Disodium 1,3,4-thiadiazole-2,5-dithiolate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017-10-23 - 2017-11-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Actually Klimisch 1, but performed on read-across substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Deviations:

no

Test guideline 3

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:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

1,3,4-thiadiazole-2,5-dithiol

EC Number:

214-014-1

EC Name:

1,3,4-thiadiazole-2,5-dithiol

Cas Number:

1072-71-5

Molecular formula:

C2H2N2S3

IUPAC Name:

1,3,4-thiadiazole-2,5-dithiol

Test material form:

solid: particulate/powder

Remarks:

white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4°C, in the dark

Method

Target gene:

tk +/-

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

L5178Y TK+/- 3.7.2c

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Methods for maintenance in cell culture if applicable: RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study.
- Properly maintained: Yes. The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air.
- Periodically checked for Mycoplasma contamination: Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically 'cleansed' against high spontaneous background: Yes. The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Metabolic activation:

with and without

Metabolic activation system:

PB/βNF induced male Sprague-Dawley rat liver S9

Test concentrations with justification for top dose:

The molecular weight of the test item was 150.24568 therefore, the maximum proposed dose level was 1500 µg/mL, the 10mM limit dose level. The purity of the test item was accounted for in the test item formulations.
Top dose was limited by toxicity and/or precipitation.
4h, -S9: 0, 12.5, 25, 50, 100, 125, 150, 175, 200 µg/ml
4h, +S9: 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 µg/ml
24h, -S9: 0, 3.13, 6.25, 12.5, 25, 37.5, 50, 75, 100 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Following solubility checks performed in-house for the Human Lymphocyte in vitro Micronucleus Test performed on the same test item, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared.

Controls

Untreated negative controls:

yes

Remarks:

solvent controls

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:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E5 cells/mL using a 24-hour exposure period without S9.
The cells were counted and processed to give 1 x 10E6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10E6 cells/mL in 10 mL cultures were established in 25 cm² tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation

DURATION
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days.
- Selection time (if incubation with a selection agent): ten to twelve days

SELECTION AGENT (mutation assays): 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures (A and B), three independent conditions

DETERMINATION OF CYTOTOXICITY
- Method: Relative Suspension Growth (%RSG)

Rationale for test conditions:

as set out in the guideline

Evaluation criteria:

Due to limitations of this free-text field, see "Any other information on materials and methods"

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

top dose was also limited by precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant pH shift noted in the evaluated concentration range
- Effects of osmolality: no relevant osmolality shift noted in the evaluated concentration range
- Precipitation: yes, at top dose

RANGE-FINDING/SCREENING STUDIES:
There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all of the three exposure groups when compared to the concurrent vehicle control groups. Test item precipitate was observed at and above 200 µg/mL in the 4-hour exposure group in the absence of metabolic activation, at and above 25 µg/ml in the 4-hour exposure group in the presence of metabolic activation, and at 400 µg/ml in the 24-hour exposure group in the absence of metabolic activation, at the end of the exposure periods. Therefore, the maximum dose levels in the subsequent Mutagenicity Test were limited by a combination of the onset of test item precipitate and test item induced toxicity in the 4-hour exposure group in the absence of metabolic activation, the onset of test item precipitate in the 4-hour exposure group in the presence of metabolic activation, and test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
For details, see tables below.

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to OECD guideline 490 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 the test item to induce mutations in mammalian cells. The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

Introduction: The GLP 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. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

 

Methods: One main Mutagenicity Test was performed. In this main test, 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 (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

 

Mutagenicity Test

Group	Concentration of DMTD (CAS No. 1072-71-5) (µg/mL) plated for viability and mutant frequency
4-hour without S9	12.5, 25, 50, 100, 125
4-hour with S9 (2%)	1.56, 3.13, 6.25, 12.5, 25, 50
24-hour without S9	3.13, 6.25, 12.5, 25, 37.5, 50

 

Results:The maximum dose levels in the subsequent Mutagenicity Test were limited by a combination of the onset of test item precipitate and test item‑induced toxicity in the 4-hour exposure group in the absence of metabolic activation, the onset of test item precipitate in the 4-hour exposure group in the presence of metabolic activation, and test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

 

Conclusion: The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.