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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3043/V9Z

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM supplemented with 10 % FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for plating efficiency: yes
- Cell cycle duration: 13-14 h
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix extracted from Aroclor activated rat livers
Test concentrations with justification for top dose:
Pretest: 1; 5; 10; 50; 100; 500; 1000; 3000 µg/ml with and without S-9 mix
1. experiment: 900; 1800; 2700 µg/ml with and without S-9 mix
2. experiment: 1800; 2250; 2700 µg/ml without S-9 mix
Vehicle / solvent:
Due to the good solubility of the test substance in water, the aqueous culture medium (MEM) was selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S-9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S-9 mix
Details on test system and experimental conditions:
DETAILS OF 1. and 2. EXPERIMENT:
METHOD OF APPLICATION: in medium

DURATION
- Attachment period: 24-30 h
- Exposure duration: 4 h with or without S-9 mix
- Expression time (cells in growth medium): 14 h
- Fixation time (start of exposure up to fixation or harvest of cells): ~ 18 h
The 2nd harvest time of 28 hours generally conducted was omitted due to the clear clastogenic effect of the pretest.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid dissolved in PBS
STAIN (for cytogenetic assays): Giemsa and Titrisol pH 7.2

NUMBER OF REPLICATIONS: 2 per dose

NUMBER OF CELLS EVALUATED: 50-200 metaphase cells depending on the incidence of chromosomally damaged cells

CHROMOSOMAL ANALYSIS
- Method: Structural and numerical chromosomal aberrations

DETERMINATION OF CYTOTOXICITY
-Method: cell counts, morphology, mitotic index

OTHER EXAMINATIONS:
- Treatment conditions: pH, osmolality, solubility of test substance (microscopically)

TEST SUBSTANCE ANALYSIS
The stability of the test substance throughout the study period will be proven by reanalysis at a later date. The homogeneity of the test substance was guaranteed by mixing before preparation of the test substance formulations. The stability of the test substance in water was determined analytically.
Statistics:
- statistical evaluation: MUCHAN program system (BASF AG).
- comparison of dose groups with control data: Fisher's exact test; correction using one-sided Bonferri-Holm test

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
chelating properties of test substance may be causative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S-9 mix at 2700 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH remained unchanged during the experiments (pH 7.4 - 7.8)
- Effects of osmolality: Osmolality remained unchanged.
- Water solubility: test substance completely soluble
- Precipitation: not observed.

RANGE-FINDING/SCREENING STUDIES:
Only the highest dose of 3,000 µg/mL exhibited chromosomal aberrations with a high proportion and a weakly toxic effect (weak suppression of the mitotic activity, decrease in the cell count, reduced cell attachment). Thus, according to the findings of the pretests and in agreement with current guidelines, 2,700 µg/mL (both with and without S-9 mix) were selected as top doses. Higher doses were not tested to avoid concentrations which may lead to artefactual chromosome breakage.

Applicant's summary and conclusion