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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:
26 March 2020 - December 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
June 2018
Deviations:
no
Remarks:
none that were considered to have not affected the integrity or validity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
no
Remarks:
none that were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
12-hydroxy-N-[2-(12-hydroxyoctadecanamido)ethyl]octadecanamide; N-(2-decanamidoethyl)-12-hydroxyoctadecanamide; N-(2-decanamidoethyl)decanamide
EC Number:
907-495-0
Cas Number:
198028-14-7
IUPAC Name:
12-hydroxy-N-[2-(12-hydroxyoctadecanamido)ethyl]octadecanamide; N-(2-decanamidoethyl)-12-hydroxyoctadecanamide; N-(2-decanamidoethyl)decanamide
Test material form:
solid
Details on test material:
Chemical name : Reaction mass of N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide) and Octadecanamide, 12-hydroxy-N-[2-[(1-oxodecyl)amino]ethyl]- and Decanamide, N,N'-1,2-ethanediylbis-
Chemical registery number : EC 907-495-0 / CAS : 198028-14-7

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The RccHan®:WIST strain was used because of the historical control data available at this laboratory.
Sex:
male/female

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
snout only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.2 - <= 3.3 µm
Geometric standard deviation (GSD):
2.38
Remarks on MMAD:
The MMAD (= 3.3 µm) values indicate the generated aerosols were respirable to the rat in all treated groups.
Details on inhalation exposure:
Please also refer to the attached "Schematic of Inhalation Exposure System"

Components of the system:
Exposure System:
- Flow through nose-only chamber
- Aluminium alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber
- For test groups the aerosol generator was attached to elutriation chamber connected to the exposure chamber via aerosol tubing

Animal Restraint:
- Plastic nose-only restraint tube

Aerosol Generation:
- Wright Dust Feed Mechanism
- The WDF mechanism is designed to produce and maintain test atmospheres containing dust by suspending material scraped from the surface of a compressed powder in a stream of dry air
- Power interrupter used for Group 2 due to the low generator settings required to achieve the target aerosol concentration

Inlet Airflow:
- From in-house compressed air system – breathing quality
- Generator flow: 18 L/minute

Extract Airflow:
- Drawn by in-house vacuum system
- Filtered locally
- Extract flow: 28-80 L/minute

Airflow Monitoring:
- High quality tapered tube flowmeters – calibrated daily
- In-line flowmeters monitored continuously

System Containment:
- Systems housed in separate ventilated cabinets

Study conduct details:
- Animals were exposed five days each week during Weeks 1 to 12 and seven days during
Week 13. Additional animal exposures were conducted during Week 14 to cover the period of necropsy.
- Duration of exposure was 6 hours each day
- Different exposure levels were achieved by varying the concentration of test item in the exposure systems, whilst keeping the duration of exposure constant
- Group 1 animals were exposed to air only at the same flow rate as the Group 4 animals
- Exposures commenced on 10 June 2020
- The animals on study were acclimatized to the method of restraint for three consecutive days immediately preceding their first exposure
- Animal placement on exposure systems altered weekly throughout the duration of the study
- System operating conditions were amended at the discretion of the Study Director to maintain achieved aerosol concentrations close to target

Concentration
Aerosol samples collected as follows:
- Filter type: Glass fiber filter, held in an open face filter holder
- Sample flow: 2-5 L/minute
- Sample volume: Measured by wet-type gas meter or electronic sampling pump
- Sample frequency: Minimum of 3 samples/group/day (Groups 2 to 4)
Group 1 samples collected on chemical analysis occasions
- Sample location: Animal exposure port
- Sample analysis: Gravimetric (all test group samples)
Chemical – please refer to "Analytical verification of doses or concentrations" (all samples for one exposure in Weeks 3, 6, 9 and 12)

Particle Size Distribution
Determined by cascade impaction – samples collected as follows:
- Impactor type: Marple 290 Series (298 configuration)
- Collection media: Stainless steel substrates and glass fiber final stage filter
- Sample flow: 3 L/minute
- Sample volume: Measured by wet-type gas meter
- Sample frequency: 13 samples/test group
- Sample location: Animal exposure port
- Sample analysis: Gravimetric
- MMAD and GSD derived

Chamber Air Temperature
Chamber air temperature was measured throughout exposure using an electronic thermometer probe. Chamber air temperature was monitored continuously and recorded at 60-minute intervals.

Chamber Relative Humidity
Chamber relative humidity was measured throughout exposure using an electronic hygrometer probe. Chamber relative humidity was monitored continuously and recorded at
60-minute intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Method
Flow diagram of the analytical procedure (Covance method DFA/M089/20) for the analysis
of compound on collection filters and stainless-steel substrates and formulation samples

Sample Assay
1) Test sample (Glass fiber filter or stainless-steel plate).
2)Extract (in water bath at 40°C for 10 mins followed by ultrasonic vibration, 10 mins) using extraction solvent.
3) Further dilute, if necessary, using extraction solvent to provide a solution containing 99422018 at a nominal concentration in the range 25 – 250 µg/mL.
4) Filter (0.20 µm).
5) Inject onto the HPLC in duplicate.

Calibration
1) Dissolve 99422018 (25 mg) in extraction solvent (50 mL) to provide primary standard (500 µg/mL).
2) Dilute the primary standard using extraction solvent to provide calibration standards at nominal
concentrations of 25, 50, 75, 100, 125 and 250 µg/mL.
3) Inject the six standards onto the HPLC for calibration.

HPLC conditions
Analytical column: Agilent Poroshell, 2.7 µm, 100 × 4.6 mm

Column temperature: 60°C

Mobile phase A: Water 100%
Mobile phase B: Ethanol 100%

HPLC Rinse Solvent: Ethanol 100%

Linear gradient:
Time (min) %A %B
0 50 50
10 0 100
15 0 100
15.5 50 50
20 50 50

Flow rate: 0.8 mL/min

Detector: ELSD,
Drift tube temperature: 60ºC
Nitrogen Flow: 1.3 mL/min
Gain: 4

Injection volume: 10 µL

Retention time: 8.0 min (approximately)

The HPLC system was calibrated using external standards. Peak area data acquired by data capture software using a quadratic 2nd order fit was subjected to least regression analysis with 1/X weighting.
Duration of treatment / exposure:
Minimum period 13 weeks followed by a 13 weeks recovery period
Weeks 1 to 12: 6 hours daily exposure, 5 days each week
Week 13: 6 hours daily exposure, 7 days
All surviving main study animals were treated throughout the necropsy period; cessation of treatment for the recovery phase animals started on the first day of the necropsy of animals allocated to the main study. Serial observations were recorded at appropriate intervals.


Training for dosing: The animals on study were acclimated to the method of restraint, over a 3 day period immediately preceding the first test item exposure.

Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
3.95 other: µg/L
Remarks:
Achieved concentration
Dose / conc.:
12.7 other: µg/L
Remarks:
Achieved concentration
Dose / conc.:
39.5 other: µg/L
Remarks:
Achieved concentration
No. of animals per sex per dose:
10 males, 10 females per treatment group for main study

5 males/ 5 females per dose group for Recovery Phase
5 males / 5 females per dose group for Lung Sampling
Control animals:
yes, concurrent vehicle
Details on study design:
Route of Administration
The inhalation route of administration was chosen to simulate the conditions of potential human exposure. Moreover this route of exposure was required by ECHA in the final decision number TPE-D-2114510121-74-01/F.

Rationale for Dose Level Selection
In a previous study (Huntingdon Life Sciences Study Number FIN0058) groups of rats were exposed by snout-only inhalation, for 6 hours per day, 5 days per week for 4 weeks, to atmospheres containing 99422018 at concentrations of 5.30, 34.4 or 328 µg/L. Treatment-related pathological changes were seen in the nasal turbinates, larynx, lungs, and tracheobronchial and mediastinal lymph nodes.

In the nasal turbinates, a concentration-related increase of eosinophilic droplets in the respiratory epithelium was evident at 34.4 and 328 µg/L. In the larynx, concentration-related squamous metaplasia and submucosal inflammation were evident at 34.4 and 328 µg/L. Necrosis of the ventral cartilage of the larynx was also evident at 328 µg/L and was considered to be adverse. In the lungs, peribronchiolar/perivascular macrophages were evident at 34.4 and 328 µg/L and increases in diffuse alveolar macrophages and bronchioloalveolar hyperplasia were evident at 328 µg/L. Alveolar inflammation, consisting of viable and degenerate neutrophils, macrophages and lymphocytes, was also evident in the lungs for males at 328 µg/L and was considered to be adverse. In the tracheobronchial lymph nodes, higher concentrations of 99422018 also resulted in concentration-dependent increases in cellularity and pigmented macrophages. In mediastinal lymph nodes, increased cellularity was evident at 328 µg/L.

On the basis of the ventral cartilage necrosis in the larynx and alveolar inflammation in the lungs, the No Observed Adverse Effect Level for the 4-week study was considered to be 34.4 µg/L.
Considering the likely progression of changes, particularly aggregates of alveolar macrophages in the lung and necrosis in the larynx, concentrations above 35 µg/L were considered unsuitable for use on studies of longer duration.

Pathological changes in the respiratory tract at 34.4 µg/L were generally of lower incidence and/or severity than those seen at 328 µg/L. Therefore for the 13 week study, a high exposure level targeted at 35 µg/L was anticipated to induce treatment related changes similar to those previously seen but was expected to be tolerated for 13 weeks. Target exposure levels of 12 and 3.5 µg/L were selected for the intermediate and low groups respectively to identify a no observed adverse effect level and to explore any possible dose relationship.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
Serial Observations
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization and recovery periods, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded on exposure days at the following times in relation to dose administration:

• Pre-exposure observation
• As each animal is returned to its home cage
• As late as possible in the working day

In addition observations were recorded on non-exposure days at the following times during the day:

• Early in the working day (equivalent to the pre-exposure observation)
• As late as possible in the working day

Clinical Signs
A detailed weekly physical examination was performed on each animal to monitor general health.

Mortality
A viability check was performed near the start and end of each working day. Animals were isolated or killed for reasons of animal welfare where necessary.

A complete necropsy was performed in all cases.

Body Weight
The weight of each animal was recorded twice in the week before treatment commenced, on the day that treatment commenced (Day 1), twice weekly for 4 weeks, weekly thereafter and before necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

Ophthalmic Examination
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope (at the discretion of the examining veterinary surgeon a slit-lamp biomicroscope may also be used) as follows:

Occasion Animals
Pretreatment All main and recovery animals
Week 13 All main and recovery animals of Groups 1 and 4

Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasions:

Occasion Animals
Week 13 All main and recovery animals
Recovery Week 13 All recovery animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:

• Hematocrit (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Absolute reticulocyte count (Retic)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Red cell distribution width (RDW)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)

* Derived values calculated in ClinAxys.

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:

• Prothrombin time (PT) - using IL PT Fibrinogen reagent.
• Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry
Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasions:

Occasion Animals
Week 13 All main and recovery animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche Cobas 6000 Analyzer in respect of:

• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Gamma-glutamyl transpeptidase (gGT)
• Total bilirubin (Bili)
• Urea
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• Triglycerides (Trig)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.
Sacrifice and pathology:
Method of kill:
Overdose of intaperitoneal pentbarbitone sodium followed by exsanguination.

Necropsy
All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

The retained tissues were checked before disposal of the carcass.

Schedule
Main study animals were killed following 13 weeks of treatment.
Recovery animals were killed following 13 weeks of treatment and 13 weeks of recovery.
Sequence To allow satisfactory inter-group comparison.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows:

Abnormalities
Adrenals
Aorta - thoracic
Bone marrow smear
Brain (including sections of cerebrum, cerebellum, and medulla/pons)
Cecum
Colon
Duodenum
Epididymides
Eyes
Femurs (femorotibial joint)
Harderian glands
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Larynx (5 sections, including the base of the epiglottis)
Liver (section from two main lobes)
Lungs (section from all major lobes including bronchi) b)
Lymph nodes - tracheo-bronchial
- left axillary
- hilar
Nasal turbinates (4 levels) c)
Oesophagus
Optic nerves
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands (submandibular/sublingual)
Sciatic nerves
Seminal vesicles
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum
Stomach
Testes
Thymus
Thyroid with parathyroids
Tongue
Trachea (including bifurcation) (at least 3 levels including 1 longitudinal section through the carina and 1 transverse section)
Ureter
Urinary bladder
Uterus with cervix
Vagina

* The right lung was used for BAL sampling and the left lung was processed for histology and light microscopy.
* Under the requirements of OECD 413, sampling of lymph nodes from the hilar region of the lung was required. The hilar lymph node was not visible at macroscopic examination and sampling of this lymph node at histology was only possible when there is an immune response (or poorly soluble particles are inhaled). The hilar lymph node was therefore only sampled if available.

Bone Marrow
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery period(s) and from animals killed prematurely during the study.

Fixation Smears were air dried and subsequently fixed in methanol.
Analysis No examinations were performed, however, the smears were retained for possible future examination.
Retention The smears were transferred to archives and will be retained for the same period as the study raw data.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals.

Bronchoalveolar Lavage (BAL)
The lung sampling schedule was as follows:

Occasion Animals
Main study termination All main study animals
Recovery study termination All recovery study animals

Procedure Following confirmation of death, the lungs (including the trachea) were removed and weighed whole. A tracheal cannula was then inserted into the trachea and secured in place.

The left lung was isolated from the right lung, via a clamp, the organ placed into a petri dish and the right lung lavaged with 3 mL of PBS (phosphate buffered saline).

The PBS was left in the airway for approximately 10 seconds whilst the lung was gently massaged before being removed and placed into a 15 mL centrifuge tube on water-ice (Tube A). This was repeated twice further using fresh 3 mL aliquots of PBS each time. In total, three aliquots of 3 mL of PBS was used to lavage the right lung.

The BAL fluid pooled from the second two lavages were placed into a second tube (Tube B). The BAL Fluid (BALF) samples were kept on water-ice after collection.

Following completion of the BAL procedure the left lung was formalin fixed and processed for histopathology.

Fate of samples The BALF samples were supplied to the department of Clinical Pathology on water ice.

Sample processing
Samples were centrifuged at 800 g for 10 minutes at approximately 4°C within 1 hour of collection of the BALF from the last animal.

The BALF supernatant from Tube A remained in the Department of Clinical Pathology for analysis.

The cell pellets from both Tube A and Tube B were transferred to the Department of Pharmacology on wet ice for analysis.

The supernatant from Tube B was refrigerated (nominally 2ºC to 8ºC) as contingency.

Sample analysis (cells)
A total and differential cell count of the BAL cells was performed using the XT-2000iV (Sysmex UK Ltd). The cell pellets from aliquots A and B were combined and resuspended in 5 mL PBS, vortexed for approximately 5 seconds and analyzed. A total and differential cell count (neutrophils, eosinophils, mononuclear cells (included monocytes and macrophages) and lymphocytes) were reported as number of cells per animal and the differential cell count also as a percentage of the total cell count. All samples were analyzed.
.
Sample analysis The BALF supernatants from Tube A were analyzed on the Cobas 6000 for the following characteristics on the day of sampling:

Lactate dehydrogenase (LDH)
Total protein (Prot)


Lung Bioanalysis and Toxicokinetics
Lung samples were obtained from lung sampling phase animals. The sampling schedule was as follows:

Occasion Animals
Main study termination Lung sampling animals only (Main)
Recovery study termination Lung sampling animals only (Recovery)

Procedure
Following confirmation of death, the lungs of each animal were exposed by excision of the sternum, the trachea and heart were removed, and the lungs freed of any connective tissue. The lungs were blotted dry with clean paper and weighed.

After weighing the lungs were wrapped in aluminium foil and snap frozen in liquid nitrogen.

The remainder of the carcass was discarded without further necropsy.

Temporary storage conditions
Lung samples were stored on solid carbon dioxide until placed into final storage conditions.

Freezer storage conditions Frozen (-60 to -80°C or below).

Fate of lung samples Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Labcorp.

Bioanalysis Performed by the Department of Bioanalysis, Labcorp.

Pharmacokinetics Performed by the Department of Metabolism, Labcorp.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List All animals killed or dying prematurely.

Main study and recovery animals of Groups 1 and 4 killed at a scheduled interval.
Nasal turbinates, larynx, trachea, left lung, tracheobronchial lymph nodes, abnormalities only All main study animals of Groups 2 and 3 killed at a scheduled interval.

Routine staining Sections were stained with hematoxylin and eosin.

Light Microscopy
Tissues preserved for examination were examined as follows:

Category Animals Tissues
Premature deaths All All animals from all groups. All specified previously

Scheduled kill Main study All animals of Groups 1 and 4. All specified previously.

All animals of Groups 2 and 3. Nasal turbinates, larynx,
trachea, left lung,
tracheobronchial lymph nodes,
abnormalities only.
Recovery All animals of Groups 1 and 4. All specified previously

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
Statistics:
Please refer to "Any other information o materials and methods"

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related clinical signs.

Clinical signs associated with the dosing procedure included salivation, wet fur and red staining of the head, nose and/or eyes, evident on return to the home cage, for rats of all groups including control, with the majority of signs resolved by the end of the working day. These signs are considered to be associated with the method of restraint.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality occurred.

Two animals died on Day 1 of dosing. Group 3 recovery female (No. 252) and Group 4 recovery female (No. 259) were both found dead within their restraint tubes during exposure. Both deaths were considered to be related to the method of restraint and not directly related to exposure to the test item. There were no macroscopic pathological findings for either early decedent. Histopathological changes for Animal 252 consisted of autolysis of the eyes and trachea and minimal apoptosis of the thymus cortex. For Animal 259 histopathological changes consisted of slight necrosis of the harderian glands and slight, multifocal mineralization of the kidney tubules.

All other animals survived to their scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on body weight or bodyweight gain after 13 weeks of treatment.

No statistically differences in body weight gain between groups were observed compared with control groups. The small differences observed were within normal intragroup variation and there was no relationship to exposure level.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item-related effects on food consumption after 13 weeks of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on ophthalmoscopy after 13 weeks of treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of exposure, neutrophil counts were higher than control for both sexes exposed to 32.2 µg/L (1.65X and 1.91X control, for males and females respectively, statistically significant). After 13 weeks of recovery, no statistically significant difference in mean neutrophil counts were observed for either sex previously exposed to 32.2 µg/L, when compared with control group. These changes were considered to be test item related, but not adverse due to the observed recovery.

All other differences from control, including those that attained a degree of statistical significance, were minor, lacked exposure relationship or were confined to one sex and were therefore attributed to normal biological variation.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on blood chemistry parameters after 13 weeks of treatment.

All other differences from control, including those that attained a degree of statistical significance, were minor, lacked exposure relationship or were confined to one sex and were therefore attributed to normal biological variation.
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of exposure, absolute and body weight adjusted lung weights were higher than control in males exposed to 32.2 µg/L and in females exposed to 12.7 or 32.2 µg/L. The weight changes were considered test item-related.

Absolute and body weight adjusted testis weights were higher than control across all treated male groups. Absolute and body weight adjusted epididymis weights were higher than control in the high dose male group. The weight changes observed for the testis and epidididymis were considered incidental, and not related to the treatment due to marked testicular degeneration associated with marked sperm decreased observed in one control animal, leading to decreased weight.

After 13 weeks of recovery period, absolute and body weight adjusted lung weights were higher than control in both sexes exposed to 32.2 µg/L. The weight changes were considered test item-related.

* PLEASE REFER TO THE SUMMARY TABLES IN "ANY OTHER INFORMATION ON RESULTS" -
A) Test Item Related Effects in Organ Weight Parameters - Terminal Sacrifice - After 13 weeks of treatment
B) Test Item Related Effects in Organ Weight Parameters - Terminal Sacrifice - After 13 weeks of recovery

All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in body weight adjusted ratios but not both; lack of an exposure concentration relationship or correlative findings; and/or the magnitude was considered small.

* PLEASE ALSO REFER TO THE ATTACHED TWO TABLES:
a) Organ Weights - Group mean absolute and adjusted values for animals sacrificed after 13 weeks of treatment"
b) Organ Weights - Group mean absolute and adjusted values for animals sacrificed after 13 weeks of recovery"
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of exposure, test item-related pale areas within the lungs were observed in all groups, including one control male and one control female, and did not show an exposure concentration-relationship. A test item-related enlargement of the tracheobronchial lymph nodes was observed in both sexes, across all test item female groups and in high and mid-dose test item male groups, with an exposure concentration-relationship.

After 13 weeks of recovery period, pale areas within the lungs were observed in all groups, including two control males and three control females. In control groups this finding inconsistently correlated with aggregates of alveolar macrophages. A test item-related enlargement of the tracheobronchial lymph nodes was observed in both sexes exposed to 32.2 ug/L after the recovery period.

* PLEASE REFER TO THE SUMMARY TABLES IN "ANY OTHER INFORMATION ON RESULTS" -
A) Incidence of Test Item-Related Macroscopic Findings - Terminal Sacrifice - After 13 weeks of treatment
B) Incidence of Test Item-Related Macroscopic Findings - Terminal Sacrifice - After 13 weeks of recovery

All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Han Wistar rats of this age; therefore, they were considered not to be test item related.

PLEASE ALSO REFER TO THE ATTACHED TABLES: -
a) Macro-pathology - Group distribution of findings for animals sacrificed after 13 weeks of treatment
b) Macro-pathology - Group distribution of findings for animals sacrificed after 13 weeks of recovery
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of exposure, test item-related microscopic findings were observed in the lungs of males exposed to 3.95, 12.7 or 32.2 µg/L and females exposed to 12.7 or 32.2 µg/L, tracheobronchial lymph nodes of both sexes exposed to 12.7 or 32.2 µg/L, and nasal turbinates and larynx of males exposed to 12.7 or 32.2 µg/L and females exposed to 32.2 µg/L.

After 13 weeks of recovery, test item -related microscopic findings were still present in the lungs, tracheobronchial lymph nodes and nasal turbinates of both sexes exposed to 32.2 µg/L, with a slight reduction in incidence and severity.

* PLEASE REFER TO THE SUMMARY TABLES IN "ANY OTHER INFORMATION ON RESULTS" -
A) Incidence and Severity of Test Item-Related Microscopic Findings - After 13 weeks of treatment
B) Incidence and Severity of Test Item-Related Microscopic Findings - Recovery sacrifice

The predominant microscopic finding in the lung was a multifocal minimal to moderate increase of perivascular/peribronchiolar foamy alveolar macrophages (correlating with test item-related macroscopic observation of pale areas) that was observed in both sexes exposed to 12.7 or 32.2 µg/L. In both sexes exposed to 32.2 µg/L and in one female exposed to 12.7 µg/L, this finding was occasionally accompanied by a minimal multifocal mixed infiltrate of inflammatory cells (composed of neutrophils and mononuclear cells), multifocal multinucleated (usually binucleated) cells and minimal alveolar eosinophilic granular material (degenerated macrophages). After 13 weeks of recovery, test item -related microscopic findings were still present in the lungs of some animals in both sexes.

Perivascular/peribronchiolar foamy alveolar macrophages were also seen in one control male and two males exposed to 3.95 µg/L, in the treated males it is considered to be incidental and unrelated to treatment based on its distribution and low incidence.

Foamy cell accumulation observed within the tracheobronchial lymph nodes in both sexes exposed to 12.7 or 32.2 µg/L correlated with the test item-related macroscopic observation of enlargement. This change is secondary to the lung findings, reflecting the drainage function of these lymph nodes. After 13 weeks of recovery, test item-related microscopic findings were still present in the tracheobronchial lymph nodes in almost all animals.

Minimal to slight eosinophilic droplets were observed within the respiratory/olfactory epithelium of the nasal turbinates of in males exposed to 12.7 µg/L and both sexes exposed to 32.2 µg/L.

This change was generally focal and confined to the nasal septum. Eosinophilic droplets can represent a response to an irritant material. After 13 weeks of recovery, test item-related microscopic findings were still present in the nasal turbinates of almost all males and one female.

Minimal focal alteration of respiratory epithelium (squamous metaplasia) in the ventral larynx was observed in males exposed to 12.7 or 32.2 µg/L and in females exposed to 32.2 µg/L. This change was not observed after 13 weeks of recovery.

All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Han Wistar rats; therefore, they were considered not to be test item related.

PLEASE ALSO REFER TO THE ATTACHED TABLES: -
a) Histopathology - Group distribution of findings for animals sacrificed after 13 weeks of treatment
b) Histopathology - Group distribution of findings for animals sacrificed after 13 weeks of recovery
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Bronchoalveolar Lavage (BAL):
Cell counts
Compared with controls, statistically significantly higher mean total white blood cell counts were evident for both sexes exposed to 12.7 µg/L (3.3X and 2.4X control, for males and females respectively) or 32.2 µg/L (7.4X and 6.5X control, respectively). Control data showed that the vast majority of cells recovered in bronchoalveolar lavage fluid (BALF) were mononuclear cells (85.41% and 76.13% for males and females respectively). Statistically significant exposure-related changes were evident in higher mean neutrophil % (up to 55.56%) and eosinophil % (up to 7.2%), in both sexes exposed to 12.7 or 32.2 µg/L. Consequently, the proportion of mononuclear cells was reduced (as low as 32.32%) in both sexes exposed to 12.7 or 32.2 µg/L.

After 13 weeks of recovery, when compared with controls, mean total cell counts were up to 3.7 fold higher for males previously exposed to 12.7 or 32.2 µg/L (statistically significant) and 2.2 fold higher for females previously exposed to 32.2 µg/L (not statistically significant). A statistically significant exposure-related change was observed with higher mean neutrophil % (up to 49.62%) in males previously exposed to 12.7 µg/L and both sexes exposed to 32.2 µg/L. Consequently, the proportion of mononuclear cells was statistically significantly reduced (as low as 40.34%) in males previously exposed to 12.7 µg/L and both sexes previously exposed to 32.2 µg/L.

PLEASE REFER TO THE SUMMARY TABLES IN "ANY OTHER INFORMATION ON RESULTS"

Lactate dehydrogenase and total protein
Mean lactate dehydrogenase was statistically significantly higher than control for males exposed to 12.7 or 32.2 µg/L (up to 4.5X) and females exposed to 32.2 µg/L (3.9X). Mean total protein concentration was also statistically significantly higher than control for males exposed to 12.7 or 32.2 µg/L (up to 2.5X) and not statistically significantly higher than control for females exposed to 32.2 µg/L (1.4X).

After 13 weeks of recovery, mean lactate dehydrogenase remained higher than control for both sexes exposed to 32.2 µg/L (up to 3.5X). Mean total protein concentration also remained statistically significantly higher than control for males exposed to 32.2 µg/L (1.7X).

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
3.95 other: µg/L
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
12.7 other: µg/L
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Organ Weights:


 


Test Item-Related Effects in Organ Weight Parameters – Terminal Sacrifice - After 13 weeks of treatment



















































































Sex



99422018



Males



Females



Exposure Level (µg/L)



0



3.95



12.7



32.2



0



3.95



12.7



32.2



Lung



 



 



 



 



 



 



 



 



Absolute Weight (g)



1.383



1.450



1.552



1.808



1.053



1.103



1.106



1.403



% Change from control



-



+5



+12



+31



-



+5



+5



+33



Body Weight Adjusted (%)



1.444



1.422



1.524



1.803**



1.036



1.096



1.118*



1.415**



% Change from control



-



-2



+6



+25



-



+6



+8



+37



* = Statistically significant difference (absolute or body weight adjusted) compared with respective control mean value



 


Test Item-Related Effects in Organ Weight Parameters – Recovery Sacrifice – After 13 Weeks of Recovery



























































 


Sex



99422018



Males



Females



Exposure Level (µg/L)



0



32.2



0



32.2



Lungs and Bronchi



 



 



 



 



Absolute Weight (g)



1.593



1.911



1.118



1.338



% Change from control



-



+20



-



+19



Body Weight Adjusted (%)



1.628



1.871*



1.130



1.297*



% Change from control



-



+15



-



+15



* = Statistically significant difference (absolute or body weight adjusted) compared with respective control mean value.


 


Macropathology


 































































































Incidence of Test Item-Related Macroscopic Findings – Terminal Sacrifice - After 13 weeks of treatment



 



99422018



Sex



Males



Females



Exposure Level (µg/L)



0



3.95



12.7



32.2



0



3.95



12.7



32.2



Lungs and Bronchi



 



 



 



 



 



 



 



 



Number Examined



10



10



10



10



10



10



10



10



  Pale areas



1



3



2



3



1



1



0



2



Lymph node, Tracheobronchial



 



 



 



 



 



 



 



 



Number Examined



10



10



10



10



10



10



10



10



  Enlarged



0



0



3



7



0



1



1



3



 


Incidence of Test Item-Related Macroscopic Findings – Recovery Sacrifice – After 13 Weeks of Recovery







































































Sex



99422018



Males



Females



 



Exposure Level (µg/L)



0



32.2



0



32.2



 



Lungs and Bronchi



 



 



 



 



 



Number Examined



5



5



5



5



 



  Pale areas



2



3



3



2



 



Lymph node, Tracheobronchial



 



 



 



 



 



Number Examined



5



5



5



5



 



  Enlarged



0



3



0



1



 




 


 Histopathology

















































































































































































































































































































































Incidence and Severity of Test Item-Related Microscopic Findings – After 13 weeks of treatment



 



99422018



Sex



Males



Females



Exposure Level (µg/L)



0



3.95



12.7



32.2



0



3.95



12.7



32.2



Lung



 



 



 



 



 



 



 



 



Number Examined



10



10



10



10



10



10



10



10



  Alveolar Macrophages, Foamy, Increased



 



 



 



 



 



 



 



 



Minimal



1



2



9



3



0



0



8



5



Slight



0



0



0



6



0



0



0



4



Moderate



0



0



0



1



0



0



0



1



  Infiltrate, Inflammatory Cells, Perivascular/Peribronchiolar



 



 



 



 



 



 



 



 



Minimal



0



0



0



9



0



0



1



9



Slight



0



0



0



1



0



0



0



0



  Multinucleated, Cells



 



 



 



 



 



 



 



 



Minimal



0



0



0



6



0



0



1



6



  Eosinophilic Granular Material



 



 



 



 



 



 



 



 



Minimal



0



0



0



6



0



0



0



5



Slight



0



0



0



1



0



0



0



0



Lymph Node, Tracheobronchial



 



 



 



 



 



 



 



 



Number Examined



9



10



10



10



10



10



10



10



  Macrophages, Foamy, Sinuses



 



 



 



 



 



 



 



 



Minimal



0



0



3



7



0



0



7



9



Slight



0



0



0



2



0



0



0



0



Nose/Turbinates



 



 



 



 



 



 



 



 



Number Examined



10



10



10



10



10



10



10



10



  Eosinophilic Droplets, Respiratory/Olfactory Epithelium



 



 



 



 



 



 



 



 



Minimal



0



0



3



8



0



0



0



3



Slight



0



0



0



1



0



0



0



0



Larynx



 



 



 



 



 



 



 



 



Number Examined



10



10



10



10



10



10



9



10



  Alteration, Respiratory Epithelium



 



 



 



 



 



 



 



 



Minimal



0



0



1



6



0



0



0



4



 


Incidence and Severity of Test Item-Related Microscopic Findings – Recovery Sacrifice















































































































































































































Sex



99422018



Males



Females



 



Exposure Level (µg/L)



0



32.2



0



32.2



 



Lung



 



 



 



 



 



Number Examined



5



5



5



5



 



  Alveolar Macrophages, Foamy, Increased



 



 



 



 



 



Minimal



0



1



0



1



 



Slight



0



4



0



2



 



  Infiltrate, Inflammatory Cells, Perivascular/Peribronchiolar



 



 



 



 



 



Minimal



0



2



0



2



 



Slight



0



1



0



0



 



  Multinucleated, Cells



 



 



 



 



 



Minimal



0



4



0



3



 



  Eosinophilic Granular Material



 



 



 



 



 



Minimal



0



2



0



1



 



Slight



0



0



0



1



 



Lymph Node, Tracheobronchial



 



 



 



 



 



Number Examined



5



5



5



4



 



  Macrophages, Foamy, Sinuses



 



 



 



 



 



Minimal



0



2



0



2



 



Slight



0



2



0



2



 



Moderate



0



1



0



0



 



Nose/Turbinates



 



 



 



 



 



Number Examined



5



5



5



5



 



  Eosinophilic Droplets, Respiratory/Olfactory Epithelium



 



 



 



 



 



Minimal



0



4



0



1



 



 


Bronchoalveolar Lavage (BAL)


Cell counts


After 13 Weeks treatment













































































Sex



Males



Females



Dose (µg/L)



0



3.95



12.7



32.2



0



3.95



12.7



32.2



Total white blood cells mean (106/animal)



2.57



3.16



8.59***



19.00***



2.21



2.56



5.24***



14.41***



Neutrophils
(% of WBC)



9.87



6.09



29.10**



53.46***



14.70



8.40



32.87***



55.56***



Lymphocytes
(% of WBC)



4.40



3.57



5.63



4.52



7.31



5.63



6.66



5.16



Mononuclear cells
(% of WBC)



85.41



89.75



62.31***



34.52***



76.13



85.21



56.22***



32.32***



Eosinophils (% of WBC)



0.32



0.36



2.96***



7.20***



1.49



0.45



3.70**



6.52***



After 13 weeks recovery













































































Sex



Males



Females



Dose (µg/L)



0



3.95



12.7



32.2



0



3.95



12.7



32.2



Total white blood cells mean (106/animal)



2.38



2.24



4.45**



8.82***



3.36



2.11



2.34



7.24



Neutrophils
(% of WBC)



6.90



15.38



21.86**



49.62***



23.28



8.28



17.93



41.90*



Lymphocytes
(% of WBC)



5.60



5.62



4.82



6.62



5.86



4.76



7.50



9.65



Mononuclear cells (% of WBC)



85.24



76.56



70.82*



40.34***



68.16



86.20



74.18



44.45**



Eosinophils
(% of WBC)



1.94



2.10



2.34



3.24



1.80



0.76



0.40



2.80



 


BIO-ANALYSIS RESULTS


(Please alse refer to the attached Figures and Tables of results from the Bio-Analysis controibuting Report)


Calibration (Linearity)


The response of the LC-MS/MS system to matrix matched calibration standard solutions of analyte 99422018 was linear over the range 25.0 to 1500 ng/g in rat lung and extended calibration range of 2500 to 150000 ng/g in rat lung.


A summary of the batches analyzed was shown in Table 1. Typical calibration standard data was presented in Table 2. Typical calibration standard curve, chromatograms of blank extracts and calibration standards were presented in Figure 1 to Figure 4.


Method validation


The analytical methods were concurrently validated in Labcorp Early Development Laboratories Ltd. Study Number 8461502. The method BM/2021/1092 was validated over the calibration range of 25.0 to 1500 ng/g in rat lung. While the method BM/2021/1119 was validated over the calibration range of 2500 to 150000 ng/g in rat lung to obtain the extended calibration range.


Limit of Quantitation (LOQ)


The LOQ of the method was defined as the lowest fortification level at which acceptable recovery data was obtained. The previous validation of the methodology (BM/2021/1119) demonstrated that analyte 99422018 can be accurately determined at a LOQ of 10000 ng/g in rat lung.


 


Limit of Detection (LOD)


The LOD of the method was defined as the concentration of the lowest calibration standard analyzed, that gave rise to a measurable chromatographic response. For this study the LOD of the method was shown to be 2500 ng/g in rat lung.


 


Fortified recovery samples


Samples were analyzed in a suitably sized batch along with recovery samples fortified with analyte 99422018, which acted as procedural recovery samples.


 


Mean procedural recoveries for analyte 99422018 were within the acceptable range of 70 to 120% (Table 3), showing the methodology to be working within its requirements on each occasion of sample analysis. 


 


A typical chromatogram of an extract of a fortified recovery sample was presented in Figure 5.


Study samples


The rat lung concentrations of analyte 99422018 following daily inhalation administration of analyte 99422018 was shown in Table 4 to Table 7. Typical chromatograms of extracts of study samples were presented in Figure 6.


 


In analytical run 1, there was a change in analyte 99422018 response during the course of the run. Therefore only the first replicate of the calibration line was accepted. The first two recovery samples were acceptable, although the third, later in the run had a low recovery value due to the change in response in this run. All analyte 99422018 dosed animals in this run had results which were ALQ. Therefore, the only data used on this run were the control group where all results were BLQ. Therefore the run was considered acceptable for the control group samples as the calibration line and overall recovery met the acceptance criteria.      


For regulatory purposes, the values in the tables are final data.


Repeat Analysis


Study samples above the limit of quantification (ALQ) in analytical run 1 were diluted 50-fold with post-extracted dilution and analyzed successfully in analytical run 5. However, nine samples (Group-2, samples 66M-70M and Group-3 samples 91M-94M) resulted in “BLQ”. Additionally, sample 95M from Group-3 showed an anomalous value upon analysis in analytical run 5 and resulted in “BLQ”.  


Hence, the study samples 66M-70M and 91M-94M were re-extracted and diluted 10-fold with matrix blank extract and the batch was injected successfully in an analytical run 6. Sample 95M from Group-3 was re-extracted and analyzed successfully in duplicate in analytical run 6. The repeat duplicate analysis confirmed the original result and reported as median of three results in the report.


Deviations from Method


There was a deviation from the method.


In analytical run 5 and 6 incorrect SST (System suitability test) solution was prepared in error and analysed by the analyst. The bioanalytical method BM/2021/1119 Section 6.4 states that SST solution should be prepared using 10 µL of working solution-1 and diluted with 240 µL of Water:Methanol:Acetic acid (30:70:0.2, v:v:v) to obtain 20.0 ng/mL solution concentration. However, in analytical run 5 and 6 SST solutions were prepared using Calibration-1 (5.00 ng/mL).


Although this is a method deviation but the lower concentration (5.00 ng/mL) was used for SST which eventually gives better assessment of the method that the system performs well even at lower concentration.


This deviation was considered to have not affected the integrity or validity of the study.


 

Applicant's summary and conclusion

Conclusions:
The test article, 99422018, was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 13 weeks at achieved aerosol concentrations of 3.95, 12.7 or 32.2 µg/L.
After 13 weeks of exposure, test item-related findings in the lungs consisted of a multifocal minimal to moderate increase of perivascular/peribronchiolar foamy alveolar macrophages for both sexes exposed to 12.7 or 32.2 µg/L. This was occasionally accompanied by a minimal multifocal mixed infiltrate. After 13 weeks of recovery, these findings were still present in the lungs of some animals of both sexes previously exposed to 32.2 µg/L. These findings were considered to be adverse. The results from the bronchoalveolar lavage fluid and haematology correlated with the histopathological findings and were suggestive of lung injury consistent with the inhalation of poorly soluble particulate matter.
Other test item-related microscopic findings consisted of foamy cell accumulation in the tracheobronchial lymph nodes of both sexes exposed to 12.7 or 32.2 µg/L, minimal to slight eosinophilic droplets in the respiratory/olfactory epithelium of the nasal turbinates of both sexes exposed to 32.2 µg/L and minimal focal alteration of respiratory epithelium in the ventral larynx in males exposed to 12.7 µg/L and both sexes exposed to 32.2 µg/L. After 13 weeks of recovery, the findings in the tracheobronchial lymph nodes and nasal turbinates were still present, but those in the larynx were not. These were considered to be secondary or adaptive changes and to be non-adverse.
On the basis of these findings, the No Observed Adverse Effect Level (NOAEL) for this study was considered to be 3.95 µg/L.

Executive summary:

The purpose of this study was the assessment of systemic and local toxic potential of 99422018 in a 13 week inhalation study in Han Wistar rats. Recovery from any effects were evaluated during a 13 week recovery period.


The study design was as follows:





































































Group



Treatment



Target exposure level (µg/L)



Number of animals



Main study



Recovery phase



Lung sampling



Male



Female



Male



Female



Main Male



Recovery Male



1



Air Control



0



10



10



5



5



5



5



2



99422018



3.5



10



10



5



5



5



5



3



99422018



12



10



10



5



5



5



5



4



99422018



35



10



10



5



5



5



5



 


Animals received the control (air) or the test item (99422018) by inhalation for 13 weeks. Recovery animals were similarly treated for 13 weeks followed by a 13 week off dose period.


During the study, lung bioanalysis and toxicokinetics, clinical condition, body weight, food consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, bronchoalveolar lavage (BAL), organ weight, macropathology and histopathology investigations were undertaken.


 


Results


Summary data are presented below:










































 



Concentration (mg/L)



Particle size



Group



Target



Achieved



MMAD (mm)



GSD



2



3.5



3.95



3.2



2.40



3



12



12.7



3.2



2.41



4



35



32.2



3.3



2.33



GSD = Geometric standard deviation; MMAD = Mass median aerodynamic diameter.



 


The mean achieved aerosol concentrations were 113, 106 and 92% of target for Groups 2, 3 and 4, respectively.  The MMAD values indicate the generated aerosols were respirable to the rat.


 


No test item-related mortality occurred.  Two recovery phase females, one from Group 3 (No.252) and one from Group 4 (No.259) were found dead, within their restraint tubes, during exposure, on Day 1 of dosing. Both deaths were considered to be related to the method of restraint and not directly related to exposure to the test item. Histopathological examination revealed no microscopic findings associated with the test item for these decedents.


 


There were no test article-related effects on clinical signs, body weight, food consumption or blood chemistry.


 


After 13 weeks of exposure, neutrophil counts were higher than control for both sexes exposed to 32.2 µg/L.  After 13 weeks of recovery, group mean neutrophil counts were broadly similar to control, for both sexes previously exposed to 32.2 µg/L.


 


At the end of the treatment period, analysis of bronchoalveolar lavage revealed higher mean total cell counts for both sexes exposed to 12.7 or 32.2 µg/L.  Control data showed that the vast majority of cells recovered in bronchoalveolar lavage fluid (BALF) were mononuclear cells.  An exposure-related trend was evident in higher mean neutrophil % and eosinophil % in both sexes exposed to 12.7 or 32.2 µg/L, and consequently, the proportion of mononuclear cells was reduced in these groups.  Higher mean lactate dehydrogenase concentrations were evident in the bronchoalveolar lavage supernatant fluid for both sexes exposed to 12.7 or 32.2 µg/L, with an exposure related-trend evident.  Mean total protein concentration was also higher than control for males exposed to 32.2 µg/L.  After 13 weeks of recovery, comparable changes were still evident for animals previously exposed to 12.7 or 32.2 µg/L.


 


After 13 weeks of exposure, lung weight parameters were higher than control for both sexes exposed to 12.7 or 32.2 µg/L. The weight changes were considered test item-related. After 13 weeks of recovery, lung weights parameters were still higher than control for both sexes exposed to 32.2 µg/L.


 


After 13 weeks of exposure, pale areas within the lungs were observed in all groups, including one control male and one control female, and did not show a dose-relationship. Enlargement of the tracheobronchial lymph nodes was observed in both sexes, across all test item female groups and in high and mid-dose test item male groups, with an exposure concentration-relationship. After 13 weeks of recovery period, pale areas within the lungs were observed in all groups, including two control males and three control females. In control groups this finding inconsistently correlated with aggregates of alveolar macrophages. Enlargement of the tracheobronchial lymph nodes was observed in both sexes exposed to 32.2 ug/L.


 


After 13 weeks of exposure, test item-related microscopic findings were observed in the lungs of males exposed to 3.95 and both sexes exposed to 12.7 or 32.2 µg/L, in tracheobronchial lymph nodes of both sexes exposed to 12.7 or 32.2 µg/L, and in nasal turbinates and larynx of males exposed to 12.7 or 32.2 µg/L and females exposed to 32.2 µg/L.


 


Findings in the lungs included increased multifocal foamy macrophages (correlated with macroscopic observations of pale areas), infiltrate of inflammatory cells, multinucleated cells and alveolar eosinophilic material.


 


Findings in the tracheobronchial lymph nodes included intrasinusoidal foamy macrophages and correlated with macroscopic observations of enlargement. Minimal to slight eosinophilic droplets were observed within the respiratory/olfactory epithelium of the nasal turbinates of the males exposed to 12.7 or 32.2 µg/L and females exposed to 32.2 µg/L. Also, alteration of respiratory epithelium was observed in males exposed to 12.7 or 32.2 µg/L and females exposed to 32.2 µg/L.


After 13 weeks of recovery, test item-related microscopic findings were still observed in the lungs, tracheobronchial lymph nodes and nasal turbinates of both sexes exposed to 32.2 µg/L, with a slight reduction in incidence and severity, and correlated with macroscopic findings of pale areas (lungs) and enlargement (tracheobronchial lymph nodes).


 


Conclusion


The test article, 99422018, was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 13 weeks at achieved aerosol concentrations of 3.95, 12.7 or 32.2 µg/L. 


 


After 13 weeks of exposure, test item-related findings in the lungs consisted of a multifocal minimal to moderate increase of perivascular/peribronchiolar foamy alveolar macrophages for both sexes exposed to 12.7 or 32.2 µg/L.  This was occasionally accompanied by a minimal multifocal mixed infiltrate. After 13 weeks of recovery, these findings were still present in the lungs of some animals of both sexes previously exposed to 32.2 µg/L, with a slight reduction in incidence and severity. These findings were considered to be adverse. The results from the bronchoalveolar lavage fluid and hematology correlated with the histopathological findings and were suggestive of lung injury consistent with the inhalation of poorly soluble particulate matter.


 


Other test item-related microscopic findings consisted of foamy cell accumulation in the tracheobronchial lymph nodes of both sexes exposed to 12.7 or 32.2 µg/L, minimal to slight eosinophilic droplets in the respiratory/olfactory epithelium of the nasal turbinates of both sexes exposed to 32.2 µg/L and minimal focal alteration of respiratory epithelium in the ventral larynx in males exposed to 12.7 µg/L and both sexes exposed to 32.2 µg/L.  After 13 weeks of recovery, the findings in the tracheobronchial lymph nodes and nasal turbinates were still present, but those in the larynx were not.  These were considered to be secondary or adaptive changes and to be non-adverse.


 


On the basis of the adverse findings, the No Observed Adverse Effect Level (NOAEL) for this study was considered to be 3.95 µg/L.