4,6-bis(octylthiomethyl)-o-cresol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4,6-bis(octylthiomethyl)-o-cresol

EC Number:

402-860-6

EC Name:

4,6-bis(octylthiomethyl)-o-cresol

Cas Number:

110553-27-0

Molecular formula:

C25 H44 O S2

IUPAC Name:

2-methyl-4,6-bis[(octylsulfanyl)methyl]phenol

Details on test material:

- Physical state: yellow liquid
- Storage condition of test material: room temperature

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO cells
- Suitability of cells: proven
- Normal cell cycle time (negative control): 16 - 20 h in stock cultures

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: twice per week
- Methods for maintenance in cell culture: The cell cultures were incubated at 37°C and 15.0 % carbon dioxide atmosphere
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: DMEM/F12 (Dulbecco's modified eagle medium; mixture 1:1) supplemented with 10 % fetal calf serum (FCS)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver induced with Aroclor 1254

Test concentrations with justification for top dose:

without and with S9 mix:
8 h: 10.0; 20.0; 30.0; 40.0; 50.0 µg/mL
24 h: 1.0; 5.0; 10.0; 20.0; 30.0; 40.0; 50.0 µg/mL
30 h: 10.0; 20.0; 30.0; 40.0; 50.0 µg/mL

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: EMS; EthyImethanesulfonate; With metabolic activation: CPA; Cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 48 h
- Exposure duration: 5, 21, 27 h
- Fixation time (start of exposure up to fixation or harvest of cells): 8, 24, 30 h
The treatment interval was 4 h.

SPINDLE INHIBITOR (cytogenetic assays): colcemid (3 h)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per slide = 200 per test group

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.
A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system.
However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the mitotic index was slightly reduced after treatment with the highest dose level

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the pre-experiment on toxicity (colony forming ability) in the absence and presence of 89 mix even after treatment with the highest concentration (50.0 ng/ml) the colony forming ability was only slightly reduced. Higher concentrations precipitated strongly in the culture medium.

STUDY RESULTS
Both, in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberrations at any dose level either to a biologically or to statistically relevant extend. The aberration rates after treatment with the test article (0.5 % - 5.0 %) are in our historical control data range (0.0 % - 5.0 %) or near to the range of the actual control values: 0.5 % - 2.0 %. In this experiment, there was a clonal effect in the CHO cell line revealing dicentric marker chromosomes distributed randomly in the treatment and control groups. Therefore, dicentric chromsomes were recorded but not included in the calculation of the aberration rates. Due to the random distribution this measure did not affect the validation of the study.

Any other information on results incl. tables

PRE-EXPERIEMNT FOR TOXICITY

In the pre-experiment the toxicity of the test article was examined with the plating efficiency (colony forming ability). The results are given below:

Table 1: Plating Efficiency Assay (PE) without metabolic activation Per flask 496 single cells were seeded.

	colonies counted
conc. per ml	flask I	flask II	mean	relative Plating Efficiency %
negative control	550	439	494.5
solvent control	383	360	371.5	100.0
0.01	457	494	475.5	128.0
0.10	490	464	477.0	128.4
0.30	428	405	416.5	112.1
1.00	389	433	411.0	110.6
3.00	332	350	341.0	91.8
10.00	314	339	326.0	87.9
25.00	253	278	265.0	71.5
50.00	286	314	300.0	80.8

Table 2: Plating Efficiency Assay (PE) with metabolic activation Per flask 496 single cells were seeded.

	colonies counted
conc. per ml	flask I	flask II	mean	relative Plating Efficiency %
negative control	356	328	342.0
solvent control	316	331	323.5	100.0
0.01	337	358	247.5	107.4
0.10	320	319	319.5	98.8
0.30	319	375	347.0	107.3
1.00	322	355	338.5	104.6
3.00	345	379	362.0	111.9
10.00	386	353	369.5	114.2
25.00	347	389	368.0	113.8
50.00	305	190	247.5	76.5

fixation interval: 8 h
Concentration (µg/mL)	Metabolic activation	Metaphases	Aberrations incl. Gaps (%)	Aberrations excl. Gaps (%)	Exchanges (%)
Vehicle Ctrl.	without	200	4.5	2.0	0.0
	with	200	3.0	2.0	0.0
50.0	without	200	1.0	0.5	0.0
	with	200	7.0	5.0	0.0
fixation interval: 24 h
Concentration (µg/mL)	Metabolic activation	Metaphases	Aberrations incl. Gaps (%)	Aberrations excl. Gaps (%)	Exchanges (%)
Negative Ctrl.	without	200	4.0	2.0	0.0
	with	200	2.5	2.0	1.0
Vehicle Ctrl.	without	200	5.5	2.0	0.0
	with	200	1.0	0.5	0.0
5.0	without	200	2.5	2.5	0.0
	with	200	5.0	3.0	0.0
20.0	without	200	2.0	2.0	0.0
	with	200	3.5	2.0	0.0
50.0	without	200	8.0	5.0	0.0
	with	200	2.0	1.0	0.0
Positive Ctrl.	without	100	28.5	26.0	13.5
	with	100	15.0	12.0	5.0
fixation interval: 30 h
Concentration (µg/mL)	Metabolic activation	Metaphases	Aberrations incl. Gaps (%)	Aberrations excl. Gaps (%)	Exchanges (%)
Vehicle Ctrl.	without	200	2.0	1.0	0.0
	with	200	2.0	0.5	0.0
50.0	without	200	2.0	0.5	0.0
	with	200	2.0	1.5	0.0

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test article did not induce structural chromosome aberrations in the CHO cell line.

Executive summary:

The test article was assessed for its potential to induce structural chromosome aberrations in CHO cells in vitro. Preparation of chromosomes was done 8 h (high dose), 24 h (low, medium and high dose) and 30 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following dose levels were evaluated both without S9 mix and with S9 mix:

8 h: 50.0 µ/ml

24 h: 5.0; 20.0; 50.0 µg/ml

30 h: 50.0 µg/ml

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment of the cells even with the highest dose level

(50.0 µg/ml) reduced only slightly the plating efficiency. Higher concentrations than 50.0 µg/ml precipitated strongly in the culture medium. Also, the mitotic index was slightly reduced with the highest concentration at each fixation interval in the absence and at interval 8 h in the presence of S9 mix. There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the CHO Chinese Hamster cell line. Therefore, the test item is considered to be non-mutagenic in this chromosomal aberration test.