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Toxicity to reproduction

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Administrative data

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)

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
138265-88-0
EC Number:
604-070-9
Cas Number:
138265-88-0
IUPAC Name:
138265-88-0
Constituent 2
Reference substance name:
Zinc borate
IUPAC Name:
Zinc borate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Zinc borate 2335
- Substance type: anorganic salt
- Physical state: White powder
- Analytical purity: >98.8% zinc borate
- Composition of test material, percentage of components: USB quality specification for zinc borate is 37.7-38.7% ZnO and 47.5-48.9% B2O3, as
determined by titration according to an ISO certified method.
- Purity test date: 2013-08-15
- Lot/batch No.: Lot no. 10F01
- Expiration date of the lot/batch: 01 June 2017
- Storage condition of test material: stored at room temperature (approximately 18°C to 24°C)
- Stability under test conditions: considered stable under this condition.

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Effect levels (P0)

open allclose all
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

Results: F1 generation

Details on results (F1)

not applicable (90-day study)

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.