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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: S. typhimurium TA98, TA100, TA1535, TA1537, with and without metabolic activation, up to 5000 µg/plate: negative (GLP, OECD 471)
HPRT assay: V79 cells, with and without metabolic activation: negative (GLP, OECD 476)
Chromosome aberration assay: CHO cells, with and without metabolic activation: negative (GLP, OECD 473)

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

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

Type of assay:

in vitro mammalian chromosome aberration test

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

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.

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

4 stains only, as required by at that time valid version of OECD 471

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat livers induced with Aroclor 1254

Test concentrations with justification for top dose:

20 - 5000 µg/plate

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see Details of test item.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

OTHER:
Positive control experiments are carried out simultaneously with the following substances without metabolic activation: 1) for strain TA 98: daunorubicin-HCl, 5 and 10 µg/plate; 2) for strain TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 µg/plate; 3) for strain TA 1535: sodium azide, 2.5 and 5.0 µg/plate; 4) for strain TA 1537: 9(5)-aminoacridine hydrochloride monohydrate, 50 and 100 µg/plate. The activation mixture is tested: 1) with strains TA 98, TA 100, TA 1537: 2-aminoanthracene, 5 µg/plate; 2) with strain TA 1535: cyclophosphamide, 250 µg/plate.

Evaluation criteria:

The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA 98, TA 1535 and TA 1537,
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by a factor of 1.5 for strain TA 100.
Generally a concentration-related effect should be demonstrable.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

At the concentrations of 1250 and 5000 µg/plate the substance precipitated in soft agar.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: At the concentrations of 1250 and 5000 p.g/0.1 ml the substance precipitated in soft agar.

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the toxicity tests treatment of the bacteria with the highest concentration (5000 µg/plate) of the test item did not lead to a growth-inhibiting effect. This concentration was therefore selected as the highest in the mutagenicity tests.

Standard plate test I (20 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	16	1.5	no	negative
	yes	34	1.1	no	negative
TA 100	no	173	1.1	no	negative
	yes	147	0.9	no	negative
TA 1535	no	15	0.9	no	negative
	yes	11	1.2	no	negative
TA 1537	no	8	1.4	no	negative
	yes	19	0.9	no	negative
Standard plate test II (20 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	39	1.1	no	negative
	yes	46	1.2	no	negative
TA 100	no	180	1.1	no	negative
	yes	147	1.1	no	negative
TA 1535	no	13	1.5	no	negative
	yes	18	0.9	no	negative
TA 1537	no	8	1.5	no	negative
	yes	23	0.9	no	negative

Reference 3

Endpoint:

in vitro gene mutation 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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

MEDIA USED
- Ham's F10, 10% pre-tested foetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat livers induced with Aroclor 1254

Test concentrations with justification for top dose:

Original experiment:
Range with activation: 25 - 500 µg/mL
Range without activation: 50 - 1000 µg/mL

Confirmatory experiment:
Range with activation: 50 - 1000 µg/mL
Range without activation: 50 - 1000 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with metabolic activation: 1.0 µL/mL of the promutagen N-nitroso-dimethylamine (DMN); without metabolic activation: 300 nL/mL of the ultimate mutagen ethylmethanesulphonate (EMS)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: six cultures each containing 100 cells were set up for every concentration.
- Number of independent experiments: 2

DURATION
- Preincubation period: 42 h
- Exposure duration: five hours by 22.5 mL treatment medium and 2.5 mL S9 activation mixture, or for 21 hours by 25 mL treatment medium alone
- Expression time (cells in growth medium): 5 days
- Selection time (if incubation with a selection agent): 7-8 days

SELECTION AGENT (mutation assays): 8 µg/mL 6-thioguanine
STAIN: Giemsa

DETERMINATION OF CYTOTOXICITY
- Method: cell viability

Evaluation criteria:

All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If this cloning efficiency is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mutant factor, which is defined as the ratio of the mutant frequency of the treated and the solvent control cultures, will be calculated.
The sensitivity of the test is restricted to a lower limit of the mutant frequency of 4x10e-6. If the mutant frequency measured is below this limit, it will be reported to be smaller than 4x10e-6 and the mutant factor will be calculated assuming a mutant frequency of 4x10e-6.

The test substance will be considered mutagenic in this test system, if either:
- The mutant frequency of the treated culture exceeds that of the solvent controls by a mutant factor of 2.5 and there is a dose-dependent increase of the mutant frequency; or:
- The mutant frequency in a treated culture exceeds that of the solvent control by a mutant factor of 3.0 at any concentration tested and reported and the absolute number of clones in the treated and untreated cultures differ by more than 20 clones per 10e6 cells plated.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Maximum concentration applicable based on solubility was 1000 µg/ml. With S9 mix, the maximum concentration was 500 µg/ml due to cytotoxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES
Due to a contamination in the first preliminary toxicity test, the experiment was repeated. The viability of the cells treated in the experiment with microsomal activation for 5 hours with the test item was reduced to a value lower than 10% of the negative control (dimethylsulfoxide) at the highest concentration of 1000 µg/ml. In the experiment without activation the viability of the cells treated for 21 hours with the test item was not decreased to a value lower than 10% of the negative control (dimethylsulfoxide) up to the highest applicable concentration of 1000 µg/ml. Therefore, the original mutagenicity test with microsomal activation was performed with concentrations ranging from 25 - 500 µg/ml and the original mutagenicity test without microsomal activation was performed with concentrations ranging from 50 - 1000 µg/ml. Because the intended toxicity was not obtained in the original experiment with microsomal activation, in the confirmatory experiment the concentrations applied were increased to 50 - 1000 µg/ml.

STUDY RESULTS
Original mutagenicity test
In the original mutagenicity test with microsomal activation, after screening with 6-thioguanine the mutant frequencies in the solvent controls were both <4.00x10e-6. This value represents the lower limit of sensitivity of the test system. The mean value used for calculation is 4.00x10e-6.
At the five lowest concentrations tested, the calculated mutant frequency values were <4.00x10e-6. Comparison of this value with the mean of the two solvent controls revealed mutant factors of 1.00. At the higher concentration up to the highest, the calculated mutant-frequency values were 5.68x10e-6 (factor 1.42) and 5.21x10e-6, resulting in a mutant factor of 1.30.
The positive control treated with 1 µL DMN/mL medium revealed a mutant-frequency of 131.27x10e-6, giving a mutant factor of 32.82.
The mutant-frequencies in the solvent controls of the cultures without microsomal activation were both <4.00x10e-6. At the six lowest concentrations tested, the calculated mutant frequency values were <4.00x10e-6. Comparison of this value with the mean of the two solvent controls revealed mutant factors of 1.00. At the highest concentration the calculated mutant-frequency value was 5.03x10e-6, resulting in a mutant factor of 1.26.
The positive control treated with 300 nL EMS/mL medium gave a mutant frequency of 1183.67x10e-6 and a corresponding mutant factor of 295.92.

Confirmatory mutagenicity test
In the confirmatory mutagenicity test with microsomal activation, after screening with 6-thioguanine the mutant-frequency values in the solvent controls were both <4.00x10e-6. At the six lowest concentrations tested, the calculated mutant frequency values were <4.00x10e-6. Comparison of this value with the mean of the two solvent controls revealed mutant factors of 1.00. At the highest concentration the calculated mutant-frequency value was 10.83x10e-6, resulting in a mutant factor of 2.71.
The positive control treated with 1 nL DMN/mL medium revealed a mutant frequency of 268.34x10e-6, giving a mutant factor of 67.09.
The mutant-frequencies in the solvent controls of the cultures without microsomal activation were both <4.00x10e-6. At all concentrations tested, the calculated mutant frequency values were <4.00x10e-6. Comparison of this value with the mean of the two solvent controls revealed mutant factors of 1.00.
The positive control treated with 300 nL EMS/mL medium gave a mutant frequency of 1179.96x10e-6 and a corresponding mutant factor of 294.99.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

RESULTS OF THE CYTOTOXICITY TEST

Experiment with microsomal activation
Treatment	mean number of clones	percent of control
negative control	74.67
negative control	94.17
1000 µg/ml	0.67	0.80%
500 µg/ml	10.00	11.80%
250 µg/ml	41.50	49.20%
125 µg/ml	43.17	51.10%
62.5 µg/ml	63.83	75.60%
31.25 µg/ml	96.00	113.70%
15.625 µg/ml	86.00	101.90%
7.8125 µg/ml	67.83	80.40%
3.90625 µg/ml	84.83	100.50%
1.95312 µg/ml	66.50	78.80%
0.97656 µg/ml	89.17	105.60%
0.48828 µg/ml	54.83	65.00%
Experiment without microsomal activation
Treatment	mean number of clones	percent of control
negative control	149.67
negative control	146.50
1000 µg/ml	102.50	69.20%
500 µg/ml	101.67	68.70%
250 µg/ml	123.67	83.50%
125 µg/ml	130.50	88.10%
62.5 µg/ml	116.83	78.90%
31.25 µg/ml	131.67	88.90%
15.625 µg/ml	127.67	86.20%
7.8125 µg/ml	136.67	92.30%
3.90625 µg/ml	134.17	90.60%
1.95312 µg/ml	137.17	92.60%
0.97656 µg/ml	133.83	90.40%
0.48828 µg/ml	134.00	90.50%

RESULTS OF THE ORIGINAL EXPERIMENT

Experiment with microsomal activation
Treatment	Mean of Survivors after expression	Mean of mutants per dish	Normalized mean of mutants per dish	Mutant Frequency (10E-6)	Mutant factor
negative control	56.83	0.17	0.29	<4.0
negative control	75.67	0.17	0.22	<4.0
positive control	37.67	4.94	13.13	131.27	32.82
500 µg/ml	53.33	0.28	0.52	5.21	1.30
400 µg/ml	29.33	0.17	0.57	5.68	1.42
300 µg/ml	58.50	0.06	0.09	<4.0	1.00
200 µg/ml	42.33	0.11	0.26	<4.0	1.00
100 µg/ml	54.00	0.00	0.00	<4.0	1.00
50 µg/ml	63.67	0.17	0.26	<4.0	1.00
25 µg/ml	54.50	0.06	0.10	<4.0	1.00
Experiment without microsomal activation
Treatment	Mean of Survivors after expression	Mean of mutants per dish	Normalized mean of mutants per dish	Mutant Frequency (10E-6)	Mutant factor
negative control	36.50	0.00	0.00	<4.0
negative control	53.17	0.00	0.00	<4.0
positive control	32.67	38.67	118.37	1183.67	295.92
1000 µg/ml	77.33	0.39	0.50	5.03	1.26
800 µg/ml	72.33	0.17	0.23	<4.0	1.00
600 µg/ml	58.50	0.11	0.19	<4.0	1.00
400 µg/ml	77.33	0.11	0.14	<4.0	1.00
200 µg/ml	55.00	0.06	0.10	<4.0	1.00
100 µg/ml	54.17	0.11	0.21	<4.0	1.00
50 µg/ml	51.50	0.17	0.32	<4.0	1.00

RESULTS OF THE CONFIRMATORY EXPERIMENT

Experiment with microsomal activation
Treatment	Mean of Survivors after expression	Mean of mutants per dish	Normalized mean of mutants per dish	Mutant Frequency (10E-6)	Mutant factor
negative control	65.67	0.11	0.17	<4.0
negative control	62.50	0.06	0.09	<4.0
positive control	26.50	7.11	26.83	268.34	67.09
1000 µg/ml	46.17	0.50	1.08	10.83	2.71
800 µg/ml	71.50	0.00	0.00	<4.0	1.00
600 µg/ml	42.83	0.06	0.13	<4.0	1.00
400 µg/ml	59.33	0.06	0.09	<4.0	1.00
200 µg/ml	37.00	0.00	0.00	<4.0	1.00
100 µg/ml	68.33	0.00	0.00	<4.0	1.00
50 µg/ml	52.33	0.00	0.00	<4.0	1.00
Experiment without microsomal activation
Treatment	Mean of Survivors after expression	Mean of mutants per dish	Normalized mean of mutants per dish	Mutant Frequency (10E-6)	Mutant factor
negative control	65.17	0.00	0.00	<4.0
negative control	66.50	0.00	0.00	<4.0
positive control	27.17	32.06	118.00	1179.96	294.99
1000 µg/ml	45.00	0.06	0.12	<4.0	1.00
800 µg/ml	43.00	0.00	0.00	<4.0	1.00
600 µg/ml	37.50	0.00	0.00	<4.0	1.00
400 µg/ml	53.33	0.06	0.10	<4.0	1.00
200 µg/ml	40.83	0.11	0.27	<4.0	1.00
100 µg/ml	42.33	0.00	0.00	<4.0	1.00
50 µg/ml	30.83	0.11	0.36	<4.0	1.00

Conclusions:

In both investigations with and without microsomal activation, a mutant factor greater than 3.0 together with a difference in the treated and untreated dishes of at least 20 clones per 10E6 cells plated was not detected and there was no indication of a concentration mutant-frequency relation in any experiment. It is concluded that, under the given experimental conditions, the test article did not induce gene mutations in this forward mutation system.

Executive summary:

The test item was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The cells were treated in the experiments with microsomal activation for 5 hours and in the experiment without microsomal activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment).

Mutagenicity test with microsomal activation

The original experiment was performed at the following concentrations: 25, 50, 100, 200, 300, 400 and 500 µg/ml. Because the intended toxicity was not obtained in the original experiment, in the confirmatory experiment the concentrations applied were increased to 50, 100, 200, 400, 600, 800 and 1000 µg/ml. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant deviations of the mutant frecjuencies as determined by the screening with 6-TG.

Mutagenicity test without microsomal activation

Both, the original and the confirmatory experiment were performed at concentrations of 50, 100, 200, 400, 600, 800 and 1000 µg/ml. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant deviations of the mutant frecjuencies as determined by the screening with 6-TG.

In conclusion, in both investigations with and without microsomal activation, a mutant factor greater than 3.0 together with a difference in the treated and untreated dishes of at least 20 clones per 10E6 cells plated was not detected and there was no indication of a concentration mutant-frequency relation in any experiment. It is concluded that, under the given experimental conditions, the test article did not induce gene mutations in this forward mutation system.

Genetic toxicity in vivo

Description of key information

Micronucleus test, Chinese hamster: negative (GLP, OECD 474)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(only 1000 erythrocytes/animal were counted)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ciba Geigy Tierfarm, Sisseln, Switzerland
- Age at study initiation: females 6- 10 weeks, males 4-9 weeks
- Weight at study initiation: females 22-32 g, males 22-34 g
- Diet: ad libitum; Standard diet: NAFAG No.924
- Water: ad libitum
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 60-64
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Arachis oil
- Source: Siegfried AG, Switzerland
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle:
- Amount of vehicle: 10 ml/kg bw
- Purity: no data

Details on exposure:

Application volume: 10 ml/kg bw

Duration of treatment / exposure:

single dose application

Frequency of treatment:

once

Post exposure period:

sacrifice was performed 16, 24 and 48 hours after gavage

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

8/sex/dose/sampling time

Control animals:

yes, concurrent vehicle

Positive control(s):

- Name: Cyclophosphamide (CPA)
- Source: ASTA-Werke, Germany
- Justification for choice of positive control(s): guideline recommended
- Number of animals: 8/sex/dose/sampling time
- Route of administration: gavage
- Vehicle: Arachis oil
- Application volume: 10 ml/kg bw
- Doses: 64 mg/kg bw
- Time for sacrifice: 24 hours after dosing

Tissues and cell types examined:

Bone marrow; erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: the dose of 5000 mg/kg was determined as the highest applicable in the mutagenicity assay

DETAILS OF SLIDE PREPARATION: Bone marrow is harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture are transferred on the end of a slide, spread out with the aid of a polished cover glass and the preparations are air-dried. Within 24 hours, the slides are stained in undiluted May-Grünwald solution for 3 min then in May-Grünwald solution/water 1/1 for 2 min. After being rinsed in distilled water, the slides are left immersed in diluted Giemsa solution (16.6%), for 10 min. After rinsing with distilled water and air-drying, the slides are cleared in Xylene and mounted.

METHOD OF ANALYSIS: Prior to analysis the slides are coded. Thereafter the quality of staining is evaluated. The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes are selected for later scoring. The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post treatment are examined. From the animals of the positive control group which are sacrificed 24 hours after application, the slides of five female and five male animals are scored. 1000 polychromatic erythrocytes per animal each are scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes is determined for each animal by counting a total of 1000 erythrocytes.

Statistics:

The significance of difference is assessed by X^2-test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose: 200, 1000, 5000 mg/kg bw
- Number of animals/sex/dose: 2 per sex per dose
- Frequency of application: once
- Rationale for exposure: Determine the highest dosage of the test substance to be applied in the mutagenicity assay
- Harvest times: 16, 24, 48 and 72 hours after gavage

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg as compared with the negative control animals.
By contrast, the positive control (cyclophosphamide, 64 mg/kg) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 1.73. In comparison with the vehicle control (0.16) this value is highly significant (p <0.05).
- Percent of polychromatic erythrocytes with micronuclei:
16 h: 0.20 for males (control: 0.20); 0.14 for females (control: 0.10)
24 h: 0.26 for males (control: 0.18); 0.14 for females (control: 0.14)
48 h: 0.30 for males (control: 0.18); 0.04 for females (control: 0.06)
pos. control, 24 h: 2.32 for males (control: 0.18); 1.14 for females (control: 0.14)
- Ratio of PCE/NCE (for Micronucleus assay):
16 h: 1.0 for males; 0.9 for females
24 h: 1.1 for males; 0.9 for females
48 h: 1.1 for males; 1.0 for females
pos. control, 24 h: 1.1 for males; 0.8 for females

SUMMARY OF EXPERIMENTAL RESULTS

Sacrifice	Treatment	Sex	polychromatic erythrocytes (average)	normochromatic erythrocytes (average)	ratio of p / n erythrocytes	number of polychromatic erythrocytes with micronuclei (average)	% of polychromatic erythrocytes with micronuclei (average)
16 h	Control	female	468	532	0.9	2.00	0.20
		male	453	547	0.8	1.00	0.10
	5000 mg/kg	female	492	508	1.0	2.00	0.20
		male	473	527	0.9	1.40	0.14
24 h	Control	female	484	516	0.9	1.80	0.18
		male	434	566	0.8	1.40	0.14
	5000 mg/kg	female	520	480	1.1	2.60	0.26
		male	460	540	0.9	1.40	0.14
48 h	Control	female	516	484	1.1	1.80	0.18
		male	493	507	1.0	0.60	0.06
	5000 mg/kg	female	526	474	1.1	3.00	0.30
		male	490	510	1.0	0.40	0.04
Positive Control
48 h	Control	female	484	516	0.9	1.80	0.18
		male	434	566	0.8	1.40	0.14
	64 mg/kg	female	514	486	1.1	23.20	2.32
		male	442	558	0.8	11.40	1.14

Conclusions:

Under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item.

Executive summary:

An in vivo micronucleus assay was performed to evaluate any mutagenic effect on polychromatic erythrocytes in bone marrow cells in vivo. Mutagenic effects present themselves in form of micronuclei in polychromatic erythrocytes in the bone marrow. These micronuclei are small particles consisting of acentric fragments of chromosomes or entire chromosomes which lag behind at anaphase stage during the mitotic process. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm. The increase in micronucleated polychromatic erythrocytes shows a clear dose dependency, comparable to the occurrence of chromosome aberrations in metaphase preparations. In this experiment the animals were treated once with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. The test article was administered by gavage. From the bone marrow smears were made.

The bone marrow smears from the animals treated with the dose of 5000 mg/kg of the test item showed no statistically significant increase (p >0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 1.73% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.16%) treated with the vehicle (arachis oil) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item.

Additional information

Gene mutation in bacteria

The test item was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium in a GLP conform standard plate test according to OECD 471. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 without and with microsomal activation with the concentrations of 20 to 5000 µg/plate. In order to confirm the results the experiments were repeated. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected; experiments were performed in which the cultures were additionally treated with an activation mixture (rat S9 mix). In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test item revealed no marked deviations. No evidence of the induction of gene mutations by the test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. As not required by the version of the OECD testing guideline valid in 1987, only four tester strains were used. This deviation is considered acceptable because a study on gene mutation in mammalian cells is available.

 

Gene mutation in mammalian cells

The test item was tested in a gene mutation study in V79 cells according to OECD guideline 476 and GLP requirements. The system allows the detection of base-pair substitutions, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested by the appearance of cells resistant to 6-thioguanine (6-TG) and can be quantified by comparison of the numbers of 6-TG-resistant colonies in the treated and control cultures. To ensure that any mutagenic effect of metabolites of the test substance found in mammals is also detected, a parallel series of experiments is performed, in which its metabolic turnover is simulated in vitro by the addition of an activation mixture containing rat-liver microsomes and co-factors to the cell cultures. The test item was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The cells were treated in the experiments with microsomal activation for 5 hours and in the experiment without microsomal activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment).

Mutagenicity test with microsomal activation:

The original experiment was performed at the following concentrations: 25, 50, 100, 200, 300, 400 and 500 µg/mL. Because the intended toxicity was not obtained in the original experiment, in the confirmatory experiment the concentrations applied were increased to 50, 100, 200, 400, 600, 800 and 1000 µg/mL. In both experiments, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant deviations of the mutant frequencies as determined by the screening with 6-TG.

Mutagenicity test without microsomal activation:

Both, the original and the confirmatory experiment were performed at concentrations of 50, 100, 200, 400, 600, 800 and 1000 µg/mL. In both experiments, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant deviations of the mutant frequencies as determined by the screening with 6-TG.

In both investigations with and without microsomal activation, a mutant factor greater than 3.0 together with a difference in the treated and untreated dishes of at least 20 clones per 10e6 cells plated was not detected and there was no indication of a concentration mutant-frequency relation in any experiment.

 

Cytogenicity in mammalian cells

The test item was assessed for its potential to induce structural chromosome aberrations in CHO cells in vitro in an OECD guideline study which followed GLP requirements. 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 item. 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 in both treatments with and without S9 mix:

8 h: 50.0 µg/mL

24 h: 5.0; 20.0; 50.0 µg/mL

30 h: 50.0 µg/mL

The concentration range of the test item 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 under the chosen experimental conditions, the test item did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the CHO Chinese Hamster cell line.

Cytogenicity in vivo

In a GLP conform Micronucleus Test according to OECD guideline 474, the test item was administered by gavage to Chinese hamsters. In this experiment the animals were treated once with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. The experiment was performed to evaluate any mutagenic effect on polychromatic erythrocytes in bone marrow cells in vivo.

The bone marrow smears from the animals treated with the dose of 5000 mg/kg showed no statistically significant increase (p >0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 1.73% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.16%) treated with the vehicle (arachis oil) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the data, classification for repeated dose toxicity is not warranted under Regulation (EC) No.1272/2008.