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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Protocol of the National Toxicology Program (NTP), performed on strains TA100, TA98, eColi pKM101

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2006

Reference 2

Reference Type:

publication

Title:

The Ames Salmonella/microsome mutagenicity assay.

Author:

Mortelmans K, Zeiger E

Year:

2000

Bibliographic source:

Mutation Research 455 (2000) 29–60

Report date:

2000

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

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

Method

Target gene:

his-

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Remarks:

cited as eColi pKM101

Metabolic activation:

with and without

Metabolic activation system:

induced male Sprague Dawley rat liver S9 (RLI)

Test concentrations with justification for top dose:

0, 25, 50, 100, 200, 250, 400, 500, 750, 1000, 2000, 3500, 5000, or 7500 µg/plate (TA100, TA98)
0, 25, 50, 100, 150, 200, 250, 400, 500, 750, or 1000 µg/plate (eColi pKM101)
Concentrations chosen based on cytotoxicity.
A minimum of five dose levels covering a range of at least three logs should be selected for the definitive test. Two or three plates should be used for each dose level and for the controls. For toxic chemicals, only the highest dose used should exhibit toxicity. For non-toxic chemicals, a high dose of 5000 or 10,000 mg/plate is acceptable.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-Nitro-o-phenylenediamine (2.5 µg/plate) -S9 / 2-Aminoanthracene (1-5 µg/plate) +S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days

SELECTION AGENT (mutation assays): his minimal agar

- OTHER: Method as cited in
K. Mortelmans, E. Zeiger / Mutation Research 455 (2000) 29–60
https://ntp.niehs.nih.gov/testing/types/genetic/index.html

Multiple sets of cultures are prepared using a range of doses and different amounts of liver enzymes and bacteria strains. Each culture is prepared in a test tube containing a suspension of one bacterial strain and either an S9 mix or a plain buffer. Then the test substance is added. Control cultures are created with the same ingredients, but without the test substance. A positive control group is created using a known mutagen. Once prepared, each culture is incubated for 20 minutes at 37º C.
After the first incubation period, agar is mixed into the cultures, and each tube is poured onto the surface of a Petri dish prepared with the standard medium. The plates are then incubated, usually for two days.

Rationale for test conditions:

as cited in K. Mortelmans, E. Zeiger / Mutation Research 455 (2000) 29–60

Evaluation criteria:

The test is only valid if the number of positive control colonies is greater than for the control cultures. Once this has been confirmed, the test cultures are compared to the control. If the substance is mutagenic, it will have caused greater bacteria growth.
• A positive response is a reproducible, dose-related increase in any set of test cultures. There is no minimum percentage of increase to define a result as positive.
• An equivocal response is any increase that is not reproducible, not dose-related, or not statistically significant.
• A negative response occurs when none of the cultures tested shows more growth than the control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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

Applicant's summary and conclusion

Conclusions:

The study was performed equivalent to OECD TG 471 with minor deficiencies in documentation and performance.
Testing was performed on bacterial strains S. typhimurium TA100, TA98, and E. coli WP2 uvr A pKM 101. Those strains detect the following mutations:
TA100: transitions and transversions, some frameshifts (base substitution at G:C basepair)
TA98: Frameshifts at (or near) GCGCGCGC (DeltaGpC frameshift)
E. coli WP2 uvr A pKM: has an AT base pair at the primary reversion site and may detect certain oxidising mutagens, cross-linking agents and hydrazines, which may not be detected by the four S. typhimurium strain in OECD TG 471 of 1981.
According to the recent OECD TG 471, the following 5 strains should be used: S. typhimurium TA1535, and S. typhimurium TA1537 or TA97 or TA97a, and S. typhimurium TA98, and S. typhimurium TA100, and E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. The lacking two strains detect the following mutations:
TA1535: Transitions and transversions (base substitution at G:C basepair)
TA1537 or TA97: Frameshifts at (or near) GGGGG (TA1537) or GGGGGG (TA97)
As obvious, the mutations detected by the lacking TA1535 strain are similar to the ones detected by TA100, which in addition detects some frameshift, so TA100 may serve as a surrogate for TA1535. Further, due to its ability to detect at least some frameshifts at the G:C basepair, the obtained negative result may give some indication that testing on TA1537 or TA97 on G-rich sites would also reveal a negative result, too. Overall, the used three strains are well-selected to cover the broadest possible spectrum of mutations with the least efforts, so the deviations from the recent OECD 471 can be considered as minimal and give a first indication on the mutagenic potential of 2,5-Dimercapto-1,3,4-thiadiazole, which will be further evaluated via a higher tier mammalian cell mutation test (OECD 490).
Positive and negative controls gave the appropriate results. Hence, the results can be considered to be sufficiently reliable to assess the potential of 2,5-Dimercapto-1,3,4-thiadiazole to induce gene mutations in bacteria. In all tested strains, 2,5-Dimercapto-1,3,4-thiadiazole did consistently not induce gene mutations, the outcome of the test can be considered negative.
This outcome is further supported by the results of the available OECD 490 study: Therein, the test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, limited by both toxicity and precipitation, in any of the three exposure groups. So there is still consistently shown that the test item is incapable to induce gene mutation, both in bacteria and mammalian cells, so the lack of the bacterial strains TA1535 and TA1537 or TA97 does clearly not underestimate the potential hazard of 2,5-Dimercapto-1,3,4-thiadiazole, and the present Ames test is sufficient to cover the endpoint gene mutation in bacteria, no further tests need to be conducted.

Executive summary:

In a reverse gene mutation assay in bacteria (similar to OECD TG 471), strains S. typhimurium TA100, TA98, and E. coli WP2 uvr A pKM 101 were exposed to 2,5-Dimercapto-1,3,4-thiadiazole in DMSO at concentrations of max. 7500 µg/plate in the presence and absence of mammalian metabolic activation (induced rat liver S9) via the pre-incubation method.

2,5-Dimercapto-1,3,4-thiadiazole was tested up to cytotoxic concentrations. The positive and negative controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable.