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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:
Sep 1992 - Jan 1993
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
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
May 26, 1983
Deviations:
yes
Remarks:
deviation from current version: only 200 metaphases scored (instead of at least 300)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
67359-57-3
EC Number:
614-052-2
Cas Number:
67359-57-3
IUPAC Name:
67359-57-3
Test material form:
liquid
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and Lot/batch No.of test material: Bayer AG; batch-no.002949
- Expiration date of the lot/batch: July 21, 1992
- Purity test date: July 21, 1992

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The batch used was analytically examined prior to study initiation and was approved for use for the test period.
- Solubility and stability of the test substance in the solvent/vehicle: A stability test in the solvent did not reveal significant degradation of the active ingredient.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dilution in ethanol

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. Sheldon Wolff, Laboratory of Radiobiology and Environmental Health, University of California, San Francisco
- Suitability of cells: chromosome number verified on August 31, 1992
- Number of passages if applicable: not specified
- Methods for maintenance in cell culture if applicable: Cells were normally grown in 20 ml of medium in 75 cm² flasks. Incubation of the cells was always performed at 37°C in a CO2-incubator (air to CO2 ratio 95:5).
- Modal number of chromosomes: 21
- Normal (negative control) cell cycle time: 14 h

MEDIA USED
- Type and identity of media: Ham´s F 12 medium supplemented with 5 or 10% fetal calf serum, 1mM L-glutamine, Penicillin-Streptomycin-Solution (50 I.U./ml; 50 µg/ml)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: chromosome number verified on August 31, 1992
Cytokinesis block (if used):
Colcemid (40 µg/ml water)
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from Aroclor 1254 treated wistar rats
Test concentrations with justification for top dose:
10, 40, 160 µg/ml (without S9)
7.2, 36, 180 µg/ml (with S9)
with harvest time 24h;
highest concentrations testet again with harvest times 8h and 30 h
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (stock solution containing 500 mg/ml in ethanol was diluted in culture medium (with 10% FCS) up to a concentration of 1mg/ml)
- Justification for choice of solvent/vehicle: solubility (tested in a pre-test)
Controls
Untreated negative controls:
yes
Remarks:
untreated control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: approx. 1x10^6 cells were seeded in 20 ml of medium per 75 cm² flasks

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 (40 µg/ml water)

STAIN (for cytogenetic assays): 5% Giemsa solution

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: The mitotic index was determined by counting 1000 cells per culture. Chromosomes of approx. 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were examined for structural changes.

DETERMINATION OF CYTOTOXICITY
- Method: determination of mitotic index (also determined in the main study) and cell survival in the presence and abscence of S9 mix (assessed in pre-tests)
Rationale for test conditions:
For the dose selection for the main study pre-tests using the following concentrations were performed: first pre-test: 1000, 750, 500, 250, 100, 50, 10 µg/ml (with and without S9 mix; 4 h exposure time; preparation time: 24 h after the beginning of the treatment, solvent DMSO); second pre-test: 250, 220, 190, 160, 130, 100 µg/ml. As indicators of cytotoxic effects, mitotic indices and numbers of surviving cells (survival index) were determined.
Based on these results, the highest doses for the main study were selected in order to be in a dose-range that induces a redcution of the mitotic index by about 50%.
Evaluation criteria:
A test was considered to be positive if a dose-dependent and statistically significant increase of aberration frequencies was observed which was outside the range of the historical solvent controls.

A test was considered to be negative if there was no evidence of increased aberration frequencies for any of the concentrations tested.

A test was considered equivocal if there was an increase which was statistically significant but not concentration-dependent, or if a concentration-dependent increase occurred which was not statistically significant.

Assay Acceptance Criteria:
An assay was acceptable if there was a biologically relevant increase in chromosome aberrations induced by the positive controls and if the numbers of aberrations for the negative controls were in the expected range based on results from the testing laboratory and from published studies.
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.
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:
at the highest test concentrations 160µg/ml, 180µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Surviving cells after treatment in the second pre-test

Concentration (µg/ml)

S9 Mix

Length of Treatment*

Harvest Time*

Survival Index1

250

220

190

160

130

100

without

4

4

4

4

4

4

24

24

24

24

24

24

0.3

3.0

22.8

58.5

99.4

94.2

250

220

190

160

130

100

with

4

4

4

4

4

4

24

24

24

24

24

24

0.2

10.7

52.0

76.4

95.3

88.2

*: in hours

1: relative to solvent control cells [in%]

Table 2: Mitotic indices in the second pre-test

Concentration (µg/ml)

S9 Mix

Length of Treatment*

Harvest Time*

Mitotic Index1

250

220

190

160

130

100

without

4

4

4

4

4

4

24

24

24

24

24

24

0

0

5.1

59.5

75.9

75.9

250

220

190

160

130

100

with

4

4

4

4

4

4

24

24

24

24

24

24

0

0

53.5

97.2

109.9

70.4

*: in hours

1:relative to solvent control cells [in%]

Applicant's summary and conclusion

Conclusions:
The test substance is not considered to be clastogenic for mammalian cells with and without metabolic activation in vitro.
Executive summary:

The clastogenic potential of the test substance was evaluated in a chromosome aberration test in vitro. Chinese hamster ovary (CHO) cells were exposed for 4 hours to concentrations of 10, 40 and 160 µg/ml medium without S9 mix and to the concentrations of 7.2, 36 and 180 µg/ml with S9 mix. Cells were harvested 8, 24 and 30 hours after the beginning of the treatment.

The test substance 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 solvent controls.

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. 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 is therefore considered to be caused by the unusually low number of cells with aberrations in the corresponding solvent control group. 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 testsystem.

Based on this test, the test substance is not considered to be clastogenic for mammalian cells with and without metabolic activation in vitro.