Reaction mass of 5,5'-{(phenylmethanediyl)bis[benzene-4,1-diyl-diazene-2,1-diyl]}bis{1-[3-(dimethylamino)propyl]-4-methyl-6-oxo-3-(pyridinium-1-yl)-1,6-dihydropyridin-2-olate} hydrochloride and 5,5’-[3,4’-(phenylmethanediyl)diphenylene]bis(diazene-2,1-diyl)bis{1-[3-(dimethylamino)propyl]-4-methyl-6-oxo-3-(pyridinium-1-yl)-1,6-dihydropyridin-2-olate} hydrochloride

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007-10-10 to 2008-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations, conducted with analoge substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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:

other: Commission Directive 2000/32/EC, L1362000, Annex 4A: ”Mutagenicity – In vitro Mammalian Chromosome Aberration Test“, dated May 19, 2000.

Deviations:

no

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:

analogue substance (refer to IUCLID chapter 13)

IUPAC Name:

analogue substance (refer to IUCLID chapter 13)

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, 64287 Darmstadt, Germany) 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.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver microsome preparations (S9 mix)

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 9.8, 19.6, 39.1, 78.1, 156.3, 312.5, 625.0, and 1250.0 µg/ml
Concentration range in the main test (without metabolic activation): 19.6, 39.1, 78.1, 156.3, 312.5, and 625.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: no justification given

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent solvent controls (deionised water) were performed.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: EMS, ethylmethane sulfonate; with metabolic activation: CPA, cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration:
Experiment I: with metabolic activation 4 hours; without metabolic activation 4 hours
Experiment II: with metabolic activation 4 hours ; without metabolic activation 18 and 28 hours, respectively
- Recovery:
Experiment I: with metabolic activation 14 hours; without metabolic activation 14 hours
Experiment II: with metabolic activation 24 hours ; without metabolic activation 0 hours
- Fixation time (start of exposure up to fixation or harvest of cells):
Experiment I: with metabolic activation 18 hours; without metabolic activation 18 hours
Experiment II: with metabolic activation 28 hours ; without metabolic activation 18 and 28 hours, respectively

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
Chromosomal aberration: 200 cells per concentration
cytotoxicity: 1000 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, and mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

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

Evaluation of Results
A test item is classified as non-clastogenic if:
the number of induced structural chromosome aberrations in all scored dose groups is in the range of the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding 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 laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps).
and
either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher’s exact test (9) (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.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:
A test item can be classified as aneugenic if the number of induced numerical aberrations is not in the range of the historical control data range (0– 5.2 % polyploid cells)

Statistics:

Statistical significance at the five per cent level (p < 0.05) was scored by means of the Fisher´s exact test. Evaluation was performed only for cells carrying aberrations excluding gaps.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 312.5 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence of the test item on the pH value
- Effects of osmolality: No relevant influence of the test item on the osmolarity was observed (solvent control 277 mOsm, pH 7.3 versus 301 mOsm and pH 7.1 at 5000 µg/mL).
- Evaporation from medium: none reported
- Water solubility: test item basically well water soluble, however at higher concentrations precipitations occurred (see below)
- Precipitation: In the range finding test precipitation of the test item in culture medium was observed after 4 hours treatment with 312.5 µg/mL and above in the absence and presence of S9 mix. In Experiment I precipitation of the test item in culture medium was observed at preparation interval 18 hours with 625 µg/mL in the absence of S9 mix and with 39.1 µg/mL and above in the presence of S9 mix. In Experiment II precipitation of the test item in culture medium was observed at preparation interval 28 hours with 156.3 µg/mL and above in the presence of S9 mix
- Other confounding effects: In this study, toxic effects indicated by reduced cell numbers of below 50 % of control were observed in Experiment I after 4 hours treatment with 625 µg/mL (46.9 % of control). In all other experimental parts concentrations showing clear cytotoxicitiy were not evaluable for cytogenetic damage.

RANGE-FINDING/SCREENING STUDIES:
Dose selection of Experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24 hours exposure with 312.5 µg/mL and above. Therefore, 625 µg/mL was chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix 1250 µg/mL was chosen as top treatment concentration with respect to the results obtained in
Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA:
The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.0 - 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory’s historical control data range: 0.0 - 4.0 % aberrant cells, excluding gaps. The validity criteria for the test therefore were being fulfilled.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In a range finding pre-test on toxicity cell numbers were scored 24 hours after start of treatment as an indicator for cytotoxicity. Concentrations between 39.1 and 5000 µg/mL were applied. Clear toxic effects were observed after 4 hours treatment with 312.5 µg/mL and above in the absence and presence of S9 mix. In addition, 24 hours continuous treatment with 312.5 µg/mL and above in the absence of S9 mix induced strong toxic effects.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the analogue substance did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the analogue substance is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to precipitating concentrations or to the highest evaluable concentrations. The same genetic toxicity properties are likely to apply to the registered substance.

Executive summary:

This study was performed to investigate the potential of the analogue substance to induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. The study followed the protocol as given in OECD Guideline 473.

The analogue 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.

The following study design was performed:

	Without S9 mix			With S9 mix
	Exp. I	Exp. II	Exp. II	Exp. I	Exp. II
Exposure period (h)	4	18	28	4	4
Recovery (h)	14	-	-	14	14
Preparation interval (h)	18	18	28	18	18

In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosome aberrations. 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 for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In Experiment I in the absence of S9 mix, clear cytotoxicity was observed at the highest applied concentration. In all other experimental parts of this study, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was 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. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to precipitating concentrations or to the highest evaluable concentrations.

Given the applicability of the proposed read across approach (see IUCLID chapter 13) the same genetic toxicity properties are likely to apply to the registered substance as well.