222 OP

Genetic toxicity: in vitro

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

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to OECD 476; GLP conform

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment for experiment I (only without metabolic activation):
0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, 10 mM
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
0.5, 1.0, 2.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 mM
Experiment I
without metabolic activation: 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM
and with metabolic activation: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 6.25, 7.5, 8.75 and 10 mM
Experiment II
without metabolic activation: 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 1.4, 1.6, 1.8 and 2.0 mM
and with metabolic activation: 0.5, 1.0, 2.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10 mM

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test item was suspended in cell culture medium and processed by ultrasound for 15 min.

Controls

Controls 1

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Migrated to IUCLID6: 300 µg/mL

Controls 2

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Migrated to IUCLID6: 1 µg/mL and 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

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:

Experiment I without S9: ≥ 2.5 mM; Experiment II without S9: ≥ 0.25 mM

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Under the test conditions described above, the test item is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The selection of the concentrations was based on data from the pre-experiments. In experiment I 10 mM (with and without metabolic activation) was selected as the highest concentration. In experiment II 2.0 mM (witout metabolic activation) and 10 mM (with metabolic activation) were selected as the highest concentrations. Experiments I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I

without metabolic activation:

0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 5.0, 7.5 and 10 mM

and with metabolic activation:

0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 6.25, 7.5, 8.75 and 10 mM

Experiment II

without metabolic activation:

0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 1.4, 1.6, 1.8 and 2.0 mM

and with metabolic activation:

0.5, 1.0, 2.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10 mM

Precipitation of the test item was noted in experiment I and II.

Biologically rlevant growth inhibition was observed in experimen I and II only without metabolic activation. In experiment I without metabolic activation the relative growth was 62.8%, with metabolic activation 130.3% for the highest concentration (10 mM) evaluated. In experiment II without metabolic activation the relative growth was 19.0%, with metabolic activation 80.4% for the highest concentration (10 mM) evaluated.

In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.