Polysulfides, di-tert-nonyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A reverse gene mutation assay in bacteria (similar to OECD TG 471, GLP; Dawkes, 1998) and a chromosome aberration study (OECD TG 473, GLP; Burman, 1998) with the test substance di-tert-nonyl polysulfide provided negative results. The results of an in vitro gene mutation study in mammalian cells (OECD TG 490, GLP; Charles River Laboratories, 2021) with the test substance di-tert-nonyl polysulfides were negative with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

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.  

Reference 2

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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 January 2021 - 09 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

TK+/--3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg) induced rat liver S9 obtained from Trinova Biochem GmbH, Giessen, Germany.
- method of preparation of S9 mix: 0.5 mL S9-fraction added to 0.5 mL S9 components which contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES, filter sterilised.
- concentration or volume of S9 mix and S9 in the final culture medium: 4% (v/v).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): no information

Test concentrations with justification for top dose:

6.3, 13, 25, 50, 100, 200, 400, 600, 800, 1000 and 1250 μg/mL
based on the dose range finding test and rejected first experiment (maximum dose in first experiment was too low). After 3 hours, the test item precipitated in the test system at concentrations of 600 μg/mL and above. The test item was not toxic and difficult to dissolve in aqueous solutions for the 3-hour exposure, so the highest concentration was determined by the solubility in the culture medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone; acetone

- Justification for choice of solvent/vehicle: none given in report. Acetone is a well-established solvent mentioned in the OECD Test Guideline.

- Justification for percentage of solvent in the final culture medium: none given in report.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single. The solvent control was tested in duplicate
- Number of independent experiments : two experiments were reported: an initial 3-hour exposure experiment with metabolic activation and a 24-hour exposure experiment without metabolic activation.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 10E06 cells (1.25 x 10E05 cells/mL for 24 hour treatment)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3-hours and 24-hours
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: microwell plates were used for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 5 μg/mL trifluorothymidine for 11 or 12 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: one cell per well in non-selective medium for determination of cloning efficiency. To determine mutant frequency, 9.6 x 10E05 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection). After incubation of 11 or 12 days, the selective plates were stained for 1.5-2 hours with 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. Plates for cloning efficiency and mutant frequency were scored with the naked eye or using a microscope.
- Criteria for small (slow growing) and large (fast growing) colonies: small colonies: morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. Large colonies: morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. Wells with two large colonies or one large and one small colony were counted as one large colony. Wells with two small colonies were counted as one small colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative suspension growth (RSG), relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER: The mutant frequency was expressed as the number of mutants per 10E06 viable cells. Small and large colony mutation frequencies were calculated in an identical manner.

Rationale for test conditions:

Standard test conditions

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutant frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

3-hour exposure

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

24-hour exposure

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The RTG was reduced to 14%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.402 at 4.9 μg/mL
- Data on osmolality: 0.347 Osm/kg at 4.9 μg/mL
- Possibility of evaporation from medium: vapour pressure is low
- Water solubility: the test substance was reported to be difficult to dissolve in aqueous solutions
- Precipitation and time of the determination: After 3 hours, the test item precipitated in the test system at concentrations of 600 μg/mL and above. After 24 hours, the test item precipitated in the test system at concentrations of 50 and/or 100 μg/mL and above.


RANGE-FINDING/SCREENING STUDIES (if applicable): a range finding study was conducted using 100 μg/mL test material in acetone. Cells were exposed for 3 hours with and without metabolic activation and for 24 hours without metabolic activation. Surviving cells of the 3-hour treatment were subcultured twice before counting, those of the 24-hour treatment were subcultured once. The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis; p-value if any : none
- Any other criteria: global evaluation factor (GEF): 126

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency RTF

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures : acceptable
o Number of cells plated in selective and non-selective medium: acceptable
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: acceptable
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls: results acceptable - see attached tables and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation: no test item exceeded the GEF. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see attached table
- Negative (solvent/vehicle) historical control data: see attached table

Conclusions:

Di-tert-nonyl polysulfide has been tested for mutagenicity to mammalian cells in a study conducted according to OECD TG 476 and in compliance with GLP. Mouse lymphoma L5178Y TK+/- cells were exposed to test substance for 3 hours at concentrations of up to 600 μg/mL in the presence and absence of metabolic activation. The experiment was repeated with a 24-hour exposure time at concentrations up to 50 μg/mL, at which concentration the Relative Total Growth (RTG) was reduced to 14%. Solvent and positive controls gave expected results No biologically relevant increase in mutant frequency was observed with the test item in the presence or absence of metabolic activation so it was concluded that di-tert-nonyl polysulfide is not mutagenic to mammalian cells under the conditions of the study

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

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

A study has been conducted to evaluate the in vitro gene mutation potential of di-tert-nonyl polysulfide in mammalian cells.

The key in vitro gene mutation study in mammalian cells study with the substance di-tert-nonyl polysulfide was conducted according to OECD TG 490 and in compliance with GLP. Mouse lymphoma L5178Y TK+/- cells were exposed to TPS 37 for 3 hours at concentrations of up to 600 μg/mL in the presence and absence of metabolic activation. The experiment was repeated with a 24-hour exposure time at concentrations up to 50 μg/mL, at which concentration the Relative Total Growth (RTG) was reduced to 14%. Solvent and positive controls gave expected results. No biologically relevant increase in mutant frequency was observed with the test item in the presence or absence of metabolic activation so it was concluded that di-tert-nonyl polysulfide is not mutagenic to mammalian cells under the conditions of the study (Charles River Laboratories, 2021).

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.