12-hydroxyoctadecanoic acid, reaction products with 1,3-benzenedimethanamine and hexamethylenediamine

Endpoint summary

Description of key information

In a subacute oral toxicity study performed in accordance with OECD guideline No 407 and in compliance with GLP, no adverse effects were observed in rats exposed to the test substance up to the limit dose-level of 1000 mg/kg b.w./day.
In a  subchronic inhalation toxicity study performed in accordance with OECD guideline No 413 and in compliance with GLP, histopathological evaluation reported no systemic effects but revealed local marked pulmonary effects. The test substance was a pneumotoxicant responsible for a sustained pulmonary inflammation that was still present after 12 weeks off dose. The cellular changes in the bronchoalveolar lavage (BAL) and haematology  were consistent with the microscopic findings and confirmed the local pulmonary toxicity of the test substance. Based on these findings, the NOAEC was set to 3.3 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

On isolated occasions slight deviations occurred resulting in an overall range of 20 to 25°C for room temperature and 36 to 74% relative humidity.

Principles of method if other than guideline:

Not applicable

Limit test:

yes

Species:

rat

Strain:

other: CD BR rats (Cri : CD® BR)

Sex:

male/female

Route of administration:

oral: gavage

Details on results:

CLINICAL CHEMISTRY: mean (± SD) ALT values (mU/mL) were significantly (p<0.01) increased compared to controls in females at 4 weeks: 33 ± 3.4, 47 ± 4.3**, 42 ± 5.7** and 39 ± 4.0** for control, 15, 150 and 1000 mg/kg bw/day groups, respectively.
Urea nitrogen was significantly (p<0.05) increased in females of the 1000 mg/kg bw/day at 4 weeks (14 ± 1.3 and 17 ± 2.2 mg/dL, for control and 1000 mg/kg bw/day groups, respectively).

ORGAN WEIGHT: adjusted mean kidneys weights (g) were significantly (**: p<0.01; *:p<0.05) decreased compared to controls in females at 4 weeks: 1.98, 1.95, 1.92 and 1.77** for control, 15, 150 and 1000 mg/kg bw/day groups, respectively. They were significantly increased after the recovery period: 1.86 and 1.98* for control and 1000 mg/kg bw/day groups, respectively. The mean adjusted kidneys weights were also significantly lower than controls for males only after the recovery period: 3.05 and 2.72** for control and 1000 mg/kg bw/day groups, respectively.

URINALYSIS: the volume of urine was significantly (p<0.05) increased in females administered 1000 mg/kg bw/day at 4 weeks (2.4 ± 1.28 and 3.8 ± 0.89 mL, for control and 1000 mg/kg bw/day groups, respectively).

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

None

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

28-day oral toxicity study complete and sufficient to fulfill the REACh annex VIII requirements

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 july 2012 to june 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

: see chapter 'any other information on materials and methods'

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (UK) Ltd.
- Age at study initiation:61 to 67 days
- Weight at study initiation: 216 to 269 g (males) and 137 to 188 g (females)
- Fasting period before study:no
- Housing: The animals were housed five of one sex per cage for the Main study, Satellite and Recovery phases, unless this number was reduced by mortality.
- Diet: Ad libitum, standard rodent diet (Rat and Mouse n°1 Maintenance 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: 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

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 mass, of particles smaller than cut-point of each stage versus the logarithm of each stage cut-point. This treatment fits the test data to a single log-normal distribution.However, as a large amount of test article on the final filter stage was apparent a different calculation regime, non-linear regression was used to achieve a better fit of the data to bimodal distributions.
The PSD data were characterised by fitting a curve to the cumulative mixture of 2 log-normal distributions and assuming that both log-normal distributions have the same geometric standard deviation. The data were plotted by the regression of the cumulative percentage, by mass, of particles smaller than cut-point of each stage versus the logarithm of each stage cutpoint.The mass median aerodynamic diameter (MMAD); geometric standard deviation of each
population of the aerosol were derived for each occasion of measurement.


Group Aerosol concentration (µg/L) Particle size
Target Achieved Achieved MMAD1 Contribution % MMAD2 Contribution % GSD
Gravimetric Chemical
mean mean
2 3 3.30 3.16 <0.21 13.0 3.00 87.0 2.23
3 20 21.2 21.4 <0.21 12.7 2.96 87.3 2.13
4 100 101 106 <0.21 10.4 2.75 89.6 2.09

MMAD Mass median aerodynamic diameter (m)
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, four animal exposure sections and a top section with an approximate interval volume of 60L.
- 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: 19L/minute - Supplementary flow: 20-60 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 daily mean chamber temperature values were within the accepted guidelines 19 to 25 °C. The few excursions above 25°C were potentially due to the positioning of the probe near animals producing an artificially high result.
Humidity was measured using an electronic hygrometer inserted into the inhalation chamber. The lowest dose group had an overall mean chamber relative humidity of 10%, the mean value for the mid-dose group was slightly higher at 13% and the control and high-dose groups values higher at 19 and 26% respectively. The low values in the lowest dose group is considered to be due to higher total airflow through the system. The higher values for control and high-dose groups are considered to be due to a greater number of animals present on the chamber.
Pressure in air chamber was not reported.
- Air flow rate: Airflow to Wright dust Feed was 19 L/minute for all groups. Supplementary airflows were 20, 60, 20 and 20 L/minute for the control, low, mid and high-dose groups respectively.
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 6-stage cascade impactor (Marple 296). 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 and 8.0 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 40, 80, 40 and 40 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 3 weeks. Ultra Performance Liquid Chromatography with UV detection analytical method 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 3 weeks, the gravimetric analysis was coupled to analytical analysis by UPLC with UV detection to check the accuracy of the gravimetric method.
The mean achieved concentrations were 3.30; 21.2 and 101 µg/L and corresponded to 110; 106 and 101% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.
The nominal concentrations (mass of the test item dispersed into the exposure system in total air flow used for exposure) showed good correlation with the mean achieved concentration allowing for 40 to 70% efficiency.

Duration of treatment / exposure:

13 weeks followed by a 12- week recovery period

Frequency of treatment:

5 days / week

Remarks:

Doses / Concentrations:
0, 3, 20 and 100 µg/L
Basis:
other: target

Remarks:

Doses / Concentrations:
0, 3.30, 21.2 and 101 µg/L
Basis:
analytical conc.

No. of animals per sex per dose:

- 3 groups, each comprising 10 male and 10 female rats received the testsubstance at target exposure levels of 3, 20 or 100 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 of the Control and high dose groups and 5 male and 5 female rats were assigned to the intermediate and low dose 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.
- A further 10 males and 10 females (with 5 males and 5 females being used for recovery) were allocated to each group and were used for bronchoalveolar lavage evaluation (BAL) only.

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, 21.3, 104 and 441 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 104 or 441 µg/L, however, the severity of the findings for animals that received 441 µg/L precluded this level being selected for this study as it was unlikely that it would be tolerated for 13 weeks. As the findings in the lung were of lower severity at 104 µg/L it was anticipated that a target exposure level of 100 µg/L would be tolerated for 13 weeks. To explore any possible dose relationship, the target exposure levels for the intermediate and low groups were selected as 20 and 3 µg/L respectively.

- 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 of the Control and high dose groups and 5 male and 5 female rats were assigned to the intermediate and low dose 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 a further 10 males and 10 females with 5 males and 5 females being used for recovery
- Post-exposure recovery period in satellite groups: 12 weeks

Positive control:

no

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.
During the recovery period, observations of the animals and their cages were recorded on a study week basis.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Weeks 1 to 4 of treatment and twice weekly during Weeks 5 to 13 (middle and end of each week), 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 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 (101 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), 3 ( 21.2 µg/L) and 4 (101 µg/L). As no treatment-related changes were observed in group 2 (3.03 µg/L), the examination was not extended to the recovery phase.
- 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 animals in Groups 1 (Control), 3 ( 21.2 µg/L) and 4 (101 µg/L).
- 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 in Week 14, following 2 days off dose after the final exposures in Week 13. Recovery phase animals were sacrificed in Week 13 of recovery following a 12 week off-dose period.
GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.
HISTOPATHOLOGY: Yes
The following tissues were examined for all main study animals of Groups 1 (Control) and 4 (101 µg/L) sacrificed on completion of the 13-week treatment period:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Femur with joint -longitudinal section including articular surface, epiphysial plate and bone marrow
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
Nasal turbinates -3 levels including nasal cavity, paranasal sinuses and nasopharynx
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Sternum - included bone marrow
Stomach - included keratinised, glandular and antrum in sections
Thyroid - included parathyroids in section where possible
Uterus - uterine body with cervix section
In addition:
- mediastinal lymph nodes showing macroscopic abnormality were examined for main study animals,
- Tracheobronchial lymph nodes, larynx and lungs which were considered to exhibit a reaction to treatment at the high dose, were examined for all main study animals of groups 2 (3.03 µg/L) and 3 (21.2 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
- Tracheobronchial lymph nodes and lungs of all recovery animals
- Larynx of recovery animals of groups 1 (Control), 3 (21.2 µg/L) and 4 (101 µg/L)

Other examinations:

- HAEMATOLOGY, BONE MARROW:
Bone marrow samples were obtained from the tibia during necropsy of all animals including early decedents.The smears from all animals of Groups 1 (Control) and 4 (101 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 at the end of treatment and all smears from the recovery phase were not examined.

- ORGAN WEIGHTS:
The following organs, taken from each animal killed after 13 weeks of treatment were weighted: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Lungs with mainstem bronchi, Ovaries, Pituitary, Prostate, Salivary glands, Seminal vesicles, Spleen, Testes, Thymus, Thyroid with parathyroids and Uterus with cervix.
Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

- BRONCHOALVEOLAR LAVAGE (BAL):
10 supplementary males and 10 supplementary females (with 5 males and 5 females being used for recovery) were allocated to each treated group for bronchoalveolar lavage evaluation (BAL) purpose only.
BAL fluid samples were examined for:

# Total and differential cell counting: Cell and differential cell counts were performed using light microscopy. Differential cell count included Macrophage number, Macrophage %, Eosinophil number, Eosinophil %, Basophil number, Basophil % , Neutrophil number , Neutrophil % , Lymphocyte number, Lymphocyte % , Monocyte number and Monocyte % . Erythrocytes and epithelial cells were ignored. The number of each cell type 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.

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

All samples from the Week 14 occasion were analysed. As no treatment-related changes were observed in group 2 (3.03 µg/L), the examination was not extended to the recovery phase. For the Week 26, recovery Week 13 occasion, only samples from Groups 1 (Control) , 3 (21.2 µg/L) and 4 (101 µg/L) were analysed.

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, 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 pathology and bronchoalveolar lavage 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 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:

no effects observed

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:

no effects observed

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 3 incidental deaths during the study, 2 animals receiving 21.2 µg/L and 1 receiving 101 µg/L:
2 main study female rats were found dead in the exposure tubes during the treatment period, 1 animal on Week/Day 7/1 from the group receiving 21.2 µg/L, and 1 animal on Week/Day 2/3 from the group receiving 101 µg/L. Histopathology examination revealed moderate congestion and alveolar haemorrhage (terminal) in the lungs of both animals.The cause of death was considered to be accidental and probably due to asphyxiation after turning in the exposure tube. No test article-related findings were seen in these two animals.
1 satellite female which received 21.2 µg/L, was sent to necropsy on welfare grounds during the recovery period, Week/Day 16/4. Clinically this animal was found to be underactive with shallow breathing and unsteady gait. Macroscopically this female had enlarged lymph nodes (mesenteric, mandibular, mediastinal, axial and lumbar) and the tracheobronchial lymph nodes were observed to be pale. In addition, there were findings in the lungs, stomach, liver, spleen and brain. The test article-related findings seen in the tracheobronchial and mediastinal lymph nodes and lungs were similar to those seen for animals that received 21.2 or 101 µg/L and were considered unlikely to have contributed to the clinical condition of this animal. The remaining macroscopic findings were unique to this animal and cannot be excluded from contributing to its clinical deterioration.

Clinical signs associated with the dosing procedure included wet fur on occasion for the majority of animals from all Groups immediately after dosing, this sign is considered to be due to the method of restraint and is not related to treatment. No other clinical signs were observed during the detailed weekly or post dosing physical examinations.

BODY WEIGHT AND WEIGHT GAIN
There were no test article-related effects.
Mean bodyweight gain after 13 weeks was higher in treated females, up to 1.20X control but not dose related. Following 12 weeks off dose mean bodyweight gain in females that received 3.30 or 21.2 µg/L was similar to control, for females that received 101 µg/L bodyweight gain during the recovery period was lower than control (0.90X). In the absence of a clear dose relationship this finding is considered not to be treatment related.
During the recovery period, Day 50, weight losses were observed in 5/5 males and 4/5 females which received 21.2 µg/L. This resulted in a reduction in the overall group means for each sex, -13g and -9g in males and females respectively. There was no corresponding effect on food consumption and bodyweightshad recovered by the next weighing occasion. In the absence of an effect in animals which received 101 µg/L and due to the transient nature of the finding thisis considered not to be test-article related.
There was no further effects in the male treated groups during the main and recovery phases of the study.

FOOD CONSUMPTION
There were no test article-related effects during the main and recovery phases of the study.

OPHTHALMOSCOPIC EXAMINATION
There were no test article-related effects.

HAEMATOLOGY
Test article-related effects were observed in haematology for both sexes during the treatment and recovery phases.
During Week 13 higher mean neutrophil counts were observed for animals receiving 21.2 or 101 µg/L, up to 1.75X and 1.76X control for males and females respectively (dose related). After 12 weeks of recovery neutrophil counts remained higher, up to 1.97X and 1.75X control for males and females respectively (dose related).
During Week 13 higher mean eosinophil counts were observed in males and females receiving 101 µg/L (1.3X and 1.4X control respectively), these remained high following 12 weeks off dose in males (1.75X). As a result of the higher neutrophil and eosinophil counts in males that received 101 µg/L, the mean white blood cell count was also higher than control during recovery Week 12 (1.29X).
During Week 13 lower mean lymphocyte counts were observed in all treated groups of both sexes, as low as 0.76X and 0.72X control for males and females respectively (not dose related), following 12 weeks off dose lymphocyte counts remained lower in females that received 21.2 or 101 µg/L, as low as 0.65X.

CLINICAL CHEMISTRY
There were no test article-related effects.
During Week 13 mean phosphorus concentrations were lower for males that received 21.2 or 101 µg/L (as low as 0.87X) compared with control. There was no dose relationship or similar effect in the females, this was therefore considered not to be an effect of treatment.
During Week 13 mean glucose concentrations were higher in all male treated groups compared with control, up to 1.19X control (not dose related), however the individual values for treated males were not consistently higher than the control range and some of the individual control values were considered to be lower than expected for animals of the age and strain used. This was therefore considered not to be an effect of treatment.
All other differences from control, including those that attained a degree of statistical significance were generally small and considered to be due to intra group variation and therefore not to be of toxicological importance.

ORGAN WEIGHTS
Following 13 Weeks of treatment mean lung and bronchi weights (unadjusted and adjusted for terminal bodyweight) were higher than control in a dose related manner in both sexes that received 21.2 or 101 µg/L; up to 2.42X and 2.37X control for males and females respectively. Following 12 Weeks off dose lung weights remained higher than control in both sexes that received 21.2 or 101 g/L (dose related), up to 2.40X and 2.50X control for males and females respectively.
At the end of the treatment period mean adrenal weights (adjusted and unadjusted for terminal bodyweight) were higher than control in females that received 101 µg/L, 1.13X. Following 12 weeks off dose, weights were similar to control.

GROSS PATHOLOGY
Macroscopic examination performed after 13 weeks of treatment revealed a dose-related increase in incidence of pale areas and accentuated lobular pattern in the lungs of animals receiving 21.2 or 101 µg/L. In addition, incomplete collapse of the lungs was observed in nearly all animals that received 101 µg/L. Enlargement and pale appearance of the tracheobronchial lymph nodes were noted also in nearly all animals receiving 21.2 or 101 µg/L .All of these findings were still evident following 12 weeks of recovery. At the end of the 13-week treatment period, enlargement and pale appearance of the mediastinal lymph nodes were also noted in nearly half of the animals receiving 21.2 or 101 µg/L. At the end of the recovery period an increased incidence of these findings were observed in the lungs of animals that received 101 µg/L.

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

In the lungs, concentrations of 101 µg/L produced slight to moderate increase of alveolar macrophages in both sexes. This change consisted of variable degrees of intra-alveolar accumulation of hypertrophic, vacuolated (foamy) macrophages, including dense aggregates adjacent to terminal bronchioles and alveolar ducts. Accompanying changes consisted of extensive alveolar proteinosis, type II pneumocyte hyperplasia and alveolar inflammatory cells. The incidence and severity of these findings showed a clear dose relationship pattern in both sexes. Similar findings were observed at lower grades of incidence and severity at 21.2 µg/L. Only minor changes were observed at 3.30 µg/L including clusters of foamy alveolar macrophages adjacent to the terminal bronchioles/alveolar ducts, occasionally associated with minor, local epithelial changes.
The microscopic changes reported in the lungs at the end of the treatment period were still present after 12 weeks off dose. In addition, foci of granulomatous inflammation were also observed in two recovery animals previously treated with 101 µg/L.

In the larynx, epithelial hyperplasia was observed at 21.2 and 101 µg/L. in the ventral part (base of the epiglottis). The epithelial changes were generally associated with inflammatory cell infiltration in the lamina propria. Complete recovery was observed in the larynx after 12 weeks off dose.

In the tracheobronchial lymph nodes, increased cellularity and macrophage aggregates were observed at 21.2 and 101 µg/L after 13-week of treatment. The severity of these findings showed a dose related pattern in both sexes. No recovery was observed following 12 weeks off dose and few animals previously receiving 3.30 µg/L showed minimal macrophage aggregates in the tracheobronchial lymph nodes.

In mediastinal lymph nodes, increased cellularity and macrophage aggregates were observed at 21.2 and 101 µg/L in both sexes. These findings were still present after 12 weeks off dose.

OTHER FINDINGS
Bronchoalveolar lavage:
Test article-related statistically significant changes were observed in the bronchoalveolar lavage fluid (BALF) in both sexes that received 21.2 or 101 µg/L, at the end of the treatment and recovery phases.
Control data shows that the vast majority of cells recovered in BALF are macrophages (> 96%). In Week 14, dose-related increases in neutrophils and lymphocytes in the BALF of rats given 21.2 or 101 µg/L led to up to 6% of the cells being lymphocytes and up to 71% of the cells being neutrophils, consequently reducing the proportion of macrophages to 44% or less. After 12 weeks off dose, the proportion of lymphocytes remained high (up to 10% in the same groups, but not dose related) as did the proportion of neutrophils (up to 60%, dose related). Cell proportional distribution was not affected at 3.30 µg/L.
Higher mean lactate dehydrogenase (LDH), phospholipid (PLIP) and total protein (Prot) concentrations were observed in both sexes receiving 21.2 or 101 µg/L during Week 14, up to 18.7X and 19.1X control for LDH, 9.5X and 9.4X control for PLIP and 7.0X and 9.5X for Prot, for males and females respectively. Concentrations remained higher than control at the end of the recovery period, up to 15.4X and 13.2X control for LDH, 8.3X and 6.0X control for PLIP and 8.4X and 8.7X for Prot, for males and females respectively. there were no effects on these parameters at 3.30 µg/L.

Bone marrow analysis:
The cellularity, distribution and morphology of the marrow was unaffected by the treatment.

Dose descriptor:

NOEC

Effect level:

3.3 other: µg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

7.5.2 /1 Macropathology results

 Animals killed after 13 weeks of treatment

Lungs

Pale areas and accentuated lobular pattern were observed at 21.2 and 101 µg/L in both sexes. Incomplete collapse was also present at 101 µg/L in males and in females.

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	3.30	21.2	101	0	3.30	21.2	101
Pale area(s)	0	0	2	10	4	3	4	8
Incomplete collapse	0	0	0	9	0	0	0	8
Lobular pattern accentuated	0	0	4	10	0	0	4	9
Number of animals examined	10	10	10	10	10	10	9	9

Tracheobronchial lymph nodes

Enlargement and pale appearance of the tracheobronchial lymph nodes were observed at 21.2 and 101 µg/L in both sexes.

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	3.30	21.2	101	0	3.30	21.2	101
Enlarged	0	0	8	9	0	0	7	9
Pale	0	0	10	10	0	0	8	9
Number of animals examined	10	10	10	10	10	10	9	9

Mediastinal lymph nodes

Enlargement and pale appearance of the mediastinal lymph nodes were observed at 21.2 and 101 µg/L in both sexes.

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	3.30	21.2	101	0	3.30	21.2	101
Enlarged	0	0	4	5	0	0	1	6
Pale	0	0	4	5	0	0	2	6
Number of animals examined	10	10	10	10	10	10	9	9

 

Animals killed after 12 weeks of recovery

Lungs

Increased incidence of pale areas, incomplete collapse and lobular pattern accentuation were observed in animals that had been treated with 101 µg/L of test article. 

Summary of findings in the lungs for animals killed after 13 weeks of treatment followed by a 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	3.30	21.2	101	0	3.30	21.2	101
Pale area(s)	3	2	4	10	5	3	4	10
Incomplete collapse	0	0	0	10	0	0	0	10
Lobular pattern accentuated	0	0	0	9	0	0	0	10
Number of animals examined	10	5	5	10	10	5	5	10

Tracheobronchial lymph nodes

Enlargement and pale appearance of the tracheobronchial lymph nodes were observed in animals previously treated with 21.2 or 101 µg/L of test article.

Summary of findings in the tracheobronchial lymph nodes for animals killed after 13 weeks of treatment followed by a 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	3.30	21.2	101	0	3.30	21.2	101
Enlarged	0	0	2	9	0	0	2	7
Pale	0	0	5	10	0	0	5	10
Number of animals examined	10	5	5	10	10	5	5	10

Mediastinal lymph nodes

Enlargement and pale appearance of the mediastinal lymph nodes were observed in animals previously treated with 21.2 or 101 µg/L of test article.

Summary of findings in the mediastinal lymph nodes for animals killed after 13 weeks of treatment followed by a 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	3.30	21.2	101	0	3.30	21.2	101
Enlarged	0	0	2	10	0	0	1	10
Pale	0	0	5	10	0	0	5	10
Number of animals examined	10	5	5	10	10	5	5	10

 

7.5.2 /2 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	3.30	21.2	101	0	3.30	21.2	101
Increased Alveolar Macrophages
Minimal	0	3	2	0	0	4	0	0
Slight	0	0	8	0	0	0	8	1
Moderate	0	0	0	10	0	0	1	8
Total	0	3	10	10	0	4	9	9
Alveolar Proteinosis
Minimal	0	0	6	0	0	0	5	0
Slight	0	0	3	0	0	0	3	0
Moderate	0	0	0	0	0	0	1	1
Marked	0	0	0	10	0	0	0	8
Total	0	0	9	10	0	0	9	9
Type II Pneumocyte Hyperplasia
Minimal	0	2	5	0	0	1	3	5
Slight	0	0	0	10	0	0	0	4
Total	0	2	5	10	0	1	3	9
Alveolar Inflammatory Cells
Minimal	1	0	8	5	0	0	8	7
Slight	0	0	0	5	0	0	0	0
Total	1	0	8	10	0	0	8	7
Number of tissues examined	10	10	10	10	10	10	9	9

 

 Larynx

Summary of treatment related findings in the larynx for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	3.30	21.2	101	0	3.30	21.2	101
Epithelial Hyperplasia, Ventral
Minimal	0	0	6	5	0	0	5	5
Slight	0	0	2	5	0	0	0	2
Total	0	0	8	10	0	0	5	7
Inflammatory Cells Lamina Propria
Minimal	0	0	7	8	0	0	6	5
Slight	0	0	0	0	0	0	0	2
Total	0	0	7	8	0	0	6	7
Number of tissues examined	10	10	10	10	10	10	9	9

Conclusions:

The test article was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 13 weeks at achieved aerosol concentrations of 3.30, 21.2 or 101 µg/L, recovery from any effects was assessed during a 12 week off dose period.
In the lungs higher concentrations of the test article produced macrophage increases, extensive alveolar proteinosis, hyperplasia of epithelial cells lining the alveoli and alveolar ducts in association with alveolar mixed inflammatory cells. The incidence and severity of these findings showed a clear dose relationship pattern in both sexes. Higher concentrations of the test article 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 and lymph nodes at the end of the treatment period were still present after 12 weeks off dose. Foci of granulomatous inflammation were also observed in two recovery animals previously treated with 101 µg/L thus suggesting a progression of the initial inflammatory changes towards chronicity. These findings in this study at 21.2 and 101 µg/L were 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.
At 3.30 µg/L, minimal macrophages aggregates were noted in few animals after 13 weeks of treatment and were still present after 12-week off dose. In the absence of any degenerative and inflammatory effects in the lungs, these findings were considered to be a normal physiological response and not adverse. The increase in macrophages correlated to the lung load with the test item and was considered to be related to pulmonary clearance.
On the basis of these findings, the exposure level of 3.30 µg/L was therefore considered to represent the no observed adverse effect level (NOAEL) for this study. A No observed Effect level (NOEL) could not be established.

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 13 weeks, followed by a 12 week recovery period. The results of this study should indicate potential target organs and provide an assessment of the in vivo pulmonary response of the test item by investigations on the bronchoalveolar lavage fluid.

Three groups, each comprising ten male and ten female rats received the test item at target exposure levels of 3, 20 or100 µg/L. A similarly constituted Control group received air only, at the same operating conditions as the high dose group. A further ten male and ten female rats were assigned to each of the Control and high dose groups and five male and five female rats were assigned to the intermediate and low dose 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. A further ten males and ten females (with five males and five females being used for recovery) were allocated to each group and were used for bronchoalveolar lavage evaluation (BAL) only. During the study, clinical condition, bodyweight, food consumption, ophthalmic examination, haematology, blood chemistry, bone marrow, bronchoalveolar lavage, organ weight, macropathology and histopathology investigations were undertaken.

The achieved aerosol concentrations were 3.30, 21.2 and 101µg/L (110, 106 and 101% of target). The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3µm) for a repeat dose inhalation study.

Test article-related effects were observed in haematology at 21.2 and 101 µg/L for both sexes during the treatment and recovery phases. Higher neutrophil counts were observed for animals receiving 21.2 or 101mg/L (up to 1.76X control). After 12 weeks of recovery neutrophil counts remained higher (up to 1.97X control). Higher eosinophil counts were observed for both sexes receiving 101mg/L (up to 1.4X control). Following 12 weeks off dose eosinophil counts remained higher than control in males (1.75X). As a result of the higher neutrophil and eosinophil counts in males that received 101mg/L, the mean white blood cell count was also higher than control during recovery Week 12 (1.29X). No abnormalities were observed in animals bone marrow.

At the end of the treatment period, mean lung and bronchi weights were higher than control in a dose related manner in both sexes that received 21.2 or 101 µg/L( up to 2.42X and 2.37X control for males and females respectively). Following 12 Weeks off dose lung weights remained higher than control in both sexes.  No effects were observed on organ weights for animals receiving 3.30 µg/L. Assessment of the bronchoalveolar lavage fluid (BALF) during Week 14, showed that the vast majority of cells recovered in the BALF of control animals were macrophages. In both sexes that received 21.2 or 101 µg/L changes in the cells included dose-related increases in percentages of neutrophils and lymphocytes with up to 6% of the cells being lymphocytes and up to 71% of the cells being neutrophils, consequently reducing the proportion of macrophages to 44% or less. After 12 weeks off dose, the proportion of lymphocytes remained high (up to 10% in the same groups, but not dose related) as did the proportion of neutrophils (up to 60%, dose related). In addition higher mean lactate dehydrogenase, phospholipid and total protein concentrations were observed in Week 14, up to 18.7X and 19.1X control for lactate dehydrogenase, 9.5X and 9.4X control for phospholipid and 7.0X and 9.5X for total protein, for males and females respectively. There was no evidence of recovery in these parameters following 12 weeks off dose. No changes were observed in bronchoalveolar lavage fluid of animals receiving 3.30 µg/L.

Macroscopic examination performed after 13 weeks of treatment revealed a dose-related increase in incidence of pale areas and accentuated lobular pattern in the lungs of animals receiving 21.2 or 101 µg/L. In addition, incomplete collapse of the lungs was observed in nearly all animals that received 101 µg/L. Enlargement and pale appearance of the tracheobronchial lymph nodes were noted also in nearly all animals receiving 21.2 or 101 µg/L. All of these findings were still evident following 12 weeks of recovery. At the end of the 13-week treatment period, enlargement and pale appearance of the mediastinal lymph nodes were also noted in nearly half of the animals receiving 21.2 or 101 µg/L. At the end of the recovery period an increased incidence of these findings were observed in the lungs of animals that received 101 µg/L. There were no particular findings at the macroscopic examination of the animals receiving 3.30 µg/L.

Treatment related histopathological changes were seen in the lungs, larynx, tracheobronchial and mediastinal lymph nodes. In the lungs slight to moderate increase of alveolar macrophages was observed at 101µg/L in both sexes. This change consisted of variable degrees of intra-alveolar accumulation of hypertrophic, vacuolated (foamy) macrophages, including dense aggregates adjacent to terminal bronchioles and alveolar ducts. Accompanying changes consisted of extensive alveolar proteinosis, type II pneumocyte hyperplasia and alveolar inflammatory cells. Similar findings were observed at lower grades of incidence and severity at 21.2µg/L. Only minor changes were observed at 3.30µg/L including clusters of foamy alveolar macrophages adjacent to the terminal bronchioles/alveolar ducts, occasionally associated with minor, local epithelial changes. After 12 weeks off dose, the changes were still present and foci of granulomatous inflammation were observed in two recovery animals previously treated with 101 µg/L.

In the ventral larynx epithelial hyperplasia was observed at 21.2 and 101µg/L. The epithelial changes were generally associated with inflammatory cell infiltration in the lamina propria. Full recovery was observed in the larynx after 12 weeks off dose. In the tracheobronchial and mediastinal lymph nodes increased cellularity and macrophage aggregates were observed at 21.2 and 101µg/L. No recovery was observed following 12 weeks off dose and few animals previously receiving 3.30µg/L showed minimal macrophage aggregates in the tracheobronchial lymph nodes.

In conclusion, higher concentrations of the test item produced macrophage increases, alveolar proteinosis, hyperplasia of epithelial cells in association with alveolar mixed inflammatory cells in the lungs. The incidence and severity of these findings showed a clear dose relationship pattern in both sexes. 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 and lymph nodes at the end of the treatment period were still present after 12 weeks off dose. Foci of granulomatous inflammation were also observed in two recovery animals previously treated with 101µg/L thus suggesting a progression of the initial inflammatory changes towards chronicity.These findings in this study at 21.2 and 101µg/L were 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. At 3.30 µg/L, minimal macrophages aggregates were noted in few animals after 13 weeks of treatment and were still present after 12-week off dose. In the absence of any degenerative and inflammatory effects in the lungs, these findings were considered to be a normal physiological response and not adverse. The increase in macrophages correlated to the lung load with the thest item and was considered to be related to pulmonary clearance.  

On the basis of  these findings, the exposure level of 3.30 µg/L was therefore considered to represent the no observed adverse effect level (NOAEL) for this study. A No observed Effect level (NOEL) could not be established.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

101 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

90-day inhalation toxicity study with 12 weeks of recovery complete and sufficient to fulfill the REACh annex IX requirements

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 july 2012 to june 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

: see chapter 'any other information on materials and methods'

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (UK) Ltd.
- Age at study initiation:61 to 67 days
- Weight at study initiation: 216 to 269 g (males) and 137 to 188 g (females)
- Fasting period before study:no
- Housing: The animals were housed five of one sex per cage for the Main study, Satellite and Recovery phases, unless this number was reduced by mortality.
- Diet: Ad libitum, standard rodent diet (Rat and Mouse n°1 Maintenance 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: 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

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 mass, of particles smaller than cut-point of each stage versus the logarithm of each stage cut-point. This treatment fits the test data to a single log-normal distribution.However, as a large amount of test article on the final filter stage was apparent a different calculation regime, non-linear regression was used to achieve a better fit of the data to bimodal distributions.
The PSD data were characterised by fitting a curve to the cumulative mixture of 2 log-normal distributions and assuming that both log-normal distributions have the same geometric standard deviation. The data were plotted by the regression of the cumulative percentage, by mass, of particles smaller than cut-point of each stage versus the logarithm of each stage cutpoint.The mass median aerodynamic diameter (MMAD); geometric standard deviation of each
population of the aerosol were derived for each occasion of measurement.


Group Aerosol concentration (µg/L) Particle size
Target Achieved Achieved MMAD1 Contribution % MMAD2 Contribution % GSD
Gravimetric Chemical
mean mean
2 3 3.30 3.16 <0.21 13.0 3.00 87.0 2.23
3 20 21.2 21.4 <0.21 12.7 2.96 87.3 2.13
4 100 101 106 <0.21 10.4 2.75 89.6 2.09

MMAD Mass median aerodynamic diameter (m)
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, four animal exposure sections and a top section with an approximate interval volume of 60L.
- 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: 19L/minute - Supplementary flow: 20-60 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 daily mean chamber temperature values were within the accepted guidelines 19 to 25 °C. The few excursions above 25°C were potentially due to the positioning of the probe near animals producing an artificially high result.
Humidity was measured using an electronic hygrometer inserted into the inhalation chamber. The lowest dose group had an overall mean chamber relative humidity of 10%, the mean value for the mid-dose group was slightly higher at 13% and the control and high-dose groups values higher at 19 and 26% respectively. The low values in the lowest dose group is considered to be due to higher total airflow through the system. The higher values for control and high-dose groups are considered to be due to a greater number of animals present on the chamber.
Pressure in air chamber was not reported.
- Air flow rate: Airflow to Wright dust Feed was 19 L/minute for all groups. Supplementary airflows were 20, 60, 20 and 20 L/minute for the control, low, mid and high-dose groups respectively.
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 6-stage cascade impactor (Marple 296). 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 and 8.0 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 40, 80, 40 and 40 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 3 weeks. Ultra Performance Liquid Chromatography with UV detection analytical method 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 3 weeks, the gravimetric analysis was coupled to analytical analysis by UPLC with UV detection to check the accuracy of the gravimetric method.
The mean achieved concentrations were 3.30; 21.2 and 101 µg/L and corresponded to 110; 106 and 101% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.
The nominal concentrations (mass of the test item dispersed into the exposure system in total air flow used for exposure) showed good correlation with the mean achieved concentration allowing for 40 to 70% efficiency.

Duration of treatment / exposure:

13 weeks followed by a 12- week recovery period

Frequency of treatment:

5 days / week

Remarks:

Doses / Concentrations:
0, 3, 20 and 100 µg/L
Basis:
other: target

Remarks:

Doses / Concentrations:
0, 3.30, 21.2 and 101 µg/L
Basis:
analytical conc.

No. of animals per sex per dose:

- 3 groups, each comprising 10 male and 10 female rats received the testsubstance at target exposure levels of 3, 20 or 100 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 of the Control and high dose groups and 5 male and 5 female rats were assigned to the intermediate and low dose 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.
- A further 10 males and 10 females (with 5 males and 5 females being used for recovery) were allocated to each group and were used for bronchoalveolar lavage evaluation (BAL) only.

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, 21.3, 104 and 441 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 104 or 441 µg/L, however, the severity of the findings for animals that received 441 µg/L precluded this level being selected for this study as it was unlikely that it would be tolerated for 13 weeks. As the findings in the lung were of lower severity at 104 µg/L it was anticipated that a target exposure level of 100 µg/L would be tolerated for 13 weeks. To explore any possible dose relationship, the target exposure levels for the intermediate and low groups were selected as 20 and 3 µg/L respectively.

- 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 of the Control and high dose groups and 5 male and 5 female rats were assigned to the intermediate and low dose 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 a further 10 males and 10 females with 5 males and 5 females being used for recovery
- Post-exposure recovery period in satellite groups: 12 weeks

Positive control:

no

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.
During the recovery period, observations of the animals and their cages were recorded on a study week basis.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Weeks 1 to 4 of treatment and twice weekly during Weeks 5 to 13 (middle and end of each week), 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 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 (101 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), 3 ( 21.2 µg/L) and 4 (101 µg/L). As no treatment-related changes were observed in group 2 (3.03 µg/L), the examination was not extended to the recovery phase.
- 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 animals in Groups 1 (Control), 3 ( 21.2 µg/L) and 4 (101 µg/L).
- 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 in Week 14, following 2 days off dose after the final exposures in Week 13. Recovery phase animals were sacrificed in Week 13 of recovery following a 12 week off-dose period.
GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.
HISTOPATHOLOGY: Yes
The following tissues were examined for all main study animals of Groups 1 (Control) and 4 (101 µg/L) sacrificed on completion of the 13-week treatment period:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Femur with joint -longitudinal section including articular surface, epiphysial plate and bone marrow
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
Nasal turbinates -3 levels including nasal cavity, paranasal sinuses and nasopharynx
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Sternum - included bone marrow
Stomach - included keratinised, glandular and antrum in sections
Thyroid - included parathyroids in section where possible
Uterus - uterine body with cervix section
In addition:
- mediastinal lymph nodes showing macroscopic abnormality were examined for main study animals,
- Tracheobronchial lymph nodes, larynx and lungs which were considered to exhibit a reaction to treatment at the high dose, were examined for all main study animals of groups 2 (3.03 µg/L) and 3 (21.2 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
- Tracheobronchial lymph nodes and lungs of all recovery animals
- Larynx of recovery animals of groups 1 (Control), 3 (21.2 µg/L) and 4 (101 µg/L)

Other examinations:

- HAEMATOLOGY, BONE MARROW:
Bone marrow samples were obtained from the tibia during necropsy of all animals including early decedents.The smears from all animals of Groups 1 (Control) and 4 (101 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 at the end of treatment and all smears from the recovery phase were not examined.

- ORGAN WEIGHTS:
The following organs, taken from each animal killed after 13 weeks of treatment were weighted: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Lungs with mainstem bronchi, Ovaries, Pituitary, Prostate, Salivary glands, Seminal vesicles, Spleen, Testes, Thymus, Thyroid with parathyroids and Uterus with cervix.
Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

- BRONCHOALVEOLAR LAVAGE (BAL):
10 supplementary males and 10 supplementary females (with 5 males and 5 females being used for recovery) were allocated to each treated group for bronchoalveolar lavage evaluation (BAL) purpose only.
BAL fluid samples were examined for:

# Total and differential cell counting: Cell and differential cell counts were performed using light microscopy. Differential cell count included Macrophage number, Macrophage %, Eosinophil number, Eosinophil %, Basophil number, Basophil % , Neutrophil number , Neutrophil % , Lymphocyte number, Lymphocyte % , Monocyte number and Monocyte % . Erythrocytes and epithelial cells were ignored. The number of each cell type 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.

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

All samples from the Week 14 occasion were analysed. As no treatment-related changes were observed in group 2 (3.03 µg/L), the examination was not extended to the recovery phase. For the Week 26, recovery Week 13 occasion, only samples from Groups 1 (Control) , 3 (21.2 µg/L) and 4 (101 µg/L) were analysed.

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, 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 pathology and bronchoalveolar lavage 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 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:

no effects observed

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:

no effects observed

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 3 incidental deaths during the study, 2 animals receiving 21.2 µg/L and 1 receiving 101 µg/L:
2 main study female rats were found dead in the exposure tubes during the treatment period, 1 animal on Week/Day 7/1 from the group receiving 21.2 µg/L, and 1 animal on Week/Day 2/3 from the group receiving 101 µg/L. Histopathology examination revealed moderate congestion and alveolar haemorrhage (terminal) in the lungs of both animals.The cause of death was considered to be accidental and probably due to asphyxiation after turning in the exposure tube. No test article-related findings were seen in these two animals.
1 satellite female which received 21.2 µg/L, was sent to necropsy on welfare grounds during the recovery period, Week/Day 16/4. Clinically this animal was found to be underactive with shallow breathing and unsteady gait. Macroscopically this female had enlarged lymph nodes (mesenteric, mandibular, mediastinal, axial and lumbar) and the tracheobronchial lymph nodes were observed to be pale. In addition, there were findings in the lungs, stomach, liver, spleen and brain. The test article-related findings seen in the tracheobronchial and mediastinal lymph nodes and lungs were similar to those seen for animals that received 21.2 or 101 µg/L and were considered unlikely to have contributed to the clinical condition of this animal. The remaining macroscopic findings were unique to this animal and cannot be excluded from contributing to its clinical deterioration.

Clinical signs associated with the dosing procedure included wet fur on occasion for the majority of animals from all Groups immediately after dosing, this sign is considered to be due to the method of restraint and is not related to treatment. No other clinical signs were observed during the detailed weekly or post dosing physical examinations.

BODY WEIGHT AND WEIGHT GAIN
There were no test article-related effects.
Mean bodyweight gain after 13 weeks was higher in treated females, up to 1.20X control but not dose related. Following 12 weeks off dose mean bodyweight gain in females that received 3.30 or 21.2 µg/L was similar to control, for females that received 101 µg/L bodyweight gain during the recovery period was lower than control (0.90X). In the absence of a clear dose relationship this finding is considered not to be treatment related.
During the recovery period, Day 50, weight losses were observed in 5/5 males and 4/5 females which received 21.2 µg/L. This resulted in a reduction in the overall group means for each sex, -13g and -9g in males and females respectively. There was no corresponding effect on food consumption and bodyweightshad recovered by the next weighing occasion. In the absence of an effect in animals which received 101 µg/L and due to the transient nature of the finding thisis considered not to be test-article related.
There was no further effects in the male treated groups during the main and recovery phases of the study.

FOOD CONSUMPTION
There were no test article-related effects during the main and recovery phases of the study.

OPHTHALMOSCOPIC EXAMINATION
There were no test article-related effects.

HAEMATOLOGY
Test article-related effects were observed in haematology for both sexes during the treatment and recovery phases.
During Week 13 higher mean neutrophil counts were observed for animals receiving 21.2 or 101 µg/L, up to 1.75X and 1.76X control for males and females respectively (dose related). After 12 weeks of recovery neutrophil counts remained higher, up to 1.97X and 1.75X control for males and females respectively (dose related).
During Week 13 higher mean eosinophil counts were observed in males and females receiving 101 µg/L (1.3X and 1.4X control respectively), these remained high following 12 weeks off dose in males (1.75X). As a result of the higher neutrophil and eosinophil counts in males that received 101 µg/L, the mean white blood cell count was also higher than control during recovery Week 12 (1.29X).
During Week 13 lower mean lymphocyte counts were observed in all treated groups of both sexes, as low as 0.76X and 0.72X control for males and females respectively (not dose related), following 12 weeks off dose lymphocyte counts remained lower in females that received 21.2 or 101 µg/L, as low as 0.65X.

CLINICAL CHEMISTRY
There were no test article-related effects.
During Week 13 mean phosphorus concentrations were lower for males that received 21.2 or 101 µg/L (as low as 0.87X) compared with control. There was no dose relationship or similar effect in the females, this was therefore considered not to be an effect of treatment.
During Week 13 mean glucose concentrations were higher in all male treated groups compared with control, up to 1.19X control (not dose related), however the individual values for treated males were not consistently higher than the control range and some of the individual control values were considered to be lower than expected for animals of the age and strain used. This was therefore considered not to be an effect of treatment.
All other differences from control, including those that attained a degree of statistical significance were generally small and considered to be due to intra group variation and therefore not to be of toxicological importance.

ORGAN WEIGHTS
Following 13 Weeks of treatment mean lung and bronchi weights (unadjusted and adjusted for terminal bodyweight) were higher than control in a dose related manner in both sexes that received 21.2 or 101 µg/L; up to 2.42X and 2.37X control for males and females respectively. Following 12 Weeks off dose lung weights remained higher than control in both sexes that received 21.2 or 101 g/L (dose related), up to 2.40X and 2.50X control for males and females respectively.
At the end of the treatment period mean adrenal weights (adjusted and unadjusted for terminal bodyweight) were higher than control in females that received 101 µg/L, 1.13X. Following 12 weeks off dose, weights were similar to control.

GROSS PATHOLOGY
Macroscopic examination performed after 13 weeks of treatment revealed a dose-related increase in incidence of pale areas and accentuated lobular pattern in the lungs of animals receiving 21.2 or 101 µg/L. In addition, incomplete collapse of the lungs was observed in nearly all animals that received 101 µg/L. Enlargement and pale appearance of the tracheobronchial lymph nodes were noted also in nearly all animals receiving 21.2 or 101 µg/L .All of these findings were still evident following 12 weeks of recovery. At the end of the 13-week treatment period, enlargement and pale appearance of the mediastinal lymph nodes were also noted in nearly half of the animals receiving 21.2 or 101 µg/L. At the end of the recovery period an increased incidence of these findings were observed in the lungs of animals that received 101 µg/L.

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

In the lungs, concentrations of 101 µg/L produced slight to moderate increase of alveolar macrophages in both sexes. This change consisted of variable degrees of intra-alveolar accumulation of hypertrophic, vacuolated (foamy) macrophages, including dense aggregates adjacent to terminal bronchioles and alveolar ducts. Accompanying changes consisted of extensive alveolar proteinosis, type II pneumocyte hyperplasia and alveolar inflammatory cells. The incidence and severity of these findings showed a clear dose relationship pattern in both sexes. Similar findings were observed at lower grades of incidence and severity at 21.2 µg/L. Only minor changes were observed at 3.30 µg/L including clusters of foamy alveolar macrophages adjacent to the terminal bronchioles/alveolar ducts, occasionally associated with minor, local epithelial changes.
The microscopic changes reported in the lungs at the end of the treatment period were still present after 12 weeks off dose. In addition, foci of granulomatous inflammation were also observed in two recovery animals previously treated with 101 µg/L.

In the larynx, epithelial hyperplasia was observed at 21.2 and 101 µg/L. in the ventral part (base of the epiglottis). The epithelial changes were generally associated with inflammatory cell infiltration in the lamina propria. Complete recovery was observed in the larynx after 12 weeks off dose.

In the tracheobronchial lymph nodes, increased cellularity and macrophage aggregates were observed at 21.2 and 101 µg/L after 13-week of treatment. The severity of these findings showed a dose related pattern in both sexes. No recovery was observed following 12 weeks off dose and few animals previously receiving 3.30 µg/L showed minimal macrophage aggregates in the tracheobronchial lymph nodes.

In mediastinal lymph nodes, increased cellularity and macrophage aggregates were observed at 21.2 and 101 µg/L in both sexes. These findings were still present after 12 weeks off dose.

OTHER FINDINGS
Bronchoalveolar lavage:
Test article-related statistically significant changes were observed in the bronchoalveolar lavage fluid (BALF) in both sexes that received 21.2 or 101 µg/L, at the end of the treatment and recovery phases.
Control data shows that the vast majority of cells recovered in BALF are macrophages (> 96%). In Week 14, dose-related increases in neutrophils and lymphocytes in the BALF of rats given 21.2 or 101 µg/L led to up to 6% of the cells being lymphocytes and up to 71% of the cells being neutrophils, consequently reducing the proportion of macrophages to 44% or less. After 12 weeks off dose, the proportion of lymphocytes remained high (up to 10% in the same groups, but not dose related) as did the proportion of neutrophils (up to 60%, dose related). Cell proportional distribution was not affected at 3.30 µg/L.
Higher mean lactate dehydrogenase (LDH), phospholipid (PLIP) and total protein (Prot) concentrations were observed in both sexes receiving 21.2 or 101 µg/L during Week 14, up to 18.7X and 19.1X control for LDH, 9.5X and 9.4X control for PLIP and 7.0X and 9.5X for Prot, for males and females respectively. Concentrations remained higher than control at the end of the recovery period, up to 15.4X and 13.2X control for LDH, 8.3X and 6.0X control for PLIP and 8.4X and 8.7X for Prot, for males and females respectively. there were no effects on these parameters at 3.30 µg/L.

Bone marrow analysis:
The cellularity, distribution and morphology of the marrow was unaffected by the treatment.

Dose descriptor:

NOEC

Effect level:

3.3 other: µg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

7.5.2 /1 Macropathology results

 Animals killed after 13 weeks of treatment

Lungs

Pale areas and accentuated lobular pattern were observed at 21.2 and 101 µg/L in both sexes. Incomplete collapse was also present at 101 µg/L in males and in females.

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	3.30	21.2	101	0	3.30	21.2	101
Pale area(s)	0	0	2	10	4	3	4	8
Incomplete collapse	0	0	0	9	0	0	0	8
Lobular pattern accentuated	0	0	4	10	0	0	4	9
Number of animals examined	10	10	10	10	10	10	9	9

Tracheobronchial lymph nodes

Enlargement and pale appearance of the tracheobronchial lymph nodes were observed at 21.2 and 101 µg/L in both sexes.

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	3.30	21.2	101	0	3.30	21.2	101
Enlarged	0	0	8	9	0	0	7	9
Pale	0	0	10	10	0	0	8	9
Number of animals examined	10	10	10	10	10	10	9	9

Mediastinal lymph nodes

Enlargement and pale appearance of the mediastinal lymph nodes were observed at 21.2 and 101 µg/L in both sexes.

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	3.30	21.2	101	0	3.30	21.2	101
Enlarged	0	0	4	5	0	0	1	6
Pale	0	0	4	5	0	0	2	6
Number of animals examined	10	10	10	10	10	10	9	9

 

Animals killed after 12 weeks of recovery

Lungs

Increased incidence of pale areas, incomplete collapse and lobular pattern accentuation were observed in animals that had been treated with 101 µg/L of test article. 

Summary of findings in the lungs for animals killed after 13 weeks of treatment followed by a 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	3.30	21.2	101	0	3.30	21.2	101
Pale area(s)	3	2	4	10	5	3	4	10
Incomplete collapse	0	0	0	10	0	0	0	10
Lobular pattern accentuated	0	0	0	9	0	0	0	10
Number of animals examined	10	5	5	10	10	5	5	10

Tracheobronchial lymph nodes

Enlargement and pale appearance of the tracheobronchial lymph nodes were observed in animals previously treated with 21.2 or 101 µg/L of test article.

Summary of findings in the tracheobronchial lymph nodes for animals killed after 13 weeks of treatment followed by a 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	3.30	21.2	101	0	3.30	21.2	101
Enlarged	0	0	2	9	0	0	2	7
Pale	0	0	5	10	0	0	5	10
Number of animals examined	10	5	5	10	10	5	5	10

Mediastinal lymph nodes

Enlargement and pale appearance of the mediastinal lymph nodes were observed in animals previously treated with 21.2 or 101 µg/L of test article.

Summary of findings in the mediastinal lymph nodes for animals killed after 13 weeks of treatment followed by a 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	3.30	21.2	101	0	3.30	21.2	101
Enlarged	0	0	2	10	0	0	1	10
Pale	0	0	5	10	0	0	5	10
Number of animals examined	10	5	5	10	10	5	5	10

 

7.5.2 /2 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	3.30	21.2	101	0	3.30	21.2	101
Increased Alveolar Macrophages
Minimal	0	3	2	0	0	4	0	0
Slight	0	0	8	0	0	0	8	1
Moderate	0	0	0	10	0	0	1	8
Total	0	3	10	10	0	4	9	9
Alveolar Proteinosis
Minimal	0	0	6	0	0	0	5	0
Slight	0	0	3	0	0	0	3	0
Moderate	0	0	0	0	0	0	1	1
Marked	0	0	0	10	0	0	0	8
Total	0	0	9	10	0	0	9	9
Type II Pneumocyte Hyperplasia
Minimal	0	2	5	0	0	1	3	5
Slight	0	0	0	10	0	0	0	4
Total	0	2	5	10	0	1	3	9
Alveolar Inflammatory Cells
Minimal	1	0	8	5	0	0	8	7
Slight	0	0	0	5	0	0	0	0
Total	1	0	8	10	0	0	8	7
Number of tissues examined	10	10	10	10	10	10	9	9

 

 Larynx

Summary of treatment related findings in the larynx for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	3.30	21.2	101	0	3.30	21.2	101
Epithelial Hyperplasia, Ventral
Minimal	0	0	6	5	0	0	5	5
Slight	0	0	2	5	0	0	0	2
Total	0	0	8	10	0	0	5	7
Inflammatory Cells Lamina Propria
Minimal	0	0	7	8	0	0	6	5
Slight	0	0	0	0	0	0	0	2
Total	0	0	7	8	0	0	6	7
Number of tissues examined	10	10	10	10	10	10	9	9

Conclusions:

The test article was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 13 weeks at achieved aerosol concentrations of 3.30, 21.2 or 101 µg/L, recovery from any effects was assessed during a 12 week off dose period.
In the lungs higher concentrations of the test article produced macrophage increases, extensive alveolar proteinosis, hyperplasia of epithelial cells lining the alveoli and alveolar ducts in association with alveolar mixed inflammatory cells. The incidence and severity of these findings showed a clear dose relationship pattern in both sexes. Higher concentrations of the test article 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 and lymph nodes at the end of the treatment period were still present after 12 weeks off dose. Foci of granulomatous inflammation were also observed in two recovery animals previously treated with 101 µg/L thus suggesting a progression of the initial inflammatory changes towards chronicity. These findings in this study at 21.2 and 101 µg/L were 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.
At 3.30 µg/L, minimal macrophages aggregates were noted in few animals after 13 weeks of treatment and were still present after 12-week off dose. In the absence of any degenerative and inflammatory effects in the lungs, these findings were considered to be a normal physiological response and not adverse. The increase in macrophages correlated to the lung load with the test item and was considered to be related to pulmonary clearance.
On the basis of these findings, the exposure level of 3.30 µg/L was therefore considered to represent the no observed adverse effect level (NOAEL) for this study. A No observed Effect level (NOEL) could not be established.

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 13 weeks, followed by a 12 week recovery period. The results of this study should indicate potential target organs and provide an assessment of the in vivo pulmonary response of the test item by investigations on the bronchoalveolar lavage fluid.

Three groups, each comprising ten male and ten female rats received the test item at target exposure levels of 3, 20 or100 µg/L. A similarly constituted Control group received air only, at the same operating conditions as the high dose group. A further ten male and ten female rats were assigned to each of the Control and high dose groups and five male and five female rats were assigned to the intermediate and low dose 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. A further ten males and ten females (with five males and five females being used for recovery) were allocated to each group and were used for bronchoalveolar lavage evaluation (BAL) only. During the study, clinical condition, bodyweight, food consumption, ophthalmic examination, haematology, blood chemistry, bone marrow, bronchoalveolar lavage, organ weight, macropathology and histopathology investigations were undertaken.

The achieved aerosol concentrations were 3.30, 21.2 and 101µg/L (110, 106 and 101% of target). The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3µm) for a repeat dose inhalation study.

Test article-related effects were observed in haematology at 21.2 and 101 µg/L for both sexes during the treatment and recovery phases. Higher neutrophil counts were observed for animals receiving 21.2 or 101mg/L (up to 1.76X control). After 12 weeks of recovery neutrophil counts remained higher (up to 1.97X control). Higher eosinophil counts were observed for both sexes receiving 101mg/L (up to 1.4X control). Following 12 weeks off dose eosinophil counts remained higher than control in males (1.75X). As a result of the higher neutrophil and eosinophil counts in males that received 101mg/L, the mean white blood cell count was also higher than control during recovery Week 12 (1.29X). No abnormalities were observed in animals bone marrow.

At the end of the treatment period, mean lung and bronchi weights were higher than control in a dose related manner in both sexes that received 21.2 or 101 µg/L( up to 2.42X and 2.37X control for males and females respectively). Following 12 Weeks off dose lung weights remained higher than control in both sexes.  No effects were observed on organ weights for animals receiving 3.30 µg/L. Assessment of the bronchoalveolar lavage fluid (BALF) during Week 14, showed that the vast majority of cells recovered in the BALF of control animals were macrophages. In both sexes that received 21.2 or 101 µg/L changes in the cells included dose-related increases in percentages of neutrophils and lymphocytes with up to 6% of the cells being lymphocytes and up to 71% of the cells being neutrophils, consequently reducing the proportion of macrophages to 44% or less. After 12 weeks off dose, the proportion of lymphocytes remained high (up to 10% in the same groups, but not dose related) as did the proportion of neutrophils (up to 60%, dose related). In addition higher mean lactate dehydrogenase, phospholipid and total protein concentrations were observed in Week 14, up to 18.7X and 19.1X control for lactate dehydrogenase, 9.5X and 9.4X control for phospholipid and 7.0X and 9.5X for total protein, for males and females respectively. There was no evidence of recovery in these parameters following 12 weeks off dose. No changes were observed in bronchoalveolar lavage fluid of animals receiving 3.30 µg/L.

Macroscopic examination performed after 13 weeks of treatment revealed a dose-related increase in incidence of pale areas and accentuated lobular pattern in the lungs of animals receiving 21.2 or 101 µg/L. In addition, incomplete collapse of the lungs was observed in nearly all animals that received 101 µg/L. Enlargement and pale appearance of the tracheobronchial lymph nodes were noted also in nearly all animals receiving 21.2 or 101 µg/L. All of these findings were still evident following 12 weeks of recovery. At the end of the 13-week treatment period, enlargement and pale appearance of the mediastinal lymph nodes were also noted in nearly half of the animals receiving 21.2 or 101 µg/L. At the end of the recovery period an increased incidence of these findings were observed in the lungs of animals that received 101 µg/L. There were no particular findings at the macroscopic examination of the animals receiving 3.30 µg/L.

Treatment related histopathological changes were seen in the lungs, larynx, tracheobronchial and mediastinal lymph nodes. In the lungs slight to moderate increase of alveolar macrophages was observed at 101µg/L in both sexes. This change consisted of variable degrees of intra-alveolar accumulation of hypertrophic, vacuolated (foamy) macrophages, including dense aggregates adjacent to terminal bronchioles and alveolar ducts. Accompanying changes consisted of extensive alveolar proteinosis, type II pneumocyte hyperplasia and alveolar inflammatory cells. Similar findings were observed at lower grades of incidence and severity at 21.2µg/L. Only minor changes were observed at 3.30µg/L including clusters of foamy alveolar macrophages adjacent to the terminal bronchioles/alveolar ducts, occasionally associated with minor, local epithelial changes. After 12 weeks off dose, the changes were still present and foci of granulomatous inflammation were observed in two recovery animals previously treated with 101 µg/L.

In the ventral larynx epithelial hyperplasia was observed at 21.2 and 101µg/L. The epithelial changes were generally associated with inflammatory cell infiltration in the lamina propria. Full recovery was observed in the larynx after 12 weeks off dose. In the tracheobronchial and mediastinal lymph nodes increased cellularity and macrophage aggregates were observed at 21.2 and 101µg/L. No recovery was observed following 12 weeks off dose and few animals previously receiving 3.30µg/L showed minimal macrophage aggregates in the tracheobronchial lymph nodes.

In conclusion, higher concentrations of the test item produced macrophage increases, alveolar proteinosis, hyperplasia of epithelial cells in association with alveolar mixed inflammatory cells in the lungs. The incidence and severity of these findings showed a clear dose relationship pattern in both sexes. 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 and lymph nodes at the end of the treatment period were still present after 12 weeks off dose. Foci of granulomatous inflammation were also observed in two recovery animals previously treated with 101µg/L thus suggesting a progression of the initial inflammatory changes towards chronicity.These findings in this study at 21.2 and 101µg/L were 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. At 3.30 µg/L, minimal macrophages aggregates were noted in few animals after 13 weeks of treatment and were still present after 12-week off dose. In the absence of any degenerative and inflammatory effects in the lungs, these findings were considered to be a normal physiological response and not adverse. The increase in macrophages correlated to the lung load with the thest item and was considered to be related to pulmonary clearance.  

On the basis of  these findings, the exposure level of 3.30 µg/L was therefore considered to represent the no observed adverse effect level (NOAEL) for this study. A No observed Effect level (NOEL) could not be established.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

3.3 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

90-day inhalation toxicity study with 12 weeks of recovery complete and sufficient to fulfill the REACh annex IX 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 substance was tested in two repeated-dose toxicity studies performed by the oral and inhalation routes:

Oral route:

In a subacute oral toxicity study performed in accordance with OECD guideline No 407 and in agreement with Good Laboratory Practice, the test substance was administered to 5 CD BR rats/sex/dose by gavage in corn oil at dose levels of 0, 15, 150 and 1000 mg/kg bw/day. Satellite groups (5/sex) were used.

No mortality was observed in any group. No signs of ill health, behavioural change or reaction to treatment were noted. At necropsy, all organs and tissues appeared normal during the macroscopic observations and there were no particular microscopic findings following 4 weeks of consecutive dosing with the test substance.

Compared to controls, a significant decrease in kidney weight was recorded in females receiving 1000 mg/kg bw/day group at the end of the treatment period. However, this change was not considered to be of toxicological importance as there were no treatment related pathology findings in the female kidneys and the opposite trend was observed at the end of the recovery period, female kidneys weights being slightly higher (+ 13%) than controls. In the absence of other effects, it was concluded that 1000 mg/kg bw/day represented the No Adverse Effect Level (NOAEL).

Inhalation:

The cumulative toxicity of the test item was assessed in a 13 -week inhalation toxicity study in rat followed by a 12 weeks recovery period. The study was performed in accordance with OECD guideline 413 and in compliance with Good Laboratory Practice. The test substance was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 13 weeks at achieved aerosol concentrations of 3.30, 21.2 or 101µg/L, recovery was assessed during a 12 week off dose period. There were no test article-related deaths or effects on bodyweight, food consumption, ophthalmoscopy, blood chemistry or bone marrow. Histopathological findings related to treatment were seen in the lungs, larynx, tracheobronchial and mediastinal lymph nodes. The findings in this study, although at an increased severity correlated with those previously reported in the 14 -day range finding toxicity (Beebe, 2013a).    

In the lungs higher concentrations of 21.2or 101µg/L of the test substance produced macrophage increases, including dense aggregates adjacent to the terminal bronchioles and alveolar ducts, extensive alveolar proteinosis, hyperplasia of epithelial cells lining the alveoli and alveolar ducts (type II pneumocytes) in association with alveolar mixed inflammatory cells. The incidence and severity of these findings showed a clear dose relationship pattern in both sexes. At the lowest dose, there was minimal accumulation of hypertrophic, vacuolated macrophages grouped in small clusters adjacent to the terminal bronchioles/alveolar ducts and alveoli, occasionally associated with minimal, local epithelial changes. The microscopic findings in the lungs were consistent with those described in the literature following inhalation of inert particles and can be attributed to macrophage overload with inhaled test material and subsequent incomplete clearance leading to macrophage accumulation in the terminal air spaces. Non-cellular material was present in the Bronchoaveolar lavage fluid (BALF) and prevented the cells counts at 21.2 and 101 µg/L. It was likely to have been test article and the persistence of this after 12 weeks off dose correlated with the persistence of vacuolated macrophages and indicated an inability of the macrophage system to clear the test article when present in high concentrations.  The microscopic changes reported in the lungs at the end of the treatment period were still present after 12 weeks off dose. Foci of granulomatous inflammation were also observed in two recovery animals previously treated with 101µg/L thus suggesting a progression of the initial inflammatory changes towards chronicity. The severity of the findings at 21.2 and 101µg/L at the end of the treatment period and the lack of regression following 12 weeks off dose at these exposure levels represented an adverse reaction to treatment.

The microscopic findings observed in the lungs correlated with necropsy observations of pale areas, incomplete collapse, lobular pattern accentuation and increased relative lung weights recorded at the end of the treatment period in animals that received 21.2 or101µg/L and in the recovery animals that previously received 101µg/L. Macrophage aggregates in the draining lymph nodes of the lungs (tracheobronchial) were present in animals receiving 21.2 or 101µg/L and correlated with pale lymph nodes observed at necropsy. Increased cellularity suggestive of lymphoid hyperplasia was observed in the groups treated with the same dose levels and correlated with enlarged lymph nodes recorded at necropsy. The changes observed in the local lymph nodes were considered a likely 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. There appeared to be no regression in the incidence or severity of these findings following a 12 week recovery period in animals that received 21.2 or 101µg/L.  Macrophage aggregates and increased cellularity were also seen in the mediastinal lymph nodes at 21.2 or101 µg/L and correlated with pallor and macroscopic enlargement recorded at necropsy. Both microscopic and macroscopic findings were still present after 12 weeks off dose and no regression in the severity of these findings was observed. In the larynx, test article related findings were seen at 21.2 or 101µg/L and consisted of epithelial hyperplasia in the ventral larynx and inflammatory cell infiltration in the lamina propria. These findings were consistent with irritation of the mucosa possibly due to the mechanical impaction and deposition of the test-article. No test article related changes were observed in the larynx after 12 weeks off dose, thus suggesting full recovery. Furthermore it is known that the larynx of the rat is particularly sensitive to inhaled substances and the ventral epithelium is a predilection site, the findings in the larynx are therefore considered to be non-adverse.

The cellular changes in the BALF were indicative of lung injury and were consistent with the findings recorded microscopically. The changes in the proportional distribution of cell types, particularly neutrophils, in the BALF of animals receiving 21.2 or101µg/L, were implicated as contributing to the lung injury incurred during the inflammatory response. The neutrophil influx played a major role in increasing the permeability of the alveolar/capillary barrier and in producing cellular toxicity during the inflammatory response. The increase in neutrophil content in the BALF was consistent with the increase in circulating neutrophils in the plasma, which were recruited into the lungs; the elevation of plasma neutrophils was therefore an indicator of the inflammatory response associated with cellular damage. Moreover, no abnormalities were observed in bone marrow thus confirming that changes in haemotology 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 21.2 or 101 µg/L, characterised by total protein and lactate dehydrogenase, further signified an inflammatory response and damage to the alveolar capillary barrier. LDH occured extracellularly in BALF only in the presence of damaged lung cells. As there was a distinct dose response in the increases of LDH, this suggested more extensive tissue damage in animals that received 101µg/L. The alveolar proteinosis seen at histopathology in both sexes that received 21.2 or 101µg/L was considered to be a result of an accumulation of protein and phospholipids (phospholipo-proteinaceous material) in the alveoli consistent with surfactant damage.    

The components in the BAL which have shown increases, neutrophils, proteins and lactate dehydrogenase, were also consistent with the expected response to the inhalation of insoluble particulate matter. In animals that received 3.30µg/L, no differences from control were noted in the BALF and haematology parameters and only minor changes were observed in the lungs at the end of the treatment period, which consisted of clusters of foamy alveolar macrophages adjacent to the terminal bronchioles/alveolar ducts, occasionally associated with minor, local epithelial changes. No recovery was observed following 12 weeks off dose and few animals previously receiving 3.30µg/L showed minimal macrophage aggregates in the tracheobronchial lymph nodes. Similar findings were not observed at the end of the treatment period in animals receiving the same dose level suggesting a progressive macrophage accumulation in the draining lymph nodes of the lungs up to a detectable level. In the absence of any degenerative and inflammatory effects in the lungs, these findings were considered to be a normal physiological response and not adverse. The increase in macrophages correlated to the lung load with the thest item and was considered to be related to pulmonary clearance.  

On the basis of  these findings, the exposure level of 3.30 µg/L was therefore considered to represent the no observed adverse effect level (NOAEL) for this study. A No observed Effect level (NOEL) could not be established.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study performed according to the OECD guideline 407 and GLP compliant.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Study performed according to the OECD guideline 413 and GLP compliant.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Study performed according to the OECD guideline 413 and GLP compliant.

Justification for classification or non-classification

According to regulation (EC) No. 1272/2008 and its subsequent amendments, the substance is classified in category 2 for target organ toxicity (repeat exposure) on the basis of the observations from the 13 -week inhalation toxicity study in rats in which significant pulmonary toxic effects, of relevance to human health, were produced at exposure concentrations as low as 0.0212 mg/L /6 hours/day.

As these pulmonary toxic effects are local , they will occur only after repeated exposure by inhalation.