6-(2-chloro-6-cyano-4-nitrophenylazo)-4-methoxy-3-[N-(methoxycarbonylmethyl)-N-(1-methoxycarbonylethyl)amino]acetanilide

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:

26 October 1998 to 10 February 1999.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP methodology followed and OCED guideline 473 used to performed the experiment.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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: EEC Directive 92/69, L 383 A, Annexe V, B 10, dated December 29, 1992

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-500-8

EC Name:

-

Cas Number:

204277-61-2

Molecular formula:

Hill formula: C23 H23 Cl N6 O8 CAS formula: C23 H23 Cl N6 O8

IUPAC Name:

methyl 2-({4-[2-(2-chloro-6-cyano-4-nitrophenyl)diazen-1-yl]-5-acetamido-2-methoxyphenyl}(2-methoxy-2-oxoethyl)amino)propanoate

Test material form:

other: solid

Details on test material:

None

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: MEM (Minimal Essential Medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm.

Test concentrations with justification for top dose:

Pre-test:
Without S9 mix (4 hours and 24 hours exposure): 3.9 / 7.8 / 15.6 / 31.3 / 62.5 / 125.0 / 250.0 / 500 .0 microgr/ml.
With S9 mix (4 hours exposure; Rounded concentration due to technical reasons): 2 / 4 / 8 / 16 / 31 / 63 / 125 / 250 microgr/ml

Experiment I:
Without S9 mix (4 hours exposure): 7.5 / 15.0 / 30.0 / 60.0 / 90.0 / 120.0 microgr/ml.
With S9 mix (4 hours exposure): 7.5 / 15.0 / 30.0 / 60.0 / 90.0 / 120.0 microgr/ml

Experiment II:
Without S9 mix (continuous exposure 18 hours): 0.63 / 1.25 / 2.50 / 5.00 / 10.0 / 20.0 microg/ml (continuous exposure 28 hours): 0.63 / 1.25 / 2.50 / 5.00 / 10.0 / 20.0 microgr/ml.
With S9 mix (4 hours exposure): 3.8 / 7.5 / 15.0 / 30.0 / 60.0 / 90.0 microgr/ml.

Vehicle / solvent:

- Vehicle /solvent used: FAT 41024/B was dissolved in DMSO
On the day of the experiment (immediately before treatment), the test article was dissolved in DMSO (E. MERCK, D-64293 Darmstadt; purity 99.5 %). The final concentration of DMSO in the culture medium did not exceed 1 % (v/v). The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.

Controls

Untreated negative controls:

yes

Remarks:

Concurrent negative (culture medium) and solvent controls (DMSO) were performed.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

EMS (without metabolic activation); CPA (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:4 / 18 and 28 hours

NUMBER OF REPLICATIONS: 2 cultures in paralell

NUMBER OF CELLS EVALUATED:
Per culture 100 metaphase plates were scored for structural chromosome aberrations.

Evaluation criteria:

A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.

A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test (10) (p < 0.05). However, both biological and statistical significance should be considered together. If the a.m. criteria for the test article 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:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The test article FAT 41024/B, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments

were performed. The chromosomes were prepared 18 h (exp. I and II) and 28 h (exp. II) after start of treatment with the test article. In experiment I the exposure period was 4 h with and without metabolic activation. In experiment II the exposure period 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 metaphase plates were scored for structural chromosome aberrations.

The applicable concentration range of the test article for the cytogenetic experiments was determined in a range finder experiment using the reduction of cell numbers 24 h after start of treatment as indicator for cytotoxicity. The highest applied concentration in this pre-test

(500 ug/ml) was chosen with regard to the solubility properties of the test article and the current OECD Guideline for in vitro mammalian cytogenetic tests. Test article concentrations between 3.9 - 500 ug/ml (without S9 mix) and 1.9 - 250 ug/ml (with S9

mix) were chosen for the assessment of the cytotoxic potential. In the range finding experiment, toxic effects indicated by reduced cell numbers below 50 % of control were observed after 4 h treatment with 125 ug/ml in the absence of S9 mix (see table 4, page 24).

In addition, strongly reduced cell numbers were observed after 24 h continuous treatment with 15.6 ug/ml. In the presence of S9 no clear dose related toxicity was observed.

Precipitation of the test article in culture medium was observed after treatment with 62.5 ug/ml and above in the absence and 31.3 ug/ml and above in the presence of S9 mix. No influence of the test article on the pH value or osmolarity was observed (solvent control:

367 mOsm, pH 7.3 versus 394 mOsm and pH 7.3 at 500 ug/ml).

Toxic effects indicated by clearly reduced mitotic indices were observed in the absence of 59 mix after continuous treatment with 20 ug/ml (18 h: 43.9 % of control) and 10 ug/ml (28 h: 49.3 % of control). In the presence of S9 mix after 4 h treatment with 90 ug/ml the mitotic index was reduced at interval 28 h (39.4 % of control). In addition, reduced cell numbers were observed in the absence of S9 mix after 4 h treatment with 15, 30 and 60 ug/ml (58, 52 and 50 % of control). However, with the next higher concentrations (90 ug/ml and 120 ug/ml) lower reductions were found (57 and 69 % of control). This observation can be explained by different sizes of the precipitate, influencing the uptake of the precipitate by phagocytosis. In the presence of S9 mix the cell numbers were reduced at

both preparation intervals after treatment with 90 ug/ml (18 h: 50 % of control; 28 h: 54 % of control). However, in experiment I at interval 18 h, the next higher concentration (120 ug/ml) revealed higher cell numbers (70 % of control).

In experiment I, at preparation interval 18 h in the absence of S9 mix, the aberration frequency after treatment with 30 ug/ml was significantly increased to 4.0 % aberrant cells exclusive gaps. Since the increase was non dose related, within our historical control data range (0 - 4 % of control) and at a concentration exhibiting precipitation, the biological relevance is questionable. However, after 28 h continuos treatment in the absence of S9 mix a clearly dose related increase was observed after treatment with 0.0, 2.5, 5 and

10 ug/ml (1.0, 1.5, 3.0 and 9.0 % aberrant cells exclusive gaps). After 18 h continuous treatment no effect was observed.

In the presence of S9 mix only in experiment I significantly increased aberration frequencies beyond our historical control data range were observed. Evaluation of cultures after treatment with 30 and 90 ug/ml revealed slightly increased aberration frequencies (6.5 and 4.5 % aberrant cells exclusive gaps). In addition, a dose related increase was observed at experimental points without reduced cell numbers. Cultures after treatment with 0.0, 7.5, 15 and 30 ug/1 revealed a dose related increase in the number of aberrant cells exclusive gaps (1.0, 1.5, 3.0 and 6.5 % aberrant cells). The decrease after treatment with 90 ug/ml (4.5 % aberrant cells) might be caused by toxicity observed at this experimental point. However, the increase in the number of cells carrying exchanges after treatment with 30 and 90 ug/ml (3.5 % and 2.0 % aberrant cells) gives additional evidence for a clastogenic potential.

At interval 28 h in the presence of S9 mix, no increase was observed. The reason might be the late preparation interval. Aberrations, leading to cell death were not detected. Another point, which should be considered is the occurrence of aberrant cells only in the presence of precipitation (with S9 mix) or at the border of precipitation (without S9 mix). However, although the increased aberration frequencies were not reproduced, the overall assessment of the test article must be weakly clastogenic.

In both experiments, EMS (600 ug/ml and 800 ug/ml) and CPA (0.71 ug/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive weakly mutagenic

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article FAT 41'024/B induced structural chromosome aberrations in V79 cells (Chinese hamster cell line).

Executive summary:

This in vitro assay was performed to assess the potential of FAT 41024/B to induce structural chromosome aberrations. Evaluation of cytogenetic damage induced in V79 cells (cell line from the lung of the Chinese Hamster) in the absence and the presence of metabolic activation was performed in two independent experiments at one preparation interval (18 h) in experiment I and two preparation intervals (18 h and 28 h) in experiment II.

The experiment was performed according to the OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) and follows GLP methodology.

The test article FAT 41024/B, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. Following study design was performed:

	Without S9 -mix	Without S9 -mix	Without S9 -mix	With S9 -mix	With S9 -mix
	Exp. I	Exp. II	Exp.II	Exp. I	Exp II
Exposure period	4 h	18 h	28 h	4 h	4 h
Recovery	14 h	-	-	14 h	24 h
Preparation interval	18 h	18 h	28 h	18 h	28 h

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the range finder was chosen with regard to the solubility properties of the test article. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

Toxic effects indicated by clearly reduced mitotic indices were observed in the absence of S9 mix after 18 h (20 ug/ml) and 28 h (10 ug/ml) continuous treatment and in the presence of S9 mix after 4 h treatment with 90 ug/ml at interval 28 h . In addition, reduced cell numbers were observed in the absence of S9 mix after 4 h treatment with 1 5 - 6 0 ug/ml and in the presence of S9 mix after treatment with 90 ug/ml.

In the absence of S9 mix, a clearly dose related increase in the number of structural aberrations was observed after 28 h continuous treatment. In the presence of S9 mix slightly but dose related increased aberration frequencies were observed at interval 18 h at concentrations exhibiting precipitation only. These effects were not observed at interval 28 h.

In this study, no increase in the frequencies of polyploid metaphases was found after treatment with the test article 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.

Conclusion

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in

vitro.

Therefore, FAT 41'024/B is considered to be mutagenic, (weakly mutagenic), in this chromosome aberration test.