Tin sulphate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

corresponding Dose-reange finder

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 August 2014 - 13 November 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Remarks:

main study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Due to the preliminary nature of this study no specific regulations or guidelines are applicable.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
- Purity, including information on contaminants, isomers, etc.:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis:
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:
- Reactivity of the test material with the incubation material used (e.g. plastic ware):

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding):
- Preliminary purification step (if any):
- Final concentration of a dissolved solid, stock liquid or gel:
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle):

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution:

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
- Description of the formulation, e.g. formulated product for foliar application; formulated product soil application; solution in organic solvent for soil application; formulated product seed treatment; solution in organic solvent seed treatment:

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added:

Species:

rat

Strain:

Wistar

Remarks:

RccHan(TM) Wistar rats

Details on species / strain selection:

Strain/Species RccHan™:WIST rat.
Supplier: Harlan (UK) Ltd

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain/Species RccHan™:WIST rat.
Supplier: Harlan (UK) Ltd
Number of animals: 15 males and 15 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatisation: 11 days before commencement of treatment.
Age of the animals at start of treatment: 61 to 67 days old.
Weight range of animals at the start of treatment: Males: 225 to 260 g, Females: 159 to 181 g

Allocation: Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Identification of animals: Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.
Identification of cages: Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal housing, diet and water supply
Environmental. control
Rodent facility Restricted entry - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between r.ooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity
Monitored and maintained within the range of 19-23 ºC and 40-70 %.
There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal accommodation: Cages Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution: The cages constituting each group were blocked together by sex on separate batteries.
Number of animals per cage: Three of the same sex.

Bedding: Wood based bedding which was changed at appropirate intervals each week.

Environmental enrichment
Aspen chew block: Provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study and replaced when necessary.

Diet supply
Diet: Rat and Mouse No. 1 Maintenance Diet.
Availability: Non-restricted (removed overnight before blood sampling for haematology or blood chemistry, urine collection and during dosing).

Water supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted (except during urine collection and dosing).

Route of administration:

inhalation: dust

Type of inhalation exposure:

snout only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 3.2 - <= 4.8 µm

Geometric standard deviation (GSD):

2.58

Remarks on MMAD:

Group 2 - Target concentration = 2.3 µg/L - Achieved Exposure level = 2.28 µg/L = MMAD = 4.8 µm - geometric standard diameter = 2.74
Group 3 - Target concentration = 9.1 µg/L - Achieved Exposure level = 10.6 µg/L = MMAD = 3.2 µm - geometric standard diameter = 2.51
Group 4 - Target concentration = 36.3 µg/L - Achieved Exposure level = 41.8 µg/L = MMAD = 3.2 µm - geometric standard diameter = 2.49

The achieved levels were 99, 116 and 115 % of the target concentrations for Groups 2, 3 and 4 respectively. The MMAD values for Groups 3 and 4 are slightly above the ideal range of 1 to 3 μm with 79 to 80 % of the particles within the respirable range (< 7 μm). The Group 2 MMAD value was higher with a lower percentage of particles within the respirable range (64 %).

Details on inhalation exposure:

The animals on study were acclimated to the method of restraint, over a 5 day period immediately preceding the first test substance exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mass Median Aerodynamic Diameter:
The MMAD values for Groups 3 and 4 are slightly above the ideal range of 1 to 3 μm with 79 +/- 80 % of the particles within the respirable range (<7 μm). A jet mill was used for Groups 3 and 4 to reduce the average particle size, however it was not practical to fit a jet mill on the Group 2
system, this is considered to be the reason for the higher MMAD value of 4.8 μm and lower percentage of particles within the respirable range (64 %).

Duration of treatment / exposure:

6 h/day

Frequency of treatment:

5

Dose / conc.:

2.28 mg/m³ air (analytical)

Remarks:

(nominal: 2.3 µg/L = 2.3 mg/m³)

Dose / conc.:

10.6 mg/m³ air (analytical)

Remarks:

(nominal: 9.1 µg/L = 9.1 mg//m²)

Dose / conc.:

41.8 mg/m³ air (analytical)

Remarks:

(nominal: 36.3 µg/L = 36.3 mg/m³)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry: yes (12 hours)
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):

This study was designed to assess the systemic toxic potential of tin monoxide in a 1 week (6 hours per day, 5 days a week) inhalation study in RccHan™;WIST rats.
The study design was as follows: Group 1 - Control - 0 µg/L (3 males & 3 females), Group 2 - Tin Monoxide - 2.3 µg/L - (3 males & 3 females), Group 3 - Tin Monoxide - 9.1 µg/L (3 males & 3 females), Group 4 - Tin Monoxide - 36.3 µg/L (3 males & 3 females)
Animals received air only or the test substance, tin monoxide by inhalation for 1 week (6 hours per day, 5 days a week).
During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), blood chemistry, urinalysis, bioavailability, blood oxygen saturation, organ weight, macropathology and histopathology investigations were undertaken.

Positive control:

none

Observations and examinations performed and frequency:

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

Signs associated with dosing:
Detailed observations were recorded daily at the following times in relation to dose administration:
- Pre-exposure observation
- During exposure however, observation is severely restricted due to tube restraint
- As each animal is returned to its home cage
- As late as possible in the working day

Clinical signs: A detailed weekly physical examination was performed on each animal to monitor general health.

Body weight: The weight of each animal was recorded daily from Day -5 (Day P3), throughout the study and before necropsy.

Food consumption: The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily from Day -5 (Day P3) throughout the study.

Haematology, peripheral blood:
Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasion: Week 1: All animals.
Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.3 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC),
Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)
Platelet count (Plt)
Morphology:,Anisocytosis, Macrocytosis, Microcytosis, Hypochromasia, Hyperchromasia
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyser. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyser and appropriate reagent in respect of:
- Prothrombin time (PT) - using IL PT-Fibrinogen reagent.
- Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Haematology, bone marrow: Bone marrow smears were prepared immediately following death on completion of the scheduled treatment period.
Fixation: Smears were air dried and subsequently fixed in methanol.
Retention Slides were retained by the Department of Biomarkers, Bioanalysis and Clinical Sciences.
Analysis: At the discretion of the pathologist, examination of smears to further evaluate other findings was not considered necessary; the smears are retained in the archives.

Blood chemistry: Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) at the following occasions:
Occasion Animals: Week 1 - All animals

Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.5 mL, Groups 2 and 3 and 0.6 mL Groups 1 and 4) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyser in respect of: Alkaline phosphatase (ALP); Alanine aminotransferase (ALT); Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (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), Albumin (Alb), Copper (CU) – Groups 1 and 4 only (non-GLP), Iron (Fe).

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

Urinalysis
Animals were placed in an individual metabolism cage overnight, without food or water. Urine samples were collected over approximately 16 hours at the following occasion: Week 1 - All animals
The individual samples were examined for the following characteristics:
Using manual methods:
- Clarity and Colour (App) - by visual assessment
- Volume (Vol) - using a measuring cylinder
- pH - using a pH meter
- Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips, interpreted using a Clinitek®500 instrument: Ketones (Keto); Bilirubin/bile pigments (Bili); Blood pigments (UBld);
Using a Roche P Modular analyser: Protein (T-Prot); Creatinine (T-Creat); Glucose (T-Gluc)

Sacrifice and pathology:

Terminal Procedures
All 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.
The retained tissues were checked before disposal of the carcass.
Schedule: Study animals were killed following treatment on Day 5.
Method of kill: Overdose of intraperitoneal pentobarbitone sodium followed by exsanguination.
Sequence: To allow satisfactory inter-group comparison.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed as detailes in the field Any other information on materials and methods

Organ weights
For bilateral organs, left and right organs were weighed together, unless specified above.
Requisite organs were weighed for study animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid.
Eyes: In Davidson’s fluid.
Bone marrow smears: See below.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. A single section was prepared from each of the tissues required.
Full List All animals killed or dying prematurely.
Study animals of Groups 1 and 4 killed at the scheduled necropsy.
Routine staining Sections were stained with haematoxylin and eosin.

Light microscopy
Tissues preserved for examination were examined as follows:
Category: Scheduled kill - Animals: All animals of Groups 1 and 4, Tissues: All specified
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Other examinations:

Bioavailability (Non-GLP)
The sampling schedule was as follows: At termination Group 1 and Group 4: Time of sampling (hours after completion of dosing): 2 hours; Parameter: Tin
Samples were collected under terminal anaesthesia.
Blood sample site: Cardiac puncture.
Anaesthetic: Isoflurane.
Anticoagulant: Lithium heparin.
Blood volume: 0.6 mL.
Treatment of samples: Gently inverted several times and then placed on an automatic mixer for at least 2 minutes continuously at ambient temperature.
Centrifugation conditions At 1300 g for 10 minutes at 21 °C.
Number of aliquots per sample: One
Temporary storage conditions: Plasma was processed at ambient temperature and stored frozen (approximately -20 ˚C) as soon as practicable following completion of blood sampling completion of blood sampling
Final storage conditions: Deep frozen (approximately -20 ºC).
Fate of plasma samples: Despatched to a Responsible Scientist on solid carbon dioxide.

Blood oxygen saturation
The sampling schedule was as follows: at Day 5 Group 1 and Group 4 animals
Blood sample site: Tail vein.
Anticoagulant: Lithium heparin.
Blood volume: 0.3 mL.
Samples were analysed without delay.
Each sample was used to measure the following parameter using a blood gas analyser system (ABL80 CO-OX FLEX).
-- Oxygen saturation (sO2)

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analysed at each timepoint separately:
- Body weight, using gains over appropriate study periods
- Haematology
- Blood chemistry
- Urinalysis
- Organ weights, absolute and adjusted for terminal body weight

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs observed during the detailed weekly physical examination.
Signs associated with the administration procedure included wet fur and red staining of the head on return to the home cage. These signs are associated with the method of restraint used.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in body weights.
There was a body weight loss for all animals on Day 4. This is considered due to all animals having food and water withdrawn overnight on Day 3 for blood and urine sampling. Body weight gains were evident for all animals on Day 5.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment related changes in food consumption.
Reduced food consumption was evident for all animals from Days 3 to 4. This is considered due to all animals having food withdrawn overnight on Day 3 for blood and urine sampling. Food consumption had recovered for all animals on Days 4 to 5.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Group mean neutrophil counts were statistically significantly high for males exposed to 41.8 μg/L (1.8X control values). Neutrophil counts were also high for males and females exposed to 2.28, 10.6 μg/L. However, a few individual values for each sex, in each group, were close to control values. Due to the lack of a dose-related response in females, it is not considered a treatment related effect in females.
Other changes from control were small, inconsistent between sexes and/or did not show clear dose relationships.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Group mean inorganic phosphorus levels where high for males exposed to 41.8 μg/L (1.3X control values) and all females exposed to tin monoxide (1.3X, 1.3X and 1.4X control values for females exposed to 2.28, 10.6 and 41.8 μg/L respectively), all attaining statistical significance.
Other changes from control including those that attained statistical significance, were small, inconsistent between sexes and/or did not show clear dose relationships.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment related effects on urinalysis parameters.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight adjusted lung and bronchi weights were statistically significantly high for females exposed to 10.6 and 41.8 μg/L (both 1.2X control values).
Group mean body weight adjusted spleen weights were higher for all males exposed to tin monoxide (1.2, 1.4 and 1.3X control values for males exposed to 2.28, 10.6 and 41.8 μg/L respectively), with males exposed to 41.8 μg/L attaining statistical significance.
All other differences from control including those that attained statistical significance, were generally small and are not considered to be of toxicological significance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed after 1 week of treatment revealed the followingbchanges in the tracheobronchial lymph nodes.
Tracheobronchial lymph nodes
Enlargement was seen in two male animals treated with 41.8 μg/L, one female animal treated with 2.28 μg/L and one female animal treated with 10.6 μg/L.
The nature and incidence of all other findings were consistent with the commonly seen background of macroscopic changes and were considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Treatment related findings
There were no microscopic findings that were considered to be related to treatment with tin monoxide.

Incidental findings
All histological changes were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Blood oxygen saturation levels appeared to be lower than control values for all males exposed to 41.8μg/L. However, the blood gas analyser reported that there were errors which may have affected the results for two of the males exposed to 41.8 μg/L (animal numbers 10 and 11). For the remaining male exposed to 41.8μg/L, the blood oxygen saturation level was lower than control (approximately 0.7X). Blood oxygen saturation levels for females exposed to 41.8 μg/L were all similar to control values.

Bioavailability (non-GLP): Tin concentrations in plasma for controls were all below the limit of quantification (< 5 ng/mL) for the test analysis. Tin concentrations in plasma for males and females exposed to 41.8 μg/L were all approximately 2-3X greater than the lower limit of quantification.

Dose descriptor:

NOAEL

Effect level:

> 41.8 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The exposure was well tolerated and resulted in no adverse findings.

Discussion

The test article, tin monoxide, was administered by snout-only inhalation administration, for 6 hours a day for 5 consecutive days, at achieved exposure levels of 2.28, 10.6 and 41.8 μg/L. There were no unscheduled deaths or treatment related effects on clinical signs, body weight, food consumption, urinalysis parameters and histopathology findings. Slightly higher body weight adjusted lung and bronchi weights were evident for females exposed to 10.6 and 41.8 μg/L. In the absence of a similar effect in males or any correlating histopathological changes, these were considered to be of no toxicological importance. Slightly higher body weight adjusted spleen weights were evident for all males exposed to tin monoxide.

Whilst histopathological correlates for these changes could not be made as spleens were not examined microscopically, the absence of any corresponding macropathology or clinical pathology changes, this is not considered to be related to treatment.

Analysis of tin monoxide concentrations in plasma for males and females exposed to 41.8 μg/L indicated that the test article was present in plasma 2 hours after the completion of exposures.

However, this work was not performed to the principles of GLP and as such, no formal conclusions can be drawn from this data.

A reduction in blood oxygen saturation levels was observed in one male exposed to 41.8 μg/L. However, due to the lack of conclusive results from the remaining two males exposed to 41.8 μg/L, a relationship to treatment cannot be confirmed for males. Blood oxygen saturation results for females were similar to control values.

An increase in neutrophil counts was evident for males exposed to 41.8 μg/L. The slight increases in inorganic phosphorous levels for males exposed to 41.8 μg/L, and all females exposed to tin monoxide, were of uncertain relationship to treatment.

Conclusion

It is concluded that snout only exposure to tin monoxide to RccHan™;WIST rats for six hours each day for five days, at achieved exposure levels of 2.28, 10.6 or 41.8 μg/L, was well tolerated and resulted in no adverse findings. The results therefore demonstrate that exposing animals to at least the same exposure levels that were used in this study would be suitable for use in a four week toxicity study.

Conclusions:

Snout only exposure to tin monoxide to RccHan™;WIST rats for six hours each day for five days, at achieved exposure levels of 2.28, 10.6 or 41.8 μg/L, was well tolerated and resulted in no adverse findings.

Executive summary:

This study was designed to assess the systemic toxic potential of tin monoxide in a 1 week (6 hours per day, 5 days a week) inhalation study in RccHan™;WIST rats. It was conducted as a Dose-range finder for the envisaged OECD 412, which was then conducted.

The study design was as follows: Group 1 - Control - 0 µg/L (3 males & 3 females), Group 2 - Tin Monoxide - nominal 2.3 µg/L = analytically verifeid 2.28 µg/L (3 males & 3 females), Group 3 - Tin Monoxide - nominal 9.1 µg/L = analytically verified 10 .6 µg/L (3 males & 3 fenales), Group 4 - Tin Monoxide - nominal 36.3 µg/L = analytically verified 41.8 µg/L (3 males & 3 females). Animals received air only or the test substance, tin monoxide by inhalation for 1 week (6 hours per day, 5 days a week).

During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), blood chemistry, urinalysis, bioavailability, blood oxygen saturation, organ weight, macropathology and histopathology investigations were undertaken.

Concernign the Mass Median aerodynamic diameter, the following is reported: Group 2 - Target concentration = 2.3 µg/L - Achieved Exposure level = 2.28 µg/L = MMAD = 4.8 µm - geometric standard diameter = 2.74, Group 3 - Target concentration = 9.1 µg/L - Achieved Exposure level = 10.6 µg/L = MMAD = 3.2 µm - geometric standard diameter = 2.51, Group 4 - Target concentration = 36.3 µg/L - Achieved Exposure level = 41.8 µg/L = MMAD = 3.2 µm - geometric standard diameter = 2.49, The achieved levels were 99, 116 and 115 % of the target concentrations for Groups 2, 3 and 4 respectively. The MMAD values for Groups 3 and 4 are slightly above the ideal range of 1 to 3 μm with 79 to 80% of the particles within the respirable range (<7 μm). The Group 2 MMAD value was higher with a lower percentage of particles within the respirable range (64 %).

Group mean body weight adjusted lung and bronchi weights high for females exposed to 10.6 and 41.8 μg/L. Group mean body weight adjusted spleen weights were higher for all males exposed to tin monoxide.

Compared with control, higher group mean neutrophil counts was evident for males exposed to 41.8 μg/L. Compared with control, higher group mean inorganic phosphorous levels were evident for males exposed to 41.8 μg/L and all females exposed to tin monoxide. Group mean body weight adjusted lung and bronchi weights were statistically significantly high

for females exposed to 10.6 and 41.8 μg/L. Group mean body weight adjusted spleen weights were higher for all males exposed to tin monoxide. Enlargement of the tracheobronchial lymph nodes was evident for two males exposed to

41.8 μg/L, one female exposed to 2.28 μg/L and one female exposed to 10.6 μg/L. Tin was present in plasma 2 hours after the completion of dosing for all male and female animals exposed to 41.8 μg/L. However, this work was not performed to GLP.

Conclusion

It is concluded that snout only exposure to tin monoxide to RccHan™;WIST rats for six hours each day for five days, at achieved exposure levels of 2.28, 10.6 or 41.8 μg/L, was well tolerated and resulted in no adverse findings. The results therefore demonstrate that exposing animals to at least the same exposure levels that were used in this study would be suitable for use in a four week toxicity study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

Adopted: 7 September 2009

Deviations:

yes

Remarks:

significant variance in dose and humidity

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

EC Methods for the determination of toxicity. Annex to Commission Directive
92/69/EC (Official Journal No. L 38 A/140), Part B, Method B.8: Repeated dose
(28 days) toxicity (inhalation).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tin monoxide

EC Number:

244-499-5

EC Name:

Tin monoxide

Cas Number:

21651-19-4

Molecular formula:

OSn

IUPAC Name:

Tin monoxide

Test material form:

other: dust

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan™:WIST rat.

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The RccHanTM:WIST strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (UK) Ltd.
- Age at study initiation: 69 to 76 days
- Weight at study initiation:
Males: 245 to 281 g
Females: 180 to 203 g
- Housing: 5 animals of the same sex/cage; Polycarbonate cages with a stainless steel mesh lid, changed at appropriate intervals; Wood based bedding which was changed at appropriate intervals each week.
- Diet (e.g. ad libitum): ad libitum (removed overnight before blood sampling for haematology or blood chemistry and during the period of exposure).
- Water (e.g. ad libitum): ad libitum (except during exposure)
- Acclimation period: At least 12 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
Diet: Rat and Mouse No. 1 Maintenance Diet.
Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
Mean value of humidity in
- Group 3: 38.6 %
- Group 4: 33.8 /(min: 27.7)

- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 2.7 - <= 3 µm

Geometric standard deviation (GSD):

2.54

Remarks on MMAD:

GSD: 2.41 - 2.54

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: snout only exposure

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6 h exposure

Frequency of treatment:

5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2.3 mg/m³ air (nominal)

Remarks:

achieved 2.44 µg/L

Doses / concentrations 2

Dose / conc.:

9.1 mg/m³ air (nominal)

Remarks:

achieved 9.19 µg/L

Doses / concentrations 3

Dose / conc.:

80 mg/m³ air (nominal)

Remarks:

achieved 87.9 µg/L

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: In a previous preliminary inhalation toxicity study (Huntingdon Life Sciences Study Number URA0001) tin monoxide was administered to rats for six hours per day, for five days, using a snout-only exposure system at achieved exposure levels of 2.28, 10.6 and 41.8 μg/L. These exposure levels were well tolerated and there were no adverse findings. Consequently, in order to establish signs of toxicity, the high target exposure level for this study has been selected as 80 μg/L. The low and intermediate target exposure levels remained the same as those used on the previous study (2.3 and 9.1 μg/L). The intermediate exposure level will investigate toxicity at the German occupational exposure limit (German TRGS 900 Sn(II) compounds). The low exposure level was not expected to cause any toxicity.
- Rationale for animal assignment (if not random): Randomly allocated on arrival.
- Fasting period before blood sampling for clinical biochemistry: removed overnight before blood sampling for
haematology or blood chemistry

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for the week before treatment started and for each week throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 4
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked:
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
Morphology:
Anisocytosis
Macrocytosis
Microcytosis
Hypochromasia
Hyperchromasia

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 4
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (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)
Albumin (Alb)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The following sequence of statistical tests was used for body weight, blood oxygen saturation, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied.
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, the non-parametric equivalent of the F1 test described above, was applied.

For clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable. For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights. Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs observed during the detailed weekly physical examination. Immediately after dosing, wet fur was observed for a majority of animals and red staining of the head was observed for a single animal on Day 1. These are both common findings associated with the method of restraint and are considered not to be treatment related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight gain was reduced for males exposed to 9.19 and 87.9 μg/L of tin monoxide (0.6X and 0.4X control values respectively). A single male exposed to 87.9 μg/L (Animal 16), experienced a body weight loss of 37 g over the 4 week exposure period. Reduced group mean body weight reduction was evident for all females exposed to tin monoxide (0.6, 0.4 and 0.8X control values, with increasing exposure concentration).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was slightly reduced for males exposed to 87.9 μg/L during the first two weeks of treatment compared to concurrent control and pretreatment values.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in group mean activated partial thromboplastin times were evident for males exposed to 9.19 and 87.9 μg/L (1.2X control values for both groups) and all females exposed to tin monoxide (1.3X control values for both groups). Calculation and statistic not traceable. Other changes from control were small, inconsistent between sexes or did not show a clear relationship to treatment and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment related effects. Calculation and statistic not traceable. Other changes from control were small, inconsistent between sexes or did not show a clear
relationship to treatment and were therefore attributed to normal biological variation.

Endocrine findings:

not examined

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):

Group mean lung and bronchi weights (adjusted for terminal body weight) were statistically significantly greater than control for all animals exposed to tin monoxide (up to 2.1X control for males and up to 2.4X control for females). Other changes from control were small, inconsistent between sexes or did not show a clear relationship to treatment and were therefore attributed to normal biological variation.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed after 4 weeks of treatment revealed changes in the lungs, tracheobronchial and mediastinal lymph nodes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with tin monoxide were present within the lungs, tracheobronchial and mediastinal lymph nodes, larynx and kidneys. In addition, one male
animal had findings within the liver, stomach, duodenum and jejunum.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Blood oxygen saturation
Group mean venous blood oxygen saturation levels were increased for females exposed to 9.19 μg/L (1.5X control values) and both sexes exposed to 87.9 μg/L (1.4 and 1.6X control values for males and females respectively). Statistical analysis was not performed for females due to the limited number of results available for females exposed to 2.44 μg/L.

Bioavailability
Two hours after the completion of exposure in Week 4, there was a dose related increase in tin concentration in the plasma of animals exposed to 9.19 and 87.9 μg/L. For animals
exposed to 87.9 μg/L, group mean tin plasma concentration values were 30.0 ng/mL for
males and 15.0 ng/mL for females. For animals exposed to 9.19 μg/L, quantifiable levels of tin were present in the plasma of a single male and female (approximately 10 ng/mL). For the remaining animals exposed to 9.19 μg/L and all animals exposed to 2.44 μg/L, tin plasma concentrations were below the lower limit of quantification. Tin was not detectable in any of the Control group samples.

Details on results:

Histopathological changes related to treatment were observed within the lungs, the tracheobronchial and mediastinal lymph nodes, larynx and kidneys. In the lungs, accumulation of pigmented and flocculent material within alveoli correlated with macroscopic discolouration. Together with pigmented and flocculent material within the epithelium and lamina propria of the larynx, this was considered to reflect the accumulation of inhaled test article, with attempted clearance by the local mononuclear-phagocyte system. This was variably accompanied by both diffuse and local aggregations of alveolar macrophages, and increased cellularity of the BALT in the lungs which was indicative of irritation by the test article. The combined findings in the lungs also correlated with increased group mean lung and bronchi weights for all animals exposed to tin monoxide. Similar changes, at a lesser severity (showing dose-dependence), were seen within the lungs of animals dosed with 2.44 or 9.19 μg/L, with the exception of increased BALT, which was absent in these animals. Histopathological changes were also observed in the tracheobronchial and mediastinal lymph nodes (increased general cellularity, with or without the accumulation of pigmented material), and kidneys (increased tubular pigment). Findings within the local lymph nodes and kidneys were considered to represent the subsequent systemic dissemination of the test article. This correlated with enlarged tracheobronchial lymph nodes, and to some extent, enlarged mediastinal lymph nodes. Histopathological findings were more pronounced, in terms of incidence and severity, amongst animals dosed with 87.9 μg/L, and displayed a clear dose response.

Effect levels

Dose descriptor:

NOAEL

Effect level:

9.19 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

After 4 weeks of inhalation exposure to tin monoxide, at concentrations up to 87.9 μg/L, led to reduced body weight gains and histopathological changes within the larynx, lungs, tracheobronchial and mediastinal lymph nodes, and kidneys. The more marked histopathological changes observed within the lungs of animals dosed with 87.9 μg/L, notably the moderate accumulation of local alveolar macrophages and accompanying increases in BALT, were indicative of irritation and considered to be adverse. Due to the low incidence and severity of findings for animals exposed to 9.19 μg/L, they were considered to be non-adverse. The no observed adverse effect level (NOAEL) was considered to be 9.19 μg/L.

Executive summary:

A GLP-compliant subacute inhalation toxicity study in rats according to OECD Guideline 412 was performed with the test item tin monoxide. The objective of this study was to assess the systemic toxic potential of tin monoxide in a 4 week (6 hours per day, 5 days per week) inhalation study in RccHanTM:WIST rats. The study included three dose groups and one control group (air control) with 5 rats/sex in each group. The analytical exposure concentrations were: 0, 2.44, 9.19 and 87.9 µg/L. The achieved levels were 106, 101 and 110% of the target concentrations for Groups 2, 3 and 4 respectively. The Mass Median Aerodynamic Diameters for Groups 2, 3 and 4 are within the ideal range (1-3 μm) for a repeat dose inhalation study. There were no treatment related clinical signs observed during the detailed weekly physical examination. Lower body weight gain was evident for males exposed to 9.19 μg/L and 87.9 μg/L and for all females exposed to tin monoxide. Food consumption was also slightly reduced for males exposed to 87.9 μg/L. Two hours after the completion of exposure in Week 4, tin was detected in the plasma of a single male and female exposed to 9.19 μg/L and in all animals exposed to 87.9 μg/L. Tin was not quantifiable in control animals or animals exposed to 2.44 μg/L. Group mean lung and bronchi weights (adjusted for terminal body weight) were greater than control for all animals exposed to tin monoxide. Histopathological changes related to treatment were observed within the lungs (the accumulation of pigmented and flocculent material within alveoli, variably accompanied by both diffuse and local aggregations of alveolar macrophages, and increased cellularity of the BALT), tracheobronchial and mediastinal lymph nodes (increased general cellularity, with or without the accumulation of pigmented material), larynx (pigment accumulation within the epithelium and lamina propria) and kidneys (increased tubular pigment). Observations within the larynx and lungs were considered to reflect the accumulation of inhaled test article, with attempted clearance by the local mononuclear-phagocyte system. Accompanying findings within the local lymph nodes and kidneys were considered to represent the subsequent systemic dissemination of test article. Findings were more pronounced, in terms of incidence and severity, amongst animals dosed with 87.9 μg/L, and displayed a clear dose-related response. Amongst animals dosed with 2.44 μg/L, findings were restricted to the lungs and tracheobronchial lymph nodes. A statistically significant increase in group mean activated partial thromboplastin times were evident for males exposed to 9.19 and 87.9 μg/L and all females exposed to tin monoxide. Group mean venous blood oxygen saturation levels were increased for females exposed to 9.19 μg/L and both sexes exposed to 87.9 μg/L.

After 4 weeks of inhalation exposure to tin monoxide, at concentrations up to 87.9 μg/L, led to reduced body weight gains and histopathological changes within the larynx, lungs, tracheobronchial and mediastinal lymph nodes, and kidneys. The more marked histopathological changes observed within the lungs of animals dosed with 87.9 μg/L, notably the moderate accumulation of local alveolar macrophages and accompanying increases in BALT, were indicative of irritation and considered to be adverse. Due to the low incidence and severity of findings for animals exposed to 9.19 μg/L, they were considered to be non-adverse. The no observed adverse effect level (NOAEL) was considered to be 9.19 μg/L.