Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro-

Endpoint summary

Description of key information

In a 28-day repeated dose oral toxicity study (acc. to OECD 421), the substance did not induce any toxicity up to the dose of 1000 mg/kg/day.

 

In a non-guideline supportive study, male rats were exposed in a nose-only set-up to dusty material at a single dose of 30 mg/m3 air for five days/6h per day with the substance and with the substance's nanoform. Examinations were performed both after exposure and at the end of a 21 -day treatment free recovery period. Parameters examined adressed systemic effects (clinical chemistry, haematology, organ weights) and local effects on the lung (broncheoalveolar lavage, histopathology) and were obtained from groups of either three or five animals. Directly after exposure and after 3 weeks recovery, no indication of systemic toxicity were observed. Histopathological findings were present and are regarded as treatment-related, but since no tissue injury was present in the examined respiratory organs, all of them are considered to be adaptive and not adverse.

In a GLP-compliant 90-days inhalation toxicity study according to OECD TG 413, rats were exposed by nose-only to 2, 8 and 40 mg/m3 (nominal). The study included a satellite group and two recovery groups (for 4 and 13 weeks) to measure lung burden and analyse the BALF. No systemic effects were observed and adverse local changes in the respiratory tract were limited to the Mid and High Concentration groups treated with test item. Based on these results, the No Observed Adverse Effect Concentration (NOAEC) for the study was 2.1 mg/m3 (Low Concentration group).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 12, 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

September 19, 1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM Kleintierfarm Madoerin AG, Fuellinsdorf/Switzerland
- Age at study initiation: 7-9 meeks
- Weight at study initiation: males: 147 - 173 g, females: 157 - 181 g
- Fasting period before study:
- Housing: Individually
- Diet (e.g. ad libitum): Pelleted standard Kliba no. 343 Batch 44/86 rat maintenance diet ('Kliba', Klingentalmuehle AG, Kaiseraugst, Switzerland), at
libitum.
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was weighed into a glass beaker on a tared Mettler PK 300 balance and the vehicle, polyethylene glycole (PEG 400), was added. The mixture was prepared daily prior to administration using a homogenizer and kept stable during application with a magnetic stirrer.
Concentrations, homogeneity, and stability were checked by RCC analytical laboratories for all preparations.

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg body weight

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days per week

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based upon the results in acute toxicity studies, which did not indicate any toxicity at limit test doses.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality were recorded twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Signs of toxicity were assessed twice daily. A description of any abnormalities were recorded and the subsequent progress was monitored.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded weekly during the acclimation and treatment period using an on-line
electronic recording system.

FOOD CONSUMPTION: Yes
- The food consumption was recorded once during the acclimation period and weekly thereafter using an online electronic recording system.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed at termination of treatment.
Ten minutes after the application of a mydriatic solution (Dispersa AG, Winterthur / Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimed light using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet. AG, Allschwil / Switzerland)
- Dose groups that were examined: Ophthalmoscopic examinations were performed on all animals.

HAEMATOLOGY: Yes, from the retro-orbital plexus
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes, under light ether anesthesia
- Animals fasted: Yes, the animals were fasted for 18 hours before blood sampling but water was provided.
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- Animals fasted: Yes, the animals were fasted for 18 hours before blood sampling but water was provided.
- How many animals: all animals
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks, over an 18-hour period into a specimen
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals were necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed
by experienced prosectors supervised by a pathologist. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination after 4 weeks.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution.
Adrenal glands, Heart, Kidneys, Liver, Spleen, Gross lesions.
Where there was a compound related alteration in an organ, this organ was histologically examined in all animals of all dose groups.

Organ weights: The following organ weights were taken from all animals necropsied at termination of treatment: Adrenal glands, Kidneys,
Liver, Testes.

HISTOPATHOLOGY: Yes
Slides of all tissues collected at terminal sacrifice from the animals of the control and high-dose groups as well as all animals which died spontaneously were examined by a pathologist. Treatment-related morphologic changes in the organs of any highdose animal required histological evaluation of the same organs in lower dose groups until a no-effect-level was determined. All abnormalities were described and included in the report.

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology were processed, embedded and cut at a thickness of 2-4 micrometers and stained with hematoxylin and eosin. Special stains were used at the discretion of the pathologist.

Other examinations:

No other examinations were performed.

Statistics:

The following statistical methods were used to analyze the body weights, food consumption and organ weights:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
- Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
- Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Starting with day 21 until termination of treatment all rats of the high dose group (1000 mg/kg bw) showed red discolored extremities. In addition red
discolored feces were observed in the same animals betmeen day 18 and termination of the test. No other symptoms related to test article treatment were observed.

Treatment-unrelated signs and symptoms:
Prior to death, one female animal of the group with 300 mg/kg showed moderate sedation (days 17-19), rales (days 18-19) and moderate ruffled fur (days 17-19).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Due to a possible intubation error, two female animals, one of the control group and one of the group 300 mg/kg died spontaneously at day 17 respectively 20 of test.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

The assessment of hematological data indicated no changes of toxicological significance at termination of the treatment.

Treatment-unrelated: All statistical differences in the results of the hematology parameters were considered to be incidental and of normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The assessment of biochemical data indicated no changes of toxicological significance at termination of the treatment.

Treatment-unrelated: All statistical differences in the results of the clinical biochemistryparameters were considered to be incidental and of normal biological variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The assessment of urinalysis data indicated no changes of toxicological significance at termination of the treatment.

Treatment-unrelated: All statistical differences in the results of the urinalysis parameters were considered to be incidental and of normal biological variation.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

HISTOPATHOLOGY:
- No treatment related microscopic findings were recorded. From the tissues examined no cause of death could be established for the two rats which died. The various spontaneous microscopic findings recorded are within the normal range observed in this age and strain of rat. They can be attributed to subclinical infections, spontaneous congenital abnormalities or physiological status.
The nephroblastoma which mas recorded in one male of the 300 mg/kg body weight group is a seldom encountered malignant kidney tumor which occurs spontaneously with a Iow incidence in many rat strains and is not related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

System:

other: none

Conclusions:

Based upon the results obtained in the study, the NOAEL for the 28-day repeated dose oral toxicity test of the substance was considered to be higher than 1000 mg/kg/day for male and female rats when administered orally by gavage.

Executive summary:

In a study according to OECD Test Guideline 407 under GLP conditions, Wistar rats were treated with three doses of the substance (100, 300 and 1000 mg/kg bw). The dose were applied by oral (gavage) daily for four weeks. Besides some clinical findings caused by the colour of the substance, no treatment-related effects were observed. Therefore, the NOAEL was considered to be higher than 1000 mg/kg/day for male and female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The quality of the whole database is determined by the only avaliable study, which is reliable and of good quality.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 2020 (study plan) to ongoing (drafting of report)
In-life phase: 14 May 2020 to 11 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

Several minor deviations, e.g. temperature relative humidity measurements outside the pre-specified ranges, were reported, but all without any effect on the study and its outcomes.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: CINIC, Lot D23508919P1
- Purity, including information on contaminants, isomers, etc.: 99.6% purity

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from humidity (tight closed container)
- expiry date: April 15, 2029
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: stable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: stable
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: none

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar Han Rat Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, SandhoferWeg 7, D-97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation:
Males: 240 – 283 g
Females: 165 – 196 g
- Fasting period before study: no
- Housing: Group caging (up to 3 animals of the same sex and dose group/cage) with additional enrichment; complying with AAALAC standards, additional enrichment (GLP Mini Fun Tunnel for hiding and chewing) was used
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Acclimation period in the study was at least 14 or 15 days. Animals were also trained to the test apparatus (restrain procedures) for 4 days and up to 6 hours prior to start of the testing in order to reduce the stress during exposure.

DETAILS OF FOOD AND WATER QUALITY:
Food: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" (Batch number: 820 61892 / 207 63596 /560 65984 / 713 70882, Expiry date: August 2020 / September 2020 / October 2020 / April 2021) produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum.
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate for the batch used. A copy of the certificate is retained in the archives of Charles River Laboratories Hungary Kft.
Water: Animals received tap water from the municipal supply, as for human consumption from 500 mL bottle ad libitum.
Water quality control analysis was performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila utca 36., Hungary). The quality control results are retained in the archives of Charles River Laboratories Hungary Kft.
The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1-24.9℃ (target range: 19-25℃)
- Humidity (%): 27-80% (target range: 30-70%)
- Air changes (per hr): at least 15 air exchanges per hour.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 May 2020 To: 11 November 2020

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 1.53 - <= 1.77 µm

Geometric standard deviation (GSD):

2.8

Remarks on MMAD:

MMAD differed slightly for the three test concentration due to the particle properties:
- 0.002 mg/L (low dose): 1.77 (GSD: 2.60)
- 0.008 mg/L (mid dose): 1.53 (GSD: 2.97)
- 0.040 mg/L (high dose): 1.71 (GSD: 2.65)

The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. Samples were collected weekly at each concentration tested and measured gravimetrically if feasible. The sampling rate was 6.0 litre/min. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data was used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and the percentages <2 µm and <4 µm (considered to be inhalable in the rat) using the corresponding software tool (Version 1.2, TSE Systems GmbH, Bad Homburg, Germany). The target ranges were < 2µm for the MMAD and 1.5 to 3 for the GSD.

Details on inhalation exposure:

Technical trials
Before animals were exposed, test item atmospheres were generated within the exposure chamber . During this period, airflow settings, test item input were varied to achieve the required atmospheric concentrations and constancy.
For the test atmosphere generation, rotating brush aerosol generators (RBG1000, Palas GmbH, Karlsruhe, Germany) were used connected to pressurized air supply. In the Low Dose group double separation glass with 2 collision metals was used, while in the Mid and High Dose groups single separator glass was used. Verification of the target concentration, particle size and particle distribution were measured gravimetrically. Technical trials included gravimetric filter analysis.

Inhalation Exposure
The atmosphere was generated according to the system and flow rates determined during the technic al trials. The exposure each day was not started until theoretical chamber concentration equilibration has been reached. During the exposure period, changes were made to airflow and test material input rates in order to achieve the required concentration.

Animal Exposure Conditions
The animals were treated by the inhalation route using a nose only exposure unit, in a TSE Rodent Exposure System with each individual concentration and control group in a dedicated tower. Modular multilevel flow – past, nose only exposure units (towers) was used. The exposure unit consisted of two, concentric stainless steel cylinders, the inner plenum and the outer chamber with 10 circularly arranged exposure ports.
The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/ relative humidity, O2 and CO2 sensors.
These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany.
The exposure units were placed in closed hoods in order to avoid cross-contamination and contam ination of the laboratory environment.
The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
Atmosphere generation was dynamic. Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where the aerosol was distributed to the individual exposure ports. After passing through the animal’s breathing zone, spent aerosol enters the outer cylinder from where it was exhausted through a suitable filter system.
Airflows and relative pressures within the system were constantly monitored and controlled by the co mputer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposu re port (breathing zone). The flow of air through each port was in the range of 1.0 L/min and 1.5 L/min.
This flow rate was considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was determined during the technical trials. However, to avoid any possible differences, the position of the animals was rotated on an exposure session basis.

Actual Test Atmosphere Concentrations
The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere (60 L/f ilter for Control and Low dose groups, 30 L/filter for Mid and High dose groups), from the exposure chamber, through 0.45 μm PVDF filters (Merck Millipore Ltd.). Samples were collected from a vacant animal exposure port (animals breathing zone) with a sampling rate of 1.0 litre/minutes. The actual sampling schedule employed was dependent on the required sample volume in order to obtain a dequate quantities of test item (exposure period) but including generally 3 samples per dose group. The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/ L) was calculated by the following formula:
CAS = M / (tS x FS),
where
M = Mass of the active substance on the filter (mg) tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere , from the exposure chamber, through 0.45 µm PVDF filters (Merck Millipore Ltd.). Samples were collected from a vacant animal exposure port (animals breathing zone) with a sampling rate of 1.0 litre/minutes. The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period) but including generally at least 3 samples per dose group

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 hours/day, 5 days/week
(with the exception of the first week which consists of 6 consecutive treatment days)

Dose / conc.:

0 mg/L air

Remarks:

control

Dose / conc.:

2.1 mg/m³ air (analytical)

Remarks:

- also refered to as low concentration
- target concentration was 2 mg/m3

Dose / conc.:

8.3 mg/L air (analytical)

Remarks:

- also refered to as mid concentration
- target concentration was 8 mg/m3

Dose / conc.:

39.2 mg/m³ air (analytical)

Remarks:

- also refered to as high concentration
- target concentration was 40 mg/m3

No. of animals per sex per dose:

15 females and 25 males per concentration

Control animals:

yes, concurrent vehicle

Details on study design:

- Main study (PEO-1): 10 females and 10 males per concentration + additional (satellite) 5 males per group (for lung burden)
- Post-exposure observation group 2 (PEO-2): 5 males per group (for lung burden after 4 weeks)
- Post-exposure observation group 3 (PEO-3): 5 females and males per group
(recovery group 13 weeks)

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Body weight of each animal was recorded with precision of 1 g at approximately one week before the first exposure, at randomization, on Day 1 (before the exposure), at least twice weekly until Day 28 and at least weekly thereafter including Day 90 (last exposure day; Day 118 for recovery), and prior to necropsy (fasted on Day 91; Day 119 for recovery). Body weight of PEO-3 animals was measured on Days 180-181.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in all animals before treatment, Day -3, and in the Control (Group 1) and High dose (Group 4) animals, during weeks 12 to 13
- Dose groups that were examined: control and high concentration group

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters in table 2 were investigated, as well the coagulation parameters Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT), both measured in seconds.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 91 (PEO-1), day 119 (PEO-2) and day 181 (PEO-3)
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the various experimental phases
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (food only)
- Parameters in table 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: YES
Time schedule for analysis: week 12/13
- Dose groups that were examined: all
- Number of animals: PEO-1: 10 males and 10 females per group,
- Parameters examined:
- sensory activity
- motor activity
- landing foot splay
- grip strength

IMMUNOLOGY: NO

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: day 91 (PEO-1) and day 180 (PEO-3)
- Dose groups that were examined: all
- Number of animals: PEO-1: 10 males and 10 females per group, PEO-3: 5 females per group
- Parameters examined:
- LDH activity (Pyruvate/NADH Kinetic (RRA) method, measured at 340/410
nm)
- Albumin (BCG staining, Endpoint (EPA) method, measured at 596/694 nm)
- total cell count and the cell viability (CountessTM II automated cell counter,
Invitrogen)
- % of alveolar macrophages (AM)
- % lymphocytes, % neutrophils
- % eosinophiles
- % other cells were determined

LUNG BURDEN: Yes
- Time schedule for analysis: day 91 (PEO-1, satellite group of 5 males), day 119 (PEO-2) and day 180 (PEO-3, males only)
- Dose groups that were examined: all
- Number of animals: PEO-1: satellite group of 5 males; PEO-2: all (i.e. 5 males); PEO-3: 5 males
- Parameters examined:
- weight of whole lungs and of right lung lobes
- analytical quantification of test substance (by HPLC-UV)
- clearance half-time

Sacrifice and pathology:

Terminally on Day 91 (on Day 119 and Day 181 for PEO-2 and PEO-3 animals, respectively) surviving animals were euthanised under sodium pentobarbital anaesthesia by exsanguination.

GROSS PATHOLOGY: Yes (see table 5)
On completion of the macroscopic examination the following tissues and organs were retained from all surviving main study (PEO-1) and all recovery animals (PEO-3 = Satellite Group 3).

HISTOPATHOLOGY:
The selected tissues (see table 5) were processed to slides in Control and High dose groups (Main Study Animals) and the animal which was found dead. In addition, full histopathology was performed on respiratory tract and associated lymph nodes in Mid and Low dose groups. Lung, tracheobronchial lymph node, mediastinal lymph node, nasal cavity and trachea from all recovery animals were proceed to microscopic evaluation. In addition, histopathology of thymus from the recovery Control and High dose females was also performed.
Any organs or tissues with macroscopic abnormalities (except minor changes) were also processed for histological examination.

Other examinations:

Lungs:
PEO-1 (Main Study Animals and Satellite Group 1):
The left lung of the Main Study Animals was preserved for histopathologic evaluation and the right lung of the Main Study Animals was used for bronchoalveolar lavage (BAL). After removal and weighing of the lungs, the right bronchus was tied off and the left lung was filled with fixative and stored for histopathology processing.
The right lung of the animals of Satellite Group 1 was used for lung burden determination. The left lung was not required but was preserved frozen (at -80oC) immediately for possible additional investigations. Accordingly, the lungs were processed as follows. After removal and weighing, the further procedure of the right lung lobes is described in section 15.3.
PEO-2 (Satellite Group 2):
The left lung of the animals was infused and preserved for possible histopathologic evaluation; the right lung of the animals was used for lung burden determination.
Accordingly, the lungs were processed as follows. After removal and weighing, the right bronchus was tied off and the left lung of the animals using for possible histopathology evaluation was inflated with formalin to ensure high quality fixation. The further procedure of the right lung lobes is described in section 15.2 below.
PEO-3 (Satellite Group 3, i.e. Recovery Animals):
The left lung of the female animals was preserved for histopathologic evaluation and the right lung of the female animals was used for bronchoalveolar lavage (BAL).
The left lung of the male animals was preserved for histopathologic evaluation and the right lung of the male animals was used for lung burden determination.
Accordingly, the lungs were processed as follows. After removal and weighing, the right bronchus was tied off and the left lung was filled with fixative and stored for histopathology processing. The further procedure of the right lung lobes is described in section 15.2 (males) and section 15.3 (females) below.
The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
Bone marrow smears (taken from the femur of all animals) were preserved, examination was not required.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 (when using Provantis).
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of continuous numeric data.
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova/Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01, as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01, as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001, as appropriate.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse clinical signs were observed in the Low, Mid or High dose groups during the observation period.
During the 90-day treatment period, fur staining by test item was observed in all treated male and female animals.
During the 4-week recovery period, alopecia (1/5 Mid dose males), fur staining by test item (all treated males), scar (1/5 Mid dose males) and wound (1/5 Mid dose males) were noted.
During the 13-weeks recovery period alopecia (1/5 Low dose and 1/5 High dose females), thin fur (2/5 Low dose females) and fur staining by test item (all treated males and females) were observed.
Observations in this study on the skin and fur were not considered to be related to test item, other than fur staining, which is a non-adverse, test item related change.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One Control female (#1503) became pregnant due to a technical error, therefore it was excluded from the study after agreement with the Sponsor (pre-terminally euthanized on Day 59).

One Low dose female (#2506) was found dead on Day 81 during exposure. Fur staining by test item on the head, nose and snout was recorded prior to death. At necropsy diffuse dark red discolouration of the non-collapsed lungs and red diffuse discolouration of the mediastinal and tracheobronchial lymph nodes were noted of this animal. Microscopically minimal multifocal pigment in the alveolar macrophages and minimal pulmonary congestion in the lung and minimal congestion in the mediastinal lymph nodes were observed. The death of this animal was not attributed to treatment (occasionally small animals can turn their head in the exposure tube, causing suffocation, although there was no clear evidence for a cause of death in this case).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related differences in the body weight or body weight gain values in any of the dose groups when compared to the controls during the 90-day treatment period and the 4-weeks and 13-weeks recovery periods. The measured values were within the range commonly recorded for this strain and age.
Some sporadic significant body weight gain and body weight differences were seen for some of the weekly evaluations, without a clear trend, therefore these changes were considered to be incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment related differences in the food consumption values in any of the treated groups when compared to the control between Day 1 and Day 90 and at the end of the 4 weeks and 3 months recovery periods. Sporadic, statistically significant changes (in Mid dose males in PEO-3 Group between Days 176-180 and in Low and Mid dose females in PEO-3 Group between Days 1-8) were considered to be incidental.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No direct test item-related adverse changes were detected in the test item treated animals (males and females) when comparing haematology parameters to control data.

In PEO-1 male animals, statistically significantly lower relative reticulocyte and lymphocyte values (in High dose Group) and higher relative neutrophil value (in Mid and High dose Groups) were observed compared to the Control Group on Day 91 (see Result Table 1 below).
The changes in the WBC distribution were considered to be a not adverse, secondary response which was correlated with the results of BAL evaluation. Despite of statistically significant changes in the relative and absolute values of reticulocytes, these changes were considered to be incidental, since the values were in the historical control ranges and there were no abnormalities in the values of red blood cells, haemoglobin and mean cell haemoglobin concentrations.

In PEO-1 females, statistically significant increased white blood cell (WBC) and absolute neutrophil granulocytes counts (by 66.1% and 104.3%, respectively) in the High dose group were observed when compared to the controls at 91 days. These changes were not considered to be a primary effect of test item and were in line with the results of BAL evaluation.

In recovery animals on Day 119, a sporadic statistically significant difference in one parameter (prothrombin time) was considered to be incidental.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing the serum chemistry parameters to the relevant Control data at the end of the treatment period on Day 91.

Sporadic statistically significant values of recovery animals measured on Day 181 were considered to be incidental.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.

Statistically significantly higher glucose value in High dose males was noted compared to Controls on Day 91, however this change was considered incidental and not treatment related. Lower urine volumes in Low and Mid dose females measured compared to Controls on Day 91 were within the normal historical control range. Therefore, these differences were considered not to be treatment related changes.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There was no adverse effect of treatment noted during the assessment of grip strength, foot splay, Irwin Test or locomotor activity in any dose group when compared with the control.

In the Mid and High dose males, an apparent dose-related decrease in grip strength of the hind limbs was measured, however the control values in this study were higher than a contemporaneous study, where the control values were similar to the treated groups of this study; hence the statistical differences in this study are not considered to be an adverse effect of the treatment.

The locomotor activity was considered to have showed a normal response in the High group, it was initially high then reduced to a plateau at approximately 30 minutes. All locomotor activity data were considered as normal; therefore the analysis of the Mid and Low dose groups of PEO-1 and PEO-2 and PEO-3 groups were considered to be not required.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Main animals (PEO-1 without satellite 1):

There was no test item related effect on the terminal body weights in test item exposed main study animals.
Significant weight changes were observed in the lungs, mediastinal lymph nodes and heart in test item exposed High dose males and females, and in the epididymis in High and Mid dose males (see Results Tables 2 and 3 below).
The decrease in the weight of hearts in both sexes and epididymis weights in test item exposed High Dose males had no correlating histopathological findings and hence no toxicological significance was assigned.

Lungs

The absolute and relative (to body and brain weights) lung weights were statistically significantly larger in High dose animals (see Table 6 below).

Mediastinal lymph nodes

The absolute and relative (to body and brain weights) mediastinal lymph node weights were statistically significantly larger in High dose animals (see Table 6 below).
The increase in lung and mediastinal lymph node weights in test item exposed animals correlated with the macroscopic and histopathological findings (see below). 

Heart

The absolute and relative (to body and brain weights) heart weights were statistically significantly lower in High dose animals.

Epididymis

The absolute and relative (to body and brain weights) epididymis weights were slightly lower in all treated males.

The decrease in the weight of hearts in both sexes and epididymis weights in test item exposed High Dose males had no correlating histopathological findings and hence no toxicological significance was assigned.




4-week recovery animals

There was no test item related effect on the terminal body weights in previously test item exposed satellite group 2 animals. When compared to the Controls, test item related weight changes were observed in the lungs of High Dose males.

Lungs

The absolute and relative (to body and brain weights) lung weights were statistically significantly larger in High Dose males (see Results Table 4 below).




13-week recovery animals

When compared to the Controls, test item related weight changes were observed in the lungs and in the mediastinal lymph node in test item exposed High Dose males and females. Weight of thymus statistically significantly decreased in High Dose females; however, this decrease did not show dose-response and it was within the normal historical control range, hence it was considered not to be test item related.

Lungs

The absolute and relative (to body and brain weights) lung weights were statistically significantly larger in High Dose animals (see Results Tables 5 and Table 6 below).
 
Mediastinal lymph nodes

The absolute and relative (to body and brain weights) mediastinal lymph node weights were statistically significantly larger in High Dose animals (ee Results Tables 5 and Table 6 below).

The increase in lung and mediastinal lymph node weights in test item exposed animals correlated with the macroscopic and histopathological findings (see below).

Thymus

The absolute and relative (to body and brain weights) thymus weights were statistically significantly lower in High Dose females.

The decrease in the weight of thymus weights in previously test item exposed females had no correlating histopathological findings and hence no toxicological significance was assigned.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Main animals

Test item-related macroscopic findings at 90 days were detected in the lungs in all dosed groups and in the mediastinal lymph node in Mid and High dose groups at necropsy.
The lungs showed a dose-related diffuse/multifocal red to dark red discoloration, the mediastinal lymph nodes were diffuse red discoloration and enlarged. The red colour is considered to be test item.
The other findings, due to very low incidence in both dosed and control groups, were considered as incidental or background.


Recovery animals (4 weeks, PEO-2)

Test item-related macroscopic findings were detected in the lungs and in the mediastinal lymph node in Mid and High Dosed groups at necropsy.
The lungs showed diffuse/multifocal red discoloration, and a diffuse red discoloration was noted for the mediastinal lymph node and it was enlarged as well.
No other finding was observed.


Recovery animals (3 months, PEO-3)

Test item-related macroscopic findings were detected in the lungs and in the mediastinal lymph node at necropsy.
The lungs showed diffuse red discoloration in all High dose animals and in 1/5 Mid dose male rat. The mediastinal lymph node showed a diffuse red discoloration in 3/5 Mid dose and 5/5 High dose males and in all Mid and High dose females. Enlargement of the mediastinal lymph node was noted in 1/5 Mid dose and 5/5 High dose males and in 2/5 Mid dose and 5/5 High dose females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Main animals (PEO-1, excluding satellite group)

Test item-related findings were observed in the lungs, trachea, nasal cavity, mediastinal lymph node and tracheobronchial lymph node.

Lungs

Minimal to marked multifocal pigments mainly in the macrophages and distributed in the alveolar lumen and wall were observed in all treated groups. The free pigments / pigmented macrophages were seen distributed throughout the lung parenchyma with predominance at the centro acinar region. Pigments were also seen in the bronchial associated lymphoid tissue (BALT). Minimal multifocal bronchiolo alveolar epithelial hyperplasia, which was considered to be regenerative, was seen around the pigmented macrophages in High Dose animals and one Mid Dose female (see Results Table 7).

Mediastinal Lymph Node

Minimal to moderate pigment and/or mild to moderate increased cellularity were seen in Mid and High Dose animals (see Results Table 8).

Tracheobronchial Lymph Node

Pigment at minimal to mild severity was seen in test item exposed Mid and High Dose animals. Increased cellularity at minimal severity was seen in High Dose animals (see Results Table 9).

Nasal cavity

Minimal amounts of pigment were seen in the nasal associated lymphoid tissue in High Dose Animals. Minimal focal/multifocal eosinophilic globules were seen in respiratory epithelium in a few High Dose animals (see Results Table 10).

Trachea

Minimal focal pigments were seen in the submucosa of the carina in 1/10 Mid Dose males, 2/10 High Dose males, 2/10 Mid Dose females and 1/10 High Dose females.

All other findings were considered incidental or background.



Recovery animals (4 weeks)

Test item-related findings were observed in the lungs, mediastinal lymph node, tracheobronchial lymph node and nasal cavity (see Results Tables 11 -14).

Lungs

Minimal to marked amounts of pigments multifocally distributed in the alveolar lumen and wall were observed in all treated groups. Pigments were also seen in the BALT. Minimal multifocal bronchiolo alveolar epithelial hyperplasia of regenerative nature, characterized by reactive macrophage type II proliferation was seen around the pigmented macrophages in Mid and High Dose males

Mediastinal Lymph Node

Minimal to moderate pigment were seen in Low, Mid and High Dose males. Increased cellularity was observed in Mid and High Dose males.

Tracheobronchial Lymph Node

Pigment at minimal to mild severity was seen in test item exposed Mid Dose animals.

Nasal cavity

Minimal focal eosinophilic globules were seen in respiratory epithelium in a few High Dose animals.



Recovery animals (3 months)

Test item-related findings were observed in the lungs, nasal cavity, mediastinal lymph node and tracheobronchial lymph node (see Results Tables 15 -18).

Lungs

Minimal to marked multifocal pigments mainly in the macrophages and distributed in the alveolar lumen and wall were observed in all treated groups. Pigments were also seen in the BALT. Minimal multifocal bronchiolo alveolar epithelial hyperplasia of regenerative nature, characterized by reactive macrophage type II proliferation, was seen in Mid and High Dose animals.

Mediastinal Lymph Node

Minimal to marked pigment were seen in Low, Mid and High Dose animals. Increased cellularity was observed in Mid and High Dose animals.

Tracheobronchial Lymph Node

Pigment at minimal to moderate severity was seen in test item exposed Low, Mid and High Dose animals. Increased cellularity was seen in Mid and High Dose males and High Dose females.

Nasal cavity

Minimal focal/multifocal eosinophilic globules were seen in respiratory epithelium in High Concentration males, Mid and High Concentration females. Although minimal eosinophilic globules were also observed in 1/5 and 1/5 males from the Low and Mid Concentration groups, respectively, these occasions were not considered as test item-related findings since similar low evidence occurred in 2/10 Control Hannover Wistar male rats in one 90-day inhalation study in this facility. Taking into consideration that there was no evidence of eosinophilic globules in Low and Mid Concentration males at the end of treatment, as well as after 4 weeks of recovery, this incidence below the High Concentration level was considered as background rather than test item-related.
At the 13-week recovery, minimal severity of eosinophilic globules was reported in 2/5 Mid and 2/5 High Concentration females. Although the incidence in 13 week recovery females was greater than background and the same as at the High Concentration (at 2/5) it is difficult to attribute the Mid Concentration eosinophilic globules to the treatment with test item after 13 weeks of recovery, although it cannot be clearly excluded.


All other findings were considered incidental or background.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar Lavage
Dose-related increase in neutrophils was observed in males and females, this change was statistically significant in the Mid and/or High dose groups. LDH was also increased in both males and females, with statistical significance in the High dose groups. These changes indicate a possible tissue injury and/or development of an inflammation-associated response in lungs. Recovery of neutrophil and LDH values were noted in females at the end of the 3-month recovery period, however increased LDH values in the High dose females were still statistically significant (see Results Table 19).


Lung Burden Determination
In the lungs, the amount of test item found at the end of treatment was very approximately proportional to the atmosphere concentrations. For the lung burden in the Low dose animals, a significant decrease of the test item was measured during the recovery (~75% clearance), while in case of the mid dose, this decrease was less significant (~57%) and thus the elimination half time was close to the limit of 90 days for normal clearance. In case of the high dose, a relatively small decrease can be seen (~23%), probably due to an overload of the lung (see Results table 20 and 21).

Key result

Dose descriptor:

NOAEC

Effect level:

2.1 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

8.3 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Result Table 1: Summary of selected haematology parameters of PEO-1 animals on Day 91

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose
Males
Reticulocyte relative (%)	2.12	2.14	1.95	1.81*	DN
HC range: 1.7-2.8	difference (%)	0.8	-8.1	-14.7
Reticulocyte absolute (K/uL)	180.64	182.34	167.01	155.37*	DN
HC range: 146.9-230.9	difference (%)	0.9	-7.5	-14.0
Neutrophil relative (%)	19.12	22.83	25.32*	27.41**	DN
HC range: 17.4-46.4	difference (%)	19.4	32.4	43.3
Lymphocyte relative (%)	74.91	71.43	68.02	65.71*	DU
HC range: 48.9-78.4	difference (%)	-4.6	-9.2	-12.3
Females
White blood cell count (K/uL)	1.551	1.544	1.834	2.577*	DN
HC range: 0.34 – 2.24	difference (%)	-0.5	18.2	66.1
Neutrophil absolute (K/uL)	0.313	0.321	0.329	0.640**	DN
HC range: 0.09 – 0.62	difference (%)	2.5	5.0	104.3

 

Notes: Data (group mean values, n=8-10) were rounded to two or three decimal places.

HC: Historical control

DN: Dunnett’s test, DU: Dunn test.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

 

 

Result Table 2: Selected organ weights in males on Day 91

Organ weight (males)	Dose groups
	Control	Low dose	Mid dose	High dose
Terminal body weight (g)	332.7	343.8	336.7	329.5	NS
	difference (%)	3.3	1.2	-1.0
Lung (absolute) (g)	1.342	1.391	1.321	1.631**	DU
	difference (%)	3.7	-1.6	21.5
Lung (relative to body weight) (%)	0.403	0.404	0.393	0.495**	DU
	difference (%)	0.3	-2.6	22.7
Lung (relative to brain weight) (%9	66.39	66.89	61.22	78.49*	DU
	difference (%)	0.8	-7.8	18.2
Lymph node mediastinal (absolute) (g)	0.0110	0.0164	0.0101	0.0383**	DU
	difference (%)	49.2	-8.0	247.8
Lymph node mediastinal (relative to body weight) (%)	0.0033	0.0048	0.0030	0.0116**	DU
	difference (%)	44.5	-8.9	251.3
Lymph node mediastinal (relative to brain weight) (%)	0.544	0.784	0.467	1.851**	DU
	difference (%)	44.1	-14.1	240.2

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

 

Result Table 3: Selected organ weights in females on Day 91

Organ weight (females)	Dose groups
	Control	Low dose	Mid dose	High dose
Terminal body weight (g)	209.0	207.6	208.7	204.9	NS
	difference (%)	-0.7	-0.1	-2.0
Lung (absolute) (g)	1.087	1.081	1.096	1.334**	DU
	difference (%)	-0.5	0.9	22.8
Lung (relative to body weight) (%)	0.519	0.522	0.525	0.652**	DN
	difference (%)	0.6	1.0	25.4
Lung (relative to brain weight) (%)	55.42	56.26	55.65	67.20**	DU
	difference (%)	1.5	0.4	21.3
Lymph node mediastinal (absolute) (g)	0.0124	0.0109	0.0120	0.0383**	DN
	difference (%)	-11.6	-3.5	209.0
Lymph node mediastinal (relative to body weight) (%)	0.0059	0.0053	0.0057	0.0187**	DN
	difference (%)	-9.6	-3.0	218.0
Lymph node mediastinal (relative to brain weight) (%)	0.630	0.571	0.609	1.926**	DN
	difference (%)	-9.4	-3.3	205.7

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

 

Results Table 4: Lung weights in males on Day 119

Organ weight (males)	Dose groups
	Control	Low dose	Mid dose	High dose
Males
Terminal body weight (g)	363.0	393.6	374.0	367.4	NS
	difference (%)	8.4	3.0	1.2
Lung (absolute) (g)	1.426	1.470	1.450	1.860**	DN
	difference (%)	3.1	1.7	30.4
Lung (relative to body weight) (%)	0.393	0.374	0.388	0.506**	DN
	difference (%)	-4.7	-1.4	28.8
Lung (relative to brain weight) (%)	66.32	69.17	67.36	89.95*	DN
	difference (%)	4.3	1.6	29.6

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

Results Table 5: Lung weights in males on Day 181

Organ weight (males)	Dose groups
	Control	Low dose	Mid dose	High dose
Terminal body weight (g)	427.0	438.2	457.4	426.2	NS
	difference (%)	2.6	7.1	-0.2
Lung (absolute) (g)	1.498	1.406	1.532	1.846**	DU
	difference (%)	-6.1	2.3	23.2
Lung (relative to body weight) (%)	0.350	0.322	0.336	0.433**	DN
	difference (%)	-8.1	-4.0	23.6
Lung (relative to brain weight) (%9	66.91	64.35	68.65	83.19**	DN
	difference (%)	-3.8	2.6	24.3
Lymph node mediastinal (absolute) (g)	0.0188	0.0145	0.0167	0.0470**	DN
	difference (%)	-22.7	-11.5	149.7
Lymph node mediastinal (relative to body weight) (%)	0.0044	0.0033	0.0037	0.0110**	DN
	difference (%)	-24.6	-15.9	149.7
Lymph node mediastinal (relative to brain weight) (%)	0.840	0.665	0.745	2.107**	DN
	difference (%)	-20.8	-11.4	150.8

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

 

Results Table 6: Lung weights in females on Day 181

Organ weight (females)	Dose groups
	Control	Low dose	Mid dose	High dose
Terminal body weight (g)	242.0	238.4	244.4	238.2	NS
	difference (%)	-1.5	1.0	-1.6
Lung (absolute) (g)	1.146	1.138	1.144	1.626**	DN
	difference (%)	-0.7	-0.2	41.9
Lung (relative to body weight) (%)	0.474	0.478	0.468	0.684**	DN
	difference (%)	1.0	-1.2	44.4
Lung (relative to brain weight) (%)	55.61	56.47	57.11	81.71**	DN
	difference (%)	1.6	2.7	46.9
Lymph node mediastinal (absolute) (g)	0.0142	0.0123	0.0161	0.0423**	DN
	difference (%)	-13.3	13.2	197.2
Lymph node mediastinal (relative to body weight) (%)	0.0058	0.0052	0.0067	0.0178**	DN
	difference (%)	-10.1	15.4	208.1
Lymph node mediastinal (relative to brain weight) (%)	0.687	0.610	0.814	2.121**	DN
	difference (%)	-11.2	18.6	208.9

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

 

Results Table 7: Incidence of test item related microscopic changes in the lungs

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	10	10	10	10	9	9	10	10
Pigment, Macrophage, alveolar, multifocal
Minimal	0	10	0	0	0	9	0	0
Mild	0	0	9	0	0	0	2	0
Moderate	0	0	1	9	0	0	8	10
Marked	0	0	0	1	0	0	0	0
Total	0	10	10	10	0	9	10	10
Pigment, Bronchial-associated lymphoid tissue, focal/multifocal
Minimal	0	0	6	9	0	1	8	10
Total	0	0	6	9	0	1	8	10
Hyperplasia, Bronchiolo-Alveolar, Regenerative focal/multifocal
Minimal	0	0	0	10	0	0	1	10
Total	0	0	0	10	0	0	1	10
Number of Tissues examined	10	10	10	10	9	9	10	10

 

Results Table 8: Incidence of test item related microscopic changes in the mediastinal lymph node

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	10	10	10	10	9	9	10	10
Pigment
Minimal	0	0	6	0	0	0	1	0
Mild	0	0	3	0	0	0	7	1
Moderate	0	0	0	10	0	0	1	9
Total	0	0	9	10	0	0	9	10
Cellularity, Increased
Mild	0	0	0	2	0	0	0	2
Moderate	0	0	0	8	0	0	0	8
Total	0	0	0	10	0	0	0	10
Number of tissues examined	10	9	10	10	9	9	10	10

 

Results Table 9: Incidence of test item related microscopic changes in the tracheobronchial lymph node

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	10	10	10	10	9	9	10	10
Pigment
Minimal	0	0	5	0	0	0	2	0
Mild	0	0	2	6	0	0	0	3
Total	0	0	7	6	0	0	2	3
Cellularity, Increased, lymphocyte
Minimal	0	0	0	4	0	0	0	0
Total	0	0	0	10	0	0	0	10
Number of tissues examined	8	10	8	7	7	6	7	6

 

 

Results Table 10: Incidence of test item related microscopic changes in the nasal cavity

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	10	10	10	10	9	9	10	10
Pigment, nasal associated lymphoid tissue, focal/multifocal
Minimal	0	0	0	5	0	0	0	4
Total	0	0	0	5	0	0	0	4
Eosinophilic globules (droplets), respiratory epithelium, focal/multifocal
Minimal	0	0	0	4	0	0	0	3
Total	0	0	0	4	0	0	0	3
Number of tissues examined	10	10	10	10	9	9	10	10

 

 

Results Table 11: Incidence of test item related microscopic changes in the lungs in satellite group 2 animals

	Males
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40
Number of animals	5	5	5	5
Pigment, Macrophage, alveolar, multifocal
Minimal	0	5	0	0
Mild	0	0	1	0
Moderate	0	0	4	4
Marked	0	0	0	1
Total	0	5	5	5
Pigment, Bronchial-associated lymphoid tissue, focal/multifocal
Minimal	0	1	2	3
Mild	0	0	0	1
Total	0	1	2	4
Hyperplasia, Bronchiolo-Alveolar, Regenerative focal/multifocal
Minimal	0	0	4	3
Total	0	0	4	3
Number of Tissues examined	5	5	5	5

 

Results Table 12: Incidence of test item related microscopic changes in the mediastinal lymph node in satellite group 2 animals

	Males
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40
Number of animals	5	5	5	5
Pigment
Minimal	0	1	1	0
Mild	0	0	4	0
Moderate	0	0	0	5
Total	0	1	5	5
Cellularity, Increased, minimal	0	0	4	0
Mild	0	0	0	4
Moderate	0	0	0	1
Total	0	0	4	5
Number of tissues examined	5	5	5	5

 

Results Table 13: Incidence of test item related microscopic changes in the tracheobronchial lymph node in satellite group 2 animals

	Males
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40
Number of animals	5	5	5	5
Pigment
Minimal	0	0	1	0
Mild	0	0	1	0
Total	0	0	2	0
Number of tissues examined	3	4	3	2

 

Results Table 14: Incidence of test item related microscopic changes in the nasal cavity

	Males
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40
Number of animals	5	5	5	5
Eosinophilic globules (droplets), respiratory epithelium, focal
Minimal	0	0	0	3
Total	0	0	0	3
Number of tissues examined	5	5	5	5

 

Results Table 15: Incidence of test item related microscopic changes in the lungs in satellite group 3 animals

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	5	5	5	5	5	5	5	5
Pigment, Macrophage, alveolar, multifocal
Minimal	0	5	0	0	0	4	0	0
Mild	0	0	3	0	0	0	3	0
Moderate	0	0	2	4	0	0	2	0
Marked	0	0	0	1	0	0	0	5
Total	0	5	5	5	0	4	5	5
Pigment, Bronchial-associated lymphoid tissue, focal/multifocal
Minimal	0	0	4	5	0	0	4	3
Mild	0	0	0	0	0	0	0	1
Total	0	0	4	5	0	0	4	4
Hyperplasia, Bronchiolo-Alveolar, Regenerative multifocal
Minimal	0	0	4	3	0	0	1	3
Mild	0	0	0	1	0	0	0	2
Total	0	0	4	4	0	0	1	5
Tissues examined	5	5	5	5	5	5	5	5

 

Results Table 16: Incidence of test item related microscopic changes in the mediastinal lymph node in satellite group 3 animals

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	5	5	5	5	5	5	5	5
Pigment
Minimal	0	1	0	0	0	3	1	0
Mild	0	0	2	0	0	0	4	0
Moderate	0	0	1	4	0	0	0	2
Marked	0	0	0	1	0	0	0	3
Total	0	1	3	5	0	3	5	5
Cellularity, Increased, lymphocytes
Minimal	0	0	2	0	0	0	4	0
Mild	0	0	1	3	0	0	0	5
Moderate	0	0	0	2	0	0	0	0
Total	0	0	3	5	0	0	4	5
Number of tissues examined	5	5	5	5	5	5	5	5

 

Results Table 17: Incidence of test item related microscopic changes in the tracheobronchial lymph node in satellite group 3 animals

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	5	5	5	5	5	5	5	5
Pigment
Minimal	0	1	0	0	0	1	1	1
Mild	0	0	3	2	0	1	0	2
Moderate	0	0	0	2	0	0	0	0
Total	0	1	3	4	0	2	1	3
Cellularity, Increased, lymphocyte
Minimal	0	0	2	0	0	0	0	1
Mild	0	0	1	3	0	0	0	0
Total	0	0	3	3	0	0	0	1
Number of tissues examined	3	3	4	4	4	3	2	5

 

Results Table 18: Incidence of test item related microscopic changes in the nasal cavity in satellite group 3 animals

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	5	5	5	5	5	5	5	5
Eosinophilic globules (droplets), respiratory epithelium, focal/multifocal
Minimal	0	1	1	3	0	0	2	2
Total	0	1	1	3	0	0	2	2
Number of tissues examined	5	5	5	5	5	5	5	5

 

Results Table 19: Summary of selected BAL parameters in males and females on Day 91

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose
Males
BAL-LDH (U/L)	312.4	123.7**	328.0	643.2**	DN
	difference (%)	-60.4	5.0	105.9
BAL-Neutrophils (10^6)	0.003	0.002	0.013*	0.285**	DU
	difference (%)	-37.3	419.1	11251.2
Total Cell Number (10^6)	0.449	0.301*	0.206**	0.527	DU
	difference (%)	-33.0	-54.1	17.4
Females
BAL-LDH (U/L)	214.4	326.8	350.6	764.8**	DN
	difference (%)	52.4	63.5	256.6
BAL-Neutrophils (10^6)	0.003	0.002	0.013	0.245**	DU
	difference (%)	-33.2	309.2	7747.1
Total Cell Number (10^6)	0.334	0.200*	0.158**	0.509	DN
	difference (%)	-40.2	-52.8	52.2

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

Results Table 20: Measured mean test item content (mg/g) in lung

Satellite group	Clearance time (week)	Measured test item content in lung (mg/g)
		Low dose	Mid dose	High dose
PEO-1	0	0.421	2.402	9.042
PEO-2	4	0.260	2.142	7.804
PEO-3	13	0.105	1.037	7.009

 

Results Table 21: Measured percentage of the clearance over time

Satellite group	Clearance time (week)	Measured clearance (%)
		Low dose	Mid dose	High dose
PEO-1	0	-	-	-
PEO-2	4	38.1	10.8	13.7
PEO-3	13	74.9	56.8	22.5

 

Conclusions:

The sub-chronic inhalation toxicity of the registered substance was investigated in a GLP-compliant 90-day study by inhalation (nose-only) in rats with 13-week recovery according to OECD TG 413. No systemic effects were observed and adverse local changes in the respiratory tract were limited to the Mid and High Concentration groups treated with test item. Based on these results, the No Observed Adverse Effect Concentration (NOAEC) for the study was 2.1 mg/m3 (Low Concentration group).

Executive summary:

The sub-chronic inhalation toxicity of the registered substance was investigated in a GLP-compliant 90-day study by inhalation (nose-only) in rats with 13-week recovery according to OECD TG 413. The objective of this 90-day study was to obtain information on the toxicity of the test item when administered to Wistar (Han) rats via inhalation route for at least 90 days with the aim of inducing toxic effects but no death or suffering at the highest of three test concentrations, and little or no evidence of toxicity at the lowest concentration. The methods used followed OECD TG 413, with the procedures that are foreseen for a test item which accumulates in the lungs. According to this, additional subgroups were used to evaluate the recovery of the animals and to investigate the clearance rate of the Test Item from the lung.

The animals were exposed to the test atmosphere at carefully determined target concentrations of 2, 8 and 40 mg/m3, as the Low, Mid and High Concentration group (also referred to as “Dose group”), respectively, using a nose-only exposure system over 90 days. 

Ten male and 10 female rats in each group (Control (filtered air), Low, Mid and High Concentration), referred to as main study animals, were treated by a 6 hour nose-only exposure to filtered air or three fixed aerosol concentrations for consecutive 5 days/week and 2 days without treatment with the exception of the first week with 6 consecutive treatment days. The main study animals were sacrificed on Day 91, i.e. on the day following the last treatment, and histopathology evaluation and BALF (Bronchoalveolar lavage fluid) analysis were performed. Additionally, 5 females per group were treated and allowed to recover for 13 weeks and sacrificed on Day 181 (histopathology evaluation and BALF analysis were performed) and 15 males per group were treated and five rats each sacrificed on Day 91 (Satellite 1), Day 119 (PEO-2 with 4-week recovery) and Day 181 (PEO-3 with 13-week recovery) for histopathology and/or lung burden evaluation.

The exposure did not cause any test item-related death or any adverse clinical signs, nor were test item-related changes in body weight, food intake, in behaviour, opththalmology evaluation,oestrus cycle, haematology, clinical chemistry and urine parameters observed.

In the bronchoalveolar lavage analysis, increased neutrophil values in Mid and High Concentration males and females and increased LDH values in High Concentration males and females were considered as treatment related.

Results of lung burden determination showed that in case of the Low Concentration group, continuous clearance was observed from the lung over time, with ~75% clearance after 3 month. In the Mid Concentration group the clearance was ~57%, while in the High Concentration group, the clearance rate was much lower (~23%) probably due to overload, considering the low rate of removal of the test item from the lung.

Microscopically, pigments in the macrophages of the lungs were seen in animals of all Concentration groups but considered not adverse. In the Mid and High Concentration groups additional test item-related changes in the lungs, nasal cavity, trachea, mediastinal lymph node and/or tracheobronchial lymph node were observed. Changes in the lungs, mediastinal and tracheobronchial lymph nodes consisted of an increase of slight to moderate cellularity and minimal bronchiolo alveolar epithelial hyperplasia of regenerative nature. This change may be considered adverse but is not consistent with a pre-neoplastic lesion. It is indicative of repair and not of an early pre-neoplastic change which is supported by the lack of formation of connective tissue and the absence of any indication for fibrosis. Eosinophilic globules seen in the nasal cavity, without nasal tissue injury following repeated inhalation exposure of the test item, represents a non-adverse adaptive response. After 4 and 13 weeks of recovery partial or full recovery was generally observed.

 

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant non-guideline study with well-characterized test material

Qualifier:

no guideline available

Principles of method if other than guideline:

5-day inhalation exposure with 21 days recovery group.
Ma-Hock et al. ( 2009) Inhal Toxicol 21, 102-118

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation: Main group day 0: control group: 246g; test group: 251g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust-free bedding, supplied by SSNIFF, Soest, Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks (Typ NGM E-022), supplied by Abedd Lab. and Vet. Service GmbH, Vienna, Austria, were added.
- Diet: Mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum.
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

other: dust aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The two measurements of particle size resulted in equal MMAD value of 0.7 μm with GSD of 2.9 and 2.5. The calculated mass fractions of particles below MMAD 3 μm was 92.2 and 94.6 %.

Figures with results of WELAS, SMPS and Cascade Impactor determinations are shown in the attachment.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. By means of dust generators the substance to be tested is generated into dust aerosols using compressed air in a mixing stage, mixed with conditioned air and passed into the inhalation systems via cyclonic separators. For each concentration, a solid particle generator (brush-generator) was used for generating the dust. The concentration were adjusted by varying the piston feed and by varying the brush rotation. For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed via the cyclonic separator into the inhalation system.
- The following test substance flow, air flows and evaporation temperatures were scheduled: see table M1

EXPERIMENTAL PROCEDURE
- Head-nose exposure systems: The inhalation atmosphere was maintained inside aerodynamic exposure systems (test group 1 in INA 60, volume V ≈ 90 L, control group in INA 120, volume V ≈ 120 L) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.
- Exposures: The head-nose exposure technique was preferably selected for this aerosol/dust/ inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult. Furthermore, by using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of the final target concentration) is shorter as compared to whole-body chambers with a higher chamber volume. A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air. In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours from Monday to Friday to reach 5 exposures. The animals did not have access to water or feed during the exposure.
- Measurements of the exposure conditions: The following exposure parameters were recorded: Supply air (conditioned), Supply air 2 (compressed), Exhaust air, Chamber humidity, Chamber temperature, Real time concentration surveillance. No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

VEHICLE
- Composition of vehicle: Conditioned supply air is activated charcoal filtered air conditioned to about 30% - 70% relative humidity and 20°C - 24°C. Compressed air is filtered air pressurized to about 6 bar.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CALCULATION OF NOMINAL CONCENTRATIONS: The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.

ANALYTICAL DETERMINATION OF CONCENTRATIONS: The concentration of the inhalation atmosphere in test group 1 was analyzed by gravimetry. This analytical method is judged to be valid because the test substance does not possess an appreciable vapor pressure. Daily means were calculated based on 3 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. In the test group, the constancy of the dust atmosphere in the chamber was continuously monitored using scattered light photometers.
- Sampling for gravimetric analyses: Equipment: Sampling equipment with probe (Millipore Corporation, Billerica, MA 01821, USA), Internal probe diameter: 7 mm, Filter: MN 85/90 BF (d = 4.7 cm), Vacuum pump (Millipore Corporation, Billerica, MA 01821, USA), Balance: Sartorius M3P-000V001 (Sartorius AG, Göttingen, Germany). Sampling: Sampling velocity: 1.25 m/s, Flow rate of sampling: 3 L/min, Sample volumes Test group 1: 90 L, Sampling site: immediately adjacent to the animals' noses at a separate spare port, Sampling frequency: as a rule, 3 samples per exposure and concentration group
- Gravimetrical method of analyses: A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust aerosol was drawn through the filter. The dust concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmosphere.
REAL TIME MONOTORING OF CONSTANCY OF CONCENTRATIONS: Scattered light photometer (VisGuard (Sigrist) in test group 1 was used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. The measurements were recorded using line recorders.

PARTICLE SIZE ANALYSIS:
- Definitions: EACD 50% (effective aerodynamic cutoff diameter 50%) defines the separation characteristic of each impactor stage. 50% of particles with the EACD given are deposited in the pertinent impactor stage; the remainder has reached one of the following stages. MMAD (mass median aerodynamic diameter) is the calculated aerodynamic diameter which divides the size distribution in half when measured by mass. Geometrical standard deviation (GSD) is the ratio of the estimated 84 percentile to the 50 percentile and indicates the slope of the cumulative particle size distribution curve.

- Equipment, sampling and method of determination: The particle size analysis was carried out with a cascade impactor. Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA), Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA), Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA), Sampling probe internal diameter 7 mm, Balance Sartorius M3P-000V001 (Sartorius AG, Göttingen, Germany), Mettler XP 205.(Mettler-Teldo AG, Greifensee, Switzerland)

- Sampling for particle size analyses: Preweighed metal collecting discs and a backup particle filter were placed into the cascade impactor and 2 samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals. A sample volume of 90 L was used in each test group. Method of analysis: Gravimetrical determination. The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the metal collecting disc and backup filter before and after sampling. The deposits in the probe and the wall losses in the impactor were also determined as difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter. Evaluation: The calculation of the particle size distribution was carried out in the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (DIN 66141: Darstellung von Korngrößenverteilungen and DIN 66161: Partikelgrößenanalyse, Beuth-Vertrieb GmbH, Berlin und Köln, Germany).
- Particle size distribution measurements with APS: Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used (TSI APS 3321). Frequency: on two days during the exposure period, with 3 repeats on each day.
- Particle size distribution measurements with Optical particle counter: For each test atmosphere measurements with an optical particle counter (WELAS 2000; (Palas® GmbH, Karlsruhe, Germany)) were performed to determine the size distribution of particles with diameters larger than 246 nm. The WELAS 2000 uses a white-light source to illuminate a measurement volume through which particles have to move singly. The measuring range of the sensor was 0.246 to 9.653 μm and the sampling flow rate 5 L/min.
- Particle size distribution measurements with scanning mobility particle sizer: To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 μm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.083 μm according to the software calculation. The duration of each measurement cycle was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily for five consecutive days

Remarks:

Doses / Concentrations:
30 mg/m³
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
31.34 mg/m³; SD = 4.49 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

3/dose/group (main group or recovery group for microscopic examination)
5/dose/group (main group or recovery group for blood sampling and BAL)

Control animals:

yes, concurrent vehicle

Details on study design:

On study day 4 after exposure and on study day 25, 3 animals per group and time point were sacrificed underwent gross necropsy. Selected organs were weighed, a broad set of organs and tissues were preserved, respiratory tract was examined histologically.
On study days 7 and 28, blood was sampled from 5 rats/group and time point. Clinical chemistry parameters, hematology parameters and acute phase proteins were examined in blood. After blood sampling the animals underwent bronchoalveolar lavage. Lavage fluid was examined for cytological and biochemical parameters including selected antigens.

Observations and examinations performed and frequency:

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

CLINICAL OBSERVATIONS: The clinical condition of the test animals was recorded once daily during the pre-exposure period and on post-exposure observation days on working days. On exposure days, clinical observation was performed at least 3 times daily, before, during and after exposure. During exposure only a group wise examination was possible.

BODY WEIGHT: The body weight of the animals was determined at the start of the pre-exposure (day -4), and then, as a rule, twice a week (Monday and Friday), as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weights from Monday to Friday. The main reason for this type of calculation is to show body weight change of the exposure week without the exposure-free weekend. It enables detection of minor decrease of body weight gain, which may be overlooked because the animals recover during the weekend. Group means were derived from the individual differences.

CLINICAL PATHOLOGY: In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The examinations for haematology and clinical chemistry were carried out in 5 animals per test group.

HAEMATOLOGY: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Prothrombin time (Hepato Quick’s test) (HQT) was measured. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

CLINICAL CHEMISTRY: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL).

ACUTE PHASE PROTEINS IN SERUM: Rat α2-macroglobulin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E-25A2M). Rat haptoglobin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E-25HPT). Both ELISA kits were measured with a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer.

BRONCHOALVEOLAR LAVAGE FLUID (BAL): The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline. The following examinations were carried out in 5 male animals per test group.
- Cytology in BAL: Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. Parameters: Total cell count (BALTCN), Macrophages (BALMPH), Polymorphonuclear neutrophils (BALPMN), Lymphocytes (BALLY), Eosinophils (BALEO), Monocytes (BALMO), Atypical cells (BALATY).
- Total Protein and enzymes in BAL: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the humoral parameters in the bronchoalveolar lavage fluid. Parameter: γ−Glutamyltransferase (GGT), Protein (MTP), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP), N-acetyl-β-Glucosaminidase (NAG)
- Antigens in BAL: The antigens were measured with MTP ELISAs at a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer. The following antigens were measured in BALF: Rat monocyte chemoattractant protein-1 (rat MCP-1) level measured with an Instant ELISA produced by Bender MedSystems, Vienna, Austria (cat. no BMS631INST), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8) measured with an ELISA produced by R&D Systems Inc., Minneapolis, US, (Quantikine rat CINC-1, cat. no. RCN100), Macrophage colony stimulating factor (M-CSF) measured with a Quantikine Mouse M-CSF ELISA produced by R&D Systems Inc., Minneapolis, USA (cat no. MMC00), Rodent osteopontin measured with an ELISA produced by R&D Systems, Inc., Minneapolis, US (Quantikine mouse osteopontin, cat. no. MOST00).

Sacrifice and pathology:

NECROPSY: All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS: The following weights were determined in all animals sacrificed on schedule: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands.

HISTOPATHOLOGY: The following organs or tissues were fixed in 4% buffered formaldehyde or modified Davidson’s solution: All gross lesions, Adrenal glands, Brain with olfactory bulb, Bone marrow (femur), Epididymides, Eyes with optic nerve and eyelids, Heart, Kidneys, Larynx/Pharynx, Liver, Lungs, Lymph nodes (tracheobronchial and mediastinal lymph nodes), Nose (nasal cavity), Oesophagus, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Stomach (forestomach and glandular stomach), Spleen, Testes, Thyroid glands, Thymus, Trachea, Urinary bladder. From the liver, one additional slice of the Lobus dexter medialis and the Lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast. The testes were fixed in modified Davidson’s solution. Fixation was followed by histotechnical processing and examination by light microscopy.
Tissues and organs examined histologically: All gross lesions (only affected animals), Nasal cavity (4 levels), Larynx (3 levels), Trachea, Lungs (5 lobes), Lymph nodes (tracheobronchial and mediastinal lymph nodes).

Statistics:

- Body weight, body weight change: A comparison of the dose group with the control group was performed using the student t-test (two-sided) for the hypothesis of equal means.
- Clinical Pathology parameters in Blood: Pair-wise comparison of the dose group with the control group was performed using Wilcoxon test (two-sided) for the equal medians.
- Clinical Pathology parameter in BALF, Pair-wise comparison of the dose group with the control group was performed using Wilcoxon test (one-sided) for the equal medians.

Throughout the sections of the report, when intergroup differences are referred to as "significant " it implies that the differences have attained statistical significance (p ≤ 0.05) when compared with the control group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute and relative weight of the adrenal glands showed an increase of +22% and +28%, respectively at the end of the exposure period. They were no longer increased at the end of the recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

An orange red discoloration was observed in the lungs of all treated males and in the fur of most of them. This finding is regarded as treatment-related and was due to the presence of the test substance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The test substance was mainly stored in the cytoplasm of macrophages as dark brown fine pigment particles.

Details on results:

The following findings were noted immediately at the end of the exposure phase:

No treatment-related changes of the acute phase protein levels in serum were observed.

Examination of bronchoalveolar lavage fluid showed no adverse effects (see tables 2)

The test substance was mainly stored in the cytoplasm of macrophages a dark brown fine pigment particles. A moderate number of pigment-laden macrophages were diffusely distributed in the lungs but were predominantly present in the lumen of terminal bronchioles, alveolar ducts and adjacent alveoli. The number of pigment particles in the macrophages was also moderate (see table 1) A minimal number of pigment-laden macrophages was observed in the bronchus-associated lymphoid tissue (BALT) and free pigment particles were found in alveoli and bronchioles. The epithelial hypertrophy and/or hyperplasia was generally observed at the level of terminal bronchioles and alveolar ducts and in more proximal located bronchiole.
Terminal bronchioles were primarily affected. This finding was characterized by an increased size and number of bronchiolar epithelial cells, accompanied by a slight cytoplasmic basophilia.

In the nasal cavity and larynx, minimal to slight amounts of free pigment particles were found admixed with the mucus secretion.
Minimal focal changes in the larynx, such as epithelial alteration in the ventral pouch of the epiglottis or lymphoid infiltrations were observed, but are considered to be background lesions that are not related to treatment. No findings were noted in the trachea.

No pigment was detected in mediastinal and tracheobronchial lymph nodes.


The following findings were noted at the end of the recovery period:
- terminal body weight (+9%)
- absolute kidneys weight (+26%) and relative (+15%)
- absolute liver weight (+18%)
Since the body weight development did not show adverse effects, the terminal body weight increase is regarded as incidental and not related to treatment. The increase of liver and kidneys weight is considered to be in relation with the terminal body weight increase. These changes likely respond to the expected standard deviations when only 3 animals per group are tested.

All males showed an orange red discoloration in the lungs, which is regarded as treatment-related.
Compared with the control group 0, all males showed slight number of pigment-laden macrophages diffusely distributed in the lung with a marked tendency to accumulate but not to aggregate in the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). The bronchus-associated lymphoid tissue (BALT) of all males showed minimal number of pigment-laden macrophages.
No treatment-related findings were observed in the nasal cavity, larynx and trachea. A minimal number of pigment-laden macrophages was detected in the mediastinal lymph node. A minimal to slight number of pigment-laden macrophages was found in the tracheobronchial lymph node.

Key result

Dose descriptor:

NOAEC

Effect level:

30 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Presence of pigment-laden macrophages in the lung

Critical effects observed:

no

Table 1: Results of histopathology findings after exposure and at the end of the recovery period

	after exposure	after exposure	after recovery	after recovery
Concentration (mg/m3)	0	30	0	30
Animals examined	3	3	3	3
Macrophages, pigment-laden	0	3 (grade 3)	0	3 (grade 2)
Macrophages, pigment-laden, in bronchus-associated lymphoid tissue	0	3 (grade 1)	0	3 (grade 1)
Free pigment particles	0	3 (grade 1)	0	0
Hypertrophy/hyperplasia, terminal bronchiole	0	3 (grade 1)	0	0

Tables 2: Findings of broncheo-alveolar lavage (days 7 and 28)

(For full names of parameters, see "Observations and examinations performed and frequecy")

BAL: Cytokines		Air control; males	30 mg/m3 males
MCP-1 BAL	Mean	14.0 x+	23.8
[pg/mL]	S.d.	0.0	16.8
day 7	N	5	5
	Median	14.0	14.0
IL-8 BAL	Mean	120.8 x+	124.7
[pg/mL]	S.d.	35.5	37.1
day 7	N	5	5
	Median	109.1	124.0
M-CSF BAL	Mean	19 x+	18
[pg/mL]	S.d.	11	8
day 7	N	5	5
	Median	14	14
OSP BAL	Mean	121.02 x+	152.33
[pg/mL]	S.d.	77.32	75.50
day 7	N	5	5
	Median	109.30	136.70
MCP-1 BAL	Mean	14.0 x+	14.0
[pg/mL]	S.d.	0.0	0.0
day 28	N	5	5
	Median	14.0	14.0
IL-8 BAL	Mean	72.7 x+	105.0
[pg/mL]	S.d.	36.6	38.0
day 28	N	5	5
	Median	77.1	94.9
M-CSF BAL	Mean	17 x+	15
[pg/mL]	S.d.	6	3
day 28	N	5	5
	Median	14	14
OSP BAL	Mean	297.68 x+	173.64
[pg/mL]	S.d.	194.53	60.56
day 28	N	5	5
	Median	198.70	144.20

BAL: cytology		Air control; males	30 mg/m3 males
TCN BAL	Mean	82.59 x+	89.82
[cn/µL BAL]	S.d.	17.82	26.42
day 7	N	5	5
	Median	75.00	94.19
MPH BAL_C	Mean	79.77 x+	84.60
[cn/µL BAL]	S.d.	17.90	27.95
day 7	N	5	5
	Median	73.69	92.08
LY BAL_C	Mean	0.29 x+	0.63
[cn/µL BAL]	S.d.	0.22	0.72
day 7	N	5	5
	Median	0.26	0.51
PMN BAL_C	Mean	2.34 x+	4.57
[cn/µL BAL]	S.d.	1.28	2.62
day 7	N	5	5
	Median	1.86	5.21
MO BAL_C	Mean	0.00 x+	0.00
[cn/µL BAL]	S.d.	0.00	0.00
day 7	N	5	5
	Median	0.00	0.00
EO BAL_C	Mean	0.19 x+	0.00
[cn/µL BAL]	S.d.	0.21	0.00
day 7	N	5	5
	Median	0.17	0.00
ATY BAL_C	Mean	0.00 x+	0.02
[cn/µL BAL]	S.d.	0.00	0.05
day 7	N	5	5
	Median	0.00	0.00
MPH BAL%	Mean	96.45 x+	92.80
[%]	S.d.	1.89	7.03
day 7	N	5	5
	Median	96.25	94.50
LY BAL%	Mean	0.35 x+	1.05
[%]	S.d.	0.29	1.57
day 7	N	5	5
	Median	0.25	0.50
PMN BAL%	Mean	2.95 x+	6.10
[%]	S.d.	1.74	5.44
day 7	N	5	5
	Median	2.75	4.50
MO BAL%	Mean	0.00 x+	0.00
[%]	S.d.	0.00	0.00
day 7	N	5	5
	Median	0.00	0.00
EO BAL%	Mean	0.25 x+	0.00
[%]	S.d.	0.31	0.00
day 7	N	5	5
	Median	0.25	0.00
ATY BAL%	Mean	0.00 x+	0.05
[%]	S.d.	0.00	0.11
day 7	N	5	5
	Median	0.00	0.00
TCN BAL	Mean	55.01 x+	50.00
[cn/µL BAL]	S.d.	8.80	12.03
day 28	N	5	5
	Median	57.84	51.40
MPH BAL_C	Mean	53.41 x+	47.99
[cn/µL BAL]	S.d.	8.38	10.59
day 28	N	5	5
	Median	53.36	48.83
LY BAL_C	Mean	0.43 x+	0.55
[cn/µL BAL]	S.d.	0.46	0.56
day 28	N	5	5
	Median	0.24	0.31
PMN BAL_C	Mean	1.08 x+	1.43
[cn/µL BAL]	S.d.	1.36	1.75
day 28	N	5	5
	Median	0.44	0.44
MO BAL_C	Mean	0.03 x+	0.03
[cn/µL BAL]	S.d.	0.06	0.07
day 28	N	5	5
	Median	0.00	0.00
EO BAL_C	Mean	0.05 x+	0.00
[cn/µL BAL]	S.d.	0.07	0.00
day 28	N	5	5
	Median	0.00	0.00
ATY BAL_C	Mean	0.00 x+	0.00
[cn/µL BAL]	S.d.	0.00	0.00
day 28	N	5	5
	Median	0.00	0.00
MPH BAL%	Mean	97.20 x+	96.40
[%]	S.d.	2.95	3.56
day 28	N	5	5
	Median	98.50	98.50
LY BAL%	Mean	0.75 x+	1.00
[%]	S.d.	0.73	0.95
day 28	N	5	5
	Median	0.50	0.75
PMN BAL%	Mean	1.90 x+	2.55
[%]	S.d.	2.33	2.65
day 28	N	5	5
	Median	1.00	1.25
MO BAL%	Mean	0.05 x+	0.05
[%]	S.d.	0.11	0.11
day 28	N	5	5
	Median	0.00	0.00
EO BAL%	Mean	0.10 x+	0.00
[%]	S.d.	0.14	0.00
day 28	N	5	5
	Median	0.00	0.00
ATY BAL%	Mean	0.00 x+	0.00
[%]	S.d.	0.00	0.00
day 28	N	5	5
	Median	0	0

Statistic Profile = Wilcoxon test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

Executive summary:

Male rats were exposed in a nose-only set-up to dusty material at a single dose of 30 mg/m3 air for five days/6h per day. Control group animals breathed the air used as vehicle. Examinations were performed both after exposure and at the end of a 21 -day treatment free recovery period. Parameters examined adressed systemic effects (clinical chemistry, haematology, organ weights) and local effects on the lung (broncheoalveolar lavage, histopathology) and were obtained from groups of either three or five animals.

Directly after exposure, treatment-related effects were observed in the adrenal gland and lungs. In the adrenal glands, an increase of the absolute (22%) and relative (28%) weight was noted. No histopathological correlate to this finding could be established, since no microscopic examination of these organs was conducted in this study. Therefore, a treatment related effect cannot be completely ruled out. The weight increase was no longer observed at the end of the recovery period. There was no indication of systemic toxicity.

In the lungs, histopathology revealed the presence of the test substance in correlation with an orange-red discoloration noted macroscopically. The test substance was predominantly observed as a dark brown mass contained predominantly in macrophages. These were diffusely distributed in the lumen of alveoli and terminal bronchioles, with a tendency to concentrate in the region of the terminal bronchioles and adjacent alveoli. Minimal amounts of free pigment particles were found lining the lumen of the alveoli without generating any observable lesions. Furthermore, a minimal hypertrophy and/or hyperplasia was predominantly observed in the terminal bronchioles and occasionally in alveolar ducts and more proximal located bronchioles. Minimal number of pigment-laden macrophages appeared also in the mediastinal and tracheobronchial lymph nodes, as part of the clearance function.

At the end of the 3-week recovery period the adrenal glands weight was within the normal range. Histopathology revealed a full regression of the hypertrophy and/or hyperplasia in the lungs. The number of luminal pigment-laden macrophages in the lungs was diminished but had not disappeared. At the same time a minimal number of pigment-laden macrophages was seen in the bronchus-associated lymphoid tissue (BALT) and in the mediastinal and tracheobronchial lymph nodes, suggesting a minimal clearance of the test substance from the lung to the regional lymph nodes.

All of these histopathological findings in the recovery group are regarded as treatment-related but since no tissue injury was present in the examined respiratory organs, all of them are considered to be adaptive and not adverse. No treatment-related findings were observed in the nasal cavity, larynx and trachea.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

The whole database is determined by two available studies, which are reliable and have consistent results..

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 2020 (study plan) to ongoing (drafting of report)
In-life phase: 14 May 2020 to 11 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

Several minor deviations, e.g. temperature relative humidity measurements outside the pre-specified ranges, were reported, but all without any effect on the study and its outcomes.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: CINIC, Lot D23508919P1
- Purity, including information on contaminants, isomers, etc.: 99.6% purity

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from humidity (tight closed container)
- expiry date: April 15, 2029
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: stable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: stable
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: none

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar Han Rat Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, SandhoferWeg 7, D-97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation:
Males: 240 – 283 g
Females: 165 – 196 g
- Fasting period before study: no
- Housing: Group caging (up to 3 animals of the same sex and dose group/cage) with additional enrichment; complying with AAALAC standards, additional enrichment (GLP Mini Fun Tunnel for hiding and chewing) was used
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Acclimation period in the study was at least 14 or 15 days. Animals were also trained to the test apparatus (restrain procedures) for 4 days and up to 6 hours prior to start of the testing in order to reduce the stress during exposure.

DETAILS OF FOOD AND WATER QUALITY:
Food: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" (Batch number: 820 61892 / 207 63596 /560 65984 / 713 70882, Expiry date: August 2020 / September 2020 / October 2020 / April 2021) produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum.
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate for the batch used. A copy of the certificate is retained in the archives of Charles River Laboratories Hungary Kft.
Water: Animals received tap water from the municipal supply, as for human consumption from 500 mL bottle ad libitum.
Water quality control analysis was performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila utca 36., Hungary). The quality control results are retained in the archives of Charles River Laboratories Hungary Kft.
The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1-24.9℃ (target range: 19-25℃)
- Humidity (%): 27-80% (target range: 30-70%)
- Air changes (per hr): at least 15 air exchanges per hour.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 May 2020 To: 11 November 2020

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 1.53 - <= 1.77 µm

Geometric standard deviation (GSD):

2.8

Remarks on MMAD:

MMAD differed slightly for the three test concentration due to the particle properties:
- 0.002 mg/L (low dose): 1.77 (GSD: 2.60)
- 0.008 mg/L (mid dose): 1.53 (GSD: 2.97)
- 0.040 mg/L (high dose): 1.71 (GSD: 2.65)

The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. Samples were collected weekly at each concentration tested and measured gravimetrically if feasible. The sampling rate was 6.0 litre/min. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data was used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and the percentages <2 µm and <4 µm (considered to be inhalable in the rat) using the corresponding software tool (Version 1.2, TSE Systems GmbH, Bad Homburg, Germany). The target ranges were < 2µm for the MMAD and 1.5 to 3 for the GSD.

Details on inhalation exposure:

Technical trials
Before animals were exposed, test item atmospheres were generated within the exposure chamber . During this period, airflow settings, test item input were varied to achieve the required atmospheric concentrations and constancy.
For the test atmosphere generation, rotating brush aerosol generators (RBG1000, Palas GmbH, Karlsruhe, Germany) were used connected to pressurized air supply. In the Low Dose group double separation glass with 2 collision metals was used, while in the Mid and High Dose groups single separator glass was used. Verification of the target concentration, particle size and particle distribution were measured gravimetrically. Technical trials included gravimetric filter analysis.

Inhalation Exposure
The atmosphere was generated according to the system and flow rates determined during the technic al trials. The exposure each day was not started until theoretical chamber concentration equilibration has been reached. During the exposure period, changes were made to airflow and test material input rates in order to achieve the required concentration.

Animal Exposure Conditions
The animals were treated by the inhalation route using a nose only exposure unit, in a TSE Rodent Exposure System with each individual concentration and control group in a dedicated tower. Modular multilevel flow – past, nose only exposure units (towers) was used. The exposure unit consisted of two, concentric stainless steel cylinders, the inner plenum and the outer chamber with 10 circularly arranged exposure ports.
The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/ relative humidity, O2 and CO2 sensors.
These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany.
The exposure units were placed in closed hoods in order to avoid cross-contamination and contam ination of the laboratory environment.
The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
Atmosphere generation was dynamic. Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where the aerosol was distributed to the individual exposure ports. After passing through the animal’s breathing zone, spent aerosol enters the outer cylinder from where it was exhausted through a suitable filter system.
Airflows and relative pressures within the system were constantly monitored and controlled by the co mputer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposu re port (breathing zone). The flow of air through each port was in the range of 1.0 L/min and 1.5 L/min.
This flow rate was considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was determined during the technical trials. However, to avoid any possible differences, the position of the animals was rotated on an exposure session basis.

Actual Test Atmosphere Concentrations
The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere (60 L/f ilter for Control and Low dose groups, 30 L/filter for Mid and High dose groups), from the exposure chamber, through 0.45 μm PVDF filters (Merck Millipore Ltd.). Samples were collected from a vacant animal exposure port (animals breathing zone) with a sampling rate of 1.0 litre/minutes. The actual sampling schedule employed was dependent on the required sample volume in order to obtain a dequate quantities of test item (exposure period) but including generally 3 samples per dose group. The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/ L) was calculated by the following formula:
CAS = M / (tS x FS),
where
M = Mass of the active substance on the filter (mg) tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere , from the exposure chamber, through 0.45 µm PVDF filters (Merck Millipore Ltd.). Samples were collected from a vacant animal exposure port (animals breathing zone) with a sampling rate of 1.0 litre/minutes. The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period) but including generally at least 3 samples per dose group

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 hours/day, 5 days/week
(with the exception of the first week which consists of 6 consecutive treatment days)

Dose / conc.:

0 mg/L air

Remarks:

control

Dose / conc.:

2.1 mg/m³ air (analytical)

Remarks:

- also refered to as low concentration
- target concentration was 2 mg/m3

Dose / conc.:

8.3 mg/L air (analytical)

Remarks:

- also refered to as mid concentration
- target concentration was 8 mg/m3

Dose / conc.:

39.2 mg/m³ air (analytical)

Remarks:

- also refered to as high concentration
- target concentration was 40 mg/m3

No. of animals per sex per dose:

15 females and 25 males per concentration

Control animals:

yes, concurrent vehicle

Details on study design:

- Main study (PEO-1): 10 females and 10 males per concentration + additional (satellite) 5 males per group (for lung burden)
- Post-exposure observation group 2 (PEO-2): 5 males per group (for lung burden after 4 weeks)
- Post-exposure observation group 3 (PEO-3): 5 females and males per group
(recovery group 13 weeks)

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Body weight of each animal was recorded with precision of 1 g at approximately one week before the first exposure, at randomization, on Day 1 (before the exposure), at least twice weekly until Day 28 and at least weekly thereafter including Day 90 (last exposure day; Day 118 for recovery), and prior to necropsy (fasted on Day 91; Day 119 for recovery). Body weight of PEO-3 animals was measured on Days 180-181.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in all animals before treatment, Day -3, and in the Control (Group 1) and High dose (Group 4) animals, during weeks 12 to 13
- Dose groups that were examined: control and high concentration group

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters in table 2 were investigated, as well the coagulation parameters Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT), both measured in seconds.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 91 (PEO-1), day 119 (PEO-2) and day 181 (PEO-3)
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the various experimental phases
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (food only)
- Parameters in table 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: YES
Time schedule for analysis: week 12/13
- Dose groups that were examined: all
- Number of animals: PEO-1: 10 males and 10 females per group,
- Parameters examined:
- sensory activity
- motor activity
- landing foot splay
- grip strength

IMMUNOLOGY: NO

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: day 91 (PEO-1) and day 180 (PEO-3)
- Dose groups that were examined: all
- Number of animals: PEO-1: 10 males and 10 females per group, PEO-3: 5 females per group
- Parameters examined:
- LDH activity (Pyruvate/NADH Kinetic (RRA) method, measured at 340/410
nm)
- Albumin (BCG staining, Endpoint (EPA) method, measured at 596/694 nm)
- total cell count and the cell viability (CountessTM II automated cell counter,
Invitrogen)
- % of alveolar macrophages (AM)
- % lymphocytes, % neutrophils
- % eosinophiles
- % other cells were determined

LUNG BURDEN: Yes
- Time schedule for analysis: day 91 (PEO-1, satellite group of 5 males), day 119 (PEO-2) and day 180 (PEO-3, males only)
- Dose groups that were examined: all
- Number of animals: PEO-1: satellite group of 5 males; PEO-2: all (i.e. 5 males); PEO-3: 5 males
- Parameters examined:
- weight of whole lungs and of right lung lobes
- analytical quantification of test substance (by HPLC-UV)
- clearance half-time

Sacrifice and pathology:

Terminally on Day 91 (on Day 119 and Day 181 for PEO-2 and PEO-3 animals, respectively) surviving animals were euthanised under sodium pentobarbital anaesthesia by exsanguination.

GROSS PATHOLOGY: Yes (see table 5)
On completion of the macroscopic examination the following tissues and organs were retained from all surviving main study (PEO-1) and all recovery animals (PEO-3 = Satellite Group 3).

HISTOPATHOLOGY:
The selected tissues (see table 5) were processed to slides in Control and High dose groups (Main Study Animals) and the animal which was found dead. In addition, full histopathology was performed on respiratory tract and associated lymph nodes in Mid and Low dose groups. Lung, tracheobronchial lymph node, mediastinal lymph node, nasal cavity and trachea from all recovery animals were proceed to microscopic evaluation. In addition, histopathology of thymus from the recovery Control and High dose females was also performed.
Any organs or tissues with macroscopic abnormalities (except minor changes) were also processed for histological examination.

Other examinations:

Lungs:
PEO-1 (Main Study Animals and Satellite Group 1):
The left lung of the Main Study Animals was preserved for histopathologic evaluation and the right lung of the Main Study Animals was used for bronchoalveolar lavage (BAL). After removal and weighing of the lungs, the right bronchus was tied off and the left lung was filled with fixative and stored for histopathology processing.
The right lung of the animals of Satellite Group 1 was used for lung burden determination. The left lung was not required but was preserved frozen (at -80oC) immediately for possible additional investigations. Accordingly, the lungs were processed as follows. After removal and weighing, the further procedure of the right lung lobes is described in section 15.3.
PEO-2 (Satellite Group 2):
The left lung of the animals was infused and preserved for possible histopathologic evaluation; the right lung of the animals was used for lung burden determination.
Accordingly, the lungs were processed as follows. After removal and weighing, the right bronchus was tied off and the left lung of the animals using for possible histopathology evaluation was inflated with formalin to ensure high quality fixation. The further procedure of the right lung lobes is described in section 15.2 below.
PEO-3 (Satellite Group 3, i.e. Recovery Animals):
The left lung of the female animals was preserved for histopathologic evaluation and the right lung of the female animals was used for bronchoalveolar lavage (BAL).
The left lung of the male animals was preserved for histopathologic evaluation and the right lung of the male animals was used for lung burden determination.
Accordingly, the lungs were processed as follows. After removal and weighing, the right bronchus was tied off and the left lung was filled with fixative and stored for histopathology processing. The further procedure of the right lung lobes is described in section 15.2 (males) and section 15.3 (females) below.
The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
Bone marrow smears (taken from the femur of all animals) were preserved, examination was not required.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 (when using Provantis).
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of continuous numeric data.
The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova/Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01, as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01, as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001, as appropriate.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse clinical signs were observed in the Low, Mid or High dose groups during the observation period.
During the 90-day treatment period, fur staining by test item was observed in all treated male and female animals.
During the 4-week recovery period, alopecia (1/5 Mid dose males), fur staining by test item (all treated males), scar (1/5 Mid dose males) and wound (1/5 Mid dose males) were noted.
During the 13-weeks recovery period alopecia (1/5 Low dose and 1/5 High dose females), thin fur (2/5 Low dose females) and fur staining by test item (all treated males and females) were observed.
Observations in this study on the skin and fur were not considered to be related to test item, other than fur staining, which is a non-adverse, test item related change.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One Control female (#1503) became pregnant due to a technical error, therefore it was excluded from the study after agreement with the Sponsor (pre-terminally euthanized on Day 59).

One Low dose female (#2506) was found dead on Day 81 during exposure. Fur staining by test item on the head, nose and snout was recorded prior to death. At necropsy diffuse dark red discolouration of the non-collapsed lungs and red diffuse discolouration of the mediastinal and tracheobronchial lymph nodes were noted of this animal. Microscopically minimal multifocal pigment in the alveolar macrophages and minimal pulmonary congestion in the lung and minimal congestion in the mediastinal lymph nodes were observed. The death of this animal was not attributed to treatment (occasionally small animals can turn their head in the exposure tube, causing suffocation, although there was no clear evidence for a cause of death in this case).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related differences in the body weight or body weight gain values in any of the dose groups when compared to the controls during the 90-day treatment period and the 4-weeks and 13-weeks recovery periods. The measured values were within the range commonly recorded for this strain and age.
Some sporadic significant body weight gain and body weight differences were seen for some of the weekly evaluations, without a clear trend, therefore these changes were considered to be incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment related differences in the food consumption values in any of the treated groups when compared to the control between Day 1 and Day 90 and at the end of the 4 weeks and 3 months recovery periods. Sporadic, statistically significant changes (in Mid dose males in PEO-3 Group between Days 176-180 and in Low and Mid dose females in PEO-3 Group between Days 1-8) were considered to be incidental.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No direct test item-related adverse changes were detected in the test item treated animals (males and females) when comparing haematology parameters to control data.

In PEO-1 male animals, statistically significantly lower relative reticulocyte and lymphocyte values (in High dose Group) and higher relative neutrophil value (in Mid and High dose Groups) were observed compared to the Control Group on Day 91 (see Result Table 1 below).
The changes in the WBC distribution were considered to be a not adverse, secondary response which was correlated with the results of BAL evaluation. Despite of statistically significant changes in the relative and absolute values of reticulocytes, these changes were considered to be incidental, since the values were in the historical control ranges and there were no abnormalities in the values of red blood cells, haemoglobin and mean cell haemoglobin concentrations.

In PEO-1 females, statistically significant increased white blood cell (WBC) and absolute neutrophil granulocytes counts (by 66.1% and 104.3%, respectively) in the High dose group were observed when compared to the controls at 91 days. These changes were not considered to be a primary effect of test item and were in line with the results of BAL evaluation.

In recovery animals on Day 119, a sporadic statistically significant difference in one parameter (prothrombin time) was considered to be incidental.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing the serum chemistry parameters to the relevant Control data at the end of the treatment period on Day 91.

Sporadic statistically significant values of recovery animals measured on Day 181 were considered to be incidental.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.

Statistically significantly higher glucose value in High dose males was noted compared to Controls on Day 91, however this change was considered incidental and not treatment related. Lower urine volumes in Low and Mid dose females measured compared to Controls on Day 91 were within the normal historical control range. Therefore, these differences were considered not to be treatment related changes.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There was no adverse effect of treatment noted during the assessment of grip strength, foot splay, Irwin Test or locomotor activity in any dose group when compared with the control.

In the Mid and High dose males, an apparent dose-related decrease in grip strength of the hind limbs was measured, however the control values in this study were higher than a contemporaneous study, where the control values were similar to the treated groups of this study; hence the statistical differences in this study are not considered to be an adverse effect of the treatment.

The locomotor activity was considered to have showed a normal response in the High group, it was initially high then reduced to a plateau at approximately 30 minutes. All locomotor activity data were considered as normal; therefore the analysis of the Mid and Low dose groups of PEO-1 and PEO-2 and PEO-3 groups were considered to be not required.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Main animals (PEO-1 without satellite 1):

There was no test item related effect on the terminal body weights in test item exposed main study animals.
Significant weight changes were observed in the lungs, mediastinal lymph nodes and heart in test item exposed High dose males and females, and in the epididymis in High and Mid dose males (see Results Tables 2 and 3 below).
The decrease in the weight of hearts in both sexes and epididymis weights in test item exposed High Dose males had no correlating histopathological findings and hence no toxicological significance was assigned.

Lungs

The absolute and relative (to body and brain weights) lung weights were statistically significantly larger in High dose animals (see Table 6 below).

Mediastinal lymph nodes

The absolute and relative (to body and brain weights) mediastinal lymph node weights were statistically significantly larger in High dose animals (see Table 6 below).
The increase in lung and mediastinal lymph node weights in test item exposed animals correlated with the macroscopic and histopathological findings (see below). 

Heart

The absolute and relative (to body and brain weights) heart weights were statistically significantly lower in High dose animals.

Epididymis

The absolute and relative (to body and brain weights) epididymis weights were slightly lower in all treated males.

The decrease in the weight of hearts in both sexes and epididymis weights in test item exposed High Dose males had no correlating histopathological findings and hence no toxicological significance was assigned.




4-week recovery animals

There was no test item related effect on the terminal body weights in previously test item exposed satellite group 2 animals. When compared to the Controls, test item related weight changes were observed in the lungs of High Dose males.

Lungs

The absolute and relative (to body and brain weights) lung weights were statistically significantly larger in High Dose males (see Results Table 4 below).




13-week recovery animals

When compared to the Controls, test item related weight changes were observed in the lungs and in the mediastinal lymph node in test item exposed High Dose males and females. Weight of thymus statistically significantly decreased in High Dose females; however, this decrease did not show dose-response and it was within the normal historical control range, hence it was considered not to be test item related.

Lungs

The absolute and relative (to body and brain weights) lung weights were statistically significantly larger in High Dose animals (see Results Tables 5 and Table 6 below).
 
Mediastinal lymph nodes

The absolute and relative (to body and brain weights) mediastinal lymph node weights were statistically significantly larger in High Dose animals (ee Results Tables 5 and Table 6 below).

The increase in lung and mediastinal lymph node weights in test item exposed animals correlated with the macroscopic and histopathological findings (see below).

Thymus

The absolute and relative (to body and brain weights) thymus weights were statistically significantly lower in High Dose females.

The decrease in the weight of thymus weights in previously test item exposed females had no correlating histopathological findings and hence no toxicological significance was assigned.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Main animals

Test item-related macroscopic findings at 90 days were detected in the lungs in all dosed groups and in the mediastinal lymph node in Mid and High dose groups at necropsy.
The lungs showed a dose-related diffuse/multifocal red to dark red discoloration, the mediastinal lymph nodes were diffuse red discoloration and enlarged. The red colour is considered to be test item.
The other findings, due to very low incidence in both dosed and control groups, were considered as incidental or background.


Recovery animals (4 weeks, PEO-2)

Test item-related macroscopic findings were detected in the lungs and in the mediastinal lymph node in Mid and High Dosed groups at necropsy.
The lungs showed diffuse/multifocal red discoloration, and a diffuse red discoloration was noted for the mediastinal lymph node and it was enlarged as well.
No other finding was observed.


Recovery animals (3 months, PEO-3)

Test item-related macroscopic findings were detected in the lungs and in the mediastinal lymph node at necropsy.
The lungs showed diffuse red discoloration in all High dose animals and in 1/5 Mid dose male rat. The mediastinal lymph node showed a diffuse red discoloration in 3/5 Mid dose and 5/5 High dose males and in all Mid and High dose females. Enlargement of the mediastinal lymph node was noted in 1/5 Mid dose and 5/5 High dose males and in 2/5 Mid dose and 5/5 High dose females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Main animals (PEO-1, excluding satellite group)

Test item-related findings were observed in the lungs, trachea, nasal cavity, mediastinal lymph node and tracheobronchial lymph node.

Lungs

Minimal to marked multifocal pigments mainly in the macrophages and distributed in the alveolar lumen and wall were observed in all treated groups. The free pigments / pigmented macrophages were seen distributed throughout the lung parenchyma with predominance at the centro acinar region. Pigments were also seen in the bronchial associated lymphoid tissue (BALT). Minimal multifocal bronchiolo alveolar epithelial hyperplasia, which was considered to be regenerative, was seen around the pigmented macrophages in High Dose animals and one Mid Dose female (see Results Table 7).

Mediastinal Lymph Node

Minimal to moderate pigment and/or mild to moderate increased cellularity were seen in Mid and High Dose animals (see Results Table 8).

Tracheobronchial Lymph Node

Pigment at minimal to mild severity was seen in test item exposed Mid and High Dose animals. Increased cellularity at minimal severity was seen in High Dose animals (see Results Table 9).

Nasal cavity

Minimal amounts of pigment were seen in the nasal associated lymphoid tissue in High Dose Animals. Minimal focal/multifocal eosinophilic globules were seen in respiratory epithelium in a few High Dose animals (see Results Table 10).

Trachea

Minimal focal pigments were seen in the submucosa of the carina in 1/10 Mid Dose males, 2/10 High Dose males, 2/10 Mid Dose females and 1/10 High Dose females.

All other findings were considered incidental or background.



Recovery animals (4 weeks)

Test item-related findings were observed in the lungs, mediastinal lymph node, tracheobronchial lymph node and nasal cavity (see Results Tables 11 -14).

Lungs

Minimal to marked amounts of pigments multifocally distributed in the alveolar lumen and wall were observed in all treated groups. Pigments were also seen in the BALT. Minimal multifocal bronchiolo alveolar epithelial hyperplasia of regenerative nature, characterized by reactive macrophage type II proliferation was seen around the pigmented macrophages in Mid and High Dose males

Mediastinal Lymph Node

Minimal to moderate pigment were seen in Low, Mid and High Dose males. Increased cellularity was observed in Mid and High Dose males.

Tracheobronchial Lymph Node

Pigment at minimal to mild severity was seen in test item exposed Mid Dose animals.

Nasal cavity

Minimal focal eosinophilic globules were seen in respiratory epithelium in a few High Dose animals.



Recovery animals (3 months)

Test item-related findings were observed in the lungs, nasal cavity, mediastinal lymph node and tracheobronchial lymph node (see Results Tables 15 -18).

Lungs

Minimal to marked multifocal pigments mainly in the macrophages and distributed in the alveolar lumen and wall were observed in all treated groups. Pigments were also seen in the BALT. Minimal multifocal bronchiolo alveolar epithelial hyperplasia of regenerative nature, characterized by reactive macrophage type II proliferation, was seen in Mid and High Dose animals.

Mediastinal Lymph Node

Minimal to marked pigment were seen in Low, Mid and High Dose animals. Increased cellularity was observed in Mid and High Dose animals.

Tracheobronchial Lymph Node

Pigment at minimal to moderate severity was seen in test item exposed Low, Mid and High Dose animals. Increased cellularity was seen in Mid and High Dose males and High Dose females.

Nasal cavity

Minimal focal/multifocal eosinophilic globules were seen in respiratory epithelium in High Concentration males, Mid and High Concentration females. Although minimal eosinophilic globules were also observed in 1/5 and 1/5 males from the Low and Mid Concentration groups, respectively, these occasions were not considered as test item-related findings since similar low evidence occurred in 2/10 Control Hannover Wistar male rats in one 90-day inhalation study in this facility. Taking into consideration that there was no evidence of eosinophilic globules in Low and Mid Concentration males at the end of treatment, as well as after 4 weeks of recovery, this incidence below the High Concentration level was considered as background rather than test item-related.
At the 13-week recovery, minimal severity of eosinophilic globules was reported in 2/5 Mid and 2/5 High Concentration females. Although the incidence in 13 week recovery females was greater than background and the same as at the High Concentration (at 2/5) it is difficult to attribute the Mid Concentration eosinophilic globules to the treatment with test item after 13 weeks of recovery, although it cannot be clearly excluded.


All other findings were considered incidental or background.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar Lavage
Dose-related increase in neutrophils was observed in males and females, this change was statistically significant in the Mid and/or High dose groups. LDH was also increased in both males and females, with statistical significance in the High dose groups. These changes indicate a possible tissue injury and/or development of an inflammation-associated response in lungs. Recovery of neutrophil and LDH values were noted in females at the end of the 3-month recovery period, however increased LDH values in the High dose females were still statistically significant (see Results Table 19).


Lung Burden Determination
In the lungs, the amount of test item found at the end of treatment was very approximately proportional to the atmosphere concentrations. For the lung burden in the Low dose animals, a significant decrease of the test item was measured during the recovery (~75% clearance), while in case of the mid dose, this decrease was less significant (~57%) and thus the elimination half time was close to the limit of 90 days for normal clearance. In case of the high dose, a relatively small decrease can be seen (~23%), probably due to an overload of the lung (see Results table 20 and 21).

Key result

Dose descriptor:

NOAEC

Effect level:

2.1 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

8.3 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Result Table 1: Summary of selected haematology parameters of PEO-1 animals on Day 91

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose
Males
Reticulocyte relative (%)	2.12	2.14	1.95	1.81*	DN
HC range: 1.7-2.8	difference (%)	0.8	-8.1	-14.7
Reticulocyte absolute (K/uL)	180.64	182.34	167.01	155.37*	DN
HC range: 146.9-230.9	difference (%)	0.9	-7.5	-14.0
Neutrophil relative (%)	19.12	22.83	25.32*	27.41**	DN
HC range: 17.4-46.4	difference (%)	19.4	32.4	43.3
Lymphocyte relative (%)	74.91	71.43	68.02	65.71*	DU
HC range: 48.9-78.4	difference (%)	-4.6	-9.2	-12.3
Females
White blood cell count (K/uL)	1.551	1.544	1.834	2.577*	DN
HC range: 0.34 – 2.24	difference (%)	-0.5	18.2	66.1
Neutrophil absolute (K/uL)	0.313	0.321	0.329	0.640**	DN
HC range: 0.09 – 0.62	difference (%)	2.5	5.0	104.3

 

Notes: Data (group mean values, n=8-10) were rounded to two or three decimal places.

HC: Historical control

DN: Dunnett’s test, DU: Dunn test.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

 

 

Result Table 2: Selected organ weights in males on Day 91

Organ weight (males)	Dose groups
	Control	Low dose	Mid dose	High dose
Terminal body weight (g)	332.7	343.8	336.7	329.5	NS
	difference (%)	3.3	1.2	-1.0
Lung (absolute) (g)	1.342	1.391	1.321	1.631**	DU
	difference (%)	3.7	-1.6	21.5
Lung (relative to body weight) (%)	0.403	0.404	0.393	0.495**	DU
	difference (%)	0.3	-2.6	22.7
Lung (relative to brain weight) (%9	66.39	66.89	61.22	78.49*	DU
	difference (%)	0.8	-7.8	18.2
Lymph node mediastinal (absolute) (g)	0.0110	0.0164	0.0101	0.0383**	DU
	difference (%)	49.2	-8.0	247.8
Lymph node mediastinal (relative to body weight) (%)	0.0033	0.0048	0.0030	0.0116**	DU
	difference (%)	44.5	-8.9	251.3
Lymph node mediastinal (relative to brain weight) (%)	0.544	0.784	0.467	1.851**	DU
	difference (%)	44.1	-14.1	240.2

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

 

Result Table 3: Selected organ weights in females on Day 91

Organ weight (females)	Dose groups
	Control	Low dose	Mid dose	High dose
Terminal body weight (g)	209.0	207.6	208.7	204.9	NS
	difference (%)	-0.7	-0.1	-2.0
Lung (absolute) (g)	1.087	1.081	1.096	1.334**	DU
	difference (%)	-0.5	0.9	22.8
Lung (relative to body weight) (%)	0.519	0.522	0.525	0.652**	DN
	difference (%)	0.6	1.0	25.4
Lung (relative to brain weight) (%)	55.42	56.26	55.65	67.20**	DU
	difference (%)	1.5	0.4	21.3
Lymph node mediastinal (absolute) (g)	0.0124	0.0109	0.0120	0.0383**	DN
	difference (%)	-11.6	-3.5	209.0
Lymph node mediastinal (relative to body weight) (%)	0.0059	0.0053	0.0057	0.0187**	DN
	difference (%)	-9.6	-3.0	218.0
Lymph node mediastinal (relative to brain weight) (%)	0.630	0.571	0.609	1.926**	DN
	difference (%)	-9.4	-3.3	205.7

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

 

Results Table 4: Lung weights in males on Day 119

Organ weight (males)	Dose groups
	Control	Low dose	Mid dose	High dose
Males
Terminal body weight (g)	363.0	393.6	374.0	367.4	NS
	difference (%)	8.4	3.0	1.2
Lung (absolute) (g)	1.426	1.470	1.450	1.860**	DN
	difference (%)	3.1	1.7	30.4
Lung (relative to body weight) (%)	0.393	0.374	0.388	0.506**	DN
	difference (%)	-4.7	-1.4	28.8
Lung (relative to brain weight) (%)	66.32	69.17	67.36	89.95*	DN
	difference (%)	4.3	1.6	29.6

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

Results Table 5: Lung weights in males on Day 181

Organ weight (males)	Dose groups
	Control	Low dose	Mid dose	High dose
Terminal body weight (g)	427.0	438.2	457.4	426.2	NS
	difference (%)	2.6	7.1	-0.2
Lung (absolute) (g)	1.498	1.406	1.532	1.846**	DU
	difference (%)	-6.1	2.3	23.2
Lung (relative to body weight) (%)	0.350	0.322	0.336	0.433**	DN
	difference (%)	-8.1	-4.0	23.6
Lung (relative to brain weight) (%9	66.91	64.35	68.65	83.19**	DN
	difference (%)	-3.8	2.6	24.3
Lymph node mediastinal (absolute) (g)	0.0188	0.0145	0.0167	0.0470**	DN
	difference (%)	-22.7	-11.5	149.7
Lymph node mediastinal (relative to body weight) (%)	0.0044	0.0033	0.0037	0.0110**	DN
	difference (%)	-24.6	-15.9	149.7
Lymph node mediastinal (relative to brain weight) (%)	0.840	0.665	0.745	2.107**	DN
	difference (%)	-20.8	-11.4	150.8

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

 

Results Table 6: Lung weights in females on Day 181

Organ weight (females)	Dose groups
	Control	Low dose	Mid dose	High dose
Terminal body weight (g)	242.0	238.4	244.4	238.2	NS
	difference (%)	-1.5	1.0	-1.6
Lung (absolute) (g)	1.146	1.138	1.144	1.626**	DN
	difference (%)	-0.7	-0.2	41.9
Lung (relative to body weight) (%)	0.474	0.478	0.468	0.684**	DN
	difference (%)	1.0	-1.2	44.4
Lung (relative to brain weight) (%)	55.61	56.47	57.11	81.71**	DN
	difference (%)	1.6	2.7	46.9
Lymph node mediastinal (absolute) (g)	0.0142	0.0123	0.0161	0.0423**	DN
	difference (%)	-13.3	13.2	197.2
Lymph node mediastinal (relative to body weight) (%)	0.0058	0.0052	0.0067	0.0178**	DN
	difference (%)	-10.1	15.4	208.1
Lymph node mediastinal (relative to brain weight) (%)	0.687	0.610	0.814	2.121**	DN
	difference (%)	-11.2	18.6	208.9

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

 

Results Table 7: Incidence of test item related microscopic changes in the lungs

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	10	10	10	10	9	9	10	10
Pigment, Macrophage, alveolar, multifocal
Minimal	0	10	0	0	0	9	0	0
Mild	0	0	9	0	0	0	2	0
Moderate	0	0	1	9	0	0	8	10
Marked	0	0	0	1	0	0	0	0
Total	0	10	10	10	0	9	10	10
Pigment, Bronchial-associated lymphoid tissue, focal/multifocal
Minimal	0	0	6	9	0	1	8	10
Total	0	0	6	9	0	1	8	10
Hyperplasia, Bronchiolo-Alveolar, Regenerative focal/multifocal
Minimal	0	0	0	10	0	0	1	10
Total	0	0	0	10	0	0	1	10
Number of Tissues examined	10	10	10	10	9	9	10	10

 

Results Table 8: Incidence of test item related microscopic changes in the mediastinal lymph node

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	10	10	10	10	9	9	10	10
Pigment
Minimal	0	0	6	0	0	0	1	0
Mild	0	0	3	0	0	0	7	1
Moderate	0	0	0	10	0	0	1	9
Total	0	0	9	10	0	0	9	10
Cellularity, Increased
Mild	0	0	0	2	0	0	0	2
Moderate	0	0	0	8	0	0	0	8
Total	0	0	0	10	0	0	0	10
Number of tissues examined	10	9	10	10	9	9	10	10

 

Results Table 9: Incidence of test item related microscopic changes in the tracheobronchial lymph node

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	10	10	10	10	9	9	10	10
Pigment
Minimal	0	0	5	0	0	0	2	0
Mild	0	0	2	6	0	0	0	3
Total	0	0	7	6	0	0	2	3
Cellularity, Increased, lymphocyte
Minimal	0	0	0	4	0	0	0	0
Total	0	0	0	10	0	0	0	10
Number of tissues examined	8	10	8	7	7	6	7	6

 

 

Results Table 10: Incidence of test item related microscopic changes in the nasal cavity

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	10	10	10	10	9	9	10	10
Pigment, nasal associated lymphoid tissue, focal/multifocal
Minimal	0	0	0	5	0	0	0	4
Total	0	0	0	5	0	0	0	4
Eosinophilic globules (droplets), respiratory epithelium, focal/multifocal
Minimal	0	0	0	4	0	0	0	3
Total	0	0	0	4	0	0	0	3
Number of tissues examined	10	10	10	10	9	9	10	10

 

 

Results Table 11: Incidence of test item related microscopic changes in the lungs in satellite group 2 animals

	Males
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40
Number of animals	5	5	5	5
Pigment, Macrophage, alveolar, multifocal
Minimal	0	5	0	0
Mild	0	0	1	0
Moderate	0	0	4	4
Marked	0	0	0	1
Total	0	5	5	5
Pigment, Bronchial-associated lymphoid tissue, focal/multifocal
Minimal	0	1	2	3
Mild	0	0	0	1
Total	0	1	2	4
Hyperplasia, Bronchiolo-Alveolar, Regenerative focal/multifocal
Minimal	0	0	4	3
Total	0	0	4	3
Number of Tissues examined	5	5	5	5

 

Results Table 12: Incidence of test item related microscopic changes in the mediastinal lymph node in satellite group 2 animals

	Males
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40
Number of animals	5	5	5	5
Pigment
Minimal	0	1	1	0
Mild	0	0	4	0
Moderate	0	0	0	5
Total	0	1	5	5
Cellularity, Increased, minimal	0	0	4	0
Mild	0	0	0	4
Moderate	0	0	0	1
Total	0	0	4	5
Number of tissues examined	5	5	5	5

 

Results Table 13: Incidence of test item related microscopic changes in the tracheobronchial lymph node in satellite group 2 animals

	Males
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40
Number of animals	5	5	5	5
Pigment
Minimal	0	0	1	0
Mild	0	0	1	0
Total	0	0	2	0
Number of tissues examined	3	4	3	2

 

Results Table 14: Incidence of test item related microscopic changes in the nasal cavity

	Males
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40
Number of animals	5	5	5	5
Eosinophilic globules (droplets), respiratory epithelium, focal
Minimal	0	0	0	3
Total	0	0	0	3
Number of tissues examined	5	5	5	5

 

Results Table 15: Incidence of test item related microscopic changes in the lungs in satellite group 3 animals

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	5	5	5	5	5	5	5	5
Pigment, Macrophage, alveolar, multifocal
Minimal	0	5	0	0	0	4	0	0
Mild	0	0	3	0	0	0	3	0
Moderate	0	0	2	4	0	0	2	0
Marked	0	0	0	1	0	0	0	5
Total	0	5	5	5	0	4	5	5
Pigment, Bronchial-associated lymphoid tissue, focal/multifocal
Minimal	0	0	4	5	0	0	4	3
Mild	0	0	0	0	0	0	0	1
Total	0	0	4	5	0	0	4	4
Hyperplasia, Bronchiolo-Alveolar, Regenerative multifocal
Minimal	0	0	4	3	0	0	1	3
Mild	0	0	0	1	0	0	0	2
Total	0	0	4	4	0	0	1	5
Tissues examined	5	5	5	5	5	5	5	5

 

Results Table 16: Incidence of test item related microscopic changes in the mediastinal lymph node in satellite group 3 animals

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	5	5	5	5	5	5	5	5
Pigment
Minimal	0	1	0	0	0	3	1	0
Mild	0	0	2	0	0	0	4	0
Moderate	0	0	1	4	0	0	0	2
Marked	0	0	0	1	0	0	0	3
Total	0	1	3	5	0	3	5	5
Cellularity, Increased, lymphocytes
Minimal	0	0	2	0	0	0	4	0
Mild	0	0	1	3	0	0	0	5
Moderate	0	0	0	2	0	0	0	0
Total	0	0	3	5	0	0	4	5
Number of tissues examined	5	5	5	5	5	5	5	5

 

Results Table 17: Incidence of test item related microscopic changes in the tracheobronchial lymph node in satellite group 3 animals

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	5	5	5	5	5	5	5	5
Pigment
Minimal	0	1	0	0	0	1	1	1
Mild	0	0	3	2	0	1	0	2
Moderate	0	0	0	2	0	0	0	0
Total	0	1	3	4	0	2	1	3
Cellularity, Increased, lymphocyte
Minimal	0	0	2	0	0	0	0	1
Mild	0	0	1	3	0	0	0	0
Total	0	0	3	3	0	0	0	1
Number of tissues examined	3	3	4	4	4	3	2	5

 

Results Table 18: Incidence of test item related microscopic changes in the nasal cavity in satellite group 3 animals

	Males				Females
Target test atmosphere concentration (mg/m^3)	0	2.0	8.0	40	0	2.0	8.0	40
Number of animals	5	5	5	5	5	5	5	5
Eosinophilic globules (droplets), respiratory epithelium, focal/multifocal
Minimal	0	1	1	3	0	0	2	2
Total	0	1	1	3	0	0	2	2
Number of tissues examined	5	5	5	5	5	5	5	5

 

Results Table 19: Summary of selected BAL parameters in males and females on Day 91

Parameters	Dose groups
	Control	Low dose	Mid dose	High dose
Males
BAL-LDH (U/L)	312.4	123.7**	328.0	643.2**	DN
	difference (%)	-60.4	5.0	105.9
BAL-Neutrophils (10^6)	0.003	0.002	0.013*	0.285**	DU
	difference (%)	-37.3	419.1	11251.2
Total Cell Number (10^6)	0.449	0.301*	0.206**	0.527	DU
	difference (%)	-33.0	-54.1	17.4
Females
BAL-LDH (U/L)	214.4	326.8	350.6	764.8**	DN
	difference (%)	52.4	63.5	256.6
BAL-Neutrophils (10^6)	0.003	0.002	0.013	0.245**	DU
	difference (%)	-33.2	309.2	7747.1
Total Cell Number (10^6)	0.334	0.200*	0.158**	0.509	DN
	difference (%)	-40.2	-52.8	52.2

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s test, DU: Dunn test, NS: Statistically not significant when compared to the control.

 

Results Table 20: Measured mean test item content (mg/g) in lung

Satellite group	Clearance time (week)	Measured test item content in lung (mg/g)
		Low dose	Mid dose	High dose
PEO-1	0	0.421	2.402	9.042
PEO-2	4	0.260	2.142	7.804
PEO-3	13	0.105	1.037	7.009

 

Results Table 21: Measured percentage of the clearance over time

Satellite group	Clearance time (week)	Measured clearance (%)
		Low dose	Mid dose	High dose
PEO-1	0	-	-	-
PEO-2	4	38.1	10.8	13.7
PEO-3	13	74.9	56.8	22.5

 

Conclusions:

The sub-chronic inhalation toxicity of the registered substance was investigated in a GLP-compliant 90-day study by inhalation (nose-only) in rats with 13-week recovery according to OECD TG 413. No systemic effects were observed and adverse local changes in the respiratory tract were limited to the Mid and High Concentration groups treated with test item. Based on these results, the No Observed Adverse Effect Concentration (NOAEC) for the study was 2.1 mg/m3 (Low Concentration group).

Executive summary:

The sub-chronic inhalation toxicity of the registered substance was investigated in a GLP-compliant 90-day study by inhalation (nose-only) in rats with 13-week recovery according to OECD TG 413. The objective of this 90-day study was to obtain information on the toxicity of the test item when administered to Wistar (Han) rats via inhalation route for at least 90 days with the aim of inducing toxic effects but no death or suffering at the highest of three test concentrations, and little or no evidence of toxicity at the lowest concentration. The methods used followed OECD TG 413, with the procedures that are foreseen for a test item which accumulates in the lungs. According to this, additional subgroups were used to evaluate the recovery of the animals and to investigate the clearance rate of the Test Item from the lung.

The animals were exposed to the test atmosphere at carefully determined target concentrations of 2, 8 and 40 mg/m3, as the Low, Mid and High Concentration group (also referred to as “Dose group”), respectively, using a nose-only exposure system over 90 days. 

Ten male and 10 female rats in each group (Control (filtered air), Low, Mid and High Concentration), referred to as main study animals, were treated by a 6 hour nose-only exposure to filtered air or three fixed aerosol concentrations for consecutive 5 days/week and 2 days without treatment with the exception of the first week with 6 consecutive treatment days. The main study animals were sacrificed on Day 91, i.e. on the day following the last treatment, and histopathology evaluation and BALF (Bronchoalveolar lavage fluid) analysis were performed. Additionally, 5 females per group were treated and allowed to recover for 13 weeks and sacrificed on Day 181 (histopathology evaluation and BALF analysis were performed) and 15 males per group were treated and five rats each sacrificed on Day 91 (Satellite 1), Day 119 (PEO-2 with 4-week recovery) and Day 181 (PEO-3 with 13-week recovery) for histopathology and/or lung burden evaluation.

The exposure did not cause any test item-related death or any adverse clinical signs, nor were test item-related changes in body weight, food intake, in behaviour, opththalmology evaluation,oestrus cycle, haematology, clinical chemistry and urine parameters observed.

In the bronchoalveolar lavage analysis, increased neutrophil values in Mid and High Concentration males and females and increased LDH values in High Concentration males and females were considered as treatment related.

Results of lung burden determination showed that in case of the Low Concentration group, continuous clearance was observed from the lung over time, with ~75% clearance after 3 month. In the Mid Concentration group the clearance was ~57%, while in the High Concentration group, the clearance rate was much lower (~23%) probably due to overload, considering the low rate of removal of the test item from the lung.

Microscopically, pigments in the macrophages of the lungs were seen in animals of all Concentration groups but considered not adverse. In the Mid and High Concentration groups additional test item-related changes in the lungs, nasal cavity, trachea, mediastinal lymph node and/or tracheobronchial lymph node were observed. Changes in the lungs, mediastinal and tracheobronchial lymph nodes consisted of an increase of slight to moderate cellularity and minimal bronchiolo alveolar epithelial hyperplasia of regenerative nature. This change may be considered adverse but is not consistent with a pre-neoplastic lesion. It is indicative of repair and not of an early pre-neoplastic change which is supported by the lack of formation of connective tissue and the absence of any indication for fibrosis. Eosinophilic globules seen in the nasal cavity, without nasal tissue injury following repeated inhalation exposure of the test item, represents a non-adverse adaptive response. After 4 and 13 weeks of recovery partial or full recovery was generally observed.

 

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant non-guideline study with well-characterized test material

Qualifier:

no guideline available

Principles of method if other than guideline:

5-day inhalation exposure with 21 days recovery group.
Ma-Hock et al. ( 2009) Inhal Toxicol 21, 102-118

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation: Main group day 0: control group: 246g; test group: 251g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust-free bedding, supplied by SSNIFF, Soest, Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks (Typ NGM E-022), supplied by Abedd Lab. and Vet. Service GmbH, Vienna, Austria, were added.
- Diet: Mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum.
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

other: dust aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The two measurements of particle size resulted in equal MMAD value of 0.7 μm with GSD of 2.9 and 2.5. The calculated mass fractions of particles below MMAD 3 μm was 92.2 and 94.6 %.

Figures with results of WELAS, SMPS and Cascade Impactor determinations are shown in the attachment.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. By means of dust generators the substance to be tested is generated into dust aerosols using compressed air in a mixing stage, mixed with conditioned air and passed into the inhalation systems via cyclonic separators. For each concentration, a solid particle generator (brush-generator) was used for generating the dust. The concentration were adjusted by varying the piston feed and by varying the brush rotation. For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed via the cyclonic separator into the inhalation system.
- The following test substance flow, air flows and evaporation temperatures were scheduled: see table M1

EXPERIMENTAL PROCEDURE
- Head-nose exposure systems: The inhalation atmosphere was maintained inside aerodynamic exposure systems (test group 1 in INA 60, volume V ≈ 90 L, control group in INA 120, volume V ≈ 120 L) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.
- Exposures: The head-nose exposure technique was preferably selected for this aerosol/dust/ inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult. Furthermore, by using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of the final target concentration) is shorter as compared to whole-body chambers with a higher chamber volume. A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air. In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours from Monday to Friday to reach 5 exposures. The animals did not have access to water or feed during the exposure.
- Measurements of the exposure conditions: The following exposure parameters were recorded: Supply air (conditioned), Supply air 2 (compressed), Exhaust air, Chamber humidity, Chamber temperature, Real time concentration surveillance. No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

VEHICLE
- Composition of vehicle: Conditioned supply air is activated charcoal filtered air conditioned to about 30% - 70% relative humidity and 20°C - 24°C. Compressed air is filtered air pressurized to about 6 bar.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CALCULATION OF NOMINAL CONCENTRATIONS: The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.

ANALYTICAL DETERMINATION OF CONCENTRATIONS: The concentration of the inhalation atmosphere in test group 1 was analyzed by gravimetry. This analytical method is judged to be valid because the test substance does not possess an appreciable vapor pressure. Daily means were calculated based on 3 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. In the test group, the constancy of the dust atmosphere in the chamber was continuously monitored using scattered light photometers.
- Sampling for gravimetric analyses: Equipment: Sampling equipment with probe (Millipore Corporation, Billerica, MA 01821, USA), Internal probe diameter: 7 mm, Filter: MN 85/90 BF (d = 4.7 cm), Vacuum pump (Millipore Corporation, Billerica, MA 01821, USA), Balance: Sartorius M3P-000V001 (Sartorius AG, Göttingen, Germany). Sampling: Sampling velocity: 1.25 m/s, Flow rate of sampling: 3 L/min, Sample volumes Test group 1: 90 L, Sampling site: immediately adjacent to the animals' noses at a separate spare port, Sampling frequency: as a rule, 3 samples per exposure and concentration group
- Gravimetrical method of analyses: A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust aerosol was drawn through the filter. The dust concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmosphere.
REAL TIME MONOTORING OF CONSTANCY OF CONCENTRATIONS: Scattered light photometer (VisGuard (Sigrist) in test group 1 was used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. The measurements were recorded using line recorders.

PARTICLE SIZE ANALYSIS:
- Definitions: EACD 50% (effective aerodynamic cutoff diameter 50%) defines the separation characteristic of each impactor stage. 50% of particles with the EACD given are deposited in the pertinent impactor stage; the remainder has reached one of the following stages. MMAD (mass median aerodynamic diameter) is the calculated aerodynamic diameter which divides the size distribution in half when measured by mass. Geometrical standard deviation (GSD) is the ratio of the estimated 84 percentile to the 50 percentile and indicates the slope of the cumulative particle size distribution curve.

- Equipment, sampling and method of determination: The particle size analysis was carried out with a cascade impactor. Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA), Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA), Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA), Sampling probe internal diameter 7 mm, Balance Sartorius M3P-000V001 (Sartorius AG, Göttingen, Germany), Mettler XP 205.(Mettler-Teldo AG, Greifensee, Switzerland)

- Sampling for particle size analyses: Preweighed metal collecting discs and a backup particle filter were placed into the cascade impactor and 2 samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals. A sample volume of 90 L was used in each test group. Method of analysis: Gravimetrical determination. The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the metal collecting disc and backup filter before and after sampling. The deposits in the probe and the wall losses in the impactor were also determined as difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter. Evaluation: The calculation of the particle size distribution was carried out in the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (DIN 66141: Darstellung von Korngrößenverteilungen and DIN 66161: Partikelgrößenanalyse, Beuth-Vertrieb GmbH, Berlin und Köln, Germany).
- Particle size distribution measurements with APS: Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used (TSI APS 3321). Frequency: on two days during the exposure period, with 3 repeats on each day.
- Particle size distribution measurements with Optical particle counter: For each test atmosphere measurements with an optical particle counter (WELAS 2000; (Palas® GmbH, Karlsruhe, Germany)) were performed to determine the size distribution of particles with diameters larger than 246 nm. The WELAS 2000 uses a white-light source to illuminate a measurement volume through which particles have to move singly. The measuring range of the sensor was 0.246 to 9.653 μm and the sampling flow rate 5 L/min.
- Particle size distribution measurements with scanning mobility particle sizer: To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 μm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.083 μm according to the software calculation. The duration of each measurement cycle was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily for five consecutive days

Remarks:

Doses / Concentrations:
30 mg/m³
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
31.34 mg/m³; SD = 4.49 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

3/dose/group (main group or recovery group for microscopic examination)
5/dose/group (main group or recovery group for blood sampling and BAL)

Control animals:

yes, concurrent vehicle

Details on study design:

On study day 4 after exposure and on study day 25, 3 animals per group and time point were sacrificed underwent gross necropsy. Selected organs were weighed, a broad set of organs and tissues were preserved, respiratory tract was examined histologically.
On study days 7 and 28, blood was sampled from 5 rats/group and time point. Clinical chemistry parameters, hematology parameters and acute phase proteins were examined in blood. After blood sampling the animals underwent bronchoalveolar lavage. Lavage fluid was examined for cytological and biochemical parameters including selected antigens.

Observations and examinations performed and frequency:

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

CLINICAL OBSERVATIONS: The clinical condition of the test animals was recorded once daily during the pre-exposure period and on post-exposure observation days on working days. On exposure days, clinical observation was performed at least 3 times daily, before, during and after exposure. During exposure only a group wise examination was possible.

BODY WEIGHT: The body weight of the animals was determined at the start of the pre-exposure (day -4), and then, as a rule, twice a week (Monday and Friday), as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weights from Monday to Friday. The main reason for this type of calculation is to show body weight change of the exposure week without the exposure-free weekend. It enables detection of minor decrease of body weight gain, which may be overlooked because the animals recover during the weekend. Group means were derived from the individual differences.

CLINICAL PATHOLOGY: In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The examinations for haematology and clinical chemistry were carried out in 5 animals per test group.

HAEMATOLOGY: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Prothrombin time (Hepato Quick’s test) (HQT) was measured. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

CLINICAL CHEMISTRY: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL).

ACUTE PHASE PROTEINS IN SERUM: Rat α2-macroglobulin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E-25A2M). Rat haptoglobin was measured with an ELISA produced by Immmunology Consultants Laboratory Inc., Newberg, OR, USA (cat. no. E-25HPT). Both ELISA kits were measured with a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer.

BRONCHOALVEOLAR LAVAGE FLUID (BAL): The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline. The following examinations were carried out in 5 male animals per test group.
- Cytology in BAL: Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. Parameters: Total cell count (BALTCN), Macrophages (BALMPH), Polymorphonuclear neutrophils (BALPMN), Lymphocytes (BALLY), Eosinophils (BALEO), Monocytes (BALMO), Atypical cells (BALATY).
- Total Protein and enzymes in BAL: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the humoral parameters in the bronchoalveolar lavage fluid. Parameter: γ−Glutamyltransferase (GGT), Protein (MTP), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP), N-acetyl-β-Glucosaminidase (NAG)
- Antigens in BAL: The antigens were measured with MTP ELISAs at a Sunrise MTP Reader, Tecan AG, Switzerland, by using the Magellan Software provided by the instrument producer. The following antigens were measured in BALF: Rat monocyte chemoattractant protein-1 (rat MCP-1) level measured with an Instant ELISA produced by Bender MedSystems, Vienna, Austria (cat. no BMS631INST), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8) measured with an ELISA produced by R&D Systems Inc., Minneapolis, US, (Quantikine rat CINC-1, cat. no. RCN100), Macrophage colony stimulating factor (M-CSF) measured with a Quantikine Mouse M-CSF ELISA produced by R&D Systems Inc., Minneapolis, USA (cat no. MMC00), Rodent osteopontin measured with an ELISA produced by R&D Systems, Inc., Minneapolis, US (Quantikine mouse osteopontin, cat. no. MOST00).

Sacrifice and pathology:

NECROPSY: All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS: The following weights were determined in all animals sacrificed on schedule: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands.

HISTOPATHOLOGY: The following organs or tissues were fixed in 4% buffered formaldehyde or modified Davidson’s solution: All gross lesions, Adrenal glands, Brain with olfactory bulb, Bone marrow (femur), Epididymides, Eyes with optic nerve and eyelids, Heart, Kidneys, Larynx/Pharynx, Liver, Lungs, Lymph nodes (tracheobronchial and mediastinal lymph nodes), Nose (nasal cavity), Oesophagus, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Stomach (forestomach and glandular stomach), Spleen, Testes, Thyroid glands, Thymus, Trachea, Urinary bladder. From the liver, one additional slice of the Lobus dexter medialis and the Lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast. The testes were fixed in modified Davidson’s solution. Fixation was followed by histotechnical processing and examination by light microscopy.
Tissues and organs examined histologically: All gross lesions (only affected animals), Nasal cavity (4 levels), Larynx (3 levels), Trachea, Lungs (5 lobes), Lymph nodes (tracheobronchial and mediastinal lymph nodes).

Statistics:

- Body weight, body weight change: A comparison of the dose group with the control group was performed using the student t-test (two-sided) for the hypothesis of equal means.
- Clinical Pathology parameters in Blood: Pair-wise comparison of the dose group with the control group was performed using Wilcoxon test (two-sided) for the equal medians.
- Clinical Pathology parameter in BALF, Pair-wise comparison of the dose group with the control group was performed using Wilcoxon test (one-sided) for the equal medians.

Throughout the sections of the report, when intergroup differences are referred to as "significant " it implies that the differences have attained statistical significance (p ≤ 0.05) when compared with the control group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute and relative weight of the adrenal glands showed an increase of +22% and +28%, respectively at the end of the exposure period. They were no longer increased at the end of the recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

An orange red discoloration was observed in the lungs of all treated males and in the fur of most of them. This finding is regarded as treatment-related and was due to the presence of the test substance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The test substance was mainly stored in the cytoplasm of macrophages as dark brown fine pigment particles.

Details on results:

The following findings were noted immediately at the end of the exposure phase:

No treatment-related changes of the acute phase protein levels in serum were observed.

Examination of bronchoalveolar lavage fluid showed no adverse effects (see tables 2)

The test substance was mainly stored in the cytoplasm of macrophages a dark brown fine pigment particles. A moderate number of pigment-laden macrophages were diffusely distributed in the lungs but were predominantly present in the lumen of terminal bronchioles, alveolar ducts and adjacent alveoli. The number of pigment particles in the macrophages was also moderate (see table 1) A minimal number of pigment-laden macrophages was observed in the bronchus-associated lymphoid tissue (BALT) and free pigment particles were found in alveoli and bronchioles. The epithelial hypertrophy and/or hyperplasia was generally observed at the level of terminal bronchioles and alveolar ducts and in more proximal located bronchiole.
Terminal bronchioles were primarily affected. This finding was characterized by an increased size and number of bronchiolar epithelial cells, accompanied by a slight cytoplasmic basophilia.

In the nasal cavity and larynx, minimal to slight amounts of free pigment particles were found admixed with the mucus secretion.
Minimal focal changes in the larynx, such as epithelial alteration in the ventral pouch of the epiglottis or lymphoid infiltrations were observed, but are considered to be background lesions that are not related to treatment. No findings were noted in the trachea.

No pigment was detected in mediastinal and tracheobronchial lymph nodes.


The following findings were noted at the end of the recovery period:
- terminal body weight (+9%)
- absolute kidneys weight (+26%) and relative (+15%)
- absolute liver weight (+18%)
Since the body weight development did not show adverse effects, the terminal body weight increase is regarded as incidental and not related to treatment. The increase of liver and kidneys weight is considered to be in relation with the terminal body weight increase. These changes likely respond to the expected standard deviations when only 3 animals per group are tested.

All males showed an orange red discoloration in the lungs, which is regarded as treatment-related.
Compared with the control group 0, all males showed slight number of pigment-laden macrophages diffusely distributed in the lung with a marked tendency to accumulate but not to aggregate in the bronchiolo-alveolar junction (terminal bronchiole, alveolar ducts and adjacent alveoli). The bronchus-associated lymphoid tissue (BALT) of all males showed minimal number of pigment-laden macrophages.
No treatment-related findings were observed in the nasal cavity, larynx and trachea. A minimal number of pigment-laden macrophages was detected in the mediastinal lymph node. A minimal to slight number of pigment-laden macrophages was found in the tracheobronchial lymph node.

Key result

Dose descriptor:

NOAEC

Effect level:

30 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Presence of pigment-laden macrophages in the lung

Critical effects observed:

no

Table 1: Results of histopathology findings after exposure and at the end of the recovery period

	after exposure	after exposure	after recovery	after recovery
Concentration (mg/m3)	0	30	0	30
Animals examined	3	3	3	3
Macrophages, pigment-laden	0	3 (grade 3)	0	3 (grade 2)
Macrophages, pigment-laden, in bronchus-associated lymphoid tissue	0	3 (grade 1)	0	3 (grade 1)
Free pigment particles	0	3 (grade 1)	0	0
Hypertrophy/hyperplasia, terminal bronchiole	0	3 (grade 1)	0	0

Tables 2: Findings of broncheo-alveolar lavage (days 7 and 28)

(For full names of parameters, see "Observations and examinations performed and frequecy")

BAL: Cytokines		Air control; males	30 mg/m3 males
MCP-1 BAL	Mean	14.0 x+	23.8
[pg/mL]	S.d.	0.0	16.8
day 7	N	5	5
	Median	14.0	14.0
IL-8 BAL	Mean	120.8 x+	124.7
[pg/mL]	S.d.	35.5	37.1
day 7	N	5	5
	Median	109.1	124.0
M-CSF BAL	Mean	19 x+	18
[pg/mL]	S.d.	11	8
day 7	N	5	5
	Median	14	14
OSP BAL	Mean	121.02 x+	152.33
[pg/mL]	S.d.	77.32	75.50
day 7	N	5	5
	Median	109.30	136.70
MCP-1 BAL	Mean	14.0 x+	14.0
[pg/mL]	S.d.	0.0	0.0
day 28	N	5	5
	Median	14.0	14.0
IL-8 BAL	Mean	72.7 x+	105.0
[pg/mL]	S.d.	36.6	38.0
day 28	N	5	5
	Median	77.1	94.9
M-CSF BAL	Mean	17 x+	15
[pg/mL]	S.d.	6	3
day 28	N	5	5
	Median	14	14
OSP BAL	Mean	297.68 x+	173.64
[pg/mL]	S.d.	194.53	60.56
day 28	N	5	5
	Median	198.70	144.20

BAL: cytology		Air control; males	30 mg/m3 males
TCN BAL	Mean	82.59 x+	89.82
[cn/µL BAL]	S.d.	17.82	26.42
day 7	N	5	5
	Median	75.00	94.19
MPH BAL_C	Mean	79.77 x+	84.60
[cn/µL BAL]	S.d.	17.90	27.95
day 7	N	5	5
	Median	73.69	92.08
LY BAL_C	Mean	0.29 x+	0.63
[cn/µL BAL]	S.d.	0.22	0.72
day 7	N	5	5
	Median	0.26	0.51
PMN BAL_C	Mean	2.34 x+	4.57
[cn/µL BAL]	S.d.	1.28	2.62
day 7	N	5	5
	Median	1.86	5.21
MO BAL_C	Mean	0.00 x+	0.00
[cn/µL BAL]	S.d.	0.00	0.00
day 7	N	5	5
	Median	0.00	0.00
EO BAL_C	Mean	0.19 x+	0.00
[cn/µL BAL]	S.d.	0.21	0.00
day 7	N	5	5
	Median	0.17	0.00
ATY BAL_C	Mean	0.00 x+	0.02
[cn/µL BAL]	S.d.	0.00	0.05
day 7	N	5	5
	Median	0.00	0.00
MPH BAL%	Mean	96.45 x+	92.80
[%]	S.d.	1.89	7.03
day 7	N	5	5
	Median	96.25	94.50
LY BAL%	Mean	0.35 x+	1.05
[%]	S.d.	0.29	1.57
day 7	N	5	5
	Median	0.25	0.50
PMN BAL%	Mean	2.95 x+	6.10
[%]	S.d.	1.74	5.44
day 7	N	5	5
	Median	2.75	4.50
MO BAL%	Mean	0.00 x+	0.00
[%]	S.d.	0.00	0.00
day 7	N	5	5
	Median	0.00	0.00
EO BAL%	Mean	0.25 x+	0.00
[%]	S.d.	0.31	0.00
day 7	N	5	5
	Median	0.25	0.00
ATY BAL%	Mean	0.00 x+	0.05
[%]	S.d.	0.00	0.11
day 7	N	5	5
	Median	0.00	0.00
TCN BAL	Mean	55.01 x+	50.00
[cn/µL BAL]	S.d.	8.80	12.03
day 28	N	5	5
	Median	57.84	51.40
MPH BAL_C	Mean	53.41 x+	47.99
[cn/µL BAL]	S.d.	8.38	10.59
day 28	N	5	5
	Median	53.36	48.83
LY BAL_C	Mean	0.43 x+	0.55
[cn/µL BAL]	S.d.	0.46	0.56
day 28	N	5	5
	Median	0.24	0.31
PMN BAL_C	Mean	1.08 x+	1.43
[cn/µL BAL]	S.d.	1.36	1.75
day 28	N	5	5
	Median	0.44	0.44
MO BAL_C	Mean	0.03 x+	0.03
[cn/µL BAL]	S.d.	0.06	0.07
day 28	N	5	5
	Median	0.00	0.00
EO BAL_C	Mean	0.05 x+	0.00
[cn/µL BAL]	S.d.	0.07	0.00
day 28	N	5	5
	Median	0.00	0.00
ATY BAL_C	Mean	0.00 x+	0.00
[cn/µL BAL]	S.d.	0.00	0.00
day 28	N	5	5
	Median	0.00	0.00
MPH BAL%	Mean	97.20 x+	96.40
[%]	S.d.	2.95	3.56
day 28	N	5	5
	Median	98.50	98.50
LY BAL%	Mean	0.75 x+	1.00
[%]	S.d.	0.73	0.95
day 28	N	5	5
	Median	0.50	0.75
PMN BAL%	Mean	1.90 x+	2.55
[%]	S.d.	2.33	2.65
day 28	N	5	5
	Median	1.00	1.25
MO BAL%	Mean	0.05 x+	0.05
[%]	S.d.	0.11	0.11
day 28	N	5	5
	Median	0.00	0.00
EO BAL%	Mean	0.10 x+	0.00
[%]	S.d.	0.14	0.00
day 28	N	5	5
	Median	0.00	0.00
ATY BAL%	Mean	0.00 x+	0.00
[%]	S.d.	0.00	0.00
day 28	N	5	5
	Median	0	0

Statistic Profile = Wilcoxon test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

Executive summary:

Male rats were exposed in a nose-only set-up to dusty material at a single dose of 30 mg/m3 air for five days/6h per day. Control group animals breathed the air used as vehicle. Examinations were performed both after exposure and at the end of a 21 -day treatment free recovery period. Parameters examined adressed systemic effects (clinical chemistry, haematology, organ weights) and local effects on the lung (broncheoalveolar lavage, histopathology) and were obtained from groups of either three or five animals.

Directly after exposure, treatment-related effects were observed in the adrenal gland and lungs. In the adrenal glands, an increase of the absolute (22%) and relative (28%) weight was noted. No histopathological correlate to this finding could be established, since no microscopic examination of these organs was conducted in this study. Therefore, a treatment related effect cannot be completely ruled out. The weight increase was no longer observed at the end of the recovery period. There was no indication of systemic toxicity.

In the lungs, histopathology revealed the presence of the test substance in correlation with an orange-red discoloration noted macroscopically. The test substance was predominantly observed as a dark brown mass contained predominantly in macrophages. These were diffusely distributed in the lumen of alveoli and terminal bronchioles, with a tendency to concentrate in the region of the terminal bronchioles and adjacent alveoli. Minimal amounts of free pigment particles were found lining the lumen of the alveoli without generating any observable lesions. Furthermore, a minimal hypertrophy and/or hyperplasia was predominantly observed in the terminal bronchioles and occasionally in alveolar ducts and more proximal located bronchioles. Minimal number of pigment-laden macrophages appeared also in the mediastinal and tracheobronchial lymph nodes, as part of the clearance function.

At the end of the 3-week recovery period the adrenal glands weight was within the normal range. Histopathology revealed a full regression of the hypertrophy and/or hyperplasia in the lungs. The number of luminal pigment-laden macrophages in the lungs was diminished but had not disappeared. At the same time a minimal number of pigment-laden macrophages was seen in the bronchus-associated lymphoid tissue (BALT) and in the mediastinal and tracheobronchial lymph nodes, suggesting a minimal clearance of the test substance from the lung to the regional lymph nodes.

All of these histopathological findings in the recovery group are regarded as treatment-related but since no tissue injury was present in the examined respiratory organs, all of them are considered to be adaptive and not adverse. No treatment-related findings were observed in the nasal cavity, larynx and trachea.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2.1 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The whole database is determined by two available studies, which are reliable and have consistent results..

Justification for classification or non-classification

The comparison of the experimental evidence with various PSLT (poor solubility and low toxicity) criteria leads to the conclusion that PR254 is a poor soluble substance with low toxicity. The detailed assessment is attached under 'Additional information'. Furthermore, in a 90-day rat inhalation study no adverse effects were observed at concentrations not overburdening the lungs. Effects seen at concentrations leading to lung overload, i.e., the increase in neutrophiles and the hyperplasia of regenerative nature, are local effects commonly observed for particles tested at concentrations leading to lung overload. Therefore, the adverse effects observed were substance-unspecific particle effects, which do not warrant a classification.