Tetrakis(phenylmethyl)thioperoxydi(carbothioamide)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro studies (Ames test, Chromosomal aberration test, HPRT) were performed on Tetrabenzylthiuram disulfide (TBzTD) and showed negative results (no mutagenic activity).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted on 26 May 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Human lymphocytes

Cytokinesis block (if used):

Fixation time: 24 hours including a 2 hr treatment with colcemid

Metabolic activation:

with and without

Metabolic activation system:

Arochlor-induced rat liver

Test concentrations with justification for top dose:

prelim study : with and without metabolic activation: 4 to 100 µg/mL
main study : 3.7 to 100 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Exposure period (with metabolic activation) 2 hours ; Exposure period (without metabolic activation) 24 hours
Duplicates were used rather than an independent repeat (the study was independently repeated later).
Chromosome aberrations were counted on four slides of each culture.

Fixation time:
24 hours including a 2 hr treatment with colcemid

200 metaphases were scored per concentration

Rationale for test conditions:

In accordance with the OECD Testing Guideline 473 adopted on 26 May 1983.

Evaluation criteria:

The major criterion to designate the results of a chromosome aberration test as positive is a dose-related, statistically significant increase in the number of cells with structural chromosome aberrations. However, a clear dose-response relationship can be absent because the yield of chromosome aberrations can vary markedly with post-treatment sampling time of an asynchronous population and because increasing doses of clastogens can induce increasing degrees of mitotic delay. A test substance producing neither a dose-related, statistically significant increase in the number of cells with structural chromosome aberrations, nor a statistically significant and reproducible positive response at any of the doses is considered non-clastogenic in this system.

Statistics:

Different types of aberrations (chromatid-type and chromosome-type) have been listed with their numbers and frequencies for all test and control cultures. As gaps are usually considered as unreliable end points for induced chromosome damage, this type of aberration was recorded separately and was not included in the final assessment of clastogenic activity. Data were analysed, when appropriate, by Fisher's exact probability test using the number of cells with aberrations to determine significant differences between test and control cultures.

Species / strain:

other: human lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 100 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 33.3 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The highest dose level was determined to by the limit of solubility of the test substances in the solvent DMSO.
The positive control substances, mitomycin C (in the absence of the S-9 mix) and cyclophosphamid (in the prensense of the S-9 mix) showed the expected statistically significant increase in structural chromosomal abberations.

Conclusions:

The test substance did not induce a significant increase in the number of cells with structural chromosome aberrations at any of the concentrations used, either in the absence or in the presence of the S-9 mix.

Executive summary:

Tetrabenzylthiuram disulfide (TBzTD) was examined for its potential to induce chromosome aberrations in human lymphocytes, both in the absence and in the presence of a metabolic activation system (S-9 mix), in compliance with OECD guideline 473. The dose levels used in the chromosome aberration assay were established on the basis of the results of a preliminary toxicity test carried out with 6 concentrations of the test substance (ranging from 0.41 to 100.0 µg/ml), both in the absence and in the presence of the metabolic activation system (S-9 mix). The highest dose level for the toxicity test was determined by the limit of solubility of the test substance in the chosen solvent (DMSO). For the chromosome aberration assay in the absence of the S-9 mix, the cells were exposed to 4 concentrations of the test substance (3.70, 11.11, 33.33 and 100.0 µg/ml) for 24 hours. In the presence of the S-9 mix, the cells were exposed for only 2 hours (because of the toxicity of the S-9 mix for the cells) to the same concentrations. As positive controls, mitomycin C (in the absence of the S-9 mix) and cyclophosphamide (in the presence of the S-9 mix) were used, while the vehicle (DMSO) was used as negative control. The test substance did not induce a significant increase in the number of cells with structural chromosome aberrations at any of the concentrations used, either in the absence or in the presence of the S-9 mix.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted on 26 May 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Arochlor-induced rat liver

Test concentrations with justification for top dose:

During the first and second experiments, five doses were used: 24.69, 74.07, 222.22, 666.66, and 2000.00 µg/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 2000 µg/plate

Rationale for test conditions:

In accordance with the OECD Testing Guideline 471, adopted on 26 May 1983.
A preliminary toxicity test was carried out in order to the select the doses to be investigated during the first and second experiments.

Species / strain:

S. typhimurium TA 1538

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 98

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:

No signs of toxicity at any concentration tested.
No reduction of background bacterial lawn at any concentration.
Positive controls gave the expected strong increase in the number of S9+ and S9- revertants.

Conclusions:

It was concluded that tetrabenzylthiuram disulfide did not show mutagenic activity in any of the strains of typhimurium, either in the absence or in the presence of the S-9 mix.

Executive summary:

Tetrabenzylthiuram disulfide was examined for mutagenic activity in the Ames test using the histidine requiring Salmonella typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and a liver microsome fraction of Aroclor-induced rats for metabolic activation (S-9 mix). It was concluded that tetrabenzylthiuram disulfide did not show mutagenic activity in any of the strains of typhimurium, either in the absence or in the presence of the S-9 mix.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September-December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as frozen stocks in liquid nitrogen.

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with ß-Naphthoflavone/Phenobarbital. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Treatments were carried out both in the absence and presence of S-9 by addition of either 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test system was 1% (v/v).

Test concentrations with justification for top dose:

Range-finder (with and without S9) : 4.688 to 150 µg/ml
Mutation experiment (without S9) : 0.25 to 10 µg/ml
Mutation experiment (with S9) : 0.5 to 100 µg/ml

Vehicle / solvent:

DMSO.

Preliminary solubility data indicated that TbzTD was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to 40 mg/mL. The solubility limit in culture medium was in the range of 25 to 50 µg/mL, as indicated by precipitation at the higher concentration which persisted for 3 hours after test article addition, with warming at 37°C. A maximum concentration of 150 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a precipitating concentration. Concentrations selected for the Mutation Experiment were based initially on the results of this cytotoxicity Range Finder Experiment.

Test article stock solutions were prepared under subdued lighting by formulating TbzTD in DMSO, with the aid of vortex mixing, warming at 37°C and ultrasonication for 2-4 minutes where required, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 2.5 hours of initial formulation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO ; diluted 100 fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

4-nitroquinoline 1-oxide (NQO) : without S9 : 0.15 and 0.20 µg/ml
Benzo[a]pyrene (B[a]P) : with S9 : 4 and 6 µg/ml

Details on test system and experimental conditions:

Range finder study:
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment culture volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10.
Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, for 9 days. Wells containing viable clones were identified by eye using background illumination and counted.

Mutation Experiments
At least 107 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours in an incubator set to 37°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10 medium.
Cell densities were determined using a Coulter counter and the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies (Brusick, 1986; Scott et al., 1991). Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.

Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.

Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask.

At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance.
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (10 days). Wells containing viable clones were identified by eye using background illumination and counted.

At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 µg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (13 days). Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:

Acceptance Criteria
The assay was considered valid if all of the following criteria were met:
1. The MF in the concurrent vehicle control cultures was considered acceptable for addition to the laboratory historical vehicle control database
2. The MF in the concurrent positive controls induced responses that were comparable with those generated in the historical positive control database and gave a clear, unequivocal increase in MF over the concurrent vehicle control
3. The test was performed with and without metabolic activation
4. Adequate numbers of cells and concentrations were analysable.

Evaluation Criteria
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the vehicle control (p=0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p=0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical vehicle control database (mean
MF +/- 2 standard deviations).

The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Toxicity
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 4.688 to 150 µg/mL (limited by solubility of the formulated test article in culture medium). Upon addition of the test article to the cultures and following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations in the absence and presence of S-9 (37.5 to 150 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. The highest concentration to give >10% RS was 37.5 µg/mL in the absence and presence of S-9, which gave 12% and 34% RS, respectively. No marked changes in osmolality or pH were observed in the Range-Finder at the highest three concentrations analysed (9.375, 18.75 and 37.5 µg/mL), compared to the concurrent vehicle controls.

In the Mutation Experiment twelve concentrations, ranging from 0.25 to 10 µg/mL in the absence of S-9 and from 0.5 to 100 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations tested in the presence of S-9 (37.5 to 100 µg/mL). Following the 3 hour treatment incubation period, no precipitation was observed in the absence and presence of S-9.
Seven days after treatment all concentrations in the absence and presence of S-9, with the exception of cultures tested at 3 and 10 µg/mL (at which one or both cultures were excessively toxic), were selected to determine viability and 6TG resistance.
The highest concentrations analysed were 7.5 µg/mL in the absence of S-9 and 100 µg/mL in the presence of S-9, which gave 14% and 18% RS, respectively. It should be noted that marked cytotoxicity was also observed at lower concentrations under both treatment conditions: in the absence of S-9, cultures analysed at 1.5 to 7.5 µg/mL gave 12 to 15% RS and in the presence of S-9, cultures analysed at 8 to 100 µg/mL gave 17 to 27% RS. All intermediate concentrations were analysed to determine viability and 6TG resistance under both treatment conditions.

Mutation results
When tested up to toxic concentrations for 3 hours in the absence of S-9, no statistically significant increases in mean MF, compared to the mean vehicle control value, were observed at any concentration analysed and there was no statistically significant linear trend. The mean MF values for the vehicle control and all test article treated cultures were within the range generated by the last 20 experiments (mean ± 2 standard deviations) performed in this laboratory (1.13 to 7.48 mutants/106 viable cells, mean value 4.31) with the exception of cultures analysed at 0.5 µg/mL (the second lowest concentration), where the MF value (7.59 mutants/106 viable cells) marginally exceeded the range. However, this value was not significantly different from the vehicle control MF of 6.40 mutants/106 viable cells and there was no statistically significant linear trend, therefore this isolated observation was considered not biologically relevant.
When tested up to toxic concentrations for 3 hours in the presence of S-9, no statistically significant increases in mean MF, compared to the mean vehicle control value, were observed at any concentration analysed. The mean MF values for the vehicle control and the large majority of test article treated cultures were within the range generated by the last 20 experiments (mean ± 2 standard deviations) performed in this laboratory (0.567 to 8.06 mutants/106 viable cells, mean value 4.31), with the exception of cultures analysed at two intermediate concentrations of 20 and 37.5 µg/mL, where the MF values (8.78 and 8.68 mutants/106 viable cells, respectively) marginally exceeded the range. However, both values were not significantly different from the vehicle control MF of 6.74 mutants/106 viable cells. There was a statistically significant linear trend (p=0.01) but the MF values observed at the highest three concentrations (50, 75 and 100 µg/mL) were all within the current vehicle historical control range. The evaluation criteria for a positive result were not fulfilled and these observations were considered not biologically relevant.

It should be noted that the data presented in this report for the Mutation Experiment were derived from a repeat experiment. In the initial experiment, final concentration ranges of 0.25 to 75 µg/mL were tested in the absence and presence of S-9. An acceptable level of cytotoxicity (10% relative survival, RS) was observed in the absence of S-9 but insufficient toxicity was observed in the presence of S-9 and no concentration achieved 10-20% RS, as required by the regulatory test guidelines. Furthermore, extreme heterogeneity for mutation between replicate cultures was observed at 1 and 2 µg/mL in the absence of S-9 and the data set was therefore unreliable. The vehicle and positive control data in the absence and presence of S-9 were acceptable and were added to the historical control ranges but the test article data were invalidated and the experiment repeated. Results from the initial experiment are not further reported here.

Conclusions:

It is concluded that TbzTD did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Executive summary:

TbzTD was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by a β-Naphthoflavone/Phenobarbital-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 4.688 to 150 µg/mL (limited by solubility of the formulated test article in culture medium). The highest concentration to give >10% relative survival (RS) was 37.5 µg/mL, which gave 12% and 34% RS in the absence and presence of S-9, respectively.

In the Mutation Experiment, twelve concentrations, ranging from 0.25 to 10 µg/mL in the absence of S-9 and from 0.5 to 100 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 7.5 µg/mL in the absence of S-9 and 100 µg/mL in the presence of S-9, which gave 14% and 18% RS, respectively. It should be noted that marked cytotoxicity was also observed at lower concentrations under both treatment conditions: in the absence of S-9, cultures analysed at 1.5 to 7.5 µg/mL gave 12 to 15% RS and in the presence of S-9, cultures analysed at 8 to 100 µg/mL gave 17 to 27% RS. All intermediate concentrations were analysed to determine viability and 6TG resistance under both treatment conditions.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore, the study was accepted as valid.

When tested up to toxic concentrations for 3 hours in the absence of S-9, no statistically significant increases in mean MF, compared to the mean vehicle control value, were observed at any concentration analysed and there was no statistically significant linear trend. The mean MF values for the vehicle control and all test article treated cultures were within the range generated by the last 20 experiments (mean ± 2 standard deviations) performed in this laboratory (1.13 to 7.48 mutants/10⁶ viable cells, mean value 4.31) with the exception of cultures analysed at 0.5 µg/mL (the second lowest concentration), where the MF value (7.59 mutants/10⁶ viable cells) marginally exceeded the range. However, this value was not significantly different from the vehicle control MF of 6.40 mutants/10⁶ viable cells and there was no statistically significant linear trend, therefore this isolated observation was considered not biologically relevant.

When tested up to toxic concentrations for 3 hours in the presence of S-9, no statistically significant increases in mean MF, compared to the mean vehicle control value, were observed at any concentration analysed. The mean MF values for the vehicle control and the large majority of test article treated cultures were within the range generated by the last 20 experiments (mean ± 2 standard deviations) performed in this laboratory (0.567 to 8.06 mutants/10⁶ viable cells, mean value 4.31), with the exception of cultures analysed at two intermediate concentrations of 20 and 37.5 µg/mL, where the MF values (8.78 and 8.68 mutants/10⁶ viable cells, respectively) marginally exceeded the range. However, both values were not significantly different from the vehicle control MF of 6.74 mutants/10⁶ viable cells. There was a statistically significant linear trend (p≤0.01) but the MF values observed at the highest three concentrations (50, 75 and 100 µg/mL) were all within the current vehicle historical control range. The evaluation criteria for a positive result were not fulfilled and these observations were considered not biologically relevant.

It is concluded that TbzTD did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus study in rats were performed on Tetrabenzylthiuram disulfide (TBzTD) and showed negative results. Tetrabenzylthiuram disulfide is considered to be not mutagenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted on 26 May 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

Corn oil

Duration of treatment / exposure:

one administration

Frequency of treatment:

one administration

Post exposure period:

72h

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 72 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 72 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice time: 72 hours
Female 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Female 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female 2000 mg/kg; No. of animals: 5; Sacrifice time: 72 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

yes, cylophosphamide 50 mg/kg

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Bone marrow was sampled at 24, 48 and 72 hours after dosing.

Statistics:

yes

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A small, but statistically significant increase in the number of micronuclei was observed in females at the 48-hour sampling time only. As the increase is borderline it is concluded that this result might not be biologically significant.

Positive control (cylophosphamide 50 mg/kg) showed toxic effects and significant increase in number of micronuclei.

Conclusions:

It is concluded that this test is valid and that TBzTD induced a small but statistically significant increase in the number of micronuclei, under the experimental conditions described in this report. However, as the increase is just borderline, this increase might be not biologically significant.

Executive summary:

Tetrabenzylthiuram disulfide (TBzTD) was tested in the Micronucleus Test in mice. Three groups, each comprising 5 males and 5 females, received a single oral dose of 2000 mg/kg body weight. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups (A to C) served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide (CP) at 50 mg/kg body weight, was harvested at 48 hours after dosing only. The test substance induced a statistically significant increase in the number of micronuclei in polychromatic erythrocytes in female mice at the 48 hour sampling time. It is concluded that this test is valid and that TBzTD induced a small but statistically significant increase in the number of micronuclei, under the experimental conditions described in this report. However, as the increase is just borderline, this increase might be not biologically significant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Ames test (OECD 471)

Tetrabenzylthiuram disulfide was examined for mutagenic activity in the Ames test using the histidine requiring Salmonella typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and a liver microsome fraction of Aroclor-induced rats for metabolic activation (S-9 mix). It was concluded that tetrabenzylthiuram disulfide did not show mutagenic activity in any of the strains of typhimurium, either in the absence or in the presence of the S-9 mix.

 

In vitro chromosomal aberrations study (OECD 473)

Tetrabenzylthiuram disulfide (TBzTD) was examined for its potential to induce chromosome aberrations in human lymphocytes, both in the absence and in the presence of a metabolic activation system (S-9 mix), in compliance with OECD guideline 473. The dose levels used in the chromosome aberration assay were established on the basis of the results of a preliminary toxicity test carried out with 6 concentrations of the test substance (ranging from 0.41 to 100.0 µg/ml), both in the absence and in the presence of the metabolic activation system (S-9 mix). The highest dose level for the toxicity test was determined by the limit of solubility of the test substance in the chosen solvent (DMSO). For the chromosome aberration assay in the absence of the S-9 mix, the cells were exposed to 4 concentrations of the test substance (3.70, 11.11, 33.33 and 100.0 µg/ml) for 24 hours. In the presence of the S-9 mix, the cells were exposed for only 2 hours (because of the toxicity of the S-9 mix for the cells) to the same concentrations. As positive controls, mitomycin C (in the absence of the S-9 mix) and cyclophosphamide (in the presence of the S-9 mix) were used, while the vehicle (DMSO) was used as negative control. The test substance did not induce a significant increase in the number of cells with structural chromosome aberrations at any of the concentrations used, either in the absence or in the presence of the S-9 mix.

in vitro gene mutation study (Lloyd, 2021)

TbzTD was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by a β-Naphthoflavone/Phenobarbital-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 4.688 to 150 µg/mL (limited by solubility of the formulated test article in culture medium). The highest concentration to give >10% relative survival (RS) was 37.5 µg/mL, which gave 12% and 34% RS in the absence and presence of S-9, respectively.

In the Mutation Experiment, twelve concentrations, ranging from 0.25 to 10 µg/mL in the absence of S-9 and from 0.5 to 100 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 7.5 µg/mL in the absence of S-9 and 100 µg/mL in the presence of S-9, which gave 14% and 18% RS, respectively. It should be noted that marked cytotoxicity was also observed at lower concentrations under both treatment conditions: in the absence of S-9, cultures analysed at 1.5 to 7.5 µg/mL gave 12 to 15% RS and in the presence of S-9, cultures analysed at 8 to 100 µg/mL gave 17 to 27% RS. All intermediate concentrations were analysed to determine viability and 6TG resistance under both treatment conditions.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore, the study was accepted as valid.

When tested up to toxic concentrations for 3 hours in the absence of S-9, no statistically significant increases in mean MF, compared to the mean vehicle control value, were observed at any concentration analysed and there was no statistically significant linear trend. The mean MF values for the vehicle control and all test article treated cultures were within the range generated by the last 20 experiments (mean ± 2 standard deviations) performed in this laboratory (1.13 to 7.48 mutants/10⁶ viable cells, mean value 4.31) with the exception of cultures analysed at 0.5 µg/mL (the second lowest concentration), where the MF value (7.59 mutants/10⁶ viable cells) marginally exceeded the range. However, this value was not significantly different from the vehicle control MF of 6.40 mutants/10⁶ viable cells and there was no statistically significant linear trend, therefore this isolated observation was considered not biologically relevant.

When tested up to toxic concentrations for 3 hours in the presence of S-9, no statistically significant increases in mean MF, compared to the mean vehicle control value, were observed at any concentration analysed. The mean MF values for the vehicle control and the large majority of test article treated cultures were within the range generated by the last 20 experiments (mean ± 2 standard deviations) performed in this laboratory (0.567 to 8.06 mutants/10⁶ viable cells, mean value 4.31), with the exception of cultures analysed at two intermediate concentrations of 20 and 37.5 µg/mL, where the MF values (8.78 and 8.68 mutants/10⁶ viable cells, respectively) marginally exceeded the range. However, both values were not significantly different from the vehicle control MF of 6.74 mutants/10⁶ viable cells. There was a statistically significant linear trend (p≤0.01) but the MF values observed at the highest three concentrations (50, 75 and 100 µg/mL) were all within the current vehicle historical control range. The evaluation criteria for a positive result were not fulfilled and these observations were considered not biologically relevant.

It is concluded that TbzTD did not induce biologically relevant increases in mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

In vivo micronucleus study (OECD 474)

Tetrabenzylthiuram disulfide (TBzTD) was tested in the Micronucleus Test in mice. Three groups, each comprising 5 males and 5 females, received a single oral dose of 2000 mg/kg body weight. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups (A to C) served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide (CP) at 50 mg/kg body weight, was harvested at 48 hours after dosing only. The test substance induced a statistically significant increase in the number of micronuclei in polychromatic erythrocytes in female mice at the 48 hour sampling time. It is concluded that this test is valid and that TBzTD induced a small but statistically significant increase in the number of micronuclei, under the experimental conditions described in this report. However, as the increase is just borderline, this increase might be not biologically significant.

Justification for classification or non-classification

Based on the available data, no classification of Tetrabenzylthiuram disulfide (TBzTD) is required for genetic toxicity according to the Regulation EC 1272/2008.