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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid, powder yellow
Details on test material:
content: 99.1 %

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
other: Chinese hamster lung fibroblasts (V79): the cells have a stable karyotype with a modal chromosome number of 22
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
Pretest on toxicity
4 h in the presence and 4 and 24 h in the absence of a metabolic activation system:
10.2 µg - 1300 µg/ml
The highest concentration was based on the solubility properties of the test item
GENE MUTATION ASSAY
--EXPERIMENT I
4 h without S9 mix:
1.8, 5.4, 16,0 (P), 48.1 (P), 144.3 (P), 433.0(P) µg/ml
4 h with S9 mix:
1.8, 5.4, 16,0 (P), 48.1 (P), 144.3 (P), 433.0(P) µg/ml
--EXPERIMENT II
24 h without S9 mix:
1.8, 5.4, 16,0, 48.1 (P), 144.3 (P), 433.0(P) µg/ml
4 h with S9 mix:
1.8, 5.4, 16,0 (P), 48.1 (P), 144.3 (P), 433.0 (P) µg/ml

P = Precipitation at the end of treatment visible to the unaided eye
Vehicle / solvent:
DMSO
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:
The assay was performed in 2 independent experiments, using 2 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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
the positive controls were functional
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

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

The study was performed to investigate the potential of Macrolex Gelb G to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD TG 476 under GLP conditiions. 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. The used concentrations ranged between 1.8 and 433.0 µg/ml.Precipitation occurred at 16.0 µg/ml and above in experiment I with and without metabolic activation and in experiment II with metabolic activation. In experiment II without metabolic activation precipitation was observed at 48.1 µg/ml. No relevant cytotoxic effects indicated by a relative cloning efficientcy 1 or cell density below 50% was observed in experiment I and II after 4 and 24 hours treatment with and without metabolic activation..

No relevant and reproducible dose dependent increase in mutamt colony numbers/10 (exp 6) 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 did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Macrolex Gelb G is considered to be non-mutagenic in this HPRT assay.