Reaction mass of dodecyl methacrylate and tridecyl methacrylate

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:

16 February 2018 to 14 March 2018 (Study initiation to experimental completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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 mass of dodecyl methacrylate and tridecyl methacrylate

EC Number:

907-961-3

Molecular formula:

C16H30O and C17H32O

IUPAC Name:

Reaction mass of dodecyl methacrylate and tridecyl methacrylate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Kyoeisha Chemical Co., Ltd-
- Lot No. of test material: 7061301
- Expiration date of the lot: 13 June 2019
- Purity test date: 3 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In original container as supplied by the Sponsor, at room temperature (Ambient). container kept tightly closed and away from heat or sunlight
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in diimethyl sulphoxide (DMSO) at 50 µL/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Solublised in DMSO at 50 µL/mL

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Initial Toxicity-Mutation Assay: 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5, 1.5, 5 µl/plate
Confirmatory Mutation Assay: 0.16, 0.31, 0.63, 1.25, 2.5, 5 µl/plate
In accordence with OECD 471, the recommended maximum test concentration for soluble non-cytotoxic substances is 5 µl/plate. This was the highest selected concentration for testing in the absence and presence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Soluble in DMSO at 50 µL/mL to permit the recommended maximum test concentration of 5 µl/plate.

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene (+S9) to verify the efficiency of the S9 fraction

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar using the plate incorporation method
- Cell density at seeding (if applicable): Cell densities (OD at 660 nm) of all tester strains were within the required range to produce cultures with approximately 1 - 2 x 109 bacteria/mL, demonstrating appropriate numbers of bacteria were plated.

DURATION
- Exposure duration: Petri plates were incubated at 37 ± 1 °C for 48 hours

NUMBER OF REPLICATIONS: Duplicate plates (Initial Toxicity Mutation Assay) and Triplicate plates (Confirmatory Mutation Test)


DETERMINATION OF CYTOTOXICITY
- Method: other: In the confirmatory mutation assay, six analysable doses were available to evaluate assay data. Cytotoxicity is detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn.
- Any supplementary information relevant to cytotoxicity: Cytotoxicity was characterised by inhibition of the background bacterial lawn and/or reduction in the number of revertant colonies. In the initial Toxicity Mutation Assay, a normal bacterial background lawn and no increase in the number of revertant colonies was observed up to 5 µL/plate both in the absence and presence of metabolic activation.

Rationale for test conditions:

Standard test conditions, ie for the solvent/vehicle, exposure concentrations and controls, were performed in accordence with OECD 471 guidence.

Evaluation criteria:

Once the criteria for a valid assay had been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were: there should be a dose-related increase in the mean revertants per plate in at least one tester strain over the range tested and/or at one or more doses of the test item either in the absence or presence of the metabolic activation system. The biological relevance of the results was considered:

Strains TA1535 and TA1537 and Escherichia coli WP2uvrA:
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.

Strain TA98, TA100:
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.
Statistical analysis was used as an aid in the evaluation of a dose response.
A response that did not meet all three of the above criteria (magnitude, concentration responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation assay were confirmed by a confirmatory mutation assay, using the same method as specified above, with an alteration in concentration spacing.

Statistics:

Simple linear regression analysis was performed for tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA, separately, to assess the dose dependent nature of any increase in revertant colonies.

In the Confirmatory Mutation Assay, statistical analysis did not reveal any significant effect.

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None specified
- Effects of osmolality: None specified
- Precipitation: No precipitation observed at the limit test concentration of 5 µL/plate.
- Other confounding effects: None

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

Applicant's summary and conclusion

Conclusions:

Under the specified experimental conditions, C12-13 alkyl methacrylate is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA.