Reaction products of 2,5-dimercapto-1,3,4-thiadiazole, sodium salt, with 1-octanethiol and hydrogen peroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

The test material was negative for the induction of micronuclei in the presence of the exogenous metabolic activation system.

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 September 2017 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Specific details on test material used for the study:

-Purity: > 99% (UVCB)
-Description: Translucent blackish yellow liquid
-Storage conditions:Room temperature, protected from light

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the intial preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL. Visible precipitate was observed at a concentration of ≥1250 µg/mL by the end of treatment. Due to excessive toxicity in all treatment conditions, the preliminary toxicity assay was repeated with the following concentrations 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL.

In the preliminary toxicity retest, no visible precipitate was observed at the beginning or end of treatment. Relative suspension growth (RSG) was 36, 8 and 78% at concentrations of 5 µg/mL (4-hour treatment with S9), 2.5 µg/mL (4-hour treatment without S9) and 1.25 µg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 0.38, 0.75, 1.5, 2.5, 4.5, 6 and 8 µg/mL (4-hour treatment with S9), and 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 and 10.0 µg/mL (4 and 24-hour treatments without S9).

Vehicle / solvent:

Tetrahydrofuran

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

L5178Y/TK+/- cells were exposed to the vehicle alone in duplicate cultures and nine to ten concentrations of test substance using single cultures. The maximum concentration evaluated was based on toxicity of the test substance in the vehicle. The pH of the treatment medium was measured, and no pH adjustment was necessary to maintain neutral pH. Osmolality of the vehicle control, the highest concentration, the lowest precipitating concentration and the highest soluble concentration also was measured at the beginning of treatment for the A1. Precipitation was determined with the unaided eye at the beginning and end of treatment. Dose levels for the definitive assay were based upon post-treatment cytotoxicity (growth inhibition relative to the vehicle control).

Evaluation criteria:

The cytotoxic effects of each treatment condition were expressed relative to the vehicle-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency for each treatment condition was calculated by dividing the mean number of colonies on the TFT-plates by the mean number of colonies on the VC-plates and multiplying by the dilution factor (2 x 10 4), and was expressed as TFT-resistant mutants/106 surviving cells. The induced mutant frequency (IMF) was defined as the mutant frequency of the treated culture minus the mutant frequency of the vehicle control cultures. The International Workshop on Genotoxicity established a Global Evaluation Factor (GEF) for a positive response at an IMF of ≥90 mutants/106 clonable cells at the Aberdeen meeting in 2003 (Moore et al., 2006).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Definitive re-test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test material was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Executive summary:

The test material was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cellsin the presence and absence of an exogenous metabolic activation system. Tetrahydrofuran (THF) was used as the vehicle.

In the intial preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay.  Visible precipitate was observed at concentrations ≥313 µg/mL at the beginning of treatment and at concentrations ≥1250 µg/mL by the end of treatment. Due to excessive toxicity in all treatment conditions, the preliminary toxicity assay was repeated with the following concentrations 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL.

In the retest of preliminary toxicity assay, no visible precipitate was observed at the beginning or end of treatment. Relative suspension growth (RSG) was 36, 88 and 78% at concentrations of 5 µg/mL (4-hour treatment with S9), 1.25 µg/mL (4-hour treatment without S9) and 1.25 µg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at all higher concentrations using all treatment conditions.  Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 0.38, 0.75, 1.5, 2.5, 4.5, 6 and 8 µg/mL (4-hour treatment with S9), and 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 and 10.0 µg/mL (4 and 24-hour treatments without S9).

In the initial definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 0.38, 1.5, 2.5, 4.5 and 6 µg/mL (4-hour treatment with S9) exhibited 12 to102 % RSG, and were cloned. Relative total growth of the cloned cultures ranged from 10 to 94% (4‑hour treatment with S9). No increases in induced mutant frequency ≥90 mutants/10⁶clonable cells were observed under 4-hour treatment with S9.   

Cultures treated at 0.25, 0.5, 1, 2, 3, 4, 6, 8 and 10 µg/mL had no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 0.25, 0.5, 1 and 2 µg/mL (4-hour treatment without S9) and 0.25, 0.5, 1 and 2 µg/mL (24-hour treatment without S9) exhibited 30 to 91% and 38 to 85% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 33 to 102% (4-hour treatment without S9) and 41 to 81% (24‑hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/10⁶clonable cells were observed under any treatment condition. 

These results indicate Reaction products of 2,5-dimercapto-1,3,4-thiadiazole, sodium salt, with 1-octanethiol and hydrogen peroxide was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 August 2017 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

-Purity: > 99% (UVCB)
-Description: Translucent blackish yellow liquid
-Storage conditions:Room temperature, protected from light

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:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Based upon the results of the preliminary toxicity assay, the dose levels selected for the mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.

No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate with all conditions.

The maximum dose of 5000 µg per plate was achieved using a concentration of 100 mg/mL and a 50.0 µL plating aliquot.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

The preliminary toxicity assay was used to establish the dose range over which the test substance would be assayed. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone and ten dose levels of the test substance, with a single plate/condition, on selective minimal agar in the presence and absence of Aroclor induced rat liver S9. Dose levels for the mutagenicity assay were based upon post-treatment toxicity.

Rationale for test conditions:

The mutagenicity assay was used to evaluate the mutagenic potential of the test substance. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and six dose levels of test substance, in triplicate, in the presence and absence of Aroclor induced rat liver S9.

Evaluation criteria:

All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
Based on historical control data (95% control limits), all tester strain cultures must exhibit characteristic numbers of spontaneous revertants per plate with the vehicle controls.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitate was observed at 1500 µg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitate was observed at 1500 µg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitate was observed at 1500 µg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitate was observed at 1500 µg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitate was observed at 1500 µg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Executive summary:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test material did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor‑induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 July 2017 to *****

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

-Purity: > 99% (UVCB)
-Description: Translucent blackish yellow liquid
-Storage conditions:Room temperature, protected from light

Species / strain / cell type:

lymphocytes:

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 µg/mL, which was the maximum dose tested based upon OECD criteria for UVCB. Cytotoxicity [>= 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses >= 500 µg/mL in the non activated 4-hour exposure group, and at doses >= 150 µg/mL in the S9 activated 4-hour and the non-activated 24-hour exposure groups. At the conclusion of the treatment period, visible precipitate was observed at doses >= 150 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 5 to 150 µg/mL for all three exposure groups.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Vinblastine

Details on test system and experimental conditions:

Target cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9, by incorporation of the test substance vehicle mixture into the treatment medium.

Rationale for test conditions:

HPBL were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose, lowest precipitating dose, and the highest soluble dose was measured. Dose levels for the micronucleus assay were based upon post-treatment toxicity (cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) or visible precipitate in the treatment medium at the conclusion of the treatment period.

Evaluation criteria:

The slides from at least three test substance treatment groups were coded using random numbers by an individual not involved with the scoring process and scored for the presence of micronuclei based on cytotoxicity. A minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei. Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
• the micronucleus should have the same staining characteristics as the main nucleus
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus

Statistics:

Statistical analysis was performed using the Fisher's exact test (p <= 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

lymphocytes: 4 hr

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

>= 150 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: 24 hr

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

> = 150 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test material was positive for the induction of micronuclei in the absence of the exogenous metabolic activation system after 24 hours of continuous exposure.

Executive summary:

The test material was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Acetonewas used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 µg/mL, which was the maximum dose tested based upon OECD criteria for UVCB. Cytotoxicity [³50%cytokinesis-blocked proliferation index(CBPI) relative to the vehicle control] was observed at doses >=500 µg/mL in the non‑activated 4-hour exposure group, and at doses >= 150 µg/mL in the S9‑activated 4-hour and the non-activated 24-hour exposure groups. At the conclusion of the treatment period, visible precipitate was observed at doses >= 150 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 5 to 150 µg/mL for all three exposure groups.

In the micronucleus assay, cytotoxicity (55± 5% CBPI relative to the vehicle control) was observed at doses> = 100 µg/mL in all three exposure groups. At the conclusion of the treatment period, visible precipitate was observed at doses >=100 µg/mL in all three exposure groups. The doses selected for evaluation of micronuclei were 5, 50, and 100 µg/mL for all three exposure groups.

In the non-activated and S9-activated 4-hour exposure groups, no significant or dose‑dependent increases in micronuclei induction were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the non-activated 24-hour exposure group, statistically significant and dose-dependent increases in micronuclei induction (1.05%) were observed at 100 µg/mL (p <= 0.01; Fisher’s Exact test and p <= 0.05; Cochran-Armitage test). The micronuclei induction was also outside the historical 95% control limit of 0.00% to1.01%.

These results indicate the test material was positive for the induction of micronuclei in the absence of the exogenous metabolic activation system. The test material was negative for the induction of micronuclei in the presence of the exogenous metabolic activation system. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)