[8-[(p-aminophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

[8-[(p-aminophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride estimated to be non genotoxic

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

calculation (if not (Q)SAR)

Remarks:

Migrated phrase: estimated by calculation

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from modelling database developed by the National Food Institute, Technical University of Denmark.

Principles of method if other than guideline:

Data is predicted using the Danish (Q)SAR Database

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

no data available

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):negativeBased on the QSAR prediction done using the Danish (Q)SAR Database, the genetic toxicity was estimated to be negative during Chromosome Aberrations in Chinese Hamster Ovary (CHO) Cells. Thus it can be concluded that the substance [8-[(p-aminophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride is non genotoxic and based on the CLP criteria for classification it can be not classified for genetic toxicity.

Executive summary:

Based on the QSAR prediction done using the Danish (Q)SAR Database, the genetic toxicity was estimated to be negative during Chromosome Aberrations in Chinese Hamster Ovary (CHO) Cells. Thus it can be concluded that the substance [8-[(p-aminophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride is non genotoxic and based on the CLP criteria for classification it can be not classified for genetic toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro gene toxicity

Based on the QSAR prediction done using the Danish (Q) SAR Database (2016), in vitro genetic toxicity was estimated to be negative during Chromosome Aberrations in Chinese Hamster Ovary (CHO) Cells. Thus it can be concluded that the substance [8-[(p-aminophenyl)azo]-7-hydroxy-2-naphthyl]trimethylammonium chloride is non genotoxic

In a opinion given by Scientific Committee on Consumer Safety for Basic Brown 16 (2013), in vitro genetic toxicity was evaluated by using Basic Brown 16 in Salmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 in the concentration of 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix for TA98 and TA100 and 0, 1, 3, 10, 33, 100, 333, 1000 and 2500 μg/plate in deionized water by direct plate incorporation. No reductions in the number of revertants were observed at 1000 μg/plate. Therefore, Basic Brown 16 was considered to be negative when tested on Salmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 strain.

Similar to above reference, in vitro genetic toxicity was evaluated by using Basic Brown 16 in Salmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 in the concentration of 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix for TA98 and TA100 and 0, 1, 3, 10, 33, 100, 333, 1000 and 2500 μg/plate in deionized water by direct plate incorporation. More or less concentration dependent increase in the number of revertants was seen up to the toxic concentration in TA98 and TA1537 in the presence of metabolic activation. However, No reductions in the number of revertants were observed in TA98, TA100, TA102, TA1535 and TA1537 without and in TA100, TA102 and TA1535 with metabolic activation. Therefore, Basic Brown 16 was considered to be ambiguous when tested on SalmonellatyphimuriumTA98, TA100, TA102, TA1535 and TA1537 strain.

Similar to above reference, in vitro genetic toxicity was evaluated by using Basic Brown 16 were expose to the V79 cells in the concentration of0, 5, 19, 20, 50 and 100 μg/ml without S9-mix and 0, 100, 300, 1000, 3000 and 6000 μg/plate with S9-mix in phosphate buffered saline. Biologically relevant and concentration dependent increase in the mutant frequency was not observed, neither in the presence nor in the absence of metabolic activation. Occasionally, an increase in mutant frequency was found; these were not reproducible and considered not biologically relevant. Therefore, Basic Brown 16 was considered to be Negative (with and without) when tested on V79 cells. 

Similar to above reference, in vitro genetic toxicity was evaluated by using Basic Brown 16 by expose to the Epiderm^TMreconstructed human skin tissue in the concentration of 3.13, 6.25, 12.5, 25 and 50 μg/tissue in 3 independent experiments. DNA was stained with the fluorescence dye SYBR Gold and Basic Brown 16 added in 10 μl in 100% ethanol on top of the tissue for 3 hours. In fist experiment, biologically relevant increase in the % tail DNA was not observed. In second experiment, statistically significant increase in the % tail DNA at 12.5 and 50 μg/tissue were observed. However, in third experiment, same results were observed as in fist experiment. The % tail DNA values again fell within the range of the concurrent solvent control. Therefore, Basic Brown 16 was considered to be Negative when tested on Epiderm^TMreconstructed human skin tissue in Single cell gel electrophoresis assay.

Similar to above reference, in 2011 for read across Basic Red 76, in vitro genetic toxicity was evaluated by using Basic Red 76 onSalmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 for the AMES assay in the concentration of experiment I: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in direct plate incorporation with 48 h incubation without and with S9-mix, experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate by pre-incubation method with 60 minutes pre-incubation without and with S9-mix and at least 48 h incubation without and with S9-mix. In the main tests toxic effects evident as clearing of the bacterial background lawn were observed in experiment I in TA98 and TA100 and in experiment II in all strains predominantly at higher concentrations. Toxic effects evident as a reduction in the number of revertants were observed at higher concentrations without and with metabolic activation in nearly all strains tested. A biologically relevant increase in revertant colonies was not observed in any of the strains tested at any dose level in the absence or presence of S9-mix in both experiments. Therefore, Basic Red 76 was considered to be not mutagenic toSalmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 with and without S9 metabolic activation.

Similar to above reference, in 2011 for read across Basic Red 76, in vitro genetic toxicity was evaluated by using Basic Red 76 on mouse lymphoma cell line L5178Y both in the absence and presence of S9 metabolic activation in the concentrations for experiment I: 26.6, 53.1, 106.3, 212.5 and 318.8 μg/ml without and with S9-mix for 4 h; expression period 72 h, selection growth 10-15 days and for experiment II: 53.1, 106.3, 212.5, 318.8 and 425.0 μg/ml without S9-mix 24 h; expression period 48 h, selection growth 10-15 days. In the pre-test after 4 h treatment, precipitation was observed at 425 μg/ml in the absence and presence of S9-mix and at 212.5 μg/ml after 24 h treatment in the absence of S9-mix. No relevant increase of the osmolarity and pH-value was observed at the maximum concentration. In both experiments in the absence and presence of S9-mix the appropriate level of toxicity (about 10-20% survival after the highest dose) was reached; only in experiment II in one culture treated with the highest concentration of the test chemical, the appropriate level of toxicity was not reached. Both in the absence and presence of metabolic activation, a biologically relevant increase in the mutant frequency due to exposure to the test chemical was not reported. Therefore, under the experimental conditions used, Basic Red 76 was considered to be not mutagenic in this mouse lymphoma assay using the tk locus.

Similar to above reference, in 2011 for read across Basic Red 76, in vitro genetic toxicity was evaluated by using Basic Red 76 on Salmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 for the AMES assay in the Concentration of experiment I: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix by direct plate incorporation with 48 h incubation and experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate by pre-incubation method with 60 minutes pre-incubation and at least 48 h incubation without and with S9-mix. In the main tests toxic effects evident as clearing of the bacterial background lawn were observed in experiment I in TA98 and TA100 and in experiment II in all strains predominantly at higher concentrations. Toxic effects evident as a reduction in the number of revertants were observed at higher concentrations without and with metabolic activation in nearly all strains tested. A biologically relevant increase in revertant colonies was not observed in any of the strains tested at any dose level in the absence or presence of S9-mix in both experiments. Therefore, Basic Red 76 was considered to in the absence and the presence of S9 metabolic activation to Salmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537.

Similar to above reference, in 2008 for another read across (8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride, in vitro genetic toxicity was evaluated by using Mouse Lymphoma assay (tk locus) in the concentration of Experiment I: 8.1, 16.3, 32.5, 65.0 and 97.5 μg/ml without S9-mix and 16.3, 32.5, 65.0, 130.0 and 195.0 μg/ml with S9-mix and Experiment II: 8.0, 16.0, 32.9, 64.0, 128.0 and 192.0 μg/ml without in deionised water. In the main test, cells were treated for 4 h or 24 h (without S9 in experiment II only) followed by an expression period of 72 or 48 h to fix the DNA damage into a stable tk mutation. In experiment I, precipitation was noted at 97.5 and 130.0 μg/ml without S9-mix and at 195.0 and 260.0 μg/ml with S9-mix; in experiment II precipitation occurred at 128.0 and 192.0 μg/ml. Both in experiment I and II no biological relevant and dose dependent increase in the number mutant colonies was observed independent of the presence or absence of S9-mix. Therefore, it can be concluded that the test substance has negative genetic toxicity effects on Mouse Lymphoma assay (tk locus).

Similar to above reference, in 2008 for another read across (8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride, in vitro genetic toxicity was evaluated by using Chinese hamster V79 cells. In the concentration for experiment IA: 128.1, 256.3, 2050.0 and 4100.0 μg/ml without S9-mix, Experiment IA: 128.1, 256.3, 1025.0 and 2050.0 μg/ml with S9-mix, Experiment IB: 31.3, 62.5, and 125.0 without S9-mix, Experiment IIA: 128.1, 256.3 and 512.5 μg/ml without S9-mix, Experiment IIA: 128.1, 256.3, 512.5 and 1025.0 μg/ml with S9-mix, Experiment IIB: 100.0, 150.0, 200.0, 250.0, 300.0 and 350.0 μg/ml without S9-mix, Experiment IIB: 100.0, 200.0, 400.0, 600.0 and 800.0 μg/ml with S9-mix in deionised water. The treatment period in the main test was 4 h in experiment I (without and with S9-mix) and in experiment II (with S9-mix) or 20 h in experiment II (without S9-mix). Harvest time was 24 h or 48 h (experiment II with S9-mix only) after the beginning of culture. For assessment of cytotoxicity a XTT test was additionally carried out in parallel to the main micronucleus test. Therefore, (8-((4-Amino-3-nitrophenyl)azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride did not induce an increase in micro nucleated cells and thus, the test was considered to be negative.

Similar to above reference, in 2014 for another read across (8-((4-Amino-3-nitrophenyl) azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride, in vitro genetic toxicity was evaluated by using L5178Y Mouse lymphoma cells in the concentration for Experiment I: 3.1, 6.2, 12.4, 24.8, 49.6 and 99.2 μg/ml without S9-mix and 6.2, 12.4, 24.8, 49.6, 99.2 and 198.4 μg/ml with S9-mix, Experiment II 3.1, 6.2, 12.4, 24.8, 37.2 and 49.6 μg/ml without S9-mix and 3.1, 6.2, 12.4, 24.8, 49.6 and 99.2 μg/ml with S9-mix in deionised water. In the main tests, cells were treated for 4h without and with S9-mix or for 24h without S9-mix followed by an expression period of 48h to fix the DNA damage into stable hprtmutations. A serum concentration of 3% was used. . A biological relevant increase of the mutant frequency was not observed in both experiments with and without metabolic activation. An isolated increase exceeding the limit of three times the mutant frequency of the untreated control was observed in one culture of experiment II with metabolic activation at the highest concentration (99.2 μg/ml). However, precipitation occurred at this concentration and the increase was not concentration dependent. Therefore, this isolated effect was considered not biologically relevant. Thus, (8-((4-Amino-3-nitrophenyl) azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride considered to be non-mutagenic when tested on hprt locus in theL5178Y Mouse lymphoma cells(with and without metabolic activation system).

Thus, on the basis of above available information for the target Basic Brown 16 and both read across Basic Red 76 and (8-((4-Amino-3-nitrophenyl) azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride. It can be concluded that the test substance has negative genetic toxicity effects and based on the CLP criteria for classification, it cannot be classified as genotoxic.

Justification for selection of genetic toxicity endpoint
Estimated to be negative during Chromosome Aberrations in Chinese Hamster Ovary (CHO) Cells.

Justification for classification or non-classification

The target Basic Brown 16 and both read across Basic Red 76 and (8-((4-Amino-3-nitrophenyl) azo)-7-hydroxy-2-naphthyl)trimethylammonium chloride were non mutagenic. Therefore, It can be concluded that the test substance has negative genetic toxicity effects and based on the CLP criteria for classification, it cannot be classified as genotoxic.