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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

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:

- Name of the test material: 3-(5,5,6-Trimethylbicyclo(2.2.1)hept-2-yl)cyclohexanol
- Common Name: Isobornyl cyclohexanol
- IUPAC name: 3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexanol
- Molecular formula: C16H28O
- Molecular weight: 236.396 g/mol
- Substance type: Organic
- Smiles: O[C@@H]1CCC[C@@H]([C@@H]2[C@@H]3C[C@@H](C(C)(C)[C@@H]3C)C2)C1

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Aroclor 1254-induced S9 was procured from Defence Research and Development Organization and stored at -60°C to -80°C inside the deep freezer. The protein concentration in the S9 fraction was 36.2 mg/mL.
- source of S9 : Aroclor 1254-induced S9 was procured from Defence Research and Development Organization and stored at -60°C to -80°C inside the deep freezer.
- method of preparation of S9 mix : An appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final concentration of approximately 10 % v/v in the S9 mix. Cofactor solution contains the following quantity of chemicals in 500 mL of Distilled Water.
D-glucose-6-phosphate= 0.8 g
β-NADP = 1.75 g
MgCl2 = 1.0 g
KCl = 1.35 g
Na2HPO4 = 6.4 g
NaH2PO4.H2O = 1.4 g
During the experiment, the S9 mix was prepared freshly.
- concentration or volume of S9 mix and S9 in the final culture medium : The protein concentration in the S9 fraction was 36.2 mg/mL. Each batch of S9 mix was tested with 2-Aminoanthracene as well as benzo (a) pyrene for its efficiency. Thus, requirements of Ames were fulfilled, and the results of efficiency testing were archived at RCC Laboratories India Private Limited.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):Each batch of S9 mix was tested with 2-Aminoanthracene as well as benzo (a) pyrene for its efficiency. Thus, requirements of Ames were fulfilled, and the results of efficiency testing were archived at RCC Laboratories India Private Limited.

Test concentrations with justification for top dose:

0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).

Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5.0 mg/plate based on the solubility and precipitation test.
There was no reduction in colony count but reduction in bacterial background lawn was observed in treated concentrations 5.0 mg/plate (T8), 1.582 mg/plate (T7) and no reduction in colony count as well as in bacterial background lawn in treated concentrations (0.501 (T6) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0, 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate , both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

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

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

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	108	20	110	22
	R2	116	17	114	16
	R3	110	22	117	17
VC (0.00)	R1	128	27	135	29
	R2	130	24	132	28
	R3	122	28	128	26
T1 (0.002)	R1	110	18	112	19
	R2	111	19	114	17
	R3	115	20	117	18
T2 (0.005)	R1	109	21	115	21
	R2	113	20	119	20
	R3	114	19	120	20
T3 (0.016)	R1	119	22	122	22
	R2	117	22	120	21
	R3	116	21	123	24
T4 (0.050)	R1	119	18	124	23
	R2	120	21	124	22
	R3	118	24	125	25
T5 (0.158)	R1	121	22	126	24
	R2	123	23	127	25
	R3	119	24	125	24
T6 (0.501)	R1	124	25	128	25
	R2	125	24	130	26
	R3	122	24	129	27
T7 (1.582)	R1	123	25	129	24
	R2	125	24	127	26
	R3	126	26	131	27
T8 (5)	R1	125	27	130	28
	R2	127	25	129	27
	R3	126	26	126	25
PC	R1	1320	956	1376	1544
	R2	1236	972	1392	1608
	R3	1448	1012	1352	1428

NC           =     Negative control

VC           =   Vehicle Control

PC            =     Positive control             

R              =     Replicate

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

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

Sodium azide [10μg/plate]: TA 100,

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

 

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL I)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	4	9	22	110	244
	R2	4	11	16	114	229
	R3	3	11	17	117	237
VC (0.00)	R1	7	16	29	135	294
	R2	7	15	28	132	278
	R3	8	15	26	128	294
T1 (0.005)	R1	4	12	21	115	235
	R2	4	11	20	119	236
	R3	4	10	20	120	240
T2 (0.016)	R1	5	13	22	122	241
	R2	4	11	21	120	248
	R3	4	12	24	123	237
T3 (0.050)	R1	4	13	23	124	248
	R2	5	13	22	124	254
	R3	5	14	25	125	261
T4 (0.158)	R1	6	14	24	126	265
	R2	5	13	25	127	269
	R3	6	15	24	125	275
T5 (0.501)	R1	5	14	25	128	268
	R2	8	16	26	130	283
	R3	7	15	27	129	274
PC	R1	148	502	1544	1376	1590
	R2	164	428	1608	1392	1648
	R3	167	474	1428	1352	1674

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	4	10	20	108	237
	R2	5	9	17	116	228
	R3	4	12	22	110	240
VC (0.00)	R1	8	16	27	128	290
	R2	7	14	24	130	264
	R3	6	15	28	122	282
T1 (0.005)	R1	5	11	21	109	234
	R2	4	10	20	113	236
	R3	5	11	19	114	240
T2 (0.016)	R1	6	10	22	119	244
	R2	5	11	22	117	241
	R3	4	12	21	116	247
T3 (0.050)	R1	5	13	18	119	260
	R2	6	12	21	120	251
	R3	5	11	24	118	247
T4 (0.158)	R1	6	13	22	121	271
	R2	6	12	23	123	259
	R3	5	14	24	119	263
T5 (0.501)	R1	6	13	25	124	274
	R2	8	15	24	125	269
	R3	6	14	24	122	277
PC	R1	158	1016	956	1320	1672
	R2	172	1200	972	1236	1548
	R3	164	1158	1012	1448	1624

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                     2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                           Sodium azide [10μg/plate]: TA 1535, TA 100                                              

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

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

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	3.67	0.58	10.33	1.15	18.33	3.21	113.67	3.51	236.67	7.51
VC (0.00)	7.33	0.58	15.33	0.58	27.67	1.53	131.67	3.51	288.67	9.24
T1 (0.005)	4.00	0.00	11.00	1.00	20.33	0.58	118.00	2.65	237.00	2.65
T2 (0.016)	4.33	0.58	12.00	1.00	22.33	1.53	121.67	1.53	242.00	5.57
T3 (0.050)	4.67	0.58	13.33	0.58	23.33	1.53	124.33	0.58	254.33	6.51
T4 (0.158)	5.67	0.58	14.00	1.00	24.33	0.58	126.00	1.00	269.67	5.03
T5 (0.501)	6.67	1.53	15.00	1.00	26.00	1.00	129.00	1.00	275.00	7.55
PC	159.67	10.21	468.00	37.36	1526.67	91.24	1373.33	20.13	1637.33	43.00

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.33	0.58	10.33	1.53	19.67	2.52	111.33	4.16	235.00	6.24
VC (0.00)	7.00	1.00	15.00	1.00	26.33	2.08	126.67	4.16	278.67	13.32
T1 (0.005)	4.67	0.58	10.67	0.58	20.00	1.00	112.00	2.65	236.67	3.06
T2 (0.016)	5.00	1.00	11.00	1.00	21.67	0.58	117.33	1.53	244.00	3.00
T3 (0.050)	5.33	0.58	12.00	1.00	21.00	3.00	119.00	1.00	252.67	6.66
T4 (0.158)	5.67	0.58	13.00	1.00	23.00	1.00	121.00	2.00	264.33	6.11
T5 (0.501)	6.67	1.15	14.00	1.00	24.33	0.58	123.67	1.53	273.33	4.04
PC	164.67	7.02	1124.67	96.42	980.00	28.84	1334.67	106.76	1614.67	62.52

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

 

 

TABLE 5 -    MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.00	1.00	10.33	0.58	15.67	0.58	90.33	5.51	243.00	7.00
VC (0.00)	7.33	0.58	15.00	1.00	27.67	0.58	114.67	3.51	279.67	4.73
T1 (0.005)	4.67	1.15	10.67	1.15	16.00	1.00	93.67	7.57	247.33	6.11
T2 (0.016)	5.00	0.00	12.00	1.00	16.67	1.53	100.00	2.65	254.67	6.11
T3 (0.050)	5.67	0.58	11.67	0.58	19.00	2.65	104.67	6.03	262.33	6.11
T4 (0.158)	6.67	0.58	13.00	1.00	23.00	2.00	107.33	4.51	271.33	5.51
T5 (0.501)	7.00	0.00	14.67	0.58	26.33	0.58	109.67	7.02	278.00	2.65
PC	155.33	5.69	390.67	28.10	1118.00	24.98	1382.67	42.39	1426.00	36.17

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.00	0.00	10.67	1.53	15.33	1.53	90.00	6.00	236.33	4.04
VC (0.00)	7.33	0.58	15.33	0.58	26.33	1.53	115.00	5.57	281.00	7.00
T1 (0.005)	4.33	0.58	11.00	1.00	15.67	0.58	94.67	5.51	239.00	5.00
T2 (0.016)	4.67	0.58	12.00	1.00	16.33	1.53	99.33	2.52	247.00	6.00
T3 (0.050)	5.33	0.58	13.00	1.00	20.00	1.00	104.00	1.00	252.00	9.54
T4 (0.158)	6.33	0.58	13.67	1.53	23.00	1.00	107.00	3.00	264.67	11.50
T5 (0.501)	7.00	1.00	15.00	1.00	26.00	1.00	110.67	4.04	279.67	5.13
PC	162.67	7.77	1424.00	44.54	801.33	28.38	1274.67	33.31	1593.33	46.01

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz.,0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plates were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data.The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.