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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:
2003-11-22 (test substance received) to 2004-09-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
: Limited information provided on test substance and methodology and only 100 cells were analyzed for each exposure group
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): JNJ-1580774-AAA (T000835)
- Physical state: solid powder
- Appearance: slight beige
Specific details on test material used for the study:
Description: Extremely pale beige solid
Purity: 100%
Label: Code No : 054906 T 835
Date received:2003-12-22
Storage conditions: Room temperature in the dark

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2 % rat liver homogenate metabolizing system (S9)
Test concentrations with justification for top dose:
- Preliminary toxicity test: 0, 1247, 29.94, 49.88, 99.75, 199.5, 399, 798, 1596 and 3192 µg/mL
- Group 1 (4(20)-hour without S9): 0, 50, 100, 200, 400, 600 and 800 g/mL (0, 200, 400, 600 and 800 µg/mL were selected for metaphase analysis in the absence of S9 and 0, 200, 400 and 800 µg/mL were selected for metaphase analysis in the presence of S9 mix.)
- Group 2 (4(20)-hour with S9): 0, 50, 100, 200, 400, 600 and 800 g/mL (0, 200, 400, 600 and 800 µg/mL were selected for metaphase analysis in the absence of S9 and 0, 200, 400 and 800 µg/mL were selected for metaphase analysis in the presence of S9 mix.)
- Group 3 (24-hour without S9): 0, 12.5, 25, 50, 100, 150 and 200 µg/mL (0, 25, 50 and 100 µg/mL were selected for metaphase analysis.)

The selection of the dose range for the preliminary toxicity test was on a 10mM maximum dose level.
The selection of the dose range for the chromosome aberration test was based on toxicity.
Dose selection for metaphase analysis was based on toxicity for all exposure groups.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: An unspecified vehicle was used.
- Justification for choice of solvent/vehicle: no data
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9; at 0.4 µg/mL for 4 hour exposure and at 0.2 or 0.4* µg/mL for 24 hour exposure (Groups 1 and 3)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9; at 10 µg/mL (Group 2)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (Groups 1 and 2); 24 hours (Group 3)
- Expression time: 20 hours (Groups 1 and 2) was provided in the study report as the expression period. However, no information was given on the time of addition of a spindle inhibitor ; 0 hours (Group 3).
- Fixation time: 24 hours (all groups)

SPINDLE INHIBITOR: no data
STAIN: no data

NUMBER OF REPLICATIONS:
2 (Except where there was a need to clarify an equivocal response, only one of the duplicate cultures was assessed for the incidence of cells with chromosome aberrations.)

NUMBER OF CELLS EVALUATED:
100 cells per culture used for metaphase analysis were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: Determination of endoreplication: investigated.
Evaluation criteria:
- A positive response was recorded for a particular treatment if the % of cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency, a dose response relationship was generally required and appropriate statistical tests may have been applied in order to record a positive response.
Statistics:
- Statistics were used in the study, but no data were provided on the tests performed.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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: no data
- Precipitation: In the range-finding test, a precipitate of the test substance was observed at and above 199.5 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
- Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 798 µg/mL in the pulse exposure groups and 99.75 µg/mL in the continuous exposure group.
- In the absence of metabolic activation the data showed clear dose-related test substance induced toxicity in both exposure groups. In the presence of metabolic activation there was a decrease in toxicity with increased dose level into the precipitation dose range.
- In the absence of metabolic activation there was a mitotic inhibition greater than 50% achieved at 798 µg/mL in the 4(20) hours exposure (Group 1), but only 32% inhibition at 99.75 µg/mL, with no metaphases at 199.5 µg/mL in the 24-hour exposure group.
- Therefore, the selection of the dose range for the chromosome aberration test was based on toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.
- The positive control substances induced statistically significant increases in the frequency of cells with aberrations. It was therefore considered that the metabolic activation system was shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- A microscopic assessment of the slides showed that adequate scorable metaphase cells were present at up to 800 µg/mL in both the 4(20) hour exposure groups and up to 100 µg/mL in the 24 hour continuous exposure group. The test substance induced approximately 80% mitotic inhibition at 800 µg/mL in both of the 4-hour pulse exposure groups.
- In the 24-hour exposure without S9 there was a steep toxicity curve with approximately 60% mitotic inhibition at 100 µg/mL and 96% inhibition at 150 µg/mL.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

The test substance did not induce statistically significant increases in the frequency of cells with aberrations at any dose level in any of the three exposure conditions. It was considered that adequate toxicity had been achieved in all exposure conditions under the conditions of the test method.

 

The test substance did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.

Applicant's summary and conclusion

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

The test substance was evaluated for induction of chromosome aberrations in primary human lymphocytes in the presence and absence of metabolic activation. The test substance did not induce statistically significant increases in the frequency of cells with chromosome aberrations in the absence or presence of metabolic activation after 4(20) hour exposures or 24 hour continuous exposure. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.