1,3-bis(2,4,4-trimethylpentan-2-yl)trisulfane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It was concluded that the test item showed no evidence of mutagenic activity in the bacterial system under the test conditions.

The test item was considered to be non-clastogenic to human lymphocytesin vitro.

The test item did not induce any increases in the mutant frequency, consequently it is considered to be non-mutagenic in the test assay.

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:

01 Feb - 03 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Remarks:

human

Details on mammalian cell type (if applicable):

- Sex, age and number of blood donors if applicable:
Preliminary Toxicity Test: male, aged 25 years
Main Experiment: male, aged 26 years

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Preliminary Toxicity Test
Concentratin (μg/mL): 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL.
The maximum dose was the maximum recommended dose level.

Main Study
4(20)-hour without S9 (μg/mL): 7.81, 15.63, 31.25, 46.87, 62.5, 125
4(20)-hour with S9 (2%) (μg/mL): 7.81, 15.63, 31.25, 46.87, 62.5, 125
24-hour without S9 (μg/mL): 7.81, 15.63, 31.25, 46.87, 62.5, 125
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, and toxicity also being considered in the 24-hour exposure group.

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DURATION
- Exposure duration:
1. 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period,
2. 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and
3. a 24-hour exposure in the absence of metabolic activation.

NUMBER OF REPLICATIONS: Duplicate cultures of human lymphocytes

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
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. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

Statistics:

Fisher's Exact test.

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 examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

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 examined

Positive controls validity:

valid

Conclusions:

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Jul - 24 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Test concentrations with justification for top dose:

1.95, 3.91, 7.81, 15.63, 31.25, 62.5 μg/mL
The maximum dose level used was limited by precipitate.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4-hour and 24-hour exposure
- Expression time (cells in growth medium):
1 x 10^6 cells/mL for the 4-hour exposure groups in both the absence and presence of metabolic activation,
0.3 x 10^6 cells/mL in 10 mL for the 24-hour exposure group in the absence of metabolic activation.

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: Relative Suspension Growth (%RSG), Viability (%V) data a Relative Total Growth (RTG).
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

4-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 examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

24-Hour Exposure

Metabolic activation:

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 examined

Positive controls validity:

valid

Conclusions:

The test item, did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October 2015 - 23 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD and EC guidelines and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Batch number: L002
- Expiry: 15 August 2016
- Appearance: Pale yellow liquid
- Storage conditions: Room temperature, in the dark
- Purity/Assay: 95.4%

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Test 1: 5, 15, 50, 150, 1500, 5000 µg/plate
Test 2: 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle used: Ethanol
- Justification for choice: The solubility of DAILUBE IS-30 was assessed at 50 mg/mL in DMSO and ethanol. It was found to be insoluble in DMSO, however in ethanol it formed a colourless solution.

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In the absence of S9 mix, 2 μg/plate for strains TA100 and TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9 mix, 50 μg/plate for strain TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S9 mix, 2 μg/plate for strain TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

In the absence of S9 mix, 2 μg/plate for strain WP2 uvrA (pKM101)

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

In the presence of S9 mix, 5 μg/plate for strains TA100 and TA1535 10 μg/plate for strain WP2 uvrA (pKM101)

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

In the presence of S9 mix, 5 μg/plate for strains TA98 and TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Positive control substance:

other: Ethanol

Details on test system and experimental conditions:

First test

Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate), positive control or vehicle control were placed in glass tubes. The vehicle control was ethanol. S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37C for ca 72 hours. After
this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer). Some plates were scored manually because of toxicity.

Second test

As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The pre-incubation procedure is not suitable for ethanol, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.

Evaluation criteria:

Any toxic effects of the test substance may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both. In the absence of any toxic effects, the maximum concentration selected for use in the second test is the same as that used in the first. If toxic effects are observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate is observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be included in the second test, unless otherwise justified by the Study Director.

Statistics:

None stated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First test

Toxicity (observed as thinning of the background lawn of non-revertant colonies) was seen in strains TA1535 following exposure to DAILUBE IS-30 at 5000 μg/plate in the absence of S9 mix in the first mutation test. Toxicity (observed as thinning of the background lawn of non-revertant colonies, and/or a reduction in revertant colony numbers) was seen in strains TA100, TA1535 and TA1537 following exposure to DAILUBE IS-30 at 5000 μg/plate in the presence of S9 mix in the first mutation test.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to DAILUBE IS-30 at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Second test

No evidence of toxicity was obtained following exposure to DAILUBE IS-30.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to DAILUBE IS-30 at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Conclusions:

It was concluded that the test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A Bacterial Reverse Mutation Test (Envigo CRS Limited, 2016, CXP0018) was conducted to examine the potential for Dailube-IS to cause gene mutation. The study was conducted according to OECD test guideline 471, EU Method B.13/14 and in compliance with GLP.

 

Mutant strains of Salmonella Typhimurium (TA1535, TA1537, TA98, and TA100) and Escherichia Coli (WP2 uvrA pKM101) were exposed to Dailube-IS as a solution in ethanol in triplicates at concentrations up to 5000 μg/plate in both the presence and absence of metabolic activation. Toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen at concentration of 5000 μg/plate in strain TA1535 in the absence of S9 mix and in strains TA100, TA1535 and TA1537 in the presence of S9 mix. Dailube-IS showed no signs of mutagenic activity in any of the tester strains used, either in the presence or the absence of metabolic activation.

A Chromosome Aberration Test in Human Lymphocytes was conducted (Envigo Research Limited, 2017, VL63RT) do determine the potential for Dailube-IS to cause chromosomal aberrations in Human Lymphocytes culturedin vitro. The study was conducted according to OECD Test Guideline 473, and in compliance with GLP.

 

Duplicate cultures were treated with Dailube-IS at concentration up to 125 μg/mL for 4 hours in the presence and absence of metabolic activation followed by 20 hours incubation, as well as 24 hour continuous exposure without metabolic activation. The maximum test concentration was limited by the test item precipitation. No significant increases in the incidence of chromosomal aberrations relative to the concurrent controls were observed at any dose level tested. It was concluded that Dailube-IS had not shown any evidence of clastogenic activity in this study.

 

L5178Y +/- Mouse Lymphoma Assay was conducted (Envigo Research Limited, 2017, BQ83KC) to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016 and was conducted and in compliance with GLP.

 

A single mutagenicity test was performed in which L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels (from 0.98 to 125μg/mL) in duplicate, together with vehicle (acetone), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation. The maximum dose level used was limited by precipitate. At the end of the treatment period, the cells were washed twice. The cultures were incubated and subcultured every 24 hours for the expression period of two days. On Day 2 of the experiment, the cells were counted. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

 

A precipitate was observed at and above 62.5µg/mL in all exposure groups. The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, there was no evidence of reductions in viability (%V), and thus indicating that residual toxicity had not occurred. consequently it is considered to be non-mutagenic in this assay.

Justification for classification or non-classification

Bacterial reverse mutation (in vitro) - Negative with and without metabolic activation.

Chromosome aberration (in vitro) - No evidence of clastogenic activity in the presence or absence of metabolic activation.

Mouse Lymphoma Assay (in vitro) - Not considered to be mutagenic in this assay.

 

Endpoint Conclusion: No adverse effect observed (negative)

Of the three studies mentioned above, none indicated any evidence of mutagenic or clastogenic activity. On this basis it is concluded that there is no basis on which to classify Dailube-IS for mutagenic effects.