Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 March 2003 - 2 July 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(1997)
Deviations:
yes
Remarks:
minor modifications cytogenetic assay as described by Evans, 1984
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Directive 2000/32/EC, Part B
Deviations:
yes
Remarks:
minor modifications cytogenetic assay as described by Evans, 1984
GLP compliance:
yes
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): XTJ 568
- Physical state: clear colourless liquid
- Analytical purity: 97.8%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: 8191-34
- Expiration date of the lot/batch: 01 January 2004
- Stability under test conditions: no data
- Storage condition of test material: at room temperature in the dark. Stable under storage conditions
- Other: specific gravity: > 0.94 g/cm3
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 568
- Physical state: clear colourless liquid
- Analytical purity: 97.8%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: 8191-34
- Expiration date of the lot/batch: 01 January 2004
- Stability under test conditions: no data
- Storage condition of test material: at room temperature in the dark. Stable under storage conditions
- Other: specific gravity: > 0.94 g/cm3

Method

Species / strain
Species / strain:
lymphocytes: human peripheral
Details on mammalian cell lines (if applicable):
- Type and identity of media: F10 complete culture medium consisted of Ham's F10 medium with thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heatinactivated (56°C; 30 min) foetal calf serium (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively), sodium bicarbonate (1.2 g/L) and 30 U/mL heparin
- Properly maintained: yes: all incubations were carried out in a humid atmosphere (80-100%) containing 5± 0.5% CO2 in air in the dark at 37 ± 1°C. The temperature, humidity and CO2- percentage were monitored throughout the experiment
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9-mix of liver rat induced with Aroclor-1254.
Test concentrations with justification for top dose:
Concentration range in the first main test (with metabolic activation): 333, 666 and 1000 µg/ml (3h exposure, 24h fixation)
Concentration range in the second main test (with metabolic activation): 100, 666 and 1250 µg/ml (3h exposure, 48h fixation)
Concentration range in the first main test (without metabolic activation): 333, 666 and 1000 µg/ml (3h exposure, 24h fixation)
Concentration range in the second main test (without metabolic activation): 100, 500 and 800 µg/ml (24h exposure, 24h fixation)
Concentration range in the second main test (without metabolic activation): 700, 850 and 875 µg/ml (48h exposure, 48h fixation)
Vehicle:
F10 medium buffered with 20 mM HEPES
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
F10 medium buffered with 20 mM HEPES
True negative controls:
no
Positive controls:
no
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Negative controls:
no
Solvent controls:
yes
Remarks:
F10 medium buffered with 20 mM HEPES
True negative controls:
no
Positive controls:
no
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and conditions:
Experiment 1:
With/without S9: 3h exposure and 24h fixation time.
Experiment 2:
With S9: 3h exposure and 48h fixation time.
Without S9: 24h and 48h exposure time, 24h and 48h fixation
time.

SPINDLE INHIBITOR (cytogenetic assays): colchicine

NUMBER OF REPLICATIONS: duplicates
NUMBER OF CELLS EVALUATED: at least 100 metaphase chromosome spreads per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells.
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
b) a statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P< 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of solvent control using Chi-square statistics:
X2=[(N-1) x (ad-bc)^2]/[(a+b) (c+d) (a+c) (b+d)]
b= total number of aberrant cells in control cultures
d = total number of non aberrant cells in control cultures
n0 = total number of cells scored in the control cultures
a = total number of aberrant cells in treated cultures to be compared with the control
c = total number of non aberrant cells in treated cultures to be compared with the control
n1 = the total number of cells scored in the treated cultures
N = sum of n0 and n1

If P [X2> [(N-1) x (ad-bc)^2]/[(a+b) (c+d) (a+c) (b+d)] (two-tailed) is small (P<0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.

Results and discussion

Test results
Species / strain:
lymphocytes: human peripheral
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Observations: In the first cytogenetic assay the test substance increased
the number of polyploid cells both in the absence and
presence of S9-mix. This may indicate that the test
substance has the potential to disturb mitotic processes and
cell cycle progression, thereby inducing numerical
chromosome aberrations.

The pH and osmolarity of a concentration of 100 µg/mL were 7.73 and 291 mOsm/kg respectively (compared to 7.51 and 290 mOsm/kg in the solvent control). The pH and osmolarity of this concentration was not measured in the first cytogenetic assay.

RANGE-FINDING/SCREENING STUDIES: The pH and osmolarity of a concentration of 5000 µg/mL were 10.01 and 305 mOsm/kg respectively (compared to 7.42 and 284 mOsm/kg in the solvent control). In the dose range finding test blood cultures were treated with 100, 333, 1000, 3330 and 5000 µg XTJ 568/mL culture medium with and without S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control culture were within the laboratory historical control data range {min=0, max=6 (mean=1.1, standard deviation: 1.1) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min=0, max=5 (mean=0.9, standard deviation=1.0) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded; for n=660 and 437 respectively}.

Applicant's summary and conclusion

Conclusions:
The substance is not clastogenic in human lymphocytes under the experimental conditions tested. In the first cytogenetic assay, the substance increased the number of polyploid cells (with and without S9) indicating a potential to disturb mitotic processes and cell cycle progression, thereby inducing numerical chromosome aberrations.