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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:
2006-11-06 to 2007-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) and EU Method A.3 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test) without deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Remarks:
The testing facility indicated that the protocol was followed without deviation.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Remarks:
The testing facility indicated that the protocol was followed without deviation.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate from "The Department of Health of the Government of the United Kingdom"
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: VRT-753136 hcl
- Molecular formula: Not applicable
- Molecular weight: Not applicable
- Smiles notation: Not applicable
- InChl: Not applicable
- Structural formula attached as image file: Not applicable
- Substance type: active
- Physical state: white powder
- Analytical purity: 100 area %
- Impurities: 0.03 area % of unknowns
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: 2006-05-25
- Lot/batch No.: batch 25515, lot AF6-001
- Expiration date of the lot/batch: 2008-05
- Stability under test conditions: no data
- Storage condition of test material: room temperature
- Other: no data

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
-Not applicable
Additional strain / cell type characteristics:
other: In the testing laboratory the cell cycle time for human lymphocytes in whole blood culture is approximately 13-15 hours.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-benzoflavone induced rat liver S9 (2 % v/v for experiment 1 and 5 % v/v for experiment 2)
Test concentrations with justification for top dose:
Experiment 1 and Experiment 2 with and without S9 activation- 14.55, 29.10, 58.20, 116.41, 232.81, 465.63, 931.25 and 1862.5 ug/mL (465.63, 931.25 and 1862.5 ug/mL were selected for metaphase analysis.)


Concentrations with high ionic strength and osmolality may cause chromosomal aberrations. Therefore, concentrations greater than 5000 ug/mL or 10 mM are not used in this test system. In this case, the highest final concentration used or subsequent testing was 1862.5 ug/mL (10 mM, based on a molecular weight of 186.25).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance in sterile water was assessed. The test substance was found to be soluble in sterile water at 50 mg/mL.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation

Migrated to IUCLID6: 0.2 ug/mL (3 hour exposure) and 0.1 ug/mL (continuous exposure)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation

Migrated to IUCLID6: 5 ug/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- In medium

DURATION
- Preincubation period: Not applicable
- Exposure duration: 3 hours (experiment 1 with and without metabolic activation and experiment 2 with metabolic activation) and 21 hours (experiment 2 without metabolic activation)
- Expression time: 16 hours (experiment 1 with and without metabolic activation and experiment 2 with metabolic activation), 0 hours (experiment 2 without metabolic activation)
- Selection time: Not applicable
- Fixation time: ~21 hours (with Colcemid present during the final 2 hours)


SELECTION AGENT :
not applicable
SPINDLE INHIBITOR:
Colcemid
STAIN:
10 % Giemsa


NUMBER OF REPLICATIONS:
2


NUMBER OF CELLS EVALUATED:
100 per replication


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: no data


OTHER:
Blood collected aseptically from two healthy male non-smoking donors was pooled and diluted with RPMI 1640 culture medium supplemented with 10 % foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin/20 ug/mL streptomycin and 2.0 mM L-glutamine. Aliquots (0.4 mL blood: 4.5 mL medium: 0.1 mL phytohaemagglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37 deg C for approximately 48 hours before treatment. The cultures were gently shaken daily to resuspend the cells.
Evaluation criteria:
The assay was considered acceptable if the vehicle and positive control values laid within the current historical control range.
The test substance was considered to cause a positive response if the following conditions were met:
1. Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) were observed at one or more test concentrations.
2. The increases exceeded the negative control range of the laboratory, taken at the 99 % confidence limit.
3. The increases were reproducible between replicate cultures.
4. The increases were not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
5. Evidence of a dose-relationship was considered to support the conclusion.
A negative response was claimed if no statistically significant increase in the number of aberrant cells above concurrent control frequencies was observed, at any dose level.
A further evaluation may have been carried out if the above criteria for a positive or a negative response were not met.
Statistics:
The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using the one-tailed Fisher exact test.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only in experiment 2 after continuous exposure in the absence of metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: The test substance was soluble in water at 50 mg/mL.
- Precipitation: no data
- Other confounding effects: no data


RANGE-FINDING/SCREENING STUDIES:
A range finding test was not performed.


COMPARISON WITH HISTORICAL CONTROL DATA:
All solvent control and positive control values for cells with aberrations excluding and including gaps in the presence and absence of metabolic activation were within historical control value ranges for both tests.
1. Experiment 1- In the absence of S9 mix, the test substance caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 465.63 ug/mL, including gap-type aberrations only, when compared with the solvent control (p<0.01). This increase was not dose-related and the significance of gap-type aberrations was questionable, therefore this was not considered to be biologically relevant.
In the presence of S9 mix, the test substance caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 1862.5 ug/mL, including gap-type aberrations only, when compared with the solvent control (p<0.001). This increase was not considered to be biologically relevant since the significance of gap-type aberrations was questionable.
Both positive control substances caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.
2. Experiment 2-in the absence of S9 mix, following a continuous exposure, the test substance caused no statically significant increases in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent control.
Following a three-hour exposure in the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent control.
Both positive control substances caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.
No increases in polyploid metaphases were observed during metaphase analysis, in either test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
1. Experiment 1-Following a three-hour exposure in the absence and presence of S9 mix, the test substance did not cause a reduction in the mitotic index compared to the solvent control value at any dose level. The dose levels selected for the metaphase analysis were 465.63, 931.25 and 1862.5 ug/mL.
2. Experiment 2-Following a continuous exposure, in the absence of S9 mix, the test substance caused a reduction in the mitotic index to 45 % of the solvent control value at 1862.5 ug/mL. The dose levels selected for the metaphase analysis were 465.63, 931.25 and 1862.5 ug/mL. In the presence of S9 mix, the test substance did not cause a reduction in the mitotic index at 1862.5 ug/mL. The dose levels select for the metaphase analysis were 465.63, 931.25 and 1862.5 ug/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

Conclusions:
The test substance was evaluated for its ability to induce chromsomal aberration in human lymphocytes cultured in vitro in the presence and absence of metabolic activation. It was concluded that the test substance had shown no evidence of clastogenic activity in this in vitro cytogenetic test system, under the experimental conditions described.
Executive summary:

Not applicable