3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

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:

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

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

Method

Species / strain

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)

Controls

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

Results and discussion

Test results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.