Ammonium benzoate

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:

20. Mar. 2017 - 07. Apr. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium benzoate

EC Number:

217-468-9

EC Name:

Ammonium benzoate

Cas Number:

1863-63-4

Molecular formula:

C7H6O2.H3N

IUPAC Name:

ammonium benzoate

Test material form:

solid: crystalline

Details on test material:

- State of aggregation: solid
- Particle size distribution: median particle size D50 = 98 µm
- Mass median aerodynamic diameter (MMAD): not determined
- Geometric standard deviation (GSD): not determined
- Shape of particles: not determined
- Surface area of particles: not determined
- Crystal structure: not determined
- Coating: none
- Surface properties: none known
- Density: 1.26
- Moisture content: ca. 1.5 %
- Residual solvent: none known
- Activation: none
- Stabilisation: none
- Other: none

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
608031
- Expiration date of the lot/batch:
25. Oct. 2018
- Purity test date: not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature, in a tightyl closed vessel
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle:
not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

Method

Target gene:

histidine deficiency

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 5

Species / strain / cell type:

S. typhimurium, other: TA 97a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

concentrations for experiment I:
5000, 1500, 500, 150 and 50 µg/plate
concentrations for experiment II:
5000, 2500, 1250, 625, 313, 156 and 78 µg/plate

top dose according to guideline

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L (nominal) in demineralized water, dimethyl sulfoxide and ethanol. Demin. Water was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro,1,2-phenylene Diamine, 2-Amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;
- Cell density at seeding (if applicable): not applicable

DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS:3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable


NUMBER OF CELLS EVALUATED: not applicable


NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable


CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: visual inspection of bacterial background, reduction of revertant colonies
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
not applicable

Rationale for test conditions:

according to Guideline

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

except for calculation of mean values, no statistical analysis performed.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

only in Exp. II in highest concentration tested, cytotoxicity was noted

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Ammonium Benzoate is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

Executive summary:

Two valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item Ammonium Benzoate was tested in the Salmonella typhimurium reverse mu-tation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in two experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

In the first experiment, Ammonium Benzoate (dissolved in demin. water) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

Ammonium Benzoate showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no rele-vant decrease in the number of revertants was observed in all bacteria strains. The test item Ammonium Benzoate showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

On the base of the first experiment, Ammonium Benzoate was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in all bacteria strain using the pre-incubation method. Ammonium Benzoate showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not present at the highest concentration (5000 μg/plate) and no bacterial growth of revertant colonies was visible.

The results of this experiments showed that the test item Ammonium Benzoate caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item Ammonium Benzoate did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Based on the results of this study it is concluded that Ammonium Benzoate is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study