Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione
EC Number:
276-763-0
EC Name:
5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione
Cas Number:
72676-55-2
Molecular formula:
C4-H2-N4-S6
IUPAC Name:
5-[(5-sulfanylidene-4,5-dihydro-1,3,4-thiadiazol-2-yl)disulfanyl]-2,3-dihydro-1,3,4-thiadiazole-2-thione
Test material form:
solid: particulate/powder

Method

Target gene:
Not applicable.
Species / strain
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.
Controlsopen allclose all
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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.