Allyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Allyl methacylate was shown to be negative in two Salmonella typhimurium Ames tests. In addition, the substance proved to be negative in two chromosome aberration tests and in one HPRT assay using mammalian cell lines. These in vitro results are further confirmed by a negative in vivo mouse bone marrow micronucleous test.

Link to relevant study records

Reference

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

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

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.

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.

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'.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

For the assessment of the mutagenic potential of Allyl methacrylate several studies are available: two reverse gene mutation assays in bacteria (Ames test), two mammalian cell chromosome aberration assays , one cell gene mutation assay (HPRT test), a mammalian cell gene mutation assay (TK test) s and an in vivo micronucleus test.

Ames tests:

The in vitro potential mutagenic activity of the test substance allyl methacrylate was investigated by the Ames test using 5 strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 1538, TA 98 and TA 100. After a preliminary assay to define the concentrations to be used for the mutagenicity study, the test substance was tested on two independent assays. Each assay was carried out both in the absence and in the presence of a metabolie activation system, the S9 mix, prepared from a liver microsomal fraction S9 of rats treated with Aroclor 1254. The method used was the direct plate incorporation method. The concentrations were:

- 100, 500, 1000, 1750 and 2500 µg/plate for the first assay with and without S9 mix and for the second assay without 59 mix

- 10, 50, 100, 500 and 1000 µg/plate for the second assay with S9 mix.

The test substance allyl methacrylate did not induce a significant increase in the revertant number with or without S9 mix in any of the 5 strains. The negative and solvent control results were equivalent to those usually obtained in our Laboratory. The number of revertants induced by the positive controls was higher than the spontaneous one, which demonstrated the sensitivity of this test and the efficacy of the S9 mix throughout this study. In conclusion, under our experimental conditions, the test substance allyl methacylate is not mutagenic in the Ames test (C.I.T., 1991).

In a second study, allyl methacrylate was tested for mutagenic activity in the Salmonella/Mammalian-Microsome Reverse Mutation Assay (Ames Test), Preincubation Method. This assay evaluates the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimurium tester strains both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclorw-induced rat liver. The doses tested in the mutagenicity assay were selected based on the results of a dose range finding study using tester strain TA100 and ten dose levels of test article ranging from 5000 to 6.67 µg per plate, one plate per dose, both in the presence and absence of microsomal enzymes. The tester strains used in this study were TA98, TA100, TA1535 and TA1537. The assay was conducted using three plates per dose level in the presence and absence of microsomal enzymes. Six dose levels of the test article were tested, ranging from 1000 to 10.0 µg per plate in the presence of S9 and from 3330 to 66.7 µg per plate in the absence of S9. The results of the initial mutagenicity assay were confirmed in an independent experiment. The results of this Ames Test, Preincubation Method, indicate that under the conditions of this study, in both an initial and a confirmatory assay, the test article, allyl methacrylate, did not cause positive increases in the number of histidine revertants per plate with and of the tester strains in either the presence or absence of microsomal enzymes prepared from Aroclor-induced rat liver (S9) (Hazleton, 1992).

Chromosome aberration tests:

Allyl methacrylate was also found to be negative in an in vitro mammalian chromosome aberration test with human lymphocytes, when tested at concentration of 2 - 200 µg/mL with and without metabolic activation by liver S9 mix of rats induced with Aroclor 1254 (C.I.T., 1994). The test was conducted in compliance with GLP according to OECD 473.

In a second test, the clastogenic potential of allyl methacrylate was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after establishing whole blood cultures, the cells were treated for 4 hours in the presence and absence of an external metabolic activation system (S-9) with 0 (negative control), 0.501, 1.67, 5.01, 16.7, 50.1, 167, and 501 µg allyl methacrylate per mL culture medium. Cultures treated with 0.4 µg/mL mitomycin C and 5.0 µg/mL cyclophosphamide served as positive controls for the non-activation and activation assays, respectively. The cultures were harvested 24 h after termination of treatment. Based upon the mitotic indices, cultures treated with 50.1, 167, and 501 µg/mL from the nonactivation assay and with 16.7, 50.1, and 167 µg/ml from the activation assay were selected for determining the incidence of chromosomal aberrations. No significant increase in the aberration frequency was noticed at any of the treatment levels in this assay when compared to the negative controls. Cultures treated with the positive control chemicals had significantly higher incidences of aberrations. Results of the initial assay , were confirmed in an independent repeat experiment utilizing similar dose levels and an additional culture harvest time (48 h after treatment). It was concluded that allyl methacrylate did not exhibit clastogenic activity in cultured rat lymphocytes under the experimental conditions used.

HPRT test:

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 metabolie 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 7,12dimethylbenz(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 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.

Mouse lymphoma test:

Allyl Methacrylate was tested both in the presence and absence of S9-mix in two independent experiments and in the presence of S9-mix only in a further two experiments. Negative results were observed in both the presence and absence of S9-mix with maximum concentrations of 64~g/ml and 750µg/ml respectively. Higher concentrations of Allyl Methacrylate (up to 125µg/ml and 1000µ/ml in the presence and absence of S9-mix respectively) gave rise to excessive toxicity.

It is therefore concluded that, Allyl Methacrylate is non-mutagenic to L5178Y TK+/- cells in either the presence or absence of S9-mix when tested to concentrations (64µg/ml and 750µg/ml in the presence and absence of S9-mix respectively) limited by the toxicity of the test sample.

Mouse micronucelous test in vivo:

Allyl methacrylate was evaluated in the mouse bone marrow micronucleus test. The micronucleus test is capable of detecting agents causing chromosomal aberrations and spindle malfunction. The test material was administered to CD-1 mice by single oral gavage at dose levels of 0 (negative control), 75, 150, and 300 mg/kg body weight (bw). The dose levels for the micronucleus assay were based on the outcome of a range finding assay. The concentrations of the test material in the dosing solutions were verified analytically. Groups of animals were sacrificed at three intervals, viz., 24, 48, and 72 hours after treatment. Mice treated with 80 mg/kg bw cyclophosphamide and sacrificed at 24 hours served as positive controls. There were five animals per sex per dose level per sacrifice time. One thousand polychromatic erythrocytes (PCE) were evaluated from each surviving animal and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) were recorded. There were no significant increases in the frequencies of MN-PCE in groups treated with the test material as compared to negative controls. The positive control mice showed significant increases in MN-PCE. Under the experimental conditions used, the test material was considered to be negative in the mouse bone marrow micronucleus test.

Justification for selection of genetic toxicity endpoint
GLP and guideline study with mammalian cells.

Justification for classification or non-classification

Based on the available data, there is no indication for a mutagenic potential of the test substance. Thus, no classification is warranted according to 67/548/EEC and Regulation (EC) No 1272/2008 (GHS, CLP), respectively.