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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Remarks:
90-Day Inhalation Toxicity Study with 6-Week and 3-Month Recovery (Nose-only) in the Rat
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 March 2020 (study plan )
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study
Version / remarks:
adopted 25 June 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tin disulphide
EC Number:
215-252-9
EC Name:
Tin disulphide
Cas Number:
1315-01-1
Molecular formula:
S2Sn
IUPAC Name:
tin disulphide
Test material form:
solid: particulate/powder
Remarks:
ochre

Test animals

Species:
rat
Strain:
other: HanWistar, Crl:WI(Han)
Details on species / strain selection:
Rats are the preferred species of choice as historically they have been used for safety evaluation studies and they are specified by the appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, SandhoferWeg 7, D-97633 Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 8 weeks old at the initiation of treatment
- Weight at study initiation: Males: 204 - 254 g; Females: 144 g - 182 g
- Fasting period before study:
- Housing: Rodents were housed in room number 229 in Polycarbonate solid floor cages (type III) with stainless steel mesh lids. SAFE 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Nest building material SAFE crinklets natural (produced by J. Rettenmaier & Söhne GmbH & Co.KG, Germany or similar)) was also added to the cages. Additional enrichment (GLP Mini Fun Tunnel for hiding and chewing) was also used during the study. Rodents were housed up to 3 animals of the same sex and group/cage. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities. The bedding and the nest building material and the enrichment device were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum while in their home cages. No food was supplied during the 6-hour exposure period in treatment days.
- Water (e.g. ad libitum): Animals received tap water from municipal supply, as for human consumption from a 500 ml bottles, ad libitum while in their home cages. No water was supplied during the 6-hour exposure period in treatment days.
- Acclimation period: 8-9 days

DETAILS OF FOOD AND WATER QUALITY:
The supplier provided analytical certificates for the batches used, which are archived with the study raw data.
Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8200 Veszprém, József Attila utca 36., Hungary). The quality control results are archived with the study raw data.
The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.6 – 25.2°C
- Humidity (%): 20 – 80 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours of continuous artificial light in each 24-hour period (from 6.00 a.m. to 6.00 p.m.)

IN-LIFE DATES:
Animal arrival: 19 March 2020
Start of experiment: 27 March 2020
Start of treatment (Day 1): 27/28 March 2020 (m/f)
End of dosing (Day 90): 24/25 June 2020 (m/f)
Necropsy of PEO-1Animals (Day 91): 25/26 June 2020 (m/f)
Start of post-exposure period (Day 91): 25/26 June 2020 (m/f)
Necropsy of PEO-2 Animals (Day 132): 05/06 August 2020 (m/f)
Necropsy of PEO-3 Animals (Day 174): 16 September 2020 (m)
End of post-exposure period (Day 174): 16 September 2020
End of experiment (in-life phase): 16 September 2020

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks:
with oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%
Mass median aerodynamic diameter (MMAD):
>= 2.01 - <= 2.53 µm
Geometric standard deviation (GSD):
2
Remarks on MMAD:
The GSD is 2.01-2.05
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: TSE Rodent Exposure System. The exposure unit consisted of three, concentric stainless steel cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports
- Method of holding animals in test chamber: Nose only exposure units (towers) were used. The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: The flow of air through each port was approximately 0.5 L/min
- Method of conditioning air: Before animals were exposed, test item atmospheres were generated within the exposure chamber. During this period, airflow settings, brush speed and test item input were varied to achieve the required atmospheric concentrations and constancy. The exposure on each day was not started until theoretical chamber concentration equilibration has been reached. During the exposures, the brush speed and test item input rate was varied to achieve the target concentration.
- System of generating particulates/aerosols: Test item was aerosolised using rotating brush generators, Palas RBG 1000 Systems (TSE Systems GmbH, Bad Homburg, Germany; Serial Number: 1719) located at the top of the exposure chamber.
- Temperature, humidity, pressure in air chamber: The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/relative humidity, O2 and CO2 sensors.
Chamber airflow rates, Test atmosphere temperature, Test atmosphere relative humidity, Test atmosphere carbon dioxide concentration and Test atmosphere oxygen concentration were monitored during each exposure period by the TSE-DACO monitoring system integrated into the exposure system.
The temperature, humidity, oxygen and carbon dioxide values were considered to be satisfactory for this type of study.
- Air flow rate: Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min. This flow rate was considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
The airflow through the chamber was adequate to keep dynamic flow and to avoid re-breathing of the test atmospheres.
- Air change rate: Atmosphere generation was dynamic. Fresh test atmosphere from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where the aerosol was distributed to the individual exposure ports.
- Method of particle size determination: The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges .
- Treatment of exhaust air: After passing through the animal’s breathing zone, spent aerosol entered the outer cylinder from where it was exhausted through a suitable filter system.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port (animals breathing zone).
The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period).
The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/L) was calculated by the following formula:
C(AS) = M / (tS x FS)
Where
M = Mass of the active substance on the filter (mg)
tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)
From the samples collected during the animal exposure for concentration, supporting analysis was occasionally performed using validated X-ray fluorescent analysis (19/106-901ANA) by the Principal Investigator of the Test Site. The filter analysis was performed at least four times during the exposure period (Week 1, Week 2, Week 3 and Week 4). The non-specific gravimetric method, with backup from this robust, specific analysis is considered to meet the guideline and GLP aspects of atmosphere confirmation.
The filter samples to be analysed were sent to the Test Site:
János Török, Ph.D.
FumoPrep Ltd.
H-1044 Budapest, Ipari Park u. 10.
Hungary

Nominal concentrations were calculated by dividing the total weight of test item in the disseminated into the test chamber by the total volume of air used during the same period.
The test atmospheres were sampled from the breathing zone during the 6-hour exposure periods, 3 times at approximately equal intervals during all exposure days of the study.
The mean achieved actual concentrations based on the gravimetric analysis of the GF10 glass fibre filters and nominal concentrations are presented below:

-Group 1 Air Control
Target Concentration : 0 mg/L
Mean Achieved Concentration: 0 mg/L
Standard Deviation of Achieved Concentration: 0 mg/L
Actual Nominal Concentration: 0.00 mg/L
-Group 2 Low dose
Target Concentration : 0.02 mg/L
Mean Achieved Concentration: 0.020 mg/L
Standard Deviation of Achieved Concentration: 0.001 mg/L
Actual Nominal Concentration: 0.08 mg/L
-Group 3 Mid dose
Target Concentration : 0.2 mg/L
Mean Achieved Concentration: 0.21 mg/L
Standard Deviation of Achieved Concentration: 0.01 mg/L
Actual Nominal Concentration: 0.76 mg/L
-Group 4 High dose
Target Concentration : 1.0 mg/L
Mean Achieved Concentration: 1.03 mg/L
Standard Deviation of Achieved Concentration: 0.03 mg/L
Actual Nominal Concentration: 2.69 mg/L

Nominal concentrations were higher than actual measured concentrations; this is normal since a fraction of the test item is usually lost in the separators, tubing or in the towers.

Based on the study sample analysis and the validation data, the concentrations of the test item in the atmospheres were considered to be fully satisfactory.

- Samples taken from breathing zone: yes

VEHICLE: no (aerosoliation in air)
- Concentration of test material in vehicle: The exposure on each day was not started until theoretical chamber concentration equilibration has been reached. During the exposures, the brush speed and test item input rate was varied to achieve the target concentration.
Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min. This flow rate was considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
The atmosphere concentrations of 0.020 mg/L, 0.21 mg/L and 1.03 mg/L were achieved in the respective groups, the target concentrations being 0.02 mg/L, 0.20 mg/L and 1.0 mg/L.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port (animals breathing zone).
The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period).

From the samples collected during the animal exposure for concentration, supporting analysis was occasionally performed using validated X-ray fluorescent analysis (19/106-901ANA) by the Principal Investigator of the Test Site. The filter analysis was performed at least four times during the exposure period (Week 1, Week 2, Week 3 and Week 4). The non-specific gravimetric method, with backup from this robust, specific analysis is considered to meet the guideline and GLP aspects of atmosphere confirmation.
The filter samples to be analysed were sent to FumoPrep Ltd.H-1044 Budapest, Ipari Park u. 10.
Hungary
Duration of treatment / exposure:
13 weeks
(+ satellite group: 6-weeks recovery: males and females)
(+ satellite group: 3-month recovery: males only)
Frequency of treatment:
6 hours per day; 5 days/week basis with the exception of the first week which consists of 6 consecutive treatment days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/L air
Dose / conc.:
0.02 mg/L air
Dose / conc.:
0.2 mg/L air
Dose / conc.:
1 mg/L air
No. of animals per sex per dose:
Main study: 10 animals/sex/group
Satellite group 1 (males only): 5 animals/group
Satellite group 2: 6 week recovery: 5 animals/sex/group
Satellite group 3: 3-months recovery (males only): 5 animals/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The concentration levels (0.02, 0.2 and 1.0 mg/L as Low, Mid and High Dose, respectively) were suggested by the Study Director based on previous data available, including the results of a preliminary non-GLP dose range finding (DRF) studies in the rat (Charles River Laboratories Hungary Kft. study code: 19/106-028P and 19/106-212PE). The dose selection was approved by the Study Monitor and by the Sponsor; it was considered that higher atmosphere concentrations would have caused a lung dust overload within the 90-day study period.
The High dose was intended to cause significant effects which may be adverse, without resulting in life-threatening effects; the only anticipated adverse effects expected based on the preliminary study work, were the local effects in the respiratory system associated with a non-toxic dust (a serious lung overload of a non-toxic dust will cause serious damage and even death). The High dose was set as high as possible, avoiding a high risk of serious adverse effects of dust overload, based on all available information at study initiation. The study was designed to identify if test item has any unexpected toxic properties other than overload.
The Mid and Low dose were set at suitable intervals with the aim of providing data on No Adverse Effect Levels.
Preliminary conclusions from previous studies with the test item were as follows:
•Daily administration of Tin Disulphide to Wistar rats during a 5-day period, at 0.5, 1.0, 2.5 and 5.0 mg/L caused no clinical signs, had no effect on body weight, caused no macroscopic changes at necropsy (other than local respiratory effects) and no clear test item related effect was seen in clinical pathology parameters. During the histopathological examination, the presence of the test item in the lungs were noted in the alveolar/bronchiolar lumen, in the mediastinal lymph node and in the lumen of the larynx, the trachea and in the nasal cavity. The changes did not show a clear dose relation (Study code 19/106-028P).
•Daily administration of Tin Disulphide to Wistar rats during a 28-day period, at 0.2, 1.0, 5.0 mg/L concentration caused no clinical signs, had no effect on body weight, caused no macroscopic changes at necropsy (other than local respiratory effects) and no clear test item related effects were seen in clinical pathology parameters. Test item-related yellow discolouration of all lobes of lungs and mediastinal lymph nodes was observed in all test item exposed animals. The enlargement of these organs was also observed. In the lavage fluid following bronchoalveolar lavage, the level of LDH showed dose dependent increase. During the histopathological examination, the accumulation of the test item in the lungs, in the mediastinal lymph node and in the trachea and the degeneration of the olfactory epithelium in the rostral part of the nasal cavity were noted. (Study code 19/106-212PE).
•Comparison of pictures after the 5-day period and 28-day period (1 mg/L and 5 mg/L) indicated that the increase in pigment in lung alveoli was more than proportional between 1 and 4 weeks dosing (i.e. prolonged exposure increased the lung deposition significantly).
- Rationale for animal assignment (if not random): All animals were allocated to study groups based on their most recent body weights. There was an equal number of animals from each weight group in each of the experimental groups assigned by randomisation. The grouping was controlled by software PROVANTIS v.9, according to the actual body weight verifying the homogeneity and variability among the groups. Males and females were randomized separately.
- Fasting period before blood sampling for clinical biochemistry: overnight period of food deprivation
- Rationale for selecting satellite groups:
Ten male and 10 female Wistar Hannover rats Crl:WI(Han) in each main group were treated by a 6 hour nose-only exposure to filtered air or three fixed aerosol concentrations for 5 days/week. The main animals were sacrificed on the day following the last exposure on Day 91 (histopathology evaluation and BALF† analysis were performed). Additionally, 5 females per group were treated and allowed to recover for 6 weeks and sacrificed on Day 132 (histopathology evaluation and BALF analysis were performed) and 15 males per group were treated and allowed to recover and sacrificed on Day 91, Day 132 (6-week recovery) and Day 174 (approximately 3-month recovery) for histopathology and/or lung burden evaluation.
- Post-exposure recovery period in satellite groups:
Satellite group 1: Day 91: 1 day recovery (males only)
Satellite group 2: Day 132: 6 week recovery (males & females)
Satellite group 3: Day 174: 3 month recovery (males only)
See tables in 'Any other information on Materials & Methods'.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: As a minimum, individual, clinical observations were performed once daily. On exposure days, prior to exposure and during exposure whilst the animals were still restrained (limited observation). Following exposure, clinical observation was performed twice (as soon as practicable after removal from restraint, and approximately one hour after completion of the exposure).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena once a week. General observation was not performed on days when detailed observation is made (except on Day 1, whereas detailed and general observation was also performed). During the recovery period(s), clinical observations was performed in the same way as during the treatment period.
Observations included changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
All observations were recorded, except on some study days during the recovery period. Due to oversight, routine health checks were made for the recovery animals (PEO2, PEO3) but clinical observation were not recorded into Provantis between Days 91-95 (males) and Days 91-94 (females). None of these animals had significant clinical changes in the days before or after this period.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g at randomization, on Days 1 (before the exposure), and weekly thereafter. Body weight of PEO-1 animals was also recorded on Day 90 (last exposure day) and prior to necropsy (fasted on Day 91). Body weight of PEO-2 and PEO-3 animals was also recorded once a week during the recovery period(s) and on Days 131 and 173 and prior to necropsy (fasted on Day 132 for PEO-2 animals and fasted on Day 174 for PEO-3 animals).


FOOD CONSUMPTION AND COMPOUND INTAKE (no feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes: Food consumption was recorded with precision of 1 g weekly. The remaining, non-consumed food was weighed weekly from Day 8. Daily and weekly food consumption is reported.

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: Not specified

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examination was conducted in all animals before treatment and in the Control (Group 1) and High dose (Group 4) main animals on Week 11/12.
Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL cyclopentolate hydrochloride; Batch No.: 180824 and 190819, expiry: August 2023 and August 2024) into the conjunctival sac. The evaluation was performed by external examination and using a OMEGA 500 ophthalmoscope.

HAEMATOLOGY: Yes
-Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91, blood samples for clinical pathology evaluation (haematology, coagulation, clinical biochemistry) were collected by heart puncture under pentobarbital anaesthesia from each PEO-1 animal.
At the end of the recovery period(s), prior to scheduled necropsy on Days 132 and 174, blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected by heart puncture under pentobarbital anaesthesia from each PEO-2 animal and PEO-3 animal.
- Anaesthetic used for blood collection: Yes: pentobarbital anaesthesia
- Animals fasted: Yes: overnight period of food deprivation
- How many animals: all
- Parameters examined:
The following parameters were evaluated in all animals:
RBC Red Blood Cell (erythrocyte) count (1012/L) M/µL
HgbC Haemoglobin Concentration (g/dL)
Hct Haematocrit (relative volume of erythrocytes) (%)
MCV Mean Corpuscular (erythrocyte) Volume (fL)
MCH Mean Corpuscular (erythrocyte) Haemoglobin (pg)
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration (g/dL)
RDW Red Cell (erythrocyte) width (%)
Plt Platelet (thrombocyte) count (109/L) K/µL
MPV Mean Platelet Volume (fL)
RETIC abs, % Reticulocyte count (absolute and %)
WBC White Blood Cell (leukocyte) count (109/L) K/µL
NE abs, % Neutrophil (absolute and %)
LY abs, % Lymphocyte (absolute and %)
MO abs, % Monocyte (absolute and %)
EO abs, % Eosinophil (absolute and %)
BA abs, % Basophil (absolute and %)
LUC abs,% Large Unstained Cells (absolute and %)
The following blood clotting parameters were evaluated in all animals:
APTT Activated Partial Thromboplastin Time (sec)
PT Prothrombin Time (sec)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91, blood samples for clinical pathology evaluation (haematology, coagulation, clinical biochemistry) were collected by heart puncture under pentobarbital anaesthesia from each PEO-1 animal.
At the end of the recovery period(s), prior to scheduled necropsy on Days 132 and 174, blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected by heart puncture under pentobarbital anaesthesia from each PEO-2 animal and PEO-3 animal.
- Animals fasted: Yes: overnight period of food deprivation
- How many animals: all
- Parameters examined:
The following parameters were evaluated in all animals:
Na+- Sodium (mmol/L)
K+ - Potassium (mmol/L)
Cl- - Chloride (mmol/L)
Ca++ - Calcium (mmol/L)
Mg++ - Magnesium (mmol/L)
Phos. - Inorganic Phosphorus (mmol/L)
TBIL - Total Bilirubin (µmol/L)
AST/GOT - Aspartate Aminotransferase activity (U/L)
ALT/GPT - Alanine Aminotransferase activity (U/L)
GGT - Gamma Glutamyl Tranferase activity (U/L)
ALKP - Alkaline Phosphatase activity (U/L)
LIPA - Lipase activity (U/L)
AMYL – α-Amylase activity (U/L)
LDH - Lactate-dehydrogenase activity (U/L)
CK - Creatin Kinase activity (U/L)
Urea/BUN - Urea/Blood Urea Nitrogen (mmol/L)
Crea. – Creatinine (µmol/L)
GLU – Glucose (mmol/L)
TRIG – Triglycerides (mmol/L)
Chol. – Cholesterol (mmol/L)
dHDL - High Density Lipoprotein (mmol/L)
dLDL - Low Density Lipoprotein (mmol/L)
BA - Bile Acid (µmol/L)
TP - Total Protein (g/L)
ALB – Albumin (g/L)
GLOB – Globulin (g/L)
A/G - Albumin/Globulin (ratio)
hsCRP - C-Reactive Protein (high-sensitivity) (mg/L)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine collection was conducted over approximately 16 hours, during an overnight period of food but not water deprivation, from each animal by placing the animals in metabolic cages.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes: an overnight period of food but not water deprivation
- Parameters examined:
The evaluation of the urine samples was performed by observation (e.g. colour, appearance) or test strips as applicable. The following parameters were evaluated:
LEU / Leukocyte
NIT / Nitrite
pH
PRO / Protein
GLU / Glucose
UBG / Urobilinogen
BIL / Bilirubin
KET / Ketones
BLD/ERY / Blood/Erythrocytes
SG / Specific Gravity
SED / Sediment
VOL / Volume
Colour/appearance

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Towards the end of the treatment period, during Week 11/12, all main animals were subjected to the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment.
- Dose groups that were examined: all main animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted, and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
A detailed assessment for neurotoxicity effects was made on the basis of these measurements.
Parameters such as body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.
To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured. The fore paws of the rat were also painted, but they were not evaluated.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of Test Item. The rats were held appropriately such that the fore limbs are allowed to grip the support bar and pulled back until they release the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on the test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 min, under dim-light and undisturbed conditions. The DVD was analysed with "SMART" software after all recordings were made to produce the appropriate parameters. Data from each dose and control group were evaluated for distance travelled in 5 minutes segments. The data from the 5 minutes segments were presented graphically with the intention of showing plateau activity in controls and comparing the treatment groups.


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis:
On completion of the macroscopic examination the following tissues and organs were retained from all animals. The right lung of the assigned animals was used for bronchoalveolar lavage and subsequent BAL fluid (BALF) analysis.
- Dose groups that were examined:
PEO-1 (Main Study Animals):
The right lung of the Main Study Animals was used for bronchoalveolar lavage (BAL).
PEO-2 (Satellite Group 2):
The right lung of the female animals was used for bronchoalveolar lavage (BAL).
- Number of animals:
Main study animals: 10 animals/sex/group
Satellite group 2: 5 animals/group
- Parameters examined:
The following parameters were determined:
-A non-frozen aliquot of the BAL fluid was used for LDH activity and Albumin measurement.
-A known aliquot of cell suspension was used to determine cells/volume by the cell counter (CountessTM automated cell counter, Invitrogen).
-A known aliquot of cell suspension was used for smear preparation on microscopic slide that was stained with modified Wright-Giemsa stain; and % of alveolar macrophages (AM) and % neutrophils was determined.
The details of the procedure are documented in raw data.


LUNG BURDEN: Yes
- Time schedule for analysis: The lungs of 5 males per lung burden group from PEO-1, PEO-2 and PEO-3 were analysed and reported in a Phase Report. The right lung of the assigned animals was used for lung burden analysis.
- Dose groups that were examined:
PEO-1 (Satellite group 1):
The right lung of the animals of Satellite Group 1 was used for lung burden.
PEO-2 (Satellite Group 2):
The right lung of the male animals was used for lung burden.
PEO-3 (Satellite Group 3):
If it is necessary, based on the findings of PEO-1 and PEO-2 groups; the right lung of the animals may be used for lung burden.
- Number of animals:
Satellite group 1: 5 male animals/sex/group
Satellite group 2: 5 male animals/group
Satellite group 3: 5 male animals/group
- Parameters examined:
In order to investigate the pulmonary deposition of the Test Item, lungs of the animals in lung burden subgroup were analysed using a specific analytical method (to be defined and validated for lung tissue) to determine the Test Item concentration in the lung. The lungs were removed and weighed, the lobes were prepared and preserved by freezing (-20℃) until shipment. Care was taken to remove as much cartilaginous tissue from the lung lobes as possible. All samples to be analysed were sent (frozen, <-20℃) to FumoPrep Ltd.H-1044 Budapest, Ipari Park u. 10. Hungary
The samples were identified appropriately and accompanied by a sample list.
The determination of the test item in the samples was performed at the Test Site for using a suitable analytical method.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy was performed on each animal irrespective of the date of death. Terminally on Day 91 for PEO-1 animals (on Day 132 and Day 174 for PEO-2 and PEO-3 animals, respectively) all animals were euthanized under pentobarbital anaesthesia by exsanguination.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
The following organs were trimmed of fat and weighed in all animals (not for lung burden animals):
With precision of at least 0.01g
Brain
Epididymides
Heart
Kidneys
Liver
Prostate
Lungs
Seminal vesicles with
coagulating glands
Spleen
Testes
Thymus
Uterus including cervix
With precision of at least 0.001g
Adrenal glands
Ovaries
Thyroid with parathyroid glands
Pituitary
Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.
For Lung Burden Animals, the terminal body weights and the lung weights (plus brain weights to allow organ weight to brain weight ratios to be calculated, brains and other organs (except the left lungs) were not retained).

HISTOPATHOLOGY: Yes
On completion of the macroscopic examination the following tissues and organs were retained from all animals.
Animal identification (Fixation and preservation only-
Aorta (Aorta thoracic and abdominal)
Brain (7 section according to the NTP recommendations)
Bone marrow (smear)
Epididymis
Eye with optic nerve (Parathyroids and optic nerves were examined histologically only if present in routine sections)
Femur with joint
Gross findings
Heart (Section including both ventricles and atria, septum with papillary muscle)
Kidneys
Large intestine (Caecum, colon and rectum)
Lachrymal gland (extraorbital) and Harderian gland
Larynx (3 levels, Base of the Epiglottis, Ventral pouch, Cricoid cartilage)
Liver
Lungs with bronchi (Lungs after weighing were infused with formalin at a pressure of 20-30 cm of water.)
Lymph nodes (Mediastinal, submandibular and mesenteric)
Nasal cavity (At least 4 levels, 1 level to include the nasopharyngeal duct and Nasal Associated Lymphoid Tissue (NALT))
Ovary with oviduct
Oesophagus
Pancreas
Pituitary
Prostate
Salivary gland (including
mandibular and sublingual)
Sciatic nerve
Seminal vesicles & coagulating glands
Skeletal muscle (quadriceps)
Skin, subcutis and mammary gland area (inguinal)
Small intestine (Duodenum, ileum and jejunum with Peyer’s patches)
Spinal cord (cervical, mid-thoracic, and lumbar)
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland (Parathyroids and optic nerves were examined histologically only if present in routine sections.)
Tongue
Trachea (Two levels, transverse section in the middle part, longitudinal horizontal including the carina of the bifurcation)
Urinary bladder
Uterus including cervix
Vagina

-PEO-1 (Main Study Animals and Satellite Group 1):
The left lung of the Main Study Animals was preserved for histopathologic evaluation and the right lung of the Main Study Animals was used for bronchoalveolar lavage (BAL).
The right lung of the animals of Satellite Group 1 was used for lung burden. The left lung was not required but was preserved the same way as for histopathology tissues, in case there is any unexpected need to make further evaluations at a later date.
-PEO-2 (Satellite Group 2):
The left lung of the female animals was preserved for histopathologic evaluation and the right lung of the female animals was used for bronchoalveolar lavage (BAL).
The left lung of the male animals was preserved for histopathologic evaluation and the right lung of the male animals was used for lung burden.
-PEO-3 (Satellite Group 3):
The left lung of the animals was preserved for histopathologic evaluation, if it is necessary, based on the findings of PEO-1 and PEO-2 groups; the right lung of the animals may be used for lung burden.
The left lung of the animals using for histopathology evaluation was inflated with formalin to ensure high quality fixation.

In case when the right lung of the animals was used for bronchoalveolar lavage (BAL) evaluation, and the left lung for histopathology, the lung was processed as follows. After removal and weighing of the lungs, the right bronchus was tied off for the BAL procedures, and the left lung was filled with fixative and stored for histopathology processing.
The eyes with the optic nerve and the testes with epididymides were preserved in modified Davidson’s fixative; all other organs in 10% buffered formalin solution.
Bone marrow smears (taken from the femur of all animals) were preserved, examination was not required.
Full histopathology was performed in Control and High dose groups (Main animals). In addition, respiratory tract and associated lymph nodes were investigated in Mid and Low dose groups.
Any organs or tissues with macroscopic abnormalities (except minor changes) were subjected to histological examination.
The retained tissues and organs for histological examination were embedded in paraffin wax, sections were cut at 4-6µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
Statistics:
see under “any information on materials and methods incl. tables”

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The exposure did not cause any test item-related adverse clinical signs.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The exposure did not cause any test item-related adverse change in body weight/weight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The exposure did not cause any test item-related adverse change in food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted on ophthalmoscopy examination.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test-item related changes in haematology were noted.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test-item related changes in clinical chemistry were noted.
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no test item-related observations in the oestrus cycle.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test-item related changes in urine parameters were noted.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The functional observation battery (FOB) and neuromuscular observations showed no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the 91 day necropsy, yellow discoloration (test item) and enlargement of the lungs and mediastinal lymph node were seen with a dose relationship in all dosed groups, as were lung weights.
After the 6 weeks and 3 month recovery period, the lung weight changes showed signs of recovery in Low/Mid in both sexes, but the High dose males showed no evidence of recovery of lung weight at 3 months (correlating with the slow clearance of lung burden).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the 91 day necropsy, yellow discoloration (test item) and enlargement of the lungs and mediastinal lymph node were seen with a dose relationship in all dosed groups, as were lung weights.
All treated groups showed respiratory related changes due to test item. But there were no significant test item-related macroscopic changes in any organs or tissues outside the respiratory system.
Outside the respiratory tract, some test item in the intestinal lumen was related to swallowing of the test item. Some very occasional observations of pigment in the circulatory system were considered to be exposure via the lymphatic system, with no indication of any adverse effect.
Macroscopic findings in the respiratory tract showed limited signs of recovery at up to 13 weeks (but all observations were related to the presence of test item and responses to dust).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
All treated groups showed respiratory related changes due to test item. But there were no significant test item-related microscopic changes in any organs or tissues outside the respiratory system.
Microscopic findings in the respiratory tract showed limited signs of recovery at up to 13 weeks (but all observations were related to the presence of test item and responses to dust).
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Results of lung burden determination showed ~2.5, ~14 and ~30 mg test item per gram of lung at 91 days. A continuous clearance was observed from the lung over time in all dose groups, however clearance at the High dose was slow and limited.
In the BAL, a dose-dependent increase of LDH and albumin values, and live and dead cell numbers were seen in both males and females after 91 days. At histology, there was a massive, dose-related presence of brown pigment (test item) in the alveolar/bronchiolar lumen, bronchio-alveolar macrophages and in the regional lymphoid tissues. Despite the amount of test item present, there were no evident toxic effects (no degenerative or inflammatory changes). Pigmented macrophages were also seen in nasal mucosa and trachea subserosa, without degenerative or inflammatory changes. An exacerbation of eosinophil droplets in the respiratory epithelium of the nasal cavity was correlated with treatment (dust), but is not considered to be a degenerative change or a specific toxic effect of the test item. All changes seen in the respiratory tract and related tissues were considered to be a response to inhaled dust. Test concentrations higher than 1 mg/L would be above the maximum tolerated dose.
BAL measurements at 6 weeks showed signs of recovery in the Low and Mid dose, with some parameters returning to normal levels. It was considered that test concentrations higher than 1 mg/L would be above the maximum tolerated dose.
Details on results:
-Mortality:
No mortality was observed during the study.
-Clinical signs:
No adverse clinical signs were observed in the Control, Low, Mid and High Dose Groups during the observation period.
During the 90-day treatment period, fur staining by test item (in all treated animals), and a low incidence of background minor observations (such as areas of alopecia in 2/25 males) was seen in all groups at a similar frequency.
During the 6-weeks recovery period, alopecia and scar in 1/5 female, during the 3-months recovery period, alopecia in 1/5 Mid dose male and 1/5 High dose male and scar in 1/5 Low dose male were recorded, however these changes were not considered to be test item related.
-Body weight and body weight gain measurements
There were no test item-related differences in the body weight or body weight gain values in any of the dose groups when compared to the controls at the end of the treatment and recovery periods. The measured values did not show a clear dose response and were within the range commonly recorded for this strain and age.
Some sporadic statistically significant decreases of body weight gain in males were seen for some of the weekly evaluations, without a clear trend, therefore these changes were considered to be incidental.
-Food consumption:
There were no treatment related differences in the food consumption values in males or females of the Low, Mid and High Dose Groups when compared to the control between Day 1 and Day 90 and at the end of the 6 weeks and 3 months recovery periods.
Minimal, statistically non-significant decrease of food consumption was recorded in Mid and High dose males on Day 131, however this change did not show a clear dose-response and was not observed in females. Sporadic, statistically significant changes were not an indication of a treatment effect.
-Neurological assessment (functional observation battery) and smart:
There was no adverse effect of treatment noted during the assessment of grip strength, foot splay, Irwin Test or locomotor activity in any dose group when compared with the control.
The 1-hour locomotor activity of all groups showed a normal response, overall, initially high then reducing to a plateau by about 30 minutes; all data was considered to be normal.
-Ophthalmology evaluation:
In the Control and High dose groups, no test item related changes were noted at ophthalmoscopy examination in Week 11 (females) and Week 12 (males) compared to pre-treatment .
PEO-2 and PEO-3 animals were not examined, as no treatment related alterations were observed in main study animals.
-Haematology:
In males, statistically significantly lower platelet count (in Low and Mid Dose Groups) and relative monocytes values (in Mid and High Dose Groups) were observed compared to the Control Group on Day 91. All values were within the historical control range and they did not show clear dose dependence, therefore these changes were not considered to be test item-related.
In females, no statistically significant changes were not observed for the above mentioned parameters on Day 91.
In recovery animals on Days 132 and 174, sporadic statistically significant differences in some parameters (haemoglobin concentration, mean cell haemoglobin, red blood cell count, absolute neutrophil granulocyte value) were considered to be incidental.
In females, decreased PTT was noted in all treated groups compared to the Control Group on Day 91, without a dose dependence. In the Mid Dose Group this change was statistically significant, but it did not have toxicological relevance.
In males, no statistically significant change in PTT values were observed for the above mentioned parameters on Day 91.
In recovery females on Day 132 decrease in PTT was not observed
The white blood cell (WBC) count was in the normal range for all animals, an apparent increase by 75% in High dose females compared to the controls at 91 days was not statistically significant and was not considered to be an effect of test item.
Statistically non-significantly increased relative neutrophil values were measured in females in all dose groups on Day 132. However, these values did not show dose-response and they were within the historical control range, therefore this change was considered to be incidental and not test item related.
-Clinical chemistry:
Statistically significant higher chloride in High Dose males and in Low and High Dose females and sodium in Low Dose females were seen compared to the control on Day 91. These changes were very small, did not show dose dependence, they were considered to be incidental.
Similar changes were not observed in recovery animals.
Other sporadic statistically significant differences in recovery animals were considered to be incidental.
In addition to changes in electrolyte values, apparently decreased bile acid was measured in females of all treatment groups (statistically significant in the High Dose Group, p<0.05) compared to the Control Group on Day 91; however the Control mean was unusually high (compared with other control data) the High dose group values were all normal and in the same range as the Low and Mid animals. In the absence of any indication of an altered liver function in other clinical pathology or histopathology results, the differences in bile acid values were not considered to be test item-related.
No statistically significant changes in bile acid values were observed in males on Day 91.
Results in recovery animals were all considered to be normal.
-Urinalysis:
No test item related effects were noted on the urinalysis parameters evaluated in males and females on Day 91.
Statistically significant increase in urine crystals of Mid and High Dose males did not show a clear dose response and was considered to be incidental.
Sporadic statistically significant differences in some parameters (urine bacteria, glucose, urinary ketones and protein) in recovery male animals on Days 132 and 174 were considered to be incidental.
-Examination of vaginal smears:
There were no test item-related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.
-Macroscopic findings:
Main animals (males & females):
Test item-related macroscopic findings at 91 days were detected in the lungs and in the mediastinal lymph node in all dosed groups at necropsy.
The lungs showed diffuse/multifocal yellow discoloration in all dosed groups, without dose relation and enlargement in all dosed groups with dose relation in the males, the mediastinal lymph nodes were yellow in all dosed groups, without dose relation and enlarged in all dosed groups with dose relation in the males. The yellow colour is considered to be test item.
The other findings, due to low incidence in both dosed and control groups, were considered as incidental or background.

Recovery animals (males & females;6 weeks):
Test item-related macroscopic findings were detected in the lungs and in the mediastinal lymph node in every Dosed groups at necropsy.
The lungs showed diffuse/multifocal yellow discoloration and enlargement in all dosed groups with dose relation, the mediastinal lymph node was yellow and enlarged as well in all dosed groups without dose relation.
The other findings, due to very low incidence through dosed and control groups were considered as incidental or background.

Recovery animals (males; 3 months):
Test item-related macroscopic findings were detected in the lungs and in the mediastinal lymph node in Dosed groups at necropsy.
The lungs showed diffuse/multifocal yellow discoloration and enlargement in every Mid and High dose animals and in 4/5 and 3/5 Low dose rats, respectively. The mediastinal lymph node was yellow and enlarged in 5/5 Low Dose and in 4/5 Mid and High dose rats, respectively.
-Bronchoalveolar lavage:
Main animals (males & females)
The LDH and albumin concentrations increased in a dose related manner in both male and female dosed animals with statistical significance (except Low Dose females), which indicates a possible tissue injury in lungs. This possible tissue injury can be explained by the increased total-, live- and dead cell numbers seen in the BAL samples. However white cell distributions could not be evaluated due to presence of high amount of test item powder in the BAL fluid and on all treated group slides, including in the recovery groups.

Recovery animals (females;6 weeks)
Partial recovery was seen in the females examined at 6 weeks. Albumin returned to normal in Low and Mid groups, while the High group showed a good improvement (reduced by almost 50%). LDH showed slight but inconsistent recovery. The number of live cells showed good recovery, with near-normal levels in Low and Mid females, but the numbers of dead cells only showed a relatively slight recovery.
-Lung burden determination:
In the lungs, the amount of test item found at the end of treatment was very approximately proportional to the atmosphere concentrations. For the lung burden in the Low dose animals, a significant decrease of the test item was measured during the 6 weeks recovery (~60% clearance), while in case of the Mid dose, this decrease was less significant (~24%). In case of the High dose, a relatively small decrease can be seen (~11%), probably due to an overload of the lung. After 3 months recovery, the amount of test item further decreased, the clearance (6 weeks to 3 months) was ~7%, ~25% and 26% for Low, Mid and High dose groups, respectively.
-Organ weight data:
There was no statistically significant difference in terminal body weights of the dosed animals compared to the controls at 91 days or in recovery groups.
There were test item-related statistically significant differences among groups in the weights of the lungs when compared to controls in the study.
Main animals (males & females):
At 91 days, in males, the absolute organ weight of the lungs was higher by 103.7% in the Low Dose group, by 129.0% in the Mid Dose group and by 183.2% in the High Dose group. The relative to body weight of the lungs was higher by 109.0% in the Low Dose group, by 137.5% in the Mid Dose group and by 186.8% in the High Dose group. The relative to brain weight of the lungs was higher by 105.3% in the Low Dose group, by 136.8% in the Mid Dose group and by 185.9% in the High Dose group.
In females, the absolute organ weight of the lungs was higher by 102.1% in the Low Dose group, by 133.2% in the Mid Dose group and by 231.1% in the High Dose group. The relative to body weight of the lungs was higher by 107.0% in the Low Dose group, by 136.1% in the Mid Dose group and by 244.5% in the High Dose group. The relative to brain weight of the lungs was higher by 99.8% in the Low Dose group, by 132.5% in the Mid Dose group and by 227.5% in the High Dose group.
These lung organ weight changes were correlated with necropsy and microscopic findings.

The other statistically significant organ weight changes did not correlate to any microscopic changes, did not show dose relation and were considered as incidental.
Sporadic statistically significant differences in some other cases were considered to be incidental.

Recovery animals (males & females;6 weeks):
Partial recovery of lung weight relative to body weight was observed in Low dose males and in Low, Mid and High dose females. However, in High dose males increased lung weight was not recovered after the 6 weeks recovery periods.

Recovery animals (male; 3 months)
Recovery of lung weight was observed after 3 months recovery period in Low and Mid dose males. In High dose males increased lung weight was not recovered after Day 174.
-Histopathological examinations:
Main animals (males & females)
At 90 days, microscopically, test item-related findings were confined to the respiratory tract and associated lymphatic system (in all treated groups) other than the apparent presence of test item inside the intestine and circulatory system. In the upper respiratory tract, observations were limited to the presence of test item.
5 point scoring system was used in histopathological evaluation:
Minimal
Mild
Moderate
Marked
Severe
At 91 days, in the lungs presence of brown pigment (test item) in the alveolar/bronchiolar lumen, pigment/dust/inert material (test item) in the bronchio-alveolar macrophages, aggregates, macrophage increased and pigment (test item) in the bronchial-associated lymphoid tissue was seen in all dosed animals. There was a clear dose response with increasing severity, in the Low dose most of these findings were mild-minimal, whereas at the High dose they were generally marked-severe. The findings were with multifocal/diffuse appearance.
In recovery animals at 6 weeks, presence of brown pigment (test item) in the alveolar/bronchiolar lumen, pigment/dust/inert material (test item) in the bronchio-alveolar macrophages, aggregates, macrophage increased and pigment (test item) in the bronchial-associated lymphoid tissue was seen in all dosed animals with multifocal/diffuse appearance, and minimal to severe severity correlated with necropsy findings. The severity of the findings was mainly severe or marked in the High Dose animals, mainly moderate in Mid Dose and mainly minimal in Low Dose animals.
The incidence and severity of these test item-related findings were similar to main animals, did not show signs of recovery.
After 3 months recovery, in the lungs presence of brown pigment (test item) in the alveolar/bronchiolar lumen, pigment/dust/inert material (test item) in the bronchio-alveolar macrophages, aggregates, macrophage increased and pigment (test item) in the bronchial-associated lymphoid tissue was seen in all dosed animals with multifocal/diffuse appearance, and minimal to severe severity correlated with necropsy findings. The severity of the findings was mainly severe or marked in the High Dose animals, mainly moderate in Mid Dose and mainly minimal in Low Dose animals; at the High dose there was no reduction compared to the main necropsy animals, with some evidence of increased severity, at the Mid dose there was some evidence of recovery, at the Low dose, there were signs of reduced pigment.
The incidence and severity of the macrophage aggregates after 3 months recovery were similar to main animals, but they did not show signs of recovery, in fact the severity increased in the Low, Mid and High dose groups by 3 months. The severity of the macrophage aggregates increased from moderate to marked in High dose males (5/5 at 3 months) this progressive effect is probably a local adverse effect, but directly related to the lung burden. In the Mid and Low dose males at 3 months, the severity had increased to moderate in all animals, but did not progress beyond a moderate effect; in the absence of any degenerative changes such as fibrosis the effects at the Mid and Low dose were considered to be adaptive rather than adverse.
At 91 days, in the mediastinal lymph node pigment macrophages (test item) were present in every dosed animal and increased cellularity (mainly sinusoidal cells) were seen in all (10/10) High and Mid dose rats (both males and females) with moderate to severe severity, also in 9/10 Low Dose males and 7/10 Low Dose females with minimal/mild severity.
In recovery animals at 6 weeks, pigment macrophages (test item) were present in every dosed animals and increased cellularity (mainly sinusoidal cells) were seen in every (5/5) High and Mid dose rats (both males and females) with mild to severe severity and in 4/5 Low Dose males and every Low Dose females with minimal to moderate severity.
The incidence and severity of these test item-related findings were similar to main animals, did not show signs of recovery.
After 3 months recovery, pigment macrophages (test item) and increased cellularity (mainly sinusoidal cells) were present in every dosed males, the severity of these findings increased in the recovery groups, compared to main animals.
At 91 days, in the nasal cavity eosinophilic globules (droplets) were seen in the respiratory epithelium of every (10/10) High Dose animal and in all 10/10 Mid Dose males and 8/10 Mid Dose females, also in 6/10 Low Dose males and in 7/10 Low Dose females with minimal severity. Pigmented exogenous/dust (test item) macrophages were present in the nasal mucosa in 6/10 High Dose males, in 10/10 High Dose females and in 1/10 Mid Dose male with minimal/mild (mainly minimal) severity.
In recovery animals at 6 weeks and 3 months, no test item related findings were noted in the nasal cavity.
At 91 days, in the trachea pigmented exogenous/dust (test item) were seen in the subserosa of 8/10 High Dose males, 10/10 High Dose females with minimal/mild severity and in 4/10 Mid Dose males, 6/10 Mid Dose females, 1/10 Low Dose males and 1/10 Low Dose females with minimal severity.
In recovery animals at 6 weeks and 3 months, no test item related findings were noted in the trachea.
At 91 days, in the intestinal lumen of the High Dose animals (in the cecum of 3/10 females, in the colon of 7/10 males and 8/10 females, in the ileum of 2/10 females) presence of pigment exogenous/dust (test item) was observed with minimal severity.
In recovery animals at 6 weeks and 3 months, no test item related findings were noted in the intestinal lumen.
At 91 days, in the heart, in the lumen of the ventricle brown pigment particles (test item) was present in 4/10 High Dose males.
In recovery animals at 6 weeks and 3 months, no test item related findings were noted in the heart.
The other findings, without meaningful differences in severity and incidence between Control and dosed groups were considered to be incidental or a common background.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEC
Remarks:
systemic
Effect level:
1 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No systemic toxicity
Key result
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
0.2 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
other:
Remarks on result:
other: This NOAEC is mainly based on local effects in the respiratory tract. Local changes in the respiratory tract were seen in all groups treated with test item.

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
no
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 mg/L air
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Validation Report: Validation of the analytical method for quantitation of tin disulphide in lung samples by XRF spectroscopy – CRL Hungary Study code: 19/106-994ANA (non-GLP):


The purpose of this study was to validate the analytical method developed for quantitative determination of tin disulphide in lung samples by XRF spectroscopy to support a repeat dose inhalation study in rats. This validation established all the significant factors influencing the quali1ty of the method, allowing a much more efficient/rapid GLP cross validation to be made, fully meeting the GLP requirements for Charles River Laboratories.


 


Summary Table: Results of the Method Validation






























Specificity



The Sn Kα1 X-ray line is in the expected position and the calculated concentration of the blank sample is below 20% of the LOQ. Passed



Repeatability



RSD 3.03% Passed



Detector response



R2 = 0.9952; all values <15% of nominal. Passed.



Calibrated linear range



0.025 – 0.375 mg/mL of the test item Passed



Limit of Quantification



The LOQ is established as the lowest calibration point (0.025 mg/mL)



Recovery of the test item from filters



107.09%, RSD 5.44% Passed.



 


Final Study Report: Cross-validation of the analytical method for quantitation of tin disulphide in lung samples– Study code: FPBSTUDY-220-XVAL3; CRL-VES Study code: 19/106-994AN (GLP):


The purpose of this study was to cross-validate the developed and validated (study title: Validation of the analytical method for quantitation of tin disulphide in Lung Samples by XRF spectroscopy) analytical method for the quantitative determination of Tin Disulphide. The procedure was found to be suitable for the analysis of the test item in Lung Samples. The cross-validation parameters met the requirements. Summary of the cross-validated method parameters is presented in Table 1.


 


Table 1. Results of cross-validation





























































Validated parameter



Acceptance criteria



Measured values



Repeatability


(N=7)



CV% of the found concentrations not more than 10%



4.34% CV for the concentration of the Test item



Linearity (Detector response)


In 0.025 – 0.375 mg/mL concentration range



Acceptable, if a correlation coefficient of 0.99 or above is obtained



R² >= 0.9931



Deviation of the re-calculated values from the nominal concentration > 20.0% for the LOQ level



94.1 – 112.7%



Deviation of the re-calculated values from the nominal concentrations > 15.0%



90.1 – 104.3%



Limit of Quantification


(N=3)



Re-calculated concentration values at LOQ level are in 80.0 – 120.0% of the nominal concentrations



0.05 mg/mL calibration sample


99.4 – 110.5%



CV% < 20.0%



6.93%



Accuracy and precision



c TI (mg/g)



1



5



10



Accuracy (%)



The mean value should be within 15% of the nominal value.



105.4



92.4



93.5



Precision (%)



The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV).



5.6



3.17



6.60



 


Validation Report: Validation of the analytical method for quantitation of tin disulphide on inhalation filter by XRF spectroscopy - CRL Hungary Study code: 19/106-901AN (non-GLP):


The purpose of this study was to validate the analytical method developed for quantitative determination of tin disulphide captured on inhalation filters by XRF spectroscopy to support a repeat dose inhalation study in rats. This validation established all the significant factors influencing the quality of the method, allowing a much more efficient/rapid GLP cross validation to be made, fully meeting the GLP requirements for Charles River Laboratories.


 


Summary Table: Results of the Method Validation










































Specificity



The Sn Kα1 X-ray line is specific. Passed



Repeatability



RSD 2.28% Passed



Detector response



R2 = 0.9901 and 0.9923. Passed.



Calibrated linear range



0.1 – 0.4 mg/mL of the test item Passed



Limit of Quantification



The LOQ is established as the lowest calibration point (0.1 mg/mL)



Recovery of the test item from filters



98.2%, RSD 2.6% Passed.



Repeatability/Precision



RSD 2.3% Passed



Stability of the test item on filter



> 6 months Passed



PLS data analysis verified by Leave-one-out cross validation



Passed



 


Final Study Report: Cross-validation of the analytical method for quantitation of tin disulphide on inhalation filter – Study code: FPBSTUDY-220-XVAL2; CRL-VES Study code: 19/106-901ANA (GLP):


The purpose of this study was to cross-validate the developed and validated (study title: Validation of the analytical method for quantitation of tin disulphide on inhalation filter by XRF spectroscopy) analytical method for the quantitative determination of Tin Disulphide. The procedure was found to be suitable for the analysis of the test item on Inhalation Filters. The cross-validation parameters met the requirements. Summary of the cross-validated method parameters is presented in Table 1.


 


Table 1. Results of cross-validation





































































Validated parameter



Acceptance criteria



Measured values



Repeatability


(N=7)



The found concentration of the calibration sample should be RSD < 5.0%



3.96% CV for the concentration of the Test item



Linearity (Detector response)


In 0.025 – 0.375 mg/mL concentration range



Correlation coefficient is above of 0.99



R² >= 0.9985



Re-calculated concentration values at LOQ level are in the 80.0 – 120.0% of the nominal concentrations



89.3% - 95.2%



Re-calculated concentration values above LOQ level are in 85.0 – 115.0% of the nominal concentrations



94.0 – 105.3%



Limit of Quantification


(N=3)



Re-calculated concentration values at LOQ level are in 80.0 – 120.0% of the nominal concentrations



0.025 mg/mL calibration sample


84.3 – 104.8%



CV% < 20.0%



8.67%



Accuracy (Recovery)



m captured (mg)



 



4.32 – 5.89



Accuracy



90.0 – 110.0%



102.7%



CV (%)



<20%



2.5%



Statistical analysis



Equivalence of variances (F-test)



P > 0.05



P = 0.487 (the variances were equal)



Equivalence of mean values (T-test)



P > 0.05



P = 0.024*



* This is a common case when the standard deviations of the two measurement series fall into narrow ranges and the number of observations is low. It does not necessarily mean that any of the two measurements provide false or incomparable results. Although the criteria set in the validation plan is not met, it is important to highlight that the accuracy acceptance range is ±10% and the difference between the two averages is less than 5% (but with small standard deviations). It can be concluded that the results of the measurements carried out in Fumoprep match the results obtained at the Science Port within a reasonable acceptance range (both were 100 ± 3%). Therefore, the method is considered valid.

Applicant's summary and conclusion

Conclusions:
There were no significant changes outside the respiratory system. All the changes observed in the respiratory tract or associated lymph system were considered to be related to the presence of test item or normal physiological responses to the presence of dust. However, the High dose level involved a lung burden that was higher than the clearance systems could process, with little or no recovery of lung weights or of histological changes (although there was no evidence of any degeneration, inflammatory changes or progression after 13 weeks of recovery). In the Mid and Low dose groups, there was good evidence of progress towards a recovery.

In conclusion, under the conditions of this study, the No Observed Effect Concentration (NOEC) for systemic effects was the High dose level of 1 mg/L.
Local changes in the respiratory tract were seen in all groups treated with test item. Recovery was significant in the Low and Mid dose groups, hence the No Adverse Observed Effect Concentration (NOAEC, based on local effects) for the study was the Mid dose (0.2 mg/L).
Executive summary:

The objective of this 90-day study was to obtain information on the toxicity of the test item when administered to Wistar (Han) rats via inhalation route for at least 90 days with the aim of inducing toxic effects but no death or suffering or excessive lung overload at the highest concentration, and little or no evidence of toxicity at the lowest concentration level. The methods used aims to follow OECD 413, with the procedures that are for a test item which does not accumulate in the lungs. According to this, additional subgroups were used to evaluate the recovery of the animals and to investigate the clearance rate of the Test Item from the lung.


The animals were exposed to the test atmosphere at target concentrations of 0.02, 0.2 and 1.0 mg/L, as the Low, Mid and High Dose Concentration, respectively, using a nose-only exposure system over 90 days. The concentration levels, based on a preliminary dose range study, were approved by the Study Monitor and Sponsor; it was considered that higher atmosphere concentrations would have caused a lung dust overload within the 90-day study period.


Ten male and 10 female Wistar Hannover rats Crl:WI(Han) in each main group were treated by a 6 hour nose-only exposure to filtered air or three fixed aerosol concentrations for 5 days/week. The main animals were sacrificed on the day following the last exposure on Day 91 (histopathology evaluation and BALF† analysis were performed). Additionally, 5 females per group were treated and allowed to recover for 6 weeks and sacrificed on Day 132 (histopathology evaluation and BALF analysis were performed) and 15 males per group were treated and allowed to recover and sacrificed on Day 91, Day 132 (6-week recovery) and Day 174 (approximately 3-month recovery) for histopathology and/or lung burden evaluation.


Male and female Crl:WI(Han) rats were treated as follows:






































































Gr. No.



Group Designation


Target Test Atmosphere Concentration (mg/L)



PEO-1 (end of dosing)



PEO-2


(Satellite Group 2)


(recovery for 6 weeks)



PEO-3


(Satellite Group 3)


(3 months)



Main Study Animals



Satellite Group 1



Males



Females



Males



Females



Males



Males



1



Air Control



0



1001-1010



1501-1510



1011–1015



1511-1515



1016-1020



1021-1025



2



Low Dose



0.02



2001-2010



2501-2510



2011–2015



2511-2515



2016-2020



2021-2025



3



Mid Dose



0.2*



3001-3010



3501-3510



3011–3015



3511-3515



3016-3020



3021-3025



4



High Dose



1.0*



4001-4010



4501-4510



4011–4015



4511-4515



4016-4020



4021-4025



Notes:



  1. PEO-1: Post-exposure observation group 1

  2. PEO-2: Post-exposure observation group 2 (6-week recovery)

  3. PEO-3: Post-exposure observation group 3 (3-month recovery)


*: Actual concentration was very slightly above the target concentrations to ensure the classification cut-off was met. † BALF: Bronchoalveolar lavage fluid


Examinations in the study were performed as follows:



















































Gr. No.



Group Designation


Target Test Atmosphere Concentration (mg/L)



PEO-1 (end of dosing)



PEO-2


(Satellite Group 2)


recovery for 6 weeks



PEO-3


(Satellite Group 3)


3 months



Main Study Animals



Satellite Group 1



Males



Females



Males



Females



Males



Males



1



Air Control



0



HP


(left lung)


+


BALF


(right lung)



LB


(right lung)



HP


(left lung)


+


BALF


(right lung)



HP


(left lung)


+


LB


(right lung)



       HP


(left lung)


+


LB


(right lung)



2



Low Dose



0.02*



3



Mid Dose



0.2*



4



High Dose



1.0



Notes:



  1. HP: Histopathology

  2. BALF: Bronchoalveolar lavage fluid

  3. LB: Lung burden


*: Actual concentration was very slightly above the target concentrations to ensure the classification cut-off was met.


Start of the exposure for all 4 groups was Day 1. For practical reasons, the start of treatment of males and females was staggered on different days.


An Air Control Group was exposed to filtered air.


The treatment was performed in 4 parallel inhalation towers, one tower for each treatment or control group.


PEO-1 animals including control were terminated on the day following the last exposure on Day 91. The PEO-2 (Satellite Group 2) animals were terminated on Day 132, the PEO-3 (Satellite Group 3) animals were terminated on Day 174.


The actual atmospheric concentrations were:
















































 



Low Dose (mg/L)



Mid Dose (mg/L)



High Dose (mg/L)



 



gravimetric



analytic



nominal



gravimetric



analytic



nominal



gravimetric



analytic



nominal



Mean



0.020



0.03



0.08



0.21



0.46



0.76



1.03



1.20



2.69



SD



0.001



0.00



0.01



0.01



0.02



0.07



0.03



0.05



0.27



The achieved concentrations for all groups were considered to be fully acceptable for Concentration and Stability of the exposures.


Analysis of control samples for concentration showed that no test item was detected.


Results:


No mortality occurred during the study.


The exposure to the test item Tin disulphide to Hannover Wistar rats for 90 days at dose levels was measured at 0.020 mg/L (Low Dose), 0.21 mg/L (Mid Dose) and 1.03 mg/L (High Dose).


The exposure did not cause any test item-related adverse clinical signs or change in body weight/weight gain or food consumption.


The functional observation battery (FOB) and neuromuscular observations showed no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups.


No treatment-related changes were noted on ophthalmoscopy examination.


No test-item related changes in haematology, clinical chemistry or urine parameters were noted.


There were no test item-related observations in the oestrus cycle.


All treated groups showed respiratory related changes due to test item. But there were no significant test item-related macroscopic or microscopic changes in any organs or tissues outside the respiratory system.


Results of lung burden determination showed ~2.5, ~14 and ~30 mg test item per gram of lung at 91 days. A continuous clearance was observed from the lung over time in all dose groups, however clearance at the High dose was slow and limited.


At the 91 day necropsy, yellow discoloration (test item) and enlargement of the lungs and mediastinal lymph node were seen with a dose relationship in all dosed groups, as were lung weights. In the BAL, a dose-dependent increase of LDH and albumin values, and live and dead cell numbers were seen in both males and females after 91 days. At histology, there was a massive, dose-related presence of brown pigment (test item) in the alveolar/bronchiolar lumen, bronchio-alveolar macrophages and in the regional lymphoid tissues. Despite the amount of test item present, there were no evident toxic effects (no degenerative or inflammatory changes). Pigmented macrophages were also seen in nasal mucosa and trachea subserosa, without degenerative or inflammatory changes. An exacerbation of eosinophil droplets in the respiratory epithelium of the nasal cavity was correlated with treatment (dust), but is not considered to be a degenerative change or a specific toxic effect of the test item. All changes seen in the respiratory tract and related tissues were considered to be a response to inhaled dust. Test concentrations higher than 1 mg/L would be above the maximum tolerated dose.