Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

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:

2009-09-14 - 2009-10-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Guideline no. 471: "Genetic Toxicology: Salmonella typhimurium Reverse Mutation Assay". (adopted May 26, 1983)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

EC Number:

236-109-7

EC Name:

Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

Cas Number:

13170-05-3

Molecular formula:

C22H27AlO5

IUPAC Name:

aluminum hydroxide bis(4-tert-butylbenzoate)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At normal room temperature and dry in the sample storeroom.

Method

Target gene:

his (S. typhimurium)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% or 5% liver S9 in standard co-factors

Test concentrations with justification for top dose:

According to the result of the preliminary test, tetrahydrofuran was selected as the vehicle for the test. The maximum dose was 5mg/plate, followed by 2 mg/plate, 0.5 mg/plate, 0.2 mg/plate and 0.05 mg/plate.

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine for strain TA98 (200 µg/plate) -S9,

Remarks:

2-aminofluorene for strain TA97, TA98, TA100 (10 µg/plate) + S9, Danthron for strain TA102 (50 µg/plate) + S9, 2-aminoanthracene for strain TA1335 (3 µg/plate) + S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 1.1 - 1.6 E9 / ml
A frozen permanent was thawn from liquid nitrogen, a master plate was made, a well-isolated colony was selected and suspended it in 15 ml nutrient cultures, incubated for 12h in a 37°C dark incubator shaken at 120 rpm (at the time, the bacteria in the growing cultures was still at the logarithmic grow phase)
Preparation of medium and culture suspension: All the operations were performed according to SOP of in vitro mutagenicity test (Ames test) of Shanghai Institute for Preventive Medicine.

Dose selection:
Solubility in the plate: 100mg of the sample was weighed and added appropriated vehicle to 2ml, mixed extensively to form a 50 mg/ml solution. 400 µl, 100 µl, 40µl and 10 µl of the upper liquid were imbibed respectively, and appropriated vehicle was added to 1.0 ml to form solution of 20 mg/ml, 5 mg/ml, 2.0mg/ml and 0.5 mg/ml. 2 ml top agar was warmed in 45°C, added 100 µl above five different concentration sample solutions, mixed quickly and decanted onto the minimal glucose plates to make them evenly distributed and solidified on the horizontal table top. These plates were cultured in 37°C for 48h. Then dissolve condition was checked under 100x microscope

DETERMINATION OF CYTOTOXICITY
To determine the toxicity 100 mg of the test sample was weighed and added appropriated vehicle to 2ml, mixed extensively to form a 50 mg/ml solution. 400µl, 100 µl, 40 µl and 10 µl of the solution were imbibed and added appropriated vehicle to 1.0 ml to be a solution of 20 mg/ml, 5 mg/ml, 2.0 mg/ml and 0.5 mg/ml. 2 ml top agar was warmed in 45°C, 0.5 ml 0.1 mol/l PBS, 0.1 ml of the sample of five concentration, 0.1 ml enrichment solution of TA100 strain were added in turn, mixed quickly and decanted onto the minimal glucose plates. Plates were inverted, two plates per dosage group. A vehicle control was set at the same time. All the plates were cultured in 37°C for 48h. Number of bacterium colony of reversion per plate was recorded. And the bacterium colonies of background were observed, compared and described under 100* microscope to offer the evidence of determining the maximum dose of the formal test.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. EPA, OECD, EEC).

Statistics:

no details given

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: consistent in Formal Test 1 and 2

Applicant's summary and conclusion

Conclusions:

The result of this Ames Test was Negative. The substance Bis(4-(tert-butyl)benzoate-0) hydroxyaluminium (CAS No.: 13170-05-3) was considered not mutagenic.

Executive summary:

The Reverse Mutation Assay ("Ames") using Salmonella typhimurium was performed according to OECD TG 471. The plate incorporation method was applied.

Positive and negative controls gave the appropriate results. Hence, the results can be considered to be sufficiently reliable to assess the potential of Bis(4-(tert-butyl)benzoate-0) hydroxyaluminium to induce gene mutations in bacteria. Dissolving extent and reasonable dosage of solvent and medium were studied. 5 dosage groups of 0.05, 0.2, 0.5, 2 and 5 mg/plate were selected, with or without 2 concentrations (5% and 10%) of S9 mixture. The numbers of revertant colonies of 5 different strains of TA97, TA98, TA100, TA102 and TA1535 were tested.

Based on the results of this study it is concluded that Bis(4-(tert-butyl)benzoate-0) hydroxyaluminium is not mutagenic in the Salmonella typhimurium reverse mutation assay.