Registration Dossier

Administrative data

Description of key information

A fourteen day range finding study for the guideline OECD 407  was performed in compliance with GLP. No indications of systemic toxicity were recorded following repeated administraton of 250, 500 or 1000 mg/kg bw/day of a formulation of tin metal powder. These results were then confirmed in the definitive 28 day subacute study, using 1000 mg/kg bw/day as the highest dose administered.

A subacute (28 day) study was performed in compliance with GLP and to the standardised guideline OECD 407. Oral administration of the test material, tin metal powder (2-11 µm) to rats for a period of up to twenty-eight consecutive days at dose level of 30, 300 and 1000 mg/kg/day resulted in no treatment-related toxicological effects and for this reason the "No Observed Effect Level" (NOEL) was considered to be 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 October 2009 to 12 January 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: The Japanese Ministry of Econnomy Trade and Industry (METI), Ministry of Health, labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, OPPTS 870.3050 Repeated DOse 28-day oral toxicity study in Rodents, July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:HsdRccHan™:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Blackthorn, Oxon.
- Age at study initiation: circa 6-8 weeks old
- Weight at study initiation: 204-246 g for males and 161-190 g for females
- Fasting period before study: No
- Housing: groups of three (satellite animals only) or five by sex in solid floored polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet: pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories UK, Oxon, UK) provided ad libitum
- Water: mains drinking water available ad libitum via polycarbonate cage bottles
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 55 ± 15 % RH
- Air changes: at least 15 changes per hour
- Photoperiod: 12h / 12h (hrs dark / hrs light; low intensity fluorescent lighting)

In life dates: the first day of dosing was 16 October 2009 and final necropsy completed on 27 November 2009
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Tin metal powder was suspended in 1 % aq. The formulation was analysed in the preliminary study and confirmed to be stable for up to 14 days and so fresh dosing suspensions were prepared at weekly intervals and stored refrigerated in the dark until used. Stability and homogeneity were confirmed by analysis.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The dosing regimen was selected using the results of the preliminary 14 day range finding study.
- Concentration in vehicle: 3, 30 and 100 mg/mL for the low, intermediate and high doses
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Due to the limited solubility of metallic tin powder in organic and aqueous media, a substance specific quantitative method of analysis was not developed. The concentration of Tin Metal Powder (2-11 μm) in the CMC formulations was determined using a gravimetric technique.
Sampling in triplicate was performed to determine formulation homogeneity. Stability determinations were conducted after sampling and again after 14 days storage. The formulations were analysed within 3 days of preparation to determine tin metal concentration

SAMPLES
The test material formulations were weighed into tared glass beakers and then dried in an oven overnight at approximately 105 °C before allowing to cool over silica gel in a desiccators and re-weighed.

HOMOGENEITY
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. On one occasion samples were left at ~105 °C over three days and not overnight, this was considered not to impact on the scientific integrity of the study.

STABILITY DETERMINATIONS
The test material formulations were sampled and analysed initially and then after storage at approximately + 4 °C in the dark for fourteen days.

VERIFICATION OF TEST MATERIAL FORMULATION CONCENTRATIONS
The test material formulations were sampled and analysed within three days of preparation.

In addition to dose formulations, the tin content of biological samples was chemically analysed using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) with an external standard technique. Test samples and controls were digested using a 3:1 hydrochloric/nitric acid mix and a microwave digester. Tin standard solutions were prepared in a similar matrix at a range of concentrations relevant to the test samples - to produce a calibration curve.
ICP-MS conditions for analysis were:
ICP-MS: Agilent 7500 cx
Acquisition Mode: spectrum
Element and Mass: Sn - 118
Peak Pattern: full quant (3)
Repetitions: 3
Interference Equation: none
Detector Setting: auto
Integration Time: 3 seconds
Details of the method validation are given in the report. Linearity and accuracy were evaluated; calibration curve produced and fortified matrix samples prepared as tin standards. The LOQ for plasma was 0.5 ppm, mean recoveries were greater than 100 %. Mean recoveries for urine and faecal samples were between 107-112 %. The method was considered sufficiently accurate for use in the study and satisfactorily validated for linearity and accuracy.

Results and validation of the method are included in the attached pdf "Chemical Analysis of Tin Content in Biological Samples"
Duration of treatment / exposure:
28 days
Two recovery groups - 5 males and 5 females per group, dosed with vehicle alone or the high dose formulation, were retained for a 14 day treatment free period following the last administration on day 28
Two groups of three rats per sex were included to provide blood, urine and faecal samples on Day 1 and 28 for analysis of tin content
Frequency of treatment:
Daily for 28 days. 14 days without treatment during recovery phase
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Five males and five females per group. The control and high dose groups had an extra five males and five females as a recovery subgroup. A satelite group with a further three males and three females were dosed as per the main study groups and used to obtain blood urine and faecal samples on day 1 and 28 for tin analysis
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results from preliminary study and data from acute studies previously conducted.
- Rationale for animal assignment (if not random): Randomised
- Rationale for selecting satellite groups: Satellite groups in control and high dose regimen retained for treatment free period of two weeks to assess recovery from any observed toxic effects. Further satellites used to obtain biological samples (blood,urine and faeces) on Day 1 and 28 for analysis of tin content
- Post-exposure recovery period in satellite groups: two weeks
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily assessment of clinical signs - prior to dosing, 30 minutes after dosing and 1 and 5 hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. During the treatment-free period, animals were observed daily. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and on days 7, 14, 21 and 26, all non-recovery test and control animals (with the exception of satellite sub-groups) were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all non-recovery test and control animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out at least two hours after dosing on each occasion.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also performed prior to terminal kill and, in the case of recovery group animals, on Days 36 and 43 prior to terminal kill.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption determined over weekly intervals throughout the study (except for the satellite sub-groups).

FOOD EFFICIENCY: Yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food efficiency (ratio of bodyweight gain to food consumed) determined over weekly intervals throughout the study (except for the satellite sub-groups).

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was observed daily for each group (except satellite sub-groups) by visual inspection of the water bottles for any overt changes except during Weeks 3 where water intake was measured gravimetrically.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at end of treatment period for all groups not allocated to the recovery phase. Recovery phase groups sampled at end of treatment free period (two weeks later). Samples collected in on day 28 for the non-recovery animals and day 42 for the recovery group animals during the treatment free-period.
- Anaesthetic used for blood collection: No - lateral tail vein sampling method (where necessary, repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43).
- Animals fasted: No
- How many animals: All non-recovery test and control group animals (with the exception of satellite sub-groups) and on all surviving recovery group animals
- Parameters checked: Haemoglobin (Hb), erythrocyte count (RBC), haematocrit (Hct), platelet count (PLT), reticulocyte count (Retic), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), total leucocyte count (WBC) and differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos) and basophils (Bas)).
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
Using similar collection procedures, blood samples were withdrawn from satellite group animals approximately four hours following dosing on the first and final days of treatment. A sufficient volume of whole blood was collected into lithium heparin anticoagulant blood tubes. The blood samples were then centrifuged and the plasma decanted into plain tubes.
- Parameters checked: Urea; inorganic phosphorus (P); glucose; gamma glutamyltranspeptidase (γGT); total protein (Tot.Prot.), aspartate aminotransferase (ASAT), albumin, alanine aminotransferase (ALAT), albumin/globulin (A/G) ratio (by calculation), alkaline phosphatase (AP), sodium (Na+), creatinine (Creat), potassium (K+), triglycerides (Tri), chloride (Cl-), total cholesterol (Chol), calcium (Ca++), total bilirubin (Bili) and bile acids (Bile).

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalytical investigations were performed on all non-recovery test and control animals (with the exception of satellite sub-groups) during Week 4 and on all recovery group animals during Week 6. From the satellite groups, urine and faecal samples were collected from each animal on the evening of Day 1 and Day 26.
- Metabolism cages used for collection of urine: Yes. Urine samples were collected overnight by individually housing rats in metabolism cages.
- Animals fasted: Yes. Animals were maintained under conditions of normal hydration during collection but without access to food.
- Parameters checked: Volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances and blood.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional observation battery of tests completed prior to initiation of dosing and then on days 7, 14, 21 and 26
- Dose groups that were examined: all non-recovery groups and the control animals
- Battery of functions tested: sensory activity / grip strength / motor activity
- Behavioural Assessments: Detailed individual clinical observations were performed for each non-recovery test and control animal using a purpose built arena. The following were observed: Gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, piloerection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal and tail elevation.
The test was developed from the methods used by Irwin (1968) and Moser et al. (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.
- Functional Performance Tests: Motor Activity. Twenty purposes built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex (with the exception of satellite and recovery sub-groups) were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was one hour for each animal; the time in seconds each animal was active and mobile was recorded for the overall hour period and also during the final 20 % of the period (considered to be the asymptotic period, Reiter and Macphail 1979).
Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal (with the exception of satellite and recovery sub-groups) was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al. (1979).
Sensory Reactivity: Each animal (with the exception of satellite and recovery sub-groups) was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin and Moser et al. (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.
The following parameters were observed: Grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex and startle reflex.

OTHER: tin concentrations were analysed for in samples of blood, urine and faeces collected from subgroups on day 1 and 28
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Scheduled termination of all animals not allocated to the recovery groups was performed on study day 29, the recovery animals were terminated following a 14 day treatment free period. There were no treatment related mortalities although one male in the high dose group was sacrificed on humane grounds following an apparent mal-administration on Day 25. All surviving animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals (with the exception of satellite sub-groups) were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Gross examinations of thoracic, abdominal and cranial cavities were completed together with examination of the associated tissues and organs.

Organ weights: The weights of the following organs were recorded for all animals killed at the end of treatment or after the recovery phase: Adrenals, brain, epididymides, heart, kidneys, pituitary (post-fixation), prostate and seminal vesicles (with coagulating glands and fluids), liver, ovaries, spleen, testes, thymus, thyroid/parathyroid (post-fixation) and uterus with cervix.

HISTOPATHOLOGY: Yes
Microscopic examinations were limited to the control and high dose animals from the treatment phase and recovery phase.
Samples of the following tissues were removed from all animals (with the exception of satellite sub-groups) and preserved in buffered 10 % formalin except where stated: Adrenals†, ovaries†, aorta (thoracic), pancreas, bone & bone marrow (femur including stifle joint), pituitary†, bone & bone marrow (sternum)†, prostate, brain (including cerebrum, cerebellum and pons)†, salivary glands (submaxillary), rectum†, caecum†, sciatic nerve†, colon†, seminal vesicles (with coagulating glands and fluids)†, duodenum, epididymides† ♦, skin (hind limb), eyes*, spinal cord (cervical, mid thoracic and lumbar)†, gross lesions†, heart†, spleen†, ileum†, stomach†, jejunum†, testes†♦, kidneys†, thymus†, liver†, thyroid/parathyroid†, lungs (with bronchi)# †, trachea†, lymph nodes (cervical and mesenteric)†, urinary bladder†, mammary gland†, uterus & cervix†, muscle (skeletal), vagina† and oesophagus.

♦ Preserved in Bouin's fluid then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later
* Eyes fixed in Davidson's fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10 % formalin before immersion in fixative
† From all non-recovery control and 1000 mg/kg/day dose group animals prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination.

Any macroscopically observed lesions were also processed together with the liver and spleen from all 30 and 300 mg/kg/day dose group animals. In addition, sections of testes and epididymides from all control and 1000 mg/kg/day males were stained with Periodic Acid-Schiff (PASS) stain and examined.
Other examinations:
Analysis of tin concentrations in samples of blood, faeces and urine were completed for satellite animals dosed at 1000 mg/kg bw/day using samples collected on Day 1 and 28.

THYROID HORMONE ASSESSMENT
At termination, blood samples were taken grom the exsanguination procedure and the plasma from each animal was stored frozen at -20 °C. No treatment related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

STAGE OF OESTRUS
At termination a vaginal smear was taken from all females (with the exception of satelite sub-groups) and the stage of oestrus was recorded.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or steel (non-parametric) test to determine significant differences from the control group. If required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (P) are presented as follows:
P < 0.01 **
P < 0.05 *
p ≥ 0.05 (not significant)
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs were observed throughout the study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male from the 1000 mg/kg bw/day high dose group was terminated on welfare grounds following dosing on Day 25. Although previously symptom-free the rat developed signs including impaired respiration, cyanosis and red fluid around the nose. The indications suggested mal-administration of the dose resulting in pulmonary administration and possible breach of the oesophagus. Necropsy of the rat revealed a foamy, off-white liquid filling the lungs - consistent with suggestion of mal-administration. There were no indications of a treatment-related cause of death and all evidence pointed to the animals having erroneously received the dose into the lungs rather than stomach.

There were no other unscheduled mortalities during the treatment or treatment free periods.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no effects on bodyweight or weight gain for any of the treated groups throughout the treatment phase of the study. An incidental weight loss for the high dose males in the recovery phase, noted after 14 days without treatment was considered to reflect normal variation rather than any toxicologically significant event.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects of treatment were noted for any of the test groups in comparison with controls, in respect of food consumption, throughout the study.
Food efficiency:
no effects observed
Description (incidence and severity):
No effects of treatment were noted for any of the test groups in comparison with controls, in respect of food efficiency (ratio of bodyweight gain to food intake), throughout the study.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No adverse effects in water consumption between treated and control groups.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related effects were apparent for any of the parameters investigated. Isolated intergroup differences were noted for circulating erythrocytes (elevated levels for low dose females, 30 mg/kg bw/day) and elevated lymphocytes for the high dose males in the recovery group. The absence of a consistent change in these parameters meant the effects were not considered attributable to administration of metallic tin.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment effects were apparent for any of the parameters investigated.
Changes observed but not considered of any toxicological significance included plasma chloride levels for the high dose females and elevated plasma calcium and inorganic phosphorus for the high dose recovery group males.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no convincing evidence of increased tin levels in 30 or 300 mg/kg/day test animals nor 1000 mg/kg/day females between Day 1 and Day 28. Males at the 1000 mg/kg/day group did show higher levels on Day 28, however in the absence of any supporting evidence to suggest an accumulative effect this variance was considered attributable to individual variability and of no toxicological significance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The functional observation battery investigated performance in a series of tests to evaluate neurobehavioural changes. No treatment-related effects were noted in any of these tests.
The behavioural assessments and clinical sign evaluations showed no inter or intragroup differences that were not attributable to normal variation for rats of this age and strain.
There were no treatment-related adverse changes detected in the functional performance parameters measured. The incidental findings were considered to be of no toxicological significance.
There were no treatment-related changes detected in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used, and of no toxicological importance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant or treatment related effects were noted. Actual and relative organ weights for treated groups showed no significant differences from controls. It was noted that thymus and adrenal weights at the end of the treatment period showed slight intergroup differences - particularly for the high dose males but the changes were not considered attributable an effect of tin.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were evident for the treated groups examined at the end of treatment or at the end of the recovery phase.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related microscopic changes were noted in any group. Histopathological changes were either common to treated and control groups or within the background range of effects commonly observed in this strain of rat. There were no toxicologically significant differences in incidence or severity for any of the histopathological observations.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Tin content was analysed in biological samples (plasma, urine and faeces) collected over the initial and final 24 hours of the treatment period.
Very high tin levels were apparent in plasma and urine samples for the low dose, 30 mg/kg bw/day group. These findings were excluded from analysis on basis of the LOQ rendering it difficult to differentiate between naturally occurring background levels of tin in these samples and tin derived from the administered dose.
The levels of tin found in the faecal samples of animals treated at dosages of 300 and 1000 mg/kg/day had increased exponentially from the levels detected in these animals at the start of the treatment supporting the views that the highest levels of clearance of tin would be in the faeces and that there was no evidence to suggest retention of tin in the animals following twenty eight days of dosing.
The plasma and faecal samples showed no indication of tin accumulation over the treatment period. There was no conclusive evidence of tin accumulation in urinary samples over the 28 day treatment phase at any dose level - the slight increase in tin content for the males dosed at 1000 mg/kg bw/day at the end of the treatment phase was considered to be natural background variation, in the absence of similar effects in females or either sex at lower doses.

Oestrous staging at the end of the treatment phase showed no treatment-related differences between treated and control females.

An assessment of thyroid hormone parameters revealed no changes considered attributable to treatment.
Key result
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no

Homogeneity and stability of test formulations was analytically confirmed. Weekly test formulations were analysed to verify concentration - the mean recovery expressed as a percentage of nominal was between 92 and 107 % in each case.

Analysis of tin in tissue samples - results for plasma, urine and faeces presented in attached tabulation document.

Conclusions:
Oral administration of the test material, tin metal powder (2-11 µm) to rats for a period of up to twenty-eight consecutive days at dose level of 30, 300 and 1000 mg/kg/day resulted in no treatment-related toxicological effects and for this reason the "No Observed Effect Level" (NOEL) was considered to be 1000 mg/kg/day.
Executive summary:

Tin metal powder, presented as a fine particulate material with a particle size in range of 2-11 µm, was administered to groups of five male and five female rats at dose levels of 30, 300 or 1000 mg/kg bw/day as a suspension in 1 % w/v CMC. Gavage administration continued for 28 consecutive days and a subgroup of the control and high dose regimen were retained for a treatment free period of two weeks to assess recovery from toxic effects.

Clinical signs, a functional observation battery of neurobehavioural changes, bodyweight development, food and water consumption were monitored during the course of the treatment phase. Clinical pathology investigations (haeamatology, biochemistry and urinalysis) were completed at the end of the dosing phase or the end of the treatment free period. In addition, samples of blood urine and faeces were obtained for analysis of tin content/concentration. All animals were subject to macroscopic examination at the scheduled termination points with organs weighed and carcasses necropsied. Tissues were preserved for histopathology but in the first instance only those from the high dose and control groups were examined due to the innocuous response observed in life at the high dose level.

The results of these investigations confirmed subacute oral administration of tin metal powder to rats elicited no changes indicative of an adverse toxicological effect and no indications of any systemic toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The overall quality of the database is high. The key study was conducted under GLP conditions and in accordance with a standardised guideline.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The key study selected for this endpoint was McRae LA (2010), the 28 day subacute study. The study was performed in compliance with GLP and the OECD guideline 407. The results of the study were considered reliable and adequate for assessment.

Metallic tin is unreactive under normal mammalian phyiological conditions, and the toxicokinetic studies (utilising extreme exposures) in the context of normal human background dietary exposure and plasma concentrations, show that metallic tin would not be absorbed to any detectable degree (see the toxicokinetics, metabolism and distribution section i.e. absorption for risk assessment purposes can be considered zero), and did not exhibit any toxic effects in the McRae (2010) gavage study in rats upto the limit dose of 1000 mg/kg bw/day. The substance has no significant solubility. Industrial working practices will ensure that the potential for exposure is extremely limited (see the CSR - highest anticipated inhalation exposure without PPE or engineering control is typically 4-5 mg/m3, peaking at 10 mg/m3, whilst dermally it is up to 9.9 mg/day. Engineering controls or PPE will be required to reduce this to comply with non-specific nuisance dust rules (the OEL for non-specific nuisance dust is frequently specified as 10 mg/m3 total inhalable and/or with a respirable fraction no greater than 3 mg/m3), hence these exposures will be considerably lower. There is no anticipated consumer exposure where tin is in solid metallic form either pure or in an alloy).

Concerning chronic toxicity testing, the registered substance does not fulfil any of the criteria that would trigger chronic (long term) further testing under column 2, point 8.6.3., of Annex X. In addition to the absence of absorption and extremely limited exposure to metallic tin (see above), there is adequate evidence of an absence of any toxicity in the 28 day rat gavage study up to the limit dose (1000 mg/kg bw/day), there are no effects anticipated from the structure of metallic tin (metallic tin is anyway an inorganic substance), and there are no other dangerous properties anticipated that would notionally only manifest after chronic toxicity testing.

Available human data also rules out any long term toxicological effects. Robertson J (1960) studied stannosis (tin oxide induced pneumoconiosis) in workers from a tin smelting works. Even with positive X-ray findings, workers with many years of exposure suffered no health effects, deposition of the dust in the lungs did not lead to a fibrous response i.e. no adverse pathology was associated with stannosis, indicating no cellular interaction. Observations were performed on workers exposed later in the production line, such as ingot casters exposed to pure molten tin, which reports that there is little dust or fume exposure during this process and changes were not observed in these workers. Though the working conditions are not comparable to today’s practices (much improved), it gives a worst case scenario of exposure during various points of the lifecycle of the registered substance.

As such no further repeat dose toxicity studies are required and the presented data is considered adequate for classification and labelling purposes.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to long-term toxicity as the presented data and the historical use of tin indicates that there is no cause for concern.