Polysulfides, di-tert-dodecyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Polysulfides, di-tert-dodecyl was not mutagenic in an in vitro bacterial reverse mutation assay (similar to OECD TG 471) or in an in vitro mammalian chromosomal aberration assay (OECD TG 473) with or without metabolic activation. In a mouse lymphoma gene mutation assay with L5178Y cells (OECD TG 46), a biologically significant mutagenic activity was observed either with or without metabolic activation. The clear increase in the number of small colonies was in favour of a clastogenic activity.

 

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

not precised in the report

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

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:

S9 fraction from liver homogenates of rats induced with 500 mg/kg bw Aroclor 1254

Test concentrations with justification for top dose:

0, 5, 150, 500, 150, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: 2-nitrofluorene (TA98 & TA1538), ENNG (TA100 & TA1535), 9-aminoacridine (TA1537). With S9: 2-aminoanthracene (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 2 (3 plates per concentration)

DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was performed to define the concentrations to be used for the mutagenicity study.

Evaluation criteria:

- negative and positive controls within the range of historical controls.
- positive: reproducible and significant dose related increase in revertants and/or reproducible doubling in the number of revertants compared with negative controls for one dose.

Species / strain:

S. typhimurium TA 100

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TPS-32 was not toxic towards the tester strains. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with TPS-32 at any dose level, either in the presence or absence of metabolic activation (S-9 mix).

Conclusions:

Not mutagenic

Executive summary:

In a study similar to OECD TG 471, a Salmonella typhimurium reverse mutation assay was performed with di-t-dodecyl polysulfides. S. typhimurium strains, TA98, TA100, TA1535, TA1537, and TA1538 were exposed to five concentrations of di-t-dodecyl polysulfides in the presence and absence of a metabolic activation system using plate incorporation at doses of 5 to 5,000 mg/plate. Exposure to five graded doses of di-t-dodecyl polysulfides in the presence and absence of metabolic activation, did not increase the reversion rates in any of the tester strains.

Reference 2

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:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction (S9) of rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

0, 300, 1000, 2500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C (0.2 µg/ml) without S9, Cyclophosphamide (50µg/ml) with S9.

Details on test system and experimental conditions:

The conditions of treatment were as follows, using 2cultures/experimental point:
. without S9 mix: the cultures were incubated with the test or control substances which remained in the culturemedium, until the appropriate harvest times*: 24 and 48 hours
. with S9 mix: the test or control substances remained inculture medium containing 15% S9 mix (10% S9/S9 mix) for 2hours.
The cells were then centrifuged, the treatment medium removed, the cells resuspended in fresh culture medium. The cultures were then incubated until the appropriate harvest times*: 24 and 48 hours. Two hours before harvesting, the cells were treated with a colcemid solution to block them at themetaphase-stage of mitosis. The chromosomal preparations were stained and screened microscopically for mitotic index and for aberrations: 200 well-spread metaphases perconcentration were read, whenever possible. The concentrations of TPS 32 for scoring were: 300, 1000 and 2500 µg/ml, 2500 µg/ml being the limit of solubility of the test substance in the culture medium.

Evaluation criteria:

The following criteria were used as an aid for determining a positive response:
. a reproducible and statistically significant increase in the aberrant cells frequency for at least one of the tested concentrations.
A test substance was considered as non-clastogenic in this test system if there is no significant increase in aberrant cells frequency at any dose above concurrent control frequencies and in both of the two harvest times.
Both biological and statistical significance was considered together in the evaluation.

Statistics:

For each harvest time, the aberrant cells frequency (exciuding gaps) in treated cultures was compared to that of the vehicle cultures. The comparison was performed using the X² test , in which p = 0.05 was used as the lowest level of significance.

Key result

Species / strain:

other: human lymphocytes

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:

TPS 32 did not show clastogenic activity in this chromosomal aberration test performed in cultured human lymphocytes.

Executive summary:

A chromosomal aberration test with human lymphocytes was performed according to OECD guideline #473 with di-t-dodecyl polysulfides (TPS 32) at the concentrations of 300, 1000 and 2500 µg/ml (2500 µg/ml being the limit of solubility in the culture medium). The test was carried out with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254. Without S9 mix, the cultures were incubated with the test or control substances, which remained in the culture medium, until the appropriate harvest times at 24 and 48 hours. With S9 mix, the test or control substances remained in a culture medium containing 15% S9 mix (10% S9/S9 mix) for 2 hours. The cells were then centrifuged, the treatment medium removed, the cells resuspended in fresh culture medium. The cultures were then incubated until the appropriate harvest times at 24 and 48 hours. Two hours before harvesting, the cells were treated with a colcemid solution to block them at the metaphase-stage of mitosis. The chromosomal preparations were stained and screened microscopically for mitotic index and for aberrations, 200 well-spread metaphases per concentration were read, whenever possible. The aberrant cells frequency in the negative and vehicle controls was within the range of the historical data (i.e. 0.5 ± 0.6%, gaps excluded). The aberrant cells frequency in the positive controls was significantly higher (p < 0.001) than that of the negative controls, indicating the sensitivity of the test system. Di-t-dodecyl polysulfides(TPS 32) did not induce any significant increase in the aberrant cells frequency, with or without S9 mix, for both of the 2 harvest times.

Reference 3

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:

1997

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 : 175, 131.25, 87.5, 43.75, 21.88, 10.94 and 5.47 µg/ml (assay 1); 175, 153.13, 131.25, 109.38, 87.5, 65.63 and 43.75 µg/ml (assay 2)
With S9 mix : 350, 175, 87.5, 43.75, 21.88 and 10.94 µg/ml (assay 1); 350, 280, 224, 179.2, 143.36, 114.69 and 91.75 µg/ml (assay 2)

Vehicle / solvent:

The di-tert-dodecyl polysulfides (TPS 32) was dissolved in dimethylsulfoxide (DMSO) at the maximum initial concentration of 70 mg/mL, corresponding to the maximum of solubility and used at 0.5% in culture medium, giving a final concentration of 350 µg/mL. At this concentration, the solution was clear. Furthermore, when added at 0.5% in the culture medium, no precipitate was noted.

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). 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) (as a positive response was obtained in the first assay using a 3-h treatment, the second assay was performed under the same experimental conditions)
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 di-tert-dodecyl polysulfides (TPS 32) as supplied
• Without S9-mix : 350 – 175 – 87.5 – 43.75 – 21.88 – 10.94 – 5.47 (3h treatment)
175 – 87.5 – 43.75 – 21.88 – 10.94 – 5.47 – 2.73 – 1.37 – 0.68 (24h treatment)
• With S9-mix : 350 – 175 – 87.5 – 43.75 – 21.88 – 10.94 – 5.47

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)

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

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 350 to 87.5 µg/mL.
- Effects of osmolality: The concentrations of 350, 175 and 87.5 µg/mL induced no variation in osmolarity higher than 3 mOsmol when compared to the solvent control.
- Precipitation: no precipitate was noted.

CYTOTOXICITY ASSAY

The results of the cytotoxicity assays without and with metabolic activation by S9-mix are summarized in Table 1.

Without metabolic activation after a 3-hour treatment, di-tert-dodecyl polysulfides (TPS 32) revealed a potent cytotoxicity in L5178Y cells, with 0.4% of adjusted RTG at the maximum concentration tested of 350 µg/mL (Table 1). The immediately lower concentration of 175 µg/mL, also induced a strong level of cytotoxicity with an adjusted RTG of 8.2% (i.e. below the lowest acceptable level of toxicity set at 10%). But this concentration was retained as the maximum concentration for the first mutagenicity test without S9-mix.

In the corresponding first mutagenicity assay (Table 15), this concentration induced a strong but acceptable toxicity with an adjusted RTG of 20.4%, i.e. close to the range 10-20% for the highest toxic concentration, and was thus assessed for mutagenicity. In the second mutagenicity assay (Table 21), this concentration induced a too strong cytotoxicity with an adjusted RTG of 5.3%. Furthermore, the immediately lower concentration of 153.1 µg/mL induced an acceptable level of cytotoxicity with an adjusted RTG of 13.7%, and was thus retained for the mutagenicity assessment. The concentration of 175 µg/mL was not retained for the interpretation of the results.

With metabolic activation, a strong but acceptable cytotoxicity was noted at the highest concentration tested of 350 µg/mL, with 21.6% of adjusted RTG (Table 1). Under these conditions, 350 µg/mL was retained as the top concentration for the mutagenicity test with S9-mix. In the corresponding mutagenicity assays (Tables 15 and 21), this concentration induced a strong but acceptable toxicity with adjusted RTGs of 23.7 and 37.9%, respectively and was thus retained for the mutagenicity assessment.

Without metabolic activation after a 24-hour treatment, di-tert-dodecyl polysulfides (TPS 32) revealed a too strong cytotoxicity with an absence of cell growth at the highest concentration tested of 350 µg/mL ; this concentration was thus discontinued (data are not shown). The 2 immediately lower concentrations of 175 and 87.5 µg/mL induced potent cytotoxicities in L5178Y cells, with 0.3 and 4.2% of adjusted RTG, respectively (Table 1). The concentration just below this induced a strong but acceptable level of cytotoxicity with an adjusted RTG of 26%. As a positive response was obtained in the first assay using a 3-h treatment, the second assay was

performed under the same experimental conditions.

MUTAGENICITY ASSAYS

Concurrently to the main assays, tests were carried out with reference mutagenic compounds (methyl methanesulfonate in the absence of metabolic activation and cyclophosphamide in the presence of metabolic activation via S9-mix).

The plating efficiency of the negative control (mean of the 2 cultures) ranged from 65 to 120 % at T2 (Tables 12, 14, 18 and 20).

The mutation frequency (MF) of the negative control is within the range of historical data of the laboratory, and ranged from 50 to 170 x10-6 mutants. In the second assay in presence of metabolic activation, the value for the mutation frequency of the negative control was above the limit previously observed in historical data (i.e. 166.8 vs. 137.7). Nevertheless, as the test item was found mutagenic in this condition, the deviation was considered as minor and did not affect the quality or the integrity of the current study.

The suspension growth value of the negative control ranged from 8 to 32.

The induced mutation frequencies (IMF) for the positive controls were significantly increased when compared to the MF for the solvent control, and demonstrated an increase above the spontaneous background MF of at least 300 x10-6 mutants, except in the 1st assay in presence of metabolic activation where the value for the IMF was of 288.1 x10-6 mutants, i.e. very close to the GEF. The observed values were within or close to the limits of historical positive controls of the laboratory. The acceptance criteria for the results were thus fulfilled.

The summaries of the test results are given in Tables 2 to 4. The individual results are shown in Appendix No. 1 (Tables 5 to 22).

In the first assay using a 3-hour treatment without metabolic activation (Tables 2, 15), statistically significant increases in the mutation frequency of total induced mutants (small and large colonies) were noted at the 2 highest concentrations tested of 175 and 131.25 µg/mL. Furthermore, a biologically significant increase was noted with an induced mutation frequency (IMF) of 189.1 x10-6 mutants at the highest dose of 175 µg/mL, that is to say above the global evaluation factor (GEF) of +126 x10-6 for a biologically significant effect (Moore et al., 2006).

This effect was reproducible as in the second assay performed under the same experimental conditions (Tables 2, 21), statistically significant increases in the mutation frequency of total induced mutants (small and large colonies) were noted. Biological significances were observedat the 3 highest analysable concentrations from 109.38 to 153.13 µg/mL with IMF ranged from 186.4 to 292.5 x 10-6 mutants.

Furthermore, dose-related increases in the mutation frequencies, and statistically significant linear trends were observed.

Furthermore, statistically significant increases in the mean number of small colonies and in the mutation frequency of small colony mutants were noted at the concentrations tested from 175 to 87.5 µg/mL (1st assay, Tables 4 and 16) or 153.1 to 87.5 µg/mL (2nd assay; Tables 4 and 22). To end, statistically linear trends were noted. When regarding the Figures 3a and 3b, it is noteworthy that small colonies are in particular responsible for the increase in the mutation frequency of total induced mutants. This is in favour of an intrinsic clastogenic activity of the test item. In the first assay with metabolic activation, no significant increase in the mutation frequency of total

induced mutants (small and large colonies, Tables 3, 15) or in the mean number of small colonies or in the mutation frequency of small colony mutants (Tables 4, 16) was noted at any of the concentrations tested. Nevertheless, an important difference between the two cultures was observed, and the statistical analysis was thus impossible.

When regarding the individual results in culture A (Table 14), an increase in the mutation frequency of total induced mutants was observed with a dose-effect relationship and an induced mutation frequency above the global evaluation factor (GEF) of +126 x10-6 mutants at least for the highest concentration tested of 350 µg/mL (256.9 x10-6 mutants vs 107.3 x10-6 mutants in the corresponding negative control, Table 14).

Moreover, a clear increase in the mean number of small colonies and in the mutation frequency of small colony mutants was noted at the concentration tested of 350 µg/mL with a mean induction ratio of 2.1 (Table 4).

The second assay was performed under the same experimental conditions.

In this assay, a statistically and biologically significant increase in the mutation frequency of total induced mutants (small and large colonies) was noted at the highest concentration tested of 350 µg/mL (Tables 3, 21). Indeed, the induced mutation frequency (IMF) reached 152.1 x10 -6 mutants at the highest dose of 350 µg/mL, that is to say above the global evaluation factor (GEF) of +126 x10-6 for a biologically significant effect (Moore et al., 2006). Furthermore, a dose-related increase in the mutation frequencies, and a statistically significant linear trend was observed.

Moreover, a statistically significant increase in the mean number of small colonies and in the mutation frequency of small colony mutants was noted at the concentration tested of 350 µg/mL (Tables 4 and 22). To end, a statistically linear trend was noted .

It is noteworthy that small colonies are in particular responsible for the increase in the mutation frequency of total induced mutants. This is in favour of an intrinsic clastogenic activity of the test item.

Conclusions:

Di-tert-dodecyl polysulfides (TPS 32) induced a biologically significant mutagenic activity being demonstrated at the TK locus in L5178Y mouse lymphoma cell culture either with or without metabolic activation, in two independent assays. The clear increase in the number of small colonies is in favour of a clastogenic activity.

Executive summary:

The potential of Di-tertio-decyl polysulfide (TPS 32) 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-decyl polysulfide (TPS 32) 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) 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. Di-tertio-decyl polysulfide (TPS 32) was dissolved in DMSO and the positive controls were methylmethane sulfonate (without S9-mix) and Cyclophosphamide (with S9-mix). In the culture medium, no precipitate was noted at the concentration of 350 µg/mL (i.e. when initial solutions are added at 0.5% in the culture medium). At this concentration, 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 175, 131.25, 87.5, 43.75, 21.88, 10.94 and 5.47 µg/ml (assay 1) and 175, 153.13, 131.25, 109.38, 87.5, 65.63 and 43.75 µg/ml (assay 2) without S9 -mix and 350, 175, 87.5, 43.75, 21.88 and 10.94 µg/ml (assay 1) and 350, 280, 224, 179.2, 143.36, 114.69and 91.75 µg/ml (assay 2) with S9-mix. Following the 3-hour treatment, cytotoxicity (decreased adjusted RTG) was observed from 153.13 µg/ml without S9-mix and at 350 µg/ml with S9-mix. A biologically significant mutagenic activity was observed either with or without metabolic activation, in the two independent assays. The clear increase in the number of small colonies is in favour of a clastogenic activity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Polysulfides, di-tert-dodecyl was not clastogenic in an in vivo micronucleus test (OECD TG 474), in male OFA Sprague Dawley rats treated twice orally with up to 2000 mg/kg/day.

The search for potential genotoxic activity of TPS 32 was assessed using the in vivo comet assay in the liver, glandular stomach and duodenum in the rat. The treatment was carried out by oral route (gavage), using 1 daily treatment for 2 consecutive days with the OCDE guideline 489 (2016) maximum recommended dose, i.e. 2000 mg/kg. Overall, under these experimental conditions, the test item does not present DNA strand breaks and/or alkali-labile sites inducer activities toward the liver and glandular stomach from OFA Sprague-Dawley male rats.

 

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August - November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

yes

Remarks:

For the duodenum, median of percentage of tail DNA in the vehicle control group was slightly lower than the minimal bound of negative historical control data : it constitutes a deviation according to OECD test guideline.

Principles of method if other than guideline:

The purpose of the in vivo Comet assay following the alkaline version (pH > 13) developed by Singh et al. (1988), is to identify those agents which induce DNA damage such as single or double DNA strand breaks 10(SSB or DSB), alkali-labile sites, DNA-DNA / DNA-protein cross-linking and SSB associated with incomplete excision repair sites in compliance with the OECD guideline 489 (2016). The advantages of the Comet assay include its demonstrated sensitivity for detecting low levels of DNA damage.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is the species recommended by OECD guideline for this test.

No difference in terms of toxicity was noted between male and female rats in the previous in vivo
genotoxicity study. At the Sponsor’s request, the study was performed in male rats.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE
- Age at study initiation: 8 weeks old
- Weight at study initiation: 190 g to 210 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in polypropylene cages
- Diet (e.g. ad libitum): A04C-10 from SAFE (batch 19330), ad libium
- Water (e.g. ad libitum): 30-70 %,
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %,
- Air changes (per hr): not precised, but ventilated system
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dose volume : 5 mL/kg b.w.
Form administered : solution

In the main genotoxicity assay, a solution of the test item at the concentrations of 400 mg/mL was prepared, giving a final dose of 2000 mg/kg, when administered at 5 mL/kg. The lowest concentrations of 224 and 82 mg/mL were obtained by successive dilution in corn oil for final dose of 1120 and 410 mg/kg.

Duration of treatment / exposure:

2 daily treatments at 24-hour intervals

Frequency of treatment:

2 daily treatments at 24-hour intervals

Post exposure period:

2-6 hours after the second treatment

Dose / conc.:

410 mg/kg bw/day

Dose / conc.:

1 120 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5 males/group, except for the high dose group : 7 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

methylmethane sulfonate (Aldrich, batch MKBX5165V) in sterile water (Fresenius, Batch 13PEP301), 100 mg/kg/day, gavage, 2 daily treatments at 24-hour intervals

Tissues and cell types examined:

Two to six hours after the second treatment, the rats are killed and cells from the selected target organs (i.e. liver, glandular stomach and duodenum) are isolated for the comet assay.

Fives males of each group were assigned for cell isolation and assessed for DNA fragmentation.
Individual animals were anaesthetized with isoflurane and exsanguined.
Single cell preparations were done within one hour after animal sacrifice.
A 'V' shaped incision was made from the centre of the lower abdomen to the rib cage. The skin and muscles was removed to reveal the abdominal cavity.
A portion of the liver, glandular stomach and duodenum was removed and washed in the cold mincing buffer until as much blood as possible has been removed. The portion was minced with a pair of fine scissors to release the cells. The cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a haemocytometer, and sufficient cells to obtain 25± 5 x 103 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.

Number of cells observed per animal : 150
Number of cells observed per dose : 750

Details of tissue and slide preparation:

After isolation, single cells are embedded in agarose on microscope slides and the obtained microgels are successively submitted to lysis, unwinding and electrophoresis in alkaline conditions and under dimmed light to prevent any additional DNA damage. After neutralization, slides are dried and could therefore be stained with a fluorescent dye (e.g. propidium iodide) before analysis and scoring. The method used for quantifying DNA migration involves a computerized image analysis system in order to collect comet data; then, the dedicated software allows indeed the calculation of metrics for DNA migration.

Determination of the cytotoxicity of the test item :
In the main assay, the frequency of hedgehogs in the control group and in the 2000, 1120 and 410
mg/kg/day treated groups was inferior to 50%. Indeed maximal values were of 1.81%, 10.55% and 5.64% in the liver, the stomach and the duodenum, respectively and comparable to the values obtained in the respective control. The test item was thus considered as not cytotoxic.

Evaluation criteria:

Expression of the results
- the median per slide of the percentage of DNA in tail for at least 50 cells
- the mean of medians of the percentage of DNA in tail per animal (i.e. 3 slides, 150 cells)
- the mean of median per concentration (i.e. 5 animals, 750 cells)
In addition, each slide was also examined for presence of hedgehogs (possible indicator of toxicity and or apoptosis). Hedgehogs were excluded from image analysis data collection. However, determining their frequency might be useful for data interpretation. Therefore, the percentage of hedgehogs was recorded for each slide per animal, and per type of treatment and per organ. The hedgehogs, also known as clouds or ghost cells, are morphological indicative of highly damaged cells often associated with severe genotoxicity, necrosis and apoptosis. A hedgehog results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum.

A study is accepted if the following criteria are fulfilled:
- Concurrent vehicle controls should be within the control limits of the distribution of the laboratory’s historical vehicle control database.
- The concurrent positive controls should induce responses that are comparable to the historical positive control data and produce a statistically significant increase compared with the concurrent vehicle control.
- The appropriate number of doses and cells must be analysed.

Moreover:
- In the vehicle group, an eventual increase in the frequency of hedgehogs, must not be >50%.
- If death(s) is(are) observed at the tested doses, the mortality rate must be less than 20 %
per group. The dead animals from the high dose treated group are replaced by those in parallel treatment.

Statistics:

In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian
distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed.
This method is based on the analysis of variance by ranks for testing equality of population medians
among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the
vehicle control in order to determine statistical significance of differences in group median values
between each group versus the vehicle control. This test was also used to compare vehicle control
and positive control to determine acceptable criteria of a valid test.

Sex:

male

Genotoxicity:

negative

Remarks:

on liver cells

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

in glandular stomach

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

in duodenum cells

Toxicity:

no effects

Vehicle controls validity:

not valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The animal demonstrated no clinical signs once dosed with the test item, whatever the dose tested.

Liver cells :
The value for the percentage of DNA in tail in the vehicle control was within the control limits of the distribution of the laboratory’s historical vehicle control database. Moreover, the concurrent positive control induced a response that was lower but comparable to the historical positive control data and produced a statistically significant increase compared with the concurrent vehicle control.
The appropriate number of doses (3) and cells (750) were analysed.
Moreover, in the vehicle group, no increase in the frequency of hedgehogs was noted.
The validity criteria for the experiment carried out in the liver were fulfilled. The experiment is thus valid.
No statistically significant increase in the mean of medians of percentage of DNA in tail per slide was observed at the any tested doses from 410 to 2000 mg/kg b.w./day of TPS 32 in hepatocytes from OFA Sprague-Dawley male rats.
Otherwise, a statistically significant decrease in the mean of medians of percentage of DNA in tail per slide were noted in the group treated with 2000 mg/kg b.w./day of TPS 32 with a value of 0.03 vs. 0.14 in the respective vehicle control. The median of the percentage of DNA in tail was close to the lowest value already observed in historical data for vehicle control i.e. 0.02, and no dose effect relationship was noted. This change was therefore considered as not biologically relevant.
Overall, it is concluded that TPS 32 did not induce any DNA strand break in hepatocytes from OFA Sprague-Dawley male rats as investigated by the in vivo Comet assay.

Glandular stomach cells :
The value for the percentage of DNA in tail in the vehicle control was within the control limits of the distribution of the laboratory’s historical vehicle control database. Moreover, the concurrent positive control induced a response that was comparable to the historical positive control data and produced a statistically significant increase compared with the concurrent vehicle control.
The appropriate number of doses (3) and cells (750) were analysed.
Moreover, in the vehicle group, no increase in the frequency of hedgehogs was noted.
The validity criteria for the experiment carried out in the glandular stomach were fulfilled. The experiment is thus valid.
No statistically significant increase in the mean of medians of percentage of DNA in tail per slide was observed at the any tested doses from 410 to 2000 mg/kg b.w./day of TPS 32 in glandular stomach from OFA Sprague-Dawley male rats.
Otherwise, statistically significant decreases in the mean of medians of percentage of tail DNA per slide were noted in the group treated with 1120 and 2000 mg/kg b.w./day of TPS 32 with values of 1.38 and 0.80, respectively vs. 5.04 in the respective vehicle control. The decrease was dose-related and the value for the mean of median of the percentage of DNA in tail at the top dose of 2000 mg/kg b.w./day was close to the lowest value already observed in historical data for vehicle control i.e. 0.80 vs. 0.74.
Overall, it is concluded that TPS 32 did not induce any DNA strand break in glandular stomach
from OFA Sprague-Dawley male rats as investigated by the in vivo Comet assay.

Duodenum :
The value for the percentage of DNA in tail in the vehicle control was outside the control limits of the distribution of the laboratory’s historical vehicle control database with a value of 0.16% vs. 0.21%. In return, the concurrent positive control induced a response that was comparable to the historical positive control data and produced a statistically significant increase compared with the concurrent vehicle control.
The appropriate number of doses (3) and cells (750) were analysed.
Moreover, in the vehicle group, no increase in the frequency of hedgehogs was noted.
The validity criteria for the experiment carried out in the duodenum was thus not completely fulfilled.
According to the OECD guideline 489, the data should be invalidated.
Scientifically, with a background of DNA fragmentation slightly lower than the minimal bound of the negative historical control, the sensitivity of the current assay could be considered as enhanced while a clear negative response was obtained meaning that the test item is not genotoxic in duodenum.

Analytical results :
The control of concentrations of TPS 32 in treatment preparations was performed in a GLP compliant laboratory following a validated method. The results are reliable.
The results of the assays for TPS 32 in treatment preparations were valid and considered as correct (only a slight deviation was noted at the lower dose). In addition, TPS 32 was not detected in the solvent control.

Conclusions:

Overall, under our experimental conditions, the test item does not present DNA strand breaks
and/or alkali-labile sites inducer activities toward the liver and glandular stomach from OFA
Sprague-Dawley male rats.
No definitive conclusion can be drawn for duodenum. For the duodenum, median of
percentage of tail DNA in the vehicle control group was slightly lower than the minimal bound
of negative historical control data. If it constitutes a deviation according to OECD guidelines
489 and doesn’t fulfil the acceptance criteria of the results, the clear negative response
observed in this organ tends to confirm the lack of genotoxicity of TPS32 in duodenum.

Executive summary:

The search for potential genotoxic activity of TPS 32 (Polysulfides, di-tert-dodecyl - CAS 68425-15-0) was assessed using the in vivo comet assay in the liver, glandular stomach and duodenum in the rat.
The treatment was carried out by oral route (gavage), using 1 daily treatment for 2 consecutive days with the OCDE guideline 489 (2016) at the maximum recommended dose, i.e. 2000 mg/kg b.w./day.

No increase in the percentage of DNA in tail was observed in liver and glandular stomach. Noteworthy, significant and dose-related decreases in the percentage of tail DNA were noted in glandular stomach but the values are included or close to the historical control data.
Overall, under our experimental conditions, the test item does not present DNA strand breaks and/or alkali-labile sites inducer activities toward the liver and glandular stomach from OFA Sprague-Dawley male rats.
No definitive conclusion can be drawn for duodenum. For the duodenum, median of percentage of tail DNA in the vehicle control group was slightly lower than the minimal bound of negative historical control data. If it constitutes a deviation according to OECD guidelines 489 and doesn’t fulfil the acceptance criteria of the results, the clear negative response observed in this organ tends to confirm the lack of genotoxicity of TPS32 in duodenum.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17/05/2010 – 06/07/2010

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:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-surl’Arbresle; FRANCE
- Age at study initiation: 5 to 10 weeks old
- Weight at study initiation: approximately 200 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: animals were housed in polypropylene cages measuring 42.5 x 26.6 x 15 cm, covered by a stainless steel netted lid, in which they will be placed in groups of 3 or 2
- Diet (ad libitum): 801175 RM1(P)DU IRR 9Kgy irradiated from Special Diets Services
(ENGLAND).
- Water (ad libitum): softened by reverse osmosis and filtered on 0.2 µm membrane
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 20 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC at 0.5% in distilled water

Details on exposure:

Dose volume : 10 mL/kg b.w.

Duration of treatment / exposure:

2 treatments

Frequency of treatment:

daily

Post exposure period:

Number of sampling times : for the negative control and the 3 treated groups: 24 hours after the second treatment for the positive control group: 24 hours after the single treatment

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5 (excepted 7 for the high dose level)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (25 mg/kg i.p.)

Tissues and cell types examined:

Bone Marrow

Details of tissue and slide preparation:

At the sampling time, the 5 animals per group were sacrificed by CO2 asphyxia; the femurs were removed, and the bone marrow was extracted with foetal calf serum (1 mL per animal). The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatant was removed. The centrifugate was spread on slides. The smears were stained using a technique, derived from the May Grunwald Giemsa technique (Schmid, 1975), which makes it possible to distinguish between polychromatic (PCE) and normochromatic erythrocytes (NCE): PCE are purple whereas NCE are red.
After coding the slides by a person not involved in the study, two slides per animal were read by two independent operators; for each animal, the number of polychromatic erythrocytes having one or more Howell-Jolly bodies (micronuclei) was determined for at least 2000 polychromatic erythrocytes. (In case of divergence, 2000 new polychromatic erythrocytes were examined; the retained value was the mean of all readings).
The polychromatic/normochromatic erythrocyte ratio will be determined by analyzing at least 1000 erythrocytes per animal.

Evaluation criteria:

For a test item to be considered negative in the micronucleus test, there must be no statistically significant increase in the number of MNPCE observed compared with negative control animals.
For a test item to be considered positive in the micronucleus test, there must be seen a statistically significant increase in MNPCE for at least one dose group and/or a statistically significant dose-related increase in MNPCE, compared to the negative control animals. Statistical significance will not be the only determinant of a positive response, and the Study Director will consider the biological relevance of the results in the final evaluation.

Statistics:

Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney U rank test. An analysis of a large number of control results has shown that the distribution of the numbers of micronuclei does not correspond to a Gaussian distribution, but to a Poisson-type distribution. This makes it necessary to use a non-parametric statistical test, and the Mann Whitney U rank test is recommended by UKEMS (LOVELL et al., 1989).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

but tested up to the limit dose of 2000 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Clinical signs of toxicity in test animals: No clinical signs were observed after up to 24 hours after the second treatment in male and female rats. As no difference between both sexes were noted, the main assay was performed in male rats only.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The results obtained on negative control animals and those treated with the positive reference substance were similar to those generally obtained in the laboratory. A statistically significant increase in the frequency of micronuclei was noted in the group treated with Cyclophosphamide, demonstrating the sensitivity of the animal strain used to a clastogenic agent. The validity criteria for the test were fulfilled and the test was validated. Regarding the frequency of micronuclei, no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with di-tert-dodecyl polysulfides (TPS 32) at any dose.
- Ratio of PCE/NCE (for Micronucleus assay): No statistically significant decrease in the ratio PCE to NCE was noted in the 3 di-tertdodecyl polysulfides (TPS 32) treatment groups when compared to the negative control group. In consequence, no proof of systemic exposure was evidenced.

Conclusions:

The validity criteria for the results were fulfilled. The study was thus considered as valid. The test item di-tert-dodecyl polysulfides (TPS 32) was investigated by means of the in vivo micronucleus test, in male OFA Sprague Dawley rats treated orally twice with 2000 – 1000 and 500 mg/kg/day, followed by one sampling time 24 hours after the last treatment. As a conclusion, di-tert-dodecyl polysulfides (TPS 32) induced no genotoxic activity under these experimental conditions.

Executive summary:

The potential clastogenic activity of di-tert-dodecyl polysulfides (TPS 32) was tested using the in vivo micronucleus test in male OFA Sprague Dawley rats, in compliance with the Commission Regulation (EC) No. 440/2008 and the OECD Guideline 474, by oral route, using 2 successive daily treatments at the maximum dose recommended by OECD guidelines, i.e. 2000 mg/kg, followed by one sampling time 24 hours after the last treatment. The two lower doses of 1000 and 500 mg/kg/day (x2) were also analysed. The validity criteria for the results were fulfilled. The study was thus considered as valid. Di-tert-dodecyl polysulfides (TPS 32) induced no genotoxic activity under these experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation (in vitro)

In a study similar to OECD TG 471 (Jones et al., 1986), polysulfides, di-tert-dodecyl was tested in a reverse mutation assay with Salmonella typhimurium strains, TA98, TA100, TA102, TA1535, and TA1537 exposed to five concentrations in the presence and absence of a metabolic activation system using plate incorporation at doses of 5 to 5,000 µg/plate. Exposure to five graded doses of polysulfides, di-tert-dodecyl in the presence and absence of metabolic activation, did not increase the reversion rates in any of the tester strains and polysulfides, di-tert-dodecyl is not considered to be a mutagen.

The potential of polysulfides, di-tert-dodecyl to induce mutations at the TK (Thymidine Kinase) locus in L5178Y mouse lymphoma cells was evaluated in a study performed according OECD TG 476 and Good Laboratory Practice (Nakab, 2010). After a preliminary toxicity test, polysulfides, di-tert-dodecyl 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) 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. Di-tert-dodecyl polysulfide (TPS 32) was dissolved in DMSO and the positive controls were methylmethane sulfonate (without S9-mix) and Cyclophosphamide (with S9-mix). In the culture medium, no precipitate was noted at the concentration of 350 µg/mL (i. e. when initial solutions are added at 0.5% in the culture medium). At this concentration, 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 175, 131.25, 87.5, 43.75, 21.88, 10.94 and 5.47 µg/ml (assay 1) and 175, 153.13, 131.25, 109.38, 87.5, 65.63 µg/ml and 43.75 (assay 2) without S9 -mix and 350, 175, 87.5, 43.75, 21.88 µg/ml and 10.94 (assay 1) and 350, 280, 224, 179.2, 143.36, 114.69 and 91.75 µg/ml (assay 2) with S9-mix. Following the 3-hour treatment, cytotoxicity (decreased adjusted RTG) was observed from 153.13 µg/ml without S9-mix and at 350 µg/ml with S9-mix. A biologically significant mutagenic activity was observed either with or without metabolic activation, in the two independent assays. The clear increase in the number of small colonies is in favour of a clastogenic activity.

Chromosomal aberration (in vitro)

A chromosomal aberration test with human lymphocytes was performed according to OECD TG 473 with polysulfides, di-tert-dodecyl at the concentrations of 300, 1000 and 2500 µg/ml (2500 µg/ml being the limit of solubility in the culture medium) (Molinier, 1994). The test was carried out with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254. Without S9 mix, the cultures were incubated with the test or control substances, which remained in the culture medium, until the appropriate harvest times at 24 and 48 hours. With S9 mix, the test or control substances remained in a culture medium containing 15% S9 mix (10% S9/S9 mix) for 2 hours. The cells were then centrifuged, the treatment medium removed, the cells resuspended in fresh culture medium. The cultures were then incubated until the appropriate harvest times at 24 and 48 hours. Two hours before harvesting, the cells were treated with a colcemid solution to block them at the metaphase-stage of mitosis. The chromosomal preparations were stained and screened microscopically for mitotic index and for aberrations, 200 well-spread metaphases per concentration were read, whenever possible. The aberrant cells frequency in the negative and vehicle controls was within the range of the historical data (i. e. 0.5 ± 0.6%, gaps excluded). The aberrant cells frequency in the positive controls was significantly higher (p < 0.001) than that of the negative controls, indicating the sensitivity of the test system. Di-tert-dodecyl polysulfides (TPS 32) did not induce any significant increase in the aberrant cells frequency, with or without S9 mix, for both of the 2 harvest times.

In vivo genotoxicity studies

The potential clastogenic activity of polysulfides, di-tert-dodecyl was tested using the in vivo micronucleus test in male OFA Sprague Dawley rats, in compliance with OECD TG 474, by oral route, using 2 successive daily treatments at the maximum dose recommended by OECD guidelines, i. e. 2000 mg/kg, followed by one sampling time 24 hours after the last treatment (Simar, 2010). The two lower doses of 1000 and 500 mg/kg/day (x2) were also analysed. The validity criteria for the results were fulfilled. The study was thus considered as valid. Di-tert-dodecyl polysulfides (TPS 32) induced no genotoxic activity under these experimental conditions.

 

The search for potential genotoxic activity of TPS 32 (Polysulfides, di-tert-dodecyl - CAS 68425-15-0) was assessed using the in vivo comet assay in the liver, glandular stomach and duodenum in the rat.
The treatment was carried out by oral route (gavage), using 1 daily treatment for 2 consecutive days with the OCDE guideline 489 (2016) at the maximum recommended dose, i.e. 2000 mg/kg b.w./day.

No increase in the percentage of DNA in tail was observed in liver and glandular stomach. Noteworthy, significant and dose-related decreases in the percentage of tail DNA were noted in glandular stomach but the values are included or close to the historical control data.
Overall, under our experimental conditions, the test item does not present DNA strand breaks and/or alkali-labile sites inducer activities toward the liver and glandular stomach from OFA Sprague-Dawley male rats.
No definitive conclusion can be drawn for duodenum. For the duodenum, median of percentage of tail DNA in the vehicle control group was slightly lower than the minimal bound of negative historical control data. If it constitutes a deviation according to OECD guidelines 489 and doesn’t fulfil the acceptance criteria of the results, the clear negative response observed in this organ tends to confirm the lack of genotoxicity of TPS32 in duodenum.

 

 

Justification for classification or non-classification

Based on the available data, no classification for genetic toxicity is required for polysulfides, di-tert-dodecyl according to the Regulation EC n°1272/2008.