Vinyl 4-(1,1-dimethylethyl)benzoate

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:

Jan. 2021 - March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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

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:

Vinyl 4-(1,1-dimethylethyl)benzoate

EC Number:

239-510-5

EC Name:

Vinyl 4-(1,1-dimethylethyl)benzoate

Cas Number:

15484-80-7

Molecular formula:

C13H16O2

IUPAC Name:

ethenyl 4-tert-butylbenzoate

Test material form:

liquid

Details on test material:

- Name: SAT 200028
- Batch No.: 19K2505
- Physical State: liquid
- Purity: 99.0%
- Expiry Date: 30 June 2021
- Storage Conditions: at room temperature, protected from light
- 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 S9 mix (rats)

Test concentrations with justification for top dose:

0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains with metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvr A without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

TA 98, TA 1537 without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 without metabolic activation

Details on test system and experimental conditions:

Four strains of S. typhimurium and one strain of E. coli WP2 uvrA (pKM101) with the following
characteristics were used:
TA98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA1537: his C 3076; rfa-; uvrB-: frame shift mutations
E. coli: WP2 uvrA (pKM101): trp-; uvrA-: base-pair substitutions

Evaluation criteria:

- Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as
"N" or “B”, respectively in the result tables) 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 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.

- 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
as compared to the reversion rate of the solvent control.

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:

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:

S. typhimurium TA 100

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 3

Key result

Species / strain:

S. typhimurium TA 98

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 4

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

Test results 5

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:

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 in experiment I and II (with and without metabolic activation).
The microbial contamination observed in two plates did not affect the quality or integrity of the results, as the microbial contamination could be clearly distinguished from the salmonella revertants and thus did not affect the evaluation.

In experiment I toxic effects of the test item were observed in tester strain TA98 at concentrations of 2.5 μL/plate and higher (with and without metabolic activation). Toxic effects of the test item were also noted in tester strain TA100 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). In tester strain TA1535 toxic effects of the test item were observed at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 2.5 μL/plate and higher (with metabolic activation). Toxic effects of the test item were also noted in tester strain E. coli WP2 uvrA (pKM101) at concentrations of 0.0316 μL/plate and higher (without metabolic activation). The reduction in the
number of revertants down to a mutation factor of ≤ 0.5 found in experiment I in tester strain E. coli WP2 uvrA (pKM101) at concentrations of 1.0 and 2.5 μL/plate (with metabolic activation) were regarded as not biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of the background lawn. No toxic effects of the test item were observed in tester
strain TA1537 (with and without metabolic activation).

In experiment II toxic effects of the test item were noted in tester strains TA98 and TA100 at concentrations of 0.0316 μL/plate and higher (without metabolic activation). Toxic effects of the test item were also noted in tester strains TA1535 and TA1537 at concentrations of 0.100 μL/plate and higher (without metabolic activation). No toxic effects of the test item were observed in tester strain E. coli WP2 uvrA (pKM101) (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 SAT 200028 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, SAT 200028 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, SAT 200028 is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item SAT 200028 was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
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:
0.00316, 0.0100, 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).
In experiment I toxic effects of the test item were observed at concentrations of 0.0316 μL/plate and higher (without metabolic activation) and at concentrations of 1.0 μL/plate and higher (with metabolic activation), depending on the particular tester strain.
In experiment II toxic effects of the test item were noted at concentrations of 0.0316 μL/plate and higher (without 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 SAT 200028 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met.