Registration Dossier

Administrative data

Description of key information

In a  subacute inhalation toxicity study performed in accordance with OECD guideline No 412 and in compliance with Good Laboratory Practice, no systemic effects were observed in rats exposed up to 328 mg/m3 air  but histopathological evaluation reported local pulmonary effects. The test substance was responsible for a mild pulmonary inflammation. The changes in haematology correlated with the microscopic findings. Based on these findings, the NOAEC was set to 34.4 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2014-08-18 to 2015-04-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Harlan (UK) Ltd.
- Age at study initiation:61 to 67 days
- Weight at study initiation: 212 to 248 g (males) and 161 to 194 g (females)
- Fasting period before study:no
- Housing: The animals were housed five of one sex per cage
- Diet: Ad libitum, standard rodent diet (Rat and Mouse n°1 Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°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.
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.2 - <= 3.4 µm
Geometric standard deviation (GSD):
2.43
Remarks on MMAD:
MMAD / GSD: The Mass Median Aerodynamic Diameters for all Groups were within the acceptable range (1-4 µm) for a repeat dose inhalation study with 80-81% of the collected particles below 7 µm; however these were slightly above the targeted range of 1 to 3 µm.
The Mass Median Aerodynamic Diameters (MMAD) of the test atmosphere were 3.2, 3.3 and 3.4 µm with Geometric Standard Deviation (GSD) of 2.43, 2.36 and 2.34 for the low, mid and high-dosed groups respectively.
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 and a top section.
- Method of holding animals in test chamber: Rats were held in polycarbonate 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: 25 L/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 concentrations of dust were altered by changing the gear ratio (and therefore the speed of rotation of the compressed powder towards the scraper blade) of the mechanism.
- Temperature, humidity, pressure in air chamber:
Temperature was measured using an electronic thermometer inserted into the inhalation chamber. The mean chamber temperatures were all within expected ranges (22.0; 22.9; 22.1 and 22.1 °C for controls, low, mid and high-dosed groups).
Humidity and pressure in air chamber were not reported.
- Air flow rate: Airflow to Wright dust Feed was 19 L/minute
- 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, 12.08 and 17.39 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 of 26, 50, 30 and 26 L/minute for the control, low, mid and high-dose groups respectively. the airflow was filtered locally.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual 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 a glass fibre filters. Sampling was performed at least three times during each exposure and for each dose-group. Samples were collected from a vacant animal exposure port (animals breathing zone). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.
In addition, the aerosol concentrations measured by gravimetric analysis were checked by a chemical analysis once every week. High Performance Liquid Chromatography with Evaporative Light Scattering Detector (ELSD) was used.

- Samples taken from breathing zone: yes, samples were collected from a vacant animal exposure port.

VEHICLE (if applicable)
- none
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. Once every week, the gravimetric analysis was coupled to analytical analysis by HPLC with ELSD detection to check the accuracy of the gravimetric method.
The mean achieved concentrations were 5.30; 34.4 and 328 µg/L and corresponded to 106, 115 and 109% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
5 day / week
Remarks:
Doses / Concentrations:
5, 30, 300 µg/L
Basis:
other: target
Remarks:
Doses / Concentrations:
5.30, 34.4, 328 µg/L
Basis:
analytical conc.
No. of animals per sex per dose:
3 groups, each comprising 5 male and 5 female rats received the test substance at target exposure levels of 5, 30 or 300 µg/L. A similarly constitutedControl group received air only, at the same operating conditions as the high dose group.
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:
In a previous preliminary inhalation toxicity study exposure levels of 0.0639, 0.315 or 1.49 mg/L, equivalent to 63.9, 315 or 1490 µg/L, were well tolerated.
However histopathological treatment related findings were evident in the larynx, lungs and tracheobronchial lymph nodes in all treated groups. Considering the likely progression of lung changes, particularly aggregates of alveolar macrophages, 1490 µg/L was considered unsuitable for longer term administration. Changes in the lungs and larynx at 315 µg/L were generally of lower incidence and/or severity than those seen at 1490 µg/L, therefore for this study, a high exposure level targeted at 300 µg/L was anticipated to induce treatment related changes similar to those previous seen but was expected to be tolerated for 4 weeks. Target exposure levels of 30 and 5.0 µg/L were selected for the intermediate and low groups respectively to identify a no-observed adverse effect level and to explore any possible dose relationship.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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 period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Weeks 1 to 4 of treatment, 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

BODY WEIGHT: Yes
- Time schedule for examinations:The weight of each animal was recorded one week before treatment commenced (Day -7), on the day that treatment commenced (Day 1), twice weekly throughout the study 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. 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: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4 of treatment and before dosing for all study animals.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40.
- 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 4 of treatment and before dosing for all study animals.
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40
- 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 study animals were sacrificed following 4 weeks of exposures.
GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. 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.
HISTOPATHOLOGY: Yes
The following tissues were examined for all study animals of Groups 1 (Control) and 4 (328 µg/L) sacrificed on completion of the 4-week treatment period:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Larynx - 5 sections
Liver - section from two main lobes
Lungs - section from all major lobes, to include bronchi
Lymph nodes - mesenteric and tracheobronchial
Nasal turbinates -4 levels including nasal cavity, paranasal sinuses, nasopharynx and nasopharynx duct and nasal associated lymphoid tissue
Oesophagus
Ovaries
Seminal vesicles
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Spleen
Stomach - included keratinised, glandular and antrum in sections
Testes
Thymus
Thyroid - included parathyroids in section where possible
Trachea - including bifurcation
Uterus - uterine body with cervix section
In addition:
- Lungs, larynx, trachea (including bifurcation), nasal turbinates and tracheobronchial lymph nodes were examined for all study animals of groups 2 (5.30 µg/L) and 3 (34.4 µg/L).
Other examinations:
- HAEMATOLOGY, BONE MARROW:
Bone marrow smears were prepared immediately following death on completion of the scheduled treatment period. The smears from all animals of Groups 1 (Control) and 4 (328 µg/L) were examined to assess the cellularity, distribution and morphology of the marrow. The smears from animals of the intermediate and low dose groups were not examined.

- ORGAN WEIGHTS:
The following organs, taken from each animal killed after 4 weeks of treatment were weighted: Adrenals, Brain, Heart, Kidneys, Liver, Lungs with mainstem bronchi, Spleen, Testes and Thymus.
Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.
Statistics:
All 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 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 pathology 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 c, as applicable.
# For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no test article-related clinical signs observed during the detailed weekly physical examination. Signs associated with the administration
procedure included wet fur, on return to the home cage. This sign is considered to be due to the method of restraint used.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight gains were lower for treated males achieving a statistical significant difference at the highest exposure concentration. Mean body weight gains were 73%, 78% and 71% of control gains for animals exposed to 5.30, 34.4 or 328 µg/L, respectively. Mean terminal body weights of treated males were therefore slightly below those of controls with values of 90.3%, 92.7% and 91.7% respectively. Lower gains were evident for females exposed to 34.4 or 328 µg/L (85% of control gains for both groups) but their mean terminal body weights remained close to those of controls (94.9% and 97% of controls respectively). In both sexes, no relationship between exposure concentration and reduction in body weight gain was observed.
The weight loss apparent on Day 25 for all groups, including control, was due to overnight food withdrawal before the blood sampling procedures for haematology and blood chemistry..

FOOD CONSUMPTION
There were no treatment related changes on food consumption.
The lower food consumption in Week 4 of treatment for all groups, including controls, was due to overnight food withdrawal before the blood sampling procedures for haematology and blood chemistry.

HAEMATOLOGY
A higher group mean neutrophil count was apparent for males exposed to 328 µg/L (1.74X control).

CLINICAL CHEMISTRY
There were no test article-related effects.

ORGAN WEIGHTS
Higher group mean lung and bronchi weights were observed for males and females exposed to 34.4 or 328 µg/L (exposure related); 1.1 and 1.8X control for males receiving 34.4 or 328 µg/L respectively (values adjusted for terminal body weight) and 1.2 and 1.7X control for females receiving 34.4 or 328 µg/L respectively (absolute values).
Lower group mean spleen weight was apparent for females exposed to 328 µg/L, 0.7X control (unadjusted and adjusted for terminal body weight). Nomicroscopic finding was identified to correlate with this lower group mean spleen weight.

GROSS PATHOLOGY
Pale areas were observed in the lungs of all males and the majority of females receiving 328 µg/L, one female receiving 5.3 µg/L and two control females.Enlargement of the tracheobronchial lymph nodes was noted in both sexes receiving 34.4 or 328 µg/L. Enlarged mediastinal lymph nodes were noted in three female animals receiving 328 µg/L and one male animal receiving 5.3 µg/L.
(See 7.5.2/1 Macropathology results in any other information on results incl.tables).

HISTOPATHOLOGY: NON-NEOPLASTIC
Changes related to treatment were seen in the nasal turbinates, larynx, lungs and tracheobronchial and mediastinal lymph nodes:

Minimal eosinophilic globules were observed in the respiratory epithelium lining the nasal septum in one male and one female receiving 328 µg/L, and in one female receiving 34.4 µg/L. This finding was also observed at a slight severity in one female receiving 328 µg/L.

A minimal inflammatory cell infiltrate was observed in the larynx of all males receiving 328 µg/L and in one male receiving 34.4 µg/L. This finding was also observed at a minimal or slight severity in the majority of females receiving 328 µg/L. Minimal or slight squamous metaplasia of the respiratory epithelium was observed in all females and males receiving 328 µg/L, and in the majority of females and two males receiving 34.4 µg/L. An increase in the severity of this finding was observed with increasing dose. Minimal necrosis of the ventral cartilage was observed in the majority of females and two males receiving 328 µg/L.

A moderate increase in diffuse alveolar macrophages and minimal or slight bronchioloalveolar hyperplasia within alveoli was observed in all animals
receiving 328 µg/L. This finding was also observed at minimal severity in one control male. Minimal or slight peribronchiolar/perivascular macrophage aggregates were observed in all animals receiving 328 or 34.4 µg/L, with an increase in severity observed with increasing dose. Minimal to moderateinflammation of alveoli was observed in the majority of males receiving 328 µg/L, but not in any females.

A minimal or slight increase in generalised cellularity was observed in the majority of tracheobronchial lymph nodes from animals receiving 328 µg/L and from some animals receiving 34.4 µg/L, with an increase in severity observed with increasing dose. A minimal increase in pigmented macrophages was observed in five males and one female receiving 328 µg/L. This finding was also observed in one male receiving 34.4 µg/L.

Microscopic examination of the mediastinal lymph nodes was only performed where an abnormality had been observed for this tissue at the macroscopic examination. In the mediastinal lymph nodes from three females receiving 328 µg/L, a slight increase in generalised cellularity was observed. No particular findings were found in the mediastinal lymph nodes of the male receiving 5.3 µg/L.

(See 7.5.2/2 Histopathology results in any other information on results incl.tables).

OTHER FINDINGS
Bone marrow analysis:
The cellularity, distribution and morphology of the marrow was unaffected by the treatment.
Key result
Dose descriptor:
NOAEC
Remarks:
Local effects
Effect level:
34.4 other: µg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Minimal necrosis of the ventral larynx, microscopic findings in lungs suggestive of alveolar hyperplasia, changes in Haematology indicative of inflammatory reaction and consistent with findings recorded microscopically.
Key result
Dose descriptor:
NOAEC
Remarks:
Systemic toxicity
Effect level:
328 other: µg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no exposure-related systemic toxicity
Key result
Critical effects observed:
no

7.5.2 / 1 Macropathology results

Lungs

Pale areas were observed in all males and the majority of females receiving 328µg/L, one

female receiving 5.3µg/L and two control females.

 

Summary of findings in the lungs for animals after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Pale area (s)

0

0

0

5

2

1

0

4

 

Tracheobronchial lymph nodes

Enlargement of the tracheobronchial lymph nodes was seen in both sexes receiving 34.4 or

328 µg/L.

 

Summary of findings in the tracheobronchial lymph nodes for animals after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Enlarged

0

0

3

4

0

0

1

5

 

Mediastinal lymph nodes

Enlarged mediastinal lymph nodes were noted in three female animals receiving 328µg/L

and one male animal receiving 5.3µg/L.

 

Summary of findings in the mediastinal lymph nodes for animals after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Enlarged

0

1

0

0

0

0

0

3

 

7.5.2 / 2 Histopathology results

Nasal Turbinates

Summary of treatment related findings in the nasal turbinates for animals killed after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Eosinophilic Globules, Respiratory Epithelium

Minimal

Slight

Total

0

0

0

0

0

0

0

0

0

1

0

1

0

0

0

0

0

0

1

0

1

1

1

2

 

Larynx

Summary of treatment related findings in the larynx for animals killed after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Inflammatory Cell Infiltrate

Minimal

Slight

Total

0

0

0

0

0

0

1

0

1

5

0

5

0

0

0

0

0

0

0

0

0

2

2

4

Squamous Metaplasia, Respiratory Epithelium

Minimal

Slight

Total

0

0

0

0

0

0

2

0

2

1

4

5

0

0

0

0

0

0

4

0

4

2

3

5

Necrosis, Ventral Cartilage

Minimal

Total

0

0

0

0

0

0

2

2

0

0

0

0

0

0

4

4

 

Lungs

Summary of treatment related findings in the lungs for animals killed after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Increased Alveolar Macrophages, Diffuse

Minimal

Moderate

Total

1

0

1

0

0

0

0

0

0

0

5

5

0

0

0

0

0

0

0

0

0

0

5

5

Macrophages Aggregates, Peribronchiolar/ Perivascular

Minimal

Slight

Total

0

0

0

0

0

0

5

0

5

0

5

5

0

0

0

0

0

0

5

0

5

1

4

5

Bronchioloalveolar Hyperplasia, Alveoli

Minimal

Slight

Total

0

0

0

0

0

0

0

0

0

2

3

5

0

0

0

0

0

0

0

0

0

5

0

5

Inflammation, Alveoli

Minimal

Slight

Moderate

Total

0

0

0

0

0

0

0

0

0

0

0

0

2

1

1

4

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Tracheobronchial lymph nodes 

Summary of treatment related findings in thetracheobronchial lymph nodes for animals killed after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Increased Cellularity, Generalised

Minimal

Slight

Total

0

0

0

0

0

0

3

0

3

0

4

4

0

0

0

0

0

0

1

0

1

1

4

5

Increased Pigmented Macrophages

Minimal

Total

0

0

0

0

1

1

5

5

0

0

0

0

0

0

1

1

 

Mediastinal lymph nodes

Microscopic examination of the mediastinal lymph nodes was only performed where an abnormality had been observed for this tissue at the macroscopic examination. In the mediastinal lymph nodes from three females receiving 328µg/L, a slight increase in generalised cellularity was observed. No particular findings were found in the mediastinal lymph nodes of the male receiving 5.3µg/L.

 

Conclusions:
The test article was administered by snout-only inhalation, for 6 hours a day, 5 days a week, for 4 weeks at achieved exposure levels of 5.30, 34.4 or 328 µg/L.
Changes related to treatment were seen in the nasal turbinates, larynx, lungs and tracheobronchial and mediastinal lymph nodes.
In the nasal turbinates, a concentration-related increase of eosinophilic droplets in the respiratory epithelium was observed at 34.4 and 328 µg/L. The eosinophilic droplets were restricted to a focal area of the epithelium and were generally of minimal severity and not associated with any other histopathological changes, so were considered as non-adverse.

Histopathological changes in the larynx of both sexes were noted in a concentration-related manner at 34.4 and 328 µg/L. These findings included squamous metaplasia, submucosal inflammation and necrosis of the ventral cartilage. The latter finding was noted only in animals receiving 328 µg/L. Minor findings in the larynx of animals receiving 34.4 µg/L were considered as non-adverse. These findings were consistent with irritation of the mucosa possibly due to the mechanical impaction and deposition of the test-article. It is known that rat shows a high susceptibility to develop squamous laryngeal metaplasia due to anatomical, airflow and epithelial pattern. The observation of necrosis in animals of both sexes in the 328 µg/L group was considered adverse.

In the lungs of all animals receiving 328 µg/L, increases in diffuse alveolar macrophages, peribronchiolar/perivascular macrophages and bronchioloalveolar hyperplasia were observed. Alveolar inflammation, consisting of viable and degenerate neutrophils, macrophages and lymphocytes, was evident for males exposed to 328 µg/L and was considered adverse. At 34.4 µg/L, minimal peribronchiolar and perivascular macrophages aggregates were noted. In the absence of any inflammatory effects in the lungs, these findings were considered to be a normal physiological response and not adverse. The increases in macrophages correlated to the lung load with the test item and were considered to be related to pulmonary clearance.

Higher concentrations of the test item also resulted in concentration-dependent increases in cellularity and pigmented macrophages in the tracheobronchial lymph nodes of both sexes. Increase in mediastinal lymph nodes cellularity was noted only in females receiving 328 µg/L. 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 airwaysand alveolar spaces and trafficking to the local draining lymph nodes.

On the basis of these findings, the No Observed Adverse Effect Level for this study was considered to be 34.4 µg/L.
Executive summary:

The cumulative toxicity of the test item was assessed when administered to Wistar rats by snout-only inhalation administration for 6 hours per day, 5 days per week, over a period of 4 weeks.The study was designated to provide a rational basis for the assessment of the toxicological risk to man. Three groups, each comprising five male and five female rats, received 99422018 at target exposure levels of 5.0, 30 or 300µg/L. A similarly constituted control group received air at the same operating conditions as the 300µg/L group. During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), haematology (bone marrow), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

The achieved gravimetric aerosol concentrations were 5.30, 34.4 and 328µg/L (106, 115 and 109% of the target concentrations). Gravimetric

results were confirmed chemically once each week. The achieved chemical aerosol concentrations were 94, 118 and 107% of the target concentrations for the low, mid and high exposure groups respectively. The Mass Median Aerodynamic Diameters for all treated groups were within the acceptable range (1-4µm) for a repeat dose inhalation study; however these were slightly above the targeted range of 1 to 3µm.

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

A decrease in mean body weight gains was observed in all treated males and at the two highest concentrations in females. In both sexes, no

relationship between exposure concentration and reduction in body weight gain was observed but the decrease in mean body weight gain was statistically significant in males receiving 328µg/L.

Haematology showed a higher mean neutrophil count in males exposed to 328µg/L (1.74X control). There were no effects on blood parameters in

animals exposed to 34.4 or 5.30µg/L.

Higher group mean lung and bronchi weights were observed for males and females exposed to 34.4 or 328µg/L (exposure related) up to 1.8X

control for males (values adjusted for terminal body weight) and up to 1.7X control for females (absolute values). Lower group mean spleen weight was apparent for females exposed to 328µg/L but did not correlate to any microscopic findings. No effects were observed on organ weights for animals receiving 5.30µg/L.

Macroscopic examination revealed pale areas in the lungs of all males and all but one female receiving 328µg/L. Enlargement of the

tracheobronchial lymph nodes was seen in both sexes receiving 34.4 or 328µg/L. Enlarged mediastinal lymph nodes were noted in the majority of females receiving 328µg/L and in one male receiving 5.3µg/L but this latter did not correlate to any microscopic findings.

Microscopically, changes related to treatment were seen in the nasal turbinates, larynx, lungs and tracheobronchial and mediastinal lymph nodes.

In the nasal turbinates, eosinophilic globules were observed at a minimal or slight severity in the respiratory epithelium lining the nasal septum in

one male and two females receiving 328µg/L, and in one female receiving 34.4µg/L.

In the larynx, inflammatory cell infiltrate was observed at a minimal or slight severity in all males and all but one females receiving 328µg/L. One

male receiving 34.4µg/L also showed a minimal inflammatory cell infiltrate. Minimal to slight squamous metaplasia of the respiratory epithelium was observed in all animals receiving 328µg/L together with a minimal necrosis of the ventral cartilage in all but one females and two males. Minimal squamous metaplasia was observed in all but one females and two males receiving 34.4µg/L.

In the lungs, moderate increases in diffuse alveolar macrophages were observed within alveoli of all animals receiving 328µg/L and correlated

with the pale areas observed at the macroscopic examination. Accompanying alveolar changes consisted of minimal to slight hyperplasia in both sexes and minimal to moderate inflammation in all but one males. High group mean neutrophil counts seen in males were likely to be related to this inflammatory response as no abnormalities were observed in bone marrow. Minimal or slight peribronchiolar/perivascular macrophage aggregates were observed in all animals receiving 328 or 34.4µg/L, with an increase in severity observed with increasing exposure level. Overall, these microscopic changes correlated with the higher group mean lung and bronchi weights observed for animals receiving 34.4 or 328µg/L.

In the tracheobronchial lymph nodes, an exposure-related increase in cellularity and pigmented macrophages were observed in both sexes that

received 34.4 or 328µg/L. These changes correlated with the enlarged lymph nodes noted macroscopically. In the mediastinal lymph nodes, slight increases in cellularity was observed for three females receiving 328µg/L, correlating with enlargement observed macroscopically.

In conclusion, changes related to treatment were seen in the nasal turbinates, larynx, lungs and tracheobronchial and mediastinal

lymph nodes.

In the nasal turbinates, a concentration-related increase of eosinophilic droplets in the respiratory epithelium was observed at 34.4 and 328µg/L.

The eosinophilic droplets were restricted to a focal area of the epithelium and were generally of minimal severity and not associated with any other histopathological changes, so were considered as non-adverse. Histopathological changes in the larynx of both sexes were noted in a concentration-related manner at 34.4 and 328µg/L. These findings included squamous metaplasia, submucosal inflammation and necrosis of the ventral cartilage. The latter finding was noted only in animals receiving 328µg/L. Minor findings in the larynx of animals receiving 34.4µg/L were considered as non-adverse. These findings were consistent with irritation of the mucosa possibly due to the mechanical impaction and deposition of the test-article. It is known that rat shows a high susceptibility to develop squamous laryngeal metaplasia due to anatomical airflow and epithelial pattern. The observation of necrosis in animals of both sexes in the 328µg/L group was considered adverse. In the lungs of all animals receiving 328µg/L, increases in diffuse alveolar macrophages, peribronchiolar/perivascular macrophages and bronchioloalveolar hyperplasia were observed. Alveolar inflammation, consisting of viable and degenerate neutrophils, macrophages and lymphocytes, was evident for males exposed to 328µg/L and was considered adverse. At 34.4µg/L, minimal peribronchiolar and perivascular macrophages aggregates were noted. In the absence of any inflammatory effects in the lungs, these findings were considered to be a normal physiological response and not adverse. The increases in macrophages correlated to the lung load with the test item and were considered to be related to pulmonary clearance. Higher concentrations of the test item also resulted in concentration-dependent increases in cellularity and pigmented macrophages in the tracheobronchial lymph nodes of both sexes. Increase in mediastinal lymph nodes cellularity was noted only in females receiving 328µg/L. 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.

On the basis of these findings, the No Observed Adverse Effect Level for this study was considered to be 34.4µg/L.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
328 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
28-day oral toxicity study complete and sufficient to fulfill the REACh annex VIII requirements

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2014-08-18 to 2015-04-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Harlan (UK) Ltd.
- Age at study initiation:61 to 67 days
- Weight at study initiation: 212 to 248 g (males) and 161 to 194 g (females)
- Fasting period before study:no
- Housing: The animals were housed five of one sex per cage
- Diet: Ad libitum, standard rodent diet (Rat and Mouse n°1 Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°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.
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.2 - <= 3.4 µm
Geometric standard deviation (GSD):
2.43
Remarks on MMAD:
MMAD / GSD: The Mass Median Aerodynamic Diameters for all Groups were within the acceptable range (1-4 µm) for a repeat dose inhalation study with 80-81% of the collected particles below 7 µm; however these were slightly above the targeted range of 1 to 3 µm.
The Mass Median Aerodynamic Diameters (MMAD) of the test atmosphere were 3.2, 3.3 and 3.4 µm with Geometric Standard Deviation (GSD) of 2.43, 2.36 and 2.34 for the low, mid and high-dosed groups respectively.
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 and a top section.
- Method of holding animals in test chamber: Rats were held in polycarbonate 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: 25 L/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 concentrations of dust were altered by changing the gear ratio (and therefore the speed of rotation of the compressed powder towards the scraper blade) of the mechanism.
- Temperature, humidity, pressure in air chamber:
Temperature was measured using an electronic thermometer inserted into the inhalation chamber. The mean chamber temperatures were all within expected ranges (22.0; 22.9; 22.1 and 22.1 °C for controls, low, mid and high-dosed groups).
Humidity and pressure in air chamber were not reported.
- Air flow rate: Airflow to Wright dust Feed was 19 L/minute
- 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, 12.08 and 17.39 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 of 26, 50, 30 and 26 L/minute for the control, low, mid and high-dose groups respectively. the airflow was filtered locally.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual 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 a glass fibre filters. Sampling was performed at least three times during each exposure and for each dose-group. Samples were collected from a vacant animal exposure port (animals breathing zone). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.
In addition, the aerosol concentrations measured by gravimetric analysis were checked by a chemical analysis once every week. High Performance Liquid Chromatography with Evaporative Light Scattering Detector (ELSD) was used.

- Samples taken from breathing zone: yes, samples were collected from a vacant animal exposure port.

VEHICLE (if applicable)
- none
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. Once every week, the gravimetric analysis was coupled to analytical analysis by HPLC with ELSD detection to check the accuracy of the gravimetric method.
The mean achieved concentrations were 5.30; 34.4 and 328 µg/L and corresponded to 106, 115 and 109% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
5 day / week
Remarks:
Doses / Concentrations:
5, 30, 300 µg/L
Basis:
other: target
Remarks:
Doses / Concentrations:
5.30, 34.4, 328 µg/L
Basis:
analytical conc.
No. of animals per sex per dose:
3 groups, each comprising 5 male and 5 female rats received the test substance at target exposure levels of 5, 30 or 300 µg/L. A similarly constitutedControl group received air only, at the same operating conditions as the high dose group.
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:
In a previous preliminary inhalation toxicity study exposure levels of 0.0639, 0.315 or 1.49 mg/L, equivalent to 63.9, 315 or 1490 µg/L, were well tolerated.
However histopathological treatment related findings were evident in the larynx, lungs and tracheobronchial lymph nodes in all treated groups. Considering the likely progression of lung changes, particularly aggregates of alveolar macrophages, 1490 µg/L was considered unsuitable for longer term administration. Changes in the lungs and larynx at 315 µg/L were generally of lower incidence and/or severity than those seen at 1490 µg/L, therefore for this study, a high exposure level targeted at 300 µg/L was anticipated to induce treatment related changes similar to those previous seen but was expected to be tolerated for 4 weeks. Target exposure levels of 30 and 5.0 µg/L were selected for the intermediate and low groups respectively to identify a no-observed adverse effect level and to explore any possible dose relationship.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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 period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Weeks 1 to 4 of treatment, 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

BODY WEIGHT: Yes
- Time schedule for examinations:The weight of each animal was recorded one week before treatment commenced (Day -7), on the day that treatment commenced (Day 1), twice weekly throughout the study 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. 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: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4 of treatment and before dosing for all study animals.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40.
- 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 4 of treatment and before dosing for all study animals.
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40
- 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 study animals were sacrificed following 4 weeks of exposures.
GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. 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.
HISTOPATHOLOGY: Yes
The following tissues were examined for all study animals of Groups 1 (Control) and 4 (328 µg/L) sacrificed on completion of the 4-week treatment period:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Larynx - 5 sections
Liver - section from two main lobes
Lungs - section from all major lobes, to include bronchi
Lymph nodes - mesenteric and tracheobronchial
Nasal turbinates -4 levels including nasal cavity, paranasal sinuses, nasopharynx and nasopharynx duct and nasal associated lymphoid tissue
Oesophagus
Ovaries
Seminal vesicles
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Spleen
Stomach - included keratinised, glandular and antrum in sections
Testes
Thymus
Thyroid - included parathyroids in section where possible
Trachea - including bifurcation
Uterus - uterine body with cervix section
In addition:
- Lungs, larynx, trachea (including bifurcation), nasal turbinates and tracheobronchial lymph nodes were examined for all study animals of groups 2 (5.30 µg/L) and 3 (34.4 µg/L).
Other examinations:
- HAEMATOLOGY, BONE MARROW:
Bone marrow smears were prepared immediately following death on completion of the scheduled treatment period. The smears from all animals of Groups 1 (Control) and 4 (328 µg/L) were examined to assess the cellularity, distribution and morphology of the marrow. The smears from animals of the intermediate and low dose groups were not examined.

- ORGAN WEIGHTS:
The following organs, taken from each animal killed after 4 weeks of treatment were weighted: Adrenals, Brain, Heart, Kidneys, Liver, Lungs with mainstem bronchi, Spleen, Testes and Thymus.
Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.
Statistics:
All 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 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 pathology 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 c, as applicable.
# For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no test article-related clinical signs observed during the detailed weekly physical examination. Signs associated with the administration
procedure included wet fur, on return to the home cage. This sign is considered to be due to the method of restraint used.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight gains were lower for treated males achieving a statistical significant difference at the highest exposure concentration. Mean body weight gains were 73%, 78% and 71% of control gains for animals exposed to 5.30, 34.4 or 328 µg/L, respectively. Mean terminal body weights of treated males were therefore slightly below those of controls with values of 90.3%, 92.7% and 91.7% respectively. Lower gains were evident for females exposed to 34.4 or 328 µg/L (85% of control gains for both groups) but their mean terminal body weights remained close to those of controls (94.9% and 97% of controls respectively). In both sexes, no relationship between exposure concentration and reduction in body weight gain was observed.
The weight loss apparent on Day 25 for all groups, including control, was due to overnight food withdrawal before the blood sampling procedures for haematology and blood chemistry..

FOOD CONSUMPTION
There were no treatment related changes on food consumption.
The lower food consumption in Week 4 of treatment for all groups, including controls, was due to overnight food withdrawal before the blood sampling procedures for haematology and blood chemistry.

HAEMATOLOGY
A higher group mean neutrophil count was apparent for males exposed to 328 µg/L (1.74X control).

CLINICAL CHEMISTRY
There were no test article-related effects.

ORGAN WEIGHTS
Higher group mean lung and bronchi weights were observed for males and females exposed to 34.4 or 328 µg/L (exposure related); 1.1 and 1.8X control for males receiving 34.4 or 328 µg/L respectively (values adjusted for terminal body weight) and 1.2 and 1.7X control for females receiving 34.4 or 328 µg/L respectively (absolute values).
Lower group mean spleen weight was apparent for females exposed to 328 µg/L, 0.7X control (unadjusted and adjusted for terminal body weight). Nomicroscopic finding was identified to correlate with this lower group mean spleen weight.

GROSS PATHOLOGY
Pale areas were observed in the lungs of all males and the majority of females receiving 328 µg/L, one female receiving 5.3 µg/L and two control females.Enlargement of the tracheobronchial lymph nodes was noted in both sexes receiving 34.4 or 328 µg/L. Enlarged mediastinal lymph nodes were noted in three female animals receiving 328 µg/L and one male animal receiving 5.3 µg/L.
(See 7.5.2/1 Macropathology results in any other information on results incl.tables).

HISTOPATHOLOGY: NON-NEOPLASTIC
Changes related to treatment were seen in the nasal turbinates, larynx, lungs and tracheobronchial and mediastinal lymph nodes:

Minimal eosinophilic globules were observed in the respiratory epithelium lining the nasal septum in one male and one female receiving 328 µg/L, and in one female receiving 34.4 µg/L. This finding was also observed at a slight severity in one female receiving 328 µg/L.

A minimal inflammatory cell infiltrate was observed in the larynx of all males receiving 328 µg/L and in one male receiving 34.4 µg/L. This finding was also observed at a minimal or slight severity in the majority of females receiving 328 µg/L. Minimal or slight squamous metaplasia of the respiratory epithelium was observed in all females and males receiving 328 µg/L, and in the majority of females and two males receiving 34.4 µg/L. An increase in the severity of this finding was observed with increasing dose. Minimal necrosis of the ventral cartilage was observed in the majority of females and two males receiving 328 µg/L.

A moderate increase in diffuse alveolar macrophages and minimal or slight bronchioloalveolar hyperplasia within alveoli was observed in all animals
receiving 328 µg/L. This finding was also observed at minimal severity in one control male. Minimal or slight peribronchiolar/perivascular macrophage aggregates were observed in all animals receiving 328 or 34.4 µg/L, with an increase in severity observed with increasing dose. Minimal to moderateinflammation of alveoli was observed in the majority of males receiving 328 µg/L, but not in any females.

A minimal or slight increase in generalised cellularity was observed in the majority of tracheobronchial lymph nodes from animals receiving 328 µg/L and from some animals receiving 34.4 µg/L, with an increase in severity observed with increasing dose. A minimal increase in pigmented macrophages was observed in five males and one female receiving 328 µg/L. This finding was also observed in one male receiving 34.4 µg/L.

Microscopic examination of the mediastinal lymph nodes was only performed where an abnormality had been observed for this tissue at the macroscopic examination. In the mediastinal lymph nodes from three females receiving 328 µg/L, a slight increase in generalised cellularity was observed. No particular findings were found in the mediastinal lymph nodes of the male receiving 5.3 µg/L.

(See 7.5.2/2 Histopathology results in any other information on results incl.tables).

OTHER FINDINGS
Bone marrow analysis:
The cellularity, distribution and morphology of the marrow was unaffected by the treatment.
Key result
Dose descriptor:
NOAEC
Remarks:
Local effects
Effect level:
34.4 other: µg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Minimal necrosis of the ventral larynx, microscopic findings in lungs suggestive of alveolar hyperplasia, changes in Haematology indicative of inflammatory reaction and consistent with findings recorded microscopically.
Key result
Dose descriptor:
NOAEC
Remarks:
Systemic toxicity
Effect level:
328 other: µg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no exposure-related systemic toxicity
Key result
Critical effects observed:
no

7.5.2 / 1 Macropathology results

Lungs

Pale areas were observed in all males and the majority of females receiving 328µg/L, one

female receiving 5.3µg/L and two control females.

 

Summary of findings in the lungs for animals after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Pale area (s)

0

0

0

5

2

1

0

4

 

Tracheobronchial lymph nodes

Enlargement of the tracheobronchial lymph nodes was seen in both sexes receiving 34.4 or

328 µg/L.

 

Summary of findings in the tracheobronchial lymph nodes for animals after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Enlarged

0

0

3

4

0

0

1

5

 

Mediastinal lymph nodes

Enlarged mediastinal lymph nodes were noted in three female animals receiving 328µg/L

and one male animal receiving 5.3µg/L.

 

Summary of findings in the mediastinal lymph nodes for animals after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Enlarged

0

1

0

0

0

0

0

3

 

7.5.2 / 2 Histopathology results

Nasal Turbinates

Summary of treatment related findings in the nasal turbinates for animals killed after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Eosinophilic Globules, Respiratory Epithelium

Minimal

Slight

Total

0

0

0

0

0

0

0

0

0

1

0

1

0

0

0

0

0

0

1

0

1

1

1

2

 

Larynx

Summary of treatment related findings in the larynx for animals killed after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Inflammatory Cell Infiltrate

Minimal

Slight

Total

0

0

0

0

0

0

1

0

1

5

0

5

0

0

0

0

0

0

0

0

0

2

2

4

Squamous Metaplasia, Respiratory Epithelium

Minimal

Slight

Total

0

0

0

0

0

0

2

0

2

1

4

5

0

0

0

0

0

0

4

0

4

2

3

5

Necrosis, Ventral Cartilage

Minimal

Total

0

0

0

0

0

0

2

2

0

0

0

0

0

0

4

4

 

Lungs

Summary of treatment related findings in the lungs for animals killed after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Increased Alveolar Macrophages, Diffuse

Minimal

Moderate

Total

1

0

1

0

0

0

0

0

0

0

5

5

0

0

0

0

0

0

0

0

0

0

5

5

Macrophages Aggregates, Peribronchiolar/ Perivascular

Minimal

Slight

Total

0

0

0

0

0

0

5

0

5

0

5

5

0

0

0

0

0

0

5

0

5

1

4

5

Bronchioloalveolar Hyperplasia, Alveoli

Minimal

Slight

Total

0

0

0

0

0

0

0

0

0

2

3

5

0

0

0

0

0

0

0

0

0

5

0

5

Inflammation, Alveoli

Minimal

Slight

Moderate

Total

0

0

0

0

0

0

0

0

0

0

0

0

2

1

1

4

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

Tracheobronchial lymph nodes 

Summary of treatment related findings in thetracheobronchial lymph nodes for animals killed after 4 weeks of treatment

Sex

Males

Females

Exposure level (µg/L)

0

5.3

34.4

328

0

5.3

34.4

328

Number of animals examined

5

5

5

5

5

5

5

5

Increased Cellularity, Generalised

Minimal

Slight

Total

0

0

0

0

0

0

3

0

3

0

4

4

0

0

0

0

0

0

1

0

1

1

4

5

Increased Pigmented Macrophages

Minimal

Total

0

0

0

0

1

1

5

5

0

0

0

0

0

0

1

1

 

Mediastinal lymph nodes

Microscopic examination of the mediastinal lymph nodes was only performed where an abnormality had been observed for this tissue at the macroscopic examination. In the mediastinal lymph nodes from three females receiving 328µg/L, a slight increase in generalised cellularity was observed. No particular findings were found in the mediastinal lymph nodes of the male receiving 5.3µg/L.

 

Conclusions:
The test article was administered by snout-only inhalation, for 6 hours a day, 5 days a week, for 4 weeks at achieved exposure levels of 5.30, 34.4 or 328 µg/L.
Changes related to treatment were seen in the nasal turbinates, larynx, lungs and tracheobronchial and mediastinal lymph nodes.
In the nasal turbinates, a concentration-related increase of eosinophilic droplets in the respiratory epithelium was observed at 34.4 and 328 µg/L. The eosinophilic droplets were restricted to a focal area of the epithelium and were generally of minimal severity and not associated with any other histopathological changes, so were considered as non-adverse.

Histopathological changes in the larynx of both sexes were noted in a concentration-related manner at 34.4 and 328 µg/L. These findings included squamous metaplasia, submucosal inflammation and necrosis of the ventral cartilage. The latter finding was noted only in animals receiving 328 µg/L. Minor findings in the larynx of animals receiving 34.4 µg/L were considered as non-adverse. These findings were consistent with irritation of the mucosa possibly due to the mechanical impaction and deposition of the test-article. It is known that rat shows a high susceptibility to develop squamous laryngeal metaplasia due to anatomical, airflow and epithelial pattern. The observation of necrosis in animals of both sexes in the 328 µg/L group was considered adverse.

In the lungs of all animals receiving 328 µg/L, increases in diffuse alveolar macrophages, peribronchiolar/perivascular macrophages and bronchioloalveolar hyperplasia were observed. Alveolar inflammation, consisting of viable and degenerate neutrophils, macrophages and lymphocytes, was evident for males exposed to 328 µg/L and was considered adverse. At 34.4 µg/L, minimal peribronchiolar and perivascular macrophages aggregates were noted. In the absence of any inflammatory effects in the lungs, these findings were considered to be a normal physiological response and not adverse. The increases in macrophages correlated to the lung load with the test item and were considered to be related to pulmonary clearance.

Higher concentrations of the test item also resulted in concentration-dependent increases in cellularity and pigmented macrophages in the tracheobronchial lymph nodes of both sexes. Increase in mediastinal lymph nodes cellularity was noted only in females receiving 328 µg/L. 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 airwaysand alveolar spaces and trafficking to the local draining lymph nodes.

On the basis of these findings, the No Observed Adverse Effect Level for this study was considered to be 34.4 µg/L.
Executive summary:

The cumulative toxicity of the test item was assessed when administered to Wistar rats by snout-only inhalation administration for 6 hours per day, 5 days per week, over a period of 4 weeks.The study was designated to provide a rational basis for the assessment of the toxicological risk to man. Three groups, each comprising five male and five female rats, received 99422018 at target exposure levels of 5.0, 30 or 300µg/L. A similarly constituted control group received air at the same operating conditions as the 300µg/L group. During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), haematology (bone marrow), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

The achieved gravimetric aerosol concentrations were 5.30, 34.4 and 328µg/L (106, 115 and 109% of the target concentrations). Gravimetric

results were confirmed chemically once each week. The achieved chemical aerosol concentrations were 94, 118 and 107% of the target concentrations for the low, mid and high exposure groups respectively. The Mass Median Aerodynamic Diameters for all treated groups were within the acceptable range (1-4µm) for a repeat dose inhalation study; however these were slightly above the targeted range of 1 to 3µm.

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

A decrease in mean body weight gains was observed in all treated males and at the two highest concentrations in females. In both sexes, no

relationship between exposure concentration and reduction in body weight gain was observed but the decrease in mean body weight gain was statistically significant in males receiving 328µg/L.

Haematology showed a higher mean neutrophil count in males exposed to 328µg/L (1.74X control). There were no effects on blood parameters in

animals exposed to 34.4 or 5.30µg/L.

Higher group mean lung and bronchi weights were observed for males and females exposed to 34.4 or 328µg/L (exposure related) up to 1.8X

control for males (values adjusted for terminal body weight) and up to 1.7X control for females (absolute values). Lower group mean spleen weight was apparent for females exposed to 328µg/L but did not correlate to any microscopic findings. No effects were observed on organ weights for animals receiving 5.30µg/L.

Macroscopic examination revealed pale areas in the lungs of all males and all but one female receiving 328µg/L. Enlargement of the

tracheobronchial lymph nodes was seen in both sexes receiving 34.4 or 328µg/L. Enlarged mediastinal lymph nodes were noted in the majority of females receiving 328µg/L and in one male receiving 5.3µg/L but this latter did not correlate to any microscopic findings.

Microscopically, changes related to treatment were seen in the nasal turbinates, larynx, lungs and tracheobronchial and mediastinal lymph nodes.

In the nasal turbinates, eosinophilic globules were observed at a minimal or slight severity in the respiratory epithelium lining the nasal septum in

one male and two females receiving 328µg/L, and in one female receiving 34.4µg/L.

In the larynx, inflammatory cell infiltrate was observed at a minimal or slight severity in all males and all but one females receiving 328µg/L. One

male receiving 34.4µg/L also showed a minimal inflammatory cell infiltrate. Minimal to slight squamous metaplasia of the respiratory epithelium was observed in all animals receiving 328µg/L together with a minimal necrosis of the ventral cartilage in all but one females and two males. Minimal squamous metaplasia was observed in all but one females and two males receiving 34.4µg/L.

In the lungs, moderate increases in diffuse alveolar macrophages were observed within alveoli of all animals receiving 328µg/L and correlated

with the pale areas observed at the macroscopic examination. Accompanying alveolar changes consisted of minimal to slight hyperplasia in both sexes and minimal to moderate inflammation in all but one males. High group mean neutrophil counts seen in males were likely to be related to this inflammatory response as no abnormalities were observed in bone marrow. Minimal or slight peribronchiolar/perivascular macrophage aggregates were observed in all animals receiving 328 or 34.4µg/L, with an increase in severity observed with increasing exposure level. Overall, these microscopic changes correlated with the higher group mean lung and bronchi weights observed for animals receiving 34.4 or 328µg/L.

In the tracheobronchial lymph nodes, an exposure-related increase in cellularity and pigmented macrophages were observed in both sexes that

received 34.4 or 328µg/L. These changes correlated with the enlarged lymph nodes noted macroscopically. In the mediastinal lymph nodes, slight increases in cellularity was observed for three females receiving 328µg/L, correlating with enlargement observed macroscopically.

In conclusion, changes related to treatment were seen in the nasal turbinates, larynx, lungs and tracheobronchial and mediastinal

lymph nodes.

In the nasal turbinates, a concentration-related increase of eosinophilic droplets in the respiratory epithelium was observed at 34.4 and 328µg/L.

The eosinophilic droplets were restricted to a focal area of the epithelium and were generally of minimal severity and not associated with any other histopathological changes, so were considered as non-adverse. Histopathological changes in the larynx of both sexes were noted in a concentration-related manner at 34.4 and 328µg/L. These findings included squamous metaplasia, submucosal inflammation and necrosis of the ventral cartilage. The latter finding was noted only in animals receiving 328µg/L. Minor findings in the larynx of animals receiving 34.4µg/L were considered as non-adverse. These findings were consistent with irritation of the mucosa possibly due to the mechanical impaction and deposition of the test-article. It is known that rat shows a high susceptibility to develop squamous laryngeal metaplasia due to anatomical airflow and epithelial pattern. The observation of necrosis in animals of both sexes in the 328µg/L group was considered adverse. In the lungs of all animals receiving 328µg/L, increases in diffuse alveolar macrophages, peribronchiolar/perivascular macrophages and bronchioloalveolar hyperplasia were observed. Alveolar inflammation, consisting of viable and degenerate neutrophils, macrophages and lymphocytes, was evident for males exposed to 328µg/L and was considered adverse. At 34.4µg/L, minimal peribronchiolar and perivascular macrophages aggregates were noted. In the absence of any inflammatory effects in the lungs, these findings were considered to be a normal physiological response and not adverse. The increases in macrophages correlated to the lung load with the test item and were considered to be related to pulmonary clearance. Higher concentrations of the test item also resulted in concentration-dependent increases in cellularity and pigmented macrophages in the tracheobronchial lymph nodes of both sexes. Increase in mediastinal lymph nodes cellularity was noted only in females receiving 328µg/L. 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.

On the basis of these findings, the No Observed Adverse Effect Level for this study was considered to be 34.4µg/L.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
34.4 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
28-day oral toxicity study complete and sufficient to fulfill the REACh annex VIII requirements

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The cumulative toxicity of the test substance was assessed in a 4 -week inhalation toxicity study in rat. The study was performed in accordance with OECD guideline 412 and in compliance with Good Laboratory Practices.

The test substance was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 4 weeks at achieved aerosol concentrations of 5.3, 34.4 or 328 µg/L. A similarly constituted control group received air at the same operating conditions as the 328 µg/L group.

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

A decrease in mean body weight gain was observed in all treated males and at the two highest concentrations in females. In both sexes, no relationship between exposure concentration and reduction in body weight gain was observed but the decrease in mean body weight gain was statistically significant in males receiving 328µg/L.

Haematology showed a higher mean neutrophil count in males exposed to 328µg/L. No abnormalities were observed in the bone marrow thus confirming that this change was secondary to the local inflammatory effects observed in the lungs.

A statistical significant increase in mean lung and bronchi weights was observed in both sexes from 34.4 µg/L. Macroscopic examination revealed pale areas in the lungs of all males and all but one female receiving 328µg/L. Enlargement of the tracheobronchial lymph nodes was seen in both

sexes receiving 34.4 or 328µg/L. Enlarged mediastinal lymph nodes were noted in the majority of females receiving 328µg/L and in one male receiving 5.3µg/L but this latter did not correlate to any microscopic findings.

Microscopically, changes related to treatment were seen in the nasal turbinates, larynx, lungs and tracheobronchial and mediastinal lymph nodes.

In the nasal turbinates, a concentration-related increase of eosinophilic droplets in the respiratory epithelium was observed at 34.4 and 328µg/L.

The eosinophilic droplets were restricted to a focal area of the epithelium and were generally of minimal severity and not associated with any other histopathological changes, so were considered as non-adverse.

In the larynx, concentration-related histopathological changes were noted in both sexes from 34.4 µg/L. These findings included squamous metaplasia and submucosal inflammation that were of minimal and slight severities at 34.4 µg/L and 328 µg/L respectively. These findings were consistent with irritation of the mucosa possibly due to the mechanical impaction and deposition of the test-article. At 328 µg/L, minimal ventral necrosis was observed in both sexes. This finding was considered to be adverse but not relevant to humans as it is a rat specific lesion related to the particular anatomical airflow and epithelial pattern of this area in rat.

In the lungs of all animals receiving 328µg/L, increases in diffuse alveolar macrophages, peribronchiolar/perivascular macrophages and bronchioloalveolar hyperplasia were observed. Alveolar inflammation was evident for males exposed to 328µg/L and was considered adverse. At 34.4µg/L, minimal peribronchiolar and perivascular macrophages aggregates were noted. In the absence of any inflammatory effects in the lungs at this concentration, these findings were considered to be a normal physiological response and not adverse. The increases in macrophages correlated to the lung load with the test item and were considered to be related to pulmonary clearance. Higher concentrations of the test item also resulted in concentration-dependent increases in cellularity and pigmented macrophages in the tracheobronchial lymph nodes of both sexes. Increase in mediastinal lymph nodes cellularity was noted only in females receiving 328µg/L. 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.

On the basis of these findings, the No Observed Adverse Effect Level for this study wasconsidered to be 34.4µg/L.


Justification for classification or non-classification

The following effects which are considered to support STOT-RE classification are addressed individually based on the results of the study:

• Morbidity or death resulting from repeated or long-term exposure : There were no deaths during the study.

• Significant functional changes in the central or peripheral nervous systems or other organ systems, including signs of central nervous system depression and effects on special senses (e.g. sight, hearing and sense of smell): There were no signs of central nervous system depression, no evidence of changes to the peripheral nervous system or special senses.

• Any consistent and significant adverse change in clinical biochemistry, haematology, or urinalysis parameters: No adverse findings were seen in the haematology parameters, the higher neutrophil count seen in males exposed to 328 µg/L were considered non-adverse and correlated with the lung inflammation seen in this group. This finding was not apparent in animals exposed to the No Observed Adverse Effect Level (NOAEL) of 34.4 µg/L. There were no effects on blood chemistry parameters. Urinalysis was not performed in this study.

• Significant organ damage that may be noted at necropsy and/or subsequently seen or confirmed at microscopic examination : Damage to the larynx was evident (ventral cartilage necrosis); however, this is considered a rat specific lesion (Osimitz et al., 2007; Kaufmann et al., 2009) and was confined to animals exposed to 328 µg/L. This finding was not apparent in animals exposed to the (NOAEL) of 34.4 µg/L.

• Multifocal or diffuse necrosis, fibrosis or granuloma formation in vital organs with regenerative capacity : No multifocal or diffuse necrosis, fibrosis or granuloma formation was evident in any of the vital organs.

• Morphological changes that are potentially reversible but provide clear evidence of marked organ dysfunction (e.g. severe fatty change in the liver) : Alveolar inflammation that was confined to males exposed to 328 µg/L was considered adverse but the level of inflammation was never graded marked or severe. The minimal grade macrophage aggregates evident for animals exposed to 34.4 mg/L were not associated with organ dysfunction.

• Evidence of appreciable cell death (including cell degeneration and reduced cell number) in vital organs incapable of regeneration: Cell death was evident in the larynx (ventral cartilage necrosis); however, this is considered a rat specific lesion not relevant to humans(Osimitz et al., 2007; Kaufmann et al., 2009) that was confined to animals exposed to 328 µg/L. This finding was not apparent in animals exposed to the (NOAEL) of 34.4 µg/L.

None of the CLP criteria which trigger the repeated dose toxicity classification (STOT-RE) is met with the substance. Therefore, no classification for repeated dose toxicity is warranted according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.

Osimitz T. G. Droege W. and Finch J. M. (2007). Toxicological significance of histologic change in the larynx of the rat following inhalation exposure. A critical review.Toxicology and Applied Pharmacology225, 229-237.

Kaufmann W, Bader R, Ernst H, Harada T, Hardisty J, Kittel B, Kolling A, Pino M, Renne R, Rittinghausen S, Schulte A, Wöhrmann T, Rosenbruch M. (2009). Larynx squamous metaplasia: A re-consideration of morphology and diagnostic approaches in rodent studies and its relevance for human risk assessment. Exp Toxicol Path,61: 591-603.