Hexaboron dizinc undecaoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro mammalian cell micronucleus test, OECD 487, GLP, zinc borate anhydrous (CAS 12767-90-7, Firebrake 500) (Geissel, 2019), positive

in vitro gene mutation studies in bacteria, OECD 471, GLP, zinc borate hydrate (CAS 138265-88-0, Firebrake ZB) (Entrup, 1981), negative

in vitro gene mutation studies in mammalian cells, OECD 476, GLP, zinc borate hydrate (CAS 138265-88-0, Firebrake ZB) (Curry, 2010), negative

Link to relevant study records

Reference

Reference 1

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)

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

Target gene:

Thymidine kinase

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.

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 JULY 1981 to 23 JULY 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Remarks:

Study pre-dates GLP; QA statement included.

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1, 10, 100, 500, 1000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Nonactivation assay: N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 9-Aminoacridine (9-AA), 2-Nitrofluorene (2-NF): Activation assay, 2-Aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS:3

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

Evaluation criteria:

The spontaneous revertant levels for each strain when used in either the direct plate assay or the activated plate assay must be within the acceptable limits as defined by the historical data
all sterility controls must be negative
all positive controls must demonstrate that the indicator strains are functional with known mutagens as evidenced by an increase of at least three times the number of revertant colonies per plate as the spontaneous revertant controls
To be considered positive for mutagenic activity, the test material should exhibit a dose response effect (increasing numbers of revertant colonies with increased amounts of the test sample)

Statistics:

Least squares linear regression analysis was used to compute the "best fit" regression line of dose response on dose level for the test sample. These regression lines represent the strength of the dose response obtained against each bacterial indicator strain. For each response line y = mx + b, the smaple coefficient, m, was tested for a statistically significant deviation from the value zero which would indicate mutagenic activity.
Significance level = 0.05

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed to strains TA 1537 and TA 100 at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed to strains TA 1537 and TA 100 at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Sample toxicity

Sample	Concentration µg/Plate	% growth inhibition
		TA 1537	TA 100
Firebrake ZB	10000	100	100
	5000	100	100
	1000	0	0
	100	0	0
	10	0	0
Controls
DMSO	0.1 ml/plate	0	0
9-AA	100µg/plate	0	-
MNNG	5µg/plate	-	0

Results of salmonella/microsomal assay with metabolic activation

Compound	Concentration/plate [µg]	Revertants per plate (mean) of bacterial strains
		TA 1535	TA 1537	TA 1538	TA 98	TA 100
Solvent control		17.7	6.7	14.0	57.3	133.0
Negative control	50µl	12.7	8.7	25.0	72.0	151.3
Positive control	2.5	197.0	223.3	1204.7	1802.7	1650.7
Firebrake ZB	1000	14.0	4.7	25.7	61.3	147.3
	500	19.3	7.3	20.0	74.3	157.3
	100	16.7	3.0	18.7	78.7	145.0
	10	15.0	5.7	20.3	73.3	141.3
	1	15.3	3.7	20.7	67.3	131.0

Results of salmonella/microsomal assay without metabolic activation

Compound	Concentration/plate [µg]	Revertants per plate (mean) of bacterial strains
		TA 1535	TA 1537	TA 1538	TA 98	TA 100
Solvent control		17.7	6.7	14.0	57.3	133.0
MNNG Positive control	5	1339.3				1461.3
2-NF Positive control	0.5			805.3	676.3
9-AA Positive control	100		404.0
Firebrake ZB	1000	18.3	3.3	15.3	56.7	131.7
	500	25.7	6.7	16.0	66.7	154.0
	100	24.3	4.3	19.0	52.0	144.0
	10	17.0	4.0	17.3	54.0	141.0
	1	23.7	5.7	15.0	50.3	146.3

Conclusions:

The test substance was considered negative under the experimental conditions.

Executive summary:

The test substance Firebrake ZB was tested in the Salmonella/microsomal mutagenicity plate incorporation assay with Salmonella strains both with and without S-9 metabolic activation. Under the conditions of this investigation, no mutagenic activity was demonstrated for the test substance.
Read-across is justified on the basis detailed in the rationale for reliability above. This study is therefore considered to be of sufficient adequate and reliable to be used as a key study.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-03-13 to 2019-06-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz, 15.05.18

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: Primary cultures of human peripheral lymphocytes

Details on mammalian cell type (if applicable):

Blood samples were obtained from a healthy donor who neither smokes nor receives medication. Primary cultures of human peripheral lymphocytes are preferred for this type of study because of their low and stable background rate of micronuclei. In addition, human cells are generally the most relevant ones for risk assessment.

Cytokinesis block (if used):

In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per culture) was determined in order to assess the cytotoxicity of the test item. From these determinations, the test item concentrations which were evaluated for scoring of micronuclei were defined.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Trinova Biochem GmbH, Gießen
- method of preparation of S9 mix: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally
- concentration or volume of S9 mix and S9 in the final culture medium: S9 mix (20 % S9) - in final culture medium - 50 μL S9 mix per mL medium

Test concentrations with justification for top dose:

3.8, 7.5, 15, 30, 60 µg/mL (cytotoxicity test)
15, 30, 60 µg/mL (genotoxicity test)
According to OECD 487, the maximum concentration of the test item should be 2 μl/mL, 2 mg/mL or 10 mM, whichever is the lowest. When cytotoxicity occurs, the highest concentration should aim to produce 55 ± 5% cytotoxicity. When the test item is a substance of unknown or variable composition, a complex reaction product or of biological origin (UVCB), testing may be started at a higher concentration to increase the amount of each of the test item components. Generally, concentration intervals of approximately 2 to 3-fold are used. When solubility is a limiting factor, the maximum analysed concentration (evaluated for micronuclei), if not limited by cytotoxicity, should be the lowest concentration at which turbidity or minimal precipitate is visible in the cultures at the end of treatment.
Due to poor solubility, for the test item Hexaboron dizinc undecaoxide, the maximum concentration according to these criteria was 60 μg/mL (please refer to section "any other information on materials and methods incl. tables").
Since the cytotoxic effects in the cytotoxicity test were not relevant, the 3 highest test item concentrations were evaluated for genotoxicity.

Vehicle / solvent:

no solvent, test item suspended in MCM (minimal culture medium); for positive controls 0.9 % NaCl was used as solvent

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.9% NaCl was used as solvent control for the positive controls Cyclophosphamide mono-hydrate (CPA) and Mitomycin C (MMC).

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: 4 h
- Harvest time after the end of treatment (sampling/recovery times): 19 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.: exposure time was 4 h, cytokinesis blocking substance cytochalasin B added after washing, the culture harvest time was 23 h after start of exposure
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
The slides were prepared by dropping the cell suspension onto a clean microscope slide. The cells were then stained with a 10 % solution of Giemsa. All slides were independently coded before microscopic analysis.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
At least 1000 binucleated cells per culture were scored for micronuclei.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
Readable binucleated cells are identified by the following criteria:
-The cell must have two main nuclei
-The two main nuclei must each have an intact and well-defined membrane
-The two main nuclei must be contained within the cytoplasm
-The cell must be visible in its entirety in the field
-The area around the cell must not contain micronucleus-like debris
-The cytoplasmic boundary should be intact and distinguishable from the boundaries of adjacent cells
Micronuclei (MN) are identified by the following criteria:
-The diameter of the MN must not exceed 1/3rd of each of the two main nuclei diameter
-The micronuclei can touch but must not overlap the two main nuclei
-Micronuclei should be large enough to discern morphological characteristics
-Micronuclei should possess a generally rounded shape with a clearly defined outline
-Micronuclei should be similar in color to the nuclei
-Should lie in the same focal plane as the cell
-Micronuclei must not be linked to the nuclei by a nucleoplasmic bridge
-Micronuclei must be within cytoplasmic boundary
-Micronuclei must be non-refractive (staining)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per culture) was determined in order to assess the cytotoxicity of the test item. From these determinations, the test item concentrations which were evaluated for scoring of micronuclei were defined. Cytotoxicity was calculated as reduction in CBPI (Cytokinesis-block proliferation index) compared to the CBPI of the concurrent solvent control.

Evaluation criteria:

The number of binucleated cells with and without micronuclei in each treatment group was compared with the solvent control value.

Statistics:

The number of binucleated cells with micronuclei in each treatment group was compared with the solvent control. Statistical significance was tested using Fisher’s exact test at the five per cent level (p < 0.05). For positive controls with high values of binucleated cells with micronuclei, the chi-square-test was used.

Species / strain:

lymphocytes: Primary cultures of human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data : -S9: negative control 0.74 % MBNC; positive control: 4.58 % MBNC; +S9: negative control 0.74 % MBNC; positive control: 3.28 % MBNC;

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship : statistically significant (-S9), no concentration-response relationship (+S9)
- Statistical analysis; p-value: p < 0.01 % at highest test item concentration (-S9 only)

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method:
-S9: mean CBPI (solvent control): 1.583 %, (positive control): 1.351 %, (test item 60, 30, 15 µg/mL): 1.483, 1.499, 1.540 %
+S9: mean CBPI (solvent control): 1.619 %, (positive control): 1.182 %, (test item 60, 30, 15 µg/mL): 1.669, 1.596, 1.549 %

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: MMC (-S9: range 1.08 - 11.49 %, mean 3.80%, 95.5 % control limits 0-8.05) ; CPA (+S9 range 1.25 - 5.14 %, mean 2.71, 95.5 % control limits 0.94 - 4.47 %)
- Negative (solvent/vehicle) historical control data: (-S9: range 0.05 - 1.06 %, mean 0.46, 95.5% control limits 0-1.0; +S9: range 0.05 - 0.90 %, mean 0.35, 95.5% control limits 0-0.73)

Acceptability

The assay is considered acceptable if it meets the following criteria:

-All experimental conditions are tested (short exposure with and without metabolic activation, extended exposure without metabolic activation) unless a positive result is achieved in any experiment.

-In each experiment, an adequate number of cells is analysable both in the controls and in at least 3 test item concentrations.

-The micronucleus induction of the solvent and positive controls is compatible with the historical laboratory control data or the literature data.

-The positive control shows a statistically significant increase of binucleated cells with micronuclei compared with the concurrent solvent control.

-The criteria for cell proliferation and for the selection of concentrations are fulfilled.

 

Classification

The test item is considered unable to induce chromosome breaks and/or loss in this test system if, in all experimental conditions, the following criteria are met:

-Neither a statistically significant nor a concentration-related increase of the number of micronucleate cells in the evaluated test concentrations is observed.

-The obtained results lie within the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5 % control limits where appropriate.

The test item is considered able to induce chromosome breaks and/or loss in this test system if, in any of the experimental conditions, all of the following criteria are met:

-At least one test concentration shows a statistically significant increase of micronucleate cells compared to the concurrent solvent control.

-In at least one experimental condition a dose-related increase of micronucleate cells can be observed using trend analysis.

-Any of the results lies outside the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5 % control limits where appropriate.

 

Results

In the experimental part without metabolic activation, the 2 highest test item concentrations (60 μg/mL and 30 μg/mL) showed statistically significant increased proportions of binucleated cells with micronuclei compared with the concurrent solvent control minimal culture medium. The values of all 3 evaluated test item concentrations lay above the historical laboratory control data (also above the 95.5% control limits) for the concurrent solvent control MCM (range: 0.05 – 1.06, 95.5% control limits: 0 - 1.00). A statistically significant dose-response relationship was also observed. In conclusion, in the experimental part without metabolic activation all criteria for a clear positive result were fulfilled.

In the experimental part with metabolic activation, the highest test item concentration (60 μg/mL) showed statistically significant increased proportions of binucleated cells with micronuclei compared with the concurrent solvent control minimal culture medium. The values of all 3 evaluated test item concentrations lay above the historical laboratory control data (also above the 95.5% control limits) for the concurrent solvent control MCM (range: 0.05 – 0.90, 95.5% control limits: 0 – 0.73). A statistically significant dose-response relationship could not be observed. Therefore, in the experimental part with metabolic activation, two out of three criteria for a positive result were fulfilled.

 

The study is considered acceptable: micronucleus induction of the solvent controls was in the range of the historical control data and the literature data (0 – 0.9%). The positive control compounds Mitomycin C (0.3 μg/mL) and CPA (30 μg/mL) showed distinct increases in the number of binucleated cells with micronuclei.

Conclusions:

In conclusion, under the experimental conditions reported, Hexaboron dizinc undecaoxide is able to induce the formation of micronuclei in human lymphocytes in vitro. The result of the micronucleus assay with the test item Hexaboron dizinc undecaoxide is considered as “positive” under the conditions of the test.

Executive summary:

This study was performed according to OECD Guideline 487 and GLP to assess the potential of Hexaboron dizinc undecaoxide to induce micronuclei in human lymphocytes cultured in vitro in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

The test item was dissolved in minimal culture medium to prepare a stock solution with a concentration of 0.6 mg/mL, corresponding to the highest concentration (60 μg/mL) in the test. A geometric series of dilutions was prepared from the stock solution. One valid experiment (4 h exposure, without and with metabolic activation) was performed. Human peripheral blood lymphocytes from whole blood culture were stimulated to divide by addition of phytohaemagglutinin and exposed to medium control, solvent control, positive controls and test item. All cell cultures were set up in duplicates. After exposure and harvesting, slides were prepared and stained. In order to assess the toxicity of the test item to the cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all cultures. On the basis of these data, the appropriate concentrations were selected to determine the proportion of binucleated cells containing micronuclei. All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

No relevant cytotoxicity was observed at any of the evaluated test item concentrations.

In the experimental part without metabolic activation, the 2 highest test item concentrations (60 μg/mL and 30 μg/mL) showed statistically significant increased proportions of binucleated cells with micronuclei compared with the concurrent solvent control minimal culture medium. The values of all 3 evaluated test item concentrations lay above the historical laboratory control data (also above the 95.5% control limits) for the concurrent solvent control MCM. A statistically significant dose-response relationship was also observed. In conclusion, in the experimental part without metabolic activation all criteria for a clear positive result were fulfilled.

In the experimental part with metabolic activation, the highest test item concentration (60 μg/mL) showed statistically significantly increased proportions of binucleated cells with micronuclei compared with the concurrent solvent control minimal culture medium. The values of all 3 evaluated test item concentrations lay above the historical laboratory control data (also above the 95.5% control limits) for the concurrent solvent control MCM. A statistically significant dose-response relationship could not be observed. Therefore, in the experimental part with metabolic activation, two out of three criteria for a positive result were fulfilled.

In conclusion, under the experimental conditions reported, Hexaboron dizinc undecaoxide is able to induce the formation of micronuclei in human lymphocytes in vitro.

The result of the micronucleus assay with the test item Hexaboron dizinc undecaoxide is considered as “positive” under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

For an evaluation of these results, please refer to the justification attached in Section 13 of this IUCLID dossier. A summary can be found in the field 'Mode of Action Analysis'.

WoE, in vivo mouse micronucleus test, OECD 474, GLP, zinc borate anhydrous (CAS 12767-90-7, Firebrake 500) (Béres, 2022), positive

WoE, in vivo mouse micronucleus test, OECD 474, GLP, zinc borate hydrate (CAS 138265-88-0, Firebrake ZB) (Herring, 2022), negative

WoE, in vivo in vivo mouse micronucleus test, GLP, ReA boric acid (CAS 10043-35-3) (O'Loughlin), negative

WoE, in vivo mouse micronucleus test, ReA zinc sulphate (CAS 7733-02-0) (Glocke, 1981), negative

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 APR 2021 to 21 MAR 2022

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 29th July, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Remarks:

Win: NMRI mice

Details on species / strain selection:

The NMRI mouse is one of the standard animals used internationally in this type of mutagenicity testing.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop zrt, Budapest, Hungary
- Age at study initiation: 9 weeks
- Weight at study initiation: 33.0 – 39.2 g
- Assigned to test groups randomly: yes, under following basis: randomly assigned using a randomization scheme; randomization was checked according to the actual body weights verifying the homogeneity and deviations between the groups
- Housing: Group caging (2 animal/cage in the pretest and in the high dose group of main tests and 5 animals/cage in the other groups of the main test); cage type: I. type polypropylene/polycarbonate; bedding: laboratory bedding. Rodents are group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities
- Diet (e.g. ad libitum): Pellet diet, ad libitum
- Water (e.g. ad libitum): Water, as for human consumption, ad libitum, from 250 mL bottles
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 40–70%
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 28 JUL, 2021 (animal receipt) To: 05 AUG, 2021

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% aqueous methylcellulose; components of vehicle: Methylcellulose and Aqua Purificata
- Concentration of test material in vehicle: 0, 50, 100, 200 mg/mL; positive control: 6 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): SLCB1319, 2012-4685

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in 1% aqueous methylcellulose for the treatment. The necessary amount of test item was weighed into a calibrated volumetric flask.
A partial volume of 1% aqueous methylcellulose was added and the formulation was stirred until homogeneity is reached. The test item was used for treatment in concentrations of 50 mg/mL, 100 mg/mL and 200 mg/mL prepared with the mentioned vehicle. The formulations were prepared fresh on day of dosing and used within 2 hours.
Cyclophosphamide (positive control) was dissolved in Aqua ad injectabilia for treatment.

Duration of treatment / exposure:

The test/vehicle items were administered orally by gavage two times at 24-hour intervals.

Frequency of treatment:

The test/vehicle items were administered orally by gavage two times at 24-hour intervals.

Post exposure period:

Treatment groups and the vehicle control group: Sampling was made once at 24 hours after the second treatment.
Positive control group (Cyclophosphamide): Sampling was performed 24 hours after the beginning of the treatment. The mice were examined regularly for visible signs of reactions to treatment, immediately after dosing, and periodically until sacrifice.

Dose / conc.:

2 000 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

No. of animals per sex per dose:

5 males/dose

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

Cyclophosphamide (CAS 6055-19-2)
- Route of administration: intraperitoneal
- Doses / concentrations: 60 mg/kg bw

Tissues and cell types examined:

Bone marrow was obtained from two exposed femurs of each animal. Blood sample of approximately 500 µL was obtained from the retro-orbital venous plexus.
Four thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 4000 PCEs counted in the optic field. The proportion of immature erythrocytes among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test/vehicle items were administered orally by gavage two times at 24-hour intervals.
The treatment volume was 10 mL/kg body weight. In the low, mid and high dose groups and the vehicle control group the sampling was made once at 24 hours after the second treatment. Five male animals per dose group were used for sampling. Cyclophosphamide (positive control) was administered intraperitoneally with a treatment volume of 10 mL/kg body weight. Sampling was performed 24 hours after the beginning of the treatment and five male animals were used for sampling. The mice were examined regularly for visible signs of reactions to treatment, immediately after dosing, and periodically until sacrifice

DETAILS OF SLIDE PREPARATION:
Bone Marrow Preparation and Staining for the micronucleus test:
The bone marrow was flushed with foetal bovine serum (5 mL). After vortex mixing, the cell suspension was concentrated by centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides. Slides were then dried at room temperature.
Subsequently the slides were stained as follows:
Fixed for a minimum of 5 minutes in methanol and allowed to air-dry.
Stained with Giemsa (10%) solution for 25 minutes.
Rinsing in distilled water.
Drying at room temperature (at least 12 hours).
Coating with EZ-MountTM

Blood sampling for plasma analysis:
Blood samples were taken into tubes containing K3EDTA at each occasion. The samples were gently flicked to mix the blood and anticoagulant. Blood samples were centrifuged at 3000 rpm, at 4 °C for 10 minutes within half an hour after sampling. The samples were transferred to an ultra-freezer and stored at –70 ± 10 °C until analysis.

Bone Marrow Preparation for analysis:
Bone marrow was flushed from both femurs with physiological saline (0.9% NaCl, 1.5 mL) and collected in Eppendorf tubes. Each sample was labelled with the following information: study no., dose, animal no., and sampling time. The samples were directly transferred to an ultra-freezer and stored at –70 ± 10 °C until analysis

METHOD OF ANALYSIS:
Examination of slides: microscopic analysis
Criteria for Identification of Micronucleated Erythrocytes
A micronucleus is defined in following way:
• A bluish mauve strongly coloured uniform circular particle in the cell.
• The particle should have a certain size and it should be located inside the cells.
• During focusing, the particle should stay uniform in colour/light refraction and shape within a large interval.
• Cells with two or more micronuclei were counted as single micronucleated cells.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, the test item is considered clearly positive if:
1. At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
2. This increase is dose-related at least at one sampling time when evaluated with an appropriate test, and
3. Any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if the following criteria had been met:
1. None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
2. There is no dose-related increase at any sampling time when evaluated by an appropriate test, 3. All results are inside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits),
4. Bone marrow exposure to the test item occurred.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data: The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative and historical control groups. The proportion of immature erythrocytes among total (immature + mature) erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative and historical control groups. The data was checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

Sex:

male

Genotoxicity:

positive

Remarks:

Induction of biologically and statistically significant increases in the frequency of micronucleated PCEs (MPCEs) at 500, 1000, 2000 mg/kg bw

Toxicity:

yes

Remarks:

2000 mg/kg bw: moderate and strong activity decrease, piloerection, incoordinated movement, decrease in grip strength, hunchback posture and narrow palpebra. In this dose group the faeces of animals were loose light brown

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: The study results are valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000, 2000 mg/kg bw
- Solubility: A non GLP Preliminary Solubility Test was performed April 07, 2021. The test item was formulated in 1% aqueous methylcellulose. Suspension suitable for treatment was obtained up to a concentration of 200 mg/mL.
- Clinical signs of toxicity in test animals:
Mortality rates: 2000 mg/kg bw: 0/4 males and 1/4 female; 1000 and 500 mg/kg bw: 0/0 males and 0/0 females
Clinical signs:
- 2000 mg/kg bw (females): Symptoms between 1–5 hours and at 24 hours: decreased activity and piloerection (slight, moderate) and loose light brown faeces. One female animal died before the second treatment. This female animal showed the following symptoms before death: decreased activity, incoordination, piloerection in heavy degree and loose light brown faeces, hunchback posture.
- 2000 mg/kg bw (males): Symptoms between 1–5 hours and at 24 hours: slight and moderate decreased activity and piloerection, expect one animal where a strong decrease in activity and piloerection was observed before and after the second treatment. In this dose group the faeces of animals were loose light brown from 24 hours after the first treatment as well as hunchback posture was observed.
- 1000 mg/kg bw (males and females): Slight and moderate piloerection (between 1–5 hours) after the first and second treatment and loose light brown faeces were observed from 24 hours after the first treatment.
- 500 mg/kg bw (males and females): Slight piloerection was observed between 1–5 hours after the first and second treatment
- Evidence of cytotoxicity in tissue analysed:
Proportion of immature erythrocytes among total (immature + mature) erythrocytes:
Two male mice: mean = 0.45
Two female mice: mean = 0.47
Historical control male mice: mean = 0.54
- Rationale for exposure: The preliminary toxicity test determined the MTD based on death and clinical signs of test item related toxicity, and whether there are differences in toxicity between the male and female animals.
- Other:
The main test was performed using male mice because the toxic effect of the test item was similar in both sexes in the preliminary acute oral toxicity test.
On the basis of the results of the preliminary toxicity test, doses for the Mouse Micronucleus Test were the following: 500, 1000 and 2000 mg/kg body weight.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The two times oral administration of 500 mg/kg body weight, 1000 mg/kg body weight and 2000 mg/kg body weight of the test item induced biologically and statistically significant increases in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male mice at 24 hours after the second treatment compared to the negative and historical control groups. These increases were dose related. The number of PCEs at 24 hours after the second treatment were outside the distribution of the historical negative control data in the dose groups of 500, 1000 and 2000 mg/kg body weight.
The frequencies of MPCEs for the negative and positive control mice were compatible with the historical control data for this laboratory. Cyclophosphamide treated mice (60 mg/kg body weight) showed a large, statistically significant increase in the MPCE number compared to the negative and historical controls. Thus, the study is considered valid.
- Ratio of PCE/NCE (for Micronucleus assay): The proportion of immature erythrocytes among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes. Compared to the negative and historical control groups the number of PCEs at 24 hours after the second treatment in the dose group of 500 mg/kg body weight was the same. In the dose groups of 1000 and 2000 mg/kg body weight a statistically significant decrease in number of PCEs was observed compared to the negative and historical control groups. This effect demonstrated exposure of the test item to the bone marrow.

- Clinical signs of toxicity in test animals:
No adverse reactions to treatment were observed in the mice of the negative and positive control groups.
In the dose group of 500 mg/kg slight piloerection was observed between 1–5 hours after the first and second treatment.
In the dose group of 1000 mg/kg moderate piloerection (between 1–5 hours) after the first and second treatment and loose light brown faeces were observed from 24 hours after the first treatment.
In the dose group of 2000 mg/kg the following symptoms were observed after the first and second treatments (between 1–5 hours and at 24 hours): moderate and strong activity decrease, piloerection, incoordinated movement, decrease in grip strength as well as hunchback posture and narrow palpebra. In this dose group the faeces of animals were loose light brown from 24 hours after the first treatment.
Two additional male mice were dosed in the highest (2000 mg/kg bw) test item treated group to replace any animals which die before the scheduled sacrifice time. No death occurred in the original population (5 animals). One of these two animals died related to the test item administration 24 hours after the first treatment. Bone marrow smears were not prepared from the survivor additional mouse. The clinical symptoms observed were as follows: strong activity decrease, strong decrease in grip strength, strong piloerection, moderate incordination and loose light brown faeces, hunchback posture and narrow palpebra.
- Body weight:
Body weight decrease was observed in animals treated with the test item. The decrease of body weight was most significant in the highest dose group, also demonstrating the toxic effect of test item.

 

Analysis of the Formulations (Test Item Concentrations)

The net signal intensity measured for the control sample was 2% of the LOQ.

Concentration of the test item was calculated based on both elements (Zinc and Boron). Mean concentrations of Firebrake 500 in the formulation samples were in the range of 97– 99% of the nominal concentrations. Deviation of the samples taken from different places (bottom, mid and top of the vessel) was < 4%, therefore the formulations can be considered homogeneous.

Bioanalysis

A group of twelve male NMRI mice were dosed once orally (by gavage) with 2000 mg/kg of the test item in 1% aqueous methylcellulose, at a dosing volume of 10 mL/kg.

Proof of exposure to the test item was demonstrated by analysis of Zinc and Boron in plasma and bone marrow of treated animals. The concentration of Boron and Zinc were measured in plasma and bone marrow samples with a validated ICP-OES method developed previously. LOQ in plasma is 0.17 µg/mL for Boron and 0.35 µg/mL for Zinc. In case of bone marrow, assuming 15 mg sample, LOQ is 1.5 µg/g for boron and 2.9 µg/g for Zinc. Concentration of the test item was calculated based on both Boron and Zinc levels.

The mean concentration of Boron in plasma was 21 µg/mL at 2 hours, 37 µg/mL at 4 hours, 40 µg/mL at 6 hours and 22 µg/mL at 24 hours after the treatment. The mean concentration of Zinc in plasma was 7.9 µg/mL at 2 hours, 14 µg/mL at 4 hours, 23 µg/mL at 6 hours and 6.8 µg/mL at 24 hours after the . The mean concentration of Zinc in vehicle control plasma was 1.1 µg/mL. The mean concentration of Boron in vehicle control plasma was 0.6 µg/mL.

The mean mass fraction of Boron in bone marrow was 48 µg/g at 2 hours, 147 µg/g at 4 hours, 163 µg/g at 6 hours and 55 µg/g at 24 hours after the treatment. The mean mass fraction of Zinc in bone marrow was 38 µg/g at 2 hours, 54 µg/g at 4 hours, 57 µg/g at 6 hours and 37 µg/g at 24 hours after the treatment. For Zinc and Boron, the highest mass fractions were measured 6 hours after treatment. The mean mass fraction of Boron and Zinc were 2.7 and 44 µg/g in the bone marrow of vehicle control animals.

It should be noted that additional exposure to Zinc and Boron results from the diet (lab chow and drinking water) which applies to all animals in this study, including controls. This should be taken into account when comparing values of treated vs. control animals.

 

MOUSE MICRONUCLEUS TEST Summary table

Groups	Sampling time: 24 hours after the second tratment	Total number of PCEs analysed	MPCE		PCE/ PCE+NCE
			mean	SD	mean	SD
Negative Control	24	20000	5.40	1.14	0.53	0.00
500 mg/kg body weight	24	20000	9.60**	1.14	0.52	0.01
1000 mg/kg body weight	24	20000	19.00**	3.54	0.46**	0.01
2000 mg/kg body weight	24	20000	23.00**	3.67	0.44**	0.02
Positive Control (60mg/kg body weight)	24	20000	138.40**	6.27	0.38**	0.01

PCE = Polychromatic Erythrocyte

NCE = Normochromatic Erythrocyte

MPCE = Number of Micronucleated Polychromatic Erythrocytes referring to 4000 PCE

Positive Control = 60 mg/kg bw Cyclophosphamide

Negative Control: 1% aqueous methylcellulose

** = p < 0.01 to the negative control (DN)

** = p < 0.01 to the historical control (U)

Kruskal-Wallis Non Parametric ANNOVA

DN=Duncan's Multiple Range

U=Mann-Whitney U-test

Conclusions:

Biologically and statistically significant increases in the frequency of MPCEs were seen in all groups of mice treated with the test item compared to the vehicle control and historical control groups.
The test item showed genotoxic activity in this Mouse Micronucleus Test.

Zinc and boron were present in mouse plasma and bone marrow samples taken at 2, 4, 6 and 24 hours after the treatment. The concentrations of both elements were higher in the plasma samples of animals treated with the test item compared to the vehicle control sample concentrations. Therefore, it is reasonable to conclude that the test item (or its respective degradation or hydrolysis products) is bioavailable. The decreased number of PCE in the higher dose groups also demonstrated the exposure to the bone marrow.

Executive summary:

The study was conducted according OECD 474 and GLP, with the objective to determine whether the Zinc Borate Anhydrous causes genotoxic effects resulting in the formation of micronuclei in erythrocytes of treated male Win:NMRI mice. Furthermore, objective of this study was to demonstrate proof of systemic exposure in the mouse after single oral (2000 mg/kg body weight) administration of the test item using a validated analytical method.

Study design:

The doses of the test item for the Micronucleus Test were determined according to a preliminary oral toxicity study. The doses selected were 500, 1000 and 2000 mg/kg bw.

A negative (vehicle) control and a positive control group were included. Treatment was carried out in 1% aqueous methylcellulose with a constant treatment volume (10 mL/kg bw). The test item and negative (vehicle) control item were administered by gavage two times at 24-hour intervals. Cyclophosphamide was dissolved in Aqua ad injectabilia (positive control) and was administered once, intraperitoneally with a treatment volume of 10 mL/kg body weight. In the low, mid and high dose groups and vehicle control group the sampling was made once at 24 hours after the second treatment. In animals treated with Cyclophosphamide (60 mg/kg bw.), the sampling was performed only at 24 hours post-treatment. Five animals per dose group were used.

Four thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells.

The suitability of the chosen vehicle for the test item was analytically verified. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation.

Measured concentrations of formulations applied in the study varied in the acceptable range (between of 97% and 99% of the nominal concentrations) and all formulations were homogenous, thereby confirming proper dosing.

In total twelve male animals/group were dosed at 2000 mg/kg body weight with the test item to demonstrate systemic circulation in NMRI mice after single oral (gavage) administration of the test item. Three male animals were treated only with the vehicle of the test item. In the test item treated groups, the sampling for plasma and bone marrow analysis was made once at 2, 4, 6 and 24 hours after the treatment (three animals per time point). In the group treated with the vehicle of the test item the sampling was performed one hour after the treatment.

Results:

The two times oral administration of 500, 1000 and 2000 mg/kg bw of the test item induced biologically and statistically significant increases in the frequency of micronucleated PCEs (MPCEs) in male mice at 24 hours after the second treatment compared to the negative and to the historical control groups. These increases were dose related. The number of PCEs at 24 hours after the second treatment were outside the distribution of the historical negative control data in the dose groups of 500, 1000 and 2000 mg/kg bw.

The proportion of immature erythrocytes among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes. Compared to the negative and historical control groups the number of PCEs at 24 hours after the second treatment in the dose group of 500 mg/kg body weight was the same. In the dose groups of 1000 and 2000 mg/kg body weight a statistically significantly decreased number of PCEs was observed compared to the negative and historical control groups. This effect has also demonstrated the exposure of the test item to the bone marrow.

The frequencies of micronucleated polychromatic erythrocytes (MPCEs) for the negative and positive control mice were within acceptable ranges and compatible with the historical control data for this laboratory. Cyclophosphamide treated mice (60 mg/kg body weight) showed a large, statistically significant increase in the MPCE number compared to the negative and historical controls. Thus, the study is considered valid.

The concentrations of the test item and bone marrow measured after treatment with 2000 mg/kg bw orally were in the linear range of the calibration curve. Test item concentrations were determined by measuring Zinc and Boron concentrations using a validated ICP-OES method. Highest concentration levels were measured six hours after the treatment: concentrations in plasma: B: 40 µg/mL, Zn: 23 µg/mL. Mass fractions in bone marrow: B: 163 µg/g, Zn: 57 µg/g. The concentrations for Zinc and Boron in the plasma of test animals were always (at all time points) higher than in the control group. In the bone marrow, mass fraction of Boron was higher in the samples of the treated animals than in the control groups, whereas the mass fraction of Zinc was in the same order of magnitude in the samples of the treated animals in comparison to the control samples. These results demonstrated the systemic exposure of the test item (and/or its corresponding degradation or hydrolysis products) and thus also the exposure of the target tissue (bone marrow).

Conclusion:

Biologically and statistically significant increases in the frequency of MPCEs were seen in all groups of mice treated with the test item compared to the vehicle control and historical control groups.

The test item showed genotoxic activity in this Mouse Micronucleus Test.

Zinc and Boron were present in mouse plasma and bone marrow samples taken at 2, 4, 6 and 24 hours after the treatment. The concentrations of both elements were higher in the plasma samples of animals treated with Firebrake 500 compared to the vehicle control sample concentrations. Therefore, it is reasonable to conclude that the test item and/or its corresponding degradation or hydrolysis products is/are bioavailable. The decreased number of PCE in the higher dose groups also demonstrated the exposure to the bone marrow.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Remarks:

Male and female mice averaged background micronucleus incidences of 0.23% and 0.25%, respectively.

Positive controls validity:

valid

Remarks:

Induced micronucleus frequency of approx. 14-fold greater than that of the vehicle control at the 24 h sampling time

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 225, 450, 900, 1800 or 3500 mg/kg/day
- Clinical signs of toxicity in test animals: All mice appeared normal throughout the range-finding assay and were sacrificed 48 hours after the last dosage administration and evaluated for specific signs of cytotoxicity.
- Evidence of cytotoxicity in tissue analysed: The percentage of PCEs among RBCs was not altered significantly by treatment with test item, which was considered to be the maximum pratical dose that could be given.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: No death occurred in any of the dosage groups in either sex. On day 2 one male mouse at 1800 mg/kg/day had rough fur and hunched position and one male mouse at 3500 mg/kg/day had rough fur.
- Induction of micronuclei (for Micronucleus assay): All treated groups had average micronucleus counts approx. equal to those of the vehicle control group.
- Ratio of PCE/NCE (for Micronucleus assay): The percentage of PCEs among RBCs in treated groups did not differ significantly from the vehicle control group.

The of the Range-finding and definitive study results are presented in attached document.

Conclusions:

The test item at dosages up to and including 3500 mg/kg/day did not induce increased incidences of micronuclei in the bone marrow erythrocytes of male and female Swiss-Webster mice. Therefore, test item was considered non-clastogenic under these test conditions.

Executive summary:

The genetic toxicity study was performed according to the guideline US-EPA-FIFRA section 158.340 Guideline 84-2 under GLP compliance.

In the definitive assay, 10 mice per sex per dosage group were orally dosed with Boric Acid in sterile deionized water at dosage levels of 900,1800, or 3500 mg/kg/day for 2 consecutive days. Five mice per sex per dosage group were sacrificed 24 hours after the final dose and the same number 48 hours after the final dose; all were evaluated for cytotoxicity and micronucleus formation in bone marrow erythrocytes. A sterile deionized water vehicle control group (10 mice per sex) and a methane positive control group (10 male mice only) were treated similarly and evaluated concurrently with the test groups.

No death occurred in any of the dosage groups in either sex. Clinical signs included rough for and a humped back. All Boric Acid-treated groups, when compared with the sterile deionized water control group, had average micronucleus counts approximately equal to that of the negative control groups and did not differ statistically from controls at p < 0.05. Background micronucleus incidences in bone marrow erythrocytes of male and  female mice treated with sterile deionized water alone averaged 033 and 035%, respectively. The percentage of PCEs among RBCs was not altered significantly by treatment with Boric Acid. The urethane positive control group had a micronucleus frequency approximately 14-fold greater than that of background at the 24-hour sampling time.

The test item at dosages up to and including 3500 mg/kg/day did not induce increased incidences of micro-nuclei in the bone marrow erythrocytes of male and female Swiss-Webster mice. Therefore, test item was considered non-clastogenic under these test conditions.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Table 1: Results of the mirconucleus test on mouse bone marrow

Compound	surviving/treated mice	Dose
		mg/kg	mmole/kg	Route of application	Micronucleated PE^a(‰)
Zinc sulfate^c	3/4	2x86.3	2x0.3	i.p.	2.9
	4/4	2x57.5	2x0.2	i.p.	1.9
	4/4	2x28.8	2x0.1	i.p.	1.7
	4/4	0		i.p.	1.9
^aPolychromatic erythocytes^cHanks' balances salt solution (HBSS)

Conclusions:

Zinc sulfate showed no genetic activity in the micronucleus test.
Read-across is justified on the basis detailed in the justification for type of information and rationale for reliability section. This study is therefore considered to be of sufficient adequacy and reliability to be used as a weight of evicence.

Executive summary:

The in vivo micronucleus test on mouse bone marrow was performed according to Schmid and international standard methods. The animals were treated at 0 and 24 h with zinc sulfate, and bone-marrow smears were prepared at 30 h. Usually, 4 mice (2 male, 2 female) were used for each of 3 doses and 1 control. Slides were coded, and 1000 polychromatic erythrocytes were scored per mouse. Significance was calculated according to the Kastenbaum-Bowman tables.

Zinc sulfate showed no genetic activity in the micronucleus test.

Read-across is justified on the basis detailed in the justification for type of information and rationale for reliability section. This study is therefore considered to be of sufficient adequacy and reliability to be used as a weight of evicence.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

Summary

(the full justification can be found attached under IUCLID Section 13)

The registered substance anhydrous zinc borate (AZB, Firebrake 500, CAS 12767-90-7) showed a genotoxic activity in the recently conducted in vivo mammalian erythrocyte micronucleus test performed according to OECD TG 474 and following GLP-principles (Béres, 2022). A dose-dependent and statistically significant increase of micronucleated blood cells (erythrocytes) compared to the control group was observed in all dose groups (500; 1000, and 2000 mg/kg bw). Due to a reduced portion of immature erythrocytes, the occurrence of clinical signs of the animals during the test, the noted genotoxicity and the results from the accompanying bioanalysis, it can be assumed as proven that the test item reached the target site (bone marrow), thereby the validity criteria are met in this study. This observed genotoxicity is supposed to be based on a thresholded effect of zinc. The rationale of this is based on the fact that zinc borate hydrolyses to zinc ion and boric acid. With regard to these two hydrolysis products, for boric acid there is high evidence from numerous existing in vivo and in vitro studies that this substance does not induce gene mutations or cytogenetic damage. Zinc salts, on the other hand, show a less clear picture for genetic toxicity endpoints. Conflicting results were found in in vitro bacteria reverse mutation, mouse lymphoma and chromosome aberration assays. Also in in vivo chromosome aberration assays negative as well as positive results were observed (EFSA 2006). These sporadically positive findings for the zinc salts, which are in line with the positive in vivo micronucleus assay for zinc borate, can be well explained from a mechanistic point of view. As zinc, as a trace element plays a crucial role in genetic and epigenetic processes, the dysregulation of zinc homeostasis can lead to epigenetic alterations (Brito et al. 2020). Based on these facts, the genetic toxicity effects observed for zinc borate are most likely to be driven by zinc. Zinc is an essential trace element. Therefore, the genotoxic effects of zinc are clearly a threshold effect caused by zinc overload conditions.

Results from a recently conducted in vivo micronucleus study with zinc borate hydrate (CAS 138265-88-0, Firebrake ZB) indicate a negative outcome (Herring 2022). The results of the bioanalysis test demonstrated that zinc and boron were present in mouse plasma and bone marrow samples 2 and/or 6 hours after treatment. The concentrations of both elements were higher in the plasma samples of animals treated with the test item compared to the vehicle control sample concentrations. Therefore, it is reasonable to conclude that the test item and/or its corresponding degradation or hydrolysis products is/are bioavailable

References

Brito et al. (2020). Zinc and Its Transporters in Epigenetics, Mol. Cells 43(4): 323-330.

EFSA (2006). Tolerable Upper Intake Levels for vitamins and minerals, Scientific Committee on Food, Scientific Panel on Dietetic Products, Nutrition and Allergies, February 2006, ISBN: 92-9199-014-0

Additional information

in vitro

A number of in vitro mutagenicity studies, including bacterial mutation assays in Salmonella typhimurium and Escherichia coli, gene mutation in mammalian cells (L5178Y mouse lymphoma, Chinese hamster cells) and chromosomal aberration in mammalian cells (Chinese hamster lung cells) have been carried out on zinc borate compounds or boric acid (Thompson, 1995; Stewart, 1993; NTP, 1987; Entrup, 1981; Curry, 2010) . No evidence of gene mutation activity was observed. 

However, a positive result was obtained in an in vitro mammalian cell micronucleus test according to OECD 487 (Geissel, 2019) indicating potential cytogenic activity. Based on these positive indications, in vivo mammalian erythrocyte micronucleus tests according to OECD Guideline 474 were conducted with zinc borate anhydrous (CAS 12767-90-7, Firebrake 500) and zinc borate hydrate (CAS 138265-88-0, Firebrake ZB). For an overall mechanistic understanding also available study data for boric acid and zinc sulphate were assessed.

in vivo: zinc borate anhydrous (CAS 12767-90-7, Firebrake 500)

A mouse micronucleus assay which has been conducted on the registered substance zinc borate anhydrous (CAS 12767-90-7, Firebrake 500) and which has been performed according to OECD TG 474 and GLP (Béres 2022). In this study the test/vehicle items were administered orally by gavage two times at 24-hour intervals to 5 males/dose. The doses were 500, 1000 and 2000 mg/kg bw, selected based on a preliminary oral toxicity study. Cyclophosphamide was used as positive control. In the low, mid and high dose groups and vehicle control group the sampling was made once at 24 hours after the second treatment. In animals treated with Cyclophosphamide (60 mg/kg bw.), the sampling was performed only at 24 hours post-treatment. Four thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The two times oral administration of 500, 1000 and 2000 mg/kg bw of the test item induced biologically and statistically significant increases in the frequency of micronucleated PCEs (MPCEs) in male mice at 24 hours after the second treatment compared to the negative and to the historical control groups. These increases were dose related. The number of PCEs at 24 hours after the second treatment were outside the distribution of the historical negative control data in the dose groups of 500, 1000 and 2000 mg/kg bw. The proportion of immature erythrocytes among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes. Compared to the negative and historical control groups the number of PCEs at 24 hours after the second treatment in the dose group of 500 mg/kg body weight was the same. In the dose groups of 1000 and 2000 mg/kg body weight a statistically significantly decreased number of PCEs was observed compared to the negative and historical control groups. This effect has also demonstrated the exposure of the test item to the bone marrow. Positive and vehicle control were considered valid. The results of the bioanalysis revealed that Zinc and Boron were present in mouse plasma and bone marrow samples taken at 2, 4, 6 and 24 hours after the treatment, it is thus reasonable to conclude that the test item and/or its corresponding degradation or hydrolysis products is/are bioavailable. The decreased number of PCE in the higher dose groups also demonstrated the exposure to the bone marrow.

in vivo zinc borate hydrate (CAS 138265-88-0, Firebrake® ZB)

A mouse micronucleus assay has also been conducted with the registered substance zinc borate hydrate (CAS 138265-88-0, Firebrake ZB) and which has been performed according to OECD TG 474 and GLP. In this study the test/vehicle items were administered orally by gavage two times at 24-hour intervals to 6 males/dose. The doses were 125, 500 and 2000 mg/kg bw. Mitomycin C  was used as positive control. Based on preliminary results the data for the concurrent vehicle control (group mean % micronucleated polychromatic erythrocytes [%MPCE]) were within the ranges determined by the laboratory historical control data (95% confidence limits). The coded positive control slides demonstrated the ability of the analyst to detect increases in micronucleated polychromatic erythrocytes as there was a statistically significant increase compared to the vehicle control (p<0.05). There were no statistically significant increases or trend observed in the group mean MPCE of male Crl:CD1 mice administered Firebrake® ZB at 125, 500 or 2000 mg/kg/day, compared to the concurrent vehicle control. The group mean %MPCE values from all groups were within the current vehicle historical control range (95% confidence limits). Based on the results presented it is concluded that FireBrake® ZB did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes in male Crl:CD1 mice when administered orally by gavage up to the standard limit dose in this in vivo test procedure. The results of the bioanalysis showed that Zinc and Boron were present in mouse plasma and bone marrow samples taken 2 and/or 6 hours after treatment. The concentrations of both elements were higher in the plasma samples of animals treated with the test item compared to the vehicle control sample concentrations. Therefore, it is reasonable to conclude that the test item and/or its corresponding degradation or hydrolysis products is/are bioavailable.

in vivo zinc sulphate

The available micronucleus test in mouse bone marrow with zinc sulphate (Gocke 1981) is a well documented study, that meets generally accepted scientific principles. In the study, animals were treated at 0 and 24 h i.p with zinc sulphate at doses of 28.8, 57.5 and 86.3 mg/kg bw, and bone-marrow smears were prepared at 30 h. Usually, 4 mice (2 male, 2 female) were used for each of 3 doses and 1 control. Slides were coded, and 1000 polychromatic erythrocytes were scored per mouse. Zinc sulphate showed no genetic activity in the micronucleus test.

in vivo boric acid

The available micronucleus test in mous bone marrow with boric acid (O'Loughlin 1991) was performed according to the guideline US-EPA-FIFRA section 158.340 Guideline 84-2 and was conducted under GLP compliance. In this study, animals (10 mice/sex/dose) were exposed to boric acid at doses of at doses of 900, 1800 and 3500 mg/kg bw via gavage for 2 consecutive days. Mice were observed daily for four days, from the start of dosing to sacrifice. No death occurred in any of the dosage groups in either sex. The test item at dosages up to and including 3500 mg/kg/day (highest dose) did not induce increased incidences of micronuclei in the bone marrow erythrocytes of male and female Swiss-Webster mice. Therefore, test item was considered non-clastogenic under these test conditions. 

Justification for classification or non-classification

Based on these results the following classification is warranted according to Regulation (EC) No 1272/2008:

zinc borate anhydrous (CAS 12767-90-7, Firebrake 500): ‘Germ cell mutagen – Category 2 (Muta. 2, H341 “Suspected of causing genetic defects”)’ is added to the existing classification.

zinc borate hydrate (CAS 138265-88-0, Firebrake ZB): not classified