Hexaboron dizinc undecaoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20/08/2010 to 28/09/2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Read-across is justified on the following basis: The family of zinc borates that include Zinc Borate 500, Zinc Borate 2335 and Zinc Borate 415 (also known as Zinc Borate 411). Zinc borate 500 is anhydrous Zinc Borate 2335 and Zinc Borate 415 has different zinc to boron ratio. Zinc borate 2335 (in common with other zinc borates such as Zinc borate 415 and 500) breaks down to Zinc Hydroxide (via Zinc oxide) and Boric Acid, therefore the family of zinc borates shares the same toxicological properties. Zinc borates are sparingly soluble salts. Hydrolysis under high dilution conditions leads to zinc hydroxide via zinc oxide and boric acid formation. Zinc hydroxide and zinc oxide solubility is low under neutral and basic conditions. This leads to a situation where zinc borate hydrolyses to zinc hydroxide, zinc oxide and boric acid at neutral pH quicker than it solubilises. Therefore, it can be assumed that at physiological conditions and neutral and lower pH zinc borate will be hydrolysed to boric acid, zinc oxide and zinc hydroxide. Hydrolysis and the rate of hydrolysis depend on the initial loading and time. At a loading of 5% (5g/100ml) zinc borate hydrolysis equilibrium may take 1-2 months, while at 1 g/l hydrolysis is complete after 5 days. At 50 mg/l hydrolysis and solubility is complete (Schubert et al., 2003). At pH 4 hydrolysis is complete.

Zinc Borate 2335 breaks down as follows: 2ZnO • 3B2O3 •3.5H2O + 3.5H2O + 4H+ ↔ 6H3BO3 + 2Zn2+ 2Zn2+ + 4OH- ↔ 2Zn(OH)2

Overall equation: 2ZnO • 3B2O3 •3.5H2O + 7.5H2O ↔ 2Zn(OH)2 + 6H3BO3

The relative zinc oxide and boric oxide % are as follows: Zinc borate 2335:zinc oxide = 37.45% (30.09% Zn) B2O3 = 48.05% (14.94% B) Water 14.5% Zinc borate 415: zinc oxide = 78.79%; (63.31% Zn) B2O3 = 16.85% (5.23% B) Water 4.36% Zinc borate, anhydrous: Zinc oxide = 45 % B2O3= 55% (17.1 % B)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

138265-88-0

EC Number:

604-070-9

Cas Number:

138265-88-0

IUPAC Name:

138265-88-0

Details on test material:

- Name of test material: Zinc borate Firebrake ZB
- Lot/batch No.: 29K16
- Storage condition of test material: Room temperature.

Method

Target gene:

Thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Clone 3.7.2.C purchased from ATCC and maintained in log phase growth by serial sub-culturing in a shaker incubator at 37 °C. To reduce the frequency of spontaneous TK-/- mutants, cell cultures were cleansed of pre-existing TK-/- mutants by exposing them to thymidine, hypoxanthine, methotrexate and glutamine (THMG) for approximately 24 h to select against the TK-/- phenotype. Cells were periodically tested for mycoplasma and were found uninfected.
The cells were cultured in RPMI-1640 supplemented with HEPES and l-glutamine, 10 % heat-inactivated horse serum, Penicillin G and streptomycin sulfate, sodium pyruvate and Pluroni F-68 (referred to as completed media). During treatment with test articles, the horse serum concentration was reduced to 5 %.
The cloning media consisted on 2.36 g/L agar in complete medium. The molten agar was added to the complete medium and dispensed into culture flasks and stored at 37 °C until use.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial S9 fraction was used. The S9 mixture consisted of 25% S9 5.0 mmol/L glucose-6-phosphate monosodium, 0.8 mmol/L NADP and RPMI-1640 media. The S9 mix was filter sterilised and kept in an ice bath until use.

Test concentrations with justification for top dose:

Initial mutagenicity assays: 0.019, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.50 and 5.00 mg/mL.
Repeat initial assay: 0.005, 0.015, 0.020, 0.030, 0.050 and 0.075 mg/mL in the S9 activated system; and 0.0001, 0.00025, 0.001, 0.005, 0.015, 0.020 and 0.040 mg/mL in the non activated system.
Confirmatory assay: 0, 0.001, 0.005, 0.015, 0.020, 0.040 and -.075 mg/mL in the S9 activated test system; and 0, 0.0001, 0.00024, 0.005, 0.015, 0.020 and 0.050 mg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1 % glycerol in ASTM Type 1 water.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

1 % glycerol in ASTM Type 1 water.

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: In the absence of metabolic activator.

Controls 2

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: In the presence of metabolic activator.

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Preincubation period:
- Exposure duration:
Initial assay: 3 h for each test system.
Confirmatory assay: 3 h for the S9-activated test system and 24 h for the non-activated test system.
- Expression time (cells in growth medium): 24 h (with and without metabolic activation) for the 3 h exposure cultures and approximately 48 h after treatment initiation for the 24 h continuous exposure cultures (without metabolic activation). The cultures were counted and diluted with fresh media and returned to the roller drum. This process was repeated approximately 24 h later.

SELECTION AGENT: Trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

Evaluation criteria:

A response induced by the test article was considered positive (mutagenic) under the following conditions:
1. mutant frequency increases in a concentration-related manner and
2. the highest achieved mutant frequency is twice that of the vehicle control with RTG not less than 10 %.
In the absence of a concentration-related increase, the response was considered positive under the following condition: At least once concentration of the test article induced twice the mutant frequency of the vehicle control with RTG above 10 %.

Statistics:

Data is presented as the number of TFT resistant mutant colonies/10E6 survivors and mean mutant frequency/10E6 survivors ± standard deviation. For analysis the mean mutant frequency/10E6 survivors of each test group was compared to the mean mutant frequency/10E6 survivors of the vehicle control group. The individual plate count data was expressed as number of mutant colonies/10E6 survivors for each concentration of test article.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity assay (repeat initial mutagenicity assay)
The initial cytotoxicity/mutagenicity assay resulted in an insufficient number of analysable dose levels and this portion of the study was aborted. The target dose levels were adjusted and the assay was repeated. In the repeat initial cytotoxicity/mutagenicity assay, in the S9-activated test system, cytotoxicity (100 - RTG) ranged fro 34 % to 60 % relative to the vehicle. In the non-activated system, cytotoxicity ranged from no-toxic to 65 %/
In the initial cytotoxicity/mutagenicity assays, no notable increase in mutation frequency was observed in the presence or absence of metabolic activation with zinc borate. In both test systems there was no evidence of a concentration responsive mutation frequency increase in the test system.

Mutageniciyt assay (confirmatory mutagenicity assay)
In the confirmatory mutagenicity assay, when testing zinc borate in the S9-activated test system, cytotoxicity (100 - RTG) ranges from non-toxic to 52 % relative to the vehicle control. In the non-activated test system, cytotoxicity ranged from 33 % to 85 %.
In the confirmatory mutagenicity assays, no notable increase in mutation frequency was observed in the presence or absence of metabolic activation with zinc borate. In both test systems there was no evidence of a concentration responsive mutation frequency increase in any test system.

Both positive controls exhibited greater than a two-fold increase in TK-/- resistant colonies as compared to the vehicle controls.

Applicant's summary and conclusion

Conclusions:

The test item was considered negative in the mouse lymphoma assay under the experimental conditions, with and without metabolic activation.

Zinc borate was insoluble and displayed excessive cytotoxicity throughout much of the dose range tested. In the analyzable dose levels of the initial assay (cytotoxicity/mutagenicity assay) and the confirmatory assay there was no significant increase in the mutation frequency in either the metabolically activated or non-activated test systems. Zinc borate is therefore considered to be negative in the mouse lymphoma assay.
Read-across is justified on the basis detailed in the rationale for reliability above. This study is therefore considered to be of sufficient adequacy and reliability to be used as a supporting study.