Polyamide wax

Endpoint summary

Description of key information

The substance was evaluated in two repeated-dose toxicity studies performed by the oral and inhalation routes.

By oral route, rats treated for 28 days showed no remarkable effects up to a dose-level of 1000 mg/kg bw/day.

By inhalation, no systemic effects were observed in rats exposed up to 26.0 mg/m3 for 90 days while local respiratory effects were noted. Histopathological evaluation performed in the subchronic toxicity revealed local pulmonary effects. The cellular changes in 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 1.55 mg/m3.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2000-04-07 to 2000-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CD® (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river (UK) Limited, Margate, Kent, England
- Age at study initiation: 39 to 43 days
- Weight at study initiation: 184 to 214g for males and 145 to 182g for female
- Fasting period before study: no
- Housing: Five of one sex per cage. The cages were made of a stainless steel body with a stainless steel mesh lid and floor.
- Diet (e.g. ad libitum): ad libitum except overnight before routine blood sampling. Rat and Mouse n°1 Maintenance Diet (Special Diets Services Limited, Witham, Essex, England)
- Water (e.g. ad libitum): ad libitum, via polycarbonates bottles fitted with sipper tubes.
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light, 12-hour dark cycle


IN-LIFE DATES: No data

Route of administration:

oral: gavage

Vehicle:

other: 1% w/v methylcellulose in purified water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared for administration as a series of graded suspensions in 1.0% w/v methylcellulose in purified water. The required amount of test material was weighed and transferred to a suitably sized mortar and ground using a pestle. A small amount of 1.0% methylcellulose was added to the mortar and test material mixed to a smooth paste. The remaining amount of 1.0% methylcellulose was added to give the require concentration and the suspension was mixed using a high-shear homogeniser to produce a homogenous suspension.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous suspensions were obtained with 1.0% w/v methylcellulose in purified water as vehicle
- Concentration in vehicle: 1.5, 15 and 100 mg/ml
- Amount of vehicle (if gavage): 10 mL/kg bw/day
- Lot/batch no. (if required): nodata
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results of the homogeneity assessment showed that at 1.5 and 100 mg/mL, E96096 was homogeneously distributed throughout the 1.0% methylcellulose formulations.
The mean concentrations of E96096 in formulations for dosing on Day 1 of treatment, ranged from 96.0 - 97.7 % of nominal concentrations and were considered satisfactory.

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days/ week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a previously conducted seven-day repeated dose oral toxicity study, CD rats were dosed with 250, 500 or 1000 mg/kg bw /day of the test substance. There were no deaths. The appareance and behaviour of the animals were unaffected by treatment. Bodyweight gain, food consumption and food conversion efficiency were unaffected by treatment. There were no organ weight changes attributable to treatment with the test material and no macroscopic findings at necropsy. Dosages of 15, 150 and 1000 mg/kg were considered suitable for the subsequent 4-week study.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
Animals were inspected at least wice daily for evidence of reaction to treatment or ill-health. Any deviations from normal were recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.


BODY WEIGHT: Yes
- Time schedule for examinations: each animal was weighed during the acclimatisation period, on the day that treatment commenced, weekly throughout the treatment period and before necropsy.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded for each week throughout the treatment period. From these records, the mean consumption per animal was calculated for each cage.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 29
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes, overnight withdrawal of food
- How many animals: 40
- Parameters examined: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), 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: on day 29
- Animals fasted: Yes, overnight withdrawal of food
- How many animals: 40
- Parameters examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase activity (gGT), 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: Yes
- Time schedule for examinations: Before commencement of treatment and weekly during the study. the FOB was performed prior to dosing during the treatment period and at a similar time of day on each occasion.
- Dose groups that were examined: All groups
- Battery of functions tested:
=> in the hand and standard arena observations performed before commencement and during each week of treatment: ease of removal from cage, salivation, lacrimation, exophthalmos, piloerection, fur condition, vocalisation on handling, reactivity to handling, arousal, gait, grooming, palpebral closure, posture, activity counts, rearing count, tremors, twitches, convulsion, defecation, urination.
=> Reflexes and responses were recorded before commencement and during week 4 of treatment: approach response, touch response, auditory qtartle reflex, tail pinch response, righting reflex, body temperature, landing footsplay, grip strength, pupil closure reflex.
=> Motor activity was measured before commencement and during week 4 of treatment. Activity measurements were recorded for ten six-minutes periods (a total of one hour).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded.
HISTOPATHOLOGY: Yes
The following tissues were examined for all study animals of Groups 1 (Control) and 4 (1000 mg/kg bw /day) sacrificed on completion of the 4-week treatment period:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Femur with - longitudinal section including bone marrow
joint
Heart - included auricular and ventricular regions
Ileum - included Peyers patch , where possible
Kidneys - included cortex, medulla and papilla regions
Liver - section from each of the five major lobes
Lungs - section from two major lobes, including bronchi
Spinal cord - transverse and longitudinal section at the cervical level
Stomach - included keratinised, glandular and antrum in sections
Thyroid - included parathyroids in section where possible
Uterus - uterus section separate from cervix section

Statistics:

For organ weights and bodyweight changes, homogeneity of variance was tested using Barlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwisecomparisons, otherwise a Dunnett's test was used.
Inter-group differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test.
For clinical pathology data, if the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of values diffrent from the mode was analysed (Fisher, 1950) followed by a test for a trend in proportion (Mantel, 1963). Otherwise, a test for heterogeneity of variance between treatments was applied (Barlett, 1937). If significant heterogeneity was found at the 1% level, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. If no significant heterogeneity was detected (or if a satisfactory transformation was found) and more than two groups were being compared, group means were compared using William's test for a dose-related response (Williams, 1971-72), or if there was evidence for a non-monotonic response, Dunnett's test (Dunnett, 1955, 1964). For separate two-group comparisons, a Student's t test was used. If significant heterogeneity of variance was present (and could not be removed by a logarithmic transformation), groups were compared using Shirley's non-parametric test for a dose-related response (Shirley, 1977), or if there was evidence for a non-monotonic response, Dunn's test (Dunn, 1964). For separate two-group comparisons, a Wilcoxon rank sum test (Wilcoxon 1945) was used.
Unless stated, group mean values or incidences for the treated groups were not significantly different from those of the Controls (p>0.05).

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:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths and no clinical signs related to the treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain was considered to be unaffected by treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was considered to be unaffected by treatment.

FOOD EFFICIENCY
Food conversion efficiency was considered to be unaffected by treatment.

HAEMATOLOGY
There were no haematologival changes which were considered to be related to treatment.
A few inter-group differences attained statistical significance but there were minor, seen in one sex only and were considered to have arisen by chance.

CLINICAL CHEMISTRY
No clinical chemistry changes which were considered to be related with treatment.

NEUROBEHAVIOUR
There were no inter-group differences in arena observations that were considered to be associated with treatment.
Landing footsplay and grip strength measurements showed some inter-group variation during week 4 of treatment and some differences achieved statistical significance. There were, however, no consistent trends and many of the differences were apparent before commencement of treatment, an association with treatment was therefore discounted.
Motor activity was unaffected by treatment.

ORGAN WEIGHTS
Organ weights were unaffected by treatment.

GROSS PATHOLOGY
Macroscopic examination of animals killed on completion of the treatment period did not reveal any treatment-related findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings which were attributable to treatment with the test material. All microscopic findings were considered to be incidental and of no toxicological importance.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

The daily administration to CD rats of the test material, at dosages of 15, 150 and 1000 mg/kg/day for four weeks was not associated with any evidence of toxicity. The No-Observe-Effect Level (NOEL) is considered to be 1000 mg/kg bw/day .

Executive summary:

In a sub-acute study performed according to OECD 407 guideline and in compliance with GLP, test material diluted in 1.0% methylcellulose in purified water was administered by oral gavage to three groups of rats (5/sex) at 15, 150 and 1000 mg/kg bw/day for four weeks. A control group received vehicle alone at the same volume-dosage (10 mL/kg bw/day).

There were no deaths and no clinical signs related to treatment, no evidence of neurotoxicity during the weekly functional battery test. Bodyweight, food consumption and food conversion efficiency were not affected by treatment. There were no haematological or blood chemistry changes related to treatment. Organ weights were unaffected by treatment and there were no macroscopic or microscopic findings which were attributable to the treatment with the test material.

The No-Observed-Effect Level (NOEL) was considered to be 1000 mg/kg bw/day.

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 is considered as a reliable study with a klimisch score of 1.

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 2019-01-18 to 2019-10-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

yes

Remarks:

: see chapter 'any other information on materials and methods' section 7.5.2 /1

GLP compliance:

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

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

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.5 - <= 2.7 µm

Geometric standard deviation (GSD):

2.45

Remarks on MMAD:

MMAD / GSD: The Particle size distribution (PSD) data was originally characterised by using linear regression of the probit of the cumulative percentage, by massof particles smaller than cut-point of each stage versus the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD); geometric standard deviation of the aerosol were derived for each occasion of measurement.

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

MMAD Mass median aerodynamic diameter (µm)
GSD Geometric standard deviation

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow through nose only chamber. This system was an aluminium alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber.
- Method of holding animals in test chamber: Rats were held in plastic tubes with their snouts protruding from the end of the tubes into the exposure chamber.
- Source and rate of air: From in-house compressed air system(breathing quality), Generator flow: 20L/minute
- System of generating particulates/aerosols: Wright Dust Feed mechanism designed to produce and maintain atmospheres of dust by suspending material scraped from the surface of a compressed powder in a stream of dry air. The aerosol exiting the WDF mechanism was supplied to a jet mill ( Jet mill supply flows: 20 to 40 L/minute) in order to reduce the particle size of the powder prior to delivery to the exposure chamber (Airflow exiting jet mill: 22 to 28 L/minute)
- Temperature, humidity, pressure in air chamber:
Temperature was measured using an electronic thermometer inserted into the inhalation chamber. The daily mean chamber temperature values were within the accepted guidelines 19 to 25 °C. The lowest and the mid-dose groups had a mean chamber temperature of 22.2°C, the mean value for the high-dose group was 23.5 °C and the mean value for the control group was 22.5°C.
Humidity was measured using an electronic hygrometer inserted into the inhalation chamber. The lowest dose group had an overall mean chamber relative humidity of 24.5%, the mean values for the mid and high-dose groups were lower at 9.7 and 15.9 % respectively. The mean value of the control group was higher at 46.7%. The low values were considered to be a result of the dried air supplied from generator.
Pressure in air chamber was not reported.
- Air flow rate: Airflow to Wright dust Feed was 20 L/minute for all groups.
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 8-stage cascade impactor (Marple 298). Samples were collected at least once during each week of exposure for each concentration tested. Samples were also collected from a vacant animal exposure port (animals breathing zone). The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, was calculated by this difference. The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than < 0.42, 0.76, 1.27, 2.86, 4.9, 8.0, 12.1 and 17.4 µm (aerodynamic diameter) was calculated. From this data, the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated.
- Treatment of exhaust air: Extract airflow was drawn by in-house vacuum system at a flow rate from 40 to 160 L/minute depending of the exposure concentration of the group . the airflow was filtered locally.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The exposure concentrations were monitored during each exposure by gravimetrical analysis of the test item deposited on a sampling filter. On 3 occasions, the gravimetric analysis was coupled to analytical analysis by LC -MS/MS to check the accuracy of the gravimetric method and ensure that the composition of the material in the test atmospheres was similar to the original material and was stable across the exposure period..
The mean achieved concentrations were 1.55; 6.50 and 26.0 µg/L and corresponded to 103; 108 and 108% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.

Duration of treatment / exposure:

13 weeks followed by a 12- week recovery period

Frequency of treatment:

5 days / week

Dose / conc.:

1.5 mg/m³ air (nominal)

Dose / conc.:

1.55 mg/m³ air (analytical)

Dose / conc.:

6 mg/m³ air (nominal)

Dose / conc.:

6.5 mg/m³ air (analytical)

Dose / conc.:

24 mg/m³ air (nominal)

Dose / conc.:

26 mg/m³ air (analytical)

No. of animals per sex per dose:

- 3 groups, each comprising 10 male and 10 female rats received the test substance at target exposure levels of 1.5, 6 or 24 µg/L. A similarly constituted Control group received air only, at the same operating conditions as the high dose group.
- A further 10 male and 10 female rats were assigned to each groups: these animals were treated for 13 weeks, which was followed by a 12 week period without treatment to assess recovery from any treatment related effect. (See in chapter 'Any other information on materials and methods' section 7.5.2 /2 Identity of treatment groups)

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
In a previously conducted two-week repeat dose inhalation toxicity study, rats were exposed 5 days/week, 6 hours/day to 0, 29.8, 108 and 499 µg/L of test substance. No animals died during the treatment period and there were no treatment related clinical findings apparent during the study. Treatment related histopathological findings in the lungs were evident for animals that received 108 or 499 µg/L and were expected to progress with prolonged exposure. For this study, a high exposure level of 24 µg/L was expected to be tolerated, and intermediate and low exposure levels of 6 and 1.5 µg/L were selected to explore any exposure-related relationship.

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

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

- Post-exposure recovery period in satellite groups: 12 weeks

Positive control:

no

Observations and examinations performed and frequency:

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


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

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

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

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

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

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

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

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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

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

The following tissues were examined for recovery animals sacrificed on completion of the 13-week treatment period followed by a 12-week off dose period:
- mediastinal lymph nodes showing macroscopic abnormality
- Axillary lymph nodes
- Nasal turbinates, larynx, trachea, left lung,tracheobronchial lymph nodes of all recovery animals

Other examinations:

- HAEMATOLOGY, BONE MARROW:
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment period for all main study animals.The smears
from animals of Groups 1 (Control) and 4 (26.0 µg/L) sacrificed on completion of the 13-week treatment period were examined to assess the cellularity,
distribution and morphology of the marrow. The smears from animals of the intermediate and low dose groups were retained but not examined.

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

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

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

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

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

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

All samples were analysed.

Statistics:

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

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item-related clinical signs during the 13 weeks of treatment or during the 12 week recovery period.
Signs associated with the administration procedure included wet fur and red staining of the head, nose and eyes on return to home cage, in which the majority had resolved by the end of the working day. These signs were seen in animals from all groups, including control, therefore they are considered to be due to the method and duration of restraint and are commonly seen on inhalation studies of this study design.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on body weight and weight gain during the 13 weeks of treatment or during the 12 week recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination did not reveal any exposure-related abnormalities

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After 13 weeks of treatment, group mean neutrophil counts were higher than control in both sexes exposed to 26.0 µg/L (up to 2.1X).
After 12 weeks of recovery, group mean neutrophil counts also remained higher than control in both sexes exposed to 26.0 µg/L (1.4X and 1.7X for males and females, respectively).
As a result of the higher mean neutrophil counts in animals exposed to 26.0 µg/L, group mean white blood cells counts of both sexes exposed to 26 µg/L were also higher than control during the treatment and recovery phases (up to 1.2 X for both phases).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 13 weeks of treatment, group mean glucose concentrations were higher than control in both sexes exposed to 26.0 µg/L (up to 1.1X or 1.6X for males and females, respectively); however, the majority of individual values were within the control range for the males and there was no exposure related trend for the females, therefore this was considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 13 weeks of treatment, group mean adjusted lung and bronchi weights were higher than control in both sexes exposed to 26.0 µg/L (1.5X and 1.4X control for males and females respectively).
After 12 weeks of recovery, group mean adjusted lung and bronchi weights remained higher than control in both sexes previously exposed to 26.0 µg/L (1.3X and 1.4X control for males and females, respectively).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

(see chapter 'any other information on results including tables' section 7.5.2 /3 for macropathology tables)
Macroscopic examination performed after 13 weeks of treatment revealed an increase in incidence of pale areas in the lungs of females receiving 26.0 µg/L. Abnormal color (dark) was also seen in 2 animals of both sexes that received 26.0 µg/L.
In addition, enlargement and pale appearance of the mediastinal and tracheobronchial lymph nodes were noted in all females and nearly all males that received 26.0 µg/L.
The majority of these findings were still evident following 12 weeks of recovery: A slight increased incidence of pale areas in the lungs was seen in some males which previously received 26.0 µg/L. Enlargement and pale appearance of the mediastinal and tracheobronchial lymph nodes were still observed in all females and a majority of males that previously received 26.0 µg/L.The tracheobronchial lymph nodes of few males previously exposed to 6.50 µg/L showed enlargement and pale appearance, the latter finding was also seen in few females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings related to treatment were seen in the lungs, tracheobronchial, axillary and mediastinal lymph nodes and nose (see chapter 'any other information on results including tables' section 7.5.2 /4 for histopathology tables) :

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

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

In hte nose, minimal hyperplasia of mucous cells was seen in the turbinates and nasopharynx of both sexes that received 6.50 or 26.0 µg/L. After 12 weeks off-dose, no evidence of recovery was observed as the test item-related finding of hyperplasia of mucous cells was still present in animals at a similar incidence and severity.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage: (see chapter 'any other information on results including tables' section 7.5.2 /5 for BAL tables)
After 13 weeks of treatment, statistically significant exposure-related increases of group mean total cell counts were observed for both sexes exposed to 6.5 or 26 µg/L.
Compared to controls, mean total cell counts were increased by 1.7 to 14.4 fold for males and 2.41 to 11.9 fold for females exposed to 6.5 and 26 µg/L respectively. Control data showed that the vast majority of cells recovered in BALF were macrophages (> or = 83%). Dose-related increases in neutrophils and eosinophils in the BALF of rats given 6.5 or 26 µg/L as well as increase in lymphocytes at the high concentration led to up to 5.7% of the cells being lymphocytes, 9.0 % of the cells being eosinophils and up to 45.3% of the cells being neutrophils, consequently reducing the proportion of macrophages to 40.7% or less.
Higher group mean lactate dehydrogenase, total protein and phospholipid concentrations were observed in the bronchoalveolar lavage supernatant fluid for both sexes exposed to 6.50 µg/L or 26.0 µg/L, up to 5.8X, 4.1X and 5.3X control for lactate dehydrogenase, total protein and phospholipids, respectively.
At 1.55 µg/L, only a marginal increase of mean total cells count was observed compared to controls (1.5 and 1.2x for males and females respectively) and cell proportional distribution was not affected.

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

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

Details on results:

Histopathological changes were evident in the lungs, lymph nodes and the nasal turbinates in animals that received 6.50 or 26.0 µg/L.

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

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

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

Key result

Dose descriptor:

NOAEC

Effect level:

1.55 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: Changes in bronchoalveolar lavage markers

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

6.5 mg/m³ air (analytical)

System:

other: pulmonary

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

7.5.2 /3 Macropathology results

 Animals killed after 13 weeks of treatment

Lungs

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

Summary of findings in the lungs for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Pale area(s)	4	1	0	3	4	1	0	7
Abnormal color (dark)	0	0	0	2	0	0	0	2
Number of animals examined	10	10	10	10	10	10	10	10

Mediastinal lymph nodes

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

Summary of findings in the mediastinal lymph nodes for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Enlarged	0	0	0	6	0	0	0	10
Pale	0	0	0	7	0	0	0	10
Number of animals examined	10	10	10	10	10	10	10	10

Tracheobronchial lymph nodes

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

Summary of findings in the tracheobronchial lymph nodes for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Enlarged	0	0	0	9	0	0	0	9
Pale	0	0	0	9	0	0	0	9
Number of animals examined	10	10	10	10	10	10	10	10

Animals killed after 12 weeks of recovery

Lungs

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

Summary of findings in the lungs for animals killed after 13 weeks of treatment followed by 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Pale area(s)	5	5	1	8	8	5	5	9
Number of animals examined	10	10	10	10	10	10	10	10

Mediastinal lymph nodes

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

Summary of findings in the mediastinal lymph nodes for animals killed after 13 weeks of treatment followed by 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Enlarged	0	0	0	7	0	0	0	6
Pale	0	0	0	5	0	0	0	10
Number of animals examined	10	10	10	10	10	10	10	10

Tracheobronchial lymph nodes

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

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

Summary of findings in the tracheobronchial lymph nodes for animals killed after 13 weeks of treatment followed by 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Enlarged	0	0	3	8	0	0	0	7
Pale	0	0	5	7	0	0	3	10
Number of animals examined	10	10	10	10	10	10	10	10

7.5.2 /4 Histopathology results

 Animals killed after 13 weeks of treatment

Lungs

Summary of treatment related findings in the lungs for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Eosinophilic Granular Material, Alveoli
Minimal	0	0	9	0	0	0	10	0
Slight	0	0	0	0	0	0	0	1
Moderate	0	0	0	7	0	0	0	7
Marked	0	0	0	3	0	0	0	2
Total	0	0	9	10	0	0	10	10
Hyperplasia, Type II Pneumocytes
Minimal	0	0	7	0	0	0	9	0
Slight	0	0	3	1	0	0	0	8
Moderate	0	0	0	7	0	0	0	2
Marked	0	0	0	1	0	0	0	0
Severe	0	0	0	1	0	0	0	0
Total	0	0	10	10	0	0	9	10
Increased Alveolar Macrophages
Minimal	0	2	4	0	0	0	2	0
Slight	0	0	6	0	0	0	7	0
Moderate	0	0	0	1	0	0	0	2
Marked	0	0	0	9	0	0	0	8
Total	0	2	10	10	0	0	9	10
Infiltrate, Inflammatory cells, Mixed, Perivascular
Minimal	5	8	9	0	7	4	10	1
Slight	0	0	0	6	0	1	0	6
Moderate	0	0	0	1	0	0	0	2
Total	5	8	9	7	7	5	10	9
Infiltrate, Inflammatory cells, Neutrophilic, Alveoli
Minimal	1	1	9	0	0	0	8	0
Slight	0	0	1	3	0	0	1	7
Moderate	0	0	0	6	0	0	0	3
Total	1	1	10	9	0	0	9	10
Agregates, Macrophage, BALT
Minimal	0	0	0	3	0	0	0	2
Slight	0	0	0	1	0	0	0	0
Total	0	0	0	4	0	0	0	2
Hemorrhage, Alveoli
Minimal	0	0	0	3	0	0	0	1
Total	0	0	0	3	0	0	0	1
Number of tissues examined	10	10	10	10	10	10	10	10

Lymph nodes (tracheobronchial, axillary and mediastinal)

Summary of treatment related findings in the lymph nodes for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Tracheobronchial Lymph Nodes
Aggregates, Macrophage
Minimal	0	0	6	0	0	0	7	1
Slight	0	0	0	5	0	0	1	8
Moderate	0	0	0	4	0	0	0	1
Total	0	0	6	9	0	0	8	10
CellularityIncreased, Paracortical
Minimal	0	1	6	6	0	0	2	3
Slight	0	0	0	2	0	0	0	5
Moderate	0	0	0	1	0	0	0	0
Total	0	1	6	9	0	0	2	8
Number of tissues examined	10	10	10	10	10	10	10	10
Left Axillary Lymph Nodes
Aggregates, Macrophage
Moderate	0	0	0	1	0	0	0	0
Total	0	0	0	1	0	0	0	0
CellularityIncreased, Paracortical
Minimal	0	0	0	1	0	0	0	1
Slight	0	0	0	3	0	0	0	1
Total	0	0	0	4	0	0	0	2
Number of tissues examined	10	10	10	10	10	10	10	10
Mediastinal Lymph Nodes
Aggregates, Macrophage
Slight	-	-	-	6	-	-	-	3
Moderate	-	-	-	0	-	-	-	7
Total	-	-	-	6	-	-	-	10
CellularityIncreased, Paracortical
Minimal	-	-	-	4	-	-	-	2
Slight	-	-	-	2	-	-	-	8
Moderate	-	-	-	1	-	-	-	0
Total	-	-	-	7	-	-	-	10
Number of tissues examined	-	-	-	7	-	-	-	10

Nose (turbinates and nasopharynx)

Summary of treatment related findings in the turbinates and nasopharynx for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Nose/turbinates
Hyperplasia,mucous cell,Dorsal
Minimal	0	0	1	4	0	0	3	7
Total	0	0	1	4	0	0	3	7
Pharynx,Nasal
Hyperplasia,mucous cell,Dorsal
Minimal	0	0	4	5	0	0	5	3
Total	0	0	4	5	0	0	5	3
Number of tissues examined	10	10	10	10	10	10	10	10

Animals killed after 12 weeks of recovery

Lungs

Summary of treatment related findings in the lungs for animals killed after 13 weeks of treatment followed by 12 weeks recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Eosinophilic Granular Material, Alveoli
Minimal	0	0	10	0	0	0	10	0
Slight	0	0	0	2	0	0	0	2
Moderate	0	0	0	6	0	0	0	3
Marked	0	0	0	2	0	0	0	5
Total	0	0	10	10	0	0	10	10
Hyperplasia, Type II Pneumocytes
Minimal	0	0	10	0	0	0	6	0
Slight	0	0	0	6	0	0	0	8
Moderate	0	0	0	2	0	0	0	2
Marked	0	0	0	2	0	0	0	0
Total	0	0	10	10	0	0	6	10
Increased Alveolar Macrophages
Minimal	0	0	2	0	0	0	5	0
Slight	0	0	8	1	0	0	5	0
Moderate	0	0	0	6	0	0	0	6
Marked	0	0	0	3	0	0	0	4
Total	0	0	10	10	0	0	10	10
Infiltrate, Inflammatory cells, Mixed, Perivascular
Minimal	8	8	9	0	8	9	10	0
Slight	0	1	1	6	0	0	0	8
Moderate	0	0	0	4	0	0	0	2
Total	8	9	10	10	8	9	10	10
Infiltrate, Inflammatory cells, Neutrophilic, Alveoli
Minimal	1	1	7	0	1	0	8	0
Slight	0	0	3	5	0	0	0	7
Moderate	0	0	0	5	0	0	0	3
Total	1	1	10	10	1	0	8	10
Agregates, Macrophage, BALT
Minimal	0	0	0	4	0	0	0	2
Slight	0	0	0	1	0	0	0	1
Moderate				1				1
Total	0	0	0	6	0	0	0	4
Number of tissues examined	10	10	10	10	10	10	10	10

Lymph nodes

Summary of treatment related findings in the lymph nodes for animals killed after 13 weeks of treatment followed by 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Tracheobronchial Lymph Nodes
Aggregates, Macrophage
Minimal	0	0	0	0	0	0	5	0
Slight	0	0	8	2	0	0	4	2
Moderate	0	0	1	6	0	0	1	8
Marked	0	0	1	2	0	0	0	0
Total	0	0	10	10	0	0	10	10
CellularityIncreased, Paracortical
Minimal	0	0	7	2	0	0	2	6
Slight	0	0	3	4	0	0	0	2
Moderate	0	0	0	4	0	0	0	0
Total	0	0	10	10	0	0	2	8
Number of tissues examined	9	10	10	10	10	10	10	10
Mediastinal Lymph Nodes
Aggregates, Macrophage
Slight	-	-	-	0	-	-	-	1
Moderate	-	-	-	7	-	-	-	9
Marked				1
Total	-	-	-	8	-	-	-	10
CellularityIncreased, Paracortical
Minimal	-	-	-	0	-	-	-	2
Slight	-	-	-	1	-	-	-	6
Moderate	-	-	-	7	-	-	-	2
Total	-	-	-	8	-	-	-	10
Number of tissues examined	-	-	-	8	-	-	-	10

 

Nose (turbinates and nasopharynx)

Summary of treatment related findings in the turbinates and nasopharynx for animals killed after 13 weeks of treatment followed by 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Nose/turbinates
Hyperplasia,mucous cell,Dorsal
Minimal	0	0	4	7	1	0	5	7
Total	0	0	4	7	1	0	5	7
Pharynx,Nasal
Hyperplasia,mucous cell,Dorsal
Minimal	0	0	4	7	1	0	6	6
Total	0	0	4	7	0	0	6	6
Number of tissues examined	10	10	10	10	10	10	10	10

 

7.5.2 /5 Bronchoalveolar lavage results

 Animals killed after 13 weeks of treatment

Dose group                     1         2              3              4

Exposure level (µg/L)      0       1.55          6.50       26.0

Group /sex		Different cell counts calculated per animal (10^6/ animal)					Different cell counts as percentage of total white blood cells (%)
		Total cells	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils
1M	Mean SD N	1.91 0.61 10	0.17 0.11 10	0.10 0.05 10	1.63 0.52 10	0.01 0.02 10	8.70 3.85 10	5.12 2.44 10	85.47 6.86 10	0.44 0.96 10
2M	Mean SD N	2.87 1.76 10	0.44 0.52 10	0.12 0.09 10	2.27 1.18 10	0.03 0.05 10	11.73 7.97 10	4.64 3.07 10	82.88 8.85 10	0.64 0.87 10
3M	Mean SD N	3.17* 1.6 10	0.82*** 0.36 10	0.27*** 0.11 10	1.95 0.76 10	0.13*** 0.11 10	25.95*** 6.65 10	9.48 5.22 10	60.64*** 7.25 10	3.64*** 2.33 10
4M	Mean SD N	27.59*** 11.12 10	12.47*** 5.40 10	1.49*** 0.64 10	11.20*** 4.39 10	2.44*** 1.07 10	44.41*** 2.96 10	5.66 1.85 10	40.69*** 1.94 10	8.89*** 2.45 10

Group /sex		Different cell counts calculated per animal (10^6/ animal)					Different cell counts as percentage of total white blood cells (%)
		Total cells	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils
1F	Mean SD N	1.38 0.39 10	0.13 0.09 10	0.07 0.04 10	1.16 0.33 10	0.01 0.02 10	9.74 4.48 10	4.72 2.84 10	84.35 5.26 10	1.19 1.99 10
2F	Mean SD N	1.70 0.63 10	0.12 0.13 10	0.07 0.08 10	1.49 0.52 10	0.01 0.02 10	6.14 6.46 10	3.70 3.94 10	89.35 10.82 10	0.81 1.36 10
3F	Mean SD N	3.33*** 0.77 10	0.77** 0.54 10	0.21** 0.08 10	2.24*** 0.55 10	0.11** 0.15 10	21.87* 12.82 10	6.39 2.72 10	68.71* 14.70 10	3.03 3.24 10
4F	Mean SD N	16.45*** 10.38 10	7.67*** 5.52 10	0.83*** 0.34 10	6.49*** 3.81 10	1.46*** 0.87 10	45.29*** 4.69 10	5.45 1.84 10	39.90*** 2.79 10	8.97*** 2.06 10

 

 Animals killed after 12 weeks recovery

Group /sex		Different cell counts calculated per animal (10^6/ animal)					Different cell counts as percentage of total white blood cells (%)
		Total cells	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils
1M	Mean SD N	1.94 0.61 10	0.16 0.11 10	0.09 0.05 10	1.65 0.61 10	0.04 0.05 10	8.87 6.32 10	5.38 4.66 10	83.70 11.34 10	2.05 2.28 10
2M	Mean SD N	1.54 0.37 10	0.11 0.09 10	0.13 0.09 10	1.26 0.23 10	0.04 0.03 10	7.02 4.78 10	7.91 4.13 10	82.99 8.52 10	2.08 1.91 10
3M	Mean SD N	2.11 0.84 10	0.47** 0.24 10	0.25*** 0.15 10	1.29 0.50 10	0.11* 0.06 10	21.42*** 5.54 10	12.07** 4.67 10	61.33*** 8.32 10	5.04* 1.78 10
4M	Mean SD N	14.66*** 7.33 10	5.39*** 2.80 10	0.95*** 0.37 10	6.61*** 3.62 10	1.70*** 0.90 10	36.82*** 4.09 10	6.98 1.87 10	44.36*** 5.31 10	11.37*** 3.78 10

 

Group /sex		Different cell counts calculated per animal (10^6/ animal)					Different cell counts as percentage of total white blood cells (%)
		Total cells	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils
1F	Mean SD N	1.25 0.48 10	0.07 0.06 10	0.04 0.02 10	1.12 0.46 10	0.01 0.02 10	5.25 5.03 10	3.89 2.96 10	89.96 6.21 10	0.90 1.51 10
2F	Mean SD N	1.21 0.51 10	0.11 0.13 10	0.09 0.10 10	0.99 0.46 10	0.02 0.03 10	8.24 7.50 10	7.74 5.76 10	82.28 12.52 10	1.74 2.51 10
3F	Mean SD N	1.98* 0.67 10	0.40*** 0.27 10	0.22** 0.15 10	1.28 0.33 10	0.08** 0.06 10	18.73*** 7.97 10	10.24* 6.47 10	66.77*** 13.65 10	3.82*** 1.33 10
4F	Mean SD N	11.66*** 7.32 10	4.36*** 2.87 10	0.90*** 0.58 10	5.41*** 3.51 10	0.99*** 0.55 10	37.14*** 3.98 10	7.95* 3.19 10	44.92*** 6.24 10	8.99*** 1.23 10

Conclusions:

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

Executive summary:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

26 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Subchronic inhalation toxicity study with 12 weeks of recovery is complete and considered as reliable (klimisch score of 1).

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 2019-01-18 to 2019-10-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

yes

Remarks:

: see chapter 'any other information on materials and methods' section 7.5.2 /1

GLP compliance:

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

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

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.5 - <= 2.7 µm

Geometric standard deviation (GSD):

2.45

Remarks on MMAD:

MMAD / GSD: The Particle size distribution (PSD) data was originally characterised by using linear regression of the probit of the cumulative percentage, by massof particles smaller than cut-point of each stage versus the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD); geometric standard deviation of the aerosol were derived for each occasion of measurement.

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

MMAD Mass median aerodynamic diameter (µm)
GSD Geometric standard deviation

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow through nose only chamber. This system was an aluminium alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber.
- Method of holding animals in test chamber: Rats were held in plastic tubes with their snouts protruding from the end of the tubes into the exposure chamber.
- Source and rate of air: From in-house compressed air system(breathing quality), Generator flow: 20L/minute
- System of generating particulates/aerosols: Wright Dust Feed mechanism designed to produce and maintain atmospheres of dust by suspending material scraped from the surface of a compressed powder in a stream of dry air. The aerosol exiting the WDF mechanism was supplied to a jet mill ( Jet mill supply flows: 20 to 40 L/minute) in order to reduce the particle size of the powder prior to delivery to the exposure chamber (Airflow exiting jet mill: 22 to 28 L/minute)
- Temperature, humidity, pressure in air chamber:
Temperature was measured using an electronic thermometer inserted into the inhalation chamber. The daily mean chamber temperature values were within the accepted guidelines 19 to 25 °C. The lowest and the mid-dose groups had a mean chamber temperature of 22.2°C, the mean value for the high-dose group was 23.5 °C and the mean value for the control group was 22.5°C.
Humidity was measured using an electronic hygrometer inserted into the inhalation chamber. The lowest dose group had an overall mean chamber relative humidity of 24.5%, the mean values for the mid and high-dose groups were lower at 9.7 and 15.9 % respectively. The mean value of the control group was higher at 46.7%. The low values were considered to be a result of the dried air supplied from generator.
Pressure in air chamber was not reported.
- Air flow rate: Airflow to Wright dust Feed was 20 L/minute for all groups.
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 8-stage cascade impactor (Marple 298). Samples were collected at least once during each week of exposure for each concentration tested. Samples were also collected from a vacant animal exposure port (animals breathing zone). The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, was calculated by this difference. The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than < 0.42, 0.76, 1.27, 2.86, 4.9, 8.0, 12.1 and 17.4 µm (aerodynamic diameter) was calculated. From this data, the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated.
- Treatment of exhaust air: Extract airflow was drawn by in-house vacuum system at a flow rate from 40 to 160 L/minute depending of the exposure concentration of the group . the airflow was filtered locally.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The exposure concentrations were monitored during each exposure by gravimetrical analysis of the test item deposited on a sampling filter. On 3 occasions, the gravimetric analysis was coupled to analytical analysis by LC -MS/MS to check the accuracy of the gravimetric method and ensure that the composition of the material in the test atmospheres was similar to the original material and was stable across the exposure period..
The mean achieved concentrations were 1.55; 6.50 and 26.0 µg/L and corresponded to 103; 108 and 108% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.

Duration of treatment / exposure:

13 weeks followed by a 12- week recovery period

Frequency of treatment:

5 days / week

Dose / conc.:

1.5 mg/m³ air (nominal)

Dose / conc.:

1.55 mg/m³ air (analytical)

Dose / conc.:

6 mg/m³ air (nominal)

Dose / conc.:

6.5 mg/m³ air (analytical)

Dose / conc.:

24 mg/m³ air (nominal)

Dose / conc.:

26 mg/m³ air (analytical)

No. of animals per sex per dose:

- 3 groups, each comprising 10 male and 10 female rats received the test substance at target exposure levels of 1.5, 6 or 24 µg/L. A similarly constituted Control group received air only, at the same operating conditions as the high dose group.
- A further 10 male and 10 female rats were assigned to each groups: these animals were treated for 13 weeks, which was followed by a 12 week period without treatment to assess recovery from any treatment related effect. (See in chapter 'Any other information on materials and methods' section 7.5.2 /2 Identity of treatment groups)

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
In a previously conducted two-week repeat dose inhalation toxicity study, rats were exposed 5 days/week, 6 hours/day to 0, 29.8, 108 and 499 µg/L of test substance. No animals died during the treatment period and there were no treatment related clinical findings apparent during the study. Treatment related histopathological findings in the lungs were evident for animals that received 108 or 499 µg/L and were expected to progress with prolonged exposure. For this study, a high exposure level of 24 µg/L was expected to be tolerated, and intermediate and low exposure levels of 6 and 1.5 µg/L were selected to explore any exposure-related relationship.

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

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

- Post-exposure recovery period in satellite groups: 12 weeks

Positive control:

no

Observations and examinations performed and frequency:

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


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

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

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

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

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

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

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

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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

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

The following tissues were examined for recovery animals sacrificed on completion of the 13-week treatment period followed by a 12-week off dose period:
- mediastinal lymph nodes showing macroscopic abnormality
- Axillary lymph nodes
- Nasal turbinates, larynx, trachea, left lung,tracheobronchial lymph nodes of all recovery animals

Other examinations:

- HAEMATOLOGY, BONE MARROW:
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment period for all main study animals.The smears
from animals of Groups 1 (Control) and 4 (26.0 µg/L) sacrificed on completion of the 13-week treatment period were examined to assess the cellularity,
distribution and morphology of the marrow. The smears from animals of the intermediate and low dose groups were retained but not examined.

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

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

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

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

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

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

All samples were analysed.

Statistics:

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

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item-related clinical signs during the 13 weeks of treatment or during the 12 week recovery period.
Signs associated with the administration procedure included wet fur and red staining of the head, nose and eyes on return to home cage, in which the majority had resolved by the end of the working day. These signs were seen in animals from all groups, including control, therefore they are considered to be due to the method and duration of restraint and are commonly seen on inhalation studies of this study design.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on body weight and weight gain during the 13 weeks of treatment or during the 12 week recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination did not reveal any exposure-related abnormalities

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

After 13 weeks of treatment, group mean neutrophil counts were higher than control in both sexes exposed to 26.0 µg/L (up to 2.1X).
After 12 weeks of recovery, group mean neutrophil counts also remained higher than control in both sexes exposed to 26.0 µg/L (1.4X and 1.7X for males and females, respectively).
As a result of the higher mean neutrophil counts in animals exposed to 26.0 µg/L, group mean white blood cells counts of both sexes exposed to 26 µg/L were also higher than control during the treatment and recovery phases (up to 1.2 X for both phases).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 13 weeks of treatment, group mean glucose concentrations were higher than control in both sexes exposed to 26.0 µg/L (up to 1.1X or 1.6X for males and females, respectively); however, the majority of individual values were within the control range for the males and there was no exposure related trend for the females, therefore this was considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 13 weeks of treatment, group mean adjusted lung and bronchi weights were higher than control in both sexes exposed to 26.0 µg/L (1.5X and 1.4X control for males and females respectively).
After 12 weeks of recovery, group mean adjusted lung and bronchi weights remained higher than control in both sexes previously exposed to 26.0 µg/L (1.3X and 1.4X control for males and females, respectively).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

(see chapter 'any other information on results including tables' section 7.5.2 /3 for macropathology tables)
Macroscopic examination performed after 13 weeks of treatment revealed an increase in incidence of pale areas in the lungs of females receiving 26.0 µg/L. Abnormal color (dark) was also seen in 2 animals of both sexes that received 26.0 µg/L.
In addition, enlargement and pale appearance of the mediastinal and tracheobronchial lymph nodes were noted in all females and nearly all males that received 26.0 µg/L.
The majority of these findings were still evident following 12 weeks of recovery: A slight increased incidence of pale areas in the lungs was seen in some males which previously received 26.0 µg/L. Enlargement and pale appearance of the mediastinal and tracheobronchial lymph nodes were still observed in all females and a majority of males that previously received 26.0 µg/L.The tracheobronchial lymph nodes of few males previously exposed to 6.50 µg/L showed enlargement and pale appearance, the latter finding was also seen in few females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings related to treatment were seen in the lungs, tracheobronchial, axillary and mediastinal lymph nodes and nose (see chapter 'any other information on results including tables' section 7.5.2 /4 for histopathology tables) :

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

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

In hte nose, minimal hyperplasia of mucous cells was seen in the turbinates and nasopharynx of both sexes that received 6.50 or 26.0 µg/L. After 12 weeks off-dose, no evidence of recovery was observed as the test item-related finding of hyperplasia of mucous cells was still present in animals at a similar incidence and severity.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage: (see chapter 'any other information on results including tables' section 7.5.2 /5 for BAL tables)
After 13 weeks of treatment, statistically significant exposure-related increases of group mean total cell counts were observed for both sexes exposed to 6.5 or 26 µg/L.
Compared to controls, mean total cell counts were increased by 1.7 to 14.4 fold for males and 2.41 to 11.9 fold for females exposed to 6.5 and 26 µg/L respectively. Control data showed that the vast majority of cells recovered in BALF were macrophages (> or = 83%). Dose-related increases in neutrophils and eosinophils in the BALF of rats given 6.5 or 26 µg/L as well as increase in lymphocytes at the high concentration led to up to 5.7% of the cells being lymphocytes, 9.0 % of the cells being eosinophils and up to 45.3% of the cells being neutrophils, consequently reducing the proportion of macrophages to 40.7% or less.
Higher group mean lactate dehydrogenase, total protein and phospholipid concentrations were observed in the bronchoalveolar lavage supernatant fluid for both sexes exposed to 6.50 µg/L or 26.0 µg/L, up to 5.8X, 4.1X and 5.3X control for lactate dehydrogenase, total protein and phospholipids, respectively.
At 1.55 µg/L, only a marginal increase of mean total cells count was observed compared to controls (1.5 and 1.2x for males and females respectively) and cell proportional distribution was not affected.

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

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

Details on results:

Histopathological changes were evident in the lungs, lymph nodes and the nasal turbinates in animals that received 6.50 or 26.0 µg/L.

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

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

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

Key result

Dose descriptor:

NOAEC

Effect level:

1.55 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: Changes in bronchoalveolar lavage markers

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

6.5 mg/m³ air (analytical)

System:

other: pulmonary

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

7.5.2 /3 Macropathology results

 Animals killed after 13 weeks of treatment

Lungs

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

Summary of findings in the lungs for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Pale area(s)	4	1	0	3	4	1	0	7
Abnormal color (dark)	0	0	0	2	0	0	0	2
Number of animals examined	10	10	10	10	10	10	10	10

Mediastinal lymph nodes

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

Summary of findings in the mediastinal lymph nodes for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Enlarged	0	0	0	6	0	0	0	10
Pale	0	0	0	7	0	0	0	10
Number of animals examined	10	10	10	10	10	10	10	10

Tracheobronchial lymph nodes

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

Summary of findings in the tracheobronchial lymph nodes for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Enlarged	0	0	0	9	0	0	0	9
Pale	0	0	0	9	0	0	0	9
Number of animals examined	10	10	10	10	10	10	10	10

Animals killed after 12 weeks of recovery

Lungs

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

Summary of findings in the lungs for animals killed after 13 weeks of treatment followed by 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Pale area(s)	5	5	1	8	8	5	5	9
Number of animals examined	10	10	10	10	10	10	10	10

Mediastinal lymph nodes

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

Summary of findings in the mediastinal lymph nodes for animals killed after 13 weeks of treatment followed by 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Enlarged	0	0	0	7	0	0	0	6
Pale	0	0	0	5	0	0	0	10
Number of animals examined	10	10	10	10	10	10	10	10

Tracheobronchial lymph nodes

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

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

Summary of findings in the tracheobronchial lymph nodes for animals killed after 13 weeks of treatment followed by 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Enlarged	0	0	3	8	0	0	0	7
Pale	0	0	5	7	0	0	3	10
Number of animals examined	10	10	10	10	10	10	10	10

7.5.2 /4 Histopathology results

 Animals killed after 13 weeks of treatment

Lungs

Summary of treatment related findings in the lungs for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Eosinophilic Granular Material, Alveoli
Minimal	0	0	9	0	0	0	10	0
Slight	0	0	0	0	0	0	0	1
Moderate	0	0	0	7	0	0	0	7
Marked	0	0	0	3	0	0	0	2
Total	0	0	9	10	0	0	10	10
Hyperplasia, Type II Pneumocytes
Minimal	0	0	7	0	0	0	9	0
Slight	0	0	3	1	0	0	0	8
Moderate	0	0	0	7	0	0	0	2
Marked	0	0	0	1	0	0	0	0
Severe	0	0	0	1	0	0	0	0
Total	0	0	10	10	0	0	9	10
Increased Alveolar Macrophages
Minimal	0	2	4	0	0	0	2	0
Slight	0	0	6	0	0	0	7	0
Moderate	0	0	0	1	0	0	0	2
Marked	0	0	0	9	0	0	0	8
Total	0	2	10	10	0	0	9	10
Infiltrate, Inflammatory cells, Mixed, Perivascular
Minimal	5	8	9	0	7	4	10	1
Slight	0	0	0	6	0	1	0	6
Moderate	0	0	0	1	0	0	0	2
Total	5	8	9	7	7	5	10	9
Infiltrate, Inflammatory cells, Neutrophilic, Alveoli
Minimal	1	1	9	0	0	0	8	0
Slight	0	0	1	3	0	0	1	7
Moderate	0	0	0	6	0	0	0	3
Total	1	1	10	9	0	0	9	10
Agregates, Macrophage, BALT
Minimal	0	0	0	3	0	0	0	2
Slight	0	0	0	1	0	0	0	0
Total	0	0	0	4	0	0	0	2
Hemorrhage, Alveoli
Minimal	0	0	0	3	0	0	0	1
Total	0	0	0	3	0	0	0	1
Number of tissues examined	10	10	10	10	10	10	10	10

Lymph nodes (tracheobronchial, axillary and mediastinal)

Summary of treatment related findings in the lymph nodes for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Tracheobronchial Lymph Nodes
Aggregates, Macrophage
Minimal	0	0	6	0	0	0	7	1
Slight	0	0	0	5	0	0	1	8
Moderate	0	0	0	4	0	0	0	1
Total	0	0	6	9	0	0	8	10
CellularityIncreased, Paracortical
Minimal	0	1	6	6	0	0	2	3
Slight	0	0	0	2	0	0	0	5
Moderate	0	0	0	1	0	0	0	0
Total	0	1	6	9	0	0	2	8
Number of tissues examined	10	10	10	10	10	10	10	10
Left Axillary Lymph Nodes
Aggregates, Macrophage
Moderate	0	0	0	1	0	0	0	0
Total	0	0	0	1	0	0	0	0
CellularityIncreased, Paracortical
Minimal	0	0	0	1	0	0	0	1
Slight	0	0	0	3	0	0	0	1
Total	0	0	0	4	0	0	0	2
Number of tissues examined	10	10	10	10	10	10	10	10
Mediastinal Lymph Nodes
Aggregates, Macrophage
Slight	-	-	-	6	-	-	-	3
Moderate	-	-	-	0	-	-	-	7
Total	-	-	-	6	-	-	-	10
CellularityIncreased, Paracortical
Minimal	-	-	-	4	-	-	-	2
Slight	-	-	-	2	-	-	-	8
Moderate	-	-	-	1	-	-	-	0
Total	-	-	-	7	-	-	-	10
Number of tissues examined	-	-	-	7	-	-	-	10

Nose (turbinates and nasopharynx)

Summary of treatment related findings in the turbinates and nasopharynx for animals killed after 13 weeks of treatment
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Nose/turbinates
Hyperplasia,mucous cell,Dorsal
Minimal	0	0	1	4	0	0	3	7
Total	0	0	1	4	0	0	3	7
Pharynx,Nasal
Hyperplasia,mucous cell,Dorsal
Minimal	0	0	4	5	0	0	5	3
Total	0	0	4	5	0	0	5	3
Number of tissues examined	10	10	10	10	10	10	10	10

Animals killed after 12 weeks of recovery

Lungs

Summary of treatment related findings in the lungs for animals killed after 13 weeks of treatment followed by 12 weeks recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Eosinophilic Granular Material, Alveoli
Minimal	0	0	10	0	0	0	10	0
Slight	0	0	0	2	0	0	0	2
Moderate	0	0	0	6	0	0	0	3
Marked	0	0	0	2	0	0	0	5
Total	0	0	10	10	0	0	10	10
Hyperplasia, Type II Pneumocytes
Minimal	0	0	10	0	0	0	6	0
Slight	0	0	0	6	0	0	0	8
Moderate	0	0	0	2	0	0	0	2
Marked	0	0	0	2	0	0	0	0
Total	0	0	10	10	0	0	6	10
Increased Alveolar Macrophages
Minimal	0	0	2	0	0	0	5	0
Slight	0	0	8	1	0	0	5	0
Moderate	0	0	0	6	0	0	0	6
Marked	0	0	0	3	0	0	0	4
Total	0	0	10	10	0	0	10	10
Infiltrate, Inflammatory cells, Mixed, Perivascular
Minimal	8	8	9	0	8	9	10	0
Slight	0	1	1	6	0	0	0	8
Moderate	0	0	0	4	0	0	0	2
Total	8	9	10	10	8	9	10	10
Infiltrate, Inflammatory cells, Neutrophilic, Alveoli
Minimal	1	1	7	0	1	0	8	0
Slight	0	0	3	5	0	0	0	7
Moderate	0	0	0	5	0	0	0	3
Total	1	1	10	10	1	0	8	10
Agregates, Macrophage, BALT
Minimal	0	0	0	4	0	0	0	2
Slight	0	0	0	1	0	0	0	1
Moderate				1				1
Total	0	0	0	6	0	0	0	4
Number of tissues examined	10	10	10	10	10	10	10	10

Lymph nodes

Summary of treatment related findings in the lymph nodes for animals killed after 13 weeks of treatment followed by 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Tracheobronchial Lymph Nodes
Aggregates, Macrophage
Minimal	0	0	0	0	0	0	5	0
Slight	0	0	8	2	0	0	4	2
Moderate	0	0	1	6	0	0	1	8
Marked	0	0	1	2	0	0	0	0
Total	0	0	10	10	0	0	10	10
CellularityIncreased, Paracortical
Minimal	0	0	7	2	0	0	2	6
Slight	0	0	3	4	0	0	0	2
Moderate	0	0	0	4	0	0	0	0
Total	0	0	10	10	0	0	2	8
Number of tissues examined	9	10	10	10	10	10	10	10
Mediastinal Lymph Nodes
Aggregates, Macrophage
Slight	-	-	-	0	-	-	-	1
Moderate	-	-	-	7	-	-	-	9
Marked				1
Total	-	-	-	8	-	-	-	10
CellularityIncreased, Paracortical
Minimal	-	-	-	0	-	-	-	2
Slight	-	-	-	1	-	-	-	6
Moderate	-	-	-	7	-	-	-	2
Total	-	-	-	8	-	-	-	10
Number of tissues examined	-	-	-	8	-	-	-	10

 

Nose (turbinates and nasopharynx)

Summary of treatment related findings in the turbinates and nasopharynx for animals killed after 13 weeks of treatment followed by 12 week recovery period
Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure level (µg/L)	0	1.55	6.50	26.0	0	1.55	6.50	26.0
Nose/turbinates
Hyperplasia,mucous cell,Dorsal
Minimal	0	0	4	7	1	0	5	7
Total	0	0	4	7	1	0	5	7
Pharynx,Nasal
Hyperplasia,mucous cell,Dorsal
Minimal	0	0	4	7	1	0	6	6
Total	0	0	4	7	0	0	6	6
Number of tissues examined	10	10	10	10	10	10	10	10

 

7.5.2 /5 Bronchoalveolar lavage results

 Animals killed after 13 weeks of treatment

Dose group                     1         2              3              4

Exposure level (µg/L)      0       1.55          6.50       26.0

Group /sex		Different cell counts calculated per animal (10^6/ animal)					Different cell counts as percentage of total white blood cells (%)
		Total cells	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils
1M	Mean SD N	1.91 0.61 10	0.17 0.11 10	0.10 0.05 10	1.63 0.52 10	0.01 0.02 10	8.70 3.85 10	5.12 2.44 10	85.47 6.86 10	0.44 0.96 10
2M	Mean SD N	2.87 1.76 10	0.44 0.52 10	0.12 0.09 10	2.27 1.18 10	0.03 0.05 10	11.73 7.97 10	4.64 3.07 10	82.88 8.85 10	0.64 0.87 10
3M	Mean SD N	3.17* 1.6 10	0.82*** 0.36 10	0.27*** 0.11 10	1.95 0.76 10	0.13*** 0.11 10	25.95*** 6.65 10	9.48 5.22 10	60.64*** 7.25 10	3.64*** 2.33 10
4M	Mean SD N	27.59*** 11.12 10	12.47*** 5.40 10	1.49*** 0.64 10	11.20*** 4.39 10	2.44*** 1.07 10	44.41*** 2.96 10	5.66 1.85 10	40.69*** 1.94 10	8.89*** 2.45 10

Group /sex		Different cell counts calculated per animal (10^6/ animal)					Different cell counts as percentage of total white blood cells (%)
		Total cells	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils
1F	Mean SD N	1.38 0.39 10	0.13 0.09 10	0.07 0.04 10	1.16 0.33 10	0.01 0.02 10	9.74 4.48 10	4.72 2.84 10	84.35 5.26 10	1.19 1.99 10
2F	Mean SD N	1.70 0.63 10	0.12 0.13 10	0.07 0.08 10	1.49 0.52 10	0.01 0.02 10	6.14 6.46 10	3.70 3.94 10	89.35 10.82 10	0.81 1.36 10
3F	Mean SD N	3.33*** 0.77 10	0.77** 0.54 10	0.21** 0.08 10	2.24*** 0.55 10	0.11** 0.15 10	21.87* 12.82 10	6.39 2.72 10	68.71* 14.70 10	3.03 3.24 10
4F	Mean SD N	16.45*** 10.38 10	7.67*** 5.52 10	0.83*** 0.34 10	6.49*** 3.81 10	1.46*** 0.87 10	45.29*** 4.69 10	5.45 1.84 10	39.90*** 2.79 10	8.97*** 2.06 10

 

 Animals killed after 12 weeks recovery

Group /sex		Different cell counts calculated per animal (10^6/ animal)					Different cell counts as percentage of total white blood cells (%)
		Total cells	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils
1M	Mean SD N	1.94 0.61 10	0.16 0.11 10	0.09 0.05 10	1.65 0.61 10	0.04 0.05 10	8.87 6.32 10	5.38 4.66 10	83.70 11.34 10	2.05 2.28 10
2M	Mean SD N	1.54 0.37 10	0.11 0.09 10	0.13 0.09 10	1.26 0.23 10	0.04 0.03 10	7.02 4.78 10	7.91 4.13 10	82.99 8.52 10	2.08 1.91 10
3M	Mean SD N	2.11 0.84 10	0.47** 0.24 10	0.25*** 0.15 10	1.29 0.50 10	0.11* 0.06 10	21.42*** 5.54 10	12.07** 4.67 10	61.33*** 8.32 10	5.04* 1.78 10
4M	Mean SD N	14.66*** 7.33 10	5.39*** 2.80 10	0.95*** 0.37 10	6.61*** 3.62 10	1.70*** 0.90 10	36.82*** 4.09 10	6.98 1.87 10	44.36*** 5.31 10	11.37*** 3.78 10

 

Group /sex		Different cell counts calculated per animal (10^6/ animal)					Different cell counts as percentage of total white blood cells (%)
		Total cells	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils	Neutrophils	Lymphocytes	Mononuclear cells	Eosinophils
1F	Mean SD N	1.25 0.48 10	0.07 0.06 10	0.04 0.02 10	1.12 0.46 10	0.01 0.02 10	5.25 5.03 10	3.89 2.96 10	89.96 6.21 10	0.90 1.51 10
2F	Mean SD N	1.21 0.51 10	0.11 0.13 10	0.09 0.10 10	0.99 0.46 10	0.02 0.03 10	8.24 7.50 10	7.74 5.76 10	82.28 12.52 10	1.74 2.51 10
3F	Mean SD N	1.98* 0.67 10	0.40*** 0.27 10	0.22** 0.15 10	1.28 0.33 10	0.08** 0.06 10	18.73*** 7.97 10	10.24* 6.47 10	66.77*** 13.65 10	3.82*** 1.33 10
4F	Mean SD N	11.66*** 7.32 10	4.36*** 2.87 10	0.90*** 0.58 10	5.41*** 3.51 10	0.99*** 0.55 10	37.14*** 3.98 10	7.95* 3.19 10	44.92*** 6.24 10	8.99*** 1.23 10

Conclusions:

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

Executive summary:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1.55 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Subchronic inhalation toxicity study with 12 weeks of recovery is complete and considered as reliable (klimisch score of 1).

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 study

In a sub-acute study performed according to OECD 407 guideline and in compliance with Good Laboratory Practices, test material diluted in 1.0% methylcellulose in purified water was administered by oral gavage to three groups of rats (5/sex) at 15, 150 and 1000 mg/kg bw/day for four weeks. A control group received vehicle alone at the same volume-dosage (10 mL/kg bw/day).

There were no deaths, no clinical signs related to treatment and no evidence of neurotoxicity during the weekly functional battery test. Bodyweight, food consumption and food conversion efficiency were not affected by treatment. There were no haematological or blood chemistry changes related to treatment. Organ weights were unaffected by treatment and there were no macroscopic or microscopic findings which were attributable to the treatment with the test material. The No-Observed-Effect Level (NOEL) was considered to be 1000 mg/kg bw/day.

 

Inhalation study

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

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

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

 

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

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

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

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

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

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

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

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

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

 

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

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

 

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 observed. At the exposure-levels achieved in this study, lung effects observed at the end of the treatment period were still present after an equivalent period of recovery but there was no trend to progress towards chronicity. Considering that histopathological markers of chronicity like granulomata or fibrosis should appear at higher exposure levels, category 2 is warranted. As these pulmonary toxic effects are local , they will occur only after repeated exposure by inhalation.