Isopropyl benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Peer-reviewed scientifically valid study, as evaluated in the expert panels in the WHO CICAD and OECD SIDS reviews.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1975

Report date:

1975

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid

EC Number:

200-618-2

EC Name:

Benzoic acid

Cas Number:

65-85-0

IUPAC Name:

benzoic acid

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Benzoic Acid, certified A.C.S., FDA Material # 73-70.
- Physical state: White Crystalline flakes

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

- Type and identity of media: The tissue homogenates and 9,000 x ~ supernatants were prepared from tissues of the following mammalian species: Mouse-ICR random bred adult males; rat-Sprague-Dawley adult males; and primate-Macaca mulatta adult males.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

- Type and identity of media: The tissue homogenates and 9,000 x ~ supernatants were prepared from tissues of the following mammalian species: Mouse-ICR random bred adult males; rat-Sprague-Dawley adult males; and primate-Macaca mulatta adult males.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

- Type and identity of media: The tissue homogenates and 9,000 x ~ supernatants were prepared from tissues of the following mammalian species: Mouse-ICR random bred adult males; rat-Sprague-Dawley adult males; and primate-Macaca mulatta adult males.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Species / strain 4

Species / strain / cell type:

Saccharomyces cerevisiae

Additional strain / cell type characteristics:

other: strain D4

Metabolic activation:

with and without

Metabolic activation system:

1. TPN (sodium salt) 6.0 µM 2. Isocitric acid 49 µM 3. Tri s buffer, pH 7.4: 28 µM 4. MgCl 2: 1.7 µM 5. Ti ssue homogenate fraction 72.0 mgs

Test concentrations with justification for top dose:

0.25, 0.50 and 1.00 percent
and in plate test: 0.50 percent

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test compound soluble under treatment conditions.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water, saline or Dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

ethylmethanesulphonate

other: Quinacrine mustard

Remarks:

Non activation assay

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

water, saline or Dimethylsulfoxide

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

N-dimethylnitrosamine

Remarks:

Activation Assay

Details on test system and experimental conditions:

The solubility, toxicity and doses for all chemicals were determined prior to screening. Each chemical was tested for survival against the specific indicator strains over a range of doses to determine the 50% survival dose. The 50% survival curve and he 1/4 and 1/2 50% doses were calculated. A maxiumum dose of 5% (w/v) was used.
Plate tests: In the nonacti vati on proced ure, approximately 10E9 cells of a log-phase culture of the bacterial indicator strains were spread over the surface of a minimal plate, and a measured amount of the test chemical was placed in the center of the test plate. In activation tests, the test chemical was added to the cells, and an aliquot of the mixture was spread on the surface of the test plate. The reaction mixture (0.1 ml ) plus tissue extract was then spotted on the surface of the plate. Positive and solvent controls were included. All plates were incubated at 37°C for four days and then scored. Each compound (test, posi tive control and solvent control) was done in duplicate.

Suspension (preincubation) tests: Log-phase bacteri a and stationary-phase yeast culures of the indicator organisms were grown in compl ete broth, washed and resuspended in 0.9% saline to densi ties of 1E9 cel ls/ml and 5E7 cells/ml , respectively. This constituted the working stock for tests of a group of test chemicals and their respecti ve controls. Tests were conducted in plastic tissue culture plates. The solvent repl aced the test chemical in the negati ve controls. Treatment was at 30°C for four hours for yeast tests and at 37°C for one hour for bacterial tests. All flasks were shaken during treatment . Following treatment, the pl ates were set on ice. Aliquots of cells were removed, diluted in sterile saline (4°C) and pl ated on the appropriate complete media. Undiluted samples from flasks containing the bacteria were pl ated on minimal selective medium in reversion experiments. Samples from a 1/10 dilution of treated cells were plated on the selected media for enumeration of gene conversion wi th strain 04. Bacterial plates were scored after incubation for 48 hours at 37°C. The yeast plates were incubated at 30°C for 3-5 days before scoring.

Activation: Bacteri a and yeast cells were grown and prepared as described in the nonactivation tests. Measured amounts of the test and control chemicals plus 0.25 ml of the stock-cell suspension were added to wells of the plate containing the appropriate tissue fraction and reaction mixture . All flasks (bacteria and yeast) were incubated at 37°C in an oxygen atmosphere with shaking. The treatment times as well as the dilutions, plating proced ures and scoring of the plates were the same as described for nonactivation tests.

Evaluation criteria:

Positive gene mutation in TA 1535, and TA 1537 and TA 1538 strains of the bacteria S. typhimurium.

Statistics:

Data recording and reporting: Following the specified incubation periods all population plates were scored by an automatic colony counter and the results from each plate of a set were recorded , in ink , on data processing forms. All minimal or other types of selective media plates were hand scored and the results recorded along with the respective population data. Other rel vant experimental data were recorded on experimental definition forms. For bacteri strains the number of colonies recorded from either the population or selective plates represents that number in 1 ml of test suspension plated. The numbers recorded for the yeast strain D4 represent the number in 0.5 ml of test suspension plated. The data were then processed and printed from a computer program.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1538

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

Species / strain:

S. typhimurium TA 1537

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 3

Species / strain:

S. typhimurium TA 1535

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 4

Species / strain:

Saccharomyces cerevisiae

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:

RANGE-FINDING/SCREENING STUDIES: The 50% survival level was determined for bacteria and yeast indicator organisms by conducting survival curves with the test compound at the following concentrations: Percent (w/v or v/v) 5.0, 0.5, 0.05, 0.005, 0.0005.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No increase in the incidence of mutations occurred at any dose of the test material in any of the strains of Salmonella or yeast. There was no increase with metabolic activation. The conclusion is that the test substance is not mutagenic.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Benzoic Acid, was not considered to be mutagenic in the Ames assays (Salmonella typhimurium strains 1535, 1537 and 1538, and Saccharomyces cervesia straom D4) used in this evaluation. This study is informative for evaluation of the toxicity and environmental fate of members of the Alkyl Benzoates category, and is adequate for filling the data requirement for the registration of this substance. It is valid for hazard classification and risk assessment.