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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
Gelbpigment E4GN
IUPAC Name:
Gelbpigment E4GN
Test material form:
other: a yellowish powder
Details on test material:
Identity: Gelbpigment E4GN
Content: 96 %

Method

Target gene:
HPRT locus in V79 cells
Species / strain
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
Controls
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.

Results and discussion

Test results
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'.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.