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REACH

Zinc oxide

EC number: 215-222-5 | CAS number: 1314-13-2

Life Cycle description
Uses advised against

Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:: sub-chronic toxicity: inhalation
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 24 November 2020 - ...June 2022
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Remarks:: The study presented herein is a guideline study without restrictions performed under GLP conditions.

Cross-referenceopen all close all

Cross-reference 1

Reason / purpose for cross-reference:: reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2020 - .. September 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was not performed under GLP conditions. Only male rats were used.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

This study is a dose-range finding study

Deviations:

yes

Remarks:

only male rats were used

Principles of method if other than guideline:

No testing guidelines exist for this type of study, which is a dose range finding study. The results of this study should facilitate the selection of appropriate concentration for the following 90-day inhalation study.
The study was conducted based on the above test guidelines pertaining to inhalation repeated dose inhalation toxicity studies.

GLP compliance:

Limit test:

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 98.2% for T0420
- Test substance No.: 20/0050-1 for T0420
- Batch identification: T0420

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: May 2022 for T0420.

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing: rats were housed together (5 animals per cage) in Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Bedding in the Polycarbonate cages were dust-free bedding. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). For enrichment wooden Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added. Wooden gnawing blocks (SAFE® block large) J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany.
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland
- Water (ad libitum): tap water
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 1.77 - <= 1.99 µm

Geometric standard deviation (GSD):

2.08

Remarks on MMAD:

MMAD / GSD: MMAD = 1.77-1.99 μm (geometric standard deviation = 1.97-2.26)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Exposure was over 14 days, at a rate of 6 hours per day for 5 days per week. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air.

Dose / conc.:

10.9 mg/m³ air

Remarks:

Test Group 1
(T 0420, 12 mg/m³)

Dose / conc.:

20.7 mg/m³ air

Remarks:

Test Group 2
(T 0420, 24 mg/m³)

Dose / conc.:

10.8 mg/m³ air

Remarks:

Test Group 3
(T 0421, 12 mg/m³)

Dose / conc.:

21.3 mg/m³ air

Remarks:

Test Group 4
(T 0421, 24 mg/m³)

Dose / conc.:

0 mg/m³ air

Remarks:

Control group

No. of animals per sex per dose:

5 animals were used per group (total of 5 groups and total of 25 animals).

Control animals:

yes

Details on study design:

- Dose selection rationale: The doses were chosen based on available data and upon approval of the sponsor. This is a dose range finding study which should facilitate the selection of appropriate concentration for a following 90-day inhalation study.
- Rationale for animal assignment (if not random): /
- Fasting period before blood sampling for clinical biochemistry: not specified
- Rationale for selecting satellite groups: /
- Post-exposure recovery period in satellite groups: /
- Section schedule rationale (if not random): /
- Other: /

Positive control:

None

Observations and examinations performed and frequency:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

No information provided

Clinical signs:

no effects observed

Description (incidence and severity):

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.

The body weight change was statistically significantly decreased in the following animals:
- Test group 2 (24 mg/m³ test item 1) from study day 0 to day 1: -7.9 g (p≤ 0.05, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 0 to day 1: -6.7 g (p≤ 0.01, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 4 to day 8: -1.7 g (p≤ 0.01, concurrent control was 5.5 g)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Due to social housing, there was only once cage (with 5 animals) per test group. No statistical evaluation was possible.

Food consumption of all exposed animals were slightly lower than the concurrent control group in a concentration-related manner. In the high concentrations, the food consumption of test group 2 (test item 1) animals was about 24 % lower than the control, while that of test group 4 animals (test item 2) was 32 % lower than in the controls. At low concentration, the food consumption was 10 % and 20 % lower than the control in test group 2 (test item 1) and test group 4 (test item 2) animals, respectively.

Food efficiency:

not specified

Description (incidence and severity):

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

not specified

Description (incidence and severity):

Ophthalmological findings:

not specified

Description (incidence and severity):

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At study day 14, in males of test group 4 (T0421, 24 mg/m3) absolute and relative neutrophil cell counts were slightly, but significantly increased, whereas relative lymphocyte counts were significantly decreased. These changes were regarded as treatment related and adverse.

Absolute and relative neutrophil cell counts were already significantly increased in rats of test group 3 (T0421, 12 mg/m3) The absolute neutrophil mean was marginally above the historical control range, whereas the mean of neutrophil relative counts was within this range (males, absolute neutrophils 0.53-1.09 Giga/L; relative neutrophils 8.5-19.0 %). Because the change was marginal and no other differential blood cell counts were altered, this change was regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).

In rats of test groups 1 and 2 (T0420, 12 and 24 mg/m3) absolute neutrophil cell counts were significantly increased, and in rats of test group 1 absolute eosinophil cell counts were significantly increased. However, the mentioned alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.

In rats of test group 4 (T0421, 24 mg/m3) relative, large unstained cell (LUC) counts were significantly decreased and hemoglobin values were significantly increased. Both parameters were within historical control ranges (males, relative LUC 0.2-0.7 %, hemoglobin 8.6-9.6 mmol/L). In males of test group 4 absolute reticulocyte counts were significantly decreased. The mean was below the historical control range (males, absolute reticulocytes 112.7-190.0 Giga/L). However, because of slightly higher hemoglobin and hematocrit values as well as red blood cell (RBC) counts compared to the controls lower reticulocyte counts were a consequence because the bone marrow wanted to adjust the circulating red blood cell counts. This effect is regarded as a physiological feedback regulation and was therefore regarded as treatment related, but non adverse.

Clinical biochemistry findings:

not specified

Description (incidence and severity):

Endocrine findings:

not specified

Description (incidence and severity):

Urinalysis findings:

not specified

Description (incidence and severity):

Behaviour (functional findings):

not specified

Description (incidence and severity):

Immunological findings:

not specified

Description (incidence and severity):

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights
When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed.
Relative changes of absolute lung weights:
Lung weights were increased in test group 1 with test item T0420 (113%) and were statistically significantly increased (p <= 0.01) in test groups 2 (135%) with test item T0420, 3 (114%) and 4 (141%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed:
Relative changes of relative lung weights
Lung weights were increased in test group 1 with test item T0420 (117%) and were statistically significantly increased (p <= 0.01) in test groups 2 (138%) with test item T0420, 3 (119%) and 4 (154%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged, gray mottled lungs with enlarged and gray coloured lung-associated lymph nodes (high dose Days 28 and 42), enlarged lungs and the lung associated lymph nodes (mid dose) and enlarged lung-associated lymph nodes
(low dose).
Incidence of gross lesions observed during necropsy
None of the animals in the control group nor in Test group 3 showed any gross lesions in the tracheobronchial lymph nodes. 2 males animals of the Test group 1 (12mg/m3) showed enlarged heobronchial lymph nodes, 1 male animal of the Test group 2 (24mg/m3) and 2 male animals of the test group 4 (24mg/m3).
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in organs listed in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the ADDITIONAL RESULTS section below with incidences and grading

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
At study day 14, rats in high concentration groups of both test items (test groups 2 and 4, T0420 and T0421, in each group 24 mg/m3) showed equivalent cell count increases in the bronchoalveolar lavage fluid (BALF): about 9fold significant increase of the total cell counts; highly, significantly increased absolute and relative neutrophil counts (about 700 fold absolute neutrophil increase) and absolute and relative monocyte counts (about 70 to 90 fold absolute monocyte increase) as well as moderately, significantly increased absolute lymphocyte counts (about 7 fold absolute lymphocyte increase). Relative macrophage cell counts were significantly decreased in both mentioned test groups.

Whereas the low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same changes as the high test item concentration groups, low concentration test group of T0421 (test group 3, 12 mg/m3) had considerable lower values: in test group 1 (T420) about 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts; in test group 3 (T0421) about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts. Relative neutrophil and monocyte counts were also significantly increased in the mentioned test groups whereas relative macrophage counts were significantly decreased.
At study day 14, total protein levels as well as enzyme activities of -Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and -N-Acetyl glucosaminidase (NAG) in BALF were significantly increased in all test groups. Quantitatively, the same trend could be observed as described in the BALF cytology. High concentration test groups of both test items (test groups 2 and 4, T0420 and T0421, each 24 mg/m3) showed equivalent changes: about 18 fold increase in total protein levels, 24 to 27 fold increase of ALP, 11 fold increase of LDH, 4 fold increase of GGT and 3 fold increase of NAG activities.

The low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same alterations in BALF compared to the high concentration test groups: 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities.

In the low concentration test group of T0421 (test group 3 (12 mg/m3) considerable lower changes could be observed: 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.

Details on results:

In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 12 and 24 mg/m³ for 6 hours daily on 5 consecutive days for 14 days. A concurrent control group was exposed to conditioned air. To examine the influence of coating, two substances T0420 and T0421 were tested at comparable concentrations. After the last exposure, animals designated for bronchoalveolar lavage, histological examinations,.

The tested atmospheric concentrations were slightly lower than the target concentration. They were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes that were close to each other. The MMADs ranged from 1.77 to 1.99 µm, which were well within the respirable range. The fraction of particles < 3 µm MMAD was higher than 70 % in all test groups. With regard to particle size distribution, there were no difference between the two substances.

During the exposure period, no clinical signs of toxicity were observed. The body weight, body weight gain was slightly lower than in the concurrent control group, although statistical significance was only in body weight change of test group 4 on single days. Although statistical evaluation could not be performed due to social housing, food consumption was apparently lower in animals exposed to both test substances than in the controls. Overall, the retarded body weight development and food consumption were slightly more severe in animals exposed to test item 2 (T0421) than those exposed to test item 1 (T0420).

Regarding clinical pathology, alterations of bronchoalveolar lavage (BAL) parameters were similar in the high concentration test groups of T0420 and T0421 (test groups 2 and 4, in each group 24 mg/m3): moderately, significantly increased total cell counts (about 9 fold), highly increased neutrophil and monocyte counts (absolute neutrophils about 700 fold, absolute monocyte about 70 to 90 fold), and slightly increased lymphocyte counts (absolute lymphocytes 7 fold). Correspondingly to the neutrophil cell count increase, alkaline phosphatase (ALP) activities were moderately, significantly increased in both test groups (24 to 27-fold). Lactate dehydrogenase (LDH) activities indicating general cell destruction were also moderately, significantly increased (about 11-fold), whereas gamma-Glutamyl-transferase (GGT) and beta-N-Acetyl glucosaminidase (NAG) activities were only marginally, but also significantly increased (GGT about 4 fold, NAG about 3 fold).

BAL values in the low concentration test group of T420 (test group 1, 12 mg/m3) were changed in the same magnitude as in the high concentration test groups: 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts, 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities

In contrast BAL values of the low concentration group of T0421 (test group 3, 12 mg/m3) showed considerable lower changes compared to controls: about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts, 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.

In addition to the mentioned local changes in the BAL, in the high concentration group of T0421 (test group 4, 24 mg/m3), a marginal increase of the absolute and relative blood neutrophil cell counts coupled with a decrease of the relative lymphocyte counts indicated a systemic acute-phase reaction.

Regarding pathology, the lungs and nasal cavity were the target organs.

In the lungs a disseminated infiltration of granulocytes and alveolar histiocytes was observed. This resulted also in an increase of the lung weight in most test groups. Some histiocytes were assumed to be destroyed. This inflammatory reaction was seen as consequence to the inhalation of the ZnO and was considered to be adverse.
In the nasal cavity in all levels, but most severely in level IV, there was degeneration and/or regeneration of the olfactory epithelium seen. This finding was regarded to be treatment-related and adverse.
The increased size of tracheobronchial lymph nodes was caused by alveolar histiocytes, transporting the phagocytosed ZnO particles from the lungs to the regional lymph nodes. As no other findings were observed, this was regarded to be treatment-related but not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Effect level:

mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Remarks on result:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Critical effects observed:

not specified

Concentration measurements in the exposure system

Study means and standard deviations of test substance concentrations:

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		Nominal concentration (mg/m³)	Effectiveness dust generation (%)
Test group	Target concentration (mg/m³)	Mean	SD	Nominal concentration (mg/m³)	Effectiveness dust generation (%)
1	12	10.94	0.41	18	59.9 %
2	24	20.67	0.89	28	74.7 %
3	12	10.76	0.37	19	56.7 %
4	24	21.28	1.02	42	50.3 %

Results of the particle size analyses

All measurements of particle size resulted in MMADs between 1.77 and 1.99 µm with GSDs between 1.97 and 2.26. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 74.2 % and 76.5 % for test item 1 (T0420) and between 71.7 % to 78.2 % for test item 2 (T0421). These values were well within the requirement of the test guidelines and showed that the generated test atmospheres contained high fraction of respirable particles. Moreover, all the measured values were close to each other, there were no significant difference between the two test items at comparable concentrations.

Cascade impactor measurements:

T 420			T 0421
	MMAD / GSD	% < 3 µm MMAD		MMAD / GSD	% < 3 µm MMAD
Test group 1 Measurement 1	1.90 µm / 2.03	74.2 %	Test group 3 Measurement 1	1.99 µm / 1.99	72.4 %
Test group 1 Measurement 2	1.79 µm / 2.04	76.5 %	Test group 3 Measurement 2	1.92 µm / 2.17	71.7 %
Test group 2 Measurement 1	1.85 µm / 2.02	75.3 %	Test group 4 Measurement 1	1.77 µm / 1.97	78.2 %
Test group 2 Measurement 2	1.77 µm / 2.17	75.2 %	Test group 4 Measurement 2	1.79 µm / 2.26	73.5 %

In the following table the data of APS measurements were presented. The APS measurement showed slightly higher MMAD. The difference between the two devices are to be explained by the mechanisms the measurements based on.

Particle size distribution measured by APS:

	Measurement date	MMAD / GSD		Measurement date	MMAD / GSD
Test group 1	26 Jun 20	2.47 µm/2.24	Test group 3	26 Jun 20	3.23 µm/1.87
		2.39 µm/2.22			3.38 µm/1.98
		2.47 µm/2.34			3.33 µm/1.91
	02 Jul 20	2.28 µm/2.37		02 Jul 20	2.93 µm/1.97
		2.13 µm/2.25			3.11 µm/2.21
		2.10 µm/2.23			3.46 µm/2.39
Test group 2	25 Jun 20	1.94 µm/2.66	Test group 4	25 Jun 20	2.41 µm/2.16
		1.82 µm/2.54			2.40 µm/2.18
		1.82 µm/2.52			2.29 µm/2.09
	03 Jul 20	2.41 µm/2.46		03 Jul 20	2.76 µm/2.25
		2.50 µm/2.37			2.67 µm/2.22
		2.50 µm/2.55			2.59 µm/2.17

BAL RESULTS

Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure):

Analyte	Gr. 1 T0420 12 mg/m³	Gr. 2 T0420 24 mg/m³	Gr. 3 T0421 12 mg/m³	Gr. 4 T0421 24 mg/m³
Total Cells	7.4*	8.9**	4.8**	8.6**
Eosinophils	4.1	16.0	6.7	11.0
Lymphocytes	4.9*	7.4**	4.0*	7.5**
Macrophages	0.6	1.1	1.1	0.7
Neutrophils	620.8*	722.3**	333.3**	734.0**
Monocytes	105.2*	91.2**	48.0**	71.4**
Epithelial cells	+	+	+	+

One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

+ increase could not be calculated because of zero activity in controls

Changes in median total protein and enzyme levels in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure). Medians instead of means were used because of high individual variation of the values with great impact on the mean values:

Analyte	Gr. 1 T0420 12 mg/m³	Gr. 2 T0420 24 mg/m³	Gr. 3 T0421 12 mg/m³	Gr. 4 T0421 24 mg/m³
Total Protein	14.4**	18.6**	5.4**	17.5**
GGT	4.6**	4.1**	2.9**	4.3**
LDH	11.0**	11.2**	4.6**	11.7**
ALP	16.6**	23.5**	7.0**	27.4**
NAG	2.7*	2.7*	1.7**	2.8**

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

Histopathology

Treatment-related findings were observed in organs listed in the tables below with incidences and grading:

Incidence and grading of histological findings in larynx:

Larynx (Level I)	Male animals
Larynx (Level I)	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Epithelial alteration	1	0	4	1	2
· Grade 1	1		4	1	2

The finding epithelial alteration is a common observation in inhalation studies and in most cases, the base of the epiglottis is affected. It shows a minimal flattening of the epithelial cells and loss of cilia. It was considered to be treatment-related but with the minimal grading it was not regarded to be adverse (Kaufmann et al., 2009).

Incidence and grading of histological findings in lungs

Lungs	Male animals
Lungs	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Infiltration, granulocytic	0	5	5	5	5
· Grade 1		4		3	2
· Grade 2		1	5	2	3
Histiocytosis, alveolar	0	5	5	5	5
· Grade 1				1
· Grade 2		4		2
· Grade 3		1	5	2	3
· Grade 4					2

In general, Lob. cran. dexter, Lob. medius dexter, and Lob. accessorius of the lungs were slightly less severely affected compared to Pulmo sinister and Lob. caudalis dexter. The finding was characterized by disseminated intra-alveolar inflammatory cells (mainly histiocytes and less number of granulocytes), intermingled by foamy roundish structures, containing occasionally a faint, bluish, roundish structures inside (interpreted as nucleus) or finely granular, eosinophilic material inside the alveoli. These structures were considered to be destroyed alveolar macrophages. These findings were regarded to be treatment-related.

Incidence and grading of histological findings in the nasal cavity:

Nasal cavity (Level IV)	Male animals
Nasal cavity (Level IV)	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Degeneration/regeneration, olfactory epithelium	0	5	5	5	5
· Grade 1		3
· Grade 2		2		3
· Grade 3			5	2	1
· Grade 4					4

Level IV of the nasal cavity was the most severely affected level and was here taken representatively for findings in the nasal cavity. There was loss, irregularity or flattening of the olfactory epithelium (interpreted as degeneration). The most affected areas were the septum, the dorsal meatus and the tips of the conchae. In some areas there was in addition an increase of nuclear size and basophilia, mainly of basal cells (interpreted as regeneration). These findings were considered as treatment-related.

Incidence and grading of histological findings in the tracheobronchial lymph nodes:

Tracheobronchial lymph nodes	Male animals
Tracheobronchial lymph nodes	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Histiocytosis (m)focal	0	3	3	4	5
· Grade 1				2	1
· Grade 2		2	3	2	4
· Grade 3		1

Histiocytes with intracytoplasmic material similar to those seen in the lungs, were found in tracheobronchial lymph nodes. This finding is regarded as treatment-related, but not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Pathology

Weight parameters

Absolute weights

When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):

Relative changes of absolute lung weights:

	Male animals
	T0420		T0421
Test group (mg/m³)	1 (12)	2 (24)	3 (12)	4 (24)
Lungs	113%	135%**	114%**	141%**

*p <= 0.05; **p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights

When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):

Relative changes of relative lung weights:

	Male animals
	T0420		T0421
Test group (mg/m³)	1 (12)	2 (24)	3 (12)	4 (24)
Lungs	117%	138%**	119%**	154%**

*p <= 0.05; **p <= 0.01

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.

The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.

Gross lesions

In some animals, the tracheobronchial lymph nodes were enlarged. This was considered as treatment-related.

Incidence of gross lesions observed during necropsy:

Tracheobronchial lymph nodes	Male animals
Tracheobronchial lymph nodes	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Enlarged	0	2	1	0	2

Conclusions:

Both Zinc oxide T420 and T421 caused lesions in nasal cavity and lung already at the low targeted concentration of 12 mg/m³ (measured concentration about 11 mg/m³). No systemic effect was observed. At comparable atmospheric concentrations, Zinc oxide T421 seemed to cause more severe effects than those caused by T420.

Executive summary:

Groups of male Wistar rats were exposed whole-body to the dust aerosols of Zinc oxide T0420 and Zinc oxide T0421 for 6 hours per day on 5 days per week for two weeks. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air. Daily clinical observation and body weight were recorded. Animals were sacrificed, assessments including hematology, bronchoalveolar lavage and histopathology (control and high concentration only) of the respiratory tract were carried out.

The following is a summary of the most relevant results:

Test group 4 (T0421, 24 mg/m³)

Impaired body weight development, reduced food consumption

Increased absolute and relative neutrophil cell counts in blood

Decrease relative lymphocyte counts in blood

Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL

Decreased relative macrophage counts in BALF

Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF

Increase of absolute and relative lung weight (141%/154%)

Minimal to slight infiltration of neutrophilic granulocytes and moderate to severe infiltration of alveolar histiocytes in the lungs in all animals

Slight to severe degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 3 (T0421 12 mg/m³)

Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL

Decreased relative macrophage counts in BALF

Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF

Increase of absolute and relative lung weight (114%/119%)

Minimal to slight infiltration of neutrophilic granulocytes and minimal to moderate infiltration of alveolar histiocytes in the lungs in all animals

Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 2 (T0420, 24 mg/m³)

Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL

Decreased relative macrophage counts in BALF

Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF

Increase of absolute and relative lung weight (135%/138%)

Slight infiltration of neutrophilic granulocytes and moderate infiltration of alveolar histiocytes in the lungs in all animals

Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 1 (T0420, 12 mg/m³)

Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL

Decreased relative macrophage counts in BALF

Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF

Minimal to slight infiltration of neutrophilic granulocytes and slight to moderate infiltration of alveolar histiocytes in the lungs in all animals

Minimal to slight degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Cross-reference 2

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 2020 - ...June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study without restrictions performed under GLP conditions.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

- adopted on 2018-06-25
- additional endpoints investigated

Deviations:

yes

Principles of method if other than guideline:

Additional endpoints (covered in IUCLID 7.8.1): reproduction toxicology , developmental toxicity, (developmental) neurotoxicity
three doses of the test substance (coated ZnO nanomaterial) were compared to one dose of 2 reference substances (non-coated microscaled ZnO and Zinc sulfate monohydrate)

GLP compliance:

yes (incl. QA statement)

Limit test:

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 93.8% for T0421.
- Test substance No.: 20/0051-1 for T0421.
- Batch identification: T0421.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: Aug 2020 for T0421.

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks:

whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 0.6 - <= 2.53 µm

Remarks on MMAD:

MMAD / GSD: MMAD = 0.60-2.53 μm (geometric standard deviation = 3.08-2.23)

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 consecutive days per week, 6 hours per day (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3).

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 (Parental animals F0) - air control; Test Group 10 (male animals for particle detection); Test Group 20 (Recovery animals) - air control

Dose / conc.:

0.52 mg/m³ air (analytical)

Remarks:

SD: 0.16 mg/m3; target concentration: 0.5 mg/m³: Test Group 4 (Parental animals F0)

Dose / conc.:

2.01 mg/m³ air (analytical)

Remarks:

SD: 0.20 mg/m3; target concentration: 2.0 mg/m³: Test Group 5 (Parental animals F0)

Dose / conc.:

10.07 mg/m³ air (analytical)

Remarks:

SD: 0.96 mg/m3, target concentration: 10 mg/m³: Test Group 6 (Parental animals F0); Test Group 16 (male animals for particle detection); Test Group 26 (Recovery animals)

No. of animals per sex per dose:

16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina

Statistics:

Statistical evaluation for test groups low, mid, high in comparison with air control group
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> DUNNETT test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+) with BONFERRONI-HOLM
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one sided-) with BONFERRONI-HOLM
-% live male day x, %live female day x
--> WILCOXON test (two-sided)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the
control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Weight parameters in pathology
-->Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided).

Clinical signs:

no effects observed

Description (incidence and severity):

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
-Exposure period, test item 2 (test groups 4, 5, 6, 16 and 26):
One male animal (No.: 82) of test group 5 showed protruding eyeball during exposure period
on study days 26 – 31. No clinical signs of toxicity were noted in any other animals of these
groups. The findings in the eye was considered incidental due to missing concentration response
relationship.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In animals exposed to low (0.5 mg/m³) and mid concentration (2 mg/m³) of test item 2 (Zinc oxide T0421), there were no statistically significant deviation from the concurrent control group was observed in body weight.

The following statistically significant body weight changes were determined in male animals of group 4 and 5:
- Test group 4: day 60 -> 67: 1.3g (p< 0.05), whereas the control group was 7.8g
- Test group 4: day 67 -> 74: 10.7g (p< 0.05), whereas the control group was 6.6g
- Test group 5: day 74 -> 81: 10.2g (p< 0.01), whereas the control group was 5.7g
- Test group 5: day 81 -> 88: 8.2g (p< 0.05), whereas the control group was 5.4g
These values were mostly higher than the control value and were of transient nature. They did not influence the mean body weight. Thus, they were considered incidental.

The following statistically significant body weight changes were determined in male animals:
- Test group 6: day 18: 329.8g (p< 0.05), whereas the control group was 345.9g
- Test group 6: day 25: 340.2g (p< 0.01), whereas the control group was 363.4g
- Test group 6: day 32: 351.3g (p< 0.01), whereas the control group was 379.4g
- Test group 6: day 39: 365.7g (p< 0.01), whereas the control group was 390.9g
- Test group 6: day 46: 373.3g (p< 0.05), whereas the control group was 394.9g

The following statistically significant body weight changes were determined in male animals:

- Test group 6: day 0 -> 4: 6.9 g (p< 0.05), whereas the control group was 10.4g
- Test group 6: day 11 -> 18: 17.2g (p< 0.05), whereas the control group was 22.0g
- Test group 6: day 18 -> 25: 10.4g (p< 0.01), whereas the control group was 17.5g
- Test group 6: day 25 -> 32: 11.1g (p< 0.05), whereas the control group was 16.1g
- Test group 6: day 81 -> 88: 8.1g (p< 0.05), whereas the control group was 5.4g

The mean body weights of test group 6 were lower than the concurrent control group animals. On study days 18, 25, 32 and 39 they co-incidence with statistically significantly lowered mean body weight change, they are likely attributed to the exposure to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421). The lower body weight change from study day 0 to study day 4 could be considered initial response to the exposure. The changes were very minor, and the mean body weight at the end of the exposure period of this group was not statistically lower than the control. Thus, they were considered not biologically relevant and not adverse.

The following statistically significant changes of body weight were determined in female
animals:
- Test group 6: day 32: 210.9g (p< 0.05), whereas the control group was 221.0g
- Test group 6: day 93: 234.4g (p< 0.01), whereas the control group was 251.8g
The following statistically significant body weight changes were determined in female
animals:
- Test group 6: day 11-> 18: 9.1 (p< 0.05), whereas the control group was 13.8g
- Test group 6: day 102 -> 109: -1.4 (p< 0.05), whereas the control group was 14.3g
- Test group 6: day 116 -> 123: 3.7 (p< 0.05), whereas the control group was -3.2g

The significant changes of body weight and body weight changes in female animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421) were all of transient nature. As there were already transient effects observed in male animals of these group, these changes in females may be also attributed to the test substance. As the mean body weight at the end of the exposure period of this group was not statistically lower than the control, these effects in body weights and body weight changes were considered not biologically relevant and not adverse.

The following significant changes were observed in recovery group male animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421):
- Test group 26: day 18: 316.6g (p< 0.01), whereas the control group was 344.0g
- Test group 26: day 25: 327.2g (p< 0.01), whereas the control group was 360.9g
- Test group 26: day 32: 338.7g (p< 0.01), whereas the control group was 375.0g
- Test group 26: day 39: 353.3g (p< 0.01), whereas the control group was 390.9g
- Test group 26: day 46: 365.0g (p< 0.01), whereas the control group was 399.8g
- Test group 26: day 53: 373.3g (p< 0.01), whereas the control group was 411.9g
- Test group 26: day 60: 378.8g (p< 0.01), whereas the control group was 414.4g
- Test group 26: day 67: 386.4g (p< 0.01), whereas the control group was 425.4g
- Test group 26: day 74: 394.6g (p< 0.01), whereas the control group was 429.7g
- Test group 26: day 81: 400.7g (p< 0.01), whereas the control group was 439.5g
- Test group 26: day 88: 404.5g (p< 0.01), whereas the control group was 446.1g
- Test group 26: day 92: 409.9g (p< 0.01), whereas the control group was 449.2g
- Test group 26: day 102: 415.4g (p< 0.01), whereas the control group was 450.4g
- Test group 26: day 109: 425.0g (p< 0.01), whereas the control group was 457.6g
Test group 26: day 116: 431.1g (p< 0.01), whereas the control group was 459.8g
- Test group 26: day 123: 437.3g (p< 0.01), whereas the control group was 465.5g
- Test group 26: day 130: 441.0g (p< 0.01), whereas the control group was 470.0g
- Test group 26: day 137: 447.8g (p< 0.05), whereas the control group was 472.3g
- Test group 26: day 144: 450.7g (p< 0.05), whereas the control group was 475.3g
- Test group 26: day 146: 452.6g (p< 0.05), whereas the control group was 475.9g

The following significant deviations from the control were observed in male recovery group animals exposed to high concentration of test item 2:
- Test group 26: day 0 -> 4: 5.5 (p< 0.01), whereas the control group was 12.6g
- Test group 26: day 11 -> 18: 10.7 (p< 0.01), whereas the control group was 21.7g
- Test group 26: day 18 -> 25: 10.6 (p< 0.01), whereas the control group was 16.9g
- Test group 26: day 67 -> 74: 8.2 (p< 0.05), whereas the control group was 4.4g
- Test group 26: day 130 -> 137: 6.8 (p< 0.01), whereas the control group was 2.3g
The mean body weights of the recovery group animals were statistically lower than the concurrent control group throughout the whole exposure and post-exposure period. The mean body weight changes were only significantly decreased during the initial period of the exposure period. This showed that the body weight development of the male animals of this groups was impaired at the initial time of the exposure. During the course of continuous exposure, as well as the recovery period, the body weight did not increase in such an extent that could compensate the initially reduced body weight gain. The retarded body weight development was also observed in main group animals exposed at the same concentration in the same chamber. Thus, the effect was considered treatment-related. As the final mean body weight was only about 5 % lower than the control, the body weight effect was considered not biologically relevant and not adverse.

Further, the following significant body weight changes were observed in male animals of test group 16 (10 mg/m³, Zinc oxide T0421)
- Test group 16: day 0 -> 4: 4.4g (p< 0.05), whereas the control group was 11.6g
- Test group 16: day 18 -> 25: 9.8g (p< 0.05), whereas the control group was 20.6g
- Test group 16: day 67 -> 7 4: -1.1g (p< 0.05), whereas the control group was 8.5g
- Test group 16: day 74 -> 81: 10.0g (p< 0.01), whereas the control group was 0.9g
As discussed above, the findings were considered treatment-related, but of no biological relevance and not adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes of mean food consumption were determined in
male animals:
• Test group 6: day 0 - 4: +22.6 g (p≤ 0.05), whereas the control group was +24.4 g
• Test group 6: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The lowered food consumption in test group 6 coincidenced with lower mean body weights and mean body weight change of these groups in the same time range. They may be related to the daily inhalation exposure to the test and reference substance. They were of transient nature, thus, they were considered not of biological relevance.

Food efficiency:

not examined

Description (incidence and severity):

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

not examined

Description (incidence and severity):

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period, in males of test group 6 (10 mg/m3 Zinc oxide T0421) total white blood cell (WBC) counts as well as absolute neutrophil and lymphocyte counts were slightly but significantly increased. These changes were regarded as treatment-related and adverse.

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges: decreased relative eosinophil cell counts in males of test groups 4 and 6 (0.5 and 10 mg/m3 Zinc oxide T0421)

The following significant changes were regarded as incidental and not treatment related, because the alteration was not dose dependent: increased hematocrit value in males of test group 5 (2 mg/m3 Zinc oxide T0421); increased absolute monocyte counts in females of test group 4 (0.5 mg/m3 Zinc oxide T0421).

After the recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) absolute large unstained cell (LUC) counts were significantly increased. This was the only change of the differential blood cell counts among these individuals. Therefore, it was regarded as if at all treatment related as non-adverse.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly increased potassium values in males of test group 6 (10 mg/m3 Zinc oxide T0421). This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).
The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test group 6 and 8 (10 mg/m3 Zinc oxide T0421
; decreased albumin values in females of test group 6 (10 mg/m3 Zinc oxide T0421);
Significantly increased alkaline phosphatase activities in females of test groups 5 and 6 (2 and 10 mg/m3 Zinc oxide T0421) were regarded as incidental and not treatment related, because the alteration was not dose dependent.
After the 8-week recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) total bilirubin values were significantly increased.

Endocrine findings:

no effects observed

Description (incidence and severity):

After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not specified

Description (incidence and severity):

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed

Overall motor activity (summation of all intervals):
Test item 2 (Zinc oxide T0421) (Test groups 4, 5 and 6):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
• Decrease of activity in the male animals of test group 6 (10 mg/m³, test item 2) at interval
9 on day 87 (p ≤ 0.01).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

Immunological findings:

not examined

Description (incidence and severity):

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with control group 0 (=100%), Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³): Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
--> These effects were observed as treatment-related, adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³):
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 10 males and 5 females
--> These effects were observed as treatment-related, adverse effects
Test group 26 (Recovery group R1, 10 mg/m³)
No treatment-related adverse findings

Neuropathological findings:

not examined

Description (incidence and severity):

neuropathological examinations of pups on PND22 (developmental neurotoxicity cohort) are examined (see IUCLID section 7.8.1)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the details on results section

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
Minimal to severe numbers of foamy macrophages in the lungs in all male and all female
animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female
animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes
(exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes
(exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium (nasal cavity,
level IV, exemplarily) in 6 males and 10 females

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV,
exemplarily) in 1 male and 1 female
Test group 4 (0.5 mg/m³)
No treatment-related adverse findings

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
Main group (F0)
The following treatment-related, adverse effects were observed:

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)

• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell
and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase
(ALP) activities in BAL of both sexes
• Increased β-Glutamyl-transferase (GGT) activity in BAL of males

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage

Details on results:

CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
-Exposure period, test item 2 (test groups 4, 5, 6, 16 and 26):
One male animal (No.: 82) of test group 5 showed protruding eyeball during exposure period on study days 26 – 31. No clinical signs of toxicity were noted in any other animals of these groups. The findings in the eye was considered incidental due to missing concentration response relationship.

BODY WEIGHT AND WEIGHT GAIN
In animals exposed to low (0.5 mg/m³) and mid concentration (2 mg/m³) of test item 2 (Zinc oxide T0421), there were no statistically significant deviation from the concurrent control group was observed in body weight.

The following statistically significant body weight changes were determined in male animals of group 4 and 5:
- Test group 4: day 60 -> 67: 1.3g (p< 0.05), whereas the control group was 7.8g
- Test group 4: day 67 -> 74: 10.7g (p< 0.05), whereas the control group was 6.6g
- Test group 5: day 74 -> 81: 10.2g (p< 0.01), whereas the control group was 5.7g
- Test group 5: day 81 -> 88: 8.2g (p< 0.05), whereas the control group was 5.4g
These values were mostly higher than the control value and were of transient nature. They did not influence the mean body weight. Thus, they were considered incidental.

The following statistically significant body weight changes were determined in male animals:
- Test group 6: day 18: 329.8g (p< 0.05), whereas the control group was 345.9g
- Test group 6: day 25: 340.2g (p< 0.01), whereas the control group was 363.4g
- Test group 6: day 32: 351.3g (p< 0.01), whereas the control group was 379.4g
- Test group 6: day 39: 365.7g (p< 0.01), whereas the control group was 390.9g
- Test group 6: day 46: 373.3g (p< 0.05), whereas the control group was 394.9g

The following statistically significant body weight changes were determined in male animals:

- Test group 6: day 0 -> 4: 6.9 g (p< 0.05), whereas the control group was 10.4g
- Test group 6: day 11 -> 18: 17.2g (p< 0.05), whereas the control group was 22.0g
- Test group 6: day 18 -> 25: 10.4g (p< 0.01), whereas the control group was 17.5g
- Test group 6: day 25 -> 32: 11.1g (p< 0.05), whereas the control group was 16.1g
- Test group 6: day 81 -> 88: 8.1g (p< 0.05), whereas the control group was 5.4g

The mean body weights of test group 6 were lower than the concurrent control group animals. On study days 18, 25, 32 and 39 they co-incidence with statistically significantly lowered mean body weight change, they are likely attributed to the exposure to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421). The lower body weight change from study day 0 to study day 4 could be considered initial response to the exposure. The changes were very minor, and the mean body weight at the end of the exposure period of this group was not statistically lower than the control. Thus, they were considered not biologically relevant and not adverse.

The following statistically significant changes of body weight were determined in female
animals:
- Test group 6: day 32: 210.9g (p< 0.05), whereas the control group was 221.0g
- Test group 6: day 93: 234.4g (p< 0.01), whereas the control group was 251.8g
The following statistically significant body weight changes were determined in female
animals:
- Test group 6: day 11-> 18: 9.1 (p< 0.05), whereas the control group was 13.8g
- Test group 6: day 102 -> 109: -1.4 (p< 0.05), whereas the control group was 14.3g
- Test group 6: day 116 -> 123: 3.7 (p< 0.05), whereas the control group was -3.2g

The significant changes of body weight and body weight changes in female animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421) were all of transient nature. As there were already transient effects observed in male animals of these group, these changes in females may be also attributed to the test substance. As the mean body weight at the end of the exposure period of this group was not statistically lower than the control, these effects in body weights and body weight changes were considered not biologically relevant and not adverse.

The following significant changes were observed in recovery group male animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421):
- Test group 26: day 18: 316.6g (p< 0.01), whereas the control group was 344.0g
- Test group 26: day 25: 327.2g (p< 0.01), whereas the control group was 360.9g
- Test group 26: day 32: 338.7g (p< 0.01), whereas the control group was 375.0g
- Test group 26: day 39: 353.3g (p< 0.01), whereas the control group was 390.9g
- Test group 26: day 46: 365.0g (p< 0.01), whereas the control group was 399.8g
- Test group 26: day 53: 373.3g (p< 0.01), whereas the control group was 411.9g
- Test group 26: day 60: 378.8g (p< 0.01), whereas the control group was 414.4g
- Test group 26: day 67: 386.4g (p< 0.01), whereas the control group was 425.4g
- Test group 26: day 74: 394.6g (p< 0.01), whereas the control group was 429.7g
- Test group 26: day 81: 400.7g (p< 0.01), whereas the control group was 439.5g
- Test group 26: day 88: 404.5g (p< 0.01), whereas the control group was 446.1g
- Test group 26: day 92: 409.9g (p< 0.01), whereas the control group was 449.2g
- Test group 26: day 102: 415.4g (p< 0.01), whereas the control group was 450.4g
- Test group 26: day 109: 425.0g (p< 0.01), whereas the control group was 457.6g
Test group 26: day 116: 431.1g (p< 0.01), whereas the control group was 459.8g
- Test group 26: day 123: 437.3g (p< 0.01), whereas the control group was 465.5g
- Test group 26: day 130: 441.0g (p< 0.01), whereas the control group was 470.0g
- Test group 26: day 137: 447.8g (p< 0.05), whereas the control group was 472.3g
- Test group 26: day 144: 450.7g (p< 0.05), whereas the control group was 475.3g
- Test group 26: day 146: 452.6g (p< 0.05), whereas the control group was 475.9g

The following significant deviations from the control were observed in male recovery group animals exposed to high concentration of test item 2:
- Test group 26: day 0 -> 4: 5.5 (p< 0.01), whereas the control group was 12.6g
- Test group 26: day 11 -> 18: 10.7 (p< 0.01), whereas the control group was 21.7g
- Test group 26: day 18 -> 25: 10.6 (p< 0.01), whereas the control group was 16.9g
- Test group 26: day 67 -> 74: 8.2 (p< 0.05), whereas the control group was 4.4g
- Test group 26: day 130 -> 137: 6.8 (p< 0.01), whereas the control group was 2.3g
The mean body weights of the recovery group animals were statistically lower than the concurrent control group throughout the whole exposure and post-exposure period. The mean body weight changes were only significantly decreased during the initial period of the exposure period. This showed that the body weight development of the male animals of this groups was impaired at the initial time of the exposure. During the course of continuous exposure, as well as the recovery period, the body weight did not increase in such an extent that could compensate the initially reduced body weight gain. The retarded body weight development was also observed in main group animals exposed at the same concentration in the same chamber. Thus, the effect was considered treatment-related. As the final mean body weight was only about 5 % lower than the control, the body weight effect was considered not biologically relevant and not adverse.

Further, the following significant body weight changes were observed in male animals of test group 16 (10 mg/m³, Zinc oxide T0421)
- Test group 16: day 0 -> 4: 4.4g (p< 0.05), whereas the control group was 11.6g
- Test group 16: day 18 -> 25: 9.8g (p< 0.05), whereas the control group was 20.6g
- Test group 16: day 67 -> 7 4: -1.1g (p< 0.05), whereas the control group was 8.5g
- Test group 16: day 74 -> 81: 10.0g (p< 0.01), whereas the control group was 0.9g
As discussed above, the findings were considered treatment-related, but of no biological relevance and not adverse.

FOOD CONSUMPTION
The following statistically significant changes of mean food consumption were determined in
male animals:
• Test group 6: day 0 - 4: +22.6 g (p≤ 0.05), whereas the control group was +24.4 g
• Test group 6: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The lowered food consumption in test group 6 coincidenced with lower mean body weights and mean body weight change of these groups in the same time range. They may be related to the daily inhalation exposure to the test and reference substance. They were of transient nature, thus, they were considered not of biological relevance.

HAEMATOLOGICAL FINDINGS:
At the end of the administration period, in males of test group 6 (10 mg/m3 Zinc oxide T0421) total white blood cell (WBC) counts as well as absolute neutrophil and lymphocyte counts were slightly but significantly increased. These changes were regarded as treatment-related and adverse.

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges: decreased relative eosinophil cell counts in males of test groups 4 and 6 (0.5 and 10 mg/m3 Zinc oxide T0421)

The following significant changes were regarded as incidental and not treatment related, because the alteration was not dose dependent: increased hematocrit value in males of test group 5 (2 mg/m3 Zinc oxide T0421); increased absolute monocyte counts in females of test group 4 (0.5 mg/m3 Zinc oxide T0421).

After the recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) absolute large unstained cell (LUC) counts were significantly increased. This was the only change of the differential blood cell counts among these individuals. Therefore, it was regarded as if at all treatment related as non-adverse.

CLINICAL CHEMISTRY:
Significantly increased potassium values in males of test group 6 (10 mg/m3 Zinc oxide T0421). This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).
The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test group 6 and 8 (10 mg/m3 Zinc oxide T0421
; decreased albumin values in females of test group 6 (10 mg/m3 Zinc oxide T0421);
Significantly increased alkaline phosphatase activities in females of test groups 5 and 6 (2 and 10 mg/m3 Zinc oxide T0421) were regarded as incidental and not treatment related, because the alteration was not dose dependent.
After the 8-week recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) total bilirubin values were significantly increased.

NEUROBEHAVIOUR:
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed

Overall motor activity (summation of all intervals):
Test item 2 (Zinc oxide T0421) (Test groups 4, 5 and 6):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
• Decrease of activity in the male animals of test group 6 (10 mg/m³, test item 2) at interval
9 on day 87 (p ≤ 0.01).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

ORGAN WEIGHTS
When compared with control group 0 (=100%), Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³): Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
--> These effects were observed as treatment-related, adverse effects

GROSS PATHOLOGY

Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³):
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 10 males and 5 females
--> These effects were observed as treatment-related, adverse effects
Test group 26 (Recovery group R1, 10 mg/m³)
No treatment-related adverse findings

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
Minimal epithelial alteration was observed in several test groups treated with test item 1 or test item 2 as well as in control animals. This finding is characterized by an increase of cell layers and replacement of respiratory epithelium by squamous epithelial cells, which may exhibit slight nuclear polymorphism and cellular atypia. The site most susceptible for this lesion, is the base of the epiglottis as it was observed in the present study. This finding was regarded to be treatment-related (inhalation).

Recovery animals: One female of test group 26 (test item 2, 10 mg/m³) revealed also a minimal epithelial alteration at the base of the epiglottis. These findings were considered to be treatment-related.

Lungs:

Parental animals:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. The finding was characterized by loss of olfactory epithelial cells and occasionally regeneration. Mainly the dorsal meatus and areas on the nasal septum were affected. This finding was regarded to be treatment-related.
One female of test group 1 (test item 1, 0.5 mg/m³) and three males and one female of test group 5 (test item 2, 2 mg/m³) showed minimal to slight degeneration of the olfactory epithelium. As this finding normally does not occur as a background lesion, it was assumed to have been most likely caused by the test substances.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period, in BAL of males and females of test group 6 (10 mg/m3 Zinc oxide T0421) total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts were significantly increased. Additionally, absolute eosinophil cell counts were increased (not significantly) in BAL of males whereas relative macrophage counts were significantly decreased in BAL of males and females of test group 6. These alterations were regarded as treatment related and adverse.
In BAL of males in test group 5 (2 mg/m3 Zinc oxide T0421) absolute and relative neutrophil cell and monocyte counts as well as relative lymphocyte counts were already significantly increased. In BAL of both sexes of test group 5 relative macrophage counts were significantly decreased, and in females of this test group relative neutrophil cell counts were already significantly increased. However, in both sexes total cell counts in BAL were not significantly altered, and the differential cell counts were only marginally changed (below 10fold).
Therefore, these alterations in BAL cytology were regarded as treatment related but nonadverse.
Recovery animals:
After the 8-week administration period, in BAL of females of test group 26 (10 mg/m3 Zinc oxide T0421) absolute epithelial cells were marginally but significantly increased. This isolated change was regarded as incidental and not treatment related.

Proteins/enzymes:
Parental animals:
After the administration period, in BAL of males and females of test group 6 (10 mg/m3 Zinc oxide T0421) total protein levels as well as lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity were moderately, significantly increased whereas γ-Glutamyltransferase (GGT) activity in males of this test group was marginally but also significantly increased. These alterations were regarded as treatment related and adverse.
Additionally, in BAL of both sexes of test group 6 (10 mg/m3 Zinc oxide T0421) β-
-N-Acetyl glucosaminidase (NAG) activity as well as in females of this test group GGT activity were significantly increased. In BAL of males of test group 5 (2 mg/m3 Zinc oxide T0421) LDH, ALP and GGT activities and in females ALP and GGT activities were significantly increased.
However, the changes were marginally (below 2fold). Therefore, these alterations were regarded as treatment related but non-adverse.
Recovery animals:
After the 8-week recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) total protein levels in BAL were still significantly increased. However, this was the only altered parameter in BAL among these individuals and the increase was only marginal (below 2fold).
Therefore, this isolated change was regarded as incidental and not treatment related.

OTHER FINDINGS:
organ burden:
ICP-OES analysis Zinc content in lungs, liver, heart and brain of parental male/female animals
As an essential element, the data showed high biological background level of zinc element (very high endogenous background). In the high concentration exposure groups, slightly increased zinc concentration was only found in lung. Conversely, statistically lower zinc content was observed in liver and heart of female animals exposed to 10 mg/m³ coated ZnO. In all other examined organs, the zinc level was comparable with the control.

- Electron microscopy:
Electron microscope analysis of particulate matter in organs and tissues: (see section overall remarks/attachments)

Dose descriptor:

NOAEC

Remarks:

local toxicity

Effect level:

0.52 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: histological findings in the nasal cavity of one male and one female rat. at the target mid concentration of 2 mg/m³

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

10.07 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Remarks on result:

other: No systemic toxicity was observed in hematology, clinical chemistry ; increased neutrophils and lymphocytes in blood at the target conc of 10 mg/m3

Critical effects observed:

yes

Lowest effective dose / conc.:

2.01 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Executive summary:

This study was a 90-Day Study (OECD test guideline (TG) 413) combined with the Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) in rat with neurotoxicity and developmental (neuro)toxicity evaluation, including detailed clinical observations addressing potential neurobehavioral effects, histological and morphological evaluations of the brains of the pups on post-natal day 22.

To compare the toxicity of uncoated and coated nano Zinc oxide, these two materials (Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated) were tested at each three concentrations. In addition, micronsize Zinc oxide T0242 and a soluble salt zinc sulfate monohydrate was tested as reference items.

Groups of male and female Wistar rats were whole-body exposed to the aerosols of ZnO nano materials, Zinc oxide T0420 and Zinc oxide T0421, for 6 hours daily, at least 90 days. Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated.

The target concentrations for Zinc oxide T0420 and T0421 were 0.5, 2 and 10 mg/m³ referring to the non-volatile fraction. For the reference item 1 microscale Zinc oxide T0242, 10 mg/m³ was tested. For the reference item 2, Zinc sulfate monohydrate a target concentration of 22 mg/m³ was tested because this is equimolar to zinc ion of the ZnO materials. Concurrent control groups were exposed to humidified air (control group 0, 10 and 20).

All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3)). Control animals were exposed to conditioned air. Male and female rats aged about 6 or 7 weeks when supplied, were used as F0 generation parental animals. The animals were exposed for 43 days before mating. The mating period were maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued until they are exposed for total minimal 90 days. After the mating period, the female F0 animals were exposed further until gestation day 19. To allow them to deliver and rearing their pups (F1 generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From PND 4 through to PND 21, the dams were exposed with their pups in exposure cages containing beddings. During the exposure food was withdrawn. Water was provided in form of hydrogel pads from PND 14 to 16 onward. The first parental female animals were in gestation stage already after the first few mating days, therefore, the post-weaning period were adjusted in such a way, that a total of minimum 90 exposure will be achieved for females.

Daily clinical observations, body weights, food consumption, ophthalmology, detailed clinical observation and FOB/MA were recorded. Moreover, male and female fertility were determined. Additional assessments including hematology and clinical chemistry in blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of exposure period. In addition, recovery groups of male and female animals were included; after an exposure period of about 90 days, these animals were kept for an additional period of ca. 60 days without exposure (control group 20, and test groups 23, 26, 27 and 28, respectively).

To assess the reproductive/developmental toxicity of the test substances (incl. reference substances), estrus cycles, male and female reproduction, delivery data were collected. In the pups, open field observations were performed on PND 13 and 21, motor activity measurements were performed on PND 13, 17 and 21. On PND 22, thyroid hormones, brain weights, neuropathology, general histopathology were examined in separate subsets of animals.

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)

• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium

Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal

Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female

Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.

No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings

Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions.
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium

Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females

Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals.
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female

Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Cross-reference 3

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 November 2020 - ...June 2022

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study presented herein is a guideline study with a major deficiency under GLP conditions. Only one concentration level was tested.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

- adopted on 2018-06-25
- additional endpoints investigated

Deviations:

yes

Principles of method if other than guideline:

Additional endpoints (covered in IUCLID 7.8.1): reproduction toxicology , developmental toxicity, (developmental) neurotoxicity
three doses of the test substance (uncoated/coated ZnO nanomaterial) were compared to one dose of 2 reference substances (non-coated microscaled ZnO and Zinc sulfate monohydrate)

GLP compliance:

yes (incl. QA statement)

Limit test:

Specific details on test material used for the study:

micro sized Zinc Oxide
Purity: 99.8%
Name of reference substance 1: Zinc oxide T0242
Reference substance No.: 20/0201-1
Batch identification: 56589
Appearance - physical state / color: Solid / white
Storage conditions: Room temperature
BET: 4.48 m2/g

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks:

whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 0.67 - <= 1.48 µm

Remarks on MMAD:

MMAD / GSD: MMAD = 0.67- 1.48 μm (geometric standard deviation = 3.71-2.40)

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 consecutive days per week, 6 hours per day (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3).

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 (Parental animals F0) - air control; Test Group 10 (male animals for particle detection); Test Group 20 (Recovery animals) - air control

Dose / conc.:

9.68 mg/m³ air (analytical)

Remarks:

SD: 1.47 mg/m3, target concentration: 10 mg/m³: Test Group 7 (Parental animals F0); Test Group 17 (male animals for particle detection); Test Group 27 (Recovery animals)

No. of animals per sex per dose:

16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Control animals:

yes

Details on study design:

Positive control:

None

Observations and examinations performed and frequency:

Sacrifice and pathology:

Statistics:

Statistical evaluation for main groups 0 (air control) versus 7 (micro ZnO) as well as 0 versus 8 (Zn sulphate), and recovery groups 20 (air control) versus 23 (T0420), 20 versus 26 (T0421), 20 versus 27 and 20 versus 28
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> Student's t-test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+)
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one-sided-)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: WILCOXON-test (two-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians
-Weight of the anesthetized animals and absolute and relative organ weights
--> WILCOXON test (two-sided)

Clinical signs:

no effects observed

Description (incidence and severity):

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
Exposure period, reference item 1 (test groups 7, 17 and 27):
One male animal of test group 7 (No. 126) showed a mass that was palpable through skin on
study days 66 - 149. No clinical signs of toxicity were noted in any other animals of these
groups.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes of body weight were determined in male
animals:
- Test group 7: day 25: 350.5g (p< 0.05), whereas the control group was 363.4g
- Test group 7: day 32: 362.2g (p< 0.05), whereas the control group was 379.4g
- Test group 7: day 39: 373.5g (p< 0.05), whereas the control group was 390.9g
- Test group 7: day 46: 377.4g (p< 0.05), whereas the control group was 394.9g
- Test group 7: day 60: 398.5g (p< 0.05), whereas the control group was 416.7g
- Test group 7: day 67: 405.2g (p< 0.05), whereas the control group was 424.6g
- Test group 7: day 94: 423.8g (p< 0.01), whereas the control group was 453.5g
- Test group 27: day 32: 361.7g (p< 0.05), whereas the control group was 375.0g
- Test group 27: day 39: 375.9g (p< 0.05), whereas the control group was 390.9g

The following statistically significant body weight changes were determined in male animals:
- Test group 7: day 25-> 32: 11.7 (p< 0.01), whereas the control group was 16.1g
- Test group 27: day 18-> 25: 12.7 (p< 0.01), whereas the control group was 16.9g
- Test group 27: day 74-> 81: 5.4 (p< 0.01), whereas the control group was 9.8g
-> Retarded body weight development in male animals as treatment-related, adverse effects

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Description (incidence and severity):

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

not examined

Description (incidence and severity):

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
decreased relative basophil counts in males of test group 7 (10 mg/m3 Zinc oxide T0242); decreased absolute and relative monocyte counts in females of test group 7 (10 mg/m3 Zinc oxide T0242); increased red blood cell (RBC) counts in females of test group 7 (10 mg/m3 Zinc oxide T0242); decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) in females of test group 7 (10 mg/m3 Zinc oxide T0242);

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased total bilirubin and sodium values in females of test group 7 (10 mg/m3 Zinc oxide T0242)(males, inorganic phosphate 1.48-1.85 mmol/L; females, albumin 35.27-40.13 g/L; total bilirubin 1.25-2.20 μmol/L; sodium 140.7-143.0 mmol/L).

Endocrine findings:

no effects observed

Description (incidence and severity):

After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not specified

Description (incidence and severity):

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.

Overall motor activity (summation of all intervals):
Reference item 1 (Test group 7):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
Decrease of activity in the female animals of test group 7 (10 mg/m³, reference item 1)
at interval 5 on day 87 (p ≤ 0.05).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

Immunological findings:

not examined

Description (incidence and severity):

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with control group 0 (=100%),
Reference item 1- Test group 7 (10mg/m3) (Zinc oxide T0242) : Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
--> These effects were observed as treatment-related, adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 7 males and 10 females
--> These effects were observed as treatment-related, adverse effects
Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female
animals

Neuropathological findings:

not examined

Description (incidence and severity):

neuropathological examinations of pups on PND22 (developmental neurotoxicity cohort) are examined (see IUCLID section 7.8.1)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the details on results section

The following treatment-related, adverse effects were observed:
Main group (F0)
Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³):
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female
animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all
female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes
(exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes
(exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV,
exemplarily) in 4 females

Test group 27 (Recovery group R1, 10 mg/m³)
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in
2 males and 3 females

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
Main group (F0)
The following treatment-related, adverse effects were observed:

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte
counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase
(ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β--N-Acetyl glucosaminidase (NAG) activity in BAL of males

Test group 27 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage

Details on results:

Dose descriptor:

LOAEC

Remarks:

local toxicity

Effect level:

9.68 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested target concentration of 10 mg/m³

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

9.68 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Remarks on result:

other: No systemic toxicity was observed in hematology, clinical chemistry ; increased neutrophils and lymphocytes in blood at the target conc of 10 mg/m3

Critical effects observed:

yes

Lowest effective dose / conc.:

9.68 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Executive summary:

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal

Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female

No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings

Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions.
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium

Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females

Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Cross-reference 4

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 November 2020 - ...June 2022

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study presented herein is a guideline study with a major deficiency under GLP conditions. Only one concentration level was tested.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

- adopted on 2018-06-25
- additional endpoints investigated

Deviations:

yes

Principles of method if other than guideline:

GLP compliance:

yes (incl. QA statement)

Limit test:

Specific details on test material used for the study:

Name of substance: Zinc sulfate monohydrate
Reference substance No.: 20/0420-1
Batch identification: 201126
Content: Zinc: 36.2 weight-%
Storage stability: Dec 2021
The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility
Storage conditions: Room temperature
Appearance - physical state / color solid / white
By Products Chlorides: 0.32 weight-%

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks:

whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 1.86 - <= 2.74 µm

Remarks on MMAD:

MMAD / GSD: MMAD = 1.86- 2.74 μm (geometric standard deviation = 1.99-2.01)

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 consecutive days per week, 6 hours per day (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3).

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 (Parental animals F0) - air control; Test Group 10 (male animals for particle detection); Test Group 20 (Recovery animals) - air control

Dose / conc.:

21.92 mg/m³ air (analytical)

Remarks:

SD: 1.30 mg/m3, target concentration: 22 mg/m³: Test Group 8 (Parental animals F0); Test Group 18 (male animals for particle detection); Test Group 28 (Recovery animals)

No. of animals per sex per dose:

16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Control animals:

yes

Details on study design:

Positive control:

None

Observations and examinations performed and frequency:

Sacrifice and pathology:

Statistics:

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20): There were no clinical signs and findings different from normal.
-Exposure period, reference item 2 (test groups 8, 18 and 28):
Eleven of the thirteen male animals of test group 8 showed salivation during exposure period on/or study days 4 - 89. In seven male animals of this group respiration sound was noted in addition. Two male animals (Nos: 136 + 138) of this group sparse fur (study day 79 – 84) were noted in addition. Salivation was also noted in three of the three male animals in test group 18.
Salivation was also noted in four of the five male animals in test group 28 during exposure period on/or study days 4 - 89. Respiration sound was observed in three of the five male rats of this group.
--> During exposure period, salivation and respiration sounds were detected in several male and female animals.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes of body weight were determined in male
animals:
- Test group 8: day 18: 329.9g (p< 0.05), whereas the control group was 345.9g
- Test group 8: day 25: 342.5g (p< 0.05), whereas the control group was 363.4g
- Test group 8: day 32: 354.2g (p< 0.01), whereas the control group was 379.4g
- Test group 8: day 39: 364.7g (p< 0.01), whereas the control group was 390.9g
- Test group 8: day 46: 370.4g (p< 0.05), whereas the control group was 394.9g
- Test group 8: day 53: 381.2g (p< 0.05), whereas the control group was 407.9g
- Test group 8: day 60: 387.4g (p< 0.01), whereas the control group was 416.7g
- Test group 8: day 67: 393.1g (p< 0.01), whereas the control group was 424.6g
- Test group 8: day 74: 397.1g (p< 0.01), whereas the control group was 431.2g
- Test group 8: day 81: 406.7g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 88: 411.8g (p< 0.01), whereas the control group was 442.2g
- Test group 8: day 92: 411.7g (p< 0.01), whereas the control group was 447.0g
- Test group 8: day 102: 383.6g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 109: 395.7g (p< 0.01), whereas the control group was 444.5g
- Test group 8: day 116: 399.7g (p< 0.01), whereas the control group was 449.3g
- Test group 8: day 123: 402.7g (p< 0.01), whereas the control group was 452.1g
- Test group 8: day 130: 404.4g (p< 0.01), whereas the control group was 459.9g
- Test group 8: day 137: 410.5g (p< 0.01), whereas the control group was 459.4g
- Test group 8: day 144: 410.6g (p< 0.01), whereas the control group was 463.8g
- Test group 8: day 146: 410.8g (p< 0.01), whereas the control group was 466.6g
- Test group 28: day 18: 327.3g (p< 0.05), whereas the control group was 344.0g
- Test group 28: day 25: 339.2g (p< 0.01), whereas the control group was 360.9g
- Test group 28: day 32: 348.7g (p< 0.01), whereas the control group was 375.0g
- Test group 28: day 39: 361.1g (p< 0.01), whereas the control group was 390.9g
- Test group 28: day 46: 371.5g (p< 0.01), whereas the control group was 399.8g
- Test group 28: day 53: 381.5g (p< 0.01), whereas the control group was 411.9g
- Test group 28: day 60: 386.6g (p< 0.01), whereas the control group was 414.4g
- Test group 28: day 67: 392.7g (p< 0.01), whereas the control group was 425.4g
- Test group 28: day 74: 401.5g (p< 0.01), whereas the control group was 429.7g
- Test group 28: day 81: 410.9g (p< 0.01), whereas the control group was 439.5g
- Test group 28: day 88: 417.2g (p< 0.01), whereas the control group was 446.1g
- Test group 28: day 92: 417.9g (p< 0.01), whereas the control group was 449.2g
- Test group 28: day 102: 423.0g (p< 0.05), whereas the control group was 450.4g
- Test group 28: day 109: 434.0g (p< 0.05), whereas the control group was 457.6g
The following statistically significant changes of body weight were determined in female
animals:
- Test group 8: day 93: 238.9g (p< 0.05), whereas the control group was 251.8g
- Test group 28: day 18: 198.4g (p< 0.01), whereas the control group was 213.1g
- Test group 28: day 25: 209.8g (p< 0.05), whereas the control group was 220.9g
- Test group 28: day 32: 212.5g (p< 0.01), whereas the control group was 228.2g
- Test group 28: day 60: 231.2g (p< 0.05), whereas the control group was 245.6g
- Test group 28: day 116: 251.6g (p< 0.05), whereas the control group was 264.2g

The following statistically significant body weight changes were determined in male animals:
- Test group 8: day 0-> 4: 4.7 (p< 0.01), whereas the control group was 10.4g
- Test group 8: day 11-> 18: 17.1 (p< 0.05), whereas the control group was 22.0g
- Test group 8: day 18-> 25: 12.6 (p< 0.01), whereas the control group was 17.5g
- Test group 8: day 25-> 32: 11.7 (p< 0.05), whereas the control group was 16.1g
- Test group 8: day 74-> 81: 9.6 (p< 0.01), whereas the control group was 5.7g
- Test group 8: day 88-> 92: -0.1 (p< 0.01), whereas the control group was 4.8g
- Test group 8: day 102-> 109: 12.1 (p< 0.05), whereas the control group was 7.6g

- Test group 18: day 74-> 81: 7.3 (p< 0.05), whereas the control group was 0.9g

The following statistically significant body weight changes were determined in female animals:
- Test group 28: day 0-> 4: 5.3 (p< 0.01), whereas the control group was 10.3g
- Test group 28: day 11-> 18: 7.3 (p< 0.01), whereas the control group was 17.7g

-> Retarded body weight development in male/female animals as treatment-related, adverse effects

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes of mean food consumption were determined in male animals:
•Test group 8: day 0 - 4: +21.3 g (p≤ 0.01), whereas the control group was +24.4 g
• Test group 8: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The following statistically significant changes of mean food consumption were determined in female animals:
Test group 8: day 4 - 11: +16.5 g (p≤ 0.01), whereas the control group was +17.6 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse. The finding in test group 8 was considered not biologically relevant due to its transient nature.

Food efficiency:

not examined

Description (incidence and severity):

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

not examined

Description (incidence and severity):

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
Increased hemoglobin values in males of test group 8 (22 mg/m3 Zinc sulfate)

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of administration in males of test group 8 (22 mg/m3 Zinc sulfate) total bilirubin values were significantly increased, but this was the only relevantly changed clinical chemistry parameter among these individuals. This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test groups 8 (22 mg/m3 Zinc sulfate)
After the 8-week recovery period, in males of test group 28 (10 mg/m3 Zinc oxide 22 mg/m3 Zinc sulfate) creatinine values were significantly lower compared to controls. However, total bilirubin values were within historical control ranges
(males, total bilirubin 1.34-2.07 μmol/L) whereas creatinine values were marginally below this range (males; creatinine 31.8-37.0 μmol/L). Therefore, total bilirubin increase was regarded as incidental and not treatment related whereas creatinine decrease was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

Endocrine findings:

no effects observed

Description (incidence and severity):

After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not specified

Description (incidence and severity):

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Immunological findings:

not examined

Description (incidence and severity):

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with control group 0 (=100%),
Reference item 2- Test group 8 (22mg/m3) (Zinc sulfate) :
Increase of absolute/relative lung weights in males (125%/138%) and females
(114%/119%)
--> These effects were observed as treatment-related, adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
•Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 5 males and 8 females--> These effects were observed as treatment-related, adverse effects
Test group 28 (Recovery group , Zinc sulfate monohydrate)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female

Neuropathological findings:

not examined

Description (incidence and severity):

neuropathological examinations of pups on PND22 (developmental neurotoxicity cohort) are examined (see IUCLID section 7.8.1)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the details on results section

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³):
Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the
epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9
females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female
animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female
animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes
(exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes
(exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium (nasal
cavity, level IV, exemplarily) in all males and all females

Test group 28 (Recovery group (Zinc sulfate monohydrate):
Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis
in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and
3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female
animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1
male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in
4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV,
exemplarily) in 1 male and 1 female

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
Main group (F0)
The following treatment-related, adverse effects were observed:
Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)

• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell
and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase
(ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes

Test group 28 (Recovery group Zinc sulfate monohydrate)
• No treatment-related adverse findings in lavage

Details on results:

CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20): There were no clinical signs and findings different from normal.
-Exposure period, reference item 2 (test groups 8, 18 and 28):
Eleven of the thirteen male animals of test group 8 showed salivation during exposure period on/or study days 4 - 89. In seven male animals of this group respiration sound was noted in addition. Two male animals (Nos: 136 + 138) of this group sparse fur (study day 79 – 84) were noted in addition. Salivation was also noted in three of the three male animals in test group 18.
Salivation was also noted in four of the five male animals in test group 28 during exposure period on/or study days 4 - 89. Respiration sound was observed in three of the five male rats of this group.
--> During exposure period, salivation and respiration sounds were detected in several male and female animals.

BODY WEIGHT AND WEIGHT GAIN
The following statistically significant changes of body weight were determined in male
animals:
- Test group 8: day 18: 329.9g (p< 0.05), whereas the control group was 345.9g
- Test group 8: day 25: 342.5g (p< 0.05), whereas the control group was 363.4g
- Test group 8: day 32: 354.2g (p< 0.01), whereas the control group was 379.4g
- Test group 8: day 39: 364.7g (p< 0.01), whereas the control group was 390.9g
- Test group 8: day 46: 370.4g (p< 0.05), whereas the control group was 394.9g
- Test group 8: day 53: 381.2g (p< 0.05), whereas the control group was 407.9g
- Test group 8: day 60: 387.4g (p< 0.01), whereas the control group was 416.7g
- Test group 8: day 67: 393.1g (p< 0.01), whereas the control group was 424.6g
- Test group 8: day 74: 397.1g (p< 0.01), whereas the control group was 431.2g
- Test group 8: day 81: 406.7g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 88: 411.8g (p< 0.01), whereas the control group was 442.2g
- Test group 8: day 92: 411.7g (p< 0.01), whereas the control group was 447.0g
- Test group 8: day 102: 383.6g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 109: 395.7g (p< 0.01), whereas the control group was 444.5g
- Test group 8: day 116: 399.7g (p< 0.01), whereas the control group was 449.3g
- Test group 8: day 123: 402.7g (p< 0.01), whereas the control group was 452.1g
- Test group 8: day 130: 404.4g (p< 0.01), whereas the control group was 459.9g
- Test group 8: day 137: 410.5g (p< 0.01), whereas the control group was 459.4g
- Test group 8: day 144: 410.6g (p< 0.01), whereas the control group was 463.8g
- Test group 8: day 146: 410.8g (p< 0.01), whereas the control group was 466.6g
- Test group 28: day 18: 327.3g (p< 0.05), whereas the control group was 344.0g
- Test group 28: day 25: 339.2g (p< 0.01), whereas the control group was 360.9g
- Test group 28: day 32: 348.7g (p< 0.01), whereas the control group was 375.0g
- Test group 28: day 39: 361.1g (p< 0.01), whereas the control group was 390.9g
- Test group 28: day 46: 371.5g (p< 0.01), whereas the control group was 399.8g
- Test group 28: day 53: 381.5g (p< 0.01), whereas the control group was 411.9g
- Test group 28: day 60: 386.6g (p< 0.01), whereas the control group was 414.4g
- Test group 28: day 67: 392.7g (p< 0.01), whereas the control group was 425.4g
- Test group 28: day 74: 401.5g (p< 0.01), whereas the control group was 429.7g
- Test group 28: day 81: 410.9g (p< 0.01), whereas the control group was 439.5g
- Test group 28: day 88: 417.2g (p< 0.01), whereas the control group was 446.1g
- Test group 28: day 92: 417.9g (p< 0.01), whereas the control group was 449.2g
- Test group 28: day 102: 423.0g (p< 0.05), whereas the control group was 450.4g
- Test group 28: day 109: 434.0g (p< 0.05), whereas the control group was 457.6g
The following statistically significant changes of body weight were determined in female
animals:
- Test group 8: day 93: 238.9g (p< 0.05), whereas the control group was 251.8g
- Test group 28: day 18: 198.4g (p< 0.01), whereas the control group was 213.1g
- Test group 28: day 25: 209.8g (p< 0.05), whereas the control group was 220.9g
- Test group 28: day 32: 212.5g (p< 0.01), whereas the control group was 228.2g
- Test group 28: day 60: 231.2g (p< 0.05), whereas the control group was 245.6g
- Test group 28: day 116: 251.6g (p< 0.05), whereas the control group was 264.2g

The following statistically significant body weight changes were determined in male animals:
- Test group 8: day 0-> 4: 4.7 (p< 0.01), whereas the control group was 10.4g
- Test group 8: day 11-> 18: 17.1 (p< 0.05), whereas the control group was 22.0g
- Test group 8: day 18-> 25: 12.6 (p< 0.01), whereas the control group was 17.5g
- Test group 8: day 25-> 32: 11.7 (p< 0.05), whereas the control group was 16.1g
- Test group 8: day 74-> 81: 9.6 (p< 0.01), whereas the control group was 5.7g
- Test group 8: day 88-> 92: -0.1 (p< 0.01), whereas the control group was 4.8g
- Test group 8: day 102-> 109: 12.1 (p< 0.05), whereas the control group was 7.6g

- Test group 18: day 74-> 81: 7.3 (p< 0.05), whereas the control group was 0.9g

The following statistically significant body weight changes were determined in female animals:
- Test group 28: day 0-> 4: 5.3 (p< 0.01), whereas the control group was 10.3g
- Test group 28: day 11-> 18: 7.3 (p< 0.01), whereas the control group was 17.7g

-> Retarded body weight development in male/female animals as treatment-related, adverse effects

FOOD CONSUMPTION
The following statistically significant changes of mean food consumption were determined in male animals:
•Test group 8: day 0 - 4: +21.3 g (p≤ 0.01), whereas the control group was +24.4 g
• Test group 8: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The following statistically significant changes of mean food consumption were determined in female animals:
Test group 8: day 4 - 11: +16.5 g (p≤ 0.01), whereas the control group was +17.6 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse. The finding in test group 8 was considered not biologically relevant due to its transient nature.

HAEMATOLOGICAL FINDINGS:
The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
Increased hemoglobin values in males of test group 8 (22 mg/m3 Zinc sulfate)

CLINICAL CHEMISTRY:
At the end of administration in males of test group 8 (22 mg/m3 Zinc sulfate) total bilirubin values were significantly increased, but this was the only relevantly changed clinical chemistry parameter among these individuals. This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test groups 8 (22 mg/m3 Zinc sulfate)
After the 8-week recovery period, in males of test group 28 (10 mg/m3 Zinc oxide 22 mg/m3 Zinc sulfate) creatinine values were significantly lower compared to controls. However, total bilirubin values were within historical control ranges
(males, total bilirubin 1.34-2.07 μmol/L) whereas creatinine values were marginally below this range (males; creatinine 31.8-37.0 μmol/L). Therefore, total bilirubin increase was regarded as incidental and not treatment related whereas creatinine decrease was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

NEUROBEHAVIOUR:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
Decrease of activity in the female animals of test group 7 (10 mg/m³, reference item 1)
at interval 5 on day 87 (p ≤ 0.05).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

ORGAN WEIGHTS
When compared with control group 0 (=100%),
Reference item 2- Test group 8 (22mg/m3) (Zinc sulfate) :
Increase of absolute/relative lung weights in males (125%/138%) and females
(114%/119%)
--> These effects were observed as treatment-related, adverse effects

GROSS PATHOLOGY

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
•Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 5 males and 8 females--> These effects were observed as treatment-related, adverse effects
Test group 28 (Recovery group , Zinc sulfate monohydrate)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
One female animal of test group 8 (reference item 2, 22 mg/m³) revealed an erosion/ulcer at the base of the epiglottis. All male and all female animals of the same test group revealed squamous metaplasia of the respiratory epithelium, mainly in the region of the base of the epiglottis. This finding is characterized by flattening of cells, increase of cellular layers, and keratinization on the surface. Furthermore, one male and nine females showed mixed (macrophages, neutrophils, lymphocytes) inflammatory cell infiltrates in this region. These findings were regarded to be treatment-related.
Recovery animals:
In the larynx mainly males and females of the reference test groups were affected. The same findings as described for the main group animals were still observed in the recovery animals.
These findings were regarded to be treatment-related.

Lungs:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

In males and females of the two reference items, similar findings were observed as described for test group 3 and 6 (test item 1 and 2, 2 mg/m³). Only the severity was slightly higher when compared with the other test items, especially in test group 7 animals.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

The males and females exposed to the reference items showed similar findings as the animals exposed to the test substances.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
Females of test group 7 (reference item 1, 10 mg/m³) and males and females of test group 8 (reference item 2, 22 mg/m³) revealed the same findings in the nasal cavity.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period in BAL of males and females in test group 8 (22 mg/m3 Zinc sulfate) total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts were significantly increased whereas relative macrophage counts were significantly decreased. Additionally, in BAL of males in this test group absolute macrophage and eosinophil cell (not significantly) counts were significantly increased. These alterations were regarded as treatment related and adverse.
Recovery animals:
After the 8-week recovery period, in BAL cytology of males and females of test group 28 (22 mg/m3 Zinc sulfate) no changes were observed.

Proteins/enzymes:

Parental animals:
After the administration period, in BAL of males and females of test group 8 (22 mg/m3 Zinc sulfate) total protein levels as well as lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity were moderately, significantly increased whereas γ -Glutamyl-transferase (GGT) activity was only marginally but also significantly increased. These alterations were regarded as treatment related and adverse. In BAL of males of test group 8 (22 mg/m3 Zinc sulfate) β -N-Acetyl glucosaminidase (NAG) activity was marginally, significantly increased in males of this test group, but the change was below 2fold. Therefore, this alteration was regarded as maybe treatment related but nonadverse.
Recovery animals:
After the 8-week recovery period, no changes of total protein and enzyme activities in BAL of males and females in test group 28 (22 mg/m3 Zinc sulfate) were observed.

OTHER FINDINGS:
organ burden:
ICP-OES analysis Zinc content in lungs, liver, heart and brain of parental male/female animals
As an essential element, the data showed high biological background level of zinc element (very high endogenous background). In the high concentration exposure groups, slightly increased zinc concentration was only found in lung. In all other examined organs, the zinc level was comparable with the control.

Dose descriptor:

LOAEC

Remarks:

local toxicity

Effect level:

21.92 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the target concentration of 22 mg/m³

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

21.92 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Remarks on result:

other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology at the target conc of 22 mg/m3

Critical effects observed:

yes

Lowest effective dose / conc.:

21.92 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Executive summary:

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal

Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female

No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals.
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female

Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Data source

Reference

Reference Type:: study report
Title:: Unnamed
Year:: 2022
Report date:: 2022

Materials and methods

Test guideline

Qualifier:: according to guideline
Guideline:: OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study
Version / remarks:: - adopted on 2018-06-25
- additional endpoints investigated
Deviations:: yes

Principles of method if other than guideline:: Additional endpoints (covered in IUCLID 7.8.1): reproduction toxicology , developmental toxicity, (developmental) neurotoxicity
three doses of the test substance (uncoated ZnO nanomaterial) were compared to one dose of 2 reference substances (non-coated microscaled ZnO and Zinc sulfate monohydrate)
GLP compliance:: yes (incl. QA statement)
Limit test:: no

Test material

Test material information

Test material form:: solid: nanoform
Details on test material:: SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 98.2% for T0420
- Test substance No.: 20/0050-1 for T0420
- Batch identification: T0420

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: May 2022 for T0420

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Specific details on test material used for the study:: SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 98.2% for T0420
- Test substance No.: 20/0050-1 for T0420
- Batch identification: T0420

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: May 2022 for T0420.

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Test animals

Species:: rat
Strain:: Wistar
Details on species / strain selection:: Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Administration / exposure

Route of administration:: inhalation: aerosol
Type of inhalation exposure:: whole body
Remarks:: whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'
Vehicle:: other: unchanged (no vehicle)
Mass median aerodynamic diameter (MMAD):: >= 0.52 - <= 2.01 µm
Remarks on MMAD:: MMAD / GSD: MMAD = 0.52-2.01 μm (geometric standard deviation = 4.04--2.28)
Details on inhalation exposure:: GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
Duration of treatment / exposure:: 90 days
Frequency of treatment:: 7 consecutive days per week, 6 hours per day (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3).

Doses / concentrationsopen all close all

Doses / concentrations 1

Dose / conc.:: 0 mg/m³ air
Remarks:: Test Group 0 (Parental animals F0) - air control; Test Group 10 (male animals for particle detection); Test Group 20 (Recovery animals) - air control

Doses / concentrations 2

Dose / conc.:: 0.52 mg/m³ air (analytical)
Remarks:: SD: 0.10 mg/m3; target concentration: 0.5 mg/m³: Test Group 1 (Parental animals F0)

Doses / concentrations 3

Dose / conc.:: 2 mg/m³ air (analytical)
Remarks:: SD: 0.20 mg/m3; target concentration: 2.0 mg/m³: Test Group 2 (Parental animals F0)

Doses / concentrations 4

Dose / conc.:: 9.97 mg/m³ air (analytical)
Remarks:: SD: 1.23 mg/m3, target concentration: 10 mg/m³: Test Group 3 (Parental animals F0); Test Group 13 (male animals for particle detection); Test Group 23 (Recovery animals)

No. of animals per sex per dose:: 16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)
Control animals:: yes
Details on study design:: - Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ±20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period
Positive control:: None

Examinations

Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina
Statistics:: Statistical evaluation for test groups low, mid, high in comparison with air control group
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> DUNNETT test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+) with BONFERRONI-HOLM
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one sided-) with BONFERRONI-HOLM
-% live male day x, %live female day x
--> WILCOXON test (two-sided)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the
control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Weight parameters in pathology
-->Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided).

Results and discussion

Results of examinations

Clinical signs:: no effects observed
Description (incidence and severity):: -During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
Exposure period, test item 1 (test groups 1, 2, 3, 13, and 23):
No clinical signs of toxicity were observed in male and female animals.
Mortality:: no mortality observed
Description (incidence):: No deaths were recorded throughout the study.
Body weight and weight changes:: effects observed, non-treatment-related
Description (incidence and severity):: The following statistically significant body weight changes were determined in male animals:
- Test group 1: day 74 -> 81: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 1: day 92 -> 93: -5.1g (p< 0.05), whereas the control group was 2.3g
- Test group 1: day 93 -> 94: 6.7g (p< 0.05), whereas the control group was -5.3g
- Test group 3: day 11 -> 18: 17.8g (p< 0.05), whereas the control group was 22.0g
- Test group 3: day 25 -> 32: 11.0g (p< 0.05), whereas the control group was 16.1g
- Test group 3: day 94 -> 95: -10.7g (p< 0.05), whereas the control group was 2.0g
- Test group 13: day 18 -> 25: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 13: day 74 -> 81: 7.5g (p< 0.01), whereas the control group was 0.9g
- Test group 23: day 0 -> 4: 4.5g (p< 0.01), whereas the control group was 12.6g
- Test group 23: day 18 -> 25: 9.7g (p< 0.01), whereas the control group was 16.9g
- Test group 23: day 53 -> 60: 9.5g (p< 0.05), whereas the control group was 2.6g
- Test group 23: day 109 -> 116: 9.6g (p< 0.01), whereas the control group was 2.2g
The following statistically significant body weight changes were determined in female
animals:
- Test group 23: day 60 -> 67: 12.1g (p< 0.05), whereas the control group was 1.0g
- Test group 23 day 102 -> 109: 3.5g (p< 0.05), whereas the control group was 9.4g

Although the deviations in body weight changes were statistically significant, they did not show any trend with the exposure-duration, as some of the means were higher than the control, on the other days lower, indicating that they were rather biological variations than substance-related changes. Moreover, the mean body weight (as well as the final body weight) did not significantly change, when compared with the concurrent control. These deviations from the control were considered not biologically relevant.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: The following statistically significant changes of mean food consumption were determined in
female animals:
• Test group 1: day 11 - 18: +19.4 g (p≤ 0.05), whereas the control group was +17.6 g
• Test group 1: day 25 - 32: +19.1 g (p≤ 0.05), whereas the control group was +17.5 g
• Test group 1: day 32 - 39: +19.3 g (p≤ 0.05), whereas the control group was +17.8 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse.
Food efficiency:: not examined
Description (incidence and severity):: /
Water consumption and compound intake (if drinking water study):: not examined
Description (incidence and severity):: /
Ophthalmological findings:: no effects observed
Description (incidence and severity):: The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: In females of test group 3 (10 mg/m3 Zinc oxide T0420) absolute and relative neutrophil cell
counts were significantly increased whereas relative lymphocyte counts were significantly
decreased. However, total white blood cell counts were not altered among these individuals,
and absolute neutrophil counts were within the historical control range (females, absolute
neutrophils 0.60-0.96 giga/L). Therefore, these changes were regarded as incidental and not
treatment related.

The following significant changes were regarded as incidental and not treatment related,
because the values were within historical control ranges: decreased relative eosinophil cell
counts in males of test groups 2 and 3 (2 and 10 mg/m3 Zinc oxide T0420) prolonged prothrombin time (HQT, i.e., Hepatoquick’s test) in females of test group 3 (10 mg/m3 Zinc oxide T0420)( males, relative eosinophils 1.4-3.1 %; relative basophils 0.1-0.4 %; hemoglobin 8.6-9.3 mmol/L; females, absolute monocytes 0.05-0.11 Giga/L; relative monocytes 1.8-2.8 %; RBC 7.55-8.84 Tera/L; MCV 50.7-55.1 fL; MCH 1.10-1.21 fmol; HQT 34.0-40.2 sec).
The following significant changes were regarded as incidental and not treatment related,
because the alteration was not dose dependent: decreased absolute and relative monocyte counts in females of test group 2 (2 mg/m3 Zinc oxide T0420) as well as absolute monocyte counts in females of test group 3 (10 mg/m3 Zinc oxide T0420).

In females of test group 23 (10 mg/m3 Zinc oxide T0420) absolute monocyte counts were
significantly decreased, but the values were within the historical control range (females,
absolute monocytes 0.05-0.07 Giga/L). Therefore this change was regarded as incidental and
not treatment related.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased inorganic phosphate levels
in males of test groups 3 (10 mg/m3 Zinc oxide T0420 )
Endocrine findings:: no effects observed
Description (incidence and severity):: After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:: not specified
Description (incidence and severity):: /
Behaviour (functional findings):: no effects observed
Description (incidence and severity):: Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
Test item T0420 (Test groups 1, 2 and 3):
there were no statistically significant deviations from the control group 0.
Immunological findings:: not examined
Description (incidence and severity):: /
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: When compared with control group 0 (=100%), Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³): Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
--> These effects were observed as treatment-related, adverse effects
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females.
•Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 6 males and 9 females
--> These effects were observed as treatment-related, adverse effects
Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
--> The foci observed in the lungs of males and females of test group 23 (test item 1, 10 mg/m³)
and test group 26 (test item 2, 10 mg/m³) were considered to be treatment-related as similar
findings were observed in the respective main groups. The same comes true for the
enlargement of the mediastinal lymph nodes in one female of test group 23 (test item 1,
10 mg/m³) and one male of test group 26 (test item 2, 10 mg/m³). These findings were regarded
to be treatment-related.
Neuropathological findings:: not examined
Description (incidence and severity):: neuropathological examinations of pups on PND22 (developmental neurotoxicity cohort) are examined (see IUCLID section 7.8.1)
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in details on results section

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV, exemplarily) in 9 males and 10 females

Test group 23 (Recovery group R1, 10 mg/m³)
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4
female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal
Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV, exemplarily) in 1 female
Histopathological findings: neoplastic:: no effects observed
Other effects:: effects observed, treatment-related
Description (incidence and severity):: BAL
Main group (F0)
Test item 1 (Zinc oxide T0420)
The following treatment-related, adverse effects were observed:
Test group 3 (10 mg/m³):
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase
(ALP) and γ -Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
Details on results:: CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:
During pre-exposure period, none of the male and female rats showed any clinical signs and findings different from normal.
Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
Exposure period, test item 1 (test groups 1, 2, 3, 13, and 23):
No clinical signs of toxicity were observed in male and female animals.

BODY WEIGHT AND WEIGHT GAIN
Body weight change:
The following statistically significant body weight changes were determined in male animals:
- Test group 1: day 74 -> 81: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 1: day 92 -> 93: -5.1g (p< 0.05), whereas the control group was 2.3g
- Test group 1: day 93 -> 94: 6.7g (p< 0.05), whereas the control group was -5.3g
- Test group 3: day 11 -> 18: 17.8g (p< 0.05), whereas the control group was 22.0g
- Test group 3: day 25 -> 32: 11.0g (p< 0.05), whereas the control group was 16.1g
- Test group 3: day 94 -> 95: -10.7g (p< 0.05), whereas the control group was 2.0g
- Test group 13: day 18 -> 25: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 13: day 74 -> 81: 7.5g (p< 0.01), whereas the control group was 0.9g
- Test group 23: day 0 -> 4: 4.5g (p< 0.01), whereas the control group was 12.6g
- Test group 23: day 18 -> 25: 9.7g (p< 0.01), whereas the control group was 16.9g
- Test group 23: day 53 -> 60: 9.5g (p< 0.05), whereas the control group was 2.6g
- Test group 23: day 109 -> 116: 9.6g (p< 0.01), whereas the control group was 2.2g
The following statistically significant body weight changes were determined in female
animals:
- Test group 23: day 60 -> 67: 12.1g (p< 0.05), whereas the control group was 1.0g
- Test group 23 day 102 -> 109: 3.5g (p< 0.05), whereas the control group was 9.4g
Although the deviations in body weight changes were statistically significant, they did not show any trend with the exposure-duration, as some of the means were higher than the control, on the other days lower, indicating that they were rather biological variations than substance-related changes. Moreover, the mean body weight (as well as the final body weight) did not significantly change, when compared with the concurrent control. These deviations from the control were considered not biologically relevant.

FOOD CONSUMPTION
The following statistically significant changes of mean food consumption were determined in
female animals:
• Test group 1: day 11 - 18: +19.4 g (p≤ 0.05), whereas the control group was +17.6 g
• Test group 1: day 25 - 32: +19.1 g (p≤ 0.05), whereas the control group was +17.5 g
• Test group 1: day 32 - 39: +19.3 g (p≤ 0.05), whereas the control group was +17.8 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse.

HAEMATOLOGICAL FINDINGS:
In females of test group 3 (10 mg/m3 Zinc oxide T0420) absolute and relative neutrophil cell
counts were significantly increased whereas relative lymphocyte counts were significantly
decreased. However, total white blood cell counts were not altered among these individuals,
and absolute neutrophil counts were within the historical control range (females, absolute
neutrophils 0.60-0.96 giga/L). Therefore, these changes were regarded as incidental and not
treatment related.

The following significant changes were regarded as incidental and not treatment related,
because the values were within historical control ranges: decreased relative eosinophil cell
counts in males of test groups 2 and 3 (2 and 10 mg/m3 Zinc oxide T0420) prolonged prothrombin time (HQT, i.e., Hepatoquick’s test) in females of test group 3 (10 mg/m3 Zinc oxide T0420)( males, relative eosinophils 1.4-3.1 %; relative basophils 0.1-0.4 %; hemoglobin 8.6-9.3 mmol/L; females, absolute monocytes 0.05-0.11 Giga/L; relative monocytes 1.8-2.8 %; RBC 7.55-8.84 Tera/L; MCV 50.7-55.1 fL; MCH 1.10-1.21 fmol; HQT 34.0-40.2 sec).
The following significant changes were regarded as incidental and not treatment related,
because the alteration was not dose dependent: decreased absolute and relative monocyte counts in females of test group 2 (2 mg/m3 Zinc oxide T0420) as well as absolute monocyte counts in females of test group 3 (10 mg/m3 Zinc oxide T0420).

In females of test group 23 (10 mg/m3 Zinc oxide T0420) absolute monocyte counts were
significantly decreased, but the values were within the historical control range (females,
absolute monocytes 0.05-0.07 Giga/L). Therefore this change was regarded as incidental and
not treatment related.

CLINICAL CHEMISTRY:
The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased inorganic phosphate levels
in males of test groups 3 (10 mg/m3 Zinc oxide T0420 )

NEUROBEHAVIOUR:
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
Test item T0420 (Test groups 1, 2 and 3):
there were no statistically significant deviations from the control group 0.

ORGAN WEIGHTS
When compared with control group 0 (=100%), Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³): Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)

GROSS PATHOLOGY
Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females.
•Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 6 males and 9 females
Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
--> The foci observed in the lungs of males and females of test group 23 (test item 1, 10 mg/m³)
and test group 26 (test item 2, 10 mg/m³) were considered to be treatment-related as similar
findings were observed in the respective main groups. The same comes true for the
enlargement of the mediastinal lymph nodes in one female of test group 23 (test item 1,
10 mg/m³) and one male of test group 26 (test item 2, 10 mg/m³). These findings were regarded
to be treatment-related.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
Minimal epithelial alteration was observed in several test groups treated with test item 1 or test item 2 as well as in control animals. This finding is characterized by an increase of cell layers and replacement of respiratory epithelium by squamous epithelial cells, which may exhibit slight nuclear polymorphism and cellular atypia. The site most susceptible for this lesion, is the base of the epiglottis as it was observed in the present study. This finding was regarded to be treatment-related (inhalation).

Recovery animals: no findings observed

Lungs:

Parental animals:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. The finding was characterized by loss of olfactory epithelial cells and occasionally regeneration. Mainly the dorsal meatus and areas on the nasal septum were affected. This finding was regarded to be treatment-related.
One female of test group 1 (test item 1, 0.5 mg/m³) and three males and one female of test group 5 (test item 2, 2 mg/m³) showed minimal to slight degeneration of the olfactory epithelium. As this finding normally does not occur as a background lesion, it was assumed to have been most likely caused by the test substances.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period, in the bronchoalveolar lavage (BAL) of males and females of
test group 3 (10 mg/m3 Zinc oxide T0420) total cell counts, as well as absolute and relative
lymphocyte, neutrophil cell (PMN) and monocyte counts as well as absolute eosinophil cell
counts (not significantly) were increased. Relative macrophage counts were significantly
decreased. These alterations were regarded as treatment related and adverse.
In the BAL of males of test group 2 (2 mg/m3 Zinc oxide T0420) absolute and relative lymphocyte, neutrophil cell and monocyte counts were already significantly increased whereas
relative macrophage counts were significantly decreased. In males of test group 1 (0.5 mg/m3
Zinc oxide T0420) absolute and relative lymphocyte counts were significantly increased. In
females of test group 2 absolute and relative monocyte counts as well as relative neutrophil
counts were significantly increased whereas relative macrophage counts were significantly
decreased. However, in the BAL of both sexes of test group 2 as well as in BAL of males of
test group 1 total cell counts were not altered, and the differential cell counts were only
marginally changed (below 10fold). Therefore, the cell count changes in BAL of both sexes in
test group 2 and in BAL of males of test group 1 were regarded as treatment related but non adverse.

Recovery animals:
After the 8-week recovery period, no significant changes in BAL cytology were observed in
BAL of both sexes of test group 23 (10 mg/m3 Zinc oxide T0420).
Proteins/enzymes:
Parental animals:
After the administration period, in BAL of males and females of test group 3 (10 mg/m3 Zinc
oxide T0420) total protein levels as well as lactate dehydrogenase (LDH) and alkaline
phosphatase (ALP) activity were moderately, significantly increased whereas β-N-Acetyl
glucosaminidathe zinc content in lungs, liver, heart and brain of parse (NAG) in males of this test group and γ-Glutamyl-transferase (GGT) activity
in both sexes were marginally but also significantly increased. These alterations were regarded
as treatment related and adverse.
Additionally, in BAL of females of test group 3 (10 mg/m3 Zinc oxide T0420) NAG activity was
significantly increased, and in BAL of males of test group 2 (2 mg/m3 Zinc oxide T0420) LDH,
ALP and GGT activities and in females of this test group ALP and GGT activities were
significantly increased. However, the changes were marginally (below 2fold). Therefore, these
alterations were regarded as treatment related but non-adverse.
Recovery animals:
After the 8-week recovery period, in BAL of males and females of test group 23 (10 mg/m3
Zinc oxide T0420) no protein level and enzyme activity changes were observed.

OTHER FINDINGS:
organ burden:
ICP-OES analysis Zinc content in lungs, liver, heart and brain of parental male/female animals
As an essential element, the data showed high biological background level of zinc element (very high endogenous background). In the high concentration exposure groups, slightly increased zinc concentration was only found in lung. In all other examined organs, the zinc level was comparable with the control.

- Electron microscopy:
Electron microscope analysis of particulate matter in organs and tissues: (see section overall remarks/attachments)

Effect levels

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1

Dose descriptor:: NOAEC
Remarks:: local toxicity
Effect level:: 0.52 mg/m³ air (analytical)
Based on:: test mat.
Sex:: male
Basis for effect level:: histopathology: non-neoplastic
Remarks on result:: other: At the target mid concentration of 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal

2

Dose descriptor:: LOAEC
Remarks:: local toxicity
Effect level:: 0.52 mg/m³ air (analytical)
Based on:: test mat.
Sex:: female
Basis for effect level:: histopathology: non-neoplastic
Remarks on result:: other: At the target low concentration of 0.5 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one female animal

3

Dose descriptor:: NOAEC
Remarks:: systemic toxicity
Effect level:: 9.97 mg/m³ air (analytical)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: clinical biochemistry; haematology; histopathology: non-neoplastic
Remarks on result:: other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology

Target system / organ toxicity

Critical effects observed:: yes
Lowest effective dose / conc.:: 2 mg/m³ air (analytical)
System:: respiratory system: lower respiratory tract
Organ:: lungs
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: not specified

Applicant's summary and conclusion

Conclusions:

Executive summary:

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)

• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium

Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal

Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female

Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.

No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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Registration Dossier

Registration Dossier

Diss Factsheets

Zinc oxide

Repeated dose toxicity: inhalation

Administrative data

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Reference

Concentration measurements in the exposure system

Results of the particle size analyses

Histopathology

Pathology

Weight parameters

Absolute weights

Gross lesions

Reference

Reference

Reference

Data source

Reference

Materials and methods

Test guideline

Test material

Constituent 1

Test animals

Administration / exposure

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Examinations

Results and discussion

Results of examinations

Effect levels

Target system / organ toxicity

Applicant's summary and conclusion

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