2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, potassium sodium salt (1:?:?), coupled with diazotized 2-[(4-amino-5-methoxy-2-methylphenyl)sulfonyl]ethyl hydrogen sulfate

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 May 2003; Experiment start date - 21 May 2003; Experiment end date - 02 July 2003; Study completion date - 10 September 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40'812/A
- Purity: Approx. 75%
- Lot/batch No.: WP 3/03
- Expiration date: 23 April 2010
- Stability in Solvent: 7 days in water, saline, polyethylene glycol, CMC, 1 day in vaseline, and FCA
- Storage conditions: At room temperature

Specific details on test material used for the study:

Identity: FAT 40812/A
Batch: WP 8/03
Purity: approx. 75 %
Appearance: Solid, dark red-brownish powder
Expiration date: 23 April 2010
Storage: At room temperature at about 20 °C

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, 0-72632 Frickenhausen). About 5 x 10E5 cells per flask were seeded into 15 ml of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PM Laboratories GmbH, D-35091 Colbe). 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:

S9 preparation:
Phenobarbital/ß-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g (supplied from RCC Ltd; Biotechnology & Animal Breeding Division, CH-4414 Fullinsdorf) 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 KCI solution (1:3 parts respectively) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -20 °C. Small numbers of the ampoules were kept at -20 °C for up to one week. The protein concentration was 26.2 mg/ml (Lot. no. 070303) in the pre-test and in the main experiment.

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 MgCl2
33 mM KCI
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 incl. tables.

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-FINDING
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 37,630 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. 24 hrs after start of treatment the cells were stained. Using a 400 fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group). The cell number of the treatment groups is given as % cells in relation to the control.

DOSE SELECTION
The highest concentration used in the cytogenetic experiments was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible. 5000 µg/mL of FAT 40812/A were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 39.1 and 5000 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hrs treatment with 2500 µg/mL and above in the absence of S9 mix and with 1250 µg/mL and above in the presence of S9 mix. In addition, 24 hours treatment with 625 µg/mL and above in the absence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 2500 µg/mL (without S9 mix) and 1500 µg/mL (with S9 mix) were chosen as top concentration in the main experiment. Since the test item was considered to be clastogenic after 4 hours treatment a second experiment was not performed.

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 1 x 10E4 - 6 x 10E4 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.
- Preparation of the Cultures: 15.5 hrs 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 additional two cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs 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 test item concentration 500 µg/mL with 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 %.
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 our 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.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. A test item can be classified as mutagenic 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. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

- In a range finding pre-test on toxicity, clear toxic effects were observed after 4 hrs treatment with 2500 µg/mL and above in the absence of S9 mix and with 1250 µg/mL and above in the presence of S9 mix. In addition, 24 hours treatment with 625 µg/mL and above in the absence of S9 mix induced strong toxic effects.

- In the pre-experiment, neither precipitation in cell culture medium nor relevant influence of the test item on the pH value or osmolarity was observed (solvent control 297 mOsm, pH 7.4 versus 315 mOsm and pH 7.4 at 5000 µg/mL).

- In the cytogenetic experiment, toxic effects indicated by reduced cell numbers of below 50 % of control were observed in after 4 hours treatment with 1500 µg/mL (42 % of control) in the absence of S9 mix and with 500 µg/mL (49 % of control) in the presence of S9 mix. In contrary, no clearly reduced mitotic indices of below 50 % of control could be observed up to the highest evaluated concentrations of the test item. Additionally, at cytotoxic test item concentrations indicated by reduced cell numbers extremely high mitotic rates were found. In detail, after 4 hours treatment with 2000 µg/mL in the absence of S9 mix and with 750, 1000, and 1500 µg/mL in the presence of S9 mix the mitotic indices were strongly increased (155 %, 308 %, 313 %, and 255 % of control, respectively). This means that surviving cells have an extremely high mitotic activity. This is no usual finding and gives an evidence for test item induced cell transformation.

- In the absence of S9 mix, the aberration rates were statistically significant (p<0.05) increased after treatment with 500 and 1500 µg/mL as compared to the corresponding solvent control (0.0 %). Besides the responses after treatment with 1500 µg/mL (9.0 % aberrant cells, exclusive gaps) was biologically relevant clearly exceeding our historical control data ratio: 0.0 – 4.0 % aberrant cells, exclusive gaps. Also, at this concentration the number of cells carrying exchanges was distinctly increased (2.5 %) as compared to the solvent control (0.0 %) and give additional evidence for a clastogenic potential of the test item. Therefore, these observations have to be regarded as being biologically relevant.
In the presence of S9 mix, a dose related increase in the number of cells carrying structural chromosome aberrations (1.0 %, 1.5 %, and 4.3 % of control) was observed in the evaluated concentration range (125, 250, and 500 µg/mL, respectively). The response of the highest concentration, confirmed in an increased sample for evaluation of 400 metaphase plates in total, slightly exceeded our historical control data range: 0.0 - 4.0 % aberrant cells, exclusive gaps. Additionally, at pre-evaluation of the slides distinct increased numbers of micronucleated cells and of cells containing fragmented nuclei were observed at the three highest test item concentrations not evaluated for cytogenetic damage (750, 1000, and 1500 µg/mL). Therefore, the sum of observations has to be regarded as biologically relevant.
No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (2.0 - 3.2 %) as compared to the rates of the solvent control (2.8 - 3.0 %). EMS (200 µg/mL) and CPA (0.7 µg/mL) was used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster V79 cells (in vitro), were exposed to the test substance, with and without metabolic activation by S9 mix. Two independent experiments were performed. The exposure period was 4 hrs with and without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the test concentration 500 µg/mL with metabolic activation where 200 metaphase plates were scored. The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data. In both experiments, clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. Clear toxic effects indicated by reduced cell numbers of below 50 % of control were observed in both experimental parts at the highest evaluated concentrations. In contrary no clearly reduced mitotic indices were observed at the test item concentrations evaluated. But after treatment with 2000 µg/mL in the absence of S9 mix and with 750, 1000, and 1500 µg/mL in the presence of S9 mix extremely high mitotic indices were observed. In the absence and presence of S9 mix, biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item at concentrations showing clear reduced cell numbers. No increase in the frequencies of polypoid metaphases were found after treatment with the test item as compared to the frequencies of the controls. In conclusion, the test substance is considered to be clastogenic in this chromosome aberration test in the absence and in the presence of S9 mix.