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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A reverse gene mutation assay in bacteria and a chromosome aberration study with the test substance di-tert-nonyl polysulfide provided negative results. Furthermore, an in vitro gene mutation study in mammalian cells with the analogue substance di-tert-butyl polysulfide was negative when not metabolically activated, however, an increase in the number of small colonies was observed with metabolic activation, indicating potential for clastogenic activity. The results of an in vitro gene mutation study in mammalian cells with the analogue substance di-tert-dodecyl polysulfides were positive with and without metabolic activation.

However, the potential for clastogenicity observed in vitro in mammalian cells was not confirmed in vivo. The results of in vivo mammalian micronucleus assays with the analogue substances di-tert-dodecyl polysulfide and di-tert-dodecyl polysulfides were negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting 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)
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-tert-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
Remarks:
Increase in number of small colonies
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-tert-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-tert-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-tert-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:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-11-03 to 1998-07-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Human Lymphocytes
Metabolic activation:
with and without
Test concentrations with justification for top dose:
122.5, 175, 250 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMF
Positive controls:
yes
Evaluation criteria:
The test article is considered as positive in this assay if:
1. a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at one or more concentrations, and
2. the proportion of cells with structural aberrations at such doses exceeds normal range
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Experiment 1: treatment in the absence and presence of S-9

S-9           

Treatment + recovery (hours)

Vehicle Control

Concentration (µg/mL)

Positive Control

-

3 + 17

DMF

122.5, 175, 250         

NQO, 2.5.µg/mL

+

(Aroclor 1254 induced S-9)

3 + 17

DMF

122.5, 175, 250         

CPA, 25 µg/mL

Experiment 2: treatment in the absence of S-9

S-9           

Treatment + recovery (hours)

Vehicle Control

Concentration (µg/mL)

Positive Control

-

20 + 0

DMF

122.5, 175, 250         

NQO, 2.5.µg/mL

+

3 + 17

DMF

122.5, 175, 250         

CPA, 25 µg/mL

Conclusions:
TPS 37 did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested in excess of its limit of solubility in both the absence and presence of S-9.
Executive summary:

In a chromosome aberration study in mammals, human lymphocyte cultures from female donors were exposed to TPS 37 in DMF at concentrations of 122.5, 175, and 250 µg/mL in the presence and absence of mammalian metabolic activation (S-9 mix) for up to 20 hours.

 

TPS 37 was tested up to concentrations of 250 µg/mL. Cultures treated with TPS 37 in the absence and presence of S-9 exhibited frequencies of cells with structural aberrations which were similar to levels seen in concurrent negative controls. One culture treated with 122.5 µg/mL for 20 hours in the absence of S-9 displayed frequencies of cells with structural aberrations that exceeded the normal range for negative controls. A replication did not produce similar results so the effect was not considered biologically relevant. All other tests exhibited frequencies of cell that were within the normal range. It is concluded that TPS 37 did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested in the presence and absence of S-9 mix.

 

This study received a Klimisch score of 1 and is classified as reliable without restriction because it provides robust results and carefully follows OECD guidelines.  

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-10-31 to 1998-05-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Cited as Directive 84/449/EEC, B.14
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Remarks:
OECD GLP
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
other: Strains: TA 98, TA 100, TA 1535, TA 1537, TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment 1: 8, 40, 200, 1000 and 5000 µg/plate
Experiment 2: 4.883, 19.531, 78.125, 312.5, 1250 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMF
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMF
True negative controls:
yes
Positive controls:
yes
Remarks:
DMSO
Details on test system and experimental conditions:
SYSTEM OF TESTING
- 2 independent trials; in the 1st and 2nd trial without metabolic activation system (MA) the direct plate incorporation method was used; this method also used in the 1st trial with MA, in the 2nd trial with MA the preincubation method (1 h, 37°C). 3 plates per concentration
- Metabolic activation system (MA): S9 fraction from liver homogenates of rats induced with 500 mg/kg Aroclor 1254
- solvent: dimethylformamide
- Controls: solvent control (with and without MA)
. Positives controls:
- Without S9
TA98: 2-nitrofluorene 5.0 µg/plate
TA100 and TA1535: Sodium azide 2.0 µg/plate
TA1537: 9-aminoacridine 50 µg/plate
TA102: glutaraldehyde 25 µg/plate
- With S9
TA98, TA100 and TA1535: 2-aminoanthracene 5.0 µg/plate
. sterility control checked during the test.
- Concentrations:
Experiment 1: 8, 40, 200, 1000 and 5000 µg/plate
Experiment 2: 4.883, 19.531, 78.125, 312.5, 1250 and 5000 µg/plate
- Cytotoxicity: A preliminary toxicity test was performed to define the concentrations to be used for the mutagenicity study. TA100 exposed to 8-5000 µg/plate with and without MA
Evaluation criteria:
The test article was considered to be mutagenic if:
1. the assay was valid
2. a dose related and reproducible increase in the number of revertants is observed, or a significant and reproducible increase in the number of revertants was induced at one or more test concentration.
Species / strain:
other: Strains: TA 98, TA 100, TA 1535, TA 1537, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: >= 1250 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Evidence of toxicity was observed at >=1250 µg/plate in only a few test strains. Precipitation of test agent was observed on all plates treated at concentrations >= 1000  µg/plate.

The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

No TPS 37 treatment of any of the test strains produced an increase in revertant numbers sufficient to be considered as indicative of mutagenic activity.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
TPS 37 did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), when tested under the conditions employed for this study, which included treatments up to 5000 µg/plate, both in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to TPS 37 in DMF at concentrations of 8, 40, 200, 1000, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the Ames test.

 

TPS 37 was tested up to limit concentrations of 5000 µg/plate. Following treatment in experiment 1, evidence of toxicity was observed at the highest dose (5000 µg/plate) treatments in only a few test strains. Precipitation of test agent was observed on all plates treated at concentrations of 1000 and 5000 µg/plate. For experiment 2, dose intervals were narrowed to more closely investigate those concentrations of TPS 37 most likely to induce mutagenic response. All treatments in the presence of S-9 employed a pre-incubation step. Evidence of toxicity was found in the higher test doses in the presence and absence of S-9 mix. Precipitation of test agent was observed on all plates at concentrations of 1250 and 5000 µg/plate. No treatment with TPS 37 of any test strains produced an increase in revertant numbers high enough to be considered characteristic of mutagenic activity. TPS 37 did not induce mutation in five strains of S. typhimurium when tested under study conditions. Conditions included treatments up to 5000 µg/plate and the absence and presence of rat liver activation system.

 

This study received a Klimisch score of 1 and is classified as reliable without restrictions because it adheres to guidelines and includes testing at the limiting concentration.

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

Genetic toxicity in vivo

Description of key information

In an in vivo mammalian somatic studies with the structural analogue substances di-tert-butyl polysulphides and  di-tert-dodecyl polysulphides, the potential for clastogenic activity was evaluated. No induced genotoxic activity was observed (Simar, 2010).

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:
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)
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

In Vitro Gene Mutation in Bacteria:

One key study was identified to evaluate the in vitro gene mutation potential of di-tert-nonyl polysulfide in bacteria.

In a key reverse gene mutation assay in bacteria (Dawkes, 1998; Klimisch score = 1), the strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to di-tert-nonyl polysulfide (TPS 37) in DMF at concentrations of 8, 40, 200, 1000, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the Ames test. TPS 37 was tested up to limit concentrations of 5000 µg/plate. Following treatment in experiment 1, evidence of toxicity was observed at the highest dose (5000 µg/plate) tested in a few test strains of S. typhimurium. Precipitation of the test material was observed on all plates at concentrations of 1000 and 5000 µg/plate. For experiment 2, dose intervals were narrowed to more closely investigate concentrations of TPS 37 most likely to induce mutagenic response. All treatments in the presence of S-9 employed a pre-incubation step. Evidence of toxicity was found in the higher test doses in the presence and absence of S-9 mix. Precipitation of test agent was observed on all plates at concentrations of 1250 and 5000 µg/plate.

Treatment with TPS 37 did not produce an increase in revertant numbers high enough to be considered characteristic of mutagenic activity in any of the strains tested. Under the conditions of this study, TPS 37 did not induce mutation in five strains of S. typhimurium in the presence or absence of metabolic activation and was therefore considered to be negative in the bacterial reverse mutation test.

Supporting studies are available for the structural analogue substances di-tert-dodecyl polysulfide and di-tert-butyl polysulfide.

An in vitro reverse mutation assay in bacteria with the test substance di-tert-dodecyl polysulfide was conducted in a similar way to OECD TG 471 and in compliance with GLP. The strains S. typhimurium TA 98, 100, 1535, 1537 and 1538 was utilised with and without metabolic activation. No reversion rates were altered in any of the test strains and was therefore concluded as not mutagenic (Jones et al., 1986).

In an in vitro gene mutation in mammalian cells which was conducted according to OECD TG 471 and in compliance with GLP, di-tert-butyl polysulfide was tested by Ames test using five different strains of the bacteria Salmonella typhimurium TA 98, 100, 1535, 1537 and 1538. No mutagenic activity was evident regardless if metabolic activation was present or absent (Molinier, 1992).

In Vitro Cytogenicity in Mammalian Cells:

One key study was identified to evaluate the in vitro cytogenicity potential of di-tert-nonyl polysulfide in mammalian cells.

In a key chromosome aberration study (Burman, 1998; Klimisch score = 1), human lymphocyte cultures from female donors were exposed to TPS 37 in dimethyl formamide (DMF) at concentrations of 122.5, 175, and 250 µg/mL in the presence and absence of mammalian metabolic activation (S-9 mix) for up to 20 hours. TPS 37 was tested up to concentrations of 250 µg/mL. Cultures treated with TPS 37 in the absence and presence of S-9 exhibited frequencies of cells with structural aberrations which were similar to levels seen in concurrent negative controls. One culture treated with 122.5 µg/mL for 20 hours in the absence of S-9 displayed frequencies of cells with structural aberrations that exceeded the normal range for negative controls. A replication did not produce similar results so the effect was not considered biologically relevant. All other tests exhibited frequencies of cells that were within the normal range.

It was therefore concluded that TPS 37 did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested in the presence and absence of S-9 mix.

Supporting studies are available for in vitro chromosome aberration test with human lymphocytes, with the structural analogue substances di-tert-dodecyl polysulfide and di-tert-butyl polysulfide. Both studies were conducted according to OECD TG 473 and in compliance with GLP. Neither of the studies evidenced any significant increase of aberrant cells frequency, regardless of presence or absence of metabolic activation (Molinier, 1994 & de Jouffrey, 1996).

In Vitro Gene Mutation in Mammalian Cells:

No key data were identified to evaluate the in vitro gene mutation potential of di-tert-nonyl polysulfide in mammalian cells. However, in vitro studies are available for the structural analogues di-tert-dodecyl polysulfides and di-tert-butyl polysulfides.

An in vitro gene mutation study in mammalian cells, with the substance di-tert-butyl polysulfide, was conducted according to OECD TG 476 and in compliance with GLP. No mutagenic effect was noted in the absence of S9-mix, nevertheless, when metabolic activation was present, a significant biological mutagenic activity was present at the TK locus in L5178Y mouse lymphoma cell culture. This mutagenic activity was confirmed in two independent assays. The clear increase in the number of small colonies suggest that clastogenic activity occurred (Simar, 2010).

An in vitro gene mutation study in mammalian cells, with the test substance di-tert-dodecyl polysulfide was conducted according to OECD TG 476 and in compliance with GLP. The test substance was tested in mouse lymphoma L5178Y TK+/- cells with and without metabolic activation. In two independent assays, a clear increase of small colonies indicating clastogenic activity was observed in both the presence and absence of metabolic activation (Nakab, 2010).

In Vivo Genetic Toxicity:

No key data were identified to evaluate the in vivo genetic toxicity potential of di-tert-nonyl polysulfide. However, data for the structural analogue substance di-tert-dodecyl polysulfide is available. The study is an in vivo mammalian somatic study, investigating the potential of clastogenic activity which was conducted according to OECD TG 474 and in compliance with GLP. Rats were treated with the maximum recommended treatment of 2000, 1000 and 500 mg/kg/day of the test material followed by sampling 24 hours after the last treatment. No induced genotoxic activity was observed (Simar, 2010).

An in vivo mammalian somatic cell study testing mammalian erythrocyte micronucleus test in mice with the test substance di-tert-butyl was conducted according to OECD TG 474 and in compliance with GLP. During two days, five mice received oral administration of the test substance at a dose level of 2000 mg/ kg/day. Five other mice received simultaneously the vehicle and acted as a control group whereas an additional group received a positive control test item (cyclophosphamide). Following the last treatment, the animals were sacrificed and bone marrow smears were prepared. The number of micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. No differences were observed between the vehicle group and the group which had been exposed to the test substance. Therefore, it was concluded that the test substance did not induce any damage to the chromosomes or the mitotic apparatus following treatment of di-tert-butyl polysulfide (Sire, 2010).

There is evidence from increase in number of small colonies observed in the in vitro mammalian gene mutation studies that di-tert-butyl polysulfides and d-tert-dodecylpolysulphides are clastogenic, as small colonies occur as a result of chromosome damage (OECD 490 paragraph 4), and these results are read across to di-tert-nonyl polysulphides. However, the in vivo results show that the effects observed vitro occur do not in vivo, so it is concluded that di-tert-nonyl polysulphides and its structural analogues are not germ cell mutagens.

Read across justification

Several criteria justify the use of the read across approach to fill data gaps for di-tert-nonyl polysulfides using di-tert-dodecyl polysulfides as an analogue. Both di-tert-nonyl polysulfides and di-tert-dodecyl polysulfides, are complex mixtures of polysulfides and have similar physiochemical properties. Hence, the toxicological properties of both these substances are also expected to be similar.

Justification for classification or non-classification

Based on the available in vitro mutation and cytogenetic data and in vivo micronucleus assays, di-tert-nonyl polysulfides does not meet the criteria for classification under CLP Regulation (EC) No 1272/2008.