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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to guideline, well conducted (GLP) and documented

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)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Chemical name: mixture of N,N-dimethylcapramide (N,N-Dimethydecan-1-amide) and
N,N-dimethylcaprylamide (N,N-Dimethyloctan-1-amide)
- Physical state: liquid
- Storage condition of test material: roomtemperature

Method

Target gene:
in vitro cytogenetic test
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from Aroclor 1254 treated wistar rats
Test concentrations with justification for top dose:
0, 10, 40, 160 µg/ml (without S9)
0, 7.2, 36, 180 µg/ml (with S9)
with harvest time 24h
highest concentration again additional with harvest time 8h and 30 h tested
Pretest with concentration up to 1000 µg/ml showed less survived cells in concentration of 160 µg/ml and above
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (stock solution containing 500mg/ml in ethanol was diluted in culture medium up to a concentration of 1mg/ml
- Justification for choice of solvent/vehicle: solubility
Controls
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
untreated control
Positive controls:
yes
Positive control substance:
other: mitomycin C and cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells): 8, 24h (main study), 30h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5% Giemsa solution

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 1 x 10e6 cells were seeded in 20ml medium (containing 0.2ml testitem in solvent, and 1mL S9 mix if metabolic activation)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell survival
Evaluation criteria:
The mitotic index was determined by counting 1000 cells per culture.
Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were
examined for structural changes.
classes of aberrations are characterized as follows:
Gaps
Break
Fragment
Deletion
Exchange
Multiple abberation
Statistics:
The statistical analysis of the results was performed by pair-wise comparison of the numbers of
metaphases with aberrations (including and excluding gaps) and of metaphases with exchanges among 200 cells for treatment
and solvent control groups.
The statistical analysis followed the recommendations outlined by Richardson et al. (1989)
Fisher exact test was used for the statistical evaluation.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
tested up to first cytotoxic concentration 160µg/ml 180µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: 2 pre studies - first pre study ranging from 10 to 1000µg/ml
- secound pre study ranging from 100 to 250 µg/ml

COMPARISON WITH HISTORICAL CONTROL DATA: historical control data were added to the study report.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Survival index determined, highest tested concentration reduces the index by a minimum of 40% (43.2 ; 160µg/ml without S9 and 66.7%; 180µg/ml with S9) harvest time of 8 h, only moderate to negligible effects (with no effects) were seen for harvest times of 24h and 30h
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Not considered to be clastogenic for mammalian cells with and without metabolic activation
Executive summary:

Chinese hamster ovary cells were treated with confidential substance name at the concentrations of 10, 40 and 160 µg/ml medium without S9 mix and at the concentrations of 7.2, 36 and 180 µg/ml with S9 mix. Confidential substance name induced cytotoxic effects in cells exposed to the highest doses with and without metabolic activation at the early harvest time of 8 hours after the beginning of the treatment. The mitotic indices were markedly reduced to 66.7 and 43.2 % relative to control cells.

With one exception, no statistically significant or biologically relevant increases of numbers of metaphases with aberrations were detected 8, 24 or 30 hours after the beginning of the four hour treatment with the test substance with and without S9 mix (Tables 2-5). A statistically significant increase of the numbers of cells with aberrations was calculated for cells exposed to the dose of 180 µg/ml with metabolic activation and the harvest time of 8 hours. However, the absolute number of cells with aberrations (3.5 %) was within the normal range as compared to the values for other solvent controls within this study and for other studies performed in this laboratory. This statistically significant increase was therefore considered to be caused by the unusually low number of cells with aberrations in the corresponding solvent control. This statistically significant result is therefore considered not to be biologically relevant.

The positive controls mitomycin C and cyclophosphamide induced clear clastogenic effects and demonstrated the sensitivity of the test system.