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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
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
other: mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium. 3 – 5E05 cells were seeded per flask (75 cm²) and incubated with "HAT" medium for 3 - 4 days. A subsequent passage in Ham's F12 medium incl. 10 % (v/v) FCS was incubated for a further 3 - 4 days.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/ß-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
- 1st Experiment, without S9 mix: 3.1, 6.3, 12.5, 25, 50, 100 µg/mL
- 1st Experiment, with S9 mix: 1.6, 3.1, 6.3, 12.5, 25, 50 µg/mL
- 2nd Experiment, without S9 mix: 4.4, 8.8, 17.5, 35, 70, 100 µg/mL
- 2nd Experiment, with S9 mix: 2.2, 4.4, 8.8, 17.5, 35, 70 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available.
- The final concentration of the vehicle DMSO in the culture medium was 1 % (v/v).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
ethylmethanesulphonate (without metabolic activation), 7,12-dimethylbenzanthracene (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Pre-incubation period: 20 - 24 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 - 9 days
- Selection time: 6 - 7 days

SELECTION AGENT: 6-thioguanine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50 % (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should be within the historical negative control data range of 0.00 – 16.43 mutants per 1E6 clonable cells.
- The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies (historical positive control data).
- At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
- Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and the historical negative control data range.
- Evidence of the reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.

Isolated increases of mutant frequencies above the historical negative control range (i.e. 15 mutants per 1E6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within the historical negative control data range.
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
MUTANT FREQUENCY
In this study, no increase in the number of mutant colonies was observed with or without S9 mix. In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.00 – 4.44 per 1E6 cells) were close to the respective vehicle control values (MFcorr.: 0.31 – 5.25 per 1E6 cells) and clearly within the range of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 16.43 per 1E6 cells; with S9 mix:
MFcorr.: 0.00 – 16.12 per 1E6 cells). No statistical significance was observed. The positive control substances EMS and DMBA induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 114.35 – 143.52 per 1E6 cells; with S9 mix: MFcorr.: 272.13 – 287.68 per 1E6 cells) were clearly within the historical positive control data range (without S9 mix: MFcorr.: 47.35 – 383.57 per 1E6 cells; with S9 mix: MFcorr.: 41.99 – 812.14 per 1E6 cells).

TEST-SPECIFIC CONFOUNDING FACTORS
- Osmolality and pH values were not influenced by test substance treatment.
- Precipitation: In this study, in the absence of S9 mix, test substance precipitation was observed in culture medium at the end of treatment at 100.0 μg/mL in the 1st and 2nd Experiment, respectively. In the presence of S9 mix precipitation was observed at 50.0 μg/mL in the 1st Experiment and at 70.0 μg/mL in the 2nd Experiment.

RANGE-FINDING/SCREENING STUDIES:
In the pre-test for toxicity based on the purity of the test substance 5500 μg/mL was used as top concentration both with and without S9 mix at 4-hour exposure time. The pre-test was performed following the method described for the main experiment. The cloning efficiency (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. In the pre-test the pH value was not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, precipitation of the test substance in the vehicle DMSO was not observed up to the highest required concentration of 5500 μg/mL. In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 171.9 μg/mL and above in the absence and presence of S9 mix. After 4 hours treatment in the absence of S9 mix cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20 % after treatment with 85.9 μg/mL and above. In addition, in the presence of S9 mix, a clearly reduced relative cloning efficiency was observed after treatment with 43.0 μg/mL and above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20 % of the respective negative control values were observed in both experiments in the presence and absence of S9 mix, at least at the highest applied concentrations. Without S9 mix, there was a decrease in the number of colonies from about 100.0 μg/mL after an exposure period of 4 hours in the 1st Experiment and from about 70.0 μg/mL onward in the 2nd Experiment. The cell densities were distinctly reduced. In addition, with S9 mix, there was a decrease in the number of colonies at 50 μg/mL in the 1st Experiment and from about 35.0 μg/mL onward in the 2nd Experiment. The cell densities were not distinctly reduced. After 4 hours of treatment the morphology and attachment of the cells treated with the highest applied concentrations was adversely influenced (grade > 2) in all experimental parts scored for gene mutations. This occurred in samples regardless of the presence of metabolic activation.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

The test is assessed as negative.

Applicant's summary and conclusion