[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 13 December 2004; Experiment start date - 06 January 2005; Experiment end date - 11 May 2005; Study completion date - 30 May 2005.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Kanpoan No. 287 - Environmental Agency

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Eisei No. 127 - Ministry of Health & Welfare

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

FAT40819/A TE

IUPAC Name:

FAT40819/A TE

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40819/A
- Purity: Approx. 77%
- Lot/batch No.: Red ROE 420 BOP 01/04
- Expiration date: 2 November 2009
- Stability in solvent: In saline, polyethylene glycol, carboxymethylcellulose at room temperature 7 days and in vaseline and FCA at room temperature 1 day.
- Storage conditions: At room temperature, in the desiccator

Specific details on test material used for the study:

Identity: FAT 40819/A
Description: Red brown powder
Batch number: Red ROE 420 BOP 01/04
Purity: approx. 77 %
Stability of test item: Stable under storage condition
Expiry date: 02 November 2009
Stability of test item dilution: Stable in PEG 300 for at least 7 days at room temperature
Storage conditions: At room temperature.

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt) were stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of standardized characteristics of the cells.
Thawed stock cultures were propagated at 37° C in 80 cm2 plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x10E5 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 1 2 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g (supplied from RCC Ltd; Laboratory Animal Services, CH- 4414 Füllinsdorf) induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D- 22335 Hamburg) and ß-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1:3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules were kept at -20 °C for up to one week. The protein concentration was 35.3 mg/mL (Lot. no. 221004) in all experimental parts.

S9 Mix
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final-protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCI2
33 mM KCl
5 mM glucose-6-phosphate
4mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

See any other information on materials and methods

Vehicle / solvent:

Deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v). The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

RANGE-FINDER
A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item. Cytotoxicity was determined using concentrations separated by no more than a factor of 2 - √10. The general experimental conditions in this pre-test were the same as described below for the cytogenetic main experiment. The following method was used: In a quantitative assessment, exponentially growing cell cultures (seeding about 40,000 cells/ slide, with regard to the culture time 48 hrs) were treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hrs and 24 hrs after start of treatment. The cells were stained 24 hrs after start of treatment. Using a 400 fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group).

EXPERIMENTAL PERFORMANCE
- Exponentially growing stock cultures more than 50 % confluent are treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then the enzymatic treatment is stopped by adding complete culture medium and a single cell suspension is prepared. The trypsin concentration for all subculturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution (Invitrogen GIBCO, D-76131 Karlsruhe). Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1E4 - 6E4 cells were seeded with regard to the preparation time. The medium was MEM with 10 % FCS (complete medium).
- Exposure duration: Exposure period 4 hours: The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent negative, solvent, and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time. Exposure period 18 and 28 hours: The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells. All cultures were incubated at 37° C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- Preparation of the cultures: 15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 µg/mL culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).
- Evaluation of Cell Numbers: For evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs and 28 hrs, respectively, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.
- Analysis of Metaphase Cells: Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetic') using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the highest evaluated concentration in Experiment II in the absence of metabolic activation, where 200 metaphase plates were scored. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

- Acceptability of the test: The chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of our historical laboratory control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.
b) The positive control substances should produce significant increases in the number of cells with structural, chromosome aberrations, which are within the range of the laboratory's historical control data.

- Evaluation of results: A test item is classified as non-clastogenic if: the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps); and/or no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if: the number of induced structural chromosome aberrations is not in the range of the historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps); and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
A test item can be classified as aneugenic if: the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

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:

RANGE FINDING STUDY: In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 39.1 and 5000 µg/mL were applied. Clear toxic effects were observed after treatment with 1250 µg/mL and above in the absence of S9 mix and with 78.1 µg/ml and above in the presence of S9 mix. In addition, 24 hrs continuous treatment with 312.5 µg/mL and above in the absence of S9 mix induced strong toxic effects.

TEST-SPECIFIC CONFOUNDING FACTORS: In the pre-experiment, neither precipitation nor relevant influence of the test item on the pH value or osmolarity was observed (solvent control 291 mOsm, pH 7.4 versus 317 mOsm and pH 7.4 at 5000 µg/mL).

MAIN TEST: In Experiment I, in the absence and the presence of S9 mix and in Experiment II, in the absence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.8 % aberrant cells, exclusive gaps) were close-to the range of the solvent control values (1.0- 3.0 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.
In Experiment II, in the absence of S9 mix, at preparation interval 28 hrs a single significant (p < 0.05) increase after evaluation of 200 metaphase plates per culture was observed after treatment with 500 µg/mL. Although this value of 3.8 % aberrant cells, exclusive gaps, was statistically significant compared to the low response (1.0 % aberrant cells) in the solvent control data, the response is within the historical control data range (0.0- 4.0 % aberrant cells). Therefore, the statistical significance has to be regarded as being biologically irrelevant. However, in Experiment II in the presence of S9 mix at preparation interval 28 hrs, a dose related increase in the number of aberrant metaphase cells was observed in the concentration range evaluated: 125, 250, and 500 µg/mL (3.0%, 8.0%, and 15.0%, respectively). The values of the two highest scored concentrations were, statistically sigificant as compared with the respective solvent control group (1.5 % aberrant cells, exclusive gaps) and clearly exceeded our historical control data range (0.0 - 4.0 aberrant cells, exclusive gaps). The dose-dependency of metaphases carrying exchanges in these three test groups (0.5 %, 2.0 %, and 9.0 %, respectively) give additional evidence for a clastogenic potential of the test item. Finally; the dose-dependencies and statistical significances observed in Experiment II in the presence of S9 mix at preparation interval 28 hrs have to be regarded as biologically relevant. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 - 3.7 %) as compared to the rates of the solvent controls (0.8 - 3.2 %). In both experiments, EMS (300 and 400 µg/mL, respectively) and CPA (1.0 and 1.4 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations, except in Experiment II in the presence of S9 mix after exposure with CPA. At this experimental part after 4 hrs treatment with 1.4 µg/mL CPA the aberration rate (2.5 %) was lower than expected and was not within our historical positive control data range: 8.5 % - 22.0 % aberrant cells, exclusive gaps. It has to be proposed that any technical error occurred in this test group. However, the observation of clastogenicity in Experiment II in the presence of S9 mix at preparation interval 28 hrs after treatment with two test item concentrations (250 and 500 µg/mL) give proof for the validity of this experimental part. Therefore, this deviation has no detrimental impact on the validity of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In both cytogenetic experiments, cytotoxicity indicated by reduced cell numbers or mitotic indices below 50 % of control was observed. In detail, in the absence of S9 mix, clearly reduced cell numbers were observed after 4 hrs treatment with 1500 µg/mL (22.0% of control) in Experiment I, and after 28 hrs treatment with 500 µg/mL (37.4 % of control) in Experiment II. In addition, in Experiment II in the presence of S9 mix, at preparation interval 28 hrs the cell numbers were distinctly reduced after treatment with 500 µg/mL (47.8 % of control). Besides, in Experiment II in the absence of S9 mix, the mitotic indices were clearly reduced after 28 hrs continuous treatment with 500 µg/mL (38.5 % of control). However, in Experiment I in the presence of S9 mix and in Experiment II after 18 hrs treatment in the absence of S9 mix, concentrations showing clear toxic effects were not scorable for cytogenetic damage.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test substance did induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the presence of S9 mix.

Executive summary:

In a GLP-compliant chromosome aberration test, performed according to OECD guideline 473, Chinese hamster V79 cells, were exposed to the test substance, with and without metabolic activation by S9 mix in two independent experiments. In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations. Dose selection for the cytogenetic experiments was performed considering the toxicity data available from the pre-test. Toxic effects indicated by reduced cell numbers or mitotic indices of below 50 % of control were observed in both cytogenetic experiments. However, in Experiment I in the presence of S9 mix and in Experiment II after 18 hrs treatment in the absence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. No biologically relevant increase of the aberration rates was observed in Experiment I and II in the absence of S9 mix and in Experiment I in the presence of S9 mix. In contrast, in Experiment II, in the presence of S9 mix, a dose-related increase in the number of cells carrying structural chromosomal aberrations with statistically significant and biologically relevant values were observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. In conclusion, it can be stated that under the experimental conditions reported, the test item did induce structural chromosome aberrations. Therefore, the test substance is considered to be clastogenic in the presence of S9 mix.