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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-dodecanethiol
EC Number:
246-619-1
EC Name:
tert-dodecanethiol
Cas Number:
25103-58-6
Molecular formula:
C11H24S to C13H28S
IUPAC Name:
2-methylundecane-2-thiol

Method

Target gene:
n/a
Species / strain
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
Controls
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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.