Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation Assay (similar to OECD Guideline 471): negative Mammalian cell gene mutation assay with chinese hamster V79 cells (according to OECD 476 and GLP): negative

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(E.coli was not included, only few details on experimental procedure, in experiments with metabolic activation a positive control was only investigated in TA1535)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine-auxotrophic
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats induced with Aroclor 1254 and a solution of co-factors
Test concentrations with justification for top dose:
25, 75, 225, 675, and 2025 µg/0.1 ml
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below for details
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37 °C in darkness

NUMBER OF REPLICATIONS: 2 independent experiments, each with 3 plates each concentration or control group

POSITIVE CONTROLS:
no metabolic activation:
- Strain TA 98: Daunorubicin-HCl (5 and 10 µg/0.1 ml phosphate buffer)
- Strain TA 100: 4-nitroquinoline-N-oxide (0.125 and 0.25 µg/0.1 ml phosphate buffer)
- Strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine (3 and 5 µg/0.1 ml phospahte buffer)
- Strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate (50 and 100 µg/0.1 ml DMSO)
with metabolic activation:
- Starin TA 1537: Cyclophophamide (250 µg/0.1 ml phosphate buffer)
Evaluation criteria:
A test substance was generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Statistics:
When the colonies had been counted, the arithmetic mean was calculated.
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 225 µg/0.1 ml and above the substance precipitated in soft agar.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Tab. 1: Salmonella/Mammalian-Microsome Mutagenicity Test: First experiment without microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -06 -25)

Compound

Concentration

TA 98

TA 100

TA 1535

TA 1537

Test substance

Control

20

152

8

2

 

25 µg/0.1 ml

16

126

11

3

 

75 µg/0.1 ml

18

131

5

2

 

225 µg/0.1 ml

13

134

10

4

 

675 µg/0.1 ml

14

123

7

2

 

2025 µg/0.1 ml

10

142

5

4

 Daunorubicin-HCl

Control

21

 

 

 

 

5 µg/0.1 ml

675

 

 

 

 

10 µg/0.1 ml

1110

 

 

 

4-Nitroquinoline-N-oxide

Control

 

147

 

 

 

0.125 µg/0.1 ml

 

661

 

 

 

0.25 µg/0.1 ml

 

1083

 

 

N-Methyl-N'-nitro- N-nitrosoguanidine

Control

 

 

10

 

 

3 µg/0.1 ml

 

 

350

 

 

5 µg/0.1 ml

 

 

1352

 

9(5)Aminoacridine hydrochloride

Control

 

 

 

3

 

50 µg/0.1 ml

 

 

 

254

 

100 µg/0.1 ml

 

 

 

866

Tab. 2: Salmonella/Mammalian-Microsome Mutagenicity Test: First experiment with microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -06 -25)

Compound

Concentration

TA 98

TA 100

TA 1535

TA 1537

Test substance

Control

28

154

6

7

 

25 µg/0.1 ml

31

145

9

5

 

75 µg/0.1 ml

33

153

17

6

 

225 µg/0.1 ml

37

137

9

3

 

675 µg/0.1 ml

23

152

9

10

 

2025 µg/0.1 ml

26

155

14

6

Cyclophosphamide

Control

 

 

6

 

 

250 µg/0.1 ml

 

 

384

 

Tab. 3: Salmonella/Mammalian-Microsome Mutagenicity Test: Second experiment without microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -07-09)

Compound

Concentration

TA 98

TA 100

TA 1535

TA 1537

Test substance

Control

21

166

11

5

 

25 µg/0.1 ml

20

147

10

3

 

75 µg/0.1 ml

26

151

12

6

 

225 µg/0.1 ml

22

152

12

7

 

675 µg/0.1 ml

20

151

14

4

 

2025 µg/0.1 ml

21

135

17

4

 Daunorubicin-HCl

Control

31

 

 

 

 

5 µg/0.1 ml

629

 

 

 

 

10 µg/0.1 ml

1025

 

 

 

4-Nitroquinoline-N-oxide

Control

 

160

 

 

 

0.125 µg/0.1 ml

 

693

 

 

 

0.25 µg/0.1 ml

 

1176

 

 

N-Methyl-N'-nitro- N-nitrosoguanidine

Control

 

 

14

 

 

3 µg/0.1 ml

 

 

489

 

 

5 µg/0.1 ml

 

 

1820

 

9(5)Aminoacridine hydrochloride

Control

 

 

 

5

 

50 µg/0.1 ml

 

 

 

164

 

100 µg/0.1 ml

 

 

 

985

Tab. 4: Salmonella/Mammalian-Microsome Mutagenicity Test: Second experiment with microsomal activation. Number of Back-Mutant Colonies per Plate (Arithmetic mean) (1981 -07-09)

Compound

Concentration

TA 98

TA 100

TA 1535

TA 1537

Test substance

Control

45

159

13

11

 

25 µg/0.1 ml

39

160

13

7

 

75 µg/0.1 ml

42

163

13

13

 

225 µg/0.1 ml

43

156

14

10

 

675 µg/0.1 ml

41

144

16

14

 

2025 µg/0.1 ml

35

165

9

9

Cyclophosphamide

Control

 

 

9

 

 

250 µg/0.1 ml

 

 

502

 

Conclusions:
No evidence of the induction of point mutations by the test article or by the metabolites of the substance formed as a result of microsomal activation was detectable.
Executive summary:

In a bacterial reverse mutation assay conducted similar to OECD Guideline 471 the test substance was analysed for mutagenicity by the detection of point mutations in bacteria (histidine-auxotrophic mutants of Salmonella typhimurium). To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were also performed with the addition of an activation mixture (rat liver microsomes and co-factors). The strains TA 98, TA 100, TA 1535, and TA 1537 were treated with the test substance dissolved in DMSO in doses of 25, 75, 225, 675, and 2025 µg/0.1 ml (plate incorporation test). A negative control (vehicle) and appropriate positive control substances were also evaluated. At concentrations of 225 µg/0.1 ml and higher the test compound precipitated in soft agar. No dose-related biologically relevant increase or doubling in the number of his+ revertants was observed in plate incorporation test in any of the tested strains with or without metabolic activation. Effects on cytotoxicity were not given. According to the results of the present study, the test substance is not mutagenic in the bacterial reverse mutation assay under the experimental conditions chosen. The positive controls yielded the expected results.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 24, 2012 to August 21, 2012
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:
(adopted July 21, 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
dated May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus

Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of 8 - 12 weeks old male Wistar rats [Hsd Cpb: WU], treated with 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days.
Test concentrations with justification for top dose:
1st Experiment: with and without S9 mix (4-hour exposure period): 5.5; 11.0 (P without S9 mix); 22.0 (P); 44.0 (P); 88.0 (P); 176.0 (P) μg/mL
2nd Experiment: without S9 mix (24-hour exposure period): 2.8; 5.5; 11.0; 22.0; 44.0 (P); 88.0 (P) µg/mL
with S9 mix (4-hour exposure period): 1.4; 2.8; 5.5; 11.0; 22.0 (P); 44.0 (P) µg/mL
P = Precipitation
In experiment I the cultures at the highest concentration of 176.0 μg/mL with and without metabolic activation were not continued to avoid analysis of too many precipitating concentrations. In experiment II the cultures at the lowest concentration of 2.8 μg/mL without and 1.4 μg/ml with metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was selected based on the solubility properties and compatibility with the cell cultures. The final concentration of DMSO in the culture medium was 0.5% (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
EMS (without S9, 0.150 mg/mL); DMBA (with S9, 1.1 µg/ml)
Remarks:
with and without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration:
1st experiment: 4 h exposure with and without S9 mix.
2nd experiment: 4 h exposure with S9 mix and 24 h without S9 mix.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

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

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item was classified as positive if it induced either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points was considered non-mutagenic in the system.
A positive response was described as follows:
- A test item was classified as mutagenic if it reproducibly induced a mutation frequency that was three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item was classified as mutagenic if there was a reproducible concentration-related increase of the mutation frequency. Such evaluation was considered also in the case that a threefold increase of the mutant frequency was not observed.
- However, in a case by case evaluation this decision depend on the level of the corresponding solvent control data. If there was by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range was discussed. The variability of the mutation rates of solvent controls within all experiments of the study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological and statistical significance was considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Linear regression analysis determined a reciprocal, single significant dose dependent trend of the mutation frequency (p <0.05) in the2nd culture of the 2nd experiment with S9-mix, going down with increasing concentration and thus biologically irrelevant.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Coarse precipitation occurred at 1400 μg/mL and above with and without metabolic activation following 4 and 24 hours treatment. Turbidity was noted at almost all of the lower concentrations. Precipitation of the test item at the end of treatment was noted in the first experiment at 22.0 μg/mL and above with and at 11.0 μg/mL and above without metabolic activation. In the second experiment precipitation occurred at 22.0 μg/mL and above with and at 44.0 μg/mL and above without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
Range finding pre-experiment: 21.9 to 2800 μg/mL, in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were spaced by a factor of 2

COMPARISON WITH HISTORICAL CONTROL DATA:
An isolated increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control and the range of historical solvent control data was observed in the first culture of experiment II at 11.0 μg/mL without metabolic activation. However, the isolated increase was judged as biologically irrelevant fluctuation as it was neither reproduced in the parallel culture under identical experimental
conditions nor dose dependent as indicated by the lacking statistical significance.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effects indicated by a relative cloning efficiency I [(mean number of colonies per flask divided by the mean number of colonies per flask of the corresponding control) x 100] below 50% in both parallel cultures was observed in both main experiments in the presence and absence of metabolic activation following 4 and 24 hours treatment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary of results

  conc.(µg/ml) P S9 mix relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies/106 cells induction factor relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies/106 cells induction factor
Experiment I / 4 h treatment      

culture I

culture II
Solvent control with DMSO     - 100.0 100.0 100.0 12.7 1.0 100.0 100.0 100.0 14.5 1.0
Positive control (EMS) 150.0   - 66.2 105.2 74.6 112.5 8.8 60.9 90.8 67.0 131.0 9.0
Test item 5.5   - 96.2 101.3 91.8 8.3 0.7 93.4 88.3 95.8 17.6 1.2
Test item 11.0 P - 98.2 92.0 96.5 16.1 1.3 92.3 112.0 95.8 5.4 0.4
Test item 22.0 P - 98.3 94.4 104.5 3.9 0.3 93.2 116.0 99.1 11.7 0.8
Test item 44.0 P - 92.3 107.9 88.8 8.7 0.7 93.4 100.5 91.0 7.9 0.5
Test item 88.0 P - 89.0 78.6 93.3 11.4 0.9 92.6 94.9 100.9 11.8 0.8
Test item 176.0 P - 89.4 culture was not continued# 92.7 culture was not continued#
Experiment I / 4 h treatment       culture I culture II
Solvent control with DMSO     + 100.0 100.0 100.0 12.5 1.0 100.0 100.0 100.0 13.1 1.0
Positive control (DMBA) 1.1   + 64.4 147.5 66.1 825.8 66.2 64.0 78.4 70.9 592.4 45.1
Test item 5.5   + 103.0 74.7 82.2 11.5 0.9 105.6 124.3 103.8 11.7 0.9
Test item 11.0   + 97.8 53.2 87.3 12.2 1.0 104.4 78.2 92.9 9.5 0.7
Test item 22.0 P + 99.7 118.6 84.9 8.0 0.6 101.8 65.1 101.1 7.4 0.6
Test item 44.0 P + 96.9 89.7 97.8 14.1 1.1 100.0 94.3 94.5 8.2 0.6
Test item 88.0 P + 96.3 79.5 86.1 11.5 0.9 97.4 152.4 102.2 6.8 0.5
Test item 176.0 P + 93.6 culture was not continued# 96.3 culture was not continued#
Experiment II / 24 h treatment       culture I culture II
Solvent control with DMSO     - 100.0 100.0 100.0 11.3 1.0 100.0 100.0 100.0 17.8 1.0
Positive control (EMS) 150.0   - 105.6 78.0 72.9 344.6 30.4 92.2 96.6 77.3 378.4 21.2
Test item 2.8   - 100.1 culture was not continued## 94.6 culture was not continued##
Test item 5.5   - 99.5 80.1 89.6 18.2 1.6 91.2 98.3 76.0 13.2 0.7
Test item 11.0   - 107.4 90.9 35.0 76.3 6.7 92.4 102.0 68.9 31.8 1.8
Test item 22.0   - 99.3 84.9 43.3 28.5 2.5 136.8 104.5 77.1 32.6 1.8
Test item 44.0 P - 102.2 87.0 82.4 18.4 1.6 92.0 103.2 75.3 14.4 0.8
Test item 88.0 P - 102.9 88.0 69.2 32.5 2.9 91.9 91.5 50.4 18.7 1.0
Experiment II / 4 h treatment          
Solvent control with DMSO     + 100.0 100.0 100.0 25.4 1.0 100.0 100.0 100.0 20.2 1.0
Positive control (DMBA) 1.1   + 20.4 60.8 62.4 1740.1 68.4 19.6 79.4 82.9 763.4 37.9
Test item 1.4   + 101.3 culture was not continued## 101.9 culture was not continued##
Test item 2.8   + 96.2 113.3 106.0 15.0 0.6 103.1 119.1 114.2 11.7 0.6
Test item 5.5   + 97.7 100.0 94.9 26.8 1.1 100.2 127.6 98.9 14.0 0.7
Test item 11.0   + 98.5 112.7 103.1 23.0 0.9 102.9 111.2 89.6 18.8 0.9
Test item 22.0 P + 95.6 112.3 108.1 22.1 0.9 104.9 120.1 100.9 0.6 0.0
Test item 44.0 P + 99.7 109.3 112.3 17.4 0.7 104.8 119.5 119.5 0.6 0.0

# culture was not continued to avoid analysis of too many precipitating concentrations

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

P precipitation visible to the naked eye at the end of treatment

Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. nd is therefore considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest applied concentration in the pre-test on toxicity (2800 μg/ml) was chosen with regard to the solubility properties of the test item. The concentration range of the main experiments was limited by precipitation of the test item in aqueous media. The test item was dissolved in DMSO. The tested concentrations of the main experiments are described above. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), 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 article 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

Micronucleus Assay (similar to OECD Guideline 474, Mammalian Erythrocyte Micronucleus Test), Chinese hamster: negative

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
(only 3 animals each sex instead of 5; no data on test material purity, animal source and acclimatisation period; proportion polychromatic erythrocytes/normochromatic erythrocytes was not documented, only 1000 cells per slide were counted)
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
hamster, Chinese
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: Males 24-32 g, females 21-31 g
- Fasting period before study: No data.
- Housing: Individual caging
- Diet: Standard diet (NAFAG No. 924)
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 59 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Arachid oil
- Amount of vehicle: 20 ml/kg bw
Duration of treatment / exposure:
2 consecutive days
Frequency of treatment:
1 application daily
Post exposure period:
24 h
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Dose / conc.:
3 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: Orally (gavage)
- Doses / concentrations: 128 mg/kg bw in 20 ml/kg bw arachid oil
Tissues and cell types examined:
Bone marrow was harvested from the shafts of both femurs.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The oral LD50 was found to be > 3000 mg/kg bw in Chinese hamsters of either sex (CIBA-Geigy Ltd., No. 820742, 1982, see chapter 7.2.1)

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs. In a siliconised pipette filled with approx. 0.5 µl 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-Grünwald solution for 2 minutes then in May-Grünwald solution/water 1/1 for 2 minutes and then in Giemsa's, 40 % for 20 minutes. After being rinsed in methanol 55 % for 5-8 seconds and washed off twice in water, they were left immersed in water for approx. 2 minutes. After rinsing with distilled water and air-drying, the slides were cleared in Xylol and mounted in Eukitt.

METHOD OF ANALYSIS:
The slides of 3 female and 3 male animals each of the negative control group, the positive control group and of the groups treated with various doses of the test substance were examined. 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.
Statistics:
The significance of difference was assessed by χ2 -test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
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) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 8.72, whereas the negative control yielded a percentage of 0.10. The difference is highly significant (p<0.05).

Tab. 1: The effect of the test substance and cyclophosphamide on bone marrow cells of Chinese hamster (animals sacrificed 24 hours after second application)

f = female, m = male

 

concentration

Animal No.

Sex

Single Jolly bodies

Fragments of nuclei in erythrocytes

Micronuclei in erythrocytes

Micronuclei in Leucopoietic cells

Polyploid cells

Total

 

 

Control (Arachid oil)

 

1

f

0.3

 

 

 

 

0.3

2

f

 

 

 

 

 

0.0

3

f

 

 

 

 

 

0.0

4

m

0.1

 

 

 

 

0.1

5

m

 

 

 

 

 

0.0

6

m

0.2

 

 

 

 

0.2

 

 

Cyclophosphamide

 

 

128 mg/kg bw

1

f

10.3

1.4

1.9

0.5

 

14.1

2

f

8.6

0.7

2.3

0.3

0.1

12.0

3

f

7.5

0.9

1.2

0.5

0.1

10.2

4

m

3.4

0.2

0.7

0.2

 

4.5

5

m

3.1

0.1

1.3

0.4

 

4.9

6

m

4.9

0.4

1.3

 

 

6.6

 

 

 

 

 

 

 

 

Test substance

 

 

750 mg/kg bw

1

f

 

 

 

 

 

0.0

2

f

0.2

 

 

 

 

0.2

3

f

0.3

 

 

 

 

0.3

4

m

0.2

 

 

 

 

0.2

5

m

0.1

 

 

 

 

0.1

6

m

 

 

 

 

 

0.0

 

 

1500 mg/kg bw

1

f

0.2

 

 

 

 

0.2

2

f

 

 

 

 

 

0.0

3

f

0.1

 

 

 

 

0.1

4

m

 

 

 

 

 

0.0

5

m

0.1

 

 

 

 

0.1

6

m

 

 

 

 

 

0.0

 

 

3000 mg/kg bw

1

f

0.1

 

 

 

 

0.1

2

f

0.2

 

 

 

 

0.2

3

f

0.1

 

 

 

 

0.1

4

m

 

 

 

 

 

0.0

5

m

0.1

 

 

 

 

0.1

6

m

0.2

 

 

 

 

0.3

Conclusions:
Under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro studies

Bacterial reverse mutation assay

In a bacterial reverse mutation assay similar to OECD Guideline 471 the test substance was analysed for mutagenicity by the detection of point mutations in bacteria (histidine-auxotrophic mutants of Salmonella typhimurium). Any mutagenic effects of the test substance is demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were also performed with the addition of an activation mixture (rat liver microsomes and co-factors). The strains TA 98, TA 100, TA 1535, and TA 1537 were treated with the test substance dissolved in DMSO in doses of 25, 75, 225, 675, and 2025 µg/0.1 ml (plate incorporation test). A negative control (vehicle) and appropriate positive control substances were also evaluated. At concentrations of 225 µg/0.1 ml and higher the test compound precipitated in soft agar. No dose-related biologically relevant increase or doubling in the number of his+ revertants was observed in plate incorporation test in any of the tested strains with or without metabolic activation. Effects on cytotoxicity were not given. According to the results of the present study, the test substance is not mutagenic in the bacterial reverse mutation assay under the experimental conditions chosen. The positive controls yielded the expected results (Ciba-Geigy, 1981).

Gene mutation in mammalian cells

In a GLP compliant mammalian cell gene mutation test according to OECD Guideline 476 the test substance was investigated for its potential to induce gene mutations at the HPRT locus in V79 cells. The assay was performed in two independent experiments, using two parallel cultures each. In the first main experiment cells were exposed for 4 hours to the test item at concentrations of 5.5; 11.0; 22.0; 44.0; 88.0 μg/mL with and without liver microsomal activation. The second experiment was performed with a treatment time of 4 hours with metabolic activation and a treatment time of 24 hours without metabolic activation, at concentrations of 5.5; 10.0; 22.0; 44.0 and 88.0 µg/mL (without metabolic activation) and 2.8; 5.5; 11.0; 22.0; and 44.0 µg/mL (with metabolic activation). The highest applied concentration in the pre-test on toxicity (2800 μg/ml) was chosen with regard to the solubility properties of the test item. The test item was dissolved in DMSO. No relevant toxic effects occurred with and without metabolic activation following 4 and 24 hours treatment. Precipitation of the test item at the end of treatment was noted in the first experiment at 22.0 μg/mL and above with and at 11.0 μg/mL and above without metabolic activation. In the second experiment precipitation occurred at 22.0 μg/mL and above with and at 44.0 μg/mL and above without metabolic activation. No relevant and reproducible increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. An isolated increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control and the range of historical solvent control data was observed in the 1st culture of the 2nd experiment at 11.0 μg/mL without metabolic activation. However, the isolated increase was judged as biologically irrelevant fluctuation as it was neither reproduced in the parallel culture under identical experimental conditions nor dose dependent as indicated by the lacking statistical significance. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the 2nd culture of the 2nd experiment with metabolic activation. This trend however, was reciprocal, going down versus increasing concentrations and thus, biologically irrelevant. Appropriate reference mutagens (EMS and DMBA), 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. According to the results of the study, the test substance does not induce gene mutations in the in vitro mammalian cell gene mutation test under the experimental conditions chosen (Harlan, 2012).

 

In vivo studies

Nucleus anomaly test in somatic interphase nuclei

In a nucleus anomaly test in somatic interphase nuclei conducted similar to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test), the test substance was analysed for mutagenicity in bone marrow cells. Single dose applications were performed to 3 male and 3 female Cricetulus griseus Chinese Hamsters each dose group once daily for two consecutive days via gavage. The test substance was analysed in the concentrations of 750, 1500, and 3000 mg/kg body weight in 20 ml/kg body weight arachid oil (vehicle). The control animals received the vehicle and positive control animals were treated with cyclophosphamide (128 mg/kg body weight in 20 ml/kg body weight arachid oil). Animals were sacrificed 24 hours after the second application and bone marrow cells were harvested. 1000 bone marrow cells were scored per animal and following parameters were evaluated: Single jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells, and polyploid cells. There was no significant increase in the frequency of micronucleated bone marrow cells after treatment with the test substance. The positive control induced the appropriate response. Hence, the test substance is not mutagenic in the nucleus anomaly test in somatic interphase nuclei under the experimental conditions chosen (Ciba-Geigy, 1981).

Sister chromatid exchange

In a sister chromatid exchange study the test substance was analysed for mutagenicity in bone marrow cells. 2 male and 2 female Cricetulus griseus Chinese Hamsters each dose group were treated orally with a single dose of the test material. The test substance was analysed in the concentrations of 750, 1500, and 3000 mg/kg body weight in 20 ml/kg body weight arachid oil (vehicle). The control animals received the vehicle and positive control animals were treated with 7, 12 – Dimethylbenz(a)anthracene (100 mg/kg body weight in 20 ml/kg body weight arachid oil). Two hours prior test substance administration a subcutaneous implantation of a 45 mg-tablet of 5-bromodeoxyuridine (BUdR) in the neck was carried out. The animals were sacrificed 24 hours after the administration of the test material and 2 hours after intraperitoneal injection of 10 mg colcemide/kg, followed by the harvest of bone marrow cells from the shafts of both femurs. The cells were transferred to slides and stained with bisbenzimide and Giemsa. The slides of two female and two male animals each of the treatment groups and of the control groups were examined. 25 differently stained metaphases of the second cell-cycle with BUdR-substitution were analysed per animal for the number of sister chromatid exchanges. In the intermediate-dose group (1500 mg/kg body weight) one female animal died after the application of the test material. There was no significant increase in the frequency of sister chromatid exchanges in bone marrow cells after treatment with the test substance. Only in the high dose group (3000 mg/kg body weight) one of the four animals showed an increased frequency of sister chromatid exchanges and this value slightly increased the mean value for this dose group. Hence, this value was significantly different from that of the concurrent control. Comparison to cumulative (negative) control data derived from analogous experiments on altogether 3500 metaphases revealed no statistically significant difference. The positive controls yielded the expected results. According to the results of the present study, the test substance is not mutagenic in the sister chromatid exchange assay under the experimental conditions chosen (Ciba-Geigy, 1982). 

Justification for classification or non-classification

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

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