N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three genotox tests are available for TBSI: a K2 in vitro mutagenicity test (Ames), a K1 in vitro mouse lymphoma assay and a K2 in vivo micronucleus test. All three tests have a negative result.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recent, well documented study according to OECD and GLP guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y mouse lymphoma (3.7.2c) cells, were obtained from American Type Culture Collection (ATCC), Virginia. These cells are heterozygous at the thymidine kinase locus, TK +/-. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock. Cell stocks are periodically checked for freedom from mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.41, 0.82, 1.63, 3.27, 6.53, 13.06, 26.13, 52.25, 104.5 and 209 µg/mL.
Main test, 3h treatment: 0, 1, 2, 4, 8, 12, 16, 20, 25, 30 and 35 µg/mL.
Main test, 24h treatment: 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14 and 16 µg/mL.

Vehicle / solvent:

Prior to commencing testing, the solubility of the test substance in vehicle compatible with this test system was assessed. Santocure TBSI was found to form a dosable suspension at 20.9 mg/mL in dimethyl sulphoxide (DMSO). A solution of 20.9 mg/mL, dosed at 1% in medium, showed precipitate in the culture medium. The osmolality of the test substance in medium was tested at 209 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control. No fluctuations in pH of the medium were observed at 209 µg/mL of more than 1.0 unit compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 209 µg/mL.

Santocure TBSI was dissolved and diluted in DMSO (ACS reagent grade), shortly before dosing. The final volume of DMSO added to the cultures was 1% v/v.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Preliminary toxicity test
Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix. For 3-hour exposures, cultures contained a total of 6 x 106 cells. The final volume of the cultures was 5 mL and the final concentration of the S9 fraction was 2% v/v, if present. For 24-hour exposures, cultures contained a total of 1.5 x 10E6 cells in a total volume of 5 mL. One culture was prepared for each concentration of the test substance for each test condition. Vehicle controls were tested in duplicate for each test condition.
The test substance was formulated and serially diluted in the solvent. Aliquots of 50 µL of test substance dilution (at 100 times the desired final concentration) or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 37°C) or 24 hours (static incubator, at 37°C, 5% (v/v) CO2). At the end of the 3-hour exposure period, the cells were washed once, resuspended in R10p to nominally 2 x 10E5 cells/mL (assuming no cell loss), incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary. At the end of the 24-hour exposure period, the cells were washed once, resuspended in 5 mL R10p and counted, to ascertain treatment growth. The cultures were then diluted to 2 x 10E5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary.
The RSG was used to determine the concentrations of test substance used in the main test; ideally the maximum concentration should reduce RTG to approximately 10 to 20% of the concurrent vehicle control value. There was evidence of toxicity in the preliminary toxicity test, so the maximum concentration tested in the 3-hour exposures in both the absence and presence of S9 mix was 35 µg/mL, and in the 24-hour exposure in the absence of S9 mix was 16 µg/mL. The formulations were added at 1% final volume in medium.

Main mutation test - 3h exposure in the absence and presence of S9 mix
The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 10E7 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls. Aliquots of 100 µL of test substance dilution (at 100 times the desired final concentration), vehicle or positive control were added, then all cultures were incubated, with continuous shaking, for 3 hours at 37°C. At least four serial dilutions of the test substance were tested.
Following the 3-hour exposure, the cells were washed once, resuspended in R10p to nominally 2 x 10E5 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 10E5 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cultures that were assessed were chosen at the Study Director’s discretion. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 10E3 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air.
After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates, the number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF). The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The maximum concentrations assessed for mutant frequency in the main test were 12 and 16 µg/mL in the absence and presence of S9 mix respectively.

Main mutation test – 24h exposure in the absence of S9 mix
A test with a 24-hour exposure in the absence of S9 mix was carried out. Duplicate 10 mL cultures containing 3 x 10E6 cells were treated for 24 hours with 100 µL of test substance or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, resuspended in 10 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to 2 x 10E5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension.
After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p, the intention being to retain at least 1 x 10E7 cells. Following this, the procedure was the same as in the 3-hour treatment. The maximum concentration assessed for mutant frequency in the main test was 12 µg/mL.

Evaluation criteria:

Acceptance criteria for test substance:
The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (ie. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 50 to 170 x 10-6. The mean cloning efficiency was between 65 to 120%. The mean suspension growth was between 8 to 32 on Day 2 following 3-hour treatments and between 32 to 180 on Day 2 following a 24-hour treatment. Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.

Acceptance criteria for positive controls:
Positive controls showed an absolute increase in mean total MF above the mean concurrent vehicle control MF of at least 300 x 10-6. At least 40% of this was due to the number of small mutant colonies. Mean RTG’s for the positive controls were greater than 10%. There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity. There was not excessive heterogeneity between replicate cultures.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989). Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
Precipitate (observed by eye at the end of treatment) was observed at concentrations of 104.5 µg/mL and greater in both the absence and presence of S9 mix following a 3-hour exposure. Exposure to Santocure TBSI at concentrations from 0.41 to 209 µg/mL in the absence and presence of S9 mix (3-hour exposure) resulted in relative suspension growth (RSG) values from 118 to 1% and from 117 to 5% respectively.
Following a continuous exposure for 24 hours, precipitation (assessed by eye at the end of treatment) was observed at concentrations of 52.25 µg/mL and greater. Exposure to concentrations from 0.41 to 209 µg/mL resulted in RSG values from 116 to 0%.
Concentrations used in the main test were based upon these data.

Main mutation test - 3h exposure in the absence of S9 mix:
Cultures were exposed to Santocure TBSI at concentrations from 1 to 35 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Santocure TBSI at concentrations from 1 to 12 µg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 106 to 20% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main mutation test - 3h exposure in the presence of S9 mix:
Cultures were exposed to Santocure TBSI at concentrations from 1 to 35 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Santocure TBSI at concentrations from 1 to 16 µg/mL were assessed for determination of mutation frequency. RTG values from 113 to 18% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants. The results obtained in response to the exposure of cultures to Santocure TBSI in the presence of S9 mix did not demonstrate mutagenic potential. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. Therefore it was considered not to be beneficial to perform a direct repeat of the assay.

Main mutation test - 24h exposure in the absence of S9 mix:
Cultures were exposed to Santocure TBSI at concentrations from 0.5 to 16 µg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Santocure TBSI at concentrations from 3 to 12 µg/mL were assessed for determination of mutation frequency. RTG values from 100 to 14% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: negative

Under the experimental conditions described, Santocure TBSI did not demonstrate mutagenic potential in this in vitro cell mutation assay.

Executive summary:

Santocure TBSI was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

Santocure TBSI was found to form a dosable suspension at 20.9 mg/mL in DMSO. A final concentration of 209 µg/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the limit of solubility.

Toxicity was observed in the preliminary toxicity test. Following a 3-hour exposure to Santocure TBSI at concentrations from 0.41 to 209 µg/mL, relative suspension growth (RSG) was reduced from 118 to 1% and from 117 to 5% in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 116 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 -hour treatment in the absence and presence of S9 mix were 12 and 16 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 20 and 18% respectively.

In the 24 -hour treatment, the maximum concentration assessed for mutant frequency was 12 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The RTG was reduced to 14%. In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded that Santocure TBSI did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Three key genotox tests are available for TBSI: a K2 in vitro mutagenicity test (Ames), a K2 in vivo micronucleus test and an in vitro K1 mutation test with Mouse Lymphoma L5178Y cells.

The Ames test has no indication on GLP, EU or US EPA protocol, but the study is documented in very much detail and performed according to the original paper by Ames et al (1975). Under the test conditions of the Ames test, the test substance is not a mutagen in Salmonella typhimurium. 

 

The in vivo micronucleus test is a K2 GLP study, but no internationally recognized protocol is mentioned. Nevertheless, the study is very well documented and the described protocol is similar to OECD 474. The results of the test substance were negative for clastogenic effect in the micronucleus test at dose level of 5000 mg/kg bw administered in single oral doses with sacrifice times of 24, 48 and 72 hours.

 

The in vitro mouse lymphoma test is performed according to GLP and internationally accepted guidelines (OECD, EU and EPA). Santocure TBSI was tested for mutagenic potential in an in vitro mammalian cell mutation assay. Under the conditions of this test, the test substance did not demonstrate mutagenic potential.

 

In conclusion, based on the available genotox studies, TBSI is believed to not exert effects of mutagenicity nor chromosome aberrations.

Justification for selection of genetic toxicity endpoint
It is to be noted that all three genotox tests are of equal importance, as they assess different types of genotoxic response to a test chemical.

Justification for classification or non-classification

Conclusive, but not sufficient for classification