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Genetic toxicity in vitro

Description of key information
Azo zinc complex pigment- melamine compound is evaluated as non-mutagenic in the available Reverse Mutation Assay in bacteria (Ames test) and in a Chromosome Aberration Test in mammalian cells in vitro. The available HPRT test using the structure analogue Azo Nickel complex pigment - melamine compound as surrogate confirms the non-mutagenic property of Azo zinc complex pigment - melamine compound.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study under GLP condition
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The study was performed to investigate the potential of Gelbpigment E4GN to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus in V79 cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
other: the cells have a stable karyotype with a modal chromosome number of 22
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from phenobarbital/ß-naphthoflavone induced liver of male Wistar rats
Test concentrations with justification for top dose:
exposure S9 concentrations in µg/mL
period mix
Experiment I
4 hours - 39.1 78.1 156.3p 312.5p ? 1250.0p
4 hours + 39.1 78.1 156.3p 312.5p ? 1250.0p
Experiment II
24 hours - 18.8 37.5 75.0p 150.0p 300.0 p 600.0 p
4 hours + 18.8 37.5 75.0p 150.0p 300.0 p 600.0 p
p = precipitation
Vehicle / solvent:
deionised water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
A pretest was performed in order to determine the concentration rannge for the mutagenicity test
The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points that is three times above the spontaneous mutation frequency.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
precipitation occurred: Exp I with and without S9 at 156.3 µg/ml and above; Exp II, without S9, 24 h treatment and with 4 h treatment at 75.0 µg/ml and above
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.

Precipitation at the end of treatment occurred in experiment I at 156.3 μg/mL and above in the presence and absence of metabolic activation. In experiment II precipitation was observed at 75.0 μg/mL and above with and without metabolic activation.

No relevant cytotoxic effect indicated by a relative cloning efficiency I and/or relative cell density below 50% in both parallel cultures was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The induction factor did not exceed the threshold of three times the corresponding solvent control at any experimental point. In the second culture of the first experiment with metabolic activation at 156.3 μg/mL the mutant frequency exceeded the historical range of solvent controls. This increase however, was judged as biologically irrelevant since the threshold of three times the corresponding solvent control was not exceeded. Furthermore, the increase was not reproduced in the parallel culture performed under identical experimental conditions and was not dose dependent as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the

experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.4 up to 29.4 mutants per 10E6 cells; the range of the groups treated with the test item was from 4.9 up to 54.9 mutants per 10E6 cells.

EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:
Interpretation of results (migrated information):
negative
Executive summary:

The study was performed to investigate the potential of Gelbpigment E4GN (used as surrogate) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD TG 476 under GLP conditions.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. No cytotoxicity was observed but the concentration range used was limited by precipitation occurring in the first experiment from 156 µg/ml onwards and in the second expriment independent from treatment time and independet of the presence of a metabolic activation system from 75 µg/ml onwards

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item was negative at the HPRT locus in V79 cells. Therefore, Gelbpigment E4GN is considered to be non-mutagenic in this HPRT assay.

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

Additional information

Additional information from genetic toxicity in vitro:

AMES TEST

Mutagenicity of the test substance, Azo zinc complex pigment - melamine compound was assessed in the bacterial reverse mutation assay using 5 bacteria strains: Salmonella typhimurium TA100, TA1545, TA98, TA1537 and Escherichia coli WP2 uvrA'. The test was conducted by the pre-incubation method according to OECD TG 471 under GLP conditions in the presence and in the absence of a metabolic activation system (liver S9 -mix).

The preliminary test with and without S9 -mix in the concentration range from 1.22 up to 5000 µg/plate yielded a negative result and no growth inhibition was observed but precipitation occurred in all tester strains from 19.5 µg/plate onwards in the absence of S9 -mix and from 1.22 µg/plate onwards in the presence of S 9 -mix. Therefore, the main study and the repeat were performed in the concentration range from 313 up to 5000 µg/plate in the absence and in the presence of the metabolic activation system and yielded negative results with no growth inhibition and precipitations in all strains at all concentrations.

Based on the results described above it was concluded that Azo zinc complex pigment-melamine compound was not mutagenic under the conditions employed in this study.

CHROMOSOME ABBERRATION TEST

An in vitro chromosomal aberration test of Azo zinc complex pigment - melamine compound was conducted with CBL/IU cells derived from the lungs of female Chinese Hamsters as indicator cells and was performed according to OECD TG 473 under GLP conditions using concentrations up to and including 5000 µg/ml in the absence and in the presence of a metabolic activation system (liver S9 mix). As results of the cell growth inhibition tests m the 50 % cell growth inhibition concentration (IC50) were 2313.9 µg/ml (-S9 mix, h-treatment), 5000 µg/ml (+S9 mix, 6 h-treatment) and 59.3 µg/ml (24 hour treatment).

As a result the incidences of both structural aberrant cells and numerical aberrant cells were less than 5 % in the -S9 mix and +S9 mix and 24 hour assays. The positive controls were functional.

In conclusion Azo zinc complex pigment - melamine compound was not considered to have the potential to induce chromosomal aberration in CHL/IU cells under the conditions employed in this study.

POINT MUTATION IN MAMMALIAN CELLS

Azo Zinc Complex Pigment – Melamine compound shall be registered according to Art 10 of Regulation EC No. 1907 / 2006 (REACH) for annual production of 10 – 100 tons (ANNEX VIII)

For mutagenicity three studies have to be provided: genotoxicity in bacteria, genotoxicity in mammalian cells and a chromosome aberration test.

For Azo Zinc Complex Pigment – Melamine compound there is no genotoxicity study in mammalian cell systems available. The respective study with Azo Nickel Complex Pigment - Melamine Compound which is performed according to OECD TG 476 under GLP conditions, yielded a negative result as no point mutations were induced. Considering the results of the available genotoxicity studies in bacteria and the available chromosome aberration tests which both do not give evidence of induced mutagenic effects for both compounds, it is likely that also Azo Zinc Complex Pigment – Melamine Compound would not induce point mutations in the HPRT locus of mammalian cells as it was shown with Azo Nickel Complex Pigment - Melamine Compound. Thus, it seems justified to take the HPRT test with Azo Nickel Complex Pigment - Melamine Compound as surrogate for the HPRT test with Azo Zinc Complex Pigment Melamine Compound.

HPRT test with Azo Nickel Complex Pigment - Melamine Compound

The study was performed to investigate the potential to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD TG 476 under GLP conditions.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. No cytotoxicity was observed but the concentration range used (37.5 - 1250 µg/ml) was limited by precipitation occurring in the first experiment from 156 µg/ml onwards and in the second experiment independent from treatment time and independent of the presence of a metabolic activation system from 75 µg/ml onwards

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item was negative at the HPRT locus in V79 cells. Therefore, Azo Nickel Complex Pigment - Melamine Compound is considered to be non-mutagenic in this HPRT assay

OVERALL CONCLUSION

Azo zinc complex pigment- melamine compound is evaluated as non-mutagenic in the available Reverse Mutation Assay in bacteria (Ames test) and in a Chromosome Aberration Test in mammalian cells in vitro. The available HPRT test using the structure analogue

Azo Nickel Complex Pigment - Melamine Compound

as surrogate confirms the non-mutagenic property of Azo Zinc Complex Pigment - Melamine Compound.

Justification for selection of genetic toxicity endpoint
There are an Ames test and a Chromosome aberration test in mammalian cells available which are performed according to the respective OECD Guidelines and therefore they are evaluated with Klimisch score 1. As surrogate for the missing HPRT test with Azo zinc complex pigment - melamine compound the respective study with Gelbpigment E4GN was taken into account. This HPRT test was performed according to the respective OECD Guideline and therefore evaluated with Klimisch score 1.

Justification for classification or non-classification

Based on the available data no classification or labelling is required.