Dichlobenil

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material was determined to be negative for genotoxicity according to studies performed in line with OECD Guideline 476, EPA Guideline 84-2 and an Ames test performed in line with sound scientific principles.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 1983 to 3 October 1983

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Target gene:

Histidine dependence

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

0, 40, 200, 1000 and 5000 µg/plate.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

other: 4-nitro-o-phenylene diamine, 2-amino-anthracene, Neutral Red,

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: five at each dose level and ten for the solvent controls

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The top concentration of 5,000 µg/plate precipitated in the top agar. The revertant colony counts per plate are summarised in Table 1. No increases in revertant colony numbers were observed after treatment of any of the five tested strains with the test material, either in the presence or absence of rat liver microsomal fraction.

Table 1: Mean revertant colony counts per plate

		Strain of Salmonella typhimurium
Concentration (µg/plate)	Metabolic activation	TA1535	TA1537	TA1538	TA98	TA100
5000	-	**	**	**	**	**
1000	-	20	5	12	13	63
200	-	14	4	9	13	81
40	-	14	7	11	14	77
0	-	13	6	13	16	84
5000	+	**	**	**	**	**
1000	+	13	3	9	13	50
200	+	14	5	12	13	69
40	+	6	6	14	23	72
0	+	11	8	17	20	76

- absence of metabolic activation

+ presence of metabolic activation

** test material precipitated in top agar

Conclusions:

Under the conditions of the test, the test material was determined to be negative in a bacterial gene mutation assay.

Executive summary:

A non-GLP compliant bacterial gene mutation assay was conducted in line with sound scientific principles similar to OECD 471 and EU Method B13/14.

No increases in revertant colony numbers were observed after treatment of any of the five tested strains with the test material, either in the presence or absence of rat liver microsomal fraction.

Under the conditions of the test, the test material was determined to be negative in a bacterial gene mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 1990 to 27 June 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

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):

- Type and identity of media: McCoy's 5a culture medium supplemented appropriately.
- Properly maintained: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

45, 60, 80 and 100 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

RANGE-FINDING ASSAYS
Cells were cultured for approximately 24 hours prior to treatment by seeding approximately 0.3 x 10E+6 cells into 5 mL of complete McCoy's 5a culture medium. The thymidine analogue 5-bromo-2'-deoxyuridine was added at 10 µM approximately two hours after the initial exposure of the cells to the test material.

ABERRATION ASSAY WITHOUT METABOLIC ACTIVATION
Cultures were initiated by seeding approximately 1.2 x 10E+6 cells (20 hour assay) and 1.0 x 10E+6 cells (30 hour assay) into 10 mL of complete McCoy's 5a medium. One day after culture initiation, the CHO cells were treated with the test substance for 17.25 and 27.5 hours. The cultures were then washed with buffered saline and complete McCoy's 5a medium containing 0.1 µg/mL Colcemid® was added to the cells. 2.5 hours later, the cells were harvested.

ABERRATION ASSAY WITH METABOLIC ACTIVATION
Cultures were initiated by seeding approximately 1.2 x 10E+6 cells (20 hour assay) and 1.5 x 10E+6 cells (10 hour assay) into 10 mL of complete McCoy's 5a medium. One day after culture initiation, the CHO cells were treated were incubated at 37 °C For two hours in the presence of the test material and the S9 mix in McCoy's 5a medium without foetal calf serum. After the two hour exposure period, the cultures were washed twice with buffered saline and refed with complete McCoy's 5a medium. The cells were incubated for an additional 7.83 and 17.75 hours with 0.1 µg/mL Colcemid® present during the last 2.5 hours. The metaphase cells were then harvested.

HARVEST PROCEDURE
Prior to harvest, visual observations of toxicity were made including an assessment of the percent confluence of the cell monolayer within the culture flasks. The cultures were also evaluated for the presence of mitotic or dead cells floating in the medium. The metaphase cells were collected by mitotic shake-off and treated to 0.075 M KCl hypotonic solution. The cultures were then fixed with an absolute methanol: glacial acetic acid (3:1 v/v), stained (with Giemsa) and slides prepared.

Evaluation criteria:

One hundred cells from each replicate culture of the four dose levels of the test material and the negative and solvent control cultures were analysed. At least 25 cells were analysed for chromosomal aberrations taking into account the following factors:
- overall chromosomal aberration frequencies;
- percentage of cells with any aberrations;
- percentage of cells with more than one aberration; and
- any evidence for increasing amounts of damage with increasing dose.

Statistics:

The Fisher's Exact Test with an adjustment for multiple comparisons was employed to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Cytotoxicity was observed at 100 µg/mL in the range finding test without metabolic activation. In all other tests no cytotoxicity was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed (see Table 1).

Table 1: Chromosome aberrations in CHO cells

	Cells with chromosome aberrations (%)
	Dosage units (µg/mL) #	Without metabolic activation		Dose units (µg/mL) ##	With metabolic activation
		20 hour	30 hours		10 hour	20 hour
Negative control	-	0.5	0.0	-	1.5	0.0
Solvent control	-	0.5	0.0	-	1.5	0.0
Test material	45 (45.1)	2.0	0.0	25.1 (25.0)	0.0	2.5
	60 (60.1)	2.5	1.0	50.1 (50.0)	0.0	1.0
	80 (80.2)	1.5	1.0	75.2 (75.0)	0.0	1.5
	100 (100)	1.0	2.5	100 (100)	0.0	0.0
Positive control (mitomycin-C)	0.040 (0.080)	14.0*	52.0*			64.0*
Positive control (cyclophosphamide)				50.0 912.5)	40.0*

# between brackets for the 30 hours

## between brackets after 20 hours

* significantly greater, p<0.01

Conclusions:

Under the conditions of the test, the test material was determined to be negative for chromosome aberrations in Chinese Hamster Ovary cells.

Executive summary:

A GLP compliant chromosome aberration assay was performed in Chinese Hamster Ovary cells and conducted in line with EPA OPP 84-2.

No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed, in the presence and absence of metabolic activation.

Under the conditions of the test, the test material was determined to be negative for chromosome aberrations in Chinese Hamster Ovary cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 September 2006 to 06 February 2007

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:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Periodically checked for karyotype stability: yes (cells from the fourth passage after cleansing were used to ensure karyotype stability)
- Periodically "cleansed" against high spontaneous background: yes (using media supplemented with hypoxanthine, aminopterin and thymidine)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced S9 mix

Test concentrations with justification for top dose:

5, 15, 25 and 50 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

MUTAGENESIS ASSAYS
CHO cells were exposed for 5 hours at 37 ± 1 °C to the vehicle alone, positive controls or the test substance in duplicate in the presence and absence of metabolic activation.

TREATMENT OF TARGET CELLS
Exponentially growing CHO-K1 cells were seeded in F12FBS5-Hx at a density of 5 x 10E+% cells/25 cm^2 surface area and incubated at 37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air for 18-24 hours.

Treatment was performed by refeeding the treatment flasks with 5 mL F12FBS5-Hx for the non-activated study or with 4 mL F12FBS5-Hx and 1 mL S9 reaction mixture for the activated study to which was added the appropriate amount of vehicle, test or control substance. Duplicate flasks were exposed to at least five concentrations of the test substance for 5 hours at 37 ± 1 °C. After the treatment period, all media were aspirated, the cells were washed with Ca++ and mg++ free Hank's balanced solution and cultured in F12FBS5-Hx for an additional 18024 hours at 37 ± 1 °C.

CYTOTOXICITY
Replicates from each treatment condition were detached using trypsin and subcultured independently in F12FBS5-Hx in triplicate at a density of 100 cells/60 mm dish. After 7-10 days incubation, the colonies were rinsed with HBSS, fixed, stained and counted.

MUTANT PHENOTYPE
Replicates from each treatment condition were trypsinised and subcultured independently in F12FBS5-Hx in duplicate at a density no greater than 10E+6 cells/100 mm dish. Subculturing by trypsinising at 2-4 day intervals was employed for the 7-9 day expression period. At the end of the expression period, selection for the mutant phenotype was performed.

Replicates from each treatment condition were trypsinized and replaced, in quintuplicate, at a density of 2 x 10E+5 cells/100 mm dish in F12FBS5-Hx containing 10 µM 6-thioguanine. For cloning efficiency determinations at the time of selection, 100 cells/60 mm dish were placed in triplicate in medium without TG. After 7-10 days incubation, the colonies were fixed, stained and counted for both cloning efficiency and mutant selection.

Evaluation criteria:

The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control. The mutant frequency for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selection, corrected for the cloning efficiency of cells prior to mutant selection and is expressed as TG-resistant mutants per 10E+6 clonable cells.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

less than 50 % cloning efficiency was noted at concentrations greater than 50 µg/mL in the preliminary toxicity test, 50 µg/mL was selected as the highest dose tested in the mutagenicity test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity assay, the maximum concentration of the test material tested was 670 µg/mL. There was visible precipitate in the treatment medium at concentrations ≥150 µg/mL. Selection of concentrations for the mutagenesis assay was based on the cloning efficiency relative to the solvent control and precipitation. Substantial toxicity, i.e., cloning efficiency 50% of the solvent control, was observed only at concentrations from 50 to 500 µg/mL with S9 activation.

Based on these findings, the concentrations chosen for the initial mutagenesis assay ranged from 5.0 to 150 µg/mL for the non-activated cultures and 1.5 to 150 µg/mL for the S9-activated cultures.

In the initial mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per 10E+6 clonable cells, were observed. Visible precipitate was observed in treatment medium at concentrations 50 µg/mL. Toxicity, i.e. cloning efficiency ≤50 % of the solvent control, was not observed at any concentration with or without S9 activation.

In the independent repeat assay, no positive responses were observed. Visible precipitate was observed in treatment medium at concentrations 50 µg/mL. Toxicity, i.e. cloning efficiency ≤50 % of the solvent control, was observed at a concentration of 50 µg/mL with S9 activation.

Table 1: CHO/HGPRT mutation test results (test 1)

	Cytotoxic effects	Mutagenicity data
Concentration (µg/mL)	Relative cloning efficiency (%)	Cloning efficiency	Mutants/10^6 clonable cells
Non-activated
0	100	0.76	2.0
5	65	0.72	0
15	82	0.67	1.5
25	100	0.81	0
50 P	62	0.78	0
Positive control*	12	0.5	358.9
Activated
0	100	0.88	0.6
1.5	87	0.69	0
15	73	0.74	0
25	56	0.56	2.7
50 P	31	0.66	6.8
Positive control**	14	0.55	174.3

Table 2: CHO/HGPRT mutation test results (test 2)

	Cytotoxic effects	Mutagenicity data
Concentration (µg/mL)	Relative cloning efficiency (%)	Cloning efficiency	Mutants/10^6clonable cells
Non-activated
0	100	0.62	3.2
1.5	109	0.59	5.1
5	105	0.56	0
15	98	0.48	4.2
25	114	0.59	0
50 P	108	0.59	0
Positive control*	40	0.46	469.2
Activated
0	100	0.64	0
1.5	104	0.66	0
5	73	0.59	0
15	84	0.57	0
25	75	0.54	4.6
50 P	96	0.56	0
Positive control**	26	0.48	218.0

*Ethyl methanesulfonate

**Benzo(a)pyrene

P = precipitating concentration

Conclusions:

Under the conditions of the test, the test material was determined to be negative in the CHO/HGPRT mutation assay in the presence and absence of metabolic activation.

Executive summary:

In a GLP compliant mammalian cell gene mutation assay (CHO/HGPRT test) conducted in line with OECD 476, the genetic toxicity of the test material was determined.

Based on the findings of the preliminary toxicity assay, the concentrations chosen for the initial mutagenesis assay ranged from 5.0 to 150 µg/mL for the non-activated cultures and 1.5 to 150 µg/mL for the S9-activated cultures.

In the initial mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per 10E+6 clonable cells, were observed. Visible precipitate was observed in treatment medium at concentrations 50 µg/mL. Toxicity, i.e. cloning efficiency ≤50 % of the solvent control, was not observed at any concentration with or without S9 activation.

In the independent repeat assay, no positive responses were observed. Visible precipitate was observed in treatment medium at concentrations 50 µg/mL. Toxicity, i.e. cloning efficiency ≤50 % of the solvent control, was observed at a concentration of 50 µg/mL with S9 activation.

Under the conditions of this study, the test material was determined to be negative in the CHO/HGPRT assay in the presence and absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test material was determined to be negative for genotoxicity according to a study performed to a methodology considered to be similar to EU Method B.12.

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

Study period:

September 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately six weeks old
- Fasting period before study: overnight before first dose administration
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature: 22-24 °C
- Air changes: 16 per hour
- Photoperiod: 12 hours light/12 hours dark

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1 % tragacanth suspension

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosages were given as two equal administrations separated by a 24 hour interval in a volume of 0.1 mL/10 g body weight prepared in 1 % tragacanth suspension

Duration of treatment / exposure:

Two doses were administered over a 24 hour period

Frequency of treatment:

Two single doses were administered separated by a 24 hour period

Post exposure period:

Six hours after the second dose, the animals were sacrificed.

Dose / conc.:

300 mg/kg bw (total dose)

Dose / conc.:

600 mg/kg bw (total dose)

Dose / conc.:

1 200 mg/kg bw (total dose)

No. of animals per sex per dose:

Five per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- mitomycin C
- Doses / concentrations: 0.4 mg/mL in sterile buffered saline

Tissues and cell types examined:

Femur bone marrow

Details of tissue and slide preparation:

Bone marrow was removed by injection of New Born calf serum into the femur. The serum and bone marrow suspension was centrifuged for 5 minutes at 800 rpm. The supernatant was removed and a bone marrow smear made from the pellet.

After air-drying overnight, the smears were fixed in methanol for 5 minutes and then successively placed in 1.25 % May-Grünwald for 15 minutes and in 3 % Giesma for 20 minutes. After rinsing in buffered distilled water, the slides were air-dried and mounted in Depex.

Evaluation criteria:

Smears were examined to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal and the ratio of normochromatic to polychromatic erythrocytes.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

All micronucleated cell counts for the test material were within the concurrent control range. The mean normochromatic/polychromatic ratio was 0.85

Toxicity:

no effects

Remarks:

No mortalities were noted in any of the doses administered

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No mortalities were observed.

At all dose levels, the mean micronucleated cell counts were in the same range as the counts obtained for the negative control group.

The mean ratios of normochromatic to polychromatic erythrocytes obtained for all dose levels were in the same range as those of the vehicle control group.

Table 1: Group mean results

		Number of micronucleated cells per 1000 erythrocytes		Ratio of normochromatic to polychromatic erythrocytes
Material	Total dose (mg/kg)	Mean	Range	Mean	Range
1% tragacanth	-	0.2	0 - 1	0.85	0.56 - 1.51
Test material	1200	0.2	0 - 1	0.92	0.58 - 1.61
	600	0.8	0 - 3	0.77	0.58 - 1.02
	300	0.5	0 - 1	0.74	0.56 - 1.09
Mitomycin C	*	32.8	12 - 54	1.91	0.90 - 2.90

* 0.16 mg/animal  

Conclusions:

Under the conditions of the test, the test material did not show any evidence of mutagenic potential for polychromatic erythrocytes of mice.

Executive summary:

In a non-GLP compliant micronucleus test considered to be equivalent or similar to EU Method B.12, the genetic toxicity of the test material was determined.

No mortalities were observed. At all dose levels, the mean micronucleated cell counts were in the same range as the counts obtained for the negative control group. The mean ratios of normochromatic to polychromatic erythrocytes obtained for all dose levels were in the same range as those of the vehicle control group.

Under the conditions of this test, the test material showed no evidence of mutagenic potential for polychromatic erythrocytes of mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A non-GLP compliant bacterial gene mutation assay was conducted in line with sound scientific principles similar to OECD 471 and EU Method B13/14.

No increases in revertant colony numbers were observed after treatment of any of the five tested strains with the test material, either in the presence or absence of rat liver microsomal fraction.

Under the conditions of the test, the test material was determined to be negative in a bacterial gene mutation assay.

 

A GLP compliant chromosome aberration assay was performed in Chinese Hamster Ovary cells and conducted in line with EPA OPP 84-2.

No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed, in the presence and absence of metabolic activation.

Under the conditions of the test, the test material was determined to be negative for chromosome aberrations in Chinese Hamster Ovary cells.

 

In a GLP compliant mammalian cell gene mutation assay (CHO/HGPRT test) conducted in line with OECD 476, the genetic toxicity of the test material was determined.

Based on the findings of the preliminary toxicity assay, the concentrations chosen for the initial mutagenesis assay ranged from 5.0 to 150 µg/mL for the non-activated cultures and 1.5 to 150 µg/mL for the S9-activated cultures.

In the initial mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per 10E+6 clonable cells, were observed. Visible precipitate was observed in treatment medium at concentrations 50 µg/mL. Toxicity, i.e. cloning efficiency ≤50 % of the solvent control, was not observed at any concentration with or without S9 activation.

In the independent repeat assay, no positive responses were observed. Visible precipitate was observed in treatment medium at concentrations 50 µg/mL. Toxicity, i.e. cloning efficiency ≤50 % of the solvent control, was observed at a concentration of 50 µg/mL with S9 activation.

Under the conditions of this study, the test material was determined to be negative in the CHO/HGPRT assay in the presence and absence of metabolic activation.

 

In a non-GLP compliant micronucleus test considered to be equivalent or similar to EU Method B.12, the genetic toxicity of the test material was determined.

No mortalities were observed. At all dose levels, the mean micronucleated cell counts were in the same range as the counts obtained for the negative control group. The mean ratios of normochromatic to polychromatic erythrocytes obtained for all dose levels were in the same range as those of the vehicle control group.

Under the conditions of this test, the test material showed no evidence of mutagenic potential for polychromatic erythrocytes of mice.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.