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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:
experimental study
Adequacy of study:
key study
Study period:
2017-10-17 to 2018-01-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
Vinylcyclohexane
EC Number:
211-779-3
EC Name:
Vinylcyclohexane
Cas Number:
695-12-5
Molecular formula:
C8H14
IUPAC Name:
vinylcyclohexane
Test material form:
liquid
Details on test material:
- Name: Vinylcyclohexane
- CAS No.: 695-12-5
- Batch No.: VCH/7/17K1
- Expiry date: 30 March 2019
- Purity: 99.8 %
- Appearance: Clear, colourless liquid

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. J. Thacker, MRC Radiobiology Unit, Harwell, UK

MEDIA USED
- Type and identity of media: EMEM Minimal medium and EMEM Complete medium (10 %)
Metabolic activation:
with and without
Metabolic activation system:
S9 liver tissue fraction
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 550, 275, 138, 68.8, 34.4, 17.2, 8.59 and 4.30 µg/mL
Main test, -S9: 275, 212, 163, 125, 96.3 and 74.1 µg/mL
Main test, +S9: 550, 458, 382, 318, 265 and 221 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- justification for choice of solvent: Solubility test and pre-test for toxicity
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
-S9
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
+S9
Details on test system and experimental conditions:
Preliminary Cytotoxicity Test:
The test item was assayed at a maximum dose level of 1100 µg/mL and at a wide range of lower dose levels: 550, 275, 138, 68.8, 34.4, 17.2, 8.59 and 4.30 µg/mL. Treatments were performed both in the absence and presence of S9 metabolism; a single culture was used at each test point and positive controls were not included. In order to evaluate baseline count, at the beginning of treatment two additional control cultures were included in the experimental scheme. These two cultures were trypsinized and cell counts were performed approximately at time 0. The baseline count was the average of the total number of cells from the two flasks. At the end of treatment, cell monolayers were washed with PBS, the cultures were trypsinised, counted, diluted and plated. After incubation for eight days, the colonies were stained with Giemsa solution and counted.

MAIN ASSAY:
A main assay was performed including negative and positive controls, in the absence and presence of S9 metabolising system. The two treatment series were assayed in separate runs. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. For each run, two additional control cultures were included in the experimental scheme, in order to evaluate baseline count. Two days before the experiment, sufficient numbers of 175 cm2 flasks were inoculated with 5 million freshly trypsinised V79 cells from a common pool, in order to have at least 20 million of cells for treatment. At the treatment time, the growth medium was removed from the flasks and replaced with treatment medium; cultures were incubated at 37 °C for three hours.

Determination of survival (Day 0):
At the end of treatment, the medium was removed and the cell monolayers were washed with PBS. The cultures were trypsinised, counted and an aliquot from each culture was diluted and plated to estimate the viability of the cells. Each cell suspension was re-plated (2 x 106 cells/F175) in order to maintain the treated cell populations. Fresh complete medium was added to the flasks which were then returned to the incubator at 37 °C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Subculturing (Day 2 and Day 5):
On Days 2 and 5, the cell populations were subcultured in order to maintain them in exponential growth. The cultures were trypsinised, counted and the number of cells taken forward was adjusted to give two million viable cells seeded in 225 cm2 flasks.

Determination of mutant frequency (Day 8):
At the expression time (Day 8), each culture was trypsinised, resuspended in complete medium and counted by microscope. After dilution, an estimated 1 x 10^5 cells were plated in each of twenty 100 mm tissue culture petri dishes containing medium supplemented with 6-thioguanine (at 7.5 µg/mL). These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants. After dilution, an estimated 200 cells were plated in each of three 60mm tissue culture petri dishes. These plates were used to estimate Cloning Efficiency (CE).
Evaluation criteria:
A test item is considered to be clearly positive if:
- At least one of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
- The increase is concentration-related.
- Any of the results are outside the distribution of the historical negative control data (95% confidence limits).

A test item is considered to be clearly negative if:
- None of the test concentrations exhibits a statistically significant increase, compared with the concurrent solvent/vehicle control.
- There is no concentration-related increase.
- All results are inside the distribution of the historical negative control data (95% confidence limits).
Statistics:
Mean of the experimental values was calculated.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No statistically significant increase over the spontaneous mutation frequency was observed at any treatment level, in the absence or presence of S9 metabolic activation. All results were inside the distribution of the historical negative control data, both in the absence and presence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:
In this study, the test item did not induce any mutation in the Chinese hamster V79 cells after in vitro treatment in the presence or absence of metabolic activation.
Executive summary:

In a mammalian cell HPRT gene mutation assay conducted according to OECD guideline 476, V79 cells cultured in vitro were exposed for 3 hours to the test item (99.8 % purity) in DMSO at concentrations of 275, 212, 163, 125, 96.3 and 74.1 µg/mL in the absence and 550, 458, 382, 318, 265 and 221 µg/mL in the presence of mammalian metabolic activation. The treated cells were maintained in growth medium for 9 days to allow phenotypic expression of induced mutation. For all tested treatment groups no dose-response relationship could be observed. The positive and negative controls did induce the appropriate response. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.