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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.

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Endpoint:
genetic toxicity in vitro, other
Type of information:
(Q)SAR
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Specific details on test material used for the study:
QSAR prediction based on algorithms implemented in OECD-Toolbox

No alert for DNA binding and Ames was found by OASIS v.1.4.

No alert for the Chromosomal Aberration Assay and the Micronucleus Test was found by OASIS v.1.1.

No alert for DNA binding was found by OECD.

No alerts for in vitro mutagenicity (Ames Assay) and in vivo mutagenicity (Micronucleus) was found by ISS.

No protein binding alerts for chromosomal aberration was found by OASIS v.1.2.

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
Remarks:
Deviations to OECD 482: slight differences in protocol, no evaluation criteria are reported
Qualifier:
no guideline followed
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:
other: Ethyl methanesulphonate ( EMS)
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.
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.
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.
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
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:
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.
Endpoint:
in vitro gene mutation study in bacteria
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
Remarks:
Deviation to OECD 471:only 4 strains were used, detailed results were not reported
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: modification of the pre-incubation test according to Yahagi et al. (1975)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 from Aroclor induced male rat and hamster
Test concentrations with justification for top dose:
If toxicity was not apparent, the highest concentration tested was 10 mg/plate, otherwise the upper limit of solubility was used. If toxicity was observed, the concentrations were chosen so that the highest concentration exhibited some degree of toxicity.
At least five concentrations (not specified) were tested in each strain with and without metabolic activation.
Vehicle / solvent:
- Solvent used: not specified (water, DMSO or ethanol)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: without S9: 4-nitro-o-phenylendiamine: TA 98 (3.3-12 µg/plate); with S9: 2-aminoanthracene: all strains (0.75-2 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: viability in complete medium and reduced number of revertant colonies and/or decrease in background lawn

- OTHER: independent experimental repeat
Evaluation criteria:
A substance was considered mutagenic if a reproducible, dose-related increase in revertant colonies was observed, wether it would be two-fold over background or not.
A substance was considered inconclusive if increased revertant colonies were found only at one concentration, if low-level responses were observed that were not reproducible or if a trend towards positive or negative was observed, but could not be decided upon.
Statistics:
performed according to Margolin et al. (1981) PNAS USA 78, 3779-3783
Key result
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
This Ames study followed a protocol comparable to the current OECD 471 guideline and only minor deviations and some reporting deficiencies were apparent. Four Salmonella typhimurium strains (TA 98, TA 100, TA 1535 and TA 1537) were investigated in two independent pre-incubation experiments for reverse mutations in the absence and presence of metabolic activation using rat and hamster liver S9. The top concentration used in the study meets the current OECD 471 requirements. Under the conditions described, the substance did not lead to an increase in revertant colonies in any of the bacterial strains. The study is considered to be sufficiently reliable.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Deviations to OECD 471: only two bacterial strains were used for testing, no detailed results are reported
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Ames spot test according to Ames et al. (1975), Mutation Research, 31, 347.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 from Aroclor induced male rats
Test concentrations with justification for top dose:
3 µmol/plate
Vehicle / solvent:
ethanol
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene
Details on test system and experimental conditions:
DETERMINATION OF CYTOTOXICITY
- Method: observation of background lawn on the plates

Evaluation criteria:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: was not observed

Conclusions:
In this non-guideline reverse mutation test, including only data on Salmonella typhimurium strains TA 98 and TA 100, no mutagenic effects were observed in the absence and presence of metabolic activation. Although no detailed results were provided, the protocol is comparable to the current OECD 471 guideline and the study is considered scientifically valid.
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 30 May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
adopted August 1998
GLP compliance:
yes (incl. certificate)
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:
other: ethyl methanesulfonate (EMS, 300µg/mL, -S9); 7,12-Dimethylbenz[a]anthracene (DMBA, 1.25µg/mL, +S9)
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 control1  -  -  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 control1  -  -  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 control2  +  -  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 control2  +  -  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.
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
Remarks:
Deviations to OECD 490: evaluation criteria
Qualifier:
no guideline followed
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:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: other: cytogenetic assay and HGPRT assay
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
test performance of HPRT assay not in accordance to current OECD test guidelines, e.g. test substance application was not performed with “log-phase cells” , cytotoxicity was assayed in an independent incubation, no details regarding test substance purity, presence of high level of cytotoxicity.
Principles of method if other than guideline:
In the present study, cyclohexanone is evaluated in terms of its ability to induce aberrations, SCEs and gene mutations (at the HPRT1 locus) in synchronized CHO cells. CHO cells were synchronized in G1.
GLP compliance:
not specified
Type of assay:
other: cytogenetic assay and HGPRT assay
Target gene:
Chromosomal aberrations, SCEs and gene mutations.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 mix
Test concentrations with justification for top dose:
2.5 - 12.5 ul/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, ethyl nitrosourea
Details on test system and experimental conditions:
In the present study, cyclohexanone is evaluated in terms of its ability to induce aberrations, SCEs and gene mutations (at the HPRT locus) in synchronized CHO cells. The tests were run with and without the S9 metabolic activation system.
Specifically, CHO cells (clone CHO-K1 provided by Dr. R. J. Preston, Oak Ridge National Laboratory) were synchronized by mitotic shake-off and plated into T25 culture flasks at 710,000 cells per flask. Approximately one hour later the cultures were treated to test or control agents, with and without the metabolic activation mixture.
After one-hour treatments, cells were washed with Ca-Mg-free saline and refed with fresh medium. One culture from each group of 3 was used to initiate the mutation assay, another was used for the aberration assay (harvest at 16 hours after start of dosing), while the
third was used for the SCE assay (harvest at 27 hours after start of dosing). Flasks for the SCE study received BrdU at a final concentration of 10 -5M, immediately after treatment and refeeding.
In the SCE and aberration assays, mitotic cells were accumulated by the addition of Colcemid to each culture 2-3 hours prior to harvest. At the time of harvest, mitotic cells that were collected by shake-off were swollen in hypotonic solution (0.075M KCl), washed 3 times in fixative (methanol/acetic acid, 3 :1 v/v) and dropped onto microslides for subsequent staining and analysis. The SCE and mutation assays without S9 were repeated using the same concentrations and procedures outlined above.
Survival data were generated by synchronizing and then exposing cells as outlined above. After one-hour treatments, cells were washed with saline, fed with fresh medium and allowed to grow undisturbed for one hour. They were then trypsinized, counted, and plated out at a density of 200 cells per 60 mm petri dish (3 dishes per treatment group). After one week the plates were stained and counted to determine plating efficiency.
Evaluation criteria:
-
Statistics:
no data
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
other: When used directly (without S9), cyclohexanone at high concentrations induced increases in the frequency of mutant cells that were not related to the concentration of the test agent. No mutant colonies were observed at 10 uL/mL, and 12.5 uL/mL proved comp
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Survival results indicate that cyclohexanone becomes toxic in the 6-10 uL/mL dose range both with and without S9. Therefore, 12.5 uL/mL was selected as the highest concentration to be employed in the multiple-endpoint study in order to guarantee a significant level of cell killing.
SCE results: When tested without S9, cyclohexanone induced small increases in SCE frequency that were statistically significant at the higher concentrations. The effect was reproducible and the increases in frequency appear to be similarly dose-related in both experiments according to linear regression analysis. It appears, therefore, that cyclohexanone is a weak inducer of SCEs when used directly. When used with S9, cyclohexanone did not induce SCEs. The S9 enzyme system apparently detoxifies this test agent so that it no longer retains its SCE-inducing capacity.
Chromosome aberration results: Cyclohexanone did not induce significant increases in aberration frequency when used either with or without S9.
Mutation results: When used directly (without S9), cyclohexanone induced increases in the frequency of mutant cells that were not related in any obvious way to the concentration of the test agent. In fact, no mutant colonies were observed at 10 uL/mL, and 12.5 uL/mL proved completely toxic as all cells died in the early stages of the assay. The absence of mutant colonies at 10 uL/mL may be due to selective killing of mutant cells, as suggested by Carrano et al. (1979). When used with S9, cyclohexanone did not induce mutations at the HPRT locus. Overall, these results are consistent with those obtained in the SCE assay in that cyclohexanone was mutagenic without S9 but not with S9.
The assay without S9 was repeated, and the second study confirmed that cyclohexanone is indeed mutagenic. Toxicity was again apparent, as cells multiplied very slowly following treatment with the two highest concentrations. The authors, however, observe colony formation on "plating efficiency" and "selection" plates at the 12.5 uL/mL concentration that had precluded such observations in the first assayl. One replicate at 12 .5 uL/mL yielded an extremely high number of mutants (558 per 106 survivors), a clearly spurious observation. It could be argued that 10-12 .5 uL/mL represents a borderline dose range wherein cells not only suffer significant genetic damage but most often die as well ; but that on some occasions sufficient numbers of heavily damaged cells may survive to produce the high yield of mutant colonies that were observed. This cculd explain the high variation of mutation frequency between the replicates at the 12.5 uL/mL dose level in Experiment 2 because a difference of 1% versus 0.1% survival would result in the tenfold difference in mutation frequency. In any case, peculiar results are known to occur under treatment conditions giving very low survival particularly when small changes in treatment concentration have such drastic effect on survival as here. Whatever the explanation for the erratic nature of these data, it is nonetheless clear the cyclohexanone does induce mutations of the HPRT locus when used directly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Cyclohexanone induced SCEs and gene mutations when used without S9 but not when used with S9 under the conditions of this assay. Under the sameconditions this test agent did not induce chromosome aberrations either with or without S9. Cyclohexanone is therefore concluded to be a weak mutagen that can be detoxified by the S9 liver enzyme system.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
major deviations to the current OECD guideline 471, not sufficiently reported, significant inconsitencies in experimental reporting
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The cyclohexanone purity was indicated to be 99% (Kafr, El-Zayat company for Insecticides, Egypt) however no details on the analytical procedure are described.
Species / strain / cell type:
other: Bacillus subtilis wild type
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
and TA 1538
Metabolic activation:
without
Test concentrations with justification for top dose:
Complementation test (Bacillus subtilis): 0, 0.1, 0.2 and 0.3 mL/1.5 mL cell suspension
Ames test (Salmonella typhimurium): 0, 0.02, 0.04, 0.06 and 0.08 mL/5ml
Details on test system and experimental conditions:
METHOD OF APPLICATION:

- Complementation test: in suspension
Loopfulls from each mutant grown on complete medium slope were suspended in 1 mL sterile distilled water in a penicillin bottle. All cultures were set up on minimal medium plates divided into 25 squares with a grid. Suspensions of similar phenotype, i.e., having the same requirement, were mixed in pairs with a loop in each square. The suspension of the second mutant was added after the first mutant cell suspension was completely settled. The plates were then incubated at 37°C for 72 hours after which they were scored and growth on minimal medium was taken as indication of complementation.

- Ames test: plate incorporation:
Samples of 0.1 mL of the tester strains cultures (grown for 24 hours) in liquid complete medium were added to small sterile test tubes each containing 5 mL of molten (45°C) top agar. Cyclohexanone was added to the tubes at concentrations of 0, 0.02, 0.04, 0.06 and 0.08 mL, then adjusted to equal volumes with distilled water. Contents of the tubes were mixed and poured onto the surface of a minimal agar plate. The plates were incubated at 37°C for 2 days, after which the numbers of revertant colonies were counted.


DETERMINATION OF CYTOTOXICITY
- Suspensions of Bacillus subtilis treated with different test item concentrations in tubes, were incubated at 30°C for 20 hours. After this period of incubation, 0.1 mL samples from each treatment with suitable dilutions were plated on complete medium. The plates were incubated at 37°C for 3 days. Then the
plates were scored and colonies were counted for survivals.
Key result
Species / strain:
other: Bacillus subtilis wild type
Metabolic activation:
without
Genotoxicity:
other: not asignable
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
other: not asignable
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
other: not asignable
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
other: not asignable
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
other: not asignable
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
without
Genotoxicity:
other: not asignable
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

Experiment with B.subtilis:

In an independent cytotoxicity experiment, B.subtilis was incubated for 20h with 0.1, 0.2 and 0.3ml cylohexanone in 1.5ml of the final treatment mixture. This resulted in high levels of cytotoxicity, which was indicated by a decrease of the survival of treated cell by 93.7, 99.8 and 99.9%, respectively (see table 1). These cytotoxicity levels are way above the acceptable level for bacterial assays, and therefore no conclusion on a specific mutagenic effect can be made from the subsequent mutation assay. The high cytotoxicity resulted in an unacceptable reduction in the total number of viable bacteria per plate, which is further questioning the scientific value of this experiment.

In a pre-experiment with incubation times of 2h, high cytotoxicity was described for the two highest concentrations (-45% in cell survival for 0.3ml and -79% for 0.2 ml cylohexanone in 1.5ml, respectively, see table 2). The number of mutants observed in the mutation assay is rather dependent on the wild type strain and the experimental conditions than of the test substance itself. This is demonstrated by the lack of dose response in the colonies and % of mutants observed. The absence of a significant number of revertants in further tests rather suggests that no specific mutagenic effect exists.

Summarized, and since wild type B.subtilis does not represent a validated test system, this assay can not be considered as relevant for the assessment of in vitro gene mutation in bacteria.

Table 1: Cell survival and mutations in B.subtilis wild type cells incubated with cyclohexanone for 20h at 30°C.

 

Cytotoxicity assay

Mutation assay

Cyclohexanone

/1.5 ml

mean No. of cells/ml

%survival

No. of colonies tested

No. of mutants

% mutants

% revertants

0

30*106

100

57

0

0

0

0.1

19*105

6.333

25

19

76

13

0.2

7*104

0.233

15

8

53

0

0.3

1*103

0.003

1

1

100

0

 

Table 2: Cell survival and mutations in B.subtilis wild type cells incubated with cyclohexanone for 2h at 30°C.

 

Cytotoxicity assay

Mutation assay

Cyclohexanone

/1.5 ml

mean No. of cells/ml

%survival

No. of colonies tested

No. of mutants

% mutants

0

2*104

100

2

0

0

0.1

19*103

95

37

15

40.45

0.2

42*103

21

130

81

62.31

0.3

11*103

55

70

22

31.45

 

Experiment in S. typhimurium tester strains:

A preliminary test to determine the adequate test concentrations is not reported for the incubations of the S. typhimurium tester strains (Ames assay). In addition, no individual cytotoxicity data is indicated in the test results. However, from high levels of lethality reported for individual tester strains it can be anticipated, that the concentrations chosen were highly cytotoxic and therefore inadequate (too high). E.g. complete lethality is reported for the TA98 strain at all 3 highest test concentrations (0.04, 0.06, 0.08 ml/5ml).

Furthermore, the rates of spontaneous revertant colonies in the control experiments are much higher than published spontaneous reversion rates, and these rates greatly vary between the individual control incubations (0 for TA1538 vs. 796 with TA1537). Based on such unreliable background data no scientifically valid conclusion can be made for in vitro gene mutation in bacteria. The experimental relevance of this experiment is additionally questioned, as such background signal in control incubations was not observed in a comparative experiment with positive controls, and entirely different incidences in revertants were observed for cyclohexanone compared to the main experiment.

Furthermore, test performance and reporting substantially differ from today's guideline, e.g. none of the incubations were repetitively carried out (duplicates, triplicates) for the respective test concentrations, as prescribed by the guideline (29).

Table3: Frequency of revertants in S. typhimurium tester strains incubated with cyclohexanone

 

Cyclohexanone concentration ml/5ml

strain

0

0.02

0.04

0.06

0.08

TA98

284

44

0

0

0

TA100

192

232

2

1

0

TA1535

0

33

65

11

440

TA1537

796

228

63

xx

0

TA1538

0

360

19

xx

348

 

Conclusions:
This older publication is reporting an Ames assay which was exclusively performed as a plate incorporation assay (OECD guideline prescribes plate incorporationand pre-incubation) with five Salmonella typhimurium tester strains (TA 98, 100, 1535, 1537, 1537) in absence of a metabolic activation (OECD guideleine additionally prescribes E. coli, and metabolic activation).
In addition a mutation assay using Bacillus subtilis is reported.
The assay performance shows major deviations to the current OECD guideline 471 and is not sufficiently reported. E.g. no details on cytotoxicity are reported for the experiments performed with Salmonalla typhimurium strains, and none of the incubations were repetitively carried out (duplicates, triplicates) for the respective test concentrations, as prescribed by the guideline (29).
Furthermore, the rates of spontaneous revertant colonies in the control experiments are much higher than published spontaneous reversion rates, and these rates greatly vary between the individual control incubations. Based on such unreliable background data no scientifically valid conclusion can be made for in vitro gene mutation in bacteria. The experimental relevance of this experiment is additionally questioned, as such background signal in control incubations was not observed in a comparative experiment with positive controls, and entirely different incidences in revertants were observed for cyclohexanone compared to the main experiment.
The read-out of the mutation assay with with wild type Bacillus subtilis does not allow a direct correlation to a specific mutagenic potential of a test substance.
Summarized there are significant concerns regarding the the adequate performance and the biological relevnce of both, the the bacterial test and the test with S.typhimurium tester strains. Therefore, this study is not considered reliable for the evaluation of mutagenicity of the test substance.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
not performed according to a validated protocol, deficiencies to current guidelines (OECD 471) and reporting deficiencies
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: determination of growth inhibition of PolAI deficient E. coli strain, application of the substance by disk-diffusion
GLP compliance:
not specified
Type of assay:
other:
Specific details on test material used for the study:
No information on the test material is provided.
Species / strain / cell type:
E. coli, other:
Additional strain / cell type characteristics:
DNA polymerase A deficient
Remarks:
and DNA polymerase proficient strain
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
No information on concentrations available.
Vehicle / solvent:
DMSO or water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
yes
Remarks:
chloramphenicol
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- standard assay: in medium and spreaded on agar or as plate incorparation, using impregnated filter disks for treatment
- modified assay: in liquid suspension with subsequent spreading on agar plates (pre-incubation assay)

DURATION
- Exposure duration: standard assay: 7-12 hours, modified assay: pre-incubation for 90 min and additional 16-18 hours on agar plates

NUMBER OF REPLICATIONS: duplicate cultures

EVALUATION:
- standard assay:
- modified assay: determination of colonies and survival index
Evaluation criteria:
Standard assay:


Modified assay:
survival index = % survivers polAI- / % survivors polAI+
A value below 1 indicates preferential killing of the polAI- strain and DNA modifying activity, a value of 1 indicates bactericidal potential to the same extent and no DNA modifying activity
Key result
Species / strain:
other: DNA polymerase A proficient and deficient strain
Metabolic activation:
not specified
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:

A DNA-polymerase deficient E. coli strain was used for this studyis not a recommended strain. Results with such strains can only give an indication on DNA damage and not a potential gene mutation reflecting potential DNA repair. This questiones the biological relevance of an effect with respect to a potential for gene mutation.
In addition, the method of application deviates to the state of the art assay performance, and information on the test substance is lacking.
As reporting of the study result for cyclohexanone is solely based on a personal communication of the author with the study owner M. Kiggins (Rhodia Inc., Hess and Clark Division) the validity of the test result can not be demonstrated. No information on the cytotoxicity and mutagenicity results is given in this publication.
Due to the deviations and insufficient reporting of results, the study is not considered reliable.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: insufficient reporting and major deviations to the current OECD guideline 487, i.e. number of cells scored too low, evaluation criteria
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: performed according to the protocol of Fenech and Morley (1985)
Deviations to the guideline:
• Incubation times 2h without and 48h with metabolic activation vs. 3-6 h with and without metabolic activation
• evaluation of 1000 binucleated cells vs. 2000
• Inappropriate parameter for cytotoxicity
• Very low sensitivity of positive controls
• Uncommon cell type
• No range of response demonstrated by historical control data
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Cyclohexanone (99.9%, Ites, Slovak Republic)
Species / strain / cell type:
lymphocytes: bovine origin
Details on mammalian cell type (if applicable):
CELLS USED
- Black cattle, whole blood
- Cell cycle length: not indicated
- 2 donors, six months

MEDIA USED
- RPMI 1640 medium supplemented with L-glutamine, HEPES, fetal calf serum, penicillin/streptomycin and phytohemagglutinin
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
0.1, 0.5, 1, 5 and 10 mmol/L
Vehicle / solvent:
- Solvent: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 2 (with S9) and 48 hours (without S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 72 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): not specified

NUMBER OF REPLICATIONS: not specified

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not specified

NUMBER OF CELLS EVALUATED: 1000 binucleated cells

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not specified

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis block proliferation index
Evaluation criteria:
Not specified
Statistics:
Chi-squared test
Key result
Species / strain:
lymphocytes: bovine origin
Metabolic activation:
with and without
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
Additional information on results:
Single slight, but significant increases in micronucleated cells were observed after treatment with the substance for 2 hours and 48 hours. This single increases incidences were found in the absence and presence of metabolic activation.

The % of binucleated cells was not significantly deviating with cyclohexanone treated cells if compared to the control. 

No concentration related increase in the number of cells containing micronuclei was observed.

Incidentally, slight (less than 2 -fold) statistically significant increases in the number of cells containing micronuclei (p<0.05) were observed. But as these increases were observed at low dose levels and were not concentration related they can neither be identified as test substance related nor as biological relevant. In addition, these increases were not observed consistently between the two different cultures (donor animal 1 and 2), and the range of response is not described by historical control data which is of particular interest as bovine peripheral lymphocytes represent an uncommon cell type for the in vitro micronucleus assay.

In addition, a very insensitive response was observed for the positive controls.

 

Table 1: Frequency of micronuclei and percent of binucleated cells in cultured bovine lymphocytes exposed to cyclohexanone.

 

Donor animal 1

Donor animal 2

Conc. [mmol/l]

% binucleated cells

# micronuclei containing BN

% binucleated cells

# micronuclei containing BN

 

48h -S9

48h -S9

Control

23.7

21

29

19

0.1

18.7

26

19.6

35*

0.5

20.2

32

16.5

26

1

22.1

31

20

27

5

14.3

31

19.1

23

10

18.7

28

18.3

24

MMC (0.4µM)

23.9

49***

27.5

48***

 

2h +S9

2h +S9

Control

19.6

23

22.8

20

0.1

21.1

30

18.4

24

0.5

19.5

39

15.2

35*

1

16.5

36

14

24

5

17

42*

17.4

22

10

14.5

24

21.6

17

CP (100µM]

24.9

51***

20.2

48***

 p<0.05

p<0.001

Conclusions:
An in vitro micronucleus assay was performed in bovine peripheral lymphocytes and at cyclohexanone concentration levels of 0.1, 0.5, 1, 5 and 10 mmol/l. The assay was performed as two independent experiments from cells of two different animals and incubation times of 48h with metabolic activation and 2 h without metabolic activation (the OECD TG 487 prescribes a treatment time of 3-6 h, with and without metabolic activation).
A total of 1000 binucleated cells was evaluated for each animal and each concentration (the OECD TG 487 prescribes the analysis of at least 2000 binucleated cells per concentration, equivalent to at least 1000 binucleated cells per culture, with two cultures per concentration).
The report of the assay is reporting the % of binucleated cells as a measure for cytotoxicity. (No CBPI (cytokinesis-block proliferation index) and RI (replicative index) were calculated as requested by the OECD TG 487.)

As a result the % of binucleated cells was not significantly deviating with cyclohexanone treated cells if compared to the control.
No concentration related increase in the number of cells containing micronuclei was observed.
Incidentally, slight (less than 2-fold) but statistically significant increases in the number of cells containing micronuclei (p<0.05) were observed. But as these increases were observed at low dose levels and were not concentration related they can neither be identified as test substance related nor as biological relevant. In addition, these increases were not observed consistently between the two different cultures (donor animal 1 and 2).
Furthermore an evaluation of the relevance of these observations is not possible, as the range of response is not described by historical control data which is of particular interest as bovine peripheral lymphocytes represent an uncommon cell type for the in vitro micronucleus assay. In addition, a very insensitive response was observed for the positive controls.
In conclusion, the reporting of the assay lacks significant detail for an appropriate interpretation of the test results. In addition, the test performance deviates from the OECD Guideline 487. Though, based on the given information in the assay no dose related and reproducible increase in in the number of cells containing micronuclei was observed, which is why it can be concluded that under the conditions of this in vitro micronucleus assay cyclohexanone is not clastogenic.

In conclusion the study shows deviations to the current OECD guideline 487. As these deviations are e.g. related to to the statistical power and incubation time, the relevance of incedental increases in micronucleated cells by less then 2-fold in comparison to the control group can not be evaluated.
Since these findings were only observed at low concentration levels in absence of a dose response, they can not be evaluated as substance related.
Due to the various methodolocical and experimental shortcomings, the study is not considered to provide adequate information for the assessment of mutagenicity of the test substance.
Endpoint:
genetic toxicity in vitro, other
Remarks:
gene mutation, chromosomal aberrations, sister chromatid exchanges
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
major deviations to current guidelines for testing of mutagenicity and cytogenetic damage, reporting deficiencies
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: performed according to Latt et. al. (1982, SCE analysis), O'Neill and Hsie (1979, CHO/HGPRT mutations), Preston et al. (1981, cytogenetic damage)
GLP compliance:
not specified
Type of assay:
other: mammalian cells: gene mutation, chromosomal aberrations, sister chromatid exchanges
Specific details on test material used for the study:
Sample No. 8245-07.
No fürther information on the test material is provided.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
clone CHO-K1
Metabolic activation:
with and without
Test concentrations with justification for top dose:
2.5, 5, 7.5, 10 and 12.5 µL/mL
concentrations selected based on cytotoxicity
Vehicle / solvent:
No information is provided on the vehicle.
Untreated negative controls:
yes
Remarks:
medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylnitrosurea
Details on test system and experimental conditions:
CHO cells (clone CHO-K1 provided by Dr. R. J. Preston, Oak Ridge National Laboratory) were synchronized in M-phase by mitotic shake-off using colcemid.
These cells were plated into T25 culture flasks at 710,000 cells per flask (SCE and abberation assay) or 1,400,000 cells per flask (mutation assay).
Plated cells rapidly re-initiate progression through the cell cycle and approximately one hour later the cultures were treated to test or control agents, with and without the metabolic activation mixture.
After one-hour treatments, cells were washed with Ca-Mg-free saline and refed with fresh medium. One culture from each group of 3 was used to initiate the mutation assay, another was used for the aberration assay (harvest at 16 hours after start of dosing), while the third was used for the SCE assay (harvest at 27 hours after start of dosing). Flasks for the SCE study received BrdU at a final concentration of 10E-5 M, immediately after treatment and refeeding.
In the SCE and aberration assays, mitotic cells were accumulated by the addition of Colcemid to each culture 2-3 hours prior to harvest. At the time of harvest, mitotic cells that were collected by shake-off were swollen in hypotonic solution (0.075 M KCl), washed 3 times in fixative (methanol/acetic acid, 3 :1 v/v) and dropped onto microslides for subsequent staining and analysis. The SCE and mutation assays without S9 were repeated using the same concentrations and procedures outlined above.
Survival data were generated by synchronizing and then exposing cells as outlined above. After one-hour treatments, cells were washed with saline, fed with fresh medium and allowed to grow undisturbed for one hour. They were then trypsinized, counted, and plated out at a density of 200 cells per 60 mm petri dish (3 dishes per treatment group). After one week the plates were stained and counted to determine plating efficiency.
Evaluation criteria:
SCE:
- With a control frequency within the 95% confidence interval, results showing r < 0.05 and statistical significance (Student's t-test, p < 0.05) at one concentration will be considered positive.
- When results are clearly non-linear, then results will be considered positive if two concentrations will show statistical significance (Dunnett's t-test).

Chromosome aberrations: for a positive outcome at least two of the following criteria have to be met:
- A signifcant r value (p < 0.05),
- at least two concentrations exceed the 95% confidence interval or
- at least two concentrations are show statistical significant increases above the control value (Dunnett's t-test, p < 0.01)

Mutation frequency: with a control frequency within the 95% confidence interval, at least two of the following criteria have to be met:
- A signifcant r value (p < 0.05),
- at least one concentration shows a 2fold increase above control or
- at least two concentrations exceed the 95% confidence interval.
Statistics:
Correlation coefficient r
Student's t-test
Dunnett's t-test
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Pre-experiment on toxicity: Moderate toxicity was observed in a concentration range of 6-10 µL/mL without S9 and at a concentration of 0.3 µL/mLwith S9. Severe toxic effects were found in a concentration range of 0.6-10 µL/mL with S9. For the main experiments, 12.5 µL/mL was selected as the highest concentration to be employed in the multiple-endpoint study in order to guarantee a significant level of cell killing.
SCE results: When tested without S9, cyclohexanone induced small increases in SCE frequency that were statistically significant at the higher concentrations. The effect was reproducible and the increases in frequency appear to be similarly dose-related in both experiments according to linear regression analysis. It appears, therefore, that cyclohexanone is a weak inducer of SCEs when used directly. When used with S9, cyclohexanone did not induce SCEs. The S9 enzyme system apparently detoxifies this test agent so that it no longer retains its SCE-inducing capacity.
Chromosome aberration results: Cyclohexanone did not induce significant increases in aberration frequency when used either with or without S9.
Mutation results: When used directly (without S9), cyclohexanone induced increases in the frequency of mutant cells that were not related in any obvious way to the concentration of the test agent. In fact, no mutant colonies were observed at 10 uL/mL, and 12.5 uL/mL proved completely toxic as all cells died in the early stages of the assay. The absence of mutant colonies at 10 uL/mL may be due to selective killing of mutant cells, as suggested by Carrano et al. (1979). When used with S9, cyclohexanone did not induce mutations at the HPRT locus. Overall, these results are consistent with those obtained in the SCE assay in that cyclohexanone was mutagenic without S9 but not with S9.
The assay without S9 was repeated, and the second study confirmed that cyclohexanone is indeed mutagenic. Toxicity was again apparent, as cells multiplied very slowly following treatment with the two highest concentrations. The authors, however, observe colony formation on "plating efficiency" and "selection" plates at the 12.5 uL/mL concentration that had precluded such observations in the first assayl. One replicate at 12 .5 uL/mL yielded an extremely high number of mutants (558 per 106 survivors), a clearly spurious observation. It could be argued that 10-12 .5 uL/mL represents a borderline dose range wherein cells not only suffer significant genetic damage but most often die as well ; but that on some occasions sufficient numbers of heavily damaged cells may survive to produce the high yield of mutant colonies that were observed. This cculd explain the high variation of mutation frequency between the replicates at the 12.5 uL/mL dose level in Experiment 2 because a difference of 1% versus 0.1% survival would result in the tenfold difference in mutation frequency. In any case, peculiar results are known to occur under treatment conditions giving very low survival particularly when small changes in treatment concentration have such drastic effect on survival as here. Whatever the explanation for the erratic nature of these data, it is nonetheless clear the cyclohexanone does induce mutations of the HPRT locus when used directly.
Remarks on result:
other: increased mutation frequencies and SCE

An independent cytotoxicity experiment indicated that cyclohexanone becomes toxic in the 6-10 µl/ml dose range following a 1h exposure without S9 and 0.3-0.6µl/ml dose range following a 1h exposure with S9 (data not shown).

Cyclohexanone did not induce significant increases in aberration frequency, with and without metabolic activation (data not shown).

In the mutation assay cyclohexanone induced increases in the frequency of mutant cells when incubated without S9 (see table 2). This observation was not related in any way to the concentration with which the cells were incubated. Even though this observation was reproduced in a confirmatory assay, it could be related to the unclear test conditions than to a test substance related intrinsic mutagenicity. When incubated with S9 no significant increase in mutant frequency was observed.

With S9, cyclohexanone did not induce SCEs, but again without S9, a statistically significant increase in SCEs was detected at the 2 highest concentration levels which did not result in complete cytotoxicity (see table 1). However, the values were within the the 95% confidence iterval of the controls and a confirmatory assay also resulted in complete cytotoxicity at these two dose levels and in absence of a reliable indicator of cytotoxicity. Therefore, the biological relevance of this observation has to be questioned.

Table 1: SCE frequencies. Indicated are means of 2 replicates. No means are presented in case one of the 2 replicates was toxic.

 

Experiment 1

Experiment 2

treatment

SCE/chromosome, -S9

SCE/chromosome, +S9

SCE/chromosome, -S9

neg. contr.

0.634

0.581

0.637

2.5 µl/ml

0.679

0.582

0.702

5 µl/ml

0.697

0.570

0.705

7.5 µl/ml

0.757*

toxic (1 replicate)

0.694

10 µl/ml

0.741*

toxic

toxic (1 replicate)

12.5 µl/ml

toxic

toxic

toxic

pos. contr. ENU, 1.5x10-3M

1.586*

-

1.398

pos. contr.

CP 10-3M

-

4.048*

4.048*

 

Table 2: HPRT mutation frequencies (duplicates)

Treatment

 

Mutants per 106plated cells

Plating efficiency

Mutants per 106survivors (mean)

 

Experiment 1, -S9

neg. contr.

2, 6

1.39, 1.24

1, 5 (3)

2.5 µl/ml

80, 35

1.65, 1.62

49, 22 (35.5*)

5 µl/ml

105, 87

1.57, 1.15

67, 76 (71.5*)

7.5 µl/ml

54, 70

1.27, 2.01

43, 35 (39*)

10 µl/ml

0, 0

1.49, 1.19

0, 0 (0)

12.5 µl/ml

-, -

-, -

-

pos. contr. ENU, 1.5x10-3M

354, 287

1.53, 1.72

233, 167 (200*)

 

Experiment 2, -S9

neg. contr.

15, 16

0.92, 0.84

16, 19 (17.5)

2.5 µl/ml

51, 38

0.87, 0.89

59, 43 (51*)

5 µl/ml

55, 38

0.99, 0.78

56, 49 (52.5*)

7.5 µl/ml

50, 65

0.90, 0.98

56, 66 (61*)

10 µl/ml

56, 42

0.86, 1.02

65, 41 (53*)

12.5 µl/ml

37, 430

0.73, 0.77

51, 558 (51*,a)

pos. contr. ENU, 1.5x10-3M

413, 379

0.74, 0.83

558, 457 (507.5*)

 

Experiment 1, +S9

neg. contr.

9, 5

1.4, 0.9

6, 6 (6)

2.5 µl/ml

34, 13

0.9, 0.89

38, 15 (26.5)

5 µl/ml

4, 5

1.45, 0.93

3, 5 (4)

7.5 µl/ml

10, 11

1.81, 1.4

6, 8 (7)

10 µl/ml

6, 19

1.09, 0.95

6, 20 (13)

12.5 µl/ml

19, 3

0.76, 0.95

25, 3 (14)

pos. contr. CP, 10-3M

52, 6

0.86, 0.88

60, 7 (33.5)

*p>0.05

a – 558 was excluded

ENU – Ethylnitrosourea

Summarized the performance of the assay is inappropriately reported and from the given information significant deviations to the guidelines can be demonstrated. Therefore the validity of the assays had to be questioned and a conclusion on genotoxicity can not be made.

Conclusions:
Three genetic endpoints (sister chromatid exchange (SCE), gene mutation at the HPRT locus, structural chromosome aberration) were assessed in one assay with CHO cells. Cells were incubated with 2.5, 5, 7.5, 10 and 12.5 µl/ml in presence and absence of a metabolic system.

Increased incidences in SCE and mutation frequencies were described for CHO cells exposed to the test substance in the absence of metabolic activation. However, increased mutation frequencies were not related to the dose of the test substance and increases in SCE were related to extensive and almost complete cytotoxicity. No effects were observed in the presence of metabolic activation. In the absence and presence of metabolic activation no increased incidences in chromosomal aberrations were present.

The reporting on the study performance lacks significant detail. There is a lack in reporting detailed information on the test substance and the assay method, such as the mean average generation time of the cells, the number of metaphases evaluated for cytogenetic damage, classification and evaluation criteria for chromosomal aberrations, evaluation criteria and historical background data of the assays.
However, based on the given information it is apparent, that the study performance shows major deviations to the to current OECD guidelines OECD 476, 473 and 479 for the SCE test (which was deleted in 2014).
E.g. for the mutation assay, no elimination of pre-existing mutant cells was performed, there is no information on the use of a selective medium (6-thioguanine, 8-azaguanine), the exposure duration of the cells was below the guideline recommendations (1h vs. 4h), cytotoxicity was not determined in the main experiment, and the treated cultures were not maintained in growth medium following incubation with the test substance (the guideline recommends 7-9 days to allow near optimal phenotypic expression of newly induced Hprt mutants). Accordingly no optimal phenotypic expression of induced mutations could have occurred.
In addition the test concentrations exceeded the maximum concentration of 5 µl/ml defined by the guideline (16.).
The assay was performed in cells which were synchronized with colcemid in the G1 phase in comparison to log-phase cultures in traditional gentox assays. This excludes any possible DNA repair ahead of entering S-phase and therefore significantly increases sensitivity and strongly questions the biological relevance of results.
In conclusion the observations might also be a secondary effect linked to the cytotoxicity of the substance. Therefore, the study is not considered suitable for the assessment of genotoxicity and mutagenicity.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
study not performed according to validated methods, reporting deficiencies
Principles of method if other than guideline:
- Principle of test: leucocyte cultivation using whole human blood, treatment of proliferating cells
- Short description of test conditions: stimulation of proliferation with phytohemagglutinin, metaphase arrest using colcemide
GLP compliance:
no
Type of assay:
other: in vitro chromosomal aberration
Specific details on test material used for the study:
No information on the test material is provided.
Species / strain / cell type:
other: human leucocytes
Metabolic activation:
without
Test concentrations with justification for top dose:
0.1 mM, 1 mM, 10 mM
Vehicle / solvent:
not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: no information available
- Fixation time (start of exposure up to fixation or harvest of cells): no information available

SPINDLE INHIBITOR (cytogenetic assays): colcemide

NUMBER OF REPLICATIONS: not specified

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: fixation with methanol/acetic acid, cellular smear on slides, staining with Unna

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not specified
12 cells were observed for each of the concentrations.
Evaluation criteria:
not specified
Statistics:
no
Key result
Species / strain:
other: human leucocytes
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified

In 12 metaphases the following was observed:

concentration 10 mM: achromatic regions (not quantified)

concentration 1 mM: achromatic regions and breaks (not quantified)

concentration 0.1 mM: achromatic regions (not quantified) and one deletion

Conclusions:
This study in primary human leucocytes was not performed according to a validated protocol and shows major reporting deficiencies. No detailed information on the test procedure and evaluation is given (exposure duration, total duration of culture, solvent and positive controls, cytotoxicity, total number of metaphases evaluated, quantification of aberrations). In addition, no criteria for evaluation of aberrations are presented and the assessed number of metaphases (12) is way below the requirement of the guideline (300). The study is therefore not considered valid for the assessment of cytogenetic damage.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
not sufficiently reported, article in russian language, missing information on performance and evaluation
Qualifier:
no guideline followed
GLP compliance:
no
Type of assay:
other: chromosome aberration study in mammalian cells
Specific details on test material used for the study:
No information on the test material is provided.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
primary human lymphocytes, originating from 15 blood donors of both sexes
Test concentrations with justification for top dose:
3h after the beginning of the cultivation the cells were exposed to cyclohexanone.
final concentration in culture medium: 0.1, 0.01 and 0.005 mg/L
Details on test system and experimental conditions:
In each culture 100 metaphases were assessed.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not examined

Chromosomal aberrations were investigated in primary human lymphocytes after exposure with the test substance (end concentration in culture medium 0.1, 0.01 or 0.005 mg/L). The exposure with the test substance resulted in increased numbers of hyperdiploid metaphases, chromosomal fragments and dicentric chromosomes. The total number of aberrations (%) increased from 1.20 ± 0.23 (control) to 3.93 ± 0.53 (0.1 mg/L), from 2.06 ± 0.61 (control) to 4.40 ± 0.61 (0.01 mg/L) and from 1.40 ± 0.27 (control) to 5.30 ± 0.97 (0.005 mg/L). No dose-dependence for the cytogenetic effects of the test substance was observed.

Table 1:

mg/l

hypoploid metaphase

hyperploid metaphase

fragments

control

3 +/-0.45

1.3 +/-0.3

1.4 +/-0.27

0.005

3.1 +/-0.62

3.6 +/-0.54

5.3 +/-0.96

control

3.66 +/-0.45

1.13 +/-0.26

2.06 +/-0.61

0.01

4.8 +/-0.45

4 +/-0.31

4.4 +/-0.61

control

3.46 +/-0.48

1.13 +/-0.27

1.2 +/-0.23

0.1

4.6 +/-0.36

3.87 +/-0.46

3.93 +/-0.53

Conclusions:
Primary human lymphocytes, originating from 15 blood donors of both sexes were treated with the substance and the ploidy of the metaphases and structural aberrations were evaluated. Statistically significant increases without a dose-related increase were observed. The study report does not provide relevant information on assay performance and evaluation, i.e. exposure time and total cultivation period of cells, 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.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: newsletter
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Specific details on test material used for the study:
No information on the test material is provided.
Species / strain / cell type:
not specified
Metabolic activation:
not specified
Key result
Genotoxicity:
negative
Remarks on result:
other: no details on results are provided
Conclusions:
In this publication the substance is listed as having no mutagenic potential in the bacterial mutagenicity assay. No detailed data on the study performance and results are provided. Thus, the reliability of the data cannot be assessed.
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.

Link to relevant study records

Referenceopen allclose all

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:
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 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).
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:
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 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
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.
Endpoint:
in vivo insect germ cell study: gene mutation
Type of information:
experimental study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)
Version / remarks:
deleted in 2014
Deviations:
no
Principles of method if other than guideline:
The basc or Müller-5 test was used (Spencer and Stern, 1948; Würgler et al. 1977). In this test, recessive lethal mutations induced in the X-chromosomes of treated male gametes are detected in the F2 generation by the absence of wild-type males in the progeny of individual gametes. F3 generation flies were also observed since this allows the detection of mosaics or delayed mutations which may not appear in the F2 generation.
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Specific details on test material used for the study:
Source: Sigma-Aldrich
Batch no.: 13975
Species:
Drosophila melanogaster
Strain:
other: wild type flies Oregon K (OrK); Müller-5 (M-5)
Sex:
male
Details on test animals and environmental conditions:
The wild-type flies were Oregon K (OrK). Two lines and maintained by shaking over to fresh medium bottles every 2-3 weeks. The Müller-5 (M-5) flies had the bas c balancer X-chromosome. Stocks were maintained in bottles containing approximately 100 mL medium. All flies on test were kept in glass vials containing approximately 8 mL medium and stoppered with cotton wool.
Route of administration:
inhalation
Duration of treatment / exposure:
40 min; 7 h
Dose / conc.:
50 ppm
Remarks:
7 h exposure
Dose / conc.:
400 ppm
Remarks:
40 min exposure
No. of animals per sex per dose:
100 flies
Control animals:
no
Positive control(s):
ethylmethanesulphonate 0.4% (v/v)
Evaluation criteria:
The untreated control frequency of lethals in the flies used was about 0.2% . True mutation frequencies can only be determined within certain limits because only integral numbers of mutations can be recorded. These frequencies strongly depend on the sizes of the test groups studied (i.e. the size of individual broods), which are relatively small.
Based upon previous experiences with this test, which is meaningful but insensitive, it is considered that, in place of a test for statistical significance, it is better to look for a reproducible increase in the frequency of lethals over the historical control value of about 0.1 %. There is, of course, no opportunity for lethals to accumulate.

Based upon the historical control values, the evaluation criteria were as follows:
(a) A compound giving frequencies below 0.5% in duplicate experiments is considered to show no evidence of mutagenic activity.
(b) A compound giving frequencies greater than 1.0% in the same brood in duplicate experiments is considered to show mutagenic potential.
(c) A compound giving frequencies between 0.5% and 1.0% shows evidence of possibly being mutagenic, but further testing might be needed.
Statistics:
-
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
not specified
Negative controls validity:
not examined
Positive controls validity:
not specified
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
A dose ranging experiment was undertaken in which flies were exposed to 50 ppm cyclohexanone for 1 h, 3 h or 6.75 h or to 400 ppm cyclohexanone for 10 min, 20 min or 70 min. At 50 ppm cyclohexanone, several of the flies were apparently moribund towards the end of the 6.75 h exposure period and activity of the remaining flies was reduced. At 400 ppm cyclohexanone, the flies were sedated within 8 min and remained so until at least the end of the exposure period. When air was drawn through the fly exposure chambers, those flies exposed to cyclohexanone for 10 min or 20 min soon recovered. Flies exposed for 70 min showed some activity after 45 min in air. An extra batch of flies was exposed to 400 ppm cyclohexanone or 50 min; these flies also were sedated in about 8 min.

Two breeding stocks were exposed in the main tests. In the 50 ppm cyclohexanone exposure groups there were no signs of toxicity, but in the 400 ppm cyclohexanone exposure groups the flies started falling to the bottom of the exposure chamber within 6 min and did not respond to being approached.

Analysis for SLRL revealed no effects in the 50 ppm cyclohexanone atmosphere exposed groups or in Stock A of the 400 ppm atmosphere group. In Stock B, however, there were 4 lethals in Brood 2, giving a frequency of 1.0%, suggesting a significant response, if reproducibility is given. The frequency, however, in Stock A Brood 2 flies was only 0 .25 % which was due to a single lethal. The response in Brood 2, therefore, was not reproducible and it cannot be concluded that cyclohexanone is a mutagen.

Conclusions:
The mutagenic activity of cyclohexanone was tested in a sex linked recessive lethal test in Drosophila melanogaster males exposed to the substance via inhalation. It is concluded, that cyclohexanone does not induce mutations in post-meiotic germ cells of male flies. The performance of this GLP compliant study is comparable to OECD guideline 477 (deleted in 2014) and is considered to be reliable.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: based on current OECD 474 recommendations for evaluation of results no clear conclusion on the mutagenic potential of the substance can be drawn
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
evaluation of only 2000 cells per animal instead of 4000 cells per animal, lack of an appropriate trend analysis for dose-relationship, lack of historical control data, analysis of only one timepoint 24h.
GLP compliance:
no
Type of assay:
other: in vivo mammalian somatic cell study: bone marrow micronucleus
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sigma-Aldrich, Cat. No. C8930
Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weeks
- Weight at study initiation: 35-39 g
Route of administration:
oral: unspecified
Vehicle:
- Vehicle/solvent used: olive oil
- Justification for choice of solvent/vehicle: chosen due results of a solubility test
Post exposure period:
24h post-treatment
Dose / conc.:
300 other: mg/kg bw
Dose / conc.:
600 other: mg/kg bw
Dose / conc.:
1 200 other: mg/kg bw
No. of animals per sex per dose:
6
Positive control(s):
mitomycin C
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
no effects
Vehicle controls validity:
not examined
Negative controls validity:
not specified
Positive controls validity:
not specified

A mouse bone marrow micronucleus assay was performed in accordance to the OECD guideline 474. 6 male animals each dose group were orally treated with cyclohexanone dose levels of 300, 600 and 1200 mg/kg bw. 24 h after treatment bone marrow cells from femurs were prepared. 2000 polychromatic erythrocytes were evaluated for micronuclei.

There were no specific symptoms among the animals orally exposed to cyclohexanone. The ranges of the body weights of the animals were 35.33 -39.02g.

No cytotoxic effects were observed in the target organ (bone marrow), but the authors report a marginal induction of the micronucleated polychromatic erythrocytes, showing statistical significance at the highest dose applied when compared to the control value.

Although the increase in MN-PCEs is reported to be statistically significant (at the top dose only) with respect to the concurrent negative control, no comparison with relevant and properly compiled negative historical control data is made or reported.  

Table: Number of micronucleated cells 

Dose group

#MNPCE

MNPCE frequency [%]

PCE/(PCE+NCE) [%]

neg contr.

3.67 +/-0.52

0.18 +/-0.03

52.21 +/-15.05

300 mg/kg

4.17 +/-1.94

0.21 +/-0.1

51.84 +/-8.61

600 mg/kg

4.33 +/-1.03

0.22 +/-0.05

49.86 +/-6.04

1200 mg/kg

7 +/-1.41*

0.35 +/-0.07*

54.29 +/-12.47

MMC, 0.5 mg/kg

27 +/-7.91

1.38 +/-0.4

48.09 +/-13.69

 

*p<0.05

MNPCE – micronucleated polychromatic erythrocytes

NCE – normochromatic erythrocytes

Conclusions:
A micronucleus assay was performed in mouse bone marrow according to the OECD guideline 474 (version 1997, 2016).
6 male animals each dose group were orally treated with cyclohexanone dose levels of 300, 600 and 1200 mg/kg bw but no justification for the choice of the dose levels is indicated.
24 h after treatment bone marrow cells from femurs were prepared and 2000 polychromatic erythrocytes were evaluated for micronuclei.
In accordance to the OECD guideline 474 samples of bone marrow should be taken at least twice, and at least 4000 immature erythrocytes per animal should be scored for the incidence of micronucleated immature erythrocytes. The use of such non-standard "dose once", "sacrifice one" protocol with a limited number of erythrocytes evaluated is not supported by the OECD test guideline for the assay.

No cytotoxic effects were observed, but the authors report a marginal induction in number of micronucleated polychromatic erythrocytes, showing statistical significance only at the highest dose applied when compared to the control value.
Although the increase in MN-PCEs is reported to be statistically significant with respect to the concurrent negative control, no comparison with relevant and properly compiled negative historical control data is made or reported.  This is a key step in the assessment of this study type, indeed the current test guideline for this assay (OECD 474 2016), states in the criteria for determining a positive response "....results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)".  Due to the lack of presentation of these data it is not possible to assess if this positive response requirement is met.

Summarized this study contains a number of deviations in study design and interpretation from those recommended by the OECD guideline 474 (version 1997, 2016). Deviations from the OECD guideline should always be supported by a robust scientific justification, which has not been presented by the authors.
Limitations in the extend of required assessments as for example a decreased number of evaluated erythrocytes (2000 instead of 4000), in the number of sampling time points (one instead of at least two), and the absence of a comparison to lab-specific historical control data, result in a significant dercrease in the statistic power of this study. Accordingly, based on this data, no conclusion on thebiological relevance of the marginal increase in the high dose in accordance to the criteria of the Report on Statistical Issues related to OECD Test Guidelelines (TGs) on Genotoxicity (OECD 2014, Series on Testing and Assessment No. 198 ENV/JM/MONO(2014)12) can be met.

No robust conclusion on the biological relevance of the study results and on the cytogenic potential of cyclohexanone in the in vivo micronucleus assay on the oral route of exposure can be taken from this study.

Therefore this study can not be applied to reliably evaluate the effects of cyclohexanone in the in vivo micronucleus assay on the oral route of exposure, and can fürthermore not be applied to evaluate the potency of cytohexanone to cause cytogenetic damage in vivo.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: subcutaneous injection of the substance to male rats, preparation of bone marrow chromosomes according to Yosida and Amano (1965)
GLP compliance:
not specified
Type of assay:
other: chromosomal aberrations in bone marrow
Specific details on test material used for the study:
Source: Kafr El-Zayat Company for Insecticides, Egypt
Species:
rat
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 100-200 g
- Assigned to test groups randomly: [no/yes, under following basis: ]

Route of administration:
subcutaneous
Vehicle:
Not specified
Duration of treatment / exposure:
for single dosing: 6, 24 and 48 hours after treatment
for repeated dosing: 6 hours after treatment
Frequency of treatment:
Single dose and repeated dose for 5 days
Dose / conc.:
100 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
100 mg/kg bw/day
Remarks:
for repeated dose experiment (5 days)
Dose / conc.:
500 mg/kg bw/day
Remarks:
for repeated dose experiment (5 days)
No. of animals per sex per dose:
5 per dose, 1 control per harvest time
Control animals:
yes
Positive control(s):
None
Tissues and cell types examined:
Bone marrow
Evaluation criteria:
Not specified
Statistics:
F-test
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not examined
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Maximum tolerated dose: 1000 mg/kg bw

RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: statistical significances increasing with time and dose

In the high dose group of the subacute study all animals died after the second injection.

A decrease in the frequency of “sticky nuclei” with time after treatment was described in the study report (see table 1). It is discussed that the decrease was greater with the low and intermediate dose than with the high dose. The highest number of “sticky nuclei” were observed in the subacute treatment.

An influence on the mitotic index was described, with a slight stimulation with the low dose and 48 h after the medium dose, while it decreased in the medium and high dose. In the subacute treatment mitosis was decreased with the low dose and unaffected by the medium dose.

Both endpoints are indicative for cell proliferation and cytotoxicity. 

 

It was interpreted that gaps were slightly increased in the high dose, but were not affected with time. Breaks were most frequent in the mid dose and decreased with time after treatment. Chromatid exchanges, centric fusions and polyploidy were more frequent in the high dose.

The study report indicates that these observations were of statistical significance. However, the statistical evaluation is not reproducible and can not be considered as valid as:

- The control group for each exposure only exists of one animal and no absolute values are reported,

- and the individual animal data and standard deviations are not indicated in the report.

 

Table 1: Effect of cyclohexanone on the frequency of “sticky nuclei” and dividing cells/1000, deducting the number observed in the control.

 

acute

subacute

mg/kg bw

6h

24h

48h

6h

“sticky nuclei”

100

8.8

6.5

1

12.4

500

9

4.2

1.2

11.6

1000

11.8

11.6

5.2

-

dividing cells

100

1.6

8.4

3.2

9.8

500

-18

-6.8

-3.8

1.4

1000

-12.6

-7

-10.2

-

 

Table 2:

 

Dose [mg/kg bw]

h after treatment

gap

break

Chromatid exchange

Centromeric fusion

Centromeric attenuation

polyploid

Abnormal nuclei

acute

100

6

2.8

3.6

8.4

3.6

4.4

0.4

23.2

24

2

3.6

2.4

3.2

4.4

0

15.6

48

2.8

1.6

2

1.6

3.6

0.4

12

500

6

2.8

13.2

4

3.6

10

0.8

34.4

24

4.8

7.6

8

6.4

0

0.2

27

48

2

7.2

6.4

4

4

3.2

26.8

1000

6

11.2

8

12.8

4.8

1.6

0.8

39.2

24

4.8

6.4

8

7.6

6

2

34.8

48

4.8

4

5.2

7.6

4.4

2

28

subacute

100

6

3.6

7.6

2.4

2.8

3.2

0.4

20

500

6

8.4

6.4

8.8

3.2

5.2

4.4

36.4

 

 

 

 

 

 

 

 

 

 

 

Conclusions:
In an acute study on chromosomal damage in the bone marrow 3 groups of 5 male rats each dose level were injected with 100, 500 and 1000 mg/kg bw. For each group one control animal was added. No further details on the type of injection are reported. From each dose level one group was sacrificed 6, 24 and 48h after injection, respectively.
In a subacute study each dose was given on five consecutive days, sacrificing the animals 6h after the last injection.
Chromosomes were prepared from the bone marrow and in 1000 cells per animal the number of dividing cells including prophase and metaphase were determined as a parameter of mitotoic activity for cyclohexanone.
In addition in every rat 50 metaphases were examined for chromosomal abnormalities.
As a result in the high dose group of the subacute study all animals died after the second injection.
A decrease in the frequency of “sticky nuclei” with time after treatment and an influence of cyclohexanone treatment on the mitotic index may be indicative for target organ cytotoxicity.
A biological relevance of statistically significant increases in the incidence of aberrations can not be evaluated due to major lack of information in the report and major deviations to the current OECD 475 guideline.
E.g. the statistical evaluation is not reproducible and can not be considered as valid. Furthermore the control group for each exposure only exists of one animal and no absolute values, no individual animal data and no standard deviations are reported.
The validity of the test can not be proven due to the lack of positive control animals. Only a reduced number of metaphases was evaluated per animal (50 metaphases), not being in line with current recommendations (200 metaphases per animal) and the observed gaps were also included in the assessment for clastogenic potential. Gaps, however, are achromatic lesions of unknown origin and should not be included in the assessment of clastogenicity. A classification according to the current evaluation criteria is not possible, due to the lack of control values and historical control data. Therefore, the study is not considered reliable for the assessment of clastogenicity.
Endpoint:
genetic toxicity in vivo, other
Remarks:
host (mammalian) mediated yeast cell gene-mutation study
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
validity of the assay cannot be assessed due to the lack of positive controls, evaluation criteria are missing
Qualifier:
no guideline available
GLP compliance:
not specified
Type of assay:
other: host mediated assay (yeast cells in mice)
Specific details on test material used for the study:
No information on the test material is provided.
Species:
mouse
Strain:
B6C3F1
Sex:
not specified
Route of administration:
oral: gavage
Details on exposure:
0.5 ml per animal per treatment
Post exposure period:
incubation time: 16 h
Dose / conc.:
300 other: mg/kg bw
Dose / conc.:
600 other: mg/kg bw
Dose / conc.:
1 200 other: mg/kg bw
No. of animals per sex per dose:
3
Control animals:
yes
Tissues and cell types examined:
Cell type examined: yeast cells (Schizosaccharomyces pombe), inoculated into the peritoneal cavity of mice
Details of tissue and slide preparation:
METHOD OF ANALYSIS: yeast cell suspension spread from peritoneal cavity of mice diluted to 1.5 * 10³ cells/mL, 0.2 mL of the dilution spread on a yeast extract agar plate, counting of forward mutants colonies by microscope.
Key result
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not examined
Negative controls validity:
not specified
Positive controls validity:
not examined
Conclusions:
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.
Endpoint:
in vivo insect germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
other information
Study period:
1 January 1986 - 7 July 1986
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:
OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)
Version / remarks:
deleted in 2014
Deviations:
yes
Remarks:
evaluation criteria are not reported
GLP compliance:
yes
Type of assay:
Drosophila SLRL assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Fisher Scientific Co., lot No.: 855621
Species:
Drosophila melanogaster
Strain:
other: male: Canton-S wild type stock, female: "Basc" stock
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Brown University
- Age at study initiation: males: mated at the age of 3-4 days, females: mated at the age of 3-10 days
Route of administration:
inhalation
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- An aliquiot (approximately 2 mL) of liquid test item (in excess of that required to produce saturation) was put into a 10 mL hypovial (A), sealed therein and allowed to evaporate. Flies were placed in similar sealed hypovials (B). Aliquots of saturated air were transferred with a gas syringe from vial A to the vials B containing the flies.


Duration of treatment / exposure:
4 h
Frequency of treatment:
once
Post exposure period:
none
Dose / conc.:
36 other: % saturation
Remarks:
approximately 1900 ppm
Control animals:
yes, concurrent no treatment
Positive control(s):
1,2 Dibromoethane
- Route of administration: inhalation
- Doses / concentrations: 25 ppm
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
not examined
Negative controls validity:
not specified
Positive controls validity:
not specified

The number of chromosomes tested was sufficient to detect an induced mutation frequency of 0.2% or greater.

The sex-linked recessive lethal results were:

Cyclohexanone: 7/9821 (0.071%)

Negative controls: 7/10,051 (0.070%)

Positive controls (DBE): 6/2585(0.232%)

It is concluded that cyclohexanone does not induce mutations in the postmeiotic germ cells of Drosophila melanogaster males when administered by inhalation.

Conclusions:
The mutagenic activity of cyclohexanone was tested in a sex linked recessive lethal test in Drosophila melanogaster males exposed to the substance via inhalation. It is concluded, that cyclohexanone does not induce mutations in post-meiotic germ cells of male flies. The performance of this GLP compliant study is comparable to OECD guideline 477 (deleted in 2014) and is considered reliable.
Endpoint:
in vivo insect germ cell study: gene mutation
Type of information:
experimental study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Remarks:
no details on methodology and no detailed results are provided
GLP compliance:
not specified
Type of assay:
not specified
Specific details on test material used for the study:
No information on the test material is provided.
Species:
Drosophila melanogaster
Sex:
male
Route of administration:
not specified
Duration of treatment / exposure:
3 days
Dose / conc.:
0.1 other: mL/100 mL
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
The available abstract on mutagenicity testing in male fruit flies does not provide any detailed information on methodology of the assay and information concerning the substance is lacking. 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 the substance.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

End point summary Genetic Toxicity

 

Endpoint conclusion:

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,

 

Gene mutation

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

The negative key study for in vitro gene mutation in bacteria was performed in accordance to the OECD guideline 471 and in compliance to GLP (BASF, 1999). This test result is supported by two negative bacterial reverse mutation assays which were well performed in accordance 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 to the OECD guideline 476 and in compliance to GLP, 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 in accordance to the current OECD guideline 490 (McGregor, 1988). 

In addition, no indication for mutagenicity was reported in asex-linked recessive lethal test which was performedin accordance to 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 a in an assay assessing the uptake of tritiated thymidine uptake into mammalian cells (Perocco, 1983). All 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 to the OECD TG 475, with an extended test protocol using 10 animals 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 administered. 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, 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).

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

 

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 4.1 (June 2015) 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 to the respective OECD guideline can therefore not be used for classification.

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 Muta is inappropriate. 

 

1.    QSAR-Models:

In a number of QSAR- Models implemented in the OECD Toolbox 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.1.4., no alert for the Chromosomal Aberration Assay and the Micronucleus Test was found by OASIS v.1.1., no alert for DNA binding was found by OECD, no alerts for in vitro mutagenicity (Ames Assay) andin vivomutagenicity (Micronucleus) was found by ISS and no protein binding alerts for chromosomal aberration was found by OASIS v.1.2.

 

2.    Genetic toxicity in vitro:

2.1 Indicator assays for DNA damage

Indicator assays provide an indication for DNA damage via effects such as DNA strandbreaks, unscheduled DNA synthesis (UDS) and sister chromatid exchange (SCE), however they may not be able to identify the manifestation of a mutation. In a number of reliable indicator assays cyclohexanone did not show any potential to directly damage DNA. In various sensitive test systems for DNA damage cyclohexanone generally resulted in negative results.

·     Cyclohexanone is negative in the DNA Damage and Repair, Unscheduled DNA Synthesis Assay performed according to the OECD guideline 482 (deleted in 2014). In this assay, human fibroblasts were incubated at concentrations up to 9.48 mg/ml, with and without metabolic activation. This result was confirmed in an additional independent experimental repeat.

(NIOSH, Report No. 210-78-0026, 1980.)

·     No DNA damage was observed in an in vitro Comet Assay using a reconstructed 3D human epidermal skin model. The assay was performed as an intra- and inter-laboratory validation in three participating laboratories. The Comet analyses were performed in compliance with the current OECD 489 guideline for the alkaline Comet assay in vivo, and the overall conclusion was that cyclohexanone is non-genotoxic.

(Reus et al. Mutagenesis 28(6), 709-720, 2013).

·     The effect of cyclohexanone on cell viability and DNA repair synthesis, determined by tritiated thymidine uptake, was investigated in human lymphocytes in vitro, in the absence and presence of metabolic activation. Cell viability was not influenced and the substance is not considered to induce DNA damage and repair synthesis.

(Perocco et al., Toxicology Letters 16(1-2), 69-76, 1983.)

 

2.2   In vitro gene mutation study in bacteria

A number of valid in vitro gene mutation assays in bacteria indicate that cyclohexanone does not result in gene mutations when incubated with or without metabolic activation.

·     The key study for in vitro gene mutation in bacteria was performed according to the OECD Guideline 471 in compliance to GLP. S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A test strains were incubated with 10 - 5000 µg cyclohexanone/plate as a Standard Plate Test and 10 - 1000 µg/plate as a Pre-Incubation Test. Each of the assays was performed with and without rat liver S-9 mix. This assay can be considered to be the most reliable data for the assessment of in vitro gene mutation in bacteria, and under these conditions it can be clearly demonstrated that cyclohexanone is not mutagenic.

(BASF SE, Study Report 40M0277/984221, 1999).

Further in vitro gene mutation studies in bacteria did not identify a mutagenic effect of cyclohexanone, however were of lower scientific quality. E.g. one assay was performed with only two (Florin et al., Toxicology, 15(3), 219-232, 1980), another with only 4 Salmonella typhimurium tester strains (Haworth et al., Environmental Mutagenesis 5 (Suppl. 1), 3–142, 1983). Nevertheless, both assays were performed with and without metabolic activation, as two independent pre-incubation assays, and in accordance to the protocol of the current OECD Test Guideline 471.

 

2.3   In vitro gene mutation study in mammalian cells

The lack of a potential to induce gene mutations identified in in vitro gene mutation assays in bacteria is confirmed by valid in vitro gene mutation studies in mammalian cells,which were performed in accordance to the respective guidelines.Therefore there is aconvincing level of evidence that cyclohexanone does not result in gene mutations in mammalian cells.

·     In the key study for in vitro gene mutation in mammalian cells cyclohexanone was assessed for its potential 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 were observed, neither with nor without the addition of a metabolizing system. This GLP and guideline compliant study clearly shows that the substance is not mutagenic in mammalian cells.

(BASF SE, Study Report, 50M0465/02M008, 2012)

 

- Cyclohexanone is negative in the mouse lymphoma assay performed according to the current OECD guideline 490. Tk+/tk- L5178Y mouse lymphoma cells were incubated at concentrations up to 5000 μg/ml, in the presence and in the absence of metabolic activation. The read-out of the mouse lymphoma assay allows to identify gene mutations and chromosomal aberrations.

(McGregor et al., Environmental and Molecular Mutagenesis 12, 85-154, 1988). 

 

2.4   In vitro cytogenicity study in mammalian cells or in vitro micronucleus study.

Cyclohexanone is negative in the mouse lymphoma assay performed according to the current OECD guideline 490. Tk+/tk- L5178Y mouse lymphoma cells were incubated at concentrations up to 5000 μg/ml, in the presence and in the absence of metabolic activation. The mouse lymphoma assay is assessing gene mutation and structural changes at the chromosomal level. Chromosomal aberrations can be identified as induced slow growing mutants which grow with prolonged doubling times (4.).

(McGregor et al., Environmental and Molecular Mutagenesis 12, 85-154, 1988). 

 

 

 

2.5   Disregarded in vitro genotoxicity studies

 

See 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.

(MassoudA.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, esults with such strains can only give an indication on DNA damage and not a potential gene mutation, and or cyclohexanone the report of this 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 to genotoxic effect which is related to the concentration of the test substance. However, due to methodological deficiencies to the OECD guideline (e.g. varying incubation times, low number of assessed cells, low sensitivity of positive controls) the study was not to 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 observe in concentrations resulting in almost complete cytotoxicity. In addition, the reporting of the studieslacks significant detail and the study performance shows major deviations to 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 detais on assay performance and results. Thus, the reliability of the data cannot be assessed.

(JETOC: Newsletter Nr. 4, 1985)

 

3.        In vivo genotoxicity

3.1   In vivo genotoxicity via inhalation

·     An in vivo chromosomal aberration assay was performed in accordance to the OECD guideline 474 with male and female rats. Animals were exposed to concentrations of 50 and 400 ppm via inhalation. 30 animals each concentration were exposed once for 7h and 10 animals each concentration repeatedly for 7h on 5 subsequent days.

The animals were sacrificed 6, 24 and 48 h following inhalation exposure and bone marrow of one femur was prepared to assess the cells on existing chromosomal aberrations.

As a result, single and repeated exposure to cyclohexanone did not result in increases of chromosomal damage in the bone marrow.

(NIOSH 1980, Tier II mutagenic screening of 13 NIOSH priority compounds; individual compound report cyclohexanone. Report No. 210-78-0026 Company Study No. PB 83-127571.)

 

·     A dominant lethal assay was performed in accordance to the OECD guideline 478 with male rats. The dominant lethal test is to investigate mutations resulting from chromosomal aberrations and gene mutations in germ cells. 10 animals each concentration were repeatedly exposed for 7h on 5 subsequent days to cyclohexanone concentrations of 50 and 400ppm via inhalation. Each treated male animal was mated to 2 untreated virgin females. Mated females were examined for pregnancy and dominant lethal effects and ovaries were examined for corpora lutea. Mating and assessment of pregnancy outcome was repeated on each of the next 9 consecutive weeks. As a result, there were no effects on pregnancy frequency, corpora lutea count, implantations or early deaths in the cyclohexanone treated groups.

(NIOSH 1980, Tier II mutagenic screening of 13 NIOSH priority compounds; individual compound report cyclohexanone. Report No. 210-78-0026 Company Study No. PB 83-127571.)

 

·     A sex-linked recessive lethal test in Drosophila melanogaster was performed in accordance to the former OECD guideline 477 (which was deleted 2014). This assay detects the occurrence of mutations, both point mutations and small deletions, in the germ line of an insect. 100 male wild type flies were exposed to 50 and 400 ppm cyclohexanone for 40 min and 7h. Exposed males were then mated individually to virgin females in the ratio 1:2, a procedure which was repeated for 3 days with the same male and 2 unmated females, respectively. Mating for F2 generation was set up by mating brothers and sisters. Assessment of F3 generation was identical to F2 generation. As a result, no mutation resulting in lethality was observed in the offspring of the exposed females.

(NIOSH 1980, Tier II mutagenic screening of 13 NIOSH priority compounds; individual compound report cyclohexanone. Report No. 210-78-0026 Company Study No. PB 83-127571.)

 

·     The mutagenic activity of cyclohexanone was tested in an additional sex linked recessive lethal test in Drosophila melanogaster males exposed to the substance via inhalation. It is concluded, that cyclohexanone does not induce mutations in post-meiotic germ cells of male flies. The performance of this GLP compliant study is comparable to OECD guideline 477 (deleted in 2014) and is considered to reliably contribute to the weight of evidence for non-mutagenicity of the substance.

(TSCATS1986, Drosophila melanogaster sex-linked recessive lethal test of cyclohexanone, Univeristy of Wisconsin, Zoology Department, OTS 0511205)

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/m3for 8h (Mraz et al. 1994; see toxicokinetics).

 

3.2     In vivo genotoxicity oral exposure

·     In a mouse micronucleus assay, 6 male animals each dose group were orally treated with cyclohexanone dose levels of 300, 600 and 1200 mg/kg bw. 24 h after treatment bone marrow cells from femurs were prepared and 2000 polychromatic erythrocytes were evaluated for micronuclei. The test performance is not in accordance to the OECD guideline 474 (version 1997, 2016), as at least 4000 immature erythrocytes per animal should be scored and samples of bone marrow should be taken at least twice. Furthermore, no justification given for the dose levels used.

As a result, no cytotoxic effects were observed, though a marginal statistically significant induction of micronucleated polychromatic erythrocytes was described at the highest dose administered. As no comparison to lab-specific historical control data is indicated the biological relevance of this marginal observation cannot be evaluated, which is now recognized as a critical aspect for the interpretation of studies of this type.

These deficiencies in study design and interpretation lead to a lack of power for the determination of the relevance of the marginal response. Therefore, no robust conclusion on the cytogenic potential of cyclohexanone in the in vivo micronucleus assay on the oral route of exposure can be taken from this study.

(Kim S. at al., Journal of Life Science, 24(7), 804-811, 2014)

 

3.3  In vivo genotoxicity disregarded 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. were injected with 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 shortcoming, 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 exists 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 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. In vitro and in vivo results with cyclohexanone were negative. Since the substance is regarded as non-genotoxic and non-mutagenic, a classification is not warranted.