3,5,5-trimethylcyclohex-2-enone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The majority of in vitro genotoxicity studies revealed clearly negative results.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Restriction: only 4 Salmonella strains tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

4 strains tested instead of 5

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:

Aroclor 1254-induced rat or hamster liver S9

Test concentrations with justification for top dose:

TA 100 and TA 1535 : 0 - 10000 μg/plate
TA 98 and TA 1537: 0 - 3333 μg/plate

Vehicle / solvent:

water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

potassium chloride

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains TA 1535 and TA 100, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

potassium chloride

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

Strain TA 98, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

potassium chloride

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Strain TA 1537, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

potassium chloride

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains, with metabolic activation

Details on test system and experimental conditions:

- Number of replicates: 3 per dose level, repeated
- Solvent: H2O
- 3 investigations were performed:
1. without metabolic activation
2. with metabolic activation (Arochlor-1254 liver rats)
3. with metabolic activation (Arochlor-1254 liver hamster)
- Cells and test compound or solvent (water) were incubated for 20 minutes at 37 ºC in the presence of either S9 or buffer. After the addition of soft agar, the contents of each tube were poured onto minimal medium, and the plates were incubated at 37 °C for 48 hours.
- The analysis was performed twice, each in triplicates.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: -S9: TA100, 1535, 98 at concentrations >=1000 µg/plate; +S9 (rat and hamster): only TA1535 at 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance was not mutagenic in strains TA100, TA1535, TA1537, or TA98 of Salmonella Typhimurium in the presence or absence of Aroclor 1254-induced rat (r.) or hamster (h.) liver S9.
Differences were not significant.

Maximum number of revertants in the standard plate test:

TA100:
Vehicle Test substance (µg/plate)
-S9 +S9(r.) +S9(h.) -S9 +S9(r.) +S9(h.)
-----------------------------------------------------------
82±4.7 92±7.5 79±2.2 88±7.4(333) 90±2.2(100) 117±2.9(333)

TA1535:
Vehicle Test substance (µg/plate)
-S9 +S9(r.) +S9(h.) -S9 +S9(r.) +S9(h.)
---------------------------------------------------------
6±2.5 7±0.9 7±1.2 4±0.9(100) 9±2.8(333) 6±1.0(100)

TA1537:
Vehicle Test substance (µg/plate)
-S9 +S9(r.) +S9(h.) -S9 +S9(r.) +S9(h.)
----------------------------------------------------------
2±0.3 3±1.5 4±0.7 4±0.7(1000) 5±1.0(100) 6±2.0(1000)

TA98:
Vehicle Test substance (µg/plate)
-S9 +S9(r.) +S9(h.) -S9 +S9(r.) +S9(h.)
----------------------------------------------------------
10±2.0 11±2.9 17±1.0 12±2.3(100) 17±1.3(100) 15±0.9(333)

The analysis was performed twice, each in triplicate; because the results were similar, data from only one experiment are shown.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1242 and 1254 (2:1 mixture) induced rat liver microsomes

Test concentrations with justification for top dose:

Without activation
0.13; 0.18; 0.24; 0.32; 0.42, 0.56; 0.75; 1.0; 1.3 µL/mL

With activation
0.067; 0.089; 0.12; 0.16; 0.21; 0.28; 0.38; 0.50; 0.67; 0.89 µL/mL

Concentration values are calculated: original data are given in µL/mL; a density factor of 0.92 g/cm3 is assumed.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 24 and 48 h

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

positive: positive dose response and >= 1 of 3 highest doses with a mutant frequency 2-fold greater than background.
equivocal: no dose response but any dose with a mutant frequency 2-fold greater than background.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

other: Clive and Spector, Mutat. Res. 44, 269-278 (1975) and Clive et al., Mutat. Res. 59, 61-108 (1979)

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Test concentrations with justification for top dose:

50 - 1600 µg/mL (details see Test Conditions)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

Experiments were performed twice, and all doses were tested in duplicate, except the solvent control (DMSO), which was tested in quintuplicate. Cells (6 x 10E+5/mL) were treated for 4 hours at 37 ºC in medium, washed, resuspended in medium, and incubated for 48 hours at 37 ºC. After expression, 3 x 10E+6 cells were plated in medium supplemented with trifluorothymidine for selection of cells that were mutant at the thymidine kinase (TK) locus, and 600 cells were plated in nonselective medium to determine the percentage of viable cells.
Trial 1: 0, 50, 100, 200, 400, 800, 1600 µg/mL
Trial 2: 0, 400, 600, 800, 1000, 1200 µg/mL
Trial 3: 0, 200, 400, 600, 800, 1000 µg/mL

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1600 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS
Without metabolic activation: mutagenic. The test substance was toxic to the cultures only at moderately to high concentrations.
Significant increases in mutant fraction occurred in all three experiments, accompanied by a reduction of relative total growth (RTG).

The lowest effective concentration was 400 µg/mL in the first experiment, where there was apparently no reduction in RTG from the vehicle control level.
In the second experiment, only at 600 µg/mL there was evidence of toxicity.
The lowest effective concentration in this experiment was 800 µg/mL. However, the cloning efficiency was low in this experiment (results questionable). In the third experiment the lowest effective concentration was 800 µg/mL.

Tennant et al. (1997) judged the test substance as positive at concentrations >= 400 µg/mL without metabolic activation.

CYTOTOXICITY
Relative total growth reduced at 800, 1000 and 1200 µg/mL.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

other: Galloway, S.M. et al., Environ Mol. Mutagen. 10 (10), 1-175, with a few modifications

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

without metabolic activation
50-1600 µg/mL

with metabolic activation
250-1500 µg/mL

Vehicle / solvent:

serum-free culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Analysis: 100 or 200 cells were scored for each dose (cells with chromosome number lower than 19 or higher than 23 were excluded).
In the absence of S9, CHO cells were incubated with test compound or solvent for 8-10 hours at 37 ºC. Cells were then washed, and fresh medium containing colcemid (0.1 µg/mL) was added. After a further 2-3 hours of incubation, cells were harvested by mitotic shake-off, fixed, and stained in 6% Giemsa. In the presence of S9, cells were incubated with test compound or solvent for 2 hours at 37 ºC. Cells were then washed, medium was added, and incubation continued for 8-10 hours. Colcemid (0.1 µg/mL) was added for the last 2-3 hours of incubation; then cells were harvested and fixed as above.

2 independent experiments:
without S9 mix
50, 160, 500, 1600 µg/mL
250, 500, 1000, 1600 µg/mL
with S9 mix
250, 500, 1000 µg/mL
750, 1000, 1250, 1500 µg/mL

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 5000 µg/mL

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not specified

True negative controls validity:

not applicable

Positive controls validity:

not specified

Additional information on results:

The test substance did not induce a significant increase of structural chromosomal aberrations in the presence or absence of S9 in Chinese hamster ovary cells.
Maximum number of cells with structural chromosomal aberrations (Abs) per 100 cells at given concentration of test substance:
Abs (µg/mL)
-S9 +S9
--------------------
2 (vehicle) 0 (vehicle)
5 (250) 2 (1500)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Negative results were seen in in vivo micronucleus tests and chromosomal abberration tests in mice.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, New York
- Age at study initiation: 6-8 weeks

Route of administration:

intraperitoneal

Vehicle:

corn oil

Details on exposure:

Single intraperitoneal application of 496.8 mg/kg of the test substance as mixture with vehicle in 10 mL/kg bw.

Duration of treatment / exposure:

12, 24, 48 hours

Frequency of treatment:

single

Post exposure period:

none

Dose / conc.:

496.8 mg/kg bw/day (nominal)

Remarks:

= Maximum Tolerated Dose/LD20

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Doses / concentrations: 0.25 mg/kg

Tissues and cell types examined:

erythrocytes from femur bone marrow

Details of tissue and slide preparation:

Sampling times: 12, 24, 48 h post dosing.
Criteria for selection of Maximum Tolerated Dose (M.T.D.): LD20 (determined in a preliminary toxicity study)

Statistics:

1-way analysis of variance and Duncan's multiple range test (P <= 0.05)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Remarks:

Maximum Tolerated Dose (LD20) was applied

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

Several tests with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without S9 were negative (Mortelmans et al., 1986; Huntingdon, 1978; Huels AG, 1988).

The mouse lymphoma tests were primarily negative. In the presence of metabolic activation one positive result was obtained in one of the two testing facilities leading to an inconclusive overall result (Honma et al., 1999). Without metabolic activation some studies gave negative results (Microbiological Associates, 1984; Honma et al., 1999a), others positive results (McGregor et al., 1988). Positive results were found only at reduced RTG (relative total growth) values (McGregor et al., 1988). In one study, the incubation time was increased to 24 h to render the test system especially sensitive for the detection of clastogens and spindle poisons. In this study, the test substance was positive in one of two trials (Honma et al., 1999b).

In a cytogenetic assay with Chinese Hamster Ovary (CHO) cells, no significant increase in chromosomal aberrations was observed (Gulati et al., 1989). A further chromosomal aberration assay performed on Chinese hamster lung (CHL) cells was positive with metabolic activation only after a modified treatment of the cells (cells were treated for 6 h and then cultured in fresh medium for another 18 h) (Matsuoka et al., 1996). Increased chromosome aberrations without metabolic activation were only observed at cytotoxic concentrations (Matsuoka et al., 1996).

Gulati et al. (1989) reported a significant increase in SCE frequency at concentrations of 500 – 1000 mg/L induced only in the absence of S9 mix (no increase in the presence of Aroclor 1254-induced rat liver S9 mix). As these high test substance concentrations were cytostatic, increased SCE frequencies could only be detected after delayed harvest.

The test substance was tested for the induction of unscheduled DNA synthesis (UDS) in rat primary hepatocytes. Concentrations ranged from 0.005 - 0.4 μL/mL, the highest concentration being toxic. No increase in the mean nuclear grain count (as compared to controls) or in the incidence of cells undergoing repair was detected at any dose level (Microbiological Associates, 1984). However, another UDS test showed positive results (Selden et al., 1994).

Furthermore, in a transformation assay the test substance was evaluated as active (Matthews et al., 1993). Two umu assays were negative with and (weakly) positive without metabolic activation (Ono et al., 1991; Yasunaga et al., 2004). Positive results were also seen in a bacillus subtilis recombination assay (Matsui et al., 1989).

 

In vivo studies

In a mouse micronucleus assay, 496.8 mg/kg (= LD20 = MTD) test substance was administered i.p. to 5 male and 5 female CD-1 mice per group. Sampling time was 12, 24, 48 hrs post dosing. Significant increases in the number of micronucleated polychromatic erythrocytes (PCE) were not observed (Microbiological Associates, 1984). Likewise, in a study with CFLP mice (5 animals/dose/sex; gavage doses of 450, 900, 1800 mg/kg bw given in 2 equal parts (i.e. half of total dose) separated by an interval of 24 hrs) a negative result was obtained 6 hours after the last dosage of the test substance (Huntingdon, 1978). The test substance was also negative a bone marrow chromosomal abberrration test in mice (NTP, 1990).

In a DNA binding study, male and female F344 rats and B6C3F1 mice were administered 500 mg/kg [1,3,5-14C]-isophorone. Livers and kidneys (target organs in the NTP carcinogenicity study) were removed after 24 hrs. There was no binding of the test substance or its metabolites to DNA of these organs and it was concluded, that it is not likely that the tumors found in male F344 rats and B6C3F1 mice following long-term oral administration (Bucher et al., 1986) were caused by genotoxic effects (Thier et al., 1990).

In a Sex Linked Recessive Lethal (SLRL) test with Drosophila melanogaster, fruit flies were fed a diet containing 2000 ppm test substance. After 72 hrs of exposure, surviving males were mated. As feeding exposure was found to be non-mutagenic, 2 - 3 day old males were then injected with a 0.7 % NaCl solution containing 12,500 ppm test substance. 24 hrs post injection, males were mated. Again there was no indication of a mutagenic effect (Foureman et al., 1994).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Some of the in vitro tests revealed inconclusive or positive results. However, all in vivo tests were clearly negative. Therefore, the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the 14th time in Regulation (EU) 2020/217.