Z-85

Genetic toxicity: in vitro

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:

Between 04 August 2006 and 14 February 2007.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to GLP in accordance with recognised guideline. The study is reliabilty K1 for the donor substance but is regarded as K2 for the target substance because it is used as a read-across. The read-across justification is included in the dossier.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

other: liquid

Details on test material:

- Physical state: tan coloured slightly viscous liquid
- Storage condition of test material: room temperature in the dark

Method

Target gene:

N/A

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,
- Properly maintained: Not applicable
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
- Periodically "cleansed" against high spontaneous background: Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1:
4(20)-hour without S9: 0, 14.61, 29.22, 58.44, 87.66, 116.88, 175.32, MMC 0.4
4(20)-hour with S9: 0, 58.44, 116.88, 233.75, 350.63, 467.5, 701.25, CP 5

Experiment 2:
4(20)-hour without S9: 0, 7.31, 14.61,29.22, 58.44, 87.66, 116.88, MMC 0.2
4(20)-hour with S9: 0, 14.61, 29.22, 58.44, 116.88, 233.75, 467.5, CP 5

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle:The test substance was insoluble in water, but was soluble in DMSO
at 37.4 mg/ml, which when dosed at 1% gave the maximum recommended dose level equivalent to 10 mM (3740 ug/ml, MW of 374).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.2 and 0.4 ug/ml without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

5.0 ug/ml with S9

Details on test system and experimental conditions:

- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,

Experiment 1:
4(20)-hour without S9: *, 14.61, 29.22*, 58.44*, 87.66*, 116.88, 175.32, MMC 0.4*
4(20)-hour with S9: 0*, 58.44, 116.88, 233.75*, 350.63*, 467.5*, 701.25, CP 5*
Experiment 2:
24-hour without S9: 0*, 7.31, 14.61*, 29.22*, 58.44*, 87.66, 116.88, MMC 0.2*
4(20)-hour with S9: 0*, 14.61, 29.22*, 58.44*, 116.88*, 233.75, 467.5, CP 5*

* = Dose levels selected for metaphase analysis

METHOD OF APPLICATION: in medium;
With Metabolic Activtion
Cultures were established approximately 48 hours prior to treatment. Cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing.

Without Metabolic Activation
Cultures were established approximately 48 hours prior to treatment. In Experiment 1, cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing. In Experiment 2, the cultures were incubated in the presence of the substance at 37°c for 24 hours
DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 24 hours
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): Mitosis was arrested by addition of democolcine two hours prior to the required harvest time and the cells were harvested and fixed

SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5% Gurrs Giemsa

NUMBER OF REPLICATIONS: Treatments performed in duplicate.

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Where possible the first 100 consecutive well-spread metaphases from each culture were counted for the number of chromosomes and scored for chromosome aberrations as appropriate.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes in comparison to controls
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated

Evaluation criteria:

A positive response was recorded for a particular treatment if the % 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 is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 14.61 to 3740 μg/ml. The maximum dose was the 10 mM concentration. An oily precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 1870 μg/ml, in the 4(20)-hour pulse exposure groups and at 3740 μg/ml in the 24 hours continuous exposure group. A cloudy precipitate was observed at and above 58.44 μg/ml and 233.75 μg/ml for the pulse and continuous exposure group respectively. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 233.75 and 58.44 μg/ml, in the 4(20 )hour exposures in the presence and absence of metabolic activation (S9) respectively. The maximum dose with metaphases present in the 24-hour continuous exposure was 58.44 μg/ml. The test material induced evidence of toxicity in all of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Chromosome Aberration Test - Experiment 1

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test for the without S9 group but was not as toxic as expected in the with S9 group. There were scorable metaphases present at up to 87.66 μg/ml in the absence of metabolic activation (S9). In the presence of metabolic activation (S9) the maximum dose level of the test material with scorable metaphases was 467.5 μg/ml. Precipitate observations in the Preliminary Toxicity Test are considered to be representative for the study.

The mitotic index data confirmed the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 58% mitotic inhibition was achieved at 87.66 μg/ml in the absence of S9. In the presence of S9 there was a steep toxicity curve and there was no mitotic inhibition up to 467.5 μg/ml but insufficient scorable metaphases at 701.25 μg/ml.

The maximum dose level selected for metaphase analysis was therefore based on toxicity and was 87.66 μg/ml for the without S9 group and 467.5 μg/ml for the with S9 group using 2% S9.

 

The chromosome aberration data are given in Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2

The qualitative assessment of the slides determined that there were scorable metaphases present at up to 116.88 μg/ml in the presence of S9. In the absence of S9 the maximum test material dose level with scorable metaphases was 58.44 μg/ml. Precipitate observations in the Preliminary Toxicity Test are considered to be representative for the study.

The mitotic index data confirmed the qualitative observations in that a dose-related inhibition of mitotic index was observed in the absence of S9 and that 67% mitotic inhibition was achieved at 58.44 μg/ml. In the presence of S9 the test material was more toxic using 1 % final concentration of S9 than was observed in Experiment 1 using 2% final concentration.

The maximum dose level selected for metaphase analysis was therefore based on toxicity and was 58.44 μg/ml for the 24 hours continuous exposure group and 116.88 μg/ml for the 4(20) hour group with 1% S9.

The chromosome aberration data are given in Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. The polyploid cell frequency data are given in Table 8. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The substance was considered to be was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction.This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method BIO of Commission Directive 2000/32/EC. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods.Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1 % final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

Results.All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells With aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced mitotic inhibition.

Conclusion.The test material was considered to be non-clastogenic to human lymphocytes in vitro.