2-(4-amino-2-nitroanilino)ethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

According to the results of the key studies, the test item HC Red No.3 induced mutagenicity in bacterial strain TA1537 and TA98 (Ames test). No genotoxic effect was observed when tested on mammalian cell lines.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

According to the results of the three key studies, the test item HC Red No. 3 did not induce mutagenicity effect when tested in in vivo tests. The genotoxicity effect observed in bacterial strains was not found in mice or rats. Hence, the test item HC Red No.3 was not classififed for mutagenicity according to REACh Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In order to assess the potential genotoxicity of the test item HC Red No. 3 :

Four in vitro key studies were availables :

- An Ames test was performed (Mecchi, OECD 471, GLP compliant, Klimisch 1, 2005) with differents bacterial strains (TA98, TA100, TA1535, TA1537 and E. Coli WP2) in presence of absence of metabolic activation system (Liver S9 fraction from Aroclor 1254 -induced rats). Toxicity was evaluated on the basis of a reduction in the number of revertant colonies and a qualitative evaluation of the bacterial lawn. The doses tested in both mutagenicity assay were 2.50, 5.00, 20.0, 50.0, 200, 500, 2000 and 5000 μg/plate in both the presence and absence of S9-mix. Both experiments were performed with the preincubation method. Under the experimental condition of the study, in both experiments precipitation was observed at 5000 μg/plate, the highest concentration tested, In both experiment a biologically relevant and statistically significant dose dependent increase in the number of revertants was observed in strain TA98 both without and with S9-mix and in strain TA1537 without S9-mix only. No increases in the mean number of revertants per plate were observed with any of the other tester strains in either the presence or absence of S9-mix.

- A Chromosome Aberration test was performed in Chinese Hamster Ovary cells (CHO) with and without metabolic activation (Murli, OECD 473, GLP compliant, Klimisch 1, 2005). In the initial toxicity assay, concentrations of 5.00, 16.7, 50.0, 167, 500, 1670, and 5000 µg/mL were tested in single cultures of CHO cells.  Treatment period was for 4 hours with and without metabolic activation or ~20 hours without metabolic activation, and cultures were harvested ~20 hours from the initiation of treatment. Doses were selected for the confirmatory assay based on the initial assay. In the confirmatory assay, the treatment period was for 4 hours with and without metabolic activation or ~18 hours without metabolic activation, and cultures were harvested ~20 hours from the initiation of treatment. Concentrations of 125, 250, 500, 750, 1000, 1250, 1500, 2000, 2500, 3250, 4000, and 5000 µg/mL for 4 hours, 62.5, 125, 250, 375, 500, 750, 1000, 1500, 2000, 2500, and 3250 µg/ mL for ~18 hours without metabolic activation, and 125, 250, 500, 1000, 1500, 2000, 2500, 3250, 4000, and 5000 µg/mL with metabolic activation were tested. Cultures treated with concentrations of 250, 750, and 1500 µg/mL for 4 hours without metabolic activation, 125, 250, and 375 µg/ mL for ~18 hours without metabolic activation, and 250, 1000, 2000, and 3250 µg/mL with metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.

- Mouse Lymphoma L5178Y cells were used to evaluate potential genotoxicity of the HC Red No.3 to induice forward mutation on the Thymidine Kinase Locus (Cifone, 2005, GLP compliant, OECD 476, Klimisch 1). Based on the results of the dose range-finding assay, three independent experiments were carried out without metabolic activation using eight concentrations in two trials ranging from 7.85 to 1000 µg/mL and 50.0 to 1000 µg/mL; and seven concentrations in one trial ranging from 50.0 to 150 µg/mL. Two experiments with metabolic activation were performed using eight concentrations ranging from 50.0-1000 µg/mL and 75.0-1000 µg/mL. In the three independent experiments (initial, repeat initial and confirmatory assays), cells were treated for 4 h or 24 h (confirmatory assay without S9-mix only) followed by an expression period of 48 h to fix the DNA damage into a stable tk mutation. Under the experimental conditions used in this study , H.C. Red #3 was not mutagenic in the mouse lymphoma assay at the tk locus. Sporadic increases at 1000 µg/mL in Confirmatory Activation Assay were observed but were not repeatable, indicating a clastogenic rather then a mutagenic effect and may have been related to the heavy precipitate observed.

- A Cell transformation study (Jones, 1988, Klimisch 2, Scientific robust method) was performed in order to evaluate HC Red n°3 for cellular transformation capability using the Syrian Hamster Embryo cell (SHE) assay.

Fibroblasts isolated from Syrian golden hamster embryos on gestational day 13 were used in a cellular transformation assay. Two independent laboratories tested HC Red n° 3 at five dosage levels up to 500 and 600 μg/ml, respectively. Duplicate assays utilizing a pH 7.4 culture protocol were performed for a duration of 7 days. Benzo[a]pyrene (B[a]P) was included as a positive control. A positive transformation response was determined using strict morphological guidelines. The transformation activity was determined by pooling the data for the same dose level in both assays. The response was first determined within each laboratory, then between the laboratories. A positive assay response was defined as a three-fold elevation over the control level of transformation at two or more doses in at least two assays. Under the experimental conditions of the study, HC Red n° 3 did not induce transformation at any dosage level in the high pH SHE assay in either laboratory.

Four in vivo key studies were availables :

-A in vivo micronucleus assay was performed in mice (Durward, 1992, OECD 474, GLP compliant, Klimisch 2). In the micronucleus study mice were treated intraperitoneally with the maximum tolerated dose level of 3000 mg/kg bw. The mice were observed 1 h after dosing and subsequently once daily as applicable. Bone marrow cells of both treated and untreated mice were collected 24, 48 and 72 h after dosing. Thousand polychromatic erythrocytes were scored for the presence of micronuclei. Toxicity and thus exposure of the target cells was determined by measuring the NCE/PCE ratio. Cyclophosphamide (50 mg/kg bw) was used as positive control. The test article HC red 3 did not induce micronuclei increase in bone marrow erythrocytes of mice which were treated intraperitoneally.

- A second micronucleus test was performed (Vasquez, 2003, Klimisch 2, comparable to OECD 474 method, GLP compliant). HC Red n° 3 was evaluated for its potential to induce micronuclei in bone marrow after oral administration of the test article to male mice. Groups of 6 mice were treated with test material dissolved in 1% CMC in normal saline, orally by gavage once daily (24 hr apart) for three consecutive days, and then were treated with a fourth dose 20 hr after the previous dose. The test article was administered by oral gavage at 250, 500, and 1000 mg/kg bw/ day. . Tissue sampling occurred 4 hours ± 19 minutes after the final treatment on Day 4. At the time of necropsy, blood samples were collected when the vena cava was cut to exsanguinate the animals. Following exsanguinations duplicate bone marrow slides were prepared for micronucleus analysis. . Analysis of micronuclei did not sustain an increase in the number of micronucleated PCEs. The % PCE present in bone marrow was not substantially changed in the treated animals indicating that HC Red n° 3 did not have cytotoxic properties in the bone marrow and that systemic distribution and thus bioavailability of HC Red n° 3 in bone marrow was not confirmed.

-A Comet assay was performed (Vasquez, 2003, Klimisch 2, comparable to OECD 486, GLP compliant). Groups of 6 mice were treated with test material dissolved in 1% CMC in normal saline, orally by gavage once daily (24 hr apart) for three consecutive days, and then were treated with a fourth dose 20 hr after the previous dose. The test article was administered by oral gavage at 250, 500, and 1000 mg/kg bw/day. Tissue sampling occurred 4 hours ± 19 minutes after the final treatment on Day 4. At the time of necropsy, blood samples were collected when the vena cava was cut to exsanguinate the animals. Following exsanguinations the liver and urinary bladder were removed for comet analysis. Comet slides were prepared from the fresh liver, urinary bladder and blood samples. The extent of DNA migration (measured as tail length, the percentage of migrated DNA, and Olive tail moment) was determined by scoring 100 cells (50 cells per each of two slides, if possible). Following oral administration of dose levels up to 1000 mg/kg bw/day administered on four consecutive days, HC Red n° 3 did not induce a biological relevant increase in DNA migration in the liver, urinary bladder or blood. No increase in the percentage of cells with Low Molecular Weight DNA was found in the liver, urinary bladder or blood of treated animals.

- A Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays was conducted (Streicker, 2005, according to OECD 474 and similar to OECD 488, GLP compliant). Male Blue Blue® B6C3F1 mice were treated daily for 8 weeks with 500 and 1000 mg/kg bw/day of test item in 1% carboxymethylcellulose in n-saline. All animals of all treatment groups were sacrificed after 8 weeks of treatment 24 ± 4 h after the last dose by CO2 asphyxiation. Necropsy included an examination of the external features of the carcass, including all external orifices, the abdominal, thoracic, and cranial cavities and all organ/tissues. Sections of all tissues from each animal were preserved in 10% NBF. Liver, urinary bladder and kidney were observed for gross or macroscopic lesions and examined microscopically. Tissue from the left lobe of the liver and the entire urinary bladder were collected from each mouse and evaluated for mutation on the cII locus.

Survival to the terminal sacrifice was 100% for  all  treatment  groups. In  comparing  the group mean body weight gain there were no statistical differences were found between the treatment groups. There were no significant treatment-related clinical observations noted during this study. At necropsy, limited gross findings were noted in any treatment group. At this time there has been no evaluation of the Micronucleus or histopathological samples. There was no statistically significant or biologically relevant increase in mutation frequency in the 500 mg/kg or 1000 mg/kg HC Red No. 3 treated groups compared to the control for either the liver or urinary bladder tissues. The positive control ENU induced large increases in mutant frequencies in both liver and urinary bladder which were statistically highly significant, demonstrating the ability of the test system to detect mutagens under the applied test conditions and in the selected target organs.

Justification for classification or non-classification

According to the results of the key studies, the test item HC Red No.3 induced mutagenicity in bacterial strain TA1537 and TA98 (Ames test). No genotoxic effect was observed when tested on mammalian cell lines.

In in vivo tests, the test item HC Red No. 3 did not induce mutagenicity effect when tested in mice or rats. The genotoxicity effect observed in bacterial strains was not found in mice or rats. Hence, the test item HC Red No.3 was not classififed for mutagenicity according to REACh Regulation.