Hexaboron dizinc undecaoxide

Endpoint summary

Description of key information

1) 28-day sub-acute study on zinc borate (hydrate) was carried out in rats (Wragg et al 1996);
2) 90-day repeated dose study of zinc borate heptahydrate, rats, oral gavage, NOAEL 100 mg/kg bw (males), 375 mg/kg bw (females), (Kirkpatrick, 2014);
3) Allen et al., 1996: BMD of 59 mg of Boric acid/kg bw (equivalent to 10.3 mg Boron/kg bw) was calculated;
4) repeated dose inhalation toxicity of zinc oxide (Placke, 1990);
5) subacute repeated dose inhalation toxicity of aerosolized zinc borate heptahydrate in rats, a minimum of ten exposures (Randazzo, 2014)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptable well documented publication which meets basic scientific principles. 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:

BMD for boron was determined.

GLP compliance:

no

Remarks:

not applicable (it is a publication)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

No details given

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

No details given

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no details given

Duration of treatment / exposure:

20 days

Frequency of treatment:

ad libitum in feed

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)

Positive control:

None.

Dose descriptor:

BMD: developmental toxicity

Effect level:

59 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

BMD: developmental toxicity

Effect level:

10.3 mg/kg bw/day (nominal)

Based on:

element

Remarks:

boron

Sex:

female

Basis for effect level:

other: see "Remarks"

Critical effects observed:

not specified

Conclusions:

The specific BMD is 59 mg/kg/day. This BMD is based on the combined results of the two studies that were similarly designed and were conducted in the same laboratory. The selected value much less than the lowest dose tested in Study A (78 mg/kg/day, which was considered to be a LOAEL) and is very close to the NOAEL determined in Study B, 55 mg/kg/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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

BMDL05

10.3 mg/kg bw/day

Species:

rat

Quality of whole database:

The key study provides BMD which is based on results of two studies and therefore is more accurate and more precise than NOAEL established in one study.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

106 mg/m³

Species:

rat

Quality of whole database:

The key study provides BMD which is based on results of two studies and therefore is more accurate and more precise than NOAEL established in one study. The oral BMD has been extrapolated to inhalation BMD of 106 mg/m³ for zinc borate anhydrous by route-to-route extrapolation (see section "DNEL derivation").

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1989-09-13 to 1989-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

equivalent to OECD 408 guideline, well documented with sufficient details 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:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Remarks:

Food and Drug Administration (FDA) Good Laboratory Practice Regulations (21 CFR, Part 58)

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 145.7 g - 152.7 g
- Fasting period before study: no, but only during the exposure period. Feed was withheld from the animals for approximately 12 hours prior to necropsy.
- Housing: individually in stainless steel wire mesh cages.
- Diet (e.g. ad libitum): ad libitum (Certified Purina Rodent Chow® (pellets)) during the quarantine and study period except during each exposure period.
- Water (e.g. ad libitum): ad libitum during the quarantine and study period except during each exposure period.
- Acclimation period: 10 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 -25 (= 67° to 77° F)
- Humidity (%): 40 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: To:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: filtered air

Remarks on MMAD:

MMAD / GSD: Microscopic analysis of commercially available zinc oxide demonstrated that the particle size of most of the bulk material was less than five microns in diameter. Therefore, it was determined that a uniform dispersion of this commercially available test material would provide a respirable aerosol of ZnO at the concentrations desired.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Exposure apparatus:
The test animals were exposed in Battelle-designed live-in exposure chambers (Model H2000, Hazleton Systems/Lab Products, Inc., Aberdeen, MD). These chambers were designed to provide good test atmosphere distribution and mixing within each tier of animals, while minimizing mixing of the atmosphere between exposure tiers. This aids in providing good test atmosphere homogeneity while rapidly purging waste products (carbon dioxide and ammonia). Air flow through the chamber is diverted at the inlet to flow vertically down the inner surfaces of the chamber (Figure 3). At each exposure level, a portion of the airstream is entrained by the edge of the excreta catch pan to form eddies which move horizontally across the tier toward the center of the chamber. The eddies provide good mixing and eliminate any stagnant zones that would otherwise exist in the chamber. The air is exhausted down the center of the chamber, between the catch pans, to the exhaust port.
The chambers contain two columns of cage batteries, each comprised of three tiers. The columns are offset vertically from each other, creating six distinct exposure shelves (Figure 3).
Sample ports are located above each shelf, at both the front and rear, permitting the chamber atmosphere to be sampled from representative locations near the breathing zone of the test animals.

- Source and rate of air: Compressed air at about 90 psig was used to deliver the zinc oxide aerosol to a single manifold (Figure 2). The concentration of ZnO in the manifold air was approximately the same as that used for the high exposure level. The target concentrations for the four lower exposure levels were achieved by diverting a metered fraction of manifold air into each exposure chamber and diluting it with HEPA/activated-charcoal-filtered room air. Control rats were exposed in a chamber that received HEPA and activated charcoal-filtered room air only. The air handling system for the control chamber was separate from the test article generation and delivery system.

- System of generating particulates/aerosols: The test material aerosol generator was designed as a two-part system; the first was a mechanism to feed test material at a constant rate into an aerosol generator, and the second was a high-energy dispersion device to aerosolize the test material. The first component of the generation system was an AccuRate Model 300 Dry Chemical Feeder (AccuRate, Inc., Whitewater, WI) which accurately delivered preset amounts of ZnO into the aerosol generator. This device employed a large capacity hopper with an Auger-type feed screw. The AccuRate feeder directed a continuous stream of ZnO past the inlet to a Fox, one-inch, Coaxial Eductor (Fox Valve Development Corp., Dover, NJ), which aspirated the material, entrained it into a high-pressure stream of air, then introduced it into a cyclone. The cyclone was designed to remove particles or aggregates of particles having aerodynamic equivalent diameters greater than 5 µm. This system dispersed the test material into a fine aerosol. The aerosol stream was passed through a 85 Kr particle charge neutralizer to achieve particle charge equilibrium prior to delivery to the distribution manifold. See Figure 1 for a diagram of the generation system.

- Air flow rate: Air flow through the chamber is diverted at the inlet to flow vertically down the inner surfaces of the chamber (Figure 3).

TEST ATMOSPHERE
- Brief description of analytical method used: see "Details on analytical verification of doses or concentrations".
- Samples taken from breathing zone: yes. Chamber aerosol concentrations were monitored by gravimetric filter analysis once per hour.

VEHICLE (if applicable)
Filtered air was used

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber aerosol concentrations were monitored by gravimetric filter analysis once per hour. Aerosol concentrations were also measured hourly in each chamber during the animal exposures. Gravimetric filter samples were collected three times during the prestudy system validation trials, and once during exposures weeks 1, 6, and 12, at each concentration level for chemical analysis by inductively coupled plasma (ICP) emission spectroscopy. The results were used to confirm the appropriateness of using gravimetric analysis to measure and report ZnO concentration in each exposure chamber.

Duration of treatment / exposure:

13 weeks, followed by a 4 week recovery period.

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
1, 3, 10, 50 and 200 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

Base-study Croup: 10 males / 10 females;
Pulaonary Function and ZnO Tissue Distribution Croup: 10 males / 10 females;
Reproductive Evaluation Group: 20 males / 40 females;
Immunotoxicity Group: 5 males / 5 females;
Post-Exposure Group: 5 males / 5 females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: This study was part of several safety evaluations and field studies designed to identify the potential exposure risk and the potential associated health hazards of inhaled zinc oxide aerosols that may be generated as part of a paper preservations process.
- Rationale for animal assignment: Ten animals per sex were randomly assigned by body weight, to each of six exposure groups using Battelle's Xybion®, Path/Tox data capture system. The programming in this system provides for statistical homogeneity of group mean body weights across all study groups. Additionally, animals were assigned to other investigation groups. Ten animals of each sex were selected at random for serological evaluation and blood was collected from each of these animals within 48 hours of receipt and again approximately 2 weeks later for titer determinations to common murine infectious agents (Pneumonia Virus of Mice, Sendai Virus, Mycoplasma Pulmonis, Kilham Rat Virus, and Sialodacryoadenitis virus/rat coronavirus). Serum was separated from each blood sample, frozen, and the samples were sent to Microbiological Associates, Bethesda, Maryland, for the serological evaluations. No significant titers were measured in any of the serum samples collected during the quarantine period. Animals excluded by the randomization process and those used for serology studies were removed from the room and killed.
- Rationale for selecting satellite groups: not reported
- Post-exposure recovery period in satellite groups: 4 weeks

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (Base-Study and Post-Exposure Groups)
- Time schedule: Twice daily, before and after each daily exposure for morbidity and mortality and once weekly for clinical evidence of toxicity or other abnormalities.

BODY WEIGHT: Yes (Base-Study and Post-Exposure Groups)
- Time schedule for examinations: Days -14, -13, -12, and -4 pretest and prior to exposure on Day 1, at weekly intervals, and prior to necropsy.

HAEMATOLOGY: Yes (Base-Study and Post-Exposure Groups)
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes (propylene glycol-free sodium pentobarbital)
- Animals fasted: No data
- How many animals: animals of the base-study group prior to necropsy (10 sex/group) and from the post-exposure group prior to necropsy (5/sex/group designated for pathology evaluation)
- Parameters examined:
• red blood cell count (10^6 cells per microliter)
• hematocrit (percent)
• hemoglobin (g/dL)
• mean corpuscular volume (cubic microns)
• mean corpuscular hemoglobin (picograms)
• mean corpuscular hemoglobin concentration (g/dL)
• platelet (10³ platelets per microliter)
• white blood cell count (10³ cells per microliter)
• white blood cell differential count (absolute and relative)
• nucleated red blood cells
• reticulocyte count (percent)
• prothrombin time (seconds)
• activated partial thromboplastin time (seconds)
• bone marrow differential count.

Smears for differential cell counts were also made from the blood samples and were stained on an Ames Hema-Tek® Slide Stainer using a modified Wright-Giemsa stain. The relative number of segmented neutrophils, band neutrophils, lymphocytes, monocytes, eosinophils, and basophils was determined for each animal. The absolute number of each cell type per milliliter was also calculated. The number of nucleated red blood cells per 100 white blood cells (nRBC/100 WBC) was also determined.
The total reticulocyte count was determined after red blood cells were pre-stained with new methylene blue and a smear prepared. The number of reticulocytes per 100 red blood cells was counted microscopically and the results reported as the percentage of red cells which were reticulocytes.

CLINICAL CHEMISTRY: Yes (Base-Study and Post-Exposure Groups)
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: No data
- How many animals: animals of the base-study group prior to necropsy (10 sex/group) and from the post-exposure group prior to necropsy (5/sex/group designated for pathology evaluation)
- Parameters examined:
• glucose (mg/dL)
• total protein (g/dL)
• albumin (g/dL)
• albumin/globulin (A:G) ratio
• blood urea nitrogen (mg/dL)
• creatinine (mg/dL)
• total bile salts (mg/dL)
• serum aspartate aminotransferase (IU/L)
• serum alanine aminotransferase (IU/L)
• alkaline phosphatase (IU/L)
• lactate dehydrogenase (IU/L)
• electrolytes (Na, K, CI, and Ca in meq/L, Mg, Cu and Phosphorus in mg/dL).

URINALYSIS: Yes
- Time schedule for collection of urine: overnight during Study Week 12 from the base~study group and during Study Week 16 from the postexposure group
- Metabolism cages used for collection of urine: No data
- Animals fasted:No data
- Parameters examined: total volume, appearance, pK, specific gravity (g/mL), glucose (mg/dL), creatinine (mg/dL), urea nitrogen (mg/dL).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Base-Study and Post-Exposure Groups): one rat per sex from each exposure group. Special attention was given to the lungs and upper respiratory tract. The time from removal of the organs until weighing was minimized (approximately five minutes). Organs weighed from all base-study animals and post-exposure group were: liver, kidneys (pair), testes or ovaries (pair), adrenals (pair), heart (excluding major vessels), thymus, brain, and lungs. Organ weights were recorded to the nearest 10 mg except for ovaries and adrenals which were recorded to the nearest 1 mg. Organ:body weight ratios and organ:brain weight ratios were also calculated. The tissues listed below were examined for gross abnormalities, dissected from the carcass and were preserved in 10 percent neutral-buffered formalin (except eyes, epididymis, and testes which were fixed in Bouin's solution) at a volume dilution of 1 part tissue to at least 15 parts of fixative. After weights of the lungs were determined, all lungs were infused through the major airways with 10 percent formalin, at 25 cm hydrostatic pressure, using a gravity filling apparatus. The trachea was ligated after infusion to ensure trapping of the fixative in airways and alveoli. The tissues collected from each animal are:
Adrenals,
Bone (femur and marrow),
Brain (three sections to include frontal cortex and basal ganglia, parietal cortex and thalamus, cerebellum and pons)
Epididymis (Bouin's fixative)
Esophagus
Eyes and optic nerve (Bouin's fixative)
Gross lesions (suspected to potentially represent a test-compound-related effect)
Harderian glands
Heart and aorta
Intestine (which included a section of duodenum, ileum, jejunum, colon, cecum, and rectum)
Kidneys
Larynx
Liver
Lungs and bronchi
Mammary gland (with overlying skin)
Mandibular lymph node
Mesenteric lymph node
Nasal cavity (four levels)
Ovaries
Pancreas
Parathyroid
Pharynx
Pituitary
Preputial or clitoral glands
(paired) Prostate Salivary glands Seminal vesicles Skeletal muscle (thigh) Skin (dorsal midline) Spinal cord (microscopically
examined only thoracic cord) Spleen
Stomach (squamous and glandular
regions) Tail for identification Testes (Bouin's fixative) Thymic lymph node Thymus
Thyroid gland Trachea
Tracheobronchial lymph nodes
Urinary bladder
Uterus
Vagina
Zymbal's glands (was not examined microscopically unless there was a gross lesion)

HISTOPATHOLOGY: Yes (Base-Study and Post-Exposure Groups: Study Day 93, 94. Base-Study Group: 5/sex/ group each day. Study Day 121, 122 Post-Exposure: 5/sex/ group (remaining 5/sex/group underwent pulmonary function testing).

The respiratory tract, defined as the lungs, nasal cavity (four sections), nasopharynx, larynx (two cross-sections), trachea (cross- and longitudinal sections), tracheobronchial lymph nodes, and thymic lymph nodes, all gross lesions suspected to be exposure-related, from animals of the base-study necropsy group and postexposure group assigned to the air control group and each zinc oxide concentration exposure group, were blocked in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and submitted for light microscopicexamination. The lungs were sectioned so as to present a maximal section of the mainstem bronchi. The nasal cavity was prepared in four sections using the landmarks described by Young (Fundam. Appl. Toxicol., 1:309-312, 1981).
In addition, all remaining tissues listed in GROSS PATHOLOGY, collected from the base-study and post-exposure animals assigned to air control group and the high (200 mg/m3) concentration group were blocked, sectioned, stained, and examined microscopically

The following tissues were collected and analyzed for total Zn: Plasma , Testis/ovaries, Red Blood Cells, Epididymis, Lung, Prostate, Liver, Pancreas, Kidney, Femur.

Other examinations:

Pulmonary Function Group:
5 Rats/sex/group from the pulmonary function and 5 rats/sex/group from the post-exposure group underwent pulmonary function testing and bronchoalveolar samples were collected from each animal.

ZnO Tissue Distribution Group:
5 Rats/sex/group from the pulmonary function groups' tissues were measured to determine the absorption, distribution, accumulation, excretion and clearance of zinc.

Reproductive Evaluation Group:
20 Males and 40 females were used for mating trials. Sperm morphology and vaginal cytologies were conducted on the animals and evaluated for possible reproduction effects of test effects.

lnwunotoxicity Group:
Cytoxan Treatment - immunization Study Day 84 and 90
Keyhole Limpet Hemocyanin Immunization - Five and thirteen days prior to the last exposure. Sera collected one day and 30 days after the last exposure from 5 rats/sex/group. Rats were boosted 30 and 38 days after exposure and sera collected 44 days post-exposure. Anti-KLH serum antibody levels were determined using the ELISA method.

Statistics:

please see "Any other information on materials and methods incl. tables".

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

less than controls

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

slight increases in several red blood cell indices

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significant increases in group mean lung weights in all exposure groups

Gross pathological findings:

no effects observed

Description (incidence and severity):

A patchy discoloration of the lung was considered to be related to ZnO exposure.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

inflammation of the lungs with several lymph nodes in the chest cavity showing reactive hyperplasia

Histopathological findings: neoplastic:

no effects observed

Details on results:

Base-Study and Post-Exposure Group:

CLINICAL SIGNS AND MORTALITY
No unscheduled deaths occurred during this study.
Observations recorded in 10 mg/m^ were considered to be short in duration and unrelated to ZnO exposure. None of the animals in the 200, 50 or 3 mg/nr group appeared clinically abnormal during the study period.


BODY WEIGHT AND WEIGHT GAIN
Male rats exposed to 50 and 200 mg/m³ of ZnO had group mean body weight values that were 11 and 16 percent, respectively, less than controls at the end of the exposure period. By the end of the recovery period the values for 50 mg/m³ was only 6 less than controls and was not significant. The female body weight values of the 3, 10, 50 and 200 mg/m³ groups were 5, 5, 6 and 12 percent less than controls at the end of exposure. The group mean body weights were also less at the end of the 4-week post-exposure period, with only the 200 mg/m³ group being significantly (p < 0.05) less than controls.

HAEMATOLOGY
There were slight increases in several red blood cell indices, (RBC, Hct, Hb) in rats of both sexes exposed to 200 mg/m³ of ZnO in the base study group. Rats from the same concentration, post-exposure group did not have any differences in red cell indices, indicating that the changes were reversible. Several rats in the higher concentrations of ZnO had increases relative to controls, in white blood count, with decreases in the relative number of lymphocytes and increases in the relative numbers of segmented neutrophils which indicated the presence of an inflammatory process.

ORGAN WEIGHTS
Significant increases in group mean lung weights in all animals exposed to 10, 50 and 200 mgJ/m³ of ZnO.

GROSS PATHOLOGY
A patchy discoloration of the lung was considered to be related to ZnO exposure.

HISTOPATHOLOGY: NON-NEOPLASTIC
The lesion of the greatest significance was an inflammation of the lungs with several lymph nodes in the chest cavity showing reactive hyperplasia.

OTHER FINDINGS
Pulmonary Function
Respiratory Physiology Evaluation:
Male and female rats exposed to greater than 10 mg/m³ ZnO aerosols developed changes in functional properties of their respiratory systems during exposure and returned to control levels after the recovery period.

Bronchoalveolar Lavage Evaluations:
Differential, total and viable cell counts of pulmonary lavage cells revealed changes indicative of lung inflammation in rats exposed to higher concentrations of ZnO.

ZnO Tissue Distribution Group
ZnO Measurements:
Exposure to 200 mg/m3 of ZnO resulted in a significantly increased total body burden of zinc. Tissue levels of zinc increased in most tissues during exposure and returned to near control levels during the recovery period (with the exception of lung and bone which showed zinc retention). Tissues with the greatest increases were lung, liver, pancreas and femur.

Reproductive Evaluation Group
Females:
No effects of ZnO exposure or estrous cycle lengths, fertility or length of gestation. No effects on clinical signs, liter size, pup survival, pup weight or sex ratio.
Males:
No significant exposure-related effects on epididymal sperm motility, concentration or morphology or testicular spermatid concentration.

Immunotoxicity Group
Anti-KLH:
Exposure to high concentrations of ZnO significantly reduces humoral immunocompetence. This immunosuppressive effect of ZnO persists following exposure.

Dose descriptor:

NOEC

Effect level:

3 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

please see summary table and figures attached to this file

Conclusions:

Based on the results of this study, all toxic effects appeared to be reversible. The no-effect concentration level was 3 mg/m³. However, the two higher concentrations of 50 and 200 mg/m³ caused many significant lesions considered health hazardous when respired.

Executive summary:

The objectives of this study were to evaluate the potential toxicity of inhaled zinc oxide (ZnO) aerosol following subchronic exposures in rats (5 days per week for 13 consecutive weeks), to define the concentration response, to identify and characterize effects on target organs, to determine the tissue distribution of zinc as a function of concentration and continued exposure, to determine the reversibility of exposure-related toxic effects, to evaluate specific toxic potential on the immune, hematopoietic, and reproductive systems and to select concentrations for a possible subsequent chronic toxicity and carcinogenicity study. This study included a base-study group of animals consisting of 10 rats/sex/exposure group, a pulmonary function/ZnO tissue distribution group of 10 rats/sex/exposure group, a reproductive evaluation group of 20 male and 40 female rats/exposure group, an immunotoxicity group of 5/rats/sex/group and a post-exposure group of 10 rats/sex/exposure group. All groups were exposed to one of five mg/m3 concentrations of ZnO aerosol (1, 3, 10, 50 and 200 mg/m3) generated from bulk powdered test article or to filtered air alone (air control group). Animals were exposed for 6 hours/day, 5 days/week for 13 weeks. The post-exposure group animals were held without further exposure for observation and additional studies for 4 weeks following the 13-week exposure period.

No unscheduled deaths occurred during this study. Clinical observations recorded in animals of 10 mg/m³ dose group were considered to be short in duration and unrelated to ZnO exposure. None of the animals in the 200, 50 or 3 mg/m³ group appeared clinically abnormal during the study period. Male rats exposed to 50 and 200 mg/rn³ of ZnO had group mean body weight values that were 11 and 16 percent, respectively, less than controls at the end of the exposure period. By the end of the recovery period the values for 50 mg/m³ was only 6 less than controls and was not significant. The female body weight values of the 3, 10, 50 and 200 mg/m³ groups were 5, 5, 6 and 12 percent less than controls at the end of exposure. The group mean body weights were also less at the end of the 4-week post-exposure period, with only the 200 mg/nr group being significantly (p≤ 0.05) less than controls.There were slight increases in several red blood cell indices, (RBC, Hct, Hb) in rats of both sexes exposed to 200 mg/m³ of ZnO in the base study group. Rats from the same concentration, post-exposure group did not have any differences in red cell indices, indicating that the changes were reversible. Several rats in the higher concentrations of ZnO had increases relative to controls, in white blood count, with decreases in the relative number of lymphocytes and increases in the relative numbers of segmented neutrophils which indicated the presence of an inflammatory process. There were significant increases in group mean lung weights in all animals exposed to 10, 50 and 200 mg/m³ of ZnO. A patchy discoloration of the lung was considered to be related to ZnO exposure. The lesion of the greatest significance was an inflammation of the lungs with several lymph nodes in the chest cavity showing reactive hyperplasia.

Regarding respiratory physiology evaluations in animals of pulmonary function exposure group, male and female rats exposed to greater than 10 mg/m3 ZnO aerosols developed changes in functional properties of their respiratory systems during exposure and returned to control levels after the recovery period. Differential, total and viable cell counts of pulmonary lavage cells revealed changes indicative of lung inflammation in rats exposed to higher concentrations of ZnO.

Exposure to 200 mg/m³ of ZnO resulted in a significantly increased total body burden of zinc. Tissue levels of zinc increased in most tissues during exposure and returned to near control levels during the recovery period (with the exception of lung and bone which showed zinc retention). Tissues with the greatest increases were lung, liver, pancreas and femur (the findings in the additional groups are not presented here).

In animals of reproductive evaluation group, no effects of ZnO exposure were observed on estrous cycle lengths, fertility or length of gestation. No effects on clinical signs, liter size, pup survival, pup weight or sex ratio. No significant exposure-related effects were observed on epididymal sperm motility, concentration or morphology or testicular spermatid concentration.

in animals of immunotoxicity group, exposure to high concentrations of ZnO significantly reduces humoral immunocompetence. This immunosuppressive effect of ZnO persists following exposure.

Based on the results of this study, all toxic effects appeared to be reversible. The no-effect concentration level was 3 mg/m³. However, the two higher concentrations of 50 and 200 mg/m³ caused many significant lesions considered health hazardous when respired.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

3 mg/m³

Study duration:

subchronic

Species:

rat

Additional information

Repeated Dose toxicity - oral:

A subacute oral repeated dose study was undertaken with Firebrake 415 (administration to rats by gavage, for a period of 28 consecutive days at dose levels of up to 1000 mg/kg/day) and resulted in toxicologically significant changes at 150, 300 and 1000 mg/kg/day (Wragg, 1996). At 150 mg/kg/day, administration of the test material produced a reduction in mean corpuscular volume, an increased plasma alkaline phosphatase concentration and parietal cell atrophy/degeneration and sub-mucosal inflammatory infiltrates in the glandular stomach. These changes were also evident at 300 mg/kg/day together with short-lived increased salivation, increased water consumption, haematological changes indicative of an anaemia and pale liver. Such changes were also observed at 1000 mg/kg/day together with clinical signs of toxicity, reduced body weight and food efficiency, an increased leucocyte and neutrophil count, haematological and blood chemical changes indicative of liver damage , urinalytical changes consistent with diuresis and an haemoglobulinuria, increased absolute and relative adrenal weight, macroscopic lesions involving adrenals, spleen and glandular stomach and histopathological abnormalities involving the adrenal glands and spleen.

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 (Kirkpatrick, 2014; OECD 408). Zinc borate was administered 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. 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. In the study of Allen et al. (1996) BMD analyses have been conducted using two existing developmental toxicity studies in rats exposed to boric acid in their diet. By considering various end points (rib XIII effects, variations of the first lumbar rib, and fetal weight changes) and various modeling approaches for those end points, the best approach for incorporating all of the information available from those studies could be determined. Particular emphasis has been placed on methods for combining data across studies and for combining potentially related effects (on rib XIII and on the first lumbar rib). The issues of study and end point selection are ones that will arise frequently in the process of estimating reference values. This example of boric acid suggests that the BMD approach provides a reasonable basis for appropriately comparing and combining study data, as opposed to ad hoc combinations of study results. Moreover, it is shown that the BMD approach can be used with combinations of end points considered to differ in severity. In this case, the preferred approach involved combining the data from the two studies, which were similarly designed and were conducted in the same laboratory, to calculate BMDs that were more accurate and more precise than those that could be derived from either study alone. It was determined that decreased fetal body weight provided the best basis for BMD calculations; BMDs calculated for fetal body weight changes were less than those for all other relevant end points. The appropriate BMD to use as the basis for boric acid reference dose calculation appears to be 59 mg/kg/day, which is very similar to the NOAEL observed in the second of the two studies (55 mg/kg/day). Although the first study failed to establish a NOAEL, the BMD approach could have been applied to that study, thereby avoiding the need for a repeat study. Similar BMD results were obtained in both studies.

Repeated dose toxicity - inhalation:

Placke et al. investigated the potential toxicity of inhalted zinc oxide following subchronic exposures in rats (Placke et al., 1990). The animals were exposed 5 days per week for 13 consecutive weeks. Additionally the concentration response was defined, the effects on target organs were identified and characterized, the tissue distribution of zinc as a function of concentration and continued exposure was determined, the reversibility of exposure-related toxic effects was determined, the specific toxic potential on the immune, haematopoietic, and reproductive systems was evaluated and the concentrations for a possible subsequent chronic toxicity and carcinogenicity study were selected.

This study included a base-study group of animals consisting of 10 rats/sex/exposure group, a pulmonary function/ZnO tissue distribution group of 10 rats/sex/exposure group, a reproductive evaluation group of 20 male and 40 female rats/exposure group, an immunotoxicity group of 5/rats/sex/group and a post-exposure group of 10 rats/sex/exposure group. All groups were exposed to one of five mg/m3 concentrations of ZnO aerosol (1, 3, 10, 50 and 200 mg/m3) generated from bulk powdered test article or to filtered air alone (air control group). Animals were exposed for 6 hours/day, 5 days/week for 13 weeks. The post-exposure group animals were held without further exposure for observation and additional studies for 4 weeks following the 13-week exposure period.

No unscheduled deaths occurred during this study. Clinical observations recorded in animals of 10 mg/m³ dose group were considered to be short in duration and unrelated to ZnO exposure. None of the animals in the 200, 50 or 3 mg/m³ group appeared clinically abnormal during the study period. Male rats exposed to 50 and 200 mg/rn³ of ZnO had group mean body weight values that were 11 and 16 percent, respectively, less than controls at the end of the exposure period. By the end of the recovery period the values for 50 mg/m³ was only 6 less than controls and was not significant. The female body weight values of the 3, 10, 50 and 200 mg/m³ groups were 5, 5, 6 and 12 percent less than controls at the end of exposure. The group mean body weights were also less at the end of the 4-week post-exposure period, with only the 200 mg/nr group being significantly (p≤ 0.05) less than controls.There were slight increases in several red blood cell indices, (RBC, Hct, Hb) in rats of both sexes exposed to 200 mg/m³ of ZnO in the base study group. Rats from the same concentration, post-exposure group did not have any differences in red cell indices, indicating that the changes were reversible. Several rats in the higher concentrations of ZnO had increases relative to controls, in white blood count, with decreases in the relative number of lymphocytes and increases in the relative numbers of segmented neutrophils which indicated the presence of an inflammatory process. There were significant increases in group mean lung weights in all animals exposed to 10, 50 and 200 mg/m³ of ZnO. A patchy discoloration of the lung was considered to be related to ZnO exposure. The lesion of the greatest significance was an inflammation of the lungs with several lymph nodes in the chest cavity showing reactive hyperplasia.

Regarding respiratory physiology evaluations in animals of pulmonary function exposure group, male and female rats exposed to greater than 10 mg/m3 ZnO aerosols developed changes in functional properties of their respiratory systems during exposure and returned to control levels after the recovery period. Differential, total and viable cell counts of pulmonary lavage cells revealed changes indicative of lung inflammation in rats exposed to higher concentrations of ZnO.

Exposure to 200 mg/m³ of ZnO resulted in a significantly increased total body burden of zinc. Tissue levels of zinc increased in most tissues during exposure and returned to near control levels during the recovery period (with the exception of lung and bone which showed zinc retention). Tissues with the greatest increases were lung, liver, pancreas and femur (the findings in the additional groups are not presented here).

In animals of reproductive evaluation group, no effects of ZnO exposure were observed on estrous cycle lengths, fertility or length of gestation. No effects on clinical signs, liter size, pup survival, pup weight or sex ratio. No significant exposure-related effects were observed on epididymal sperm motility, concentration or morphology or testicular spermatid concentration.

In animals of immunotoxicity group, exposure to high concentrations of ZnO significantly reduces humoral immunocompetence. This immunosuppressive effect of ZnO persists following exposure.

Moreover, a 14 -day repeated dose toxicity study was performed (Randazzo, 2014) and the objective of this study was to evaluate the tolerability and potential toxic effects of aerosolized zinc borate when administered to Sprague Dawley rats as 6-hour, nose-only inhalation exposures on a 5-day per week basis for 2 weeks (minimum of 10 exposures). Standard toxicity endpoints included clinical observations, body weights, macroscopic examinations and organ weight determinations at necropsy, and microscopic examinations of select tissues. The results of this study were to be used to select exposure concentrations for a subsequent 4-week inhalation toxicity study in this test system.

Zinc borate was administered as 6-hour, nose-only inhalation exposures to 5 groups (Groups 2-6) of Crl:CD(SD) rats. Each animal received 10 total exposures over a period of 2 weeks. The target exposure concentrations were 5, 10, 25, 75, and 150 mg/m³ for Groups 2, 3, 4, 5, and 6, respectively. Overall mean measured exposure concentrations were 5.1, 10.4, 24, 75, and 150 mg/m³ for Groups 2, 3, 4, 5, and 6, respectively. A concurrent control group (Group 1) was exposed to humidified filtered air on a comparable regimen. Each group consisted of 5 animals/sex. On the day following the final exposure (study day 12 or 13), up to 5 animals/sex/group were euthanized and subjected to necropsy and tissue collection.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure, at the approximate midpoint of each exposure for visible clinical signs while animals were in nose-only restraint tubes, and at approximately 1-2 hours (+ 0.25 hours) following exposure on exposure days and once daily on the non-exposure days. Detailed physical examinations were performed 1 week (± 1 day) prior to randomization, on the day of randomization, study day 0 (prior to exposure), weekly (± 1 day) during the study period, and on the day of the scheduled necropsy. Individual body weights and food consumption were recorded 1 week (± 2 days) prior to randomization, on the day of randomization, prior to exposure on study day 0 (body weight only), weekly (± 1 day) during the study period, on the day prior to the scheduled necropsy, and on the day of the scheduled necropsy (body weight only). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. The lung and main stem bronchi, nasal cavity and turbinates, mediastinal lymph nodes, and any gross lesions were examined microscopically from all animals.

All animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on body weight. Test substance-related lower food consumption was noted for the 75 mg/m³ group males and the 150 mg/m³ group males and females from study day 0 to 7; however, these differences were transient as they were only noted at this single interval. Test substance-related macroscopic findings included enlarged mediastinal lymph nodes in the 75 and 150 mg/m³ group males and females, enlarged bronchial lymph nodes in the 150 mg/m³ group males and in the 25 and 150 mg/m³ group females; and pale lung in a single 150 mg/m³ group female. Test substance-related higher lung weights were noted in all test substance-exposed groups. Test substance-related microscopic findings were noted in the lung, nose, and bronchial lymph nodes. Only bronchial lymph nodes with gross lesions were examined and these were in the 25 and 150 mg/m³ groups. Alterations in these bronchial lymph nodes were both lymphoid and reticuloendothelial hyperplasia.

In conclusion, nose-only inhalation exposure of Zinc Borate to Crl:CD(SD) rats at concentrations of 5, 10, 25, 75, and 150 mg/m³ for 6 hours per day for a total of 10 exposures over a 2-week period resulted in test substance-related lower food consumption at 75 and 150 mg/m³ and test substance-related higher lung weights at all concentrations. Test substance-related macroscopic and microscopic findings were noted at ≥ 25 mg/m³. Based on the adverse histology findings of mild to moderate degeneration of the olfactory epithelium in the nose, and the lung findings of moderate alveolar histiocytosis and moderate alveolar proteinosis, the no-observed-adverse-effect-concentration (NOAEC) was defined as 25 mg/m³.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Developmental toxicity is considered to be a critical endpoint for zinc borate due to its dissociation (please refer to read-across statement). Therefore BMD established for boric acid (Allen et al., 1996) is more appropriate than NOAEL from the repeated dose toxicity study.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No study is selected since BMD of 10.3 mg B/kg bw, established in an oral developmental study in rats (Allen et al., 1996) is more appropriate for derivation of inhalation DNEL (by route-to-route extrapolation) than NOAEL for zinc oxide or NOAEL for zinc borate from the inhalation range-finding study.

Justification for classification or non-classification

Zinc borates are sparingly soluble salts. Solubility of zinc borate was tested according to OECD 29 (TDP) based on a 100 mg zinc borate/L loading. Dissolved zinc concentrations in water were 7.33 mg Zn/L and 1.77 mg Zn/L at pH6 and pH8, respectively, after 24 hours (Appendix A). Hydrolysis under high dilution conditions leads to zinc hydroxide via zinc oxide and boric acid formation and at physiological conditions zinc borate will be hydrolyzed to boric acid and zinc hydroxide. Zinc hydroxide and zinc oxide solubility is low under neutral and basic conditions with water solubility’s of < 1.6 mg/L, slightly above the solubility of <1 mg/L generally considered as poorly soluble (ECB 2004; ATSDR 2005). Therefore, the boron (as boric acid) can be assumed to be absorbed in the lungs leaving the less soluble zinc oxide and zinc hydroxide.

In a 90-day inhalation study of ZnO at exposure levels of 1, 3, 10, 50 and 200 mg/m3, the lesion of the greatest significance was an inflammation of the lungs with several lymph nodes in the chest cavity showing reactive hyperplasia. All toxic effects appeared to be reversible (Placke et al. 1990).

In a 14-day inhalation study of zinc borate (ZB) to rats, test substance-related higher lung weights were observed at all concentrations. Test substance-related macroscopic and microscopic findings were noted at ≥ 25 mg/m³including adverse histology findings of mild to moderate degeneration of the olfactory epithelium in the nose, and the lung findings of moderate alveolar histiocytosis and moderate alveolar proteinosis.  The increased lung weight correlated with the findings of alveolar histiocytosis and proteinosis in the lung. The no-observed-adverse-effect-concentration (NOAEC) was defined as 25 mg/m³(Randazzo 2014). 

 

The effects in the lungs of ZnO and ZB treated animals is consistent with effects associated with “lung overload” in rats. Pulmonary inflammation is regarded as a key driver in the cascade of pathogenic events following repeated inhalation of poorly soluble particles (PSP). Rats are particularly sensitive to particle overload (ILSI 2000; ECETOC 2013) and a product of the experimental condition and not necessarily a true reflection on the intrinsic toxic potential of the particles to cause inflammation and fibrosis. Numerous sub-chronic or chronic experimental rat inhalation studies with PSPs such as titanium dioxide, coal dust or talc revealed that at typical particle exposure concentrations of 1 to 30 mg/m³, the conditions of lung overload were achieved and pulmonary inflammatory responses, altered morphology, epithelial hyperplasia and finally chronic disease including fibrosis observed (ILSI, 2000; ECETOC 2013).

 

Lung overload is associated with exposure to particles with low solubility of low toxicity and occurs when a threshold dose of particles is achieved within the lung. During repeated exposure to poorly soluble particles, the lung burden of particles increases until a steady state or equilibrium is achieved between deposition and clearance of particles. Below the lung overload threshold, particles are cleared via normal mechanisms at a normal clearance rate, generating no appreciable response. Once the threshold is reached, the clearance mechanisms of the lung become overloaded there is a reduction of particle clearance from the deep lung, reflecting a breakdown in alveolar macrophage-mediated dust removal due to the loss of macrophage mobility (ECHA 2012; ECETOC 2013; ILSI 2000). 

 

 Based on the solubility of zinc borate, zinc hydroxide and ZnO in aqueous media, it can be expected that small concentrations of ZB and ZnO will be in the form of a completely dissolved solution under static equilibrium conditions. This means that ZB and ZnO under physiological conditions in the lungs likely will be simultaneously present as a mixture of solubilized boric acid, Zn ions and a major part as undissolved ZnO particles – all with different diffusion and uptake characteristics.

 

The higher sensitivity of rats to non-neoplastic and uniqueness to neoplastic lung changes had raised questions on the appropriate use and interpretation of the responses of the rat as an animal model for hazard identification or quantitative extrapolation and risk characterization (ILSI, 2000). Humans are less sensitive to “lung overload” as epidemiological studies thus far have not been able to detect an association between occupational exposures to poorly soluble particles of low toxicity and an increased risk for lung cancer.The measured differences of particle retention, distribution and clearance patterns in the lungs of exposed rats vs. primates or humans, may account for both the greater sensitivity in rats and corresponding differences in pulmonary pathological responses to long-term particle exposures.

Independent of particle size, inhalation exposure to high concentrations of low soluble particles of low toxicity are eliciting comparable localized pulmonary toxicity via processes that are pro-inflammatory in nature, causing oxidative stress and an persistent pulmonary inflammatory response (ECETOC 2013).

 

STOT-RE classification depends upon the availability of reliable evidence that a repeated exposure to the substance has produced a consistent and identifiable toxic effect in humans or, that toxic effect which have been observed in experimental animals are relevant for human health. Of particular relevance for the classification of particles causing effects by inhalation is the specific recognition of the phenomenon of “lung overload” in Section 3.9 of the ECHA guidance on the application of the CLP criteria as a mechanism not relevant to humans. The ECHA guidance for the EU CLP Regulation states that “The relevance of lung overload in animals to humans is currently not clear and is subject to continued scientific debate” (ECHA, 2013). While this statement is inconclusive with regard to lung overload from a classification standpoint, it recognizes the issue of lung overload and the need for applying weight of evidence and expert judgment in the final classification decision. The effects observed in the lungs of rats exposed to zinc borate and zinc oxide via inhalation are considered to be due to lung overload of the slightly soluble zinc hydroxide and zinc oxide particles, a mechanism not relevant to humans. Furthermore, the pulmonary inflammation observed in rats exposed via inhalation to ZnO appeared to be reversible. Therefore, zinc borate is not classified as a STOT-RE for effects in the lung.