Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2019-01-18 to 2019-10-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
2009
Deviations:
yes
Remarks:
: see chapter 'any other information on materials and methods' section 7.5.2 /1
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Ltd.
- Age at study initiation:61 to 67 days
- Weight at study initiation: 222 to 275 g (males) and 150 to 205 g (females)
- Fasting period before study:no
- Housing: The animals were housed two of one sex per cage for the Main study and Recovery.
- Diet: Ad libitum, standard rodent diet (Teklad 2014C Diet) except overnight before routine blood sampling and during dosing. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C
- Humidity: 40 to 70%
- Air changes (per hr):Each animal room was supplied with filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h continuous dark/24 h.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.5 - <= 2.7 µm
Geometric standard deviation (GSD):
2.45
Remarks on MMAD:
MMAD / GSD: The Particle size distribution (PSD) data was originally characterised by using linear regression of the probit of the cumulative percentage, by massof particles smaller than cut-point of each stage versus the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD); geometric standard deviation of the aerosol were derived for each occasion of measurement.

Group Aerosol concentration (µg/L) Particle size
Target Achieved MMAD (µm) GSD
gravimetric
1 - - - -
2 1.5 1.55 2.5 2.45
3 6 6.50 2.5 2.41
4 24 26.0 2.7 2.41

MMAD Mass median aerodynamic diameter (µm)
GSD Geometric standard deviation
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow through nose only chamber. This system was an aluminium alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber.
- Method of holding animals in test chamber: Rats were held in plastic tubes with their snouts protruding from the end of the tubes into the exposure chamber.
- Source and rate of air: From in-house compressed air system(breathing quality), Generator flow: 20L/minute
- System of generating particulates/aerosols: Wright Dust Feed mechanism designed to produce and maintain atmospheres of dust by suspending material scraped from the surface of a compressed powder in a stream of dry air. The aerosol exiting the WDF mechanism was supplied to a jet mill ( Jet mill supply flows: 20 to 40 L/minute) in order to reduce the particle size of the powder prior to delivery to the exposure chamber (Airflow exiting jet mill: 22 to 28 L/minute)
- Temperature, humidity, pressure in air chamber:
Temperature was measured using an electronic thermometer inserted into the inhalation chamber. The daily mean chamber temperature values were within the accepted guidelines 19 to 25 °C. The lowest and the mid-dose groups had a mean chamber temperature of 22.2°C, the mean value for the high-dose group was 23.5 °C and the mean value for the control group was 22.5°C.
Humidity was measured using an electronic hygrometer inserted into the inhalation chamber. The lowest dose group had an overall mean chamber relative humidity of 24.5%, the mean values for the mid and high-dose groups were lower at 9.7 and 15.9 % respectively. The mean value of the control group was higher at 46.7%. The low values were considered to be a result of the dried air supplied from generator.
Pressure in air chamber was not reported.
- Air flow rate: Airflow to Wright dust Feed was 20 L/minute for all groups.
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 8-stage cascade impactor (Marple 298). Samples were collected at least once during each week of exposure for each concentration tested. Samples were also collected from a vacant animal exposure port (animals breathing zone). The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, was calculated by this difference. The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than < 0.42, 0.76, 1.27, 2.86, 4.9, 8.0, 12.1 and 17.4 µm (aerodynamic diameter) was calculated. From this data, the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated.
- Treatment of exhaust air: Extract airflow was drawn by in-house vacuum system at a flow rate from 40 to 160 L/minute depending of the exposure concentration of the group . the airflow was filtered locally.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The exposure concentrations were monitored during each exposure by gravimetrical analysis of the test item deposited on a sampling filter. On 3 occasions, the gravimetric analysis was coupled to analytical analysis by LC -MS/MS to check the accuracy of the gravimetric method and ensure that the composition of the material in the test atmospheres was similar to the original material and was stable across the exposure period..
The mean achieved concentrations were 1.55; 6.50 and 26.0 µg/L and corresponded to 103; 108 and 108% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.
Duration of treatment / exposure:
13 weeks followed by a 12- week recovery period
Frequency of treatment:
5 days / week
Doses / concentrationsopen allclose all
Dose / conc.:
1.5 mg/m³ air (nominal)
Dose / conc.:
1.55 mg/m³ air (analytical)
Dose / conc.:
6 mg/m³ air (nominal)
Dose / conc.:
6.5 mg/m³ air (analytical)
Dose / conc.:
24 mg/m³ air (nominal)
Dose / conc.:
26 mg/m³ air (analytical)
No. of animals per sex per dose:
- 3 groups, each comprising 10 male and 10 female rats received the test substance at target exposure levels of 1.5, 6 or 24 µg/L. A similarly constituted Control group received air only, at the same operating conditions as the high dose group.
- A further 10 male and 10 female rats were assigned to each groups: these animals were treated for 13 weeks, which was followed by a 12 week period without treatment to assess recovery from any treatment related effect. (See in chapter 'Any other information on materials and methods' section 7.5.2 /2 Identity of treatment groups)
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:
In a previously conducted two-week repeat dose inhalation toxicity study, rats were exposed 5 days/week, 6 hours/day to 0, 29.8, 108 and 499 µg/L of test substance. No animals died during the treatment period and there were no treatment related clinical findings apparent during the study. Treatment related histopathological findings in the lungs were evident for animals that received 108 or 499 µg/L and were expected to progress with prolonged exposure. For this study, a high exposure level of 24 µg/L was expected to be tolerated, and intermediate and low exposure levels of 6 and 1.5 µg/L were selected to explore any exposure-related relationship.

- Fasting period before blood sampling for clinical biochemistry:
Blood samples were collected after overnight withdrawal of food and prior to dosing.

- Rationale for selecting satellite groups:
# In order to assess recovery from any treatment related effect, 10 male and 10 female rats were assigned to each groups for a 12-week recovery period.
# To provide an assessment of the in vivo pulmonary response of the test item, investigations on the bronchoalveolar lavage fluid were performed in all main study and recovery phase animals.

- Post-exposure recovery period in satellite groups: 12 weeks
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupants.
Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed
condition, as appropriate.
During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily detailed observations were recorded at the following times in relation to dose administration:
* Pre-exposure observation
* As each animal is returned to its home cage
* As late as possible in the working day

In addition observations were made in the treatment period, on days without exposures, at the following times during the day:
* Early in the working day (equivalent to pre-exposure observation)
* As late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded twice weekly from Week -1 to Week 4 and weekly thereafter, and before necropsy.

FOOD CONSUMPTION: Yes
-The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and each week throughout the treatment and recovery periods. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during Week 13 of treatment. As no treatment-related changes were observed, the
examination was not extended to the recovery phase.
- Dose groups that were examined: all animals before treatment commenced and all main study animals of Groups 1 (Control) and 4 (26.0 µg/L) during week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 13 of treatment for all main study animals (before dosing) and during Week 12 of recovery for all animals
in Groups 1 (Control), 2 ( 1.55 µg/L), 3 (6.50 µg/L) and 4 (26.0 µg/L).
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: During Week 13 of treatment for all main study animals and during Week 12 of recovery for all animalsof the recovery phase.
- Parameters examined: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (RETA), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count including Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (PTP) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 13 of treatment
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: all main study animals. As no treatment-related changes were observed, the analysis was not extended to the recovery phase.
- Parameters examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No


Sacrifice and pathology:
All main study animals were sacrificed ifollowing 13 weeks of treatment. Recovery phase animals were sacrificed following 13 weeks of treatment and 12 week of recovery.
GROSS PATHOLOGY: Yes
All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue
(external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
HISTOPATHOLOGY: Yes
The following tissues were examined for all main study animals of Groups 1 (Control) and 4 (26.0 µg/L) sacrificed on completion of the 13-week treatment
period:
Adrenals
Aorta - thoracic
Brain - cerebellum, cerebrum and medulla/pons
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femur and marrow - femorotibial joint
Harderian glands
Heart - included auricular and ventricular regions
Ileum
Jejunum
Kidneys
Larynx - 5 sections
Liver - section from two main lobes
Lungs - the right lung was used for BronchoAlveolar Lavage sampling and the left lung was processed for histopathology
Lymph nodes -left axillary
Lymph nodes -tracheobronchial
Lymph nodes -hilar; the hylar lymph node is not visible at macroscopic examination and sampling of this lymph node at histology is only possible when there is an immune response. in this study, hilar lymph node was not visible and therefore not sampled.
Nasal turbinates -4 levels including nasal cavity, paranasal sinuses and nasopharynx, Nasal Associated Lymphoid Tissue and the teeth
Ovaries
Pancreas
Pituitary
Prostate
Salivary gland - submandibular/ sublingual, only one examined
Sciatic nerves -only one examined
Seminal vesicle
Skeletal muscle -only one examined
Skin with mammary glands - inguinal area
Spinal cord - transverse and longitudinal sections at the cervical, lumbar and thoracic levels
Spleen
Sternum - included bone marrow
Stomach
Testes
Thymus
Thyroid - included parathyroids
Trachea -including bifurcation and sampled at 3 levels, cranial, middle and caudal
Urinary bladder
Uterus - uterine body with cervix section
Vagina
In addition:
- mediastinal lymph nodes showing macroscopic abnormality were examined for main study animals,
- Axillary lymph nodes were considered to exhibit a reaction to treatment at the highest exposure level, and were examined for all main study and recovery animals,
- Nasal turbinates, larynx, trachea, left lung,tracheobronchial lymph nodes were examined for all main study animals of groups 2 (1.55 µg/L) and 3 (6.50 µg/L).

The following tissues were examined for recovery animals sacrificed on completion of the 13-week treatment period followed by a 12-week off dose period:
- mediastinal lymph nodes showing macroscopic abnormality
- Axillary lymph nodes
- Nasal turbinates, larynx, trachea, left lung,tracheobronchial lymph nodes of all recovery animals
Other examinations:
- HAEMATOLOGY, BONE MARROW:
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment period for all main study animals.The smears
from animals of Groups 1 (Control) and 4 (26.0 µg/L) sacrificed on completion of the 13-week treatment period were examined to assess the cellularity,
distribution and morphology of the marrow. The smears from animals of the intermediate and low dose groups were retained but not examined.

- ORGAN WEIGHTS:
The following organs, taken from each animal killed after 13 weeks of treatment and after 12 week of recovery were weighted: Brain, Epididymides, Heart,
Kidneys, Liver, Lungs, Ovaries, Spleen, Testes, Thymus, Thyroid with parathyroids.
Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

- BRONCHOALVEOLAR LAVAGE (BAL):
The right lung of all main study and recovery phase animals was used for bronchoalveolar lavage sampling and the left lung was processed for histology and light microscopy.
BAL fluid samples were examined for:

# Total and differential cell counting: Cell and differential cell counts were performed using the XT-2000iV (Sysmex UK Ltd). Differential cell count included
Mononuclear cells number (includes macrophages and monocytes), Mononuclear cells number % (includes macrophages and monocytes), Eosinophil number, Eosinophil %, Basophil number, Basophil % , Neutrophil number , Neutrophil % , Lymphocyte number, Lymphocyte %. The number of each cell type per
animal were recorded and also expressed as a percentage of the total count.

#Total protein analysis: This analysis was indicative of inflammatory processes and damage to the alveolar capillary barrier.

# Phospholipid analysis: This analysis was performed to determine disturbances in the metabolic activity of type II epithelial cells.The assay was not validated at this time and results were given for information only.

# Lactate dehydrogenase analysis: Lactate dehydrogenase analysis was performed to provide information about lung and pulmonary endothelial cell injury.

All samples were analysed.
Statistics:
All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other parameters, analyses were carried out using the individual animal as the basic experimental unit.
# The following sequence of statistical tests was used for bodyweight, organ weight, bronchoalveolar lavage and clinical pathology:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test was not significant at the 1% level, Williams' test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test was applied. If the H1 approximate test was not significant at the 1% level, Shirley's test was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.
For clinical pathology data if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups
were compared using pairwise comparisons of each dose group against the control both for i) values or =c, and for ii) values < or =c versus values >c, as applicable.
# For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item-related clinical signs during the 13 weeks of treatment or during the 12 week recovery period.
Signs associated with the administration procedure included wet fur and red staining of the head, nose and eyes on return to home cage, in which the majority had resolved by the end of the working day. These signs were seen in animals from all groups, including control, therefore they are considered to be due to the method and duration of restraint and are commonly seen on inhalation studies of this study design.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on body weight and weight gain during the 13 weeks of treatment or during the 12 week recovery period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test article-related effects during the main and recovery phases of the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination did not reveal any exposure-related abnormalities
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, group mean neutrophil counts were higher than control in both sexes exposed to 26.0 µg/L (up to 2.1X).
After 12 weeks of recovery, group mean neutrophil counts also remained higher than control in both sexes exposed to 26.0 µg/L (1.4X and 1.7X for males and females, respectively).
As a result of the higher mean neutrophil counts in animals exposed to 26.0 µg/L, group mean white blood cells counts of both sexes exposed to 26 µg/L were also higher than control during the treatment and recovery phases (up to 1.2 X for both phases).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 13 weeks of treatment, group mean glucose concentrations were higher than control in both sexes exposed to 26.0 µg/L (up to 1.1X or 1.6X for males and females, respectively); however, the majority of individual values were within the control range for the males and there was no exposure related trend for the females, therefore this was considered to be incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, group mean adjusted lung and bronchi weights were higher than control in both sexes exposed to 26.0 µg/L (1.5X and 1.4X control for males and females respectively).
After 12 weeks of recovery, group mean adjusted lung and bronchi weights remained higher than control in both sexes previously exposed to 26.0 µg/L (1.3X and 1.4X control for males and females, respectively).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
(see chapter 'any other information on results including tables' section 7.5.2 /3 for macropathology tables)
Macroscopic examination performed after 13 weeks of treatment revealed an increase in incidence of pale areas in the lungs of females receiving 26.0 µg/L. Abnormal color (dark) was also seen in 2 animals of both sexes that received 26.0 µg/L.
In addition, enlargement and pale appearance of the mediastinal and tracheobronchial lymph nodes were noted in all females and nearly all males that received 26.0 µg/L.
The majority of these findings were still evident following 12 weeks of recovery: A slight increased incidence of pale areas in the lungs was seen in some males which previously received 26.0 µg/L. Enlargement and pale appearance of the mediastinal and tracheobronchial lymph nodes were still observed in all females and a majority of males that previously received 26.0 µg/L.The tracheobronchial lymph nodes of few males previously exposed to 6.50 µg/L showed enlargement and pale appearance, the latter finding was also seen in few females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological findings related to treatment were seen in the lungs, tracheobronchial, axillary and mediastinal lymph nodes and nose (see chapter 'any other information on results including tables' section 7.5.2 /4 for histopathology tables) :

In the lungs, granular eosinophilic material in the alveoli, type II pneumocytes hyperplasia, increased alveolar macrophages, mixed perivascular inflammatory cell infiltrates and neutrophilic inflammatory cell infiltrates were observed in animals that received 6.50 or 26.0 µg/L. The severity of these findings showed a clear concentration relationship pattern in both sexes. Additionally, macrophage aggregates of the bronchus- associated lymphoid tissues (BALT) and alveolar haemorrhage was seen in some animals of both sexes that received 26.0 µg/L.
A minimal increased in alveolar macrophages was seen in 2 out of 10 males that received 1.55 µg/L. At this concentration, a minimal perivascular mixed inflammation was also observed in males but its incidence was similar to controls These findings at this exposure level were considered as a physiological response and unrelated to test item due to lack of other associated inflammatory lesions and no such lesions were observed in females at this exposure level.
After 12 week off-dose, no evidence of recovery was observed in animals which previously received 6.50 or 26.0 µg/L as test item-related findings of granular eosinophilic material in the alveoli, type II pneumocytes hyperplasia, increased alveolar macrophages, mixed perivascular inflammatory cell infiltrates, neutrophilic inflammatory cell infiltrates and/ or macrophages of BALT were still present at a similar incidence and severity. At 1.55 µg/L, the minimal increase in alveolar macrophages seen in few males at the end of the treatment period was no more present.

In the tracheobronchial, axillary and mediastinal lymph nodes, macrophage aggregates and/or increased cellularity of the paracortical region was seen in both sexes that received 26.0 µg/L. These findings were also observed in tracheobronchial lymph nodes of both sexes that received 6.50 µg/L.
After 12 weeks off-dose, no evidence of recovery was observed as test item-related findings of macrophage aggregates and/or increased cellularity of the paracortical region were still present in animals at a similar incidence and severity.

In hte nose, minimal hyperplasia of mucous cells was seen in the turbinates and nasopharynx of both sexes that received 6.50 or 26.0 µg/L. After 12 weeks off-dose, no evidence of recovery was observed as the test item-related finding of hyperplasia of mucous cells was still present in animals at a similar incidence and severity.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Bronchoalveolar lavage: (see chapter 'any other information on results including tables' section 7.5.2 /5 for BAL tables)
After 13 weeks of treatment, statistically significant exposure-related increases of group mean total cell counts were observed for both sexes exposed to 6.5 or 26 µg/L.
Compared to controls, mean total cell counts were increased by 1.7 to 14.4 fold for males and 2.41 to 11.9 fold for females exposed to 6.5 and 26 µg/L respectively. Control data showed that the vast majority of cells recovered in BALF were macrophages (> or = 83%). Dose-related increases in neutrophils and eosinophils in the BALF of rats given 6.5 or 26 µg/L as well as increase in lymphocytes at the high concentration led to up to 5.7% of the cells being lymphocytes, 9.0 % of the cells being eosinophils and up to 45.3% of the cells being neutrophils, consequently reducing the proportion of macrophages to 40.7% or less.
Higher group mean lactate dehydrogenase, total protein and phospholipid concentrations were observed in the bronchoalveolar lavage supernatant fluid for both sexes exposed to 6.50 µg/L or 26.0 µg/L, up to 5.8X, 4.1X and 5.3X control for lactate dehydrogenase, total protein and phospholipids, respectively.
At 1.55 µg/L, only a marginal increase of mean total cells count was observed compared to controls (1.5 and 1.2x for males and females respectively) and cell proportional distribution was not affected.

After 12 weeks of recovery, comparable changes of lower magnitude were still observed in animals exposed to 6.50 or 26.0 µg/L. Compared to controls, mean total cell counts were still increased by 7.6 fold for males exposed to 26.0 µg/L and 1.6 to 9.3 fold for females exposed to 6.5 and 26 µg/L respectively.
Dose-related increases in neutrophils and eosinophils in the BALF of rats given 6.5 or 26 µg/L as well as increase in lymphocytes at the high concentration led to up to 7.95% of the cells being lymphocytes 11.4 % of the cells being eosinophils and up to 37.1% of the cells being neutrophils, the proportion of macrophages remained reduced to 45% or less. Group mean lactate dehydrogenase, total protein and phospholipid concentrations remained higher than control for both sexes previously exposed to 26.0 µg/L or 6.50 µg/L, up to 5.7X, 5.8X and 2.0X control for lactate dehydrogenase, total protein and phospholipids, respectively.
At 1.55 µg/L, no statistically significant changes were observed compared to controls in mean total cells counts and cell distribution.

Bone marrow analysis:
The cellularity, distribution and morphology of the marrow was unaffected by the treatment.
Details on results:
Histopathological changes were evident in the lungs, lymph nodes and the nasal turbinates in animals that received 6.50 or 26.0 µg/L.

In the lung, changes consisted of the presence of granular eosinophilic material in the alveoli, type II pneumocytes hyperplasia, increased alveolar macrophages, mixed perivascular inflammatory cell infiltrates and neutrophilic inflammatory cell infiltrates in animals that received 6.50 or 26.0 µg/L. Furthermore, alveolar haemorrhage was seen in animals that received 26.0 µg/L. These changes correlated with increased lung weights seen at the macroscopic examination while increased alveolar macrophages and alveolar haemorrhage correlated with pale areas and dark abnormal colour respectively. The lesions were considered as a reactive inflammatory response and attempted clearance of the inhaled material. There was no evidence of recovery following 12 weeks without exposure to the test item. Major findings were still present at a similar incidence and severity than those noted after the end of the treatment period without trend to progress towards chronicity. However, The the incidence and severity of the lung findings were considered adverse for animals that received 6.50 or 26.0 µg/L.
Changes in the lymph nodes consisted of increased cellularity and macrophage aggregates in the tracheobronchial, axillary and mediastinal lymph nodes of animals that received 26.0 µg/L, which correlated with enlargement and pale areas seen in the tracheobronchial and mediastinal lymph nodes at the macroscopic examination. Similar findings were seen in the tracheobronchial of animals that received 6.50 µg/L. These lesions were considered as a secondary response to the inflammatory process and attempted clearance of insoluble material from the lungs. There was no evidence of recovery in the tracheobronchial or mediastinal lymph nodes following 12 weeks without exposure to the test item; however, complete recovery was apparent in the axillary lymph nodes.
In the nasal turbinates and nasopharynx, mucous cell hyperplasia seen in animals that received 6.50 or 26.0 µg/L was indicative of a response to an inhaled irritant. There was no evidence of recovery following 12 weeks without exposure to the test item.

The higher cell counts and percentage of cells observed in the bronchoalveolar lavage fluid (BALF) of animals exposed to 6.50 or 26 µg/L when compared with control, correlated with the inflammatory response seen histopathologically in the lungs. The cellular changes in the BALF are indicative of lung injury and are consistent with the findings recorded microscopically. The changes in the proportional distribution of cell types, particularly neutrophils, in the BALF of animals receiving 6.50 or 26.0 µg/L, are implicated as contributing to the lung injury incurred during the inflammatory response. The neutrophil influx plays a major role in increasing the permeability of the alveolar/capillary barrier and in producing cellular toxicity during the inflammatory response (Drent et al., 1996). The increase in neutrophil content in the BALF is consistent with the increase in circulating neutrophils in the blood, which are recruited into the lungs; the elevation of neutrophils in the blood is therefore an indicator of the inflammatory response associated with cellular damage. Moreover, no abnormalities were observed in bone marrow thus confirming that changes in the haematology parameters were secondary to the local inflammatory effects observed in the lungs.

The increases in the biochemical content of the BALF, in animals that received 6.50 or 26.0 µg/L, characterised by total protein and lactate dehydrogenase, further signify an inflammatory response and damage to the alveolar capillary barrier. LDH occurs extracellularly in BALF only in the presence of damaged lung cells (Henderson et al., 1979). As there was a distinct dose response in the increases of LDH this suggests more extensive tissue damage in animals that received 26.0 ¿g/L. After 12 weeks of recovery, comparable changes of generally lower magnitude except for lactate deshydrogenase and total protein concentrations were still observed in animals exposed to 6.50 or 26.0 µg/Lwhich suggested the test item persisted in the lungs during the recovery period.

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
1.55 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: Changes in bronchoalveolar lavage markers

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
6.5 mg/m³ air (analytical)
System:
other: pulmonary
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

7.5.2 /3 Macropathology results

 Animals killed after 13 weeks of treatment

Lungs

An increase in incidence of pale areas was observed in females receiving 26.0 µg/L. Abnormal color (dark) was also seen in 2 animals of both sexes that received 26.0 µg/L.

Summary of findings in the lungs for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

 

 

 

 

 

 

 

 

 

Pale area(s)

4

1

0

3

4

1

0

7

Abnormal color (dark)

0

0

0

2

0

0

0

2

 

 

 

 

 

 

 

 

 

Number of animals

examined

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

Mediastinal lymph nodes

Pale appareance and enlargement were seen in both sexes that received 26.0 µg/L.

Summary of findings in the mediastinal lymph nodes for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

 

 

 

 

 

 

 

 

 

Enlarged

0

0

0

6

0

0

0

10

Pale

0

0

0

7

0

0

0

10

 

 

 

 

 

 

 

 

 

Number of animals examined

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

Tracheobronchial lymph nodes

Pale appareance and enlargement were seen in both sexes that received 26.0 µg/L.

Summary of findings in the tracheobronchial lymph nodes for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

 

 

 

 

 

 

 

 

 

Enlarged

0

0

0

9

0

0

0

9

Pale

0

0

0

9

0

0

0

9

 

 

 

 

 

 

 

 

 

Number of animals examined

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

Animals killed after 12 weeks of recovery

Lungs

A slight increased incidence of pale areas in the lungs was seen in some males which previously received 26.0 µg/L. Abnormal color (dark) was no more observed.

Summary of findings in the lungs for animals killed after 13 weeks of treatment followed by 12 week recovery period

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

 

 

 

 

 

 

 

 

 

Pale area(s)

5

5

1

8

8

5

5

9

 

 

 

 

 

 

 

 

 

Number of animals

examined

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

Mediastinal lymph nodes

Pale appareance and enlargement were seen in both sexes which previously received 26.0 µg/L.

Summary of findings in the mediastinal lymph nodes for animals killed after 13 weeks of treatment followed by 12 week recovery period

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

 

 

 

 

 

 

 

 

 

Enlarged

0

0

0

7

0

0

0

6

Pale

0

0

0

5

0

0

0

10

 

 

 

 

 

 

 

 

 

Number of animals examined

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

Tracheobronchial lymph nodes

Pale appareance and enlargement were seen in some males and females that previously received 26.0 µg/L and some males that previously received 6.50 µg/L.

Pale appearance was also seen in some females that previously received 6.50 µg/L.

Summary of findings in the tracheobronchial lymph nodes for animals killed after 13 weeks of treatment followed by 12 week recovery period

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

 

 

 

 

 

 

 

 

 

Enlarged

0

0

3

8

0

0

0

7

Pale

0

0

5

7

0

0

3

10

 

 

 

 

 

 

 

 

 

Number of animals examined

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

7.5.2 /4 Histopathology results

 Animals killed after 13 weeks of treatment

Lungs

Summary of treatment related findings in the lungs for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

Eosinophilic Granular Material, Alveoli

 

 

 

 

 

 

 

 

Minimal

0

0

9

0

0

0

10

0

Slight

0

0

0

0

0

0

0

1

Moderate

0

0

0

7

0

0

0

7

Marked

0

0

0

3

0

0

0

2

Total

0

0

9

10

0

0

10

10

Hyperplasia, Type II Pneumocytes

 

 

 

 

 

 

 

 

Minimal

0

0

7

0

0

0

9

0

Slight

0

0

3

1

0

0

0

8

Moderate

0

0

0

7

0

0

0

2

Marked

0

0

0

1

0

0

0

0

Severe

0

0

0

1

0

0

0

0

Total

0

0

10

10

0

0

9

10

Increased Alveolar Macrophages

 

 

 

 

 

 

 

 

Minimal

0

2

4

0

0

0

2

0

Slight

0

0

6

0

0

0

7

0

Moderate

0

0

0

1

0

0

0

2

Marked

0

0

0

9

0

0

0

8

Total

0

2

10

10

0

0

9

10

Infiltrate, Inflammatory cells, Mixed, Perivascular

 

 

 

 

 

 

 

 

Minimal

5

8

9

0

7

4

10

1

Slight

0

0

0

6

0

1

0

6

Moderate

0

0

0

1

0

0

0

2

Total

5

8

9

7

7

5

10

9

Infiltrate, Inflammatory cells, Neutrophilic, Alveoli

 

 

 

 

 

 

 

 

Minimal

1

1

9

0

0

0

8

0

Slight

0

0

1

3

0

0

1

7

Moderate

0

0

0

6

0

0

0

3

Total

1

1

10

9

0

0

9

10

Agregates, Macrophage, BALT

 

 

 

 

 

 

 

 

Minimal

0

0

0

3

0

0

0

2

Slight

0

0

0

1

0

0

0

0

Total

0

0

0

4

0

0

0

2

Hemorrhage, Alveoli

 

 

 

 

 

 

 

 

Minimal

0

0

0

3

0

0

0

1

Total

0

0

0

3

0

0

0

1

Number of tissues examined

10

10

10

10

10

10

10

10

Lymph nodes (tracheobronchial, axillary and mediastinal)

Summary of treatment related findings in the lymph nodes for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

Tracheobronchial Lymph Nodes

 

 

 

 

 

 

 

 

Aggregates, Macrophage

 

 

 

 

 

 

 

 

Minimal

0

0

6

0

0

0

7

1

Slight

0

0

0

5

0

0

1

8

Moderate

0

0

0

4

0

0

0

1

Total

0

0

6

9

0

0

8

10

CellularityIncreased, Paracortical

 

 

 

 

 

 

 

 

Minimal

0

1

6

6

0

0

2

3

Slight

0

0

0

2

0

0

0

5

Moderate

0

0

0

1

0

0

0

0

Total

0

1

6

9

0

0

2

8

Number of tissues examined

10

10

10

10

10

10

10

10

Left Axillary Lymph Nodes

 

 

 

 

 

 

 

 

Aggregates, Macrophage

 

 

 

 

 

 

 

 

Moderate

0

0

0

1

0

0

0

0

Total

0

0

0

1

0

0

0

0

CellularityIncreased, Paracortical

 

 

 

 

 

 

 

 

Minimal

0

0

0

1

0

0

0

1

Slight

0

0

0

3

0

0

0

1

Total

0

0

0

4

0

0

0

2

Number of tissues examined

10

10

10

10

10

10

10

10

Mediastinal Lymph Nodes

 

 

 

 

 

 

 

 

Aggregates, Macrophage

 

 

 

 

 

 

 

 

Slight

-

-

-

6

-

-

-

3

Moderate

-

-

-

0

-

-

-

7

Total

-

-

-

6

-

-

-

10

CellularityIncreased, Paracortical

 

 

 

 

 

 

 

 

Minimal

-

-

-

4

-

-

-

2

Slight

-

-

-

2

-

-

-

8

Moderate

-

-

-

1

-

-

-

0

Total

-

-

-

7

-

-

-

10

Number of tissues examined

-

-

-

7

-

-

-

10

Nose (turbinates and nasopharynx)

Summary of treatment related findings in the turbinates and nasopharynx for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

Nose/turbinates

 

 

 

 

 

 

 

 

Hyperplasia,mucous cell,Dorsal

 

 

 

 

 

 

 

 

Minimal

0

0

1

4

0

0

3

7

Total

0

0

1

4

0

0

3

7

Pharynx,Nasal

 

 

 

 

 

 

 

 

Hyperplasia,mucous cell,Dorsal

 

 

 

 

 

 

 

 

Minimal

0

0

4

5

0

0

5

3

Total

0

0

4

5

0

0

5

3

Number of tissues examined

10

10

10

10

10

10

10

10

Animals killed after 12 weeks of recovery

Lungs

Summary of treatment related findings in the lungs for animals killed after 13 weeks of treatment followed by 12 weeks recovery period

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

Eosinophilic Granular Material, Alveoli

 

 

 

 

 

 

 

 

Minimal

0

0

10

0

0

0

10

0

Slight

0

0

0

2

0

0

0

2

Moderate

0

0

0

6

0

0

0

3

Marked

0

0

0

2

0

0

0

5

Total

0

0

10

10

0

0

10

10

Hyperplasia, Type II Pneumocytes

 

 

 

 

 

 

 

 

Minimal

0

0

10

0

0

0

6

0

Slight

0

0

0

6

0

0

0

8

Moderate

0

0

0

2

0

0

0

2

Marked

0

0

0

2

0

0

0

0

Total

0

0

10

10

0

0

6

10

Increased Alveolar Macrophages

 

 

 

 

 

 

 

 

Minimal

0

0

2

0

0

0

5

0

Slight

0

0

8

1

0

0

5

0

Moderate

0

0

0

6

0

0

0

6

Marked

0

0

0

3

0

0

0

4

Total

0

0

10

10

0

0

10

10

Infiltrate, Inflammatory cells, Mixed, Perivascular

 

 

 

 

 

 

 

 

Minimal

8

8

9

0

8

9

10

0

Slight

0

1

1

6

0

0

0

8

Moderate

0

0

0

4

0

0

0

2

Total

8

9

10

10

8

9

10

10

Infiltrate, Inflammatory cells, Neutrophilic, Alveoli

 

 

 

 

 

 

 

 

Minimal

1

1

7

0

1

0

8

0

Slight

0

0

3

5

0

0

0

7

Moderate

0

0

0

5

0

0

0

3

Total

1

1

10

10

1

0

8

10

Agregates, Macrophage, BALT

 

 

 

 

 

 

 

 

Minimal

0

0

0

4

0

0

0

2

Slight

0

0

0

1

0

0

0

1

Moderate

 

 

 

1

 

 

 

1

Total

0

0

0

6

0

0

0

4

Number of tissues examined

10

10

10

10

10

10

10

10

Lymph nodes

Summary of treatment related findings in the lymph nodes for animals killed after 13 weeks of treatment followed by 12 week recovery period

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

Tracheobronchial Lymph Nodes

 

 

 

 

 

 

 

 

Aggregates, Macrophage

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

0

5

0

Slight

0

0

8

2

0

0

4

2

Moderate

0

0

1

6

0

0

1

8

Marked

0

0

1

2

0

0

0

0

Total

0

0

10

10

0

0

10

10

CellularityIncreased, Paracortical

 

 

 

 

 

 

 

 

Minimal

0

0

7

2

0

0

2

6

Slight

0

0

3

4

0

0

0

2

Moderate

0

0

0

4

0

0

0

0

Total

0

0

10

10

0

0

2

8

Number of tissues examined

9

10

10

10

10

10

10

10

Mediastinal Lymph Nodes

 

 

 

 

 

 

 

 

Aggregates, Macrophage

 

 

 

 

 

 

 

 

Slight

-

-

-

0

-

-

-

1

Moderate

-

-

-

7

-

-

-

9

Marked

 

 

 

1

 

 

 

 

Total

-

-

-

8

-

-

-

10

CellularityIncreased, Paracortical

 

 

 

 

 

 

 

 

Minimal

-

-

-

0

-

-

-

2

Slight

-

-

-

1

-

-

-

6

Moderate

-

-

-

7

-

-

-

2

Total

-

-

-

8

-

-

-

10

Number of tissues examined

-

-

-

8

-

-

-

10

 

Nose (turbinates and nasopharynx)

Summary of treatment related findings in the turbinates and nasopharynx for animals killed after 13 weeks of treatment followed by 12 week recovery period

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure level (µg/L)

0

1.55

6.50

26.0

0

1.55

6.50

26.0

Nose/turbinates

 

 

 

 

 

 

 

 

Hyperplasia,mucous cell,Dorsal

 

 

 

 

 

 

 

 

Minimal

0

0

4

7

1

0

5

7

Total

0

0

4

7

1

0

5

7

Pharynx,Nasal

 

 

 

 

 

 

 

 

Hyperplasia,mucous cell,Dorsal

 

 

 

 

 

 

 

 

Minimal

0

0

4

7

1

0

6

6

Total

0

0

4

7

0

0

6

6

Number of tissues examined

10

10

10

10

10

10

10

10

 

7.5.2 /5 Bronchoalveolar lavage results

 Animals killed after 13 weeks of treatment

Dose group                     1         2              3              4

Exposure level (µg/L)      0       1.55          6.50       26.0

Group /sex

Different cell counts calculated per animal (106/ animal)

Different cell counts as percentage of total white blood cells (%)

 

Total cells

Neutrophils

Lymphocytes

Mononuclear cells

Eosinophils

Neutrophils

Lymphocytes

Mononuclear cells

Eosinophils

1M

Mean

SD

N

 

1.91

0.61

10

0.17

0.11

10

0.10

0.05

10

1.63

0.52

10

0.01

0.02

10

8.70

3.85

10

5.12

2.44

10

85.47

6.86

10

0.44

0.96

10

2M

Mean

SD

N

 

2.87

1.76

10

0.44

0.52

10

0.12

0.09

10

2.27

1.18

10

0.03

0.05

10

11.73

7.97

10

4.64

3.07

10

82.88

8.85

10

0.64

0.87

10

3M

Mean

SD

N

 

3.17*

1.6

10

0.82***

0.36

10

0.27***

0.11

10

1.95

0.76

10

0.13***

0.11

10

25.95***

6.65

10

9.48

5.22

10

60.64***

7.25

10

3.64***

2.33

10

4M

Mean

SD

N

 

27.59***

11.12

10

12.47***

5.40

10

1.49***

0.64

10

11.20***

4.39

10

2.44***

1.07

10

44.41***

2.96

10

5.66

1.85

10

40.69***

1.94

10

8.89***

2.45

10

Group /sex

Different cell counts calculated per animal (106/ animal)

Different cell counts as percentage of total white blood cells (%)

 

Total cells

Neutrophils

Lymphocytes

Mononuclear cells

Eosinophils

Neutrophils

Lymphocytes

Mononuclear cells

Eosinophils

1F

Mean

SD

N

 

1.38

0.39

10

0.13

0.09

10

0.07

0.04

10

1.16

0.33

10

0.01

0.02

10

9.74

4.48

10

4.72

2.84

10

84.35

5.26

10

1.19

1.99

10

2F

Mean

SD

N

 

1.70

0.63

10

0.12

0.13

10

0.07

0.08

10

1.49

0.52

10

0.01

0.02

10

6.14

6.46

10

3.70

3.94

10

89.35

10.82

10

0.81

1.36

10

3F

Mean

SD

N

 

3.33***

0.77

10

0.77**

0.54

10

0.21**

0.08

10

2.24***

0.55

10

0.11**

0.15

10

21.87*

12.82

10

6.39

2.72

10

68.71*

14.70

10

3.03

3.24

10

4F

Mean

SD

N

 

16.45***

10.38

10

7.67***

5.52

10

0.83***

0.34

10

6.49***

3.81

10

1.46***

0.87

10

45.29***

4.69

10

5.45

1.84

10

39.90***

2.79

10

8.97***

2.06

10

 

 Animals killed after 12 weeks recovery

Group /sex

Different cell counts calculated per animal (106/ animal)

Different cell counts as percentage of total white blood cells (%)

 

Total cells

Neutrophils

Lymphocytes

Mononuclear cells

Eosinophils

Neutrophils

Lymphocytes

Mononuclear cells

Eosinophils

1M

Mean

SD

N

 

1.94

0.61

10

0.16

0.11

10

0.09

0.05

10

1.65

0.61

10

0.04

0.05

10

8.87

6.32

10

5.38

4.66

10

83.70

11.34

10

2.05

2.28

10

2M

Mean

SD

N

 

1.54

0.37

10

0.11

0.09

10

0.13

0.09

10

1.26

0.23

10

0.04

0.03

10

7.02

4.78

10

7.91

4.13

10

82.99

8.52

10

2.08

1.91

10

3M

Mean

SD

N

 

2.11

0.84

10

0.47**

0.24

10

0.25***

0.15

10

1.29

0.50

10

0.11*

0.06

10

21.42***

5.54

10

12.07**

4.67

10

61.33***

8.32

10

5.04*

1.78

10

4M

Mean

SD

N

 

14.66***

7.33

10

5.39***

2.80

10

0.95***

0.37

10

6.61***

3.62

10

1.70***

0.90

10

36.82***

4.09

10

6.98

1.87

10

44.36***

5.31

10

11.37***

3.78

10

 

Group /sex

Different cell counts calculated per animal (106/ animal)

Different cell counts as percentage of total white blood cells (%)

 

Total cells

Neutrophils

Lymphocytes

Mononuclear cells

Eosinophils

Neutrophils

Lymphocytes

Mononuclear cells

Eosinophils

1F

Mean

SD

N

 

1.25

0.48

10

0.07

0.06

10

0.04

0.02

10

1.12

0.46

10

0.01

0.02

10

5.25

5.03

10

3.89

2.96

10

89.96

6.21

10

0.90

1.51

10

2F

Mean

SD

N

 

1.21

0.51

10

0.11

0.13

10

0.09

0.10

10

0.99

0.46

10

0.02

0.03

10

8.24

7.50

10

7.74

5.76

10

82.28

12.52

10

1.74

2.51

10

3F

Mean

SD

N

 

1.98*

0.67

10

0.40***

0.27

10

0.22**

0.15

10

1.28

0.33

10

0.08**

0.06

10

18.73***

7.97

10

10.24*

6.47

10

66.77***

13.65

10

3.82***

1.33

10

4F

Mean

SD

N

 

11.66***

7.32

10

4.36***

2.87

10

0.90***

0.58

10

5.41***

3.51

10

0.99***

0.55

10

37.14***

3.98

10

7.95*

3.19

10

44.92***

6.24

10

8.99***

1.23

10

Applicant's summary and conclusion

Conclusions:
The results from the bronchoalveolar lavage fluid and haematology correlated with the histopathological findings and were suggestive of lung injury consistent with the inhalation of in poorly soluble particulate matter. Since no toxicologically relevant adverse changes were observed in the low-concentration group, the NOAEC for sub-chronic inhalation exposure to was placed at 1.55 µg/L.
Executive summary:

The toxicity of the test substance upon repeated exposure by inhalation was investigated in a 90-day sub-chronic study in Wistar rats according to OECD 413 and Good Laboratory Practices.

Four main groups of ten male and ten female rats each were exposed nose-only to target concentrations of 0 (control), 1.5, 6 or 24 µg/L for 6 hours/day, 5 days/week over a 90-day period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, recovery groups – also consisting of ten male and ten female animals each – were simultaneously exposed with animals of the main groups and were sacrificed after a 12-week recovery period following the last exposure.

During the study, clinical condition, body weight, food consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, bronchoalveolar lavage (BAL), bone marrow, organ weight, macropathology and histopathology investigations were undertaken.

The achieved aerosol concentrations were 1.55, 6.50 and 26.0 µg/L (103, 108 and 108% of target). The mass median aerodynamic diameter (MMAD) for the low, mid and high-exposed Groups were within the ideal range (1 to 3mm) for a repeat dose inhalation studyChemical analysis of test atmosphere samples by LC-MS/MS indicated that the composition of the material in the test atmospheres was similar to the original material and was stable across the exposure period.

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

Test item-related effects were observed in the haematology at 26.0 µg/L for both sexes during the treatment and recovery phases. Higher mean neutrophil counts were observed for animals that received 26.0 µg/L (up to 2.1 x control). After 12 weeks of recovery, mean neutrophil counts remained higher than control (up to 1.7 x control). As a result of the higher mean neutrophil counts in animals exposed to 26.0 µg/L, mean white blood cells counts of both sexes were also higher than control during the treatment and recovery phases (up to 1.2 x for both phases).No abnormalities were observed in animals bone marrow confirming that changes in haemotology parameters were secondary to the local inflammatory effects observed in the lungs. No effects were observed in haematology for animals receiving 6.50 or 1.55 µg/L.

At the end of the treatment period, group mean adjusted lung and bronchi weights were higher than control in both sexes exposed to 26.0 µg/L (up to 1.5 x and 1.4 x control for males and females respectively).Following 12 Weeks off dose lung and bronchi weights remained higher than control by a similar magnitude. No effects were observed on organ weight for animals receiving 6.50 or 1.55 µg/L.

At the end of the treatment period, analysis of bronchoalveolar lavage revealed statistically significant exposure-related increases of mean total cell counts in both sexes exposed to 6.50 or 26.0 µg/L. Compared to controls, mean total cell counts were increased by 1.7 to 14.4 fold for males and 2.41 to 11.9 fold for females exposed to 6.50 and 26.0µg/L respectively.Control data showed that the vast majority of cells recovered in BronchoAlveolar Lavage Fluid (BALF) were macrophages (> or = 83%). Concentration-related increases in neutrophils and eosinophils in rat given 6.50 or 26 µg/L as well as increase in lymphocytes at 26.0 µg/L led to up to 5.7% of the cells being lymphocytes, 9.0 % of the cells being eosinophils and up to 45.3% of the cells being neutrophils, consequently reducing the proportion of macrophages to 40.7% or less. A concentration-related increase in total protein, phospholipids and lactate dehydrogenase activity was also observed in the bronchoalveolar lavage supernatant fluid for both sexes exposed to 6.50 or 26.0 µg/L. After 12 weeks of recovery,comparable changes of generally lower magnitude except for lactate deshydrogenase and total protein concentrations were still observed in animals exposed to 6.50 or 26.0 µg/L. No significant changes were observed in bronchalveolar lavage parameters of animals exposed to 1.55 µg/L at the end of both treatment and recovery periods.

Macroscopic examination performed after 13 weeks of treatment revealed increased incidence of pale areas seen in females that received 26.0 µg/L. Abnormal color (dark) was seen in both sexes that received 26.0 µg/L. Pale and enlarged tracheobronchial and mediastinal lymph nodes were observed in both sexes that received 26.0 µg/L. These findings were generally still apparent after 12 weeks of recovery with pale and/or enlarged tracheobronchial lymph nodes also apparent for a proportion of animals previously exposed to 6.50 µg/L. There were no particular findings at the macroscopic examination of the animals exposed to 1.55 µg/L.

Treatment related histopathological changes were seen in the lungs, nasal turbinates, nasopharynx, tracheobronchial and mediastinal lymph nodes. In the lungs, granular eosinophilic material in the alveoli, type II pneumocytes hyperplasia, increased alveolar macrophages, mixed perivascular inflammatory cell infiltrates and neutrophilic inflammatory cell infiltrates were observed in animals that received 6.50 or 26.0 µg/L. Additionally, macrophage aggregates of the bronchus-associated lymphoid tissues (BALT) and alveolar haemorrhage was seen in some animals of both sexes that received 26.0 µg/L. There was no evidence of recovery following 12 weeks without exposure. Major findings were still present at a similar incidence and severity than those noted after the end of the treatment period without trend to progress towards chronicity. At 1.55 µg/L, only a minor reversible change consisting of a minimal increase in alveolar macrophages was observed in two males.

In the lymph nodes,macrophage aggregates and/or increased cellularity of the paracortical region was seen in tracheobronchial, left axillary and mediastinal lymph nodes of both sexes that received 26.0 µg/L. These findings were also observed in tracheobronchial lymph nodes of both sexes that received 6.50 µg/L. Full recovery was seen in the axillary lymph nodes; however, no evidence of recovery was observed in the tracheobronchial or mediastinal lymph nodes.

In the nasal turbinates and nasopharynx, hyperplasia of mucous cells was seen in both sexes that received 6.50 or 26.0 µg/L; no evidence of recovery was apparent.

In conclusion, in the lungs higher concentrations of the test item produced increases in alveolar macrophages,granular eosinophilic material in the alveoli, type II pneumocytes hyperplasia, mixed perivascular inflammatory cell infiltrates and neutrophilic inflammatory cell infiltrates. The incidence and severity of these findings showed aclear dose relationship patternin both sexes. Macrophage aggregates of the bronchus- associated lymphoid tissues and alveolar haemorrhage was seen at the highest exposure level. Hyperplasia of the mucous cells was also evident in nasal turbinates and nasopharynx of animals exposed to 6.50 or 26.0 µg/L and was indicative of a response to an inhaled irritant. Higher concentrations of the test item also resulted in increased cellularity and macrophage aggregates in the tracheobronchial and mediastinal lymph nodes. The changes in the local lymph nodes were considered to be a secondary response to the increased influx of macrophages clearing the test material from the terminal airways and alveolar spaces and trafficking to the local draining lymph nodes. The microscopic changes reported in the lungs, lymph nodes and nasal turbinates at the end of the treatment period were still present after 12 weeks off dose. These findings in this study at 6.50 and 26.0 µg/Lwere considered to be adverse. The results from the bronchoalveolar lavage fluid and haematology correlated with the histopathological findings and were suggestive of lung injury consistent with the inhalation of poorly soluble particulate matter.

On the basis of these findings, the exposure level of 1.55 µg/L is was considered to represent the no observed adverse effect level (NOAEL) for this study.