Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance induced no mutants in the bacterial Ames assay. An HPRT study will be conducted in 2018.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 1981)
Deviations:
yes
Remarks:
, strain TA102 or E. coli is lacking (OECD TG 471, adopted 1997)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS
Species / strain / cell type:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from Aroclor1254-induced rats
Test concentrations with justification for top dose:
25, 75, 225, 675 and 2025 µg per plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details on test system and conditions
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Each Petri dish contained: 1) approx. 20 mL of minimum agar (Agar, Difco Laboratories, Detroit, Michigan, U.S.A., plus salts (Vogel-Bonner Medium E) and glucose), 2) 0.1 mL of the solution of the test substance or the vehicle and 0.1 mL of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8% plus 0.5% NaCl) in 2.0 mL of soft agar. The soft agar was composed of: 100 mL of 0.6% agar solution with 0.6% NaCl and 10 mL of a solution of l-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and +biotin 0.5 mM (Fluka, Buchs, Switzerland). In the experiments in which the substance was metabolically activated, 0.5 mL of an activation mixture was added also. One mL activation mixture contained: 0.3 mL S9 fraction of liver from rats induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 mL of a solution of co-factors.

DURATION
The plates were incubated for about 48 hours at 37°C in darkness.

NUMBER OF REPLICATIONS
3 plates per dose

DETERMINATION OF CYTOTOXICITY
No data

POSITIVE CONTROLS
TA 98: daunorubicin-HCl (5 and 10 µg/plate)
TA 100: 4-nitroquinoline-N-oxide (0.125 and 0.25 µg/plate)
TA 1535: N-Methyl-N'-nitro-N-nitrosoguanidine (3 and 5 µg/plate)
TA 1537: 9(5)aminoacridine hydrochloride monohydrate (50 and 100 µg/plate)
TA 1538: 2-nitrofluorene (5 and 10 µg/plate)
The activation mixture was tested with Strain TA 1535 and cyclophosphamide (250 µg/plate)
Evaluation criteria:
When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Species / strain:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 675 and 2025 µg/plate the substance precipitated in soft agar
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments without microsomal activation.

Concentration

(µg/plate)

TA98

TA100

TA1535

TA1537

TA1538

0

11

136

8

4

12

25

20

129

10

3

8

75

18

158

10

7

11

225

13

145

10

6

6

675

16

156

7

2

12

2025

17

159

7

6

9

Daunorubicin-HCl

19

(control)

 

 

 

 

5

134

 

 

 

 

10

293

 

 

 

 

4-NQO

 

156

(control)

 

 

 

0.125

 

650

 

 

 

0.250

 

>1000

 

 

 

N-methyl-N´-nitro-N-nitrosoguanidine

 

 

8

(control)

 

 

3

 

 

349

 

 

5

 

 

1470

 

 

9(5) Aminoacridine-HCl

 

 

 

6

(control)

 

50

 

 

 

85

 

100

 

 

 

520

 

2-Nitrofluorene

 

 

 

 

11

(control)

5

 

 

 

 

660

10

 

 

 

 

823

Table 2: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments with microsomal activation.

Concentration

(µg/plate)

TA98

TA100

TA1535

TA1537

TA1538

0

26

141

4

3

15

25

24

136

7

4

15

75

21

141

12

5

19

225

26

128

7

5

16

675

24

112

7

3

16

2025

21

130

8

5

17

Cyclophosphamide

 

 

 

 

 

0

 

 

13

 

 

250

 

 

214

 

 

Conclusions:
The test substance showed no mutagenic potential in bacterial cells.
Executive summary:

The mutagenic potential of the test material was assessed in an Ames assay with TA98, TA100, TA1535, TA1537 and TA1538 as tester strains. The bacteria were treated with 25, 75, 225, 675, and 2025 µg test substance per plate, with and without microsomal activation. Adequate positive controls and a solvent control were also applied. In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with the test substance revealed no marked differences. The slight increase in the number of back-mutant colonies in the experiment on Strain TA 1535 with microsomal activation is attributed to variations in the rate of spontaneously occurring back-mutants.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 April 2018 - 15 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2016
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
GLP compliance:
yes (incl. certificate)
Type of assay:
other: HPRT
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Cell cycle length, doubling time or proliferation index: doubling time of about 12 - 16 hours
Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%). All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
The doses are selected based on the data and the observations from the pretest and taking into account the current guidelines
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubilisation properties and its compatibility with cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): approximately 7 days
- Selection time (if incubation with a selection agent): approximately 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): approximately 15 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The colonies were stained with 10% methylene blue in 0.01% KOH solution.

DETERMINATION OF CYTOTOXICITY
- Cloning efficiency 1 (CE1; survival)
cloning efficiency determined immediately after treatment to measure toxicity.
- Cloning efficiency 2 (CE2; viability)
cloning efficiency determined after the expression period to measure viability of the cells without selective agent.
Evaluation criteria:
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:

a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:

a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.
Statistics:
A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together. A t-test was not performed since the 95% confidence interval was not exceeded at any experimental point.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: Precipitation was observed at 20.0 µg/mL in the presence of metabolic activation.
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
A pre-experiment was performed in order to determine the toxicity of the test item (4 hours treatment with and without metabolic activation). The range finding pre-experiment was performed using concentrations between 3.9 and 500.0 µg/mL. The highest concentration was based on the solubility properties of the test item in ethanol and with respect to OECD guideline 476. No relevant cytotoxic effects, indicated by a relative cloning efficiency of approx. 50% or below were observed after 4 hours treatment in the presence of metabolic activation. In the absence of metabolic activation cytotoxic effects were observed at 3.9 µg/mL and at 15.6 µg/mL and above. The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Precipitation occurred after 4 hours treatment at 7.8 µg/mL and above with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects indicated by an adjusted cloning efficiency I (referring to the mean values) below 50% was observed at 1.3 µg/mL and above in the absence of metabolic activation. In the presence of metabolic activation no cytotoxic effects were noted up to the highest concentration.

Summary of Results

conc (µg/ml) Precipitation S9 mix relative cloning efficiency I (%) relative cell density (%) relative adjusted cloning efficiency I (%) mutant colonies per 10^6 cells 95 % confidence interval
Main Experiment /4h treatment mean values of cultures I and II
Solvent control with ethanol - 100 100 100 17.7 2.7-31.2
Positive control (EMS) 300 - 86.0 123.2 105.9 244.0 2.7-31.2
Test item 0.2 - 89.1 103.8 92.5 14.7 2.7-31.2
Test item 0.3 - 84.0 124.8 104.0 20.4 2.7-31.2
Test item 0.6 - 86.3 134.8 115.4 11.0 2.7-31.2
Test item 1.3 - 41.4 120.5 49.8 6.2 2.7-31.2
Test item 2.5 - 23.0 127.9 29.5 8.9 2.7-31.2
Test item 5.0 - 10.4 112.4 11.7 8.6 2.7-31.2
Test item 10.0 - 0 65.4 0 # #
Test item 20.0 P - 0 38.2 0 # #
Solvent control with ethanol 100 100 100 12.0 2.9-30.9
Positive control (DMBA) 2.3 + 91.6 70.9 64.7 96.3 2.9-30.9
Test item 0.2 + 97.5 97.6 95.1 ## ##
Test item 0.3 + 93.4 95.2 89.0 ## ##
Test item 0.6 + 95.2 95.0 90.5 ## ##
Test item 1.3 + 91.8 90.1 82.6 18.3 2.9-30.9
Test item 2.5 + 89.2 98.0 87.7 11.1 2.9-30.9
Test item 5.0 + 88.7 91.0 80.4 17.3 2.9-30.9
Test item 10.0 + 93.2 90.8 84.9 22.5 2.9-30.9
Test item 20.0 P + 89.6 77.2 69.1 15.3 2.9-30.9

P = precipitation visible at the end of treatment

#    culture was not continued due to exceedingly serve cytotoxicity

##  culture was not continued as a minimum of only four analysable concentrations are required

Conclusions:
It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
Executive summary:

A GLP-compliant OECD 476 study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The main experiment was performed with a treatment period of 4 hours with and without microsomal activation. The highest applied concentration in the pre-test on toxicity (500.0 µg/mL) was chosen with regard to the solubility properties of the test item in an appropriate solvent (ethanol), and with respect to the current OECD Guideline 476. The test item was dissolved in ethanol. Cytotoxic effects indicated by a relative adjusted cloning efficiency I below 50% (mean value) were observed at 1.3 µg/mL and above without metabolic activation. No substantial and dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The tes article did not show genotoxic effects in bone marrow of Chinese hamsters.

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:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
1000 instead of 2000 cells scored per animal
Principles of method if other than guideline:
Treatment consisted of one daily dose (gavage) of 1250, 2500 or 5000 mg/kg on each of two consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made and interphase cells were analyzed for nucleus anomalies resulting from chromosomal damage.
GLP compliance:
no
Remarks:
but QAU statment included.
Type of assay:
micronucleus assay
Species:
hamster, Chinese
Strain:
other: random outbred strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln
- Age at study initiation: 6-10 weeks (females), 4-9 weeks (males)
- Weight at study initiation: 20-27 g (females), 22-30 g (males)
- Housing: single housing
- Diet: Standard diet: NAFAG No.924, ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 49-60
- Photoperiod: 12 hours light
Route of administration:
oral: gavage
Vehicle:
Arachid oil (20 mL/kg bw)
Duration of treatment / exposure:
2 days
Frequency of treatment:
Once daily for two consecutive days
Post exposure period:
24 h
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
Dose / conc.:
2 500 mg/kg bw/day (actual dose received)
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide: 128 mg/kg bw in 20 ml/kg Arachid oil.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The oral LD50 was found to be >5000 mg/kg bw in Chinese hamsters of either sex (cf. Lab.Report: GU 2, dated March 15, 1983)

TREATMENT AND SAMPLING TIMES:
Single daily dosing of animals for 2 days. 24 h after last dosing, the bone marrow was prepared.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 mL rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Gruenwald solution for 2 min then in May-Gruenwald solution/water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 sec and washed off twice in water, they were left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylol and mounted in Eukitt.

METHOD OF ANALYSIS:
The slides of three female and three male animals each of the negative control group, the positive control group and of the groups treated with various doses of test substance were examined microscopically. 1000 bone marrow cells each were scored per animal and the following anomalies were registered:
a) Single Jolly bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells, e) polyploid cells.
Evaluation criteria:
Statistic significant difference of examined cell types in control and test group.
Statistics:
The significance of difference was assessed by CHI-Square test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg bw) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 7.2, whereas the negative control yielded a percentage of 0.17. The difference is highly significant (p<0.05).

Table 1: Percent of cells with anomalies of nuclei, 24 h after second application of test substance.

 

Number of animal

Sex of animals

Single Jolly-Bodies

Fragments of nuclei in erythrocytes

Micronuclei in erythroblasts

Micronuclei in leucopoietic cells

Polyploid cells

Totals   

Vehicle control

1

F

0.2

 

 

 

 

0.2   

 

2

F

0.2

 

 

 

 

0.2   

 

3

F

0.1

 

 

 

 

0.1   

 

4

M

 

 

 

 

 

0.0   

 

5

M

0.2

 

 

 

 

0.2   

 

6

M

0.3

 

 

 

 

0.3   

Cyclophosphamide, 128 mg/kg bw

1

F

7.7

0.3

1

0.3

 

9.3   

 

2

F

6.5

0.2

1.5

0.8

0.1

9.1   

 

3

F

7.9

0.7

0.3

0.1

 

9.0   

 

4

M

4.9

0.2

0.7

0.1

0.1

6.0   

 

5

M

3.9

 

0.4

0.4

 

4.7   

 

6

M

4.6

0.2

0.3

0.1

 

5.2   

Test substance, 1250 mg/kg bw

1

F

 

 

 

 

0.0   

 

2

F

0.1

 

 

 

 

0.1   

 

3

F

0.1

 

 

 

 

0.1   

 

4

M

 

 

0.1

 

0.1   

 

5

M

 

 

 

 

0.0   

 

6

M

0.1

 

 

 

 

0.1   

Test substance,2500 mg/kg bw  1  F  0.1          0.1   
   2  F            0.0   
   3  F            0.0   
   4  M  0.1          0.1   
   5  M            0.0   
   6  M            0.0   
 Test substance, 5000 mg/kg bw  1  F  0.1          0.1   
   2  F            0.0   
   3  F  0.2          0.2   
   4  M            0.0   
   5  M  0.1          0.1   
   6  M  0.1          0.1   
Conclusions:
The test substance did not show any genotoxic potential under the test conditions chosen.
Executive summary:

The test article's clastogenic potential was determined in a nucleus anomaly test in Chinese hamsters. The test substance was administered by gavage to 6 male and female Chinese hamsters. Treatment consisted of one daily dose of 1250, 2500 or 5000 mg/kg bw on each of two consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made and interphase cells were observed for anomalies. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. Thus, the test substance did not show any genotoxic potential under the test conditions chosen.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial mutagenicity

The test article's mutagenic potential was assessed in an Ames assay with S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538. The bacteria were treated with 25, 75, 225, 675, and 2025 µg test substance per plate, with and without microsomal activation. Adequate positive controls and a solvent control were also applied. In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with the test substance revealed no marked differences. The slight increase in the number of back-mutant colonies in the experiment on strain TA 1535 with microsomal activation is attributed to variations in the rate of spontaneously occurring back-mutants. Thus, the test substance showed no mutagenic potential in bacterial cells under the conditions used.

HPRT study

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. Two parallel cultures were used throughout the assay. The main experiment was analysed for gene mutations at the following concentrations: 0.2, 0.3, 0.6, 1.3, 2.5 and 5.0 µg/l without S9 mix; 1.3, 2.5, 5.0, 10.0 and 20.0 µg/l with S9 mix. The concentration range of the main experiment was chosen according to the data generated in the pre-experiment. Precipitation was observed at 20.0 µg/mL in the presence of metabolic activation. Cytotoxic effects indicated by an adjusted cloning efficiency I (referring to the mean values) below 50% was observed at 1.3 µg/mL and above in the absence of metabolic activation. In the presence of metabolic activation no cytotoxic effects were noted up to the highest concentration. No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentration scored for gene mutations. The 95% confidence interval was not exceeded at any of the test item concentrations. The linear regression analysis (least squares) showed no significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05. The mutant frequencies obtained in the main experiment for the solvent controls (mean values) with and without S9 mix were in the range from 12.0 to 17.7 mutants per 106 cells. The values were well within the 95% confidence interval of our laboratory’s historical solvent control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mutant frequencies (mean values) of the groups treated with the test item was from 6.2 up to 22.5 mutants per 106 cells. EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test article is considered to be non-mutagenic in this HPRT assay.

Micronucleus assay in vivo

The genotoxic potential of the test substance was assessed in a nucleus anomaly test in Chinese hamsters. The test substance in arachid oil was administered by gavage to 6 male and female Chinese hamsters per dose group. Treatment consisted of one daily dose of 1250, 2500 or 5000 mg/kg body weight on each of two consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made and interphase cells were observed for anomalies. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 7.2% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (Arachid 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 article.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008.