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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro - Ames assay

Petroleum Resins (Kendex 0897) is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23. May 2018 to 30. May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997 “Bacterial Reverse Mutation Test“
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity-Reverse mutation test using bacteria”
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
No further details specified in the study report.
Target gene:
histidine
Species / strain / cell type:
S. typhimurium, other: 97a
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
First Experiment: 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate
Second Experiment: 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 µL/plate
The second experiment dose range was based on the findings from experiment one.
Vehicle / solvent:
In a non-GLP pre-test, the solubility of the test item was tested in demineralized water, dimethyl sulfox-ide (DMSO), ethanol, acetone and tetrahydrofurane (THF).
The liquid test item is sufficiently soluble in a concentration of 200 mL/L in tetrahydrofurane, only.
Based on the non-GLP pre-test, THF was chosen as vehicle, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO), for the positive controls ni-trophenylendiamine, benzo-a-pyrene and 2-amino-anthracene Demineralised water, for the positive control sodium azide Tetrahydrofurane (THF), for the test item
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9; 2-Amino-anthracene, C14H11N; CAS-No.: 613-13-8
Details on test system and experimental conditions:
Culture of Bacteria
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutrient broth. After incuba-tion for eight hours at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Conduct of Experiment
Preparations
Different media and solutions were prepared preliminary (exact production dates are documented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.

Experimental Parameters
First Experiment
Concentrations tested: 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: plate incorporation method

Second Experiment
Concentrations tested: 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 µL/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: pre-incubation method

Description of the Method
General preparation
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
The test item solutions were prepared according to chapter 6.1.3.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
25 µL test solution at each dose level, resp. 100 µL solvent (negative control) or reference mutagen solution (positive control)
500 µL S9 mix or phosphate buffer (for test without metabolic activation).
100 µL bacteria suspension
2000 µL overlay agar (top agar)

The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
25 µL test solution at each dose level, resp. 100 µL solvent (negative control) or reference mutagen solution (positive control)
500 µL S9 mix or phosphate buffer (for test without metabolic activation).
100 µL bacteria suspension

After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.

References and Validity
Genotype Confirmation
Genotype confirmation is performed for each batch of lyophilized bacteria before stock culture preparation.

Histidine requirement
Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop. The plates were incubated for 24 hours at 37 ±1 °C.

Ampicillin/Tetracycline-Resistance (pKM 101, pAQ1)
Each strain was streaked on an ampicillin agar plate and on an ampicillin-tetracycline agar plate. TA1535 was used as control strain, since it is not ampicillin resistant. The plates were incubated for 24 hours at 37 ±1 °C.

UV-sensitivity (uvrB)
Each strain was streaked on a plate, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates for the strain TA97a, TA100 and TA102 were irradiat-ed for 8 seconds, the plates for the strain TA 98 were irradiated for 10 seconds and the plates for the strain TA1535 were irradiated for 6 seconds with a germicidal lamp (254 nm, 30W).
Keeping a distance of 33 cm for the strains TA97a, TA102 and TA1535.
Keeping a distance of 66 cm for the following strains: TA98, TA100.
Incubation for 24 hours at 37 ±1 °C followed.

Crystal violet sensitivity (deep rough/rfa)
For each strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs (9 mm), each soaked with 10 µL of crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation for 24 hours at 37 ±1°C.

Spontaneous Revertants
Three replicates, with/without S9, for each solvent which was used in the test, incubation for 48 hours at 37 ±1°C.

Determination of Titre
The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of e+9 cells/mL (at the least), two replicates with and without metabolic activation.

Toxicity Control
Performed in experiment 1 only analogously to the titre control with the maximum dose of test item on maximal-soft agar, two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1°C.

Sterility Control
Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incuba-tion for 48 hours at 37 ±1°C, four replicates.

Solubility
Plates were checked for precipitation of test item at the end of the incubation by visual inspection.

Positive Controls
Using diagnostic mutagens, three replicates were prepared. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate, incubation for 48 hours at 37 ±1°C.
Rationale for test conditions:
In accordance with test guidelines.
Evaluation criteria:
The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Statistics:
Not specified
Key result
Species / strain:
S. typhimurium, other: 97a
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:
S. typhimurium TA 102
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 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
Additional information on results:
First Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the nor-mal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all (one exception) were within the historical control data ranges.

Solubility and Toxicity
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the conditions of this experiment.
To verify this result, a further experiment was performed.

Second Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory.. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

Solubility and Toxicity
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

Survey of the Findings– First Experiment

The mean revertant values of the three replicates are presented in the following table.

Mean Revertants First Experiment

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

82

105

36

40

89

103

213

316

14

18

sd

1.5

6.1

2.3

2.9

7.0

18.6

26.6

68.2

0.6

1.7

DMSO

Mean

101

101

38

42

82

96

289

249

19

20

sd

6.1

13.3

4.0

2.0

14.6

10.4

22.0

4.6

2.1

4.5

THF

Mean

88

121

39

43

80

81

281

271

18

17

sd

5.8

37.8

1.2

6.7

4.7

6.4

38.0

58.3

4.0

1.2

Positive
Controls*

Mean

536

525

77

119

387

1001

672

652

216

141

sd

90.9

33.3

4.6

25.0

22.7

0.0

12.0

18.3

50.0

26.0

f(I)

5.31

5.20

2.03

2.83

4.35

10.43

2.33

2.62

15.43

7.05

5 µL/plate

Mean

118

90

40

39

78

87

295

352

12

13

sd

17.3

9.0

1.2

9.2

5.6

4.5

36.3

26.2

2.3

3.1

f(I)

1.34

0.74

1.03

0.91

0.98

1.07

1.05

1.30

0.67

0.76

1.5 µL/plate

Mean

86

127

41

39

79

75

345

340

14

14

sd

3.5

11.4

2.0

5.1

7.1

5.5

36.3

12.0

0.6

4.4

f(I)

0.98

1.05

1.05

0.91

0.99

0.93

1.23

1.25

0.78

0.82

0.5 µL/plate

Mean

101

139

40

41

89

83

340

301

17

15

sd

16.7

8.1

1.2

9.1

11.0

8.6

48.7

15.1

2.3

4.7

f(I)

1.15

1.15

1.03

0.95

1.11

1.02

1.21

1.11

0.94

0.88

0.15 µL/plate

Mean

95

127

41

41

75

81

360

335

12

13

sd

14.7

25.5

0.0

0.0

0.6

2.5

31.7

34.5

1.0

2.0

f(I)

1.08

1.05

1.05

0.95

0.94

1.00

1.28

1.24

0.67

0.76

0.05 µL/plate

Mean

94

100

40

51

82

86

339

313

18

18

sd

16.9

17.6

1.5

2.0

0.6

21.9

22.0

72.1

3.2

1.5

f(I)

1.07

0.83

1.03

1.19

1.03

1.06

1.21

1.15

1.00

1.06

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

* Different positive controls were used

 

Survey of the Findings– Second Experiment

The mean revertant values of the three replicates are presented in the following table.

Mean Revertants Second Experiment

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin.

water

Mean

87

138

43

42

124

132

305

312

35

32

sd

7.5

19.3

6.0

10.0

5.3

5.9

18.0

21.2

4.0

3.6

DMSO

Mean

86

104

41

48

110

117

307

328

33

33

sd

11.7

13.9

9.5

6.0

6.0

8.7

22.0

26.2

3.5

1.7

THF

Mean

92

110

47

48

93

93

284

292

34

40

sd

19.8

35.6

8.1

3.5

6.4

6.1

61.6

104.8

2.6

0.6

Positive
Controls*

Mean

584

696

440

403

675

1001

729

1317

216

163

sd

77.3

54.1

13.9

101.6

41.1

0.0

18.9

102.9

35.6

9.2

f(I)

6.79

6.69

10.73

8.40

5.44

8.56

2.37

4.02

6.17

4.94

5 µL/plate

Mean

103

108

47

51

124

102

379

415

33

30

sd

1.2

6.2

9.1

10.0

4.6

12.8

32.3

32.1

1.2

2.1

f(I)

1.12

0.98

1.00

1.06

1.33

1.10

1.33

1.42

0.97

0.75

2.5 µL/plate

Mean

93

88

47

43

116

129

349

384

34

33

sd

15.7

16.5

8.5

7.2

5.3

16.2

11.5

77.1

2.9

2.1

f(I)

1.01

0.80

1.00

0.90

1.25

1.39

1.23

1.32

1.00

0.83

1.25 µL/plate

Mean

105

88

54

45

104

117

363

377

32

30

sd

35.5

10.6

5.8

6.8

7.2

7.0

54.0

24.4

1.0

3.5

f(I)

1.14

0.80

1.15

0.94

1.12

1.26

1.28

1.29

0.94

0.75

0.63 µL/plate

Mean

81

83

50

43

117

103

368

253

30

31

sd

10.1

9.5

7.8

7.8

4.2

16.8

40.0

31.1

3.1

3.5

f(I)

0.88

0.75

1.06

0.90

1.26

1.11

1.30

0.87

0.88

0.78

0.31 µL/plate

Mean

103

84

48

48

116

126

305

349

37

39

sd

19.9

11.8

4.2

8.6

10.4

3.6

20.1

46.7

4.6

7.1

f(I)

1.12

0.76

1.02

1.00

1.25

1.35

1.07

1.20

1.09

0.98

0.16 µL/plate

Mean

100

71

50

42

122

115

371

340

42

36

sd

19.6

7.0

4.7

4.0

4.0

14.0

38.0

72.1

1.7

3.5

f(I)

1.09

0.65

1.06

0.88

1.31

1.24

1.31

1.16

1.24

0.90

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

* Different positive controls were used

 

First Experiment- Positive Controls

Without metabolic activation:
4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 20 µg/plate
Sodium azide (Na-azide) in demineralized water, 1 µg/plate

 

With metabolic activation:
2-Amino anthracene (2-AA) in DMSO, 1 µg/plate
Benzo-a-pyrene (BaP) in DMSO, 20 µg/plate

 

Diagnostic Mutagens (colonies per plate)

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Substance

NPD

2-AA

NPD

BaP

Na-azide

2-AA

NPD

2-AA

Na-azide

2-AA

Repl. 1

432

552

74

120

380

1001

672

672

232

140

Repl. 2

576

488

82

94

412

1001

660

636

160

168

Repl. 3

600

536

74

144

368

1001

684

648

256

116

Mean

536

525

77

119

387

1001

672

652

216

141

sd

90.9

33.3

4.6

25.0

22.7

0.0

12.0

18.3

50.0

26.0

f(I)

5.31

5.20

2.03

2.83

4.35

10.43

2.33

2.62

15.43

7.05

Rev. abs.

435

424

39

77

298

905

383

403

202

121

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

Rev.abs. = absolute revertants

 

Second Experiment- Positive Controls

Without metabolic activation:
4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 20 µg/plate
Sodium azide (Na-azide) in demineralized water, 1 µg/plate

 

With metabolic activation:
2-Amino anthracene (2-AA) in DMSO, 1 µg/plate
Benzo-a-pyrene (BaP) in DMSO, 20 µg/plate

 

Diagnostic Mutagenes (colonies per plate)

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Substance

NPD

2-AA

NPD

BaP

Na-azide

2-AA

NPD

2-AA

Na-azide

2-AA

Repl. 1

516

640

456

344

720

1001

736

1200

244

152

Repl. 2

668

748

432

520

640

1001

708

1360

176

168

Repl. 3

568

700

432

344

664

1001

744

1392

228

168

Mean

584

696

440

403

675

1001

729

1317

216

163

sd

77.3

54.1

13.9

101.6

41.1

0.0

18.9

102.9

35.6

9.2

f(I)

6.79

6.69

10.73

8.40

5.44

8.56

2.37

4.02

6.17

4.94

Rev. abs.

498

592

399

355

551

884

422

989

181

130

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

Rev.abs. = absolute revertants

 

 

Historical Data

In the following table, the history of the spontaneous revertants and positive controls of the performed experiments with these strains up to 16. May 2018 (demin. water and DMSO) resp. 20. Mar. 2018 (THF) is stated in comparison with the experiments performed within this study. Only experiments which were performed before the performance of the study were considered.

For the historical data, the plate incorporation method and the pre- incubation method were used.

 

Historical Data of Spontaneous Revertants

Strain

 

TA97a

TA98

TA100

TA102

TA1535

Induction

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Demin. water

Mean

88

94

22

24

93

97

281

300

18

18

Min

60

63

6

8

51

64

85

67

6

7

Max

144

138

52

51

147

141

425

587

36

40

SD

17

16

12

11

16

15

57

72

6

6

Exp 1

82

105

36

40

89

103

213

316

14

18

Exp 2

87

138

43

42

124

132

305

312

35

32

DMSO

Mean

88

97

22

23

90

93

280

293

18

17

Min

58

67

7

8

44

62

79

80

8

6

Max

135

144

47

50

138

199

413

459

35

37

SD

17

16

12

11

16

17

56

60

6

6

Exp 1

101

101

38

42

82

96

289

249

19

20

Exp 2

86

104

41

48

110

117

307

328

33

33

THF

Mean

87

101

21

25

97

102

339

332

25

23

Min

69

78

10

17

61

73

201

269

18

13

Max

98

121

47

48

187

187

492

433

34

40

SD

8

15

12

11

33

33

96

57

5

8

Exp 1

88

121

39

43

80

81

281

271

18

17

Exp 2

92

110

47

48

93

93

284

292

34

40

Positive Controls*

Mean

534

524

416

127

486

777

1099

1200

264

134

Min

264

228

100

39

220

273

491

408

55

45

Max

1165

1181

1001

487

984

1912

2331

6083

515

712

SD

170

169

168

99

155

284

409

560

85

80

Exp 1

536

525

77

119

387

1001

672

652

216

141

Exp 2

584

696

440

403

675

1001

729

1317

216

163

1001 colonies per plate means the bacteria growth was too strong for counting.

* Different positive controls were used

Conclusions:
The test item Petroleum Resins (Kendex 0897) showed no increase in the number of revertants in all bacteria strains in both experiments.
All negative and strain-specific nearly all positive control values (one exception) were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that Petroleum Resins (Kendex 0897) is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Executive summary:

Determination of the mutagenic potential of Petroleum Resins (Kendex 0897) with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14

 

Findings and Results:

Two valid experiments were performed.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item Petroleum Resins (Kendex 0897) was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1:

In the first experiment, the test item (dissolved in tetrahydrofurane) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

Based on the results of the first experiment, the test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that Petroleum Resins (Kendex 0897) is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

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

Additional information

Genetic toxicity in vitro - Ames assay

Two valid experiments were performed.

The test item Petroleum Resins (Kendex 0897) was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1:

In the first experiment, the test item (dissolved in tetrahydrofurane) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

Based on the results of the first experiment, the test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that Petroleum Resins (Kendex 0897) is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

Justification for classification or non-classification