tert-dodecanethiol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tert-dodecanethiol did not produce genetic changes in standard in vitro tests using animal cells (mouse lymphoma forward mutation assay and in vitro sister chromatid exchange assay) or bacterial cells (Ames Salmonella assay), and no clastogenic activity in the recent chromosomal aberrations assay in cultured Human lymphocytes.

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:

December 2020 - March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

n/a

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes from blood collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age).

Cytokinesis block (if used):

During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium) (Acros Organics, Geel, Belgium).

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies).
The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL
S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Test concentrations with justification for top dose:

In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose-range finding test. The test item was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
The highest tested concentration was determined by the solubility of the test item in the culture medium.
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary.

Without S9-mix:10, 105 and 125 µg/mL culture medium (3 h exposure time, 24 h fixation time).
With S9-mix:31.3, 62.5 and 125 µg/mL culture medium (3 h exposure time, 24 h fixation time).

Without S9-mix:5, 15, 30, 40, 50, 60 and 70 µg/mL culture medium (24 h exposure time, 24 h fixation time).
Without S9-mix:5, 30 and 40 µg/mL culture medium (24 h exposure time, 24 h fixation time).

Vehicle / solvent:

ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

The final concentration of the solvent in the culture medium was 0.5% (v/v). The pH and the osmolarity of the culture medium containing the highest non-precipitating concentration were recorded. Test item concentrations were used within 2 hours after preparation.

Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added.

Environmental conditions
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 39 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.3 - 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Second Cytogenetic Assay
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of the test item for 24 h in the absence of S9-mix.
The cells were not rinsed after exposure but were fixed immediately after 24 h (24 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.


Chromosome Preparation
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium) (Acros Organics, Geel, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).

Preparation of Slides
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

Mitotic Index/Dose Selection for Scoring of the Cytogenetic Assay
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analyzable concentrations were used for scoring of the cytogenetic assay. For the 3 h exposure time in the absence and presence of S9-mix, the test item was not cytotoxic and difficult to dissolve in aqueous solutions. Therefore the highest concentration analyzed was determined by the solubility in the culture medium. For the 24 h exposure time in the absence of S9-mix chromosomes of metaphase spreads were analyzed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of Slides for Chromosome Aberrations
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with Charles River Den Bosch study identification number and code was placed over the marked slide. One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was = 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.

Rationale for test conditions:

ACCEPTABILITY CRITERIA
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).

Evaluation criteria:

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range

Statistics:

Graphpad Prism version 8.4.2. (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose-range Finding Test / First Cytogenetic Assay

The test item precipitated directly in the culture medium at concentrations of 31.3 µg/mL and above. The test item precipitated in the culture medium after 3 h exposure at concentrations of 125 µg/mL and above. The pH and osmolarity of a concentration of 31.3 µg/mL were 7.5 and 380 mOsm/kg respectively (compared to 7.5 and 380 mOsm/kg in the solvent control).
At the 3 h exposure time, blood cultures were treated in duplicate with 31.3, 62.5 and 125 µg test item/mL culture medium with and without S9-mix (first cytogenetic assay).
At the 24 hour exposure time single blood cultures were treated with 7.8, 15.6, 31.3, 62.5, 125 and 250 µg test item /mL culture medium without S9-mix (dose-range finding test).
In the presence of S9-mix (3h of exposure), appropriate dose levels could be selected for scoring of chromosome aberrations.
In the absence of S9-mix (3h of exposure), no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 62.5 µg/mL not enough cytotoxicity was observed (3%), whereas the next higher concentration of 125 µg/mL was too toxic for scoring (64%). So, the experiment without S9-mix (3h exposure time) was repeated in cytogenetic assay 1A. The following dose levels were selected for the cytogenetic assay:
Without S9-mix:10, 85, 95, 105, 115 and 125 µg/mL culture medium (3 h exposure time, 24 h fixation time).
The following dose levels were selected for scoring of chromosome aberrations (3h exposure):
Without S9-mix : 10, 105 and 125 µg/mL culture medium
(3 h exposure time, 24 h fixation time).
With S9-mix : 31.3, 62.5 and 125 µg/mL culture medium
(3 h exposure time, 24 h fixation time).
Both in the absence and presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations after 3h exposure.
Both in the absence and presence of S9-mix, the test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes after 3h exposure.

Second Cytogenetic Assay
To obtain more information about the possible clastogenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to the test item in the absence of S9-mix for 24 hours. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix:5, 15, 30, 40, 50, 60 and 70 µg/mL culture medium (24 h exposure time, 24 h fixation time).
Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix:5, 30 and 40 µg/mL culture medium (24 h exposure time, 24 h fixation time).
These dose levels represent an inhibition of the mitotic index of 35%, 44% and 60% respectively.
The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.


The ability of the test item to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analyzed was selected based on the solubility of the test item in the culture medium (first experiment) or toxicity with an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control (second experiment).

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database (see Table 8). The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.
Therefore it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Conclusions:

In conclusion, this test is valid and Tertiododecylmercaptan is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Executive summary:

The objective of this study was to evaluate Tertiododecylmercaptan for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The possible clastogenicity of the test item was tested in two independent experiments. The vehicle of the test item was ethanol.

In the first cytogenetic assay, the test item was tested up to 125 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix. The test item precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test item was tested up to 40 µg/mL for a 24 h continuous exposure time with a 24 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.  Therefore it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. 

In conclusion, this test is valid and Tertiododecylmercaptan is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Study not duplicated

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver microsomal fraction

Test concentrations with justification for top dose:

100, 67, 44.9, 30.1, 20.2, 13.5, 9.0, 6.1 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: EMS (-S9) and 3-MCA (+S9)

Details on test system and experimental conditions:

The assay was performed with the TK+/- phenotype of L5178Y mouse lymphoma cells from sub-line 3.7.2C using a minimum of eight test compound doses with and without metabolic activation by an Aroclor-induced rat liver microsomal fraction. Appropriate negative, solvent, and positive controls were included with each assay. 
The test compound dose levels were determined by a preliminary multidose-ranging study with the highest dose tested being selected to give approximately 50-90% inhibition of suspension cell growth depending on the solubility of the compound. Tertiary dodecyl mercaptan solubilized at approximately 100 µg/ml in dimethylsulfoxide. The maximum dose selected for the mutagenicity test was 100 µg/ml because it exhibited growth inhibition. 
Each test concentration was prepared to contain the test dose in 0.1 ml volumes. Six million precleansed TK+/- cells in 6 ml of F10 P were added to centrifuge tubes. An additional 4 of the S-9 mix were added to half of the tubes. Immediately thereafter, 0.1 ml of the 100x and 0.1 ml of the solvent were added to the appropriate tubes. Each tube was mixed, gassed with a mixture of carbon dioxide and air, and incubated at 37; 0.50 C on a revolving roller drum for 4 hours. Following this incubation the tubes were centrifuged and the treatment solutions decanted. The cells were washed twice with F10 P and resuspended in 20 ml F10 P after the second wash. The tube cultures were readjusted to 3.0 x 100,000 cells/ml as necessary. At the end of the expression period, a sample of each of the cultures was centrifuged and the cells resuspended at 500,000 viable cells/ml in F10 P. The concentrated cells were serially diluted and appropriate dilutions were plated in triplicate in cloning medium with and without TFT. Approximately 500,000 cells were plated on each of 3 selective medium plates containing 2 mg/ml TFT, and 100 cells were cloned on each of 3 non-selective plates for each test concentration and a control tube. The plates  were incubated for 12+/-2 days. The mutant colonies (TK-/-) were counted on the selective TFT containing plates and the survivors (TK+/- and TK-/-) were counted on the non-selective medium plates.

Evaluation criteria:

A chemical will be considered positive if a dose-related response is obtained in which the mutation frequencies at two or more test concentrations (in the absence of severe toxicity) are at least two to three-fold higher than the mutation frequency of the solvent control.

Statistics:

None

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Acceptable growth inhibition observed at 100 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cells were exposed to eight graded doses of the test material in the presence of and in the absence of metabolic activation. A two-fold increase in the induction of forward mutations at the T/K locus in L5178Y mouse lymphoma cells occurred only at the 13.5 µg/ml level with activation. Test considered positive if dose-related response at two or more test concentrations is at least two or three-fold higher than mutation frequency of solvent control. Therefore, tertiary dodecyl mercaptan is not considered to be mutagenic in this test system.

See the table of results in the attached document

Conclusions:

t-dodecyl mercaptan was considered to be negative in this test system

Executive summary:

In an in vitro Mammalian Cell Gene Mutation (mouse lymphoma) assay, L5178Y TK (+/-) mouse lymphoma cells were exposed to eight graded doses of t-dodecyl mercaptan (6.1-100 µg/ml) with and without metabolic activation according to OECD Test Guideline 476. A 2-fold increase in the induction of forward mutations at the T/K locus in L5178Y mouse lymphoma cells occurred only at the 13.5 µg/mL level with activation. The test is considered positive if the dose-related response at 2 or more test concentrations is at least 2- or 3-fold higher than the mutation frequency of solvent control.  Therefore, t-dodecyl mercaptan was considered to be negative in this test system.  

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No duplication of the study

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

S. typhimurium, other: Strains TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Metabolic activation system:

S-9 hepatic microsomes from Aroclor (1254)-induced adult male Sprague-Dawley rats.

Test concentrations with justification for top dose:

10,000, 3,333, 1,111, 370, 123.5 µg/plate. Highest dose of test substance used represented limit of solubility.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of t-DM, 100 mg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - S9: MNNG (TA 1535, TA 100), 9-AA (TA 1537), 2-NF (TA 1538, TA 98). + S9: 2-AA (all strains)

Details on test system and experimental conditions:

Exposure of auxotrophic bacteria was done dIrectly by the plate Incorporation assay. For the induction of reverse mutations, the chemicals was tested against the five bacterial strains with and without the metabolic activation systems. Each concentration of the compound and controls will be plated in triplicate.
Data derived from the toxicity studies wasutilized to establish test levels of the test compound. A minimum of five concentrations of the test compound was prepared to contain the test dose in 0.1 ml volumes for evaluation with and without metabolic activation. The highest dose tested was that level of the chemical exhibiting inhibition of the bacterial growth in toxicity studies, unless this is not possible due to limited solubility of the investigated agent.
To each of 2 ml of complete top agar, 0.1 ml of an overnight broth culture of each tester strain, 0.1 ml of the test compound or diluent, and 0.5 ml of the S-9 mix for the activated tests were added. The contents of the tube were mixed thoroughly and poured onto VBE minimal agar plates. Plates were gently rotated and tilted to assure untform distribution of the top agar, allaôwed to harder on an even surface for one hour, inverted and put in a dark 37 +/- 0.5°C incubator. After two days, the colonies in both test plates and controls will be counted using an electronic colony counter and the density of the background growth will be noted.

Evaluation criteria:

For a test to be considered valid, the following conditions are required:
1. Demonstration of toxicity of the chemical for the S. typhimurium strain(s), unless this is not possible due to limited solubility of the test compound.
2. The negative control responses are within the normal range of the laboratory data base.
3. Confirmation of sensitivity and responsiveness of the tester strains to mutagenic action as indicated by their responses to the positive controls.

If the above criteria are met, a chemical is considered to be mutagenic if it induces a positive response in a dose-related manner over three concentrations with the baseline increase in the number of histidine revertants equal to twice the solvent control level.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

See tables of results in the attached document.

Conclusions:

Exposure to five graded doses of the test material in the presence of and in the absence of metabolic activation did not increase the reversion to histidine prototrophy of S. typhimurium strains TA1535, TA1537, TA1538, TA98 or TA100. Therefore, tertiary dodecyl mercaptan is not considered to be mutagenic in this test system.

Executive summary:

Five Salmonella typhimurium tester strains, TA1535, TA1537, TA1538, TA98 and TA100, were utilized as the experimental organisms. Each strain was exposed to a minimum of five test compound doses both with and without metabolic activation by an Aroclor-induced rat liver microsomal fraction. The test compound dose levels were determined by a preliminary multidose-ranging study with optimal concentration allowing survival of about 50% of the cells. Tertiary dodecyl mercaptan solubilized at approximately 100 mg/ml in dimethyl sulfoxide. The maximum dose selected for the mutagenicity test was approximately 10,000 µg/plate because it represents the limits of solubility of the test material. The mutagenicity assay was done directly by the plate incorporation method. Each of 2 ml of complete top agar, 0.1 ml of an overnight broth culture of each tester strain, 0.1 ml of the test compound or diluent and 0.5 ml of the S-9 mix, for the activated tests only, were combined, mixed thoroughly, and poured onto VBE minimal agar plates. Each concentration of the compound and the positive and negative controls were plated in triplicate. Plates were gently rotated and tilted to assure uniform distribution of the top agar, allowed to harden to an even surface for 1 hour, inverted and put in a dark 37 +/- 0.50°C incubator. After 2 days, the colonies on both test and control plates were counted using an electronic colony counter and the density of the background growth was noted. Exposure to five graded doses of the test material in the presence of and in the absence of metabolic activation did not increase the reversion to histidine prototrophy of S. typhimurium strains TA1535, TA1537, TA1538, TA98 or TA100. Therefore, tertiary dodecyl mercaptan is not considered to be mutagenic in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation

In the reverse mutation assay, Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 were exposed to at least five concentrations of tert-dodecanethiol in the presence and absence of a metabolic activation system using concentrations up to 10 mg/plate according to OECD TG 471. No increase in reversion rates occurred over the dose range tested, with or without S9 mix, in any of the five (Hazleton 1983) test strains.

In an in vitro Mammalian Cell Gene Mutation (mouse lymphoma) assay, L5178Y TK (+/-) mouse lymphoma cells were exposed to eight graded doses of tert-dodecanethiol (6.1-100 µg/mL) with and without metabolic activation according to OECD TG 476. A 2-fold increase in the induction of forward mutations at the T/K locus in L5178Y mouse lymphoma cells occurred only at the 13.5 µg/mL level with activation. The test is considered positive if the dose-related response at 2 or more test concentrations is at least 2- or 3-fold higher than the mutation frequency of solvent control. Therefore, tert-dodecanethiol was considered to be negative in this test system (Hazleton 1983).

 

Chromosomal aberration

in the key study (Covance 2021), the objective of this study was to evaluate Tertiododecylmercaptan for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity of the test item was tested in two independent experiments. The vehicle of the test item was ethanol. In the first cytogenetic assay, the test item was tested up to 125 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-mix. The test item precipitated in the culture medium at this dose level. In the second cytogenetic assay, the test item was tested up to 40 µg/mL for a 24 h continuous exposure time with a 24 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.  Therefore it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in the report. In conclusion, this test is valid and Tertiododecylmercaptan is not clastogenic in human lymphocytes under the experimental conditions described in this report.

In the supporting chromosomal aberration assay (Covance 1997), human lymphocytes were exposed to tert-dodecanethiol concentrations up to 94 µg/mL, with and without metabolic activation. The study was conducted according to OECD TG 473. No effects were observed at the 20-hour sampling time. At 44 hours in the absence of S9, the number of cells with aberrations was significantly higher than in concurrent negative controls at all dose levels analyzed. The numbers of cells with aberrations observed exceeded the historical control range, but this effect was only seen in both replicates at the highest concentration tested (30.03 µg/mL) at which severe mitotic inhibition was apparent. It was concluded that tert-dodecanethiol induced chromosomal aberrations in cultured human peripheral blood lymphocytes; however, this effect was restricted to prolonged, cytotoxic treatment in the absence of S9, and is therefore, considered to be equivocal.

 

DNA damage

In an in vitro Sister Chromatid Exchange (CHO cells) assay, CHO cells were exposed to five graded doses (0.5-75 µg/mL) of tert-dodecanethiol with and without metabolic activation according to OECD TG 479. There were no dose-dependent increases in the number of SCEs/chromosome. Therefore, tert-dodecanethiol was considered to be negative in this test system (Hazleton, 1984).

Justification for classification or non-classification

Based on the available data, no classification for germ cell mutagenicity is required for Tert-dodecanethiol according to the Regulation EC n°1272/2008.