Reaction products of Glycerin formal and 2-Propenoic acid, 2-methyl-, methyl ester

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:

02.06.2017 to 10.07.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of Glycerin formal and 2-Propenoic acid, 2-methyl-, methyl ester

EC Number:

945-527-5

Cas Number:

1620329-57-8

Molecular formula:

C8H12O4

IUPAC Name:

Reaction products of Glycerin formal and 2-Propenoic acid, 2-methyl-, methyl ester

Test material form:

liquid

Specific details on test material used for the study:

Chemical Name: 5-Methacryloyloxy-1,3-dioxan (Isomer 1) 4-Methacryloyloxymethyl-1,3-dioxolan (Isomer 2)
CAS No.: 1620329-57-8 (mixture of both isomers)
4-Methacryloyloxymethyl-1,3-; CAS 132977-93-6 (Isomer 1): 77.8 %
5-Methacryloyloxy-1,3-dioxan; CAS 10525-59-6 (Isomer 2): 21.6 %
Batch No.: 1250881508
Purity: 99.6%
Expiry Date: not applicable
Storage Conditions: room temperature, protected from light
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation test): 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment 2 (pre-incubation test): 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate

Vehicle / solvent:

The test item was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Auqua dest

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

sodium azide

Remarks:

10 µg/plate for TA 100, TA 1535

Controls 2

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

other: 4-NOPD; 4-nitro-o-phenylene-diamine

Remarks:

10 µg/plate for TA 98, 40 µg/plate for TA 1537

Controls 3

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

1 µL/plate for E. coli WP2 uvrA

Controls 4

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

other: 2-AA; 2-aminoanthracene

Remarks:

2.5 µg/plate for TA 98, TA 100, TA 1535 and TA 1537; 10 µg/plate for E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 h

NUMBER OF EXPERIMENTS: 2 (one performed as pre-incubation assay, one as plate incorporation assay), 3 plates per concentration each

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

OTHER:
automatic colony count with "ProtoCOL counter" (Meintrup DWS Laborgeräte GmbH).

Evaluation criteria:

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Statistics:

no appropriate statistical method available

Results and discussion

Test results

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:

valid

Positive controls validity:

valid

Additional information on results:

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Glycerolformal methacrylate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Glycerolformal methacrylate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Glycerolformal methacrylate is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, adopted 26 July 1997, strains TA 1535, TA 1537, TA 98, TA 100 of S. typhimurium and Ecol WP2 were exposed to Glycerolformal methacrylate in DMSO at concentrations of 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate in a plate incorporation assay and pre-incubation assay in the presence and absence of mammalian metabolic activation (S9 mix).

There was no evidence of induced mutant colonies over background.

The positive controls induced the appropriate responses in the corresponding strains.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Therefore, Glycerolformal methacrylate is considered to be non-mutagenic in this bacterial reverse mutation assay.