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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Sep 2001 - 21 Feb 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Kanpoan 287 (Japan EPA), Eisei 127 (MHW) and Heisei 09/10/31 Kikyoku No. 2 (MITI)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Protectol PE
- Physical state: colorless liquid
- Analytical purity: 99.9%
- Lot/batch No.: 664287
- Stability under test conditions: The stability of the test substance in water over a period of 96 hours has been verified analytically by the data owner.
- Storage condition of test material: Room temperature, under nitrogen

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) supplemented with 10% FCS (fetal calf serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix. The S9 was prepared from 8-12 weeks old male Wistar Hanlbm rats induced by applications of 80 mg/kg b.w. Phenobarbital i.p. and ß-naphthoflavone p.o. each on three consecutive days. The livers were prepared 24 hours after the last treatment.
Test concentrations with justification for top dose:
Pre-test: 10.9, 21.9, 43.8, 87.5, 175, 350, 700 and 1400 µg/mL
Experiment I: 43.8, 87.5, 175, 350, 700 and 1400 µg/mL
Experiment II and III: 175, 350, 525, 700, 1050 and 1400 µg/mL
The highest applied concentration in the pre-test on toxicity was chosen with regard to the molecular weight of the test substance with respect to the OECD guideline.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without S9 mix: 4 and 18 hours; with S9 mix: 4 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hours

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

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: In each chamber, 1 x 10^4 to 6 x 10^4 cells were seeded (at least 2 chambers per dish and group). 100 metaphase plates per culture were scored for structural chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells was determined of each test group in a sample of 500 cells per culture (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).
Evaluation criteria:
A test substance is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of historical control data (0.0 - 4.0% aberrant cells exclusive gaps).
A test substance is classified as clastogenic if:
- the number of induced structural chromosome aberrations are not in the range of historical control data (0.0 - 4.0% aberrant cells exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test substance can be classified as aneugenic if:
- the number of induced numerical aberrations are not in the range of historical control data (0.0% - 8.5% polyploid cells)
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test substance are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
ambiguous
Remarks:
see remarks on results
Cytotoxicity / choice of top concentrations:
other: In the pre-test on toxicity cell numbers 24 hours after start of treatment were scored for cytotoxicity. Distinctly decreased cell numbers were scored with 700 µg/mL and above.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
No clear toxic effects were observed after 4 hours treatment up to the highest evaluated test substance concentration (1400 µg/mL).

Any other information on results incl. tables

Genotoxicity without metabolic activation:

A single statistically significant increase (p<0.05) of aberrant cells was observed in experiment I after 4 hours treatment with 1400 µg/mL. Although this increase of 4% aberrant cells, exclusive gaps, was statistically significant compared to the low response in the corresponding solvent control (1% aberrant cells, exclusive gaps), the value is within the historical control data range. Therefore, the statistical significance is regarded as being biologically irrelevant.

For tables of results on chromosomal analyses refer to attached background material.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative