Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537, and E. coli WP2 uvr A pKM 101 with or without addition of S9 mix (reference 7.6.1 -1). Furthermore, the substance is non-mutagenic in mammalian cells under conditions where the positive controls exerted potent mutagenic effects (reference 7.6.1-2).

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:
July 26, 2000 - January 10, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
29.Dec.1992
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
September 11, 1989
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
All these strains contain mutations in the histidine operon, thus imposing a requirement for histidine in the growth medium.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate
2nd series: 5.00, 15.8, 50.0, 158 and 500 µg per plate

The test material concentrations used were selected according to the EEC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (test item and positive controls N-Ethyl-N'-nitro-N-nitrosoguanidine, cumene hydroperoxide, 2-Aminoanthracene, Benzo[a] pyrene) and Ethanol for positive control 9-Aminoanthracene

- Justification for choice of solvent/vehicle: Preferentially distilled water or dimethyl sulfoxide (DMSO), alternatively acetone or ethanol, are used as solvents. Analysis of the historical data of the laboratory and experience of other research groups (Maron et al. 1981) showed that the amounts of the selected solvents used have no influence on the number of spontaneous revertants of any strain. For this reason only the respective solvent control was used as the negative control in this study.

- Justification for percentage of solvent in the final culture medium: Since on the one hand organic solvents may have diverse effects on e.g. gene regulation and, on the other hand, high amounts of water (added as the solvent) will dilute the top agar, usually the maximum amount of solvent is limited to 100 µl per plate for water and 10 µL per plate for DMSO, ethanol, acetone or other organic solvents. Only the highest test material concentration may be plated with either 316 µL water or 31.6 µL organic solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Daunomycin
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
3 parallel plates were used for each concentration step of the test material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Incubation of plates was performed at + 37 °C for 2 to 3 days.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
Evaluation criteria:
A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate in the 1st test series.

STUDY RESULTS :
- Signs of toxicity :
toxicity to the bacteria was observed at concentrations > 500 or 1580 µg/plate depending upon the experimental conditions used.
- Mean number of revertant colonies per plate and standard deviation : see tables 1-4

Table 1: Results / Series No.: 1

Positive Controls: Individual and Mean Number of Revertant Colonies         

Strain

Positive Control

Concentr. [µg/plate]

+ /-
S9-Mix

#

Mean revertant colonies per plate

SD*

Individual revertant colonies per plate

TA 98

DAUN

2

-

643

196

869

538

521

TA 100

ENNG

5

-

904

41

866

947

899

TA 102

CUM

67

-

396

37

421

414

354

TA 1535

ENNG

10

-

332

12

329

321

345

TA 1537

9-AA

50

-

1683

331

1727

1990

1333

WP2

ENNG

5

-

1327

83

1264

1296

1421

 

 

 

 

 

 

 

 

 

TA 98

2-AA

1

+

322

19

338

301

326

TA 100

2-AA

1

+

537

25

564

532

514

TA 102

B (a) p

10

+

806

3

804

805

809

TA 1535

2-AA

1

+

151

17

152

134

167

TA 1537

2-AA

2

+

92

34

95

124

56

WP2

2-AA

10

+

1459

IB

1477

1441

1459

 

#        Test material incubated in the presence

(+)       or absence

(-)        of S9-Mix

*          Standard deviation

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

 

Table 2: Results/ Series No.: 2

Positive Controls: Individual and Mean Number of Revertant Colonies             

Strain

Positive Control

Concentr. [µg/plate]

+ /-
S9-Mix

#

Mean revertant colonies per plate

SD*

Individual revertant colonies per plate

TA 98

DAUN

2

-

93

18

73

97

108

TA 100

ENNG

5

-

520

39

565

500

494

TA 102

CUM

150

-

661

72

703

702

578

TA 1535

ENNG

10

-

187

11

194

192

174

TA 1537

9-AA

50

-

186

40

144

191

224

WP2

ENNG

5

-

1103

12

1117

1096

1097

 

 

 

 

 

 

 

 

 

TA 98

2-AA

10

+

329

150

434

396

158

TA 100

2-AA

10

+

333

30

302

336

361

TA 102

B (a) p

10

+

775

120

790

887

649

TA 1535

2-AA

2

+

94

11

107

87

649

TA 1537

2-AA

20

+

42

10

17

49

30

WP2

2-AA

10

+

933

81

861

1020

918

 

#        Test material incubated in the presence

(+)       or absence

(-)        of S9-Mix

*          Standard deviation

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

 

Table 3: Results / Series No.: 1

Summary of the Mean Number of Revertant Colonies              

Test material

Concentr. [µg/plate]

+ /-
S9-Mix

 

Mean revertant colonies / plate

TA 98

TA 100

TA 102

TA 1535

TA 1537

WP2

Solvent control

Test item

 

-

14

97

269

12

4

128

5

-

14

91

264

12

3

119

15.8

-

17

96

268

12

6

127

50

-

15

99

249

15

4

122

158

-

16

112

242

12

7

114

500 PE

-

8

73

189

11

2

53

1580 PE

-

0

3

2

2

0

2

5000 PE

-

0

3

0

2

0

2

 

 

 

 

 

 

 

 

 

Solvent control

Test item

 

+

20

120

271

13

7

170

5

+

25

121

277

20

9

164

15.8

+

27

132

288

14

8

180

50

+

26

142

278

13

9

167

158

+

27

143

342

17

8

130

500 PE

+

22

130

214

12

5

81

1580 PE

+

2

4

2

3

0

3

5000 PE

+

0

5

0

3

0

5

 

 

 

 

 

 

 

 

 

Postive controls

Name

-

DAUN

ENNG

CUM

ENNG

9-AA

ENNG

Conc (µg/plate)

2

5

67

10

50

5

Revert. / plate

343

904

396

332

1683

1327

 

 

 

 

 

 

 

 

Name

+

2-AA

2-AA

B (a) p

2-AA

2-AA

2-AA

Conc (µg/plate)

1

1

10

1

2

10

Revert. / plate

322

537

806

151

92

1459

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

9-AA    9-Aminoacridine

Table 4 Results / Series No.: 2

Summary of the Mean Number of Revertant Colonies              

Test material

Concentr. [µg/plate]

+ /-
S9-Mix

 

Mean revertant colonies / plate

TA 98

TA 100

TA 102

TA 1535

TA 1537

WP2

Solvent control

Test item

 

-

15

93

218

11

5

130

5

-

13

87

219

7

7

130

15.8

-

12

92

220

11

8

127

50

-

13

88

222

12

4

122

158

-

16

88

221

15

7

119

500

-

11

79

206

11

6

91

 

 

 

 

 

 

 

 

 

Solvent control

Test item

 

+

25

140

275

18

6

134

5

+

22

139

254

17

5

133

15.8

+

20

153

226

20

7

151

50

+

20

156

259

16

3

144

158

+

26

176

266

18

6

128

500

+

30

153

261

12

6

115

 

 

 

 

 

 

 

 

 

Postive controls

Name

-

DAUN

ENNG

CUM

ENNG

9-AA

ENNG

Conc (µg/plate)

2

5

150

10

50

5

Revert. / plate

93

520

661

187

186

1327

 

 

 

 

 

 

 

 

Name

+

B (a) p

B (a) p

B (a) p

2-AA

B (a) p

2-AA

Conc (µg/plate)

10

10

10

2

20

10

Revert. / plate

329

333

775

94

42

1459

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

B (a) p Benzo(a)pyrene

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 03, 2004 - January 26, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) (migrated information)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
8. June 2000
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: L5178Y TK(+/-) mouse lymphoma cells

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: For the different experimental investigations, the cells were thawed rapidly, diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air.
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Exposure medium:
RPMI- 5 (RPMI 1640 with 5% horse serum) 470 mL RPMI 1640 25 mL horse serum (heat-inactivated) 5 mL penicillin/streptomycin
- Culture medium:
RPMI-10 (RPMI 1640 with 10% heat-inactivated horse serum) 445 mL RPMI 1640 50 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin
- Survivor- and selection medium:
RPMI-20 (RPMI 1640 with 20% heat-inactivated horse serum) 395 mL RPMI 1640
100 mL horse serum {heat-inactivated horse serum) 5 mL penicillin/streptomycin
Metabolic activation:
with and without
Metabolic activation system:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
without S9-Mix:
5.00, 15.8, 50.0, 158, 281 and 500 µg/ mL medium
with S9-Mix:
1.58, 5.00, 15.8,50.0, 158 and 500 µg / mL medium

The concentrations selected were modified and adapted in order to achieve a toxicity level recommended by OECD guideline 476. A dose-range finding test was performed to detect adequate concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Preferentially water (maximally 1%) or DMSO, acetone or ethanol (maximally 0.1%), are used as solvents.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration
Each treatment, in the absence or presence of S9 mix, was performed in duplicate (single cultures only used for positive control treatments).

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10E7 cells in RPMI 5 medium
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours in the presence and 24 hours (1st series) or 3 hours (2nd series) in the absence of S9 mix at 37°C.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Cultures were maintained in flasks for 2 days (until day 3 of the experiment)
- Selective agent: At the end of the expression period (day 3), the cell densities in the selected cultures were adjusted to 1 x 10E4/mL. TFT (300 µg/mL) was diluted 100-fold into these suspensions to give a final concentration of 3 µg/mL. Plates were incubated until scorable (day 10 to day 14)
- Criteria for small (slow growing) and large (fast growing) colonies: At least for the negative and positive controls and, in case of a positive test material-induced effect, also for those cultures that showed the highest test material-induced effect, the mutation frequency is determined separately for small and large colonies in addition to the total mutation frequency.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)
Evaluation criteria:
The effects of the test material upon the mutation frequency are defined as
• "No effect" or "no increase" in the mutation frequency if the mean frequency of the parallel incubations of a given test material concentration is less than 2.0-fold above the mean of the actual negative controls or the mean mutation falls within the historical range of the negative controls.
• "Clear effect" or "clear increase" in the mutation frequency if the test material induces at least a 3.0-fold increase above the mean of the actual negative controls and the mean mutation frequency for a given test material concentration is at least 1.5-fold above the highest value of the historical negative controls.
• All other results are defined as a "weak effect" or a "weak increase" of the mutation frequency.

Test materials are assessed as negative or non-mutagenic in this test system if
• the assay is considered valid and
• no effect (no increase in the mutation frequency) occurs in the two experimental series performed or
• a weak effect (weak increase) occurs in one series and no effect (no increase) in the other series of experiments.
Test materials are assessed as positive or mutagenic in this test system if
• the assay is considered valid and
• a clear effect (clear increase in the mutation frequency) occurs at similar concentrations of the test material in the two experimental series performed, or
• a clear effect (clear increase) occurs in one series and a weak effect (weak increase) in the other series of experiments at identical concentrations, or
• weak effects (weak increases) occur dose-dependently (over at least two test material concentrations) and reproducibly at identical concentrations in the two experimental series performed.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The test material did not lead to a relevant fluctuation in the pH of the cell culture medium.
- Data on osmolality: The test material did not lead to a relevant fluctuation in the osmolarity of the cell culture medium.
- Precipitation and time of the determination: No precipitation of the test material in the cell culture medium occurred

RANGE-FINDING/SCREENING STUDIES
In a preceding range finding test, the relative survival was determined after exposure to various test material concentrations ranging between 5.00 and 5000 µg/mL. A reduction in the relative survival of the cells occurred at concentrations > 158 or 1580 in the absence of S9 mix and 15.8 µg/mL in the presence of S9 mix, respectively.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: Toxicity to the cells was observed at concentrations > 5.00 or 500 µg/mL, depending upon the experimental condition.

- Genotoxicity results: see table 1

HISTORICAL CONTROL DATA : see table 2

Table 1: Summary of results

 

Without S9 mix (EZ.1571 / 1581)

1stExperimental series

Test material

[µg/mL]

RSa

(%)

RTGb

(%)

MFc

Solvent

0

100

100

121

 

 

 

 

 

Test item

5.00

91.1

121

173

15.8

83.5

111

151

50.0

71.1

50.2

137

158

1.37

0.0896

144

281

0

-

-

500

0

-

-

 

 

 

 

 

NQO

0.10

42.7

118

1050

0.20

21.1

35.2

1140

 

2ndExperimental series

Test material

[µg/mL]

RSa

(%)

RTGb

(%)

MFc

Solvent

0

100

100

89.6

 

 

 

 

 

Test item

15.8

89.8

100

120

50.0

91.3

88.4

110

158

77.2

75.8

122

500

27.8

4.31

143

889

0.622

-

-

1580

-

-

-

 

 

 

 

 

NQO

0.10

86.7

31.2

792

0.20

60.4

33.9

529

 

 

a:       Relative Survival (CE of cells after treatment)

b:        Relative Total Growth

c:        5-TFT Mutant Frequency per 106viable cells

EZ:     Internal study number

NQO:  4-Nitroquinoline N-oxide

DMBA:           7,12-dimethylbenz[a]anthracene

- :       Lost due to toxicity of test material or not plated for selection of mutants

With S9 mix(EZ 1572/ 1582/ 1594)

1stExperimental series

Test material

[µg/mL]

RSa

(%)

RTGb

(%)

MFc

Solvent

0

100

100

202

 

 

 

 

 

Test item

1.58

97.8

89.3

223

5.00

82.3

61.2

289

15.8

21.5

10.6

1110

50.0

13.5

9.04

846

158

5.56

2.09

1250

500

0

-

-

 

 

 

 

 

DMBA

2.00

62.1

64.2

1160

3.00

78.3

48.8

2000

 

2nd. Experimental series

Test material

[ug/mL]

RSa

(%)

RTGb

(%)

MFc

Solvent

0

100

100

161

 

 

 

 

 

Test item

1.58

93.0

76.3

196

5.00

91.4

35.6

344

8.89

49.7

32.6

199

15.8

23.2

19.8

256

50.0

11.0

11.6

203

158

2.02

0.165

250

 

 

 

 

 

DMBA

1.00

50.5

38.1

2120

2.00

45.9

6.53

2970

 

3rd. Experimental series

Test material

[ug/mL]

RSa

(%)

RTGb

(%)

MFc

Solvent

0

100

100

196

 

 

 

 

 

Test item

1.58

84.7

94.3

178

2.81

64.8

79.0

190

5.00

51.0

74.7

194

8.89

30.7

36.3

256

15.8

13.9

22.8

255

28.1

20.8

23.1

269

 

 

 

 

 

DMBA

2.00

66.4

40.6

2460

3.00

6.81

9.07

6640

 

a:       Relative Survival (CE of cells after treatment)

b:        Relative Total Growth

c:        5-TFT Mutant Frequency per 106 viable cells

EZ:     Internal study number

NQO:  4-Nitroquinoline N-oxide

DMBA:           7,12-dimethylbenz[a]anthracene

- :       Lost due to toxicity of test material or not plated for selection of mutants

Table 2: Historical data

Study number

Mean Mutation Frequency [x10-6]a

Solvent controlsb

Positive controlsc

- S9 mix

+ S9 mix

- S9 mix

+ S9 mix

T14248

63

123

 

95

106

 

378

794

 

488

568

 

T14277

128

179

 

168

133

 

1440

755

 

955

963

 

T14278

184

 

 

94

 

 

718

 

 

429

 

 

T14325

118

158

 

172

219

 

498

684

 

445

816

 

T14327

199

79

 

148

86

 

1181

1490

 

672

413

 

T14506

88

103

166

123

112

 

746

843

571

673

675

 

T14646

178

246

289

233

 

 

1196

869

924

518

 

 

T14690

166

251

 

174

117

 

1920

1439

 

606

478

 

T14786

103

106

 

90

53

 

1263

1529

 

554

465

 

T14862

88

122

 

90

257

 

955

645

 

484

1448

 

T14905

219

131

 

143

246

 

760

1008

 

562

2046

 

T15101

147

141

 

121

120

233

541

857

 

576

1453

1230

T15141

222

159

 

157

152

 

749

537

 

1196

1440

 

T15152

132

183

 

186

114

 

465

473

 

696

632

 

T15358

156

153

 

179

170

202

883

1244

 

1548

2172

2152

T15359

146

129

 

121

215

137

443

683

 

1050

1741

2096

T15360

136

131

 

157

187

 

358

462

 

2400

955

 

T15597

71.8

84.1

 

104

96

 

486

2755

 

1098

805

 

T15549

82.5

91.4

 

102

88

 

431

661

 

463

608

 

T15575

179

97.8

 

87.9

55.6

 

614

315

 

836

669

 

T15630

93.9

89.3

 

177

78.7

 

1050

834

 

1320

12040

 

T15596

140

103

 

106

171

 

666

524

 

1700

1200

 

T15681

98

131

 

95.2

95.2

 

704

1110

 

5150

1560

 

T15682

113

136

 

122

245

 

957

471

 

1240

2470

 

T15766

219

157

 

301

230

 

970

369

 

1660

1530

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Historical Controls

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean±SD:

141±50

146±57

847±445

1160±810

Range:

53 - 301

315 - 2755

413 - 5150

a:       Mean values from 1 to 4 Individual cultures from 1 to 3 experimental series of studies performed in the laboratory using the described protocol

b;       All solvent controls from different exposure times are pooled for each study

c:       NQO (0.10 to 0.25 µg/mL) in the absence and BaP (1.25 to 5.0 µg/mL) or DMBA (1.0 to 3.0 µg/mL) in the presence of S9 mix


 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test

A study according OECD TG 471 was performed to test for the mutagenic potential of the substance using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA pkM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively.

The substance was dissolved in dimethyl sulfoxide and tested at concentrations ranging from 5.0 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate. Toxicity to the bacteria was observed at concentrations > 500 or 1580 µg/plate depending upon the experimental conditions used.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

With and without addition of S9 mix as the external metabolizing system, the substance was not mutagenic under the experimental conditions described (reference 7.6.1-1).

In vitro gene mutation in mammalian cells

The substance was tested for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in mouse lymphoma cells according to OECD TG 476. The study consisted of two or three independent experimental series, conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pre-treated with Aroclor 1254). The exposure times were 3 and 24 hours in the absence and 3 hours in the presence of S9 mix, respectively.

Concentrations ranging from 1.58 to 1580 µg/mL were tested in the absence or presence of S9 mix. No Precipitation of the test material in the incubation medium was observed. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred at concentrations > 5.00 or < 500 µg/mL, depending upon the experimental condition used. The doses tested were selected to determine viability and mutagenicity (5-trifluorothymidine (TFT) resistance) 2 days after treatment.

Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and 7,12-dimethylbenz[a]anthracene (with S9 mix). Therefore, the study was accepted as valid.

No biologically relevant increases in mutant frequency were observed following treatment with the test item in the three experimental series performed.

It is therefore concluded that the substance is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects (reference 7.6.1-2).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.