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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

3 in vitro studies are available to estimate the mutagenic and genotoxic potency of the substance: 1 Ames test and 1 mammalian mutation assay in mouse lymphoma thymidine kinase locus assay using the cell line L5178Y indicated no mutagenic effects. In micronucleus assay, it was concluded that high concentrations of 2-amino-6-chloro-4-nitrophenol have the potential to induce micronuclei in cultured human peripheral blood lymphocytes in the presence of S9-mix.

2 Ames tests

•       Sokolowski (2004),The bacterial reverse mutation test of 4-amino-3-methylphenol was performed by following methods similar to the OECD guideline 471 (Bacterial Reverse Mutation Test).

The assay was performed by using different strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA1538), with and without metabolic activation (S9 mix (Phenobarbital/β-Naphthoflavone induced rat liver S9)) in agar media. The mutagenicity assay was performed using the preincubation method at the following dose levels: 3, 10, 33, 100, 333, 1000 and 2500 µg/plate. Reduced background growth was observed with and without metabolic activation in strain TA100 at 2500 µg/plate and in strain TA102 at 1000 and 2500 µg/plate. No substantial increase in revertant colony number was observed in the strains following treatment at any dose level in the presence or absence of S9 mix, with an exception of effects observed in tester strain TA98 at 1000 µg/plate. A moderate but dose dependent increase in revertant colony numbers was observed in strain TA 98 (with and without metabolic activation) exceeding the threshold of twice the numbers of the corresponding solvent control at 1000 µg/plate. Therefore, a confirmatory test (experiment 3) was performed using tester strain TA 98, to clarify the questionable results. The confirmatory test (experiment 3) using tester strain TA98 was conducted at 100, 333, 1000, 1754, 2500, 3750 and 5000 µg/plate. No relevant increase in the number of revertant colonies in tester strain TA98 occurred in the confirmatory experiment. Therefore, the variations noted in tester strain TA98 in experiment 2 were considered irrelevant.

2-amino-6-chloro-4-nitrophenol (Chlororange base) did not induce gene mutations in Salmonella typhimurium in any of the tester strains (TA98, TA100, TA1535, TA1537 and TA102) in the presence or absence of S9 mix.

•       Cosmital (1989), The test substance, 2-amino-6-chloro-4-nitrophenol (a nitroaromatic compound), was evaluated for potential mutagenicity in a single nitroreductase deficient strain of Salmonella typhimurium (TA98NR). The test substance was tested at concentrations of 10, 30, 100, 300, 1000, 3000 and 6000 ug/plate in triplicate without metabolic activation. Solvent and positive controls were concurrently performed. The highest two concentrations, 3000 and 6000 ug/plate, were cytotoxic to the bacteria based on a reduction of the background lawn by 50% and 100% in comparison to the solvent control, respectively. At the concentrations tested, there was no induction of revertants by a factor of 2 -3 above the solvent control at any concentration. Results for the solvent and positive control were presented. The test substance was concluded to be negative for mutagenicity in strain TA98NR in the absence of metabolic activation when tested up to cytotoxic concentrations.

2 mammalian mutation assays in mouse lymphoma thymidine kinase locus assay using the cell line L5178Y

•       Kennelly (1986), The in-vitro mammalian gene mutation test of 2-amino-6-chloro-4-nitrophenol was determined following method comparable to OECD guideline 476 (In vitro Mammalian Cell Gene Mutation Test).

The microsomal enzymes obtained from livers of Wistar rats treated with Aroclor 1254 were used as the metabolic activation system (S9).

The test substance was tested at 0, 15.8, 50, 158 and 500 µg/mL in the presence and absence of S-9 for viability and 6-thioguanine resistance.

There were no statistically significant increases in the mutation rates in the presence or absence of metabolic activation. No dose-related increases in the mutation rate were observed in the presence and absence of metabolic activation.

Based on the results, 2-amino-6-chloro-4-nitrophenol was considered non-mutagenic in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the presence or absence of metabolic activation.

•       Wollny (2003). The assay was performed in two independent experiments (both with and without liver microsomal activation) using two parallel cultures each. The cells were exposed to test substance for 4 h in Experiment I (with and without metabolic activation) and for a treatment period of 24 h in Experiment II (without metabolic activation only). Based on the pre-test toxicity (conducted in the range 14.8 – 1900 µg/mL) results, the following concentrations of the test substance, which was dissolved in DMSO, were selected for the main test:

Experiment I (4 h treatment):

Without metabolic activation: 0, 30, 60, 120, 240, 480 and 960 µg/mL

With metabolic activation: 0, 3.8, 7.5, 15, 30, 60 and 120 µg/mL

Experiment II (24 h treatment):

Without metabolic activation: 0, 15, 30, 60, 120, 180 and 240 µg/mL

In experiment I severe toxic effects occurred in the absence of metabolic activation at 960.0 µg/mL in both cultures (relative cloning efficiency 1: 12.8 to 15.3%). In the presence of metabolic activation both the relative cloning efficiency and the relative total growth were reduced to less than 50 % of the corresponding solvent control at the highest analysable concentration of 60.0 µg/mL (both cultures).

In experiment II, a steep toxicity gradient was noted with severe toxicity noted at 180 μg/mL and above for both parallel cultures (relative cloning efficiency: 1.5 and 1.9%) and only a minor effect at 120 μg/mL. Values obtained at 180 μg/mL and above were rejected as the acceptance criteria (cloning efficiency ≥ 10 %) were not fulfilled at these severely toxic concentrations.

In experiment I, no substantial and reproducible increase of the mutation frequency was observed in either culture. In Experiment II, the mutation frequency just reached the threshold of twice the corresponding solvent control at 120 µg/mL in the first culture. However, the absolute value of mutation frequency remained well within the range of negative and solvent controls; and no comparable effects occurred in the second culture under identical conditions. Therefore, the isolated increase in the first culture was judged as biologically irrelevant fluctuation.

Based on above, 2-amino-6-chloro-4-nitrophenol was considered non-mutagenic in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y, in the absence and presence of metabolic activation.

4 chromosomic aberration, SCE or micronucleus assays evaluated the potential clastogenic effect.

•       Kirkland (1986), the in vitro Mammalian Chromosome aberration test of 2-amino-6-chloro-4-nitrophenol was performed by following the method similar to the OECD guideline 473 (In vitro Mammalian Chromosome Aberration Test).

The objective of this study was to evaluate the clastogenic potential of the test substance by examining its effects on the chromosomes of CHO cells in the absence and presence of metabolic activation. Aroclor-induced rat liver S9 fraction was used as the metabolic activation system.

Doses with and without S-9: 0(DMSO), 5, 16.5, 50, 165, 500, 1650 and 5000 µg/mL.

The test substance precipitated out of solution at 1650 and 5000 µg/mL. The test substance was completely toxic at 5000 and 1650 µg/mL, but did not inhibit mitosis at 500 µg/mL (both in the absence and presence of S-9). Based on these results, the cultures treated with 0, 50, 165 and 500 µg/mL test concentration were analyzed for chromosomal aberrations.

In the absence of metabolic activation, there was no evidence of induction of structural aberrations. Small but statistically significant increases in numerical aberrations were observed at 165 and 500 µg/mL. The total number of structural and numerical aberrations did not, however, exceed the defined control range. The numbers and types of damage seen indicate that the result is probably not biologically significant. This increase in numerical aberrations was probably "background noise" rather than test substance related induction of aberrations.

In the presence of metabolic activation, all types of aberrations (structural and numerical) occurred within frequencies similar to control values and there were no significant differences.

Under the test conditions, 2-amino-6-chloro-4-nitrophenol did not induce structural chromosome aberrations in the presence and absence of metabolic activation.

•       COSMITAL (1985), the test substance, 2-amino-6-chloro-4-nitrophenol, was evaluated for its potential to induce sister chromatid exchanges (SCE) in cultures of CHO-K1 cells both in the absence and presence of metabolic activation.  Three separate experiments were performed.  Experiments 1 and 2 were performed both with and without metabolic activation at concentrations of 1e-5, 3e-5, 1e-4, 3e-4, and 1e-3 Molar.  Experiment 3 was performed only without metabolic activation at concentrations of 5e-4, 1e-3, 2e-3, and 4e-3 Molar.  Cultures were exposed to the test substance for 2 hours.  At least two replicate cultures were used for analyzing potential SCE induction at each dose level.  Results of Experiments 1 and 2 showed that the test substance produced a negative response at all concentrations tested both with and without metabolic activation.  In Experiment 3, which was conducted only without metabolic activation, a negative response was obtained at the first three test substance concentrations, while a weak positive result was obtained at the highest dose tested.  Precipitate was observed at the two highest concentrations tested in Experiment 3.  According to the study report, the solvent and positive controls were found to be in the expected range for Experiments 1 and 2, while there were no conclusions documented for the controls in Experiment 3.  Under the conditions of this assay, the test substance did not induce sister chromatid exchanges in CHO-K1 cells up to concentrations of 1000 uM both with and without metabolic activation.  When higher concentrations were tested without metabolic activation, the test substance was negative up to and including test substance concentrations of 2000 uM. Although the test substance did induce a weakly positive response at 4000 uM, this was only observed in the presence of a precipitate.

•       Marshall (1988), the in vitro Mammalian Chromosome aberration test of 2-amino-6-chloro-4-nitrophenol was performed by following the methods similar to the OECD guideline 473 (In vitro Mammalian Chromosome Aberration Test).

o       Human peripheral blood lymphocytes were cultured in vitro and treated in the presence and absence of a rat liver metabolizing system (S-9). Doses were: 0 (DMSO), 39.1, 78.13, 156.25, 312.5, 625, and 1250 µg/mL

Based on the mitotic index, the test concentrations selected for the chromosome aberration analysis were 0, 312.5, 625, and 1250 µg/mL (with and without S-9).

In the absence of metabolic activation, the number of aberrations in treated cultures was similar to those found in cells from solvent control cultures (no significant difference).

In the presence of metabolic activation, a statistically significant number of aberrations was observed at 1250 µg/mL. At this dose more aberrations were observed in the cells from the male donor than from the female donor. This paralleled the increased mitotic inhibition in the male cultures.

The number of aberrations observed in both donors at this dose level exceeded the range seen in historical controls. There was no evidence of increased chromosome damage at lower doses. Based on the results, 2-amino-6-chloro-4-nitrophenol induced chromosomal aberrations in human peripheral blood lymphocytes (in vitro) with metabolic activation when tested at 1250 µg/mL. However, test substance did not induced chromosomal aberrations in the absence of metabolic activation.

•       Clare (2005), The in-vitro micronucleus test of 2-amino-6-chloro-4-nitrophenol was conducted following methods comparable to the OECD guideline 487 (In vitro micronucleus test) using human lymphocyte cultures.

Two independent experiments were performed in this study. In experiment I, cells were treated with the test substance 24 hours after mitogen stimulation with phytohaemagglutinin (PHA). In the second experiment, the treatment started 48 hours after the mitogen stimulation. The exposure times for the test substance in the presence and the absence of S9 mix were 3 and 20 hours, respectively.

Experiment I (treatment 24 hours after mitogen stimulation):

Without S9: 0, 37.5, 75, 150, 175, 200, 225, 250, 275, 300, 350, 400, 45 and 500 µg/mL

With S9: 0, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 and 1800 µg/mL

Experiment II (treatment 48 hours after mitogen stimulation):

Without S9: 0, 50, 100, 150, 200, 225, 250, 275, 300, 350, 400, 450 and 500 µg/mL

With S9: 0, 200, 300, 400, 450, 500, 550, 600, 650, 700, 800, 900 and 1000 µg/mL  (Trial 1); 0, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600 and 1886 µg/mL (Trial 2)  

Additionally, two repeat experiments were conducted as follows:

Experiment III (repeat of Exp II (without S9) conducted to achieve a concentration closer to 60% toxicity): 0, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 425, 450, 475, 500 µg/mL

Experiment IV (repeat of Exp II (with S9) conducted to clarify the results): 0, 400, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 and 1886 µg/mL

Based on the results of the concurrent cytotoxicity assay, the following test doses were selected for micronucleus analysis:

Experiment I: Without S9: 0, 175, 225 and 275 µg/mL; With S9: 0, 1200, 1400 and 1600 µg/mL

Experiment II: Without S9: 0, 150, 275, 350 and 400 µg/mL; With S9: 0, 1100, 1300 and 1600 µg/mL

Experiment III (without S9): 0, 100, 150, 250 and 300 µg/mL

Experiment IV (with S9): 0, 1200, 1300, 1400 and 1500 µg/mL

The results of the study are as follows:

WITHOUT METABOLIC ACTIVATION:

In Experiment I, when cells were treated 24 hours after stimulation with PHA, the frequencies of micronucleated binucleated cells (MNBN) were statistically significantly elevated at each concentration analyzed. The frequency of MNBN cells exceeded the historical vehicle control range in both cultures at the lowest and intermediate doses (175 and 225 µg/mL), but not after the highest concentration (275 µg/mL). There was no obvious concentration-effect relationship.

In Experiment II, the frequencies of MNBN were similar to the concurrent controls and all the MNBN frequencies were within the historical vehicle control range.

In Experiment III, when cells were treated 48 hours after stimulation by PHA, the frequencies of MNBN cells were similar to those of the concurrent DMSO control cultures, except from cultures exposed to 300.0 µg/mL, which were slightly higher. However, all MNBN frequencies were within the historical vehicle control range

The test substance was considered to have an equivocal effect of questionable biological relevance on micronucleus induction in the absence of S-9, when lymphocytes were exposed 24 hours after, but not 48 hours after stimulation with a mitogen. Therefore, the results were considered as ambiguous.

WITH METABOLIC ACTIVATION

In Experiment I, there were increases in the frequency of MNBN cells in cultures exposed to 1400 and 1600 µg/mL test substance compared with the concurrent negative control cultures, with single cultures of the these two concentrations exceeding the historical control range. These effects were associated with high cytotoxicity of 40 and 65% respectively.

In Experiment II, there was a small increase in the frequency of MNBN cells at all concentrations tested compared with the concurrent negative control cultures. The majority of cultures showed frequencies of MNBN cells that exceeded the historical vehicle control range.

In Experiment IV, there was an increase in the frequency of MNBN cells at all concentrations tested compared with the concurrent negative controls. All cultures showed frequencies of MNBN cells that exceeded the historical vehicle control range. Therefore, the results fulfill the criteria of a positive response. It may be note-worthy that much higher concentrations were tested in the presence than in the absence of S-9.

All positive control substances induced statistically significant increases in the proportion of cells with micronuclei.

Based on above, it was concluded that high concentrations of 2-amino-6-chloro-4-nitrophenol have the potential to induce micronuclei in cultured human peripheral blood lymphocytes in the presence of S9-mix. The potential mechanism of the mutagenic action (i. e. clastogenic vs aneugenic) has not been identified.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

2 studies were available to evaluate the in vivo genotoxic effects, and no evidence for a mutagenic/clastogenic potential was noted in vivo.

•       Honarvar (2003), the in-vivo micronucleus test was performed using 2-amino-6-chloro-4-nitrophenol (Chlororange base), following the OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test). Adult male and female NMRI mice received by intraperitoneal route, the test substance at the following doses, investigated with 12 animals (6 male and 6 female)/ dose group:

24 h preparation interval: 0, 18.75, 37.50 and 75.00 mg/kg bw

48 h preparation interval: 75 mg/kg bw

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

The mean number of PCEs was not affected by the test substance at any test concentration or sampling time as compared to the mean value of PCEs in the vehicle control. Hence, 2-Amino-6-chloro-4-nitrophenol, even tested at systemically toxic doses, showed no clear cytotoxic effect in the bone marrow. However, the occurrence of discolored urine as well as the observed signs of toxicity demonstrated that the substance was systemically distributed and bio-available. The animals treated with 75 mg/kg bw expressed toxic reactions such as reduction of spontaneous activity, abdominal position, eyelid closure and ruffled fur.

Based on the results, 2-amino-6-chloro-4-nitrophenol was not mutagenic in the in vivo micronucleus test using NMRI mice after a single intraperitoneal administration up to the maximum tolerated dose of 75 mg/kg bw.

•       Volkner (1987), 2-Amino-6-chloro-4-nitrophenol was administered by gavage to 10 to 16 week old NMRI mice at dose levels of 0, 15 or 150 mg/kg bw in DMSO at a volume of 5 ml/kg bw. Bone marrow cells were investigated 24 h after administration for all three dose and the control groups as well as 48 h and 72 h after administration for the high dose and the control groups.

Femoral bone marrow was sampled from mice after sacrifice 24 hours after dosing for all groups and after 48 and 72 h for the control and the additional high dose groups. At least 1000 PCEs per animal were analysed. In addition, the ratio between polychromatic (PCE) and normochromatic (NCE) erythrocytes per animal was determined. Ten animals per test group (5 males and 5 females) were evaluated.

In the pre-test for toxicity, one of two animals died within 1h after administering doses of 2000, 1000 and 500 mg/kg bw. At 250 mg/kg bw, 2 of 6 treated animals died within 48 h. At 200 mg/kg bw, 1 of 2 animals died within 72 h. At 150 mg/kg bw, severe clinical signs as abdominal position, tremor and convulsion were noted, but no animal died. This dose was selected as the highest test dose for the main experiment, as no lethality was observed.

The ratio of PCE/NCE did not reveal any treatment related effect. Hence 2-Amino-6-chloro-4 - nitrophenol, even if tested at systemically toxic doses, showed no clear cytotoxic effect in the bone marrow. However, the occurrence of severe systemic toxic effects and the toxicokinetic study performed with rats demonstrate a good bioavailability of the test item after oral administration.

There was no statistically significant or biologically relevant increase in the number of micronuclei per 1000 PCEs in the mice of any of the 2-Amino-6-chloro-4-nitrophenol treated groups at any preparation time compared to the respective vehicle control groups.

2-Amino-6-chloro-4-nitrophenol was not mutagenic in the in vivo micronucleus test using NMRI mice after a single oral administration via gavage up to the maximum tolerated dose of 150 mg/kg bw, at which severe signs of toxicity were noted

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Several in vitro and in vivo mutagenicity tests have been performed with 2-

Amino-6-chloro-4-nitrophenol. Both the Ames test and the mouse lymphoma assays were

negative, indicating that 2-Amino-6-chloro-4-nitrophenol does not possess a potential to cause

gene mutation in vitro. 2-Amino-6-chloro-4-nitrophenol did reveal a potential to induce

micronuclei in cultured human lymphocytes. However, no evidence for a mutagenic/clastogenic potential was noted in vivo. Therefore the genotoxic effect seen in the in vitro micronucleus test with human lymphocytes does not occur under appropriate in vivo test conditions.