Benzoic acid, 2-[[3-(4-hydroxyphenyl)-1-oxopropyl]amino]-

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

Study period:

2004-04-14 to 2004-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2003-07-01

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

470-130-4

EC Name:

-

Cas Number:

697235-49-7

Molecular formula:

Hill formula: C16H15NO4 CAS formula: C16H15NO4

IUPAC Name:

2-((3-(4-Hydroxyphenyl)propanoylamino)benzoic acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical state: white powder
- Molecular formula: C16H15NO4
- Molecular weight: 285.3 g/mole

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared from Sprague-Dawley male rats (8-10 weeks old) induced with Aroclor 1254 (500 mg/kg body weight)

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Positive control with metabolic activation: dissolved in DMSO; concentrations: 0.8, 0.9 and 1.7 µg/plate; strains: all strains

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Positive control without metabolic activation: dissolved in distilled water; concentration: 50 µg/plate; strain: TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Positive control without metabolic activation: dissolved in distilled water; concentration: 0.15 µg/plate; Strain TA 102

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Positive control without metabolic activation: dissolved in DMSO; concentration: 2.5 µg/plate; strain: TA98

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control without metabolic activation: dissolved in distilled water; concentration: 0.7 µg/plate; strains: TA100 and TA1535

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Tests were performed with and without liver homogenate activation. To each culture tube containing 2 mL of top agar 0.1mL of bacteria was added followed by the test solution and 0.5 mL of S9-mix or phosphate buffer in the assays without metabolic activation. The test components were mixed thoroughly with a vortex and immediately poured onto coded minimal agar plates and carefully spread to achieve an uniform distribution of the top agar on the surface of the plate. The minimal agar plates contain 20 to 25 mL of 1.5 % agar in Vogel-Bonner medium E with 2 % glucose. Three parallel plates were prepared for each experimental point. Plates were kept for 48 to 72 h at 37°C in the dark and then examined. Besides the counting of the number of revertant colonies (his+ revertants), the plates were examined for the existence of a normal background lawn and/or precipitates and microscopically for microcolony growth. A reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of bacteriotoxicity.

The experiment was repeated in full alter an interval of at least 3 days.

Evaluation criteria:

not stated

Statistics:

Estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level was conducted using a X2-test (Mohn and Ellenberger, 1977)*.

*Reference
- Mohn G.R., and J. Ellenberger (1977), The use of E. coli K12/343/113 (lambda) as a multi-purpose indicator strain in various mutagenicity testing procedures, in: B.J. Kilbey (Ed.), Handbook of mutagenicity test procedures, Elsevier, Amsterdam, pp. 95-118

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

please refer to "Additional information on results below"

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test compound failed to induce a significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. Similarly, the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level did not reveal a significant effect at any one of the test points.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation of the test compound on the plates was not observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix the test item was bacteriotoxic towards the strains TA100 and TA1537 at 5000 µg/plate. In the presence of S9-mix the test item was bacteriotoxic towards the strain TA102 at 5000 μg/plate.

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

In conclusion, these results indicate that the test item under the experimental conditions described is not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.