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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation test: 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is non-mutagenic to bacterial cells in the Ames test (OECD 471) and non mutagenic in the in vitro micronucleus test on human lymphoma cells (OECD 487). However, the substance is weakly mutagenic to mouse lymphoma cells in the in vitro gene mutation in mammalian cells test (MLA/TK, OECD 476).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2014 - March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2014
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes: primary cells from peripheral human blood
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I (-S9): 13.0, 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000 µg/mL;
(+S9): 13, 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000 µg/mL

Experiment IIa (-S9): 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL

Experiment IIb (-S9): 50.0, 100.0, 200.0, 250.0, 300.0, 350.0, 400.0, 450.0, 500.0, 550.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Stock formulations of the test item and serial dilutions were made in DMSO. The final concentration of DMSO in the culture medium was 0.5 %. The osmolarity and pH were determined in the solvent control and the maximum concentration without metabolic activation.
- Justification for choice of solvent/vehicle: The solvent was chosen based upon the test item solubility properties and the relative non-toxicity of DMSO to the cell cultures.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S-9; 17.5 µg/mL prepared in saline (0.9 % NaCl [w/v])
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S-9; pulse treatment; 2.0 µg/mL
Positive controls:
yes
Positive control substance:
other: Demecolcin
Remarks:
without S-9; continuous treatment; 50.0 ng/mL (Exp. IIA); 125.0 ng/mL (Exp. IIB)
Details on test system and experimental conditions:
TEST SYSTEM: Human Lymphocytes
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (24 years old) for Experiment I, from a female donor (34 years old) for Experiment IIA and from a female donor (35 years old) for Experiment IIB. The lymphocytes of these donors 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.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment 1: 4 hours with S9, 4 hours without S9; Experiment 2a: 20 hours without S9; Experiment 2b: 20 hours without S9.
- Expression time (cells in growth medium): Experiment 1: 40 hours with S9, 40 hours without S9; Experiment 2a: 40 hours without S9; Experiment 2b: 40 hours without S9.
- Fixation time (start of exposure up to fixation or harvest of cells): After centrifugation and removal of the supernate the cells were resuspended in fixative and kept cold for 20 minutes. The fixation procedure was repeated one or more times prior to dropping the cell suspension onto a clean microscope slide.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: at least 1000 binucelate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. To describe a cytotoxic effect, the CBPI was determined in 500 cells per culture and cytotoxicity was expressed as % cytostasis. A CBPI of 1 (all cells are mononucleated) is equivalent to 100 % cytostasis

OTHER EXAMINATIONS:
Micronuclei in mononucleated cells were recorded when observed, since aneugenic substances are known to increase the number of micronucleated and mononucleated cells.

OTHER: Only cells containing clearly visible cytoplasm were included in the analysis.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

There is no requirement for verification of a clear positive or negative response.
Statistics:
Statistical significance was confirmed by using the Chi-squared test (a < 0.05) with the validated R Script CHI2. Rnw script for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Species / strain:
lymphocytes: primary cells from peripheral human blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects on pH were observed.
- Effects of osmolality: no effects on osmolality were observed.
- Water solubility: In Experiment I, precipitation of the test item in the culture medium was observed at 373.2 µg/mL and above in the absence of S9 mix and at 213.2 µg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment IIA and IIB in the absence of S9 mix at 500.0 µg/mL and above at the end of treatment.

RANGE-FINDING/SCREENING STUDIES:
The preliminary cytotoxicity test (Experiment I) was performed to determine the concentrations to be used in the main experiments. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described for the mutagenicity assay.

The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of v10 and a solvent and positive control. All cell cultures were set up in duplicate.
In the preliminary test, precipitation of the test item was observed at the end of treatment at 373.2 µg/mL and above in the absence of S9 mix, and at 213.2 µg/mL and above in the presence of S9 mix. Since at least three concentrations (with and without S9 mix) fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

In Experiment IIA, 1000.0 µg/mL was chosen as top treatment concentration to further clarify the effects of cytotoxicity and test item precipitation on the test system. This experimental part was repeated with a top dose of 550.0 µg/mL and narrow concentration spacing to obtain evaluable, non-precipitating concentrations in a cytotoxic range (Experiment IIB).

In Experiment I in the absence and presence of S9 mix, cytotoxicity in the targeted toxicity range was observed up to the highest evaluated concentrations. In Experiment IIA in the absence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment IIB, in the absence of S9 mix, cytotoxicity in the targeted toxicity range was observed at the highest evaluated concentrations.

In the absence and presence of S9 mix in Experiment I, no biologically relevant increases in the number of micronucleated cells were observed after treatment with the test item. The micronucleus frequencies of the cells after treatment with the test item (0.20 – 1.00 % micronucleated cells) exceeded the range of the concurrent solvent control values (0.20 – 0.65 % micronucleated cells), but were clearly within the range of the laboratory historical control data. In Experiment I in the presence of S9 mix two statistically significant increases were observed after treatment with 121.9 and 213.2 µg/mL (1.00 and 0.95 % micronucleated cells). In Experiment IIA, in the absence of S9 mix, one single statistically significant increase was observed after treatment with 125.0 µg/mL (0.70 % micronucleated cells). No statistically significant increases were observed at concentrations of 100.0, 200.0, 400.0, or 450.0 µg/mL in Experiment IIB after treatment in the absence of S9 mix. Since these statistically significant values are clearly within the range of the laboratory historical solvent control data (without S9 mix: 0.05 – 1.45 % micronucleated cells; with S9 mix: 0.15 – 1.70 % micronucleated cells) the findings were regarded as not biologically relevant.

Conclusions:
The test item, 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione (CAS RN 72676-55-2), did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and therefore is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating or the highest evaluable concentrations.
Executive summary:

The test item 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione (CAS RN 72676-55-2), dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments.

In each experimental group two parallel cultures were analysed and 1000 binucleated cells per culture were evaluated for cytogenetic damage.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487. The highest applied concentration in Experiment I of this study, 2000.0 µg/mL of the test item, was chosen based upon the recommendation of the current OECD Guideline 487.

In Experiment I in the absence and presence of S9 mix, cytotoxicity in the targeted toxicity range was observed up to the highest evaluated concentrations. In the experiments IIA and IIB in the absence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment IIB in the absence of S9 mix moderate cytotoxicity of 46.9 % cytostasis was observed at the highest evaluated concentration.

In this study in the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item at the evaluated concentrations.

However, in Experiment I in the presence of S9 mix two statistically significant increases were observed after treatment with 121.9 and 213.2 µg/mL (1.00 and 0.95 % micronucleated cells). In Experiment IIA in the absence of S9 mix one single statistically significant increase was observed after treatment with 125.0 µg/mL (0.70 % micronucleated cells).

Since these values are clearly within the range of the laboratory historical solvent control data (without S9 mix: 0.05 – 1.45 % micronucleated cells; with S9 mix: 0.15 – 1.70% micronucleated cells) the findings were regarded as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione (CAS RN 72676-55-2) is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating or the highest evaluable concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February - April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK +/-, locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- 3.7.2c mouse lymphoma cell line
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/ß-naphthoflavone (PB/ßNF) S9 from the livers of male Sprague-Dawley rats
Test concentrations with justification for top dose:
Experiment 1: Group Concentration of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione (CAS RN 72676-55-2) (µg/mL)
4-hour without S9 5, 10, 20, 30, 40, 80, 160, 320
4-hour with S9 (2%) 5, 10, 20, 40, 80, 160, 240, 320

Experiment 2. Group Concentration of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione (CAS RN 72676-55-2) (µg/mL)
4-hour without S9 5, 10, 15, 20, 30, 40, 50, 100
4-hour with S9 (1%) 10, 20, 40, 60, 80, 120, 160, 180
Vehicle / solvent:
Vehicle and positive controls were used in parallel with the test item. Solvent (dimethyl sulfoxide, DMSO) Ethylmethanesulphonate (EMS; Sigma batch BCBK5968V) at 400 µg/mL was used for Experiments 1 and 2 as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0302605 at 2 µg/mL was used as the positive control in the presence of metabolic activation in both experiments.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, treatment conditions were modified; the cells were treated with the test item at eight dose levels using 4-hour exposure groups both in the absence and presence of metabolic activation (1% S9).

The dose range of test item used in the Experiment 1 was selected following the results of a preliminary toxicity test. The preliminary toxicity test was performed using a 4-hour exposure period in the absence and presence of S9 and a 24-hour exposure period in the absence of S9 using a dose range of 19.53 to 5000 µg/ml. Additional preliminary toxicity was performed using existing toxicity data in order to achieve optimum toxicity (20-240 µg/mL) in the presence of metabolic activation (1% S9). Test item dose levels for the mutagenicity experiments were selected based upon test item-induced toxicity. The dose range was narrowed and the S9 conditions were modified for Experiment 2 based upon the results of Experiment 1.

There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.
Evaluation criteria:
For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126E-6 and demonstrates a positive linear trend should be considered positive.

However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgment should be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
yes
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The maximum dose level used in the main test was limited by test item induced toxicity. No precipitate of test item was observed. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

In Experiment 1, a modest dose-related positive result exceeding the GEF value was obtained in the absence of metabolic activation only. In addition, statistically significant, linear increases in the mutant frequency were observed at less than 25% relative survival. The response observed was considered to be a relatively weak mutagenic response.

In Experiment 2, the results confirmed the reproducible mutagenic effects from Experiment 1. A modest dose-related positive result exceeding the GEF value was obtained in the absence of metabolic activation only. In addition, statistically significant, linear increases in the mutant frequency were observed at cytotoxic dose levels. The response observed was considered to be a relatively weak reproducible mutagenic response.
Conclusions:
In an OECD 476 study, conducted according to GLP conditions, 5,5'-Dithiodi-1,3,4-thia diazole-2(3H)-thione induced reproducible, toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells in the absence of metabolic activation (4-hour exposure) and is therefore considered to be weakly mutagenic.
Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (OECD 476).

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, treatment conditions were modified; the cells were treated with the test item at eight dose levels using 4-hour exposure groups both in the absence and presence of metabolic activation (1% S9).

The dose range of test item used in the Experiment 1 was selected following the results of a preliminary toxicity test. The preliminary toxicity test was performed using a 4-hour exposure period in the absence and presence of S9 and a 24-hour exposure period in the absence of S9 using a dose range of 19.53 to 5000 µg/ml. Additional preliminary toxicity was performed using existing toxicity data in order to achieve optimum toxicity (20-240 µg/mL) in the presence of metabolic activation (1% S9). Test item dose levels for the mutagenicity experiments were selected based upon test item-induced toxicity. The dose range was narrowed and the S9 conditions were modified for Experiment 2 based upon the results of Experiment 1.

The maximum dose level used in the main test was limited by test item induced toxicity. No precipitate of test item was observed. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

In Experiment 1, a modest dose-related positive result exceeding the GEF value was obtained in the absence of metabolic activation only. In addition, statistically significant, linear increases in the mutant frequency were observed at less than 25% relative survival. The response observed was considered to be a relatively weak mutagenic response. In Experiment 2, the results confirmed the reproducible mutagenic effects from Experiment 1. A modest dose-related positive result exceeding the GEF value was obtained in the absence of metabolic activation only. In addition, statistically significant, linear increases in the mutant frequency were observed at cytotoxic dose levels. The response observed was considered to be a relatively weak reproducible mutagenic response.

The test item induced reproducible, toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells in the absence of metabolic activation (4-hour exposure) at dose levels close to optimum toxicity, and is therefore considered to be weakly mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 February 2014 to 20 March 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 Rat Liver, induced with phenobarbitone/ß-naphthoflavone
Test concentrations with justification for top dose:
Plate Incorporation method (Experiment 1): -/+S9: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate
Pre-incubation Method (Experiment 2): -/+S9: 5, 15, 50, 150, 500, 1500 and 5000 ug/plate (amended based on the results of Experiment 1 (Plate Incorporation Method))
Vehicle / solvent:
dimethyl sulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
without S9
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
with S9
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1369241 07/18) and incubated at 37 ¿C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
Evaluation criteria:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS
5. Greater than two fold increase above the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response.

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The vehicule (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mic were validated.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 1500 µg/plate in both the presence and the absence of metabolic activation (S9 -mix) in both experiments. These results were not indicative of toxicity sufficiently severe to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic acitivation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).
Conclusions:
In an OECD 471 study, conducted according to GLP, 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is negative for bacterial reverse mutation in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli strain WP2uvrA, using both the Ames plate incorporation method and pre-incubation method, with and without metabolic activation (S-9 mix). 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione, therefore, is considered non-mutagenic in bacterial cells.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli strain WP2uvrA were treated with the test tiem using both the Ames plate incorporation method (Experiment 1) and pre-incubation method (Experiment 2) at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The eight doses for Experiment 1 ranged from 1.5 to 5000 µg/plate since the test item was not deemed particularly toxic, and were spaced at half-log10 intervals. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range of the Experiment 2 was amended based on the results of Experiment 1 and was 5 to 5000 µg/plate. Seven test item dose levels were selected in Experiment 2 in order to achieve four non-toxic dose levels and the potential toxic limit of the test item following the change in the test methodology.

The vehicule (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mic were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 1500 µg/plate in both the presence and the absence of metabolic activation (S9 -mix) in both experiments. These results were not indicative of toxicity sufficiently severe to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic acitivation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test substance was evaluated for its genotoxic potential using the Comet assay to assess induction of DNA damage in liver, stomach and duodenum cells of male rats. The study was performed to the standardized guideline OECD 489 under GLP conditions.  Under the conditions of this study, the administration of the test material at doses up to and including a dose of 2000 mg/kg bw did not cause a significant increase in DNA damage in the liver, duodenum or stomach of male rats relative to the concurrent vehicle control. Therefore, the test material was concluded to be negative in the in vivo Comet Assay (2019).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 March 2018 to 13 September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Strain:
other: Sprague Dawley (Hsd:SD)
Details on species / strain selection:
Rats are used routinely in this test. This outbred strain maximises genetic heterogeneity, which tends to eliminate strain-specific responses.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Virus antibody-free (VAF) animals were acclimated for five days and were judged to be healthy prior to utilization in the study. The animals were 6 weeks of age at the start of the treatment.

Test animals and environmental conditions were as described in the OECD 489 guidelines.
Route of administration:
oral: gavage
Vehicle:
Vehicle(s)/solvent(s) used: Arachis oil (peanut oil, USP)
- Justification for choice of solvent/vehicle: Arachis oil was chosen based upon existing information regarding the limited solubility of the test material in water at high concentrations, a 1983 acute oral toxicity study using corn oil as the vehicle, and the preference of the laboratory for arachis oil over corn oil for the 14-day range finding study and OECD 422 studies using this test material.
- Concentration of test material in vehicle: The test material was prepared in arachis oil/peanut oil USP at 125 mg/mL and 250 mg/L.
- Amount of vehicle (if gavage or dermal): Low and mid dose test substance formulations and the vehicle control substance were administered at a dose volume of 4 mL/kg/dose. The high dose test substance formulation was administered at dose volumes of 8 mL/kg/dose.
- Type and concentration of dispersant aid (if powder): Not applicable
- Lot/batch no. (if required): MKBS3571V
- Purity: USP grade
Details on exposure:
Preparation of Control Substances
Neat EMS was prepared in 0.9% sodium chloride for injection (saline). The dosing formulation was prepared at a concentration of 20 mg/mL just prior to use.

Preparation of Test Substance Dose Formulations
The test substance dose formulations were prepared fresh on each day of use.

Each concentration was prepared by calibrating a suitable size amber glass vial, containing an amount of test substance appropriate to the target batch size. Approximately 70% of the total volume of vehicle was added to the vial, and the mixture was stirred magnetically. Additional vehicle was added to achieve the final target volume, and the formulation was again stirred magnetically and sonicated until uniform.
Duration of treatment / exposure:
Dose Administration
Low and mid dose test substance formulations and the vehicle control substance were administered at a dose volume of 4 mL/kg/dose. The high dose test substance formulation and positive control substance formulation were administered at dose volumes of 8 and 10 mL/kg/dose, respectively.
Frequency of treatment:
All animals in Group 1 (dosed with the vehicle), and Groups 2 through 4 (dosed with the test substance) were dosed once per day on two consecutive days (Study Days 1 and 2). The second dose occurred approximately 21 hours after the first dose.

Animals in Group 5 (dosed with the positive control) were dosed once approximately 3 to 4 hours prior to organ collection on Study Day 2.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Dose range-finding study: 3 per sex per dose at 500, 1000 and 2000 mg/kg bodyweight.

Definitive study: 6 males per dose at 0, 500, 1000 and 2000 mg/kg bodyweight; 3 males for positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate (EMS)
Tissues and cell types examined:
Liver, stomach and duodenum were evaluated for DNA damage; a section of each tissue was preserved for possble histopathology evaluation.
Details of tissue and slide preparation:
Sections of the liver, glandular stomach and duodenum were placed in chilled mincing solution and minced or scraped to release the cells. Cell suspensions were strained into pre-labeled tubes and kept on wet ice during preparation of the slides. An aliquot of the suspension was used to prepare the Comet slides.

From each suspension, an aliquot of cells was mixed with low melting agarose (0.5%) and the cell/agarose suspension was applied to commercially available pre-treated multi-well microscope slides. The slides were kept at 2 to 8°C for at least 15 minutes to allow the gel to solidify. At least two 20-well slides were prepared per animal per tissue and identified with a random code. Following solidification of the agarose, the slides were placed in jars containing lysis solution and were stored overnight at 2 to 8°C.

After lysis, slides/wells were washed with neutralization buffer and placed in the electrophoresis chamber. The chamber reservoirs were filled with alkaline buffer (300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water (pH > 13)), and remained in the buffer for 20 minutes at 2-10°C, protected from light. Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10°C and protected from light.

The slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 4 hours and stored at room temperature with desiccant. Slides were stained with a DNA stain prior to scoring.
Evaluation criteria:
Fifty randomly selected, non-overlapping cells per slide/well were assessed and measured for Comet Tail Migration, % Tail DNA, and Tail Moment in a total of 150 cells per animal; if 150 cells were not available, then the calculations were performed using the number of scorable cells.
Each slide was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was also determined when possible. “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.
Statistics:
The mean values of 150 counts of % Tail DNA, Tail moment and Tail migration were determined and presented for each animal in each treatment group for each organ. The mean and standard deviation of the median values only for % Tail DNA were presented for each treatment group. Statistical analysis was performed only for % Tail DNA.

Since the differences and variations between groups were found not to be significant for liver and stomach, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05). Levene’s test indicated heterogeneous group variances for duodenum (p = 0.05); therefore, the suitability of a transformation of the original data was evaluated (e.g. using logarithm transformed values of the original data) in an attempt to meet the normality criteria. Afterwards, statistical analysis was performed using the parametric tests described above. A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p = 0.01). A pair-wise comparison (Student’s T-test, p = 0.05) was used to compare the positive control group to the concurrent vehicle control group.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test substance gave a negative (non-DNA damaging) response in % Tail DNA for liver, duodenum and stomach of male rats. None of the test substance-treated animal slides had significant increases in % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, duodenum and stomach.
Conclusions:
Administration of the test material at doses up to and including a dose of 2000 mg/kg/dose did not cause a significant increase in DNA damage in the liver, duodenum or stomach of male rats relative to the concurrent vehicle control. Therefore, the test material was concluded to be negative in the in vivo Comet Assay.
Executive summary:

The test substance was evaluated for its genotoxic potential using the Comet assay to assess induction of DNA damage in liver, stomach and duodenum cells of male rats. The study was performed to the standardized guideline OECD 489 under GLP conditions.

 

Arachis oil (peanut oil, USP) was selected as the vehicle. Low and mid test substance formulations and vehicle control substance formulation were administered at a dose volume of 4 mL/kg/dose. The high test substance formulation and positive control substance formulation were administered at dose volumes of 8 and 10 mL/kg/dose, respectively. All formulations were administered by oral gavage.

 

In the dose range-finding assay (DRF), the dose levels tested were 500, 1000 and 2000 mg/kg/dose in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/dose, which is the highest guideline recommended dose for this assay. The definitive assay dose levels tested were 500, 1000 and 2000 mg/kg/dose, administered by oral gavage once daily for two consecutive days approximately 21 hours apart.

 

The test substance gave a negative (non-DNA damaging) response in % Tail DNA for liver, duodenum and stomach of male rats. None of the test substance-treated animal slides had significant increases in % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, duodenum and stomach.

 

Under the conditions of this study, the administration of the test material at doses up to and including a dose of 2000 mg/kg/dose did not cause a significant increase in DNA damage in the liver, duodenum or stomach of male rats relative to the concurrent vehicle control. Therefore, the test material was concluded to be negative in the in vivo Comet Assay.

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

Additional information

In vitro bacterial gene mutation test (OECD 471, 2014):

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation method (Experiment 1) and pre-incubation method (Experiment 2) at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The eight doses for Experiment 1 ranged from 1.5 to 5000 µg/plate since the test item was not deemed particularly toxic, and were spaced at half-log10 intervals. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range of the Experiment 2 was amended based on the results of Experiment 1 and was 5 to 5000 µg/plate. Seven test item dose levels were selected in Experiment 2 in order to achieve four non-toxic dose levels and the potential toxic limit of the test item following the change in the test methodology.

The vehicule (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mic were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 1500 µg/plate in both the presence and the absence of metabolic activation (S9 -mix) in both experiments. These results were not indicative of toxicity sufficiently severe to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione was considered to be non-mutagenic under the conditions of this test.

In vitro gene mutation in mammalian cells test (OECD 476, MLA/TK, 2015):

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (OECD 476).

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, treatment conditions were modified; the cells were treated with the test item at eight dose levels using 4-hour exposure groups both in the absence and presence of metabolic activation (1% S9).

The dose range of test item used in the Experiment 1 was selected following the results of a preliminary toxicity test. The preliminary toxicity test was performed using a 4-hour exposure period in the absence and presence of S9 and a 24-hour exposure period in the absence of S9 using a dose range of 19.53 to 5000 µg/ml. Additional preliminary toxicity was performed using existing toxicity data in order to achieve optimum toxicity (20-240 µg/mL) in the presence of metabolic activation (1% S9). Test item dose levels for the mutagenicity experiments were selected based upon test item-induced toxicity. The dose range was narrowed and the S9 conditions were modified for Experiment 2 based upon the results of Experiment 1.

The maximum dose level used in the main test was limited by test item induced toxicity. No precipitate of test item was observed. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

In Experiment 1, a modest dose-related positive result exceeding the GEF value was obtained in the absence of metabolic activation only. In addition, statistically significant, linear increases in the mutant frequency were observed at less than 25% relative survival. The response observed was considered to be a relatively weak mutagenic response. In Experiment 2, the results confirmed the reproducible mutagenic effects from Experiment 1. A modest dose-related positive result exceeding the GEF value was obtained in the absence of metabolic activation only. In addition, statistically significant, linear increases in the mutant frequency were observed at cytotoxic dose levels. The response observed was considered to be a relatively weak reproducible mutagenic response.

The test item induced reproducible, toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells in the absence of metabolic activation (4-hour exposure) at dose levels close to optimum toxicity, and is therefore considered to be weakly mutagenic under the conditions of the test.

In vitro micronucleus test (OECD 487, 2016):

The test item 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. In each experimental group, two parallel cultures were analysed and 1000 binucleated cells per culture were evaluated for cytogenetic damage.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487. The highest applied concentration in Experiment I of this study, 2000.0 µg/mL of the test item, was chosen based upon the recommendation of the current OECD Guideline 487.

In Experiment I in the absence and presence of S9 mix, cytotoxicity in the targeted toxicity range was observed up to the highest evaluated concentrations. In the experiments IIA and IIB in the absence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment IIB in the absence of S9 mix moderate cytotoxicity of 46.9 % cytostasis was observed at the highest evaluated concentration.

In this study in the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item at the evaluated concentrations.

However, in Experiment I in the presence of S9 mix two statistically significant increases were observed after treatment with 121.9 and 213.2 µg/mL (1.00 and 0.95 % micronucleated cells). In Experiment IIA in the absence of S9 mix one single statistically significant increase was observed after treatment with 125.0 µg/mL (0.70 % micronucleated cells).

Since these values are clearly within the range of the laboratory historical solvent control data (without S9 mix: 0.05 – 1.45 % micronucleated cells; with S9 mix: 0.15 – 1.70% micronucleated cells) the findings were regarded as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating or the highest evaluable concentrations.

In vivo Comet assay (OECD 489, 2019):

The test substance was evaluated for its genotoxic potential using the Comet assay to assess induction of DNA damage in liver, stomach and duodenum cells of male rats. The study was performed to the standardized guideline OECD 489 under GLP conditions.

Arachis oil (peanut oil, USP) was selected as the vehicle. Low and mid test substance formulations and vehicle control substance formulation were administered at a dose volume of 4 mL/kg/dose. The high test substance formulation and positive control substance formulation were administered at dose volumes of 8 and 10 mL/kg/dose, respectively. All formulations were administered by oral gavage.

In the dose range-finding assay (DRF), the dose levels tested were 500, 1000 and 2000 mg/kg/dose in 3 animals/sex. Based upon the results, the high dose for the definitive assay was 2000 mg/kg/dose, which is the highest guideline recommended dose for this assay. The definitive assay dose levels tested were 500, 1000 and 2000 mg/kg/dose, administered by oral gavage once daily for two consecutive days approximately 21 hours apart.

The test substance gave a negative (non-DNA damaging) response in % Tail DNA for liver, duodenum and stomach of male rats. None of the test substance-treated animal slides had significant increases in % Tail DNA compared to the respective vehicle controls. The vehicle control % Tail DNA was within the Testing Facility’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the vehicle control. Thus, all criteria for a valid assay were met for liver, duodenum and stomach.

Under the conditions of this study, the administration of the test material at doses up to and including a dose of 2000 mg/kg/dose did not cause a significant increase in DNA damage in the liver, duodenum or stomach of male rats relative to the concurrent vehicle control. Therefore, the test material was concluded to be negative in the in vivo Comet Assay.

Justification for classification or non-classification

Based on the available data, no classification for mutagenicity is required for 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione according to the Regulation EC N°1272/2008.

Justification: The mutagenic alerts observed in the in vitro gene mutation assay were considered to be not relevant based on the negative results obtained in the in vivo Comet assay. Based on the weight of evidence, the substance is not consider to be mutagenic.