Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: negative;

HPRT performed with a structural analogue: 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
Study period:
1984-07-17 to 1984-07-27
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)
Deviations:
yes
Remarks:
fifth strain is missing in order to detect cross-linking mutagens
GLP compliance:
no
Remarks:
but QAU statement included
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
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
20, 80, 320, 1280, and 5120 µg/0.1 mL with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see in "Any other informations on materials and methods"
Details on test system and experimental conditions:
METHOD OF APPLICATION: agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Statistics:
Arithmetic mean values and standard deviation were 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:
no cytotoxicity
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 320 µg/0.1 ml and above the substance precipitated in soft agar.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was carried out with concentrations ranging from 0.1 to 5000 µg/0.1 ml. Thereupon, the concentration of 5120 µg/0.1 ml was used as the highest in the mutagenicity test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Plate incorportation assay - First experiment:

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

 

Frameshift type

 

TA 100

TA1535

TA98

TA1537

0

121 ± 10

14 ± 10

32 ± 10

6 ± 1

20

103 ± 18

8 ± 3

32 ± 6

7 ± 1

80

128 ± 4

19 ± 2

23 ± 3

7 ± 3

320

118 ± 28

13 ± 5

24 ± 4

8 ± 5

1280

128 ± 23

11 ± 5

19 ± 2

6 ± 2

5120

107 ± 8

17 ± 1

17 ± 8

7 ± 2

Positive controls, –S9

Name

4NQO

SA

DR

9AA

Concentrations (μg/plate)

0.125

2.5

5

50

0.25

5

10

100

Mean No. of colonies/plate (average of 3 ± SD)

851 ± 81

1426 ± 105

366 ± 82

100 ± 22

1456 ± 141

1965 ± 181

841 ± 79

1531 ± 801

+

0

121 ± 6

11 ± 6

45 ± 3

17 ± 3

+

50

108 ± 10

11 ± 6

54 ± 10

16 ± 2

+

150

116 ± 6

13 ± 4

50 ± 6

18 ± 4

+

500

94 ± 9

14 ± 4

45 ± 3

15 ± 5

+

1500

101 ± 10

14 ± 7

53 ± 1

17 ± 3

+

5000

89 ± 13

13 ± 7

47 ± 5

17 ± 2

Positive controls, –S9

Name

 

CP

 

 

Concentrations (μg/plate)

 

250

 

 

Mean No. of colonies/plate (average of 3 ± SD)

 

547 ± 103

 

 

4NQO: 4-nitroquinoline-N-oxide

SA: sodium azide

DR: daunorubicin

9AA: 9(5)aminoacridine hydrochloride monohydrate

CP: cyclophosphamide

Table 2: Plate incorportation assay - Second experiment

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

 

Frameshift type

 

TA 100

TA1535

TA98

TA1537

0

139 ± 10

12 ± 4

27 ± 6

5 ± 3

20

120 ± 23

16 ± 6

23 ± 5

10 ± 6

80

148 ± 19

14 ± 6

27 ± 6

9 ± 4

320

136 ± 14

15 ± 4

26 ± 7

12 ± 5

1280

126 ± 10

12 ± 7

23 ± 3

9 ± 4

5120

127 ± 9

12 ± 3

23 ± 2

11 ± 4

Positive controls, –S9

Name

4NQO

SA

DR

9AA

Concentrations (μg/plate)

0.125

2.5

5

50

0.25

5

10

100

Mean No. of colonies/plate (average of 3 ± SD)

992 ± 26

865 ± 65

806 ± 69

66 ± 21

1617 ± 139

1343 ± 61

1186 ± 188

973 ± 349

+

0

127 ± 17

15 ± 1

47 ± 9

10 ± 4

+

50

130 ± 10

13 ± 7

39 ± 4

10 ± 6

+

150

123 ± 24

17 ± 2

43 ± 5

13 ± 5

+

500

129 ± 9

12 ± 4

46 ± 15

9 ± 4

+

1500

114 ± 5

15 ± 2

33 ± 6

13 ± 5

+

5000

118 ± 20

12 ± 4

35 ± 8

6 ± 3

Positive controls, –S9

Name

 

CP

 

 

Concentrations (μg/plate)

 

250

 

 

Mean No. of colonies/plate (average of 3 ± SD)

 

369 ± 48

 

 

4NQO: 4-nitroquinoline-N-oxide

SA: sodium azide

DR: daunorubicin

9AA: 9(5)aminoacridine hydrochloride monohydrate

CP: cyclophosphamide

The slight increase in the number of back-mutant colonies observed in the second experiment without microsomal activation on strain TA 1537 at the concentrations of 20, 320 and 5120 µg/0.1 mL is attributed to fluctuations in the rate of spontaneously occurring back-mutants.

Conclusions:
No evidence of the induction of point mutations by the test article or by the metabolites of the substance was detectable in the strains of S. typhimurium used in these experiments.
Executive summary:

In an Ames test similar to OECD guideline 471, the test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium, using the plate incorporation method. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 80, 320, 1280 and 5120 µg/0.1 mL. In order to confirm the results the experiments were repeated. 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 substance revealed no marked deviations. No evidence of the induction of point mutations by the test substance 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
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 supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1, without S9: (79.4), 158.8, 317.5, 635.0, 1270,0, 2540.0 µg/ml
Experiment 1, with S9: 79.4, 158.8, 317.5, 635.0, 1270,0, (2540.0) µg/ml
Experiment 2, with and without S9: 79.4, 158.8, 317.5, 635.0, 1270,0, (2540.0) µg/ml
numbers in parantheses: these cultures were discontinued.
Vehicle / solvent:
DMSO; the final concentration of DMSO in the culture medium was 0.5 % v/v.
- Justification for choice of solvent/vehicle: The solvent was selected based on solubility properties and tolerance by the 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 h (with and without S9), 24h (without S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7-10 days
- Fixation time (start of exposure up to fixation or harvest of cells): 17 - 22 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: two independent cultures were used

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density
Evaluation criteria:
A test item is classified as positive if it induces 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 is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is 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 has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this 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 is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance was considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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 and osmolarity: There was no shift of the pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: Turbidity was noted in the absence of metabolic activation at 158.8 and 317.5 μg/mL in experiment I and at 158.8, 317.5, and 635.0 μg/mL in experiment II. As in the preexperiment such a broad range of turbidity may be based on denatured protein rather than test item precipitation. Precipitation of the test item was noted at 635 μg/mL and above in the first experiment with and without metabolic activation. In the second experiment precipitation was noted at 635 μg/mL and above with and at 1270 μg/mL and above without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 39.7 to 3907.7 μg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant cytotoxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal of the test item. Precipitation occurred at 635.0 μg/mL and above with and without metabolic activation following 4 hours treatment. Following 24 hours treatment precipitation was noted at 2540 μg/mL and above. In the
absence of metabolic activation turbidity was observed from 39.7 to 317.5 μg/mL following 4 hours treatment and from 317.5 to 1270 μg/mL after 24 hours treatment. Such a broad range of turbidity indicates that the turbidity is most likely not based on test item particles but rather denatured protein. Based on the occurrence of precipitation in the pre-experiment the individual concentrations were selected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures was noted in both main experiments with and without metabolic activation following 4 and 24 hours treatment.

Summary of Results

concentration (µg/ml) T/P S9 Mix relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor
Experiment I / 4h treatment culture I culture II
solvent control (DMSO) - 100 100 100 3 1 100 100 100 17.7 1
positive control (EMS) 150 - 113 76.5 98.2 88.7 29.6 112.2 84.1 113.1 113.1 6.4
test item 79.4 T - 66.1 culture was discontinued# 80.1 culture was discontinued#
test item 158.8 T - 60.5 64.5 97.4 4.5 1.5 63.3 66.2 112.8 2.5 0.1
test item 317.5 T - 21.5 28.5 88.4 15.2 5.1 29.4 34 109.8 25.1 1.4
test item 635 P - 60.9 42.5 99.5 7.3 2.4 63.9 55.5 98.3 32.7 1.8
test item 1270 P - 77.3 57.2 93.4 13.8 4.6 78.7 71.1 96.5 6.9 0.4
test item 2540 P - 78.3 50.7 97.6 9.6 3.2 82.9 70.3 89 13.8 0.8
solvent control (DMSO) + 100 100 100 37.9 1 100 100 100 11.5 1
positive control (DMBA) 1.1 + 86.4 120.2 66 1014.8 26.8 82.2 109 93.3 626.1 54.4
test item 79.4 + 100.8 102 74.6 62.8 1.7 103.3 96 118.7 7.8 0.7
test item 158.8 + 105.3 114.3 107.9 30 0.8 110.3 100.4 96.2 21.7 1.9
test item 317.5 + 107.2 105.5 111.6 32.6 0.9 106.7 100.8 88.5 33 2.9
test item 635 P + 108.3 108.6 86.2 11.9 0.3 109.5 111.4 102.2 27.3 2.4
test item 1270 P + 78.9 112.8 112.6 34.3 0.9 99.2 100 87.8 19.9 1.7
test item 2540 P + 86.7 culture was discontinued## 100 culture was discontinued##
Experiment II / 24h treatment culture I culture II
solvent control (DMSO) - 100 100 100 16.1 1 100 100 100 30.2 1
positive control (EMS) 150 - 102.7 113.5 90.4 394.8 24.5 97.9 91.4 81.9 496.7 16.5
test item 79.4 - 104.6 113.5 85.7 24.1 1.5 101.9 67.2 134.8 17.5 0.6
test item 158.8 T - 102.7 71.8 99.3 31.1 1.9 99.1 68.5 140.2 18.5 0.6
test item 317.5 T - 104.5 56.5 98 20.7 1.3 100.5 59.4 105.2 15.1 0.5
test item 635 T - 100.4 72.7 100.3 20.4 1.3 98.1 50.9 111.5 12.1 0.4
test item 1270 P - 102.4 95.9 97 14.2 0.9 97.6 107.2 117.1 36.2 1.2
test item 2540 P - 100 culture was discontinued## 96.9 culture was discontinued##
Experiment II / 4h treatment
solvent control (DMSO) + 100 100 100 14.5 1 100 100 100 31.5 1
positive control (DMBA) 1.1 + 80.6 118.3 75.4 757.6 52.3 69.4 47.3 81.3 606.4 19.2
test item 79.4 + 115.6 126.7 93.7 17.6 1.2 96.5 79.6 81.9 26.2 0.8
test item 158.8 + 112.6 130.1 73.2 31.4 2.2 92.9 68.6 81.3 27 0.9
test item 317.5 + 108.4 130.9 82.6 31 2.1 93.2 118.9 107.7 31.4 1
test item 635 P + 108.1 142.3 89.4 21.7 1.5 91.3 125 87.3 20 0.6
test item 1270 P + 109 117.6 87 36.3 2.5 94.3 85.9 91.6 38.2 1.2
test item 2540 P + 112.3 culture was discontinued## 91.4 culture was discontinued##

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

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

T Turbidity

P Preparation visible 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 and is therefore considered to be non-mutagenic in this HPRT assay.
Executive summary:

A GLP-compliant mammalian cell mutagenicity test according to OECD guideline 476 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 study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The tested concentrations ranged from 79.4 to 1270 µg/ml.

Turbidity was noted in the absence of metabolic activation at 158.8 and 317.5 μg/mL in experiment I and at 158.8, 317.5, and 635.0 μg/mL in experiment II. As in the preexperiment such a broad range of turbidity may be based on denatured protein rather than test item precipitation. Precipitation of the test item was noted at 635 μg/mL and above in the first experiment with and without metabolic activation. In the second experiment precipitation was noted at 635 μg/mL and above with and at 1270 μg/mL and above without metabolic activation. No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures was noted in both main experiments with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. In experiment I the induction factor exceeded the threshold of 3.0 at 317.5, 1270, and 2540 μg/mL in the first culture without metabolic activation (induction factors of 5.1, 4.6, and 3.2). These isolated effects were biological irrelevant as they were based on the rather low solvent control of just 3.0 colonies per 106 cells and were not confirmed in the parallel culture II performed under identical experimental conditions or in the second experiment. In the first culture of experiment I the mutant colonies/106 cells exceeded the range of the historical solvent control data at 79.4 μg/mL with metabolic activation. Another increase exceeding the range of the historical solvent control data was observed in the second culture of experiment II at 1270 μg/mL with metabolic activation. However, the threshold of three times the mutation frequency of the corresponding solvent control was not reached or exceeded. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.0 up to 37.9 mutants per 106 cells; the range of the groups treated with the test item was from 2.5 up to 62.8 mutants per 106 cells. The highest solvent control of 37.9 colonies per 106 cells slightly exceeded the historical range of solvent controls with metabolic activation (3.4 - 36.6 mutant colonies per 106). The mean value of both parallel cultures (37.9 and 11.5 equal to a mean of 24.7) remained well within the historical range of solvent controls with metabolic activation. EMS (150 μg/mL) and DMBA (1.1 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 and is therefore 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 substance was negative in a micronucleus assay.

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:
July 16, 1984 - November 15, 1984
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)
Version / remarks:
21. July 1997
Deviations:
yes
Remarks:
only one time point observed
Principles of method if other than guideline:
The test was conducted according to the procedures of Boller et al. (1970, Humangenetik 11, 35-54), Matter et al. (1970, Mutation Res. 12, 417-425) and Müller et al. (1972, Verh. Dtsch. Ges. Path. 56, 381-384).
GLP compliance:
no
Remarks:
but QAU statement included
Type of assay:
micronucleus assay
Species:
hamster, Chinese
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln
- Age at study initiation: females: 6-10 weeks, males 4-9 weeks
- Weight at study initiation: females: females: 22-30 g, males: 22-33 g
- Fasting period before study: no data
- Housing: single
- Diet: NAFAG No.924
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 24
- Humidity (%): 59 - 68
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% aqueous solution of CMC (carboxymethyl cellulose)
Duration of treatment / exposure:
two days
Frequency of treatment:
daily one application on 2 consecutive days
Post exposure period:
24 hours
Dose / conc.:
875 mg/kg bw/day (actual dose received)
Dose / conc.:
1 750 mg/kg bw/day (actual dose received)
Dose / conc.:
3 500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Doses / concentrations: 128 mg/kg
Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
Bone marrow cells 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 Xylene 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. 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 X² -test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Table 1: Effects of the test substance and cyclophosphamide on bone marrow cells of chinese hamser

 

 

 

Percent of cells with anomalies of nuclei

 

# of animals

sex

single jolly bodies

Fragment of nuclei in erythrocytes

Micronuclei in erythrocyblasts

Micronuclei in leucopoietic cells

Polyploid cells

Total

Control

1

female

0.2

 

 

 

 

0.2

2

female

 

 

 

 

 

0

3

female

 

 

 

 

 

0

4

male

 

 

 

 

 

0

5

male

0.1

 

 

 

 

0.1

6

male

 

 

 

 

 

0

Cyclophosphamide (128 mg/kg bw)

1

female

10.4

2.8

2

 

 

15.2

2

female

9.8

3

1.7

0.2

 

14.7

3

female

9.8

1.7

1.7

0.2

 

13.4

4

male

7.8

1.3

1.1

0.1

 

10.3

5

male

6.1

1.2

2.6

0.1

 

10

6

male

8.5

1.6

1.3

0.1

 

11.5

Test substance (875 mg/kg bw)

1

female

 

 

 

 

 

0

2

female

 

 

 

 

 

0

3

female

0.2

 

 

 

 

0.2

4

male

 

 

 

 

 

0

5

male

 

 

 

 

 

0

6

male

 

 

 

 

 

0

Test substance (1750 mg/kg bw)

1

female

0.1

 

 

 

 

0.1

2

female

0.1

 

 

 

 

0.1

3

female

0.2

 

 

 

 

0.2

4

male

0.1

 

 

 

 

0.1

5

male

0.1

 

 

 

 

0.1

6

male

0.1

 

 

 

 

0.1

Test substance (3500 mg/kg bw)

1

female

 

 

 

 

 

0

2

female

 

 

 

 

 

0

3

female

 

 

 

 

 

0

4

male

 

 

 

 

 

0

5

male

 

 

 

 

 

0

6

male

 

 

 

 

 

0

The bone marrow smears from animals treated with various doses of the test substance showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 12.5% cells with anomalies of nuclei. This is significantly different from the controls (0.05%) treated with the vehicle (0.5% CMC) alone.

Conclusions:
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.
Executive summary:

The in vivo genotoxic potential of the test substance was evaluated in a micronucleus test in bone marrow erythrocytes of young, male and female chinese hamsters similar in designs to OECD guideline 474. Treatment consisted of one daily dose of 875, 1750 or 3500 mg/kg on each of two consecutive days. The animals were sacrificed 24 h after the second application and bone marrow smears were prepared. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a positive control experiment with cyclophosphamide (128 mg/kg) yielded 12.5% cells with anomalies of nuclei. This is significantly different from the controls (0.05%) treated with the vehicle (0.5% CMC) 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 substance.

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

Additional information

In vitro:

Gene mutation in bacteria

The test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium, using the plate incorporation method. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 80, 320, 1280 and 5120 µg/0.1 ml. In order to confirm the results the experiments were repeated. 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 substance revealed no marked deviations. No evidence of the induction of point mutations by the test substance 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. The study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data with the restriction of missing a fifth strain in order to detect cross-linking mutagens. The study was performed under GLP-like quality control.

 

Cytogenicity in mammalian cells

In accordance with Annex VIII (8.4.2) of the REACH legislation, the test does not need to be conducted if adequate data from a reliable in vivo cytogenicity assay is available. In this case, a valid micronucleus study in vivo is available.

Gene mutation in mammalian cells

A GLP compliant in vitro mammalian cell gene mutation test (HPRT-Test) according to OECD Guideline 476 was performed with a structural analogue (see attached read across justification document). The test substance was investigated in two independent experiments, using two parallel cultures each. In the main experiment I cells were exposed for 4 hours to the test item at concentrations of 79.4; 158.8; 317.5; 635.0; 1270.0; 2540.0 μg/ml with and without liver microsomal activation, whereas 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 the same concentrations. The test item was dissolved in DMSO. The maximum concentration of the pre-experiment and the main experiments was 2540.0 μg/ml. 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. Precipitation of the test item was noted at 635 μg/ml and above. No relevant cytotoxic effect was found. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. According to the results of the study, the test substance did not induce gene mutations in the in vitro mammalian cell gene mutation test under the experimental conditions chosen.

In vivo:

Micronucleus test in the hamster

The in vivo genotoxic potential of the test substance was evaluated in a micronucleus test in bone marrow erythrocytes of young, male and female chinese hamsters. Treatment consisted of one daily dose of 875, 1750 or 3500 mg/kg on each of two consecutive days. The animals were sacrificed 24 h after the second application and bone marrow smears were prepared. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a positive control experiment with cyclophosphamide (128 mg/kg) yielded 12.5% cells with anomalies of nuclei. This is significantly different from the controls (0.05%) treated with the vehicle (0.5% CMC) 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 substance. The study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenicity (micronucleus test) data with the restriction of observing only one time point after treatment. The study was performed under GLP-like quality control.

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 (EC) No.1272/2008. Based on the present data, classification for genotoxicity is not warranted under Regulation (EC) No.1272/2008.