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

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In-life phase: 14 May 2020 to 11 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 Jule 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

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

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)
- 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

Test animals

Species:

rat

Strain:

Wistar

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
- 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

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 1.53 - <= 1.77

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.

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)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2.1 mg/m³ air (analytical)

Remarks:

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

Doses / concentrations 2

Dose / conc.:

8.3 mg/m³ air (analytical)

Remarks:

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

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Results of examinations

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.

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.

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

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

Pigment at minimal severity was 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 multifocal pigments 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, regenerative 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, regenerative 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 Low, Mid and High Dose animals.

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).


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, in the 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 increased cellularity and bronchiolo alveolar epithelial hyperplasia of regenerative nature. This change may be considered adverse but is not consistent with a preneoplastic lesion. The term ‘bronchiolo alveolar hyperplasia’ is used based on the limited pathological standard “INHAND” nomenclature. 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. After 4 and 13 weeks of recovery partial or full recovery was observed.

Details on results:

Discussion:
During the in-life phase, there was one death Day 81 (Low Concentration female). The death of this animal may have been related to the technical procedures of restraint for 6 hours per day.
Body weight, food consumption, clinical observations and neuromuscular assessments were all normal in all groups.
At haematology and clinical chemistry, there were no indications of any treatment related
effects.
At necropsy, significantly increased lung and mediastinal lymph node weights were seen in High Concentration male and female groups, but there were no systemic organ weight or macroscopic effects identified any other organ or tissue.
Bronchoalveolar lavage analysis reflected the lung organ weight and microscopic findings; increased neutrophil values in Mid and High Concentration males and females and increased LDH values in High Concentration males and females, indicating an inflammatory response. Lung burden determination showed ~75% clearance from the Low Concentration lung samples after 3 months, ~57% in the Mid Concentration and ~23% clearance from the High Concentration samples. Clearance was significantly lower in the High Concentration group.
At histopathology, test item related changes were observed in the lungs, nasal cavity, trachea, mediastinal and tracheobronchial lymph nodes in the main study animals exposed to the test item for 90 days.
In the lung at 90 days, macrophages loaded with a pigment, which is considered to be test item, was seen distributed throughout the parenchyma with a predominance around the centro acinar region in High Concentration animals. Pigment was also seen outside the macrophages within the alveolar lumen/alveolar wall. Alveolar pigments and pigmented macrophages correlated with macroscopic red discolouration in the lung observed at necropsy and increase in the lung weight observed in High Concentration animals. There was no degenerative or inflammatory reaction around the pigmented macrophages. Thus, the pigmented macrophages in the lung observed in the current study was considered as a result of normal non adverse physiological response to phagocytose the inhaled test item. In addition, minimal bronchiolo alveolar epithelial hyperplasia of regenerative nature characterised by increase in the size/number of epithelial cells in the alveolar wall around the centro acinar region with pigmented macrophages were also seen in 10/10 High Concentration males, 10/10 High Concentration females and in 1/10 Mid Concentration females. This change may be considered adverse (Nikula et al 2014) but is not consistent with a pre-neoplastic lesion. The term ‘bronchiolo alveolar hyperplasia’ is used based on the limited pathological standard “INHAND” (International Harmonization of Nomenclature and Diagnostic Criteria) nomenclature. It indicates repair and not an early pre-neoplastic change which is supported by the lack of formation of connective tissue and the absence of any indication for fibrosis. Although the BAL data indicated an inflammatory response, the severity was probably below the level detectable by histopathology.
Pigment was also seen in the BALT, NALT, tracheobronchial and mediastinal lymph nodes, indicating drainage of the test item into the lymphatic system. The lymph nodes also showed increase in cellularity in majority of High Concentration animals in both sexes. The presence of pigment and increase in the cellularity correlated with the macroscopic observation in High Concentration animals. These findings are considered to reflect normal clearance of the test item.
Minimal focal/multifocal eosinophilic globules seen in respiratory epithelium in the nasal cavity of a few High Concentration animals was considered as not adverse.
After 4 weeks of recovery period (PEO-2 males), the test item related changes were still present in lungs and mediastinal lymph node at all concentration levels, nasal cavity in High Concentration animals, tracheobronchial lymph node in Mid Concentration animals. Tracheal changes were not present in High Concentration PEO-2 males indicating full recovery. The histopathological changes observed in the lung and mediastinal lymph node correlated with macroscopic observations and organ weight changes.
After 13 weeks of recovery period (satellite group 3 males and females), test item related changes were still present in the lungs, mediastinal lymph node, tracheobronchial lymph node and nasal cavity at all concentration levels, except Low Concentration females. Bronchiolo alveolar epithelial hyperplasia of regenerate nature appeared to have partially reversed; it was still present in the Mid and High Concentration 4-week and 13-week recovery animals, however Low Concentration animals did not show this finding at the end of the recovery periods. Despite the relatively low percentage clearance of test item after 3 months recovery, the histopathological pulmonary appearance did not indicate a worsening of the effects, the appearance was of a normal though overloaded clearance. Tracheal changes as observed in the main study animals were not present in High Concentration PEO-3 animals indicating full recovery. The histopathological changes observed in the lung, mediastinal and tracheobronchial lymph nodes correlated with macroscopic observations and organ weight changes in lungs and mediastinal lymph node.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

39.2 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse systemic effects observed up to the highest tested concentration.

2

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

2.1 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

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<0.01DN: 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/m3)	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/m3)	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/m3)	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/m3)	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/m3)	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/m3)	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/m3)	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/m3)	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/m3)	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/m3)	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/m3)	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/m3)	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

 

Applicant's summary and conclusion

Conclusions:

In conclusion, under the conditions of this study 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 the actual findings, the No Observed Adverse Effect Concentration (NOAEC) for the study was 2.1 mg/m3 (Low Concentration group).

Executive summary:

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 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. The concentration levels were set by the Sponsor in consultation with the Study Director based on information from previous experimental work, including the results of a preliminary dose range finding study in the rat and published literature.

Ten male and 10 female Wistar Hannover rats Crl:WI(Han) in each group (Control, 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 sacrificed on Day 91 (Satellite 1), Day 119 (PEO-2 with 4-week recovery) on Day 181 (PEO-3 with 13-week recovery) for histopathology and/or lung burden evaluation.

Start of the exposure for all 4 groups was Day 1. For practical reasons, the start of treatment of males and females was staggered by one day An Air Control Group was exposed to filtered air. The treatment was performed in 4 parallel inhalation towers, one tower for each treatment or control group. Surviving PEO-1 animals including control were terminated on the day following the last exposure on Day 91. The PEO-2 animals were terminated on Day 119, the PEO-3 animals were terminated on Day 181.

The actual atmospheric concentrations (gravimetric determination) were 2.1 ± 0.2 (low dose), 8.3 ± 0.5 (mid dose) and 39.2 ± 1.9 (high dose), respectively.  The achieved concentrations for all groups were considered to be sufficiently precise for Concentration and Stability of the exposures. Analysis of control samples showed that no test item was detected. A Low Concentration female animal was found dead on Day 81, and one Control female was excluded from the study; these findings were not considered to be test item related. The exposure did not cause any test item-related adverse clinical signs or change in body weight or food intake. The functional observation battery (FOB) and neuromuscular observations showed no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups. No treatment-related changes were noted on ophthalmoscopy examination. No toxicologically significant effects were observed in haematology, clinical chemistry or urine parameters. There were no test item-related observations in the oestrus cycle. 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, in the 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 hanges 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 increased cellularity and bronchiolo alveolar epithelial hyperplasia of regenerative nature. This change may be considered adverse but is not consistent with a pre- neoplastic lesion. The term ‘bronchiolo alveolar hyperplasia’ is used based on the limited pathological standard “INHAND” nomenclature. 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. After 4 and 13 weeks of recovery partial or full recovery was observed.

In conclusion, under the conditions of this study 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 the actual findings, the No Observed Adverse Effect Concentration (NOAEC) for the study was 2.1 mg/m³ (Low Concentration group).