Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.03.2010 - 19.05.2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
according to guideline
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
GLP compliance:
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Methacrylic acid, monoester with propane-1,2-diol
EC Number:
EC Name:
Methacrylic acid, monoester with propane-1,2-diol
Cas Number:
Molecular formula:
1-hydroxypropan-2-yl 2-methylprop-2-enoate; 2-hydroxypropyl 2-methylprop-2-enoate
Test material form:
Details on test material:
The chemical 2-propenoic acid, 2-methyl-, monoester with 1,2-propanediol (CAS No. 27813-02-1) consists of two isomers. This occurs due to the method of industrial production, which is usually either the reaction between methacrylic acid and propylene oxide, or methacrylic acid esterification of 1,2-propanediol. The predominant isomer is the secondary alcohol 2-propenoic acid, 2-methyl, 2-hydroxypropyl ester, which is assigned the CAS No. 923-26-2, and the isomer 2-propenoic acid, 2-methyl, 2-hydroxy- 1-methylethyl ester, the other chemical in this group, for which CAS 4664-49-7 is assigned. The purification of the two isomers requires extensive effort and it is supposed no such effort has been taken during data collection otherwise noted. Therefore toxicity information for both CAS No. 923-26-2 and CAS No. 27813-02-1 is considered to be applicable to both chemicals..

Specific details on test material used for the study:
- Supplier: Rohm and Haas Company,
- Analytical purity: 98.96%
- Lot/batch No.: 0005398178


Target gene:
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 nutrient mix (GIBCO, Grand Island, New York) supplemented with 5% (v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum (GIBCO), antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 25 µg/ml; GIBCO) and an additional 2 mM L-glutamine (GIBCO).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mixS9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
45.1, 90.1, 180.3, 360.5, 721, and 1442 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: Ethyl methanesulfonate and 20-methylcholanthrene
Details on test system and experimental conditions:
Each dose level was set up in duplicate from the time of treatment until the completion of the assay. The cultures were trypsinized at the end of the treatment and replated at a density of 1 x 10e6 cells/100 mm dish (at least two dishes/replicate) for phenotypic expression (O'Neill et al., 1977a; O'Neill and Hsie, 1979). In addition, 200 cells/60 mm dish (three dishes/replicate) were also plated to determine the toxicity and incubated for approximately 6-8 days to permit colony formation. During the phenotypic expression period (7-9 days), cells in the larger petri dishes were subcultured every 2-3 days and plated (at least two dishes/replicate) at a density of about 1 x 10e6 cells/100 mm petri dish.

At each subculture, cells from various dishes within each replicate were pooled prior to replating. At the end of the expression period, the cultures were trypsinized and plated at a density of 2 x 105 cells/100 mm dish (a total of 10 dishes/treatment) in the selection media (Ham's F-12 without hypoxanthine and with 6-thioguanine) for the determination of HGPRT- mutants and 200 cells/60 mm dish (three dishes/treatment) in Ham's F-12 medium without hypoxanthine for determination of cloning efficiency. The dishes were incubated for about 7-9 days and the colonies were fixed/stained with methanol/crystal violet. The mutation frequency (expressed as mutants per 106 clonable cells) for each replicate were calculated.
Evaluation criteria:
For an assay to be acceptable, the mutant frequency in positive controls should have been significantly higher than the solvent controls. An additional criteria, was that the mutant frequency in the solvent controls should have been within reasonable limits of the laboratory historical control values and literature values. The test chemical was considered positive if it induced a statistically significant, dose related, reproducible increase in mutant frequency. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the solvent controls.
The frequency of mutants per 106 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutation frequency variance. The actual plate counts are assumed to follow a Poisson distribution therefore the mean plate count was used as an estimate of variance (Kirkland, 1989). If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the solvent control yielded significant differences.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:

There was no appreciable change in either the pH or osmolality at this concentration as compared to the culture medium with solvent alone.

RANGE-FINDING/SCREENING STUDIES: Concentration levels of 0 (solvent control), 45.1, 90.1, 180.3, 360.5, 721, and 1442 µg/ml of the test material were selected in the absence and presence of S9. In the absence of S9, little to no toxicity was observed with RCS values ranging from 74.7 to 113.8%. In the presence S9, cultures displayed moderate to no toxicity with RCS values ranging from 52.9 to 88.4%. The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 were not significantly different from the concurrent solvent control values.

All mutant frequencies were within a reasonable range of historical background values.
A second confirmatory assay with treatment of cultures in the absence or the presence of S9 was not considered necessary since the results of the initial test yielded clearly negative results.

Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

Result table see under attachments

Applicant's summary and conclusion

Interpretation of results: negative

The results of the in vitro CHO/HGPRT forward gene mutation assay with hydroxypropyl methacrylate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.
Executive summary:

In the in vitro Chinese hamster ovary cell/hypoxanthineguanine- phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay, Hydroxypropyl methacrylate (2-methyl-2-propenoic acid monoester with 1,2- propanediol) was evaluated in concentrations between 45.1 and 1442 µg/ml in the absence and presence of an externally supplied metabolic activation (S9) system. The highest concentration was based on the assay system limit of 10 mM. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assay in the absence of S9 and 20- methylcholanthrene for assay in the presence of S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e. distilled water). The results of the in vitro CHO/HGPRT forward gene mutation assay with hydroxypropyl methacrylate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.