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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:
experimental study
Adequacy of study:
key study
Study period:
2006-02-24 to 2006-05-31
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4A: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test", dated May 19, 2000.
Deviations:
no
GLP compliance:
no
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): T1310
- Physical state: white solid
- Analytical purity: 100 %
- Purity test date: no data
- Lot/batch No.: batch 00465047 RT001310G4A851
- Expiration date of the lot/batch: 2006-06-30
- Storage condition of test material: at room temperature
Specific details on test material used for the study:
Description: white solid
Batch number: 00465047 RT001310G4A851
Purity: 100 %
Stability in solvent: not indicated by sponsor
Storage conditions: Room temperature
Expiry date: 2003-06-30

Method

Species / strain
Species / strain:
lymphocytes: human
Details on mammalian cell lines (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
The highest applied concentration in this study (1660 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473.

- Experiment 1 (with and without S9): 10.8, 18.9, 33.0, 57.8, 101.1, 177.0, 309.7, 542.0, 948.6 and 1660.0 µg/mL (542.0, 948.6 and 1660.0 µg/mL were selected for metaphase analysis in the presence of metabolic activation)
- Experiment 1 in the absence of metabolic activation was repeated use to missing clastogenic effects in the positive control.
- Experiment 1 (without S9 - repeat): 101.1, 177.0, 309.7, 542.0, 948.6 and 1660.0 µg/mL (542.0, 948.6 and 1660.0 µg/mL were selected for metaphase analysis)

- Experiment 2 (without S9): 101.1, 177.0, 309.7, 542.0, 948.6 and 1660.0 µg/mL (542.0, 948.6 and 1660.0 µg/mL were selected for metaphase analysis)
Vehicle:
- Vehicle(s)/solvent(s) used: deionised water (the final concentration of deionised water in the culture medium was 10 % (v/v))
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its relative non-toxicity to the cell cultures.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and conditions:
METHOD OF APPLICATION: in medium
- In Experiment 1 (4-hour pulse treatment), about 80 hrs after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test substance. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and
positive controls were performed. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test item was discarded and the cells were resuspended in "saline G". The washing procedure was repeated once. After washing the cells were resuspended in complete culture medium and cultured until preparation.
- After washing the cells were resuspended in complete culture medium and cultured until preparation.
- In Experiment 2 (22-hour continuous treatment), about 80 hrs after seeding for each test group 2 blood cultures (10 mL each) are set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with complete medium (with 10 % FCS) containing the test substance without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.
- All cultures were incubated at 37 °C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

DURATION
- Exposure duration: Experiment I: 4 hours; Experiment II: 22 hours
- Expression time: Experiment I: 15 hours; Experiment II: 0 hours
- Fixation time: Experiments I and II: 22 hours

SPINDLE INHIBITOR:
- Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37° C for 20 minutes. After removal of the hypotonic solut ion by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slides.

STAIN:
- The cells used for evaluation of cytogenetic damage were stained with Giemsa or according to the Fluorescent plus Giemsa technique.

NUMBER OF REPLICATIONS: All cell cultures were set up in duplicate.

NUMBER OF CELLS EVALUATED:
- One hundred well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive controls in experiment 2, where only 50 metaphase plates were scored. Only metaphases with 46 +/- 1 centromere regions were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: At least one thousand cells per culture were counted for determination of mitotic index.

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.
- Determination of endoreplication: yes

OTHER:
- Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 µg/mL) to reassure the replication time of the cultured lymphocytes for each experiment.
Evaluation criteria:
- The test substance was classified as non-mutagenic if 1) the number of induced structural chromosome aberrations in all evaluated dose groups was in the range of the historical control data (0.0-4.0 % aberrant cells, exclusive gaps); and 2) no significant increase of the number of structural chromosome aberrations was observed.
- The test substance was classified as mutagenic if 1) the number of induced structural chromosome aberrations was not in the range of the historical control data (0.0-4.0 % aberrant cells, exclusive gaps); and 2) either a concentration related or a significant increase of the number of structural chromosome aberrations was observed.
- If the above mentioned criteria for the test substance were not clearly met, the classification with regard to the historical data and the biological relevance was discussed and/or a confirmatory experiment was performed.
- The test substance could be classified as aneugenic if the number of induced numerical aberrations was not in the range of the historical control data (0.0-1.5 % polyploid cells).
Statistics:
- Statistical significance was confirmed by means of the Fisher's exact test (p<0.05); however, both biological and statistical significance was considered together.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In Experiment 1, in the absence of S9 mix, at 309.7 µg/mL and above and in the presence of S9 mix, at 948.6 µg/mL and above, the pH was adjusted to physiological value using small amounts of 2 N NaOH (Exp.1: solvent control: pH 7.3 versus pH 7.0 at 1600 µg/mL). In addition, in Experiment 2, at 542.0 µg/mL and above, the pH was also adjusted before application by using 2 N NaOH.
- Effects of osmolality: No relevant increase in the osmolarity was observed (i.e. Exp. 1: solvent control: 278 mOsm versus 306 mOsm at 1600 µg/mL).
- Evaporation from medium: no data
- Water solubility: 320 g/L
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES:
- Since the cultures with S9 mix fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment 1 as described above in the materials and methods section.
- The experimental part without S9 mix was repeated with concentrations between 101.1 and 1660 µg/mL due to the missing clastogenicity in the positive control.

COMPARISON WITH HISTORICAL CONTROL DATA:
- In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test substance (0.0-2.0 % aberrant cells, exclusive gaps) were close to within the range of the solvent control values (1.0-1.5 % aberrant cells, exclusive gaps) and clearly within the range of the historical control data: 0.0-4.0 % aberrant cells, exclusive gaps

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In this study, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicted by clearly reduced mitotic indices could be observed up to the highest applied test substance concentration.

Any other information on results incl. tables

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test substance (0.0-0.4 %) as compared to the rates of the solvent controls (0.0-0.2 %).

Both of the positive controls showed distinct increases in cells with structural chromosome aberrations.

The proliferation index of the lymphocytes in solvent control cultures in the 22 hour preparation interval with and without S9 mix (4 hour treatment; 1.06 and 1.43, respectively), in the 22 hours preparation interval without S9 mix (continuous treatment; 1.67), was checked by analyzing the proportion of mitotic cells in the 1st, 2nd, and 3rd metaphase (M1, M1 +, M2 and M3) indicating that the lymphocytes divided about a 1.5 times within the early preparation interval. This was also proven by the occurrence of sufficient numbers of mitotic cells and by a clear clastogenicity observed after treatment with the positive control substances.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with and wihtout metabolic activation

The test substance was evaluated for induction of chromosome aberrations in human lymphocytes in the presence and absence of S9 metabolic activation. Under the conditions of the study, it was concluded that the test substance was negative for induction of chromosome aberrations in the presence and absence of metabolic activation.