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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key study. Method according to OECD 471 and Guidelines for Screening Mutagenicity Testing Of Chemicals (Japan), GLP study. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Therefore, the test item was not mutagenic under test conditions.

Key study. Method according to OECD 473 and Guidelines for Screening Mutagenicity Testing Of Chemicals (Japan), GLP study.

No chromosome structural abnormality or inducing effect of polyploid cells was observed in any of the tests at any concentration, with or without metabolic activation. Therefore, the test item was not mutagenic under test conditions.

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:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-requiring gene in four strains of S. typhimurium, and tryptophan requiring gene in E. coli WP2.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: National Institute of Public Health (obtained December 19, 1994)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (PB) and 5,6-benzoflavone (BF) induced rat liver S9.
Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 100 - 5000 μg/plate; no bacterial toxicity up to highest dose level.
- Main tests 1 and 2: 0, 312.5, 625, 1250, 2500 and 5000 μg / plate.
- Justification: In order to determine the appropriate concentration of the test substance in this test, tests were conducted by the same experimental method as this test using six concentrations of 100, 200, 500, 1000, 2000 and 5000 μg/plate for all indicator strains. The test was performed on one plate of each concentration. As a result, regardless of the presence or absence of metabolic activation, no growth inhibition was observed at any concentration in any strain.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was reported to be soluble in DMSO, slightly soluble in water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2-aminoanthracene (2-AA)
Remarks:
see table 4 in 'Any other information on materials and methods'
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Plate incorporation method, with and without metabolic activation. 0.1 mL of the solvent, test item solution or positive control substance solution to were put in a test tube; then, 0.5 ml of phosphate buffer solution (or 0.5 ml of S9 mixture solution in metabolic activation test) and 0.1 ml of assay bacterial solution were mixed in, and cultured with shaking at 37ºC for 20 minutes. Next, a mixture containing 2 ml of top agar (kept at 45ºC) was overlaid on the plate. After solidification, plates were incubated at 37ºC for 48 hours. After the incubation period, the number of revertant colonies was counted, and at the same time, the presence or absence of growth inhibition of the indicator strain was observed using a stereomicroscope.
- Cell density at seeding (if applicable): see table 3.

DURATION
- Preincubation period: no.
- Exposure duration: 48h

SELECTION AGENT (mutation assays): No specific selection agent was used. The lack of amino-acid in the medium allowed only mutants to grow because of their ability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.
Evaluation criteria:
The results were judged as positive if the following three criteria were satisfied based on the average number of revertant colonies on the plate at each concentration.
1) In the test substance treatment group, the number of revertant colonies was increased more than twice the solvent control value.
2) The number of revertant colonies increases with increasing test substance concentration (concentration dependence).
3) The repeatability of the increase in the number of revertant colonies is observed from the results of the two tests.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: none reported

RANGE-FINDING/SCREENING STUDIES: in the preliminary test, performed in the range of 100 to 5000 μg/plate, no growth inhibition by the test substance was observed in any of the strains regardless of the presence or absence of metabolic activation. Therefore, the top dose for the main tests was 5000 μg/plate, and the next doses were 2500, 1250, 625 and 312.5 μg/plate (common ratio 2).

HISTORICAL CONTROL DATA: not specified.

Table 1. Results of reverse mutation test of o-TSA on bacteria (1st trial) without metabolic activation (direct method).

Test substance

concentration

(µg/plate)

Number of revertant colonies per plate (Mean ± S.D.)

TA100

TA1535

WP2 uvrA

TA98

TA1537

0

124

136

126

8

8

7

10

17

7

16

19

24

9

11

14

(129 ± 6)

(8 ± 1)

(11 ± 5)

(20 ± 4)

(11 ± 3)

312.5

116

127

145

7

8

18

15

19

13

23

15

26

12

13

8

(129 ± 15)

(11 ± 6)

(16 ± 3)

(21 ± 6)

(11 ± 3)

625

144

138

105

9

6

9

10

13

14

21

22

24

10

3

10

(129 ± 21)

(8 ± 2)

(12 ± 2)

(22 ± 7)

(11 ± 2)

1250

137

121

103

10

5

10

18

16

6

13

17

19

7

10

7

(120 ± 17)

(8 ± 3)

(13 ± 6)

(16 ± 3)

(8 ± 2)

2500

114

125

101

11

7

7

12

14

18

21

21

22

5

9

8

(113 ± 12)

(8 ± 2)

(15 ± 3)

(21 ± 1)

(7 ± 2)

5000

119

111

130

7

8

7

15

14

10

17

22

15

14

7

11

(120 ± 10)

(7 ± 1)

(13 ± 3)

(18 ± 4)

(11 ± 4)

Posit.

control

803

847

873

340

313

321

1125

1177

1106

292

298

308

978

906

641

(841 ± 35)

(325 ± 14)

(1136 ± 37)

(299 ± 8)

(842 ± 177)

Table 2. Results of reverse mutation test of o-TSA on bacteria (1st trial) with metabolic activation (S9 mix).

Test substance

concentration

(µg/plate)

Number of revertant colonies per plate (Mean ± S.D.)

TA100

TA1535

WP2 uvrA

TA98

TA1537

0

120

152

123

8

11

8

11

20

25

32

46

37

13

10

13

(132 ± 18)

(0 ± 2)

(19 ± 7)

(38 ± 7)

(12 ± 2)

312.5

154

128

138

11

10

10

15

8

19

37

34

37

15

8

0

(140 ± 13)

(10 ± 1)

(14 ± 6)

(36 ± 2)

(11 ± 4)

625

136

129

142

13

11

11

14

17

21

51

45

44

14

18

0

(145 ± 11)

(9 ± 2)

(13 ± 2)

(41 ± 7)

(13 ± 4)

1250

157

135

143

8

11

8

15

11

13

35

49

38

11

10

17

(132 ± 11)

(11 ± 4)

(17 ± 5)

(37 ± 7)

(16 ± 4)

2500

139

137

119

8

15

11

21

17

38

30

44

 

16

13

20

(132 ± 11)

(11 ± 4)

(17 ± 5)

(37 ± 7)

(16 ± 4)

5000

120

130

126

16

12

9

16

18

12

36

43

37

18

14

19

(125 ± 5)

(12 ± 4)

(15 ± 3)

(39 ± 4)

(17 ± 3)

Posit.

control

548

589

604

127

128

120

331

1251

1307

324

337

316

108

94

105

(580 ± 29)

(125 ± 4)

(1297 ± 42)

(326 ± 11)

(102 ± 7)

Table 3. Results of reverse mutation test of o-TSA on bacteria (2nd trial) without metabolic activation (direct method).

Test substance

concentration

(µg/plate)

 

Number of revertant colonies per plate (Mean ± S.D.)

 

TA100

TA1535

WP2 uvrA

TA9S

TA1537

0

117

134

114

10

10

11

9

13

11

16

20

22

7

6

3

(122 ± 11)

(10 ± 1)

(11 ± 2)

(19 ± 3)

( 5 ± 2)

312.5

139

119

139

6

10

9

23

14

14

31

20

28

5

6

3

(132 ± 12)

( S ± 2)

(17 ± 5)

(26 ± 6)

( 5 ± 2)

625

107

121

126

6

7

7

14

10

11

27

14

19

7

8

3

(118 ± 10)

( 7 ± 1)

(12 ± 2)

(20 ± 7)

( 6 ± 3)

1250

105

107

103

10

6

6

15

S

9

13

20

19

7

5

7

(105 ± 2)

( 7 ± 2)

(11 ± 4)

(17 ± 4)

( 6 ± 1)

2500

125

93

100

12

4

12

19

12

14

20

19

14

3

6

12

(103 ± 17)

( 9 ± 5)

(15 ± 4)

(18 ± 3)

( 7 ± 5)

5000

102

91

101

7

7

6

15

12

16

17

16

19

5

8

5

( 99 ± 4)

( 7 ± 1)

(14 ± 2)

(17 ± 2)

( 6 ± 2)

Positive

control

797

793

795

301

311

293

961

1109

993

264

262

305

606

654

610

(795 ± 2)

(303 ± 9)

(1028 ± 71)

(277 ± 2-1)

(621 ± 26)

Table 4. Results of reverse mutation test of o-TSA on bacteria (2nd trial) with metabolic activation (S9 mix).

Test substance

concentration

(µg/plate)

 

Number of revertant colonies per plate (Mean ± S.D.)

 

TA100

TA1535

WP2 uvrA

TA9S

TA1537

0

154

144

136

8

6

15

20

20

25

35

42

39

11

13

9

(145 ± 9)

(10 ± 5)

(22 ± 3)

(39 ± 4)

(11 ± 2)

312.5

165

157

156

7

11

10

22

20

13

31

41

41

6

7

9

(159 ± 5)

(9 ± 2)

(18 ± 5)

(38 ± 6)

(7 ± 2)

625

157

155

144

13

9

18

12

23

11

37

37

41

13

10

8

(152 ± 7)

(13 ± 5)

(15 ± 7)

(38 ± 2)

(10 ± 3)

1250

158

131

120

12

7

13

23

16

10

42

30

43

9

12

10

(136 ± 20)

(11 ± 3)

(16 ± 7)

(38 ± 7)

(10 ± 2)

2500

122

111

123

11

7

7

17

18

11

46

39

43

12

7

17

(119 ± 7)

(8 ± 2)

(15 ± 4)

(43 ± 3)

(12 ± 5)

5000

134

125

110

8

8

14

10

13

18

38

27

32

15

10

8

( 123 ± 12)

(10 ± 3)

(14 ± 4)

(32 ± 6)

(11 ± 4)

Positive

control

504

499

472

127

160

132

1207

1170

1290

357

373

341

106

136

101

(492 ± 17)

(140 ± 18)

(1222 ± 61)

(357 ± 16)

(114 ± 19)

 

Conclusions:
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Therefore, the test item was not mutagenic under test conditions.
Executive summary:

The ability of the test item to induce mutation was studied in a bacterial reverse mutation test (Ames test) according to OECD Guidelines No. 471 and the Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) (GLP study). Four histidine requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and tryptophan requiring Escherichia coli WP2 uvrA were tested in triplicate at six concentrations of test item in up to 5000 μg/plate, with and without metabolic activation (S9 mix), based on the results of a preliminary range-finding test. The plate incorporation method was used. Vehicle (DMSO) and positive controls were run in parallel. No cytotoxicity was observed at any dose tested, the vehicle control plates gave counts of revertant colonies within the normal range, and all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. All validity criteria were met. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Therefore, the test item was not mutagenic under test conditions.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: CHL cells (Chinese Hamster lung-derived fibroblast cells)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: National Institute of Health Sciences (NIHS), National Institute of Health Food Research Institute, originating from National Institute of Health Research Institute, mutagenicity unit, January 13, 1985)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle MEM culture medium (Gibco Laboratories, lot No. 73K2362, 1003745) supplemented with 10% fetal bovine serum (Gibco Laboratories, lot No. 1002839, 1004615)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver, induced with phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
To determine the treatment concentration used in the chromosomal aberration test, a preliminary cell growth inhibition test was performed in the range of 93.75 - 3000 μg/mL for both the continuous treatment method and the short treatment method, and the dose capable of inhibiting 50% of growth was selected as the maximum dose. With the continuous method, inhibition >50% was observed at 3000 μg/mL both at 24 hours and 48 hours treatment, and in the short treatment method, 50% inhibition was not observed at any concentration with or without S9 mix.
Based on the preliminary test, the concentration in the chromosomal aberration test is 375, 750, 1500, 2250 and 3000 μg/mL for 24 hours and 48 hours for the continuous treatment method, and 375, 750. 1500 and 3000 μg/mL for the absence and presence of S9 mix for the short treatment method.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (Wako Pure Chemical Industries, Ltd., Lot No. TPK 7807, TPR7212, purity 99.9%)
- Justification for choice of solvent/vehicle: DMSO was used as a solvent because the test substance was soluble in it, while only slightly soluble in water.
Untreated negative controls:
yes
Remarks:
untreated control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: N'-methyl-N-nitro-N'-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 4 × 10^3 CHL cells were seeded in a Petri dish (diameter 6 cm, Becton Dickinson Co.) containing 5 ml of the culture solution and cultured in a 37°C incubator (5% CO 2).
- Direct method / continuous treatment method (24 and 48h): cells were cultured for 3 days, then, the culture solution was discarded, and culture broth with the test item was added, and cultured for 24/48h.
- Metabolic activation method / short treatment method (6h treatment, 18h post-exposure): cells were cultured for 3 days, then, the culture solution was discarded, and exposed to medium with test item for 6h, in the presence and absence of S9 mix. After processing, fresh culture medium was added and further cultured for 18h. Two petri dishes were used for each concentration.

DURATION
- Preincubation period: 3 days
- Exposure duration: for the direct method (long treatment assay): 24 and 48 hours; for the metabolic activation method (short treatment assay, with and without metabolic activation): 6h.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Two hours before the end of the culture, colcemid (Gibco laboratories) was added to a final concentration of 0.2 μg/ml. The cells were detached by trypsinization and harvested by centrifugation. After hypotonic treatment with an aqueous solution of 75 mM potassium chloride, cells were fixed with a mixture of cold methanol / acetic acid (3: 1) prepared at the time of use. After preparing chromosome specimens by air drying, they were stained with 1.4 vol% Giemsa solution in Sørensen buffer (pH 6.8, Jatron, lot No. 1478) for about 15 minutes. Three slide specimens were prepared for each dish.

NUMBER OF CELLS EVALUATED: 200

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 metaphase images of 100 dishes per dish, i.e. 2 dishes per concentration, were observed under a microscope with a total magnification of 600 ×. All specimens were coded and observed blindly. Chromosome analysis is carried out based on classification method by the Japan Society for Environmental Mutagen Society / Mammalian Testing Subcommittee (MMS), and structural abnormality such as chromosomal type or chromatid type gap, truncation, exchange and polyploid cells.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
According to the criteria of Ishidate et al (1987), it was considered that :
- the test substance induced chromosomal abnormality if at least 10 % of cells presented chromosomal aberrations (positive result)
- the test substance did not induce chromosomal aberrations if less than 5 % presented chromosomal aberrations (negative result)
Results between 5 to 10% were considered as false positives.
Key result
Species / strain:
other: Chinese Hamster cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the highest dose caused 50% inhibition.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
To determine the concentrations to be used in the chromosomal aberration test, the inhibitory effect of the test substance on the proliferation of CHL cells was measured. The proliferation degree was measured using a monolayer cultured cell densitometer (Monocellater -Olympus). The ratio between the degree of proliferation for each concentration of the test substance and the degree of proliferation in the control group (only solvent) was determined. In the continuous method, inhibition >50% was observed at 3000 μg/mL both at 24 hours and 48 hours treatment, and in the short treatment method, 50% inhibition was not observed at any concentration with or without S9 mix. Based on the preliminary test, the concentration in the chromosomal aberration test is 375, 750, 1500, 2250 and 3000 μg/mL for 24 hours and 48 hours for the continuous treatment method, and 375, 750. 1500 and 3000 μg/mL for the absence and presence of S9 mix for the short treatment method.

TEST-SPECIFIC CONFOUNDING FACTORS: none reported.

SUMMARY OF RESULTS:
- In any concentration group treated with o-toluenesulfonamide for 24 hours and 48 hours, no structural abnormality of chromosome and induction of ploidy cells were observed. At 2250 μg/mL and above, no observable metaphase image was observed due to toxicity to cells.
- No structural abnormality of chromosome and induction of ploidy cells were observed in any concentration group treated for 6 hours in the absence or presence of S9 mix.

Table 1 – Chromosome analysis of Chinese hamster cells (CHL) continuously treated with o-TSA without S9 mix.

 

Group

 

Concen-

tration

(µg/mL)

Time of exposure (hr)

No. of cells analysed

No. of structural aberrations

No. of cells with aberrations

Polyploid2)

(%)

Judgement3)

gap

ctb

cte

csb

cse

oth

total

-g (%)

+g (%)

SA

NA

Solvent1)

0

24

200

2

1

1

0

0

0

4

2(1.0)

4(2.0)

0.5

-

-

OTSA

375

24

200

1

1

1

0

0

0

3

2(1.0)

3(1.5)

0

-

-

 

750

24

200

1

0

1

0

0

0

2

1(0.5)

2(1.0)

0

-

-

 

1500

24

200

1

0

4

0

0

0

5

4(2.0)

5(2.5)

0.5

-

-

 

2250

24

Toxic

 

 

 

 

 

 

 

 

 

 

 

 

 

3000

24

Toxic

 

 

 

 

 

 

 

 

 

 

 

 

MNNG

2.5

24

200

6

56

198

4

2

0

266

198(99.0)

198(99.0)**

0

+

-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Solvent

0

48

200

0

0

1

1

0

0

2

2(1.0)

2(1.0)

0

-

-

OTSA

375

48

200

0

0

1

0

0

0

1

1(0.5)

1(0.5)

0

-

-

 

750

48

200

0

0

1

0

0

0

1

1(0.5)

1(0.5)

0.5

-

-

 

1500

48

200

1

2

3

0

0

0

6

4(2.0)

5(2.5)

0

-

-

 

2250

48

Toxic

 

 

 

 

 

 

 

 

 

 

 

 

 

3000

48

Toxic

 

 

 

 

 

 

 

 

 

 

 

 

MNNG

2.5

48

200

6

57

182

14

5

0

264

191(95.5)

191(95.5)**

0.5

+

-

Abbreviations: gap: chromatid gap and chromosome gap, ctb: chromatic break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring), oth: others. -g: total no. of cell with aberrations except gap, +g: total no. of cells with aberrations, SA: structural aberration, NA: numerical aberration. OTSA: o-toluenesulfonamide, MNNG: 1-methyl-3-nitro-1-nitrosoguanidine.

1) Dimenthyl sulfoxide was used as solvent. 2) Two hundred cells were analysed in each group. 3) Multi-sample χ2 test was done at p<0.05.

**: Significantly different from solvent group data at p<0.01 by Ficher’s exact test.

 

Table 2 – Chromosome analysis of Chinese hamster cells (CHL) treated with o-TSA with and without S9 mix

 

Group

 

Concen-

tration

(µg/mL)

 

S9

mix

Time of exposure (hr)

No. of cells analysed

No. of structural aberrations

No. of cells with aberrations

Polyploid2)

(%)

Judgement3)

gap

ctb

cte

csb

cse

oth

total

-g (%)

+g (%)

 

SA

NA

Solvent1)

0

-

6-(18)

200

2

0

2

0

0

0

4

2(1.0)

4(2.0)

0

-

-

OTSA

375

-

6-(18)

200

0

2

0

0

0

0

2

2(1.0)

2(1.0)

0

-

-

 

750

-

6-(18)

200

0

0

1

0

0

0

1

1(0.5)

1(0.5)

0.5

-

-

 

1500

-

6-(18)

200

0

0

0

0

0

0

0

0(0.0)

0(0.0)

0

-

-

 

3000

-

6-(18)

200

0

2

3

1

0

0

6

4(2.0)

4(2.0)

0

 

 

BP

10

-

6-(18)

200

0

1

2

1

0

0

4

3(1.5)

3(1.5)

0

-

-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Solvent

0

+

6-(18)

200

0

0

0

0

1

0

1

1(0.5)

1(0.5)

1.5

-

-

OTSA

375

+

6-(18)

200

0

0

1

0

0

0

1

1(0.5)

1(0.5)

0

-

-

 

750

+

6-(18)

200

0

0

1

1

1

0

3

2(1.0)

2(1.0)

0.5

-

-

 

1500

+

6-(18)

200

0

1

3

0

0

0

4

3(1.5)

3(1.5)

0

-

-

 

3000

+

6-(18)

200

1

0

0

0

0

0

1

0(0.0)

1(0.5)

0

 

 

BP

10

+

6-(18)

200

6

29

129

2

1

0

167

133(66.5)

136(68.0)**

0.5

+

-

Abbreviations: gap: chromatid gap and chromosome gap, ctb: chromatic break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring), oth: others. -g: total no. of cell with aberrations except gap, +g: total no. of cells with aberrations, SA: structural aberration, NA: numerical aberration. OTSA: o-toluenesulfonamide, BP: benzo(a)pyrene.

1) Dimenthyl sulfoxide was used as solvent. 2) Two hundred cells were analysed in each group. 3) Multi-sample χ2 test was done at p<0.05 and then Ficher’s exact test was done at p<0.05 or P<0.01. **: Significantly different from solvent group data at p<0.01 by Ficher’s exact test.

Conclusions:
The test item did not induce chromosomal abnormalities in CHL cells under the test conditions. Therefore, it is not mutagenic.
Executive summary:

The ability of the test item to induce chromosomal aberrations in cultured Chinese hamster cells (CHL) was examined according to the Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD Guideline 473 (GLP study). Two different types of assays were carried out: a long-term treatment (direct method) of 24 and 48 hours of continuous treatment with the test substance and a short-term treatment consisting of culture for 18 hours after treatment for 6 hours (metabolic activation method), with and without metabolic activation (phenobarbital and 5,6-benzoflavone induced rat liver S9 mix). Two hours before the end of the culture, Colcemid was added and metaphase cells were collected, fixed and stained with Giemsa. For structural abnormalities, 200 cells were analyzed per group. Negative, solvent, and positive controls were included. The maximum dose for each assay was selected based on a cytotoxicity preliminary study: the dose capable of inhibiting 50% growth was selected as the maximum dose (3000 µg/ml). No chromosome structural abnormality or inducing effect of polyploid cells was observed in any of the tests at any concentration. Therefore, the test item is not mutagenic under test conditions.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available data (negative results in vitro, in OECD 471 and 473), the test item is not classified for genetic toxicity according to CLP, Regulation (EC) No. 1272/2008.