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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study, GLP compliant, conducted according to OECD Guideline 473, with several minor protocol deviations of no detrimental impact on the 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:
yes
Remarks:
Revised CA-Mg free salt solution; updated culture preparation information; only scored 50 metaphase plates due to high response from new lot of EMS; updated EMS concentration range; updated results evaluation criteria for aneugenic response.
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, Annex 4A, dated May 19, 2000
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): T002113
- Molecular weight (if other than submission substance): 400.7 g/mol
- Substance type: white solid
- Physical state: solid
- Analytical purity: 92.9%
- Lot/batch No.: RT002113 G3A231
- Expiration date of the lot/batch: 2001-12-01
- Storage condition of test material: Room temperature
- Other:
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: RT002113 G3A231
- Expiration date of the lot/batch: 2001-12-01
- Purity test date: not indicated
- Purity: 92.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

Method

Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
- Type and identity of media: Frozen in liquid nitrogen, propogated into Minimal Essential Medium suplemented with 10% fetal calf serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9, from male Wistar Hanlbm rats
Test concentrations with justification for top dose:
Pre-test concentrations: 0, 34, 68, 136, 272, 544, 1088, 2175 and 4350 µg/mL, with and without S9 activation.
The highest applied concentration in the pre-test on toxicity (4350 µg/ml = 10 mM of the active ingredient) was chosen with regard to the molecular weight (Mw: 400.7 g) and the purity (92.9 %) of the test item with respect to the current OECD Guideline 473.
Main experiment concentrations: 5, 10, 20, 30, 40 and 60 µg/mL without S9 (30, 40 and 60 µg/mL were scored for the metaphase analysis); 12.5, 25, 50, 75, 100 and 150 mg/mL with S9 (50, 75, 100 and 150 µg/mL were scored for the metaphase analysis).
Vehicle:
- Vehicle(s)/solvent(s) used: Deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
- Other: On the date of the experiment (immediately before treatment), the test item was dissolved in deionized water. The final concentration of deionised water in the culture medium was 10% (v/v).
Controlsopen allclose all
Negative controls:
yes
Remarks:
culure medium
Solvent controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation, dissolved in nutrient medium, 0.7 µg/mL
Negative controls:
yes
Remarks:
culure medium
Solvent controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation, dissolved in nutrient medium, 1000 µg/mL
Details on test system and conditions:
METHOD OF APPLICATION: in medium
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9) with 10 % FCS (v/v) containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium were added.
Concurrent negative, solvent and positive controls were performed. After 4 hours the cultures were whashed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium):15.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h

SPINDLE INHIBITOR (cytogenetic assays): 15.5 h after the start of the treatment colcemid was added (0.2 μg.mL culture medium) to the cultures. 2.5 hrs after the addition of colcemid, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37°C. After incubation in the hypotonic solution, the cells were fixed with a mixture of methanol and glacial acetic acid (3:1). Per experiment two slides per group were prepared. After preparation the cells were stained.
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control cultures without S9 mix (deviation: 50 metaphase plates scored due to the use of a new lot of EMS, with unusually high response of the positive substance). Only metaphases with characteristic chromosome numbers of 22 +/-1 were included in the analysis).

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index was determined in a sample of 1000 cells per culture of each test group in %.

OTHER EXAMINATIONS:
- Evaluation of the cultures was performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but non included in the calculation of the aberration rates.
- Determination of polyploidy: the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype)
- Determination of endoreplications: yes

OTHER:
Prior to treatment the cells were washed with Ca-Mg-free salt solution and treated for 5 minutes with trypsin. Additionally, two cultures per test substance and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The cell number of the treated groups was given in percentage compared to the respective solvent control.
Evaluation criteria:
A test item is classified as non-clastogenic if:
- The number of induced structural chromosome aberrations in all evaluated dose groups are in the range of the historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations are not in the range of the 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

A test item can be classified as aneugenic if:
- The number of induced numerical aberrations are not in the range of the historical control data (0.0 - 8.5 % polyploid cells)
Statistics:
Fisher's exact test (p<0.05) was used to determine statistical significance.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: In the pre-experiment, no relevant influence on the pH value or osmolarity was observed (solvent control 275 mOsm, pH 7.4 versus 297 mOsm and pH 7.1 at 4350 µg/ml).
- Evaporation from medium: no data
- Precipitation: In the pre-experiment, precipitation of the test item in culture medium was observed after 4 hrs treatment with 1088 µg/ml and above in the absence and the presence of 59 mix.

RANGE-FINDING/SCREENING STUDIES: In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 34 and 4350 µg/ml were applied. Clear toxic effects were observed after treatment with 68 µg/ml and above in the absence of 59 mix and with 136 µg/ml and above in the presence of 59 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: In both experiments, EMS (1000 µg/ml) and CPA (0.7 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In the absence of S9 mix the response of the positive control substance EMS was unusually high due to the use of a new lot. Therefore, in deviation to the standard conditions only 50 metaphase plates per positive control culture were evaluated in this experimental part.This modification was without relevance for the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Clear toxic effects indicated by reduced cell numbers about 50% of control were observerd after 4 h treatment with 60 µg/mL (46 % of control) in the absence of S9 mix and with 150 µg/mL (52 % of control) in the presence of S9 mix. Neither in the absence nor in the presence of S9 mix decreased mitotic indices were observed up to the highest test item concentrations.

Any other information on results incl. tables

In the main experiment, in the absence of S9 mix, no increase in the number of cells carrying structural chromosomal aberrations was observed.

The aberration rates of the cells after treatment with the test item (2.0-3.0% aberrant cells, excl gaps) were near the solvent control value (2.0%) and within the range of the historical control data (0.0-4.0% ).

However, in the presence of S9 mix, the aberration rates (3.0, 6.0, 9.5, 10.5% aberrant cells excl gaps, respectively) were dose dependent and significantly increased after treatment with 50, 75, 100 and 150 µg/mL as compared to the corresponding solvent control. In addition, the values of the three highest evaluated test item concentrations were biologically relevant increased compared to the historical control data. Also, the number of cells carrying exchanges was distinctly increased (2.5%, 3.5%, 8.5 and 5.0 % respectively) as compared to the solvent control (0.0%) give additional evidence for a clastogenic potential of the test item. These observations have been regarded as being biologically relevant.

In the main experiment, no biologically relevant increase in the number of polyploid metaphases was found after treatment with the test item (2.1 % - 4.6 %) as compared to the rates of the solvent control.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative without metabolic activation
positive with metabolic activation

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, T002113 is considered to be clastogenic in this chromosome aberration test in the presence of S9 mix.