Boric acid, zinc salt

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 (Geissel, 2019), positive

in vitro gene mutation studies in bacteria, OECD 471, GLP (Entrup, 1981), negative

in vitro gene mutation studies in mammalian cells, OECD 476, GLP (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:

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

Adequacy of study:

key study

Study period:

20/08/2010 to 28/09/2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study. 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with 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:

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

Adequacy of study:

key study

Study period:

16/07/1981 to 23/07/1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study with acceptable restrictions. 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 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 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 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:

Interpretation of results:
negative

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 adequacy and reliability to be used as a key study.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

supporting study

Study period:

29-01-1993 to 03-08-1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study. 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:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

not specified

GLP compliance:

yes

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

10, 50, 100, 500, 1000 and 5000 μg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 9-Aminoacridine hydrochloride, 2-Anthramine, 2-Nitrofluorene, Sodium azide

Statistics:

Bartlett's t-test, Dunnett's t-test

Species / strain:

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

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

Results from the first mutagenicity test (4% S-9)

Without S9
Dose/plate [µg]	Revertants/plate (mean)
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
0	15	13	25	25	115
10	15	11	24	18	136
50	14	10	21	25	131
100	12	9	19	27	129
500	12	14	24	26	119
1000	14	8	19	20	135
5000	15	8	31	20	135
With S9
0	12	12	21	30	121
10	14	15	25	25	114
50	12	7	20	33	131
100	16	11	20	30	118
500	13	11	22	27	126
1000	11	12	21	27	131
5000	13	10	20	27	122

Results of the second mutagenicity test (10% S-9)

Without S9
Dose/plate [µg]	Revertants/plate (mean)
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
0	20	10	26	24	145
10	18	9	20	26	138
50	20	11	20	22	154
100	16	13	20	22	140
500	16	12	22	25	139
1000	10	8	22	22	141
5000	17	11	23	27	145
With S9
0	13	13	22	27	140
10	11	12	26	25	162
50	13	14	23	27	152
100	17	10	25	26	151
500	11	8	24	29	158
1000	13	8	22	27	155
5000	14	5	26	40	132

Conclusions:

Interpretation of results:
negative

Firebrake 415 was found to be not mutagenic as tested according to the procedures used in these assays.
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 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
For comparative purposes, exposures to borates are often expressed in terms of boron (B) equivalents based on the fraction of boron in the source substance on a molecular weight basis. Read-across between zinc metaborate and the different boron compounds can be done on the basis of boron (B) equivalents. Conversion factors are given in the following table.

Conversion factors to boron equivalents:
Substance Formula Conversion factor for equivalent dose of B (multiply by)
Boric acid H3BO3 0.1748
Boric oxide B2O3 0.311
Disodium tetraborate
anhydrous Na2B4O7 0.2149
Disodium tetraborate
pentahydrate Na2B4O7.5H2O 0.1484
Disodium tetraborate
decahydrate Na2B4O7.10H2O 0.1134
Disodium octaborate
tetrahydrate Na2B8O13.4H2O 0.2096
Zinc Borate anhydrous 2ZnO ·3 B2O3 0.1745
Zinc Borate heptahydrate 2ZnO ·3B2O3·3.5H2O 0.1494
Zinc Metaborate anhydrous 2ZnO.2B2O3 0.1432
Zinc Metaborate trihydrate  2ZnO.2B2O3.3H2O 0.1215

The family of zinc borates includes zinc metaborate anhydrous and zinc metaborate trihydrate. Zinc metaborate anhydrous is the anhydrous form of zinc metaborate trihydrate. Zinc metaborate and zinc metaborate anhydrous 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 solubilizes. Therefore, it can be assumed that at physiological conditions and neutral and lower pH zinc borate will be hydrolyzed to boric acid, zinc oxide and zinc hydroxide (Schubert et al., 2003).

Zinc metaborate trihydrate breaks down as follows:
2ZnO 2B2O3 3H2O + H2O + 4H+ <-> 4H3BO3 + 2Zn2+
2Zn2+ + 4OH- <-> 2Zn(OH)2
____________________________________________________________
Overall equation
2ZnO 2B2O3 3H2O + 5H2O <-> 2Zn(OH)2 + 4H3BO3

The relative zinc oxide and boric oxide % are as follows:

Zinc metaborate trihydrate: Zinc oxide = 45.71 % (36.73 % Zn)
B2O3 = 39.11 % (12.15 % B)
Water 15.18 %
Zinc metaborate, anhydrous: Zinc oxide = 53.89 % (43.3 % Zn)
B2O3= 46.11 % (14.32 % B)

The results of an ADME study in rats conducted with zinc borate show that significant amounts can be taken up into the systemic circulation via oral absorption (Muzzio and Johnson, 2010). Therefore, oral absorption of boron is set to 100 % (worst-case) for the purposes of hazard assessment. The oral absorption of boron is considered to be the same in animals and in humans (worst-case). Because of the homeostatic control of zinc, the amount of zinc absorbed and bioavailable after repeated oral dosing of zinc borate is likely limited.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
source substance: Boric acid, CAS No 10043-35-3, Purity 99.7 %
target substance: Zinc metaborate, CAS No 1332-07-6, purity. > 90 %

3. ANALOGUE APPROACH JUSTIFICATION
Based on the results of a 90-day oral toxicity study, oral administration of zinc borate 2335 to rats for 90 days resulted in no adverse effects for males and females at dosage levels of 50 (7.5 mg B/kg) and 100 mg/kg/day (14.9 mg B/kg) and for females at 200 (29.8 mg B/kg) and 375 mg/kg/day (56 mg B/kg). For males, a dosage level of 375 mg/kg/day (56 mg B/kg) resulted in adverse effects on male reproductive organs, including effects on spermatogenic parameters with corresponding lower organ weights and gross and microscopic findings. Adverse effects on spermatogenic parameters were also noted at 200 mg/kg/day (29.9 mg B/kg) although there were no correlating microscopic findings. Therefore, the no-observed-adverse-effect level (NOAEL) was 100 mg/kg/day, equivalent to 14.9 mg boron/kg/day, for males and 375 mg/kg bw/day
(equivalent to 56 mg B/kg bw/day) for females. The NOAEL and LOAEL of zinc borate based on fertility effects in males is similar to studies of boric acid and sodium borates that demonstrated NOAEL and LOAEL of 17.5 mg boron/kg bw/day and 26 mg boron/kg bw/day, respectively.

The potential of zinc borate for developmental toxicity was evaluated based on the results of the Developmental Toxicity Test (Edwards, 2014). Three treatment groups of 25 Sprague Dawley rats were administered the test article via oral gavage at dose levels of 100, 125 or 150 mg/kg/day. Higher mean litter proportions of reduced ossification of the 13th rib(s) and sternebra(e) nos. 5 and/or 6 unossified were noted in the 100 (14.9 mg B/kg bw/day), 125 (18.7 mg B/kg bw/day), and 150 (22.4 mg B/kg bw/day) mg/kg/day groups compared to the control group. These findings were considered secondary to the reduced fetal weights noted in these groups. In addition, higher mean litter proportions of 7th cervical ribs and lower mean litter proportions of 14th rudimentary rib(s) were noted in the 100, 125, and 150 mg/kg/day groups and higher mean litter proportions of 25 presacral vertebrae were noted in the 125 and 150 mg/kg/day groups compared to the control group. No test substance-related fetal malformations were observed in the test substance-treated groups. Based on these results, a dosage level of 150 mg/kg bw/day (equivalent to 22.4 mg boron/kg bw/day) was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. A NOAEL was not established for zinc borate in this study. A dosage level of 100 mg/kg bw/day (equivalent to 14.9 mg boron/kg bw/day) was considered to be the LOAEL for embryo/fetal development when zinc borate was administered orally by gavage to rats. The LOAEL and expected NOAEL based on reduced fetal weight and fetal variations in this study for zinc borate corresponds to the developmental toxicity LOAEL of 13.3 mg B/kg bw/day and NOAEL of 9.6 mg B/kg bw/day for boric acid.

Based on the chemical behavior of zinc borate and the critical toxicity endpoints that correlate with boron dose levels the read across to boric acid and sodium borates for zinc borate toxicity endpoints is supported. For zinc metaborate no such data is available for comparison, but it is reasonable to assume that the results would be similar.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative

Test substance is non genotoxic.
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 supporting study.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

supporting study

Study period:

1995-02-23 to 1995-05-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study. 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:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) guidelines.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 1994-01-31; Date of signature 1994-03-16

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine synthesis for Salmonella typhimurium
- Tryptophan synthesis for Escherichia coli

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9

Test concentrations with justification for top dose:

- Preliminary toxicity study: 0, 50, 150, 500, 1500 and 5000 μg/plate
- Range finding study: 0, 50, 150, 500, 1500 and 5000 μg/plate
- Main study: 0, 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle/suspending agent control.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation Migrated to IUCLID6: 2 ug/plate for WP2uvrA; 3 ug/plate for TA100 and 5 ug/plate for TA135

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation Migrated to IUCLID6: 80 ug/plate for TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation Migrated to IUCLID6: 0.2 ug/plate for TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 1ug/plate for TA100; 2 ug/plate for TA1535 and TA1537; 10 ug/plate for WP2uvrA and 0.5 ug/plate for TA98

Remarks:

With metabolic activation

Details on test system and experimental conditions:

TESTER STRAINS

The strains were obtained from the British Industrial Biological Research Association on 14 August 1987 and were stored at minus 196 degrees Centigrade in a Statebourne liquid nitrogen freezer (model SXR 34). Prior to use, characterisation checks were carried out to determine the amino acid requirement, presence of rfa, R factors, uvr mutation and the spontaneous reversion rate. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 degrees Centigrade for approximately 10 hours.

PREPARATION OF TEST AND CONTROL MATERIALS

The test material was accurately weighed and approximate half-log suspensions prepared in sterile distilled water using an autovortex mixer and a 10 minute exposure in an ultrasonic bath on the day of each experiment. Analysis for concentration, homogeneity and stability of the test material formulations was not a requirement of the test guideline and was not carried out.

MICROSOMAL ENZYME FRACTION

S9 was prepared in house on 08 March 1995 from the livers of male Sprague-Dawley rats weighing approximately 200 g. The animals had received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg) five days before S9 preparation. The S9 was stored at minus 196 degrees Centigrade in a Statebourne liquid nitrogen freezer (Model SXR 34).

S9 MIX AND AGAR

The S9 mix was prepared at 4 degrees Centigrade as follows: S9 (5.0 mL); 1.65 M potassium chloride/0.4 M magnesium chloride (1.0 mL); 0.1 M glucose-6-phosphate (2.5 mL); 0.1 M NADP (2.0 mL); 0.2 M sodium phosphate buffer pH 7.4 (25.0 mL); sterile distilled water (14.5 mL).

Top agar was prepared using 0.6 % Difco Bacto agar and 0.5 % sodium chloride with 5 mL of 1.0 mM histidine/1.0 mM biotin and 1.0 mM tryptophan solution added to each 100 mL of top agar. Base agar plates were prepared using 1.2 % Oxoid Agar Technical No. 3 with Vogel-Bonner Medium E and 20 mg/mL D-glucose.

PRELIMINARY TOXICITY STUDY

A mixture of 0.1 mL bacterial suspension (TA100 or WP2uvrA), 0.1 mL of test solution, 0.5 mL phosphate buffer and 2 mL of molten, trace histidine/tryptophan supplemented media was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Five doses of the test material suspension and a vehicle/suspending agent control (sterile distilled water) were tested in duplicate. After approximately 48 hours incubation at 37 degrees Centigrade the plates were scored for revertant colonies and examined for a thinning of background lawn.

MUTATION STUDY - EXPERIMENT 1 (RANGE FINDING)

Five concentrations of test material suspension were assayed in triplicate against each tester strain using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.

TEST MATERIAL AND VEHICLE CONTROLS

Known aliquots (0.1 mL) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 mL of molten trace histidine/tryptophan supplemented top agar at 45 degrees Centigrade, 0.1 mL of the appropriately diluted test material suspension or vehicle control and either 0.5 mL of S9 liver microsome mix or 0.5 mL of pH 7.4 buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material with and without S9-mix.

POSITIVE CONTROLS WITHOUT ACTIVATION

A known aliquot (0.1 mL) of one of the positive control solutions (ENNG, 9AA or 4NQO) was added to a test tube containing 2.0 mL of molten trace histidine/tryptophan supplemented top agar and 0.1 mL of the appropriate bacterial suspension. Finally, 0.5 mL of pH 7.4 buffer was added to the tube, the contents mixed and poured onto an agar plate. This procedure was then repeated, in triplicate, for each tester strain. The plates were incubated at 37 degrees Centigrade for approximately 48 hours and the number of revertant colonies counted.


POSITIVE CONTROLS WITH ACTIVATION

A known aliquot (0.1 mL) of 2AA solution was added to a test tube containing 2.0 mL of molten trace histidine/tryptophan supplemented top agar and 0.1 mL of the appropriate bacterial suspension. Finally, 0.5 mL of S9-mix was added to the tube, the contents mixed and poured onto an agar plate. This procedure was then repeated, in triplicate, for each tester strain. The plates were incubated at 37 degrees Centigrade for approximately 48 hours and the number of revertant colonies counted.

MUTATION STUDY - EXPERIMENT 2 (MAIN STUDY)

The second experiment was performed using the same methodology described for Experiment 1. Fresh bacterial cultures, test material suspensions and control solutions were used in triplicate.

Evaluation criteria:

For the substance to considered positive, it should have induced a dose-related and statistically significant increase in mutation rate (at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results, or equivocal results are obtained, then a third experiment may be used to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility, or up to the maximum recommended dose of 5000 μg/plate. In this study the limiting factor was the maximum recommended dose.

Statistics:

As recommended in Kirkland DJ (Ed), Statistical Evaluation of Mutagenicity Test Data, UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report Part III (1989), Cambridge University Press.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to maximum recommended dose of 5000 ug/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to maximum recommended dose of 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Checks for characteristics, viability and spontaneous reversion rate for each tester strain were found to be satisfactory. Results for negative controls (spontaneous reversion rates) are presented in Table 1 of Appendix I (attached).

Individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls (with and without activation) are presented in Appendix I (attached). Results are also presented graphically in Appendix III (attached).

No toxicity was exhibited in any of the bacterial strains used. A precipitate was observed at and above 1500 μg/plate but did not interfere with the scoring of revertant colonies.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material either with or without metabolic activation.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

 Mean numbers of strain TA100 and WP2uvrA revertant colonies for the toxicity assay

 

Strain	Dose (μg/plate)
	Zero	50	150	500	1500	5000
TA100	115	115	119	117	113P	115P
WP2uvrA	41	33	34	34	36P	35P
P = precipitate

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

No significant increase in the frequncy of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was found to be non-mutagenic under the conditions of the test.
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 6

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

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:
- Cell density at seeding (if applicable):
- 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 afer 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

Link to relevant study records

Reference

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:

key study

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:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The family of zinc borates includes zinc metaborate anhydrous and zinc metaborate trihydrate. Zinc metaborate anhydrous is the anhydrous form of zinc metaborate trihydrate. Zinc metaborate and zinc metaborate anhydrous 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 solubilizes. Therefore, it can be assumed that at physiological conditions and neutral and lower pH zinc borate will be hydrolyzed to boric acid, zinc oxide and zinc hydroxide (Schubert et al., 2003).

Zinc metaborate trihydrate breaks down as follows:
2ZnO 2B2O3 3H2O + H2O + 4H+ <-> 4H3BO3 + 2Zn2+
2Zn2+ + 4OH- <-> 2Zn(OH)2
____________________________________________________________
Overall equation
2ZnO 2B2O3 3H2O + 5H2O <-> 2Zn(OH)2 + 4H3BO3

The relative zinc oxide and boric oxide % are as follows:

Zinc metaborate trihydrate: Zinc oxide = 45.71 % (36.73 % Zn)
B2O3 = 39.11 % (12.15 % B)
Water 15.18 %
Zinc metaborate, anhydrous: Zinc oxide = 53.89 % (43.3 % Zn)
B2O3= 46.11 % (14.32 % B)

The results of an ADME study in rats conducted with zinc borate show that significant amounts can be taken up into the systemic circulation via oral absorption (Muzzio and Johnson, 2010). Therefore, oral absorption of boron is set to 100 % (worst-case) for the purposes of hazard assessment. The oral absorption of boron is considered to be the same in animals and in humans (worst-case). Because of the homeostatic control of zinc, the amount of zinc absorbed and bioavailable after repeated oral dosing of zinc borate is likely limited.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
source substance: Zinc sulphate, CAS No 7733-02-0
target substance: Zinc metaborate, CAS No 1332-07-6, purity. > 90 %

3. ANALOGUE APPROACH JUSTIFICATION
Based on the chemical behavior of zinc borate and the critical toxicity endpoints that correlate with boron dose levels the read across to boric acid and sodium borates for zinc borate toxicity endpoints is supported. For zinc metaborate no such data is available for comparison, but it is reasonable to assume that the results would be similar.
In addition the behavior of the zinc compound is adequately covered with the read-across to zinc sulphate. Hydrolysis of zinc metaborate 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 metaborate hydrolyses to zinc hydroxide, zinc oxide and boric acid at neutral pH quicker than it solubilizes. Therefore, it can be assumed that at physiological conditions and neutral and lower pH zinc (meta)borate will be hydrolyzed to boric acid, zinc oxide and zinc hydroxide (Schubert et al., 2003). Toxicity of zinc oxide and zinc hydroxide is mediated via the zinc ion (Zn2+). The read-across compound zinc sulphate (ZnSO4) will also undergo partial hydrolysis in aqueous solutions forming zinc oxide, so that the chemical behavior of zinc metaborate and the critical toxicity endpoints are adequately covered by a read-across approach to zinc sulphate.

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:

no study available (further information necessary)

Additional information

supporting studies

in vitro gene mutation studies in bacteria (Thompson, 1995 and Stewart, 1993), negative

in vitro mammalian chromosome aberration studies (NTP, 1987), Read-Across to Boric acid, negative

in vivo erythrocyte micronucleus assay in mice (Gocke, 1981), Read-Across to zinc sulphate, negative

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. No evidence of mutagenic activity was observed. A positive result was obtained in an in vitro mammalian cell micronucleus test (OECD 487) indicating potential cytogenic activity. Based on this positive result an in vivo mammalian erythrocyte micronucleus test according to OECD Guideline 474 is considered appropriate to fulfil data requirements of Annex IX of the REACH Regulation.

Justification for classification or non-classification

A positive result was observed in an in vitro micronucleus test with the test item hexaboron dizinc undecaoxide according to OECD 487 (Geissel, 2019), while available studies on gene mutations in bacteria (OECD 471, Entrup, 1981) and mammalian cells (OECD 476, Curry, 2010) were negative. According to the Health Risk Assessment Guidance for Metals (HERAG Fact Sheet 05, 2007), appropriate in vivo follow-up testing is recommended prior to hazard classification due to the high prevalence of positive in vitro data for metals such as zinc. Therefore, to draw a final conclusion on C&L for mutagenicity, an in vivo micronucleus test according to OECD Guideline 474 is proposed in the lead dossier of the read-across substance zinc borate.