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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 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.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol
EC Number:
222-294-1
EC Name:
3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol
Cas Number:
3407-42-9
Molecular formula:
C16H28O
IUPAC Name:
3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexanol
Test material form:
liquid
Details on test material:
- Name of the test material: 3-(5,5,6-Trimethylbicyclo(2.2.1)hept-2-yl)cyclohexanol
- Common Name: Isobornyl cyclohexanol
- IUPAC name: 3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexanol
- Molecular formula: C16H28O
- Molecular weight: 236.396 g/mol
- Substance type: Organic
- Smiles: O[C@@H]1CCC[C@@H]([C@@H]2[C@@H]3C[C@@H](C(C)(C)[C@@H]3C)C2)C1

Method

Target gene:
Histidine
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
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Aroclor 1254-induced S9 was procured from Defence Research and Development Organization and stored at -60°C to -80°C inside the deep freezer. The protein concentration in the S9 fraction was 36.2 mg/mL.
- source of S9 : Aroclor 1254-induced S9 was procured from Defence Research and Development Organization and stored at -60°C to -80°C inside the deep freezer.
- method of preparation of S9 mix : An appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final concentration of approximately 10 % v/v in the S9 mix. Cofactor solution contains the following quantity of chemicals in 500 mL of Distilled Water.
D-glucose-6-phosphate= 0.8 g
β-NADP = 1.75 g
MgCl2 = 1.0 g
KCl = 1.35 g
Na2HPO4 = 6.4 g
NaH2PO4.H2O = 1.4 g
During the experiment, the S9 mix was prepared freshly.
- concentration or volume of S9 mix and S9 in the final culture medium : The protein concentration in the S9 fraction was 36.2 mg/mL. Each batch of S9 mix was tested with 2-Aminoanthracene as well as benzo (a) pyrene for its efficiency. Thus, requirements of Ames were fulfilled, and the results of efficiency testing were archived at RCC Laboratories India Private Limited.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):Each batch of S9 mix was tested with 2-Aminoanthracene as well as benzo (a) pyrene for its efficiency. Thus, requirements of Ames were fulfilled, and the results of efficiency testing were archived at RCC Laboratories India Private Limited.
Test concentrations with justification for top dose:
0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO
Controls
Untreated negative controls:
yes
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
Key result
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
True 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, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 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 0.002 – 5.0 mg/plate based on the solubility and precipitation test.
There was no reduction in colony count but reduction in bacterial background lawn was observed in treated concentrations 5.0 mg/plate (T8), 1.582 mg/plate (T7) and no reduction in colony count as well as in bacterial background lawn in treated concentrations (0.501 (T6) mg/plate – 0.002 (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, 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate , both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The spontaneous reversions in the solvent, negative and positive controls were within the range of the inhouse historical control data

Ames test:
- Mean number of revertant colonies per plate and standard deviation: No substantial increase in the number of revertant colonies of any tester strains were observed following treatment with the test chemical at any dose level in both confirmatory tests neither in the presence nor in the absence of metabolic activation system. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)

R

Without metabolic activation (-S9)

With metabolic activation (+S9)

TA100

TA 98

TA100

TA 98

NC

(0.00)

R1

108

20

110

22

R2

116

17

114

16

R3

110

22

117

17

VC

(0.00)

R1

128

27

135

29

R2

130

24

132

28

R3

122

28

128

26

T1

(0.002)

R1

110

18

112

19

R2

111

19

114

17

R3

115

20

117

18

T2

(0.005)

R1

109

21

115

21

R2

113

20

119

20

R3

114

19

120

20

T3

(0.016)

R1

119

22

122

22

R2

117

22

120

21

R3

116

21

123

24

T4

(0.050)

R1

119

18

124

23

R2

120

21

124

22

R3

118

24

125

25

T5

(0.158)

R1

121

22

126

24

R2

123

23

127

25

R3

119

24

125

24

T6

(0.501)

R1

124

25

128

25

R2

125

24

130

26

R3

122

24

129

27

T7

(1.582)

R1

123

25

129

24

R2

125

24

127

26

R3

126

26

131

27

T8

(5)

R1

125

27

130

28

R2

127

25

129

27

R3

126

26

126

25

PC

R1

1320

956

1376

1544

R2

1236

972

1392

1608

R3

1448

1012

1352

1428

NC           =     Negative control

VC           =   Vehicle Control

PC            =     Positive control             

R              =     Replicate

T              =     Test concentration (T8: Highest, T1: Lowest)

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

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

 

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

4

9

22

110

244

R2

4

11

16

114

229

R3

3

11

17

117

237

VC

(0.00)

R1

7

16

29

135

294

R2

7

15

28

132

278

R3

8

15

26

128

294

T1

(0.005)

R1

4

12

21

115

235

R2

4

11

20

119

236

R3

4

10

20

120

240

T2

(0.016)

R1

5

13

22

122

241

R2

4

11

21

120

248

R3

4

12

24

123

237

T3

(0.050)

R1

4

13

23

124

248

R2

5

13

22

124

254

R3

5

14

25

125

261

T4

(0.158)

R1

6

14

24

126

265

R2

5

13

25

127

269

R3

6

15

24

125

275

T5

(0.501)

R1

5

14

25

128

268

R2

8

16

26

130

283

R3

7

15

27

129

274

PC

R1

148

502

1544

1376

1590

R2

164

428

1608

1392

1648

R3

167

474

1428

1352

1674

 

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

20

108

237

R2

5

9

17

116

228

R3

4

12

22

110

240

VC

(0.00)

R1

8

16

27

128

290

R2

7

14

24

130

264

R3

6

15

28

122

282

T1

(0.005)

R1

5

11

21

109

234

R2

4

10

20

113

236

R3

5

11

19

114

240

T2

(0.016)

R1

6

10

22

119

244

R2

5

11

22

117

241

R3

4

12

21

116

247

T3

(0.050)

R1

5

13

18

119

260

R2

6

12

21

120

251

R3

5

11

24

118

247

T4

(0.158)

R1

6

13

22

121

271

R2

6

12

23

123

259

R3

5

14

24

119

263

T5

(0.501)

R1

6

13

25

124

274

R2

8

15

24

125

269

R3

6

14

24

122

277

PC

R1

158

1016

956

1320

1672

R2

172

1200

972

1236

1548

R3

164

1158

1012

1448

1624

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 [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

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

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

10.33

1.15

18.33

3.21

113.67

3.51

236.67

7.51

VC

(0.00)

7.33

0.58

15.33

0.58

27.67

1.53

131.67

3.51

288.67

9.24

T1

(0.005)

4.00

0.00

11.00

1.00

20.33

0.58

118.00

2.65

237.00

2.65

T2

(0.016)

4.33

0.58

12.00

1.00

22.33

1.53

121.67

1.53

242.00

5.57

T3

(0.050)

4.67

0.58

13.33

0.58

23.33

1.53

124.33

0.58

254.33

6.51

T4

(0.158)

5.67

0.58

14.00

1.00

24.33

0.58

126.00

1.00

269.67

5.03

T5

(0.501)

6.67

1.53

15.00

1.00

26.00

1.00

129.00

1.00

275.00

7.55

PC

159.67

10.21

468.00

37.36

1526.67

91.24

1373.33

20.13

1637.33

43.00

 

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

10.33

1.53

19.67

2.52

111.33

4.16

235.00

6.24

VC

(0.00)

7.00

1.00

15.00

1.00

26.33

2.08

126.67

4.16

278.67

13.32

T1

(0.005)

4.67

0.58

10.67

0.58

20.00

1.00

112.00

2.65

236.67

3.06

T2

(0.016)

5.00

1.00

11.00

1.00

21.67

0.58

117.33

1.53

244.00

3.00

T3

(0.050)

5.33

0.58

12.00

1.00

21.00

3.00

119.00

1.00

252.67

6.66

T4

(0.158)

5.67

0.58

13.00

1.00

23.00

1.00

121.00

2.00

264.33

6.11

T5

(0.501)

6.67

1.15

14.00

1.00

24.33

0.58

123.67

1.53

273.33

4.04

PC

164.67

7.02

1124.67

96.42

980.00

28.84

1334.67

106.76

1614.67

62.52

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-INCUBATION METHOD (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)

4.00

1.00

10.33

0.58

15.67

0.58

90.33

5.51

243.00

7.00

VC

(0.00)

7.33

0.58

15.00

1.00

27.67

0.58

114.67

3.51

279.67

4.73

T1

(0.005)

4.67

1.15

10.67

1.15

16.00

1.00

93.67

7.57

247.33

6.11

T2

(0.016)

5.00

0.00

12.00

1.00

16.67

1.53

100.00

2.65

254.67

6.11

T3

(0.050)

5.67

0.58

11.67

0.58

19.00

2.65

104.67

6.03

262.33

6.11

T4

(0.158)

6.67

0.58

13.00

1.00

23.00

2.00

107.33

4.51

271.33

5.51

T5

(0.501)

7.00

0.00

14.67

0.58

26.33

0.58

109.67

7.02

278.00

2.65

PC

155.33

5.69

390.67

28.10

1118.00

24.98

1382.67

42.39

1426.00

36.17

 

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

0.00

10.67

1.53

15.33

1.53

90.00

6.00

236.33

4.04

VC

(0.00)

7.33

0.58

15.33

0.58

26.33

1.53

115.00

5.57

281.00

7.00

T1

(0.005)

4.33

0.58

11.00

1.00

15.67

0.58

94.67

5.51

239.00

5.00

T2

(0.016)

4.67

0.58

12.00

1.00

16.33

1.53

99.33

2.52

247.00

6.00

T3

(0.050)

5.33

0.58

13.00

1.00

20.00

1.00

104.00

1.00

252.00

9.54

T4

(0.158)

6.33

0.58

13.67

1.53

23.00

1.00

107.00

3.00

264.67

11.50

T5

(0.501)

7.00

1.00

15.00

1.00

26.00

1.00

110.67

4.04

279.67

5.13

PC

162.67

7.77

1424.00

44.54

801.33

28.38

1274.67

33.31

1593.33

46.01

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, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plates were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). 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.

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.