17-acetoxy-1β,2β-methanopegna-4,6-diene-3,20-dione

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:

Mar to May 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

not specified

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:

17-acetoxy-1β,2β-methanopegna-4,6-diene-3,20-dione

EC Number:

427-820-5

EC Name:

17-acetoxy-1β,2β-methanopegna-4,6-diene-3,20-dione

Cas Number:

2701-50-0

Molecular formula:

C24H30O4

IUPAC Name:

(1S,2S,3R,5S,11R,12S,15R,16S)-15-acetyl-2,16-dimethyl-6-oxopentacyclo[9.7.0.0²,⁸.0³,⁵.0¹²,¹⁶]octadeca-7,9-dien-15-yl acetate

Test material form:

solid: bulk

Method

Target gene:

not specified

Species / strain

Species / strain / cell type:

lymphocytes: human primary cells

Details on mammalian cell type (if applicable):

- Type and identity of media: MCCoy's 5a medium
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver homogenate
Type and composition of metabolic activation system:
S9 (batch no. 110997), derived from male Wistar rats pretreated with phenobarbital and ß-naphthoflavone, protein content 26.8 mg/mi, was prepared by RCC-CCR (Cytotest Cell Research, Roßdorf, Germany). The components of the standard 89 mix were 12.2% (viv) 89, 4 mmol/l NADP, 5 mmol/l glucose-6-phosphate, 8 mmol/I MgCI2, 34.3 mmol/l KCI and 50 mmol/l sodium phosphate buffer, pH 7.4.

Test concentrations with justification for top dose:

Assay without S9 mix:
1st harvesting: 1, 2.5, 5, 7.5, 10, 25, 50, 75, 100, 125 µg/ml ; 2nd harvsting: 50, 75, 100, and 125 µg/ml
Assay with S9 mix:
5, 10, 25, 50, 75, 100 and 125 µg/ml (first harvesting time) and 75, 100 and 125 µg/ml (second harvesting time).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of ZK 5690 was tested in DMSO, yielding a solubility of > 12.5 mg/ml. But when this stock solution was added at 1% to tissue culture medium containing 15% (vIv) FCS precipitates of the test compound were visible starting at ca. 50 µg/ml.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: -S9 mix: triaziquone

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: One

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

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 h
- Exposure duration/duration of treatment: without S9 mix: 21h first harvesting, 44 h second harvesting; with S9 mix: 24 h first harvesting, 44h second harvesting


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. To arrest the cells in the metaphase, colcemide (final concentration 0.08 µg/mL) was added ca. 3.5 h before the cells were harvested.

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells were collected by centrifugation, exposed to 1 % (w/v) tri-sodiumcitrate-2-hydrate hypotonie solution (for swelling) and fixed in glacial acetic acid/methanol, 1 +3. Drops of concentrated cell suspension were placed on slides, which were allowed to air-dry before being stained with 10% (v/v) Giemsa and mounted with Eukitt. For assessment of cell cycle kinetics, the slides were stained using a modified fluorescence-plus-Giemsa technique (FPG-technique). The slides were stained for 15 minutes with Hoechst 33258 (final concentration 4.5 µg/mL, rinsed and covered with phosphate butter (pH 6.9) during irradiation with ultra-violet light (e.g. Heraeus Sterisol®, 30 watt). After 90 min incubation in 12 x SSC at 60°C the slides were stained with 7% (v/v) Giemsa for ca. 10min, then rinsed with water and air-dried. After drying, the slides were mounted with Eukitt. On the basis of a "BUdR-control-culture", which was harvested after 2 days incubation with BUdR, the success of the differential staining was demonstrated in each instance.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For the analysis of chromosomal aberrations, 100 cells per replicate culture were scored if possible, i.e. 200 cells per concentration level, except for the positive control in which only 100 cells were scored to prove the positive response. The number of chromosomes per metaphase was determined on the
television monitor. Only metaphases with 2 n = 46 chromosomes were included in the analysis.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Using the vernier scale at the microscope stage, the coordinates of all metaphase spreads with structural aberrations were recorded.
All metaphase spreads were examined for both chromatid and chromosome aberrations, notably achromatic lesions (gaps), breaks, acentric fragments, deletions and exchange figures, with the following exception: an achromatic lesion (AL) may show a non-staining region greater than the diameter of the chromatid if there is no dislocation of the apparently detached part.
The incidence of cells with numerical alterations in chromosome number (excl. aneuploidy) such as endoreduplication and polyploidy was also recorded.
Endoreduplicated cells (endopolyploidy) were classified as quasitetraploid metaphases
exhibiting chromosome pairing, whereas a polyploid cell is any metaphase containing multiple copies of the haploid number (n) of the chromosome complement, e.g. 3 n, 4 n etc.
All structural chromosome aberrations were assigned artificial lesion (or break) scores as folIows:
chromatid and chromosome (isochromatid) breaks, acentric fragments and deletions were scored as one lesion each
exchanges, ringsand dicentrics were designated 2 lesions each.
The clastogenic potential of the compound was evaluated by calculating the breakage rate and the percentage of aberrant cells excluding ALs. The aberration frequency (% cells showing breaks excluding cells with ALs) in untreated or solvent-treated human peripheral Iymphocyte cultures should be in the range of 0 - 3%. A break incidence of up to 3% is therefore classified as a negative response, especially if a dose-dependent increase does not exist.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI); other: cell cycle progression
- Any supplementary information relevant to cytotoxicity: For determination of the cell cycle progression, 100 metaphase spreads per culture, treated with BUdR and stained with the FPG-technique, were analyzed for the occurrence of first, second and third mitosis in order to demonstrate that the first harvesting time chosen corresponds to approximately one to one and a half cell cycles after start of treatment.

Evaluation criteria:

please refer to any other information on materials and methods incl. tables

Statistics:

The positive control group and the dose groups were compared to the appropriate negative control using Fisher's exact test, each at the level of significance α = 0.05.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human primary cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 75 µg/ml onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, ZK 5690 showed no biologically relevant clastogenic potential in the chromosomal aberration assay with cultured human Iymphocytes from the peripheral blood whether in the absence or in the presence of rat liver S9 mix when investigated up to a recommended maximum concentration of 125 IJg/ml at which precipitates of the test compound could be observed. Therefore, ZK 5690 has to be considered as non-mutagenic in the chromosomal aberration assay on cultured human peripheral blood Iymphocytes under the experimental conditions described.

Executive summary:

There was no increase in the percentage of aberrant cells in the cultures treated with ZK 5690 at final concentrations of 10, 75 and 125 µg/ml (first harvesting time) and 125 µg/ml (second harvesting time) as compared with the solvent control. Additionally, there was no relevant reduction of the mitotic index at any concentration scored for chromosomal aberrations but visible precipitates of the test compound occurred from 75 µg/ml onwards.
Since the results were obviously negative in this case a statistical analysis was not performed.
Cyclophosphamide, the positive control, proved to be clearly clastogenic (p < 0.05).

Number of polyploid (including endoreduplicated) cells per concentration level observed in the course of scoring 200 metaphases for structural chromosomal aberrations:
First harvesting time
1/solvent; 0/10, 0/75 and 0/125 polyploid cells/µg ZK 5690/ml
Second harvesting time
0/solvent; 0/125 polyploid cells/µg ZK 5690/ml
The observed polypoidy rates do not arouse any suspicion of an aneugenic potential of the test compound.

In conclusion, ZK 5690 showed no biologically relevant clastogenic potential in the chromosomal aberration assay with cultured human lymphocytes from the peripheral blood whether in the absence or in the presence of rat liver S9 mix when investigated up to a recommended maximum concentration of 125µg/ml at which precipitates of the test compound could be observed. Therefore, ZK 5690 has to be considered as non-mutagenic in the chromosomal aberration assay on cultured human peripheral blood lymphocytes under the experimental conditions described.