2,2-bis(methylthio)propane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation assays

The potential of 2,2-bis(methylthio) propane to induce reverse mutation was evaluated inSalmonella typhimurium(Haddouk, 2007). The study was performed according to the OECD guideline 471 and in compliance with the Principles of Good Laboratory Practice Regulations. A preliminary toxicity test was performed to define the dose-levels of 2,2-Bis(methylthio)propane to be used for the mutagenicity study. 2,2-Bis(methylthio)propane was then tested in two independent experiments, 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. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes,). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. 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, 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. 2,2-Bis(methylthio)propane was dissolved in dimethylsulfoxide (DMSO). Since 2,2-Bis(methylthio)propane was freely soluble and not severely toxic in the preliminary test, the highest dose-level for the main test was 5000 µg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels ranged from 21 to 5000 µg/plate, for all tester strains with and without S9 mix. No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. Without S9 mix, a moderate to strong toxicity was noted at dose-levels = 556µg/plate. With S9 mix, a moderate to strong toxicity was noted at 5000 µg/plate in the direct plate incorporation method and at dose-levels = 185µg/plate in the preincubation method. 2,2-Bis(methylthio)propane did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains in the first experiment. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. 2,2-bis(methylthio) propane did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

The potential of 2,2-bis(methylthio) propane to induce mutations at the TK (Thymidine Kinase) locus was evaluated in L5178Y TK^+/-mouse lymphoma cells (Sarlang, 2012a). The study was performed according to international guidelines (OECD guideline No. 476and Council Regulation (EC) No. 440/2008. After a preliminary toxicity test, 2,2-Bis(methylthio)propane was tested in two independent experiments, 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 (3-hour treatment) or cultures of 50 mL at 2 x 10⁵cells/mL (24-hour treatment) 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% (3-hour treatment) or 10% (24-hour treatment) in a, 5% CO₂humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. 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. 2,2-Bis(methylthio)propane was dissolved in dimethylsulfoxide (DMSO). 

The dose-levels for the positive controls were as follows.

. without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL, (3-hour treatment) or 5 µg/mL (24-hour treatment).

. with S9 mix: cyclophosphamide (CPA), used at a final concentration of 3 µg/mL.

With one exception which was not considered to impact the validity of the results, all acceptance criteria were of the vehicle and positive controls were met in this study. The study was therefore considered as valid. Since 2,2-Bis(methylthio)propane was toxic in the preliminary test, the choice of the highest dose-level for the main experiments was 500 µg/mL, based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG). 

Experiments without S9 mix: using a treatment volume of 1%, the selected dose-levels were 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the first experiment (3-hour treatment) and for the second experiment (24-hour treatment). Following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels = 125 µg/mL, as shown by a 40-100% decrease in Adj. RTG. Following the 24-hour treatment, a slight to severe toxicity was induced at dose-levels = 15.6 µg/mL, as shown by a 35-100% decrease in Adj. RTG. No relevant increase in the mutation frequency was induced in the first experiment (3-hour treatment), up to the dose-level of 250 µg/mL, which showed a 69% decrease in the Adj. RTG. No relevant increase in the mutation frequency was induced in the second experiment (24-hour treatment), up to the dose-level of 250 µg/mL, which showed a recommended 88% decrease in the Adj. RTG. In both experiments, a statistical analysis performed on the individual mutation frequencies of total induced mutants obtained at dose-levels up to 250 µg/mL did not demonstrate a linear trend between the mutation frequency and the dose. Thus these results did not meet the criteria of a positive response.

Experiments with S9 mix: using a treatment volume of 1%, the selected dose-levels were 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the first experiment, and 5.6, 31.3, 62.5, 125, 250, 375 and 500 µg/mL for the second experiment. Following the first experiment, a slight to severe toxicity was induced at dose-levels = 31.3 µg/mL, as shown by a 28-100% decrease in Adj. RTG.

Following the second experiment, a moderate to severe toxicity was induced at dose-levels = 125 µg/mL, as shown by a 43-100% decrease in Adj. RTG. No noteworthy increase in the mutation frequency which could be considered as biologically relevant was noted in comparison to the vehicle control in either experiment. In both experiments, a statistical analysis performed on the individual mutation frequencies of total induced mutants obtained at dose-levels up to 250 µg/mL did not demonstrate a linear trend between the mutation frequency and the dose.

Thus, these results did not meet the criteria of a positive response.

2,2-bis(methylthio) propane did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Chromosomal aberration assays

The potential of 2,2-bis(methylthio) propane to induce chromosome aberrations was evaluated in cultured human lymphocytes (Sarlang, 2012b). The study was performed according to the international guidelines (OECD No. 473 and Council Regulation (EC) No. 440/2008 of 30 May 2008, Annex, Part B.10) and in compliance with the Principles of Good Laboratory Practice. 2,2-Bis(methylthio)propane was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of Mitotic Index (MI) in the first experiment was also taken into account. For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at 37°C, for about 48 hours. In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. In the second experiment without S9 mix, cells were exposed continuously to the test or control items until harvest, and with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively. Three hours before harvest, each culture was treated with a Colcemid® solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. 2,2-Bis(methylthio)propane was dissolved in dimethylsulfoxide (DMSO).

With a treatment volume of 55 µL/5.5 mL culture medium, the dose-levels used for treatment were 20.58, 61.73, 185.2, 555.6, 1667 and 5000 µg/mL for the first experiment, both with and without S9 mix, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the second experiment with S9 mix, and 31.25, 62.5, 125, 250, 500 and 1000 µg/mL for the second experiment without S9 mix. At the end of the 3-hour treatment periods, an emulsion was observed at dose-levels = 1667 µg/mL in the first experiment and at dose-levels = 250 µg/mL in the second experiment. At the end of the 20- and 44-hour treatment periods, no precipitate or emulsion was observed up to the highest dose-level tested of 1000 µg/mL. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered to be valid.

Experiments without S9 mix: following the 3-hour treatment in the first experiment, a slight to severe toxicity was observed at dose-levels = 185.2 µg/mL as shown by a 38 to 100% decrease in the MI. Following the 20-hour treatment in the second experiment, a moderate to severe toxicity was noted at dose-levels = 500 µg/mL as shown by a 42 to 100% decrease in the MI. Following the 44-hour treatment in the second experiment, a moderate to severe toxicity was observed at dose-levels = 500 µg/mL as shown by a 45 to 100% decrease in the MI. The dose-levels selected for metaphase analysis were 61.73, 158.2 and 555.6 µg/mL for the 3-hour treatment, the latter inducing a 58% decrease in the MI, 125, 250 and 500 µg/mL for the 20-hour treatment, the latter inducing a 42% decrease in the MI and higher dose-levels being too cytotoxic, and 500 µg/mL for the 44-hour treatment, the latter inducing a 45% decrease in the MI and the higher dose-level being too cytotoxic. No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments. These results met the criteria for a negative response. 

Experiments with S9 mix: at the 20-hour harvest time in the first experiment, a severe toxicity was observed at dose-levels = 555.6 µg/mL, as shown by a 100% decrease in the MI. At the 20-hour harvest time in the second experiment, a marked toxicity was noted at 500 µg/mL, as shown by a 66% decrease in the MI. At the 44-hour harvest time, a marked toxicity was observed at 500 µg/mL, as shown by a 72% decrease in the MI. The dose-levels selected for metaphase analysis were 20.58, 61.73 and 185.2 µg/mL for the 20-hour harvest time in the first experiment, higher dose-levels being too cytotoxic, 62.5, 125 and 250 µg/mL for the 20-hour harvest time in the second experiment, higher dose-levels being too cytotoxic, and 250 µg/mL for the 44-hour harvest time, higher dose-levels being too cytotoxic. No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment, at either harvest time. These results met the criteria for a negative response.

2,2-bis(methylthio) propane did not induce chromosome aberrations in cultured human lymphocytes, in the presence or the absence of a rat 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No chemical analysis of the dosage forms was performed as part of this study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium, other: 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

liver post mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

21, 62, 185, 556, 1667 and 5000 µg/plate, for all tester strains with and without S9 mix

Vehicle / solvent:

dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: 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.

Evaluation criteria:

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 number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

Not applicable

Species / strain:

S. typhimurium, other: 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL.
Consequently, with a treatment volume of 50 µL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at any dose-level.
A moderate to strong toxicity (mainly thinning of the bacterial lawn) was noted towards the three strains used, either with or without S9 mix at dose-levels = 1000 µg/plate.

MUTAGENICITY EXPERIMENTS
No precipitate was observed in the Petri plates when scoring the revertants at any dose-level.
Without S9 mix, a moderate to strong toxicity was noted at dose-levels = 556 µg/plate.
With S9 mix, a moderate to strong toxicity was noted at 5000 µg/plate in the direct plate incorporation method and at dose-levels = 185 µg/plate in the preincubation method.
The test item did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains in the first experiment.

Conclusions:

2,2-BIS(METHYLTHIO)PROPANE did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

The potential of 2,2-BIS(METHYLTHIO)PROPANE (BMTP) to induce reverse mutation was evaluated in Salmonella typhimurium. The study was performed according to the OECD guideline 471 and in compliance with the Principles of Good Laboratory Practice Regulations. A preliminary toxicity test was performed to define the dose-levels of BMTP to be used for the mutagenicity study. BMTP was then tested in two independent experiments, 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. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes,). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. 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, 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. BMTP was dissolved in dimethylsulfoxide (DMSO). Since BMTP was freely soluble and not severely toxic in the preliminary test, the highest dose-level for the main test was 5000 µg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels ranged from 21 to 5000 µg/plate, for all tester strains with and without S9 mix. No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. Without S9 mix, a moderate to strong toxicity was noted at dose-levels = 556 µg/plate. With S9 mix, a moderate to strong toxicity was noted at 5000 µg/plate in the direct plate incorporation method and at dose-levels = 185 µg/plate in the preincubation method. BMTP did not induce any noteworthy increase in the number of revertants, either with or without S9 mix, in any of the five strains in the first experiment. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. 2,2-BIS(METHYLTHIO)PROPANE did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 March 2012 - 21 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable (not a gene mutation assay)

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin
(100 µg/mL) and phytohemagglutinin (PHA: a mitogen to stimulate lymphocyte division)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

20.58, 61.73, 185.2, 555.6, 1667 and 5000 µg/mL for the first experiment, both with and without S9 mix,
15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the second experiment with S9 mix,
31.25, 62.5, 125, 250, 500 and 1000 µg/mL for the second experiment without S9 mix

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide
- Justification for choice: test item was soluble in the vehicle at 500 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
see Executive summary

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level

EVALUATION OF THE TEST ITEM CYTOTOXICITY
The cytotoxicity of the test item was evaluated using the mitotic index (number of cells in mitosis/number of cells examined), which indicates whether a substance causes mitotic inhibition. The number of cells in mitosis is scored on a total of 1000 cells per culture. Scoring of MI (Mitotic Index) was undertaken without blinding.

METAPHASE ANALYSIS
The following structural aberrations were recorded for each metaphase: gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations). In addition, the following numerical aberrations were recorded when encountered: polyploidy, endoreduplication.

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Experiments without S9 mix
Cytotoxicity
Following the 3-hour treatment in the first experiment, a slight to severe toxicity was observed at dose-levels = 185.2 µg/mL as shown by a 38 to 100% decrease in the MI.
Following the 20-hour treatment in the second experiment, a moderate to severe toxicity was noted at dose-levels = 500 µg/mL as shown by a 42 to 100% decrease in the MI.
Following the 44-hour treatment in the second experiment, a moderate to severe toxicity was observed at dose-levels = 500 µg/mL as shown by a 45 to 100% decrease in the MI.
 
Metaphase analysis
The dose-levels selected for metaphase analysis were as follows:
.           61.73, 158.2 and 555.6 µg/mL for the 3-hour treatment, the latter inducing a 58% decrease in the MI,
.           125, 250 and 500 µg/mL for the 20-hour treatment, the latter inducing a 42% decrease in the MI and higher dose-levels being too cytotoxic,
.           500 µg/mL for the 44-hour treatment, the latter inducing a 45% decrease in the MI and the higher dose-level being too cytotoxic.
 
No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments. These results met the criteria for a negative response.
 
Experiments with S9 mix
Cytotoxicity
At the 20-hour harvest time in the first experiment, a severe toxicity was observed at dose-levels = 555.6 µg/mL, as shown by a 100% decrease in the MI.
At the 20-hour harvest time in the second experiment, a marked toxicity was noted at 500 µg/mL, as shown by a 66% decrease in the MI.
At the 44-hour harvest time, a marked toxicity was observed at 500 µg/mL, as shown by a 72% decrease in the MI.
 
Metaphase analysis
The dose-levels selected for metaphase analysis were as follows:
.           20.58, 61.73 and 185.2 µg/mL for the 20-hour harvest time in the first experiment, higher dose-levels being too cytotoxic,
.           62.5, 125 and 250 µg/mL for the 20-hour harvest time in the second experiment, higher dose-levels being too cytotoxic,
.           250 µg/mL for the 44-hour harvest time, higher dose-levels being too cytotoxic.
 
No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment, at either harvest time. These results met the criteria for a negative response.

Conclusions:

BMTP did not induce chromosome aberrations in cultured human lymphocytes, in the presence or the absence of a rat metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce chromosome aberrations in cultured human lymphocytes.

The study was performed according to the international guidelines (OECD No. 473 and Council Regulation (EC) No. 440/2008 of 30 May 2008, Annex, Part B.10) and in compliance with the Principles of Good Laboratory Practice.

 

Methods

The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254.

 

The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of Mitotic Index (MI) in the first experiment was also taken into account.

 

For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at 37°C, for about 48 hours.

In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

 

The second experiment was performed as follows:

.           without S9 mix, cells were exposed continuously to the test or control items until harvest,

.           with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.

 

Three hours before harvest, each culture was treated with a Colcemid® solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

With a treatment volume of 55 µL/5.5 mL culture medium, the dose-levels used for treatment were as follows:

.           20.58, 61.73, 185.2, 555.6, 1667 and 5000 µg/mL for the first experiment, both with and without S9 mix,

.           15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the second experiment with S9 mix,

.           31.25, 62.5, 125, 250, 500 and 1000 µg/mL for the second experiment without S9 mix.

 

At the end of the 3-hour treatment periods, an emulsion was observed at dose-levels = 1667 µg/mL in the first experiment and at dose-levels = 250 µg/mL in the second experiment.

At the end of the 20- and 44-hour treatment periods, no precipitate or emulsion was observed up to the highest dose-level tested of 1000 µg/mL.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered to be valid.

 

Experiments without S9 mix

Cytotoxicity

Following the 3-hour treatment in the first experiment, a slight to severe toxicity was observed at dose-levels = 185.2 µg/mL as shown by a 38 to 100% decrease in the MI.

Following the 20-hour treatment in the second experiment, a moderate to severe toxicity was noted at dose-levels = 500 µg/mL as shown by a 42 to 100% decrease in the MI.

Following the 44-hour treatment in the second experiment, a moderate to severe toxicity was observed at dose-levels = 500 µg/mL as shown by a 45 to 100% decrease in the MI.

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.           61.73, 158.2 and 555.6 µg/mL for the 3-hour treatment, the latter inducing a 58% decrease in the MI,

.           125, 250 and 500 µg/mL for the 20-hour treatment, the latter inducing a 42% decrease in the MI and higher dose-levels being too cytotoxic,

.           500 µg/mL for the 44-hour treatment, the latter inducing a 45% decrease in the MI and the higher dose-level being too cytotoxic.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments. These results met the criteria for a negative response.

 

Experiments with S9 mix

Cytotoxicity

At the 20-hour harvest time in the first experiment, a severe toxicity was observed at dose-levels = 555.6 µg/mL, as shown by a 100% decrease in the MI.

At the 20-hour harvest time in the second experiment, a marked toxicity was noted at 500 µg/mL, as shown by a 66% decrease in the MI.

At the 44-hour harvest time, a marked toxicity was observed at 500 µg/mL, as shown by a 72% decrease in the MI.

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.           20.58, 61.73 and 185.2 µg/mL for the 20-hour harvest time in the first experiment, higher dose-levels being too cytotoxic,

.           62.5, 125 and 250 µg/mL for the 20-hour harvest time in the second experiment, higher dose-levels being too cytotoxic,

.           250 µg/mL for the 44-hour harvest time, higher dose-levels being too cytotoxic.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment, at either harvest time. These results met the criteria for a negative response.

 

Conclusion

The test item did not induce chromosome aberrations in cultured human lymphocytes, in the presence or the absence of a rat metabolizing system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 March 2012 - 21 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

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

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Without S9: 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL ( first and second experiments)
With S9: 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the first experiment,
            15.6, 31.3, 62.5, 125, 250, 375 and 500 µg/mL for the second experiment.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide
- Justification for choice: test item was soluble in the vehicle at 500 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.

Evaluation criteria:

Positive result defined as:
- 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 E-6
- A dose-related trend is demonstrated by a statistically significant trend test
- Unless clearly positive, the reproducibility should be confirmed

Negative results defined as:
- No evidence of mutagenicity at concentrations inducing moderate cytotoxicity (10% < Adj. RTG <20%), or
- If there is no culture with 10% < Adj. RTG <20%:
¿ at least one negative data point with 20% < Adj. RTG <25% + negative data from 20% < Adj. RTG <100%, or
¿ at least one negative data point with 1% < Adj. RTG <10% + negative data from 25% < Adj. RTG <100%

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

With one exception which was not considered to impact the validity of the results, all acceptance criteria were of the vehicle and positive controls were met in this study. The study was therefore considered as valid.
 
Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main experiments was 500 µg/mL, based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).
 
Experiments without S9 mix
Using a treatment volume of 1%, the selected dose-levels were 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the first experiment (3-hour treatment) and for the second experiment (24-hour treatment).

Cytotoxicity
Following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels = 125 µg/mL, as shown by a 40-100% decrease in Adj. RTG.
Following the 24-hour treatment, a slight to severe toxicity was induced at dose-levels = 15.6 µg/mL, as shown by a 35-100% decrease in Adj. RTG.
 
Mutagenicity
No relevant increase in the mutation frequency was induced in the first experiment (3-hour treatment), up to the dose-level of 250 µg/mL, which showed a 69% decrease in the Adj. RTG.
No relevant increase in the mutation frequency was induced in the second experiment (24-hour treatment), up to the dose-level of 250 µg/mL, which showed a recommended 88% decrease in the Adj. RTG.
In both experiments, a statistical analysis performed on the individual mutation frequencies of total induced mutants obtained at dose-levels up to 250 µg/mL did not demonstrate a linear trend between the mutation frequency and the dose.
Thus these results did not meet the criteria of a positive response.
 
Experiments with S9 mix
Using a treatment volume of 1%, the selected dose-levels were as follows:
.           7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the first experiment,
.           15.6, 31.3, 62.5, 125, 250, 375 and 500 µg/mL for the second experiment.
 
Cytotoxicity
Following the first experiment, a slight to severe toxicity was induced at dose-levels = 31.3 µg/mL, as shown by a 28-100% decrease in Adj. RTG.
Following the second experiment, a moderate to severe toxicity was induced at dose-levels = 125 µg/mL, as shown by a 43-100% decrease in Adj. RTG.
 
Mutagenicity
No noteworthy increase in the mutation frequency which could be considered as biologically relevant was noted in comparison to the vehicle control in either experiment.
In both experiments, a statistical analysis performed on the individual mutation frequencies of total induced mutants obtained at dose-levels up to 250 µg/mL did not demonstrate a linear trend between the mutation frequency and the dose.
Thus, these results did not meet the criteria of a positive response.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

BMTP did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item 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. 476and Council Regulation (EC) No. 440/2008 of 30 May 2008) and in compliance with the principles of Good Laboratory Practice.

 

Methods

After a preliminary toxicity test, the test item was tested in two independent experiments, 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 (3-hour treatment) or cultures of 50 mL at 2 x 10⁵cells/mL (24-hour treatment) 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% (3-hour treatment) or 10% (24-hour treatment) in a, 5% CO₂humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

 

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.

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

The dose-levels for the positive controls were as follows:

.           without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL, (3-hour treatment) or 5 µg/mL (24-hour treatment),

.           with S9 mix: cyclophosphamide (CPA), used at a final concentration of 3 µg/mL.

 

Results

With one exception which was not considered to impact the validity of the results, all acceptance criteria were of the vehicle and positive controls were met in this study. The study was therefore considered as valid.

 

Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main experiments was 500 µg/mL, based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

 

Experiments without S9 mix

Using a treatment volume of 1%, the selected dose-levels were 7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the first experiment (3-hour treatment) and for the second experiment (24-hour treatment).

Cytotoxicity

Following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels = 125 µg/mL, as shown by a 40-100% decrease in Adj. RTG.

Following the 24-hour treatment, a slight to severe toxicity was induced at dose-levels = 15.6 µg/mL, as shown by a 35-100% decrease in Adj. RTG.

 

Mutagenicity

No relevant increase in the mutation frequency was induced in the first experiment (3-hour treatment), up to the dose-level of 250 µg/mL, which showed a 69% decrease in the Adj. RTG.

No relevant increase in the mutation frequency was induced in the second experiment (24-hour treatment), up to the dose-level of 250 µg/mL, which showed a recommended 88% decrease in the Adj. RTG.

In both experiments, a statistical analysis performed on the individual mutation frequencies of total induced mutants obtained at dose-levels up to 250 µg/mL did not demonstrate a linear trend between the mutation frequency and the dose.

Thus these results did not meet the criteria of a positive response.

 

Experiments with S9 mix

Using a treatment volume of 1%, the selected dose-levels were as follows:

.           7.81, 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL for the first experiment,

.           15.6, 31.3, 62.5, 125, 250, 375 and 500 µg/mL for the second experiment.

 

Cytotoxicity

Following the first experiment, a slight to severe toxicity was induced at dose-levels = 31.3 µg/mL, as shown by a 28-100% decrease in Adj. RTG.

Following the second experiment, a moderate to severe toxicity was induced at dose-levels = 125 µg/mL, as shown by a 43-100% decrease in Adj. RTG.

 

Mutagenicity

No noteworthy increase in the mutation frequency which could be considered as biologically relevant was noted in comparison to the vehicle control in either experiment.

In both experiments, a statistical analysis performed on the individual mutation frequencies of total induced mutants obtained at dose-levels up to 250 µg/mL did not demonstrate a linear trend between the mutation frequency and the dose.

Thus, these results did not meet the criteria of a positive response.

 

Conclusion

The test item did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The potential of 2,2-bis(methylthio) propane to induce structural or numerical damage was evaluated in bone marrow cells of rat (Sire, 2008). The study was performed according to the OECD guideline 474 and in compliance with the Principles of Good Laboratory Practice Regulations. Several preliminary toxicity tests were performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female Sprague-Dawley rats received two oral treatments of 2,2-Bis(methylthio)propane at dose-levels of 500, 1000 and 2000 mg/kg/day for males and 375, 750 and 1500 mg/kg/day for females, at a 24-hour interval. One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test item (Cyclophosphamide) once by oral route at the dose-level of 15 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

The dose-level of 2000 mg/kg/day for males and 1500 mg/kg/day for females were considered to be the maximum tolerated dose and were retained as the highest dose-levels for the main test. The two other selected dose-levels were 1000 and 500 mg/kg/day for males and 750 and 375 mg/kg/day for females. In either males or females, the mean values of MPE as well as the PE/NE ratio in the groups treated with BMTP, were equivalent to those of the vehicle control group. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data. Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

2,2-bis(methylthio) propane did not induce any damage to chromosomes or mitotic apparatus of rat bone marrow cells after two oral administrations separated by a 24-hour interval at dose-levels of 500, 1000 and 2000 mg/kg/day for males and 375, 750 and 1500 mg/kg/day for females.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: 218 g for males (ranging from 174 to 261 g) and 184 g for females (ranging from 147 to 221 g)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: by groups in Individually Ventilated Cages (IVC) (polycarbonate 900 cm2, Tecniplast) containing sawdust
- Diet : free access to SsniffR/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 22
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 28 January 2008 to 21 March 2008

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The Sponsor supplied the dosing solution ready to use. All the preparations were carried out under nitrogen atmosphere. The preparations were sent to the laboratory CIT for dosing of animals purposes. An aliquot from each preparation was kept by the sposonr to determine the concentration and stability.

Duration of treatment / exposure:

2 administrations

Frequency of treatment:

24-hour interval

Post exposure period:

24 h

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg/day (males)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
375, 750 and 1500 mg/kg/day (females)
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, once by oral route at the dose-level of 15 mg/kg

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose-level of 2000 mg/kg/day for males and 1500 mg/kg/day for females were considered to be the maximum tolerated dose and were retained as the highest dose-levels for the main test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.

DETAILS OF SLIDE PREPARATION:
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.

Statistics:

Normality and homogeneity of variances will be tested using a Kolmogorov Smirnov test and a Bartlett test.
If normality and homogeneity of variances were demonstrated, the statistical comparisons was performed using a Student t-test (two groups) or a one-way analysis of variance (¿ three groups) followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (¿ three groups) was performed followed by a Dunn test (if necessary).

All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05 for all tests.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
At 2000 mg/kg/day, no mortality was observed in the two treated males. Sedation, hypoactivivty, half-closed eyes, unsteady gait and rhinorrea were noted.
One of the two females treated at 2000 mg/kg/day was found dead 24 hours following the first treatment. The other female showed clinical signs following the first treatment and was sacrificed without any second administration of the test item.

At 1500 or 1000 mg/kg/day, no mortality was induced in the treated females. Hypoactivity, unsteady gait, half closed eyes, and in addition ocular secretion at 1500 mg/kg/day, were observed in treated animals.

The dose-level of 2000 mg/kg/day for males and 1500 mg/kg/day for females were considered to be the maximum tolerated dose and were retained as the highest dose-levels for the main test.
The two other selected dose-levels were 1000 and 500 mg/kg/day for males and 750 and 375 mg/kg/day for females.


RESULTS OF DEFINITIVE STUDY
In males, no clinical signs were noted at 500 mg/kg/day, hypoactivity and unsteady gait were noted following the first treatment at 1000 mg/kg/day. At 2000 mg/kg/day, one male out of 8 was found dead 24 hours following the second treatment. Clinical signs were noted in all treated animals (hypoactivity, unsteady gait, sedation, lateral recumbency and sometimes half-closed eyes).

In females, no clinical signs were noted at 375 mg/kg/day, unsteady gait was observed following the first treatment at 750 mg/kg/day. At 1500 mg/kg/day, one female out of 8 was found dead 24 hours following the first treatment. Clinical signs were noted in all treated animals (hypoactivity, unsteady gait, sedation, lateral recumbency, half-closed eyes and ocular secretions).

In either males or females, the mean values of MPE as well as the PE/NE ratio in the groups treated with the test item BMTP, were equivalent to those of the vehicle control group.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data.
Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Conclusions:

BMTP did not induce any damage to chromosomes or mitotic apparatus of rat bone marrow cells after two oral administrations separated by a 24-hour interval at dose-levels of 500, 1000 and 2000 mg/kg/day for males and 375, 750 and 1500 mg/kg/day for females.

Executive summary:

The potential of BISMETHYLTHIOPROPANE (BMTP) to induce structural or numerical damage was evaluated in bone marrow cells of rat. The study was performed according to the OECD guideline 474 and in compliance with the Principles of Good Laboratory Practice Regulations. Several preliminary toxicity tests were performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female Sprague-Dawley rats received two oral treatments of BMTP at dose-levels of 500, 1000 and 2000 mg/kg/day for males and 375, 750 and 1500 mg/kg/day for females, at a 24-hour interval. One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test item (Cyclophosphamide) once by oral route at the dose-level of 15 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

The dose-level of 2000 mg/kg/day for males and 1500 mg/kg/day for females were considered to be the maximum tolerated dose and were retained as the highest dose-levels for the main test. The two other selected dose-levels were 1000 and 500 mg/kg/day for males and 750 and 375 mg/kg/day for females. In either males or females, the mean values of MPE as well as the PE/NE ratio in the groups treated with BMTP, were equivalent to those of the vehicle control group. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data. Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

BISMETHYLTHIOPROPANE did not induce any damage to chromosomes or mitotic apparatus of rat bone marrow cells after two oral administrations separated by a 24-hour interval at dose-levels of 500, 1000 and 2000 mg/kg/day for males and 375, 750 and 1500 mg/kg/day for females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to the available genotoxicity data, no classication for germ cell mutations is warranted for 2,2-bis(methylthio)propane following Regulation (EC) No 1272/2008.