Octadecyl 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:

Octadecyl 3-mercaptopropionate

EC Number:

250-801-6

EC Name:

Octadecyl 3-mercaptopropionate

Cas Number:

31778-15-1

Molecular formula:

C21H42O2S

IUPAC Name:

octadecyl 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, 1600, 500, 160, 50 and 16 μg/plate (tested up to limit dose)

Vehicle / solvent:

acetone (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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Microdrops (colloid chemical phenomenon) were observed following the plate incorporation and pre-incubation procedures, in all examined strains, at 5000 μg/plate in the absence and presence of exogenous metabolic activation (±S9). The microdrops did not disturb the scoring in any case.

Any other information on results incl. tables

The spontaneous revertant colony numbers of the acetone vehicle control plates showed mostly the characteristic mean numbers agreed with the actual historical control data ranges in the investigated strains in all experimental phases of the study. However, the actual vehicle control values were below the corresponding historical control data ranges in the initial mutation test, in the case of Salmonella typhimurium TA100, in the absence and presence of exogenous metabolic activation (±S9), (actual values, −S9: 71; +S9: 75, the corresponding ranges: 78-101 and 82-119); furthermore, in the confirmatory mutation test in Salmonella typhimurium TA98, in the absence of S9 (actual value: 14, the corresponding range: 16-30); furthermore, in TA100, in the absence of S9 (actual value: 68). The slightly lower acetone control mean values were considered acceptable, remained within the biological variability range (no extreme outliers, remained well within the corresponding historical control data ranges of the parallel investigated untreated control).

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 ODMP has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item ODMP 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 acetone and the following  concentrations of the test item were prepared and investigated in the main experiments: ±S9: 5000, 1600, 500, 160, 50 and 16 μg/plate.
For the selected concentration range, the noticed cytotoxicity (absent), 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, acetone plates with and without S9 Mix demonstrated the characteristic mean numbers of spontaneous revertants that were in line with the corresponding historical control data ranges. The outlier values noticed in the initial and confirmatory mutation tests remained well within the acceptable range, evaluated as reflecting on the biological variability of the applied test system, have no effect on the validity and results of this study.

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:
Microdrops (colloid chemical phenomenon) were observed following the plate incorporation and pre-incubation procedures, in all examined strains, at 5000 μg/plate in the absence and presence of exogenous metabolic activation (±S9). The microdrops did not disturb the scoring in any case.

Cytotoxicity results:
In the initial and confirmatory mutation tests, inhibitory effects of the test item on bacterial growth were not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained within the range of the  biological variability of the applied test system and the background lawn development was not affected in any case.

Mutagenicity results:
No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Evabochem® 318 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 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.