Cyclohexanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative, in vitro gene mutation study in mammalian cells, OECD 476, Wollny (2013).

Negative, in vitro gene mutation study in bacteria, OECD 471, DuPont (1976).

Negative, in vitro gene mutation study in bacteria, OECD 471, DuPont (1992).

Ambiguous, in vitro gene mutation study in bacteria, OECD 471 Haworth (1983).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 December 2012 to 15 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

from 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

from 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

from 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Complete culture medium: RPMI 1640 medium supplemented with 15 % horse serum, 100 U/ 100 µg/mL penicillin / streptomycin, 220 µg/mLsodium pyruvate and 0.5 - 0.75 U/mL amphotericin.Cloning medium: As for complete culture medium.Selective medium: Complete culture medium with the addition of 5 µg/mL TFT.- Properly maintained: Yes.- Periodically checked for Mycoplasma contamination: Yes- Periodically checked for karyotype stability:Yes- Periodically "cleansed" against high spontaneous background: YesPrior to mutagenicity testing, the amount of spontaneous mutants was reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with 1.0E-04 M hypoxanthine, 2.0E-07 M aminopterin and 1.6E-05M thymidine. The incubation of the cells in HAT-medium was followed by a recovery period of two days in RPMI 1640 medium containing 1.0E-04 M hypoxanthine and 1.6E-05 thymidine. After this incubation the cells were returned to normal RPMI 1640 medium. Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell back of Harlan CCR allowing the repeated use of the same cell culture batch in many experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability.Thawed stock cultures are propagated in plastic flasks in RPMI 1640 complete culture medium. The cells are sub-cultured two times prior to treatment. The cell cultures were incubated at 37 ± 5 °C in humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

- Experiment I, without S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
- Experiment I, with S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
- Experiment II, without S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
- Experiment II, with S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubilisation properties and its nontoxicity to the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 - 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium supplemented with 15 % horse serum (HS), 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 U/mL Amphotericin B used as antifungal agent plus 5 µg/mL TFT.

NUMBER OF REPLICATIONS: 2NUMBER OF CELLS EVALUATED: 10^7

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growthIn the pre-test screen, 10^7 cells were exposed to each concentration of the test substance for four hours with and without metabolic activation. Following treatment the cells were washed twice by centrifugation (425 g, 10 mins) and re-suspended in “Saline G”. Subsequently the cells were re-suspended in 30 mL complete culture medium for a 2-day growth period. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3E+05 cells/mL if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period.

- POSITIVE CONTROL: Methyl methane sulfonate in nutrient medium to a final concentration of 0.18 mM (19.5 µg) without metabolic activation. Cyclophosphamide in 0.9 % saline to a final concentration of 10.7 µM (3.0 µg/mL) and 16.1 µM (4.5 µg/mL) with metabolic activation.

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively. A relevant increase of the mutation frequency should be dose-dependent. A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls and the mutation rates of all negative and vehicle controls of this study are taken into consideration. Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled. Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively.A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using a statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

All strains/cell types tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant test substance related effect on pH was noted.
- Effects of osmolality: No relevant test substance related effect on osmolarity was noted.
- Precipitation: No precipitation of the test item was noted in experiment I and II with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
- A pre-test was performed at doses of 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0 µg/mL in order to determine the concentration range of the mutagenicity experiments. Both pH value and osmolarity were determined at the maximum concentration of the test item and in the solvent control without metabolic activation. 10^7 cells were exposed to each concentration of the test item for 4 hours with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The mutation rates in the vehicle controls lie within the historical range.

OTHER:
- In both main experiments no precipitation was noted up to the maximum concentration with and without metabolic activation.No relevant cytotoxic effects indicated by a relative cloning efficiency 1 (survival) or a relative total growth of less than 50 % in both cultures occurred in experiment I and II with and without metabolic activation.No substantial and reproducible concentration dependent increase of the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed in the main experiments up to the maximum concentration tested with and without metabolic activation. A linear regression analysis (least squares) was performed to asses a possible concentration dependent increase of mutant frequencies using SYSTAT® 11 statistics software. No significant concentration dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups.In this study, the range of the solvent control values was from 64 up to 158 mutant colonies per 10^6 cells; the range of the acceptable group values treated with the test substance was from 29 up to 226 mutant colonies per 10^6 cells. The highest value of 226 slightly exceeded the historical range of solvent controls (42 – 216 colonies per 10^6 cells) but there was no concentration dependent increase as indicated by the lack of statistical significance and the mutation frequency in the parallel culture remained well within the historical range of solvent controls under identical conditions, hence these data are considered acceptable.The positive controls showed a distinct increase in induced total mutant colonies at acceptable levels of cytotoxicity.

Summary of results, Experiment I and II

			Relative	Relative	Mutant		Relative	Relative	Mutant
	Conc. µg per mL	S9 mix	cloning efficiency	total growth	colonies/  10^6cells	threshold	cloning efficiency	total growth	colonies/  10^6cells	thresholds
Column	1	2	3	4	5	6	7	8	9	10
Experiment I/ 4 h treatment			Culture I				Culture II
Solv. control with water		-	100.0	100.0	64	190	100.0	100.0	77	203
Pos. control with MMS	19.5	-	70.8	43.7	362	190	84.2	46.3	302	203
Test item	31.3	-	70.8	Culture was not continued #			88.9	Culture was not continued #
Test item	62.5	-	67.0	163.9	29	190	96.0	107.2	82	203
Test item	125.0	-	51.5	111.3	53	190	92.3	126.3	56	203
Test item	250.0	-	85.4	132.1	54	190	70.2	106.6	77	203
Test item	500.0	-	50.0	108.3	69	190	96.0	130.1	57	203
Test item	1000.0	-	61.4	88.9	88	190	88.9	73.1	97	203
Solv. control with water			100.0	100.0	81	207	100.0	100.0	74	200
Pos, control with CPA	3.0	+	29.4	27.1	665	207	18.6	26.6	928	200
Pos, control with CPA	4.5	+	25.5	14.4	1295	207	23.4	14.9	1355	200
Test item	31.3	+	124.2	Culture was not continued #			72.8	Culture was not continued #
Test item	62.5	+	91.6	80.8	95	207	97.8	94.0	103	200
Test item	125.0	+	104.6	137.8	92	207	75.3	101.4	87	200
Test item	250.0	+	103.0	84.8	109	207	80.6	116.4	88	200
Test item	500.0	+	82.9	54.4	142	207	74.0	103.6	56	200
Test item	1000.0	+	74.0	127.1	115	207	88.4	105.3	90	200
Experiment II / 4 h treatment			Culture I				Culture II
Solv. control with water		-	100.0	100.0	117	243	100.0	100.0	88	214
Pos. control with MMS	19.5	-	73.4	33.9	447	243	69.3	27.4	576	214
Test item	31.3	-	74.7	Culture was not continued #			89.6	Culture was not continued #
Test item	62.5	-	69.7	121.3	104	243	79.7	96.0	59	214
Test item	125.0	-	77.5	151.2	114	243	76.0	86.3	74	214
Test item	250.0	-	68.5	133.4	97	243	81.0	68.3	97	214
Test item	500.0	-	76.1	142.1	87	243	86.6	60.3	79	214
Test item	1000.0	-	70.9	133.6	106	243	77.2	51.0	125	214
Solv. control with water			100.0	100.0	150	276	100.0	100.0	158	284
Pos, control with CPA	3.0	+	51.0	29.2	744	276	41.7	34.8	1434	284
Pos, control with CPA	4.5	+	21.3	4.1	1652	276	12.8	4.7	3153	284
Test item	31.3	+	96.6	Culture was not continued #			98.1	Culture was not continued #
Test item	62.5	+	126.2	100.5	106	276	118.9	155.1	146	284
Test item	125.0	+	90.4	80.6	146	276	110.9	127.9	208	284
Test item	250.0	+	95.0	133.4	106	276	87.9	155.1	181	284
Test item	500.0	+	86.2	91.1	92	276	927	77.7	150	284
Test item	1000.0	+	67.8	62.2	129	276	86.4	83.2	226	284

threshold = number of mutant colonies per 10^6 cells of each solvent control plus 126

# culture was not continued since a minimum of only four analysable concentrations is required

Conclusions:

The test material did not induce mutations in the absence and presence of metabolic activation.

Executive summary:

The mutagenic potential of the test material was assessed according to OECD Guideline 476. This GLP-study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in three independent experiments, using two parallel cultures each. Experiments I and II were performed with and without liver microsomal activation and a treatment period of 4 hours.

The concentration range of the main experiments went up to approximately 10 mM. No cytotoxic effects occurred in the main experiments up to the maximum concentration in the absence and presence of metabolic activation. No substantial and reproducible dose dependent increase in mutant colony numbers was observed up to the maximum concentrations tested with and without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion, it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test item is considered to be non mutagenic in this mouse lymphoma assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Limited documentation, no data on substance purity, prior to GLP, aminoanthracene was used as sole indicator of the efficacy of S9 mix, no E.coli or TA 102 and no TA 100 srains tested.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Limited description of test substance, no E. coli strain tested, TA 102, and TA 100 strain not tested, 2-aminoanthracene was sole indicator used to provide information of the efficacy of S9.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His Operon

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented post-mitochondrial fraction (S9), prepared from the livers of rats.

Test concentrations with justification for top dose:

500, 1000, 2500, 5000, 7500, 10000, 15000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol 100 %

Positive controls:

yes

Positive control substance:

other: WITH S9: 2-aminoanthracene (2AA) for TA1538, TA98, TA1535 and 1537. WITHOUT S9: N-Methyl-N'-nitro-H-nitrosoguanidine (MNNG) for TA1535; 2-Nitrofluorene (2-NF) for TA1538 and TA 98; 9-aminoacridine (9AAD) for T1537.

Remarks:

In the non-activated system: negative, or solvent control, and positive controls. A second negative control (-S9 control) is included in the activated assay to measure any activity of the compound in the absence of the S9 activator mixture.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- In the absence of metabolic activation, 0.1 mL of a solution of the test compound and approximately 10^8 bacteria were added to 2 mL of top agar (0.6% agar, 0.6% NaCl, 0.05 mM L-histidine, 0.05 mM biotin).
- The metabolic activation system involved the addition of 0.5 mL of S9 mixture to the chemical-top agar.
- The S9 mix contains per ml: 0.3 mL of the 9000 x g supernatant of homogenized rat liver, 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4mM NADP and 100 mM sodium phosphate (pH 7.4).

DURATION
- Exposure duration: 48 h
- Temperature: 37 °C

SELECTION AGENT (mutation assays): L-histidine

NUMBER OF DUPLICATES: 2DETERMINATION OF CYTOTOXICITY Prior to testing for mutagenicity, the compound was tested for toxicity to the tester strains.
- Method: no data

POSITIVE CONTROLSWITH S9: 2-aminoanthracene (2AA) 10µg/plate (TA1538 and TA98), 100µg/plate (TA1535 and 1537).

WITHOUT S9: M-Methyl-N'-nitro-H-nitrosoguanidine (MNNG) 2µg/plate (TA1535); 2-nitrofluorene (2NF) 25 µg/plate (TA1538 and TA 98); 9-aminoacridine (9AAD) 50 µg/plate (TA1537).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- With S9: ≥7500 µg/plate in TA1537 and ≥10000 µg/plate for TA98;
- Without S9: ≥7500 µg/plate in TA1537 and TA 98; and ≥10000 µg/plate for TA1538

Table of Revertant Factors based on an assessment using OECD 471 guideline criteria:

Strain tested	Metabolic activation system	Duplicates	Revertant factor range	Revertant factor Positive control	Dose dependency	Cytotoxicity	Assessment
TA 1535	No	2	0.81-1.4	93.8	No	No	Negative
	Yes	2	0.6-1.2	25.4	No	No	Negative
TA 1537	No	2	1.1-1.4	36.2		≥7500 µg/plate	Negative
	Yes		0.8-1.2	26	No	≥7500 µg/plate	Negative
TA 1538	No	2	0.7-1.0	131.6		No	Negative
	Yes		0.8-1.1	93.8	No	No	Negative
TA 98	No	2	0.92-1.0	108	No	≥7500 µg/plate	Negative
	Yes	2	0.95-1.3	90.9	No	≥10000 µg/plate	Negative

Conclusions:

The compound was not mutagenic in the microbial assays in either the presence or absence of a liver microsomal activation system (S9 mixture), i.e. it did not significantly increase the spontaneous, or background, mutation frequency.

Executive summary:

This study was performed to investigate the potential of test substance to induce gene mutations in the plate incorporation test using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA1538.

The plates incubated with the test substance showed normal background growth in all strains used. A cytotoxic effect was observed in the presence of S9 mix for the strains TA1537 and TA98 at respectively ≥7500 µg/plate and ≥10000 µg/plate. In the abscence of S9 mix, cytotoxicity was observed above 7500 µg/plate for the strains TA1537 and TA 98 and above ≥10000 µg/plate for TA1538.

No increase in revertant colony numbers of any of the four tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). Reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base-pair changes or frame-shifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Negative, in vivo chromosome aberration in mouse, OECD 474, BASF (1991).

Negative, in vivo micronucleus in mouse, Poelloth (1997).

Negative, in vivo insect germ cell study, Gonchavora (1970).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(16 h for highest dose is performed 2 hours earlier than guideline recommendation, only 1000 MN PCE evaluated /animal)

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, D-W8741 Sulzfeld, FRG
- Weight at study initiation: 26.4 g
- Assigned to test groups randomly?: yes
- Housing: individua
l- Diet: ad libitum; (Kliba Haltungsdiaet, Klingentalmuehle AG, CH-4303 Kaiseraugst, Switzerland)
- Water: ad libitum, feed and water analysis indicated that the contaminants occuring in these medium would not alter result outcome
- Acclimation period: 1 wk

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s): aqueous 0.5% CMC (carboxymethyl cellulose)
- Justification for choice: no data
- Concentration of test material in vehicle: 5, 10 and 15%
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

- Justification for choice of concentrations: based on preliminary toxicity test

Duration of treatment / exposure:

- Negative and positive control: 24h
- 500 mg/kg bw: 24h
- 1000 mg/kg bw: 24h
- 1500 mg/kg bw: 16, 24, 48h

Frequency of treatment:

single dose application

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 020 mg/kg bw/day (actual dose received)

Dose / conc.:

1 550 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

- Negative control: 5
- 500 mg/kg bw: 5
- 1000 mg/kg bw: 5
- 1500 mg/kg bw: 15 (5 animals/sex for 16, 24, 48h sacrifice time point)

Control animals:

yes, concurrent vehicle

Positive control(s):

For clastogenicity: Cyclophosphamide
- No. of animals: 2M/3F
- Dose: 20 mg/kg bw
- Vehicle: aqua dest
- Route of exposure: orally
- Frequency: single dose application
- Volume of application: 10 mL/kg bw

For spindle poison effect: Vincristine
- No. of animals: 3M/3M
- Dose: 0.15 mg/kg bw
- Vehicle: aqua dest
- Route of exposure: ip
- Frequency: single dose application
- Volume of application: 10 mL/kg bw

Tissues and cell types examined:

Bone marrow
- Number of polychromatic erythrocytes (PCE) examined: 1000/animal. Following parameters were measured: Number of polychromatic erythrocytes, number of polychromatic erythrocytes containing micronuclei, number of normochromatic erythrocytes, number of normochromatic erythrocytes containing micronuclei, ratio of polychromatic to normochromatic erythrocytes, number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A pretest was performed to determine the oral acute toxicity of cyclohexanol after single oral administration of 2000 mg/kg bw. This dose caused no mortality, however the severe toxical signs were seen. The mice exhibited; irregular respiration, piloerection, staggering and squatting posture directly after test substance treatment. After about 15 minutes, abdominal position and a narcotic-like state was additionally observed. The general state of the animals was poor. In the main study however, 11 mortalities out of 30 animals occured at this dose. Hence the study protocol was amended. The highest dose was reduced to 1500 mg/kg bw

SACRIFICE AND SAMPLING TIMES:
- Hours after application: 16h & 48h (1500 mg/kg bw), 24h (all dose groups)

DETAILS OF SLIDE PREPARATION:
- Preparation of the bone marrow were based on the method of SCHMID, W. 1976 & 1977- Schmid W. (1976). The micronucleus test for cytogenetic analysis. In : Hollaender, A . (ed), Chemical Mutagens, Principles and Methods for their Detection, Volume 4, Plenum Press, New YorK- Schmid W., (1977) In: Kilbey et al . (eds), Handbook of Mutagenicity Test Procedures, Elsevier Scientific Publishing Company, Amsterdam - New York - Oxford.

Statistics:

According to the authors, perfoming a statistical evaluation was not necessary since the number of polychromatic micronucleated erythrocytes from the dosed groups was within/nearly the range of the actual control value and within the historical values.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CLINICAL SIGNS
- Control: Animals of the negative control group as well as both positive control groups did not show any clinical signs of toxicity.
- 500 mg/kg bw: about 30 minutes - 2 hours after test substance administration; irregular respiration, piloerection and squatting posture. After about 4 hours all symptoms had disappeared
- 1000 mg/kg bw: about 30 minutes - 4 hours after treatment; irregular respiration, piloerection, staggering, squatting posture and the general state of the animals was poor. The day after treatment no clinical signs were exhibited by the animals1500 mg/kg bw: about 30 - 60 minutes after treatment; irregular respiration, piloerection, abdominal position, narcotic-like state, staggering and the general state of the animals was poor. Some of these signs were still present two days after administration.

MISCROSPCOPIC EVALUATIONS (1000 PCE/dose were investigated)

TEST SUBSTANCE
- Percentage of PCE with micro nuclei after 24h: 1.2% (control), 1.5% (500 mg/kg bw), 1.2% (1000 mg/kg bw), 0.8% (1500 mg/kg bw)
- Percentage of PCE with micro nuclei after 16 and 48h in 1500 mg/kg bw dose group: 1.5% (16h), 1.1% (48h)
- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals
- Percentage of NCE with micro nuclei after 24h: 1.59% (control), 0.97% (500 mg/kg bw), 0.88% (1000 mg/kg bw), 1.65% (1500 mg/kg bw), 1.54% (CP), 1.86% (Vincristine)- Percentage of NCE with micro nuclei after 16 and 48h in 1500 mg/kg bw dose group: 2.11% (16h), 0.24% (48h)
- Ratio normocytes/total amount of polychromatic erythrocytes after 24 hours: 6301 (control), 5134 (500 mg/kg bw), 4520 (1000 mg/kg bw), 5467 (1500 mg/kg bw), 3255 (CP), 4848 (Vincristine)
- Ratio normocytes/total amount of polychromatic erythrocytes aster 16 and 45h in the 1500 mg/kg bw dose group: 5203 (16h) and 4191 (24h)

POSITIVE CONTROLS
- Cyclophosphamide (20 mg/kg bw): With 8.2%, the positive control substance cyclophosphamide for clastogenicity, produced polychromatic erythrocytes containing exclusively small micronuclei
- Vincristine (0.15 mg/kg bw): With 84.0%, the positive control vincristine for spindle poisoning effects producd a clearly enhanced number of micronuclei containing polychromatic erythrocytes with the expected amount of large micronuclei, i.e. 13.2%.

The number of NCE/total amount of PCE did not show a clear dose-dependent effect decrease. However, the values of the treated groups are below that of control animals. In addition the time-dependent decrease of the value from 5467 to 4191 in the high dose indicates that the test substance has reached the bone marrow.

Conclusions:

The test substance did not show any clastogenic or aneugenic activity in this test system in vivo.

Executive summary:

In a standard in vivo micronucleus test with cyclohexanol in mice conducted to GLP and OECD Testing guideline 474, there were no biologically relevant, significant differences in the frequency of polychromatic erythrocytes containing micronuclei either between the solvent control and the three dose groups (1500, 1000 and 500 mg/kg bw) at any sacrifice intervals time (16, 24 and 48 hours). Thus, under the experimental conditions chosen, the test substance cyclohexanol did not show any chromosome-damaging (clastogenic) effects nor did it lead to any impairment of chromosome distribution in the course of mitosis.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Reliable in vitro mutagenicity studies (Ames test, gene mutation study in mammalian cells) with cyclohexanol are available, which do not indicate a genotoxic potential of cyclohexanol. The result in one available Ames test was "ambiguous" due to a not dose-dependent increase in the mutant frequency (up to 2.0) in one strain (TA 98). The positive chromosomal aberration effects in human leukocytes described in one publication should be considered as inconclusive and inadequate for the assessment of mutagenicity of cyclohexanol. In a reliable in vivo micronucleus test performed to OECD guideline 474, negative results were found, which are adequate for the assessment of chromosomal aberration effects.

In vitro gene mutation

Bacterial reverse gene mutation

A bacterial reverse gene mutation assay (DuPont 1976, equivalent to OECD guideline 471 with the limitation that no E.coli or TA102 strain was tested) is available. Bacteria strains of S. typhimurium TA 98, TA 1535, TA 1537, TA 1538 were exposed to cyclohexanol (no data on analytical purity) in ethanol at concentrations of 0, 500, 1000, 2500, 5000, 7500, 10000, 15000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method. Cyclohexanol was tested up to the limit concentration of 5000 µg/plate and beyond. Cytotoxicity was seen at concentrations ≥7500 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

Haworth (1983) performed another bacterial gene mutation assay (equivalent to OECD guideline 471 with the limitation that no E.coli or TA102 strain was tested). The mutagenic potential of cyclohexanol (no data on purity) was determined using the preincubation method in S. typhimurium strains TA 98, TA 1535, TA 1537, TA 100 at the concentrations of 0, 33, 100, 333, 1000, 3333 µg/plate with (S9) and without metabolic activation. Cyclohexanol was dissolved in water and was not tested up to the limit concentration of 5000 µg/plate probably due to limited solubility. In the strain TA 98, an increase of revertants was attained in the presence of metabolic activation, peaking at 333 µg/plate with 94 revertants vs. 48 revertants in the control in trial 1 and 57 revertants vs. 37 revertants in control in trial 2. No dose-dependent increase was observed. The positive controls induced the appropriate responses in the corresponding strains. The study results were considered as ambiguous, as no dose-dependency in the strain showing an increase in mutation frequency was observed.

Mammalian cell gene mutation

Wollny (2013) carried out a GLP-study to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The method followed OECD TG 476. The assay was performed in three independent experiments, using two parallel cultures each. Experiments I and II were performed with and without liver microsomal activation using a treatment period of four hours. The concentration range of the main experiments went up to approximately 10 mM. No cytotoxic effects occurred in the main experiments up to the maximum concentration in the absence and presence of metabolic activation. No substantial and reproducible dose-dependent increase in mutant colony numbers was observed up to the maximum concentrations tested with and without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. During the mutagenicity test described and under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test item was considered to be non-mutagenic in this mouse lymphoma assay.

In vitro cytogenetic assays

In a publication providing little experimental details, Collin (1971) reported an in vitro assay measuring chromosomal aberration where human leukocytes were tested at concentrations between 0.1-10 mM without metabolic activation. Gaps, but no increase in breaks were observed without any dose response relationship. There was no information with respect to cytotoxicity or presence of a control group. Only a statement on observations from 12 cells per concentration was given, but the total number of cells studied was not specified. The study led to an inconclusive result on the mutagenicity potential of the substance due to clastogenic effects and is considered inappropriate for the assessment of the mutagenic potential of cyclohexanol.

In vivo gene mutation

In vivo cytogenetic assays 

The potential of cyclohexanol to induce chromosomal aberration in-vivo was examined in a bone marrow micronucleus assay (BASF AG 1991, according to OECD TG 474). Groups of NMRI mice (5/sex/dose) were treated by oral gavage with a single dose of cyclohexanol (98.8%) in aqueous carboxymethylcellulose (CMC) at doses of 0, 500, 1000, 1500 mg/kg bw. Bone marrow cells were harvested at 24 hours post-treatment in all dose groups and 16 and 48 hours post treatment in the highest dose group. 

There were signs of toxicity during the study. Clinical signs of toxicity included irregular respiration, piloerection, squatting posture, abdominal position, narcotic-like state, staggering and a poor general state. In the lowest dose group, remission was within 4 hours, in the mid dose within 24 hours and in the highest dose group symptoms were still present 48 hours post administration. There were no biologically relevant, significant differences in the frequency of polychromatic erythrocytes containing micronuclei between the solvent control and the dosed groups at any time (16, 24 and 48 hours). The ratio of NCE/PCE did not show a clear dose-dependent effect, however, the values of the treated groups were below that of control animals. Thus, it can be assumed that the test substance has reached the target organ. Based on the results of the study, cyclohexanol was considered to be non-mutagenic in this in vivo assay.

The mutagenicity of cyclohexanol was studied in vivo in the mouse according to the micronucleus assay (Poelloth and Mangelsdorf 1997). The substance was not mutagenic in this study. However, the lack of experimental details does not allow to conclude on the validity of this result.

The potential mutagenic effects of a low dose of cyclohexanol via inhalation exposure were studied in mouse bone marrow cells including chromosome aberrations (CA) and micronucleus (MN) assays (Sivikova et al. 2007). Capillaries with cyclohexanol (purity not available) were placed in an experimental chamber. These tubes were evaporating the substance continuously over a period of six weeks. No clastogenic and/or aneugenic effect of cyclohexanol was observed. Only one unknown dose was tested (”low dose”). No positive control substance was tested. Only male test animals were used. There are little details on the method of administration, and the method of analysis of the test concentration consisted in measuring the remaining level of test material in the capillaries (no analytical confirmation of the test concentration). Due to these major methodological deficiencies, the validity of the test cannot be confirmed. This study is therefore disregarded and not used in the conclusion on the mutagenicity of cyclohexanol.

Sister chromatid exchange assay

The potential mutagenic effects of a low dose of cyclohexanol via inhalation exposure were studied in mouse bone marrow cells using the sister chromatid exchange (SCE) assay (Sivikova et al. 2007). Capillaries with cyclohexanol (purity not available) were placed in an experimental chamber. These tubes were evaporating the substance continuously over a period of six weeks. A statistically significant elevation of the frequency of SCE in the animals exposed to cyclohexanol was detected (average of 5.67 ± 2.34 compared to average of 3.96 ± 1.67 in the control group). The authors of the study concluded that the test material demonstrated a genotoxic potential under the conditions of the study. However, the study has a number of methodological deficiencies:

•       the chemical identity and the purity of the test substance were not confirmed

•       only one unknown dose was tested (”low dose”)

•       there are little details on the method of administration, and the method of analysis of the test concentration consisted in measuring the remaining level of test material in the capillaries (no analytical confirmation of the test concentration)

•       there is no information on a potential dose-response relationship of the effects observed in this limited study

•       no positive control substance was tested to demonstrate the adequacy of the test system

•       only male test animals were used

Due to these major methodological deficiencies and the absence of a dose-response relationship, the validity of the test cannot be confirmed. This study is therefore disregarded and not used in the conclusion on the mutagenicity of cyclohexanol. Furthermore, the OECD deleted the guideline for the (in vitro) sister chromatoid exchange assay (OECD TG 479), because of a lack of understanding of the mechanism(s) of action of the effect detected by the test.

Sex-linked Recessive Lethal test in Drosophila melanogaster

The mutagenicity of cyclohexanol was studied in vivo according to the Drosophila SLRL assay (EFSA 2004). The substance was not mutagenic. However, the complete lack of experimental details does not allow to conclude on the reliability and validity of this result.

 

Justification for classification or non-classification

In accordance with Regulation (EC) No. 1272/2008, Annex I, 3.5.2, based on the available data the test substance is considered to be not mutagenic. Therefore, cyclohexanol is not classified for mutagenicity.