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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Polysulfides, di-tert-butyl was not genotoxic in an in vitro bacterial reverse mutation assay (OECD TG 471) and an in vitro Mammalian Chromosomal Aberration assay (OECD TG 473) with or without metabolic activation A positive response was observed in the mouse lymphoma assay (OECD TG 476). In this assay, it is noteworthy that small colonies are in particular responsible for the increase in the mutation frequency of total induced mutants. This observation is in favour of an intrinsic clastogenic activity of the substance.

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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:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Human Lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254
Test concentrations with justification for top dose:
. for treatment:
- 3, 10, 30, 100, 300, 500 µg/ml with and without S9 mix.
As very strong toxicity was seen at 300 and 500 µg/ml and as the requested toxicity was not obtained in this experiment for the lower dose-levels without S9 mix, it was repeated using the following dose-levels: 50, 100, 150, 200, 250 µg/ml.
. for chromosome aberrations scoring:
- without S9 mix: 150, 200, 250 µg/ml
- with S9 mix: 3, 10, 30 µg/ml.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9 mix, mitomycin C: 0.2 µg/ml. With S9 mix, cyclophosphamide: 50 µg/ml.
Details on test system and experimental conditions:
No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used and dose-levels for scoring of chromosome aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI), when possible.

For each culture (two cultures/dose level):, 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.

Treatment was as follows:
. 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 and then rinsed.
Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. 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.

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.
Evaluation criteria:
Acceptance criteria
The study was considered valid since the following criteria were met:
. the frequency of celas with structural chromosome aberrations in the vehicle controls was within the range of our historical data,
. the frequency of cells with structural chromosome aberrations in the positive controls was significantly higher than that of the controls and within the range of our historical data.

Evaluation criteria
A statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was 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 findings.
Statistics:
For each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle 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:
other: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 300 µg/ml without S9, >= 100 µg/ml with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TPS 44 was poorly soluble in the vehicle (DMSO), the limit of solubility being approximately 100 mg/ml.

Consequently, with a maximum dose-volume of 100 µl/5.5 ml culture medium, the dose levels were for treatment:
- 3, 10, 30, 100, 300, 500 µg/ml.

A moderate emulsion was observed in the culture medium at 500 µg/ml.

The top dose-level for scoring chromosome aberrations was selected according to criteria specified in the international regulations: since the test substance was toxic, the top doselevel was based on the level of toxicity, i.e. when possible a reduction greater than 50% of mitotic index.

Without S9 mix, marked cytoxicity was noted after treatment:
- at 500 µg/ml, haemolysis was observed and, therefore, no harvest was performed;
- at 300 µg/ml, the mitotic index was in mean 19 and 22% of control cultures, for the first and the second harvest times respectively. At lower dose-levels, no toxicity was seen.

As the request degree of cytotoxicity was not achieved, this experiment was consequently considered as a preliminary toxicity test (only mitotic index was scored). A new experiment was performed at the following treatment-levels:
- 50, 100, 150, 200, 250 pg/ml.
However, no marked reduction of the mitotic index was noted.

The highest dose-level of this new experiment (250 µg/ml) being very closed to the previous dose-level of the preliminary toxicity test (300 µg/ml) which induced marked toxicity (approximately 80% reduction of the mitotic index), the requested toxicity was considered as satisfactory. The slides corresponding to the three highest dose-levels of the experiment were consequently scored for chromosome aberrations.

With S9 mix, cytotoxicity of the test substance was shown by the reduction of the mitotic index of treated cultures when compared to control cultures:
. at 100, 300 and 500 µg/ml, the mitotic index was between 0 and 12% of the control, for the two harvest times.
at 30 µg/ml, it was 47% or 56% of the control for the first or second harvest times respectively,
at 10 and 3 µg/ml, it was quite equivalent to the control.

Therefore, chromosome aberrations were scored on the slides corresponding to the following dose-levels: 3, 10, 30 pg/ml.

The test substance did not induce chromosome aberrations both with and without S9 mix for the two harvest times.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria and within the range of our historical data for both harvest times.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
TPS 44 did not induce chromosome aberrations in cultured human lymphocytes.
Executive summary:

The potential of di-tert-butyl polysulfides (TPS44) to induce chromosome aberrations was evaluated in cultured human lymphocytes. TPS 44 was tested, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used and dose-levels for scoring of chromosome aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI), when possible. 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. Without S9 mix, cells were exposed continuously to the test or control substance(mitomycin C: 0.2 µg/ml), with S9 mix, cells were exposed to the test or control substance (cyclophosphamide: 50 µg/ml) for three hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. 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. After hypotonic treatment (KC1 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. TPS 44 was dissolved in dimethylsulfoxide (DMSO). The dose-levels of TPS44 scored for chromosomal aberrations (two cultures/dose level) were 150, 200, 250 µg/ml, without S9mix and 3, 10, 30 µg/ml, with S9mix. The frequency of cells with structural chromosome aberrations in the vehicle and positive controls was as specified in the acceptance criteria and within the range of the historical data. TPS 44 did not induce any significant increase in the frequency of cells with structural chromosome aberrations, with and without S9 mix for the two harvest times. TPS44 did not induce chromosome aberrations in cultured human lymphocytes.

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:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine reversion
Species / strain / cell type:
S. typhimurium, other: strains: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
a liver microsomal fraction S9 of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
- without S9 mix:
. 31.25, 62.5, 125, 250 and 500 µg/plate for the first assay,
. 3.90, 7.81, 15.63, 31.25 and 62.5 µg/plate for the second assay,
. 3.125, 6.25, 12.5, 25 and 50 µg/plate for the third assay.
- with S9 mix:
. 31.25, 62.5, 125, 250 and 500 µg/plate for both assays.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: NaN3 (TA 1535 & TA 100), 9AA (TA 1537) and 2-NF (TA 1538 & TA98). +S9: 2-anthramine (all strains)
Details on test system and experimental conditions:
Toxicity test:
In order to determine the maximum concentration of the test substance which does not modify the bacterial growth, 5 concentrations were tested in the TA 100 strain. If toxicity occurred, the background lawn was sparse compared to control plates and/or the number of colonies had decreased. The sterility of the test substance was checked during this assay.

Mutagenicity tests:
In 2 independent assays, 5 concentrations of the test substance (3 plates/concentration) were tested on the 5 strains with and without metabolic activation. Simultaneously, spontaneous revertants of each strain alone (negative control) or in the presence of the solvent (solvent control) were performed. The strains sensitivity and the metabolic activation ability of S9 mix were checked in the presence of known mutagens (positive controls). The sterility of the S9 mix was checked during each assay: before the beginning and at the end of the experiment.
The methods used were:
- the direct plate incorporation method for the 2 assays without S9 mix and for the first assay with S9 mix,
- the preincubation method (1 h, 37'C) for the second assay with S9 mix.
Evaluation criteria:
- a test substance is considered as mutagenic if, for each test, it induces a doubling in the number of revertants when compared to that in the negative and/or solvent controls, for at least one of the tested strains and at one or more of the tested concentrations. In this case, a statistically significant dose relationship is investigated, using a linear regression analysis, and considered as significant if p 0.05 (for n = 18 values, the correlation coefficient r must be >= 0.47).

- a test substance is considered as non-mutagenic if the above two criteria are not fully met.
Statistics:
None
Species / strain:
S. typhimurium, other: strains: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 25 µg/plate without S9|>= 500 µg/plate with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TPS 44 did not induce a significant increase in the revertant number with or without S9 mix in any of the 5 strains. The negative and solvent control results were equivalent to those usually obtained in our Laboratory. The number of revertants induced by the positive controls was higher than the spontaneous one, which demonstrated the sensitivity of this test and the efficacy of the S9 mix throughout this study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
TPS 44 did not show mutagenic activity in the Ames test.
Executive summary:

The in vitro potential mutagenic activity of di-tert-butyl polysulfides (TPS 44) was investigated by the Ames test using 5 strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 1538, TA 98 and TA 100. After a preliminary assay to define the concentrations to be used for the mutagenicity study, the test substance was tested on two independent assays. Each assay was carried out both in the absence and in the presence of a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction S9 of rats treated with Aroclor 1254. The direct plate incorporation method for the 2 assays without S9 mix and for the first assay with S9 mix, and the preincubation method (1 h, 37'C) for the second assay with S9 mix were used.

The concentrations were 31.25, 62.5, 125, 250 and 500 µg/plate for the first assay, 3.90, 7.81, 15.63, 31.25 and 62.5 µg/plate for the second assay, and 3.125, 6.25, 12.5, 25 and 50 µg/plate for the third assay, without S9mix and 31.25, 62.5, 125, 250 and 500 µg/plate for both assays with S9mix. TPS 44 did not induce a significant increase in the revertant number with or without S9 mix in any of the 5 strains. The negative and solvent control results were equivalent to those usually obtained in the Laboratory. The number of revertants induced by the positive controls was higher than the spontaneous one, which demonstrated the sensitivity of this test and the efficacy of the S9 mix throughout this study. In conclusion, TPS 44 did not show mutagenic activity in the Ames test.

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:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- mouse lymphoma cells were obtained from ATCC (American Type Culture Collection - Rockville, MD 20852 - USA). A stock of these cells is maintained and stored frozen in liquid nitrogen.
Contamination by mycoplasma is checked using Mycoalert Mycoplasma Detection kit (Cambrex Bio Science Rockland, inc) for each batch of the cells. Only the batches, which contain no mycoplasma, are used in the mutagenicity test.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of rat liver induced by Aroclor 1254
Test concentrations with justification for top dose:
Without S9 mix : 40, 30, 20, 15 and 10 µg/ml (assay 1: 3-hour treatment); 50, 41.6, 34.7, 28.9, 24.1 and 20.1 µg/ml(assay 2: 24-hour treatment)
With S9 mix : 80, 60, 40, 30, 20, 15 and 10 µg/ml (assay 1); 80, 60, 40, 30 and 20 µg/ml (assay 2)
Vehicle / solvent:
As TPS 44 was not soluble in aqueous solvent, the test item was dissolved in DMSO. TPS 44 was soluble at 500 mg/mL DMSO without any micelles.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9-mix : methyl methanesulfonate 10 µg/mL (3-h treatment); methyl methanesulfonate 2 µg/mL (24-h treatment). With S9-mix : cyclophosphamide, 2 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration:
Without S9-mix : 3 hours (short treatment); 24 hours (continuous treatment)
With S9-mix : 3 hours
- Expression time (cells in growth medium): 2 days after treatment
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): TFT (3 µg/mL)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Concentrations tested expressed as µg/mL pure Di-tertio-butyl polysulfide (TPS 44)
Without S9-mix : 75 – 37.5 – 18.75 – 9.38 – 4.70 – 2.40 – 1.20 (3-h treatment)
50 – 25 – 12.5 – 6.25 – 3.13 – 1.56 – 0.78 (24-h treatment)
With S9-mix : 150 - 75 – 37.5 – 18.75 – 9.38 – 4.70 – 2.40
Evaluation criteria:
A test item is considered as mutagenic in this system if the following conditions are fulfilled:
1. The induced mutation frequency for at least one tested concentration is higher than the mutation frequency in the vehicle control cultures by at least the global evaluation factor of 126 x10-6 (Moore et al., 2006).
2. A statistical trend test demonstrates a positive dose related increase in the mutation frequency (Moore et al., 2006).
3. The results have to be reproducible in an independent study, at least from a qualitative point of view.
If none of the three criteria mentioned above is fulfilled, the tested test item is considered as not mutagenic in this study system.
In all other cases, the results are discussed case by case, and the results obtained on other study systems are taken into account.
All these criteria are not absolute: however, they give help when a decision has to be taken, making a conclusion possible in the majority of the cases.
Statistics:
Statistical evaluation of data for the total number of mutants and for small colony mutants is performed using the method proposed by Robinson et al. (Statistical evaluation of bacterial/mammalian fluctuation tests. In Statistical evaluation of mutagenicity test data. KIRKLAND D.J. (Ed). Cambridge University Press, Cambridge- New York, (1990) 102-140)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH was comprised in the acceptable range of 6-8 at the highest concentrations tested from 2500 to 78.13 µg/mL.
- Effects of osmolality: The concentrations of 1250, 625, 312.5, 156.25 µg/mL induced no variation in osmolarity higher than 50 mOsmol when compared to the solvent control.
- Precipitation: The concentrations of 1250, 625, 312.5, 156.25 µg/mL (i.e. when initial solutions are added at 0.5% in the culture medium), were still cloudy but no particles were visible anymore (unaided eye).
Conclusions:
Di-tertio-butyl polysulfide (TPS 44) induced a biologically significant mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture only in presence of metabolic activation, in two independent assays. The clear increase in the number of small colonies is in favour of a clastogenic activity. Conversely, no mutagenic effect was noted in absence of S9-mix.
Executive summary:

The potential of Di-tertio-butyl polysulfide (TPS 44) to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated in a study performed according to the international guidelines OECD No. 476 and Good Laboratory Practice. After a preliminary toxicity test, Di-tertio-butyl polysulfide (TPS 44) was tested in two independent experiments, with and without a metabolic activation system, the S9-mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 5 x 10e6 (3-hour treatment) or 1.25 x 10e6 (24-hour treatment) cells/mL in 10 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9-mix (final concentration of S9 fraction 5%), at 37°C. Cytotoxicity was measured by assessment of plating efficiency, Relative Survival Growth (RSG), and Relative Total Growth (RTG), after treatment (T0) and 48 hours after treatment (PE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. The Di-tertio-butyl polysulfide (TPS 44) was dissolved in DMSO and the positive controls were methylmethane sulfonate (without S9-mix) and Cyclophosphamide (with S9-mix). In the culture medium, the concentration of 1250, 625, 312.5, 156.25 µg/mL (i.e. when initial solutions are added at 0.5% in the culture medium), were still cloudy but no particles were visible anymore. At these concentrations, the pH and the osmolality values were comparable to those of the vehicle control culture. The cloning efficiencies and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. The selected concentrations were 40, 30, 20, 15 and 10 µg/ml (assay 1: 3-hour treatment) and 50, 41.6, 34.7, 28.9, 24.1 and 20.1 µg/ml (assay 2: 24-hour treatment) without S9 -mix and 80, 60, 40, 30, 20, 15 and 10 µg/ml (assay 1) and 80, 60, 40, 30 and 20 µg/ml (assay 2) with S9 -mix. Following the 3-hour treatment, cytotoxicity ( decreased adjusted RTG) was observed at 40 µg/ml without S9 -mix and from 60 µg/ml with S9 -mix. In the 24-hour treatment without S9-mix, cytotoxicity was observed from 34.7 µg/ml. A biologically significant mutagenic activity was observed only in presence of metabolic activation, in the two independent assays. The clear increase in the number of small colonies is in favour of a clastogenic activity. Conversely, no mutagenic effect was noted in absence of S9-mix.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Polysulfides, di-tert-butyl was not genotoxic in an in vivo bone marrow micronucleus assay in mice (OECD TG 474), in the presence of a significant bone marrow toxicity.

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:
06 September 2010 - ...........................................
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate coherence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: on the dfay of treatment, the animals were approximately 6 weeks old
- Weight at study initiation: 33.6 g (ranging from 31.8 to 37.5 g)
- Housing: the animals were housed by 2 or 3, in Individually Ventilated Cages (IVC) (polysulfone 900 cm2, Tecniplast) containing autoclaved sawdust
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered with a 0.22 µm filter) contained in bottles
- Acclimation period: at least 5 days before the day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%,
- Air changes (per hr): at least 12 cycles/hour of filtered non-recycled fresh air
- Photoperiod (hrs dark / hrs light): 12 h / 12 h
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: 0.5% aqueous carboxymethylcellulose
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Lot/batch no.: 060M0017
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was mixed with the required quantity of vehicle, under magnetic stirring
and until the obtention of a satisfactory homogenization.
For the preliminary test then for the main experiment, the test item was prepared in the vehicle at the concentration of 200 mg/mL.
The dosage forms were prepared daily by the CIT Pharmacy, within the 4 hours before use, and delivered to the study room in brown flasks, at room
temperature.
Duration of treatment / exposure:
A period of 2 days (2 treatments).
Frequency of treatment:
Two treatments separated by 24 hours.
Post exposure period:
24 hours
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
8 males.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control was Cyclophosphamide.
- Route of administration: dissolved in distilled water
- Concentration: 5 mg/mL.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In order to select the top dose-level for the cytogenetic study, the dose-level of 2000 mg/kg/day was administered twice 24 hours apart, to three males and three females.
At this dose-level, neither mortality, nor clinical signs were observed in the animals throughout the observation period.
Despite that the PE/NE ratio of the females given the test item at 2000 mg/kg/day was slightly lower than the corresponding vehicle control, all ratios remained consistent with the historical data.
The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no signs of toxicity were observed at 2000 mg/kg/day and in the absence of any clear differences between sexes, the main experiment was undertaken as a limit test at 2000 mg/kg/day using only male mice.

DETAILS OF SLIDE PREPARATION:
At the time of sacrifice, all the animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital, then killed by cervical dislocation. The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS:
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
The analysis of the slides was performed at CIT and the scoring was performed "blind".
Evaluation criteria:
For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the
concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the
evaluation of data obtained.
Statistics:
Normality and homogeneity of variances will be tested using a Kolmogorov Smirnov test and a Bartlett test.
When normality and homogeneity of variances were demonstrated, the statistical comparisons was performed using a Student t-test (two groups) or a one-way analysis of variance (¿ 3 groups) followed by a Dunnett test (when necessary).
When normality or homogeneity of variances was not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (¿ 3 groups) was performed followed by a Dunn test (when necessary).
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
PE/NE ratio decreased
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Neither mortality, nor clinical signs were observed in any animals given 2000 mg/kg/day throughout the observation period.

The mean values of MPE of the vehicle control and positive control groups were within the corresponding historical control ranges. The cyclophosphamide induced a significant increase (p < 0.05) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

The PE/NE ratio of the vehicle control was found above the historical data range. This value is mainly due one out of five animals and it is considered not to impact on the validity of the test.
When compared to the vehicle control, the PE/NE ratio of the test item treated group decreased significantly (p < 0.05), showing that the bone marrow cells were effectively exposed to the test item.

The mean value of MPE in the group treated with the test item was found not different from that of the vehicle group.
Conclusions:
The test item Di-tertio-butyl polysulfides (TPS 44) did not induce damage to the chromosomes or the mitotic apparatus of male mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-level of 2000 mg/kg/day.
Executive summary:

The potential of the test item Di-tertio-butyl polysulfides (TPS 44) to induce structural or numerical damage was evaluated in bone marrow cells of mice. The study was performed according to the international guidelines (OECD 474 and Commission Directive No. B12) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no signs of toxicity were observed at 2000 mg/kg/day and in the absence of any clear differences between sexes, the main experiment was undertaken as a limit test at 2000 mg/kg/day using only male mice. In the main study, one group of five maleSwiss Ico: OF1 (IOPS Caw) mice received oral administrations of Di-tertio-butyl polysulfides (TPS 44) at the dose-level of 2000 mg/kg/day, over a 2-day period. One group of five males received the vehicle (0.5% aqueous carboxymethylcellulose) under the same experimental conditions, and acted as control group. One group of five males received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg/day. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Neither mortality, nor clinical signs were observed in any animals given 2000 mg/kg/day throughout the observation period. The mean values of MPE of the vehicle control and positive control groups were within the corresponding historical control ranges. The cyclophosphamide induced a significant increase (p < 0.05) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid. When compared to the vehicle control, the PE/NE ratio of the test item treated group decreased significantly (p < 0.05), showing that the bone marrow cells were effectively exposed to the test item. The mean value of MPE in the group treated with the test item was found not different from that of the vehicle group.

Di-tertio-butyl polysulfides (TPS 44) did not induce damage to the chromosomes or the mitotic apparatus of male mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-level of 2000 mg/kg/day.

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

Additional information

Bacterial gene mutation assay

The in vitro mutagenic activity of polysulfides, di-tert-butyl was investigated by the Ames test using 5 strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 1538, TA 98 and TA 100 (Molinier, 1992). After a preliminary assay to define the concentrations to be used for the mutagenicity study, the test substance was tested on two independent assays. Each assay was carried out both in the absence and in the presence of a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction S9 of rats treated with Aroclor 1254. The direct plate incorporation method for the 2 assays without S9 mix and for the first assay with S9 mix, and the preincubation method (1 h, 37'C) for the second assay with S9 mix were used.

The concentrations were 31.25, 62.5, 125, 250 and 500 µg/plate for the first assay, 3.90, 7.81, 15.63, 31.25 and 62.5 µg/plate for the second assay, and 3.125, 6.25, 12.5, 25 and 50 µg/plate for the third assay, without S9 mix and 31.25, 62.5, 125, 250 and 500 µg/plate for both assays with S9 mix. Di-tert-butyl polysulfides did not induce a significant increase in the revertant number with or without S9 mix in any of the 5 strains. The negative and solvent control results were equivalent to those usually obtained in the Laboratory. The number of revertants induced by the positive controls was higher than the spontaneous one, which demonstrated the sensitivity of this test and the efficacy of the S9 mix throughout this study. In conclusion, di-tert-butyl polysulfides did not show mutagenic activity in the Ames test.

Mammalian gene mutation assay

The potential of polysulfides, di-tert-butyl to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated in a study performed according to the guideliness OECD No. 476 and in compliance with GLP (Simar, 2010). After a preliminary toxicity test, polysulfides, di-tert-butyl was tested in two independent experiments, with and without a metabolic activation system, the S9-mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Approximately 5 x 10e6 (3-hour treatment) or 1.25 x 10e6 (24-hour treatment) cells/mL in 10 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9-mix (final concentration of S9 fraction 5%), at 37°C. Cytotoxicity was measured by assessment of plating efficiency, Relative Survival Growth (RSG), and Relative Total Growth (RTG), after treatment (T0) and 48 hours after treatment (PE2). The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype. Polysulfides, di-tert-butyl was dissolved in DMSO and the positive controls were methylmethane sulfonate (without S9-mix) and Cyclophosphamide (with S9-mix). In the culture medium, the concentration of 1250, 625, 312.5, 156.25 µg/mL (i. e. when initial solutions are added at 0.5% in the culture medium), were still cloudy but no particles were visible anymore. At these concentrations, the pH and the osmolality values were comparable to those of the vehicle control culture. The cloning efficiencies and the mutation frequencies of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. The selected concentrations were 40, 30, 20, 15 and 10 µg/ml (assay 1: 3-hour treatment) and 50, 41.6, 34.7, 28.9, 24.1 and 20.1 µg/ml (assay 2: 24-hour treatment) without S9 -mix and 80, 60, 40, 30, 20, 15 and 10 µg/ml (assay 1) and 80, 60, 40, 30 and 20 µg/ml (assay 2) with S9 -mix. Following the 3-hour treatment, cytotoxicity (decreased adjusted RTG) was observed at 40 µg/ml without S9 -mix and from 60 µg/ml with S9 -mix. In the 24-hour treatment without S9-mix, cytotoxicity was observed from 34.7 µg/ml. A biologically significant mutagenic activity was observed only in presence of metabolic activation, in the two independent assays. The clear increase in the number of small colonies is in favour of a clastogenic activity. Conversely, no mutagenic effect was noted in absence of S9-mix.

Chromosomal aberration assay

In vitro

The potential of polysulfides, di-tert-butyl to induce chromosome aberrations was evaluated in cultured human lymphocytes. Polysulfides, di-tert-butyl was tested, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254 (de Jouffrey, 1996). No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used and dose-levels for scoring of chromosome aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI), when possible. 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. Without S9 mix, cells were exposed continuously to the test or control substance(mitomycin C: 0.2 µg/ml), with S9 mix, cells were exposed to the test or control substance (cyclophosphamide: 50 µg/ml) for three hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. 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. After hypotonic treatment (KC1 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. Polysulfides, di-tert-butyl was dissolved in dimethylsulfoxide (DMSO). The dose-levels of polysulfides, di-tert-butyl scored for chromosomal aberrations (two cultures/dose level) were 150, 200, 250 µg/ml, without S9 mix and 3, 10, 30 µg/ml, with S9 mix. The frequency of cells with structural chromosome aberrations in the vehicle and positive controls was as specified in the acceptance criteria and within the range of the historical data. Polysulfides, di-tert-butyl did not induce any significant increase in the frequency of cells with structural chromosome aberrations, with and without S9 mix for the two harvest times. Polysulfides, di-tert-butyl did not induce chromosome aberrations in cultured human lymphocytes.

In vivo

The potential of polysulfides, di-tert-butyl to induce cytogenetic damage was evaluated in bone marrow cells of mice (Sire, 2010). The study was performed according to the guidelines (OECD 474 and Commission Directive No. B12) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. The top dose-level for the cytogenetic test was selected according to the criteria specified in the test guidelines; since no signs of toxicity were observed at 2000 mg/kg/day and in the absence of any clear differences between sexes, the main experiment was undertaken as a limit test at 2000 mg/kg/day using only male mice. In the main study, one group of five maleSwiss Ico: OF1 (IOPS Caw) mice received oral administrations of polysulfides, di-tert-butyl at the dose-level of 2000 mg/kg/day, over a 2-day period. One group of five males received the vehicle (0.5% aqueous carboxymethylcellulose) under the same experimental conditions, and acted as control group. One group of five males received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg/day. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Neither mortality, nor clinical signs were observed in any animals given 2000 mg/kg/day throughout the observation period. The mean values of MPE of the vehicle control and positive control groups were within the corresponding historical control ranges. The cyclophosphamide induced a significant increase (p < 0.05) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid. When compared to the vehicle control, the PE/NE ratio of the test item treated group decreased significantly (p < 0.05), showing that the bone marrow cells were effectively exposed to the test item. The mean value of MPE in the group treated with the test item was found not different from that of the vehicle group.

Polysulfides, di-tert-butyl did not induce damage to the chromosomes or the mitotic apparatus of male mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-level of 2000 mg/kg/day.


Justification for classification or non-classification

According to the available data and criteria of Regulation (EC) No 1272-2008, no classification is warranted for polysulfides, di-tert-butyl as a germ cell mutagen..