4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 16 January 2001. Experimental completion date: 6 February 2001.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

EC Number:

260-152-0

EC Name:

4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Cas Number:

56396-10-2

Molecular formula:

C25H20N4O4

IUPAC Name:

4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Test material form:

solid: particulate/powder

Remarks:

(magenta)

Details on test material:

For all studies conducted on substance, where identifier was ST463, purity was stated as >98 - 99.9% (depending on batch)

Specific details on test material used for the study:

Identity: ST 463
Chemical name: 2-Naphthalenecarboxamide, 3-hydroxy-4[[2-
methoxy-5-[(phenylamino)carbonyl] phenyl]
azo]-
Appearance: Magenta powder
Storage conditions: Room temperature
Lot number: 6611
Expiry date: 1 August 2001
Purity: 99.9%
Date received: 30 November 2000

Method

Target gene:

histidine locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, rat livers stimulated by Aroclor 1254, was used as the metabolic activation system

Test concentrations with justification for top dose:

First test (range finding): 5, 15, 50, 150, 500, 1500, 5000 µg/plate. (This is the standard limit concentration recommended in the regulatory guidelines this assay follows).
Second test: 50, 150, 500, 1500, 5000 µg/plate.

No visible thinning of the background lawn of non-revertant cells was obtained following exposure to ST 463 in the range-finding test. A maximum exposure concentration of 5000 pg/plate was, therefore, selected for use in the second test.

Vehicle / solvent:

The test substance was reported to be insoluble in solvents compatible with the test system.
Suspensions of the test substance were, therefore, prepared in purified water (obtained by reverse osmosis) containing 0.15% agar.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strains TA1535 and TA100

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

30 µg/plate for strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for strain TA98

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.05 µg/plate for strain WP2uvrA/pKM101 (CM891)

Positive control substance:

other: 2-2(-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

without metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for strain WP2uvrA/pKM101 (CM891). 2 µg/plate for strain TA1535.

Positive control substance:

other: 2-Aminioanthracene

Remarks:

with metabolic activation

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate for strains TA1537, TA98 and TA100

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

MUTATION TEST PROCEDURE:
First test (range-finding):
The test substance was added to cultures of the five tester strains at seven concentrations separated by ca half-log10 intervals. The highest concentration of ST 463 tested was 50 mg/ml in the chosen solvent, which provided a final concentration of 5000 µg/plate. This is the standard limit concentration recommended in the regulatory guidelines this assay follows. The negative control was the chosen solvent, purified water containing 0.15% agar. The appropriate positive controls were also included.

An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S9 mix or 0.5 ml 0.1 Msodium phosphate buffer (pH 7.4) were placed in glass tubes. An aliquot of 0.1 ml of the test dilution, positive or negative control was added, followed immediately by 2 ml of molten agar containing 0.5mM histidine/biotin/tryptophan. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Each petri dish was individually labelled with a unique code corresponding to a sheet, identifying the dish’s contents. Three petri dishes were used for each concentration. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at 37°C for ca 72 hours. After this period the appearance of the background bacterial lawn was examined and revertant colonies counted using a Domino automated colony counter.

Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration normally used in the second test would be the same as that used in the first. If toxic effects were observed a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at the top concentration. Ideally a minimum of three non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating dose levels should be obtained, unless otherwise justified by the Study Director.

Second test:
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. 5000 µg/plate was again chosen as the top concentration, but only five concentrations were used.

Evaluation criteria:

Refer to any other information on materials and methods 'assessment of results'.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results obtained with ST 463 and positive control compounds are presented in Tables 1-10 (attached background material).

The absence of colonies on sterility check plates confirmed the absence of microbial contamination.

The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.

The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory (except TA98 and TA100, test 2, and TA1537, where slightly higher counts were obtained; this was not considered to affect the integrity of the study). Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

FIRST TEST (RANGE-FINDING):
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to ST 463 at any concentration in either the presence or absence of S9 mix.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to ST 463. A maximum exposure concentration of 5000 pg/plate was, therefore, selected for use in the second test.

SECOND TEST
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to ST 463 at any concentration in either the presence or absence of S9 mix.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to ST 463

Applicant's summary and conclusion

Conclusions:

It is concluded that, under the test conditions employed, ST 463 showed no evidence of mutagenic activity in this bacterial system.

Executive summary:

This report describes a study designed to assess the mutagenic potential of ST 463 in a bacterial system.

In this in vitro assessment of the mutagenic potential of ST 463, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to the test

substance diluted in purified water containing 0.15% agar. Purified water containing 0.15% agar was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). The first (range-finding) was a standard plate incorporation assay; the second involved a pre-incubation stage.

Concentrations of ST 463 up to 5000 µg/plate were tested in the mutation tests. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log₁₀ dilutions of the highest concentration. No signs of

toxicity were observed towards the tester strains in either mutation test.

No evidence of mutagenic activity was seen at any concentration of ST 463 in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions employed, ST 463 showed no evidence of mutagenic activity in this bacterial system.