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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 October 2017 - 07 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD guideline No. 471, adopted on 21st July 1997

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Propane-2-thiol
EC Number:
200-861-4
EC Name:
Propane-2-thiol
Cas Number:
75-33-2
Molecular formula:
C3H8S
IUPAC Name:
propane-2-thiol
Constituent 2
Chemical structure
Reference substance name:
Propane-1-thiol
EC Number:
203-455-5
EC Name:
Propane-1-thiol
Cas Number:
107-03-9
Molecular formula:
C3H8S
IUPAC Name:
propane-1-thiol
Test material form:
liquid

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
n/a
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
n/a
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500 and 5000 µg/plate, in both experiments without S9 mix and in the first experiment with S9 mix
78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate, in the second experiment with S9 mix
Vehicle / solvent:
According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control plates was dimethylsulfoxide (DMSO).
Since the test item was found to be freely soluble in the final treatment medium and non-toxic in the preliminary test, the highest dose level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the pre-incubation method (60 minutes, 37°C, in tightly sealed tubes due to the volatile characteristic of the test item).

DURATION
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn

NUMBER OF REPLICATIONS: three plates/dose level
Evaluation criteria:
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose levels,
- nor any evidence of a dose-response relationship is noted.
Statistics:
no

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-1st experiment with S9 mix (direct plate incorporation method): no noteworthy toxicity, at any dose levels, in any strains. -2nd experiment with S9 mix (pre-incubation method): toxicity, at dose levels = 1250 µg/plate in the 5 strains.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: No precipitate was observed in the Petri plates when scoring the revertants, at any dose levels.

RANGE-FINDING STUDY:
The selected dose levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains, in both experiments without S9 mix and in the first experiment with S9 mix.
The selected dose levels were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the five strains, in the second experiment with S9 mix.

RESULTS OF CYTOTOXICITY and GENOTOXICITY:
Using the direct plate incorporation method (both experiments without S9 mix and first experiment with S9 mix), no noteworthy toxicity was noted at any dose-levels, towards the five strains used.
Using the pre-incubation method (second experiment with S9 mix), a moderate to strong toxicity (thinning of the bacterial lawn) was noted at dose levels = 1250 µg/plate in the five strains.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment with or without S9 mix. These results met the criteria for a negative response.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached




Applicant's summary and conclusion

Conclusions:
ISOPROPYLMERCAPTAN did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.
Executive summary:

The potential of ISOPROPYLMERCAPTAN to induce reverse mutations was evaluated in Salmonella typhimurium. The study was performed according to the international guidelines (OECD guideline No. 471 and Council Regulation) and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose levels of the test item, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C, in tightly sealed tubes due to the volatile characteristic of the test item). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C (in sealed jars due to the volatile characteristic of the test item), the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Since the test item was found to be freely soluble in the final treatment medium and non-toxic in the preliminary test, the highest dose level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose levels for each strain and test condition. The study was therefore considered to be valid. The selected dose levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains, in both experiments without S9 mix and in the first experiment with S9 mix. The selected dose levels were 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the five strains, in the second experiment with S9 mix. No precipitate was observed in the Petri plates when scoring the revertants, at any dose levels. Using the direct plate incorporation method (both experiments without S9 mix and first experiment with S9 mix), no noteworthy toxicity was notedat any dose-levels,towards the five strains used. Using the pre-incubation method (second experiment with S9 mix), a moderate to strong toxicity (thinning of the bacterial lawn) was noted at dose levels = 1250 µg/plate in the five strains. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, in either experiment with or without S9 mix. These results met the criteria for a negative response.

Under the experimental conditions of this study, ISOPROPYLMERCAPTAN did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.