Di-tert-dodecyl disulphide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutations

The potential of DI-tert-DODECYL DISULFIDE to induce a reverse mutation was evaluated in bacteria Salmonella typhimurium (Molinier, 1995). A preliminary toxicity test was performed to define the doses to be used for the mutagenicity study. The test substance was then tested in two independent tests, with or without a metabolic activation system, the S9 mix, prepared from a liver micrasomal fraction (S9) of rats induced with Araclor 1254. The tests were perf ormed according to the direct plate incorporation method except the second test with S9 mix, according to the preincubation method (one hour, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five doses of the test substance (three plates/dose). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. DI-tert-DODECYL DISULFIDE was dissolved in dimethylsulfoxide (DMSO). The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria and within the range of the historical data. The top dose was selected according to the criteria specified in the international regulations. Since the test substance was non-toxic, freely soluble, the top dose was 5000 µg/plate. The selected range dose was: 312.5, 625, 1250, 2500 and 5000 µg/plate. The test substance did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the five strains. Under these experimental conditions, DI-tert-DODECYL DISULFIDE did not show mutagenic activity in this bacterial reverse mutation assay on Salmonella typhimurium.

The potential of DI-tert-DODECYL DISULFIDE to induce mutations at the TK (thymidine kinase) locus was evaluated in L5 l 78Y mouse lymphoma cells (Molinier, 1996). After a preliminary toxicity test, DI-tert-DODECYL DISULFIDE was tested in two independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Araclor 1254. Approximately 0.5.10e6 cells/ml in 20 ml culture medium with 5% horse serum were exposed to the test or control substances, in the presence or absence of S9 mix (final concentration of S9 fraction 2% ), for three hours at 37°C. Cytotoxicity was then determined using cloning efficiency (CE0) before expression of the mutant phenotype. Cells viability (using cloning efficiency CE1 ) and number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. The test substance was dissolved in ethanol. The dose-levels for the positive contrals were as follows: without S9 mix: 25 µg/ml of methylmethane sulfonate, with S9 mix: 3 µg/ml of cyclophosphamide.

The cloning efficiencies CE0 and CE2 and mutation frequencies of the vehicle and positive controls were within the range of our historical data. The treament-levels were for both mutagenicity experiments, with and without S9 mix: 200, 400, 800 and 1600 µg/ml; 1600 µg/ml was the highest treatment-level above the limit of solubility in the final culture medium at the end of the treatment-period. The cloning efficiencies CE0 and CE2 were considered equivalent to that of the control cultures. DI-tert-DODECYL DISULFIDE did not induce any significant increase in the mutation frequency, with and without S9 mix. Under our experimental conditions, DI-tert-DODECYL DISULFIDE did not show mutagenic activity in this mouse lymphoma assay.

Chromosomal aberrations

The potential of DI-tert-DODECYL DISULFIDE to induce chromosome aberrations was evaluated in cultured human lymphocytes (Molinier, 1996). The test substance, dissolved in ethanol, was tested in two independent tests, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254. For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemogglutinin) and incubated at 37°C in a humidified atmosphere of 5% CO2/95% air, for 48 hours.

First test: cells were then exposed to the test or control substances, with or without S9 mix, for three hours then rinsed. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/ml) to black cells at the metaphase-stage of mitosis. Harvest time was 20 hours from the beginning of treatment, i.e. approximately 1.5 cell cycle times. As this test gives negative results, both with or without S9 mix, an additional test was performed as follows:

Second test:

. without S9 mix, cells were exposed continuously to the test or control substances.

. with S9 mix, cells were exposed to the test or control substances for three hours then rinsed.

One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/ml) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours from the beginning of treatment, i.e. approximately 1.5 cell cycle times and 24 hours after.

After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

The doses of DI-tert-DODECYL DISULFIDE were as follows:

First test with and without S9 mix: 50, 100, 200, 400, 600 and 800 µg/rnl for treatment and 400, 600 and 800 µg/ml for chromosomal aberrations scoring. 800 µg/ml being the lowest precipitating dose.

Second test with and without S9 mix: 200, 400, 600 and 800 µg/ml for treatment and 400, 600 and 800 µg/ml for chromosomal aberrations scoring. The doses of the positive controls were 3 µg/ml mitomycin C (MMC) without S9 mix (3 hours of treatment) or 0.2 µg/ml (continuous treatment) and 50 µg/ml of cyclophosphamide (CPA) with S9 mix.

The frequency of cells with structural chromosome aberrations in the negative, vehicle and positive controls was as specified in the acceptance criteria and within the range of the historical data. The test substance did not induce any significant increase in the frequency of cells with structural chromosome aberrations, with or without S9 mix, for both of the two tests and for the two harvest times. Under these experimental conditions, DI-tert-DODECYL DISULFIDE did not induce chromosome aberrations in cultured human lymphocytes.

Link to relevant study records

Reference

Reference 1

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:

26th May 1983 and revised Draft document of December 1994 .

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

liver micrasomal fraction (S9) of rats induced with Araclor 1254

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

To assess the toxicity of the test substance to the bacteria, six doses (one plate/dose) were tested in the TA 98, TA 100 and TA 102 strains, with or without S9 mix.

The tests were peïf ormed according to:
. direct plate incorporation method (both tests without S9 mix, first test with S9 mix): 0.05 to 0.1 ml of the test substance solution, 0.5 ml of S9 mix when required and 0.1 ml of the strain were mixed with 2 ml of overlay agar containing traces of the relevant amino-acid and biotin and maintained at 45°C. After rapid homogenization, the mixture was spread out on a Petri plate containing minimum medium.
. preincubation rnethod (second test with S9 mix): 0.05 to 0.1 ml of the test substance solution, 0.5 ml of S9 mix and 0.1 ml of the strain were incubated for 60 minutes at 37°C prior adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Artek counter, model 880, O.S.I., 75015 Paris, France).

Evaluation criteria:

Biological relevance of the results was considered first. In addition, the following criteria may be used as an aid for determining a positive response:
. a dose-related increase in the number of revertants,
and/or
. a reproducible increase in the number of revertants (i.e. a doubling in at least one strain when compared to that of the controls) for at least one of the doses.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

DI-tert-DODECYL DISULFIDE did not show mutagenic activity in this bacterial reverse mutation assay on Salmonella typhimurium.

Executive summary:

The potential of DI-tert-DODECYL DISULFIDE to induce a reverse mutation was evaluated in bacteria Salmonella typhimurium. A preliminary toxicity test was performed to define the doses to be used for the mutagenicity study. The test substance was then tested in two independent tests, with or without a metabolic activation system, the S9 mix, prepared from a liver micrasomal fraction (S9) of rats induced with Araclor 1254. The tests were perf ormed according to the direct plate incorporation method except the second test with S9 mix, according to the preincubation method (one hour, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five doses of the test substance (three plates/dose). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. DI-tert-DODECYL DISULFIDE was dissolved in dimethylsulfoxide (DMSO). The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria and within the range of the historical data. The top dose was selected according to the criteria specified in the international regulations. Since the test substance was non-toxic, freely soluble, the top dose was 5000 µg/plate. The selected range dose was: 312.5, 625, 1250, 2500 and 5000 µg/plate. The test substance did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the five strains. Under these experimental conditions, DI-tert-DODECYL DISULFIDE did not show mutagenic activity in this bacterial reverse mutation assay on Salmonella typhimurium.

Reference 2

Endpoint:

in vitro gene mutation 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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

4th April 1984 and revised Draft document of September 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y cells, originally obtained through ATCC, were kindly supplied by Dr. Oudelkhim-Diot
(Vitry-sur-Seine, France).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Araclor 1254

Test concentrations with justification for top dose:

200, 400, 800 and 1600 µ/ml
1600 µg/ml was the highest treatment-level above the limit of solubility in the final culture medium at the end of the treatment-period.

Vehicle / solvent:

ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

- Treatment
The day before treatment, the cells were seeded in 50 ml of RPMI 1640 medium containing 10% horse serum, L-Glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml) and sodium pyruvate (200 µg/ml). The cells were then incubated at 37°C in a humidified atmosphere of 5% CO2/95% air. After 24-hour incubation, culture medium was removed and cells were counted.
Approximately 0.5 10e6 cells/ml in 20 ml culture medium with 5% horse serum, were exposed to the test or control substances, in the presence or absence of S9 mix (final concentration of S9 fraction: 2% ), for three hours at 37°C. The treatment medium was then removed and the cells were counted and seeded as follows:
Cytotoxicity plates
1.6 cells/well (one 96-well plate/culture = two plates/dose-level) to determine cytotoxicity using cloning efficiency (CE0 ). After 12 ± 1 days of incubation at 37°C, the clones were counted.
To enable the expression of the mutant phenotype, 2.10e5 cells/ml were reincubated at 37°C and replated on day 1 at 2.10e5 cells/ml. After two days, the cells were seeded as follows:
Viability plates
1.6 cells/well (one 96-well plate/culture = two plates/dose-level) to de fine the number of viable cells (CE,). After 12 ± 1 days of incubation at 37°C, the clones were counted.
Mutant plates
2000 cells/well (two 96-well plates/culture = four plates/dose-level) to select the TFT (trifluorothymidine resistant) mutant cells (for determination of CEmutant). After 12 ± 1 days of incubation at 37°C in the presence of 4 µg TFT/ml of culture medium, the clones were counted, differentiating small and large colonies:
- size of small colonies, < 25% of the diameter of the well,
- size of large colonies, > 25% of the diameter of the \vell.
- Preliminary toxicity test
To assess the cytotoxicity of the test substance, four dose-levels of DI-tert-DODECYL DISULFIDE (one culture/dose-level) were tested both with and without metabolic activation.
- Mutagenicity experiments In two independent experiments, four dose-levels of DI-tert-DODECYL DISULFIDE (two cultures/dose-level) were tested both with and without metabolic activation. In each experiment, the following controls were included using at least duplicate cultures:
vehicle controls: cultures treated with the vehicle (100 µl ethanol/20 ml culture medium)
positive controls: cultures treated with:
- MMS, in the absence of S9 mix,
- CPA, in the presence of S9 mix.

Evaluation criteria:

A reproducible two-fold increase in the mutant frequency when compared with the vehicle controls, at any dose-level and/or evidence a dose-relationship were considered as a positive result. Reference to historical data, or other considerations of biological relevance might be also taken into account in the evaluation of the data obtained.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The test substance was poorly soluble in the vehicle (ethanol), the limit of solubility in this vehicle being approximately 300 mg/ml. Consequently, with a maximum dose volume of 100 µl/20 ml culture medium, the highest treatment-level feasible was 1500 µg/ml; at this dose-level, a moderate emulsion was observed in the culture medium. The treatment-levels were: 150, 500, 1000 and 1500 µg/ml. No cytoxicity was noted after treatment, with and without S9 mix: the cloning efficiencies CE0 of treated cultures were considered equivalent to that of control cultures.

MUTAGENICITY EXPERIMENTS
The cloning efficiencies CE0 and CE2 and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria and within the range of our historical data. The top dose was selected according to the criteria specified in the international regulations: since the test substance was non-toxic and poorly soluble, the top dose-level was a dose-level above the limit of solubility in the final culture medium at the end of the treatment period. Using an initial solution at 320 mg/ml vehicle and a treatment-volume of 100 µl/20 ml culture medium, the treament-levels were for both mutagenicity experiments with and without S9 mix: 200, 400, 800 and 1600 µg/ml. The cloning efficiencies CE0 and CE2 were considered equivalent to that of the control cultures: RCE0 was at least 72% and RCE2 67%. The test substance did not induce any significant increase in the mutation frequency, with and without S9 mix.

Conclusions:

DI-tert-DODECYL DISULFIDE did not show mutagenic activity in this mouse lymphoma assay

Executive summary:

The potential of DI-tertDODECYL DISULFIDE to induce mutations at the TK (thymidine kinase) locus was evaluated in L5 l 78Y mouse lymphoma cells. After a preliminary toxicity test, DI-tert-DODECYL DISULFIDE was tested in two independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Araclor 1254. Approximately 0.5.10e6 cells/ml in 20 ml culture medium with 5% horse serum were exposed to the test or control substances, in the presence or absence of S9 mix (final concentration of S9 fraction 2% ), for three hours at 37°C. Cytotoxicity was then determined using cloning efficiency (CE0) before expression of the mutant phenotype. Cells viability (using cloning efficiency CE1 ) and number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. The test substance was dissolved in ethanol. The dose-levels for the positive contrals were as follows: without S9 mix: 25 µg/ml of methylmethane sulfonate, with S9 mix: 3 µg/ml of cyclophosphamide.

The cloning efficiencies CE0 and CE2 and mutation frequencies of the vehicle and positive controls were within the range of our historical data. The treament-levels were for both mutagenicity experiments, with and without S9 mix: 200, 400, 800 and 1600 µg/ml; 1600 µg/ml was the highest treatment-level above the limit of solubility in the final culture medium at the end of the treatment-period. The cloning efficiencies CE0 and CE2 were considered equivalent to that of the control cultures. DI-tert-DODECYL DISULFIDE did not induce any significant increase in the mutation frequency, with and without S9 mix. Under our experimental conditions, DI-tert-DODECYL DISULFIDE did not show mutagenic activity in this mouse lymphoma assay.

Reference 3

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:

26th May 1983 and revised draft document of September 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Mammalian Chromosome Aberration Test

Species / strain / cell type:

primary culture, other: human lymphocytes

Details on mammalian cell type (if applicable):

human lymphocytes will corne from whole blood sampled into heparinised sterile tubes obtained from two healthy donors.

Cytokinesis block (if used):

colcemid

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Araclor 1254

Test concentrations with justification for top dose:

50, 100, 200, 400, 600 and 800 µg/ml
The highest treatment-level feasible was 800 µg/ml; at this dose-level, a moderate emulsion was observed in the culture medium.

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

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:

- Treatment
At least three doses of the test substance (two cultures/dose) were tested, with or without metabolic activation. For treatment with S9 mix, 1.5% S9 (i.e. 15% S9 mix) is added to the culture medium. During each test, the following controls were made using at least duplicate cultures:
. negative control: untreated culture,
. vehicle control: culture treated with the vehicle (15 µl ethanol/5.5 ml culture medium),
. positive contrais: culture treated (only for the first harvest time) with:
- MMC, without S9 mix,
- CPA, with S9 mix.
For each culture, 0.5 ml of heparinised whole blood was added to 5 ml of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml) and 3.6% of a mitogen (phytohaemagglutinin, PHA, to stimulate the lymphocytes to divide) in a tube. The tubes were then placed at 37°C for 48 hours. First test Cells were then exposed to the test or control substances, with or without S9 mix, for three hours then rinsed. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/ml) to block cells at the metaphase-stage of mitosis. Harvest time was 20 hours from the beginning of treatment, i.e. approximately 1.5 cell cycle times. As this test gives negative results, both with or without S9 mix, an additional test was performed as follows:·
Second test
. without S9 mix, cells were exposed continuously to the test or control substances
. with S9 mix, cells were exposed to the test or control substances for three hours then rinsed.
One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/ml) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours from the beginning of treatment, i.e. approximately 1.5 cell cycle times and 24 hours after.
After hypotonie treatment (KCI 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

Evaluation criteria:

Biological relevance of the results was considered first. In addition, the following criteria may be used as an aid for deterrnining a positive response: a reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at Ieast one of the doses and one of the two harvest times.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the control cultures. The comparison was performed using the X² test, in which p = 0.05 was used as the lowest level of significance.

Species / strain:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First test:
The test substance was poorly soluble in the vehicle (ethanol), the limit of solubility in this vehicle being approximately 300 mg/ml. Consequently, with a maximum dose volume of 15µl/5.5ml culture medium, the highest treatment-level feasible was 800 µg/ml; at this dose-level, however, a moderate emulsion was observed in the culture medium.
The treatment-levels were: 50, 100, 200, 400, 600, 800 µg/ml. No cytotoxicity was noted after treatment, with or without S9 mix: the mitotic index of treated cultures was considered equivalent to that of contrai cultures. The top dose-level for scoring was selected according to the criteria specified in the international regulations: since the test substance was non-toxic, poorly soluble, the top dose was a dose above the limit of solubility in the final culture medium at the end of the treatment period: 800 µg/ml. Therefore, chromosome aberrations were scored on the slides corresponding to the following three doses: 400, 600, 800 µg/ml.
The test substance did not induce chromosome aberrations both with and without S9 mix after three hours of exposure. Therefore a second test was performed using a continuous exposure without S9 mix until harvest and a later harvest time both with and without S9 mix.
Second test:
The doses for treatment were: 200, 400, 600, 800 µg/ml. The mitotic index of treated cultures was considered equivalent to that of the controls. Therefore the slides corresponding to the three following doses were scored for chromosome aberrations: 400, 600, 800 µg/ml. The test substance did not induce chromosome aberrations both with and without S9 mix for both harvest times. The frequencies of cells with structural chromosome aberrations of the negative, vehicle and positive controls were as specified in acceptance criteria and within the range of our historical data for both tests and both harvest times.

Conclusions:

DI-tert-DODECYL DISULFIDE did not induce chromosome aberrations in cultured human lymphocytes

Executive summary:

The potential of DI-tertDODECYL DISULFIDE to induce chromosome aberrations was evaluated in cultured human lymphocytes. The test substance, dissolved in ethanol, was tested in two independent tests, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254. For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemogglutinin) and incubated at 37°C in a humidified atmosphere of 5% CO2/95% air, for 48 hours.

First test: cells were then exposed to the test or control substances, with or without S9 mix, for three hours then rinsed. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/ml) to black cells at the metaphase-stage of mitosis. Harvest time was 20 hours from the beginning of treatment, i.e. approximately 1.5 cell cycle times. As this test gives negative results, both with or without S9 mix, an additional test was performed as follows:

Second test:

. without S9 mix, cells were exposed continuously to the test or control substances.

. with S9 mix, cells were exposed to the test or control substances for three hours then rinsed.

One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/ml) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours from the beginning of treatment, i.e. approximately 1.5 cell cycle times and 24 hours after.

After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

The doses of DI-tert-DODECYL DISULFIDE were as follows:

First test with and without S9 mix: 50, 100, 200, 400, 600 and 800 µg/rnl for treatment and 400, 600 and 800 µg/ml for chromosomal aberrations scoring. 800 µg/ml being the lowest precipitating dose.

Second test with and without S9 mix: 200, 400, 600 and 800 µg/ml for treatment and 400, 600 and 800 µg/ml for chromosomal aberrations scoring. The doses of the positive controls were 3 µg/ml mitomycin C (MMC) without S9 mix (3 hours of treatment) or 0.2 µg/ml (continuous treatment) and 50 µg/ml of cyclophosphamide (CPA) with S9 mix.

The frequency of cells with structural chromosome aberrations in the negative, vehicle and positive controls was as specified in the acceptance criteria and within the range of the historical data. The test substance did not induce any significant increase in the frequency of cells with structural chromosome aberrations, with or without S9 mix, for both of the two tests and for the two harvest times. Under these experimental conditions, DI-tert-DODECYL DISULFIDE did not induce chromosome aberrations in cultured human lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

DI-TERT-DODECYL DISULPHIDEt is not classified for germ cell mutations according to CLP and GHS Regulations.