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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity / in vitro


Bacterial reverse mutation test:


The registered substance, i.e.,  3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol  (CAS No. 3407-42-9) has been tested non-mutagenic (negative) in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 both in the presence and absence of the S9 metabolic activation system. The test was performed as per OECD TG 471 (adopted: on 21st July 1997, corrected on 26th June 2020) and GLP. 


 


In vitro mammalian chromosome aberration test:


The test material, 3-(5,5,6-trimethylbicyclo[2.2.1] hept-2-yl)cyclohexan-1-ol (CAS No. 3407-42-9), has been tested non-clastogenic (negative)  both in the presence (1% and 2%) and the absence of metabolic activation in an invitro mammalian chromosomal aberration test using primary culture of human peripheral lymphocytes. The study was performed according to OECD 473 and GLP.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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:
The study contains experimental data of the registered substance
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
adopted on July 29 2016
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Identification : 3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol
Description : Colourless viscous liquid
Chemical Name : 3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol
Batch Number : KC2007203
CAS No. : 3407-42-9
Purity : 98.10 % (GC)
Molecular Weight : 236.396
Molecular Formula : C16H28O
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Human Peripheral Blood Lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented S9 microsomal fraction, the cofactor contained D-glucose-6-phosphate(0.80), MgCl2(1g), KCl(1.35g), NaHPO4(6.40g), NaH2PO4.H20(1.40g), beta-NADP(1.75g) in 500ml of Reverse Osmosis (RO) water. The S9 microsomal fraction was prepared from the liver of Aroclor1254-induced rats
Test concentrations with justification for top dose:
Test concentrations: 0.004, 0.008 and 0.016 mg/mL
Justification: In preliminary cyto toxicity test, the test concentration 0.016 (T9) mg/mL produced a 52.72 % and 53.01% decrease in the mitotic index compared to VC in the absence (-S9) and presence of metabolic activation (+S9), respectively. The test concentration 0.008 (T8) mg/mL produced 23.91% and 26.26% decreases in the mitotic index compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively. The test concentration 0.004 (T7) mg/mL produced 17.31% and 16.62 % decreases in the mitotic index (cytotoxicity) compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively.
The dose level that results in approximately 55 ± 5 % reduction in the mitotic index was selected as the highest test concentration in the main test. Hence, the concentration of 0.016 mg/mL was chosen as the highest test item concentration for the main study, both in the presence and absence of metabolic activation. Test concentration were spaced by factor 2.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:Ethyl alcohol

- Justification for choice of solvent/vehicle: Test item was soubilized in ethyl alcohol.

- Justification for percentage of solvent in the final culture medium: 2 mg/mL (recommended maximum test concentration for non-cytotoxic substances)
Untreated negative controls:
yes
Remarks:
Distille water
Negative solvent / vehicle controls:
yes
Remarks:
Ethyl alcohol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentratons : Duplicate
- Number of independent experiments : Two independent experiments (Phase I and II)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): NA
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: 4 hrs (Phase I with and without S9, and Phase II with S9), 24 hrs (Phase II, without S9)
- Harvest time after the end of treatment (sampling/recovery times): 24 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays):Colcemid (final concentration: 0.3 µg/mL) was added three hours before harvesting to stop cell proliferation
- If cytokinesis blocked method was used for micronucleus assay: NA
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells were fixed by Carnoy's fixative. The slides were prepared by dropping the cell suspension onto a clean pre-chilled microscope slide. The slides were dried on the slide warmer and labelled. Two slides were made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX. All slides, including positive, vehicle, and negative controls, were independently coded before microscopic analysis.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): : A minimum of 1000 cells was counted in different fields of slide per culture, and the number of metaphases was recorded for mitotic index calculation. Three hundred well-spread metaphases per concentration and control were scored for cytogenetic damage on coded slides.The evaluation was performed using microscopes with 100 x oil immersion objectives.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): No micronucleated cells were detected as it is a CA test.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Chromosomal and chromatid gaps, breaks, acentric, fragments, exchanges, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46  2 centromere regions were included in the analysis. The Mitotic Index (% cells in mitosis) was determined to describe a cytotoxic effect
- Determination of polyploidy: yes
- Determination of endoreplication: Yes


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Cytotoxicity was determined by a reduction in the mitotic index in comparison with vehicle control data
Evaluation criteria:
A test item was classified as clastogenic if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control.
- if the increase is dose-dependent when evaluated with the appropriate trend test
- any of the results are outside the historical vehicle control range.
A test item was classified as non-clastogenic if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control.
- if there is no dose-dependent increase.
- all results are inside the historical vehicle control range.
Statistics:
Statistical significance was confirmed using the non-parametric Mann-Whitney test. However, both biological and statistical significance were considered together
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In Phase I, 0.016 mg/mL of test item produced a 53.61% (-S9) and 51.29% (+S9) decrease in MI compared to VC. In Phase II, 0.016 mg/ml of test item produced a 52.86% (-S9) and 52.42% (+S9) decrease in MI compared to VC.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Phase I : The mean percentages of aberrant cells were 0.333 (NC), 0.333 (VC), 0.667 (at 0.004 mg/mL), 0.333 (0.008 mg/mL), 0.667 (0.016 mg/mL) and 10.333 (at 600 µg/mL EMS - PC) in the absence of metabolic activation. In the presence of metabolic activation, the mean percentage of aberrant cells were 0.333 (NC), 0.667 (VC), 0.333 (at 0.004 mg/mL), 0.667 (0.008 mg/mL), 0.667 (0.016 mg/mL) and 10.000 (at 30 µg/mL CPA - PC).

Phase II : In the absence of metabolic activation, the mean percent aberrant cells were 0.333 (NC), 0.333 (VC), 0.337 (at 0.004 mg/mL), 0.667 (0.008 mg/mL), 1.000 (0.016 mg/mL) and 10.333 (at 600 µg/mL EMS - PC). In the presence of the metabolic activation, the mean percent aberrant cells were 0.333 (NC), 0.667 (VC), 0.333 (at 0.004 mg/mL), 0.667 (0.008 mg/mL), 1.000 (0.016 mg/mL) and 11.000 (at 30 µg/mL CPA - PC).

SOLUBILITY, PRECIPITATION AND pH CHECK:
Based on the solubility, precipitation and pH tests, Ethyl alcohol was selected as vehicle control for the study.
Solubility Details
Solvent used Distilled water DMSO Acetone Ethyl alcohol
Quantity of test item 80.01 mg 80.02 mg 80.03 mg 80.01 mg
Volume of vehicle added 400 µL 400 µL 400 µL 400 µL
Final Concentration 200 mg / mL 200 mg / mL 200 mg / mL 200 mg / mL
Sample ID A B C D
Solubility status Insoluble Insoluble Insoluble Soluble

As mentioned in the above table, the solubility of the test item was checked in Distilled water, Dimethyl Sulphoxide (DMSO), and Acetone, and the test substance was found insoluble at 200 mg/mL. The solubility of the test item was checked in Ethyl alcohol, and it was found soluble at 200 mg/mL to give the final treatment concentration of 2 mg/mL culture (recommended maximum test concentration for non-cytotoxic substances). Therefore, Ethyl alcohol was selected as a solvent for the study.

Precipitation Details
Sample ID Volume of Volume of Concentration mg/ml Result
Test item RPMI0
preparation
D 80 µL of D 7.92 mL 2 mg/mL Precipitation
D 80 µL of D 7.92 mL 1 mg/mL Slight Precipitation

Precipitation was observed in 2 mg/mL concentration. Slight precipitation was observed at 1 mg/mL, which was judged not to interfere with the conduct of the test. Hence, the concentration of 1 mg/mL was selected as the high dose for the cytotoxicity experiment.
To assess the effect of the test item on the pH of medium, the pH of the culture medium (with the test item added) was measured following 0 and 4 hours of exposure at 37°C. No significant changes in pH were observed at 0 or 4 hours when compared with the Vehicle control.

pH Details
Sample Time pH Mean ± SD
S1 0th hour 7.33 7.31 ± 0.03
S2 7.35
S3 7.41
CT1 0th hour 7.28 7.36 ± 0.04
CT2 7.33
CT3 7.31
S1 4th hour 7.31 7.31 ± 0.04
S2 7.35
S3 7.28
CT1 4th hour 7.38 7.35 ± 0.03
CT2 7.33
CT3 7.34

S = sample, CT = Control



Remarks on result:
other: Non-mutagenic potential

MITOTIC INDEX - CYTOTOXICITYEXPERIMENT III

Treatment

R

Mitotic Index (%)

Absence of

Metabolic Activation (-S9)

Presence of

Metabolic Activation (1% S9)

Mitotic Index

Mean

SD

Percent

Reduction vs. NC

Mitotic Index

Mean

SD

Percent Reduction vs. NC

NC

(0.0 mg/mL)

R1

10.38

10.27

0.16

-

10.20

10.10

0.15

-

R2

10.16

9.99

VC 

(0.0 mg/mL)

R1

9.36

9.17

0.27

-

9.97

9.86

0.15

2.32

R2

8.98

9.75

T7
(0.004 mg/mL)

R1

7.69

7.58

0.15

17.31

7.96

8.22

0.37

16.62

R2

7.48

8.48

T8
(0.008 mg/mL)

R1

7.07

6.98

0.13

23.91

7.36

7.27

0.13

26.26

R2

6.89

7.18

T9
(0.016 mg/mL)

R1

4.39

4.34

0.07

52.72

4.79

4.63

0.21

53.01

R2

4.29

4.48

PC

R1

7.98

8.04

0.08

12.37

8.68

8.43

0.35

14.48

R2

8.09

8.18

Key:R = Replicate,NC = Negative control,VC = Vehicle control,PC = Positive control,SD = Standard Deviation

SUMMARY OF MITOTIC INDEX

Mitotic Index (%)

Phase I

Treatment

 Absence of

Metabolic Activation (-S9)

Treatment

Presence of

Metabolic Activation (+S9) (1%)

Mean

SD

Percentage reduction vs. VC

Mean

SD

Percentage reduction

vs. VC

NC

10.23

0.20

-

NC

10.63

0.22

-

VC 

9.69

0.13

-

VC

9.53

0.07

10.37

T1

(0.004 mg/mL)

7.78

0.28

19.63

T1

(0.004 mg/mL)

7.83

0.21

17.80

T2

 (0.008 mg/mL)

7.03

0.23

27.39

T2

 (0.008 mg/mL)

6.79

0.29

28.79

T3

 (0.016 mg/mL)

4.49

0.42

53.61

T3

 (0.016 mg/mL)

4.64

0.35

51.29

PC

8.28

0.44

14.47

PC

8.28

0.15

13.13

Mitotic Index (%)

Phase II

Treatment

Absence of

Metabolic Activation (-S9)

Treatment

Presence of

Metabolic Activation (+S9) (2%)

Mean

SD

Percentage reduction vs. VC

Mean

SD

Percentage reduction vs. VC

NC

10.13

0.22

-

NC

10.37

0.29

0

VC

9.84

0.21

-

VC

9.33

0.21

10.05

T1

(0.004 mg/mL)

7.88

0.14

19.92

T1

(0.004 mg/mL)

7.98

0.14

14.48

T2

 (0.008 mg/mL)

7.49

0.28

23.90

T2

 (0.008 mg/mL)

7.13

0.21

23.57

T3

 (0.016 mg/mL)

4.64

0.21

52.86

T3

 (0.016 mg/mL)

4.44

0.06

52.41

PC

8.48

0.14

13.79

PC

8.03

0.22

13.95

Key:NC = Negative control VC = Vehicle control,,PC = Positive control, MI = Mitotic Index, -S9 = Absence of metabolic activation, + S9 = Presence of metabolic activation

SUMMARY OF PERCENT ABERRANT CELLS

Percent Aberrant Cells

Phase I

Treatment

Absence of

Metabolic Activation (-S9)

Treatment

Presence of

Metabolic Activation (+S9) (1%)

Mean

SD

Mean

SD

NC

0.333

0.471

NC

0.333

0.471

VC

0.333

0.471

VC

0.667

0.000

T1

(0.004 mg/mL)

0.667

0.000

T1

(0.004 mg/mL)

0.333

0.471

T2

 (0.008 mg/mL)

0.333

0.471

T2

 (0.008 mg/mL)

0.667

0.000

T3

 (0.016 mg/mL)

0.667

0.000

T3

 (0.016 mg/mL)

0.667

0.000

PC

10.333

0.471

PC

10.000

0.943

Percent Aberrant Cells

Phase II

Treatment

Absence of

Metabolic Activation (-S9)

Treatment

Presence of

Metabolic Activation (+S9) (2%)

Mean

SD

Mean

SD

NC

0.333

0.471

NC

0.333

0.471

VC

0.333

0.471

VC

0.667

0.000

T1

(0.004 mg/mL)

0.333

0.471

T1

(0.004 mg/mL)

0.333

0.471

T2

 (0.008 mg/mL)

0.667

0.000

T2

(0.008 mg/mL)

0.667

0.000

T3

 (0.016 mg/mL)

1.000

0.471

T3

(0.016 mg/mL)

1.000

0.471

PC

10.333

0.471

PC

11.000

0.471

Key:NC = Negative control VC = Vehicle control,PC = Positive control, MI = Mitotic Index, -S9 = Absence of metabolic activation, + S9 = Presence of metabolic activation.

INDIVIDUAL OBSERVATION OF SLIDES FOR MITOTIC INDEX AND CHROMOSOME ABERRATIONS

Phase I [In the Absence of Metabolic Activation, (-S9)]

Treatment

Culture No.

Mitotic Index

Frequencies of Aberration

Total No. of Aberration

Number of aberrated cells

Percentage of Aberrated Cells

NC

R1

10.09

-

0

0

0.00

R2

10.38

1 cse

1

1

0.67

VC

R1

9.59

1 cte

1

1

0.67

R2

9.78

-

0

0

0.00

T1

(0.004 mg/mL)

R1

7.98

1 cse

1

1

0.67

R2

7.58

1 ctb

1

1

0.67

T2

 (0.008 mg/mL)

R1

6.87

-

0

0

0.00

R2

7.19

1 ctb, 1 AC

2

1

0.67

T3

 (0.016 mg/mL)

R1

4.79

1 csb

1

1

0.67

R2

4.20

1 cse

1

1

0.67

PC

R1

8.59

4 ctb, 4 cte, 3 ctg, 4 csb, 3 cse, 2 csg, 2 AC, 06 fragments

23

15

10.00

R2

7.98

5 ctb, 6 cte, 3 ctg, 4 csb, 4cse, 4 csg, 2 AC, 06 fragments

27

16

10.67

Phase I [In the Presence of Metabolic Activation (1% S9)]

Treatment

Culture No.

Mitotic Index

Frequencies of Aberration

Total No. of Aberration

Number of aberrated cells

Percentage of Aberrated Cells

NC

R1

10.48

-

0

0

0.00

R2

10.79

1 ctb

1

1

0.67

VC

R1

9.58

1 cse

1

1

0.67

R2

9.48

1 cte, 1 csb

2

1

0.67

T1

(0.004 mg/mL)

R1

7.68

1 cte

1

1

0.67

R2

7.98

-

0

0

0.00

T2

 (0.008 mg/mL)

R1

6.99

1 ctb, 1 cte

2

1

0.67

R2

6.58

1 csb,

1

1

0.67

T3

 (0.016 mg/mL)

R1

4.89

1 cte, 1 ctb

2

1

0.67

R2

4.40

1 cse, 1 ctb

2

1

0.67

PC

R1

8.38

5 ctb, 3 cte, 2 ctg, 5 csb, 5 cse, 1 csg, 2 AC, 05 fragments

25

16

10.67

R2

8.18

4 ctb, 4 cte, 2 ctg, 3 csb, 3 cse, 3 csg, 1 AC, 07 fragments

22

14

9.33

Key: MI = Mitotic Index, ctg = Chromatid gap, ctb = Chromatid break, csg = Chromosomal gap, csb = Chromosomal break, DC = Dicentric,AC = Acentric, cte = Chromatid exchange, cse = Chromosome exchange, NC = Negative Control, VC = Vehicle Control, PC = Positive Control

Phase II [In the Absence of Metabolic Activation (-S9)]

Treatment

Culture No.

Mitotic Index

Frequencies of Aberration

Total No. of Aberration

Number of aberrated cells

Percentage of Aberrated Cells

NC

R1

10.29

-

0

0

0.00

R2

9.98

1 cse

1

1

0.67

VC

R1

9.99

-

0

0

0.00

R2

9.69

1 csb, 1 AC

2

1

0.67

T1

(0.004 mg/mL)

R1

7.78

1 cte

2

1

0.67

R2

7.98

-

0

0

0.00

T2

 (0.008 mg/mL)

R1

7.68

1 cte, 1 AC

2

1

0.67

R2

7.29

1csb

1

1

0.67

T3

 (0.016 mg/mL)

R1

4.49

1 cte

1

1

0.67

R2

4.79

1 csb, 1 cte, 1 AC

3

2

1.33

PC

R1

8.38

4ctb,6cte,2ctg,4csb,4cse,1csg,1 DC, 3AC,07fragments

29

16

10.67

R2

8.58

5ctb,3cte,2ctg,2csb,5cse,1csg,1 DC, 3AC,05fragments

24

15

10.00

Phase II [In the Presence of Metabolic Activation (2% S9)]

Treatment

Culture No.

Mitotic Index

Frequencies of Aberration

Total No. of Aberration

Number of aberrated cells

Percentage of Aberrated Cells

NC

R1

10.58

1cte

1

1

0.67

R2

10.17

-

0

0

0.00

VC

R1

9.18

1 csb

1

1

0.67

R2

9.48

1 ctb

1

1

0.67

T1

(0.004 mg/mL)

R1

7.88

1 cte, 1 csb

2

1

0.67

R2

8.08

-

0

0

0.00

T2

 (0.008 mg/mL)

R1

7.28

1ctb, 1 cte

2

1

0.67

R2

6.99

1 csb

1

1

0.67

T3

 (0.016 mg/mL)

R1

4.40

1 ctb, 1 cse

2

2

1.33

R2

4.49

1 cte

1

1

0.67

PC

R1

8.18

5 ctb, 3 cte, 1 ctg, 4 csb, 5 cse, 1 csg, 1 AC, 04 fragments

22

16

10.67

R2

7.88

4 ctb, 5 cte, 2 ctg, 5 csb, 3 cse, 1 csg, 1 DC, 2 AC, 05 fragments

25

17

11.33

Key: MI = Mitotic Index, ctg = Chromatid gap, ctb = Chromatid break, csg = Chromosomal gap, csb = Chromosomal break, AC = Acentric, cte = Chromatid exchange, cse = Chromosome exchange, NC = Negative Control, VC = Vehicle Control, PC = Positive Control

HISTORICAL DATA

HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H)
RCC LABORATORIES INDIA PVT LTD, HYDERABAD, INDIA.

S.No.

Study No.

Vehicle

Phase I

Phase II

Absence of S9

Presence of S9

Absence of S9

Presence of S9

Vehicle Control

Positive Control

Vehicle Control

Positive Control

Vehicle Control

Positive Control

Vehicle Control

Positive Control

1

1151

DMSO

0.000

9.000

0.000

10.500

0.000

9.000

0.000

8.000

2

1333

DMSO

0.000

8.000

0.000

7.500

0.500

8.500

0.500

9.000

3

2060

DMSO

0.500

8.000

0.000

7.000

1.500

6.500

0.000

9.000

4

2450

DMSO

0.000

10.000

0.000

10.500

0.000

11.500

0.000

12.000

5

2452

DMSO

0.000

10.000

0.000

8.500

0.000

9.500

0.000

8.500

6

3000

PBS

0.000

7.500

0.000

8.500

0.000

11.000

0.000

10.000

7

3313

DMSO

0.000

8.000

0.000

10.500

0.500

9.500

0.000

9.500

8

3422

DMSO

0.000

9.000

0.500

10.000

1.000

9.500

1.000

8.500

9

3665

RPMI

0.500

8.500

0.000

7.500

0.000

8.500

0.500

8.000

10

3801

Sodium Phosphate Buffer

1.500

9.500

1.000

9.000

1.000

9.500

0.500

9.500

11

3862

DMSO

1.500

9.500

1.000

9.000

1.000

9.500

0.500

9.500

12

4792

PBS

0.500

7.500

0.500

8.500

0.500

8.500

0.000

8.000

13

4938

DMSO

0.500

8.500

1.000

8.500

0.500

8.000

1.000

8.000

14

5123

DMSO

0.333

9.000

0.667

8.667

0.333

9.667

0.333

9.000

15

5739

Ethyl alcohol

0.333

10.333

0.333

10.000

0.333

9.667

0.333

10.667

16

5824

PBS

0.333

10.000

0.333

11.000

0.333

9.333

0.333

10.000

17

6461

PBS

0.333

10.000

0.333

9.667

0.333

9.000

0.333

10.000

18

6196

RPMI

0.333

11.000

0.333

10.000

0.333

10.667

0.333

10.000

19

6121

DMSO

0.667

8.667

0.667

9.667

0.667

9.667

0.667

9.333

20

6678

DMSO

0.333

10.333

0.333

10.000

0.333

9.667

0.333

10.667

21

6687

DMSO

0.333

11.333

0.333

11.333

0.333

12.333

0.333

12.000

22

6221

DMSO

0.333

9.667

0.333

10.667

0.333

9.667

0.333

10.333

23

6834

DMSO

0.333

10.333

0.333

11.333

0.333

11.333

0.333

10.667

24

6759

PBS

0.667

10.667

0.000

10.000

0.333

12.000

0.333

11.333

25

6430

DMSO

0.333

9.000

0.333

10.000

0.667

9.667

0.667

9.667

26

7703

DMSO

0.333

10.000

0.333

10.000

0.333

10.333

0.333

10.333

27

7576

RPMI

0.333

10.000

0.333

10.667

0.333

10.333

0.333

10.333

28

7572

DMSO

0.667

10.333

0.667

10.000

0.667

9.667

0.333

10.000

29

7574

Ethyl alcohol

0.333

10.333

0.333

10.333

0.333

10.000

0.333

10.333

30

7434

DMSO

0.667

10.000

0.333

10.667

0.333

9.667

0.333

11.000

31

7708

DMSO

0.333

9.667

0.333

10.333

0.333

9.333

0.333

9.667

32

7263

DMSO

0.333

10.667

0.333

10.333

0.333

10.667

0.333

9.667

33

8072

DMSO

0.333

10.333

0.333

10.000

0.333

10.333

0.333

10.333

HISTORICAL DATA FOR IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST (CA-H)
RCC LABORATORIES INDIA PVT LTD, HYDERABAD, INDIA.

S.No.

Study No.

Vehicle

Phase I

Phase II

Absence of S9

Presence of S9

Absence of S9

Presence of S9

Vehicle Control

Positive Control

Vehicle Control

Positive Control

Vehicle Control

Positive Control

Vehicle Control

Positive Control

34

4825

DMSO

0.667

9.000

0.667

9.333

0.333

10.000

0.667

9.000

35

8112

DMSO

0.333

9.667

0.333

10.000

0.333

10.667

0.333

10.333

36

8142

DMSO

0.333

10.000

0.333

10.333

0.333

10.000

0.333

10.333

37

8091

DMSO

0.333

10.333

0.333

10.333

0.333

10.000

0.333

10.000

38

8174

RPMI

0.333

11.000

0.333

10.000

0.333

9.667

0.333

10.667

39

7657

DMSO

0.333

10.333

0.333

11.333

0.333

10.000

0.333

11.000

40

8176

DMSO

0.333

11.333

0.333

8.667

0.333

10.667

0.333

10.000

41

8541

DMSO

0.667

9.667

0.333

10.000

0.667

9.667

0.333

10.000

42

8064

DMSO

0.333

10.667

0.667

9.667

0.333

10.333

0.333

10.000

43

8486

DMSO

0.333

10.000

0.333

10.333

0.333

10.333

0.667

11.333

44

8660

RPMI

0.333

11.000

0.333

10.000

0.333

10.333

0.333

10.000

45

8722

DMSO

0.667

10.000

0.667

10.667

0.667

9.333

0.667

10.666

46

8670

DMSO

0.333

10.000

0.333

11.000

0.333

10.667

0.667

10.000

47

8680

DMSO

0.333

10.333

0.667

10.333

0.333

10.000

0.333

10.000

48

8658

DMSO

0.333

10.000

0.333

11.000

0.333

10.667

0.667

10.000

49

9845

DMSO

0.333

10.000

0.333

11.000

0.333

10.667

0.333

10.667

50

9861

DMSO

0.333

10.667

0.333

10.333

0.333

10.333

0.667

10.000

51

9862

DMSO

0.667

10.000

0.333

9.000

0.333

10.000

0.333

10.667

52

9911

DMSO

0.333

10.667

0.333

11.333

0.333

9.333

0.333

10.000

53

9925

DMSO

0.333

11.333

0.333

11.000

0.333

11.333

0.333

11.000

54

10049

DMSO

0.333

10.000

0.333

11.000

0.333

9.667

0.667

11.000

55

9939

DMSO

0.333

9.667

0.333

11.000

0.333

8.000

0.333

9.000

56

10679

DMSO

0.333

11.667

0.333

10.667

0.333

13.333

0.333

15.000

57

10807

DMSO

0.333

10.333

0.667

11.000

0.333

9.000

0.333

10.667

58

10858

RPMI

0.667

10.333

0.667

11.000

0.333

11.000

0.333

10.667

Mean

0.396

9.874

0.379

10.009

0.406

9.948

0.385

10.083

SD

0.277

0.941

0.241

1.007

0.254

1.079

0.215

1.124

Mean + 2SD

0.951

11.755

0.861

12.023

0.914

12.106

0.815

12.330

Mean - 2SD

-0.158

7.992

-0.104

7.995

-0.103

7.791

-0.045

7.836

Conclusions:
The test material, 3-(5,5,6-trimethylbicyclo[2.2.1] hept-2-yl)cyclohexan-1-ol (CAS No. 3407-42-9) was tested as non-clastogenic (negative) both in the presence (1% and 2%) and the absence of metabolic activation in an invitro mammalian chromosomal aberration test using primary culture of human peripheral lymphocytes. The study was performed according to OECD 473 and GLP.
Executive summary:

The study was performed to determine the clastogenic potential of the test material, 3-(5,5,6-trimethylbicyclo[2.2.1] hept-2-yl)cyclohexan-1-ol (CAS No. 3407-42-9) on primary culture of human peripheral blood lymphocytes. The study was performed according to OECD test guideline No. 473, adopted on July 29, 2016. The study was performed in two independent assay (Phase I-II) in the presence and absence of S9 metabolic activation. The solubility of the test item was checked in Distilled water, Dimethyl Sulphoxide (DMSO), and Acetone, and the test substance was found insoluble at 200 mg/mL. The solubility of the test item was checked in Ethyl alcohol, and it was found soluble at 200 mg/mL to give the final treatment concentration of 2 mg/mL culture (recommended maximum test concentration for non-cytotoxic substances). Therefore, Ethyl alcohol was selected as a solvent for the test substance during the study. Precipitation was observed at 2 mg/mL concentration. Slight precipitation was observed at 1 mg/mL, which was judged not to interfere with the conduct of the test. Hence, the concentration of 1 mg/mL was selected as the high dose for the cytotoxicity experiment. Cytotoxicity was assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.25, 0.50 and 1.00  mg/ml in treated culture media. All the tested concentrations in the initial cytotoxicity experiment were cytotoxic both in the presence and absence of the S9 metabolic activation system. Next, cytotoxicity was assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.031, 0.063 and 0.125 mg/ml of culture media in a second cytotoxicity experiment (Experiment II). All the tested concentrations in the cytotoxicity experiment (II) were cytotoxic both in the presence and absence of the metabolic activation system. The cytotoxicity was then assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.004, 0.008  and 0.016 mg/mL in treated culture media in the third cytotoxicity experiment (Experiment III). The test concentration 0.016 mg/mL produced a 52.72 % and 53.01% decrease in the mitotic index compared to VC in the absence (-S9) and presence of metabolic activation (+S9), respectively. The test concentration 0.008 (T8) mg/mL produced 23.91% and 26.26% decreases in the mitotic index compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively. The test concentration 0.004 (T7) mg/mL produced 17.31% and 16.62 % decreases in the mitotic index (cytotoxicity) compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively. The dose level that results in approximately 55 ± 5 % reduction in the mitotic index was selected as the highest test concentration in the main test. Hence, the following test concentrations were assessed in the main study: 0.0 (NC), 0.0 (VC), 0.004, 0.008 and 0.16 mg/ml along with positive control substances in the presence and absence of metabolic activation. The main study was performed in two independent phases (Phase I-II). In Phase I, cells were exposed to the test concentrations of 0.004, 0.008 and 0.016 mg/ml along with negative control (NC: Distilled water) and vehicle control (VC: Ethyl alcohol) for 4 hours both in the presence and absence of S9 metabolic activation (1% v/ v). In Phase II, cells were treated for 4 hours in the presence of increased metabolic activation (2% v/v) and 24 hours in the absence of metabolic activation. Three hours before cell harvesting, colcemid (final concentration: 0.3 µg/ml) was added to each culture tube to stop cell proliferation. The cultures were harvested 24 hours after the beginning of the treatment, fixed then stained with 5% fresh Giemsa stain. A minimum of 1000 cells were counted in different fields of slide per culture, and the number of metaphases was recorded for mitotic index calculation. Three hundred well-spread metaphases per concentration and control were scored for cytogenetic damage on coded slides. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in calculation of the aberration rates. Only metaphases with 46±2 centromere regions were included in the analysis. Results: There was no significant increase in the percent of aberrant cells at any concentrations tested when compared to the vehicle control in both phases and the presence and absence of S9 metabolic activation. In Phase I, the mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.667 (at 0.004 mg/ml), 0.333 (0.008 mg/ml), 0.667 (0.016 mg/ml) and 10.333 (PC: 600 µg/mL EMS) in the absence of metabolic activation. In the presence of metabolic activation, the mean percentage of aberrant cells was 0.333 (NC), 0.667 (VC), 0.333 (at 0.004 mg/ml), 0.667 (0.008 mg/ml), 0.667 (0.016 mg/ml) and 10.000 (PC: 30 µg/mL CPA). The observed mean mitotic index in the absence of metabolic activation was 10.23 (NC), 9.69 (VC), 7.78, 7.03, 4.49 and 8.28 (PC) at 0.0 (NC), 0.0 (VC), 0.004, 0.008 and0.016 mg/ml, and 600 µg/mL EMS (PC), respectively. In the presence of metabolic activation, the mean mitotic index was 10.63 (NC), 9.53 (VC), 7.83, 6.79, 4.64 and 8.28 (PC) at 0.0(NC), 0.0 (VC), 0.004, 0.008 and0.016 mg/ml and 30 µg/mL CPA (PC), respectively. In the Phase II experiment, in the absence of metabolic activation, the mean percent aberrant cells were 0.333 (NC), 0.333 (VC), 0.337 (at 0.004 mg/ml), 0.667 (0.008 mg/ml), 1.000 (0.016 mg/ml) and 10.333 (PC: 600 µg/mL EMS). In the presence of the metabolic activation, the mean percent aberrant cells were 0.333 (NC), 0.667 (VC), 0.333 (at 0.004 mg/ml), 0.667 (at 0.008 mg/ml), 1.000 (at 0.016 mg/ml) and 11.000 (PC: at 30 µg/mL CPA). The observed mean mitotic index in the absence (-S9) of metabolic activation was 10.13 (NC), 9.84 (VC),7.88 (at 0.004 mg/ml),7.49 (at 0.008 mg/ml), 4.64 (0.16 mg/ml) and 8.48 (PC: 600 µg/mL EMS).In the presence (+S9) of metabolic activation, the mean mitotic index was 10.37 (NC), 9.33 (VC), 7.98 (at 0.004 mg/ml), 7.13 (at 0.008 mg/ml),4.44 (at 0.16 mg/ml) and 8.03 (PC: 30 µg/mL CPA). The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system and the suitability of the methods and conditions employed in the experiment. Conclusion: Based on the above results, the test material, 3-(5,5,6-trimethylbicyclo[2.2.1] hept-2-yl)cyclohexan-1-ol (CAS No. 3407-42-9), was tested as non-clastogenic (negative)  both in the presence (1% and 2%) and the absence of cofactor supplemented S9 metabolic activation system using primary culture of human peripheral lymphocytes. The study was conducted according to OECD TG 473 and GLP.

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted 21st July 1997, corrected 26th June 2020
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Purity: 98.10% (GC)
Appearance: Colourless Viscous Liquid
Lot No. KC2007023
Molecular Weight 236.396 g/mol
Molecular Formula: C16H28O
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Cofactor-supplemented liver S9 microsomal fraction was used. The S9 fraction derived from Aroclor 1254-induced rats.
The cofactor solution contained the followers:
D-glucose-6-phosphate: 0.8 g
β-NADP: 1.75 g
MgCl2: 1.0 g
KCl: 1.35 g
Na2HPO4.H2O: 6.4 g
NaH2PO4.H2O: 1.4 g
Test concentrations with justification for top dose:
Test concentrations:
0.0 (NC), 0.0(VC), 0.313, 0.625, 1.250, 2.500 and 5.0 mg/plate

Justification:
Test concentrations were selected based on the solubility and precipitation check and a preliminary cytotoxicity test. The test substance was soluble in Dimethyl sulfoxide up to 50 mg/ml; slight precipitation was observed at 50 mg/ml, which was considered not to interfere with the counting process. Therefore, a preliminary cytotoxicity test was performed with test substance concentrations of 0.0 (negative control; NC), 0.0 (vehicle control; VC), 0.039, 0.078, 0.156, 0.313, 0.625, 1.250, 2.500 and 5.0 mg/plate in the absence and presence of S9 metabolic activation using triplicate plates of TA98 and TA100 tester strains. There was no reduction in revertant colonies but moderate inhibition of background lawn was observed at 5.0 mg/plate both in the presence and absence of S9 metabolic activation. No reduction in colony count or diminished background lawn was observed at concentrations of 2.500-0.039 mg/plate neither in the presence (+S9) nor the absence (-S9) of metabolic activation.
Vehicle / solvent:
Solvent/vehicle control: DMSO
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2--Aminoanthracene, 4-Nitro-o-phenylenediamine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates were used.
- Number of independent experiments: Two (Experiment I-II)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1-2 X 109 cells/mL
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: Experiment I: in medium (plate incorporation), Experiment II: in suspension (preincubation)
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 minutes
- Exposure duration/duration of treatment:48 hours
- Harvest time after the end of treatment (sampling/recovery times): 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Cytotoxicity was determined by the growth inhibition of the bacterial background lawn and/or reduction in the number of revertant colonies
- Any supplementary information relevant to cytotoxicity:No
Rationale for test conditions:
Solubility/precipitation: The test substance was soluble in Dimethyl sulfoxide up to 50 mg/ml; slight precipitation was observed at 50 mg/ml, which was considered not to interfere with the counting process.

Preliminary cytotoxicity test: It was performed with strains TA98 and TA100. Eight test concentrations with spacing factor 2 were tested for toxicity with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for Trial I.
Evaluation criteria:
A test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding vehicle/solvent control was observed.
A dose-dependent increase was considered biologically relevant if the threshold of biological significance was exceeded at more than one concentration.
An increase exceeding the threshold of biological significance at only one concentration was judged as biologically relevant if it was reproduced in an independent second experiment.
A dose-dependent increase in the number of revertant colonies below the threshold of biological significance was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and vehicle control, the increase was not considered biologically relevant.
Statistics:
The data were presented as the number of revertant colonies per plate. Individual plate counts and the mean number of revertant colonies with the standard deviation for each concentration of the test item and positive and negative (untreated) controls were reported. The mean number of revertant colonies and the standard deviation of test item-treated samples were compared to the spontaneous reversion rates of the solvent control. Microsoft Office Excel-based calculation was used for descriptive statistical analysis.
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
- Data on osmolality:
- Possibility of evaporation from medium:
- Water solubility: The test item was not soluble in water.
- Precipitation and time of the determination: The test item was dissolved din DMSO at 50 mg/ml and checked for precipitation on an agar plate. Slight precipitation was observed at 5 mg/plate concentration, which was considered non-interfering with the colony count.
- Definition of acceptable cells for analysis: Cultures with regular background growth in the negative and solvent (vehicle) control was accepted for the test.
- Other confounding effects:
RANGE-FINDING/SCREENING STUDIES (if applicable):
Test concentrations were selected based on a preliminary cytotoxicity test.This pre-test was performed with strains TA98 and TA100. Eight test concentrations with spacing factor 2 were tested for toxicity with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for Trial I.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: The spontaneous reversion rates in the negative and vehicle control samples were within the respective ranges of the in-house historical data.
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: Non-mutagenic
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA
Ames test:
- Signs of toxicity: No cytotoxicity was observed, but it tested up to the recommended limit concentration
- Individual plate counts: See “Any other information on results incl. tables.”
- Mean number of revertant colonies per plate and standard deviation: See “Any other information on results incl. tables.”
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Available, See “Any other information on results incl. tables.”
- Negative (solvent/vehicle) historical control data: Available, See “Any other information on results incl. tables.”
Remarks on result:
other: Non-mutagenic

Table 1: Revertant counts for pre-experiment





































































































































































































































































Dose (mg/plate)



R



Absence of Metabolic Activation


 (-S9)



Presence of Metabolic Activation (+S9)



TA100



TA 98



TA100



TA 98



NC


(0.00)



R1



102



20



114



19



R2



98



21



100



23



R3



114



19



108



22



VC


(0.00)



R1



118



26



114



25



R2



108



23



124



27



R3



120



25



118



26



T1


(0.039)



R1



112



26



116



24



R2



118



19



120



21



R3



104



22



104



25



T2


(0.078)



R1



118



19



118



21



R2



112



20



112



23



R3



110



24



114



22



T3


(0.156)



R1



104



24



108



22



R2



114



23



120



20



R3



118



22



110



23



T4


(0.313)



R1



104



22



106



24



R2



102



21



112



21



R3



116



25



114



22



T5


(0.625)



R1



116



23



110



23



R2



108



19



124



25



R3



114



20



108



26



T6


(1.250)



R1



116



23



122



25



R2



108



20



108



22



R3



104



21



118



24



T7


(2.500)



R1



114



24



114



23



R2



106



22



116



22



R3



110



20



106



24



T8


(5.0)



R1



104 (+++)



21 (+++)



116 (+++)



24 (+++)



R2



116 (+++)



24 (+++)



120 (+++)



22 (+++)



R3



112 (+++)



20 (+++)



110 (+++)



22 (+++)



PC



R1



1232



848



1288



1312



R2



1344



864



1424



1224



R3



1120



768



1328



1288



NC= Negative control;  VC =Vehicle ControlPC =Positive control


R =Replicate, T=Test concentration (T8: Highest, T1: Lowest), Background Lawn: +++ = Moderate Inhibition, 4-Nitro-o-phenylenediamine [10μg/plate]: TA 98, Sodium azide [10μg/plate]: TA 100, 2-Aminoanthracene [2.5μg/plate]: TA 98, TA 100


Table 2: Revertant counts in plate incorporation method (Trial I)



























































































































































































































Dose (mg/plate)



R



Presence of Metabolic Activation (+S9)



TA 1537



TA 1535



TA 98



TA 100



TA 102



NC


(0.00)



R1



4



13



19



114



240



R2



3



11



23



100



236



R3



5



12



22



108



244



VC


(0.00)



R1



7



14



25



114



268



R2



5



16



27



124



252



R3



6



15



26



118



256



T1


(0.313)



R1



6



15



24



106



256



R2



6



13



21



112



240



R3



5



14



22



114



252



T2


(0.625)



R1



5



13



23



110



252



R2



6



13



25



124



240



R3



5



15



26



108



244



T3


(1.250)



R1



4



16



25



122



240



R2



4



13



22



108



252



R3



6



14



24



118



236



T4


(2.500)



R1



4



13



23



114



248



R2



5



14



22



116



240



R3



4



12



24



106



252



T5


(5.0)



R1



5



12



24



116



244



R2



6



14



22



120



252



R3



4



14



22



110



236



PC



R1



180



480



1312



1288



1332



R2



156



456



1224



1424



1440



R3



168



368



1288



1328



1308




























































































































































































































Dose (mg/plate)



R



Absence of Metabolic Activation (-S9)



TA 1537



TA 1535



TA 98



TA 100



TA 102



NC


(0.00)



R1



3



11



20



102



244



R2



4



13



21



98



228



R3



3



11



19



114



236



VC


(0.00)



R1



5



14



26



118



252



R2



5



13



23



108



260



R3



6



15



25



120



248



T1


(0.313)



R1



5



12



22



104



248



R2



4



13



21



102



236



R3



5



14



25



116



252



T2


(0.625)



R1



4



13



23



116



256



R2



6



15



19



108



232



R3



5



12



20



114



240



T3


(1.250)



R1



4



15



23



116



232



R2



5



14



20



108



244



R3



4



12



21



104



240



T4


(2.500)



R1



3



12



24



114



256



R2



5



11



22



106



244



R3



3



13



20



110



232



T5


(5.0)



R1



4



11



21



104



248



R2



4



14



24



116



232



R3



4



13



20



112



240



PC



R1



160



1104



848



1232



1584



R2



188



1080



864



1344



1392



R3



172



1332



768



1120



1512



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 3: Revertant count in the preincubation 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



5



11



22



110



240



R2



4



14



23



102



252



R3



4



13



21



116



236



VC


(0.00)



R1



6



16



25



122



272



R2



7



15



27



124



252



R3



6



17



26



118



268



T1


(0.313)



R1



6



15



24



114



248



R2



6



16



25



120



264



R3



6



13



23



112



256



T2


(0.625)



R1



6



15



23



118



256



R2



4



14



26



108



264



R3



5



16



24



116



252



T3


(1.250)



R1



7



13



24



114



252



R2



4



15



22



110



244



R3



6



13



23



116



260



T4


(2.500)



R1



6



14



25



116



244



R2



5



15



21



112



264



R3



5



13



24



110



252



T5


(5.0)



R1



5



14



26



118



264



R2



4



14



22



110



256



R3



5



15



23



116



244



PC



R1



176



352



1236



1404



1464



R2



180



400



1140



1356



1346



R3



156



448



1344



1548



1380




























































































































































































































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



13



21



104



248



R2



3



12



20



112



240



R3



3



14



23



108



232



VC


(0.00)



R1



5



14



25



116



260



R2



7



17



23



112



244



R3



5



15



24



120



256



T1


(0.313)



R1



5



14



22



110



256



R2



4



13



24



106



240



R3



6



15



25



118



244



T2


(0.625)



R1



7



13



25



104



256



R2



4



15



23



116



240



R3



5



13



22



110



248



T3


(1.250)



R1



5



16



22



106



232



R2



3



13



23



116



252



R3



4



15



21



114



244



T4


(2.500)



R1



4



13



23



108



236



R2



6



12



22



106



252



R3



4



15



24



112



248



T5


(5.0)



R1



5



15



24



114



236



R2



4



15



21



110



244



R3



4



13



23



108



252



PC



R1



208



1284



732



1308



1368



R2



172



1140



828



1164



1596



R3



184



1200



984



1284



1464



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 [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)



4.00



1.00



12.00



1.00



21.33



2.08



107.33



7.02



240.00



4.00



VC


(0.00)



6.00



1.00



15.00



1.00



26.00



1.00



118.67



5.03



258.67



8.33



T1


(0.313)



5.67



0.58



14.00



1.00



22.33



1.53



110.67



4.16



249.33



8.33



T2


(0.625)



5.33



0.58



13.67



1.15



24.67



1.53



114.00



8.72



245.33



6.11



T3


(1.250)



4.67



1.15



14.33



1.53



23.67



1.53



116.00



7.21



242.67



8.33



T4


(2.500)



4.33



0.58



13.00



1.00



23.00



1.00



112.00



5.29



246.67



6.11



T5


(5.0)



5.00



1.00



13.33



1.15



22.67



1.15



115.33



5.03



244.00



8.00



PC



168.00



12.00



434.67



58.97



1274.67



45.49



1346.67



69.90



1360.00



70.31






































































































































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)



3.33



0.58



11.67



1.15



20.00



1.00



104.67



8.33



236.00



8.00



VC


(0.00)



5.33



0.58



14.00



1.00



24.67



1.53



115.33



6.43



253.33



6.11



T1


(0.313)



4.67



0.58



13.00



1.00



22.67



2.08



107.33



7.57



245.33



8.33



T2


(0.625)



5.00



1.00



13.33



1.53



20.67



2.08



112.67



4.16



242.67



12.22



T3


(1.250)



4.33



0.58



13.67



1.53



21.33



1.53



109.33



6.11



238.67



6.11



T4


(2.500)



3.67



1.15



12.00



1.00



22.00



2.00



110.00



4.00



244.00



12.00



T5


(5.0)



4.00



0.00



12.67



1.53



21.67



2.08



110.67



6.11



240.00



8.00



PC



173.33



14.05



1172.00



139.08



826.67



51.43



1232.00



112.00



1496.00



96.99



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)



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)



4.33



0.58



12.67



1.53



22.00



1.00



109.33



7.02



242.67



8.33



VC


(0.00)



6.33



0.58



16.00



1.00



26.00



1.00



121.33



3.06



264.00



10.58



T1


(0.313)



6.00



0.00



14.67



1.53



24.00



1.00



115.33



4.16



256.00



8.00



T2


(0.625)



5.00



1.00



15.00



1.00



24.33



1.53



114.00



5.29



257.33



6.11



T3


(1.250)



5.67



1.53



13.67



1.15



23.00



1.00



113.33



3.06



252.00



8.00



T4


(2.500)



5.33



0.58



14.00



1.00



23.33



2.08



112.67



3.06



253.33



10.07



T5


(5.0)



4.67



0.58



14.33



0.58



23.67



2.08



114.67



4.16



254.67



10.07



PC



170.67



12.86



400.00



48.00



1240.00



102.06



1436.00



99.92



1396.67



60.74






































































































































Dose


(mg/plate)



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)



3.33



0.58



13.00



1.00



21.33



1.53



108.00



4.00



240.00



8.00



VC


(0.00)



5.67



1.15



15.33



1.53



24.00



1.00



116.00



4.00



253.33



8.33



T1


(0.313)



5.00



1.00



14.00



1.00



23.67



1.53



111.33



6.11



246.67



8.33



T2


(0.625)



5.33



1.53



13.67



1.15



23.33



1.53



110.00



6.00



248.00



8.00



T3


(1.250)



4.00



1.00



14.67



1.53



22.00



1.00



112.00



5.29



242.67



10.07



T4


(2.500)



4.67



1.15



13.33



1.53



23.00



1.00



108.67



3.06



245.33



8.33



T5


(5.0)



4.33



0.58



14.33



1.15



22.67



1.53



110.67



3.06



244.00



8.00



PC



188.00



18.33



1208.00



72.33



848.00



127.18



1252.00



77.15



1476.00



114.47



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


HISTORICAL CONTROL DATA
























































































































































































































































Trial I (Plate Incorporation Method)



Strains



Metabolic Activation



Treatment



Mean



SD



Max



Min



TA 1537



S9 +



Negative control



6



2



10



2



S9 -



6



2



10



2



S9 +



Solvent control



6



2



10



2



S9 -



6



2



10



2



S9 +



Positive control



168



38



245



92



S9 -



175



43



261



89



TA 1535



S9 +



Negative control



12



3



18



7



S9 -



12



3



18



7



S9 +



Solvent control



13



3



18



7



S9 -



13



3



18



7



S9 +



Positive control



336



211



757



86



S9 -



1200



263



1726



674



TA 98



S9 +



Negative control



24



6



36



11



S9 -



23



6



35



11



S9 +



Solvent control



25



6



37



13



S9 -



23



5



33



13



S9 +



Positive control



1099



312



1722



476



S9 -



815



284



1383



248



TA 100



S9 +



Negative control



117



28



173



61



S9 -



114



26



166



62



S9 +



Solvent control



116



28



172



60



S9 -



113



26



165



61



S9 +



Positive control



1488



390



2268



709



S9 -



1311



298



1906



715



TA 102



S9 +



Negative control



274



42



358



190



S9 -



271



55



382



161



S9 +



Solvent control



279



65



409



150



S9 -



277



82



442



112



S9 +



Positive control



1648



305



2258



1037



S9 -



1896



364



2624



1168



 
























































































































































































































































Trial II (Pre-Incubation Method)



Strains



Metabolic Activation



Treatment



Mean



SD



Max



Min



TA 1537



S9 +



Negative control



6



2



10



2



S9 -



6



2



10



2



S9 +



Solvent control



6



2



10



3



S9 -



6



2



10



2



S9 +



Positive control



170



39



249



91



S9 -



182



43



268



96



TA 1535



S9 +



Negative control



13



3



18



7



S9 -



12



3



18



7



S9 +



Solvent control



13



3



18



8



S9 -



13



3



18



7



S9 +



Positive control



299



197



694



145



S9 -



1244



260



1765



724



TA 98



S9 +



Negative control



24



6



35



13



S9 -



23



5



33



13



S9 +



Solvent control



24



5



35



14



S9 -



23



5



32



14



S9 +



Positive control



1269



275



1819



719



S9 -



740



210



1160



320



TA 100



S9 +



Negative control



117



25



166



67



S9 -



113



23



159



66



S9 +



Solvent control



116



22



159



73



S9 -



112



20



151



73



S9 +



Positive control



1469



347



2163



775



S9 -



1352



263



1878



827



TA 102



S9 +



Negative control



281



32



345



218



S9 -



276



28



331



220



S9 +



Solvent control



281



34



350



212



S9 -



276



34



344



207



S9 +



Positive control



1595



287



2168



1022



S9 -



1753



248



2248



1258



Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD

Conclusions:
The registered substance, i.e., 3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol (CAS No. 3407-42-9) was non-mutagenic (negative) in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 both in the presence and absence of the S9 metabolic activation system. The test was performed as per OECD TG 471 (adopted: on 21st July 1997, corrected on 26th June 2020) and GLP.
Executive summary:

The potential of the registered substance, i.e.,  3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol (CAS number: 3407-24-9) to induce gene mutation in the histidine operon was tested in five Salamomella Typhimurium strains (TA98, TA100, TA1535, TA1537 and TA102) in the absence and presence of an exogenous metabolic activation system. The test was conducted according to OECD TG 471 (adopted: on 21st July 1997, corrected on 26th June 2020) and GLP. Cofactor-supplemented liver S9 microsomal fraction was used, and the S9 fraction was obtained from Aroclor-1254-induced rats. Test concentrations were selected based on the solubility and precipitation checks and a preliminary cytotoxicity test. The test substance was insoluble in water but was soluble in Dimethyl sulfoxide (DMSO) up to 50 mg/ml; slight precipitation was observed at 50 mg/ml, which was considered not to interfere with the counting process. Therefore, a preliminary cytotoxicity test was performed with test substance concentrations of 0.0 (negative control; NC), 0.0 (vehicle control; VC), 0.039, 0.078, 0.156, 0.313, 0.625, 1.250, 2.500 and 5.0 mg/plate in the absence and presence of S9 metabolic activation using triplicate plates of TA98 and TA100 tester strains. There was no reduction in revertant colonies, but moderate inhibition of background lawn was observed at 5.0 mg/plate both in the presence and absence of S9 metabolic activation. No reduction in colony count or diminished background lawn was observed at concentrations of 2.500-0.039 mg/plate, neither in the presence (+S9) nor the absence (-S9) of metabolic activation. Hence, the following test concentrations were selected for the bacterial mutagenicity test: 0.0 (NC, water), 0.0(VC, DMSO), 0.313, 0.625, 1.250, 2.500 and 5.0 mg/plate. The methods applied were the plate incorporation method (Trial I) and the pre-incubation method (Trial II). Trial I and II were performed in two independent experiments, both with and without metabolic activation. Each test substance concentration, including negative, vehicle and strain-specific positive controls, was tested in triplicates in the absence and presence of S9 metabolic activation. The following positive control substances were applied during the test: Sodium azide (without S9 mix, TA1535, TA100), 4-Nitro-o-phenylenediamine (without S9 mix, TA98, TA1537), Methyl methane sulfonate (without S9 mix, TA102), 2-Aminoanthracene (with S9 mix, TA98, TA100, TA1535, TA1537 and TA102). The plates were treated and incubated at 37°C for 48 hours. Results: The results from Trial I and II revealed no significant or biologically relevant increase in the number of revertant colonies at any concentrations tested either in the presence or absence of metabolic activation. Further, no trend of an increased number of revertant colonies with increased dosing of the test item was observed. The spontaneous reversion rates in the negative and vehicle control samples were within the respective ranges of the in-house historical data. Each strain-specific positive control in Trial I and II showed a significant increase in the number of revertant colonies. Conclusion: The registered substance  (CAS No. 3407-42-9) did not induce gene mutation by base-pair change or frameshift in  Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 both in the presence and absence of S9 metabolic activation system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation test: 


The potential of the registered substance, i.e.,  3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol (CAS number: 3407-24-9) to induce gene mutation in the histidine operon was tested in five Salamomella Typhimurium strains (TA98, TA100, TA1535, TA1537 and TA102) in the absence and presence of an exogenous metabolic activation system. The test was conducted according to OECD TG 471 (adopted: on 21st July 1997, corrected on 26th June 2020) and GLP. Cofactor-supplemented liver S9 microsomal fraction was used, and the S9 fraction was obtained from Aroclor-1254-induced rats. Test concentrations were selected based on the solubility and precipitation checks and a preliminary cytotoxicity test. The test substance was insoluble in water but was soluble in Dimethyl sulfoxide (DMSO) up to 50 mg/ml; slight precipitation was observed at 50 mg/ml, which was considered not to interfere with the counting process. Therefore, a preliminary cytotoxicity test was performed with test substance concentrations of 0.0 (negative control; NC), 0.0 (vehicle control; VC), 0.039, 0.078, 0.156, 0.313, 0.625, 1.250, 2.500 and 5.0 mg/plate in the absence and presence of S9 metabolic activation using triplicate plates of TA98 and TA100 tester strains. There was no reduction in revertant colonies, but moderate inhibition of background lawn was observed at 5.0 mg/plate both in the presence and absence of S9 metabolic activation. No reduction in colony count or diminished background lawn was observed at concentrations of 2.500-0.039 mg/plate, neither in the presence (+S9) nor the absence (-S9) of metabolic activation. Hence, the following test concentrations were selected for the bacterial mutagenicity test: 0.0 (NC, water), 0.0(VC, DMSO), 0.313, 0.625, 1.250, 2.500 and 5.0 mg/plate. The methods applied were the plate incorporation method (Trial I) and the pre-incubation method (Trial II). Trial I and II were performed in two independent experiments, both with and without metabolic activation. Each test substance concentration, including negative, vehicle and strain-specific positive controls, was tested in triplicates in the absence and presence of S9 metabolic activation. The following positive control substances were applied during the test: Sodium azide (without S9 mix, TA1535, TA100), 4-Nitro-o-phenylenediamine (without S9 mix, TA98, TA1537), Methyl methane sulfonate (without S9 mix, TA102), 2-Aminoanthracene (with S9 mix, TA98, TA100, TA1535, TA1537 and TA102). The plates were treated and incubated at 37°C for 48 hours. Results: The results from Trial I and II revealed no significant or biologically relevant increase in the number of revertant colonies at any concentrations tested either in the presence or absence of metabolic activation. Further, no trend of an increased number of revertant colonies with increased dosing of the test item was observed. The spontaneous reversion rates in the negative and vehicle control samples were within the respective ranges of the in-house historical data. Each strain-specific positive control in Trial I and II showed a significant increase in the number of revertant colonies. Conclusion: The registered substance  (CAS No. 3407-42-9) did not induce gene mutation by base-pair change or frameshift in  Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of S9 metabolic activation system.


 


In vitro mammalian chromosome aberration test:


The study was performed to determine the clastogenic potential of the test material, 3-(5,5,6-trimethylbicyclo[2.2.1] hept-2-yl)cyclohexan-1-ol (CAS No. 3407-42-9) on primary culture of human peripheral blood lymphocytes. The study was performed according to OECD test guideline No. 473, adopted on July 29, 2016. The study was performed in two independent assay (Phase I-II) in the presence and absence of S9 metabolic activation. The solubility of the test item was checked in Distilled water, Dimethyl Sulphoxide (DMSO), and Acetone, and the test substance was found insoluble at 200 mg/mL. The solubility of the test item was checked in Ethyl alcohol, and it was found soluble at 200 mg/mL to give the final treatment concentration of 2 mg/mL culture (recommended maximum test concentration for non-cytotoxic substances). Therefore, Ethyl alcohol was selected as a solvent for the test substance during the study. Precipitation was observed at 2 mg/mL concentration. Slight precipitation was observed at 1 mg/mL, which was judged not to interfere with the conduct of the test. Hence, the concentration of 1 mg/mL was selected as the high dose for the cytotoxicity experiment. Cytotoxicity was assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.25, 0.50 and 1.00  mg/ml in treated culture media. All the tested concentrations in the initial cytotoxicity experiment were cytotoxic both in the presence and absence of the S9 metabolic activation system. Next, cytotoxicity was assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.031, 0.063 and 0.125 mg/ml of culture media in a second cytotoxicity experiment (Experiment II). All the tested concentrations in the cytotoxicity experiment (II) were cytotoxic both in the presence and absence of the metabolic activation system. The cytotoxicity was then assessed at test item concentrations of 0.0 (NC), 0.0 (VC), 0.004, 0.008  and 0.016 mg/mL in treated culture media in the third cytotoxicity experiment (Experiment III). The test concentration 0.016 mg/mL produced a 52.72 % and 53.01% decrease in the mitotic index compared to VC in the absence (-S9) and presence of metabolic activation (+S9), respectively. The test concentration 0.008 (T8) mg/mL produced 23.91% and 26.26% decreases in the mitotic index compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively. The test concentration 0.004 (T7) mg/mL produced 17.31% and 16.62 % decreases in the mitotic index (cytotoxicity) compared to VC in the absence (-S9) and presence (+S9) of metabolic activation, respectively. The dose level that results in approximately 55 ± 5 % reduction in the mitotic index was selected as the highest test concentration in the main test. Hence, the following test concentrations were assessed in the main study: 0.0 (NC), 0.0 (VC), 0.004, 0.008 and 0.16 mg/ml along with positive control substances in the presence and absence of metabolic activation. The main study was performed in two independent phases (Phase I-II). In Phase I, cells were exposed to the test concentrations of 0.004, 0.008 and 0.016 mg/ml along with negative control (NC: Distilled water) and vehicle control (VC: Ethyl alcohol) for 4 hours both in the presence and absence of S9 metabolic activation (1% v/ v). In Phase II, cells were treated for 4 hours in the presence of increased metabolic activation (2% v/v) and 24 hours in the absence of metabolic activation. Three hours before cell harvesting, colcemid (final concentration: 0.3 µg/ml) was added to each culture tube to stop cell proliferation. The cultures were harvested 24 hours after the beginning of the treatment, fixed then stained with 5% fresh Giemsa stain. A minimum of 1000 cells were counted in different fields of slide per culture, and the number of metaphases was recorded for mitotic index calculation. Three hundred well-spread metaphases per concentration and control were scored for cytogenetic damage on coded slides. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in calculation of the aberration rates. Only metaphases with 46±2 centromere regions were included in the analysis. Results: There was no significant increase in the percent of aberrant cells at any concentrations tested when compared to the vehicle control in both phases and the presence and absence of S9 metabolic activation. In Phase I, the mean percentage of aberrant cells was 0.333 (NC), 0.333 (VC), 0.667 (at 0.004 mg/ml), 0.333 (0.008 mg/ml), 0.667 (0.016 mg/ml) and 10.333 (PC: 600 µg/mL EMS) in the absence of metabolic activation. In the presence of metabolic activation, the mean percentage of aberrant cells was 0.333 (NC), 0.667 (VC), 0.333 (at 0.004 mg/ml), 0.667 (0.008 mg/ml), 0.667 (0.016 mg/ml) and 10.000 (PC: 30 µg/mL CPA). The observed mean mitotic index in the absence of metabolic activation was 10.23 (NC), 9.69 (VC), 7.78, 7.03, 4.49 and 8.28 (PC) at 0.0 (NC), 0.0 (VC), 0.004, 0.008 and0.016 mg/ml, and 600 µg/mL EMS (PC), respectively. In the presence of metabolic activation, the mean mitotic index was 10.63 (NC), 9.53 (VC), 7.83, 6.79, 4.64 and 8.28 (PC) at 0.0(NC), 0.0 (VC), 0.004, 0.008 and0.016 mg/ml and 30 µg/mL CPA (PC), respectively. In the Phase II experiment, in the absence of metabolic activation, the mean percent aberrant cells were 0.333 (NC), 0.333 (VC), 0.337 (at 0.004 mg/ml), 0.667 (0.008 mg/ml), 1.000 (0.016 mg/ml) and 10.333 (PC: 600 µg/mL EMS). In the presence of the metabolic activation, the mean percent aberrant cells were 0.333 (NC), 0.667 (VC), 0.333 (at 0.004 mg/ml), 0.667 (at 0.008 mg/ml), 1.000 (at 0.016 mg/ml) and 11.000 (PC: at 30 µg/mL CPA). The observed mean mitotic index in the absence (-S9) of metabolic activation was 10.13 (NC), 9.84 (VC),7.88 (at 0.004 mg/ml),7.49 (at 0.008 mg/ml), 4.64 (0.16 mg/ml) and 8.48 (PC: 600 µg/mL EMS).In the presence (+S9) of metabolic activation, the mean mitotic index was 10.37 (NC), 9.33 (VC), 7.98 (at 0.004 mg/ml), 7.13 (at 0.008 mg/ml),4.44 (at 0.16 mg/ml) and 8.03 (PC: 30 µg/mL CPA). The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system and the suitability of the methods and conditions employed in the experiment. Conclusion: Based on the above results, the test material, 3-(5,5,6-trimethylbicyclo[2.2.1] hept-2-yl)cyclohexan-1-ol (CAS No. 3407-42-9), was tested as non-clastogenic (negative)  both in the presence (1% and 2%) and the absence of cofactor supplemented S9 metabolic activation system using primary culture of human peripheral lymphocytes. The study was conducted according to OECD TG 473 and GLP.

Justification for classification or non-classification

A combination of in vitro genotoxicity tests was conducted with the registered substance, i.e., 3-(5,5,6-trimethylbicyclo[2.2.1] hept-2-yl)cyclohexan-1-ol (CAS: 3407-42-9), to assess its genotoxic properties. The substance tested non-mutagenic (negative) in Salmonella Typhimurium TA98, TA100, TA1535, TA1537 and TA102 according to OECD TG 471 and GLP both in the presence and absence of cofactor-supplemented liver S9 microsomal fraction. It has also been tested non-clastogenic (negative) in the primary culture of human peripheral blood cells according to OECD TG 473 and GLP both in the presence and absence of cofactor-supplemented liver S9 microsomal fraction. An in vitro gene mutation study with the Substance according to OECD TG 476 and GLP is ongoing. Justification for germ cell mutagenicity will be provided after receiving the data from the OECD 476 study.