Fd Chem. Toxic. Vol. 29, No. 2, pp. 119-124, 1991
0278-6915/91 $3.00+ 0.00 Pergamon~ pie
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CARCINOGENICITY OF p-CHLOROANILINE IN RATS A N D MICE R. S. CHHABRA,J. E. HUFF,J. K. HASEMANand M. R. ELWELL National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 and A. C. PETERS Battelle Memorial Institute, Columbus, OH 43201, USA (Received 4 September 1990; revisions received 21 November 1990)
Abstract--p-Chloroaniline (PCA), a dye intermediate, was evaluated for potential long-term toxicity and carcinogenicity. Groups of 50 F344/N rats of each sex were given by gavage PCA hydrochloride in deionized water at doses of 0, 2, 6 or 18 mg/kg body weight, 5 days/wk for 103 wk. Groups of 50 male and female B6C3Ft mice of each sex were given 0, 3, 10 or 30 mg/kg on the same schedule. In general, body weights and survival were unaffected by PCA administration. In rats the group given 18 mg/kg had mild haemolytic anaemia and slight increases in methaemoglobin at various times during the study. Fibrosis of the spleen was significantly increased in all PCA-treated groups of male rats and in the 18-mg/kg group of female rats. Sarcomas of the spleen occurred in male rats, their incidence being 0/49, 1/50, 3/50 and 38/50 in control low-, mid- and high-dose groups, respectively. There was a slightly increased incidence of pheochromocytomas of the adrenal gland in both male and female rats. Dosed groups of male mice had increased incidences of hepatocellular adenomas or carcinomas (11/50, 21/49, 20/50 and 21/50 in controls, low- mid- and high-dose groups, respectively). Haemangiosarcomas of the liver or spleen were also increased in the high-dose group (incidences of 4/50, 4/49, 1/50 and 10/50 in controls, low-, mid- and high-dose groups, respectively). In conclusion, PCA was carcinogenic in male rats and male mice.
INTRODUCTION p-Chloroaniline (PCA) is an aromatic amine widely used in the dye, textile and rubber industries (Beard and Hoe, 1981). It is used as an intermediate in the manufacture of more than l0 dyes and pigments (Colour Index, 1971) and has been detected as a degradation product in some pharmaceutical preparations (Ciarlone et al., 1976). PCA was identified as a degradation product of 4,4'-dichloro-3-(trifluoromethyl) carbanilide, an active component of deodorant soap bars (Demers and Yates, 1977). In rats, PCA is one of the metabolites of the urea herbicides monuron, buturon and monolinuron (Ernst, 1969; Hargesheimer et al., 1981). The primary toxic effect of many aromatic amines is methaemoglobin formation. Specific information on PCA toxicity in humans is limited. Short-term exposure of humans to PCA results in cyanosis, a manifestation of methaemoglobin formation that could develop with or without loss of haemoglobin (Beard and Hoe, 1981). Toxicological studies in rats and mice have shown that the haemopoietic system is the major target for PCA and a number of other aromatic amines. Methaemoglobin formation and the accompanying haemolytic anaemia, extramedullary haematopoiesis and splenomegaly were indicative of erythrocyte toxicity and regenerative
Abbre~'iation: PCA = p-chloroaniline.
anaemia induced by PCA in a 90oday study (Chhabra et al., 1990). Carcinogenesis studies on PCA in feed were conducted by the National Cancer Institute in Fischer 344 rats and B6C3F~ mice of each sex (NCI, 1979). Concentrations of 250 or 500 ppm for rats and 2500 or 5000 ppm for mice were administered for 78 wk and then the animals were observed for a further 24 wk (rats) or 13 wk (mice). In that study rare splenic neoplasms occurred in dosed male rats; however, the number of neoplasms was insufficient to establish unequivocally the carcinogenicity of PCA (NCI, 1979). The widespread exposure of workers to PCA in the dye, chemical and pharmaceutical manufacturing industries and the structural resemblance of PCA to known carcinogens such as aniline and its analogues (Bus and Popp, 1987; Weisburger, 1983) were the reasons for re-evaluating this chemical for carcinogenicity in laboratory animals. The studies described in this paper were conducted in F344/N rats and B6C3F~ mice of each sex by gavage. The details of this study have been reported in a comprehensive technical report prepared by the National Toxicology Program (NTP, 1989). MATERIALS AND METHODS
Chemicals. PCA ($2 flakes, technical grade) was obtained in one lot from E.I. DuPont de Nemours and Co, Inc. (Wilmington, DE, USA). PCA was
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R.S. CHHABRAet al.
identified as p-chloroaniline by infra-red, ultraviolet/visible, and nuclear magnetic resonance spectra. Purity was determined by elemental analysis, Karl Fischer water analysis, titration of the amine component with perchloric acid, thin-layer chromatography and gas chromatography. Cumulative data indicated that PCA was 99.1% pure. All doses administered to animals in these studies were prepared from weighed quantities of PCA dissolved in water containing hydrochloric acid. All doses refer to the PCA content of the solution. To prepare a standard solution, PCA and molar equivalents of 1.0N-hydrochloric acid were mixed in a calibrated container. The resulting PCA hydrochloridc solution was diluted to the desired concentration with dcionizcd water. The solution had a pH of approximately 2. Quantitative analysis of aqueous solutions of PCA hydrochloridc showed that the dose solutions were stable for at least 14 days at room temperature. The dose solutions were prepared weekly, placed in foil-wrapped containers, and refrigerated until the day of dosing. Animals. Male and female F344/N rats were obtained from Charles River Breeding Laboratories (Kingston, NY, USA) and B6C3F~ mice were purchased from the Frederick Cancer Research Center (Frederick, MD, USA). The rodents were shipped to the study laboratory at 5 ~ wk of age. The rats were quarantined for 20 days and mice for 12 days. Thereafter, a complete autopsy was performed on five animals of each sex and species in order to assess their health status. The rodents were placed on study at 7-8 wk of age. They were housed five to a cage with absorb-dri hardwood chip as bedding, and given NIH 07 feed and tap-water ad lib. Cages and racks were rotated. The animal room temperature was 70-74°F and the relative humidity was in the range 40 to 60%. Fluorescent lights were on for 12 hr/day and room air was changed 15 times/hr. Experimental design. Groups of 49 or 50 rats of each sex were given by gavagc PCA HCI in deionizcd water at doses of 2, 6 or 18 mg/kg body weight for 103 wk. Groups of 50 mice of each sex were given 3, 10 or 30 mg/kg on the same schedule. Vehicle controls were given deionized water by gavagc. Blood samples were collected in Vacutaincrs containing EDTA by rctro-orbital puncture from 15 randomly selected rats from each group at 6, 12, 18 and 24 months. Hacmatological analyses were per-
formed with an Ortho ELT-8 Hematology Analyzer. Tbe methaemoglobin concentration was determined by the method of Evelyn and Malloy (1938). All animals were observed twice a day. Clinical signs were recorded once a week for 13 wk and then at least once every 4 wk. Individual body weights were recorded once a week for the first 13 wk and once a month thereafter. Mean body weights were calculated for each group. Animals found moribund and those surviving to the end of the studies were humanely killed. An autopsy was performed on all animals. At autopsy, all organs and tissues were examined for grossly visible lesions. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and slides made were stained with hacmatoxylin and eosin. Histopathological examination of tissues was carried out according to an inverse pyramid design (McConncll, 1983a,b); complete histopathological examinations were performed on tissues from all animals in the high-dose and vehicle control groups and on tissues from animals in lower dose groups that died during the study. In addition, samples from all grossly visible lesions in animals of all dose groups were examined histopathologically. Potential target organs for chemically related neoplastic and non-neoplastic effects were identified; these target organs]tissues from animals in the lower dose groups wcrc examined histopathologicaily. Statistical analysis. The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Taronc's (1975) life-table test for dose-related trend. Data on turnout incidence were analysed by life-table tests (appropriate for fatal tumours) and logistic regression analysis (appropriate for turnouts observed in animals dying of an unrelated cause). For further discussion of these statistical methods, see Haseman (1984) and Dinse and Haseman (1986). RESULTS
Rats The mean body weights of treated groups were generally within 5-6% of those of vehicle controls (Table 1). The survival rates of groups exposed to
Table I. Survival and average final body weights of rats in the 2-yr garage studies of PCA-hydrochloride Dose of PCA (mg/kg body weight) Parameter No. of rats initially in study No. of rats at termination Survival P value* Final body weight (g) Animals initially in study Animals at termination Survival P value* Final body weight (g)
0 (Controls)
2
6
18
M~I~ 49 18 0.793 442
50 32 0.007 462
50 32 0.005 453
50 20 0.367 462
F~m~dcs 50 27 0.244 323
50 39 0.011 333
50 36 0.075 334
50 37 0.043 310
PCA = p-chloroaniline *The result of the life-table trend test is in the vehicl© control column, and the results of the life-table pairwisc comparisons with the vehicle controls are in the dosed columns.
Carcinogenicity study of p-chloroaniline PCA were generally higher than those of the vehicle controls (Table l). This was possibly due to a decreased incidence of leukaemia in PCA-exposed animals. Animals given the mid- or high-dose had blue extremities, indicative of cyanosis. Haematological evaluations after 6, 12, 18 and 24 months of treatment revealed mild haemolytic anaemia in rats given 18 mg/kg (data not shown), and a slight elevation of methaemoglobin in treated groups. In treated rats 0.34-4.0% of haemoglobin was converted to methaemoglobin in comparison with 0.26-1.67% in vehicle controls. The incidence of proliferative mesenchymal lesions in the spleen was increased in PCA-treated rats (Table 2). Fibrosis of the spleen was increased in dosed males and females, and when extensive in the red pulp it replaced many or all of the haematopoietic cells. The incidence of sarcomas (fibrosarcoma, osteosarcoma, haemangiosarcoma) was significantly increased in high-dose males. Many of the splenic sarcomas metastasized to one or more sites including most of the major abdominal organs, lungs and lymph nodes. One fibrosarcoma was observed in the spleen of a mid-dose female, and one splenic osteosarcoma occurred in a high-dose female. In rats in the high-dose group, metaplasia of the stroma of the spleen consisted of multiple foci of lipocytes in the fibrotic stroma; these foci were also present in some of the splenic sarcomas. Pheochromocytomas or malignant pheochromocytomas (combined) in male rats occurred with a significant positive trend; the incidence in the highdose group was significantly greater than that in the vehicle controls. Adrenal gland medullary hyperplasia was increased in high-dose female rats; pheochromocytomas were slightly increased in high-
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dose females (Table 2). In PCA-dosed rats, hypcrplasia of the bone marrow was increased in incidence but not in average severity. The incidence of mononuclear cell leukaemia was significantly lower in all dosed groups of males and females than in vehicle controls.
Mice The mean body weights of high-dose male and female mice were generally within 5% of those of vehicle controls throughout the studies (Table 3). No compound-related clinical signs of toxicity were noticed. The survival of the mid-dose group of male mice was significantly lower than that of the vehicle controls after wk 99. No other significant differences in survival were observed between groups (Table 3). The incidences of hepatocellular carcinomas in mid- and high-dose males and of hepatocellular adenomas or carcinomas (combined) in low-, midand high-dose males were significantly greater than those in vehicle controls (Table 4). Hepatocellular carcinomas metastasized to the lungs in 1/50 vehicle control, 1/49 low-dose, 2/50 mid-dose and 9/50 highdose male mice. Hepatocellular adenoma and carcinoma (considered separately or combined) were not significantly increased in female mice. The incidence of haemangiosarcomas in high-dose male mice was slightly increased relative to that in vehicle controls. Haemangiosarcomas occurred primarily in the liver and spleen. There were no treatment-related differences in the incidence of haemangiosarcoma in female mice. The incidence of lymphoma was significantly decreased in male mice given the low or high dose of PCA and in female mice in mid- and high-dose group (Table 4).
Table 2. Summary of treatment-related lesions in rats exposed to p-PCA hydrochloride for 2 yr Incidence (no. of rats affected/no, examined) Site and lesion
PCA dose (rag~ kg body weight)...
0 (Controls)
2
6
18
Males Spleen Fibrosis Stromal metaplasia Fibroma SarcomaS" Bone marrow Hyperplasia Adrenal medulla Hygerplasia Pheochromocytoma Haematopoietic system Mononuclear cell leukaemia
3/49 0/49 0/49 0/49
11/50* 0/50 0/50 1/50
12/50' 0/50 0/50 3/50
41/50"* 24/50** 2/50 38/50**
26/49
36/50*
35/49*
46/50**
15/49 13/49
21/48 14/48
15/48 15/48
17/49 26/49*
21/49
3/50**
2/50**
3/50**
1/50 0/50 0/50
2/50 0/50 0/50
3/50 0/50 1/50
42/50** I 1/50"* 1/50
1I/S0
12/48
21/50
37/47**
4/50 2/50
4/50 3/50
7/50 l/50
24/50** 6/50
2/50*s
1/50**
Females Spleen Fibrosis Stromal metaplasia Sarcomat Bone marrow Hypcrplasia Adrenal medulla Hyperplasia Pheochromocytoma Haematopoietic system Mononuclear cell leukaemia
10/50
1/50"*
PCA - p-chloroaniline ~'Fibrosarcoma, osteosarcoma or haemangiosarcoma. Values marked with asterisks differ significantly from the corresponding values for the controls (*P < 0.05; **P < 0.01).
FCT 2~.:2-- D
R . S . C H 1 4 A a ~ e t al.
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Table 3. Survival and average final body weights of mice in the 2-yr gavage studies of
PCA-hydrochloride Dose of PCA (mg/kg body weight) Parameter
0 (Controls)
No. of mice initially in study No. of mice at termination Survival P value* Final body weight (g) Animals initially in study Animals at termination Survival P value* Final body weight (g)
3
10
Mal¢~ 50 43 0.295 42.9
30
50 36 0.211 42.2
50 29 0.005 41.8
50 35 0. 110 41.1
Female~ 50 39 0.875 44.4
50 42 0.298 43.9
50 44 0.408 45.2
50 41 0.966 43.5
PCA = p-chloroaniline *The result of the life-table trend test is in the vehicle control column, and the results of the life table pairwis¢ comparisons with the vehicle controls are in the dosed columns. DISCUSSION
Earlier 2-yr studies had strongly indicated the carcinogenicity of PCA because fibromas and sarcomas were observed in the spleens of treated male rats (NCI, 1979). These mesenchymal neoplasms (six fibromas and four sarcomas) occurred in the spleens of high-dose male rats (fed PCA at 500 ppm in the diet). Haemangiomatous neoplasms were the principal neoplastic lesions seen in mice in the NCI study. They were observed in 2/20, 10/50 and 14/50 males and 0/18, 3/49 and 8/42 females given PCA at 0, 2500 or 5000 ppm in the feed, respectively. The findings of these NCI studies have been confirmed by the results of the studies reported here. PCA was considered to be carcinogenic in male rats, because of the marked increased incidence of sarcomas in the spleen (38/50) in the high-dose group. The results of the two studies do differ in the incidences of neoplasms in the spleen. The reasons for these differences were not specifically studied but could be related to dose and route of administration. It is possible that in the NCI studies PCA was
administered at less than the targeted concentrations because of the instability of the chemical mixed in feed. The target concentrations in feed were approximately equivalent to 15 and 30 mg/kg body weight for rats, compared with 2, 6 and 18 mg/kg in our studies in which PCA was administered by gavage. The different modes of oral administration (single dose per day by gavage v. continuous dosing by feed) could have resulted in differences in metabolism of the chemical and this could have been responsible for the quantitative differences between the NCI studies and those reported here. The dose response for the development of splenic tumours in male rats in the current study was non-linear with respect to dose. However, because of their rare occurrence in controls, the few sarcomas in the female rats in the 1ow(!/49) and mid-dose (3/50) groups were considered most likely to be due to PCA administration. The doses used in these studies apparently did not saturate the metabolic and excretory pathways, as determined by disposition studies on PCA (NTP, 1989). The female rats appeared to be less sensitive than the males to the induction of splenic neoplasms by
Table 4. Summary of treatment-related neoplastic lesions in mice exposed to p-PCA hydrochloride for 2 yr Incidence (no. of mice affected/no, examined) Site and lesion
PCA dose rag/ kg body weight)...
O(Controls)
2
6
18
Mal¢~ Liver Adenoma Carcinoma Adenoma/carcinoma Haemangiosarcoma Spleen Haemangiosarcoma Liver and/or spleen Haemangiosarcoma Haematopoietic system Lymphoma
9/50 3/50 I I/50t 2/50
15/49 7•49 21/49t 2/49
10/50 l I/50* 20/50"~f 1/50
4/50 17/50"* 21/50* 6/50
3/50
2/49
0/50
5/50
4/50~
4/49
1/50
I 0/50~
10/50
3/49*
9/50
3/50*
6/50
9/50
8/50
I I/50
2/50
1/50
1/50
I/50
19/50
12/50
Femal~ Liver Adenoma/carcinoma Liver and/or spleen Haemangiosarcoma Haematopoietic system Lymphoma
5/50**
PCA = p-chloroaniline *One mouse had adenoma and carcinoma. :~Onc mouse had haemangiosarcoma in liver and spleen. Values marked with asterisks differ significantly from the corresponding control values (*P < 0.05; **P < 0.01).
10/50"
Carcinogenieity study of p-chloroaniline PCA; neoplasms of the spleen were seen in only one mid-dose and one high-dose female rat. This sex difference in induction of splenic neoplasms is consistent with results obtained in studies in rats of other aniline dyes. However, the incidences of splenic fibrosis in high-dose male (41/50) and female (42/50) rats were similar. Fibrosis and stromal metaplasia (lipocyte or fat infiltration) have been observed in the spleens of rats administered aromatic amines or their derivatives. No splenic fibrosarcomas or osteosarcomas were observed in mice of either sex dosed with PCA. Such a lack of response was expected because of the results of studies with other anilines (Weisburger, 1983). None of the aromatic amines studied have increased the incidences of splenic tumours in mice. However, induction of hepatic neoplasms by those chemicals was frequently observed in mice, and in the present study the incidences of hepatocellular carcinomas were increased in PCA-treated male mice. Haemangiosarcomas of the liver and/or spleen were found in male mice and the number was significantly increased in the high-dose group in comparison with the controls. Mice responded differently from rats with respect to the spleen as a target organ for PCA toxicity. Although in the present study no non-neoplastic splenic effects were observed in mice, in a 90-day study (Chhabra et al., 1990) splenic weights were increased and increased extramedullary haematopoiesis was observed in the spleens of mice given 30 mg PCA/kg body weight. The differences between rats and mice with regard to the development of splenic and liver neoplasms could be due to the differences in metabolism and disposition of PCA. PCA was cleared from blood at a much faster rate in mice than in rats. Perry et al. (1981) studied the disposition of [~C]PCA in F344 rats, dogs and A/J and Swiss Webster mice. They reported that the initial decay constants for PCA clearance from whole blood in both strains of mice were 10 times greater than those in dogs and rats. The PCA clearance in mice was too rapid to permit calculation of kinetic parameters. The reports of development of tumours of the spleen in F344 rats treated with structurally related aniline compounds led some workers to study the pathogenesis of splenic lesions and the disposition of radioactive aniline in the rat in order to elucidate the possible mechanism(s) of action of these chemicals (Bus and Popp, 1987; Goodman et al., 1984). These studies indicated that splenic tumours in rats induced by aniline and related compounds are most likely to be the result of a non-genotoxic mechanism. Erythrocyte toxicity, as evidenced by methaemoglobin formation, is the major toxic effect of aniline and related compounds in various species. A toxic effect of PCA on erythrocytes was demonstrated in the 13-wk study (Chhabra et al., 1990). In the present study the increased incidence of hyperplasia of the bone marrow observed after 2 years of treatment may have been related to the mild haemolytic anaemia or secondary to the decrease in the normal extramedullary haematopoiesis in the spleen of rats as a result of the extensive fibrosis present. It is possible that the methaemoglobin bound to aniline com-
123
pounds or their reactive metabolites was broken down in the red pulp of the spleen. When released from the haemoglobin these reactive metabolites could bind to the splenic mesenchymal tissues and stimulate the formation of turnouts. However, it is possible that a direct-acting genotoxic mechanism was involved in the induction of the neoplasms; McCarthy et al. (1985) found that in rats and mice [~4C]aniline binds to a greater extent to the kidney, small intestine, large intestine and spleen than to other tissues. Protein and RNA were the major macromolecular targets for [~4C]aniline binding; DNA binding occurred to a lesser extent. The possibility for involvement of genotoxic mechanisms is further supported by data reported by Parodi et al. (1982a,b). In their studies aniline induced DNA damage in vivo in the liver and kidney of rats. Also, aniline was positive in the induction of sister chromatid exchanges in vivo in male Swiss mice. The splenic toxicity and carcinogenicity induced by PCA in male rats seem to be similar to those produced by aniline in rats. Whether the mechanism of carcinogenesis was mediated through genotoxic or non-genotoxic events remains unresolved; either or both may influence the carcinogenic responses in male rats (spleen) and male mice (liver). NTP studies (NTP. 1989) have shown that in vitro PCA was clearly genotoxic in the Salmonella assay and in tests for chromosomal aberrations in Chinese hamster ovary cells. However, it was mutagenic in the presence of S-9 activating system and thus, the in r'itro genotoxic activity of PCA appears to be dependent on metabolism for its full expression. Experimental evidence indicates that PCA undergoes oxidative transformations as a reactive amine (Smith and Gorrod, 1978; Uehleke and Hellmer, 1971), with the probable formation of electrophilic intermediates that could be stabilized by chlorine. For example, p-chloro-Nhydroxyaniline (Von Jagow et al., 1966), which may be formed through a nitronium ion intermediate in the presence of oxidative enzymes, and p-aminophenol (Ichikawa et al., 1969), which may be formed by an arene oxide intermediate possibly in the absence of metabolizing enzymes, have been reported as metabolites of PCA in the rabbit. Both of these intermediates are potential electrophiles and could covalently bind macromolecules. In conclusion, PCA was considered to be carcinogenic in male rats on the basis of the increased incidences of sarcomas of the spleen. These neoplasms occur rarely in control rats (Solleveld et al., 1981). Pheochromocytomas of the adrenal medulla may also have been increased by PCA administration. Evidence of carcinogenicity in female rats was slight, as indicated by the presence of sarcomas of the spleen in one mid- and one high-dose animal and the slightly increased incidence of pheochromocytoma of the adrenal gland. PCA was considered to be carcinogenic in male mice on the basis of increased incidences of hepatocellular neoplasms and of haemangiosarcomas of the liver or spleen. REFERENCE~ Beard R. R. and Noe J. T. (1981) Aromatic nitro and amino compounds. In Patty's Industrial Hygiene and
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CARCINOGENICITY OF p-CHLOROANILINE IN RATS A N D MICE R. S. CHHABRA,J. E. HUFF,J. K. HASEMANand M. R. ELWELL National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 and A. C. PETERS Battelle Memorial Institute, Columbus, OH 43201, USA (Received 4 September 1990; revisions received 21 November 1990)
Abstract--p-Chloroaniline (PCA), a dye intermediate, was evaluated for potential long-term toxicity and carcinogenicity. Groups of 50 F344/N rats of each sex were given by gavage PCA hydrochloride in deionized water at doses of 0, 2, 6 or 18 mg/kg body weight, 5 days/wk for 103 wk. Groups of 50 male and female B6C3Ft mice of each sex were given 0, 3, 10 or 30 mg/kg on the same schedule. In general, body weights and survival were unaffected by PCA administration. In rats the group given 18 mg/kg had mild haemolytic anaemia and slight increases in methaemoglobin at various times during the study. Fibrosis of the spleen was significantly increased in all PCA-treated groups of male rats and in the 18-mg/kg group of female rats. Sarcomas of the spleen occurred in male rats, their incidence being 0/49, 1/50, 3/50 and 38/50 in control low-, mid- and high-dose groups, respectively. There was a slightly increased incidence of pheochromocytomas of the adrenal gland in both male and female rats. Dosed groups of male mice had increased incidences of hepatocellular adenomas or carcinomas (11/50, 21/49, 20/50 and 21/50 in controls, low- mid- and high-dose groups, respectively). Haemangiosarcomas of the liver or spleen were also increased in the high-dose group (incidences of 4/50, 4/49, 1/50 and 10/50 in controls, low-, mid- and high-dose groups, respectively). In conclusion, PCA was carcinogenic in male rats and male mice.
INTRODUCTION p-Chloroaniline (PCA) is an aromatic amine widely used in the dye, textile and rubber industries (Beard and Hoe, 1981). It is used as an intermediate in the manufacture of more than l0 dyes and pigments (Colour Index, 1971) and has been detected as a degradation product in some pharmaceutical preparations (Ciarlone et al., 1976). PCA was identified as a degradation product of 4,4'-dichloro-3-(trifluoromethyl) carbanilide, an active component of deodorant soap bars (Demers and Yates, 1977). In rats, PCA is one of the metabolites of the urea herbicides monuron, buturon and monolinuron (Ernst, 1969; Hargesheimer et al., 1981). The primary toxic effect of many aromatic amines is methaemoglobin formation. Specific information on PCA toxicity in humans is limited. Short-term exposure of humans to PCA results in cyanosis, a manifestation of methaemoglobin formation that could develop with or without loss of haemoglobin (Beard and Hoe, 1981). Toxicological studies in rats and mice have shown that the haemopoietic system is the major target for PCA and a number of other aromatic amines. Methaemoglobin formation and the accompanying haemolytic anaemia, extramedullary haematopoiesis and splenomegaly were indicative of erythrocyte toxicity and regenerative
Abbre~'iation: PCA = p-chloroaniline.
anaemia induced by PCA in a 90oday study (Chhabra et al., 1990). Carcinogenesis studies on PCA in feed were conducted by the National Cancer Institute in Fischer 344 rats and B6C3F~ mice of each sex (NCI, 1979). Concentrations of 250 or 500 ppm for rats and 2500 or 5000 ppm for mice were administered for 78 wk and then the animals were observed for a further 24 wk (rats) or 13 wk (mice). In that study rare splenic neoplasms occurred in dosed male rats; however, the number of neoplasms was insufficient to establish unequivocally the carcinogenicity of PCA (NCI, 1979). The widespread exposure of workers to PCA in the dye, chemical and pharmaceutical manufacturing industries and the structural resemblance of PCA to known carcinogens such as aniline and its analogues (Bus and Popp, 1987; Weisburger, 1983) were the reasons for re-evaluating this chemical for carcinogenicity in laboratory animals. The studies described in this paper were conducted in F344/N rats and B6C3F~ mice of each sex by gavage. The details of this study have been reported in a comprehensive technical report prepared by the National Toxicology Program (NTP, 1989). MATERIALS AND METHODS
Chemicals. PCA ($2 flakes, technical grade) was obtained in one lot from E.I. DuPont de Nemours and Co, Inc. (Wilmington, DE, USA). PCA was
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R.S. CHHABRAet al.
identified as p-chloroaniline by infra-red, ultraviolet/visible, and nuclear magnetic resonance spectra. Purity was determined by elemental analysis, Karl Fischer water analysis, titration of the amine component with perchloric acid, thin-layer chromatography and gas chromatography. Cumulative data indicated that PCA was 99.1% pure. All doses administered to animals in these studies were prepared from weighed quantities of PCA dissolved in water containing hydrochloric acid. All doses refer to the PCA content of the solution. To prepare a standard solution, PCA and molar equivalents of 1.0N-hydrochloric acid were mixed in a calibrated container. The resulting PCA hydrochloridc solution was diluted to the desired concentration with dcionizcd water. The solution had a pH of approximately 2. Quantitative analysis of aqueous solutions of PCA hydrochloridc showed that the dose solutions were stable for at least 14 days at room temperature. The dose solutions were prepared weekly, placed in foil-wrapped containers, and refrigerated until the day of dosing. Animals. Male and female F344/N rats were obtained from Charles River Breeding Laboratories (Kingston, NY, USA) and B6C3F~ mice were purchased from the Frederick Cancer Research Center (Frederick, MD, USA). The rodents were shipped to the study laboratory at 5 ~ wk of age. The rats were quarantined for 20 days and mice for 12 days. Thereafter, a complete autopsy was performed on five animals of each sex and species in order to assess their health status. The rodents were placed on study at 7-8 wk of age. They were housed five to a cage with absorb-dri hardwood chip as bedding, and given NIH 07 feed and tap-water ad lib. Cages and racks were rotated. The animal room temperature was 70-74°F and the relative humidity was in the range 40 to 60%. Fluorescent lights were on for 12 hr/day and room air was changed 15 times/hr. Experimental design. Groups of 49 or 50 rats of each sex were given by gavagc PCA HCI in deionizcd water at doses of 2, 6 or 18 mg/kg body weight for 103 wk. Groups of 50 mice of each sex were given 3, 10 or 30 mg/kg on the same schedule. Vehicle controls were given deionized water by gavagc. Blood samples were collected in Vacutaincrs containing EDTA by rctro-orbital puncture from 15 randomly selected rats from each group at 6, 12, 18 and 24 months. Hacmatological analyses were per-
formed with an Ortho ELT-8 Hematology Analyzer. Tbe methaemoglobin concentration was determined by the method of Evelyn and Malloy (1938). All animals were observed twice a day. Clinical signs were recorded once a week for 13 wk and then at least once every 4 wk. Individual body weights were recorded once a week for the first 13 wk and once a month thereafter. Mean body weights were calculated for each group. Animals found moribund and those surviving to the end of the studies were humanely killed. An autopsy was performed on all animals. At autopsy, all organs and tissues were examined for grossly visible lesions. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and slides made were stained with hacmatoxylin and eosin. Histopathological examination of tissues was carried out according to an inverse pyramid design (McConncll, 1983a,b); complete histopathological examinations were performed on tissues from all animals in the high-dose and vehicle control groups and on tissues from animals in lower dose groups that died during the study. In addition, samples from all grossly visible lesions in animals of all dose groups were examined histopathologically. Potential target organs for chemically related neoplastic and non-neoplastic effects were identified; these target organs]tissues from animals in the lower dose groups wcrc examined histopathologicaily. Statistical analysis. The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Taronc's (1975) life-table test for dose-related trend. Data on turnout incidence were analysed by life-table tests (appropriate for fatal tumours) and logistic regression analysis (appropriate for turnouts observed in animals dying of an unrelated cause). For further discussion of these statistical methods, see Haseman (1984) and Dinse and Haseman (1986). RESULTS
Rats The mean body weights of treated groups were generally within 5-6% of those of vehicle controls (Table 1). The survival rates of groups exposed to
Table I. Survival and average final body weights of rats in the 2-yr garage studies of PCA-hydrochloride Dose of PCA (mg/kg body weight) Parameter No. of rats initially in study No. of rats at termination Survival P value* Final body weight (g) Animals initially in study Animals at termination Survival P value* Final body weight (g)
0 (Controls)
2
6
18
M~I~ 49 18 0.793 442
50 32 0.007 462
50 32 0.005 453
50 20 0.367 462
F~m~dcs 50 27 0.244 323
50 39 0.011 333
50 36 0.075 334
50 37 0.043 310
PCA = p-chloroaniline *The result of the life-table trend test is in the vehicl© control column, and the results of the life-table pairwisc comparisons with the vehicle controls are in the dosed columns.
Carcinogenicity study of p-chloroaniline PCA were generally higher than those of the vehicle controls (Table l). This was possibly due to a decreased incidence of leukaemia in PCA-exposed animals. Animals given the mid- or high-dose had blue extremities, indicative of cyanosis. Haematological evaluations after 6, 12, 18 and 24 months of treatment revealed mild haemolytic anaemia in rats given 18 mg/kg (data not shown), and a slight elevation of methaemoglobin in treated groups. In treated rats 0.34-4.0% of haemoglobin was converted to methaemoglobin in comparison with 0.26-1.67% in vehicle controls. The incidence of proliferative mesenchymal lesions in the spleen was increased in PCA-treated rats (Table 2). Fibrosis of the spleen was increased in dosed males and females, and when extensive in the red pulp it replaced many or all of the haematopoietic cells. The incidence of sarcomas (fibrosarcoma, osteosarcoma, haemangiosarcoma) was significantly increased in high-dose males. Many of the splenic sarcomas metastasized to one or more sites including most of the major abdominal organs, lungs and lymph nodes. One fibrosarcoma was observed in the spleen of a mid-dose female, and one splenic osteosarcoma occurred in a high-dose female. In rats in the high-dose group, metaplasia of the stroma of the spleen consisted of multiple foci of lipocytes in the fibrotic stroma; these foci were also present in some of the splenic sarcomas. Pheochromocytomas or malignant pheochromocytomas (combined) in male rats occurred with a significant positive trend; the incidence in the highdose group was significantly greater than that in the vehicle controls. Adrenal gland medullary hyperplasia was increased in high-dose female rats; pheochromocytomas were slightly increased in high-
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dose females (Table 2). In PCA-dosed rats, hypcrplasia of the bone marrow was increased in incidence but not in average severity. The incidence of mononuclear cell leukaemia was significantly lower in all dosed groups of males and females than in vehicle controls.
Mice The mean body weights of high-dose male and female mice were generally within 5% of those of vehicle controls throughout the studies (Table 3). No compound-related clinical signs of toxicity were noticed. The survival of the mid-dose group of male mice was significantly lower than that of the vehicle controls after wk 99. No other significant differences in survival were observed between groups (Table 3). The incidences of hepatocellular carcinomas in mid- and high-dose males and of hepatocellular adenomas or carcinomas (combined) in low-, midand high-dose males were significantly greater than those in vehicle controls (Table 4). Hepatocellular carcinomas metastasized to the lungs in 1/50 vehicle control, 1/49 low-dose, 2/50 mid-dose and 9/50 highdose male mice. Hepatocellular adenoma and carcinoma (considered separately or combined) were not significantly increased in female mice. The incidence of haemangiosarcomas in high-dose male mice was slightly increased relative to that in vehicle controls. Haemangiosarcomas occurred primarily in the liver and spleen. There were no treatment-related differences in the incidence of haemangiosarcoma in female mice. The incidence of lymphoma was significantly decreased in male mice given the low or high dose of PCA and in female mice in mid- and high-dose group (Table 4).
Table 2. Summary of treatment-related lesions in rats exposed to p-PCA hydrochloride for 2 yr Incidence (no. of rats affected/no, examined) Site and lesion
PCA dose (rag~ kg body weight)...
0 (Controls)
2
6
18
Males Spleen Fibrosis Stromal metaplasia Fibroma SarcomaS" Bone marrow Hyperplasia Adrenal medulla Hygerplasia Pheochromocytoma Haematopoietic system Mononuclear cell leukaemia
3/49 0/49 0/49 0/49
11/50* 0/50 0/50 1/50
12/50' 0/50 0/50 3/50
41/50"* 24/50** 2/50 38/50**
26/49
36/50*
35/49*
46/50**
15/49 13/49
21/48 14/48
15/48 15/48
17/49 26/49*
21/49
3/50**
2/50**
3/50**
1/50 0/50 0/50
2/50 0/50 0/50
3/50 0/50 1/50
42/50** I 1/50"* 1/50
1I/S0
12/48
21/50
37/47**
4/50 2/50
4/50 3/50
7/50 l/50
24/50** 6/50
2/50*s
1/50**
Females Spleen Fibrosis Stromal metaplasia Sarcomat Bone marrow Hypcrplasia Adrenal medulla Hyperplasia Pheochromocytoma Haematopoietic system Mononuclear cell leukaemia
10/50
1/50"*
PCA - p-chloroaniline ~'Fibrosarcoma, osteosarcoma or haemangiosarcoma. Values marked with asterisks differ significantly from the corresponding values for the controls (*P < 0.05; **P < 0.01).
FCT 2~.:2-- D
R . S . C H 1 4 A a ~ e t al.
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Table 3. Survival and average final body weights of mice in the 2-yr gavage studies of
PCA-hydrochloride Dose of PCA (mg/kg body weight) Parameter
0 (Controls)
No. of mice initially in study No. of mice at termination Survival P value* Final body weight (g) Animals initially in study Animals at termination Survival P value* Final body weight (g)
3
10
Mal¢~ 50 43 0.295 42.9
30
50 36 0.211 42.2
50 29 0.005 41.8
50 35 0. 110 41.1
Female~ 50 39 0.875 44.4
50 42 0.298 43.9
50 44 0.408 45.2
50 41 0.966 43.5
PCA = p-chloroaniline *The result of the life-table trend test is in the vehicle control column, and the results of the life table pairwis¢ comparisons with the vehicle controls are in the dosed columns. DISCUSSION
Earlier 2-yr studies had strongly indicated the carcinogenicity of PCA because fibromas and sarcomas were observed in the spleens of treated male rats (NCI, 1979). These mesenchymal neoplasms (six fibromas and four sarcomas) occurred in the spleens of high-dose male rats (fed PCA at 500 ppm in the diet). Haemangiomatous neoplasms were the principal neoplastic lesions seen in mice in the NCI study. They were observed in 2/20, 10/50 and 14/50 males and 0/18, 3/49 and 8/42 females given PCA at 0, 2500 or 5000 ppm in the feed, respectively. The findings of these NCI studies have been confirmed by the results of the studies reported here. PCA was considered to be carcinogenic in male rats, because of the marked increased incidence of sarcomas in the spleen (38/50) in the high-dose group. The results of the two studies do differ in the incidences of neoplasms in the spleen. The reasons for these differences were not specifically studied but could be related to dose and route of administration. It is possible that in the NCI studies PCA was
administered at less than the targeted concentrations because of the instability of the chemical mixed in feed. The target concentrations in feed were approximately equivalent to 15 and 30 mg/kg body weight for rats, compared with 2, 6 and 18 mg/kg in our studies in which PCA was administered by gavage. The different modes of oral administration (single dose per day by gavage v. continuous dosing by feed) could have resulted in differences in metabolism of the chemical and this could have been responsible for the quantitative differences between the NCI studies and those reported here. The dose response for the development of splenic tumours in male rats in the current study was non-linear with respect to dose. However, because of their rare occurrence in controls, the few sarcomas in the female rats in the 1ow(!/49) and mid-dose (3/50) groups were considered most likely to be due to PCA administration. The doses used in these studies apparently did not saturate the metabolic and excretory pathways, as determined by disposition studies on PCA (NTP, 1989). The female rats appeared to be less sensitive than the males to the induction of splenic neoplasms by
Table 4. Summary of treatment-related neoplastic lesions in mice exposed to p-PCA hydrochloride for 2 yr Incidence (no. of mice affected/no, examined) Site and lesion
PCA dose rag/ kg body weight)...
O(Controls)
2
6
18
Mal¢~ Liver Adenoma Carcinoma Adenoma/carcinoma Haemangiosarcoma Spleen Haemangiosarcoma Liver and/or spleen Haemangiosarcoma Haematopoietic system Lymphoma
9/50 3/50 I I/50t 2/50
15/49 7•49 21/49t 2/49
10/50 l I/50* 20/50"~f 1/50
4/50 17/50"* 21/50* 6/50
3/50
2/49
0/50
5/50
4/50~
4/49
1/50
I 0/50~
10/50
3/49*
9/50
3/50*
6/50
9/50
8/50
I I/50
2/50
1/50
1/50
I/50
19/50
12/50
Femal~ Liver Adenoma/carcinoma Liver and/or spleen Haemangiosarcoma Haematopoietic system Lymphoma
5/50**
PCA = p-chloroaniline *One mouse had adenoma and carcinoma. :~Onc mouse had haemangiosarcoma in liver and spleen. Values marked with asterisks differ significantly from the corresponding control values (*P < 0.05; **P < 0.01).
10/50"
Carcinogenieity study of p-chloroaniline PCA; neoplasms of the spleen were seen in only one mid-dose and one high-dose female rat. This sex difference in induction of splenic neoplasms is consistent with results obtained in studies in rats of other aniline dyes. However, the incidences of splenic fibrosis in high-dose male (41/50) and female (42/50) rats were similar. Fibrosis and stromal metaplasia (lipocyte or fat infiltration) have been observed in the spleens of rats administered aromatic amines or their derivatives. No splenic fibrosarcomas or osteosarcomas were observed in mice of either sex dosed with PCA. Such a lack of response was expected because of the results of studies with other anilines (Weisburger, 1983). None of the aromatic amines studied have increased the incidences of splenic tumours in mice. However, induction of hepatic neoplasms by those chemicals was frequently observed in mice, and in the present study the incidences of hepatocellular carcinomas were increased in PCA-treated male mice. Haemangiosarcomas of the liver and/or spleen were found in male mice and the number was significantly increased in the high-dose group in comparison with the controls. Mice responded differently from rats with respect to the spleen as a target organ for PCA toxicity. Although in the present study no non-neoplastic splenic effects were observed in mice, in a 90-day study (Chhabra et al., 1990) splenic weights were increased and increased extramedullary haematopoiesis was observed in the spleens of mice given 30 mg PCA/kg body weight. The differences between rats and mice with regard to the development of splenic and liver neoplasms could be due to the differences in metabolism and disposition of PCA. PCA was cleared from blood at a much faster rate in mice than in rats. Perry et al. (1981) studied the disposition of [~C]PCA in F344 rats, dogs and A/J and Swiss Webster mice. They reported that the initial decay constants for PCA clearance from whole blood in both strains of mice were 10 times greater than those in dogs and rats. The PCA clearance in mice was too rapid to permit calculation of kinetic parameters. The reports of development of tumours of the spleen in F344 rats treated with structurally related aniline compounds led some workers to study the pathogenesis of splenic lesions and the disposition of radioactive aniline in the rat in order to elucidate the possible mechanism(s) of action of these chemicals (Bus and Popp, 1987; Goodman et al., 1984). These studies indicated that splenic tumours in rats induced by aniline and related compounds are most likely to be the result of a non-genotoxic mechanism. Erythrocyte toxicity, as evidenced by methaemoglobin formation, is the major toxic effect of aniline and related compounds in various species. A toxic effect of PCA on erythrocytes was demonstrated in the 13-wk study (Chhabra et al., 1990). In the present study the increased incidence of hyperplasia of the bone marrow observed after 2 years of treatment may have been related to the mild haemolytic anaemia or secondary to the decrease in the normal extramedullary haematopoiesis in the spleen of rats as a result of the extensive fibrosis present. It is possible that the methaemoglobin bound to aniline com-
123
pounds or their reactive metabolites was broken down in the red pulp of the spleen. When released from the haemoglobin these reactive metabolites could bind to the splenic mesenchymal tissues and stimulate the formation of turnouts. However, it is possible that a direct-acting genotoxic mechanism was involved in the induction of the neoplasms; McCarthy et al. (1985) found that in rats and mice [~4C]aniline binds to a greater extent to the kidney, small intestine, large intestine and spleen than to other tissues. Protein and RNA were the major macromolecular targets for [~4C]aniline binding; DNA binding occurred to a lesser extent. The possibility for involvement of genotoxic mechanisms is further supported by data reported by Parodi et al. (1982a,b). In their studies aniline induced DNA damage in vivo in the liver and kidney of rats. Also, aniline was positive in the induction of sister chromatid exchanges in vivo in male Swiss mice. The splenic toxicity and carcinogenicity induced by PCA in male rats seem to be similar to those produced by aniline in rats. Whether the mechanism of carcinogenesis was mediated through genotoxic or non-genotoxic events remains unresolved; either or both may influence the carcinogenic responses in male rats (spleen) and male mice (liver). NTP studies (NTP. 1989) have shown that in vitro PCA was clearly genotoxic in the Salmonella assay and in tests for chromosomal aberrations in Chinese hamster ovary cells. However, it was mutagenic in the presence of S-9 activating system and thus, the in r'itro genotoxic activity of PCA appears to be dependent on metabolism for its full expression. Experimental evidence indicates that PCA undergoes oxidative transformations as a reactive amine (Smith and Gorrod, 1978; Uehleke and Hellmer, 1971), with the probable formation of electrophilic intermediates that could be stabilized by chlorine. For example, p-chloro-Nhydroxyaniline (Von Jagow et al., 1966), which may be formed through a nitronium ion intermediate in the presence of oxidative enzymes, and p-aminophenol (Ichikawa et al., 1969), which may be formed by an arene oxide intermediate possibly in the absence of metabolizing enzymes, have been reported as metabolites of PCA in the rabbit. Both of these intermediates are potential electrophiles and could covalently bind macromolecules. In conclusion, PCA was considered to be carcinogenic in male rats on the basis of the increased incidences of sarcomas of the spleen. These neoplasms occur rarely in control rats (Solleveld et al., 1981). Pheochromocytomas of the adrenal medulla may also have been increased by PCA administration. Evidence of carcinogenicity in female rats was slight, as indicated by the presence of sarcomas of the spleen in one mid- and one high-dose animal and the slightly increased incidence of pheochromocytoma of the adrenal gland. PCA was considered to be carcinogenic in male mice on the basis of increased incidences of hepatocellular neoplasms and of haemangiosarcomas of the liver or spleen. REFERENCE~ Beard R. R. and Noe J. T. (1981) Aromatic nitro and amino compounds. In Patty's Industrial Hygiene and
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