Octadecanoic acid, 12-hydroxy-, reaction products with decanoic acid and ethylenediamine

Repeated dose toxicity: inhalation

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 guideline

Test guideline 1

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.

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Reaction mass of Octadecanamide, 12-hydroxy-N-[2-[(1-oxodecyl)amino]ethyl]- and N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide) and Decanamide, N,N'-1,2-ethanediylbis-

EC Number:

907-495-0

Cas Number:

198028-14-7

Molecular formula:

C90H180N6O9

IUPAC Name:

Reaction mass of Octadecanamide, 12-hydroxy-N-[2-[(1-oxodecyl)amino]ethyl]- and N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide) and Decanamide, N,N'-1,2-ethanediylbis-

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 / concentrations

Doses / concentrations 1

Dose / conc.:

3.95 other: µg/L

Remarks:

Achieved concentration

Doses / concentrations 2

Dose / conc.:

12.7 other: µg/L

Remarks:

Achieved concentration

Doses / concentrations 3

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 12.7 or 32.2 µg/L and in females exposed to 3.95, 12.7 or 32.2 µg/L, reaching statistical significance for males exposed to 32.2 µg/L (only for adjusted) and females exposed to 12.7 or 32.2 µg/L (only for adjusted). The weight changes were considered test item-related.
After 13 weeks of exposure, 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. These intergroup differences had no histological correlation and were considered unrelated to treatment.
After 13 weeks of recovery absolute and body weight adjusted lung weights were higher than control in both sexes previously exposed to 32.2 µg/L, reaching statistical significance for both sexes previously exposed to 32.3.µg/L (only for adjusted). The weight changes were considered test item-related. Partial recovery in the absolute and body weight adjusted lung weight was observed.
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.

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 treated groups, did not show an exposure concentration-relationship and was inconsistently correlated with histopathological findings of increased foamy alveolar macrophages. A test item-related enlargement of the tracheobronchial lymph nodes was observed in females exposed to 3.95 µg/L and both sexes exposed to 12.7 µg/L or 32.2 µg/L, with an exposure concentration-relationship.
After 13 weeks of recovery, pale areas within the lungs were observed only in groups previously exposed to 12.7 µg/L (males) or 32.2 µg/L (both sexes), correlated with increased of foamy alveolar macrophages and did not show reversibility. The same finding was observed in two control males and three control females and inconsistently correlated with aggregates of alveolar macrophages. A test item-related enlargement of the tracheobronchial lymph nodes was observed in both sexes previously exposed to 32.2 µg /L.
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.

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 of males previously 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 males previously exposed to 12.7 or 32.2 µg/L and females exposed to 3.95, 12.7 or 32.2 µg/L and nasal turbinates of males previously exposed to 3.95 or 32.2 µg/L and females exposed to 12.7 or 32.2 µg/L. No effect on the larynx was observed after 13 weeks of recovery.
The predominant microscopic finding in the lungs was a multifocal minimal to moderate increase of foamy alveolar macrophages, that was observed in the males exposed to 3.95, 12.7 or 32.2 µg/L and females exposed to 12.7 or 32.2 µg/L. This finding was associated in both sexes exposed to 32.2 µg/L with minimal alveolar eosinophilic granular material (degenerate macrophages). In males exposed to 32.2 µg/L and female exposed to 12.7 µg/L and 32.2 µg/L, this finding was accompanied by a minimal multifocal mixed infiltrate of inflammatory cells (composed of neutrophils and mononuclear cells) and minimal multifocal multinucleated (usually binucleated) cells. These findings, taken together, are considered to be adverse. Two males exposed to 3.95 µg/L showed minimal increased foamy alveolar macrophages without any other histological changes, and this is considered to be non adverse.
After 13 weeks of recovery, a minimal to slight increase of foamy alveolar macrophages were present in the lungs of males previously exposed to 3.95, 12.7 or 32.2 µg/L and females previously exposed to 12.7 or 32.2 µg/L. Perivascular/peribronchiolar infiltrate of inflammatory cells, multinucleated cells and eosinophilic granular material were seen in both sexes previously exposed to 32.2 µg/L. The reduction in severity represents a partial recovery.
Minimal to slight foamy macrophages accumulation, observed within the tracheobronchial lymph nodes in both sexes exposed to 12.7 or 32.2 µg/L, generally 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 of both sexes previously exposed to 12.7 and 32.2 µg/L and one female previously exposed to 3.95 µg/L. The degree of incidence and severity was similar with main phase, indicating no clear recovery over this period.
Minimal to slight eosinophilic droplets were observed within the respiratory epithelium of the nasal turbinates in males exposed to 12.7 µg/L and both sexes exposed to 32.2 µg/L. 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 2/5 males previously exposed to 3.95 µg/L and 4/5 males previously exposed to 32.2 µg/L. In females, this finding was observed in 1/4 and 1/4 animals previously exposed to 12.7 or 32.2 µg/L, respectively. This finding is considered to be adaptative and non-adverse. The degree of incidence and severity was slightly lower than main phase suggesting partial recovery.
Minimal focal alteration of respiratory epithelium 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. This change can also represent a low-grade response to minimal irritation and is considered adaptative.
All other microscopic findings (including minimal mineralization of tubules in the kidney observed more in females) 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.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung Bioanalysis
After 13 weeks of exposure, the test item was present in lung samples taken from all animals exposed to the test item, with lung sample concentrations increasing with increasing aerosol concentration.
After 13 weeks of recovery, the test item was still present in lung samples taken from all animals previously exposed to the test item, again with a concentration relationship, However the mean concentrations were approximately 6%, 30% and 22% of the equivalent main study values for Groups 2, 3 and 4 respectively. This indicates ongoing but incomplete clearance of the test item after 13 weeks off-exposure.


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.

Lactate dehydrogenase and total protein
Mean lactate dehydrogenase was higher than control for both sexes exposed to 12.7 or 32.2 µg/L (up to 4.5X and 3.9X control for males and females respectively). With exception of the females exposed to 12.7 µg/L, these changes were statistically significantly and an exposure related-trend was evident. Mean total protein concentration was also higher than control for males exposed to 12.7 or 32.2 µg/L (up to 2.5X control, statistically significant) and higher than control for females exposed to 32.2 µg/L (1.4X, not statistically significant).
After 13 weeks of recovery, mean lactate dehydrogenase remained statistically significantly higher than control for both sexes exposed to 32.2 µg/L (3.5X and 2.2X control for males and females respectively). Mean total protein concentration also remained statistically significantly higher than control for males exposed to 32.2 µg/L (1.7X control).

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

32.2 other: µg/L

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

ophthalmological examination

2

Key result

Dose descriptor:

NOAEC

Remarks:

local effects

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 (10^6/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 (10^6/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.

 

Group	Achieved Concentration (mg/L)	Mean Concentration (ng/g)
		Main Study	Recovery Phase
1	0	BLQ	BLQ
2	3.95	310000	17900
3	12.7	1332000	401000
4	32.2	2950000	653600

BLQ – Below limit of quantification <100 ng/g.

 

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity is considered to be 32.2 µg/L, based on the lack of systemic effect. The No Observed Adverse Effect Concentration for local effects is considered to be 3.95 µg/L, based on the histopathological findings seen in the lungs.

Executive summary:

The test item 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 in males exposed to 3.95 and both sexes exposed to 12.7 or 32.2 µg/L. This was occasionally accompanied by a minimal multifocal mixed infiltrate of inflammatory cells, minimal multifocal multinucleated cells and minimal alveolar eosinophilic granular material. These findings, taken together, are considered to be adverse at 12.7 or 32.2 µg/L. Males exposed to 3.95 µg/L showed minimal increased foamy alveolar macrophages without any other histological changes, and this is considered to be non-adverse. After 13 weeks of recovery, only perivascular/peribronchiolar foamy alveolar macrophages were still present in the lungs of males previously given ≥3.95 µg/L and females previously given ≥12.7 µg/L, representing a partial recovery. 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. Lung Bioanalysis showed the test item was present in all exposed animals with levels increasing with aerosol concentration. After 13 weeks of recovery, the test item was still present in the lungs of previously exposed to the test item, however the levels were greatly reduced, indicating ongoing, but incomplete, clearance.
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, with a slight reduction in incidence and severity, indicating partial and ongoing recovery. No effect on the larynx was observed after 13 weeks of recovery. These were considered to be secondary or adaptive changes and to be non-adverse.
The No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity is considered to be 32.2 µg/L, based on the lack of systemic effect. The No Observed Adverse Effect Concentration for local effects is considered to be 3.95 µg/L, based on the histopathological findings seen in the lungs.