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Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
07 APR 2021 to 21 MAR 2022
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Adopted 29th July, 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Hexaboron dizinc undecaoxide
EC Number:
235-804-2
EC Name:
Hexaboron dizinc undecaoxide
Cas Number:
12767-90-7
Molecular formula:
B6O11Zn2
IUPAC Name:
dizinc(2+) tris(oxo[(oxoboranyl)oxy]borane) dioxidandiide
Test material form:
solid

Test animals

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

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

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 1% aqueous methylcellulose; components of vehicle: Methylcellulose and Aqua Purificata
- Concentration of test material in vehicle: 0, 50, 100, 200 mg/mL; positive control: 6 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): SLCB1319, 2012-4685
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in 1% aqueous methylcellulose for the treatment. The necessary amount of test item was weighed into a calibrated volumetric flask.
A partial volume of 1% aqueous methylcellulose was added and the formulation was stirred until homogeneity is reached. The test item was used for treatment in concentrations of 50 mg/mL, 100 mg/mL and 200 mg/mL prepared with the mentioned vehicle. The formulations were prepared fresh on day of dosing and used within 2 hours.
Cyclophosphamide (positive control) was dissolved in Aqua ad injectabilia for treatment.
Duration of treatment / exposure:
The test/vehicle items were administered orally by gavage two times at 24-hour intervals.
Frequency of treatment:
The test/vehicle items were administered orally by gavage two times at 24-hour intervals.
Post exposure period:
Treatment groups and the vehicle control group: Sampling was made once at 24 hours after the second treatment.
Positive control group (Cyclophosphamide): Sampling was performed 24 hours after the beginning of the treatment. The mice were examined regularly for visible signs of reactions to treatment, immediately after dosing, and periodically until sacrifice.
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
No. of animals per sex per dose:
5 males/dose
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
Cyclophosphamide (CAS 6055-19-2)
- Route of administration: intraperitoneal
- Doses / concentrations: 60 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow was obtained from two exposed femurs of each animal. Blood sample of approximately 500 µL was obtained from the retro-orbital venous plexus.
Four thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 4000 PCEs counted in the optic field. The proportion of immature erythrocytes among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test/vehicle items were administered orally by gavage two times at 24-hour intervals.
The treatment volume was 10 mL/kg body weight. In the low, mid and high dose groups and the vehicle control group the sampling was made once at 24 hours after the second treatment. Five male animals per dose group were used for sampling. Cyclophosphamide (positive control) was administered intraperitoneally with a treatment volume of 10 mL/kg body weight. Sampling was performed 24 hours after the beginning of the treatment and five male animals were used for sampling. The mice were examined regularly for visible signs of reactions to treatment, immediately after dosing, and periodically until sacrifice

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

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

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

METHOD OF ANALYSIS:
Examination of slides: microscopic analysis
Criteria for Identification of Micronucleated Erythrocytes
A micronucleus is defined in following way:
• A bluish mauve strongly coloured uniform circular particle in the cell.
• The particle should have a certain size and it should be located inside the cells.
• During focusing, the particle should stay uniform in colour/light refraction and shape within a large interval.
• Cells with two or more micronuclei were counted as single micronucleated cells.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, the test item is considered clearly positive if:
1. At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
2. This increase is dose-related at least at one sampling time when evaluated with an appropriate test, and
3. Any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if the following criteria had been met:
1. None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
2. There is no dose-related increase at any sampling time when evaluated by an appropriate test, 3. All results are inside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits),
4. Bone marrow exposure to the test item occurred.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data: The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative and historical control groups. The proportion of immature erythrocytes among total (immature + mature) erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative and historical control groups. The data was checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

Results and discussion

Test results
Sex:
male
Genotoxicity:
positive
Remarks:
Induction of biologically and statistically significant increases in the frequency of micronucleated PCEs (MPCEs) at 500, 1000, 2000 mg/kg bw
Toxicity:
yes
Remarks:
2000 mg/kg bw: moderate and strong activity decrease, piloerection, incoordinated movement, decrease in grip strength, hunchback posture and narrow palpebra. In this dose group the faeces of animals were loose light brown
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: The study results are valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000, 2000 mg/kg bw
- Solubility: A non GLP Preliminary Solubility Test was performed April 07, 2021. The test item was formulated in 1% aqueous methylcellulose. Suspension suitable for treatment was obtained up to a concentration of 200 mg/mL.
- Clinical signs of toxicity in test animals:
Mortality rates: 2000 mg/kg bw: 0/4 males and 1/4 female; 1000 and 500 mg/kg bw: 0/0 males and 0/0 females
Clinical signs:
- 2000 mg/kg bw (females): Symptoms between 1–5 hours and at 24 hours: decreased activity and piloerection (slight, moderate) and loose light brown faeces. One female animal died before the second treatment. This female animal showed the following symptoms before death: decreased activity, incoordination, piloerection in heavy degree and loose light brown faeces, hunchback posture.
- 2000 mg/kg bw (males): Symptoms between 1–5 hours and at 24 hours: slight and moderate decreased activity and piloerection, expect one animal where a strong decrease in activity and piloerection was observed before and after the second treatment. In this dose group the faeces of animals were loose light brown from 24 hours after the first treatment as well as hunchback posture was observed.
- 1000 mg/kg bw (males and females): Slight and moderate piloerection (between 1–5 hours) after the first and second treatment and loose light brown faeces were observed from 24 hours after the first treatment.
- 500 mg/kg bw (males and females): Slight piloerection was observed between 1–5 hours after the first and second treatment
- Evidence of cytotoxicity in tissue analysed:
Proportion of immature erythrocytes among total (immature + mature) erythrocytes:
Two male mice: mean = 0.45
Two female mice: mean = 0.47
Historical control male mice: mean = 0.54
- Rationale for exposure: The preliminary toxicity test determined the MTD based on death and clinical signs of test item related toxicity, and whether there are differences in toxicity between the male and female animals.
- Other:
The main test was performed using male mice because the toxic effect of the test item was similar in both sexes in the preliminary acute oral toxicity test.
On the basis of the results of the preliminary toxicity test, doses for the Mouse Micronucleus Test were the following: 500, 1000 and 2000 mg/kg body weight.


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

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

Any other information on results incl. tables

 


Analysis of the Formulations (Test Item Concentrations)


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


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


Bioanalysis


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


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


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


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


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


 


MOUSE MICRONUCLEUS TEST Summary table
































































Groups



Sampling
time: 24 hours after the second tratment



Total number of
PCEs analysed



MPCE



PCE/
PCE+NCE



mean



SD



mean



SD



Negative Control



24



20000


5.40

1.14



0.53



0.00



500 mg/kg body weight



24



20000



9.60**



1.14



0.52



0.01



1000 mg/kg body weight



24



20000



19.00**



3.54



0.46**



0.01



2000 mg/kg body weight



24



20000



23.00**



3.67



0.44**



0.02


Positive Control (60mg/kg body weight)

24



20000



138.40**



6.27



0.38**



0.01



PCE = Polychromatic Erythrocyte


NCE = Normochromatic Erythrocyte


MPCE = Number of Micronucleated Polychromatic Erythrocytes referring to 4000 PCE


Positive Control = 60 mg/kg bw Cyclophosphamide


Negative Control: 1% aqueous methylcellulose


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


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


Kruskal-Wallis Non Parametric ANNOVA


DN=Duncan's Multiple Range


U=Mann-Whitney U-test

Applicant's summary and conclusion

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

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

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


Study design:


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


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


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


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


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


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


Results:


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


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


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


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


Conclusion:


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


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


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