Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The available data from three in vitro assays show that the substance does not have a genotoxic potential.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2009-10-09 to 2009-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable, chromosome aberration
Species / strain / cell type:
lymphocytes: human lymphocyte cultures
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced animals
Test concentrations with justification for top dose:
First experiment:
3+17 hour -S-9: 15.1, 130 and 200 µg/mL
3+17 hour +S-9: 54.9, 130 and 200 µg/mL

Second experiment:
20+0 hour -S-9: 125, 150, 175 and 200 µg/mL
3+17 hour +S-9: 140, 160, and 180 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:the test substance was soluble in DMSO at concentrations up to approximately 80.10 mg/mL when heated to 80ºC with the aid of vortex mixing and ultrasonication.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4 Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA)
Remarks:
no remarks
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in suspension

DURATION
- Preincubation period: no
- Exposure duration:
First experiment: 3 hours
Second experiment: 20 hours and 3 hours
- Expression time (cells in growth medium): no data
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SELECTION AGENT (mutation assays): no data
SPINDLE INHIBITOR (cytogenetic assays): no data
STAIN (for cytogenetic assays): the cells were stained for 5 minutes in filtered 4% (v/v) Giemsa in pH 6.8 buffer.

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and percentage of cells in mitosis

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: hyperdiploidy and structural aberrations
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range was observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p < 0.05)
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result [ ]. Biological relevance was taken into account, for example consistency of response within and between concentrations and/or between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations.
Statistics:
No data
Key result
Species / strain:
lymphocytes: human lymphocyte cultures
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes in pH compared to the concurrent vehicle controls, were observed
- Effects of osmolality: No marked changes in osmolality compared to the concurrent vehicle controls, were observed
- Evaporation from medium: no data
- Water solubility: not soluble
- Precipitation: yes
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the proportion of cells with structural aberrations in these cultures fell within current historical vehicle control (normal) ranges

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Table 7.6.1/2: Experiment 1 – Results summary

Treatment

Concentration (mg/mL)

Cytotoxicity (%)

% Cells with Chromosome Aberrations (Excluding Gaps)

Historical(%)#

Statistical significance

 

 

 

 

 

 

 

 

 

 

3+17 hour -S-9

Vehiclea

-

2.00

0-3

-

 

 

 

15.1

10

1.50

 

NC

 

 

 

130

30

1.50

 

NC

 

 

 

200

39

1.50

 

NC

 

 

 

*NQO, 2.50

ND

20.17

 

p=0.001

 

 

 

 

 

 

 

 

 

 

3+17 hour +S-9

Vehiclea

-

0.50

0-3

-

 

 

 

54.9

23

1.50

 

NC

 

 

 

130

4

0.00

 

NC

 

 

200

17

1.00

 

NC

 

 

*CPA, 12.5

ND

15.76

 

p=0.001

 

 

 

 

 

 

 

 

 

aVehicle control,DMSO

* Positive control

#95thpercentile of the observed range

NC = Not calculated

ND = Not determined

Table 7.6.1/3: Experiment 2 – Results summary

Treatment

Concentration (mg/mL)

Cytotoxicity (%)

% Cells with Chromosome Aberrations (Excluding Gaps)

Historical(%)#

Statistical significance

 

 

 

 

 

 

 

 

20+0 hour -S-9

Vehiclea

-

0.00

0-3

-

 

 

125

9

1.00

 

NC

 

 

150

17

0.50

 

NC

 

 

175

47

0.50

 

NC

 

 

200

34

0.50

 

NC

 

 

*NQO, 2.50

ND

40.82

 

p=0.001

 

 

 

 

 

 

 

 

3+17 hour +S-9

Vehiclea

-

1.50

0-3

-

 

 

140

ND

9.00

 

NC

 

 

160

0

0.50

 

NC

 

 

180

1

1.50

 

NC

 

 

*CPA, 12.5

ND

42.55

 

p=0.001

 

 

 

 

 

 

 

 

 

aVehicle control,DMSO

* Positive control

#95thpercentile of the observed range

NC = Not calculated

ND = Not determined

Conclusions:
The test substance did not induce chromosome aberration in cultured human peripheral blood lymphocytes in the absence and in the presence of activation system.
Executive summary:

The test substance was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors. This study was performed in accordance with OECD Guideline 473 and Good Laboratory Practices.

A range-finding study was performed to select the test article concentrations for chromosome analysis, by evaluating the effect of the test substance on mitotic index. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals.

In the first experiment, human lymphocyte cultures were treated with the test substance at concentrations of 15.1, 130 and 200 µg/mL in the absence of S9 and 54.9, 130 and 200 µg/mL in the presence of S9. In the second experiment, human lymphocyte cultures were treated with the test substance at concentrations of 125, 150, 175 and 200 µg/mL in the absence of S9 and 140, 160, and 180 µg/mL in the presence of S9.

Appropriate negative (vehicle) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within current historical vehicle control (normal) ranges. 4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9 respectively.

Treatment of cells with the test substance in the absence and presence of S-9 in both experiments resulted in frequencies of cells with structural aberrations that were generally similar to those observed in concurrent vehicle control cultures for all concentrations analysed.

The only exception to this was observed in one culture at the lowest concentration (140 µg/mL) analysed in the presence of S-9 in Experiment 2, where the number of aberrant cells (excluding gaps) was markedly higher than the concurrent control values. There was therefore no evidence of reproducibility of this isolated effect between cultures or between experiments and the observation was not considered biologically relevant.

No increases in the total frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were observed in cultures treated with the test substance in the absence and presence of S-9 in Experiments 1 and 2.

Therefore, the test substance did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to the limit of solubility in culture medium both in the absence and the presence of S-9.

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:
From 2009-10-09 to january 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
The thymidine kinase (tk) gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 10
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9: Aroclor 1254 induced rat liver post mitochondrial fraction
Test concentrations with justification for top dose:
See table 7.6.1/1
In experiment 1, ten concentrations, ranging from 4.14 to 200 µg/mL, were tested in the absence and presence of S 9.
In Experiment 2, ten concentrations, ranging from 15.0 to 150 µg/mL in the absence of S-9 and from 15.0 to 200 ¿g/mL in the presence of S 9, were tested.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Methyl methane sulphonate (MMS); Benzo[a]pyrene (B[a]P)
Remarks:
no remarks
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no data
For the cytotoxicity range-finder experiment, in the absence and presence of S 9, a 3 hour treatment incubation period was used.

- Exposure duration: 3 hours incubation at 37±1°C
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of 2 days during which the tk-/- mutation would be expressed.
- Selection time (if incubation with a selection agent): 12 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): 5 trifluorothymidine, TFT
SPINDLE INHIBITOR (cytogenetic assays): no data
STAIN (for cytogenetic assays): no data

NUMBER OF REPLICATIONS: in duplicate

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

OTHER EXAMINATIONS:
no data
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF
2. The linear trend test was positive.
The test article was considered as positive in this assay if both of the above criteria were met.
The test article was considered as negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
No data
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: yes, fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies
- Effects of osmolality: yes, changes in osmolality of more than 50 mOsm/kg may be responsible for an increase in mutant frequencies
- Evaporation from medium: no data
- Water solubility: no soluble
- Precipitation: yes, in the cytotoxicity Range-Finder Experiment, following the treatment incubation period, precipitate was observed at the highest concentration tested in the absence of S-9 (50.0 µg/mL) and at the highest three concentrations tested in the presence of S-9 (12.5 to 50.0 µg/mL).

- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Table 7.6.1/2: Experiment 1 (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RTG

MF§

 

%RTG

MF§

0

100

82.9

0

100

66.9

4.14

105

80.4

4.14

95

90.0

6.37

107

79.8

6.37

100

65.6

9.80

108

80.5

9.80

104

81.7

15.1

98

62.6

15.1

108

64.6

23.2

128

62.9

23.2

104

72.9

35.7

118

53.3

35.7

100

67.4

54.9

103

68.5

54.9

114

75.0

84.5 P, PP

82

109

84.5 P

80

81.5

 

 

 

 

 

130 P, PP

106

63.8

Linear trend

 

NS

Linear trend

 

NS

MMS

 

 

 

B[a]P

 

 

 

15.0

56

714

2.00

55

800

20.0

47

847

3.00

22

1220

 

 

 

 

 

 

 

 

 

 

 

 

Table 7.6.1/3: Experiment 2 (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RTG

MF§

 

%RTG

MF§

0

100

61.0

0

100

71.4

40.0

99

52.7

45.0

106

42.4

50.0

99

62.4

60.0

108

53.9

60.0

103

49.9

75.0

99

67.3

70.0

107

49.1

90.0 P

115

45.3

80.0 P

94

58.9

105 P

132

54.2

90.0 P

101

65.3

120 P

119

55.2

120 P,PP

103

53.3

150 P, PP

105

64.0

Linear trend

 

NS

Linear trend

 

NS

MMS

 

 

 

B[a]P

 

 

 

15.0

44

190

2.00

84

448

20.0

32

364

3.00

43

675

 

 

 

 

 

 

 

 

 

 

 

 

MF: Mutant frequency

§: 5-TFT resistant mutants/106viable cells 2 days after treatment

% RTG: % Relative total growth

P: Precipitation observed at time of treatment

PP: Precipitation observed following treatment incubation period.

NS: Not significant

Conclusions:
The test substance did not induce mutation at the tk locus in the L5178Y mouse lymphoma cells in the presence and in the absence of activation system.
Executive summary:

In an in vitro mammalian cell gene mutation test, the test substance was assessed for its ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. This GLP study was performed according to OECD guideline 476 and the UKEMS guidelines.

The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9).

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 1.56 to 50 mg/mL (primarily limited by solubility in DMSO, heated to 37°C and using 1% v/v test article additions in culture medium).

In Experiments 1 and 2, the mutant frequencies of the concentrations plated were all less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF, 126 mutants per 106viable cells), indicating a negative result. There were no statistically significant linear trends.

In Experiment 1, ten concentrations ranging from 4.14 to 200 mg/mL, were tested in the absence and presence of S-9. Two days after treatment, the highest concentrations selected to determine viability and TFT resistance were 84.5 mg/mL in the absence of S-9 and 130 mg/mL in the presence of S-9 (both limited by the appearance of post-treatment precipitate), which gave 82% and 106% RTG, respectively.

In Experiment 2, ten concentrations ranging from 15.0 to 150 mg/mL, in the absence of S-9 and from 15.0 to 200 mg/mL in the presence of S-9, were tested. Two days after treatment, the highest concentrations selected to determine viability and TFT resistance were 120 mg/mL in the absence of S-9 and 150 mg/mL in the presence of S-9 (both limited by the appearance of post-treatment precipitate), which gave 103% and 105% Relative Total Growth, respectively.

In Experiments 1 and 2, the Mutant Frequency (MF) values of the concentrations plated were all less than the sum of the mean control MF plus the GEF, indicating a negative result. There were no statistically significant linear trends.

Positive and negative controls were valid for both experiments.

Therefore, the test substance did not induce mutation at the tk locus of L5178Y mouse lymphoma. These conditions included treatments up to precipitating concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 01-25-2010 to 03-16-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
The histidine gene for strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
The tryptophan gene for strains E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
other: Histidine deficient
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Tryptophan deficient
Metabolic activation:
with and without
Metabolic activation system:
liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
1, 3, 10, 30, 100, 300 and 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was insoluble in water, ethanol and acetone
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: sodium azide; 9-Aminoacridine; 2-Nitrofluorene; 4-Nitroquinoline-1-oxide
Remarks:
In absence of S9 Mix
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene; Benzo[a]pyrene
Remarks:
In presence of S9 Mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period:
First test: no preincubation period
Second test: at 37°C for 30 minutes
- Exposure duration: . all plates were incubated at approximately 37°C for ca 72 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays):
- histidine production for S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- tryptophane production for E. coli WP2 uvr A pKM 101

NUMBER OF REPLICATIONS: Three Petri dishes were used for each treatment.

OTHER: no data
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.
Statistics:
Dunnett's test
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Precipitate was observed on all plates containing 99422018 at 300 and 1000 µg/plate in both tests.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data
Remarks on result:
other:

Table 7.6.1/1: Results obtained in the presence of metabolic activation: test 1

With metabolic activation

 

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard Deviation

Fold increase relative to vehicle

Individual revertant
colony counts

 

 

 

 

 

 

 

 

TA98

DMSO

-

47.0

1.7

-

46, 49, 46

 

test substance

1 µg

52.7

12.7

1.1

39, 64, 55

 

 

3 µg

61.3

7.0

1.3

68, 62, 54

 

 

10 µg

43.3

9.2

0.9

38, 38, 54

 

 

30 µg

52.7

3.2

1.1

54, 49, 55

 

 

100 µg

40.0

3.6

0.9

39, 44, 37

 

 

300 µg

40.7

4.7

0.9

46 P, 39 P, 37 P

 

 

1000 µg

31.7

2.1

0.7

31 P, 34 P, 30 P

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA100

DMSO

-

170.7

8.1

-

180, 166, 166

 

test substance

1 µg

192.0

8.7

1.1

202, 186, 188

 

 

3 µg

190.7

23.5

1.1

214, 167, 191

 

 

10 µg

163.3

16.1

1.0

145, 175, 170

 

 

30 µg

166.7

39.1

1.0

170, 126, 204

 

 

100 µg

175.7

17.6

1.0

174, 194, 159

 

 

300 µg

166.0

25.2

1.0

189 P, 170 P, 139 P

 

 

1000 µg

132.3

10.5

0.8

122 P, 143 P, 132 P

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA1535

DMSO

-

24.0

3.5

-

26, 26, 20

test substance

1 µg

22.0

4.0

0.9

22, 26, 18

 

 

3 µg

21.7

2.1

0.9

21, 20, 24

 

 

10 µg

24.7

4.0

1.0

20, 27, 27

 

 

30 µg

20.7

1.2

0.9

20, 22, 20

 

 

100 µg

19.3

1.2

0.8

18, 20, 20

 

 

300 µg

15.3

3.1

0.6

16 P, 18 P, 12 P

 

 

1000 µg

17.7

1.5

0.7

19 P, 18 P, 16 P

 

 

 

 

 

 

 

 

 

Key to Plate Postfix Codes

 

 

 

 

 

 

P

Precipitate

With metabolic activation

 

Strain

Addition

Concentration per plate

Mean revertants per plate

Standard Deviation

Fold increase relative to vehicle

Individual revertant
colony counts

 

 

 

 

 

 

 

 

TA1537

DMSO

-

34.3

4.6

-

29, 37, 37

 

test substance

1 µg

32.0

10.6

0.9

44, 24, 28

 

 

3 µg

33.7

5.0

1.0

33, 39, 29

 

 

10 µg

34.0

8.7

1.0

38, 40, 24

 

 

30 µg

39.7

2.1

1.2

39, 38, 42

 

 

100 µg

32.0

9.5

0.9

23, 31, 42

 

 

300 µg

24.7

3.8

0.7

22 P, 23 P, 29 P

 

 

1000 µg

22.7

11.6

0.7

35 P, 21 P, 12 P

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

WP2 uvrA

DMSO

-

163.0

11.4

-

176, 158, 155

(pKM101)

test substance

1 µg

165.0

15.1

1.0

170, 148, 177

 

 

3 µg

174.0

25.2

1.1

147, 178, 197

 

 

10 µg

151.0

27.6

0.9

122, 177, 154

 

 

30 µg

138.0

28.9

0.8

117, 171, 126

 

 

100 µg

153.0

20.0

0.9

148, 175, 136

 

 

300 µg

151.3

21.2

0.9

148 P, 174 P, 132 P

 

 

1000 µg

133.0

9.8

0.8

136 P, 141 P, 122 P

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA98

B[a]P

5 µg

406.7

45.7

8.7

431, 435, 354

TA100

AAN

5 µg

878.0

48.9

5.1

923, 826, 885

TA1535

AAN

5 µg

348.0

22.3

14.5

368, 352, 324

TA1537

B[a]P

5 µg

177.0

7.0

5.2

180, 182, 169

WP2 uvrA (pKM101)

AAN

10 µg

476.7

14.5

2.9

460, 484, 486

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

 

 

B[a]P

AAN

Benzo[a]pyrene

2-Aminoanthracene

P

Precipitate

Conclusions:
The test substance showed no evidence of mutagenic activity in the bacterial system using Salmonella typhimurium and Escherichia coli.
Executive summary:

The test substance was tested in a bacterial reverse mutation test according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) and Good Laboratory Practices.

The strains S. typhimurium TA1535, TA1537, TA98 and TA100, and E. coli WP2 uvrA (pKM101) were treated with the test substance at concentrations of 1, 3, 10, 30, 100, 300 and 1000 µg in DMSO, with and without activation with liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone.

A first test was performed without preincubation period whereas in a second test there was a preincubation period of 30 minutes. Vehicle and positive controls were performed in both tests.

In the first test, without preincubation period, no evidence of toxicity was obtained following exposure to the test substance. Precipitate was observed on all plates containing the test substance at 300 and 1000 µg/plate in both tests. A maximum exposure concentration of 1000 µg/plate was, therefore, selected for use in the second test.

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test substance at any concentration up to and including 1000 µg/plate in either the presence or absence of S9 mix.

In the second test, with 30 minutes of preincubation period, no evidence of toxicity was obtained following exposure to the test substance. Precipitate was observed on all plates containing the test substance at 300 and 1000 µg/plate in both tests.

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test substance at any concentration up to and including 1000 µg/plate in either the presence or absence of S9 mix.

the test substance showed no mutagenic activity in the bacteria system in the presence and in the absence of system of activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Reverse gene mutation assay

In a reverse gene mutation assay in bacteria performed according to the OECD test guideline No. 471 and in compliance with Good Laboratory Practice, strains TA 1535, TA 97, TA 98 and TA 100 of S. typhimurium and E. coli WP2 were exposed to the test substance in DMSO at concentrations of 0, 1, 3, 10, 30, 100, 300 and 1000 µg/plate in the presence and absence of mammalian metabolic activation (S9 mix) . In this study, there was no evidence of induced mutant colonies over background.

In vitro cytogenicity study in mammalian cells

In an in vitro chromosome aberration test performed according to the OECD guideline No. 473 and in compliance with Good Laboratory Practice, the human lymphocyte cultures were treated with the test substance in two experiments. In the first experiment the human lymphocyte cultures were treated with the test substance at concentrations of 0, 15.1, 130 and 200 µg/mL without metabolic activation and 0, 54.9, 130 and 200 µg/mL with metabolic activation (rat liver homogenate metabolising system). In the second experiment human lymphocyte cultures were treated with the test substance at concentrations of 125, 150, 175 and 200 µg/mL in the absence of S9 and 140, 160, and 180 µg/mL in the presence of S9. Two treatment conditions were used for the study: 4-hour exposure followed by a 17-hour expression period (with and without S9) and 20-hour exposure (without S9 only). No chromosome aberrations were induced by treatment with the test substance in cultured peripheral blood lymphocytes when tested to the limit of solubility in culture medium, both in the absence and the presence of S9. In this study, there was no evidence of in vitro cytogenicity of the test substance in mammalian cells.

In vitro gene mutation study in mammalian cells

In a mammalian cell gene mutation assay performed similarly to OECD 476, the test substance diluted in water was tested in the agar version of L5178Y mouse lymphoma assay. The test substance was tested in the mutagenesis assay over a range of concentrations from 4.14 to 200 mg/mL in the presence and absence of the S-9 activation. Both the non-activated and S-9 activated cultures did not produce significant increases in mutant frequency in comparison with that of the solvent control cultures.The test substance did not induce mutation at the tk locus of L5178Y mouse lymphoma cells.


Justification for classification or non-classification

Based on the results from three in vitro guideline compliant assays, the substance is not classified for genotoxicity according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.