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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
Study period:
13 November 2018 - 14 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test method according OECD Guideline 471. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver.
Test concentrations with justification for top dose:
0.001, 0.0032, 0.01, 0.032 and 0.1 µL/plate.

Initial cytotoxicity test: TA100 tester strain was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 µL /plate. There was cytotoxicity in all tested concentrations.
Follow up cytotoxicity test: Test was performed at 0.4, 0.3, 0.2, 0.1, 0.05, 0.025 and 0.0125µL/plate. The test item showed cytotoxicity at 0.2, 0.3 and 0.4 µL/plate with lawn intensity 2+ (Thin lawn) and 0.1 µL/plate with lawn intensity 3+ (Slightly thin lawn). The test item did not result in cytotoxicity at 0.05, 0.025 and 0.0125 µL/plate with bacterial lawn 4+ intensity. Hence, 0.1 µL/plate was selected as the highest concentration for mutation test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle:
A solubility test was carried out with Distilled Water and Dimethyl Sulphoxide (DMSO). The test item was found miscible in DMSO at a concentration of 50 µL/mL. Subsequently, in a precipitation test in DMSO, the test item resulted in no precipitation at and up to 5 µL/plate tested concentration.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
2 µg/plate.
Positive control substance:
2-nitrofluorene
Remarks:
Salmonella TA 98, without metabolic activation.
Positive controls:
yes
Remarks:
1 µg/plate.
Positive control substance:
sodium azide
Remarks:
Salmonella TA 100 and TA 1535, without metabolic activation.
Positive controls:
yes
Remarks:
50 µg/plate.
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA 1537, without metabolic activation.
Positive controls:
yes
Remarks:
5 µg/plate.
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
E.coli WP2 uvrA (pKM 101), without metabolic activation.
Positive controls:
yes
Remarks:
4 µg/plate.
Positive control substance:
other: 2-aminoanthracene
Remarks:
All Salmonella strains, with metabolic activation.
Positive controls:
yes
Remarks:
30 µg/plate.
Positive control substance:
other: 2-aminoanthracene
Remarks:
E.coli WP2 uvrA (pKM 101), with metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Plate incorporation (initial mutation test): Test item, vehicle or positive control (100 µL) and the tester strains (100 µL) along with S9/phosphate buffer saline (500 µL) were mixed with 2 mL of molten soft agar and poured on to minimal glucose agar plates.

Pre-incubation (confirmatory mutation test): Test item, vehicle or positive control (100 µL) and the tester strains (100 µL) along with S9/Phosphate Buffer Saline (500 µL) were incubated in an incubator shaker at 100±5 rpm. Post incubation, the test constituents were mixed with 2 mL molten soft agar and poured on to minimal glucose agar plates.

- Cell density at seeding (if applicable): 18×108 cells/mL.

DURATION
- Preincubation period: 30 minutes (Pre-incubation test)
- Exposure duration: 65 hours and 50 minutes (Plate incorporation test) and 65 hours (Pre-incubation test)

NUMBER OF REPLICATIONS: 3 replicates per dose and controls.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Bacterial background lawn intensity
- Any supplementary information relevant to cytotoxicity:
The condition of the bacterial background lawn was evaluated for evidence of the test item toxicity using the code system as follows:
0: No lawn (Absent): Distinguished by a complete lack of any background lawn compared to solvent control plates.
1+: Very thin lawn (Extremely reduced): Distinguished by an extreme thinning of the background lawn compared to solvent control plates.
2+: Thin lawn (Moderately reduced): Distinguished by a marked thinning of the background lawn compared to solvent control plates.
3+: Slightly thin lawn (Slightly reduced): Distinguished by a noticeable thinning of the background lawn compared to solvent control plates.
4+: Thick lawn (Normal): Distinguished by a healthy background lawn comparable to solvent control plates.



Evaluation criteria:
Positive result: there is a concentration related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system, AND this increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in S. typhimurium strains TA98, TA100 and E. coli WP2 uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
Equivocal response: biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited.
Negative result: neither positive nor equivocal.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
3+ (Slightly thin lawn) at 0.1 µL/plate.
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:
cytotoxicity
Remarks:
3+ (Slightly thin lawn) at 0.1 µL/plate.
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:
cytotoxicity
Remarks:
3+ (Slightly thin lawn) at 0.1 µL/plate.
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:
cytotoxicity
Remarks:
3+ (Slightly thin lawn) at 0.1 µL/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Remarks:
3+ (Slightly thin lawn) at 0.1 µL/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A solubility test was carried out with Distilled Water and Dimethyl Sulphoxide (DMSO). The test item was found miscible in DMSO at a concentration of 50 µL/mL. Subsequently, in a precipitation test in DMSO, the test item resulted in no precipitation at and up to 5 µL/plate tested concentration.
Initial cytotoxicity test: TA100 tester strain was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 µL /plate. There was cytotoxicity in all tested concentrations.
Follow up cytotoxicity test: Test was performed at 0.4, 0.3, 0.2, 0.1, 0.05, 0.025 and 0.0125µL/plate. The test item showed cytotoxicity at 0.2, 0.3 and 0.4 µL/plate with lawn intensity 2+ (Thin lawn) and 0.1 µL/plate with lawn intensity 3+ (Slightly thin lawn). The test item did not result in cytotoxicity at 0.05, 0.025 and 0.0125 µL/plate with bacterial lawn 4+ intensity. Hence, 0.1 µL/plate was selected as the highest concentration for mutation test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 5 below
- Negative (solvent/vehicle) historical control data: see table 5 below

The observed numbers of revertant colonies for vehicle and positive controls were within the historical control ranges except for positive control with TA1535 in the plate incorporation method with and without S9 which were slightly below the range and for positive control with E. coli in the preincubation method with S9 which were slightly above the range. These observations are considered without impact on the final conclusions of this assay.

Any other information on results incl. tables

Table1. Summary of initial cytotoxicity test - Salmonella typhimurium TA100 tester strain

Test Item Concentration (µL/plate)

No. of Revertants/plate

With S9

Without S9

R1

R2

R3

Average

±SD

Bacterial Lawn Intensity

R1

R2

R3

Average

±SD

Bacterial Lawn Intensity

Vehicle Control

121

106

119

115

8.1

4+

108

111

102

107

4.6

4+

0.5

39

35

39

38

2.3

2+

36

36

34

35

1.2

2+

0.6

24

28

30

27

3.1

2+

26

25

21

24

2.6

2+

0.7

25

20

23

23

2.5

2+

19

22

24

22

2.5

2+

0.8

20

19

16

18

2.1

1+

18

15

18

17

1.7

1+

0.9

15

18

12

15

3.0

1+

15

17

14

15

1.5

1+

1

8

11

9

9

1.5

1+

7

10

8

8

1.5

1+

2

0

0

0

0

0.0

0

0

0

0

0

0.0

0

3

0

0

0

0

0.0

0

0

0

0

0

0.0

0

4

0

0

0

0

0.0

0

0

0

0

0

0.0

0

5

0

0

0

0

0.0

0

0

0

0

0

0.0

0

Table 2. Summary of follow up initial cytotoxicity test - Salmonella typhimurium TA100 tester strain

Test Item Concentration (µL/plate)

No. of Revertants/plate

With S9

Without S9

R1

R2

R3

Average

±SD

Bacterial Lawn Intensity

R1

R2

R3

Average

±SD

Bacterial Lawn Intensity

Vehicle Control

113

103

116

111

6.8

4+

102

101

114

106

7.2

4+

0.0125

109

110

114

111

2.6

4+

106

99

113

106

7.0

4+

0.025

106

107

112

108

3.2

4+

105

100

110

105

5.0

4+

0.05

108

99

104

104

4.5

4+

102

106

100

103

3.1

4+

0.1

70

72

76

73

3.1

3+

77

75

69

74

4.2

3+

0.2

60

56

49

55

5.6

2+

61

57

55

58

3.1

2+

0.3

43

52

56

50

6.7

2+

41

46

44

44

2.5

2+

0.4

46

50

38

45

6.1

2+

42

48

39

43

2.9

2+

Lawn intensity:

4+= Thick lawns: Distinguished by a healthy (Normal) background lawn comparable to vehicle control plates.    

3+=Slightly thin lawn: Distinguished by a noticeable thinning of the background lawn compared to solvent/vehicle control plates.

2+= Thin lawn: Distinguished by a marked thinning of the background lawn compared to solvent/vehicle control plates.

1+= Very thin lawn: Distinguished by an extreme thinning of the background lawn compared to solvent/vehicle control plates.

0 = No lawn : Distinguished by a complete lack of any background lawn compared to solvent/vehicle control plates

Table 3. Summary of colony counts of revertants of trial-I. Plate Incorporation Method

Treatment

Test Concentration  

(µL/plate)

No. of Revertants (Mean of 3 Plates)

With S9

 

Without S9

Salmonella typhimurium

E.coli WP2 uvrA

(pKM 101)

Salmonella typhimurium

E.coli WP2 uvrA

(pKM 101)

TA 98

TA 100

TA 1535

TA 1537

TA 98

TA 100

TA 1535

TA 1537

Vehicle Control

100 µL of Dimethyl

Sulphoxide

Mean

25.0

109.0

21.0

9.3

167.0

23.0

104.7

19.7

8.3

169.7

±SD

2.0

6.6

3.0

2.5

9.6

2.6

4.2

2.5

1.5

10.7

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

PINE OIL 50%

0.001

Mean

23.7

110.3

21.0

8.7

167.0

21.7

103.0

20.0

7.0

160.0

±SD

2.5

6.1

2.0

2.5

6.2

1.5

5.0

2.0

1.0

3.0

Fold Increase

0.9

1.0

1.0

0.9

1.0

0.9

1.0

1.0

0.8

0.9

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.0032

Mean

24.0

108.7

20.3

9.3

166.7

21.7

107.3

19.7

8.3

158.0

±SD

1.7

8.3

2.5

3.8

5.1

2.9

3.2

2.1

2.1

8.5

Fold Increase

1.0

1.0

1.0

1.0

1.0

0.9

1.0

1.0

1.0

0.9

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.01

Mean

22.0

103.3

20.3

6.7

155.0

21.0

100.0

19.0

6.7

152.7

±SD

2.0

5.5

1.5

0.6

5.0

2.0

5.0

1.7

2.1

5.7

Fold Increase

0.9

0.9

1.0

0.7

0.9

0.9

1.0

1.0

0.8

0.9

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.032

Mean

18.7

88.7

18.0

6.0

148.0

17.7

88.3

16.7

5.3

144.3

±SD

2.1

1.5

1.0

1.0

4.6

1.5

3.5

0.6

0.6

4.0

Fold Increase

0.7

0.8

0.9

0.6

0.9

0.8

0.8

0.8

0.6

0.9

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.1

Mean

14.7

68.7

15.0

4.0

126.0

14.0

68.3

14.7

4.3

125.7

±SD

0.6

5.1

1.0

1.0

6.6

1.0

3.5

0.6

0.6

5.9

Fold Increase

0.6

0.6

0.7

0.4

0.8

0.6

0.7

0.7

0.5

0.7

Lawn Intensity

3+

3+

3+

3+

3+

3+

3+

3+

3+

3+

Positive Control

100 µL of respective Positive Control

Mean

364.0

417.0

111.0

106.3

404.7

359.3

403.0

106.3

103.3

407.3

±SD

6.6

7.5

17.1

5.7

15.2

9.5

9.5

7.4

6.7

10.1

Fold Increase

14.6

3.8

5.3

11.4

2.4

15.6

3.9

5.4

12.4

2.4

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Table 4. Summary of colony counts of revertants of trial-II. Preincubation Method

Treatment

Test Concentration 

 (µL/plate)

No. of Revertants (Mean of 3 Plates)

With S9

Without S9

Salmonella typhimurium

E.coli WP2 uvrA

(pKM 101)

Salmonella typhimurium

E.coli WP2 uvrA

(pKM 101)

TA 98

TA 100

TA

1535

TA 1537

TA 98

TA 100

TA

1535

TA 1537

Vehicle Control

100 µL of

Dimethyl

Sulphoxide

Mean

26.7

113.7

22.7

8.0

170.7

22.7

108.0

20.7

7.7

165.3

±SD

1.2

5.9

3.5

2.0

6.0

2.5

7.5

3.1

3.1

6.7

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

PINE OIL 50%

0.001

Mean

24.0

105.0

21.0

8.0

174.0

21.0

109.0

21.0

7.3

169.0

±SD

2.6

4.4

3.6

3.0

5.6

2.6

3.6

2.0

1.5

5.6

Fold Increase

0.9

0.9

0.9

1.0

1.0

0.9

1.0

1.0

1.0

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.0032

Mean

24.3

115.3

21.3

7.0

172.3

22.3

108.3

20.3

6.7

161.0

±SD

0.6

7.4

2.3

1.0

6.7

3.2

8.1

3.1

2.1

8.0

Fold Increase

0.9

1.0

0.9

0.9

1.0

1.0

1.0

1.0

0.9

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.01

Mean

22.7

108.3

22.0

7.7

156.3

21.3

99.7

20.7

7.0

153.3

±SD

2.5

2.1

3.6

3.8

6.5

2.1

12.4

3.5

2.0

4.5

Fold Increase

0.9

1.0

1.0

1.0

0.9

0.9

0.9

1.0

0.9

0.9

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.032

Mean

20.7

92.0

18.3

5.7

158.0

19.0

89.3

16.7

6.0

147.3

±SD

2.9

6.6

1.5

0.6

14.8

2.6

4.5

1.2

1.0

5.7

Fold Increase

0.8

0.8

0.8

0.7

0.9

0.8

0.8

0.8

0.8

0.9

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.1

Mean

16.7

68.7

15.7

4.3

128.7

16.0

68.3

15.3

3.7

123.7

±SD

0.6

6.5

0.6

0.6

2.5

1.0

1.5

0.6

0.6

7.1

Fold Increase

0.6

0.6

0.7

0.5

0.8

0.7

0.6

0.7

0.5

0.7

Lawn Intensity

3+

3+

3+

3+

3+

3+

3+

3+

3+

3+

Positive Control

100 µL of respective Positive Control

Mean

357.7

413.7

127.7

107.3

413.3

355.3

405.7

115.7

103.0

406.0

±SD

9.6

10.7

8.5

14.3

4.5

5.0

9.5

7.0

11.8

8.5

Fold Increase

13.4

3.6

5.6

13.4

2.4

15.7

3.8

5.6

13.4

2.5

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Table 5. Historical data

Plate Incorporation Method

Metabolic Activation

With Metabolic Activation

 

 

 

 

 

 

 

 

 

Without Metabolic Activation

Tester strain

Salmonella typhimurium

E.coli uvrA           

(pKM 101)

Salmonella typhimurium

E.coli

uvrA           

(pKM 101)

TA 98

TA 100

TA 1535

TA 1537

TA 98

TA 100

TA 1535

TA 1537

Vehicle Control (DMSO)

Mean

21.3

103.4

21.2

9.2

169.8

21.2

102.8

21.2

9.4

171.1

±SD

3.2

5.5

2.2

1.8

7.6

2.7

4.7

2.3

1.9

6.8

Min

16

96

13

6

156

15

91

15

7

157

Max

40

130

26

14

191

32

126

27

15

191

Positive Control

Mean

401.7

383.6

140.1

119.2

383.5

396.6

379.7

141.1

120.1

383.1

±SD

23.4

16.9

8.0

6.6

9.8

27.4

18.3

9.9

7.3

10.4

Min

256

246

118

100

320

290

270

110

98

371

Max

440

419

182

149

421

430

446

190

158

416

 

 

Preincubation Method

Metabolic Activation

With Metabolic Activation

 

 

 

 

 

 

 

 

 

Without Metabolic Activation

Tester strain

Salmonella typhimurium

E.coli

uvrA           

(pKM 101)

Salmonella typhimurium

E.coli

uvrA           

(pKM 101)

TA 98

TA 100

TA 1535

TA 1537

TA 98

TA 100

TA 1535

TA 1537

Vehicle Control (DMSO)

Mean

21.2

102.9

20.9

9.4

171.7

20.9

102.1

21.1

9.6

171.3

±SD

2.8

4.6

2.2

2.0

7.2

2.8

4.3

2.3

2.6

6.8

Min

16

89

14

4

153

15

88

16

6

152

Max

32

128

28

17

198

32

129

28

23

198

Positive Control

Mean

402.5

383.9

140.6

118.6

382.9

398.3

381.3

141.3

119.5

382.8

±SD

18.5

16.1

9.3

6.3

10.4

24.5

16.2

11.0

6.4

10.3

Min

290

293

103

99

330

281

312

110

97

321

Max

429

425

187

151

412

428

450

200

173

418

Applicant's summary and conclusion

Conclusions:
The test item PINE OIL 50% is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.1 µL/plate under the test conditions.

Executive summary:

The test item PINE OIL 50% was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471, following the Principles of GLP. The experiments were carried out in triplicate using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA (pKM101) in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). Based on the results of the solubility test, the test item was formulated in DMSO as vehicle. In an initial cytotoxicity test, TA100 tester strain was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 µL /plate. There was cytotoxicity in all tested concentrations, thus a follow up cytotoxicity test was performed at 0.4, 0.3, 0.2, 0.1, 0.05, 0.025 and 0.0125 µL/plate. Based on the results of these cytotoxicity tests, concentrations of 0.001, 0.0032, 0.01, 0.032 and 0.1 µL/plate were finally selected for testing in the mutation test. Vehicle control and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, 4-nitroquinoline N-oxide for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. Two trials were carried out for this study in triplicate. Trial I was carried out as plate incorporation method and Trial II as pre incubation method. Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials. The mean values of revertant colonies of the negative (vehicle) control plates were within the historical control range. The number of revertant colonies in the positive controls resulted in 2.4 to 15.7 fold increase under identical conditions. Based on the results of the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.1 µL/plate under the test conditions.