Butyl 3-mercaptopropionate

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:

June 21, 2022 - June 30, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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, corrected 26th June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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:

Butyl 3-mercaptopropionate

EC Number:

240-343-5

EC Name:

Butyl 3-mercaptopropionate

Cas Number:

16215-21-7

Molecular formula:

C7H14O2S

IUPAC Name:

butyl 3-sulfanylpropanoate

Method

Target gene:

his (S. typhimurium) / trp (E. coli)

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:

Rat Liver S9 Fraction

Test concentrations with justification for top dose:

±S9: 5000, 2500, 800, 250, 80 and 25 μg/plate (tested up to limit dose)

Vehicle / solvent:

DMSO (based on solubility and concentration range finding tests)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA98, without S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2; initial mutation test (plate incorporation test) and a confirmatory mutation test (pre-incubation test)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min at 37ºC
- Exposure duration/duration of treatment: 48 h


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs

Evaluation criteria:

A result is considered positive if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2
uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the 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.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

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

Positive controls validity:

valid

Test results 2

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

Positive controls validity:

valid

Test results 3

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

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the initial and confirmatory mutation tests, unequivocal inhibitory effect of the test item on bacterial growth was observed.
The inhibitory effect was indicated by affected background lawn development: reduced or slightly reduced background lawn and/or decreased revertant colony counts (absent revertants or revertants below the corresponding historical control data ranges and/or actual vehicle control ranges).
In general, 800 μg/plate, noticed following the pre-incubation procedure (confirmatory mutation test) in all examined strains in absence of exogenous metabolic activation (−S9), in Salmonella typhimurium TA100 also in the presence of exogenous metabolic activation (+S9)) was considered as the lowest concentration showing unequivocal cytotoxicity.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Any other information on results incl. tables

In the performed experiments, occasional increases in the revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest mean revertant colony number increases were observed in the confirmatory mutation test (pre-incubation test) in Salmonella typhimurium TA1537 strain at 250 μg/plate, in the absence of metabolic activation (−S9). The revertant colony numbers at this treatment remained well within the corresponding historical control data range, the higher counts were unique, did not accompany with concentration relationship; and remained far below the genotoxicological threshold for being positive. The mutation rate at this treatment was 1.572.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.
In conclusion, the test item BuMP has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item BuMP was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.
Bacterial strains, exogenous metabolic activation:
Salmonella typhimurium TA98, target mutation: hisD3052; mutation type: frameshift;
Salmonella typhimurium TA100, target mutation: hisG46; mutation type: base pair substitution;
Salmonella typhimurium TA1535, target mutation: hisG46; mutation type: base pair substitution;
Salmonella typhimurium TA1537, target mutation: hisC3076; mutation type: frameshift;
Escherichia coli WP2 uvrA, target mutation: trpE; mutation type: base pair substitution.
Exogenous metabolic activation:
The experiments were carried out in the presence and absence of a cofactor supplemented post-mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The S9 mix contained 10 % (v/v) S9.

Experimental phases:
The study included preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Vehicle, test item concentrations, rationale for dose selection:
Based on the results of the solubility test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO) and the following concentrations of the test item were prepared and investigated in the main experiments: ±S9: 5000, 2500, 800, 250, 80 and 25 μg/plate.
For the selected concentration range, the noticed cytotoxicity (noticed in the informatory toxicity test), the solubility (adequately soluble in the applied test system), and the laboratory’s experience with used strains (for concentration choice at TA1535, TA1537 and Escherichia coli WP2 uvrA strains) were taken into consideration based on the recommendations in OECD 471 guideline.
At the preparation of the test item solutions any correction (multiplier) factor was not taken into consideration, the concentrations were based on the final formulation as is.

Validity of the Study:
In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled.
The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with and without S9 Mix demonstrated the characteristic mean numbers of spontaneous revertants that were mostly in line with the corresponding historical control data ranges. The slightly outlier value in the initial mutation test case of Escherichia coli WP2 uvrA strain (−S9) was considered acceptable, remained within the biological variability range (no extreme outlier).

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases.

Solubility, precipitation:
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Cytotoxicity results:
In the initial and confirmatory mutation tests, unequivocal inhibitory effect of the test item on bacterial growth was observed. The inhibitory effect was indicated by affected background lawn development: reduced or slightly reduced background lawn and/or
decreased revertant colony counts (absent revertants or revertants below the corresponding historical control data ranges and/or actual vehicle control ranges).
In general, 800 μg/plate, noticed following the pre-incubation procedure (confirmatory mutation test) in all examined strains in absence of exogenous metabolic activation (−S9), in Salmonella typhimurium TA100 also in the presence of exogenous metabolic activation (+S9)) was considered as the lowest concentration showing unequivocal cytotoxicity.

Mutagenicity results:
No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Evabopol® 394 at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Conclusion:
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli
WP2 uvrA.
In conclusion, the test item Evabopol® 394 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.