Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity in vitro

AMES (non-GLP). Key CRL 2019

OECD 471

In conclusion, based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 2019 - 11 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC Guideline No. 440/2008. Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Physical Description: Light orange liquid
Purity/Composition: Not indicated
Storage Conditions: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum foil
Solubility in vehicle: Dimethyl sulfoxide: Not indicated
Stability in vehicle: Dimethyl sulfoxide: Not indicated

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-Fraction
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per
10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

Test concentrations with justification for top dose:

Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

Dose-range fnding test
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
The highest concentration of test item used in the subsequent mutation assays was 5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Vehicle / solvent:

The vehicle of the test item was dimethyl sulfoxide (Merck, Darmstadt, Germany).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
Number of cultures per concentration: triplicate
Number of independent experiments : eight concetrations

METHOD OF TREATMENT/ EXPOSURE:
Cell density at seeding: optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL)
Test substance added in agar

TREATMENT AND HARVEST SCHEDULE:
Preincubation period: In the Pre-Incubation Assay, solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C
Exposure duration/duration of treatment: 48 h

FOR GENE MUTATION:
Method used: selective agar plate

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC).

Evaluation criteria:

4.7.4. Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Signs of toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Precipitation of C894 on the plates was observed at the start of the incubation period at concentrations of 164 µg/plate and upwards and at 5000 µg/plate at the end of the incubation period.

First Experiment:  Direct Plate Assay

The test item was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix.  Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix:  52, 164, 512, 1600 and 5000 μg/plate.  The results are shown in Table 1 and Table 2 (in appendix 1, attached).  The individual data are presented in Appendix 3 (attached).

Precipitate

Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 164 µg/plate and upwards and at 5000 µg/plate at the end of the incubation period.

Toxicity

To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.  The definitions are stated in Appendix 2 (attached).

No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

In strain TA1537 (absence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed.  However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item.  It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.    

Mutagenicity

In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Second Experiment:  Pre-Incubation Assay

To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of S9-mix.  Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.  The results are shown in Table 3 (appendix 1, attached), the individual data are presented in Appendix 3 (attached).

Precipitate

Precipitation of the test item on the plates was observed at the concentration of 5000 µg/plate at the end of the incubation period in all tester strains.  In addition the test item also precipitated at the dose level of 1600 µg/plate in the strains TA1535, TA98 and TA100 in the absence of S9-mix.  

Toxicity

There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

 

Mutagenicity

In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested. 

DISCUSSION

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.  

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Conclusions:

In conclusion, based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of the test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).  

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item was a light orange liquid.  The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay.  In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98.  In the second mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay.  In all three experiments, the test item precipitated on the plates at the top dose of 5000 μg/plate.  In addition, in the second mutation experiment, the test item also precipitated at the dose level of 1600 µg/plate in the strains TA1535, TA98 and TA100 in the absence of S9-mix.  The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.  

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.  These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.   

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Studies conducted to recognised testing guidelines with GLP certification.

Justification for classification or non-classification

The test item does not have to be classified and has no obligatory labelling requirement for genetic toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).