Cyclohexanone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is non-mutagenic with and without metabolic activation in a bacterial reverse mutation assay (Ames test), according to OECD 471, and in a mammalian cell forward gene mutation assay (HPRT), according to 476. Due to a further mammalian cell forward mutation assay (TK) the test substance was also considered to be non-mutagenic with and without metabolic activation. Furthermore, the test substance was considered to be non-mutagenic with and without metabolic activation in a chromosomal aberration assay (UDS) in human fibroblasts.

Well-documented and applicable (Q)SAR data are available demonstrating the absence of a chemical structure activity relationship for cyclohexanone to known germ cell mutagens.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

slight differences in protocol, no evaluation criteria are reported

Principles of method if other than guideline:

Test performance comparable to OECD 482 (deleted in 2014)

GLP compliance:

not specified

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Specific details on test material used for the study:

Source: Sigma-Aldrich
Batch no.: 13975

Target gene:

human fibroblasts

Species / strain / cell type:

other: human fibroblasts (diploid)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix

Test concentrations with justification for top dose:

up to 9.48 mg/ml of culture medium

Vehicle / solvent:

dimethylsulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Key result

Species / strain:

other: human fibroblasts (diploid)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the initial assay involving tritiated thymidine incorporation into non-S phase cells, there was no indication of any significant increase in the number if silver grain per nucleus at any concentration of cyclohexanone. The highest concentration used in this test was 10 µl, or 9.478 mg/mL. The positive control substances used, 4-nitroquinoline- N-oxide and 2-aminoanthracene, induced significant responses in unscheduled DNA synthesis in these cells. Some of the mean grain counts per nucleus were rather high in this first experiment, so, it was repeated At a dose level of 74 µg/mL in the presence of S9 mix there was a high result, but the standard deviation also was very high. These experiments gave no indication of UDS induction. These positive control substances, however, are not appropriate for the demonstration of short patch repair when measured by Method 2. The tritiated deoxyguanosine incorporation assay was used to confirm the results of the first assay. During the course of these experiments, the permeability of both cell lines to deoxyguanosine decreased greatly, this reduction being aggravated by the addition of S9 mix to the incubation medium. In consequence, the measured incorporation of radioactivity was insufficient to provide any reasonable analysis of data produced.

Conclusions:

Only minor deviations to the OECD guideline 482 (deleted in 2014) were obvious in the NIOSH study. A negative result in an UDS assay employing human fibroblasts in concentrations up to 9.48 mg/mL (with and without metabolic activation) was reported. This negative outcome was confirmed in an independent experimental repeat.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

Comet assay in reconstructed 3D human epidermal skin (EpiDerm TM tissue): intra- and inter-laboratory reproducibility study, joining: 3 labs
Cell isolation procedure according to: Curren, R.D. et al. (2006), Mutat. Res. 607, 39-51
Comet assay procedure according to: Singh, N.P. et al. (1988), Exp. Cell Res. 175, 184-191; Burlinson, B. (2012), Methods Mol. Biol. 817, 143-163; Tice, R.R. et al. (2000) Environ. Mol. Mutagen., 35, 206-221; in compliance with current OECD 489 (Comet vivo)

GLP compliance:

not specified

Type of assay:

comet assay

Specific details on test material used for the study:

Source: Sigma-Aldrich

Test concentrations with justification for top dose:

Top dose 1600 µg/cm2, at least three dose levels (serial dilutions with maximal 3.16-fold spacing)

Vehicle / solvent:

Acetone
- Justification for choice of solvent: no effect on background tail DNA values, facilitation of absorption in the epidermis and quick evaporation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: moisture was removed with a cotton tip, topical application of 10 µL of the dose solutions directly on the surface of the skin

DURATION
- Tissue acclimatisation: overnight in 6-well plates containing 1 ml maintenance medium in a humidified incubator at 37°C and 5% carbon dioxide, skin models were placed in 1 ml fresh medium on the day of treatment
- Exposure duration: 3 hours in a humidified incubator (37°C, 5% carbon dioxide)

NUMBER OF REPLICATIONS: 4 skin models per dose group, 2 independent experiments

NUMBER OF CELLS EVALUATED: 100 nuclei on duplicate slides (50/slide) per dose, total of 400 nuclei per dose

DETERMINATION OF CYTOTOXICITY
- in cell suspension after preparation: trypan blue exclusion or ethidium bromide/acridine orange staining

Evaluation criteria:

A test compound was considered positive for genotoxicity if it had at least one study with two or more (consecutive) dose levels producing statistically significant increases in % tail DNA, or if the highest concentration produced a statistically significant increase in % tail DNA in the absence of a relevant cytotoxic effect (>30%) and a significant effect was reproduced in an independent study. If a test compound induced a significant increase only at a dose other than the highest dose, it was considered positive only if the trend test was positive, and the effect was reproducible. A test compound that did not demonstrate a relevant increase of the % tail DNA was considered non-genotoxic.

Statistics:

Dunnett pairwise comparison of each treatment to the control, test for dose-response trend based upon simple linear regression

Key result

Species / strain:

other: human epidermal skin model

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

In general, the reproducibility of the assay was considered good since all carcinogens were correctly identified by the participating labs. For the non-carcinogens only one lab did not identify the substance correctly.

A statistically significant increase in % tail DNA compared to the concurrent solvent control at all dose levels tested was observed in one of the three testing labs. The maximum increase in % tail DNA compared to the solvent control was 3-fold at the highest applied dose. However, no dose related increase was observed at the tested dose range. In the second lab, a statistically significant increase at the top dose was observed in one experiment. The % tail DNA values observed in this experiment were relatively low (group means % tail DNA of tissues ranged from 2–5% vs. 1% in the solvent control). The increased % tail DNA value could not be confirmed in the experimental repeat. Therefore, the observed response was considered not biologically relevant. In the third lab, no relevant increase in % tail DNA was observed at the dose range applied. In conclusion, the substance was considered to be non-genotoxic due to the negative outcome in two labs and the lack of dose-relationship in the first lab where statistically significant increases were observed.

Conclusions:

An intra- and inter-laboratory Comet study using a reconstructed 3D human epidermal skin model was performed with the test substance in three participating laboratories. This non-guideline study was performed according to a scientifically valid protocol. The Comet analysis was in compliance with the current OECD 489 guideline for the alkaline Comet assay in vivo. In two of the participating labs the outcome of the assay was non-genotoxic, whereas in one lab significant increases in % tail DNA were observed after treatment with the test substance. However, the overall conclusion was non-genotoxic, since no dose-response relationship could be observed.

Reference 3

Endpoint:

genetic toxicity in vitro, other

Remarks:

[³H]TdR uptake study in mammalian cells

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

GLP compliance:

no

Type of assay:

other: [³H]TdR uptake study in mammalian cells

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

healthy adult donors

Metabolic activation:

with and without

Metabolic activation system:

rat liver phenobarbital-induced S9

Test concentrations with justification for top dose:

0.01, 0.001 and 0.001 mol/L

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent/vehicle: low water solubility of most of the substances tested

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

chloroform

Positive controls:

yes

Positive control substance:

other: chloromethyl methyl ether

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- human lymphocytes
- Cells density: 100000 - 200000 / well of a micro test plate

DURATION
- Exposure duration: 4 h

NUMBER OF REPLICATIONS: 6

DETERMINATION OF CYTOTOXICITY
- Method: trypan-blue staining technique

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

100 % cell viability, but reduced [3H]TdR uptake at 0.01 mol/L

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

No toxicity was determined by the trypan-blue staining technique, but the test item reduced the [³H]TdR uptake at a concentration of 0.01mol/L in the presence of S9 mix. No increased [³H]TdR uptake was observed at any concentration in the absence or presence of the S9 mix.

Conclusions:

The effect of cyclohexanone on cell viability and DNA synthesis, determined by tritiated thymidine uptake, were investigated in human lymphocytes in vitro in the absence and presence of metabolic activation. Cell viability was not influenced by the substance and the DNA synthesis was slightly decreased. Since no increase in DNA synthesis was observed, the substance is not considered to induce DNA damage and repair synthesis. This non-guideline study is scientifically valid and appropriate for the assessment of non-genotoxicity.

Reference 4

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)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium strains and E. coli

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix

Test concentrations with justification for top dose:

10 - 5000 µg/plate (Standard Plate Test); 10 - 1000 µg/plate (Pre-Incubation Test)

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: with S-9 mix: 2-aminoanthracene; without S-9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine and 4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

Bacterial test strains were used, both in the standard plate test and in the preincubation test, both in the presence and absence of metabolic activation (+S9, -S9, respectively). DMSO was used as solvent. Three test plates per dose or per control were run in each of the 3 experiments.

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Statistics:

-

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY: No precipitation of the test substance was found.
TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 100 µg - 2,500 µg/plate onward.
MUTAGENICITY: An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Conclusions:

A GLP-compliant Ames test performed according to OECD guideline 471 with concentrations ranging from 10 - 5000 µg/plate showed negative results with and without metabolic activation in all recommended tester strains. The assay was performed as standard plate and pre-incubation assay. The study demonstrated the non-mutagenicity of the substance in bacterial test strains.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 April 2012 - 18 Sept 2012

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:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 30 May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

adopted August 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Cyclohexanone rein K2A Ex2187 Q209
- Expiration date of the lot/batch: stability of the test substance is guaranteed until February 2012

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: all media were supplemented with 1% (v/v) penicillin/streptomycin, 1% (v/v) amphotericine B
- Treatment medium (4 hours): Ham's F12 medium containing stable glutamine and hypxanthine
- Treatment medium (24 hours)/culture medium: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10%(v/v) fetal calf serum (FCS)
- Pretreatment medium (HAT medium): Ham's F12 with hypoxanthine, aminopterin, thymidine and 10% (v/v) fetal calf serum (FCS)
- Selection medium (TG medium): Hypoxanthine free Ham's F12 with 6-thioguanine, 1% (v/v) stable glutamine, 10% (v/v) fetal calf serum (FCS)
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

pre-test
with and without S9 mix (4 + 24 hours): 3.8, 7.7, 15.3, 30.6, 61.3, 122.5, 245.0, 490.0, 980.0 µg/mL

1st Experiment
without S9 mix (4-hour exposure period): 0; 122.5; 245.0; 490.0; 980.0 μg/mL
with S9 mix (4-hour exposure period): 0; 122.5; 245.0; 490.0; 980.0 μg/mL

2nd Experiment
without S9 mix (24-hour exposure period): 0; 61.3; 122.5; 245.0; 490.0; 980.0 μg/mL
with S9 mix (4-hour exposure period): 0; 100.0; 200.0; 400.0; 980.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: medium
- Justification for choice of solvent/vehicle: due to the good solubility of the test substance in water, medium was chosen as vehicle

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 1st experiment - 4 hours exposure with and without S9 mix, 2nd experiment - 24 hours exposure without and 4 hours with S9 mix
- Expression time (cells in growth medium): 6-7 days with passage of cells after 3-4 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): after 16 days

SELECTION AGENT (mutation assays): 10µg/mL 6-thioguanine
STAIN (for cytogenetic assays): giemsa

NUMBER OF REPLICATIONS: 2 replicates in 2 independent experiments each

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 1000000 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: not influenced
- Precipitation: no precipitation

RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the purity and the molecular weight of the test substance 980 μg/mL (approx. 10 mM) was used as top concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. No effects on pH and osmolality were observed, no precipitation up to the highest concentration tested was seen and no cytotoxicity (reduced cloning efficiency - about or below 20% survival) was found up to the highest concentration under all test conditions.

COMPARISON WITH HISTORICAL CONTROL DATA: the results were in range with historical control data

The substance cyclohexanone was assessed for its potential to induce gene mutations at HPRT locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats. The following doses were tested based on a preliminary cytotoxicity test.

1st Experiment

without S9 mix (4-hour exposure period) 0; 122.5; 245.0; 490.0; 980.0 μg/mL

with S9 mix (4-hour exposure period) 0; 122.5; 245.0; 490.0; 980.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period) 0;122.5; 245.0; 490.0; 980.0 μg/mL

with S9 mix (4-hour exposure period) 0; 100.0; 200.0; 400.0; 980.0 μg/mL

Cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours or in the presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6- thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation no cytotoxicity or precipitation was observed up to the highest required concentration evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any increase in the mutant frequencies neither without S9 mix nor after the addition of a metabolizing system in two independent experiments.

Table1: summary of results of experiments 1 and 2

Experiment	Exposure period	Test groups	S9 mix	Precipitation*	Genotoxicity**	Cytotoxicity***
	[h]	[µg/mL]			MF corr.	CE1	CE2
					[per E+06]	[%]	[%]
1	4	negative control	-	-	1.99	100.0	100.0
		122.5	-	-	3.67	121.2	107.8
		245.0	-	-	0.00	99.0	107.1
		490.0	-	-	4.59	119.1	99.1
		980	-	-	3.76	87.8	96.2
		positive control^1	-	-	108.86	107.1	89.7
2	24	negative control	-	-	1.90	100.0	100.0
		61.3	-	-	n.c.^1	101.5	n.c.^1
		122.5	-	-	1.71	104.8	105.0
		245.0	-	-	6.65	97.9	99.8
		490.0	-	-	0.79	103.2	94.0
		980.0	-	-	0.79	103.2	94.0
		Positive control^1	-	-	668.91	93.0	67.6
1	4	Negative control	-	-	0.00	100.0	100.0
		122.5	+	-	0.74	103.1	93.4
		245.0	+	-	1.18	92.3	94.1
		490.0	+	-	1.04	98.4	103.3
		980.0	+	-	0.75	93.9	100.7
		Positive control^2	+	-	327.55	57.9	76.8
2	4	Negative control	+	-	5.46	100.0	100.0
		100.0	+	-	1.60	98.4	92.9
		200.0	+	-	4.80	96.4	98.3
		400.0	+	-	2.53	96.3	89.6
		980.0	+	-	5.02	85.1	88.9
		Positive control^2	+	-	347.85	74.7	72.0

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per E+06 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

n.c.¹ Culture was not continued since a minimum of only 4 analysable concentrations are required

¹ EMS 300 µg/mL

² DMBA 1.25 µg/mL

Conclusions:

To assess the potential of cyclohexanone to induce forward gene mutations at the HPRT locus, two independent experiments with test substance concentrations ranging from 100 - 980 μg/mL (approx. 10 mM) were performed in Chinese hamster ovary (CHO) cells. No increase in the mutant frequencies was observed, neither with nor without the addition of a metabolizing system.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

evaluation criteria

Principles of method if other than guideline:

The experimental procedures used were based upon those described by Clive and Spector [1975] and Clive et al. [1979], but certain small differences were incorporated.

GLP compliance:

not specified

Type of assay:

other: in vitro mammalian cell gene mutation assay

Specific details on test material used for the study:

Source: Radian Corporation, Austin, TX 78766

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

other: tk+/tk- -3 .7 .2C heterozygote of L5178Y

Metabolic activation:

with and without

Metabolic activation system:

Uninduced and Aroclor induced rat liver S9

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500, 5000 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

L5178Y mouse lymphoma cells (free from mycoplasma) were cultured in Fischer's medium (designated F0), supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/ml, 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (designated F10P). Within 1 week of an experiment start, cultures were purged of tk-/tk- mutants by exposure for 1 day to F10P containing THMG (thymidine 6 μg/mL, hypoxanthine 5 μg/mL, glycine 7.5 μg/mL and methotrexate 0.1 µg/mL), then for 3 days to F10P containing THG only (i .e., THMG without methotrexate).
S9 mix was prepared by dissolving preweighed cofactors in Fischer's medium containing 5% heat-inactivated horse serum, 9 parts of this solution mixed with 1 part S9. The concentrations of the cofactors in S9 mix were NADP, 4 mM and glucose-6-phosphate, 25 mM. If required, S9 mix was added to constitute 10% of the incubation mixture, i.e., the S9 concentration in the final incubation mixture was 10 µL/mL.
Each mutagenicity experiment normally consisted of the following groups: vehicle control (4 cultures), positive control (2 cultures) and at least five test compound concentrations (2 cultures per concentration). A pre-test on toxicity was performed in which cell population expansion was measured. Ten-fold differences in test compound concentrations were used in the pre-test, the highest being 5 mg/mL. This test was followed by two mutagenicity experiments in the absence and in the presence of S9 mix. Test compound concentrations were primarily two-fold dilutions from the highest testable concentration, as estimated from the toxicity test.

Exposure: Each exposed culture consisted of 6 x 10E6 cells in a final volume of 10 mL F5P. The tube was incubated for 4 hours under rotation. At the end of the incubation time, the cells were sedimented by centrifugation at 500 x g for 10 min, washed, and finally resuspended in 20 mL F10P. These cell suspensions (3 x 10E5 cells/mL) were incubated for a 2-day expression period, the cell population density being adjusted back to 20 mL of 3 x 10E-5 cells/mL after 24 hours. After 48 hours, the cell population densities were estimated and culture volumes containing 3 x 10E-6 cells adjusted to 15 mL with F10P, giving a cell population density of 2 x 10E-5 cells/mL.

Cloning efficiency: A 0.1-mL sample of the cell suspension was withdrawn and diluted 1:100. Three 0.1-mL samples (200 cells) of the diluted cultures were transferred to 30-mL tubes, mixed with 25-mL cloning medium (Fischer's medium containing 20% heat-inactivated horse serum, i.e. F20P) containing 0.35% Noble agar and poured into 90-mm Petri plates.

Mutant selection: Three aliquots (each containing 10E6 cells) of the remaining culture were distributed to 30-mL tubes, mixed with 20-mL cloning medium to give final concentrations of 0.35% Noble agar and 3 μg trifluorothymidine/mL, then poured into 90-mm Petri plates.

Incubation: The agar was gelled at 4°C for 5-10 min, then the plates were incubated for 11-14 days in 5% carbon dioxide/95% air at 37°C.

Colony counting: Colonies were counted using an Artek 880 Automated Colony Counter, with the colony size discriminator control in the "off" position.

Evaluation criteria:

Not specified

Statistics:

The statistical analysis was based upon the mathematical model proposed for this system [Lee and Caspary, 1983] and consisted of a dose-trend test [Barlow et al., 1972] and a variance analysis of pair-wise comparisons of each dose against the vehicle control.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Conclusions:

Cyclohexanone at concentrations up to 5000 µg/mL did not lead to a significant increase in mutation frequencies, neither in the presence nor in the absence of S9 mix. Therefore, the substance is considered non-mutagenic. Although this supporting study was not performed according to the current OECD guideline 490, the minor deviations to the guideline are acceptable.
If colonies in a tk mutation are scored using the criteria of normal (large) and slow growth (small) colonies, gross structural chromosome aberrations (i.e. clastogenic effects) can be detected, since mutant cells that have suffered damage to both e the tk gene and the growth genes situated close to the tk gene have prolonged doubling times and are more likely to form small colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In a chromosomal aberration assay the test substance did not lead to relevant increases in chromosomal aberrations in bone marrow of rats. In a dominant lethal assay no effects attributable to the test substance were observed. In a mouse micronucleus assay, cyclohexanone did not induce micronuclei up to the highest dose tested.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

50 metaphases instead of 200 metaphases per animal were scored, mitotic index was not determined, evaluation criteria are not reported

GLP compliance:

not specified

Type of assay:

other: chromosome aberration

Specific details on test material used for the study:

Source: Sigma-Aldrich
Batch no.: 13975

Species:

rat

Strain:

other: CD (a remote Sprague-Dawley-derived strain)

Sex:

male/female

Details on test animals or test system and environmental conditions:

All the animals were located in a room which was separate from but adjacent to the area where the exposures were conducted. They were housed individually in cages in a room with a light intensity of approximately 200 lux, a 12 h light-dark cycle, approximately 10 air changes per hour, temperature maintained at ca 22°C with extreme limits of 17°C and 24°C, and relative humidity ca 50%, with extreme limits of 32% and 52%. Food and water were freely available to the rats at all times. The animals to be dosed were individually identified using brass ear tags bearing the aninal number and the suffix letter showing the compound designation. Each rat was ascribed a cage card which identified that animal by project number, animal niunber, sex and treatment group.

Route of administration:

inhalation: vapour

Vehicle:

none

Details on exposure:

Animals were sacrificed 6, 24, and 48 hours following inhalation exposure to vapors of 50 or 400 ppm for 1 or 5 days.
Each rat was injected i.p. with 3 mg/kg colchicine dissolved in Hank's Balanced Salt Solution (HBSS) 4 h after the last dose was given. Two hours later the rats were killed by neck dislocation. One femur from each animal was dissected out, cleaned of adherent tissue and the marrow aspirated into a 10 ml plastic blood sample tube containing 4 ml HBSS at ambient temperature and lithium hepari. Each tube was labelled with the appropriate random number from a slide coding sheet. Hence, from this time until the completed result sheets were de-coded, the rat number and group were unknown to the scientists and technicians. The cell suspension was centrifuged at 1,500 r.p.m. for 5 min, the supernatant fluid discarded and replaced with 4 ml fresh HBSS. The cells were suspended, then centrifuged again and the supernatant fluid discarded. 4-5 ml 0 .075 M-KC1 pre-heated to 37°C was added to the cells while they were agitated on a vortex mixer. Following incubation for 20 min in a 37°C water bath, the cells were centrifuged, the supernatant fluid decanted and the cells fixed in 4 ml freshly prepared fixative (methanol : glacial acetic acid; 3:1). The fixative was removed after centrifugation and replaced with 2 ml fresh fixgtive. Tubes containing fixed cells were stored in a 4°C refrigerator overnight. The following morning (or later, up to 3 days) the fixative was changed and cell suspensions dropped onto clean slides labelled with the same number as the tube and allowed to dry thoroughly. Slides were stained in a bath of Giemsa R66 (Gurr) diluted with 10 parts distilled water for 30 min, rinsed briefly in distilled water, dehydrated in alcohol, cleared in xylene and mounted in DePeX.
Slide Reading: Leitz binocular microscopes were used for this purpose. Magnification was nominaily x 1,000 using x 10 magnification eye pieces and x 100 objectives. Wherever possible, for each animal 50 cells with a minimum of 41 well spread chromosomes were examined and scored. The location of all spreads examined were recorded using the microscope stage vernier. The slide number was always located on the right hand side. Abnormalities looked for were: gaps, breaks, fragments, dicentrics, translocations (within the limitations of the staining methods) and pulverisation.

Duration of treatment / exposure:

single dose 1 x 7 h or 1 x 7 h repeated dosing 5 days

Frequency of treatment:

7 h daily for repeated dosing

Post exposure period:

no data

Dose / conc.:

50 ppm

Dose / conc.:

400 ppm

No. of animals per sex per dose:

30 for single dosing
10 for repeated dosing

Control animals:

yes, concurrent no treatment

Positive control(s):

ethyl methanesulphonate

Tissues and cell types examined:

All animals were inspected for clinical signs/mortality twice daily except at weekends when they were observed once only. During the dosing period, all animals subjected to the exposure manipulations were weighed upon removal from the exposure chambers.

Details of tissue and slide preparation:

Dosing solutions were prepared daily 5 min before administration to the animals was started. The desired amount of ethyl methanesulphonate was weighed into a volumetric flask and diluted with distilled water to obtain the correct concentration.
Positive control animals were not allowed access to food or water whilst the remaining test groups were being exposed. Ethyl methanesulphonate was administered orally by gavage to the rodents at a constant dose volume of 10 mL/kg at around 16.00 h on each day that dosing was required.

Evaluation criteria:

Not specified

Statistics:

A one-sided Student's t test was used on the transformed values.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the multiple exposure cytogenetic test, there were no indications of induction of chromosomal damage in either the male or female rats exposed to 50 ppm or 400 ppm cyclohexanone atmospheres. Responses to the positive control substance, ethyl methanesulphonate, were significant in the females, but not in the males. The single exposure test male rats showed a significant increase in gap frequency at the 6 h sample time in the 400 ppm cyclohexanone atmosphere exposed group (p < 0.005). No similar increase in aberration frequency was observed in the female rats at this or any other sampling time. Also, aberrations excluding gaps were not increased in any of the male rat groups eNposed to cyclohexanone. Responses to ethyl methanesulphonate were not uniformly significant. Frequencies of all aberrations were increased in male rats at the 6 h and 24 h sampling times and in female rats at the 24 h and 48 h sampling times. Aberrations excluding gaps were increased in male rats at the 24 h and 48 h sampling times and in female rats at the 24 h and 48 h sampling times.

Conclusions:

In a chromosomal aberration assay doses of 50 and 400 ppm were applied to male and female rats via the inhalation route (single dose 7 h/day or 7 h/d for 5 days). The assay followed in general the current OECD guideline 474, showing minor deviations, but the studies are well documented and considered scientifically valid. Single and repeated dosing of cyclohexanone did not lead to relevant increases in chromosomal aberrations in the bone marrow of the animals. The study is considered to sufficiently cover the endpoint investigated.
The bioavailability of cyclohexanone following inhalation exposure is very good. This was demonstrated by a significant increase in cyclohexanone plasma levels in rats exposed to 400 and 1600 ppm for 6h (Industrial Health Foundation Inc., 1987; see toxicokinetics) and a significant increase in urinary cyclohexanone metabolites following exposure of human volunteers to 200 mg/m3 for 8h (Mraz et al. 1994; see toxicokinetics).

Reference 2

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

evaluation criteria are not reported

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

evaluation criteria are not reported

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

Source: Sigma-Aldrich
Batch no.: 13975

Species:

rat

Strain:

other: CD (a remote Sprague-Dawley-derived strain)

Sex:

male/female

Details on test animals or test system and environmental conditions:

All the animals were located in a room which was separate from but adjacent to the area where the exposures were conducted. They were housed individually in cages in a room with a light intensity of approximately 200 lux, a 12 h light-dark cycle, approximately 10 air changes per hour, temperature maintained at ca 22°C with extreme limits of 17°C and 24°C, and relative humidity ca 50%, with extreme limits of 32% and 52%. Food and water were freely available to the rats at all times. The animals to be dosed were individually identified using brass ear tags bearing the aninal number and the suffix letter showing the compound designation. Each rat was ascribed a cage card which identified that animal by project number, animal niunber, sex and treatment group.

Route of administration:

inhalation: vapour

Vehicle:

none

Duration of treatment / exposure:

1 x 7 h repeated dosing 5 days

Frequency of treatment:

7 h daily

Dose / conc.:

50 ppm

Dose / conc.:

400 ppm

No. of animals per sex per dose:

10 males

Control animals:

yes, concurrent no treatment

Positive control(s):

ethylmethanesulphonate
- Route of administration: orally by gavage
- Doses / concentrations: 100 mg/kg bw for 5 consecutive days

Evaluation criteria:

Not specified

Statistics:

Chi-square test
beta-binominal model

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Pregnancy frequency was calculated in 2 ways: firstly, by considering as pregnant females with corpora lutea graviditatis and secondly and more reliably by considering is pregnant only females with implantations. With neither method was there any effect upon pregnancy frequency due to cyclohexanone treatment, but there were reductions in Weeks 2 and 3 and, to lesser extents, weeks 1 and 4 in the positive control group.
Corpora lutea graviditatis counts were not reduced in either of the cyclohexanone treated groups; these counts were reduced, however, in Weeks 1-3 of the positive control group.
Implantations per pregnancy were unaffected by cyclohexanone treatment, but were reduced in Weeks 1-4 by the positive control group.
The frequencies of live implantations and live implantations and late deaths followed very closely the pattern of total implantations per pregnancy.
An exception was in Week 8 of the 50 ppm cyclohexanone exposure group where live implantations and live implantations and late deaths frequencies were low compared with the other groups. A review of the data showing pregnancies with either one or more early deaths or two or more early deaths did not indicate any increase in these frequencies in the cyclohexanone treated groups, when compared with the air control group.
Analysis of the proportions of early deaths by various statistical methods did not indicate any effects attributable to cyclohexanone treatment in Week 8 of the 50 ppm cyclchexanone exposure group the proportion of early de-iths was particularly high, but no sustained increase was seen in the 400 ppm cyclohexanone exposure group.
It is considered that the high, sporadic frequencies - totally unrelated to treatment - of early deaths may have been due to the effects of the sialodacryoadenitis virus, clinical signs of which were definitely seen in Week 8 and may have been present earlier.
The bioavailability of cyclohexanone following inhalation exposure is very good. This was demonstrated by a significant increase in cyclohexanone plasma levels in rats exposed to 400 and 1600 ppm for 6h (Industrial Health Foundation Inc., 1987; see toxicokinetics) and a significant increase in urinary cyclohexanone metabolites following exposure of human volunteers to 200 mg/m3 for 8h (Mraz et al. 1994; see toxicokinetics).

Conclusions:

In a dominant lethal assay doses of 50 and 400 ppm were applied to male rats via the inhalation route (7 h/d for 5 days). The assay followed in general the current OECD guideline 478, showing minor deviations, but the study is well documented and considered scientifically valid. There were no effects attributable to the test substance in the dominant lethal assay. The study is considered to sufficiently cover the endpoint investigated.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Supplier: Sigma-Aldrich Chemie GmbH
Batch: BCCB1352
Purity: 100.0%
Molecular weight: 98.14 g/mol
Physical state / Appearance: Colourless liquid
Retest Date: 30 June 2023
Storage Conditions: At room temperature
Stability in Solvent: Not indicated by the Sponsor

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations, which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies.

Sex:

male

Details on test animals or test system and environmental conditions:

Strain: Mouse (NMRI)
Source: Charles River Laboratories Research Models and Services Germany GmbH Sandhofer Weg 7, 97633 Sulzfeld, Germany
Number of Animals in the pre-test: 2 males and 2 females
Number of Animals in the main study: 43 males (no gender differences in pre study)
Initial Age at Start of Experiment: 6 – 10 weeks (main study)
Acclimation: minimum 5 days
Body Weight at Start of Treatment: mean value 33.2 g (SD ± 1.4 g); range 30.1 – 35.4 g
Body Weight at End of Treatment: mean value 33.8 g (SD ± 1.6 g); range 30.8 – 37.7 g
According to the supplier’s assurance, the animals were in healthy condition. The animals were under acclimatisation in the animal house of ICCR-Roßdorf GmbH for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.
Housing: Single
Cage Type: Macrolon Type II / III, with wire mesh top
Bedding: Granulated soft wood bedding
Feed: Pelleted standard diet, ad libitum
Water: Tap water, ad libitum
Environment: Temperature: 22 ± 2°C
Relative humidity: 45 – 65%, except for deviations (with the aim of 50 – 60%)
Artificial light: 6.00 a.m. – 6.00 p.m.
Ventilation: at least eight air changes per hour
Environmental enrichment: nesting material, wooden chewing blocks

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

The animals received the test substance, the negative or the positive control substance once by oral gavage, using a stainless steel feeding needle with rounded tip (1.2 Gauge) and disposable syringe at a dose volume of 10 mL/kg b.w.

Duration of treatment / exposure:

24h (negative control, low, mid and high dose, positive control)/48h (negative control and high dose)

Frequency of treatment:

once

Post exposure period:

Sampling of the bone marrow was done 24 h and 48 h after treatment, respectively.

Dose / conc.:

312.5 mg/kg bw (total dose)

Remarks:

7 male animals

Dose / conc.:

625 mg/kg bw (total dose)

Remarks:

7 male animals

Dose / conc.:

1 250 mg/kg bw (total dose)

Remarks:

14 male animals (2 x 7 males per test group)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: Cyclophosphamide (CPA)
Supplier: Acros Organics
Batch: A0406976
Expiry Date: 14 August 2020
Dissolved in: sterile water
Batch No.: 19NCB170
Expiry Date: February 2022
Dosing: 40 mg/kg b.w.
Route and Frequency of Administration: orally, once
Volume Administered: 10 mL/kg b.w.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

The animals were sacrificed using CO2 followed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum using a disposable syringe. The cell suspension was centrifuged at 1500 rpm (3900 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 4000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined from the same slide by counting until 500 PCEs had been determined among total erythrocytes and expressed as polychromatic erythrocytes per total erythrocytes counted. The analysis was performed with coded slides. Immature and mature erythrocytes were identified by their pale and blue to green colour, respectively. Micronuclei are distinguished by being small nuclei separate from and additional to the main nuclei of the cells.

Evaluation criteria:

A test substance is classified as positive in the assay if
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
b) This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
c) Any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations.
A test item that fails to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes, applying the above mentioned criteria, is considered negative in this system, given that there is evidence for bone marrow exposure.

Statistics:

nonparametric Mann-Whitney test, linear regression analysis

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

hunched posture, decreased activity

Vehicle controls validity:

valid

Positive controls validity:

valid

An additional study has been carried out to demonstrate proof of systemic exposure in the mouse after oral (gavage) administration of the test item. Therefore, three groups of 3 male mice of the Crl:CD-1 strain were dosed with 312.5, 625 or 1250 mg/kg cyclohexanone in olive oil on one occasion, by gavage, at a dose volume of 10 mL/kg body weight.

All animals were observed for any visible signs of reaction to treatment and body weights were recorded on Days 1 and 2. Blood samples were taken at 1, 4 and 24 hours after dosing for bioanalysis.

There were no clinical signs observed following administration of cyclohexanone at 312.5 mg/kg/day (Group 1) or 625 mg/kg/day (Group 2). Clinical signs observed following administration at 1250 mg/kg/day (Group 3) included excessive salivation, piloerection, partially closed eyes and coldness to the touch. There was minor body weight loss in all 3 groups.

Exposure to cyclohexanone was confirmed in all 3 groups by detectable concentrations of the test item in circulating plasma at 1 hour and 4 hours after dosing using a validated bioanalysis method. Cyclohexanone was below the limit of quantification 24 hours after dosing.

In summary, proof of exposure was demonstrated by detectable plasma concentrations of cyclohexanone in male Crl:CD-1 mice after a single administration of cyclohexanone at 312.5, 625 and 1250 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation

 

All well conducted and sufficiently validated in vitro tests for gene mutation yielded negative results.

The negative key study for in vitro gene mutation in bacteria was performed in accordance with the OECD guideline 471 and in compliance with GLP requirements (BASF, 1999). This test result is supported by two negative bacterial reverse mutation assays which were well performed according to scientific standards, however not with the complete number of Salmonella typhimurium tester strains requested by the guideline (Florin, 1980; Haworth, 1983).

The key study for in vitro gene mutation in mammalian cells was performed in accordance with the OECD guideline 476 and in compliance with GLP requirements, which did not result in an induction of gene mutations in the hprt gene in CHO cells (BASF, 2012). This test result is supported by the absence of an induction of gene mutation in the tk gene of the L5178Y mouse lymphoma cell line, in an assay which was performed according to OECD guideline 490 (McGregor, 1988). 

In addition, no indication for mutagenicity was reported in a sex-linked recessive lethal test which was performed in accordance with the former OECD TG 477, and in which Drosophila melanogaster were exposed to atmospheres of cyclohexanone (NIOSH, 1980).

 

Structural chromosome aberrations in vitro

 

As the mouse lymphoma assay can identify gene mutations and structural chromosome aberrations, this assay also constitutes a valuable and scientifically reliable information for the absence of a clastogenic potential of cyclohexanone in mammalian cells (McGregor, 1988).

This test result is supported by the absence of DNA damage and repair in an unscheduled DNA synthesis assay (NIOSH, 1980), the absence of DNA strandbreaks in an in vitro Comet assay (Reus, 2013), and the absence of DNA repair synthesis in an assay assessing the uptake of tritiated thymidine into mammalian cells (Perocco, 1983). All these studies are relevant and sensitive indicators for the potential occurrence of structural chromosome aberrations.

 

Structural (and numerical) chromosome aberrations in vivo

 

No structural chromosome aberrations were identified in somatic cells in an in vivo rat bone marrow chromosome aberration assay. This assay was performed via inhalation and in accordance with the OECD TG 475, with an extended test protocol using 10 animals for each test concentration, an acute and a subacute exposure, and 3 sampling times (6, 24, 48 h) for the acute exposures (NIOSH, 1980).

Furthermore, no dominant lethal effects indicative for structural and numerical chromosome aberrations in germ cells were identified in an inhalation dominant lethal test with rats using an acute and a subacute exposure protocol (NIOSH, 1980).

 

In an in vivo mouse micronucleus assay in which cyclohexanone was applied orally, a marginal, statistically significant increase in micronucleus frequency was reported at the highest dose of 1200 mg/kg bw administered to mice. However, from the report of the study significant deficiencies in test performance and interpretation in comparison to the OECD 474 (2016) test guideline can be identified (e.g., no justification of dose level, use of only a single sampling time in conjunction with a single dose regime, lower number of erythrocytes examined than required by the test guidance, no comparison to historical negative control data). This leads to a lack of statistical and scientific power for the determination of the biological relevance of the marginal response. On the basis of these study design and interpretation deficiencies no robust conclusion can be taken from this study (Kim, 2014).

 

To scrutinise these results, an in vivo mouse micronucleus assay has been performed according to the current OECD guideline 474 and GLP principles (Dony, 2020). 7 animals per dose group received 312.5, 625, or 1250 mg/kg bw per gavage. 24 hours after treatment (and additionally 48h after treatment of the high dose group), animals were sacrificed, and bone marrow taken from the femurs. Per animal 4000 polychromatic erythrocytes (PCE) were analysed for micronuclei. Cyclohexanone did not induce micronuclei up to the highest dose tested. In an additionally performed experiment, it could be shown that the test substance is available systemically under the test conditions chosen.

Considering these results, the marginal increase in micronuclei in the high dose seen in the study by Kim et al. seem to be coincidental.

 

Taken together, the lack of reported in vitro chromosome damage or aneugenic effects and the lack of activity seen in reliable and valid in vivo studies provide a convincing weight of evidence for the absence of any potential of cyclohexanone to induce structural (or numerical) chromosomal aberrations.

 

For a more detailed description of the in vitro and in vivo genotoxicity data available for cyclohexanone, please refer to the overview report attached to IUCLID chapter 13.

 

Other data

In several QSAR- Models implemented in the OECD Toolbox v. 4.4, no alert for a potential genotoxic mode of action was identified for cyclohexanone. The predictions are based on the implementation of a range of profilers connected with genotoxicity and carcinogenicity, and the incorporation of numerous databases with results from experimental studies into a logical workflow.

E.g., no alert for DNA binding and Ames was found by OASIS v.1.7., no alert for the Chromosomal Aberration Assay and the Micronucleus Test was found by OASIS v.2.8., no alert for DNA binding was found by OECD, no alerts for in vitro mutagenicity (Ames Assay) and in vivo mutagenicity (Micronucleus) were found by ISS and no protein binding alert for chromosomal aberration was found by OASIS v.1.6.

 

 

Classification for Germ Cell Mutagenicity

 

According to Regulation (EC) No 1272/2008 (3.5.2.3.9), “…the classification of individual substances shall be based on the total weight of evidence available, using expert judgement. In those instances, where a single well conducted test is used for classification, it shall provide clear and unambiguously positive results.” In addition, “The relevance of the route of exposure used in the study of the substance compared to the most likely route of human exposure shall also be taken into account.”

The ECHA Guidance on the application of CLP Criteria Version 5.0 (July 2017) further specifies, that the hazard class of Germ Cell Mutagenicity “… is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny.” (3.5.2.1).

In conclusion, the total weight of evidence of in vitro and in vivo data generated and reported in accordance to accepted scientific standards clearly demonstrates the absence of a genotoxic potential for cyclohexanone. Negative in vivo assays were performed via inhalation as the relevant route of exposure for cyclohexanone (see exposure assessment). An isolated inconclusive observation described in an oral assay which was not performed in accordance with the respective OECD guideline has been rebutted by the results of a newly performed in vivo mouse micronucleus assay, which was performed according to the current OECD guideline and in accordance with GLP principles.

 

Furthermore, cyclohexanone does not have the potential to induce mutations in germ cells of rodents and insects in vivo.

In conclusion, there are sufficient data available from reliable and scientifically valid in vitro and in vivo studies to demonstrate that cyclohexanone does not exhibit a genotoxic or mutagenic potential, and classification for mutagenicity is inappropriate. 

 

 

Disregarded studies

Disregarded in vitro genotoxicity studies

Please refer to the respective IUCLID study summaries for details.

 

An older publication is reporting an Ames assay and an assay with suspensions of a B. subtilis wild type strain. For both assays test performance and reporting substantially differ from today's guideline, e.g., none of the incubations were repetitively carried out (duplicates, triplicates). High levels of cytotoxicity in the assay with the not validated B. subtilis does not allow reliable conclusions. The same applies for the Ames assay for which no cytotoxicity was determined within the assay and high and inconsistent spontaneous revertant colonies in the negative control were reported. Only some of the indications why results of these studies cannot be applied for a reliable assessment of the mutagenicity of cylohexanone are mentioned here.

(Massoud A.A. et al., Egyptian Journal of Microbiology, 18, No. 1-2, pp. 213-224, 1983; Massoud A.A., Mutation Research 74(3) 174 1980)

 

A publication is reporting a positive result from a DNA-polymerase deficient E. coli strain. However, results with such strains can only give an indication on DNA damage and not a potential gene mutation, and for cyclohexanone the report of a positive finding is exclusively based on a personal communication of the author with the study owner M. Kiggins (Rhodia Inc., Hess and Clark Division). As no further details are available, e.g., on individual values and levels of cytotoxicity, this study cannot be considered valid for the assessment of in vitro gene mutation in bacteria.

(Rosenkranz, H. S. and Leifer, Z. Chemical Mutagens. Principles and Methods for their Detection, 6, 109, 1980.)

 

Results of an in vitro micronucleus assay performed in bovine peripheral lymphocytes did not indicate a genotoxic effect which is related to the treatment with cyclohexanone. However, due to methodological deviations from the OECD guideline (e.g., varying incubation times, low number of assessed cells, low sensitivity of positive controls) the study was considered to not provide reliable information for the assessment of genotoxicity of cyclohexanone.

(Piesova E. et al. (2003), Folia Veterinaria 47, 3: 161-163.)

 

Three genetic endpoints (sister chromatid exchange, gene mutation, structural chromosome aberration) were assessed in one assay with CHO cells.

Increases in mutation frequencies without metabolic activation were not correlated to the concentration of cyclohexanone and increases in SCE without metabolic activation were only observed at concentrations resulting in almost complete cytotoxicity. In addition, the reporting of the studies lacks significant details, and the study performance shows major deviations from the respective OECD guidelines (e.g. no mutant selection, no phenotype selection, too high test concentrations, synchronization of cells in the G1 phase with colcemid). Therefore, this study is not considered suitable for the assessment of genotoxicity of cyclohexanone.

(Aaron, C. S. et al.Environmental Mutagenesis, 7 Suppl.3, 60-61, 1985)

 

A study with primary human lymphocytes was not performed according to a validated protocol and shows major reporting deficiencies. E.g. no details on exposure duration, solvent and positive controls, cytotoxicity is indicated.

The total number of metaphases evaluated (12) is way below the requirement of the guideline (300). The study is therefore not considered valid for the assessment of cytogenetic damage.

(Collin J. P., Diabetes, 19(4), 215-221, 1971)

 

Increases in hyperploid and fragmented metaphases in a concentration independent manner were described in a study with primary human lymphocytes, originating from 15 blood donors of both sexes. The study report is written in Russian and does not provide any relevant information on assay performance and evaluation, i.e., exposure time total cultivation period, cytotoxicity data and a positive control to show the validity of the test system. Therefore, the study is not considered reliable for the evaluation of genotoxic effects.

(Dyshlovoi V. D. et al., Gigiena i Sanitariya, 46(5), 76-77, 1981)

 

No mutagenic potential in the bacterial mutagenicity assay is reported for cyclohexanone in an abstract without any further technical details on assay performance and results. Thus, the reliability of the data cannot be assessed.

(JETOC: Newsletter Nr. 4, 1985)

 

Disregarded in vivo genotoxicity studies

A subcutaneous study consisted of an acute study with 3 groups of 5 male rats each dose level and a subacute study in which each dose was given on five consecutive days. Animals were injected subcutaneous doses of 100, 500 and 1000 mg/kg bw. For each group, only one control animal was added. Animals were sacrificed 6, 24 and 48h after injection in the acute and 6h after the last injection in the subacute study, respectively. 

All animals in the high dose group of the subacute study died after the second injection and target organ toxicity was described by a decrease in the frequency of “sticky nuclei” with time and an influence of cyclohexanone treatment on the mitotic index. Due to a major lack of information in the report and major experimental shortcomings, the biological relevance of statistically significant increases in the incidence of aberrations cannot be evaluated. E.g., the negative control group for each exposure only consists of one animal and no positive control was included at all. Furthermore, no individual values and standard deviations are reported. Only a reduced number of metaphases was evaluated per animal (50 metaphases), not being in line with current OECD recommendations (200 metaphases per animal) and the observed gaps were also included in the assessment for clastogenic potential. Since in addition subcutaneous application is not a recommended route of exposure, the study cannot be considered reliable for the assessment of clastogenicity.

(De Hondt H. A. et al., Egyptian Journal of Genetics and Cytology, 12(1), 31-40, 1983)

 

In a host mediated assay the mutagenicity of cyclohexanone was investigated using yeast cells (Schizosaccharomyces pombe) inoculated into the intraperitoneal cavity of mice. The mice were treated with the substance orally and the recovered yeast cells were spread on agar plates for a forward mutation assay. No mutagenic effect was observed in yeast attributable to the substance application in mice. This study with few methodological deficiencies (i.e., positive control was not included to demonstrate assay validity, no evaluation criteria) is considered not to be sufficiently reliable and provides only a minor contribution to the assessment of mutagenicity.

TSCATS 1982, In vivo mutagenicity studies with trichloroethylene and other solvents (preliminary results), Doc I.D. 878211294.

 

The available abstract on mutagenicity testing in male fruit flies does not provide any detailed information on methodology of the assay. The outcome of the testing is non-mutagenic, however, no details on the results are reported. This publication does not provide reliable information for the assessment of the mutagenicity of cyclohexanone.

(Goncharova 1970, Genet. Tsitol. 137-142; cited in: Chem.Abstr. 76, 68 1972)

 

 

Justification for classification or non-classification

The available experimental test data with the substance cyclohexanone are reliable and suitable for classification purposes under Regulation 1272/2008. All reliable in vitro and in vivo tests with cyclohexanone gave negative results. Since the substance is regarded as non-genotoxic and non-mutagenic, a classification is not warranted.