4-methylcyclohexanone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three reliable in vitro studies addressing different aspects of potential mutagenicity of 4 -methylcyclohexanone are available:

A bacterial reverse mutation assay was conducted with Salmonella typhimurium strains TA1535, TA100, TA1537, TA98 and Escherichia coli strains WP2 uvrA pKM101, and WP2 pKM101, according to OECD 471 (Chang, 2016). During the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this assay. The study was conducted under GLP according to a guideline method, and was thus assigned a Klimisch reliability rating 1.

A gene mutation assay in Chinese Hamster V79 cells (V79/HPRT), according to test guideline OECD 476 (Wollny, 2017). Under the experimental conditions reported, the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in the HPRT assay. The study was conducted under GLP according to a guideline method, and was thus assigned a Klimisch reliability rating 1.

A micronucleus test in human lymphocytes was conducted according to test guideline OECD 487 (Chang, 2016). Under the experimental conditions reported, the test substance did not induce micronuclei. Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration. The study was conducted under GLP according to a guideline method, and was thus assigned a Klimisch reliability rating 1.

The results from all three in vitro studies report 4-methylcyclohexanone as non-mutagenic, and in accordance with the standard testing approach for mutagenicity, further in vivo studies are not triggered. The available data is considered to be relevant, reliable, and adequate for the purposes of classification and risk assessment.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June 2016 to 08 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations.
Reverse mutation assays determine the frequency with which an agent reverses or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains, or a single strain with multiple markers are necessary to assure reliable detection of mutagens that may be specific to one tester strain or locus. The reversion of bacteria from growth-dependence on a particular amino acid to growth in the absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely used marker.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The S. typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively. The S. typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA1535, TA100, WP2 uvrA pKM101, and WP2 pKM101) and frameshift (TA1537, TA98) mutations.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Expriment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
In the pre-experiment, the concentration range of the test substance was 3 - 5000 μg/plate. The pre-experiment is reported as experiment I. Since no cytotoxic effects were observed in experiment I, 5000 μg/plate was chosen as the maximal concentration for experiment II.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 2.5 µg/plate in DMSO for TA1535, TA1537, TA98 and TA100; 10 µg/plate in DMSO for WP2 uvrA (pKM101) and WP2 (pKM101) with S9

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 60 mins
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant.

Statistics:

Not applicable

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

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

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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

Table 1: Summary of results Experiment I without S9 - Revertant Colony Counts (Mean ±SD)

Group	Concn. (per plate)	TA1535	TA1537	TA98	TA100	WP2 pKM101	WP2 uvrA pKM101
DMSO	-	11 ± 3	8 ± 1	35 ± 4	186 ± 13	217 ± 17	350 ± 14
Untreated	-	7 ± 3	14 ± 5	36 ± 5	199 ± 13	224 ± 21	362 ± 23
CA5110	3 µg	10 ± 4	8 ± 2	25 ± 3	169 ± 11	188 ± 5	336 ± 14
CA5110	10 µg	13 ± 3	9 ± 2	24 ± 7	167 ± 19	199 ± 12	331 ± 19
CA5110	33 µg	10 ± 1	11 ± 4	25 ± 5	165 ± 13	198 ± 10	311 ± 17
CA5110	100 µg	13 ± 3	8 ± 3	26 ± 9	163 ± 5	201 ± 9	337 ± 24
CA5110	333 µg	12 ± 5	8 ± 2	29 ± 8	178 ± 9	192 ± 9	337 ± 8
CA5110	1000 µg	10 ± 2	11 ± 5	35 ± 7	178 ± 10	197 ± 7	316 ± 15
CA5110	2500 µg	7 ± 2	9 ± 2	35 ± 2	176 ± 6	191 ± 17	323 ± 23
CA5110	5000 µg	8 ± 1	13 ± 5	39 ± 3	91 ± 9	166 ± 9	331 ± 7
NaN3	10 µg	1060 ± 26	-	-	1693 ± 110	-	-
4-NOPD	10 µg	-	-	426 ± 37	-	-	-
4-NOPD	50 µg	-	63 ± 10	-	-	-	-
MMS	2.0 µL	-	-	-	-	3807 ± 415	3555 ± 69

Table 2: Summary of results Experiment I with S9 - Revertant Colony Counts (Mean ±SD)

Group	Concn. (per plate)	TA1535	TA1537	TA98	TA100	WP2 pKM101	WP2 uvrA pKM101
DMSO	-	12 ± 4	14 ± 3	34 ± 12	164 ± 17	230 ± 13	393 ± 10
Untreated	-	15 ± 4	15 ± 2	47 ± 6	192 ± 21	259 ± 20	436 ± 10
CA5110	3 µg	11 ± 1	16 ± 5	36 ± 4	153 ± 23	229 ± 22	404 ± 9
CA5110	10 µg	9 ± 3	18 ± 3	41 ± 4	142 ± 27	224 ± 16	401 ± 23
CA5110	33 µg	13 ± 3	12 ± 2	40 ± 6	122 ± 16	195 ± 27	379 ± 24
CA5110	100 µg	13 ± 3	13 ± 3	42 ± 6	150 ± 13	214 ± 18	396 ± 15
CA5110	333 µg	12 ± 0	15 ± 3	45 ± 10	163 ± 20	225 ± 35	402 ± 21
CA5110	1000 µg	10 ± 3	17 ± 6	45 ± 3	171 ± 9	208 ± 4	371 ± 5
CA5110	2500 µg	14 ± 3	17 ± 3	45 ± 8	175 ± 11	198 ± 15	345 ± 20
CA5110	5000 µg	12 ± 3	14 ± 2	37 ± 7	137 ± 8	170 ± 21	359 ± 26
2-AA	2.5 µg	381 ± 25	189 ± 12	4418 ± 246	4830 ± 406	-	-
2-AA	10.0 µg	-	-	-	-	1082 ± 26	2285 ± 128

Table 3: Summary of results Experiment II without S9 - Revertant Colony Counts (Mean ±SD)

Group	Concn. (per plate)	TA1535	TA1537	TA98	TA100	WP2 pKM101	WP2 uvrA pKM101
DMSO		11 ± 5	12 ± 2	21 ± 5	158 ± 17	201 ± 30	350 ± 9
Untreated		14 ± 3	13 ± 4	28 ± 10	205 ± 23	241 ± 21	369 ± 10
CA5110	33 µg	16 ± 5	8 ± 3	20 ± 5	157 ± 5	209 ± 22	338 ± 10
CA5110	100 µg	12 ± 3	8 ± 3	23 ± 2	139 ± 11	189 ± 10	355 ± 15
CA5110	333 µg	16 ± 3	10 ± 1	25 ± 5	137 ± 16	200 ± 4	331 ± 18
CA5110	1000 µg	17 ± 4	9 ± 1	30 ± 2	120 ± 15	211 ± 5	351 ± 11
CA5110	2500 µg	16 ± 3	10 ± 4	34 ± 3	123 ± 8	199 ± 8	330 ± 7
CA5110	5000 µg	14 ± 3	7 ± 2	20 ± 2	68 ± 10	110 ± 20	303 ± 25
NaN3	10 µg	1053 ± 27			2256 ± 119
4-NOPD	10 µg			334 ± 33
4-NOPD	50 µg		81 ± 3
MMS	2.0 µL					3232 ± 332	2779 ± 377

 

Table 4: Summary of results Experiment II - with S9 - Revertant Colony Counts (Mean ±SD)

Group	Concn. (per plate)	TA1535	TA1537	TA98	TA100	WP2 pKM101	WP2 uvrA pKM101
DMSO		15 ± 1	15 ± 6	45 ± 6	129 ± 14	249 ± 26	394 ± 10
Untreated		12 ± 2	19 ± 3	37 ± 4	216 ± 12	296 ± 20	483 ± 3
CA5110	33 µg	13 ± 2	11 ± 3	39 ± 9	144 ± 19	240 ± 38	396 ± 20
CA5110	100 µg	15 ± 3	13 ± 3	36 ± 8	141 ± 19	245 ± 18	417 ± 16
CA5110	333 µg	16 ± 6	16 ± 6	44 ± 15	135 ± 18	241 ± 29	407 ± 5
CA5110	1000 µg	11 ± 1	12 ± 4	38 ± 9	113 ± 3	239 ± 14	400 ± 22
CA5110	2500 µg	15 ± 1	15 ± 5	43 ± 11	87 ± 7	183 ± 8	363 ± 6
CA5110	5000 µg	17 ± 3	10 ± 2	32 ± 10	53 ± 4	157 ± 2	341 ± 27
2 AA	2.5 µg	380 ± 45	159 ± 16	3865 ± 655	4594 ± 579
2 AA	10.0 µg					1055 ± 88	2035 ± 113

NaN3 sodium azide 2-AA 2-aminoanthracene 4-NOPD 4-nitro-o-phenylene-diamine MMS methyl methane sulfonate

 

Conclusions:

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

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101.

The plates incubated with the test substance showed normal background growth up to 5000 μg/plate in all strains with and without metabolic activation.

In experiment II, cytotoxicity (evident as a reduction in the number of revertants (below the indication factor of 0.5)) was observed in strain TA100 at 5000 μg/plate both with and wihout metabolic activation. No other cytotoxic effects were observed in any other strain both with and without metabolic activation.

No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations and all mutation rates were within the range of normal biological variability.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

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

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 July 2016 to 11 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. V. Theodor (64380 Rossdorf, Germany).
- Suitability of cells: Human lymphocytes have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Cell cycle length, doubling time or proliferation index: Treatments started after a 48 hour stimulation period with phytohemeagglutinine (PHA) when cells were actively proliferating and the cells were prepared at approx. 2 – 2.5 fold of the normal cell cycle time.
- Sex, age and number of blood donors if applicable: Healthy non-smoking donors not receiving medication. For this study, blood was collected from a female donor (30 years old) for Experiment IA, from a female donor (27 years old) for Experiment IB and from a male donor (22 years old) for Experiment II.
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood
- Culture conditions: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 h after blood collection

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (phytohemagglutinin) (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
- Properly maintained: [yes]

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

The highest applied concentration in this study (1141 μg/mL of the test substance, approx. 10 mM) was chosen with regard to the molecular weight and the purity (98.3%) of the test substance and with respect to the current OECD Guideline 487.
Exp. IA with/without S9: 7.4, 13.0, 22.7, 39.7, 69.5, 122, 213, 373, 652, 1141 µg/mL (only 213, 373, 652, 1141 µg/mL evaluated)
Exp. II without S9: 39.7, 69.5, 122, 213, 373, 652, 1141 µg/mL (only 213, 373, 652, 1141 µg/mL evaluated)
Exp. IB with S9 mix: 69.5, 122, 213, 373, 652, 1141 µg/mL (only 213, 373, 652, 1141 µg/mL evaluated)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 0.5% acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Positive controls:

yes

Positive control substance:

cyclophosphamide

Positive controls:

yes

Positive control substance:

other: demecolcin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

DURATION
- Stimulation period: 48h
- Exposure duration: 4 h (Exp. IA -S9); 20h (Exp II -S9); 4h (Exp. IB +S9)
- Recovery time: 16h (Exp IA and Exp IB; 0h (Exp II)
- Cytochalasin B exposure: 20 h
- Total culture period: 88h:

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 μg/mL)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were harvested by centrifugation 40 h after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in saline G (approximately 5 mL) and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in KCl solution (5 mL, 0.0375 M) and incubated at 37 °C for 20 minutes. Ice-cold fixative mixture of methanol and glacial acetic acid (1 mL, 19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, were mounted after drying and were covered

NUMBER OF CELLS EVALUATED: 500 cells per culture in duplicate

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Micronuclei only be evaluated in cells that have completed mitosis during exposure to the test substance or during post-exposure period and thus a cytokinesis blocker, cytochalasin B, is added to the cell culture to ensure that there are binucleate cells to be evaluated for micronuclei.

DETERMINATION OF CYTOTOXICITY
- Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in binucleated cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not be more than one third of the area of the main nucleus. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture. Cytotoxicity is expressed as cytostasis, calculating the CBPI, and used therefore as a cut off criteria. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Rationale for test conditions:

Assessed in three independent experiments with one preparation interval (40 hours).
Micronuclei only be evaluated in cells that have completed mitosis during exposure to the test substance or during post-exposure period and thus a cytokinesis blocker, cytochalasin B, was added to the cell culture to ensure that there were binucleate cells to be evaluated for micronuclei.
Treatments started after a 48 hour stimulation period with phytohemeagglutinine (PHA) when cells were actively proliferating and the cells were prepared at approx. 2 – 2.5 fold of the normal cell cycle time.
For validation of the test, control mutagens were tested in parallel to the test substance.
Concentration selection was performed according to the current OECD Guideline for the in vitro micronucleus test. The highest test substance concentration should be 10 mM, 2 mg/mL, or 2 μL/mL, whichever is the lowest. At least three test substance concentrations should be evaluated for cytogenetic damage.
With regard to the molecular weight and the purity (98.3%) of the test substance, 1141 μg/mL (approx. 10 mM) were applied as the top concentration for treatment of the cultures in the pre-test. Test substance concentrations ranging from 7.4 to 1141 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Since the cultures fulfilled the requirements for cytogenetic evaluation in the absence of S9 mix, this test was designated Experiment IA. The experimental part with S9 mix was repeated with the same top dose (Exp. IB) due to invalid solvent control.
No cytotoxicity was observed in the experiments IA and IB after 4 h treatment with or without S9 mix up to the highest concentrations. Therefore, the same concentration (1141 μg/mL) was chosen as top concentration for Experiment II. This experiment was rerun with same concentrations (except 39.7 μg/mL)) due to invalid solvent control -number of micronuclei outside historical control range.

Evaluation criteria:

Test substance considered negative if in all of the experimental conditions examined:
− None of the test item concentrations exhibits a stati sig increase compared to concurrent solvent control.
− No concentration-related increase when evaluated with an appropriate trend test.
− The results in all evaluated test item concentrations are within the range of the laboratory historical solvent control data (95% control limit).
The test substance is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Test substance is considered positive if, in any of the experimental conditions examined:
− At least one of the test substance concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)
When all of the criteria are met, the test substance is then considered able to induce chromosome breaks and/or gain or loss in this test system.

In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. narrow concentration spacing, other metabolic activation conditions, i.e. S9 concentration or S9 origin) could be useful.
In rare cases, even after further investigations, the data set maynot allow a conclusion of positive or negative response and will therefore be concluded as equivocal.

Statistics:

Statistical significance was confirmed by using the Chi-squared test (α < 0.05) using a validated R Script for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1: Summary of results

a) Experiment 1A Exposure period 4 h without S9

Preparation interval	Test Substance concentration	Proliferation index CBPI	Cytostasis (%) a	Micronucleated cells (%) b
40h	Solvent control	1.77		0.45
40h	MMC 1.5 µg/mL	1.33	56.8	13.75*
40h	213 µg/mL	1.76	1.0	0.70
40h	373 µg/mL	1.75	2.0	0.70
40h	652 µg/mL	1.72	6.0	0.65
40h	1141 µg/mL	1.78	nc	0.75

b) Experiment II Exposure period 20 h without S9

Preparation interval	Test Substance concentration	Proliferation index CBPI	Cytostasis (%) a	Micronucleated cells (%) b
40h	Solvent control	1.94		0.85
40h	Demecolcin 125 ng/mL	1.49	48.4	3.20*
40h	213 µg/mL	1.94	0.5	0.50
40h	373 µg/mL	1.90	4.5	0.40
40h	652 µg/mL	1.96	nc	0.30
40h	1141 µg/mL	1.95	nc	0.55

c) Experiment 1B Exposure period 4 h with S9

Preparation interval	Test Substance concentration	Proliferation index CBPI	Cytostasis (%) a	Micronucleated cells (%) b
40h	Solvent control	2.07		0.60
40h	CPA 17.5 µg/mL	1.82	23.2	5.30*
40h	213 µg/mL	2.02	5.1	0.45
40h	373 µg/mL	2.00	6.3	0.80
40h	652 µg/mL	2.01	5.9	1.05
40h	1141 µg/mL	2.00	6.7	0.85

a For the positive control groups and the test item treatment groups the values are related to the solvent controls

b The number of micronucleated cells was determined in a sample of 2000 binucleated cells

*The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

Solvent control Acetone 0.5% v/v

MMC Mitomycin C

CPA Cyclophosphamide

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Executive summary:

The test substance, dissolved in acetone, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments.

In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (1141 μg/mL of the test substance, approx. 10 mM) was chosen with regard to the molecular weight and the purity (98.3%) of the test substance and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In the absence and presence of S9 mix for all exposures, no cytotoxicity was observed up to the highest applied concentration (1141 μg/mL, approx. 10mM).

For all experiments, no relevant increase in the number of micronucleated cells was observed after treatment with the test substance.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June 2016 to 08 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

This in vitro test is an assay for the detection of forward gene mutations in mammalian cells. Gene mutations are discussed as an initial step in the carcinogenic process.
The V79 cells are exposed to the test item both with and without exogenous metabolic activation. At a defined time interval after treatment the descendants of the treated original population are monitored for the loss of functional HPRT enzyme.
HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expression period is terminated by adding 6-TG to the culture medium. The mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. After a suitable period the colonies are counted. Mutant frequencies are calculated from the number of mutant colonies corrected for cell survival

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian cell gene mutation assay (HPRT)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a sufficiently high cloning efficiency of untreated cells (as a rule more than 50 %) are both necessary properties for the appropriate performance of the study, both attributes that are present in this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
- Thawed stock cultures are propagated at 37 °C in 75 cm^2 plastic flasks. About 2-3×10^6 cells are seeded into each flask with MEM (minimal essential medium, 15 mL) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells are sub-cultured once or twice weekly. The cell cultures are incubated at 37°C in a 1.5 % carbon dioxide atmosphere (98.5 % air).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium, 15 mL) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%).
- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Periodically checked for karyotype stability: [yes}
- Periodically 'cleansed' against high spontaneous background: [yes]

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

The maximum concentration of the pre-experiment and the main experiments (1141 μg/mL, equal to 10 mM) was chosen with respect to the OECD Guideline 476 (2015) regarding the purity of the test substance (98.3% w/w).
The main experiments were evaluated at the following concentrations:
Experiment I (with and without metabolic activation):
142.6; 285.3; 570.5; 855.8; and 1141.0 μg/mL
Experiment II (with metabolic activation):
142.6; 285.3; 570.5; 855.8; and 1141.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: According to its solubilisation properties and its compatibility with cell cultures

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2-3×10^6 cells are seeded into each flask with MEM (minimal essential medium, 15 mL)

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 h

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Three or four days after first sub-cultivation approximately 2.0×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of approximately 7 days five 75 cm² cell culture flasks were seeded with about 4 - 5×105 cells each in medium containing 6-TG (11 μg/mL). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
After 7 – 10 days the colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Colonies with more than 50 cells were counted. If in doubt the colony size was checked with a preparation microscope.

NUMBER OF CELLS EVALUATED: Approximately 500 single cells (duplicate cultures per concentration level)

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
d) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
e) there is no concentration-related increase when evaluated with an appropriate trend test,
f) all results are inside the distribution of the historical negative control data
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

A linear regression (least squares, using a validated Excel sheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
A t-test was performed to evaluate an isolated increase of the mutation frequency exceeding the 95% confidence interval in the first experiment with metabolic activation (culture I, 1141.0 μg/mL). Again, a t-test is judged as significant if the p-value (probability value) is below 0.05.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1: Summary of results Experiment I (without and with metabolic activation)

a) Culture 1

Treatment	Conc. (µg/mL)	S9 mix	Relative cloning efficiency (%)	Relative cell density (%)	Relative adjusted cloning efficiency (%)	Mutant colonies / 10^6cells	95% control limit
Solvent control	-	no	100.0	100.0	100.0	19.2	0.2-29.7
EMS	300.0	no	105.7	89.3	94.4	165.9	0.2-29.7
CA5110	71.3	no	100.3	94.7	95.0	#	#
CA5110	142.6	no	90.1	73.9	66.6	22.0	0.2-29.7
CA5110	285.3	no	108.6	89.7	97.4	10.0	0.2-29.7
CA5110	570.5	no	110.4	87.5	96.6	15.5	0.2-29.7
CA5110	855.8	no	84.1	76.1	64.0	13.0	0.2-29.7
CA5110	1141.0	no	16.0	61.2	9.8	4.9	0.2-29.7
Solvent control	-	yes	100.0	100.0	100.0	7.1	0.2-29.7
DMBA	2.3	yes	79.8	74.8	59.7	196.2	0.2-29.7
CA5110	71.3	yes	88.0	92.0	80.9	#	#
CA5110	142.6	yes	82.0	97.9	80.3	7.1	0.6-29.7
CA5110	285.3	yes	79.4	88.3	70.1	28.8	0.6-29.7
CA5110	570.5	yes	78.3	71.6	56.1	23.9	0.6-29.7
CA5110	855.8	yes	70.6	82.6	58.4	24.9	0.6-29.7
CA5110	1141.0	yes	65.0	62.9	40.9	57.7	0.6-29.7

# culture was not continued as a minimum of only four analysable concentrations is required by the test guideline

b) Culture 2

Treatment	Conc. (µg/mL)	S9 mix	Relative cloning efficiency (%)	Relative cell density (%)	Relative adjusted cloning efficiency (%)	Mutant colonies / 10^6cells	95% control limit
Solvent control	-	no	100.0	100.0	100.0	11.4	0.2-28.7
EMS	300.0	no	91.8	57.4	52.7	154.8	0.2-28.7
CA5110	71.3	no	112.9	59.0	66.6	#	#
CA5110	142.6	no	98.4	68.3	67.2	9.2	0.2-28.7
CA5110	285.3	no	105.6	55.2	58.3	9.6	0.2-28.7
CA5110	570.5	no	112.5	58.0	65.2	12.2	0.2-28.7
CA5110	855.8	no	89.2	71.3	63.6	12.1	0.2-28.7
CA5110	1141.0	no	30.2	46.1	13.9	12.1	0.2-28.7
Solvent control	-	yes	100.0	100.0	100.0	12.7	0.6-28.7
DMBA	2.3	yes	99.0	87.7	86.8	160.3	0.6-28.7
CA5110	71.3	yes	76.2	110.3	84.1	#	#
CA5110	142.6	yes	86.1	107.5	92.6	17.9	0.6-28.7
CA5110	285.3	yes	91.4	91.4	83.5	24.4	0.6-28.7
CA5110	570.5	yes	80.9	92.5	74.9	14.6	0.6-28.7
CA5110	855.8	yes	95.4	88.1	84.1	15.0	0.6-28.7
CA5110	1141.0	yes	86.3	77.6	66.9	20.7	0.6-28.7

Table 2: Summary of results Experiment II (with metabolic activation)

a) Culture 1

Treatment	Conc. (µg/mL)	S9 mix	Relative cloning efficiency (%)	Relative cell density (%)	Relative adjusted cloning efficiency (%)	Mutant colonies / 10^6cells	95% control limit
Solvent control	-	yes	100.0	100.0	100.0	11.4	0.6-28.7
DMBA	2.3	yes	78.7	87.9	69.2	151.8	0.6 -28.7
CA5110	71.3	yes	104.2	93.2	97.1	#	#
CA5110	142.6	yes	79.1	82.3	65.1	17.6	0.6-28.7
CA5110	285.3	yes	71.7	94.8	68.0	10.0	0.6-28.7
CA5110	570.5	yes	83.7	46.2	8.7	12.2	0.6-28.7
CA5110	855.8	yes	82.9	35.1	10.9	12.1	0.6-28.7
CA5110	1141.0	yes	30.2	10.3	5.9	12.1	0.6-28.7

# culture was not continued as a minimum of only four analysable concentrations is required by the test guideline

b) Culture 2

Treatment	Conc. (µg/mL)	S9 mix	Relative cloning efficiency (%)	Relative cell density (%)	Relative adjusted cloning efficiency (%)	Mutant colonies / 10^6cells	95% control limit
Solvent control		yes	100.0	100.0	100.0	19.7	0.6-28.7
DMBA	2.3	yes	89.4	89.4	80.0	132.0	0.6-28.7
CA5110	71.3	yes	94.8	62.6	59.3	#	#
CA5110	142.6	yes	91.7	78.1	71.6	10.7	0.6-28.7
CA5110	285.3	yes	108.1	56.8	61.4	10.1	0.6-28.7
CA5110	570.5	yes	98.6	60.6	59.8	13.9	0.6-28.7
CA5110	855.8	yes	75.4	46.7	35.2	14.4	0.6-28.7
CA5110	1141.0	yes	31.8	24.6	7.8	10.6	0.6-28.7

# culture was not continued as a minimum of only four analysable concentrations is required by the test guideline

Conclusions:

In conclusion it can be stated that under the experimental conditions reported, the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in the HPRT assay.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. Two parallel cultures were used throughout the assay.

The assay was performed in two independent experiments, using two parallel cultures each. Experiment I was performed with and without liver microsomal activation and a treatment period of 4 hours. Experiment II was performed with liver microsomal activation and a treatment time of 4 hours.

The maximum concentration of the pre-experiment and the main experiments (1141 μg/mL, equal to 10 mM) was chosen with respect to the OECD Guideline 476 (2015) regarding the purity of the test substance (98.3% w/w).

The main experiments were evaluated at the following concentrations:

Experiment I (with and without metabolic activation):

142.6; 285.3; 570.5; 855.8; and 1141.0 μg/mL

Experiment II (with metabolic activation):

142.6; 285.3; 570.5; 855.8; and 1141.0 μg/mL

Relevant cytotoxic effects, indicated by an adjusted relative cloning efficiency I below 50%, with both cultures, occurred at 1141.0 μg/mL in the first experiment without metabolic activation and at 855.8 μg/mL and above in the second experiment with metabolic activation.

At 1141.0 μg/mL in culture I of experiment I with metabolic activation the mutant frequency exceeded both, the historical range of solvent controls and the 95% control limit.

This increase was not reproduced in the parallel culture under identical conditions but a t-test comparing combined parallel cultures in Experiment I to the mean concurrent control values indicated a statistically significant increase in the mean mutation frequency.

Due to the non-reproducible but statistically significant increase seen in Culture I with metabolic activation and also the positive trend observed in the trend test it was not possible to conclude this experimental part of the study to be “clearly negative”. Therefore, a second experiment with metabolic activation was performed. No comparable increase occurred in the repeat experiment and consequently, the effect was judged as non-reproducible and biologically irrelevant.

No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration.

For all experiments, a linear regression analysis (least squares) was performed on individual cultures to assess any possible dose dependent increase in mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in culture I of experiment I with metabolic activation, but not in the parallel culture. This part of the study was repeated as experiment II with metabolic activation under identical experimental conditions. A significant trend occurred in the first culture with metabolic activation however, this was reciprocal, going down versus increasing concentrations and thus, biologically irrelevant.

In both experiment I and II, the number of mutants in the negative control treated cultures were within the historical control 95% control limit.

EMS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies confirming the correct functioning of the assay.

Based on these results the test substance did not induce gene mutations at the HPRT locus in V79 cells.

In conclusion it can be stated that under the experimental conditions reported, the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in the HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test substance produced negative results in an in vitro bacterial gene mutation test, an in vitro gene mutation assay in Chinese Hamster V79 cells, and an in vitro micronucleus test in human lymphocytes. As a result, and in accordance with Regulation (EC) No. 1272/2008, Annex I, Part 3, 3.5.2, 4-methylcyclohexanone is not considered to be classified for germ cell mutagenicity.