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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Genetic toxicity in vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98 and TA 100: negative with and without metabolic activation (similar to OECD 471) Genetic toxicity in vitro: Chromosome aberration (in vitro mammalian cell micronucleus test): human lymphcytes: negative with and without metabolic activation (OECD 487, GLP) Genetic toxicity in vitro: Gene mutation (mammalian cell gene mutation assay / HPRT assay): V79 cells: negative with and without metabolic activation (OECD 476, GLP)
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-11 - 2015-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented GLP OECD Guideline study without deviations on the registered substance itself
Qualifier:
according to
Guideline:
other: OECD 487 “In vitro Mammalian Cell Micronucleus Test”, adopted 26 September 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Str. 80, D65189 Wiesbaden, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a female donor (32 years old) for Experiment I and for Experiment II. The lymphocytes of the respective donor have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
8.65, 15.14, 26.50, 46.37, 81.15, 142.02, 248.54, 434.94, 761.14, 1332.00 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (test item), deionised water (MMC, Demecolcin), saline (CPA)
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcin
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 or 20 hours
- Expression time (cells in growth medium): 16h or none plus 20 hours with Cytochalasin B
- Fixation time (start of exposure up to fixation or harvest of cells): 40 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: other: % cytostasis
Evaluation criteria:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976) (COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.
Statistics:
Chi square test (α < 0.05), using the validated R Script CHI2.Rnw
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
10 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence on pH value was observed
- Effects of osmolality: No relevant influence on osmolarity value was observed
- Precipitation: none noted

COMPARISON WITH HISTORICAL CONTROL DATA:
The micronucleus rates of the cells after treatment with the test item (0.45 – 0.90 % micronucleated cells) did not exceed the range of the solvent control values (0.60 – 1.05 % micronucleated cells) and were within the range of the laboratory historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis could be observed up to the highest applied concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test item 3-Methyl-thiazolidin-2-thion, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.

The highest treatment concentration in this study, 1332.00 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

No relevant influence on osmolarity or pH value was observed.

No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis could be observed up to the highest applied concentration.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.45 – 0.90 % micronucleated cells) did not exceed the range of the solvent control values (0.60 – 1.05 % micronucleated cells) and were within the range of the laboratory historical control data.

In both experiments, either Demecolcin (75.0 µg/mL), MMC (2.0 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

See attachment for tables

Conclusions:
The study was conducted under GLP according to OECD guideline 487 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 3-Methyl-thiazolidin-2-thion to induce micronuclei in mammalian cells. Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, 3-Methyl-thiazolidin-2-thion is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.
Executive summary:

In a mammalian cell cytogenetics assay, an in vitro micronucleus test (OECD 487), human primary lymphocyte cultures were exposed to 3-Methyl-thiazolidin-2-thion in DMSO at concentrations of 0, 8.65, 15.14, 26.50, 46.37, 81.15, 142.02, 248.54, 434.94, 761.14, and 1332.00 µg/ml with and without metabolic activation (induced rat liver S9). In two independent experiments, cells were either exposed to the test item over 4h (±S9) or 20h (-S9).

In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1332.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

In Experiment I and II in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. 3-Methyl-thiazolidin-2-thion is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Appropriate mutagens (Demecolcin (75.0 ng/mL), MMC (2.0 μg/mL) or CPA (17.5 μg/mL)) were used as positive controls. They induced statistically significant increases in cells with micronuclei.

This study is classified as acceptable. This study satisfies the requirement for an in vitro micronucleus test (OECD 487) for in vitro cytogenetic mutagenicity data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

There are all three stipulated (Annex VIII) genetic toxicity studies available, being of high quality and sufficiently reliable to assess the respective endpoints. All studies are on the registered substance itself and consistently reveal that 3-Methyl-thiazolidin-2-thion is negative for genetic toxicity, i.e. gene mutation in bacteria and mammalian cells and micronucleus induction in human lymphocytes. There is no indication given that these results are not relevant to assess the risk of genetic damage in humans, as both gene and chromosome mutation is covered and due to the addition of induced rat liver S9 mix in vivo metabolism is sufficiently mimicked. Hence, no data gaps were identified, no additional testing is required and the available data suffices to exclude the hazard of 3-Methyl-thiazolidin-2-thion towards genetic material.

Justification for classification or non-classification

All available in vitro studies on 3-Methyl-thiazolidin-2-thion covering the endpoints gene mutation in bacteria and mammalian cells and micronucleus induction in human lymphocytes gave negative results. These cover the main causes for genetic damage sufficiently and mimic due to the addition of S9 in vivo metabolism to a wide extent. Based on the available data, there is no indication given that 3-Methyl-thiazolidin-2-thion should be classified as mutagen.