Aniline

Endpoint summary

Additional information

Irrespective of the route of administration, aniline causes toxicity in erythrocytes and the hematopoietic system with corresponding effects on the spleen, bone marrow, liver and kidney. Cyanosis with increased MetHb levels, erythrocyte lesions with formation of Heinz bodies and hemolytic anemia are characteristic observations. Reticulocytosis and increased bone marrow and extramedullary erythropoiesis are compensatory reactions of the red blood cell toxicity. The damaged erythrocytes are scavenged mostly in the spleen. Accumulation of hemosiderin in the spleen and sometimes also in liver and kidney, spleen congestion, dark coloration and increased spleen weight are observed after repeated exposure to aniline. Further signs of toxicity after repeated exposure are splenitis, spleen hyperplasia and fibrosis.

Except for black discoloration of the spleen no histological evidence of hematotoxicity was found in mice.

In a subacute oral study on the mode of action of aniline toxicity, aniline hydrochloride was fed with the diet to male F344 rats for 28 days. 12 animals per group received dietary (weekly adjusted) nominal dose levels of 0, 10, 30 and 100 mg/kg bw/d. Because of the instability of the test substance in the diet the actual ingested dose levels were corresponding to 0, 4, 12, 41 mg/kg bw/d of aniline. Hemoglobin adducts of aniline were detectable at all dose levels after 1 and 4 weeks of exposure (Zwirner-Baier et al., 2003). Heinz bodies sporadically observed in the at the lowest dose, but were significantly increased in the mid and high dose groups after 1 and 4 weeks. Furthermore, in the mid and high dose group hematological findings (increased MCV, MCH and reticulocytosis; decreased MCHC and erythrocyte count) indicated anemia. Hemolytic anemia in the high dose group was accompanied by an increase in serum transferrin and iron binding in the blood, and neutrophil leucocytosis as an indication for an inflammatory process in the spleen. Minimal vascular congestion was observed in the low dose group (2 and 4 animals after week 1 and 4, respectively). In addition, spleen weight was increased after 1 and 4 weeks in the mid and high dose group with an dose dependent increase in severity. In the high dose group focal perisplenitis with moderate vascular congestion and hemosiderin deposition in Kupffer cells of the liver was observed. A no effect level could not be derived from this study, thus the LOAEL for aniline was 4 mg/kg bw/d (BASF, 2001; Zwirner-Baier et al., 2003).

In a carcinogenicity study rats (130/dose group) received aniline in food at body doses of approx. 0, 7, 22, 72 mg/kg bw. Non-neoplastic findings observed in the spleen were fatty metamorphosis, stromal hyperplasia and chronic capsulitis of the spleen, mostly in all three dose groups. Hematological effects, hemosiderosis and splenic hematopoiesis were observed in all dose groups, including the lowest applied dose of 7 mg/kg bw (CIIT, 1982).

In a subacute inhalation study male rats (30/group) were exposed nose-only to aniline concentrations of 9.2, 32.4, 96.5, and 274.9 mg/m3 for 6 h/d, 5 d/week for 2 weeks followed by a 2 week observation period without exposure to aniline. Serial sacrifices were performed on 4 days during the exposure period and at the end of the observation period. No mortality was observed in any of the dose groups during the study. Cyanosis was seen at the two highest concentrations and did not progress during the exposure period. MetHb formation and erythrotoxicity were the main signs of toxicity. In the two highest doses anemia, Heinz bodies, decreased hemoglobin and hematocrit, reticulocytosis and effects on the spleen (splenomegaly, hemosiderin accumulation, and hematopoiesis) were observed. A borderline increase in splenic extramedullary hematopoiesis observed in the 32.4 mg/m3 dose group was judged by the author a homeostatic response rather than an adverse effect. At 9.2 mg/m3 (2.4 ppm) no significant effects were observed (Pauluhn, 2004a, 2004b).

In rats exposed head-only to 0, 38, 115, 192, 578 mg/m3 aniline vapor for 8 h/d for 5 d or 12 h/d for 4 d MetHb levels were increased above control levels at the beginning of the second day of exposure to the two highest concentrations with a tendency to accumulate. A slight increase in MetHb levels but no cumulative effect was observed at 115 mg/m3. The NOAEC of this study was 38 mg/m3 (10 ppm) for both exposure periods (Kim and Carlson, 1986).

In a subchronic study 9 male rats and two dogs were exposed for 26 weeks, 20 female mice and 10 guinea pigs were exposed for 20 weeks to 19 mg/m3 (5 ppm) aniline vapor (whole body) for 6 h/d. The validity of this study is limited because only one concentrations was tested, histopathology and statistics were incomplete and autopsy was carried out only in a small number of animals. All rats showed a mild bluish skin coloration of their extremities, tail, eyes and nose during the last 4 weeks of exposure. Only in rats, blood analysis revealed a marginal increase in MetHB levels of 0.6% (week 23). There were no substance related pathological alterations in any of the tested species. Based on the absence of a significant increase in MetHb levels and the absence of spleen toxicity the tested concentration of 19 mg/m3 (5 ppm) can be considered a NOAEC (Oberst et al., 1956).

Further studies on the toxicity of aniline after repeated oral exposure summarized in the EU risk assessment report on aniline (ECB, 2004) revealed similar effects in the erythrocytes and the hematopoietic system in rats (Ciccoli et al. 1999, Khan et al. 1997). In most cases a NOAEL was not derived in these studies and/or the LOAELs were higher than those in the studies cited above.

Justification for classification or non-classification

Studies with repeated administration have been carried out with rats and, to a limited extend, with mice and dogs.

Irrespective of the route of administration,the toxicity of aniline in rats manifests in effects of the red blood cells and the haematopoietic system with corresponding effects on the spleen, bone marrow, liver and kidney. Cyanosis with increased MetHb levels, erythrocyte lesions with formation of Heinz bodies and haemolytic anaemia are characteristic observations.

The LOAEL for repeated oral administration of anline to rats is 4 mg/kg bw (28 day study, rat) and 7 mg/kg bw (2 year feeding study, rat). No NOAEL was determined.

After inhalation exposure of rats to aniline for up to 2 weeks the NOAEC was about 10 ml/m³ (about 0.038 mg/l).

No dermal study with repeated exposure is available, however, since aniline is well absorbed via the dermal route this route is included in the classification.

Aniline uptake (oral, dermal, inhalative) leds to MetHb formation in humans. The NOAEL for single oral uptake was determined with 35 mg, corresponding to about 0.5 mg/kg bw.

Thus, information on toxicity observed after repeated exposure to aniline fulfills the criteria in Annex VI to 67/548/EEC as toxic and labelling with T, R 48/23/34/25. On the basis of hemolytic anemia observed in animal studies, and the limited evidence of hematotoxicity in humans following repeated exposure aniline is classified into category 1 for specific target organ toxicity - repeated exposure (STOT RE) according to the classification criteria in Annex I to the CLP regulation (EC) 1272/2008. This classification is in accordance with the entries for harmonized classification and labelling in the Annex VI to the CLP regulation.

Furthermore, for toxicity after repeated exposure the following specific concentration limit has been set in Annex VI to the CLP regulation:

 

Concentration ≥ 1% =>  STOT RE category 1 (Annex VI to CLP, table 3.1) and  T; R48/23/24/25 (Annex VI to CLP, table 3.2)

0.2% ≤ C < 1% => STOT RE category 2 (Annex VI to CLP, table 3.1) and  Xn; R48/20/21/22 (Annex VI to CLP, table 3.2)