Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not cause a relevant or dose dependent increase in the number of revertants colonies at any dose level with the Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA either in the presence or absence of S9 metabolic activation system in the plate incorporation assay performed and hence it is not likely to classify as a gene mutant in vitro.

 

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

In vitro gene mutation study in mammalian cells

Test chemical did not induced mutation in mouse lymphoma assay (TK locus) in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

 

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:
27-10-2004 - 19-11-2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
other: EC Directive 2000/32/EC, L 136, Annex 4D, B.13/B.14
Qualifier:
according to
Guideline:
other: Japanese Substance Control Law (JSCL) Test Guideline III. 1 Gene Mutation Test With Bacteria
Principles of method if other than guideline:
Salmonella/microsome mutagenicity test (Ames test) (plate incorporation) was performed to assess the mutagenic nature of the test chemical
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella typhimurium strains and tryptophan for E.coli strains
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Salmonella typhimurium strains TA98: hisD3052 rfa uvr B+R TA100: hisG46 rfa uvr B+R Ta1535: hisG46 rfa uvrB TA1537: hisC3076 rfa uvrB Escherichia coli WP2uvrA: pKM101
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: pKM101
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
9000g S9 metabolic activation system was prepared from liver of male Sprague Dawley rat homogenate
Test concentrations with justification for top dose:
Plate incorporation assay:
With: 0, 50, 160, 500, 1600 or 5000 µg/plate
Without: 0, 50, 160, 500, 1600 or 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solube in DMSO
Untreated negative controls:
yes
Remarks:
Untreated control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: 2-nitrofluorene (TA98, -S9), 2-aminoanthracene (All strains, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): No data

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes
- Any supplementary information relevant to cytotoxicity: Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occuring mutants compared to the corresponding solvent control value

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The test chemical was considered positive if-
- it produces atleast 2-fold increase in the mean number of revertants/plate of atleast one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
- it induces a dose related increase in the mean number of revertants per plate of at least one of the test strains over the mean number of revertants per plate of the appropriate vehicle in atleast two or three concentrations of the test item at complete bacterial background lawn

If the aboce critera is not achieved, it is considered to show no mutagenic activity in the system
Statistics:
No data
Species / strain:
S. typhimurium, other: 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:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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: Visible precipitation was observed on plates at 160 µg/plate and above
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential

Table: Results of the mutagenicity test for the test chemical

TA100

S9

Dose levelsµg/plate

Number of revertants/plate

Mean

SD

Ratio dose/control

p

Plate 1

Plate 1

Plate 3

+

2-Aminoanthracene

1703

1512

1460

1558.3

128.0

12.7

 

+

Negative control

124

128

119

123.7

4.5

-

 

+

DMSO

111

134

122

122.3

11.5

-

 

+

50

135

116

126

125.7

9.5

1.0

 

+

160

137

130

151

139.3

10.7

1.1

P

+

500

135

125

127

129.0

5.3

1.1

P

+

1600

129

127

139

131.7

6.4

1.1

P

+

5000

122

104

110

112.0

9.2

0.9

p

-

Sodium azide

586

539

620

581.7

40.7

5.4

 

-

Negative control

123

115

119

119.0

4.0

-

 

-

DMSO

109

119

98

108.7

10.5

-

 

-

50

109

109

104

107.3

2.9

1.0

 

-

160

109

103

130

114.0

14.2

1.0

P

-

500

109

98

109

105.3

6.4

1.0

P

-

1600

103

108

115

108.7

6.0

1.0

P

-

5000

109

98

79

95.3

15.2

0.9

P

P: Precipitated

TA1535

S9

Dose levelsµg/plate

Number of revertants/plate

Mean

SD

Ratio dose/control

p

Plate 1

Plate 1

Plate 3

+

2-Aminoanthracene

440

439

456

445.0

9.5

47.7

 

+

Negative control

4

7

10

7.0

3.0

-

 

+

DMSO

10

9

9

9.3

0.6

-

 

+

50

8

8

4

6.7

2.3

0.7

 

+

160

11

7

5

7.7

3.1

0.8

P

+

500

11

5

4

6.7

3.8

0.7

P

+

1600

7

10

8

8.3

1.5

0.9

P

+

5000

12

6

7

8.3

3.2

0.9

p

-

Sodium azide

193

1889

143

175.0

37.8

32.8

 

-

Negative control

13

6

11

10.0

3.6

-

 

-

DMSO

6

4

6

5.3

1.2

-

 

-

50

7

4

5

5.3

1.5

1.0

 

-

160

6

7

5

6.0

1.0

1.1

P

-

500

10

7

3

6.7

3.5

1.3

P

-

1600

6

8

5

6.3

1.5

1.2

P

-

5000

8

5

10

7.7

2.5

1.4

P

P: Precipitated

 

TA1537

S9

Dose levelsµg/plate

Number of revertants/plate

Mean

SD

Ratio dose/control

p

Plate 1

Plate 1

Plate 3

+

2-Aminoanthracene

99

102

93

98.0

4.6

5.9

 

+

Negative control

19

17

12

16.0

3.6

-

 

+

DMSO

18

16

16

16.7

1.2

-

 

+

50

28

22

17

22.3

5.5

1.3

 

+

160

20

23

26

23.0

3.0

1.4

P

+

500

24

24

20

22.7

2.3

1.4

P

+

1600

19

23

24

22.0

2.6

1.3

P

+

5000

14

17

27

19.3

6.8

1.2

p

-

9-aminoacridine

270

222

153

215.0

58.8

23.0

 

-

Negative control

12

15

8

11.7

3.5

-

 

-

DMSO

7

9

12

9.3

2.5

-

 

-

50

13

11

16

13.3

2.5

1.4

 

-

160

9

11

8

9.3

1.5

1.0

P

-

500

8

7

12

9.0

2.6

1.0

P

-

1600

15

13

10

12.7

2.5

1.4

P

-

5000

10

13

11

11.3

1.5

1.2

P

P: Precipitated

 

TA98

S9

Dose levelsµg/plate

Number of revertants/plate

Mean

SD

Ratio dose/control

p

Plate 1

Plate 1

Plate 3

+

2-Aminoanthracene

1054

1120

1076

1083.3

33.6

54.2

 

+

Negative control

18

23

23

21.3

2.9

-

 

+

DMSO

19

21

20

20.0

1.0

-

 

+

50

21

18

14

17.7

3.5

0.9

 

+

160

28

27

21

25.3

3.9

1.3

P

+

500

30

21

28

26.3

4.7

1.3

P

+

1600

32

26

34

30.7

4.2

1.5

P

+

5000

20

34

34

29.3

8.1

1.5

p

-

2-nitrofluorene

301

299

301

300.3

1.2

26.5

 

-

Negative control

12

14

9

11.7

2.5

-

 

-

DMSO

14

13

7

11.3

3.8

-

 

-

50

14

14

8

12.0

3.5

1.1

 

-

160

8

14

13

11.7

3.2

1.0

P

-

500

16

13

12

13.7

2.1

1.2

P

-

1600

13

4

12

9.7

4.9

0.9

P

-

5000

7

11

10

9.3

2.1

0.8

P

P: Precipitated

 

E.coli WP2uvrA

S9

Dose levelsµg/plate

Number of revertants/plate

Mean

SD

Ratio dose/control

p

Plate 1

Plate 1

Plate 3

+

2-Aminoanthracene

125

97

88

103.3

19.3

7.0

 

+

Negative control

15

18

11

14.7

3.5

-

 

+

DMSO

16

11

17

14.7

3.2

-

 

+

50

17

19

17

17.7

1.2

1.2

 

+

160

16

15

15

15.3

0.6

1.0

P

+

500

15

11

18

14.7

3.5

1.0

P

+

1600

14

16

14

14.7

1.2

1.0

P

+

5000

16

13

12

13.7

2.1

0.9

p

-

9-NQO

529

578

572

559.7

26.7

31.7

 

-

Negative control

17

17

23

19.0

3.5

-

 

-

DMSO

26

14

13

17.7

7.2

-

 

-

50

21

15

15

17.0

3.5

1.0

 

-

160

18

14

17

16.3

2.1

0.9

P

-

500

15

20

20

18.3

2.9

1.0

P

-

1600

19

17

15

17.0

2.0

1.0

P

-

5000

12

19

18

16.3

3.8

0.9

P

P: Precipitated

 

Conclusions:
The test chemical did not cause a relevant or dose dependent increase in the number of revertants colonies at any dose level with the Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA either in the presence or absence of S9 metabolic activation system in the plate incorporation assay performed and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Salmonella/microsome mutagenicity test (Ames test) (plate incorporation) was performed to assess the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA in the presence and absence of S9 metabolic activation system in the plate incorporation assay. The test chemical was dissolved in DMSO and used at dose level of 0, 50, 160, 500, 1600 or 5000 µg/plate. The plates were incubated for 48 hrs and observed for revertant colonies. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not cause a relevant or dose dependent increase in the number of revertants colonies at any dose level with the Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA either in the presence or absence of S9 metabolic activation system in the plate incorporation assay performed and hence it is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on the various test chemicals.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
5. No data
6. No data
Species / strain / cell type:
mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)
Remarks:
5
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle MEM culture medium supplemented with 10% fetal bovine serum
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)
Remarks:
6
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
5. No data
6. No data
Metabolic activation:
with and without
Metabolic activation system:
5. S9 mix
6. S9 mix
Test concentrations with justification for top dose:
5. Continuous treatment: 0, 0.3, 0.7, 1.3 µg/mL
Short term treatment method: 0, 06, 1.1, 2.2 µg/mL
6. Continuous treatment: 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL
Short term treatment method: With S9: 0, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL
Without S9: 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL
Vehicle / solvent:
5. - Vehicle(s)/solvent(s) used: 0.5% CMC Na
- Justification for choice of solvent/vehicle: The test chemical was soluble in CMC Na
6. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
CMC Na
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
5
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
6
Details on test system and experimental conditions:
5. METHOD OF APPLICATION: in medium
Cells at the start of the experiment: 20000 cells

DURATION
- Preincubation period: No data
- Exposure duration: Direct method: 24 and 48 hrs
Short term treatment method with S9: 6 hrs
- Expression time (cells in growth medium):
Direct method: 24 and 48 hrs
Short term treatment method with S9: 18 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: For structural abnormalities, 200 metaphase cells per group and 800 division metastatic cells for multiplicative cells were analyzed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
6. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: Direct method: 24 and 48 hrs
Short term treatment method with S9: 6 hrs
- Expression time (cells in growth medium):
Direct method: 24 and 48 hrs
Short term treatment method with S9: 18 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: For structural abnormalities, 200 metaphase cells per group were analyzed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
5. No data
6. No data
Evaluation criteria:
5. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was also observed.
6. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was also observed.
Statistics:
5. A significant difference test (p <0.05) between the negative control group and the test substance treated group and between the negative control group and the positive control group was performed on the occurrence frequency of cells having chromosomal abnormality by Fisher's exact probability test method. According to the judgment criteria of Ishikan et al, the frequency of cells with chromosomal abnormality is negative, negative 5% or higher and less than 10% positive false positive 10% or higher Respectively.
6. No data
Species / strain:
mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)
Remarks:
5
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mammalian cell line, other: Chinese hamster cultured cells (CHL/IU)
Remarks:
6
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
5. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To determine the treatment concentration of the test substance used for the chromosomal aberration test, the influence of the test substance on cell proliferation was investigated. The growth inhibitory action of the test substance on CHL / IU cells was determined by measuring the proliferation degree of each group using a monolayer culture cell densitometer, and the negative control group was used as an index.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
6. No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.
Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL/IU) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in 0.5% CMC Na and used at dose level of 0, 0.3, 0.7, 1.3 µg/mL in continuous treatment method and 0, 06, 1.1, and 2.2 µg/mL in short term treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Giemsa stained six slide specimens were prepared for each petri dish. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, the presence or absence of cells (polyploid) was also observed.The test chemical did not induce chromosome aberration in Chinese hamster cultured cells (CHL/IU) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

In another genetic toxicity in vitro study, the given test chemical was used to detect the mutagenic nature. The study was performed using Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL without S9 and 0, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL with S9 in the short term treatment method and 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450µg/mL in continuous treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method. The test chemical did not induce chromosome aberration in Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data of read across substances
Justification for type of information:
Data for the target chemical is summarized based on the structurally similar read across chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
WoE derived based on the experimental data from structurally and functionally similar read across chemicals i.e. WoE-8, WoE-9 and WoE-10.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
8. Thymidine Kinase Locus (TK+/-)
9. TK locus
10. tk locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
8, 9 and 10
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
8. S9-mix prepared from the livers of β-naphthoflavone and sodium phenobarbitone-treated rats.
9. No data
10. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system.
Test concentrations with justification for top dose:
8. 0, 5.3, 10.7, 21.4, 42.8, 85.5 and 171 μg active dye/ml; with and without metabolic activation.
9. 0, 21.4, 42.8, 85.5, 171 and 342 μg active dye/ml; without metabolic Activation
10. Experiment I: 0, 78.1, 156.3, 312.5, 625 and 1250 μg/ml without S9-mix; 312.5, 625, 1250, 2000 and 2500 μg/ml with S9-mix
Experiment II: 0, 312.5, 625, 1250 and 2500 μg/ml without S9-mix
Vehicle / solvent:
8. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical is soluble in DMSO
9. DMSO is used as a solvent.- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical is soluble in DMSO
10. - Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The given test chemical was dissolved in deionised water.
Untreated negative controls:
yes
Remarks:
Negative controls consisted of untreated cultures and cultures treated with the solvent alone (DMSO).
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Remarks:
8
Untreated negative controls:
yes
Remarks:
Negative controls consisted of untreated cultures and cultures treated with the solvent alone (DMSO).
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
9
Untreated negative controls:
yes
Remarks:
In accordance with the OECD guideline
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: In accordance with the OECD guideline
Remarks:
10
Details on test system and experimental conditions:
8. DURATION
- Exposure duration: 4-hour
- Expression time (cells in growth medium): 4-hour

STAIN (for cytogenetic assays):No

NUMBER OF REPLICATIONS: Duplicates
9. DURATION
- Exposure duration: 24-hour
- Expression time (cells in growth medium): 24-hour
NUMBER OF REPLICATIONS: Duplicates
10. NUMBER OF REPLICATIONS:
- Number of cultures per concentration : duplicate
- Number of independent experiments - 2 (Experimental I and II)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h treatment without and with S9-mix (Experimental I); 24 h treatment without S9-mix (Experimental II)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): expression period 72 h (Experimental I); expression period
48 h (Experimental II)
- Selection time (if incubation with a selective agent): selection period of 10-15 days (Experimental I); selection period of 10-15 days (Experimental II)
- Criteria for small (slow growing) and large (fast growing) colonies: To discriminate between large (indicative for mutagenic effects) and small colonies (indicative for a clastogenic effect) colony sizing was performed.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)
Rationale for test conditions:
No data
Evaluation criteria:
8. The cell line was observed for gene mutation at the TK locus
9. Number of mutant colonies were observed.
10. Toxicity was measured in the main experiments as percentage relative total growth of the treated cultures relative to the total growth of the solvent control cultures. To discriminate between large (indicative for mutagenic effects) and small colonies (indicative for a clastogenic effect) colony sizing was performed.
Statistics:
No data
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
8
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Strong cytotoxicity was observed at 171 μg active dye/ml after 4-hour treatment
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
9
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Relevant toxic effects were observed at the highest concentration of 342μg active dye/ml under precipitation after 24-hour treatment.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
10
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: precipitation occurred at 2000 μg/ml and above
Remarks:
experiment I
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
10
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: precipitation occurred at 2500 μg/ml and above at both treatment intervals
Remarks:
experiment II
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
8. No data
9. No data
10. - Precipitation and time of the determination: In the pre-test and experiment II precipitation occurred at 2500 μg/ml and above at both treatment intervals without and with S9-mix; in experiment I at 2000 μg/ml and above.

RANGE-FINDING/SCREENING STUDIES (if applicable):
Test concentrations were based on the results of a pre-test on toxicity with exposure up to the prescribed maximum concentration of 5000 μg/ml measuring relative suspension growth. In the pre-test, precipitation occurred at 2500 μg/ml and above at both treatment intervals without and with S9-mix.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency - In both experiment a reproducible, biologically relevant increase in the number of mutant colonies was not observed independent of the presence or absence of S9-mix. An isolated increase of the mutant frequency was observed in the second experiment at 1250 μg/ml in one out of two parallel cultures.
Remarks on result:
other: No mutagenic potential
Conclusions:
Test chemical did not induction mutation in mouse lymphoma assay (TK locus) in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

The In Vitro Mammalian Cell Gene Mutation Test was conducted by using the given test chemical in Mouse lymphoma cell line L5178Y on Thymidine Kinase Locus (TK+/-). The test was performed in duplicate cultures at the doses 5.3, 10.7, 21.4, 42.8, 85.5 and 171 μg active dye/ml with and without metabolic activation extracted from the livers of β-naphthoflavone and sodium phenobarbitone-treated rats. Known mutagens in the presence (3-methylcholanthrene) or absence of S9 (methyl methane sulfonate) were used as positive controls. Negative controls consisted of untreated cultures and cultures treated with the solvent alone (DMSO). The experiment used a pulse (4-hour) treatment procedure. The cell line was observed for gene mutation at the TK locus. Precipitation was observed at 342 μg active dye/ml in the absence and presence of S9 mix. Strong cytotoxicity was observed at 171 μg active dye/ml after 4-hour treatment. The test chemical was not mutagenic in the mouse lymphoma assay (TK locus) in the presence and absence of metabolic activation as no increased number of mutant colonies where observed up to the maximal concentrations of the substance and hence it is not likely to classify as a gene mutant in vitro.

 

In another study, the In Vitro Mammalian Cell Gene Mutation Test was conducted by using the given test chemical in Mouse lymphoma cell line L5178Y on Thymidine Kinase Locus (TK+/-). The test was performed in duplicate cultures at the doses 21.4, 42.8, 85.5, 171 and 342 μg active dye/ml without metabolic activation. Known mutagen in the absence of S9 (methyl methane sulfonate) was used as positive controls. Negative controls consisted of untreated cultures and cultures treated with the solvent alone (DMSO). The experiment used a pulse (24-hour treatment) treatment procedure. The cell line was observed for gene mutation at the TK locus. Relevant toxic effects were observed at the highest concentration of 342 μg active dye/ml under precipitation after 24-hour treatment. There was no increase in mutant colonies were observed and therefore the given test chemical was non-mutagenic without activation in mouse lymphoma assay (TK locus) and hence it is not likely to classify as a gene mutant in vitro.

 

Both the above results were supported with the In vitro mammalian cell gene mutation test conducted by using the given test chemical at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation. Test concentrations were based on the results of a pre-test on toxicity with exposure up to the prescribed maximum concentration of 5000 μg/ml measuring relative suspension growth. In the main test, cells were treated for 4 h or 24 h (without S9-mix experiment II) followed by an expression period of 72 or 48 h (experiment II) to fix the DNA damage into a stable tk mutation. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. Toxicity was measured in the main experiments as percentage relative total growth of the treated cultures relative to the total growth of the solvent control cultures. To discriminate between large (indicative for mutagenic effects) and small colonies (indicative for a clastogenic effect) colony sizing was performed. Negative and positive controls were in accordance with the OECD guideline. In the pre-test and experiment II precipitation occurred at 2500 μg/ml and above at both treatment intervals without and with S9-mix; in experiment I at 2000 μg/ml and above. The appropriate level of toxicity (10-20% survival after the highest concentration) was not reached in experiment I without S9-mix. In both experiment a reproducible, biologically relevant increase in the number of mutant colonies was not observed independent of the presence or absence of S9-mix. An isolated increase of the mutant frequency was observed in the second experiment at 1250 μg/ml in one out of two parallel cultures. As no increased mutant frequencies was observed at a higher concentration (2500 μg/ml) in the same test and no mutagenic effect was observed in the first experiment, this isolated positive effect was considered not biologically relevant. Under the experimental conditions used, the given test chemical was not mutagenic in the mouse lymphoma assay using the tk locus as reporter gene.

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in mouse lymphoma assay (TK locus) in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Data available from various sources was reviewed to determine the mutagenic nature of the given test chemical. The studies are as mentioned below:

Ames assay:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

Salmonella/microsome mutagenicity test (Ames test) (plate incorporation) was performed to assess the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA in the presence and absence of S9 metabolic activation system in the plate incorporation assay. The test chemical was dissolved in DMSO and used at dose level of 0, 50, 160, 500, 1600 or 5000 µg/plate. The plates were incubated for 48 hrs and observed for revertant colonies. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not cause a relevant or dose dependent increase in the number of revertants colonies at any dose level with the Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA either in the presence or absence of S9 metabolic activation system in the plate incorporation assay performed and hence it is not likely to classify as a gene mutant in vitro.

 

In another study, Salmonella/microsome mutagenicity test (Ames test) (pre-incubation) was performed to assess the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA in the presence and absence of S9 metabolic activation system in the pre-incubation assay. Polysynthrene Blue R was dissolved in DMSO and used at dose level of 0, 50, 160, 500, 1600 or 5000 µg/plate in the main study, 0, 0.5, 1.6, 5, 16 or 50 µg/plate (TA1537, -S9 and +S9) and 0, 500, 1000, 1600, 3000 or 5000 µg/plate (TA98, +S9) in the first repeat preincubation test and at dose levels of 0, 16, 50, 160, 500, 1600, 3000 or 5000 µg/plate (TA98; +S9) in the second repeat preincubation test. The plates were pre-incubated for 20-30 mins and incubated for 48 hrs to observe for revertant colonies. Concurrent solvent and negative control chemicals were also included in the study. A slight increase in the number of revertants was noted in strain TA98 with S9 mix at precipitating doses of 500 and 1600 µg/plate. However, individual values overlap in the historical solvent control range and the historical negative control range, no clear dose-dependency occurred and rations were only 1.6 and 2.0 compared with negative control. This increase was therefore considered to be equivocal and a repeat experiment with a narrower dose range was performed. No increase in the number of reveratants was observed with all doses in the form of reduced bacteria lawn. Therefore, a second repeat experiment was also performed using doses down to 16 µg/plate. Slight toxicity starting with 160 µg/plate was confirmed, but again no relevant increase in revertants was noted. A dose/control ration of 1.9 was noted with the precipitating dose 500 µg/plate, but mean value of revertants was within the historical negative range and no clear dose-dependency occurred in combination with slight toxic effects on the bacterial lawn. The criteria for a positive response were not fulfilled. Taken together, the marginal increase in revertants with strain TA98 cannot be considered to be biologically relevant. The test chemical did not cause any biological relevant mutagenic effect in Salmonella typhimurium tester strains TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Both the above studies were supported with the gene mutation toxicity study conducted to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli WP2uvrA. The test chemical was dissolved in DMSO and used at dose level of 0.8 -2500 µg/plate. The doses for the main study were based on the toxicity study conducted at dose level of 4-10000 µg/plate and proved to be toxic at 2500 µg/plate. Visible precipitation on the plates was observed at 500 µg/plate. Thinning of the bacterial lawn was noted at 2500 - 10000 µg/plate. Based on this experiment, 2500 µg/plate was chose as the highest dose for main study. Concurrent solvent and positive control plates were also included in the study. Control plates without mutagen showed that the spontaneous revertant colonies were similar to that described in literature. All the positive control chemicals gave the expected increase in the number of revetants colonies. In the absence of metabolic activation system, the test chemical did not show any influence on the number of revertants/plate in any of the bacterial tester strains tested. In the presence of S9 metabolic activation system, treatment with the test chemical resulted in a dose related increase in the number of revertants colonies with the strains TA1538 and TA98.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in the Salmonella typhimurium and Escherichia coli strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

 

In vitro mammalian chromosome aberration study:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster cultured cells (CHL/IU) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in 0.5% CMC Na and used at dose level of 0, 0.3, 0.7, 1.3 µg/mL in continuous treatment method and 0, 06, 1.1, and 2.2 µg/mL in short term treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method. Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Giemsa stained six slide specimens were prepared for each petri dish. The presence or absence of structural abnormality such as chromosome type or chromosome type gap, the presence or absence of cells (polyploid) was also observed.The test chemical did not induce chromosome aberration in Chinese hamster cultured cells (CHL/IU) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

In another genetic toxicity in vitro study, the given test chemical was used to detect the mutagenic nature. The study was performed using Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL without S9 and 0, 38.3, 76.6, 153, 306, 613, 1230, 2450 µg/mL with S9 in the short term treatment method and 0, 19.1, 38.3, 76.6, 153, 306, 613, 1230, 2450µg/mL in continuous treatment method. The doses for the main study were based on data from preliminary dose range finding study. Concurrent solvent and positive control plates were also included in the study. The cells were exposed to the test chemical for 24 or 48 hrs in the continuous treatment method and for 6 hrs in the short term treatment method. The test chemical did not induce chromosome aberration in Chinese hamster cultured cells (CHL) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

In vitro gene mutation study in mammalian cells

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

The In Vitro Mammalian Cell Gene Mutation Test was conducted by using the given test chemical in Mouse lymphoma cell line L5178Y on Thymidine Kinase Locus (TK+/-). The test was performed in duplicate cultures at the doses 5.3, 10.7, 21.4, 42.8, 85.5 and 171 μg active dye/ml with and without metabolic activation extracted from the livers of β-naphthoflavone and sodium phenobarbitone-treated rats. Known mutagens in the presence (3-methylcholanthrene) or absence of S9 (methyl methane sulfonate) were used as positive controls. Negative controls consisted of untreated cultures and cultures treated with the solvent alone (DMSO). The experiment used a pulse (4-hour) treatment procedure. The cell line was observed for gene mutation at the TK locus. Precipitation was observed at 342 μg active dye/ml in the absence and presence of S9 mix. Strong cytotoxicity was observed at 171 μg active dye/ml after 4-hour treatment. The test chemical was not mutagenic in the mouse lymphoma assay (TK locus) in the presence and absence of metabolic activation as no increased number of mutant colonies where observed up to the maximal concentrations of the substance and hence it is not likely to classify as a gene mutant in vitro.

 

In another study, the In Vitro Mammalian Cell Gene Mutation Test was conducted by using the given test chemical in Mouse lymphoma cell line L5178Y on Thymidine Kinase Locus (TK+/-). The test was performed in duplicate cultures at the doses 21.4, 42.8, 85.5, 171 and 342 μg active dye/ml without metabolic activation. Known mutagen in the absence of S9 (methyl methane sulfonate) was used as positive controls. Negative controls consisted of untreated cultures and cultures treated with the solvent alone (DMSO). The experiment used a pulse (24-hour treatment) treatment procedure. The cell line was observed for gene mutation at the TK locus. Relevant toxic effects were observed at the highest concentration of 342 μg active dye/ml under precipitation after 24-hour treatment. There was no increase in mutant colonies were observed and therefore the given test chemical was non-mutagenic without activation in mouse lymphoma assay (TK locus) and hence it is not likely to classify as a gene mutant in vitro.

 

Both the above results were supported with the In vitro mammalian cell gene mutation test conducted by using the given test chemical at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation. Test concentrations were based on the results of a pre-test on toxicity with exposure up to the prescribed maximum concentration of 5000 μg/ml measuring relative suspension growth. In the main test, cells were treated for 4 h or 24 h (without S9-mix experiment II) followed by an expression period of 72 or 48 h (experiment II) to fix the DNA damage into a stable tk mutation. Liver S9 fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. Toxicity was measured in the main experiments as percentage relative total growth of the treated cultures relative to the total growth of the solvent control cultures. To discriminate between large (indicative for mutagenic effects) and small colonies (indicative for a clastogenic effect) colony sizing was performed. Negative and positive controls were in accordance with the OECD guideline. In the pre-test and experiment II precipitation occurred at 2500 μg/ml and above at both treatment intervals without and with S9-mix; in experiment I at 2000 μg/ml and above. The appropriate level of toxicity (10-20% survival after the highest concentration) was not reached in experiment I without S9-mix. In both experiment a reproducible, biologically relevant increase in the number of mutant colonies was not observed independent of the presence or absence of S9-mix. An isolated increase of the mutant frequency was observed in the second experiment at 1250 μg/ml in one out of two parallel cultures. As no increased mutant frequencies was observed at a higher concentration (2500 μg/ml) in the same test and no mutagenic effect was observed in the first experiment, this isolated positive effect was considered not biologically relevant. Under the experimental conditions used, the given test chemical was not mutagenic in the mouse lymphoma assay using the tk locus as reporter gene.

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in mouse lymphoma assay (TK locus) in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

 

Justification for classification or non-classification

Based on the data available and applying weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and In vitro gene mutation study in mammalian cells. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.