Benzoic acid, 4-[2-[2-oxo-1-[(phenylamino)carbonyl]propyl]diazenyl]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study, well documented

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Validity criteria were taken from the literature and DIN guideline, since in OECD and EC guideline no validity criteria are described.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-[(1E)-2-[2-oxo-1-(phenylcarbamoyl)propyl]diazen-1-yl]benzoic acid

EC Number:

603-014-0

Cas Number:

124772-04-9

Molecular formula:

C17 H15 N3 O4

IUPAC Name:

4-[(1E)-2-[2-oxo-1-(phenylcarbamoyl)propyl]diazen-1-yl]benzoic acid

Method

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

Metabolic activation:

with and without

Metabolic activation system:

S9-fraction from livers of male Wistar rats

Test concentrations with justification for top dose:

TA 1537, TA98, TA 100, TA 1535: 0.016-0.05-0.16-0.5-1.6 mg/plate
EC WP2 (uvrA): 0.05-0.16-0.5- 1.6-5.0 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

other: 4-Nitro-o-phenylene-diamine, 2-Aminoanthracene, Nitrofurantoine, ICR 191

Details on test system and experimental conditions:

TEST SYSTEM: Salmonella typhimurium strains TA 1537, TA 98, TA 100 and TA 1535 and Escherichia coli EC WP2 (uvrA) with (+) and without (-) metabolic activation system (S9). Salmonella typhimurium strains Escherichia coli EC WP2 and were selected according to OECD / EC guideline.

Origin:
University of California, Berkeley Divison of Biochemistry & Molecular Biology, CA 94720 USA (TA 98, TA 100)
TRINOVA BIOCHEM GMBH, Kerkrader Strafte 10, 35394 Gieften (E coli WP2 (uvrA), TA1535, TA1537) derived from master culture obtained directly from University of California)
Storage at test facility: Under liquid nitrogen (< -80 °C)
Inoculum: An overnight culture was prepared. Incubation was performed for 18 h at 37 °C. Titer of the overnight culture was > 1 x 108 cells/mL.

Metabolic activation system (S9):
As metabolic activation system a post-mitochondrial (S9) fraction from livers of male Wistar rats, which were induced with Phenobarbital intraperitoneally and ß-Naphtoflavone orally was used. The S9 fraction was received from CCR (In den Leppsteinwiesen 19, D-64380 Roftdorf). At test start an aliquot of the S9-fraction was thawed and enriched with cofactors according to DIN Guideline 38415 part 4 (S9-Mix). Batch number of each S9-fraction was 040205 (34.7 mg protein/mL S9).

Media: According to DIN Guideline 38415 Part 4 (December 1999)

Test container: Petri dishes with cams (0 94 mm, 16 mm high) with about 25 mL. VOGEL-BONNER minimal medium (for strain TA 97a with 0.4 % D+ Glucose, for alt other strains with 2 % D+ Glucose)

Application / Composition of test media: 2.0 mL top agar with 0.5 mM Histidin-/Biotin-solution for plates without metabolic activation system 1.5 mL top agar with 0.5 mM Histidin-/Biotin-solution for plates with metabolic activation system 0.5 mL S9-Mix for plates with metabolic activation system 0.1 mL test item-solution, reference item-sotution and aqua bidest. for controls, respectively 0.1 mL bacteria (overnight culture)

Titer control.: 2.0 mL top agar with 5 mM Histidin- / 0.5 mM Biotin-soiution, 0.5 mL S9-Mix, 0.1 mL bacteria (10"5 dilution from overnight culture)

Replicates: Three replicates per concentration level and control. Titer control replicates were prepared 2-fold.

Genotypes: The genotypes of the tested strains will be checked in the course of every study: histidine and tryptophan auxotrophy, respectively, ampiciliin resistance, UV-sensitivity and growth inhibition by crystal violet.

Based on the results of a preliminary test (NON-GLP) the test concentrations were selected. Stock solutions were freshly prepared with DMSO. Each inoculum was an overnight culture with a titer > 1 -10s cells/mL. Two independent studies were conducted as described above. Evaluations were performed as described below.

KIND AND FREQUENCY OF MEASUREMENT AND OBSERVATION

Genotypes were evaluated for each study. Plates of the mutagenicity test were inspected for present and reduced background lawn after an incubation time of 48 h. Colonies per plate (revertants) were counted, if no reduced background lawn was observed.
Plates with colonies which correspond not with the typical shape and colour of Salmonella typhimurium and Escherichia coli were regarded as contaminated and were not included in calculations.

Evaluation criteria:

Since a reduced background lawn is regarded to be a cytotoxic effect, plates with reduced background lawn were not included into evaluationprocedures. Arithmetic mean values and standard deviations were calculated from colonies per plate of three replicates.
For evaluation of the results the induction rate of the mean values was calculated (1):

Induction rate = revertant colonies of test item / revertant colonies of the corresponding control
The test item is to be interpretated mutagenic if a concentration effect relationship occurred and the induction rate is > 2.

The following genotypes of the tested strains had to be confirmed:
- Histidine auxotrophy / tryptophan auxotrophy
- Ampicillin resistance
- UV-sensitivity (except TA 102)
- Growth inhibition with crystal violet (rfa-mutation)

Titer of the overnight culture had to be > 1 • 108 cells/mL

Spontaneous revertants/plate (negative controls) should be within the following ranges:
-TA1537 ±S9 : 2-40
-TA 98 + S9 :15-50
-TA100 ±S9 :60 - 200
-TA 1535±S9 : 5-30
-EC WP2 + S9 : 3-100
The induction rates of the positve controls had to be > 2.

Results and discussion

Test results

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Revertant colonies of the test item plates are given in tables 5 - 24. Spontaneous revertants are listed in tables 3 - 4. The arithmetic mean value and the standard deviation were calculated out of the three replicates of the revertant colony plates. For evaluation of the results the induction rates of the mean values were calculated.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In this study Polyalkylene Yellow Monoazo was found to have no mutagenic effects on Salmonella typhimurium strains TA 1537, TA 98, TA 100 and TA 1535 Escherichia coli EC WP2 with (+) and without (-) the metabolic activation system S9 from male Wistar rats.