Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, equivalent or similar to OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO cells were propagated in Harn's F-12 nutrient medium supplemented with 10% heat-inactivated fetal bovine serum (HIFBS) and 2mM L-glutarnine (antibiotic-free culture medium) to obtaina sufficient number of cells for freezing a large number of stock ampules. The cells were cryopreserved in Harn's F-12 culture medium with 8% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen. Prior to using the stock cultures for the test, representative ampules were tested for contarninating microorganisms, including mycoplasma. Stock ampules free of contarninating organisms were used to initiate the stock cultures for the test. The cell cultures obtained from the stock ampules were maintained by subculturing for a maximum period of 1 month and used to initiate cultures for the assays.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix
Test concentrations with justification for top dose:
Test 1:
without S9 mix: 125, 250, 500, 750 and 1000 mg/mL
with S9 mix: 6.3, 12.5, 25, 50 and 75* µg/mL

Test 2:
without S9 mix: 125, 250, 500, 750 and 1000 mg/mL
with S9 mix: 6.3, 12.5, 25 and 50 µg/mL

* not evaluated due to high cytotoxicity
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: DMSO was shown to be a proper vehicle for the assay in a solubility pre-test.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: Ethyl methanesulfonate (EMS); with S9: 7,12-Dimethylbenz(a)anthracene (DMBA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 h
- Expression time: 18-24 h
- Selection time: 8 d

SELECTION AGENT: 6-thioguanine
STAIN: Giemsa

NUMBER OF REPLICATIONS: 3
NUMBER OF INDEPENDENT ASSAYS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative cell survival
Evaluation criteria:
The test article is considered positive in the assay if it induces a statistically significant and reproducible increase in mutation frequency at more than one of the dose levels tested. The final interpretation of the data also will take into consideration such factors as the mutation frequency and cloning efficiencies in the negative controls and dose-response relationships.
Statistics:
The frequencies of mutants per 1E06 clonable cells are analyzed with a pairwise t-test, with the pairs being the replications for each dose. In the pairwise analysis, the square roots of the frequencies are used to correct for heterogeneity of variance, and the test is conducted at an alpha level of 0.05. Comparisons to the negative control are made with an approximate Chi-square statistic at an alpha level of 0.01 (one-sided). Examination of trends is conducted with least squares regression using an alpha level of 0.025, one-sided.

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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
There was no change in the pH of the culture medium after the addition of the test article dosing solution.
- Effects of osmolality:
The osmolality readings were below the upper limit for the CHO/HGPRT Gene Mutation Assay (500 mOs/kg).
- Solubility:
The solubility of the test article in DMSO and ethanol was determined. 99 mg of the test artiele was completely soluble in 0.1 mL of DMSO, and 96 mg of the test article was soluble in 0.1 mL of ethanol. It was decided to use DMSO as the solvent for the test article.

RANGE-FINDING/SCREENING STUDIES:
In the S-9 activated system, the test article was completely toxic in the highest three concentrations of 100, 500 and 1000 µg/mL. The next lower coneentration of 50 mg/mL had a Relative Cell Survival (RCS) of 54%. All the other concentrations, 0.05, 0.1, 0.5, 1.0, 5.0 and 10 µg/mL, were completely nontoxic. In the non-activated system, the test article was completely non-taxic at all concentrations, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 500 and 1000 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
There was no significant change in the number of mutants per 1E06 surviving cells between the test article treated cultures and the solvent control cultures, in both the non-activated as weIl as activated systems. As anticipated, the positive controls EMS and DMBA caused significant increases in the number of mutants per 1E06 surviving cells. The mutant frequencies of the solvent controls were within historical negative control values.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the activated system, the 75 µg/mL concentration was completely toxic and 50 µg/mL had an RCS of 13%. All the other concentrations were nontoxic.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

The results of the HGPRT Mutation Assays indicated that Allyl methacrylate did not cause a significant increase in the mutant frequency at the HGPRT locus in the presence or absence of S-9 activation.
Under the conditions of the study, Allyl Methacrylate; did not induce gene mutation in Chinese hamster ovary cells.

Executive summary:

Allyl methacrylate was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase(CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two separate assays.  The test material was assayed at concentrations ranging from 125 to 1000 µg/ml and from 6.3 to 75 µg/ml in the absence and presence of an externally supplied metabolic activation (S-9) system, respectively. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals (ethyl methane sulfonate for assay without S-9 and 7,12-dimethylbenz(a)anthracene for assays with S-9). Negative control cultures were treated with the solvent used to dissolve the test material. Based upon the frequency of TGr mutants recovered in cultures treated with the test material, it was concluded that the test material did not induce a mutagenic response in the assay system employed.