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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2 August - 24 October 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and methods similar to OECD 473.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Internal Method No. 449.1, Main Dept. Exp. Toxicology.
Deviations:
not specified
Principles of method if other than guideline:
Internal Method No. 449.1, Main Dept. Exp. Toxicology.
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): ZK 22.663 (DBTCl)

Method

Species / strain
Species / strain / cell type:
lymphocytes: whole blood culture from the peripheral blood
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood cultures were prepared by adding 0.4 ml heparinized blood, obtained by venipuncture from a healthy adult male, to 4.67 ml of culture medium containing McCoy's 5a medium, 15% (v/v) fetal calf serum (FCS), antibiotics and PHA.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Assay without S9 mix:
- First assay: 0.001, 0.003, 0.006, 0.01, 0.03, 0.06, 0.1, 0.3, 0.6, 1.0 and 3.0 µg/ml
- Second assay: 0.006, 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2 and 0.4 µg/ml

Assay with S9 mix:
- First assay: 0.050, 0.075, 0.10, 0.25, 0.50, 0.75, 1.0, 2.5, 5.0 and 7.5 µg/ml
Second assay: 0.05, 0.10, 0.25, 0.50, 0.75, 1.0, 2.0 and 3.0 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: With S9 mix: cyclophosphamide; without S9 mix: triaziquone
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 26 hours
- Exposure duration: After 3 hours incubation at 37°C, the cells were washed three times with McCoy's 5a culture medium and re-incubated for approximately 24 hours.
- Expression time (cells in growth medium): nda
- Selection time (if incubation with a selection agent): To arrest the cells in the metaphase, colcemide (final concentration 0.08 µg/ml) was added ca. 3.5 hours before harvesting the cells.
The cells were collected by centrifugation, exposed to 1 % (w/v) tri-sodiumcitrate hypotonic solution (for swelling) and fixed in glacial acetic acid/methanol.


STAIN (for cytogenetic assays): The slides were stained for 15 minutes with Hoechst 33258 (final concentration 4.5 µg/ml) in phosphate buffer (pH 6.9), mounted in the same buffer and exposed at 60°C to "black-light-blue" UV from 2x20 Watt tubes (e.g. Philips, Type TL 20W/08,F20T12BLB) for the time required for differentiation between chromatids (ca. 10 min.). Finally, slides were stained with 5 % (v/v) Giemsa for 4 - 8 min. and air dried.


NUMBER OF REPLICATIONS:
Duplicate


DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
Evaluation criteria:
All structural chromosome aberrations were assigned artifical lesion (or break) scores as follows:
- Chromatid and chromosome (isochromatid) breaks, acentric fragments and deletions were scored as one lesion each.
- Exchanges, rings and dicentrics were designated 2 lesions each.
Statistics:
To assess the difference between the positive control vs. solvent control, Fisher's exact test (α = 0.05) will be performed. The treatment effect will be analyzed in a Chi-squared test (exact, α = 0.05) including the solvent control and all treatment groups. In case of significance the global test will be followed by comparisons of the individual groups with the solvent control using Fisher's exact test (least significant difference test).

Results and discussion

Test results
Species / strain:
lymphocytes: whole blood culture from the peripheral blood
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Nor reported
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The data of the study indicate that without and with an added metabolizing system ZK 22.663 induces chromosome aberrations in human lymphocytes in vitro. From the four assays conducted in two seperate studies one assay without and one assay with S9 mix gave statistically significant (P < 0.05) positive results, whereby in the remaining assays the response was borderline negative. Especially in this case a mutagenic response could only be achieved at distinctly cytotoxic concentrations. The clastogenic effects of ZK 22.663 started only at a concentration range where the mitotic index was reduced by approximately 50 %. On the other hand, because the toxicity curve of ZK 22.663 is very steep, it was very difficult and not always possible to achieve an optimum concentration range where the mitotic index was reduced by at least 50 % at the highest concentration evaluable for chromosomal aberrations. Due to this critical situation it was not possible to reproduce the positive response unequivocally in two independent assays. However, considering the clear-cut positive results in two assays which are supported by the borderline negative results in the remaining assays, and taking into account that the human lymphocyte test is succeeded by a mouse bone-marrow micronucleus test in which the in vivo clastogenic potential of ZK 22.663 is examined carefully, no further efforts should be undertaken to optimize the in vitro investigations.

In conclusion, despite the inconsistencies discussed above, it is justified to classify ZK 22.663 as a clastogen in the human lymphocyte test in vitro.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

Evaluation of the data indicates a clastogenic potential of the test material in the human lymphocyte test in vitro at clearly cytotoxic concentrations.
Executive summary:

An evaluation of the clastogenic potential in the human lymphocyte test (Schering report number: IC 1-90) indicates a clastogenic potential of the test material in the human lymphocyte test in vitro at clearly cytotoxic concentrations.

From the four assays conducted without and with an extrinsic metabolizing system in two independent studies, one assay without and one with S9 mix gave statistically significant (P < 0.05) increases in the frequency of chromosomal aberrations at the highest concentrations evaluated, whereby in the remaining assays the results were borderline negative. In each assay of this investigation, the test material was tested up to cytotoxic concentrations as indicated by an obvious reduction of the mitotic index.