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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

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)

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990
Reference Type:
study report
Title:
Unnamed
Year:
1989

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc oxide
EC Number:
215-222-5
EC Name:
Zinc oxide
Cas Number:
1314-13-2
Molecular formula:
ZnO
IUPAC Name:
oxozinc
Test material form:
aerosol dispenser: not specified
Remarks:
migrated information: aerosol
Details on test material:
- Name of test material (as cited in study report): Zinc oxide (ZnO)
- Physical state: fine white powder
- Purity: 97.4 ± 0.8%
- Lot No.: TV02229H
- Expiration date of the lot/batch: not listed
- Storage condition of test material: in sealed containers, protected from light and moisture, at room temperature

Test animals

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:

Administration / exposure

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
Doses / concentrations
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

Examinations

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".

Results and discussion

Results of examinations

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.

Effect levels

Dose descriptor:
NOEC
Effect level:
3 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

please see summary table and figures attached to this file

Applicant's summary and conclusion

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.