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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

TBzTD did not show mutagenic activity in the reverse mutation assay (Ames test), performed according to OECD guideline 471 and GLP, in any of the strains of Salmonella typhimurium, either in the absence or in the presence of S-9 mix. In three independent mammalian cytogenicity tests performed according to OECD guideline 473 and GLP it is concluded that TBzTD (purified and technical) did not induce structural chromosome aberrations in cultured human lymphocytes, either in the absence or in the presence of S-9 mix.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 27, 1987 to July 3, 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tetrabenzylthiuramdisulphide (TBeTD; same as TBzTD)
- Substance type: Mono-constituent substance
- Physical state: Off-white powder
- Purity test date: > 95%, technically pure
- Lot/batch No.: G 1542
Species / strain / cell type:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Arochlor-induced rat liver
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): CA 24.69 ... CA 2000 µg/plate
Concentration range in the main test (without metabolic activation): CA 24.69 ... CA 2000 µg/plate
Vehicle / solvent:
Solvent: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation: 2000 µg/plate
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and 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 applicable
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:
Tetrabenzylthiuram disulfide (TBzTD) did not show mutagenic activity in any of the strains of Salmonella typhimurium, either in the absence or in the presence of the S-9 mix.
Executive summary:

1. 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).

2. 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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 28, 1988 to October 24, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Chromosome Aberration assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Tetrabenzylthiuramdisulphide (TBeTD; same as TBzTD)
- Substance type: Mono-constituent substance
- Stability under test conditions: TBzTD is stable from pH 3 to 13.
- Storage condition of test material: 20°C
- Physical state: Light-yellow (wet) powder
- Lot/batch No.: 880526/5
- Purity test date: Purified substance
Species / strain / cell type:
lymphocytes: human
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Arochlor-induced rat liver
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 3.70, 1.11, 3.33 and 100.0 µg/ml
Concentration range in the main test (without metabolic activation): 3.70, 1.11, 3.33 and 100.0 µg/ml
Vehicle / solvent:
- vehicle: DMSO

The test substance could only be dissolved in DMSO at a concentration of 10.0 mg/ml under gentle heating in a waterbath at 50°C. Upon addition of 50 µI of this solution to 5.0 ml of culture medium (final concentration of the test substance: 100.0 µg/ml), the test substance flocculated in the culture medium. At concentrations of 50.0 and 25.0 pg/ml, slight flocculation occurred in the medium. In view of these observations, it was decided to use 100.0 µg/ml as highest concentration for the toxicity test.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
in the absence of the S-9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in the presence of the S-9 mix
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 2 hours
Exposure period (without metabolic activation): 24 hours

Fixation time:
24 hours including a 2 hr treatment with colcemid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(≥ 100 µg/ml; limit of solubility)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(≥ 33.3 µg/ml; slight toxicity)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations: The positive control substances, mitomycin C (in the absence of the S-9 mix) and cyclophosphamid (in the presence of the S-9 mix) showed the expected statistically significant increase in structural hromosomal abberations.
Remarks on result:
other: not genotoxic
Conclusions:
From the reported findings it is concluded that tetrabenzylthiuram disulfide did not induce structural chromosome aberrations in cultured human lymphocytes, either in the absence or in the presence of the S-9 mix, under the conditions employed in this examination.
Executive summary:

1. 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 ("Genetic Toxicology: in vitro mammalian cytogenetic test").

2. 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).

3. 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.

4. 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.

5. 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo micronucleus test in mice performed according to OECD guideline 474 and GLP, a small but statistically significant increase in the number of micronuclei was observed. However, as the increase is just borderline, this increase might be not biologically significant. In summary it is concluded that TBzTD showed no genotoxic effects in the tests performed.

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
Study period:
April 22, 1988 to September 28, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(1983)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(Directive 84/449/EEC)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test
Species:
mouse
Strain:
Swiss
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
Corn oil
Dose / conc.:
0 mg/kg bw/day (actual dose received)
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 times: 24 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 72 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 72 hours
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): -
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg body weight
Tissues and cell types examined:
Bone marrow
Statistics:
The Wilcoxon rank-sum test was used to assess significant differences between the numbers of micronuclei in the treatment and control groups, in which P
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
Doses producing toxicity: No signs of toxicity at 2000 mg/kg (maximum dose based on solubility of the test substance in corn oil.
Vehicle controls validity:
valid
Positive controls validity:
valid
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
Doses producing toxicity: No signs of toxicity at 2000 mg/kg (maximum dose based on solubility of the test substance in corn oil.
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The small but statistically significant increase in the number of micronuclei in bone marrow of female mice at the 48-hour sampling time is considered to be not biologically significant, as the increase is just borderline.
Additional information on results:
Observations:
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.
Control (cylophosphamide 50 mg/kg) showed toxic effects and significant increase in number of micronuclei.

RESULTS OF RANGE-FINDING STUDY
- Dose range: In a preliminary study 6 animals (3 males and 3 females) were dosed orally with 2000 mg/kg body weight.
- Solubility: Higher concentrations could not be dosed because of aggregates of the test substance in the suspension.
- Clinical signs of toxicity in test animals: The mice did not show any signs of reaction to treatment.
- Other: Based on the results of this pilot study 2000 mg/kg body weight was selected as an appropriate dose for the Micronucleus Test.

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 (D to F), 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)

Additional information

Salmonella typhimurium, Reverse Mutation Assay (Ames test)

Tetrabenzylthiuram disulfide (TBzTD) was examined for mutagenic activity in the Salmonella typhimurium, Reverse Mutation Assay (Ames test) according to OECD guideline 471 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 TBzTD did not show mutagenic activity in any of the strains of Salmonella typhimurium, either in the absence or in the presence of the S-9 mix.

Mammalian cytogenetic test (in vitro)

The test according to OECD guideline 473 ("Genetic Toxicology: in vitro mammalian cytogenetic test") was conducted twice, one time with purified and one time with technical TBzTD. TBzTD (purified and technical) 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 ("Genetic Toxicology: in vitro mammalian cytogenetic test"). In the absence of the S-9 mix, the cells were exposed for 24 hours to 6 concentrations of the TBzTD (purified and technical) (ranging from 0.4 to 100.0 µg/ml). 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. The highest concentration used in the assay (100.0 µg/ml) was determined by the limit of solubility of the test substance in the culture medium. Four out of 6 concentrations of the test substance (3.7, 11.1, 33.3 and 100.0 µg/ml) were selected for scoring of chromosome aberrations. The test substance did not induce a statistically 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. The positive control substances, mitomycin C (in the absence of the S-9 mix) and cyclophosphamide (in the presence of the S-9 mix), induced the expected increase in the incidence of structural chromosome aberrations. It was concluded that TBzTD (purified and technical) did not induce structural chromosome aberrations in cultured human lymphocytes, either in the absence or in the presence of the S-9 mix, under the conditions used in the present assay.

Micronucleus Test in Mice (in vivo)

Tetrabenzylthiuram disulfide (TBzTD) was tested in the in vivo Micronucleus Test in mice according to OECD guideline no. 474. Three groups (D to F), 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 results of the genetic toxicity studies, the substance does not need to be classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.