Propane-2-thiol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471 study

The potential of the registered substance, ISOPROPYLMERCAPTAN, to induce reverse mutations was evaluated in Salmonella typhimurium (Chevalier, 2018a). 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 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.

Under the experimental conditions of this study, the registered substance, 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.

 

OECD 490 study

The objective of this study was to evaluate the potential of the registered substance, ISOPROPYLMERCAPTAN, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells (Chevallier, 2018b). The study was performed according to international guidelines (OECD guideline No. 490 and Council Regulation) and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, ISOPROPYLMERCAPTAN, diluted in dimethylsulfoxide (DMSO), was tested in a single experiment (3-hour treatment), with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Cultures of 20 mL at 5 x 105cells/mL were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a, 5% CO2humidified incubator. Due to the volatile characteristic of the test item, tightly sealed tubes were used during the 3-hour treatment period. Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype. Since the test item was found freely soluble in the final treatment medium and induced a severe cytotoxicity only at the highest recommended dose level (i.e.10 mM) in the preliminary cytotoxicity test, 10 mM was selected as the highest dose level for the main experiment, according to the criteria specified in the international guidelines. 

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%.The study was therefore considered to be valid. The selected dose levels were 0.31, 0.63, 1.25, 2.5, 3.8, 5, 7.5 and 10 mM, both with and without S9 mix. 

No precipitate was observed in the culture medium at the end of the 3-hour treatment period, at any dose levels. 

Cytotoxicity

In the absence of S9 mix, a 72 to 89% decrease in the Adj. RTG was observed at dose levels = 3.8 mM. The recommended level of cytotoxicity was reached at dose levels = 5 mM (means Adj. RTG between 11 and 19%).

In the presence of S9 mix, a 46 to 100% decrease in the Adj. RTG was observed at dose levels = 3.8 mM. None of the selected dose levels induced the recommended level of cytotoxicity. However, considering the narrow dose levels spacing used, this selection was considered as suitable to allow a reliable interpretation.

Mutagenicity

No noteworthy increase in the mutation frequency was noted relative to the corresponding vehicle control, at any of the tested dose levels. In the absence of S9 mix only, a dose-response relationship in the MF was demonstrated by the linear regression(p<0.005).Since all the Induced Mutation Frequencies (IMF) remained substantially below the GEF of 126 x 10-6, this linear trend was considered as meaningless and these results were considered to be a negative response. These overall results did not meet the criteria of a positive response.

Under the experimental conditions of this study, the registered substance, ISOPROPYLMERCAPTAN, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

 

OECD 487 study

The potential of the registered substance, ISOPROPYLMERCAPTAN, to induce an increase in the frequency of micronucleated cells was evaluated in the mouse cell line L5178Y TK+/-(Chevallier, 2018c). The study was performed according to the OECD guideline No. 487 and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, the test item diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

	First experiment	Second experiment
Without S9 mix	3 h treatment + 24 h recovery 24 h treatment + 0 h recovery	24 h treatment + 0 h recovery
With S9 mix	3 h treatment + 24 h recovery

 

Due to the volatile characteristic of the test item, specific gas permeable sealing membranes were applied on the 24-well plates during the treatment periods (3h and 24h).Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. After the final cell counting, the cells were washed and fixed. Then, cells from at least three dose levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation (”blind” scoring). When the slide analysis was undertaken, micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

Since the test item was found cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the level of cytotoxicity, according to the criteria specified in the international regulations. The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid. First of all, no emulsion or precipitation was observed in the culture medium at the end of the treatment periods, in any experiments, up to the highest tested dose level of 3.5 mM. Using a test item concentration of 53.32 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatmentS were 0.09, 0.19, 0.38, 0.75, 1, 1.5, 2 and 3.5 mM.

Short treatment without S9 mix: 3 h treatment + 24 h recovery

A moderate to severe cytotoxicity was induced at dose levels = 0.75 mM, as shown by a 47 to 100% decrease in the PD. The dose levels selected for the micronucleus analysis were 0.38, 0.75 and 1 mM, the latter inducing a 56% decrease in the PD (i.e.the recommended level of cytotoxicity of 55 ± 5% cytotoxicity).

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression andnone of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. These results met the criteria of a negative response.

Continuous treatments without S9 mix: 24 h treatment + 0 h recovery

A moderate to severe cytotoxicity was induced at dose levels = 0.38 mM, as shown by a 64 to 100% decrease in the PD. Since the cytotoxicity obtained was higher than expected (based on the results of the preliminary cytotoxicity test), not enough analyzable dose levels were available for the analysis of micronuclei, according to the recommendations of the OECD Guideline. Thus, the corresponding slide analysis was not performed and a second experiment was undertaken under the same experimental conditions but using the following lower range of dose levels:0.05, 0.09, 0.19, 0.38, 0.5, 0.75, 1 and 1.5 mM.

A slight to severe cytotoxicity was induced at dose levels = 0.75 mM, as shown by a 38 to 100% decrease in the PD. The dose levels selected for the micronucleus analysis were 0.38, 0.5 and 0.75 mM, the latter inducing only a 38% decrease in the PD but higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. A statistically significant dose-response relationship was observed (demonstrated by the linear regression). Since all frequencies remained within the corresponding vehicle historical range and in the absence of statistically significant difference with the vehicle control, the statistically significant linear trend was considered as meaningless in terms of genotoxicity. As a consequence, these results were considered to meet the criteria of a negative response.

Although none of the selected dose levels induced the recommended level of cytotoxicity in this second experiment, the results were considered as suitable to allow a reliable interpretation considering the narrow dose levels spacing used.

Experiment with S9 mix

A slight to severe cytotoxicity was induced at dose levels = 0.75 mM, as shown by a 36 to 100% decrease in the PD. The dose levels selected for the micronucleus analysis were 0.19, 0.38 and 0.75 mM, the latter inducing only a 36% decrease in the PD but higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression and all frequencies remained within the vehicle control historical range. These results met the criteria of a negative response.

Although none of the selected dose levels induced the recommended level of cytotoxicity, the results were considered as suitable to allow a reliable interpretation considering the narrow dose levels spacing used.

Under the experimental conditions of the study, the registered substance, ISOPROPYLMERCAPTAN, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

Histidine operon

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.

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2017 - 06 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD guideline No. 490, adopted on 28 July 2015

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

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:

0.005, 0.01, 0.04, 0.12, 0.37, 1.11, 3.33 and 10 mM

Vehicle / solvent:

According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control cultures was dimethylsulfoxide (DMSO).
Since the test item was found freely soluble in the final treatment medium and induced a severe cytotoxicity only at the highest recommended dose level (i.e. 10 mM) in the preliminary cytotoxicity test, 10 mM was selected as the highest dose level for the main experiment.
Using a test item concentration of 152.32 mg/mL and a treatment volume of 0.5 % (v/v) in the culture medium (i.e. 100 µL/20 mL culture medium), the highest recommended dose level of 10 mM (corresponding to 761.6 µg/mL) was achievable.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h treatment + 24 h recovery (with and without S9 mix) and 24 h treatment + 0 h recovery (without S9 mix)

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

Rationale for test conditions:

Since the test item was found freely soluble in the final treatment medium and induced a severe cytotoxicity only at the highest recommended dose level (i.e. 10 mM) in the preliminary cytotoxicity test, 10 mM was selected as the highest dose level for the main experiment, according to the criteria specified in the international guidelines.

Evaluation criteria:

In all cases, biological relevance is taken into consideration when evaluating the results.

Evaluation of a positive response:
Based on IWGT recommendations (d, e, f), a test item is considered clearly positive if, in any of the experimental conditions examined:
- at least at one dose level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10 6,
- a dose-response relationship is demonstrated by a statistically significant trend test.

Evaluation of a negative response:
A test item is considered clearly negative if, in all experimental conditions, no dose-response relationship is demonstrated or, if there is an increase in MF, it does not exceed the GEF.

Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose levels with Adj. RTG between 10 and 20%, are not considered as positive results.

A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if (g):
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.

Statistics:

To assess the dose-response relationship, a linear regression was performed between dose levels and individual mutation frequencies obtained at dose levels showing a mean Adj. RTG = 10% (see Appendix 4). This statistical analysis was performed using SAS Enterprise Guide software.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.8 mM

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: none
- Precipitation: no emulsion or precipitation was observed in the culture medium at the end of the treatment periods, in any experiments, up to the highest tested dose level of 3.5 mM.

RANGE-FINDING STUDIES:
Using a test item concentration of 152.32 mg/mL and a treatment volume of 0.5 % (v/v) in the culture medium (i.e. 100 µL/20 mL culture medium), the highest recommended dose level of 10 mM (corresponding to 761.6 µg/mL) was achievable. Thus the dose levels selected for the treatment of the preliminary test were: 0.005, 0.01, 0.04, 0.12, 0.37, 1.11, 3.33 and 10 mM.
At the highest tested dose level of 10 mM, the pH of the culture medium was approximately 7.7 (7.4 for the vehicle control) and the osmolality was 365 mOsm/kg H2O (380 mOsm/kg H2O for the vehicle control). Thus none of the selected dose levels was considered to produce extreme culture conditions and the highest recommended dose level of 10 mM could be selected as the highest dose level for the main mutagenicity experiment.
No precipitate was observed in the culture medium at the end of the 3-hour treatment period, at any dose levels.
A 40 to 93% decrease in the Adj. RTG was observed at dose levels = 3.33 mM, both with and without S9 mix.
Since the test item was found freely soluble in the final treatment medium and induced a severe cytotoxicity only at the highest recommended dose level (i.e. 10 mM) in the preliminary cytotoxicity test, 10 mM was selected as the highest dose level for the main experiment, according to the criteria specified in the international guidelines.
The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.
For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.
No precipitate was observed in the culture medium at the end of the 3-hour treatment period, at any dose levels.

Cytotoxicity
In the absence of S9 mix, a 72 to 89% decrease in the Adj. RTG was observed at dose levels = 3.8 mM (Table 2). The recommended level of cytotoxicity was reached at dose levels = 5 mM (means Adj. RTG between 11 and 19%).
In the presence of S9 mix, a 46 to 100% decrease in the Adj. RTG was observed at dose levels = 3.8 mM (Table 4). None of the selected dose levels induced the recommended level of cytotoxicity. However, considering the narrow dose levels spacing used, this selection was considered as suitable to allow a reliable interpretation.

Mutagenicity
No noteworthy increase in the mutation frequency was noted relative to the corresponding vehicle control, at any of the tested dose levels (Table 3 and 5).
In the absence of S9 mix only, a dose-response relationship in the MF was demonstrated by the linear regression (p<0.005, Appendix 4). Since all the Induced Mutation Frequencies (IMF) remained substantially below the GEF of 126 x 10-6, this linear trend was considered as meaningless and these results were considered to be a negative response.

These overall results did not meet the criteria of a positive response.

Conclusions:

ISOPROPYLMERCAPTAN did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of ISOPROPYLMERCAPTAN to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK^+/-mouse lymphoma cells. The study was performed according to international guidelines (OECD guideline No. 490 and Council Regulation) and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, ISOPROPYLMERCAPTAN, diluted in dimethylsulfoxide (DMSO), was tested in a single experiment (3-hour treatment), with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Cultures of 20 mL at 5 x 10⁵cells/mL were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a, 5% CO₂humidified incubator. Due to the volatile characteristic of the test item,tightly sealed tubeswere used during the 3-hour treatment period. Cytotoxicity wasmeasured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) andCloning Efficiency following the expression time (CE₂). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype. Since the test item was found freely soluble in the final treatment medium and induced a severe cytotoxicity only at the highest recommended dose level (i.e.10 mM) in the preliminary cytotoxicity test, 10 mM was selected as the highest dose level for the main experiment, according to the criteria specified in the international guidelines. 

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%.The study was therefore considered to be valid. The selected dose levels were 0.31, 0.63, 1.25, 2.5, 3.8, 5, 7.5 and 10 mM, both with and without S9 mix. 

No precipitate was observed in the culture medium at the end of the 3-hour treatment period, at any dose levels. 

Cytotoxicity

In the absence of S9 mix, a 72 to 89% decrease in the Adj. RTG was observed at dose levels = 3.8 mM. The recommended level of cytotoxicity was reached at dose levels = 5 mM (means Adj. RTG between 11 and 19%).

In the presence of S9 mix, a 46 to 100% decrease in the Adj. RTG was observed at dose levels = 3.8 mM.None of the selected dose levels induced the recommended level of cytotoxicity. However, considering the narrow dose levels spacing used, this selection was considered as suitable to allow a reliable interpretation.

Mutagenicity

No noteworthy increase in the mutation frequency was noted relative to the corresponding vehicle control, at any of the tested dose levels. In the absence of S9 mix only, a dose-response relationship in the MF was demonstrated by the linear regression(p<0.005).Since allthe Induced Mutation Frequencies (IMF) remained substantially below the GEF of 126 x 10^-6, this linear trend was considered as meaningless andthese results were considered to be a negative response. These overall results did not meet the criteria of a positive response.

Under the experimental conditions of this study, ISOPROPYLMERCAPTAN, did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2017 - 28 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD guideline No. 487, adopted 29 July 2016

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable (not a gene mutation assay).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10% inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

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:

0.09, 0.19, 0.38, 0.75, 1, 1.5, 2 and 3.5 mM.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: using a test item concentration of 152.32 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium, the highest recommended dose level of 10 mM (corresponding to 761.6 µg/mL) was achievable. Thus, the dose levels selected for the treatment of the preliminary test were 0.005, 0.01, 0.04, 0.12, 0.37, 1.11, 3.33 and 10 mM.
- Since the test item was found cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the level of cytotoxicity, according to the criteria specified in the international regulations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
Preliminary cytotoxicity test
Without S9 mix
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix
3 h treatment + 24 h recovery

Main cytogenetic experiments
Without S9 mix
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix
3 h treatment + 24 h recovery

NUMBER OF CELLS EVALUATED: 2000/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- for at least one dose level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

Statistics:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the ki2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 1 mM

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: none

RANGE-FINDING STUDIES:
Using a test item concentration of 152.32 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium, the highest recommended dose level of 10 mM (corresponding to 761.6 µg/mL) was achievable. Thus, the dose levels selected for the treatment of the preliminary test were 0.005, 0.01, 0.04, 0.12, 0.37, 1.11, 3.33 and 10 mM.

At the highest dose level of 10 mM, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 380 mOsm/kg H2O (392 mOsm/kg for the vehicle control). Therefore, none of the tested dose levels was considered to produce extreme culture conditions and 10 mM could be selected as the highest dose level for the main experiment.

At the end of the treatment periods, no emulsion or precipitation was observed in the culture medium at any dose levels.

A slight to severe cytotoxicity was observed at dose levels >= 1.11 mM in all experimental conditions, as shown by a 30 to 100% decrease in the PD.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see Tables enclosed
- Indication whether binucleate or mononucleate where appropriate: mononucleates cells 'L5178Y cell line).

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

RESULTS OF CYTOTOXICITY:
Since the test item was found cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the level of cytotoxicity, according to the criteria specified in the international regulations.

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

First of all, no emulsion or precipitation was observed in the culture medium at the end of the treatment periods, in any experiments, up to the highest tested dose level of 3.5 mM.

Short treatment without S9 mix: 3 h treatment + 24 h recovery
Using a test item concentration of 53.32 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatment were 0.09, 0.19, 0.38, 0.75, 1, 1.5, 2 and 3.5 mM.
A moderate to severe cytotoxicity was induced at dose levels = 0.75 mM, as shown by a 47 to 100% decrease in the PD (Table 2).
The dose levels selected for the micronucleus analysis were 0.38, 0.75 and 1 mM, the latter inducing a 56% decrease in the PD (i.e. the recommended level of cytotoxicity of 55 ± 5% cytotoxicity).
No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control (Table 3; p>0.05). Moreover, no dose-response relationship was demonstrated by the linear regression (p>0.05) and none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. These results met the criteria of a negative response.

Continuous treatments without S9 mix: 24 h treatment + 0 h recovery
Using a test item concentration of 53.32 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatment were 0.09, 0.19, 0.38, 0.75, 1, 1.5, 2 and 3.5 mM.
A moderate to severe cytotoxicity was induced at dose levels = 0.38 mM, as shown by a 64 to 100% decrease in the PD (Table 4). Since the cytotoxicity obtained was higher than expected (based on the results of the preliminary cytotoxicity test), not enough analyzable dose levels were available for the analysis of micronuclei, according to the recommendations of the OECD Guideline. Thus, the corresponding slide analysis was not performed and a second experiment was undertaken under the same experimental conditions but using the following lower range of dose levels: 0.05, 0.09, 0.19, 0.38, 0.5, 0.75, 1 and 1.5 mM.
A slight to severe cytotoxicity was induced at dose levels = 0.75 mM, as shown by a 38 to 100% decrease in the PD (Table 5).
The dose levels selected for the micronucleus analysis were 0.38, 0.5 and 0.75 mM, the latter inducing only a 38% decrease in the PD but higher dose levels being too cytotoxic.
No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control (Table 6; p>0.05). A statistically significant dose response relationship was observed (demonstrated by the linear regression (Appendix 4; p<0.05). Since all frequencies remained within the corresponding vehicle historical range and in the absence of statistically significant difference with the vehicle control, the statistically significant linear trend was considered as meaningless in terms of genotoxicity. As a consequence, these results were considered to meet the criteria of a negative response.
Although none of the selected dose levels induced the recommended level of cytotoxicity in this second experiment, the results were considered as suitable to allow a reliable interpretation considering the narrow dose levels spacing used.

Experiment with S9 mix
Using a test item concentration of 53.32 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatment were 0.09, 0.19, 0.38, 0.75, 1, 1.5, 2 and 3.5 mM.
A slight to severe cytotoxicity was induced at dose levels = 0.75 mM, as shown by a 36 to 100% decrease in the PD (Table 7).
The dose levels selected for the micronucleus analysis were 0.19, 0.38 and 0.75 mM, the latter inducing only a 36% decrease in the PD but higher dose levels being too cytotoxic.
No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control (Table 8; p>0.05). Moreover, no dose-response relationship was demonstrated by the linear regression (p>0.05) and all frequencies remained within the vehicle control historical range. These results met the criteria of a negative response.
Although none of the selected dose levels induced the recommended level of cytotoxicity, the results were considered as suitable to allow a reliable interpretation considering the narrow dose levels spacing used.

Conclusions:

ISOPROPYLMERCAPTAN did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The potential of ISOPROPYLMERCAPTAN to induce an increase in the frequency of micronucleated cells was evaluated in the mouse cell line L5178Y TK^+/-. The study was performed according to the OECD guideline No. 487 and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, the test item diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

	First experiment	Second experiment
Without S9 mix	3 h treatment + 24 h recovery 24 h treatment + 0 h recovery	24 h treatment + 0 h recovery
With S9 mix	3 h treatment + 24 h recovery

 

Due to the volatile characteristic of the test item, specific gas permeable sealing membranes were applied on the 24-well plates during the treatment periods (3h and 24h). Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. After the final cell counting, the cells were washed and fixed. Then, cells from at least three dose levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). When the slide analysis was undertaken, micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

Since the test item was found cytotoxic in the preliminary test, the highest dose levels selected for the main experiments were based on the level of cytotoxicity, according to the criteria specified in the international regulations. The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid. First of all, no emulsion or precipitation was observed in the culture medium at the end of the treatment periods, in any experiments, up to the highest tested dose level of 3.5 mM. Using a test item concentration of 53.32 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatmentS were 0.09, 0.19, 0.38, 0.75, 1, 1.5, 2 and 3.5 mM.

Short treatment without S9 mix: 3 h treatment + 24 h recovery

A moderate to severe cytotoxicity was induced at dose levels = 0.75 mM, as shown by a 47 to 100% decrease in the PD. The dose levels selected for the micronucleus analysis were 0.38, 0.75 and 1 mM, the latter inducing a 56% decrease in the PD (i.e. the recommended level of cytotoxicity of 55 ± 5% cytotoxicity).

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression andnone of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle historical range. These results met the criteria of a negative response.

Continuous treatments without S9 mix: 24 h treatment + 0 h recovery

A moderate to severe cytotoxicity was induced at dose levels = 0.38 mM, as shown by a 64 to 100% decrease in the PD. Since thecytotoxicity obtained was higher than expected (based on the results of the preliminary cytotoxicity test), not enough analyzable dose levels were available for the analysis of micronuclei,according to the recommendations of the OECD Guideline.Thus, the corresponding slide analysis was not performed and a second experiment was undertaken under the same experimental conditions but using the following lower range of dose levels:0.05, 0.09, 0.19, 0.38, 0.5, 0.75, 1 and 1.5 mM.

A slight to severe cytotoxicity was induced at dose levels = 0.75 mM, as shown by a 38 to 100% decrease in the PD. The dose levels selected for the micronucleus analysis were 0.38, 0.5 and 0.75 mM, the latter inducing only a 38% decrease in the PD but higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. A statistically significant dose-response relationship was observed (demonstrated by the linear regression). Sinceall frequencies remained within thecorresponding vehicle historical range and in the absence of statistically significant difference with the vehicle control, the statistically significant linear trend was considered as meaningless in terms of genotoxicity. As a consequence, these results were considered to meet the criteria of a negative response.

Although none of the selected dose levels induced the recommended level of cytotoxicity in this second experiment, the results were considered as suitable to allow a reliable interpretation considering the narrow dose levels spacing used.

Experiment with S9 mix

A slight to severe cytotoxicity was induced at dose levels = 0.75 mM, as shown by a 36 to 100% decrease in the PD. The dose levels selected for the micronucleus analysis were 0.19, 0.38 and 0.75 mM, the latter inducing only a 36% decrease in the PD but higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. Moreover, no dose-response relationship was demonstrated by the linear regression and all frequencies remained within the vehicle control historical range. These results met the criteria of a negative response.

Although none of the selected dose levels induced the recommended level of cytotoxicity, the results were considered as suitable to allow a reliable interpretation considering the narrow dose levels spacing used.

Under the experimental conditions of the study, ISOPROPYLMERCAPTAN did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK^+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Not classified according to CLP and GHS criteria.