[No public or meaningful name is available]

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:

August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

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 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate

Molecular formula:

C15 H20 O3

IUPAC Name:

Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate

Test material form:

liquid

Details on test material:

- Name: 2-Propenoic acid,2-[[3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5(or 6)-yl]oxy]ethyl ester
- CAS No.: 68169-12-0 (outside EU)
- EC Name: Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7ahexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate
- EC Number: 951-956-9
- Batch No.: OX8J0799
- Molecular Weight: 248.32 g/mol
- Physical State: liquid
- Colour: clear
- Active Components: 98.52% active components (different isomers), 1.48% 2-Cyclopenta-1,3-dienyloxyethan-1-ol
- Purity: 98.52%
- Expiry Date: 17 September 2023
- Storage Conditions: room temperature
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/β-naphthoflavone induced rat S9 Mix

Test concentrations with justification for top dose:

0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0 and 2.5 μL/plate
The recommended maximum dose was reached during the experiment as toxicity was observed in the highest concentration used.

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:

A. dest

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD (4-nitro-o-phenylene-diamine)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

A. dest

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-AA (2-aminoanthracene)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

In order to investigate the potential of 2-Propenoic acid,2-[[3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5(or 6)-yl]oxy]ethyl ester; EC Name: Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-6-yloxy)ethyl acrylate for its ability to induce gene mutations the plate incorporation test was performed with the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).

In the experiment several concentrations of the test item were used. The 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 experiment:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0 and 2.5 μL/plate

Using a correction factor of 1.015, the highest concentration of 2.5 μL/plate corresponds to 2.46 μL/plate active component. This difference in concentration can be regarded as negligible. The recommended maximum dose was reached during the experiment as toxicity was observed in the highest concentration used. Therefore, the test item was tested up to a cytotoxic concentration.

Rationale for test conditions:

The OECD Guideline for Testing of Chemicals, Section 4, No. 471 – Bacterial Reverse Mutation Test - recommends using a combination of S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101).
The S. typhimurium histidine (his) reversion system and the E. coli tryptophan (trp) reversion system measures his- -> his+ reversions and trp- -> trp+.
The S. typhimurium strains are constructed to differentiate between base pair (TA100, TA1535) and frameshift (TA98, TA1537) mutations.
The E. coli strain detects only base substitution mutagens.

Evaluation criteria:

CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

VALIDITY
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA (pKM101))
- the negative control plates (A. dest.) with and without S9 mix are within the following historical control data range
- corresponding background growth on negative control, solvent 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.

MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

No precipitation of the test item was observed in any tester strain used (with and without metabolic activation).

Toxic effects of the test item were noted in all tester strains used.

Toxic effects of the test item were noted in tester strain TA98 at concentrations of 0.100 μL/plate and higher (without metabolic activation) and at concentrations of 1.0 μL/plate and higher (with metabolic activation). Toxic effects of the test item were observed in tester strains TA100, TA1535 and TA1537 at concentrations of 0.316 μL/plate and higher (without metabolic activation) and at concentrations of 1.0 μL/plate and higher (with metabolic activation). In tester strain E. coli WP2 uvrA (pKM101) toxic effects of the test item were noted at concentrations of 0.316 μL/plate and higher (with and without metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation.

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, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay, when tested up to a cytotoxic concentration.

Executive summary:

In order to investigate the potential of 2-Propenoic acid,2-[[3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-5(or 6)-yl]oxy]ethyl ester; EC Name: Reaction mass of 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7 -methanoinden-5-yloxy)ethyl acrylate and 2-(3a,4,5,6,7,7a-hexahydro-1H-4,7-methanoinden-6 -yloxy)ethyl acrylate for its ability to induce gene mutations the plate incorporation test was performed with the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).

In the experiment several concentrations of the test item were used. The 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 experiment:

0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0 and 2.5 μL/plate

Using a correction factor of 1.015, the highest concentration of 2.5 μL/plate corresponds to 2.46 μL/plate active component. This difference in concentration can be regarded as negligible. The recommended maximum dose was reached during the experiment as toxicity was observed in the highest concentration used. Therefore, the test item was tested up to a cytotoxic concentration.

Results

No precipitation of the test item was observed in any tester strain used (with and without metabolic activation).

Toxic effects of the test item were noted in all tester strains used:

Toxic effects of the test item were observed at concentrations of 0.1 μL/plate and higher (without metabolic activation) and at concentrations of 0.316 μg/plate and higher (with metabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in the experiment.

All criteria of validity were met.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay, when tested up to a cytotoxic concentration.