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

Endpoint summary

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

Reference

Reference 1

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.

Reference 2

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 Control;  PC =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 21^st July 1997, corrected on 26^th 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 21^st July 1997, corrected on 26^th 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.