Preussman, 1976; Lijinsky & Taylor, 1976) and, because formation of nitrosocarbaryl from ingested carbaryl and nitrite is the most likely mode of human exposure, it was decided to investigate its formation in this way in rats. Since the amount formed in a rat’s stomach was likely to be small, the transplacental route of exposure for the rats was chosen. This *Research supported by the National Cancer Institute under contract with the Energy Research and Development Administration and Union Carbide Corporation. fPresent address: Chemical Carcinogenesis Prog&, Frederick Cancer Research Center, Frederick, Maryland 21701, USA. IPresent address: Department of Pathology and Parasitology, School of Veterinary Medicine, Auburn University, Auburn, Alabama 36830, USA. 229
is considered the most sensitive mammalian test system, and has responded to very small doses of nitroso compounds formed in this way in uiuo (Ivankovic & Preussmann, 1970).
Experimental Technical-grade carbaryl (‘Sevin’, kindly supplied by Union Carbide Corp., Bound Brook, NJ) was crystallized from acetone, after insoluble dark material had been filtered off, colourless crystals, m.p. 143-144°C being obtained. These were finely powdered in a mortar and mixed with water to give a fairly uniform suspension which would pass through the needle of a small syringe. The suspensions given to the animals by gavage contained carbaryl (30mg/ml) in water or in sodium nitrite solution, (40 mg NaN02/ml). Twenty-four female Sprague-Dawley rats, 1214 wk old, were mated by leaving them with males for a week-end, in which time the majority would normally be expected to become pregnant. They were divided into three groups of eight animals. Those in group 1 were given 1 ml of a suspension of 30 mg carbaryl in water on each of 10 days between day 4 and 18 of gestation (weekends omitted). One animal died during the treatment and three of the remainder gave birth to a total of nine female and 13 male rats. Group II rats were given 1 ml of a suspension of 30 mg carbaryl in 4% sodium nitrite solution on days 4, 5 and 6 of gestation (first trimester). At that time six of the eight animals died of nitrite poisoning and the treatment was discontinued. Only one of the remaining animals gave birth, to a litter of seven females and three males. Group III rats were given only 0.6 ml of the suspension given to group II, to avoid nitrite poisoning. Treatment was given on each of days 14-18 of gestation (last trimester). No animals died and six gave birth to litters totalling 32 females and 32 males. To provide additional numbers for the significance of tumour incidences in the animals given carbaryl with nitrite to be assessed, an additional
.
Carbaryl + NaN02 Carbaryl + NaN02
Carbaryl + NaN02
II, III, IV II
III
Transpiacental
Intragastric Transplacental
Route Intragastric Transplacental
14-18
14-18t 4-6
Time of exposure* 4-18 4-18
of rats treated
*Days of gestation on which test solution was administered to dams. Non-pregnant tOr days 4-6 (group II).
Treatment Carbaryl Carbaryl
Group no. I I
Table 1. Survival
60 I 9 12 22 7 3 30 32
or carbaryl
IO 7 9 12 20 I 3 28 32
and
9 11 20 I 3 28 31
1
80
nitrite
90 5 9 10 18 6 3 26 21
13 5 3 23 24
100 3 l
110 2 6 5 9 4 2 15 16
120 2 3 2 7 2 1 10 5
t!l 0 2 0 5 2
130 1
No. of animals surviving at week
females of group IV were treated on an equivalent no. of days.
No. of rats/group 7F 9F 13M 22F 7F 3M 32F 32M
with carbaryl
3 0
1
0
1
1
140 - 150 1 0
0
0
160
e F 2 9 !i ii r el 5 i
Transplacental
Carbaryl + NaNO*
III
1418
4-6
1418t
4-18
4-18
treated
1 22 4
3M 32F 32M
13
ii
4
Breast
rats
5
in
7F
22F
9F 13M
6F
rats/ w-w
No. of
Tumours
*Days of gestation on which test solution was administered to dams. Non-pregnant tOr days 46 (group II).
Transplacental
Carbaryl + NaNO,
II
Intragastric
Carbaryl + NaNO,
Transplacental
Carbaryl
I
II, III, IV
Intragastric
Carbaryl
I
Route
Treatment
Group no.
Time of exposure*
Table 2.
carbaryl
nitrite
6
1 1
1 4 6
0
Pancreas
1
Adrenal
and
0
2
0
0
Liver
0
1
0
0
Uterus
No. of animals with turnours of
or carbaryl
Stomach papilloma (1) Lymphosarcoma (1) Mesothelioma (1) Myeloid leukaemia (2) Osteoadenocarcinoma of gut (1) Adenocarcinoma of gut (1) Lymphosarcoma (1) Myeloid leukaemia (1) Mesothelioma (1) Zymbal gland carcinoma (1) Skin trichoepithelioma (1)
Lymphosarcoma (1) Oesophageal papilloma (1) Lymphangioma (1) Lymphosarcoma (1) Myeloid leukaemia (1) Thyroid carcinoma (1)
(1)
(1)
Other tissues Lymphosarcoma Leukemia (1) Adenocarcinoma
females of group IV were treated on an equivalent no. of days.
4
1 17
2
11
7 2
3
Pituitary
with
232
W.
LUINSKY
and
twelve rats (group IV) were given the same treatment as group III on five successive days. ‘All the adults and offspring, were observed until natural death except a few that were killed when moribund. Complete autopsy was performed and all tumours and other lesions were fixed for histological examination, together with most grossly normal major organs.
Results and Discussion As seen in Table 1, survival of all of the rats was good (with the exception of those killed by nitrite poisoning) and there was little difference between the groups when account was taken of the longer period of experimental observation of the offspring which were treated in utero. In each group at least half of the animals were alive after 2 years and a number survived beyond 2.5 years. Therefore, the dose of carbaryl given, with or without nitrite, had no apparent life-shortening effect. The distribution of tumours in the rats of the various groups is shown in Table 2. The common tumours in all groups were those normally seen in Sprague-Dawley rats, notably mammary fibroadenomas and tumours of the pituitary (predominantly in females) and adrenal tumours (mostly cortical adenomas). There were a few islet-cell adenomas of the pancreas and some hepatocellular carcinomas. The distribution of these tumours did not suggest any treatment-related effect on their incidence. In the females treated with carbaryl alone, and in their offspring, there were very few tumours other than those mentioned, and it appears that the dose of carbaryl administered, 300 mg or 1.5 mmol was without tumorigenic effect. Although this is by no means an exhaustive test of the carcinogenicity of carbaryl, the lack of response of the transplacentally exposed rats suggests that the tumorigenic effect of carbaryl, if any, is exceedingly weak. There is a small possibility that the compound does not cross the placenta, but it has been shown to be readily absorbed from the stomach (C&per, Pekas & Dinusson, 1973). The offspring of the females receiving carbaryl plus nitrite in the latter part of pregnancy gave no indication of a transplacental carcinogenic effect of the nitrosocarbaryl formed in the stomach. Nitrosocarbaryl is a fairly potent carcinogen when administered to rats, producing squamous carcinomas of the stomach when given intragastrically (Eisenbrand et al., 1976; Lijinsky & Taylor, 1976) and sarcomas when given SC (Eisenbrand, Ungerer & Preussmann, 1975), and can be formed to some extent in vitro even at
H. W. TAYLOR
low concentrations of carbaryl and nitrite (Eisenbrand et al. 1975). We conclude that either insufficient nitrosocarbaryl was formed in the rats in uiuo during the 5 days of treatment with 18mg carbaryl plus 24mg sodium nitrite, or that unlike methylnitrosourea and ethylnitrosurea a significant proportion of the nitrosocarbaryl formed does not cross the placenta of rats. While transplacental treatment of rats with larger quantities of nitrosocarbaryl might establish this point, the results of these experiments demonstrate no overt transplacental carcinogenic hazard from ingestion of carbaryl and nitrite together. Neither do they establish conclusively the safety of a combination of carbaryl and nitrite when ingested by humans, who might be a more sensitive species to nitrosocarbaryl (received directly or transplacentally). However, an experiment of this kind with its negative results does give a little more comfort than results that are clearly positive. REFERENCES
Casper, H. H., Pekas, J. C. & Dinusson, W. E. (1973). Gastric absorption of a pesticide (I-napthyl-N-methylcarbamate) in the fasted rat. Pesric. Biochem. Pkysiol. 2, 391. Eisenbrand, G., Schmlhl, D. & Preussman, R. (1976). Carcinogenicity in rats of high oral doses of N-nitrosocarbaryl, a nitrosated pesticide. Cancer Letters 1, 281. Eisenbrand, G., Ungerer, 0. & Preussmann, R. (1975). The reaction of nitrite with pesticides. II. Formation, chemical properties and carcinogenic activity of the N-nitroso derivative of N-methyl-1-naphthyl carbamate (carbaryl). Fd Cosmet. Toxicol. 13, 365. Elespuru, R. K. & Lijinsky, W. (1973). The formation of carcinogenic nitroso compounds from nitrite and some types of agricultural chemicals. Fd Cosmet. Toxicol. II, 807. Elespuru, R., Lijinsky, W. & Setlow, J. K. (1974). Nitrosocarbaryl as a potent mutagen of environmental significance. Nature, Lond. 241, 386. Ivankovic, S. u. Preussmann, R. (1970). Transplazantare Erzeugung maligner Tumoren nach oraler Gabe von Athylharnstoff und Nitrit an Ratten. Naturwissenschafterl 57, 460. Lijinsky, W. & Elespuru, R. K. (1976). Mutagenicity and carcinogenicity of N-nitroso derivatives of carbamate insecticides. 1; Enuironmental N-Nitroso Compounds: Analvsis and Formation. Edited bv E. A. Walker, P. Bog&ski and L. Griciute. IARC Scientific Publns.‘no. 14, p. 425. International Agency for Research on Cancer Lyon. Lijinsky, W. & Taylor, H. W. (1976). Carcinogenesis in Sprague-Dawley rats of N-nitroso-N-alkylcarbamate esters. Cancer Letters 1, 275. Regan, J. D., Setlow, R. B., Francis. A. A. & Lijinsky, W. (1976). Nitrosocarbaryl: its effect on human DNA. Mutation
Res. 38, 293.
