N-(4-aminophenyl)aniline

Endpoint summary

Additional information

In vitro data

Several Ames test results for 4-ADPA have been reported. Although the study results are reliable the test design of the studies does not comply with the current guideline with regard to the number and kind of tester strains. However, three assays are comparable to a guideline study (Mortelmans 1986, Monsanto Co 1986a, 1993). In these studies no biologically relevant and dose dependent increases in revertants was evaluated in any of the tester strains evaluated with and without metabolic activation. Whereas in a very limited documented Ames screening test, the test substance 4-ADPA was assessed to be positive in the TA98 tester strain in the presence of metabolic activation (Yoshikawa 1976). An ambiguous finding was reported by Freeman (1987). However, the studies have some shortcomings and are limited reported, thus it is considered that these studies are inadequate and not suitable for evaluation.

The test substance 4-ADPA was evaluated in several mammalian cell test systems. The mutagenic potential of 4-ADPA was evaluated in a HGPRT test in CHO cells at dose levels of 5, 10, 33, 67 and 100 µg/ml with and without metabolic activation. The detailed description of the test method confirmed that the study complies with the requirements of current guidelines (OECD TG 476, 1997). There was no statistically significant increase in the mutation frequencies of the 4-ADPA treated cultures when compared to the solvent control (Monsanto Co. 1986b). However, a positive response in a mouse lymphoma assay (MLA) which was conducted in the frame of the National Toxicology Program is documented for 4-ADPA (NTP, 2003).

The clastogenic potential of 4-ADPA was evaluated in two in vitro chromosome aberration and an in vitro micronucleus assay. No significant increases in chromosome aberrations were reported in a study with Chinese hamster ovary (CHO) cells (Monsanto Co. 1986c). The detailed description of the test method showed, however, that the study deviates in various parameters from the requirements of the current guideline. The study is limited by the lack of an independent repeat experiment with longer treatment time to confirm the negative result, the small number of metaphases evaluated per concentration, and the fact that only non- cytotoxic concentrations were evaluated for chromosome aberrations. The results of the pre-test for cytotoxicity are described, and based on these results the concentrations for the main study was selected. 4-ADPA was tested in concentrations of 5 to 75 mg/ml without S9 (75 µg/ml was toxic and not scored) and of 5 to 20 µg/ml with S9 (no data available on cytotoxicity; in a pre-test 25 µg/ml caused a 91% increase in average proliferation time at 50 µg/ml and only 13 scorable metaphases were available). In comparison to the solvent control 4-ADPA produced no significant increase in chromosome aberrations at any dose level. The positive controls induced clastogenic effects and demonstrated biologically relevant or statistically significant increased numbers of aberrant metaphases (Monsanto Co.1986). In a NTP study (2003) which was conducted in the frame of the National Toxicology Program, a positive response with regard to the induction of chromosome aberrations and sister chromatid exchanges is documented for 4-ADPA. However, the study has several shortcomings. It is unclear whether the results are included or excluded gaps. Moreover, data on cytotoxicity are lacking. However, clastogenic effects were noted in a current OECD guideline study (TG 487). In this study the induction of micronuclei was evaluated in human lymphocytes. Three concentrations were microscopically evaluated. The highest dose group showed cytotoxcity, indicated by a reduction of the replication index (RI) about 60 %. The lower doses were chosen such that a range from maximum to little or none cytotoxicity was covered. Under the experimental conditions used 4-ADPA induced micronuclei in the presence of S9 metabolic activation and, thus was assessed as genotoxic in human lymphocytes (SCCP 2007).

In addition, 4-ADPA was examined for genotoxicity in the in vitro DNA repair assay in primary rat hepatocytes according to international standard methods. Cytotoxicity was observed at 100, 500 and 1000 µg/ml in the preliminary experiment and at 50 and 100µg/ml in the replicate experiment. Unscheduled DNA synthesis was measured at concentrations between 0.5 and 100µg/ml in the replicate assay. The net grain counts were negative at each 4-ADPA concentration, in the solvent control, and in the medium control, in contrast to the strong positive response produced in both experiments by the positive control. An apparent increase in net grains per nucleus was observed with increasing concentrations of 4-ADPA in both experiments but there were no net grain counts greater than zero and no increases in the percentage of cells in repair. These results indicate that 4-ADPA was not a genotoxic agent in the in vitro rat hepatocyte DNA repair assay (Monsanto Co.1986d).

In vivo data

The genotoxic potential of 4-ADPA was evaluated in two in vivo UDS assays. The test substance 4-ADPA induced no significant increase of unscheduled DNA synthesis in hepatocytes of Fischer-344 rats that had received single dosage of 50, 100 and 250 mg/kg bw by gavage. It induced DNA replication at 250 mg/kg bw (2.68 % compared to vehicle control 0.06%). The study was performed according to current international standard methods but no information is available on systemic toxicity in the high dose animals except the information that the highest dose had to be reduced to 250 mg/kg bw because at 400 mg/kg bw one of the two rats died prior to the 16-hour sacrifice and another yielded unscorable slides (Monsanto Co. 1986e). The findings from the Monsanto study were confirmed by a more recent in vivo UDS. Sprague Dawley rats treated with 187.5, 375, and 750 mg/kg bw test substance were prepared 2 to 4 or 12 to 16 hours after treatment and showed no biologically relevant induction in unscheduled DNA synthesis. However, clinical signs were indicated at the 2 to 4 hour and at the 12 to 16 hour time point (highest dose group only) (SCCP 2007).

The clastogenic potential of 4-ADPA was evaluated in an in vivo micronucleus assay. Male ICR mice were treated with 0, 50, 100 or 200 mg/kg per day on two consecutive days. In the high-dose group, 1 animal died, and reduced activity was observed for 4 hours after administration of the test substance. Apart from the significant body weight reduction in the highest dose group (ca. 13%), no other signs of toxicity were reported. There were no significant differences between the treatment groups and the vehicle control in the ratio of polychromatic to normochromatic erythrocytes in the bone marrow, and therefore no indication of cytotoxicity in the bone marrow. There was also no indication that the test substance reached the target tissue (toxicokinetic investigations were not performed). Although the number of micronucleated cells in the highest dose group (3.1%) was evaluated compared to the vehicle control (1.83%), there was no statistically significant difference between the vehicle control group and all treatment groups in bone marrow cells containing micronuclei. The authors concluded that the treatment with the test substance did not induce micronuclei under the conditions of this test (NIER 2001). This finding was confirmed by a more recent in vitro MNA study, which was done under GLP and in accordance to TG 474 (SCCP 2007). Male Sprague Dawley rats were administered to single i.p. doses of 0, 25, 50 and 100 mg/kg bw. 24 h or 48 h (highest dose group and current vehicle control only) after dosing bone marrow cells were colleted. Toxicity and thus exposure of the target cells was determined by measuring the ratio between polychromatic and total erythrocytes (PCE/EC). Two satellite groups of 3 male rats, treated with the vehicle and 100 mg/kg bw, were used in a toxicokinetic study. These animals were bled 1, 2, 8, and 24 h after treatment and plasma was collected. In this study no mortality occurred. Clinical signs following exposure were lethargy at all doses and piloerection and prostration at 100 mg/kg bw indicating systemic toxicity in exposed animals. Measurements of plasma levels in the toxicokinetics study confirmed the systemic exposure. The decrease of 13% in the PCE/EC ratio found in the highest dose group compared to the concurrent controls also indicates exposure of bone marrow cells. A statistically significant increase in the number of micronucleated PCEs compared to the concurrent vehicle controls was only found at the 24 h sampling time for the highest dose group. However, this increase was clearly within the range of the historical controls and is, therefore considered not biologically relevant. At the 48 h sampling time (highest dose only) no increase in the number of micronucleated PCEs compared to the concurrent vehicle controls was found (SCCP 2007).

Short description of key information:
The test substance 4-ADPA was investigated for its potency to induce gene mutation in bacteria and mammalian cells. However, the studies, especially the Ames assays, have limitation and thus can only be used for supportive evidence. 4-ADPA was negative in three bacterial gene mutation studies and did not induce gene mutations at the hprt locus and did not cause unscheduled DNA synthesis in primary hepatocytes. A positive result was reported in one mammalian gene mutation assay (mouse lymphoma assay). However, the quality of this study was poor and the results are not suitable for evaluation. In one of two in vitro chromosome aberration tests 4-ADPA was negative (Monsanto Co. 1986c). The second one scored positive and this findings confirmed in a sister chromatid exchange assay (NTP 2003); however these NTP studies are limited documented and are at most supportive evidence. In an in vitro micronucleus assay guideline study (TG 487) an increase in cells with micronuclei was found in presence of metabolic activation (SCCP 2007). Under in vivo conditions the potency to induce genotoxicity could not be confirmed. In an in vivo micronucleus assay done with Fischer-344 rats 4-ADPA did not induced a biologically relevant increase in micronucleated bone marrow cells in rats (NIER 2001). This finding is supported by an in vivo micronucleus assay performed under current guidelines (SCCP 2007). Moreover, exposure to 4-ADPA did not induced unscheduled DNA synthesis (Monsanto Co. 1986e, SCCP 2007).
In conclusion, based on the findings discussed above 4-ADPA itself can be considered to have no in vivo genotoxic potential.

Endpoint Conclusion:

Justification for classification or non-classification

In conclusion, based on the findings discussed above 4-ADPA itself can be considered to have no in vivo genotoxic potential.