bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity:

In vitro: Gene mutation test in bacteria (Ames Test): S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, 0, 50, 150, 500, 1500, 5000 µg/plate, plate incorporation, ±S9: negative ± S9 (OECD 471, GLP)

In vitro: Chromosome aberration test in mammalian cells (micronucleus test): human lymphocytes, 0, 2.5, 5, 10, 20, 40, 80 µg/ml, ±S9: negative ± S9 (OECD 487, GLP)

In vitro: Gene mutation test in mammalian cells (HPRT assay): V79 cells, 2.5; 4.9; 9.8; 19.5 µg/mL, ±S9: negative ± S9 (OECD 476, GLP)

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

Study period:

1996-03-26 - 1996-04-09 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-documented study according to OECD 471 with minor deviations: only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended (OECD 471 version of May 1983) S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 471: Genetic Toxicology: Salmonella typhimurium, Reverse Mutation Assay, 26 May 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, (OJ No. L383A, 29.12.92), Part B, Method B.14. Other effects - Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency, Method: HG-Gene Muta ― S. typhimurium: The Salmonella typhimurium reverse mutation assay, 1984

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

Target gene:

his-

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The strains were obtained from Professor B.N. Ames, University of California, California, USA.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Top Dose: 5000 µg/plate, as demanded by the guideline, no toxic effects in the pre-test noted. Ideally the concentrations chosen for the mutation tests should include a minimum of three non-toxic concentrations.
Doses: 0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: At 50 mg/ml the test item was immiscible with water and dimethyl sulphoxide, and miscible with ethanol.

Untreated negative controls:

yes

Remarks:

solvent controls

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Remarks:

Solvent control for positive controls: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: none
- Exposure duration: 3 days
- Expression time (cells in growth medium): = exposure duration

SELECTION AGENT (mutation assays): histidine-minimal agar

NUMBER OF REPLICATIONS: Three petri dishes were used for each dose level.

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance can be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration used in the main tests is the same as that used in the preliminary toxicity test.

Rationale for test conditions:

As stipulated by the guideline

Evaluation criteria:

ASSESSMENT OF RESULTS
The mean number of revertant colonies for all treatment groups is compared with the solvent control groups. The mutagenic activity is assessed by applying the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989) and will usually be analysis of variance followed by Dunnett's test.

Statistics:

see above

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (1st experiment), at 1500 & 5000 µg/plate (2nd experiment)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate and at 1500 µg/plate -S9 only (1st experiment), at 1500 & 5000 µg/plate (2nd experiment)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Testing for mutagenicity in bacteria was performed according to OECD TG 471 of 1983. Here, only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended (OECD 471 version of May 1983) S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible. Also, the testing was sufficiently documented, positive and negative controls gave the appropriate response. Hence, the results can be considered as sufficiently reliable to assess the mutagenic potential of the test item. In both presence and absence of metabolic activation, the test item did not induce a significant increase in the number of revertant colonies per plate over those of the negative control plates. In consequence, the test item is considered to be non-mutagenic under the conditions of this test.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test item according to OECD TG 471 (1983) under GLP, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) were exposed to the test substance, diluted in ethanol which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary toxicity test with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150 and 50 µg/plate.

No evidence of mutagenic activity was seen at any dose level of the test item in either mutation test.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, when tested in ethanol, the test item shows no evidence of mutagenic activity in this bacterial system.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-08 - 2017-10-10 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

OECD Guidelines for Testing of Chemicals (2014) No. 487 "In Vitro Mammalian Cell Micronucleus Test", adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU method B.47

Version / remarks:

Commission Regulation (EU) No 2017/735 B49 “In vitro Mammalian Cell Micronucleus Test”, dated 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

Target gene:

n/a

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index: Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
- Sex, age and number of blood donors if applicable:
Preliminary Toxicity Test: female, aged 26 years
Main Experiment: female, aged 27 years
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Cytokinesis block (if used):

Cytochalasin B at a final concentration of 4.5 µg/mL

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / beta-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

The test item was considered to be a UVCB and therefore the maximum recommended dose was initially set at 5000 µg/mL. However, due to formulation difficulties the maximum dose that could be achieved was 2500 µg/mL. No purity correction was required.
Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed at 0.5%. Therefore, the maximum achievable concentration was 2500 µg/mL.
The dose range for the Preliminary Toxicity Test was 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL.
The dose levels of the test item in the main test were 0*, 2.5, 5, 10*, 20*, 40*, 80 µg/ml (*Dose levels selected for analysis of micronucleus frequency in binucleate cells)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in dimethyl sulphoxide at 500 mg/mL but was soluble in acetone at 500 mg/mL in solubility checks performed in house. Prior to each experiment, the test item was accurately weighed, dissolved in acetone and serial dilutions prepared.

Untreated negative controls:

yes

Remarks:

solvent controls

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine (DC)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48h
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): 24h
- Fixation time (start of exposure up to fixation or harvest of cells): 28h or 48h

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF REPLICATIONS: Duplicate lymphocyte cultures (A and B), three independent conditions

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After Cytochalasin B treatment the cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making. The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

NUMBER OF CELLS EVALUATED:
Minimum of approximately 500 cells per culture for Cytokinesis Block Proliferation Index (CBPI)
The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The criteria for identifying micronuclei were that they were round or oval in shape, non refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)
- Any supplementary information relevant to cytotoxicity: Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Rationale for test conditions:

as set out in the guideline

Evaluation criteria:

Due to limitations of this free-text field, see "Any other information on materials and methods"

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Key result

Species / strain:

lymphocytes: human

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no differences to control
- Effects of osmolality: no differences to control
- Evaporation from medium: none
- Precipitation: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 39.06 µg/mL and 78.13 µg/mL in the 4-hour exposure groups in the absence and presence of S9, respectively and at and above 39.06 µg/mL in the 24-hour continuous exposure group.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL. The maximum dose was the maximum achievable dose level due to formulation difficulties.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 39.06 µg/mL and 78.13 µg/mL in the 4-hour exposure groups in the absence and presence of S9, respectively and at and above 39.06 µg/mL in the 24-hour continuous exposure group.
Hemolysis was observed following exposure to the test item at and above 1250 µg/mL in the 4-hour exposure groups and at 1250 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 1250 µg/mL in all three exposure groups. However, since precipitate occurred at lower dose levels this was the main factor considered in the selection of dose levels for CPI analysis. The test item induced no marked evidence of toxicity in any of the exposure groups in the dose range selected.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and was 80 µg/mL for all three exposure groups.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: similar to control

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: The test item did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see tables below

Conclusions:

The study was conducted under GLP according to OECD guideline 487 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of the test item to induce micronuclei in mammalian cells. Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, Bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Executive summary:

Introduction:

This report describes the results of an in vitro study according to OECD 487 under GLP for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

 

Methods: Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. The dose levels selected for the Main Test were as follows:

Exposure Group	Final concentration of the test item (µg/mL)
4-hour without S9	0, 2.5, 5, 10, 20, 40, 80
4-hour with S9 (2%)	0, 2.5, 5, 10, 20, 40, 80
24-hour without S9	0, 2.5, 5, 10, 20, 40, 80

 

Dose formulation analysis was performed on the top dose formulation (16 mg/mL) of the main test, the results indicated that the average concentration was 103% of nominal. Homogeneity and stability of the test item in acetone was confirmed for a 24-hour period.

 

Results:All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the lowest precipitating dose level. The test item did demonstrate some toxicity in the Preliminary Toxicity Test but at dose levels above the lowest precipitating dose level and so had no influence the dose selection for the main test.

The dose formulation analysis demonstrated accurate formulation of the test item at 80 mg/mL.

 

Conclusion: The test item, Bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-07 - 2018-01-08 (experimental phase)

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:

OECD Guidelines for the Testing of Chemicals No. 476 “In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes“ (adopted 29 July 2016)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008 B.17: ”Mutagenicity – In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

United States Environmental Protection Agency „Health Effects Test Guidelines, OPPTS 870.5300, In vitro Mammalian Cell Gene Mutation Test“, EPA 712-C-98-221, August 1998.

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Japanese Guidelines: “Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable: Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of the laboratory allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3×10E6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: doubling time 12 - 16 h in stock cultures

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Phase separation occurred at 156.3 μg/mL and above after 4 hours treatment with and without metabolic activation.
The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0.
Doses were 0, 2.5, 4.9, 9.8, 19.5, 39.1, 78.1, 156.1 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

acetone, purity 99.98%, 0.5% final concentration

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Approximately 0.7 to 1.2×10E7 were seeded in plastic flasks

DURATION
- Preincubation period: 24h
- Exposure duration: 4h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): ca. 8 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine

NUMBER OF REPLICATIONS: duplicate cultures per concentration level

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:

as indicated by the guideline

Evaluation criteria:

Evaluation of Results
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

Statistical Analysis
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.
Linear Regression
experimental group p-values of the mean values
without S9 mix 0.359
with S9 mix 0.496

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none, Solvent control: 383 mOsm, 2500 μg/mL test item: 359 mOsm
- Effects of osmolality: none, Solvent control: 7.37, 2500 μg/mL test item: 7.35
- Precipitation: phase separation was observed at the highest evaluated dose

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 19.5 μg/mL and 2500 μg/mL were used. The highest concentration of the pre-experiment was based on the solubility of the test item in acetone of the test item.
In the pre-experiment a relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 625.0 μg/mL and above without metabolic activation. In the presence of metabolic activation strong cytotoxic effects were determined at 1250.0 μg/mL and above.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Phase separation occurred at 156.3 μg/mL and above after 4 hours treatment with and without metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see respective table

Conclusions:

The study was conducted under GLP according to OECD guideline 476 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of the test item to induce gene mutations in mammalian cells. Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed according to OECD TG 476 under GLP to investigate the potential of Bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment was 2500 μg/mL. According to the OECD guideline it should be 5mg/mL for UVCB’s, but the maximum concentration was limited by the solubility properties of the test item in acetone. The concentration range of the main experiment was limited by phase separation of the test item.

No substantial and dose dependent increase of the mutation frequency was observed in the main experiment.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

As all available and required in vitro genotoxicity tests, i.e. testing for gene mutations in both bacteria and mammalian cells as well as chromosome mutations in mammalian cells, revealed negative results, no conclusion on a mode of action for genotoxic events can be drawn. No indication is given that the obtained results are not relevant for humans, as in vivo metabolism of the test item is sufficiently mimicked by addition of S9 mix, human lymphocytes were also tested and direct genotoxins act commonly species-independent.

Justification for classification or non-classification

All available test results for gene mutation and chromosome aberrations (micronucleus test) in vitro are consistently negative, and no need for classification as mutagen or directly genotoxic carcinogen was identified. Gene and chromosome mutations are considered initial steps in rather complex carcinogenesis. As the substance does not induce those, no need to consider the substance as carcinogen is evident.

According to Regulation 1272/2008 and amendments, regarding “3.5. Germ cell mutagenicity“, this hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class. Further, substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens. Last but not least, to arrive at a classification, test results are considered from experiments determining mutagenic and/or genotoxic effects in germ and/or somatic cells of exposed animals. Mutagenic and/or genotoxic effects determined in in vitro tests shall also be considered.

In consequence, consistent negative results in three different in vitro test systems addressing three different events related to genetic damage allow to conclude that the substance does not need to be classified as germ cell mutagen.