Methyl 2-((allyloxy)methyl)acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jul 2008 - 25 Aug 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Ministry of Labor Ordinance No. 76, 1988 and Ordinance No.13, 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

methyl 2-[(prop-2-en-1-yloxy)methyl]prop-2-enoate

EC Number:

819-688-6

Cas Number:

219828-90-7

Molecular formula:

C8H12O3

IUPAC Name:

methyl 2-[(prop-2-en-1-yloxy)methyl]prop-2-enoate

Test material form:

liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose-finding study:
Test substance groups: Selected in accordance with the “Guidelines”.
Positive control groups: Selected in accordance with the concentrations for positive control substances in “Guide to Mutagenicity Studies under the Industrial Safety and Health Act”
Concentrations: 0.00305, 0.0122, 0.00488, 0.195, 0781, 3.13, 12.5 and 50 mg/mL.

Main study:
In the dose-finding study, growth inhibitions were observed at 5000μg/plate for five tester strains in the absence and presence of the metabolic activity system. Therefore, the composition of groups in the main study included 6 dose levels by a common ratio of 2 both in the absence and presence of the metabolic activity system; from the highest dose of 5000 μg/plate to the lowest dose of 156 μg/plate.
Concentrations: 1.56, 3.13, 6.25, 12.5 and 50 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance is insoluble in water, but soluble at 50 mg/mL or more in DMSO. Therefore, DMSO was selected as the solvent.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Additive volume of the prepared solution 100 μL per plate. Reason for the additive volume of the prepared solution 100 µL, which is the maximal possible volume without inhibition of bacterial growth by DMSO, was selected for positive control substances. Also, the same volume was selected for the test substance and negative control solutions.

45 μL of each tester strain were directly inoculated into 20 mL of 2.5% nutrient broth. The resulting mixture was cultured with shaking at 50 rpm for 9 hr at 37°C.
After incubation, the optical density of bacterial suspensions was measured to confirm that the viable bacterial count was not less than 1.0 × 10E9/mL.

In the absence of the metabolic activation system (without S9 mix):
500 μL of 100 mM Na-phosphate buffer (pH 7.4) and 100 μL of bacterial suspension were added to 100 μL of the test solution in each culture tube and the suspending solution was pre-incubated with shaking for 20 minutes at 37°C. Subsequently, 2 mL of a mixture of 0.5 mM biotin-0.5 mM histidine solution (in case of E. coli: 0.5 mM tryptophan solution) and 0.6% soft agar fluid (volume ratio 1:10) was added to each of the suspending solutions, and then poured onto the minimal glucose agar plate (Tesmedia AN Medium, Oriental Yeast Co., Ltd.,). All the plates were incubated for 48 hours at 37°C.
In the presence of metabolic activation system (With S9 mix):
500 µL of S9 mix and 100 µL of bacterial suspension were added to 100 µL of the test solution in each culture tube and the suspending solution was pre-incubated with shaking for 20 minutes at 37°C. Subsequently, 2 mL of a mixture of 0.5 mM biotin-0.5 mM histidine solution (in case of E. coli: 0.5 mM tryptophan solution) and 0.6% soft agar fluid (volume ratio 1:10) was added to each of the suspending solutions, and then poured onto the minimal glucose agar plate. All the plates were incubated for 48 hours at 37°C.

After incubation, the presence or absence of precipitation was checked visually for all dose levels. Moreover, the background lawn was observed using a microscope to confirm the presence or absence of bacterial growth inhibition. Thereafter, the numbers of revertant colonies were counted, twice for each plate, using a colony counter (PC-10NII, Toyo Sokki Co., Ltd.). The number of colonies was corrected and the mean of the corrected values was the number of revertant colonies for each plate.

Rationale for test conditions:

Guidelines

Evaluation criteria:

Acceptance criteria
From the number of revertant colonies on each plate, the mean values (rounded to integers) are obtained. If all of the following criteria are met, the results are considered positive.
1) The number of revertant colonies in the test substance group is equal to or greater than twice the mean negative control value.
2) Dose-dependency is observed.
3) Reproducibility is observed when comparing the dose-finding study and main study results.

Statistics:

not specified

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

For five tester strains, the number of revertant colonies in the test substance groups did not increase greater than twice that in the negative control group in the absence or presence of the metabolic activation system.
Bacterial growth inhibition was observed at 2500 µg/plate and more for five tester strains in the absence and presence of the metabolic activation system.
Precipitation of the test substance on the plates was not observed in five tester strains in the absence or presence of the metabolic activation system.

Applicant's summary and conclusion

Conclusions:

In the results of the dose-finding study and main study, Methyl α-(Allyloxymethyl)acrylate did not induce increases in the number of revertant colonies for five tester strains in the absence or presence of the metabolic activation system, and the number did not reach more than twice that of the negative control group.
Therefore, Methyl α-(Allyloxymethyl)acrylate was judged not to be mutagenic agent, under the conditions of this study.