4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide

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:

From 24 OCT 2007 to 28 FEB 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 473), GLP compliant

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, dated May 19, 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide

EC Number:

220-509-3

EC Name:

4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide

Cas Number:

2786-76-7

Molecular formula:

C26H22N4O4

IUPAC Name:

4-[(4-carbamoylphenyl)diazenyl]-N-(2-ethoxyphenyl)-3-hydroxy-2-naphthamide

Test material form:

solid: bulk

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: supplemented MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver S9 extract from phenobarbital/beta-naphthoflavone induced Wistar rats

Test concentrations with justification for top dose:

without metabolic activation (P: precipitation occurred)
Experiment 1: 2.0, 3.9, 7.8 (P), 15.6 (P), 31.3 (P), 62.5 (P), 125.0 (P), 250.0 (P) µg/ml
Experiment 2a: 0.13, 0.25, 0.5, 1.0 (P), 2.0 (P), 3.9 (P) µg/ml
Experiment 2b: 0.5, 1.0 (P), 2.0 (P), 3.9 (P) µg/ml

with metabolic activation (P: precipitation occured)
Experiment 1: 2.0, 3.9, 7.8 (P), 15.6 (P), 31.3 (P), 62.5 (P), 125.0 (P), 250.0 (P) µg/ml
Experiment 2: 1.0, 2.0, 3.9, 7.8 (P), 15.6 (P), 31.3 (P), 62.5 (P), 125.0 (P) µg/ml
Not all test groups were evaluated (for evaluation points see executive summary)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and low toxicity to 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:

Two independent experiments were performed:
Experiment 1 with an exposure duration of 4 h without and with metabolic activation and chromosome preparation 18 h after start of treatment
Experiment 2 with an exposure duration of 18 and 28 h (each without metabolic activation) and chromosome preparation 18 h and 28 h after start of treatment, respectively as well as an exposure duration of 4 h (with metabolic activation) and chromosome preparation at 28 h after start of treatment. Two parallel cultures were analysed per group.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without S9 mix: 4, 18 and 28 h; with S9 mix: 4 h
- Fixation time (start of exposure up to fixation or harvest of cells): without S9 mix: 18 and 28 h; with S9 mix: 18 and 28 h

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

NUMBER OF CELLS EVALUATED: 100 metaphase cells per slide (200 per dose from two independent cultures)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell numbers

Evaluation criteria:

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.

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 criteria is valid:
A test item can be classified as aneugenic if:
-the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 5.2.% polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant effect
- Effects of osmolality: no relevant effect
- Precipitation: observed at concentrations >=7.8 µg/mL (with S9 mix, Experiment I & II), >=1.0 µg/mL (without S9 mix, Experiment II) and >=7.8 µg/mL (without S9 mix, Experiment I)

RANGE-FINDING/SCREENING STUDIES:
No clear cytotoxicity was observed in the range-finding pre-test (test concentrations up to 250 µg/mL).

COMPARISON WITH HISTORICAL CONTROL DATA:
Results were within the range of historical control values

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was not observed up to the highest concentrations tested (250 µg/mL)

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

The test item did not induce structural chromosome aberrations in V79 Chinese Hamster lung cells in vitro without or with metabolic activation in this study.

Executive summary:

The test item (suspended 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 according to OECD TG 473. The highest applied concentration was determined in a pre-test on cytotoxicity and technical aspects (precipitation).

The following study design was performed with 2 independent cultures per group:

	without S9 mix			with S9 mix
	exp. I	exp. II		exp. I	exp. II
Exposure period	4 hrs	18 hrs	28 hrs	4 hrs	4 hrs
Preparation interval	18 hrs	18 hrs	28 hrs	18 hrs	28 hrs

The following concentrations were tested (evaluated experimental points in bold letters):

without metabolic activation:

Experiment 1: 2.0, 3.9, 7.8 (P), 15.6 (P), 31.3 (P), 62.5 (P), 125.0 (P), 250.0 (P) µg/ml

Experiment 2a: 0.13, 0.25 , 0.5, 1.0 (P), 2.0 (P), 3.9 (P) µg/ml

Experiment 2b: 0.5, 1.0 (P), 2.0 (P), 3.9 (P) µg/ml

with metabolic activation:

Experiment 1: 2.0, 3.9, 7.8 (P), 15.6 (P), 31.3 (P), 62.5 (P), 125.0 (P), 250.0 (P) µg/ml

Experiment 2: 1.0, 2.0, 3.9, 7.8 (P), 15.6 (P), 31.3 (P), 62.5 (P), 125.0 (P) µg/ml

P: precipitation occured

No toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item, but precipitation occured as indicated above.

100 metaphase per culture (i.e. 200 metaphase per concentration) were scored for structural chromosome aberrations. In both independent experiments, no 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.

Therefore, the test item was not clastogenic in this in vitro assay.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.