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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
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From December 16, 1997 to March 26, 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Source study has reliability 1. Details on the read across are available in section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
numbers of mutant colonies but no detrimental impact
Qualifier:
according to
Guideline:
other: EPA; 40 CFR; Ch.I; Part 798; Detection of gene mutation in somatic cells in culture; pp. 717720 (7-1-86 Edition)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cell gene mutation tests, forward gene mutation

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT enzyme)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
mammalian microsomal fraction S9 mix (liver) (50 µl/ml)
Test concentrations with justification for top dose:
- At least four concentration levels were tested. These concentration levels should yield a concentration related toxic effect. The highest concentration level should induce a reduced level of survival.
Two independent experiments were run:
- experiment I:
without S9 mix: 3.0; 5.0; 10.0; 25.0; and 50.0 µg/ml
with S9 mix: 3.0; 5.0; 10.0; 25.0; and 50.0 µg/ml
- experiment II:
without S9 mix: 0.5*; 1.0; 3.0; 5.0; and 10.0 µg/ml
with S9 mix: 3.0; 5.0; 10.0; and 50.0 µg/ml
The limit of solubility was 10 µg/ml in both experiments as indicated by a slight perturbation. Visible precipitation occurred above this concentration.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Remarks:
cells were cultivated without interruption
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
cells cultivated the same without treatment
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
The assay was performed in two independent experiments with and without liver microsomal activation. Treatment duration was 4 h in the first experiment and in the second experiment with metabolic activation. In the second experiment without metabolic activation, treatment duration with test substance was extended to 24 h.
Rationale for test conditions:
V79 cells: high proliferation rate, good cloning efficiency of negative control cells, and stable karyotype with a modal chromosome number of 22.
Evaluation criteria:
A test substance is classified as positive if it induces either a concentration-related increase of mutant frequency or a reproducible and positive response at one of test points. A test substance producing neither a concentration-related increase of mutant frequency nor a reproducible positive response at any of test points is considered non-mutagenic in this system.
A significant response is described as follows: a test substance is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
Test substance is classified as mutagenic if there is a reproducible concentration-related increase of mutation frequency. Such evaluation may be considered also in the case that a threefold increase of mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5-33.1 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed.
Statistics:
Stained (with 10 % methylene blue in 0.01 % KOH solution) colonies with more than 50 cells were counted.
Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only at maximal concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Cytotoxicity
Strong toxic effects occurred in the first experiment in both cultures at the maximal concentration without metabolic activation. Only minor toxic effects were observed up to the maximal concentration with metabolic activation. In the second experiment strong toxic effects occurred only in one out of two parallel cultures at the maximal concentration without metabolic activation. This deviation between parallel cultures is probably based upon erratic toxic effects caused by precipitation during long term exposure (24 h).

- Genotoxicity
No relevant and reproducible increase in mutant colony numbers was observed up to the highest investigated concentration, neither in the presence nor in the absence of metabolic activation.
Taking into account mutation rates found in groups treated with test substance compared to negative and solvent controls, it can be concluded that no relevant increase of gene mutations was observed. Test substance did not induce a reproducible concentration-related increase in mutant colony numbers. Mutant values of groups treated with test substance remained in the range of historical negative controls. The highest value of mutant colonies (30.0 colonies per 10^6 cells) occurred at the lowest concentration in culture II in the second experiment with metabolic activation. This increase was considered as biologically irrelevant since it was not reproduced in the parallel culture in experiment II nor in both cultures of experiment I under identical conditions. In this study in both experiments (with and without S9 mix) the range of the negative controls was from 2.5 up to 22.0 mutants per 10^6 cells; the range of groups treated with test article was from 3.0 up to 30.0 mutants per 10^6 cells. EMS (0.6 mg/ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

Applicant's summary and conclusion

Conclusions:
Not mutagenic.
Executive summary:

Method

An in vitro mammalian cell gene mutation test (forward gene mutation HPRT) was run according to OECD guideline 476. Chinese hamster V79 cells were exposed to test substance at concentrations ranging from 0.5 to 50.0 µg/plate with or without metabolic activation (S9 mix). The assay was performed in two independent experiments with and without liver microsomal activation. Treatment duration was 4 h in the first experiment and in the second experiment with metabolic activation. In the second experiment without metabolic activation, treatment duration was extended to 24 h. Positive and negative controls were valid.

Results

Strong cytotoxic effects occurred in the first experiment in both cultures at the maximal concentration without metabolic activation. Only minor toxic effects were observed up to the maximal concentration with metabolic activation.

In the second experiment strong cytotoxic effects occurred only in one out of two parallel cultures at the maximal concentration without metabolic activation. No relevant and reproducible increase in mutant colony numbers was observed up to the highest investigated concentration, neither in the presence nor in the absence of metabolic activation. Under study conditions, test substance did not induce gene mutations at the HPRT locus in V79 cells and thus was not considered as genotoxic.