Reaction mass of 1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one and 1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June, 2015 - 31 August, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(2014)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one and 1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one

EC Number:

944-482-9

Molecular formula:

C13H20O

IUPAC Name:

Reaction mass of 1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one and 1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected from healthy adult male volunteers by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Average Generation Time (AGT): 12.8 - 13.0 h
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight)

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix, 3hr exposure; 24 hr fixation: 5.4, 17, 52, 164 and 512 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 5.4, 17, 52, 164 and 512 µg/mL
First cytogenetic test:
With and without S9-mix, 3 h exposure time, 24 h fixation time: 5, 50, 75, 100, 125, 150, 175 and 200 µg/mL
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix, 3 h exposure, 24 h fixation time: 5, 100 and 125 µg/ mL
With S9-mix, 3 h exposure, 24 h fixation time: 5, 100 and 150 µg/ mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 50, 75, 100, 125 and 150 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 5, 10, 20, 30, 40, 50, 75 and 100 µg mL
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 75 and 100 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 10, 30 and 75 µg mL

Vehicle / solvent:

- Vehicle used: dimethyl sulfoxide
- Justification for choice of vehicle: A solubility test was performed to select the appropriate vehicle. The substance did not form a homogeneous suspension in culture medium. Consequently, GALBASCONE was dissolved in dimethyl sulfoxide of spectroscopic quality and dimethyl sulfoxide is accepted and approved by authorities and international guidelines. GALBASCONE concentrations were used within 2.5 hours after preparation.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Mitomycin C (in HBSS) was used at a final concentration of 0.5 and 0.75 μg/mL for a 3 h exposure period, 0.2 and 0.3 μg/mL for a 24 h exposure period and 0.1 and 0.15 μg/mL for a 48 h exposure period.

Remarks:

without S9

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide (in HBSS) was used at a final concentration of 10 μg/mL for a 3 h exposure period (24 h fixation time).

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

ENVIRONMENTAL CONDITIONS:
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 65 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 -37.4°C).

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 150 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.

A test substance is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Fisher’s exact test.
In case the Fisher’s exact test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 200 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 164 µg/mL and above in the absence and presence of S9, 3 hr treatment/24 hr fixation; at dose levels of 52 µg/mL and above in the absence of S9 for the continuous treatment of 24 hr and at dose levels of 17 µg/mL and above for the continuous treatment of 48 hr.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

Applicant's summary and conclusion

Conclusions:

A chromosome aberration study with the substance was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that the substance is not clastogenic in human lymphocytes.

Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles.

In the first cytogenetic assay, the substance was tested up to and including cytotoxic concentrations of 125 and 150 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9 -mix, respectively. In the second cytogenetic assay, the substance was tested up to and including the cytotoxic concentration of 100 μg/mL for a 24 h exposure time with a 24 h fixation time and up to and including the cytotoxic concentration of 75 μg/mL for a 48 h exposure time with a 48 h fixation time in the absence of S9 -mix.

Reliable positive and negative controls were included. The substance Galbascone did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. Although in the second cytogenetic assay at the highest dose of 100 μg/ml at the 24 h exposure time the number of chromosomal aberrations was statistically different compared to the control, the number of aberrations was within the historical data range (7 cells with aberrations per 300 metaphases compared with control limits (+ gaps) -1.79 – 3.51 per 100 metaphases and (- gaps) -1.51 – 2.84 per 100 metaphases) and therefore this increase was considered not biologically relevant. No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

Finally, it is concluded that the substance is not clastogenic in human lymphocytes.