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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed to investigate the potential of the test chemical to induce gene muta¬tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
12H-phthaloperin-12-one
EC Number:
230-049-5
EC Name:
12H-phthaloperin-12-one
Cas Number:
6925-69-5
Molecular formula:
C18H10N2O
IUPAC Name:
12H-phthaloperin-12-one
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material : 12H-phthaloperin-12-one
- Common name : C.I. Solvent Orange 60
- Molecular formula : C18H10N2O
- Molecular weight : 270.29 g/mol
- Smiles notation : O=C1N2c3c4c(cccc4N=C2c2ccccc12)ccc3
- InChl : 1S/C18H10N2O/c21-18-13-8-2-1-7-12(13)17-19-14-9-3-5-11-6-4-10-15(16(11)14)20(17)18/h1-10H
- Substance type : Organic
- Physical state : Solid

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other:
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Aroclor 1254 induced S9 metabolic activation system was procured from Defence Research and Developement Establishment
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was mixed with S9 cofactor solution which contains D-glucose-6-phosphate 0.8 g, β-NADP 1.75 g, MgCl2 1.0 g, KCl 1.35g, Na2HPO4 6.4 g, NaH2PO4.H2O 1.4 g in 500 ml of distilled water
- concentration or volume of S9 mix and S9 in the final culture medium : 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) : No data available
Test concentrations with justification for top dose:
0.0 (N.C), 0.0 (V.C), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate. Based on the results of pre-experiment, the doses were selected.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO
Controls
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
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- 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: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

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: No data
- Any supplementary information relevant to cytotoxicity: No data

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:
A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.
Statistics:
No data

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
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:
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
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.001 – 2.5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count but reduction in background lawn was observed in treated concentrations 2.5 mg/plate (T8), 0.791 mg/plate (T7) and no reduction in colony count as well as in background lawn in treated concentrations (0.250 (T6) mg/plate – 0.001 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: (0.050, 0.158, 0.501, 1.582 and 5 mg/plate , both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

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: Please refer the table in remark section.
- Negative (solvent/vehicle) historical control data: Please refer the section in remark section

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

Any other information on results incl. tables

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL I)

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

3

11

23

110

236

R2

4

10

22

118

248

R3

4

12

24

112

250

VC

(0.00)

R1

5

15

28

120

264

R2

6

16

26

129

288

R3

8

14

27

124

274

T1

(0.050)

R1

4

12

24

123

240

R2

4

13

25

124

248

R3

5

13

23

122

252

T2

(0.158)

R1

5

14

25

123

260

R2

4

13

24

125

256

R3

4

13

24

123

248

T3

(0.501)

R1

5

14

26

124

254

R2

6

13

25

125

266

R3

4

14

23

124

274

T4

(1.582)

R1

6

15

26

125

280

R2

6

14

24

123

268

R3

5

13

25

126

272

T5

(5)

R1

7

14

27

126

284

R2

6

15

26

125

276

R3

5

15

26

127

264

PC

R1

176

632

1128

1592

1944

R2

164

608

1088

1608

1832

R3

182

666

1264

1678

2116

 

Dose (mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

4

10

22

98

220

R2

5

12

20

108

234

R3

3

10

25

114

248

VC

(0.00)

R1

6

14

27

112

252

R2

5

14

23

120

268

R3

7

16

25

124

282

T1

(0.050)

R1

5

12

24

112

228

R2

4

14

21

114

236

R3

4

13

24

106

244

T2

(0.158)

R1

5

13

23

115

240

R2

5

12

23

108

254

R3

4

12

24

117

238

T3

(0.501)

R1

6

14

24

118

250

R2

5

13

25

114

242

R3

4

12

23

120

260

T4

(1.582)

R1

6

14

25

121

262

R2

5

15

23

122

244

R3

5

12

26

122

258

T5

(5)

R1

6

14

26

123

268

R2

6

16

25

124

274

R3

6

15

25

124

262

PC

R1

180

1308

788

1304

1692

R2

194

1368

816

1432

1832

R3

202

1434

942

1460

1868

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10
μg/plate]:TA 102                                             Sodium azide [10μg/plate]: TA 1535, TA 100                                             

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

3

11

18

114

242

R2

4

12

20

110

238

R3

3

11

24

107

256

VC

(0.00)

R1

6

16

24

128

292

R2

7

15

25

126

288

R3

5

16

29

132

290

T1

(0.050)

R1

3

11

23

120

252

R2

5

12

22

118

256

R3

4

14

24

99

262

T2

(0.158)

R1

4

13

25

109

256

R2

4

12

26

119

264

R3

5

14

21

112

268

T3

(0.501)

R1

6

14

24

119

258

R2

3

15

25

120

258

R3

5

13

24

113

262

T4

(1.582)

R1

5

14

24

121

276

R2

7

14

26

123

264

R3

4

15

25

119

280

T5

(5)

R1

6

16

28

120

284

R2

7

14

27

124

274

R3

5

15

28

118

268

PC

R1

198

496

1524

1976

1808

R2

206

526

1478

1884

1872

R3

178

570

1596

1964

1944

 

 

Dose

(mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

4

10

23

98

246

R2

4

12

22

108

244

R3

5

13

20

112

230

VC

(0.00)

R1

6

16

27

122

280

R2

7

15

29

130

296

R3

7

14

30

136

304

T1

(0.050)

R1

4

13

24

112

234

R2

5

12

23

100

248

R3

4

14

23

119

241

T2

(0.158)

R1

5

13

25

116

246

R2

4

14

24

118

238

R3

5

13

23

107

252

T3

(0.501)

R1

5

15

26

128

242

R2

4

14

23

116

256

R3

6

13

25

108

262

T4

(1.582)

R1

7

15

24

120

266

R2

5

16

26

128

258

R3

5

13

25

116

240

T5

(5)

R1

6

14

27

128

254

R2

7

15

26

121

266

R3

6

16

28

121

274

PC

R1

176

1540

920

1696

1824

R2

163

1572

984

1740

1768

R3

181

1612

1016

1704

1920

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10
μg/plate]:TA 102                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

3.67

0.58

11.00

1.00

23.00

1.00

113.33

4.16

244.67

7.57

VC

(0.00)

6.33

1.53

15.00

1.00

27.00

1.00

124.33

4.51

275.33

12.06

T1

(0.050)

4.33

0.58

12.67

0.58

24.00

1.00

123.00

1.00

246.67

6.11

T2

(0.158)

4.33

0.58

13.33

0.58

24.33

0.58

123.67

1.15

254.67

6.11

T3

(0.501)

5.00

1.00

13.67

0.58

24.67

1.53

124.33

0.58

264.67

10.07

T4

(1.582)

5.67

0.58

14.00

1.00

25.00

1.00

124.67

1.53

273.33

6.11

T5

(5)

6.00

1.00

14.67

0.58

26.33

0.58

126.00

1.00

274.67

10.07

PC

174.00

9.17

635.33

29.14

1160.00

92.26

1626.00

45.74

1964.00

143.05

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.00

1.00

10.67

1.15

22.33

2.52

106.67

8.08

234.00

14.00

VC

(0.00)

6.00

1.00

14.67

1.15

25.00

2.00

118.67

6.11

267.33

15.01

T1

(0.050)

4.33

0.58

13.00

1.00

23.00

1.73

110.67

4.16

236.00

8.00

T2

(0.158)

4.67

0.58

12.33

0.58

23.33

0.58

113.33

4.73

244.00

8.72

T3

(0.501)

5.00

1.00

13.00

1.00

24.00

1.00

117.33

3.06

250.67

9.02

T4

(1.582)

5.33

0.58

13.67

1.53

24.67

1.53

121.67

0.58

254.67

9.45

T5

(5)

6.00

0.00

15.00

1.00

25.33

0.58

123.67

0.58

268.00

6.00

PC

192.00

11.14

1370.00

63.02

848.67

82.03

1398.67

83.17

1797.33

92.98

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

 

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD
(TRIAL II)

Dose

(mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

3.33

0.58

11.33

0.58

20.67

3.06

110.33

3.51

245.33

9.45

VC

(0.00)

6.00

1.00

15.67

0.58

26.00

2.65

128.67

3.06

290.00

2.00

T1

(0.050)

4.00

1.00

12.33

1.53

23.00

1.00

112.33

11.59

256.67

5.03

T2

(0.158)

4.33

0.58

13.00

1.00

24.00

2.65

113.33

5.13

262.67

6.11

T3

(0.501)

4.67

1.53

14.00

1.00

24.33

0.58

117.33

3.79

259.33

2.31

T4

(1.582)

5.33

1.53

14.33

0.58

25.00

1.00

121.00

2.00

273.33

8.33

T5

(5)

6.00

1.00

15.00

1.00

27.67

0.58

120.67

3.06

275.33

8.08

PC

194.00

14.42

530.67

37.22

1532.67

59.48

1941.33

50.01

1874.67

68.04

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.33

0.58

11.67

1.53

21.67

1.53

106.00

7.21

240.00

8.72

VC

(0.00)

6.67

0.58

15.00

1.00

28.67

1.53

129.33

7.02

293.33

12.22

T1

(0.050)

4.33

0.58

13.00

1.00

23.33

0.58

110.33

9.61

241.00

7.00

T2

(0.158)

4.67

0.58

13.33

0.58

24.00

1.00

113.67

5.86

245.33

7.02

T3

(0.501)

5.00

1.00

14.00

1.00

24.67

1.53

117.33

10.07

253.33

10.26

T4

(1.582)

5.67

1.15

14.67

1.53

25.00

1.00

121.33

6.11

254.67

13.32

T5

(5)

6.33

0.58

15.00

1.00

27.00

1.00

123.33

4.04

264.67

10.07

PC

173.33

9.29

1574.67

36.07

973.33

48.88

1713.33

23.44

1837.33

76.87

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

Trial I:

Trial I was performed with five concentration of test item along with the negative, vehicle and concurrent positive contros with the remaining three strains i.e TA 1535, TA 1537 and TA 102 by the plate incorporation method . For TA 98 and TA 100 revertant colony counts were directly incorporated in the trial I from the pre experiment upto the required five concentrations. [T4 (0.050 mg/plate) to T8 (5 mg/plate)].

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.