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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013-01-04 to 2013-03-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD Guideline study. For justification of read across see section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Methyl benzoate
EC Number:
202-259-7
EC Name:
Methyl benzoate
Cas Number:
93-58-3
IUPAC Name:
methyl benzoate

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbitone/ß-naphthoflavone induced rat livers
Test concentrations with justification for top dose:
Experiment 1
4-hour without S9: 0, 85, 170, 340, 680, 906.67, 1133.33, 1360 µg/mL
4-hour with S9 (2 %): 0, 85, 170, 340, 680, 906.67, 1133.33, 1360 µg/mL
Experiment 2
24-hour without S9: 0, 85, 170, 340, 680, 906.67, 1133.33, 1360 µg/mL
4-hour with S9(1%): 0, 42.5, 85, 170, 340, 680, 1020, 1360 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Ethyl methane sulphonate (EMS) was used at 500 and 750 µg/mL as the positive control in the 4-hour cultures without S9 and at 250 and 500 |ug/mL for the 24-hour cultures without S9.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours and 24 hours
- Expression time (cells in growth medium): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): after the 7 day expression period cells were counted and incubated for another 6 days before fixation and staining

STAIN: Giemsa

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 200 (in triplicate)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
The test item is classified as mutagenic if there is a reproducible dose-related increase in the mutation frequency where at least a threefold increase in the mutant frequency over the vehicle control value is observed. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. A test item producing neither a dose-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
Statistics:
not required

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:
Marked toxicity was observed at the maximum dose in the 4 h and 24 h exposure groups in the absence of S9. Less toxicixty was observed in the 4 h exposure group in the presence of S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A greasy/oily precipitate of the test item was observed at the end of exposure at and above 680 µg/mL and 340 µg/mL in the 4-hour exposure groups in the absence and presence of S9 respectively. In the 24-hour exposure group greasy/oily precipitate was observed at 1360 µg/mL at the end of the exposure period.


RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed with the dose levels 5.31, 10.63, 21.25, 42.5, 85, 170, 340, 680 and 1360 µg/mL. Exposure was for 4 hours or 24 hours at 37 °C. Results from a preliminary cytotoxicity test were used to select the test item dose levels for the mutagenicity experiments.
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

The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. Methyl benzoate was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.
Executive summary:

This study was conducted according to OECD Guideline 476 to assess the potential mutagenicity of methyl benzoate on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. Chinese hamster (V79) cells were treated with the test item at up to seven dose levels, in duplicate, together with vehicle (solvent) and positive controls in the presence and absence of an S9 metabolic activation system. Four treatment conditions were used for the test. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose range of the test item was selected based on the results of a preliminary cytotoxicity test and were as follows:

4 hour without S9: 85, 170, 340, 680, 906.67, 1133.33, 1360 µg/mL

4 hour with S9 (2 %): 85, 170, 340, 680, 906.67, 1133.33, 1360 µg/mL

24 hour without S9: 85, 170, 340, 680, 906.67, 1133.33, 1360 µg/mL

24 hour with S9 (1 %): 42.5, 85, 170, 340, 680, 1020, 1360 µg/mL

The vehicle (solvent) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system. The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. As a conclusion, the test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.