Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed as per the OECD guideline 471 using Salmonella typhimurium strains TA100, TA1535, TA98, TA1535 and E. coli WP2 uvrA with and without of S9 metabolic activation system. The study was performed at various dose levels and in triplicate plates/dose. Concurrent solvent and positive control chemicals were incorporated in the study. The plates were observed for dose dependent increase in the number of revertants/plate. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA100, TA1535, TA98, TA1535 and E. coli WP2 uvrA in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHL/IU cell line. The duration of treatment was short term from 6-18 hrs and continuous treatment for 24 and 14 hrs. The doses were dissolved in DMSO and the dose levels were Short term treatment (With S9: 0, 0.90, 1.81, 3.61, 7.23 or 14.5µg/mL, Without S9: 0, 7.23, 14.5, 28.9, 57.8 or 116µg/mL), for 24 hrs and 48 hrs treatment (Without S9: 0, 14.5, 28.9, 57.8 or 116µg/mL) and retest dose level was 0, 5.71, 8.56, 12.8, 19.3 or 28.9µg/mL with S9. Concurrent solvent and vehicle control chemicals were included in the study. The test chemical did not induce chromosomal aberrations in CHL/IU cell line in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
genetic toxicity in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from J check
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
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:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
All strains: 0, 1.22, 4.88, 19.5, 78.1, 313, 1250 or 5000 µg/plate

TA100:
Without S9: 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate
With S9: 0, 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate

TA1535: With and without S9: 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate

TA98: With and without S9: 0, 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate

TA1535: With and without S9: 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate

E.coli WP2 uvrA: With and without S9: 0, 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
other: 2- (2- Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, WP2uvrA, TA98, -S9), 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl (TA1537, -S9), 2-Aminoanthracene (TA1535, WP2uvrA, +S9), Benzo[a]pyrene (TA100, TA1537, TA98, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- 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 plates were used/dose level and the study was performed in triplicates

NUMBER OF CELLS EVALUATED: No data

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:
No data
Evaluation criteria:
The plates were observed for a dose dependent increase in the number of revertants/plate
Statistics:
No data
Species / strain:
other: TA100, TA1535, TA98, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential

Table 1:

Metabolic activation system

Dose (µg/plate)

TA100

TA1535

WP2uvrA

TA98

TA1537

Without S9

DMSO

98

11

18

27

10

113

11

17

32

7

 

4.88

119

1

15

26

7

19.5

112

5

15

25

6

78.1

109

7

15

23

8

313

0

0

0

0

0

1250

0

0

0

0

0

5000

0

0

0

0

0

With S9

DMSO

166

16

21

53

12

1.22

195

14

20

53

7

4.88

914

17

20

49

8

19.5

229

10

21

49

5

78.1

302

14

27

61

9

313

0

0

0

0

0

1250

0

0

0

0

0

5000

0

0

0

0

0

Positive control

 

AF-2

SAZ

AF-2

AF-2

ICR-191

 

535

331

81

436

1550

Positive control

 

BAP

2AA

2AA

BAP

BAP

 

835

299

1153

292

118

 

Table 2:

Metabolic activation system

Dose (µg/plate)

TA100

TA1535

WP2uvrA

TA98

TA1537

Without S9

DMSO

125±17.1

14±3.5

28±1.0

16±3.6

8±2.5

2.44

119±15.5

7±4.0

NT

NT

7±1.0

4.88

120±4.6

11±2.5

NT

NT

8±2.5

9.77

125±2.3

10±5.1

26±6.8

18±2.9

5±1.5

19.5

117±7.5

8±3.8

26±4.0

20±11.9

6±2.3

39.1

121±2.1

12±5.1

25±6.0

17±4.0

5±0.6

78.1

112±10.4

7±0.6

20±8.1

15±2.5

6±2.0

156

NT

NT

0±0.0

0±0.0

NT

313

NT

NT

0±0.0

0±0.0

NT

 

 

Table 3:

Metabolic activation system

Dose (µg/plate)

TA100

TA1535

WP2uvrA

TA98

TA1537

Without S9

DMSO

143±9.0

8±2.5

25±7.1

51±11.3

11±2.9

2.44

NT

13±2.1

NT

NT

14±3.0

4.88

NT

8±3.1

NT

NT

9±3.5

9.77

151±5.3

15±2.6

28±13.1

57±8.2

9±3.2

19.5

166±7.5

9±1.0

28±3.0

44±7.0

10±1.5

39.1

188±12.9

18±4.2

30±4.5

47±5.7

17±4.0

78.1

237±29.5

12±3.2

25±5.9

53±10.7

11±3.2

156

127±10.5

NT

29±6.2

31±2.9

NT

313

0 0.0

NT

0 0.0

0 0.0

NT

 

Table 4:

Metabolic activation system

Dose (µg/plate)

TA100

TA1535

WP2uvrA

TA98

TA1537

Without S9

DMSO

107±18.0

14±3.8

12±1.7

19±9.5

9±0.6

2.44

121±5.1

15±1.5

NT

NT

6±1.5

4.88

118±7.0

9±3.6

NT

NT

4±0.0

9.77

121±9.9

10±2.3

14±5.2

19±1.7

5±2.5

19.5

123±12.3

7±1.2

15±1.7

19±3.2

9±2.3

39.1

112±13.5

8±3.6

21±5.5

19±3.5

5±1.0

78.1

94±3.5

7±1.0

16±4.5

20±5.2

7±3.1

156

NT

NT

12±4.6

0±0.0

NT

313

NT

NT

0 0.0

0±0.0

NT

 

Table 5:

Metabolic activation system

Dose (µg/plate)

TA100

TA1535

WP2uvrA

TA98

TA1537

Without S9

DMSO

124±15.1

9±3.2

19±6.0

46±7.5

11±2.6

2.44

NT

9±2.0

NT

NT

9±3.5

4.88

NT

8±1.5

NT

NT

7±1.2

9.77

142±11.4

9±1.2

15±1.0

39±3.1

8±2.9

19.5

151±13.3

8±3.8

16±3.0

42±7.6

10±2.5

39.1

186±12.7

16±5.0

16±4.7

47±13.1

10±2.1

78.1

212±13.3

11±4.0

16±2.6

44±6.1

11±2.6

156

115±22.1

NT

17±3.2

27±4.6

NT

313

0±0.0

NT

0±0.0

0±0.0

NT

Conclusions:
The test chemical did not induce gene mutation in Salmonella typhimurium strains TA100, TA1535, TA98, TA1535 and E. coli WP2 uvrA in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed as per the OECD guideline 471 using Salmonella typhimurium strains TA100, TA1535, TA98, TA1535 and E. coli WP2 uvrA with and without of S9 metabolic activation system. The study was performed at various dose levels and in triplicate plates/dose. Concurrent solvent and positive control chemicals were incorporated in the study. The plates were observed for dose dependent increase in the number of revertnats/plate. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA100, TA1535, TA98, TA1535 and E. coli WP2 uvrA in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from J-check
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
other: CHL/IU
Details on mammalian cell type (if applicable):
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimal essential medium
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 14.5, 28.9, 57.8, 116, 231, 463, 925 or 1850 µg/mL

Short term treatment:
With S9: 0, 0.90, 1.81, 3.61, 7.23 or 14.5 µg/mL
Without S9: 0, 7.23, 14.5, 28.9, 57.8 or 116 µg/mL

24 hrs and 48 hrs treatment:
Without S9: 0, 14.5, 28.9, 57.8 or 116 µg/mL

Retest:
With S9: 0, 5.71, 8.56, 12.8, 19.3 or 28.9 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: Short term (6-18 hrs), 24 and 48 hrs
- Expression time (cells in growth medium): Short term (6-18 hrs), 24 and 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: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, cell growth inhibition caused by the test material was noted

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

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The slides were observed for chromatid and chromosome gaps, chromatid and chromosome breaks, chromatid and chromosome exchange and fragmentation
Statistics:
No data
Species / strain:
other: CHU/IU
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential

Table: Cell growth ratio in the cell growth inhibition test in cultured Chinese hamster cells treated with β- bromostyrene (Short term treatment- +S9)

Cell growth inhibition test

Study type

Treatment and conc. (µg/mL)

Cell growth ratio

Observations

S9

Time (hr)

Plate

Mean (%)b

Condition of cells

Color of medium

Preciptates/Crystals

1

2

+

6-18

0

100a

100

-

-

-

-

87

-

-

-

-

14.5

49

46

++

-

-

-

37

++

-

-

-

28.9

24

33

++

-

-

-

37

++

-

-

-

57.8

12

13

+++

-

-

-

12

+++

-

-

-

116

0

6

+++

-

-

-

12

+++

-

-

-

231

0

0

+++

-

-

-

0

+++

-

-

+

463

0

6

g

-

-

+

12

g

-

+

+

925

0

6

g

-

+

+

12

g

-

+

+

1850

12

13

g

-

+

+

12

g

-

+

+

Concentration of 50% cell growth inhibition: below 14.5µg/mL

Table: Cell growth ratio in the cell growth inhibition test in cultured Chinese hamster cells treated with β- bromostyrene (Short term treatment- -S9)

Cell growth inhibition test

Study type

Treatment and conc. (µg/mL)

Cell growth ratio

Observations

S9

Time (hr)

Plate

Mean (%)b

Condition of cells

Color of medium

Preciptates/Crystals

1

2

-

6-18

0

100a

100

-

-

-

-

100

-

-

-

-

14.5

71

71

+

-

-

-

71

+

-

-

-

28.9

57

57

++

-

-

-

57

++

-

-

-

57.8

71

57

++

-

-

-

42

++

-

-

-

116

14

14

++

-

-

-

14

++

-

-

-

231

14

7

+++

-

-

-

0

+++

-

-

+

463

0

0

g

-

+

+

0

g

-

+

+

925

0

0

g

-

+

+

0

g

-

+

+

1850

0

0

g

-

+

+

0

g

-

+

+

Concentration of 50% cell growth inhibition: belo0w 67.3µg/mL

Table: Cell growth ratio in the cell growth inhibition test in cultured Chinese hamster cells treated with β- bromostyrene (Continuous treatment: 24 hrs)

Cell growth inhibition test

Study type

Treatment and conc. (µg/mL)

Cell growth ratio

Observations

S9

Time (hr)

Plate

Mean (%)b

Condition of cells

Color of medium

Preciptates/Crystals

1

2

-

24

0

100a

100

-

-

-

-

100

-

-

-

-

14.5

59

59

+

-

-

-

59

+

-

-

-

28.9

59

59

+

-

-

-

59

+

-

-

-

57.8

59

59

+

-

-

-

59

+

-

-

-

116

0

0

+

-

-

-

0

+

-

-

-

231

0

0

TOX

-

-

-

0

TOX

-

-

+

463

0

0

g

-

+

+

0

g

-

+

+

925

0

0

g

-

+

+

0

g

-

+

+

1850

0

0

g

-

+

+

0

g

-

+

+

Concentration of 50% cell growth inhibition: below 66.7µg/mL

Table: Cell growth ratio in the cell growth inhibition test in cultured Chinese hamster cells treated with β- bromostyrene (Continuous treatment: 48 hrs)

Cell growth inhibition test

Study type

Treatment and conc. (µg/mL)

Cell growth ratio

Observations

S9

Time (hr)

Plate

Mean (%)b

Condition of cells

Color of medium

Preciptates/Crystals

1

2

-

48

0

100a

100

-

-

-

-

99

-

-

-

-

14.5

84

80

++

-

-

-

76

++

-

-

-

28.9

69

65

++

-

-

-

61

++

-

-

-

57.8

69

65

++

-

-

-

61

++

-

-

-

116

7

7

++

-

-

-

7

++

-

-

-

231

0

0

TOX

-

-

-

0

TOX

-

-

+

463

0

0

g

-

+

+

0

g

-

+

+

925

0

0

g

-

+

+

0

g

-

+

+

1850

0

0

g

-

+

+

0

g

-

+

+

Concentration of 50% cell growth inhibition: below 72.9µg/mL

Table: Cell growth ratio in the chromosome aberration test in cultured Chinese hamster cells treated with β- bromostyrene (Short term treatment: +S9)

Cell growth inhibition test

Study type

Treatment and conc. (µg/mL)

Cell growth ratio

Observations

S9

Time (hr)

Plate

Mean (%)b

Condition of cells

Color of medium

Preciptates/Crystals

1

2

-

6-18

0

100a

100

-

-

-

-

100

-

-

-

-

0.90

100

93

-

-

-

-

85

-

-

-

-

1.81

100

100

-

-

-

-

100

-

-

-

-

3.61

100

93

-

-

-

-

85

-

-

-

-

7.23

85

85

-

-

-

-

85

-

-

-

-

14.5

71

73

+

-

-

-

85

+

-

-

-

 

 

PC

100

100

-

-

-

-

100

-

-

-

-

a: the plate in the negative control was regarded as 100% growth

b: the mean showed as a growth ration against the negative control value

c: Condition of cells was observed at the end of the treatment. Color of medium was observed immediately after the addition of test solution, precipitates and crystals were observed immediately after the addition of test solution1and at the end of treatment2

d:         -: most of the cells were attached to the surface of the plates and their shape was normal

+: there was discontinuity amons a small number of surviving cells

++: there was discontinuity among approximately half of the surviving cells

+++: there was discontinuity among most of the surviving cells

e:          - : No changes of color

f:           - : Absence of precipitates /crystals

+: presence of precipitated

g: condition of cells could not be observed due to sever precipitates of the test article

Conclusions:
The test chemical did not induce chromosomal aberrations in CHL/IU cell line in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.
Executive summary:

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHL/IU cell line. The duration of treatment was short term from 6-18 hrs and continuous treatment for 24 and 14 hrs. The doses were dissolved in DMSO and the dose levels were Short term treatment (With S9: 0, 0.90, 1.81, 3.61, 7.23 or 14.5µg/mL, Without S9: 0, 7.23, 14.5, 28.9, 57.8 or 116µg/mL), for 24 hrs and 48 hrs treatment (Without S9: 0, 14.5, 28.9, 57.8 or 116µg/mL) and retest dose level was 0, 5.71, 8.56, 12.8, 19.3 or 28.9µg/mL with S9. Concurrent solvent and vehicle control chemicals were included in the study. The test chemical did not induce chromosomal aberrations in CHL/IU cell line in the presence and absence of S9 metabolic activation system and hence 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 for the target chemical was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed as per the OECD guideline 471 using Salmonella typhimurium strains TA100, TA1535, TA98, TA1535 and E. coli WP2 uvrA with and without of S9 metabolic activation system. The study was performed at various dose levels and in triplicate plates/dose. Concurrent solvent and positive control chemicals were incorporated in the study. The plates were observed for dose dependent increase in the number of revertnats/plate. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA100, TA1535, TA98, TA1535 and E. coli WP2 uvrA in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using CHL/IU cell line. The duration of treatment was short term from 6-18 hrs and continuous treatment for 24 and 14 hrs. The doses were dissolved in DMSO and the dose levels were Short term treatment (With S9: 0, 0.90, 1.81, 3.61, 7.23 or 14.5µg/mL, Without S9: 0, 7.23, 14.5, 28.9, 57.8 or 116µg/mL), for 24 hrs and 48 hrs treatment (Without S9: 0, 14.5, 28.9, 57.8 or 116µg/mL) and retest dose level was 0, 5.71, 8.56, 12.8, 19.3 or 28.9µg/mL with S9. Concurrent solvent and vehicle control chemicals were included in the study. The test chemical did not induce chromosomal aberrations in CHL/IU cell line in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the data available for the target chemical, it does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification