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Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: sub-chronic study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-04-01 - 2013-10-08
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:
other: OECD Guideline 408; OPPTS Guideline 870.3100
Deviations:
no
Principles of method if other than guideline:
Effects on reproductive organs, including effects on spermatogenic parameters were studied
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS Crl:CD(SD) rats
- Source: Charles River Laboratories, Inc., Raleigh, NC, received on 02 April 2013
- Age at study initiation: approximately 37 days old at receipt, approximately 7 weeks old at the initiation of dose administration
- Weight at study initiation: 177 g to 236 g for males and from 148 g to 182 g for females at randomization
- Fasting period before study: no, but fasting prior to clinical pathology blood collection
- Housing: housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly
- Diet (e.g. ad libitum): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal) - this basal diet had a zinc content of 82.6 ppm and a boron content of 10.2 ppm - ad libitum except during the period of fasting prior to clinical pathology blood collection when food, but not water, was withheld
- Water (e.g. ad libitum): Reverse osmosis-treated (on-site) drinking water - ad libitum
- Acclimation period: 14-day acclimation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 71 ± 5°F = 22 ± 3°C)
- Humidity (%): 50 ± 20%
Actual mean daily temperature ranged from 70.3°F to 72.1°F (21.3°C to 22.3°C) and mean daily relative humidity ranged from 38.5% to 62.7% during the study.
- Air changes (per hr): minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod

IN-LIFE DATES: From: 2013-04-02 To: 2013-10-08
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% sodium carboxymethylcellulose [CMC; medium viscosity] in deionized water
Details on exposure:
REPARATION OF DOSING SOLUTIONS:
The vehicle suspension was prepared every 5 to 10 days for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored refrigerated (approximately 2°C to 8°C). The vehicle was stirred continuously throughout the preparation, sampling, and dose administration procedures.
The test substance formulations were prepared every 7 to 8 days as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated (approximately 2°C to 8°C). The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
1% sodium carboxymethylcellulose [CMC; medium viscosity] in deionized water
- Justification for use and choice of vehicle (if other than water): commonly recognised vehicle
- Concentration of test substance in vehicle: 0, 10, 20, 40 and 75 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Purity: 1% in deionised water

The vehicle and test substance formulations were administered orally by gavage via an appropriately sized flexible Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily for 91-92 consecutive days, through the day prior to the primary necropsy.
Details on mating procedure:
Not applicable (it is 90-day study)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of dose administration, samples for homogeneity and concentration determinations, and used as time-zero for stability evaluation, were collected from the top, middle, and bottom strata of the 10, 20, 40, and 75 mg/mL dosing formulations; samples were also collected from the middle stratum of the control group formulation. After sampling for homogeneity and concentration, a single aliquot from each concentration, sufficient for dosing a group of animals for 1 day, was stored refrigerated for 8 days. Following 8 days of refrigerated storage, samples were collected from the top and bottom strata of each test substance aliquot and analyzed for stability and resuspension homogeneity. Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared during study weeks 1-12. For the study week 0, 6, and 12 samples, 1 set of samples was analyzed; for the remaining samples, 1 set was stored refrigerated (approximately 2°C to 8°C) and 1 set was stored frozen (-10°C to -30°C) as backup samples. The samples collected for study weeks 1-5 and 7-11 were stored frozen (-10°C to -30°C) for possible future analysis. All analyses were conducted using an analytical method based on colorimetric titrations, assessing both zinc oxide and boric oxide concentrations for the bulk homogeneity assessment, the 8-day refrigerated homogeneity/stability assessment of the 15 April formulations, and the 7- and 13-day frozen stability assessment of the 28 May formulations, and assessing zinc oxide concentration only for the concentration assessment of the 28 May formulations.
The analyzed dosing formulations were found to contain 91.2% to 105% of the test substance. The test substance was not detected in the vehicle formulation that was administered to the control group (Group 1).
Duration of treatment / exposure:
92 days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
actual ingested
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
actual ingested
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
actual ingested
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Remarks:
actual ingested
No. of animals per sex per dose:
15 animals/sex/group (Groups 1 and 5)
10 animals/sex/group (Groups 2-4)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were determined from the results of a previous study and were provided by the Sponsor. In a previous study (Kirkpatrick, 2013, WIL-946001), it was concluded that oral administration of zinc borate 2335 for 28 days at dosages of 125, 250, and 500 mg/kg/day was generally well tolerated, but treatment-related changes were noted in the kidneys, testes, and epididymides in the 500 and 1000 mg/kg/day groups and in the pancreas and glandular stomach in the 250, 500, and 1000 mg/kg/day groups, and haematology, coagulation, and serum chemistry effects were noted in the 125, 250, 500, and 1000 mg/kg/day groups. However, all mean haematology and serum chemistry values for the 125 and 250 mg/kg/day groups were within the normal reference ranges. On study day 27, body weights in the 1000 mg/kg/day male and female dose groups were 21% and 9% lower than the control group, respectively. Though priority was given to detecting a dose-related trend, it was expected that the low dosage level would be the no-observed-adverse-effect level in the current study.
Positive control:
None.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Clinical examinations were performed at the time of dose administration and approximately 1 to 2 hours following dose administration. During the recovery period, the animals were observed once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations performed weekly (± 2 days)
On 12 April (4 days prior to the initiation of dose administration), all available rats were weighed and examined in detail for physical abnormalities. Detailed physical examinations were conducted on all animals 1 week (± 2 days) prior to randomization, on the day of randomization, weekly (± 2 days) during the study period, and on the day of the scheduled necropsies.

BODY WEIGHT: Yes
- Time schedule for examinations: body and food weights recorded weekly (± 2 days)
On 12 April (4 days prior to the initiation of dose administration), all available rats were weighed. Individual body weights were recorded 1 week (± 2 days) prior to randomization, on the day of randomization, on study day 0, weekly (± 2 days) during the dosing and recovery periods, and on the day prior to the first day of the scheduled necropsies. During study week 12, body weights were recorded twice weekly. The second weekly body weights are identified in the text and on the report tables as study week 13. Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsies.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
Individual food weights were recorded 1 week (± 2 days) prior to randomization, on the day of randomization, and weekly (± 2 days) during the dosing and recovery periods. Food consumption was calculated as g/animal/day for the corresponding body weight intervals. During study week 12, food consumption was recorded on the last day of the week, which was the day prior to the primary necropsy. This second weekly interval during study week 12 is identified in the text and on the report tables as study week 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ocular examinations were conducted on all animals during acclimation (05 April; study week -2), near the end of the dosing period (09 July; study week 12), and near the end of the recovery period (09 August; study week 16). All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study week 13 and 17
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLATELET), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count, Percent (RETIC), Absolute (RETIC ABSOLUTE), Mean platelet volume (MPV),

Differential leukocyte count Percent and absolute of Neutrophil (NEU) /Lymphocyte (LYMPH) / Monocyte (MONO) / Eosinophil (EOS) /Basophil (BASO) /Large unstained cell (LUC), in addition: Red cell distribution width (RDW) / Haemoglobin distribution width (HDW) /Platelet estimate / Red cell morphology (RBC Morphology)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study week 13 and 17
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Sorbitol dehydrogenase (SDH), Triglycerides (Triglyceride), Appearance

URINALYSIS: Yes
- Time schedule for collection of urine: study week 13 and 17
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Color (COL), Clarity (CLA), Protein (PRO), Glucose (GLU), Ketones (KET), Bilirubin (BIL), Occult blood (BLD), Leukocytes (LEU), Nitrites (NIT), Microscopy of sediment

Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected from all animals assigned to the scheduled necropsies (study week 13 and 17). The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected for haematology and serum chemistry evaluation via the retro-orbital sinus of animals anaesthetized by inhalation of isoflurane. Blood was collected for coagulation parameters at the time of euthanasia via the vena cava of animals euthanized by inhalation of carbon dioxide. Blood was collected into tubes containing potassium EDTA (haematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: FOB assessments were recorded for all animals during study weeks 12 (conducted prior to dose administration) and 17 (recovery period).
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Parameters examined in all male animals:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology]
MOTILITY/VIABILITY ASSESSMENT
Immediately following euthanasia and exsanguination, the reproductive tract of each male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a 10-minute incubation period, a sample of sperm was loaded into a 100-micron slide for determination of sperm motility. Because sperm motility can be affected by temperature shock, all cannule s and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported.
MORPHOLOGY ASSESSMENT
A sample of sperm for morphology assessment was obtained from the right cauda epididymis of each male. Sperm morphology was evaluated by light microscopy using a modification of the wet-mount technique. Abnormal forms of sperm (double heads, double tails, micro- or megacephalic, etc.) were recorded from a differential count of 200 spermatozoa/animal, if possible.
ENUMERATION OF EPIDIDYMAL AND TESTICULAR SPERM NUMBERS AND SPERM PRODUCTION RATE
The left testis and cauda epididymis from each male were weighed, stored frozen, homogenized, and evaluated for determination of homogenization-resistant spermatid count and calculation of sperm production rate (testis only). An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye. For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or 20 fields were counted for each sample. Sperm production rate was calculated using the testicular concentration
Litter observations:
Not applicable (90-day study)
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Necropsies performed on 10 animals/sex/group during study week 13; selected organs weighed; bone marrow smears collected for cytology evaluation (examined for Groups 1 and 5); male spermatogenic evaluations performed; selected tissues examined microscopically from all animals.
Necropsies performed on remaining 5 animals/sex in Groups 1 and 5 following a 29-day recovery period; selected organs weighed; bone marrow smears collected; male spermatogenic evaluations performed; selected tissues examined microscopically.

A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Clinical findings that were confirmed macroscopically were designated CEO on the individual macroscopic data tables. The following tissues and organs were collected and placed in 10% neutral-buffered formalin (except as noted):
Adrenal glands (2)*, Animal ID, Aorta*, Bone with marrow, Femur with joint, Sternum*, Bone marrow smear (from femur)a, Brain b*, Cervix*, Epididymides (2)c*, Eyes with optic nerve (2)d, Gastrointestinal tract, Esophagus*, Stomach*, Duodenum*, Jejunum*, Ileum*, Cecum*, Colon*, Rectum* Gross lesions (per WIL Research SOPs)*, Heart*, Kidneys (2)*, Lacrimal glands (2), Larynx, Liver (sections of 2 lobes)*, Lungs (including bronchi, fixed by, inflation with fixative [2])*, Lymph nodes (Axillary (2)*, Mandibular (2)*, Mesenteric*), Nasal cavity, Ovaries (2) with oviducts e*, Pancreas*, Peripheral nerve (sciatic)*, Peyer’s patches*, Pharynx, Pituitary*, Prostate*, Salivary glands (mandibular [2])*, Seminal vesicles (2)*, Skeletal muscle (quadriceps), Skin (with mammary gland)f*, Spinal cord*, Cervical, Lumbar,Thoracic, Spleen*, Testes (2)c*, Thymus*, Thyroid (with parathyroids [2])e*, Trachea*, Trachea bifurcation, Uterus*, Urinary bladder*, Vagina*

* = Examined microscopically from all animals in Groups 1 and 5 euthanized at the primary necropsy.
a = Bone marrow smears were obtained at scheduled necropsies but not placed in formalin; slides were examined from animals in Groups 1 and 5 at the primary necropsy.
b = Following collection of the appropriate protocol-specified tissues, the entire head was removed and preserved (olfactory bulbs were severed from the brain and remained in the skull).
c = Right testis, entire right epididymis, and left epididymis corpus and caput were placed in modified Davidson’s solution.
d = Fixed in Davidson’s solution.
e = Parathyroids and oviducts were examined if in the plane of section and in all cases where a gross lesion of the organ was present.
f = For females; a corresponding section of skin was taken from the same anatomical area for males.

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled necropsies:
Adrenals, Brain, Epididymides (total and cauda)*, Heart, Kidneys, Liver, Lungs, Ovaries with oviducts, Spleen, Testes*, Thymus, Uterus, Paired organs were weighed together. Designated organs (*) were weighed separately (left and right) after fixation. Organ to final body weight and organ to brain weight ratios were calculated.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION
After fixation, protocol-specified tissues were trimmed. Trimmed tissues were processed into paraffin blocks, sectioned, mounted on glass microscope slides, and stained with hematoxylin and eosin.
Microscopic examination was performed on all asterisk-designated tissues from all animals in the control and 375 mg/kg/day groups euthanized at the primary necropsy. In addition, the stomach (glandular and non-glandular), pancreas, kidneys, testes, epididymides, prostate, and gross lesions were examined from all animals in the 50, 100, and 200 mg/kg/day groups euthanized and the primary necropsy and all animals in the control and 375 mg/kg/day groups euthanized at the recovery necropsy. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.
Postmortem examinations (offspring):
Not applicable (90-day study)
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was 2 or less. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. The percentage of motile spermatozoa and the percentage of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Dunn’s test was used to compare the test substance-treated groups to the control group.
Reproductive indices:
Not examined (90-day study)
Offspring viability indices:
Not applicable (90-day study)
Clinical signs:
no effects observed
Description (incidence and severity):
All animals survived to the scheduled necropsies. Test substance-related clear material around the mouth was noted for males and females in the 200 and 375 mg/kg/day groups at the time of dosing and/or 1-2 hours following dose administration during study.
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights were unaffected by test substance administration.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean body weights were unaffected by test substance administration.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
germ cell degeneration in the 375 mg/kg/day group; acute and chronic inflammation (focal or multifocal in all instances) of the prostate in the 200 and 375 mg/kg bw group.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: Food consumption was unaffected by test substance administration
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
test substance-related lower percentages of motility, progressive motility, and morphologically normal sperm in the 200 and 375 mg/kg/day groups; lower sperm production and sperm conc. in the 375 mg/kg bw group.
Reproductive performance:
not examined
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: based on the effects on the male reproductive organs including effects on spermatogenic parameters with corresponding lower organ weights and gross and microscopic findings
Dose descriptor:
NOAEL
Effect level:
374 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no significant observations were found
not applicable (90-day study)
Remarks on result:
not measured/tested
Remarks:
this values are not available as the entry is based on a subacute und a subchronic repeated dose toxicity study in rats
Reproductive effects observed:
not specified

MACROSCOPIC EXAMINATION

Test substance-related findings in 375 mg/kg/day group males at the primary necropsy were small epididymides in 3 of 10 males, yellow area in the prostate in 1 of 10 males, and soft/small testes in 2 of 10 males. One 200 mg/kg/day group male had a gross observation of yellow area in the prostate. Test substance-related findings in 375 mg/kg/day group males at the recovery necropsy were soft/small testes in 1 of 5 males and prostate with yellow areas in 1 of 5 males.

ORGAN WEIGHTS

Test substance-related lower epididymis and testes weights were noted in the 375 mg/kg/day group males, and (epididymis only) in the 100 and 200 mg/kg/day groups. Statistically significant, lower weights (actual and relative to brain weight or body weight) for epididymides (left and right cauda) were noted at the primary necropsy and at the recovery necropsy in the 375 mg/kg/day group. Weights for epididymides (left and right cauda) were also slightly lower in the 100 and 200 mg/kg/day groups, but the changes were not statistically significant in these 2 groups. Testes weights (actual, not statistically significant) were lower in the 375 mg/kg/day group at the primary and recovery necropsies, and are considered test substance-related based on microscopic changes. Testes and epididymis weights in the 100 and 200 mg/kg/day groups were within range of values in the WIL Research historical control data.

The left cauda epididymis mean weight was lower by 44% in the 375 mg/kg/day group at the primary necropsy. The actual mean weight (0.18 grams compared to 0.32 grams for the control group) was barely within the lowest recorded value range in the WIL Research historical control database, but was considered to be treatment-related based on consistency of the change among the dose groups, similarity to weight change in the right cauda epididymis, which was lower by a similar percentage but below the historical control range, and correlation with microscopic findings. There were no other weight changes considered to be test substance-related. However, some statistically significant differences were observed when the control and test substance-treated groups were compared.

MICROSCOPIC EXAMINATION

Microscopic findings, considered to be directly or indirectly related to the test substance, were observed in the stomach (glandular and non-glandular), kidney, pancreas (please see "Repeated dose toxicity section"), and male reproductive organs.

Microscopic examination of the right testis showed germ cell degeneration in the 375 mg/kg/day group. Decreased size of the right epididymis, and the left epididymis generally correlated with germ cell degeneration in the testes. The severe-graded epididymis was from the same animal with germ cell degeneration in the testis graded severe. The testis finding was not detected in dose groups lower than 375 mg/kg/day.

One 375 mg/kg/day group male at the recovery necropsy had a gross observation of soft/small testis. Microscopically, this testis had germ cell degeneration at a grade of severe. Another male in the 375 mg/kg/day recovery group had testis germ cell degeneration at a grade of moderate. Based strictly on the presence of these 2 animals at the recovery necropsy, testicular germ cell degeneration was not completely recovered at the 375 mg/kg/day dose level. However, the presence of the finding at the recovery necropsy may represent loss of germ cells during the dosing period to the extent that recovery was not possible, rather than continued loss/degeneration of germ cells.

There were no findings of testes germ cell degeneration in dose groups lower than 375 mg/kg/day at the primary necropsy.

Microscopic examination of the prostate at the primary necropsy showed acute and chronic inflammation in the 200 and 375 mg/kg/day groups. The one 200 mg/kg/day group male which had chronic inflammation also had a gross observation involving the prostate (yellow areas, irregularly shaped). Three males were detected with inflammation of the prostate from the 375 mg/kg/day recovery necropsy, indicating no recovery at this dosage level.

In addition to acute and chronic inflammation (focal or multifocal in all instances) of the prostate, a common finding was cell debris and/or granular, condensed, secretions in the prostate acini. This change in the nature of the prostatic secretions likely accounted for the gross observation of yellow areas, in most instances. Incidence of cell debris and/or granular, condensed, secretions in the prostate at the primary necropsy.

Prostatic cellular debris/granular, condensed secretions were detected in 2 animals fromthe 375 m/kg/day dose group at the recovery necropsy. One was an animal with severe germ cell degeneration; the other had normal testis morphology.

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

SPERMATOGENIC EVALUATIONS

Test substance-related effects on spermatogenic parameters were noted in the 200 and 375 mg/kg/day groups.

At the study week 13 necropsy, test substance-related lower percentages of motility, progressive motility, and morphologically normal sperm were noted for males in the 200 and 375 mg/kg/day groups compared to the control group; the differences were statistically significant for the 200 mg/kg/day group males. In addition, the 375 mg/kg/day group males were noted with a lower sperm production rate and lower mean left testis and epididymis weights and sperm concentrations at the study week 13 necropsy compared to the control group; the differences were generally statistically significant.

Several of these test substance-related effects persisted to the study week 17 recovery necropsy and included statistically significantly lower left cauda epididymis weight and sperm concentration and statistically significantly lower percentages of progressive motility and morphologically normal sperm for males in the 375 mg/kg/day group compared to the control group.

Conclusions:
Based on the results of this study, oral administration of zinc borate 2335 to Crl:CD(SD) rats for a minimum of 90 consecutive days resulted in no adverse effects for males and females at dosage levels of 50 and 100 mg/kg/day and for females at 200 and 375 mg/kg/day. For males, a dosage level of 375 mg/kg/day 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 although there were no correlating microscopic findings. Therefore, the no-observed-adverse-effect level (NOAEL) was 100 mg/kg/day for males and 375 mg/kg/day for females.
Executive summary:

In order to investigate the repeated dose toxicity of Zinc borate 2335, it was administered in the vehicle (1% sodium carboxymethylcellulose in deionized water) orally by gavage to rats. Zinc borate was adminstered once daily for a minimum of 90 consecutive days to 4 groups (Groups 2-5) at dosage levels of 50, 100, 200 and 375 mg/kg bw (Kirkpatrick, 2014; OECD 408). A concurrent control group (Group 1) received the vehicle on a comparable regimen. Groups 1 and 5 each consisted of 15 animals/sex and Groups 2-4 each consisted of 10 animals/sex.

Following up to 92 days of dose administration, 10 rats/sex/group were euthanized; the remaining 5 rats/sex in the control and high-dose groups were euthanized following a 29-day nondosing (recovery) period. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily. Detailed physical examinations, individual body weights and food consumption were performed and recorded weekly throughout the dosing and recovery periods, and on the day of the scheduled necropsies. Individual body weights were also recorded on the day prior to the first day of the scheduled necropsies (non-fasted) and on the day of the scheduled necropsies (fasted). Functional observational battery (FOB) and locomotor activity data were recorded for all animals during study weeks 12 and 17. Ophthalmic examinations were performed prior to the initiation of dosing (study week -2) and during study weeks 12 and 16. Clinical pathology parameters (haematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals assigned to the primary (study week 13) and recovery (study week 17) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues, including gross lesions, were examined microscopically from all animals in the control and 375 mg/kg/day groups euthanized at the primary necropsy. In addition, the stomach (glandular and nonglandular), pancreas, kidneys, testes, epididymides, prostate, and gross lesions were examined from all animals in the 50, 100, and 200 mg/kg/day groups euthanized at the primary necropsy and all animals in the control and 375 mg/kg/day groups euthanized at the recovery necropsy. Bone marrow smears were collected from all animals for cytology evaluation and were

examined from the control and 375 mg/kg/day group animals euthanized at the primary necropsy. Spermatogenic endpoints were evaluated for all males at the scheduled necropsies.

There were no test substance-related effects on survival, body weight, food consumption, FOB parameters, locomotor activity, or haematology and coagulation. In addition, there were no test substance-related ophthalmic findings. Clinical pathology findings attributed to test substance administration included slightly lower total protein, globulin, and/or higher A/G ratios at ≥200 mg/kg/day for females and at 375 mg/kg/day for males; minimally lower calcium secondary to lower total protein and globulin at 375 mg/kg/day for males; lower cholesterol at ≥100 mg/kg/day for males and at ≥200 mg/kg/day for females; lower triglyceride values at ≥50 mg/kg/day for males; and higher urine pH at ≥100 mg/kg/day for males and at ≥200 mg/kg/day for females at the study week 13 evaluation. There were no meaningful differences between controls and the 375 mg/kg/day group at the study week 17 evaluation.

Test substance-related macroscopic findings were noted for the 375 mg/kg/day group males and consisted of small epididymides, a yellow area in the prostate, and soft/small testes at the study week 13 necropsy; soft/small testes and the yellow area in the prostate were also observed at the study week 17 recovery necropsy. In addition, 1 male in the 200 mg/kg/day group was noted with test substance-related yellow area in the prostate. Test substance-related lower epididymis (entire and cauda) and testes weights were noted in the 375 mg/kg/day group males at the study week 13 necropsy. Epididymis weights in the 100 and 200 mg/kg/day groups were not statistically significantly different from the control group and were within the range of the historical control data. Furthermore, no microscopic findings were seen in the epididymis of the 100 and 200 mg/kg/day groups. Testicular weights were lower in the 375 mg/kg/day group compared to the control group at both the primary (up to 13% lower) and recovery (up to 18% lower) necropsies; these differences did not reach statistical significance but did correlate with lower testicular sperm concentration and sperm production rate as well as germ cell degeneration observed microscopically at this same dosage level. The lower epididiymis (entire and cauda) and testicular weights persisted to the recovery necropsy for the 375 mg/kg/day group males and were considered adverse at this dosage level. Adverse test substance-related microscopic findings were noted in the 375 mg/kg/day group males at the primary and recovery necropsies and consisted of germ cell degeneration in the testes, decreased size of the epididymides, inflammation of the prostate, and debris in the prostate.

Based on the results of this study, oral administration of zinc borate 2335 to rats for a minimum of 90 consecutive days resulted in no adverse effects for males and females at dosage levels of 50 and 100 mg/kg/day and for females at 200 and 375 mg/kg/day. For males, a dosage level of 375 mg/kg/day 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 although there were no correlating microscopic findings. Therefore, the no-observed-adverse-effect level (NOAEL) was 100 mg/kg/day for males and 375 mg/kg/day for females.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
There are a lot of studies available on reproduction toxicity of boron and zinc.
Additional information

Protective Effects of Zinc against Boric Acid Related Effects on Fertility

It has been shown that zinc, essential for spermatogenesis, protects the testes of male mice against chromium-and cobalt-induced testicular damages (Afonne, 2002; Anderson, 1993).

In the first study, male mice (CD-1) were exposed to 150 ppm potassium dichromate and 500 ppm zinc chloride either individually or in combination for 12 weeks (Afonne, 2002). After the exposure and sacrifice, the epididymal sperm number counted. It was shown, that zinc had no effect on food intake and epididymal sperm number compared to control, while it significantly reduced the fluid intake, body weight gain and testis weight of mice. Chromium VI on the other hand, significantly decreased the body weight gain, food and fluid intake, and epididymal sperm number, but had no effect on testis weight compared to control. Concomitant exposure to both metals significantly increased the epididymal sperm number compared to chromium. Histological examination showed that chromium exposure induced severe pathologic changes on the mouse testis, which were reduced significantly by the combined metal exposure. Zinc had no deleterious effect on the testicular histology. Therefore it can be concluded, that chronic exposure to chromium VI produces a marked testicular toxicity, which can be prevented by concomitant zinc administration.

In the second study, CD-1 male mice were administered one of the following in their drinking water: 1) 400 ppm CoC12, 2) 800 ppm ZnCl2, 3) 400 ppm CoCI2 + 800 ppm ZnCl2, or 4) distilled water for 13 weeks (Anderson, 1993). Comparison of testicular weights revealed no difference between the control and zinc-treated groups, while there was a small but significant reduction in the zinc/cobalt-treated group, and a large reduction in the cobalt-treated group. Histologic evaluation of testes confirmed the degenerative effects of cobalt, as well as the normal morphology in the zinc-treated group. Furthermore, 90 % of the animals in the zinc/cobalt-treated group exhibited complete or partial protection as demonstrated by tubular morphology. This study indicates that zinc prevents cobalt-induced testicular damage.

To investigate the effect of zinc on boric acid related toxicity on fertility effects an in vitro spermatogenesis study with boric acid in the presence of varying amounts of zinc was conducted (Durand, 2013). Seminiferous tubules were cultured in the presence of 32 µg/mL boric acid and varying concentrations of zinc chloride equivalent to 0.48, 1.2, 2.4 and 4.8 µg Zn/mL to determine the effects on the number of 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), and post-meiotic cells (round spermatids)) after 1 and 2 weeks of culture. An absence of boric acid related effects on spermatogenesis was observed in the presence of zinc (see also Appendix D, Figure 15). All germ cells populations were decreased by boric acid at 32μg/mL. At Day 14, 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, and a dose dependent increase of round spermatids. Details of this study are presented in Appendix G.

In addition to the in vitro study, 28-day and 90-day oral (gavage) toxicity studies of zinc borate in Sprague-Dawley Rats were completed (Kirkpatrick, 2013, 2014, for full summary of study results please refer to the section “Repeated dose toxicity: oral”). Dosage levels tested in the 28-day study were 125, 250, 500, 1000 mg ZB/kg bw equivalent to 18.65, 37.3, 74.6 and 149.2 mg Boron/kg bw. The 500 mg/kg/day dose equivalent to74.6 mg B/kg bw was determined to be the no-observed-adverse-effect level (NOAEL) for male fertility effects based on the small magnitude or minimal to mild changes in the 125, 250, and 500 mg/kg/day groups. No microscopic changes were noted in the 125 mg/kg/day group. No microscopic findings were noted in the ovaries. The male fertility NOAEL for boric acid is 17.5 mg B/kg bw.

Dosage levels tested in the 90-day study were 50, 100, 200, and 375 mg ZB/kg bw equivalent to 7.46, 14.92, 29.84 and 55.95 mg Boron/kg bw. The objectives of this study were to evaluate the potential toxicity of zinc borate when administered daily by oral gavage to Sprague Dawley rats for a minimum of 90 consecutive days and to assess recovery from such effects. Adverse test substance-related microscopic findings were noted in the 375 mg/kg/day group males and consisted of germ cell degeneration in the testes, decreased size of the epididymides, inflammation of the prostate, and debris in the prostate. Test substance-related effects on spermatogenic parameters were noted in the 200 and 375 mg/kg/day group males at the study week 13 necropsies, as indicated by lower percentages of motility, progressive motility, and normal sperm at 200 and 375 mg/kg/day and a lower sperm production rate at 375 mg/kg/day. No microscopic findings were found in the testes or epididymis in the 200 mg/kg/day dose group. These effects for the 375 mg/kg/day group were considered adverse due to correlating microscopic findings. Based on the results of this study, oral administration of zinc borate to Sprague Dawley rats for a minimum of 90 consecutive days resulted in no adverse effects for females at dosage levels of 50, 100, 200, and 375 mg/kg/day. For males, a dosage level of 375 mg/kg/day 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 although there were no correlating microscopic findings. Therefore, no-observed-adverse-effect level (NOAEL) was 100 mg/kg/day for males and 375 mg/kg/day for females.

All these results suggest that zinc interacts with boric acid reducing boric acid induced toxicity on spermatogenesis. Of note, even with high exposures to zinc, tissue concentrations in the testes, epididymis, and ovaries of rats remain well below normal zinc levels found in comparative human tissues. In a 90-day inhalation study of ZnO, exposure to 200 mg/m³ of ZnO resulted in a significantly increased total body burden of zinc with an increase in zinc levels in most tissues with exception of testes, epididymis, ovaries, and RBC during exposure. Tissues with no increase in zinc levels were testes, epididymis, ovaries, and RBC (Placke, 1990, please refer to the section “Repeated dose toxicity: inhalation”). A greater degree of protection from boric acid related fertility effects would be expected in human tissues with substantially greater concentrations of zinc.


Short description of key information:
1) In vitro spermatogenesis study investigating toxicity of boric acid to reproduction in the presence of varying amounts of zinc; Durand, 2013
2) Kirkpatrick, 2014 - 90-day oral repeated dose toxicity study in rats.

Justification for selection of Effect on fertility via oral route:
Adverse effects on male reproductive organs, including effects on spermatogenic parameters with corresponding lower organ weights and gross and microscopic findings were observed.

Effects on developmental toxicity

Description of key information
- OECD 414, oral administration, rats, NOAEL for parental toxicity 150 mg/kg and NOAEL for developmental toxicity < 100 mg/kg (=LOAEL) (Edwards, 2014);
- Allen et al., 1996: BMD of 10.3 mg/kg bw/day for boron has been derived;
- Embryonic Stem cell Test (EST): investigation of the embryotoxic potential of boric acid in the presence of varying concentrations of zinc in vitro (Hofman-Huther, 2013).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Meets acceptable scientific standards 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:
no guideline required
Principles of method if other than guideline:
Developmental toxicity risk assessment has typically relied on the estimation of reference doses or reference concentrations based on the use of NOAELs divided by uncertainty factors. The benchmark dose (BMD) approach has been proposed as an alternative basis for reference value calculations. In this analysis of the developmental toxicity observed in rats exposed to boric acid in their diet, BMD analyses have been conducted using two existing studies. By considering various endpoints (rib XIII effects, variations of the first lumbar rib) and fetal weight changes and various modelling approaches for those endpoints, the best approach for incorporating all of the information available from the studies was determined. In this case, the approach involved combining data from two studies which were similarly designed and were conducted in the same laboratory to calculate BMDs that were more accurate and more precise than from either study alone.
GLP compliance:
not specified
Remarks:
not applicable (it is a publication)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
oral: feed
Vehicle:
not specified
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
20 days
Frequency of treatment:
continuously in diet
Remarks:
0, 0.025, 0.05, 0.075, 0.1 & 0.2% equivalent to 0, 19, 36, 55, 76 & 143 mg Boric acid/kg bw (Price et al., 1994, 1995) - Study B
Basis: nominal in diet
Remarks:
0, 0.1, 0.2, 0.4 & 0.8 % equivalent to 0, 78, 163, 330 & 539 mg Boric acid/kg bw (Heindel et al., 1992) - Study A
Basis: nominal in diet
No. of animals per sex per dose:
- 29 time-mated females/group (study A);
- 60 time-mated females/group (study B).
Control animals:
yes, plain diet
Details on study design:
The studies consist of two phases:
- Phase I: developmental toxicity termination on gd 20;
- Phase II: Postnatal recovery termination on pnd 21 (has not been considered in the analyses dicussed in the publication)
Dose descriptor:
BMD:
Effect level:
59 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: not specified
Dose descriptor:
BMD:
Effect level:
10.3 mg/kg bw/day
Based on:
element
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- incidence of total malformations, enlarged lateral ventricles in the brain, agenesis or shortening of rib XIII , and variations of the first lumbar rib, as well as decreased fetal weights.
Dose descriptor:
other: not specified
Based on:
not specified
Sex:
not specified
Basis for effect level:
other: see "Remarks"
Remarks on result:
other: not specified
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Developmental toxicity risk assessment has typically relied on the estimation of reference doses or reference concentrations based on the ues of NOAELs divided by uncertainty factors. The benchmark dose approach has been proposed as an alternative basis for reference value calculations. In the analysis presented of the developmental toxicity of rats exposed to boric acid in their diet, BMD analyses have been conducted using two existing studies. By considering various endpounts (rib XIII effects, variations of the first lumbar rib) and fetal weight changes and various modelling approachesfor those endpoints the best approach for incorporating all the information was determined. Decreased foetal body weight provided the best basis for BMD calculations. The BMD was calculated as 59 mg/kg bw/day.
Executive summary:

A benchmark dose developed by Allen et al. (1996) was based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996). The benchmark dose is defined as the 95 % lower bound on the dose corresponding to a 5 % decrease in the mean fetal weight (BMDL05). The BMDL05of 10.3 mg/kg body weight per day as boron is close to the Price et al. (1996) NOAEL of 9.6 mg/kg body weight per day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
BMDL05
10.3 mg/kg bw/day
Species:
rat
Quality of whole database:
There are a lot of studies available on developmental toxicity of boron and zinc.
Additional information

The potential of zinc borate for developmental toxicity was evaluated based on the results of the Developmental Toxicity Test (OECD 414, Edwards, 2014, Company Study No. WIL-946004). Three treatment groups of 25 Sprague Dawley [Crl:CD(SD)] rats were administered the test article via oral gavage at dose levels of 100, 125 or 150 mg/kg/day. The dosage levels were determined in an oral gavage dose range-finding prenatal developmental toxicity study (Edwards 2013).Compared to boric acid, a 55 % increase in the developmental NOAEL based on lower fetal body weights was demonstrated in the GLP developmental dose range finder study of zinc borate (Edwards 2013). One additional group of 25 animals/sex served as the control and received the vehicle, 1% carboxymethylcellulose in deionized water) via gavage. The maximal dose volume of 10 mL/kg. Dosing began on Gestation Day 6 and was continued up to Gestation Day 20.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 21, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The foetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

The oral administration of Zinc Borate to female rats at 100, 125 and 150 mg/kg/day produced no test article-related findings, as no remarkable clinical or macroscopic findings were noted. All paternal animals survived to scheduled necropsy without significant test article-related effects on body weight, food consumption. The lower mean body weight gains during the latter part of gestation and lower mean gravid uterine weights at 100, 125, and 150 mg/kg/day were attributed to lower mean foetal weights noted in all test substance-treated groups. Mean male, female, and combined foetal weights in the 100, 125, and 150 mg/kg/day groups were significantly (p < 0.01) lower (up to 5.5 %, 10.9 %, and 10.9 %, respectively) than the concurrent control group; the values in the 125 and 150 mg/kg/day groups were also below the minimum mean values in the historical control data. Intrauterine survival was unaffected by test substance administration at dosage levels of 100, 125, and 150 mg/kg/day. Parameters evaluated included postimplantation loss, live litter size, and foetal sex ratios.

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant. No test substance-related external malformations were noted for foetuses in the test substance-treated groups. One foetus in the 150 mg/kg/day group was noted with a cleft palate (skeletally, the rostral portion of the palatine plates were not joined). Because this finding occurred in a single foetus, and the mean litter proportion was within the WIL Research historical control data range for gestation day 20 foetuses, it was not considered test substance-related. There were no external developmental variations noted for foetuses in this study.

No test substance-related visceral developmental variations were noted in the 100, 125, and 150 mg/kg/day groups. Renal papilla(e) not fully developed and/or distended ureter(s) was noted for 3 and 5 foetuses in the 100 and 150 mg/kg/day groups, respectively; this finding was also noted for 18 foetuses in the control group. Accessory lobule(s) of the liver were noted for 3, 3, and 2 foetuses in the control, 100, and 125 mg/kg/day groups, respectively. These findings occurred infrequently or at a frequency similar to the control group, did not occur in a dose-related manner, and/or the mean litter proportions were within the WIL historical control data ranges; therefore, they were not considered test substance-related. Two and 6 foetuses in the control and 100 mg/kg/day groups, respectively, were noted with renal papilla(e) not fully developed (Woo and Hoar Grade 1). In the 100 mg/kg/day group, 7 foetuses in a single litter were noted with dark red areas on the adrenal glands. In the control group, 1 foetus was noted with yellow areas on the liver. These findings were not classified as either a malformation or developmental variation, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

There were no test substance-related skeletal malformations noted for foetuses in the test substance-treated groups. In the 150 mg/kg/day group, one foetus was noted with only 12 pairs of ribs present. Sternoschisis (sternal bands nos. 1 through 6 not joined, bilateral) was observed for 1 foetus in the 125 mg/kg/day group. One foetus in the 100 mg/kg/day group was noted with a costal cartilage anomaly (costal cartilage arising from right 7th cervical rib fused to right costal cartilage no. 1 and associated with sternum in normal no. 1 position). Because these malformations were noted for single foetuses, did not occur in a dose-related manner, and/or the mean litter proportions were within the WIL historical control data ranges, they were not considered test substance-related. Test substance-related skeletal developmental variations were noted in the 100, 125, and 150 mg/kg/day groups. Higher mean litter proportions of reduced ossification of the 13th rib(s), sternebra(e) nos. 5 and/or 6 unossified, and 7th cervical ribs were noted in all test substance-treated groups compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). In addition, higher (not statistically significant) mean litter proportions of 25 presacral vertebrae were noted in the 125 and 150 mg/kg/day groups compared to the control group. Significantly (p<0.01) lower mean litter proportions of 14th rudimentary rib(s) were noted at 100, 125, and 150 mg/kg/day compared to the control group.

Skeletal developmental variations that were noted in all groups, including the control group, consisted of sternebra(e) malaligned (slight or moderate), vertebral centra not fully ossified, and bent rib(s). Other skeletal developmental variations noted in the test substance-treated groups were noted in single foetuses, occurred infrequently, did not occur in a dose-related manner, and/or the mean litter proportions were within the WIL historical control data ranges.Therefore, these findings were not considered test substance-related.

In conclusion, when the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. 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, 125, and 150 mg/kg/day groups compared to the control group. These findings were considered secondary to the reduced foetal 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 foetal malformations were observed in the test substance-treated groups.

Based on these results, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and a dosage level of <100 mg/kg/day was considered to be the NOAEL for embryo/foetal development when zinc borate 2335 was administered orally by gavage to bred Crl:CD(SD) rats.

Protective Effects of Zinc against Boric Acid Related Developmental Effects

Comparative zinc concentrations in humans and rat indicate that the protective effects of zinc are present early in the developing human fetus. Significantly higher concentrations (3x) of zinc in the human fetus compared to laboratory animals have been reported (see Appendix G, Figure 15).

To investigate the effect of zinc on boric acid related toxicity on developmental effects, a GLP-compliant Embryonic Stem cell Test (EST) conducted according to INVITTOX Protocol No. 113 was conducted to investigate the embryotoxic potential of boric acid in the presence of varying concentrations of zinc in vitro (Hofman-Huther, 2013). No greater sensitivity of embryonic stem cells compared to fully differentiated cells was observed and no concern for in vivo embryotoxicity is triggered for boric acid at various concentrations of zinc. A reduction in the boric acid inhibition of differentiation of D3 embryonic stem cells was observed with increasing concentrations of zinc (see Appendix G, Figure 17) (Hofman-Huther, 2013).

Developmental effects have been observed in three species, rats, mice and rabbits (Allen et al., 1996). The most sensitive species being the rat with a NOAEL of 9.6 mg B/kg bw/day. This is based on a reduction in mean foetal body weight/litter, increase in wavy ribs and an increased incidence in short rib XIII at 13.3 mg B/kg bw/day. The reduction in foetal body weight and skeletal malformations had reversed, with the exception of short rib XIII, by 21 days postnatal. At maternally toxic doses, visceral malformations observed included enlarged lateral ventricles and cardiovascular effects. The NOAEL for this endpoint is 9.6 mg B/kg bw/day corresponding to 55 mg boric acid/kg bw/day; 85 mg disodium tetraborate decahydrate/kg, 65 mg disodium tetraborate pentahydrate/kg and 44.7 mg disodium tetraborate anhydrous/kg. The critical effect is considered to be decreased fetal body weight in rats, for which the NOAEL was 9.6 mg/kg body weight per day. A benchmark dose developed by Allen et al. (1996) was based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996) (cited in Allen et al., 1996). The benchmark dose is defined as the 95% lower bound on the dose corresponding to a 5% decrease in the mean fetal weight (BMDL05). The BMDL05of 10.3 mg/kg body weight per day as boron is close to the Price et al. (1996, cited in Allen et al., 1996) NOAEL of 9.6 mg/kg body weight per day.

While developmental effects of boron have been observed in rodent bioassays that include fetal body weight reduction and minor skeletal variations, there is no evidence of developmental effects in humans attributable to boron in studies of populations with high exposures to boron (Tuccar et al 1998; Col et al. 2000; Chang et al 2006). Three epidemiological studies evaluating high environmental exposures to boron and developmental effects in humans have been conducted. Epidemiological studies of human developmental effects have shown an absence of effects in exposed borate workers and populations living in areas with high environmental levels of boron. A more detail discussion of these studies is presented in Appendix B.

These data show that humans are likely less sensitive to the reproductive and developmental effects of boric acid than laboratory animals due to the comparative high zinc stores in target tissues in humans compared to laboratory animals. A greater degree of protection from boric acid related developmental effects would be expected in human tissues with substantially greater concentrations of zinc.


Justification for selection of Effect on developmental toxicity: via oral route:
BMD established for boric acid is more accurate than the NOAEL from the developmental toxicity study (OECD 414).

Justification for classification or non-classification

Adverse treatment related effects on gonads were observed in the 90-day study in male rats (OECD 408; Kirkpatrick, 2014) and in the developmental study in rats (OECD 414; Edwards, 2014). In the 90-day study, effects on spermatogenic parameters with corresponding lower organ weights and gross and microscopic findings were observed in the highest dose group (375 mg/kg bw). Adverse effects on spermatogenic parameters were also noted at 200 mg/kg/day although there were no correlating microscopic findings. The no-observed-adverse-effect level (NOAEL) for reproductive toxicity was 100 mg/kg/day for males and 375 mg/kg/day for females. In the developmental study, decreased fetal body weight and fetal skeletal developmental variations (higher mean litter proportions of reduced ossification of the 13th rib[s], sternebra[e] nos. 5 and/or 6 unossified, 7th cervical ribs, and 25 presacral vertebrae and lower mean litter proportions of 14th rudimentary rib[s]) were observed. The lowest dose level of 100 mg/kg bw is considered to be a LOAEL for developmental effects. Based on these results, additional study (two-generation (OECD 416) or extended study (OECD 443) will not provide additional results. Therefore, the substance meets the criteria for classification and labelling for reproductive toxicity (Cat 2, H361d) in accordance with European regulation (EC) No. 1272/2008.

Additional information