4-(1,4-dioxa-spiro[4.5]dec-8-yl)-cyclohexanone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The reverse mutation assay in bacteria (Ames test) according to OECD TG 471 was negative (reference 7.6.1 -1).

The in vitro chromosomal aberration assay according to OECD TG 473 was negative (reference 7.6.1 -2).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 November 1997 - 30 March 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1983

Deviations:

yes

Remarks:

Deviations to current guideline version (2016): no pulse treatment without S9, reduced number of metaphases evaluated, historical control data: 95% confidence interval lacking

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human lymphocytes, blood samples were drawn from healthy volunteers
- Normal cell cycle time (negative control): average generation time 13.8 hours (experiment 1) and 14.1 hours (experiment 2)

For lymphocytes:
- Sex, age and number of blood donors: 31-year old male (experiment 1), 25-year old male (experiment 2)
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: non-pooled
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and composition of media: Ham's F10 without thymidine and hypoxanthine, supplemented with 20% (v/v) heatinactivated fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL / 50 µg/mL), sodium bicarbonate (1.2 g/L) and heparin (30 U/mL)
- CO2 concentration: 5%
- humidity level: 80 - 100%
- temperature: 37°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : rat liver, prepared from adult male Wistar rats
- method of preparation of S9 mix : rats were injected intraperitoneally with solution (20% w/v) of Aroclor 1254 (500 mg/kg bw) in corn oil, five days after injection rats were sacrificed and livers removed, livers were homogenised in sodium phosphate buffer containing Na2-EDTA, the homogenate was centrifuged at 9000 x g for 15 minutes and supernatant transferred and stored in liquid nitrogen
- concentration or volume of S9 mix and S9 in the final culture medium : S9 mix contained 1.63 mg MgCl2 x 6H2O, 2.46 mg KCl, 1.7 mg glucose-6-phosphate, 3.4 mg NADP and 4 µmol HEPES per mL, to 0.5 mL S9 mix 0.5 mL S9 fraction was added; 0.2 mL S9 mix were added to 5.3 mL exposition medium (concentration in medium was 1.8% (v/v))
- quality controls of S9: not specified

Test concentrations with justification for top dose:

Experiment 1:
- without S9 mix 24 hours treatment time, 24 hours fixation time: 100, 180, 333, 420, 560 and 750 µg/mL
- without S9 mix 48 hours treatment time, 48 hours fixation time: 180, 333, 420 and 560 µg/mL
- with S9 mix 3 hours treatment time, 24 hours fixation time: 100, 333 and 1000 µg/mL
- with S9 mix 3 hours treatment time, 48 hours fixation time: 1000 µg/mL

Experiment 2:
- without S9 mix 24 hours treatment time, 24 hours fixation time: 100, 333, 420, 560, 750 and 1000 µg/mL
- with S9 mix 3 hours treatment time, 24 hours fixation time: 100, 333 and 1000 µg/mL

Vehicle / solvent:

- Solvent: Dimethylsulfoxid
- Justification for choice of solvent/vehicle: not specified
- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: without S9 24 and 48 hours, with S9 3 hours
- Harvest time after the end of treatment (sampling/recovery times): without S9 24 and 48 hours, with S9 24 and 48 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: colchicine 0.5 µg/mL during last 3 hours of culture
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): pelleted cells were swollen by hypotonic treatment (KCl 0.56%) and fixed (3 changes) with methanol:acetic acid (3:1 v/v), fixed cells were dropped onto slides and immersed in 1:1 mixture of 96% (v/v) ethanol/ether, slides were allowed to dry and stained for 10 - 30 minutes with 5% (v/v) Giemsa, dried slides were cleared by dipping in xylene and embedded in MicroMount
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 100 metaphases/per culture, total 200 metaphases
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): only metaphases containing 46 chromosomes were analysed, for definitions of chromosome aberrations see Table 1
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- mitotic index (MI): number of metaphases per 1000 cells
- Any supplementary information relevant to cytotoxicity: metaphases were analysed of cultures with inhibition of the mitotic index of about 50% or greater whereas the mitotic index of the lowest analysed concentration was approximately the same as the mitotic index of the solvent control

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- all slides were randomly coded before scoring of chromosomal damage
- the number of cells with aberrations and the number of aberrations were calculated

Rationale for test conditions:

a pre-experiment on cytotoxicity was performed with 10, 33, 100, 333 and 1000 µg/mL with and without S9 mix

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Chi-squared test

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.64 at concentration 750 µg/mL, 7.51 in solvent control
- Data on osmolality: 414 mOsm/kg at concentration 750 µg/mL, 426 mOsm/kg in solvent control
- Possibility of evaporation from medium: not specified
- Water solubility: not specified
- Precipitation and time of the determination: at 1000 µg/mL

RANGE-FINDING/SCREENING STUDIES: a pre-experiment on cytotoxicity was performed with 10, 33, 100, 333 and 1000 µg/mL with and without S9 mix

STUDY RESULTS
- Concurrent vehicle negative and positive control data : the number of cells with chromosome aberrations in the solvent controls were within the laboratory historical control data range, the positive controls showed statistically significant increases in the frequency of aberrant cells

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis: no statistical significance in treatment groups, positive controls showed statistically significant increase

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: with S9 no cytotoxicity was observed, without S9 mix at least in the highest applied concentration cytotoxicity was observed
- Genotoxicity results: 100 metaphases per culture were scored for chromosomal damage (total 200), neither a statistically significant nor a biologically relevant increase in the number of cells with chromosome aberrations was observed

HISTORICAL CONTROL DATA
- Positive historical control data: not specified
- Negative (solvent/vehicle) historical control data: range (min-max) see Acceptability criteria

Conclusions:

Based on the results of this chromosome aberration assay, the test item is not considered to be clastogenic in the absence and presence of metabolic activation.

Executive summary:

The test item was assessed for its genotoxic potential as determined in the chromosomal aberration assay in cultured human lymphocytes in the presence and absence of metabolic activation (Aroclor 1254 induced rat liver S9).

In the absence of S9 mix the test item was tested up to 750 µg/mL for a 24 hour treatment time with a 24 hour fixation time and up to 420 µg/mL for a 48 hour treatment time with a 48 hour fixation time in the first experiment. In the second experiment the test item was tested up to 750 µg/mL for a 24 hour treatment time with a 24 hour fixation time. In the presence of S9 mix the test item was tested up to 1000 µg/mL for 3 hours treatment time with a 24 hour fixation time and for a 3 hour treatment time with a 48 hour fixation time inthe first experiment. In the second experiment the test item was tested up to 1000 µg/mL for a 3 hour treatment time with a 24 hour fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicatiing that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of metabolic activation in two independently repeated experiments. It is concluded, that the test item is not clastogenic in human lymphocytes under the experimental conditions of this study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 December 1996 - 04 February 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Proposal for replacement of Guidelines 471 and 472, revised draft document, OECD

Version / remarks:

1995

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from Aroclor 1254 induced male Wistar rats (single intraperitoneal injection, 500 mg/kg bw). 5-7 days after administration of Aroclor 1254, rats were sacrificed and livers collected. The liver homogenate was centrifuged at 9000 x g for 10 min and the supernatant was transferred and stored frozen in liquid nitrogen. The S9 batch was tested for its metabolic activity using reference mutagens. For details on S9 mix composition see Table 1. In the experiment parts including metabolic activation 0.5 mL S9 mix were added to 2 mL top agar.

Test concentrations with justification for top dose:

1st series with/without S9: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate
2nd series with/without S9: 50, 158, 500, 1580, 5000 µg/plate

Vehicle / solvent:

Solvent: Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

cumene hydroperoxide

other: Daunomycin, 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate s
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2-3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- revertant colonies were either scored automatically using a colony counter or manually

Evaluation criteria:

The assessment of test item induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test item concentration which shows the highest number of colonies. Criteria based on historical laboratory control data and statistical considerations are shown in Table 2.
A test material is defined as non-mutagenic in this assay if
- ‘no’ or ‘weak increases’ occur in the first and second series of the main experiment.
(‘Weak increases’ randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a ‘clear increase’, and the effects are reproduced at similar concentration levels in the same test system;
- ‘clear increases’ occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
In all further cases should a third test series with the bacterial strain in question be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Statistics:

not mandatory for this test

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not specified
- Precipitation: 5000 µg/plate

Conclusions:

UInder the conditions of this bacterial reverse mutation test, the test item was not mutagenic in Salmonella typhimurium strains in the absence and presence of metabolic activation.

Executive summary:

The test item was examined for mutagenic activity in two series of in vitro microbial assays employing Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, and TA 1537 as indicator organisms. The test material was tested directly and after metabolic activation by liver S9-Mix obtained from rats pretreated with Aroclor 1254. Precipitation of the test material on the agar plates was macroscopically visible at the highest concentration tested (5000 µg/plate). Toxicity to the bacteria was observed in the range between 500 and 5000 µg/plate, depending upon the experimental condition.

The validity of the mutation assay can be assessed by the results obtained for the negative and positive controls. The negative control mutant frequencies were all in the regular range. The strain specific positive control compounds in the absence of S9-Mix yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene and benzo[a]pyrene, which require metabolic activation, were strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9-Mix) was functioning.

In both series of experiments, each performed with and without the addition of rat liver S9-Mix as the external metabolizing system, the test item showed no increase in the number of revertants of any bacterial strain. Thus, it can be concluded that the test item was not mutagenic under the described experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation test (Ames)

The test item was examined for mutagenic activity in two series of in vitro microbial assays employing Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, and TA 1537 as indicator organisms. The test material was tested directly and after metabolic activation by liver S9-Mix obtained from rats pretreated with Aroclor 1254. Precipitation of the test material on the agar plates was macroscopically visible at the highest concentration tested (5000 µg/plate). Toxicity to the bacteria was observed in the range between 500 and 5000 µg/plate, depending upon the experimental condition.

The validity of the mutation assay can be assessed by the results obtained for the negative and positive controls. The negative control mutant frequencies were all in the regular range. The strain specific positive control compounds in the absence of S9-Mix yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene and benzo[a]pyrene, which require metabolic activation, were strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9-Mix) was functioning.

In both series of experiments, each performed with and without the addition of rat liver S9-Mix as the external metabolizing system, the test item showed no increase in the number of revertants of any bacterial strain. Thus, it can be concluded that the test item was not mutagenic under the described experimental conditions (reference 7.6.1 -1).

In vitro chromosomal aberration assay

The test item was assessed for its genotoxic potential as determined in the chromosomal aberration assay in cultured human lymphocytes in the presence and absence of metabolic activation (Aroclor 1254 induced rat liver S9).

In the absence of S9 mix the test item was tested up to 750 µg/mL for a 24 hour treatment time with a 24 hour fixation time and up to 420 µg/mL for a 48 hour treatment time with a 48 hour fixation time in the first experiment. In the second experiment the test item was tested up to 750 µg/mL for a 24 hour treatment time with a 24 hour fixation time. In the presence of S9 mix the test item was tested up to 1000 µg/mL for 3 hours treatment time with a 24 hour fixation time and for a 3 hour treatment time with a 48 hour fixation time inthe first experiment. In the second experiment the test item was tested up to 1000 µg/mL for a 3 hour treatment time with a 24 hour fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicatiing that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of metabolic activation in two independently repeated experiments. It is concluded, that the test item is not clastogenic in human lymphocytes under the experimental conditions of this study (reference 7.6.1 -2).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genotoxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.