Yellow PE 3260

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:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Revised Chemical Substance Law (1987) according to the notification of December 9, 1986 by EA, Environmental Agency (no. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014), Japan.

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

1

Reference substance name:

bis[2-hydroxy-N,N-bis(2-hydroxyethyl)ethanaminium] 7-{[4-(4-{[2-(cyanoamino)-4-hydroxy-6-oxo-1,6-dihydropyrimidin-5-yl]diazenyl}benzamido)-3-methoxyphenyl]diazenyl}naphthalene-1,3-disulfonate

Cas Number:

115811-45-5

Molecular formula:

C29H19N9O10S2.2C6H16NO3

IUPAC Name:

bis[2-hydroxy-N,N-bis(2-hydroxyethyl)ethanaminium] 7-{[4-(4-{[2-(cyanoamino)-4-hydroxy-6-oxo-1,6-dihydropyrimidin-5-yl]diazenyl}benzamido)-3-methoxyphenyl]diazenyl}naphthalene-1,3-disulfonate

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (Minimal Essential Medium) supplemented with 10 % fetal calf serum (FCS).
- Properly maintained: yes (Cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 4.5
% carbon dioxide (95.5 % air))
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 mix from Aroclor 1254-induced rat liver
- source of S9: obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm which received a single i.p. injection of 500 mg Aroclor 1254 per kg body weight in olive oil 5 days previously.
- method of preparation of 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 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.
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µl/ml S9 mix contained in serum-free medium together with different conc. of the test article
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

Concentration range in the main test (experiment I, with metabolic activation): 10, 30, 100, 1000 µg/ml;
Concentration range in the main test (experiment II, with metabolic activation): 10, 30, 100, 3000 µg/ml;
Concentration range in the main test (experiment I, without metabolic activation): 3, 10, 30 µg/ml;
Concentration range in the main test (experiment II, without metabolic activation): 10, 30, 50 µg/ml.

Based on a dose range finding pre-test.

Vehicle / solvent:

- Deionised water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1x10^4 -6x10^4 cells per chamber
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 18 and 28 hours
Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hours
All cultures were incubated at 37° C in a humidified atmosphere with 4.5 % CO2 (95.5 % air).

PREPARATION OF THE CULTURES:
15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.5 h 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 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).
Additionally, two cultures per treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the substance is given as reduction of % cells as compared to the negative control.


METHODS FOR MEASUREMENT OF CYTOTOXICITY/GENOTOXICITY
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" (9)) 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 metaphases per culture were scored for cytogenetic damage on coded slides. 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 was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosome aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosome aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test. 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:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations: - Precipitation occured 4 hours after treatment at concentrations >= 100 µg/ml with S9-mix and at >= 30 µg/ml without S9-mix.


RANGE-FINDING/SCREENING STUDIES (if applicable):
The highest concentration used in the pre-test was 5000 µg/ml (highest guideline­recommended concentration. Severe toxic effects (no surviving cells) were observed in a concentration range from 300 - 5000 µg/ml in the absence of S9 mix.
With S9 mix 1000 and 5000 µg/ml reduced the cell number to 29.4 % and 12.6 %, respectively, of the solvent control.
Precipitation of the test article in the culture medium was observed starting at concen­trations of 30 µg/ml (without S9 mix) and 100 µg/ml (with S9 mix).


HISTORICAL CONTROL DATA
The aberration rates of the cells after treatment with the test article (0.0 % - 2.0 %) were in the range of the solvent control values (0.5 % -3.0 %) and in the range of the laboratory historical control data: 0.0 %-4.0 %.

Applicant's summary and conclusion

Conclusions:

As determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro, it can be stated that in the study described and under the experimental conditions reported, the substance induced reproducibly structural chromosome aberrations in the presence of S9 mix within a concentration range where precipitation of the test article was observed.
Therefore, the substance is considered to be mutagenic in this chromosome aberration test.

Executive summary:

The substance, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments according to First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 473, adopted May 26, 1983, „ In vitro Mammalian Cytogenetic Test" and EEC Directive 92/69, L 383 A, Annex V, B 10, dated December 29,1992.

The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosome aberrations.

Substantial reductions of the mitotic indices were observed in the absence of S9 mix, in experiment I after treatment with 10 and 30 µg/ml at preparation interval 18 hours. In the presence of S9 mix the mitotic indices were reduced after treatment with the highest evaluated concentrations (1000 µg/ml in experiment I and 3000 μg/ml in experiment II). In the presence of S9 mix, there were biologically relevant and statistically significant increases in cells carrying structural chromosome aberrations after treatment with 1000 μg/ml of the test article at fixation interval 28 h in experiment I as well as after treatment with 3000 μg/ml (18 h and 28 h fixation interval) in experiment II.

In the absence of S9 mix, no biologically relevant increase in cells carrying structural chromosome aberrations was observed.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells carrying structural chromosome aberrations.

In conclusion, as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro, it can be stated that in the study described and under the experimental conditions reported, the registration substance induced reproducibly structural chromosome aberrations in the presence of S9 mix within a concentration range where precipitation of the test article was observed.

Therefore, the substance is considered to be mutagenic in this chromosome aberration test.