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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available in vitro data on the registered and the analogue substance, the registered substance is considered to be "not mutagenic/genotoxic".

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-12-07 to 2010-08-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expression period is terminated by adding 6-TG to the culture medium.

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; Technical University Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Thawed stock cultures are propagated at 37 °C in 80 cm2 plastic flasks. About 15 ml MEM (minimal essential medium; SEROMED, Berlin, Germany) supplemented with 10 % fetal bovine serum (FBS; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % neomycin.
- 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:

other: cells deficient in HPRT

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

Concentrations in µg/ml

Experiment I
Without S9 mix*: 95, 190, 380, 760, 1140, 1520
With S9 mix*: 190, 380, 760(p), 1140(p), 1520(p), 1900(p)

Experiment II
Without S9 mix**: 50, 100, 200, 400, 600, 800
With S9 mix*: 100, 200(p), 400(p), 800(p), 1200(p), 1400(p)
* 4 hours treatment ** 24 hours treatment p = precipitation

In the first main experiment the cultures at the lowest concentration of 95 µg/mL without metabolic activation and 190 µg/mL with metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures at the highest concentration of 1900 µg/mL in the presence of metabolic activation were not continued due to exceedingly severe toxic effects. In experiment II the cultures at 1400 µg/mL with metabolic activation were not continued to avoid analysis of too many precipitating concentrations. The cultures at 50 µg/mL without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: EMS, ethylmethane sulfonate; With metabolic activation: DMBA, 7,12-dimethylbenz(a)anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours (serum free medium with test item); Experiment II: 24 hours (test item in complete medium)
- Expression time (cells in growth medium): 15 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)

NUMBER OF REPLICATIONS: 2 replicates per test item concentration

NUMBER OF CELLS EVALUATED: Cell suspensions were seeded into plastic culture flasks. Approximately 1.5×10^6 (single culture) and 5×10^2 cells (in duplicate) were seeded in MEM with 10 % FBS (complete medium) for the determination of mutation rate and toxicity, respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxic effects as indicated by a relative cloning efficiency of less than 50 % in both parallel cultures occurred in experiment I at 1140 µg/mL and above without metabolic activation and at 1520 µg/mL and above with metabolic activation. In experiment II cytotoxic effects as described above were noted at 800 µg/mL without metabolic activation (24 hours treatment).

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: mutation rates, cloning efficiency survival, cloning efficiency viability

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 106 cells found in the solvent controls fall within the laboratory historical control data range.
- the positive control substances must produce a significant increase in mutant colony frequencies.
- the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria (as demonstrated in the annexes of attached to the study report).

Evaluation of Results
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

experimental group: p-value
experiment I, culture I without S9 mix: 0.975
experiment I, culture II without S9 mix: 0.073
experiment I, culture I with S9 mix: 0.333
experiment I, culture II with S9 mix: 0.555
experiment II, culture I without S9 mix: 0.354
experiment II, culture II without S9 mix: 0.080
experiment II, culture I with S9 mix: 0.265
experiment II, culture II with S9 mix: 0.328

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of 3.0.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: at 1140 µg/mL and above without metabolic activation, at 1520 µg/mL and above with metabolic activation. Experiment II: at 800 µg/mL without metabolic activation (24 hours treatment)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no relevant shift of pH of the medium even at the maximum concentration of the test item.
- Effects of osmolality: There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.
- Evaporation from medium: no observations reported
- Water solubility: test item basically well soluble in water
- Precipitation: Precipitation of the test item was noted at 760 µg/mL and above in the first experiment with metabolic activation. In experiment II precipitation was noted at 200 µg/mL and above with metabolic activation.
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency slightly exceeded the historical range of solvent controls at some of the test points. Neither one of those minor increases was dose dependent as indicated by the lacking statistical significance.

ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.7 up to 25.9 mutants per 10^6 cells; the range of the groups treated with the test item was from 7.9 up to 40.7 mutants per 10^6 cells.

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported the substance registered did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The registered substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study followed the protocol as given in OECD Guideline 476.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I: without S9 mix: 190; 380; 760; 1140; and 1520 µg/mL; with S9 mix: 190; 380; 760; 1140; and 1520 µg/mL Experiment II: without S9 mix: 100; 200; 400; 600; and 800 µg/mL; with S9 mix: 100; 200; 400; 800; and 1200 µg/mL

Precipitation of the registered substance was noted at 760µg/mL and above in the first experiment with metabolic activation. In experiment II precipitation was noted at 200µg/mL and above with metabolic activation.

Cytotoxic effects as indicated by a relative cloning efficiency of less than 50 % in both parallel cultures occurred in experiment I at 1140 µg/mL and above without metabolic activation and at 1520 µg/mL and above with metabolic activation. In experiment II cytotoxic effects as described above were noted at 800 µg/mL without metabolic activation (24 hours treatment).

No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of 3.0.

The mutation frequency slightly exceeded the historical range of solvent controls at some of the test points. Neither one of those minor increases was dose dependent as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.7 up to 25.9 mutants per 10^6 cells; the range of the groups treated with the test item was from 7.9 up to 40.7 mutants per 10^6 cells.

EMS (225 µg/mL in experiment I and 150 µg/mL in experiment II) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that under the experimental conditions reported the registered substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the registered substance is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Two in-vitro mutagenicity studies on the registered substance are available. These are supplemented by two in-vitro genotoxicity studies conducted on the analogue substance. A full justification for the appropriateness of the performed read-across approach is given as an appendix in chapter 13 of this dossier.

Two valid reverse bacterial mutation assays have been conducted, one guideline-conform Ames Test on the registered substance and one guideline-conform Ames Test on the analogue substance. Both gave a negative result in the presence and in the absence of exogenous metabolic activation. In a valid chromosomal aberration assay with and without metabolic activation conducted on the analogue substance, the test result was "not genotoxic". Furthermore, the valid mammalian gene mutation assay (HPRT) conducted on the registration substance, likewise revealed a negative result with and without S9-mix. Overall, a very consistent database of negative mutagenicity / genotoxicity test data is available for both, the analogue as well as the registration substance, which further substantiate the read-across approach used. Based hereupon, it is concluded that the registration substance is not mutagenic / genotoxic with and without exogenous metabolic activation.

Justification for selection of genetic toxicity endpoint
Guideline-conform study under GLP without deviations

Justification for classification or non-classification

The available in-vitro genotoxicity studies on the registered and the analogue substance revealed all negative results. On this basis the substance registered is not to be classified as to its genotoxic properties.