Cyclopropanemethanol, 1-methyl-2-[(1,2,2-trimethylbicyclo[3.1.0]hex-3-yl)methyl]-

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:

From 13 October 1999 to 24 February 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Cyclopropanemethanol, 1-methyl-2-[[(1S,3R,5R)- 1,2,2-trimethylbicyclo[3.1.0]hex-3-yl]methyl]-, (1S,2S)-, rel-

Molecular formula:

C15H26O

IUPAC Name:

Cyclopropanemethanol, 1-methyl-2-[[(1S,3R,5R)- 1,2,2-trimethylbicyclo[3.1.0]hex-3-yl]methyl]-, (1S,2S)-, rel-

Constituent 2

Reference substance name:

Cyclopropanemethanol, 1-methyl-2-[[(1S,3R,5R)- 1,2,2-trimethylbicyclo[3.1.0]hex-3-yl]methyl]-, (1R,2R)-, rel-

Molecular formula:

C15H26O

IUPAC Name:

Cyclopropanemethanol, 1-methyl-2-[[(1S,3R,5R)- 1,2,2-trimethylbicyclo[3.1.0]hex-3-yl]methyl]-, (1R,2R)-, rel-

Test material form:

liquid

Specific details on test material used for the study:

Substance identity: Javanol
Appereance: Liquid

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation by RCC Cytotest Cell Research):

The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (BRL, CH-4414 Fuillinsdorf; weight approx. 220 - 320 g) which received daily applications of 80 mg/kg b.w. phenobarbital i.p. dissolved in deionised water (Desitin; D-22335 Hamburg) and B-naphthoflavone orally dissolved in corn oil (Aldrich, D- 89555 Steinheim) on three subsequent days. The livers were prepared 24 hours after the last treatment.

After decapitation of the anaesthetised animals, the livers of the animals were removed, washed in 150 mM KCI and homogenized. The homogenate, diluted 1+3 with KCl was centrifuged at 9000 g for 10 minutes (4° C). Aliquotes of the supernatant containing the microsomal fraction were frozen and stored in ampoules at -80° C. Small numbers of the ampoules were kept at -20°C for up to one week. The protein content was determined using the analysis kit of Bio-Rad Laboratories, D-80939 Miinchen: Bio-Rad protein assay, Catalogue No. 5000006.

The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml. The protein concentration was 30.3 mg/ml (Lot. No.: 240999) in the pre-test and experiment I 23.9 mg/ml (Lot. No.: 121199) in experiment II.


S9 Mix:

An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8mM MgCl,
33 mM KCl
5 mM_glucose-6-phosphate
4mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. (1).

Test concentrations with justification for top dose:

Range-Finder/Pre-test:
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. With respect to the molecular weight of Javanol, 2230 microgram/mL was applied as the top concentration for treatment of the cultures in the pre-test. Test item concentrations between 17.4 and 2230 microg/ml (with and without S9-mix) were chosen for the evaluation of cytotoxicity.

Using reduced cell numbers as an indicator for toxicity in the pre-test, strong toxic effects were observed after 4 and 24h treatment with 34.9 microgram/mL and above in the absence of S9 mix and with 69.7 microg/ml and above in the presence of S9 mix. Considering the toxicity data of the pre-test, 50 microgram/mL (without S9 mix) and 75 microgram/mL(with S9 mix) were chosen as top concentrations for 4h treatment in experiment 1. For the 24h continuous treatment, 40 microg/ml were chosen as top concentration in the absence of S9 mix.

Dose selection of experiment 2 was influenced by the results of experiment 1. In the presence of S9 mix in experiment 1 reduced cell numbers were observed after 4h treatment with 45 microgram/mL. Therefore, 60 microg/ml were chosen as top treatment concentration in experiment 2. In the absence of S9 mix 18h treatment with 10 microg/ml induced strong toxicity indicated by reduced miotic indices. Therefore, 15 microgram/mL were chosen as top treatment concentration for 28h continuous exposure. The applied concentrations in the cytogenetic experiments are presented in table 2 page 15.

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Treatment

Exposure period 4 hours
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 ul/ml S9 mix per ml culture medium were added. Concurrent negative, solvent and positive controls were performed. After 4 h the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
The "Saline G" solution was composed as follows (per litre):
NaCl 8000 mg KCl 400 mg Glucose 1100 mg Na,HP0x7H,0 290 mg KH,PO, 150 mg
pH was adjusted to 7.2
Exposure period 18 and 28 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium (10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.
All cultures were incubated at 37° C in a humidified atmosphere with 4.5 % CO, (95.5 % air).

Preparation of the Cultures
16 h and 26 h, respectively after the start of the treatment colcemid was added (0.2 ug/ml culture medium) to the cultures. 2 h later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).
Additionally, two cultures per test item and solvent control treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the test item is given as reduction of % cells as compared to the solvent control.

Analysis of Metaphase Cells
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" (9)) using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 + 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Data Recording
The data generated were recorded in the raw data file. The results are presented in tabular form, including experimental groups with the test item, negative, solvent and positive controls.

Evaluation criteria:

A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data ( 0.0 - 4.0% aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are not in the range of our historical control data ( 0.0 - 4.0% aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

In experiment I, in the absence of S9 mix, reduced mitotic indices were observed after 4 h treatment with 20 µg/ml (35 % of control) and after  18  h continuous  treatment  with 10 µg/ml (54 % of control).

In the presence of S9 mix after 4 h treatment with 45 µg/ml reduced mitotic indices (37% of control) and cell numbers (39 % of control) were observed.

In experiment II the mitotic indices were reduced after 28 h continuous treatment with  15 µg/ml (56 % of control) in the absence of S9 mix. In addition the cell numbers were reduced at this experimental point (41 % of control). In the presence of S9 mix reduced cell numbers were observed after 4 h treatment with 45 µg/ml (38 % of control).

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test item (exp.I:0.0 % - 2.0 %; exp. II: 0.5 % - 3.0 %) near to the range of the solvent control values (exp. I: 0.0 % - 1.5 %; exp. II: 1.5 % - 2.0 %) and within the range of our historical control data: 0.0% -4.0%. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (exp.I:1.8 % - 4.6 %; exp. II: 1.8 % - 5.1 %) as compared to the rates of the solvent controls (exp.I: 3.3 % - 4.9 %; exp. II: 2.7 % - 3.0 %).

In both experiments, EMS (600 and 1000 µg/ml, respectively) and CPA (0.71 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test item did not induce statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of S9. Javanol was therefore considered to be non-clastogenic in this chromosomal aberration test.

Executive summary:

In an in vitrochromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured V79 Chinese hamster cells were exposed to Javanol both the absence and the presence of a metabolic activation system (S9-mix) in two independent experiments. Two independent chromosome aberration assays were conducted. The substance was dissolved in DMSO prior to testing.Following study design was performed:

 

without S9-mix				with S9-mix
	exp. I		exp. 1I	exp. I	exp. 1I
Exposure period	4h	18 h	28 h	4h	4h
Recovery	14 h	-	-	14 h	24 h
Preparation interval	18 h	18 h	28 h	18 h	28 h

 

Doses applied were:

Without S9

Experiment 1: 4hr- 5, 10, 20, 30, 40, 50 µg/mL; 18hr- 2.5, 5, 10, 20, 30, 40 µg/ml

Experiment 2: 28hr- 1.4, 2.5, 5, 7.5, 10, 15 µg/mL

With S9

Experiment 1: 4hr- 7.5, 15, 30, 45, 60, 75 µg/mL

Experiment 2: 4hr- 3.8, 7.5, 15, 30, 45, 60 µg/mL

 

Toxic effects indicated by clearly reduced cell numbers and/or mitotic indices were observed in all experimental parts of the cytogenecity study. At the highest concentration which was evaluated for cytogenetic damage, clear toxicity was observed. In both independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls and they induced statistically significant increases in cells with structural chromosome aberrations indicating the validity of the test.

Based on these results, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells in vitro.

Therefore, Javanol is considered to be non-clastogenic in this chromosome aberration test.