2-[(2-nitrophenyl)amino]ethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

According to the results of the three key studies, the test item HC Yellow No. 2 induced genotoxicity in in vitro bacteria strains and mammalian cells as mouse lymphoma cells (L5871Y) and CHO cells.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

According to the results of the two key studies, the test item HC Yellow No.2 did not induce genotoxicity and clastogenicity in Micronucleus Assay and Uscheduled DNA Synthesis performed in mice and rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Three in vitro key studies were available to assess genotoxicity of the test item HC Yellow No. 2 :

- Wagner, 2005, Klimisch 1, GLP compliant, OECD 471: The test item HC Yellow No. 2 was investigated for the induction of gene mutations in Salmonella typhimurium (TA98, TA100, TA1537, TA 1535) and Escherichia coli (WP2uvrA) (Ames test) according to OECD 471 method. Bacterias were exposed to differents doses : - initial experiment: 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg/plate without and with S9-mix and - confirmatory experiment: 25, 75, 200, 600, 1200, 1800 and 5000 μg/plate without and with S9-mix. After incubation preincubation and/or incubation period, revertant colonies and toxicity were evaluated. . A biologically relevant and more or less dose dependent increase in the number of revertant colonies was observed in strain TA98 after exposure to the test item in the presence of S9-mix.

- Ramadevi Gudi, Klimisch 1, GLP compliant, OECD 473 : HC Yellow No.2 has been investigated in the absence and presence of metabolic activation for the induction of chromosomal aberrations in CHO cells.

HC Yellow has been investigated in the absence and presence of metabolic activation for the induction of chromosomal aberrations in CHO cells. The highest dose selected for the main experiment was the lowest dose which induced substantial, the selected doses were : -4 h treatment: 250, 500 and 1000 μg/ml without S9-mix, 500, 875 and 1000 μg/ml with S9-mix and -20 h treatment: 180, 375 and 750 μg/ml without S9-mix. In the main test cells were treated for 4 hours and harvested 20 hours after the start of treatment or treated for 20 hours and harvested immediately after the end of treatment. Two hours before harvest, colcemid  was added to each plate. Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. Toxicity was determined by measuring relative cell growth inhibition and by the decrease in the mitotic index. Chromosome (metaphase) preparations were stained with 5% Giemsa and examined microscopically for chromosomal aberrations and the mitotic index.

Biological relevant and dose-dependent increases in the number of cells with chromosome aberrations were found after 4 hours treatment with the test item with S9-mix and after 20 hours treatment without S9-mix.

- San, Klimisch 1, GLP compliant, OECD 476 : The test item was assayed for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation. Selected doses for the study were : - initial assay: 25, 50, 75, 100, 200, 300, 400, 500, 600 and 700 μg/ml without S9-mix, 1.5, 3, 4, 5, 6, 7, 8, 9, 10, 15 and 50 μg/ml with S9-mix ; -extended treatment assay: 50, 75, 100, 125, 150, 200, 300, 400, 500 and 600 μg/ml without S9 -mix ; - independent repeat assay: 1, 2.5, 7.5, 10, 12.5, 60 and 75 μg/ml with S9-mix ; -confirmatory extended treatment assay: 25, 50, 100, 150, 200, 300, 400, 500, 600 and 650 μg/ml without S9-mix. In the main test, cells were treated for 4 h (initial assay:) or 24 h (extended treatment assay and confirmatory extended treatment assay, without S9-mix), followed by an expression period of 48 h to fix the DNA damage into a stable tk mutation and a selection growth of 10-14 days. Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. To discriminate between large (indicative for mutagenic effects) and small colonies (indicative for a clastogenic effect) colony sizing was performed. Toxicity was measured in the main experiments as percentage relative total growth of the treated cultures relative to the total growth of the solvent control cultures.

Dose related increase in mutant frequency was observed in the initial assay (4 h exposure) without S9-mix and at the second highest dose only with S9-mix. In the extended treatment assay (24 h treatment) a mid dose (200 μg/ml) and the highest dose showed an increase in mutant frequency. The independent repeat assay demonstrated a dose dependent increase in the mutant frequency. Finally a dose dependent increase in mutant frequency was also found in the confirmatory extended treatment assay without S9-mix. The data on colony size distribution pointed to an increase in the frequency of small colonies when the treated cultured were compared to the solvent control cultures indicating to a clastogenic rather then a mutagenic potential of the test item.

Two in vivo key studies were performed :

- Krsmanovic, 2005, Klimish 1, OECD 473, GLP compliant : In the definitive experiment mice were exposed by oral gavage to doses of 0, 250, 500 and 1000 mg/kg bw. Bone marrow cells were collected 24 h or 48 h (control and high dose only) after dosing. Toxicity and thus exposure of the target cells was determined by measuring the ratio between polychromatic and total erythrocytes (PCE/EC). In the main study, reductions up to 18% in the ratio of PCEs to total erythrocytes were observed in the Test item -treated groups relative to the vehicle control. Biological relevant increases in the number of micronucleated PCEs compared to the concurrent vehicle controls were not found at any dose tested, neither 24 or 48 h after treatment and neither for male and females.

- Kamala, 2005, Klimisch 1, OECD 486, GLP compliant : This study was conducted to evaluate DNA damage caused by the test substance HC Yellow 2, in the In Vivo Unscheduled DNA Synthesis (UDS) Test in Rats. In the Main UDS Test, animals were treated for either 2 to 4 hours or 12 to 16 hours. The test Item was administered to 5 male rats per dose per exposure time at doses of 750 and 1500 mg/kg bw. Two additional groups of 5 rats each received a single oral gavage dose of PEG-400 or 35 mg/kg bw dimethylnitrosamine (DMN) and served as the vehicle and positive control, respectively.

Animals were observed after dose administration and at time of harvest for clinical signs of chemical effect. Hepatocytes for UDS analysis were collected 2-4 h and 12-16 h after administration of the test substance. Cells were incubated with radiolabelled thymidine. Evaluation of autoradiography was done after 3 days. UDS was reported as net nuclear grain, neither a biological relevant increase in mean net nuclear grain count nor in the percentage of cells in repair as compared to the untreated control was found in hepatocytes of any treated animal both for the 2-4 h and the 12-16 h treatment time.

Justification for classification or non-classification

According to the results of the three key studies, the test item HC Yellow No. 2 induced genotoxicity in in vitro on bacteria strains and mammalian cells as mouse lymphoma cells and CHO cells. However, this genotoxic effect was not observed in differents in vivo tests performed on rats and mice (Micronucleus Assay and Uscheduled DNA synthesis). Hence, the test substance was not considered as genotoxic according to the results of the three in vitro key studies and two in vivo key studies.The HC Yellow No. 2 was not classified according to CLP criterias.