Hexaboron dizinc undecaoxide

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

in vitro study (no guideline followed, no GLP) Read-across is justified on the following basis: The family of zinc borates that include Zinc Borate 500, Zinc Borate 2335 and Zinc Borate 415 (also known as Zinc Borate 411). Zinc borate 500 is anhydrous Zinc Borate 2335 and Zinc Borate 415 has different zinc to boron ratio. Zinc borate 2335 (in common with other zinc borates such as Zinc borate 415 and 500) breaks down to Zinc Hydroxide (via Zinc oxide) and Boric Acid, therefore the family of zinc borates shares the same toxicological properties. Zinc borates are sparingly soluble salts. Hydrolysis under high dilution conditions leads to zinc hydroxide via zinc oxide and boric acid formation. Zinc hydroxide and zinc oxide solubility is low under neutral and basic conditions. This leads to a situation where zinc borate hydrolyses to zinc hydroxide, zinc oxide and boric acid at neutral pH quicker than it solubilises. Therefore, it can be assumed that at physiological conditions and neutral and lower pH zinc borate will be hydrolysed to boric acid, zinc oxide and zinc hydroxide. Hydrolysis and the rate of hydrolysis depend on the initial loading and time. At a loading of 5% (5g/100ml) zinc borate hydrolysis equilibrium may take 1-2 months, while at 1 g/l hydrolysis is complete after 5 days. At 50 mg/l hydrolysis and solubility is complete (Schubert et al., 2003). At pH 4 hydrolysis is complete. Zinc Borate 2335 breaks down as follows: 2ZnO • 3B2O3 •3.5H2O + 3.5H2O + 4H+ ↔ 6H3BO3 + 2Zn2+ 2Zn2+ + 4OH- ↔ 2Zn(OH)2 ____________________________________________________________ Overall equation 2ZnO • 3B2O3 •3.5H2O + 7.5H2O ↔ 2Zn(OH)2 + 6H3BO3 The relative zinc oxide and boric oxide % are as follows: Zinc borate 2335:zinc oxide = 37.45% (30.09% Zn) B2O3 = 48.05% (14.94% B) Water 14.5% Zinc borate 415: zinc oxide = 78.79%; (63.31% Zn) B2O3 = 16.85% (5.23% B) Water 4.36% Zinc borate, anhydrous: Zinc oxide = 45 % B2O3= 55% (17.1 % B)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of the study was to investigate the combined effects of zinc chloride with boric acid on spermatogenesis. A primary culture of seminiferous tubules from 21 days old Sprague Dawley rats was performed. Then, the combined effects of Zinc Chloride at 4 concentrations (1, 2.5, 5 and 10 μg/mL equivalent to 0.48, 1.2, 2.4 and 4.8 μg Zn/mL) with Boric Acid at 32 μg/mL on the number of each of the following cell types after 1 and 2 weeks of culture: somatic cells (sertoli and myoid cells) and germ cells (pre-meiotic cells (spermatogonia), meiotic cells (young spermatocytes, middle to late pachytene spermatocytes and secondary spermatocytes), post-meiotic cells (round spermatids)) were determined.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Boric acid

EC Number:

233-139-2

EC Name:

Boric acid

Cas Number:

10043-35-3

Molecular formula:

H3BO3

IUPAC Name:

Boric acid

Details on test material:

- Name of test material (as cited in study report): Boric acid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

-

Administration / exposure

Route of administration:

other: in vitro 3D cell culture

Details on exposure:

The Bio-Alter® technology is a proprietary 3D cell culture process which allows achieving 80% of spermatogenesis in vitro in rats in a serum free culture medium. This culture process ensures the maintenance (or reformation) of the blood-testis barrier and allows to study the mitotic and meiotic phases of spermatogenesis and the beginning of spermiogenesis. The three-dimensional organization of cells in the culture system preserves the polarization of the Sertoli cells. Thus, the introduction of the chemical in the basal medium can mimic the access constraints of the substance to meiotic germ cells, as it occurs, in vivo, in the testis.
Endpoints:
- Total Cell number & viability (on total cells):
The total cell number and viability was assessed by cell counter (TC-20, BioRad) and trypan blue exclusion on day 7 and on day 14.
- Survival / death of somatic and germ cells, proliferation of Sertoli cells and spermatogonia (stem cells of spermatogenesis):
The number of each cell type (somatic cells, spermatogonia, young spermatocytes, late pachytene spermatocytes, secondary spermatocytes and round spermatids) were measured by FACS analysis and compared to control.

Duration of treatment / exposure:

14 Days

Frequency of treatment:

continuously in culture

Details on study schedule:

The objective of the study was to investigate the combined effects of zinc chloride with boric acid on spermatogenesis. A primary culture of seminiferous tubules from 21 days old Sprague Dawley rats was performed. The combined effects of Zinc Chloride at 4 concentrations (1, 2.5, 5 and 10 µg/ml equivalent to 0.48, 1.2, 2.4 and 4.8 µg Zn/ml) with Boric Acid at 32 µg/ml on the number of each of the following cell types after 1 and 2 weeks of culture: Somatic cells (sertoli and myoid cells) and Germ cells: Pre-meiotic cells (spermatogonia), Meiotic cells (young spermatocytes, middle to late pachytene spermatocytes and secondary spermatocytes), Post-meiotic cells (round spermatids).
The concentration of boric acid is based on the boron concentration found in testis at the fertility LOAEL (established by Gilleron et al.) and based on a spermatogenesis toxicity evaluation of 4 concentrations of boric acid using Bio-Alter® technology.
The concentrations of zinc chloride were based on a dose range finder study up to cytoxic levels.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Medium control
Basis:
other: in vitro culture

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Boric Acid 32 μg/ml
Basis:
other: in vitro culture

Doses / concentrations 3

Remarks:

Doses / Concentrations:
Boric Acid + Zinc Chloride (1, 2.5, 5.0 and 10.0 μg/ml)
Basis:
other: in vitro cultur

Details on study design:

- Dose selection rationale: The concentration of boric acid is based on the boron concentration found in testis at the fertility LOAEL (Gilleron et al, No year) and based on a spermatogenesis toxicity evaluation of 4 concentrations of boric acid using Bio-Alter® technology.

Reference: Gilleron J, Carette D, Durand P, Pointis G, Segretain D. Connexin 43 a potential regulator of cell proliferation and apoptosis within the seminiferous epithelium. Int J Biochem Cell Biol. 41(6):1381-90.

Examinations

Statistics:

For every condition, cell number and viability were determined from 3 pooled wells at D7 and from 4 pooled wells at D14. As a consequence, it was not possible to compare statistically 2 conditions.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

All germ cells populations were decreased by boric acid at 32 μg/ml. It was also observed that, at D14, in the presence of boric acid increasing concentrations of zinc chloride lead to:

- A dose dependent increase in the number of germ cells,
- An increased number of spermatogonia,
- A dose dependent increase in the number of young spermatocytes,
- A dose dependent increase of the number of middle to late pachytene spermatocytes,
- A dose dependent increase of secondary spermatocytes,
- A rather dose dependent increase of round spermatids.

These results suggest that zinc interacts with boric acid reducing boric acid induced toxicity on spermatogenesis.

Effect levels (P0)

Dose descriptor:

conc. level:

Effect level:

32 other: mg/mL

Based on:

test mat.

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: germ cell opulations (migrated information)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Total cell number

At D7 and D14, boric acid at 32 μg/ml had no clear cut effect on the number of total cells. However, at day D7 and D14, boric acid with varying concentrations of zinc chloride increased in a dose dependent manner the number of total cells.

Total cell viability

Boric acid at 32 μg/ml had no effect on cell viability at any day of culture. Decreased cell viability was observed at all concentrations of zinc chloride on D14, with the greatest decrease seen in cultures treated with 10 μg/ml zinc chloride.

Total somatic cell number

No clear cut effect of boric acid was observed on the number of somatic cells at D7 and D14. A dose dependent increase of somatic cells was observed at D14 with boric acid in the presence of increasing concentrations of zinc chloride.

Total germ cell number

In the presence of 32 μg/ml boric acid, a small decrease in the number of total germ cells was observed. A dose dependent increase in the number of germ cells was observed at D7 and D14 in the presence of boric acid with increasing concentration of zinc chloride.

Spermatogonia

Boric acid decreased slightly the number of spermatogonia. This could result from a direct effect of boric acid on spermatogonia, which are located before the Sertoli cell barrier (tight junctions between the Sertoli cells: the main component of the blood-testis barrier). In that location the boric acid can reach the spermatogonia directly without the necessity to cross the Sertoli cell barrier. At D7, the addition of zinc chloride maintained the number of spermatogonia to the number observed in the control (without boric acid). At D14, the number of spermatogonia in the presence of zinc chloride was always higher than in the control or boric acid in the absence of zinc.

Young spermatocytes

The number of young spermatocytes was decreased on D7 with boric acid at 32 μg/ml. Zinc chloride (in the presence of boric acid) induced a dose dependent increase in the number of young spermatocytes with increasing concentrations of zinc chloride. This effect was particularly marked at D7.

Middle to late pachytene spermatocytes

The number of middle to late pachytene spermatocytes was decreased on D7 with boric acid at 32 μg/ml. Compared to boric acid alone, boric acid with increasing concentrations of zinc chloride induced a dose dependent increase of the number of middle to late pachytene spermatocytes, both at D7 and D14.

Secondary spermatocytes

Boric acid at 32 μg/ml decreased the number of secondary spermatocytes at D7 and D14. This confirms that the meiotic phase is impaired by boric acid. At D7, zinc chloride, only at 10μg/ml, increased the number of secondary spermatocytes, as compared to the effect of boric acid alone. However, this number remained lower than the number observed in the control (without boric acid). At D14, zinc chloride increased the number of secondary spermatocytes in a dose dependent manner. Moreover, the lower concentrations of zinc chloride were sufficient to raise the number of secondary spermatocytes to the number observed in the control.

Round sprematids

Boric acid at 32 μg/ml decreased, but slightly, the number of round spermatids at D7 and D14. Usually the number of round spermatids is a very useful parameter since these cells have completed the two meiotic divisions and that the differentiation of round spermatids in elongated spermatids and spermatozoa involves only cytological differentiation, without DNA synthesis (this is an “end-product”). At D7, the addition of zinc chloride 10μg/ml increased the number of round spermatids as compared to the effect of boric acid alone. At D14, the presence of zinc chloride increased the number of round spermatids in a rather dose dependent manner. This result fits the effects of zinc chloride on the number of secondary spermatocytes. Taken together, these results suggest that zinc interacts with boric acid reducing its toxicity on the meiotic divisions.

Applicant's summary and conclusion

Conclusions:

Using the Bio-Alter® technology, we observed that boric acid at 32 μg/ml altered the spermatogenetic process in cultures of rat seminiferous tubules for 7 and 14 days. These results demonstrate that zinc interacts with boric acid reducing its toxicity on spermatogenesis.

Executive summary:

Spermatogenesis toxicity of compoundscan be evaluated using an innovative assay that mimics the in vivo situation: the Bio-Alter® technology. The objective of this study was to investigate the combined effects of zinc chloride with boric acid on spermatogenesis. A culture of seminiferous tubules from juvenile male Sprague Dawley rats was performed.

After 7 and 14 days of culture +/- boric acid (32 μg/ml) +/- zinc chloride (1, 2.5, 5 and 10 μg/ml), cells were recovered and analyzed:

- Total cell number and viability was assessed by cell counter.

- Number of somatic cells, pre-meiotic cells (spermatogonia), meiotic cells (young

spermatocytes, late pachytene spermatocytes, secondary spermatocytes) and postmeiotic

cells (round spermatids) were measured by FACS analysis.

All germ cells populations were decreased by boric acid at 32μg/ml. We also observed that, at D14, in the presence of boric acid increasing concentrations of zinc chloride lead to:

- A dose dependent increase in the number of germ cells,

- An increased number of spermatogonia,

- A dose dependent increase in the number of young spermatocytes,

- A dose dependent increase of the number of middle to late pachytene spermatocytes,

- A dose dependent increase of secondary spermatocytes,

- A rather dose dependent increase of round spermatids.

These results suggest that zinc interacts with boric acid reducing boric acid induced toxicity on spermatogenesis. However, at D14, zinc chloride might have induced some features of testicular toxicity including an increase of the number of somatic cells and a decrease of total cell viability. Alternatively, this decrease could result from the higher number of germ cells (the viability of which is lower than that of somatic cells in culture) in the wells receiving zinc chloride.