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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registered substance did not cause genotoxic effects in bacteria (OECD 471,Ames-Prival) and mammalian cells (OECD 476, HGPRT & OECD 473, chromosomal aberration test in V79 cells)

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

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

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

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).

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'.

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.

Reference 2

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:

From30 OCT 2007 to 16 JAN 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 476), GLP compliant

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

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

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
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from phenobarbital/beta-naphthoflavone pretreated Wistar rats

Test concentrations with justification for top dose:

without S9 mix:
Experiment I: 2.0, 3.9, 7.8, 15.6, 31.3 (P), 250 (P) µg/ml
Experiment II: 3.9, 7.8, 15.6 (P), 31.3 (P), 62.5 (P), 125 (P) mg/ml

With S9 mix:
Experiment I: 2.0, 3.9, 7.8, 15.6 (P), 31.3 (P), 250 (P) µg/ml
Experiment IA: 7.8, 15.6, 31.3 (P), 62.5 (P), 125 (P), 250 (P) µg/ml

Not all test groups were evaluated (for evaluation points see executive summary)

Vehicle / solvent:

- DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I and Experiment IA: 4 h, experiment II: 24 h
- Expression time (cells in growth medium): 24 h
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 3+7+8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2 experiments, treatments performed in duplicate, both with and without metabolic activation

NUMBER OF CELLS EVALUATED: colonies with more than 50 cells

DETERMINATION OF CYTOTOXICITY
- Method: colony formation

Evaluation criteria:

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 spontanous 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 negative control data. If there is by chance a low spontanous mutation rate in the range normally found (0.5-31.8 mutants per 10 to the power of 6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into considration.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only in experiment I without metabolic activation at the maximum concentration (250 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment I: from 31.3 to 250.0 µg/ml without metabolic activation, from 15.6 to 250.0 µg/ml with metabolic activation (Exp I) and from 31.3 to 250 µg/ml with metabolic activation (Exp IA)
Experiment II: from 15.6 to 125 µg/ml

RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed with concentrations ranging from 2.0 to 250.0 µg/ml

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test substance did not increase the mutant frequency at the HPRT locus in Chinese hamster V79 cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD TG 476.

Chinese hamster V79 cells were treated with the test substance in the following concentrations:

without S9 mix:

Experiment I: 2.0, 3.9, 7.8, 15.6, 31.3 (P), 250 (P) µg/ml

Experiment II: 3.9, 7.8, 15.6 (P), 31.3 (P), 62.5 (P), 125 (P) µg/ml

With S9 mix:

Experiment I: 2.0, 3.9, 7.8, 15.6 (P), 31.3 (P), 250 (P) µg/ml

Experiment IA: 7.8, 15.6, 31.3 (P), 62.5 (P), 125 (P), 250 (P) µg/ml

Marked in bold: cultures which were not continued.

In experiment I and IA the treatment period was 4 h with and without metabolic activation. The second experiment was performed in the absence of metabolic activation with a treatment time of 24 h.

A precipitation of the test substance was noted in experiment I from 15.6 or 31.3 to 250 µg/ml and in experiment II from 15.6 to 125 µg/ml. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, indicating the satisfactory performance of the test system. The test substance demonstrated no statistically significant or dose related increases in mutant frequency at any dose level, either with or without metabolic activation, thus indicating that the test item is not mutagenic under the conditions tested.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 NOV 2004 to 07 DEC 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG471) with Prival modification for azo-dyes, GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 or hamster liver S9

Test concentrations with justification for top dose:

Experiment I (Plate incorporation method): 0, 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment II (Pre-incubation method):0, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle/ Solvent used: DMSO
- Justification for choice of solvent/ vehicle: solubility properties and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (rat liver S9 mix)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (TA 1535, TA 100, TA 1537 and WP2 uvrA), congo red (TA98)

Remarks:

with metabolic activation (hamster liver S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: plate incorporation method without and with induced rat liver S9 mix (induction with phenobarbital/beta-naphthoflavone)
Experiment II: pre-incubation method without and with non-induced hamster liver S9 mix.

DURATION:
-preincubation period (experimentII): 30 °C, for 30 min
- exposure duration: 48 h at 37 °C in the dark

NUMBER OF REPLICATIONS: 3 plates per strain and dose level including the controls

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the thresshold is regarded as an indication of mutagenic potential if reproduced in an independent second experiment. however, whenever the colony counts remain within the historical range of negative annd solvent controls such as an increase is not considered biologically relevant.

Statistics:

Arithmetic mean, standard deviation and the ratio of treated versus solvent were calculated .

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

minor toxic effects at concentrations of 2500 and 5000 µg/plate (for details see below)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

minor toxic effects at concentrations of 2500 and 5000 µg/plate (for details see below)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Did not occurr in experiment I at all, but in experiment II without activation (1000-5000 mg/plate) and with activation (2500-5000 µg/plate for T1535, TA 1537, TA 98, WP2uvrA, and 1000-5000 µg/plate for TA 100).
Nevertheless undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of colonies is slightly above the historical control range in strains TA 1535 (negative control, exp.I) and WP2 uvrA (solvent control, exp.I) without S9 mix and in strain WP2 uvrA (negative and solvent control, exp.I) with S9 mix. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
The historic range of positive controls was exceeded with metabolic activation in strains TA 1535 (exp.II) and WP2 uvrA (expI). This effect indicates the sensitivity of the strains rather than compromising the assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- minor toxic effects at concentrations of 2500 and 5000 µg/plate: reduced background growth in experiment I in all strains with and without metabolic activation; in experiment II in strain TA1535 and 1537 with metabolic activation

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and pre-incubation assay according to Prival) with and without metabolic activation.

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay.

Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the following concentrations: 0, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

The registered substance did not cause genotoxic effects in bacteria or mammalian cells in vitro.