Aniline

Carcinogenicity

Justification for classification or non-classification

In two carcinogenicity studies on F344 rats aniline caused a non-linear increase in the incidence of spleen tumors in males and a few splenic tumors in female rats. Re-examination of splenic tumors showed that the occurrence of non-neoplastic splenic lesions was strongly correlated with tumor incidence. It was concluded that sarcomas arise from pre-existing fibrotic areas and splenic fibrosis and hyperplasia are likely to present pre-neoplastic lesions.

B6C3F1 mice no aniline dependent increase in tumors was observed in males and females.

Aniline showed positive responses in genotoxicity assays in vivo at high doses in rats and in mice, but overall the genotoxicity appears to be very low, if any (SCOEL 2010). Since aniline induced positive responses in genotoxicity tests in both, rats and mice but mice failed to develop tumors there is no plausible scientific proof that the underlying mechanism of the carcinogenicity in rats is based on the observed genotoxic responses. Many investigations indicate that the development of spleen tumours is connected to erythrocyte toxicity, which is indicated by the formation of MHb and Heinz bodies with the histopathological effects in the spleen being a consequence of this effect (MAK 2007). On the basis of this hypothesis it can be argued that repetitive toxic effects play a decisive role for the development of tumours, and that no increased tumour risk should be expected in the absence of an increased erythrocyte turnover. Aniline is therefore categorised by SCOEL (2010) and MAK (2007) as carcinogen with a practical threshold. From the limited human database a final assessment of human cancer risk is not possible.

In accordance to the EU criteria for classification and labelling of carcinogens in Annex VI to 67/548/EEC, aniline is classified as carcinogenic, category 3 and labelled with R 40 “limited evidence of a carcinogenic effect”. According to the GHS-based criteria for carcinogenicity in the CLP regulation classifiaction as carcinogenic category 2 is warranted. This classification in accordance with the entries for harmonized classification and labelling in the Annex VI to the CLP regulation.

Additional information

Carcinogenicity studies are available for aniline applied via the diet as aniline hydrochloride in rats and mice. Most information is available from feeding studies with rats over 2 years.

Dietary aniline hydrochloride was administered to male and female F344 rats (130 animals/sex/group) at body doses of 0, 10, 30 and 100 mg/kg/d corresponding to concentrations of 0, 200, 600 and 1600 ppm in the diet. At 26 and 52 weeks, ten rats of each sex in each group, and at week 78, twenty rats of each sex in each group were sacrificed intermittently. The remaining 90 animals per sex and group were treated until the termination of the study. No aniline-related effect on the survival rate was observed. In males and the high dose group females decreased blood parameters hematocrit, hemoglobin, erythrocyte count were observed. In the high dose group animals, the liver weights were increased. No primary spleen tumors were found in the control and low dose males. Spleen tumors developed in the high dose males were 3 fibrosarcomas, 21 stromal sarcomas, 1 capsular sarcoma, 6 haemangiosarcomas and 3 osteogenic sarcomas. In the mid dose group only one male showed stromal sarcoma. Females had no spleen tumor other than one hemangiosarcoma in the high dose group. Treatment related non neoplastic findings were mainly observed in high dose animals. Most frequent non neoplastic lesion was chronic capsulitis observed in high-dose males and females (62/130 and 70/130, respectively). Fatty metamorphosis of the splenic red pulp was noticed in high dose males only. Stromal hyperplasia and fibrosis of the splenic red pulp in high dose males and, to a lesser degree, in the high dose females. Stromal hyperplasia characterized by morphologic similarities to the cell type of the stromal sarcoma possibly represents pre-neoplastic lesions. Also, increased deposits of hemosiderin and severity of extramedullary hematopoiesis in high-dose males, incidence of splenic atrophy in high-dose males and females were reported to be increased (CIIT, 1982)

Fischer 344 rats were given 3000 or 6000 ppm aniline hydrochloride in the diet for 103 weeks. Survival was not affected by treatment and body weight gain was slightly reduced in the high dose groups. There were no splenic tumors in control males and females. Mesenchymal tumors, mostly of the spleen alone but also of multiple organs of the body cavity, were found. The incidence of haemangiosarcomas of the spleen in low dose and high dose males was 19/50 and 20/46 and in the body cavity 0/50 and 1/50, respectively. The incidence of fibrosarcomas in males was 3/50 and 7/46 in the spleen and 2/50 and 8/48 in the body cavity. The growth of the fibrosarcomas was frequently invasive. In the females, the tumor incidence was markedly lower but the values for fibrosarcomas and sarcomas together were significantly elevated to the control group (1/50, 7/50). In addition in the males, the incidence of phaeochromocytoma was increased in a dose-dependent manner (2/24, 6/50, 12/44), capsular fibrosis and papillary hyperplasia developed in the spleen and hemosiderosis was observed in the renal tubules and the liver. Survival, even of the high dose animals was not reduced compared to control values. Blood parameters were not recorded in this study (NCI, 1978).

Review of the splenic sections of the NCI study (Weinberger, 1985) in rats revealed a statistically significant increase in splenic tumors (mainly fibrosarcoma and fibroma) in the low and high dose males (13/48 and 28/47, respectively). A total of two splenic tumors was evident in low-dose females (4%) and 5 (10%) tumors were seen in high-dose females. Furthermore, non neoplastic lesions in spleen as fibrosis, capsular hyperplasia and hemorrhage were seen with statistically significantly higher incidence in males and females of each dose group. The incidence of fatty metamorphosis was significantly increased in male groups. Occasionally fibrosarcomas are described to occur within areas of fibrosis and well differentiated areas of fibroblasts were found adjacent to less differentiated areas of tumor cells. Splenic fibrosis and fatty metamorphosis showed a strong statistical correlation with tumor incidence. Due to the morphologic similarity of splenic fibrosis and capsular hyperplasia to the induced splenic sarcomas it was suggested that these lesions are pre-neoplastic and sarcomas arise from areas of pre-existing fibrosis

In a carcinogenicity study with B6C3F1 mice (NCI, 1978) no treatment related increase in the incidence of tumors or non-neoplastic changes were observed. Groups of 50 B6C3F1 mice were supplied with feed containing aniline hydrochloride at concentrations of 0, 0.6 and 1.2 percent for 103 weeks. Test substance concentrations are equivalent to body doses of 737 and 1510 mg/kg bw/d in male mice and 733 and 1560 mg/kg bw/d in female mice. No treatment related effect on survival rates was observed when comparing dose and control groups. In the males, body weight gain was reduced and hair loss was observed. With respect to the spleen, the target organ in rats, there was a single hemangioma in high-dose males (1/49), a hemangiosarcoma in a low-dose male (1/49) and a malignant lymphoma in a control male (1/38). In the spleen of females, one control animal (1/45) had a hemangiosarcoma and malignant lymphomas were seen in three low-dose females (3/48).

In humans occupationally exposed to aniline, o-toluidine, hydroquinone and other chemicals an increased incidence of bladder cancer had been reported and analysed (NIOSH Alert, 1990; Ward et al., 1991, Soharan, 2008, Carreon, 2010). Air concentration of aniline and o-toluidine measured by NIOSH was each below 1 ppm. Since the workers were exposed to a number of different substances bladder cancer reported in these studies cannot be attributed to aniline.Exposure to o-toluidine is likely associated with this risk because it is a more potent carcinogen than aniline in animals and was found in the urine of workers at higher levels than aniline. It was, however, not possible to determine worker’s exposure to o-toluidine and aniline separately.

The differences in toxicity between rats and mice with respect to aniline treatment seem to indicate a quantitative higher susceptibility of rats, especially male rats, which may be explainable by differences in the metabolic pathways. In both species aniline is metabolized primarily by N-acetylation followed by aromatic ring hydroxylation. In mice, the main elimination pathway of N-acetylaniline requires glucuronidation whereas in rats elimination of N-acetaniline occurs after sulphate conjugation which becomes saturated at higher dose levels leading to an increased excretion of unaltered aniline. McCarthy et al. (1985) observed a 5-fold lower Km and 1.5-fold higher V_maxfor mouse aniline p-hydroxylase than for the rat enzyme which suggests that the mouse is more efficient in metabolizing aniline than the rat. Furthermore, the amount of p-aminophenol relative to o-aminophenol conjugates in the urine was 8:1 in rats and 1:6 in mice. No species difference was observed in the hepatic enzyme activity of N-hydroxylase. N-hydroxylation leads to formation of phenylhydroxylamine the principal route by which aniline induces formation of MetHb (Harrison and Jollow, 1987). MetHb is reduced back to hemoglobin by erythrocyte NADH-dependent MetHb reductase. Under normal conditions MetHb reductase maintains MetHb level at or below 1% and is the rate limiting enzyme controlling the toxicokinetics of MetHb reduction. Species specific differences with a five and ten time higher activity of erythrocyte MetHb reductase from rats and mice than in human have been observed.

A comprehensive overview and discussion of aniline mode-of action is given in MAK (2007). Methemoglobin (MHb) formation of aniline, linked with toxic effects on the hematopoietic system with erythrocyte toxicity and effects on the spleen must be taken into account. Aniline is not mutagenic in standard bacterial tests, whereas in cultured mammalian cells positive effects were observed with respect to structural chromosomal aberrations at high concentrations. In vivo, induction of micronucleated erythrocytes in the bone marrow was observed in both, rats and mice, however, the doses in these studies were high and were associated with MetHb formation and toxicity (Bomhard and Herbold, 2005). In general, the genotoxicity of aniline appears to be very low, if any (SCOEL, 2010). Since positive results in in vivo micronucleus tests have been observed in both, rats and mice, the relevance of this finding for the tumor development remains questionable since mice did not develop tumors after lifetime exposure to aniline. Repeated dose toxicity studies demonstrated that splenic toxicity of aniline is clearly associated with damage of erythrocytes in that the spleen is the organ responsible for the erythrocyte clearance.

Data on carcinogenicity of aniline in humans are inadequate for an evaluation. Aniline caused mesenchymal tumors in the spleen of rats but not in mice after dietary chronic exposure. Tumors were mainly observed in males and the tumor incidence was non-linear (CIIT, 1982). Tumor development could be connected to erythrocyte toxicity - indicated by the formation of MetHb and Heinz bodies - and the resulting splenic histopathological changes (fibrosis, fatty metamorphosis, capsulitis, hyperplasia and hematopoiesis) (MAK, 2007). Subacute exposure of male rats to aniline in the diet caused hemolytic anemia, resulting in inflammatory reaction in the spleen and perturbations in iron metabolism (BASF, 2001). Continuous dietary exposure to aniline elicits repetitive erythrotoxicity and related effects on hematopoiesis and spleen in male rats, which could play a decisive role for the development of tumors. Repetitive toxic effects play a decisive role for the development of tumours, and therefore no increased tumour risk should be expected in the absence of an increased erythrocyte turnover (MAK, 2007). This view could experimentally be supported by Mellert et al. (2004), corroborating the contention that experimentally carcinogenic doses of aniline cause early effects on haematological parameters, inflammatory reaction in the spleen and perturbations in iron metabolism as a result of haemolytic anaemia. Accordingly, the experimental carcinogenicity of aniline can reasonably be linked to a defined threshold-related process. Aniline is categorised by SCOEL (2010) and MAK (2007) as carcinogen with a practical threshold.