(3E)-1,7,7-trimethyl-3-(4-methylbenzylidene)bicyclo[2.2.1]heptan-2-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 March 1986 to 25 April 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1982

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

(3E)-1,7,7-trimethyl-3-(4-methylbenzylidene)bicyclo[2.2.1]heptan-2-one

EC Number:

701-394-3

Cas Number:

1782069-81-1

Molecular formula:

C18H22O

IUPAC Name:

(3E)-1,7,7-trimethyl-3-(4-methylbenzylidene)bicyclo[2.2.1]heptan-2-one

Method

Target gene:

Chinese hamster cell line V79

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory stock
- Suitability of cells: The V79 cell line has been used for many years in in vitro experiments with success. The high proliferation rate and plating efficiency of untreated cells are necessary for the appropriate performance of the study.
- Cell cycle length, doubling time or proliferation index: Doubling time 12 to 16 hours in stock cultures.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37°C in plastic flasks. Seeding was done with 100000 to 300000 cells per flask in 5 mL of MEM-medium supplemented with 10% fetal calf serum. Cells were subcultured twice a week.
- Modal number of chromosomes: 22

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

The highest concentration should reduce the mitotic index to about 50 % as compared to the solvent control.
7 hour preparation time: 10 μg/mL (without S9) and 36 μg/mL (with S9)
18 hour preparation time: 1, 5 and 11 μg/mL (without S9) and 3, 15 and 36 μg/mL (with S9)
28 hour preparation time: 11 μg/mL (without S9) and 36 μg/mL (with S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (1%)
- Justification for choice of solvent/vehicle: Ethanol is relatively non-toxic in the concentrations applied.

Controls

Untreated negative controls:

yes

Remarks:

untreated cultures

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethyl methanesulfonate (without metabolic activation), cyclophosphamide (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding: Two days old logarithmically growing stock cultures more than 50% confluent were trypsinised and a single cell suspension was prepared. The cells were seeded into four parallel plastic tissue culture flasks at a concentration of 300000 cells per flask (7 hour interval) and 150000 cells per flask (18 and 28 hour intervals).

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 5, 15.5 and 25.5 hours after the start of treatment
- Fixation time: 2.0 (7 hour interval) or 2.5 (18 and 28 hour intervals) hours from addition of colcemid

STAIN: Aceto-orcein

NUMBER OF REPLICATIONS: 4

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were trypsinised and suspended in a hypotonic solution (37.5 mM KCl) for 10 minutes at 37°C. After centrifugation (800 to 1000 rpm) the cell pellets were fixed in three steps with glacial acetic acid/ methanol 1 + 3 and stored overnight at 4°C. The next day the cells were spread on wet and ice cold slides and stained.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100 metaphases/flask were scored per test concentration.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index
- Any supplementary information relevant to cytotoxicity: Mitotic index was determined in 1000 cells in each replicate.

Rationale for test conditions:

The test item concentrations were determined in a preliminary experiment using the mitotic index and plating efficiencies as an indicator for toxicity response.

Evaluation criteria:

Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural aberrations.
1. The test substance is classified as mutagenic if it induces with one of the concentrations tested a significantly increased aberration rate as compared with the negative control. The significance is obvious either by an enhancement of the rate clearly over the control range or it is proven by adequate biometry.
2. The test substance is classified as mutagenic if there is a concentration related increase in the aberration rates as compared with the solvent controls.
3. The test substance is considered negative when 1 and 2 do not apply.

Statistics:

No statistical analysis performed as there is no indication of a dose-response relationship.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: See Table 2.

The sensitivity of the test system was demonstrated by the clearly enhanced structural aberration rates found in the groups treated with the positive control substances.

In only one group of cells treated with the lowest concentration of the test item, a higher number of cells with structural chromosome aberrations as compared to the range of the negative controls was found in the presence of S9 mix at preparation interval 18 h. In all other groups treated with the test item, aberration rates within or near the control range were found. This single higher value alone is not regarded as being sufficient to prove mutagenicity.

Any other information on results incl. tables

Table 1. Mutagenicity data and mitotic index

Preparation time	Treatment group	Total cells analysed	Number of aberrant cells including gaps	% of aberrant cells including gaps	Number of aberrant cells excluding gaps	% of aberrant cells excluding gaps	% of aberrant cell exchanges	% mitotic index (absolute)	% mitotic index (relative)
18 hours	Untreated control -S9	400	9	2.25	5	1.25	0.25	6.8	100.0
18 hours	Untreated control +S9	400	24	6.00	7	1.75	0.00	6.0	100.0
18 hours	Solvent control -S9	400	17	4.25	4	1.00	0.00	4.8	100.0
18 hours	Solvent control +S9	400	14	3.50	9	2.25	0.25	5.4	100.0
18 hours	Positive control -S9	200	60	60.00	52	52.00	45.00	7.7	113.2
18 hours	Positive control +S9	200	69	69.00	65	65.00	39.00	4.3	71.6
18 hours	1 μg/mL test item -S9	400	23	5.75	10	2.50	0.00	4.6	95.6
18 hours	3 μg/mL test item +S9	400	37	9.25	19	4.75	0.00	4.5	83.6
18 hours	5 μg/mL test item -S9	400	19	4.75	5	1.25	0.25	5.6	115.6
18 hours	15 μg/mL test item +S9	400	13	3.25	4	1.00	0.50	2.9	54.2
18 hours	11 μg/mL test item -S9	400	17	4.25	11	2.75	0.50	3.2	65.8
18 hours	36 μg/mL test item +S9	400	21	5.25	5	1.25	0.00	7.5	139.8
7 hours	Solvent control -S9	400	16	4.00	4	1.00	0.00	5.9	100.0
7 hours	Solvent control +S9	400	10	2.50	1	0.25	0.00	5.9	100.0
7 hours	10 μg/mL test item -S9	400	14	3.50	8	2.00	0.00	2.8	47.7
7 hours	36 μg/mL test item +S9	400	28	7.00	13	3.25	0.00	5.2	86.9
28 hours	Solvent control -S9	400	17	4.25	6	1.50	0.25	4.4	100.0
28 hours	Solvent control +S9	400	10	2.50	2	0.50	0.00	1.9	43.1
28 hours	11 μg/mL test item -S9	400	11	2.75	6	1.50	0.5	5.7	100.0
28 hours	36 μg/mL test item +S9	400	25	6.25	10	2.50	0.00	5.7	99.1

Table 2. Preliminary study results

Treatment group	Number of seeded cells per flask	Mean number of found cells per flask	% PE (absolute)	% PE (relative)
Untreated control -S9	496	300.0	60.5	100.0
Untreated control +S9	498	435.0	87.3	100.0
Solvent control -S9	496	311.5	62.8	100.0
Solvent control +S9	498	387.0	77.7	100.0
10.0 μg/mL test item -S9	496	224.0	45.2	71.9
11.0 μg/mL test item -S9	496	155.5	31.4	49.9
12.0 μg/mL test item -S9	496	20.0	4.0	6.4
13.0 μg/mL test item -S9	496	17.0	3.4	5.5
14.0 μg/mL test item -S9	496	0.0	0.0	0.0
15.0 μg/mL test item -S9	496	0.0	0.0	0.0
15.0 μg/mL test item +S9	498	356.5	71.6	92.1
20.0 μg/mL test item +S9	498	377.5	75.8	97.5
25.0 μg/mL test item +S9	498	350.5	70.4	90.6
30.0 μg/mL test item +S9	498	342.0	68.7	88.4
35.0 μg/mL test item +S9	498	233.5	46.9	60.3
40.0 μg/mL test item +S9	498	2.0	0.4	0.5

Applicant's summary and conclusion

Conclusions:

The test item was not mutagenic to V79 Chinese hamster cell line in vitro in the presence and absence of metabolic activation.

Executive summary:

The potential of the test item to induce chromosomal aberrations was determined in vitro with cells of the V79 Chinese hamster cell line according to OECD Guideline 473. Chinese hamster lung fibroblasts V79 were treated with the test item at concentrations of 1, 5, 10 and 11 µg/mL without S9-activation and 3, 15 and 36 µg/mL with S9-activation. Cells were incubated with the test item for 4 hours, followed by a 5, 15.5 or 25.5 -hour incubation period in growth medium. Colcemid was added as a fixing agent and the cells were incubated for a further 2.0 or 2.5 hours. The study included negative, solvent and positive (ethyl methanesulfonate and cyclophosphamide) controls . Mutagenicity was evaluated by determining the number of gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations in 400 metaphases per test concentration. Cytotoxicity was evaluated by determination of mitotic index in 1000 cells in each replicate. The test item was not mutagenic to V79 Chinese hamster cell line in the presence and absence of metabolic activation. This study is considered reliable without restriction (Klimisch 1).