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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A recently conducted in vitro gene mutation study in bacteria (Thompson, 2018), in vitro gene mutation study in mammalian cells (Brown, 2018) and an in vitro cytogenicity study (Lacey, 2019) concluded that boric oxide is not genotoxic and non-clastogenic under the conditions of the studies.

Same results were obtained from studies on boric acid.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 APR 2018 - 04 JUN 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted June 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Expiry Date: The Sponsor has confirmed that an expiration date is not applicable for the Test Item
Storage Conditions: Room temperature in the dark over silica gel
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa-; uvrB- (all); R-factor (only TA98 and TA100)
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: tryp-; uvrA-
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 29 March 2018 was used in this study.
- method of preparation of S9 mix : The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9: 5.0 mL
1.65 M KCl/0.4 M MgCl2: 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
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): sterility, sensitivity to diagnotic mutagens confirmed
Test concentrations with justification for top dose:
The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqueous solvents (sterile distilled water)

- Justification for choice of solvent/vehicle: recommended in guideline

- Justification for percentage of solvent in the final culture medium: maximum solubility of the test item
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: approx. 0.9 to 9 x 10^9 bacteria per mL.
- Test substance added: in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48h
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies
- Method used: agar
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: automated colony counting system

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Any supplementary information relevant to cytotoxicity: The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that are statistically significant but are within the in-house historical vehicle/untreated control range are not reported in the tables section.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in the same vehicle at 25 mg/mL in solubility checks performed in-house.
- Precipitation and time of the determination: No test item precipitate was observed on the plates at the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Ames test:
- Signs of toxicity : There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

Conclusions:
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item diboron trioxide (B203) did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test diboron trioxide (B203) was considered to be non-mutagenic.
Executive summary:

The study was performed according to the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test in compliance with GLP.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method both with and without the addition of a metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix) in the second experiment.

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1.

Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. Minor statistical values were noted in Experiment 2 (WP2uvrA at 15, 150 and 500 μg/plate in the absence of S9-mix), however these responses were within the in-house historical vehicle/untreated control ranges and were, therefore considered of no biological relevance.

In conclusion, diboron trioxide (B2O3) was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 APR 2018 - 14 JUN 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Expiry Date: The Sponsor has confirmed that an expiration date is not applicable for the Test Item
Storage Conditions: Room temperature in the dark over silica gel
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Normal cell cycle time (negative control): approximately 12 hours

For cell lines:
- Absence of Mycoplasma contamination: Master stocks of cells were tested and found to be free of mycoplasma.
- Methods for maintenance in cell culture: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air.
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media); 37°C with 5% CO2 in air; RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Lot No. PB/βNF S9 29/03/2018 was used in this study and was pre-prepared in-house (outside the confines of this study) following standard procedure.
- method of preparation of S9 mix : The S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.
- concentration or volume of S9 mix and S9 in the final culture medium: 20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.
- quality controls of S9:: Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test (sterility, sensitivity to diagnotic mutagens confirmed)
Test concentrations with justification for top dose:
The test item had a molecular weight of 69.62 therefore the maximum recommended dose level was set at 696 μg/mL (approximately 10mM). 2.72, 5.44, 10.88, 21.75, 43.5, 87, 174, 348, 696 µg/mL was tested
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqueous solvents (culture medium without serum)

- Justification for choice of solvent/vehicle: recommended in guideline
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
The positive controls were formulated in DMSO.
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10^6 cells/mL in 10 mL (4 h), 0.3 x 106 cells/mL in 10 mL (24 h)
- Test substance added: in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 or 24 hours
- Harvest time after the end of treatment (sampling/recovery times): 4 or 24 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time: ten to twelve days
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used, indicate its identity, its concentration and, duration and period of cell exposure:
4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies: Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data, a Relative Total Growth (RTG) value.
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37°C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item

- Any supplementary information relevant to cytotoxicity:
Percentage Relative Suspension Growth (%RSG):
The cell counts obtained immediately post exposure and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

Viability (%V)
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = number of negative wells/ total wells plated
%V = -ln P(0) x 100 / number if cells/well

Relative Total Growth (RTG)
For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = %V / Mean Solvent Control

Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100

Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS

- OTHER:
Evaluation criteria:
Plate count data from the viability and mutation frequency plates and the daily cell count data will be analyzed using a dedicated computer program (Mutant 2.40 York Electronics) which applies the statistical analysis mothod. The Historical Vehicle and Positive Control data is generated by the Mutant 240C program on a rolling system of the last twenty sets of archived data. The program combines the 4-hour and 24-hour data in the absence of metabolic activation as the acceptability criteria is the same for all three of the exposure groups. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance. An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Statistics:
the statistical analysis method recommended by the UKEMS (Robinson et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear trend events.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There was no marked change in pH when the test item was dosed into media
- Data on osmolality: the osmolality did not increase by more than 50 mOsm
- Precipitation and time of the determination: No precipitate was observed.

RANGE-FINDING/SCREENING STUDIES:
There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three exposure groups when compared to the concurrent vehicle control groups. A plateau of toxicity was observed at and above 174 μg/mL in both 4-hour exposure groups. The toxicity was more pronounced in the 24-hour exposure group, however acceptable toxicity was observed all the way up to the maximum recommended dose level of 696 μg/mL. No precipitate was observed. Therefore, the dose levels selected for the main test was up to the maximum recommended dose level of 696 μg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
There was evidence of marked dose related toxicity following exposure to the test item in all three exposure groups which was more pronounced in the 24-hour exposure group. Optimum toxicity was achieved at 696 μg/mL in the 24-hour exposure group. There was evidence of modest reductions in viability (%V) in the 24-hour exposure group only, indicating that residual toxicity had occurred.
The test item did not induce any toxicologically significant increases in the mutant frequency x 10-6 per viable cell at any of the dose levels, in any of the three exposure groups. The GEF value was not exceeded in all three of the exposure groups.
Conclusions:
The test item diboron trioxide (B2O3) did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

The study was performed according to the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline in compliance with GLP.

In one main mutagenicity test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (R0 culture media), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test at a concentration range of 2.72 to 696 μg/mL. The maximum dose level used in the Mutagenicity Test was up to the maximum recommended 10mM dose level of 696 μg/mL. The dose levels plated for viability and expression of mutant colonies were as follows:

4 hour with and without S9 mix and 24 h without S9 mix: 21.75, 43.5, 87, 174, 348, 696 µg/mL

The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency within the historical control data range, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in either of the three exposure groups. The GEF value was not exceeded at any test item concentration.

In conclusion, the test item, diboron trioxide (B2O3) did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10 ^-6, consequently it is considered to be non-mutagenic in this assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 APR 2018 - 29 AUG 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Expiry Date: The Sponsor has confirmed that an expiration date is not available for the Test Item
Storage Conditions: Room temperature in the dark over silica gel
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human peripheral lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35)
- Suitability of cells: screened for suitability
- Normal cell cycle time: approximately 16 hours

For lymphocytes:
- Sex, age and number of blood donors: Preliminary Toxicity Test: female, aged 27 years; Main Experiment: male, aged 30 years
- Whether whole blood or separated lymphocytes were used: C ells (whole blood cultures) were grown and lymphocytes of fresh heparinized whole blood were stimulated to divide
- Whether blood from different donors were pooled or not: blood from single donors was used
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 29/03/18 was used in this study.
- method of preparation of S9 mix: The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
- quality controls of S9: sterility and sensitivity to diagnostic mutagens confirmed
Test concentrations with justification for top dose:
The dose range of test item used was 0, 2.72, 5.44, 10.88, 21.75, 43.5, 87, 174, 348 and 696 μg/mL. In a preliminary toxicity test, the test item induced no evidence of toxicity in the short period exposure groups but in the 24-hour continuous exposure group, there was evidence of toxicity present. The selection of the maximum dose level for the Main Experiment was, therefore, based on the maximum recommended dose level (696 μg/mL) for all three exposure groups.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqueous solvents (culture medium: Eagle's minimal essential medium with HEPES buffer (MEM))

- Justification for choice of solvent/vehicle: The test item was insoluble in dimethyl sulphoxide at 69.6 mg/mL but was soluble in MEM at 6.96 mg/mL in solubility checks performed in-house

Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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: duplicate
- Number of independent experiments: 1

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest.
- Harvest time after the end of treatment (sampling/recovery times): 24 h after treatment

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985)
- Determination of polyploidy: cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported
- Determination of endoreplication: Endoreduplicated cells were recorded separately and are included in the polyploid cell total number

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: Mitotic index data was used to estimate test item toxicity
Evaluation criteria:
The following criteria were used to determine a valid assay:
- The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range
- All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix
- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
- The required number of cells and concentrations were analyzed
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Species / strain:
lymphocytes: human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
4 hour exposure
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: human peripheral lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
24 hour exposure
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There was no significant change in pH when the test item was dosed into media
- Data on osmolality: osmolality did not increase by more than 50 mOsm
- Precipitation and time of the determination: no precipitate observations were made at the end of exposure in any of the exposure groups tested.

RANGE-FINDING/SCREENING STUDIES: No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the exposure groups tested.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 696 μg/mL in the all three exposure groups.
The test item induced no evidence of toxicity in the short period exposure groups but in the 24-hour continuous exposure group, there was evidence of toxicity present.
The selection of the maximum dose level for the Main Experiment was, therefore, based on the maximum recommended dose level (696 μg/mL) for all three exposure groups.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range. All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis: The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: In the 4(20)-hour exposure group in the presence and absence of S9, no mitotic inhibition or precipitate was observed at any dose level tested where the maximum dose level selected for metaphase analysis was the Maximum Recommended Dose level (696 μg/mL). In the 24-hour exposure group, 10%, 50%, 82% and 93% inhibition of mitotic index was observed at 87, 174, 348 and 696 μg/mL, respectively. Therefore, the maximum dose level selected for metaphase analysis was 174 μg/mL because this dose achieved the 55±5% optimum toxicity as determined in the OECD 473 test guideline.
- Genotoxicity results
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps : 300 cells scored (150 per culture/concentration), The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes): The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.
Conclusions:
Diboron trioxide (B2O3) did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The study was performed according to the OECD Guidelines for Testing of Chemicals No. 473 "In Vitro Mammalian Chromosome Aberration Test" adopted 29 July 2016 and the Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011 in compliance with GLP. The purpose of the study was to assess the potential chromosomal mutagenicity of the test item, diboron trioxide (B2O3), on the metaphase chromosomes of normal human lymphocytes.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited on toxicity in the 24-hour exposure group but tested up to the Maximum Recommended Dose for the short exposure groups. The dose levels selected for the Main Experiment were as follows: 0, 10.88, 21.75, 43.5, 87, 174, 348, 696 µg/mL

All vehicle (Eagle's minimal essential medium with HEPES buffer (MEM)) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic to human lymphocytes in the short exposures but was toxic in the 24-hour exposure group. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced 55 ± 5% mitotic inhibition or was the maximum recommended dose level, depending on exposure group.

Thus, the test item, diboron trioxide (B2O3) was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In addition the results of an in vivo bone marrow cytogenetic assay (chromosome aberration assay, O’Loughlin 1991) also showed boric acid to be non genotoxic.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
other: US-EPA-FIFRA section 158.340 Guideline 84-2
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
Distilled water.
Details on exposure:
Mice given two doses (in 10 mL distilled water) by gavage.

Duration of treatment / exposure:
2 days.
Frequency of treatment:
Animals dosed once per day.
Post exposure period:
No data
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Dose / conc.:
900 mg/kg bw/day (actual dose received)
Dose / conc.:
1 800 mg/kg bw/day (actual dose received)
Dose / conc.:
3 500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
No data
Control animals:
not specified
Tissues and cell types examined:
No data
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Clinical signs included rough fur and haunched position.
Conclusions:
Interpretation of results: negative
The test substance was not genotoxic.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

No classification according to Regulation (EC) No 1272/2008 is required for boric oxide regarding genotoxicity as all results for boric acid and recent studies on boric oxide were negative in the tests.

Please also refer to the read-across statement attached to section 13.