reaction mass of 3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 1-(2,6,6-trimethylcyclohex-2-en-1-yl)pent-1-en-3-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From July 30 to November 07, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted according to OECD test Guideline No. 476 without any deviation. The study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across. The supporting substance is considered adequate for read-across purpose (see Iuclid section 13 for additional justification).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(E)-1-(2,6,6-trimethyl-1,3-cyclohexadien-1-yl)-2-buten-1-one

EC Number:

245-844-2

EC Name:

(E)-1-(2,6,6-trimethyl-1,3-cyclohexadien-1-yl)-2-buten-1-one

Cas Number:

23726-93-4

IUPAC Name:

1-(2,6,6-trimethylcyclohexa-1,3-dien-1-yl)but-2-en-1-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): (E)-1-(2,6,6-TRIMETHYL-1,3-CYCLOHEXADIEN-1-YL)-2-BUTEN-1-ONE
- Physical state: Pale yellow liquid
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cells: CHO-K1 cells were obtained from ECACC, Salisbury, Wiltshire.
- Type and identity of media: The stocks of cells were stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal bovine serum (FBS) and antibiotics (Penicillin/Streptomycin at 100 units/100 μg per mL) at 37 °C with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, the cells were supplied mycoplasma free.
- Periodically checked for karyotype stability: no, assumed to be stable
- Periodically "cleansed" against high spontaneous background: yes. Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM), Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (2% final S9 concentration)

Test concentrations with justification for top dose:

Preliminary toxicity test: 7.43, 14.87, 29.73, 59.46, 118.93, 237.85, 475.7, 951.4 and 1902.8 μg/mL (up to 10 mM). The 24-hr was repeated : 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 16 μg/mL.
Main test 1 (3hr -S9 mix): 1.75, 3.5, 7, 10.5, 14, 21, 28 µg/mL.
Main test 1 (3hr+S9 mix): 1.88 3.75, 7.5, 15, 20, 25, 30 µg/mL.
Main test 2 (24hr -S9 mix): 0.63, 1.25, 2.5, 5, 7.5, 10, 12 µg/mL.
Main test 2 (3hr +S9 mix): 1.25, 2.5, 5, 10, 20, 25, 30, µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the substance was found to be soluble in dimethyl sulphoxide (DMSO).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

500 and 750 µg/mL (4 hr), 200 and 300 µg/mL (24hr)

Positive control substance:

ethylmethanesulphonate

Remarks:

in the absence of S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 and 1 µg/mL

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

in the presence of S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours 24 hours
- Expression time (cells in growth medium): 7 days, at 37°C, in a humidified atmosphere of 5% CO2 in air.

Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.


NUMBER OF REPLICATIONS: duplicate cultures for each concentration of the test compound, positive and negative controls.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (200 cells/plate)

OTHER: ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10-6 will not be used for the evaluation of a test item.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test item clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test item must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/mL) or twice the solubility limit of the test item in culture medium. Where a test item is excessively toxic, with a steep response curve, a concentration that is
at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes/flasks for mutant colony count and three flasks for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.

Evaluation criteria:

See "any information on materials and methods incl. tables"

Statistics:

SPSS program or a suitable alternative.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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: There was no significant change in pH when the test item was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm at the dose levels investigated
- Evaporation from medium: not expected
- Water solubility: not soluble in water
- Precipitation: No precipitate of the test item was seen at the end of the exposure period in either exposure group in the 2 main experiments.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A dose range of 7.43, 14.87, 29.73, 59.46, 118.93, 237.85, 475.7, 951.4 and 1902.8 μg/mL was used in the preliminary cytotoxicity test for all three exposure groups. The maximum dose level tested was the maximum recommended dose level, the 10 mM concentration. Due to excessive toxicity being demonstrated in the 24-hour exposure group, with only one surviving dose level, this exposure group was repeated with a dose range of 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 16 μg/mL.
A greasy/oily precipitate was seen at the end of the exposure period at and above 237.85 μg/mL in the 4-hour exposure in the absence of S9 and at and above 118.93 μg/mL in the 4-hour exposure in the presence of S9 and in the 24-hour exposure. Cloudy precipitate was also seen at 951.4 μg/mL in the 4-hour exposure in the absence of S9 and at and above 951.4 μg/mL in the presence of S9. A precipitate of the test item was observed at the end of exposure in the initial 24-hour experiment at and above 951.4 μg/mL. In the repeat of the 24-hour exposure group, no precipitate was observed at the end of the exposure period.
The results of the individual flask counts and their analysis are presented in Table 1 (see "attached background material). It can be seen that the test item was very toxic and there was a dose-related reduction in the cloning efficiency (CE) in all three exposure groups. The dose levels at and above 29.73 μg/mL and 59.46 μg/mL in the 4-hour exposure groups in the absence and presence of S9, respectively, had no viable cells remaining at the end of the exposure period. The 24-hour exposure group had only one surviving dose level and was therefore repeated with a revised dose range. The results of the repeat of the 24-hour exposure are presented in Table 2 (see "attached background material).
The selection of the maximum dose level for the main experiments was based on toxicity and was 28 μg/mL and 30 μg/mL for the 4-hour exposure groups in the absence and presence of S9, respectively, in Experiment 1. In Experiment 2 the maximum dose selected was 12 μg/mL for the 24-hour exposure and 30 μg/ml for the 4-hour exposure in the presence of S9 (2%).

COMPARISON WITH HISTORICAL CONTROL DATA:
It can be seen that the vehicle control values were all considered to be within an acceptable range, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test 1: The Day 0 and Day 7 cloning efficiencies for the 4-hour exposure with and without metabolic activation are presented in Table 3 and Table 4. The Day 0 toxicity in the absence and presence of S9 was similar to that seen in the Preliminary Toxicity Test with a dose related reduction in cloning efficiency. In the absence of S9 a reduction in the Day 0 cloning efficiency of 44% and 90% was achieved at 10.5 μg/mL and 14 μg/mL, respectively. There were no colonies surviving from the Day 0 cultures at 21 μg/mL and 28 μg/mL in the absence of S9 and these dose levels were not plated for mutant frequency or Day 7 viability. In the presence of S9 a dose related response was demonstrated and a reduction in the Day 0 cloning efficiency of 87% was achieved at 30 μg/mL. The 4-hour exposure group in the absence of S9 demonstrated a modest reduction in the Day 7 cloning efficiency of 28% at 14 μg/mL but no reduction was seen in the 4-hour exposure in the presence of S9.
The Day 0 and Day 7 vehicle control cloning efficiencies in the absence and presence of S9 did not achieve 70%, in all replicates, however, since they achieved at least 50% this was considered to be acceptable.
The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 3 and Table 4 (see "attached background document"). There were no increases in mutation frequency per survivor that exceeded the vehicle control value by 20 x 10-6 at any dose level in either exposure group.
- Test 2: The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation exposure groups are presented in Tables 5 and 6. It can be seen that, as in Experiment 1, there was a dose related reduction in cloning efficiency at Day 0 in both exposure groups. The 24-hour exposure group achieved ideal maximum toxicity at 7.5 μg/mL with a reduction in cloning efficiency of 87%. The dose levels of 10 μg/mL and 12 μg/mL in the absence of S9 were too toxic for plating for mutant frequency. In the 4-hour exposure in the presence of S9 a reduction in cloning efficiency at Day 0 of 88% was achieved at 25 μg/mL which was within the ideal upper range of 80 to 90% toxicity. The dose level of 30 μg/mL in the presence of S9 had no surviving cells at the end of the exposure period and was therefore not maintained to be plated for Day 7 viability.
The Day 0 vehicle control cloning efficiencies in the absence of S9, did not achieve 70% in both replicates, however, since they achieved at least 50% this was considered to be acceptable. The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 5 and Table 6. There were no increases in mutation frequency per survivor that exceeded the vehicle control value by 20 x 10-6 at any dose level in the absence or presence of S9.

Remarks on result:

other: strain/cell type: HPRT gene locus

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation, in both experiments.

Executive summary:

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, Chinese hamster ovary (CHO) cells were treated with the test item at up to eight dose levels, in duplicate, together with vehicle (dimethyl sulphoxide) and positive controls. Four treatment conditions were used for the test. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure in the presence of S9 was repeated (using a 2% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose range of the test item was selected based on the results of a preliminary cytotoxicity test and were as follows:

- 4-hour without S9: 1.75, 3.5, 7, 10.5, 14, 21, 28 µg/mL

- 4-hour with S9 (2%): 1.88, 3.75, 7.5, 15, 20, 25, 30 µg/mL

- 24-hour without S9: 0.63, 1.25, 2.5, 5, 7.5, 10, 12 µg/mL

- 4-hour with S9 (2%): 1.25, 2.5, 5, 10, 20, 25, 30 µg/mL

The vehicle (dimethyl sulphoxide) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Under the test conditions, the test material is not classified according to the annex VI of the Regulation EC No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

The supporting substance is considered adequate for read-across purpose (see Iuclid section 13 for additional justification).