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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:
The experimental phases of the study were performed between 11 March 2008 and 12 June 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
The study was conducted according to the requirements of the Japanese New Chemical Law (METI).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 21/08/07 Date of Signature: 15/10/07
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): Barium di(bistridecylsulfosuccinate) in mixture with Barium hydrogen phosphate
- Substance type: Pale yellow solid
- Physical state: Solid
- Lot/batch No.: Y-T-1
- Storage condition of test material: Room temperature, in the dark

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and beta-naphthoflavone induced rat live, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: The dose range of test material used was 11.72 to 3000 µg/ml.

Experiment 1:
Without S9-mix: 0*, 11.88, 23.75*, 47.5*, 95*, 142.5, 190, MMC 0.4*
With S9-mix: 0*, 23.75, 47.5, 95*, 142.5*, 190*, 237.5 CP 4*

Experiment 2:
Without S9-mix: 0*, 11.88, 23.75, 47.5, 95*, 142.5*, 190*, MMC 0.2*
With S9-mix: 0*, 11.88, 23.75, 47.5, 95*, 142.5*, 190*, CP 5*

* Dose levels selected for metaphase analysis
Vehicle / solvent:
The test material was accurately weighed, suspended in MEM and serial dilutions prepared.
(MEM) = Eagle's minimal essential medium with HEPES buffer (MEM).
The test material was considered to be a mixture and therefore the maximum dose level was 5000 µg/ml, which was the maximum recommended dose level. However, due to formulation practicalities the maximum dose was limited to 3000 µg/ml. There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm. Chemical analysis of the test material formulations was not performed because it is not a requirement of the test method.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9

Migrated to IUCLID6: (CP)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9

Migrated to IUCLID6: (MMC)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 48 hrs
- Exposure duration: Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.
- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure.
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs.


SELECTION AGENT (mutation assays): No selection agent.
SPINDLE INHIBITOR (cytogenetic assays): Demecolcine
STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Gurrs Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS: Duplicate cultures


NUMBER OF CELLS EVALUATED: 100/culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - 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.
-Scoring of Chromosome Damage: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy: Frequency of polyploid cells


OTHER: None.
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: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Refer to information on results and attached tables.
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: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material suspended in MEM
- Precipitation:
Premlinary toxictiy test: A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure period, at and above 187.5 µg/ml, in both the 4(20)-hour exposure groups and in the 24-hour continuous exposure group.
Experiment 1: A precipitate of the test material was found to persist onto the microscope slides at and above 95 µg/ml in both the absence and presence of S9.
Experiment 2: A precipitate of the test material was found to persist onto the microscope slides at 190 µg/ml in both the absence and presence of S9.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 11.72 to 3000 µg/ml. The maximum dose was limited by formulation practicalities. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure period, at and above 187.5 µg/ml, in both the 4(20)-hour exposure groups and in the 24-hour continuous exposure group. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 93.75 µg/ml in the 4(20)-hour treatment in the absence of metabolic activation (S9) and 24-hour continuous exposure groups. The maximum dose with metaphases present in the 4(20)-hour exposure group with metabolic activation was 187.5 µg/ml. It should be noted that test material precipitate was observed on the microscope slides at and above 46.88 µg/ml in the 4(20)-hour exposure groups, and at and above 93.75 µg/ml in the 24-hour exposure group.
The mitotic index data are presented in Appendix 1 (5) and (6) (please see attached background material). It can be seen that the test material showed clear evidence of toxicity in all three exposure groups and the test material exhibited a very steep toxicity curve.
Dose selection for Experiment 1 and 2 was based on toxicity for all exposure groups used.


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 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.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
EXPERIMENT 1:

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at 95 µg/ml in the absence of metabolic activation (S9). In the presence of metabolic activation (S9) the maximum test material dose level with metaphases suitable for scoring was 190 µg/ml.

The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 1, Appendix 2 (please see attached background material). These data show an approximate 50% mitotic inhibition was achieved at 190 µg/ml in the presence of S9. In the absence of metabolic activation no clear evidence of mitotic inhibition was observed in the test material dose levels that had metaphases present for analysis.

A precipitate of the test material was found to persist onto the microscope slides at and above 95 µg/ml in both the absence and presence of S9.

The maximum dose level selected for metaphase analysis was based on toxicity in both exposure groups, and was 95 µg/ml in the absence of S9 and 190 µg/ml in the presence of S9.

The chromosome aberration data are given in Appendix 2, Form 1 (please see attached background material). 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 in either the absence or presence of metabolic activation (S9).

The polyploid cell frequency data are given in Appendix 2, Form 1 (please see attached background material). The test material did not induce any statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

EXPERIMENT 2:
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 190 µg/ml in the absence and presence of S9.

The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 2, Appendix 2 (please see attached background material). These data show an approximate 59% mitotic inhibition was achieved at 190 µg/ml in the absence of S9, and in the presence of S9 25% mitotic inhibition was seen at 190 µg/ml. Whilst 50% mitotic inhibition was not achieved in the presence of S9 it was considered that the test material had been adequately tested. With no clastogenic response being recorded in the first experiment and 100% toxicity being observed at higher dose levels in the preliminary toxicity test and Experiment 1 it was considered unnecessary to repeat this exposure group.

A precipitate of the test material was found to persist onto the microscope slides at 190 µg/ml in both the absence and presence of S9.

The maximum dose level selected for metaphase analysis was the same for both exposure groups, the maximum dose level tested of 190 µg/ml.

The chromosome aberration data are given in Appendix 2, Form 2 (please see attached background material). 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 in either the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Appendix 2, Form 2 (please see attached background material). The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

For the appendixes and forms mentioned above please refer to the attached backgrouind material section for the following:

Appendix 1 (5): SHORT TERM TREATMENT TEST

(1): Test condition of Cell Growth Inhibition Test

(2): Results of Cell Growth Inhibition Test

(3): Test condition of Chromosome Aberration Test

Appendix 1 (6): CONTINOUS TREATMENT TEST

(1): Test condition of Cell Growth Inhibition Test

(2): Results of Cell Growth Inhibition Test

(3): Test Condition of Chromosome Aberration Test

Appendix 2, Form 1:-

Short Term Treatment Test - Experiment 1 Without Metabolic Activation (S9)

Short Term Treatement Test - Experiment 1 With Metabolic Activation (S9)

Appenddix 2, Form 2:-

Continous Treatment Test - Experiment 2

Short Term Treatment Test - Experiment 2

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore 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). This study was conducted according to the requirements of the Japanese New Chemical Substance Law (METI).

Methods.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. 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 an 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9), with a 20-hour expression period. In Experiment 2, in the absence of metabolic activation the exposure time was increased to 24 hours and the 4(20)-hour with S9 group was repeated with a final S9 concentration of 1%.

Results.

All vehicle (solvent) control groups 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 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 dose levels that induced approximately 50% mitotic inhibition.

Conclusion.

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