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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 based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
11 May 2004 - 16 November 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and OECD guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
Duplicate cultures of human lymphocytes were exposed to methyl-2-mercaptobenzimidazole, zinc salt at concentrations of 0, 31.25, 62.5, 125, 250, 375, and 500 µg/ml for 4 hours (with and without metabolic activation) and then examined for chromosomal aberrations.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: off-white powder
Date received: 11 March 2004
Storage: Room temperature in the dark.

Method

Target gene:
no data
Species / strain
Species / strain / cell type:
human lymphoblastoid cells (TK6)
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. The cell-cycle time for the lymphocytes from the donors used in this study were determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculated the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.

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, at 37°C with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutin (PHA) at 90 µg/ml final concentration.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Without S9: 31.25, 62.5, 125, 250, 375, 500 µg/ml
With S9: 31.25, 62.5, 125, 250, 375, 500 µg/ml
Vehicle / solvent:
Dimethyl sulphoxide (DMSO)
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
Parallel with test material
Positive controls:
yes
Remarks:
mitomycin C 0.4 and 0.2 microg/ml
Positive control substance:
mitomycin C
Remarks:
Without S9
Positive controls:
yes
Remarks:
cyclophosphamide 5.0 microg/ml
Positive control substance:
cyclophosphamide
Remarks:
With S9
Details on test system and experimental conditions:
The test material was accurately weighed, dissolved in dimethyl sulphoxide (DMSO) and serial dilutions prepared. The molecular weight of the test material was 393.9 and the maximum dose level in the preliminary toxicity test would normally have been the 10mM maximum recommended dose level of 3940 µg/ml. However, the maximum dose level was limited to 2000 µg/ml because the test material precipitate was observed to aggregate into a single mass at and above 1970 µg/ml and, therefore, effectively reduce its maximum exposure to the cells. The maximum dose levels selected for the main experiments were further reduced due to test material toxicity. 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.

Culture conditions:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispersed into sterile plastic flasks for each culture:

9.05 ml MEM, 15% (FCS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin
0.75 ml heparinised whole blood

With Metabolic Activation (S9) Treatment:
After approximately 48 hours incubation at 37°C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 ml of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 ml of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 ml of the appropriate solution was added to the cultures. 1 ml of 20% S9 mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test of Experiment 1.

In Experiment 2, 1 ml of 10% S9 mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 ml.

After 4 hours at 37°C, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at 37°C in 5% CO2 in humidified air.

Without Metabolic Activation (S9) Treatment:
In Experiment 1, after approximately 48 hours incubation at 37°C with 5% CO2 in humidified air in cultures were decanted into tubes and centrifuged. Approximately 9 ml of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 ml of the appropriate vehicle control, test material solution or positive control solution. The total volume for each culture was a nominal 10 ml.

After 4 hours at 37°C, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 ml. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 ml of vehicle control, test material dose solution or positive control solution. The nominal final volume of each culture was 10 ml. The cultures were then incubated at 37°C for 24 hours.

Preliminary Toxicity Test:
A preliminary toxicity test was performed on cell cultures using a 4 hours exposure time with and without metabolic activation followed by a 20 hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test material used was 15.6 to 2000 µg/ml. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Using a quantitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test material toxicity and for selection of the dose levels for the main study.

- Experiment 1:
i) 4 hour exposure to the test material without S9 mix followed by 20 hour culture in treatment-free media prior to cell harvest.

ii) 4-hour exposure to the test material with S9 mix followed by 20 hour culture in treatment free media prior to cell harvest.

- Experiment 2:
i) 24 hour continuous exposure to the test material without S9 mix prior to cell harvest.

ii) 4 hour exposure to the test material with S9 mix followed by 20 hour culture in treatment free media prior to cell harvest.

Cell Harvest:
Mitosis was arrested by addition of demecolcine two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in 0.075M hypotonic KCl. After approximately fourteen minutes including centrifugation, most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was charged at least three times and the cells stored at 4°C for at least four hours to ensure complete fixation.

Preparation of Metaphase Spreads:
The lymphocytes were resuspended in several ml of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining:
When the slides were dry they were stained in 5% Gurrs Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.

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.
Evaluation criteria:
Where possible the first 100 consecutive well spread metaphases from each culture were counted, where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the International System for Chromosome Nomenclature (1985) as described by Scott et al and compatible and equitable 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.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, the concurrent vehicle value using Fisher's Exact test

Results and discussion

Test results
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Additional information on results:
Toxicity Test:
The dose range for the Preliminary Toxicity Test was 15.6 to 2000 µg/ml. The maximum dose was based on the lowest acceptable precipitating dose level. A precipitate of the test material was observed in the parallel blood free cultures at the end of the exposure, at and above 250 µg/ml, in the 4(2) hour pulse exposure groups and at and above 125 µg/ml in the continuous exposure group. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 250 µg/ml in all three of the treatment groups. The test material showed no clear evidence of toxicity, as indicated by dose related reductions in mitotic index in the surviving dose levels, in any of the exposure groups.

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 and that there were scorable metaphases present up to 375 µg/ml in the absence and presence of metabolic activation (S9).
The mitotic index data confirms the qualitative observations in that a dose related inhibition of mitotic index was observed, and that 65% mitotic inhibition was achieved at 375 µg/ml in the presence of S9. In absence of S9 the dose relationship was not as clear cut, and the maximum mitotic inhibition of 41% was achieved at 375 µg/ml.
The maximum dose level selected for metaphase analysis, 375 µg/ml, was based on toxicity in both the absence and presence of S9.
All 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. It should be noted that in presence of S9 at 375 µg/ml it was only possible to score 36 metaphase cells for the A culture. This was due to the level of test material toxicity observed in this culture. However, with no evidence of a response being observed in this experiment and in Experiment 2, it was, therefore, considered that the overall result and integrity of the study was not affected.
The test material did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosom Aberration Test - Experiment 2:
The qualitative assessment of the slides determined that there were scoreable metaphases present up to 125 µg/ml in the absence of S9. In the presence of S9 the maximum test material dose level with scoreable metaphases was 375 µg/ml.
The mitotic index data confirm the qualitative observations in that a dose related inhibition of mitotic index was observed, and that 39% mitotic inhibition was achieved at 125 µg/ml in the absence of S9. In the presence of S9 52% mitotic inhibition was achieved at 375 µg/ml.
The maximum dose levels selected for metaphase analysis, 125 and 375 µg/ml in the absence and presence of S9 respectively were based on toxicity.
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 test material did not induce any statistically significant increases 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 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.