Potassium metaborate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Remarks:

reported in a publication

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

esting protocol based on the publications by Ames (1975), modified acc. Yahagi (1975), performed on strains TA100, TA98 only

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Absence of mutagenic activity of sodium borate (borax) and boric acid in the Salmonella pre-incubation test.

Author:

Benson W H, Birge W J, Dorough H W

Year:

1984

Bibliographic source:

Environmental Toxicology and Chemistry 3: 209-214.

Reference 2

Reference Type:

publication

Title:

Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test.

Author:

Ames, B.N.; et al.

Year:

1975

Bibliographic source:

Mutation Research 31, 347-364

Reference 3

Reference Type:

publication

Title:

Mutagenicity of carcinogenic azo dyes and their derivatives

Author:

Yahagi T, Degawa M, Seino Y, Matsushima T, Nagao M, Sugimura T, Hashimoto Y

Year:

1975

Bibliographic source:

Cancer Letters, 1 (1975) 91-96

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983 / only 2 strains tested

Principles of method if other than guideline:

The mutagenicity of borax and boric acid was examined in Salmonella typhimurium strains TA98 and TA100 by the preincubation method.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Boric acid

EC Number:

233-139-2

EC Name:

Boric acid

Cas Number:

10043-35-3

Molecular formula:

H3BO3

IUPAC Name:

Boric acid

Constituent 2

Reference substance name:

1303-96-4

EC Number:

603-411-9

Cas Number:

1303-96-4

IUPAC Name:

1303-96-4

Specific details on test material used for the study:

Sodium tetraborate (borax) (Na2B407- 10 H20) and boric acid (H3B03) were certified ACS-grade (Fisher Scientific, Fair Lawn, NJ).

Method

Target gene:

his-

Species / strain

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

The bacterial strains (TA98 and TA 100) were acquired from Dr. B. N. Ames (Biochemistry Department, University of California, Berkeley, CA). They are histidine-requiring mutants that lack an excision repair system and the lipopolysaccharide barrier, making them more sensitive and permeable to foreign compounds. These two strains contain the R-factor plasmid pKm 101 that codes for an error-prone DNA repair system, which increases their sensitivity for mutagenic expression. Strain TA98 is a frameshift mutant, whereas TA100 is a base-pair substitution mutant.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

In preliminary assays, borax and boric acid showed mutagenic activity at a level of 1 µg/plate; therefore, a range of doses from 0.01 to 100 µg/plate was chosen for study.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

The liquid preincubation procedures used to determine mutagenesis were similar to those described by Yahagi et al. Some mutagens that cannot be detected efficiently in the standard plate incorporation assay can be tested using a slight modification of the standard assay, wherein the test chemical is incubated with the S-9 mixture and bacterial culture before incorporation into the soft agar overlay. The modified procedure involved mixing 0.1 ml of the test solution(s), 0.5 ml of S-9 mixture or 0.2 M sodium phosphate buffer, and 0.1 ml of bacterial test strain culture (10E9 bacteria/ml), in the order given, to sterile glass tubes placed in an ice bath. The tubes were then incubated for 30 min at 37°C in an incubator-shaker. Following incubation, the tubes were again placed on ice.
Top agar containing just enough histidine to support several cell divisions was dispensed into each tube, in 2-ml aliquots, from a Fisher lsotemp Dry Bath (model 145) maintained at 37°C. Cellular toxicity was determined using 0.1 ml of a diluted nutrient broth culture (10E3 bacteria/ml) and top agar containing an excess of histidine (3 µmol/tube). The contents were mixed and poured on minimal glucose bottom agar previously added to a 15 x 100 mm petri plate. The plates were incubated for 48 h at 37°C and the mutagenicity was assayed by counting the number of histidine-revertant colonies per plate using a Biotran II Automated Colony Counter.
The S-9 fraction (9,000 x g supernatant of rat liver) used in the mutagenesis assays was obtained from male Sprague-Dawley rats (200-250 g body weight). Induction of the liver enzyme system was accomplished by injecting the animals intraperitoneally with Aroclor 1254 (500 mg/kg) 5 d before sacrifice. The resulting S-9 liver preparation was stored at -70°C prior to use. All the components of the Salmonella mutagenicity test were prepared as described by Ames et al.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The study was performed equivalent to OECD TG 471 with minor deficiencies in documentation and performance, positive and negative controls gave the appropriate response, but only 2 strains were tested. The results were negative, and the test item is so considered not to induce gene mutations in bacteria, and the results may be used as supporting information.

Executive summary:

In a reverse gene mutation assay in bacteria (similar to OECD 471 of 1983), strains TA98 and TA100, of S. typhimurium were exposed to Borax and Boric acid at concentrations of up to 100 µg/plate in the presence and absence of mammalian metabolic activation (induced rat liver S9) via pre-incubation. 

The positive controls induced the appropriate responses in the corresponding strains. There was no clear evidence of induced mutant colonies over background.