N,N'-(2-chloro-1,4-phenylene)bis[4-[(2,5-dichlorophenyl)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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

published in O.J. L 142, 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-(2-chloro-1,4-phenylene)bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

EC Number:

226-106-9

EC Name:

N,N'-(2-chloro-1,4-phenylene)bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Cas Number:

5280-78-4

Molecular formula:

C40H23Cl5N6O4

IUPAC Name:

N,N'-(2-chloro-1,4-phenylene)bis{4-[(2,5-dichlorophenyl)diazenyl]-3-hydroxy-2-naphthamide}

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: red powder
- Analytical purity: ca. 99%
- Impurities (identity and concentrations): benzene, 1,2-dichloro- (ca. 0.2%), water (ca. 0.8%)
- Lot/batch No.: U7006/2007
- Expiration date of the lot/batch: 02/2023
- Storage condition of test material: ambient temperature

Method

Target gene:

his- / trp-gene (Salmonella/ E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Delor 106 (mixture of PCBs) induced rat liver S9 mix

Test concentrations with justification for top dose:

- 1st experiment: 50, 150, 500, 1500, 5000 µg/plate
- 2nd experiment: 15, 50, 150, 500, 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance not soluble in water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see Details on test system and conditions

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

100 µl of the test substance of required concentration, 0.1 ml 16-18h culture of the tester strain, 0.5 ml relevant buffer and 30 or 100 µl of S9 postmitochondrial fraction (in case of test with metabolic activation) were added to the 2 ml top agar (with trace of histidine or tryptophan) kept in a test tube at 45 ± 3°C. After shaking the mixture was poured into a minimal glucose agar plate. The number of revertant colonies on the plate was counted manually or by using an AccuCount 1000 after 48 - 72 h incubation at 37 ± 1°C.

NUMBER OF REPLICATIONS: Triplicate

Positive controls:

- without metabolic activation:

Sodium azide (AS), 1.5 µg per plate
4-nitro-o-phenylenediamine (NPD), 20 µg per plate
9-aminoacridine hydrochloride monohydrate (AAc), 100 µg per plate
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 20 µg per plate

- with metabolic activation:

2-aminoanthracene (2-AA), 1.0 µg per plate -TA 1535, 2.5 µg per plate - TA 1537, 25 µg per plate - E.coli
2-aminofluorene (AF), 10 µg per plate

Results and discussion

Test results

Test results 1

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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(see Additional information on results for details)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Following information is available for the preliminary toxicity test: From 500 µg per plate, the test substance made precipitates in top agar, which was homogenized by shaking on Vortex shaker. Sometimes, "maps" of the test substance were observable on dishes. Bacterial background was not evaluable from 500 µg per plate due to thick layer of the test substance in background, but according to the normal number of revertants, colony size and distribution it was assumed, that the test substance is not toxic in any dose. At the highest dose, evaluation was difficult, because of the coloured layer was too thick and non transparent. Colony counting had to be made from the upper side of Petri dishes.
For the main test it is reported that all tubes with top agar and the test substance were shaken on Vortex shaker before pouring onto plates. The test substance made homogenous layer on plates, but at various concentrations sometimes occurred dishes with "maps" of the test substance in the background. The test substance was not toxic in any dose.

RANGE-FINDING/SCREENING STUDIES: At first, the test substance (insoluble) was suspended in water for injections. As it was badly wettable it was not possible to achieve homogenous suspension in the highest concentration recommended in the guidelines (5000 µg per 0.1 mL). Therefore, the test substance was suspended in dimethylsulfoxide (DMSO) till the required concentration. In the DMSO, the test substance was weIl suspended. Concentration range generated from the maximum concentration by dilution was then tested for toxicity in strain TA 100.

Any other information on results incl. tables

1st experiment (standard plate test; 50 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	17	1.1	no	negative
	yes	29	0.9	no	negative
TA 100	no	113	1.0	no	negative
	yes	125	1.0	no	negative
TA 1535	no	20	1.5	no	negative
	yes	19	1.2	no	negative
TA 1537	no	18	1.2	no	negative
	yes	18	1.1	no	negative
WP2 uvr A	no	24	1.2	no	negative
	yes	25	1.1	no	negative
2nd experiment (standard plate test; 15 - 1500 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	27	1.0	no	negative
	yes	39	1.1	no	negative
TA 100	no	104	1.1	no	negative
	yes	112	1.1	no	negative
TA 1535	no	21	1.3	no	negative
	yes	19	1.3	no	negative
TA 1537	no	10	1.2	no	negative
	yes	15	1.1	no	negative
WP2 uvr A	no	28	1.0	no	negative
	yes	29	1.1	no	negative

Applicant's summary and conclusion

Conclusions:

negative