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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
7 January 2002 - 27 May 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted according to OECD guideline 473 and under GLP conditions. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of this endpoint study record.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Cyclobutanate
- Molecular formula (if other than submission substance): C14H20O2
- Molecular weight (if other than submission substance): 220.31
- Smiles notation (if other than submission substance): C12C(CC(C\3C1C\C=C3)C2)OC(=O)CCC
- InChl (if other than submission substance): 1/C14H20O2/c1-2-4-14(15)16-13-8-9-7-12(13)11-6-3-5-10(9)11/h3,5,9-13H,2,4,6-8H2,1H3
- Storage condition of test material: room temperature in the dark

Method

Target gene:
Not relevant
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagles Minimal Essential Media
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Male Sprague-Dawley rat liver S9 induced by Phenobarbitone/β-naphthoflavone
Test concentrations with justification for top dose:
Short Term Treatment Test - Experiment 1:
6(18)h without S9 mix: 0*, 4.35, 8.7*, 17.3*, 34.5*, 51.7, 68.9 µg/ml
6(18)h with S9 mix:: 0*, 68.9, 137.7*, 275.4*, 550.8*, 826.15, 1101.5 µg/ml

Continuous and Short Term Treatment Test - Experiment 2:
24-hours without S9 mix: 0*, 2.175, 4.35, 8.7*, 17.3*, 34.5*, 51.7 µg/ml
48-hours without S9 mix: 0*, 2.175, 4.35*, 8.7*, 17.3*, 34.5, 51.7 µg/ml
6(18)h with S9 mix:: 0*, 68.9, 137.7*, 275.4*, 550.8*, 661.0, 826.15 µg/ml

* Dose levels selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material soluble at 10 mM concentration
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With S9 mix: Cyclophosphamide (promutagen); without S9 mix: Mitomycin C (direct-acting mutagen)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration:
Experiment 1 (Short Term Treatment Test): 6-hour exposure, both with and without S9 mix.
Experiment 2 (Continuous and Short Term Treatment Test): 24-hour continuous exposure without S9 mix; 48-hour continuous exposure without S9 mix, and a repeat of the 6-hours exposure with S9 mix.

- Fixation time (start of exposure up to fixation or harvest of cells):
Experiment 1 (Short Term Treatment Test): 18 hours
Experiment 2 (Continuous and Short Term Treatment Test): 24 and 48 hours (continuous treatment), and 18 hours (repeat of short term treatment test with S9 mix).

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.1 μg/ml., last 2 hours of incubation.
STAIN (for cytogenetic assays): Giemsa solution

NUMBER OF REPLICATIONS: duplicate cultures for each treatment.

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads from each culture.

DETERMINATION OF CYTOTOXICITY
- Method: Cell Growth Inhibition Test: a preliminary toxicity test was performed on cell cultures using 24 and 48-hour continuous exposure times without S-9 mix, and a 6-hour exposure period both with and without S-9 mix, followed by an 18-hour recovery period. The dose range used was 8.7 to 2203 ug/ml. Precipitate observations were noted at the beginning and end of the treatment period. Growth inhibition was estimated by counting the number of cells at the end of the culture period on an electronic cell counter, and expressing the cell count as a percentage of the concurrent vehicle control value. Slides were also prepared to check for the presence, number and quality of cells in metaphase. Selected dose levels were scored for mitotic index.

OTHER EXAMINATIONS:
- Determination of polyploidy: Cells with 38 or more chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported.
- Determination of endoreplication: Endoreduplicated cells are recorded and are included in the polyploid cell total number. If there was a dose-related increase in endoreduplicated cells then they are reported separately.
Evaluation criteria:
Where increases in the frequency of cells with aberrations were seen, statistical comparisons were made with the vehicle. A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test or Chi-squared test if the control frequency of cells with aberrations was 5 or greater. If the study gives a positive response then a D20 value will be calculated, which is the presumed dose level of the test substance that is required to induce aberrations in 20% of metaphases.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Cell Growth Inhibition Test: in all cases Cyclobutanate showed evidence of cell toxicity. A precipitate of the test material was seen at and above 275.4 ug/ml. Metaphases were present at dose levels up to 34.5 ug/ml in the 6(18)-hour without-S9 exposure group, and at up to 550.8 ug/ml in the 6(18)-hour with-S9 exposure group. The maximum dose with metaphases present in the 24-hour and 48-hour continuous exposures was 34.5 ug/ml. Due to the toxic nature of Cyclobutanate, cultures were discarded at and above 137.7 ug/ml in the groups without metabolic activation and at and above 1101.5 ug/ml in the groups with metabolic activation. The dose selection for the main experiments was based on toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: The aberration levels in the negative and solvent controls were normal for the laboratory. The frequencies of breaks were within the normal historical control level for this laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In both Experiment I and II Cyclobutanate demonstrated similar toxicity to that observed in the Cell Growth Inhibition Test and the data demonstrates a steep toxicity curve.
Short Term Treatment Test - Experiment 1:
The cell count data show that more than 50% growth inhibition was achieved at 34.5 ug/ml in the absence of S9, and an approximate 50% growth inhibition was achieved at 550.8 ug/ml in the presence of S9. Therefore, it was considered that adequate toxicity had been achieved.
Continuous and Short Term Treatment Test - Experiment 2:
Cyclobutanate did not achieve an approximate 50% growth inhibition in the 24-hour exposure group but the maximum scorable dose level was 34.5 ug/ml and there were no scorable metaphases at 51.7 ug/ml. In the 48-hour exposure group an approximate 60% reduction in growth index was observed in the cell count data and again at 34.5 ug/ml, but there were no scorable metaphases. In the 6(18)-hour treatment, dose selection was as in Experiment 1. The inclusion of additional intermediate dose levels was used to attempt to achieve an approximate 50% inhibition ideal. A steep toxicity curve was observed, with 44% growth inhibition of the cell count data. It was considered that adequate toxicity had been achieved for all exposure groups in Experiment 2.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Cyclobutanate did not induce any statistically significant increases in the number of polyploid cells at any dose level in either treatment case.

Applicant's summary and conclusion

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

Cyclobutanate did not induce any toxicologically significant, dose-related increases in the frequency of cells with chromosome aberrations, either in the presence or absence of a liver enzyme metabolising system, or after various exposure times. Cyclobutanate was therefore considered to be non-clastogenic to CHL cells in vitro.
Executive summary:

This study was conducted according to a method which was designed to assess the potential chromosomal mutagenicity of Cyclobutanate on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line according to the requirements of OECD Guideline 473 and the updated Annex V EU B10 Method. Duplicate cultures of CHL cells were treated with Cyclobutanate at several dose levels, together with vehicle (DMSO) and positive controls. Five exposure groups were used: Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolising system; Experiment 2 included a 24-hour continuous exposure without metabolic activation, a 48-hour continuous exposure without metabolic activation and a repeat of the 6(18)-hours exposure with metabolic activation. The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 8.70 to 34.5 ug/ml for the 6(18)-hour exposure, without S9, 137.7 to 550.8 ug/ml for the with-S9 exposure, in both Experiment 1 and 2, and 4.35 to 34.5 ug/ml for the 24 and 48-hour treatments. The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. Cyclobutanate did not induce any toxicologically significant increases in the frequency of cells with aberrations in any of the exposure groups. Cyclobutanate was shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all exposure groups. Cyclobutanate was shown to be non-clastogenic to CHL cells in vitro.