Dimethylbis(oleoyloxy)stannane

Endpoint summary

Description of key information

Repeated Dose Toxicity Oral: Read-across to DMTC (Dimethyltin dichloride CAS No. 753 -73 -1)

- Appel (2000): 90 day  NOAEL (rats) = 15 ppm (equivalent to 0.62 and 0.65 mg/kg/day for males and females, respectively, when adjusted for DMTC purity). Neurotoxicity at 200 ppm

- Beyrouty (1997): 90 day LOAEL (rats) = 25 ppm (equivalent to 1.44 and 1.98 mg/kg bw/day for males and female, respectively, when adjusted for DMTC purity). Reduced food (males only) and water intake and neuropathological lesions. Vacuolisation of white matter in the brain and spinal cord was also observed for animals in the 25 ppm group.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10th July - 26th October 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

other: read across: target

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Dose levels: 25, 75, 200 ppm

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc., St. Constant, Quebec
- Age at study initiation: 65-68 days of age
- Weight at study initiation: 308 to 414 g (males); 181 to 268 g (females).
- Housing: All rats were housed individually in stainless-steel mesh-bottomed cages equipped with a water bottle.
- Diet (e.g. ad libitum): A standard certified commercial pelleted laboratory diet (PMI Certified Rodent Chow 5002) was provided through a feeding box ad libitum.
- Water (e.g. ad libitum): Water (with test article incorporated, where appropriate) which had been further treated by reverse osmosis and ultraviolet sterilization was provided ad libitum.
- Acclimation period: approx. 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10th July 1996 To: 26th October 1996

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A solution of the test material in drinking water was prepared daily (rather than weekly as indicated in the protocol) for each group by direct dilution. Appropriate amounts of the test material were added to drinking water and mixed until dissolved.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF TEST MATERIAL
An appropriate sample of test material was analysed using GC/MS prior to and at the end of the treatment period. The purity of the test material was assessed against analytical standards of certified purity. Confirmation of purity was established by comparison of the retention times of the reference standards and the major components of the test material. Characterisation was performed by inspecting the MS of each of the eluted compounds and checking for characteristic mass-to-charge ratio.

ANALYSIS OF DOSE PREPARATION
Homogeneity of the treated drinking water was evaluated prior to treatment initiation.

Stability of the test material in drinking water was evaluated prior to the commencement of treatment by analysis of samples obtained immediately after preparation and after storage for one day at room temperature.

The accuracy of mixing of the treated drinking water was checked during the treatment period by analysis of a sample taken from each group after preparation as follows during the study:

Group 1 - Days 0, 8, 19, 47 and 91
Group 2 - Days 0, 1, 6, 12, 19, 47 and 91
Group 3 - Days 0, 1, 6, 7, 12, 19, 47 and 91
Group 4 - Days 0, 1, 8, 12 and 19

Concentrations were considered acceptable when the mean of duplicate analysis (and subsequent triplicate analysis if deemed necessary) of a sample was ± 20% of the nominal concentration.

Duration of treatment / exposure:

Up to 90 days

Frequency of treatment:

Daily

Dose / conc.:

0 ppm

Remarks:

(nominal in water)

Dose / conc.:

25 ppm

Remarks:

(nominal in water)

Dose / conc.:

75 ppm

Remarks:

(nominal in water)

Dose / conc.:

200 ppm

Remarks:

(nominal in water) (up to week 5 of treatment)

Dose / conc.:

150 ppm

Remarks:

(nominal in water) (200 ppm dose reduced to 150 ppm during 5th week of treatment and terminated during 6th week)

No. of animals per sex per dose:

15 per sex per dose in main study; 15 per sex per dose in neurotoxicity study

Control animals:

yes

Details on study design:

Approximately 4 weeks before treatment commenced, all animals were weighed and 30 males and 30 females were randomly assigned to each of the 4 groups using a computer-based randomisation procedure which ensured the homogeneity of group means and variances for body weight. Males and females were randomised separately. The animals within each group were split into several replicates of approximately equal numbers of animals, and treatment initiation and evaluations were staggered over 3 days.

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
All animals were checked twice daily for mortality and signs of ill health or reaction to treatment. In addition, a physical examination of each animal was conducted at least once weekly.

BODY WEIGHT:
The animals were individually weighed on a weekly basis. Neurotoxicity component animals were also weighed on each day of behavioural testing. Fasted body weights were recorded prior to terminal sacrifice.

FOOD CONSUMPTION:
Individual food consumption was determined weekly for all animals.

WATER CONSUMPTION:
Individual water consumption was determined daily for all animals.

OPHTHALMOSCOPIC EXAMINATION:
Funduscopic (indirect ophthalmoscopy) and biomicroscopic (slit lamp) examinations were performed on all animals during the prestudy period and on the main study animals during the 13th week of treatment.

HAEMATOLOGY:
Prior to the commencement of treatment, hematological examinations were performed on the 10 male and 10 female health screen animals. These same investigations were performed for the main study animals after 4 weeks of treatment (Day 29) on 5/sex/group (except Group 4 females), preterminals (where possible) prior to sacrifice, and after 13 weeks of treatment (Day 92) on 10/sex/group. Food was removed overnight from animals to be sampled. For all animals, blood samples were obtained from the abdominal aorta immediately following sodium pentobarbital anesthesia.

Parameters examined:
hematocrit; hemoglobin; red blood cell count; red cell distribution width; blood cell morphology; platelet count; mean platelet volume; white blood cell counts (total and differential); Wintrobe's constants (calculated); prothrombin time; activated partial thromboplastin time; reticulocyte count

CLINICAL CHEMISTRY:
Prior to the commencement of treatment, blood chemistry examinations were performed on the 10 male and 10 female health screen animals. These same investigations were performed for the main study animals after 4 weeks of treatment (Day 29) on 5/sex/group (except Group 4 females), preterminals (where possible) prior to sacrifice, and after 13 weeks of treatment (Day 92) on 10/sex/group. Food was removed overnight from animals to be sampled. For all animals, blood samples were obtained from the abdominal aorta immediately following sodium pentobarbital anesthesia.

Parameters examined:
blood urea nitrogen; total protein; albumin; globulin; A/G ratio; alkaline phosphatase; alanine aminotransferase; aspartate aminotransferase; glucose; sodium; potassium; calcium; chloride; creatinine; total bilirubin; phosphorus

URINALYSIS:
Prior to the commencement of treatment, urinalysis examinations were performed on the 10 male and 10 female health screen animals. These same investigations were performed for the main study animals after 4 weeks of treatment (Day 29) on 5/sex/group (except Group 4 females), preterminals (where possible) prior to sacrifice, and after 13 weeks of treatment (Day 92) on 10/sex/group. Urine samples were collected from individual animals placed in metabolism cages overnight, during which time they were deprived of food and water.

Parameters examined:
colour and appearance; pH; glucose; ketones; urobilinogen; blood; nitrite; volume; specific gravity; protein; bilirubin; microscopy of centrifuged deposit

NEUROBEHAVIOURAL EXAMINATION:
Functional Observed Battery:
All neurotoxicity component animals (15/sex/group) were tested prior to the initiation of treatment (prestudy) and once during the 4th, 8th and 13th weeks of treatment. In addition, neurotoxicity component recovery animals (10/sex/group) were tested approximately 2 weeks after treatment cessation. Testing was performed by the same trained technicians who were unaware of the animals treatment.

Observations in Home Cage:
body position; tremors, twitches, convulsions; bizarre/stereotypic behaviour

Removal from Home Cage:
ease of removal; vocalization

Observations in Arena:
rearing; ataxic, hypotonic and impaired gait; overall gait incapacity; bizarre/stereotypic behaviour; palpebral closure; tremors, twitches, convulsions; piloerection; respiratory rate/pattern; locomotor activity level arousal; grooming; defecation; urination; olfactory response

Handling Observations:
lacrimation; pupil size; salivation; urinary staining; diarrhea; body tone; extensor thrust; corneal reflex; pinna reflex; toe and tail pinch; visual placing

On Surface:
auricular startle; air righting reflex

On Top of Box:
positional passivity

Grip Strength:
Calibration:
Before the start of testing and following completion of testing on each day, the Chatillon strain gauges were checked using calibration weights and the readings recorded.

Forelimb:
The dial on the gauge was set to "0". The rat was held by the body and/or tail and allowed to grip the mesh, and then was pulled slowly and steadily until it released its grip. Maximum strain was recorded two times alternating with hindlimb grip testing.

Hindlimb:
The dial on the gauge was set to "0". The rat was allowed to set its hindpaws against the mesh and was pulled backward by the base of the tail until it released its grip. The maximum strain was recorded two times alternating with forelimb grip testing.

Kindlimb splay:
Landing foot spread was measured using inking of the hind feet. Hindlimb splay was recorded twice. The ink was wiped off the feet after testing using a paper towel dampened with water.

Body temperature:
The rectal probe was gently inserted, the reading allowed to stabilize and the temperature then recorded.

The functional observational battery was performed with equipment built for this purpose. The arena was a 2' square of plexiglass placed on a raised platform. The tests were conducted in the room housing the animals, where temperature, humidity and photoperiod are monitored. Odors in the room were minimised by maintaining adequate air changes and cleaning of equipment, as necessary.

Motor Activity:
The neurotoxicity component animals (15/sex/group) were transferred (following FOB evaluations) to a testing room where activity levels were measured individually in figure 8 enclosures. Animals were tested prior to treatment (prestudy), and once during the 4th, 8th and 13th weeks of treatment. In addition, neurotoxicity component recovery animals (10/sex/group) were tested approximately 2 weeks after treatment cessation. Animals from the control and treated groups were balanced across enclosures, where possible, using a preassigned distribution. The sessions were of 1 hour duration and activity counts were recorded by a microcomputer in 6 successive 10 minute intervals.

In the testing room, temperature and humidity were monitored, and a background sound level of approximately 70 dBA and an illumination of approximately 800-1200 Lux maintained throughout testing. Light levels in the testing room were measured before the start of testing and following completion of testing on each day. The sound level was recorded on a continuous basis throughout testing on each day.

In addition to the "diagnostic" function in the system, a check of each beam was made by manually "breaking" each beam a predetermined number of times and verifying that the "breaks" were properly recorded. These checks were made at least prior to the start of testing and at the completion of testing on each day.

Sacrifice and pathology:

GROSS PATHOLOGY:
Prior to commencement of treatment, the 10 male and 10 female rats designated for health screen purposes were anesthetized by intraperitoneal injection of sodium pentobarbital (35 mg/kg or as required). A blood sample was collected from the abdominal aorta after which the animal was euthanised by exsanguination from the abdominal aorta and then subjected to necropsy. Abnormal tissue samples were retained in neutral buffered 10% formalin.

In order to avoid autolytic change, a complete necropsy was conducted immediately on any animal euthanised during the study or at its conclusion (except those perfused). For animals found dead during the study, the necropsy was completed as soon as possible after death. Prior to necropsy, the carcass was stored in a refrigerator at circa 4°C.

All animals found dead or euthanised for humane reasons during the study were subjected to necropsy and tissue samples were preserved. Animals euthanised for humane reasons were killed by an intraperitoneal injection of sodium pentobarbital (35 mg/kg or as required) followed by exsanguination from the abdominal aorta.

After 4 or 13 weeks of treatment, the main study animals were anesthetized by an intraperitoneal injection of sodium pentobarbital (35 mg/kg or as required). A blood sample was collected from the abdominal aorta. The animals were then euthanised by exsanguination from the abdominal aorta and subjected to necropsy. A similar proportion of animals from each treatment group and/or sex, as appropriate, were euthanised on any one day unless otherwise specified.

Following completion of the recovery period, all neurotoxicity component recovery animals not perfused were anesthetized by an intraperitoneal injection of sodium pentobarbital (35 mg/kg or as required). The animals were then euthanised by exsanguination from the abdominal aorta and subjected to necropsy. Abnormal tissue samples were retained in neutral buffered 10% formalin.

For each animal, necropsy consisted of an external examination, including identification of all clinically recorded lesions, as well as a detailed internal examination.

All animals were fasted before scheduled euthanasia.

ORGAN WEIGHTS:
For each main study animal euthanised after 4 or 13 weeks of treatment, the following organs were dissected free of fat and weighed:

adrenals; brain; heart; kidneys; liver; ovaries; pituitary; spleen; testes; thymus

Paired organs were weighed together. Organ weight ratios relative to body weight were calculated.

TISSUE PRESERVATION:
On completion of the necropsy of each main study animal euthanised after 4 or 13 weeks of treatment (and preterminals), the following tissues and organs were retained. Neutral buffered 10% formalin was used for fixation and preservation unless otherwise indicated

abnormal tissues; animal identification++; adrenals; aorta (thoracic); bone and marrow (femur and sternum); brain (cerebrum, cerebellum and medulla oblongata); cecum; colon; duodenum; epididymides*; esophagus; eyes*; heart; ileum; jejunum; kidneys; larynx; liver (sample of 2 lobes); lungs (sample of 2 lobes)**; lymph nodes (cervical/mandibular and mesenteric); mammary gland (inguinal+); nasal cavity and sinuses; optic nerves*+; ovaries; pancreas; pharynx; pituitary; prostate; rectum; salivary gland; sciatic nerve; seminal vesicles; skeletal muscle; skin; spinal cord (cervical, thoracic and lumbar); spleen; stomach; testes*; thymus; thyroid lobes (and parathyroids+); tongue; trachea; urinary bladder; uterus; vagina

*Fixed in Zenker's fluid (sacrificed animals only).
** Lungs were infused with neutral buffered 10% formalin (sacrificed animals only).
+ Only examined histopathologically when present in routine sections of eyes (optic nerves), thyroid lobes (parathyroids) or mammary gland (inguinal).
++Retained but not processed.

For each sacrificed animal, three femoral bone marrow smears were prepared, one of which was stained with May-Grinwald-Giemsa. All smears were retained for possible future examination.

HISTOPATHOLOGY:
Histopathological examination was performed on the aforementioned tissues following embedding in paraffin wax, sectioning and staining with hematoxylin and eosin as follows:
a) All animals in the control and mid dose groups.
b) Lungs, liver, kidneys, thymus, spleen, lymph nodes, brain and spinal cord (selected regions) and any gross lesions for animals in the low dose group.

Other examinations:

TISSUE PRESERVATION - NEUROTOXICITY COMPONENT
After 13 weeks of treatment and at the end of the recovery period, 5 rats/sex/group from Group 1-3 were deeply anesthetized by intraperitoneal injection of sodium pentobarbital (approximately 35 mg/kg or as required). When anesthesia was deep, the thorax was opened. A 16-gauge needle was inserted into the left ventricle and the right atrium was opened. Perfusion with lactated Ringer's solution containing heparin (1000 IU/liter) and sodium nitrite (0.02 g/L) was initiated and continued until the auricular effluent was essentially free of blood. The perfusion fluid was then changed to a mixture of 3% glutaraldehyde, 3% paraformaldehyde, 0.05% calcium chloride and 0.1% picric acid in 0.1 M cacodylate buffer (pH 7.3 to 7.5). Six males and five females from Group 4 were perfused during Week 5 or 6.

NEUROPATHOLOGY - NEUROTOXICITY COMPONENT
On completion of the perfusion, the calvarium and the dorsal vertebral column were exposed by removing the skin and underlying muscle. The skin on the lateral surface of the hindlimbs was also removed. The skin and tissues on the ventral surface of the abdomen and thorax were removed and discarded. The thoracic and abdominal organs were removed as two groups of tissues and placed in a separate tissue bag in neutral buffeted 10% formalin. The remaining carcass containing the brain, spinal cord and limbs was placed in another tissue bag containing neutral buffered 10% formalin.

The following tissues listed were sampled for all animals. Tissues from the control, mid dose and high dose animals were processed for neuropathological evaluation. Tissues of brain and spinal cord from the low dose group were also processed for neuropathological examination. The remaining trimmed tissues from the low dose group were kept in neutral buffered 10% formalin. The remaining carcasses from all animals were retained. The nervous system tissues of animals were retained. The nervous system tissues of animals in all groups were grossly examined at the time of sampling and any pathology observed was recorded and reported.

TISSUES FOR PARAFFIN EMBEDDING, SECTIONING AND STAINING
Brain (6 levels) - forebrain (through the septum), center of the cerebrum (through the hypothalamus), midbrain, cerebellum and pons, midcerebellum and medulla oblongata, and medulla oblongata.
Spinal cord - cervical, thoracic, lumbar (cross-section)
Skeletal muscle - gastrocnemius (longitudinal and cross-sections)
Grossly abnormal central nervous system tissues.

Note: Brain weight (excluding olfactory bulbs), length and maximum coronal width were recorded prior to trimming.

The tissues listed above for the control, low (limited tissues), mid and high dose animals were prepared for examination by embedding in paraffin wax and sectioning at 6 microns. The skeletal muscle was stained with hematoxylin and eosin. Adjacent sections of brain and spinal cord were stained with hematoxylin and eosin, Klüver Barrera, Holmes and PTAH (or alternate) stains and then examined by light microscopy.

TISSUES FOR EPOXY EMBEDDING, SECTIONING AND STAINING
Peripheral Nervous System (pNs):
Sciatic nerve (mid-thigh region) (cross-section)
Sciatic nerve (at sciatic notch) (longitudinal and cross-sections)
Sural nerve (at knee) (cross-section)
Tibial nerve (at knee) (longitudinal and cross-sections)

Central Nervous System (CNS):
Gasserian ganglion - left (cross-section)
Lumbar dorsal root ganglion (L4) (cross-section)
Lumbar dorsal root (L4) (cross-section)
Lumbar ventral root (L4) (longitudinal and cross-sections)
Cervical dorsal root ganglion (C5) (cross-section)
Cervical dorsal root (C5) (cross-section)
Cervical ventral root (C5) (cross-section)

Grossly abnormal central or peripheral nervous system tissues.

For the control, mid dose and high dose group animals, the tissues listed above were rinsed in 0.1 M sodium cacodylate buffer and placed in 2% osmium tetroxide for 2 hours. Each piece of tissue was then rinsed in buffer and stained in a 1% aqueous solution of uranyl acetate for 2 hours. The tissues were then rinsed in distilled water, dehydrated in ascending concentrations of ethyl alcohol and embedded in a mixture of Jembed and Araldite. Epoxy sections (0.5 µm) were obtained with a glass knife, stained with borate-buffered 1% toluidine blue, cover slipped and examined by light microscopy.

The tibial branches to the calf musculature were retained for possible future examination.

Statistics:

See 'Any other information on materials and methods incl. tables'.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clear treatment-related clinical signs were noted for the 200 ppm group. Findings for animals found dead and sacrificed were tremors, convulsions, aggression/hypersensitivity/difficulty (when handled), appearance of dehydration/thin/weak, cold to touch, lying on side, and decreased home-cage activity levels. An increased incidence of fur staining at the head, body and limbs was also noted. Few abnormal clinical signs were noted for the 75 ppm group. Findings included tremors, hypersensitivity (difficulty when handled) and a thin dehydrated body condition for the male that died, a thin and dehydrated appearance at the beginning of the treatment period for another male and abnormal gait/behaviour at the beginning of the treatment period for a third male. One female from this group had findings including hypersensitivity, convulsions and reduced activity towards the end of treatment period. No abnormal clinical signs were clearly evident for the 25 ppm group.

Mortality:

mortality observed, treatment-related

Description (incidence):

A number of 200 ppm animals (7 males and 21 females) either died or were sacrificed (due to poor condition) between Days 18 and 34 of the treatment period. The remaining 200 ppm animals were sacrificed by Day 36 of the study as their survival to study completion was doubtful. One 75 ppm male (No. 3025) was found dead on Day 41. No other deaths occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of the 200 ppm group (males and females) were significantly (P<0.05 or P<0.01) lower than the control group for all intervals measured following treatment initiation. For the last 2 or 3 body weight occasions prior to termination of this group, these animals showed a weight loss with several animals having large body weight reductions. Body weights were significantly (P<0.05 or P<0.01) reduced for 75 ppm males on most occasions when compared to the control group. No significant differences were noted between the control and 75 ppm females or 25 ppm group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake values were significantly (P<0.01 or P<0.001) lower for the 200 ppm group (males and females) during all intervals measured after treatment start. Food consumption values for the 75 ppm males were also significantly (P4.05 or P<0.01) reduced on most occasions when compared to the control group. Occasional significant (P4.05 or P<0.01) reductions in food consumption were noted for the 75 ppm females and 25 ppm males when compared to the control group. No significant differences were noted between the control and 25 ppm females.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was significantly (P<0.05, P<0.01 or P<0.001) decreased for males and females in all treated groups on most daily assessments during the study. Following treatment termination, water consumption values were generally comparable between the control and treated groups.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes attributed to treatment with the test material.

Haematological findings:

no effects observed

Description (incidence and severity):

No significant differences were detected between the control and treated groups for hematological parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

On Day 29, males in the 200 ppm group showed significant (P<0.05 or P<0.01) increases in BUN, creatinine and phosphorus. Potassium levels were significantly (P<0.01) decreased for 200 ppm males. In addition, slight increases in the mean AST and ALT levels were observed for 200 ppm males. Females in the 75 ppm group showed a significant (P<0.05) decrease in total protein, and a decrease in albumin was observed for the 25 and 75 ppm group females. This latter finding (decreased total protein/albumin) was most probably attributable to intergroup variation as the values were comparable to those collected for health screen animals prior to treatment initiation and/or they were within historical control ranges. Many of the 200 ppm animals that were sacrificed preterminally showed marked changes in various blood biochemical parameters including increases in BUN, creatinine, AST, ALT and phosphorus. At the Day 92 assessment, the biochemical parameters were essentially the same between the control and treated groups.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis data was generally comparable between the control and treated groups for the Day 29 and 92 assessments. However, males in the 200 ppm group did show an elevated urine pH at the Day 29 assessment.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional Observational Battery (FOB):
Males:
At the Week 4 assessment, one 200 ppm male showed slight ataxia resulting in a slight overall gait incapacity, slight tremors at the limbs and clonus of the jaws (chomping). One 75 ppm male had a severe ataxia including falling over which resulted in a severe overall incapacity. This animal also showed bizarre behaviour in the form of unusual hindlimb movements. Several 200 ppm males did show a rapid response to the toe pinch test.

At the Week 8 assessment, the same 75 ppm male was observed with a swaying movement in the home cage and continued to exhibit ataxic gait. The number of fecal boli (defecation) was significantly (P<0.05) increased for the 75 ppm group but the interpretation of this finding is unclear.
At the Week 13 assessment, the same 75 ppm male showed side to side body shaking together with the ataxia noted earlier. Defecation was again significantly (P<0.01) increased for the 75 ppm group. At the recovery assessment, this animal continued to show the same findings.
A significantly (P<0.01) easier removal from the home cage for the 25 ppm males at the prestudy assessment was due to intergroup variation.

Females:
At the Week 4 assessment, three females in the 200 ppm group showed slight or moderate tremors at the limbs or head. Clonic convulsions were also observed for these animals. Two animals had a hunched posture and one of these animals, a severe overall gait incapacity attributed to a severe ataxia was noted as was red liquid material at the urogenital region. The incidence of a rearing body position in the home cage as well as the average number of rearing incidents in the arena were significantly (P<0.01) reduced for the 200 ppm group. In addition, hindlimb grip strength was significantly (P<0.05) decreased and body temperature was significantly (P<0.01) reduced for the 200 ppm females. The 75 ppm group showed a significantly (P<0.05) lower body temperature.
At the Week 8 assessment, 75 ppm females showed a significant (P<0.01) decrease in rearing incidents in the arena.
At the Week 13 assessment, 75 ppm females showed a significantly (P<0.001) lower number of rearing incidents in the arena. The body temperature was also significantly (P<0.01) reduced for 75 ppm females. Defecation was significantly (P<0.01) increased for 75 ppm females.
At the recovery assessment, the number of rearing incidents in the arena was significantly (P<0.05) reduced for the 75 ppm group.

Motor Activity:
No significant differences were detected between the control and treated males on any occasion. Females in the 75 ppm group showed significant (P<0.01 or P<0.001) reductions in total activity counts at the Week 4, 8 and 13 assessments. In addition, at the Week 13 assessment, the linear constructed variable was also significantly (P<0.01) different between the control and 75 ppm group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Non-Perfused Animals:
Several statistically significant (P<0.05 or P<0.01) differences in absolute weights were noted for various organs between control and treated groups. At the interim sacrifice, 200 ppm males showed decreases in spleen, heart, pituitary and thymus weights while 75 ppm females had reduced heart weight, and 25 ppm females had reduced heart and liver weights. At the terminal evaluation, 75 ppm males showed decreases for heart and thymus weights, and the kidney weights were increased for the 25 and 75 ppm females. At the recovery period, 25 ppm males showed an increase in heart weight.

Most of these differences were no longer statistically significant once the organ weights were expressed relative to body weight. When this was done, however, the thymus weight continued to be reduced for 200 ppm males at the interim sacrifice and 75 ppm males at the terminal sacrifice, 25 and 75 ppm females continued to have increased kidney weights at the terminal evaluation, and the 25 ppm males had increased heart weight at the recovery assessment. In addition, the relative (to body weight) kidney, brain and adrenals weights of the 200 ppm males and spleen weight of 25 ppm males were increased at the interim evaluation, as was the relative kidney weight of the 75 ppm males and the relative heart weight of the 25 ppm males at the recovery evaluation.

Perfused Animals:
The brain weight of the 75 ppm males was significantly (P<0.05) increased at the terminal evaluation. The significance of this finding is unclear as no significant difference was detected for the 75 ppm females, and the brain weight of the 75 ppm males at the end of the recovery period was comparable to that of the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Non-Perfused Animals:
Pre-terminal animals in the 200 ppm group showed various findings. Several animals had a small thymus and/or spleen, fluid was noted in the brain on occasion, and other findings included emaciated carcass, dilatation of stomach/intestine, discoloured digestive material, dark/depressed areas in the stomach, and dark areas of the lungs.

Perfused Animals:
Dilatation of the brain ventricles was noted for some pre-terminal animals in the 200 ppm group. At the terminal evaluation, brain ventricular dilatation was seen for one male in each of the 25 and 75 ppm groups and for 2 females in the 75 ppm group and 1 control group female

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Non-Perfused Animals:
At the interim evaluation, no obvious treatment-related histopathological findings were noted. Terminal evaluations indicated clear changes in brain tissue characterised by neuronal necrosis and vacuolization of white matter, together with possible treatment-related lymphoid atrophy of the thymus, for animals in the 75 ppm group. Vacuolization was also noted in brain tissue for 25 ppm animals.

Perfused Animals:
Pre-terminal 200 ppm animals showed treatment-related lesions in the brain and spinal cord in the form of neuronal necrosis and vacuolization of white matter. Axonal degeneration of spinal cord tissue was also seen as was degeneration of peripheral nerves but the significance of this latter finding is unclear. At the terminal evaluation, neuronal necrosis and vacuolization of white matter in brain and spinal cord tissue were seen for animals in the 75 ppm group. Vacuolization of white matter in the brain and spinal cord was also observed for animals in the 25 ppm group.

Histopathological findings: neoplastic:

not examined

Details on results:

The range (average) of achieved intakes for the males were 1.2 to 2.2 (1.6), 4.6 to 6.2 (5.2) and 13.1 to 17.4 (15.5) mg/kg/day for the 25, 75 and 200 ppm groups, respectively. The range (average) achieved intakes for the females were 1.9 to 2.8 (2.2), 6.3 to 7.1 (6.7) and 17.4 to 20.5 (19.4) mg/kg/day for the 25, 75 and 200 ppm groups, respectively.

Dose descriptor:

NOAEL

Effect level:

< 1.6 other: mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No mortality and reduced food (males only) and water intake and neuropathological lesions. Vacuolisation of white matter in the brain and spinal cord was also observed for animals in the 25 ppm group.

Dose descriptor:

NOAEL

Effect level:

< 2.2 other: mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No mortality and reduced food (males only) and water intake and neuropathological lesions. Vacuolisation of white matter in the brain and spinal cord was also observed for animals in the 25 ppm group.

Dose descriptor:

LOAEL

Effect level:

25 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: 1.6 mg/kg bw/day (males); 2.2 mg/kg bw/day (females)

Critical effects observed:

not specified

Conclusions:

Under the conditions of the test, male and female rats treated with a methyltin chloride mixture, administered via the drinking water, resulted in several deaths, reduced body weight, decreased food and water intake, blood biochemical changes, behavioural effects such as aggressiveness and neuropathological lesions at 200 ppm (equivalent to 15.5 and 19.4 mg/kg/day for males and females, respectively). For the 75 ppm group (equivalent to 5.2 and 6.7 mg/kg/day for males and females, respectively), one male died, body weights were reduced (males only), food and water intake were decreased, and behavioural changes (such as reduced motor activity for females) and neuropathological lesions were noted. At 25 ppm (equivalent to 1.6 and 2.2 mg/kg/day for males and females, respectively), no mortality occurred and treatment-related findings were limited to reduced food (males only) and water intake and neuropathological lesions. The no observed effect level was considered to be below 25 ppm.

Executive summary:

The objective of this study was to evaluate the toxicity of a methyltin chloride mixture (~90% dimethyltin dichloride and ~10% methyltin trichloride) when administered via the drinking water to male and female Sprague-Dawley rats for 13 weeks.

Groups of 30 male and 30 female Sprague-Dawley rats (15/sex forming the main-study component and 15/sex forming the neurotoxicity component) were treated for up to 13 weeks, via the drinking water, with a methyltin chloride mixture at concentrations of 25, 75 or 200 ppm. A control group comprised of the same number of animals was handled in an identical manner, except that it received untreated drinking water. Ten rats/sex from the control, low and mid dose groups of the neurotoxicity component were retained for a two week recovery period after 13 weeks of treatment.

During the study, body weights and food consumption were recorded at least once weekly, water consumption was measured daily, food intake was assessed weekly and any abnormal clinical signs were recorded each day. In addition, a functional observational battery (FOB) and motor activity test were conducted on neurotoxicity component animals prestudy and once during each of Weeks 4, 8 and 13, and for recovery animals during week 15 as well.

After 4 weeks of treatment, 5 rats/sex/group (males only for 200 ppm group) of the main-study component were sacrificed and given a necropsy (including organ weight measurements) and then a full histopathological evaluation was conducted on animals in the control and mid dose groups and a limited histopathological examination was performed on animals in the low dose group. Identical procedures were conducted on the remaining 10 rats/sex from the control, low and mid dose groups of the main-study component following 13 weeks of treatment.

Animals in the 200 ppm group were sacrificed by week 6 of the study due to poor condition (see below). At least 5 rats/sex of these were given a whole-body glutaraldehyde/paraformaldehyde perfusion and later they underwent a full neuropathological examination. The remaining 200 ppm animals were subjected to necropsy with standard tissues retained for possible future histopathological examination.

After 13 weeks of treatment, 5 rats/sex from the control, low and mid dose groups of the neurotoxicity component were given a whole-body perfusion. Those animals in the control and mid dose groups underwent a full neuropathological examination while animals in the low dose group were given a limited examination (brain and spinal cord only). At the end of the study, all remaining animals were euthanised and given a gross pathological evaluation.

Seven males and twenty-one females in the 200 ppm group died or were sacrificed due to poor condition during the first 5 weeks of treatment. All remaining animals in this group were sacrificed by week 6 as it was doubtful that they would survive to study completion. One male in the 75 ppm group was found dead during week 6 but no other deaths occurred.

Animals in the 200 ppm group showed clinical signs such as tremors, convulsions, aggression/hypersensitivity/difficulty (when handled), appearance of dehydration/thin/weak, cold to touch, lying on side and decreased home-cage activity levels. An increased incidence of fur staining was also noted for the 200 ppm group. Abnormal clinical signs for the 75 ppm group were limited to tremors, hypersensitivity (difficulty when handled) and a thin dehydrated body condition for the male that died, a transitory dehydrated appearance for another male and hypersensitivity, convulsions and reduced activity for one female. No clearly abnormal clinical signs were evident for the 25 ppm group.

The body weights were significantly lower for the the 200 ppm group on all intervals and for the 75 ppm males on most intervals measured following treatment initiation. No significant differences were noted between the control and 75 ppm females or 25 ppm group.

Food intake values were significantly lower for the 200 ppm group on all intervals and for the 75 ppm males on most intervals measured after treatment start. Occasional significant reductions in food consumption were noted for the 75 ppm females and the 25 ppm males when compared to the control group. No significant differences were noted between the control and 25 ppm females.

Water consumption was significantly decreased for all treated groups on most daily assessments measured during the treatment period. Following treatment termination, water consumption values were generally comparable between the control and treated groups. The average achieved intakes of the methyltin chloride mixture were 1.6, 5.2 and 15.5 mg/kg/day for males, and 2.2, 6.7 and 19.4 mg/kg/day for females in the 25, 75 and 200 ppm groups, respectively.

The FOB indicated several findings primarily affecting the 200 ppm group. At the week 4 assessment, 200 ppm females showed significantly reduced rearing, lower hindlimb grip strength and decreased body temperature. Ataxic gait was observed for one 200 ppm male along with tremors and clout's of jaws (chomping). Three females in the 200 ppm group showed tremors and clonic convulsions. A hunched posture was noted for one 200 ppm male, and for one 200 ppm female ataxia was noted as was red liquid material at the urogenital region. Findings for the 75 ppm group at this time were limited to a significantly lower body temperature for females, and one male showed ataxia and bizarre behaviour in the form of unusual hindlimb movements which was also seen for this animal on subsequent testing occasions. At all other assessments (Weeks 8, 13 and recovery), rearing was significantly decreased for the 75 ppm females and the body temperature was significantly decreased for these females at the Week 13 assessment. No obvious treatment-related findings were noted between the control and 25 ppm groups on any occasion.

No significant differences were detected for motor activity levels between the control and treated males on any occasion. Females in the 75 ppm group showed significant reductions in total activity counts at the week 4, 8 and 13 assessments. In addition, at the week 13 assessment, the linear constructed variable (rate of linear decrease) was significantly lower for the 75 ppm females when compared to the control group.

No significant differences were detected between the control and treated groups for hematological parameters at the interim or terminal evaluations.

Blood biochemical changes were observed for males in the 200 ppm group at the week 4 assessment. Significant increases in BUN, creatinine and phosphorus were noted and potassium levels were significantly decreased. In addition, slight increases in the mean AST and ALT levels were observed for 200 ppm males. Many of the 200 ppm animals that were sacrificed preterminally showed marked changes in various blood biochemical parameters including increases for BUN, creatinine, AST, ALT and phosphorus.

Urinalysis data was generally comparable between the control and treated groups. However, males in the 200 ppm group did show an elevated urine pH at the week 4 assessment.

Significant decreases in absolute and relative (to terminal body weight) thymus weights were noted for the 200 ppm males at the interim sacrifice and 75 ppm males at the terminal sacrifice, and kidney weights (absolute and relative) were significantly increased for 25 and 75 ppm females at the terminal sacrifice.

Gross pathological evaluations on preterminal animals indicated a small thymus and/or spleen, emaciated carcass, dilatation of digestive tract/discoloured digestive material, and dark areas on the stomach and/or lungs. A small thymus was also seen at the interim evaluation for 200 ppm males and at the terminal evaluation for the 75 ppm group.

Histopathological examination indicated clear treatment-related nervous system lesions for preterminal 200 ppm animals in various regions of the brain and spinal cord, characterised by ventricular dilatation, neuronal necrosis and white matter vacuolization. Similar treatment-related nervous system changes were observed at the terminal examination for 75 ppm animals and possible treatment-related lesions in the thymus (lymphoid atrophy) for this group. The 25 ppm group showed vacuolization in brain and spinal cord tissue at the terminal evaluation.

In conclusion, treatment of male and female rats with a methyltin chloride mixture, administered in the drinking water, resulted in several deaths, reduced body weight, decreased food and water intake, blood biochemical changes, behavioural effects such as aggressiveness and neuropathological lesions at 200 ppm (equivalent to 15.5 and 19.4 mg/kg/day for males and females, respectively). For the 75 ppm group (equivalent to 5.2 and 6.7 mg/kg/day for males and females, respectively), one male died, body weights were reduced (males only), food and water intake were decreased, and behavioural changes (such as reduced motor activity for females) and neuropathological lesions were noted. At 25 ppm (equivalent to 1.6 and 2.2 mg/kg/day for males and females, respectively), no mortality occurred and treatment-related findings were limited to reduced food (males only) and water intake and neuropathological lesions. The no observed effect level was considered to be below 25 ppm.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance dimethyltin dichloride (DMTC) (EC Number 212-039-2, CAS Number 753-73-1), see attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

< 1.6 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

food consumption and compound intake

histopathology: non-neoplastic

neuropathology

water consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

< 2.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

neuropathology

water consumption and compound intake

Critical effects observed:

not specified

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7th June 1999 - 11th August 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

other: read across: target

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

neurobehavioural testing and neuropathology - according to OECD Guideline for the Testing of Chemicals 424 (adopted 21 July 1997)

Deviations:

no

Principles of method if other than guideline:

Dose levels of 0, 1, 6, 15 ppm for 13 weeks or 200 ppm for 1 month

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks old; pre-test neurobehavioural testing was to be conducted on animals of 5 weeks of age.
- Weight at study initiation: 149.8 g to 191.5 g (mean 174.3 g) for males and from 123.4 g to 154.2 g (mean 137.7 g) for females
- Housing: under conventional conditions in one room, in macrolon cages, type 3.5 (males) or type 4 (females) with sterilized wood shavings (Woody Clean, Type 3/4) as bedding material, five (main groups) or three (satellite groups) rats per cage, separated by sex. In the last week of the treatment period, the rats of the main groups were placed in stainless-steel metabolism cages (one rat per cage) for 16 hours to collect urine.
- Diet (e.g. ad libitum): Rat & Mouse No. 3 Breeding Diet, RM3 ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): At least 30%. The relative humidity exceeded 70% on most days of the study during 2 to 24 hours per day, to a maximum of 95%.
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness.



IN-LIFE DATES: From: 7th June 1999 To: 20th September 1999

Route of administration:

oral: feed

Vehicle:

other: diet

Details on oral exposure:

DIET PREPARATION
The test substance was incorporated in the basal diet by mixing in a mechanical blender. Four batches of test diets were prepared, viz. on 31 May, 28 June, 20 July and 20 August 1999. A 10,000 ppm premix was prepared first. The high-dose (200 ppm) diet was prepared by diluting the premix with the appropriate amount of RM3 diet. Subsequently, the 6 and 15 ppm diets were prepared by diluting the high-dose diet with the appropriate amount of RM3 diet. The 1 ppm diet was prepared by diluting the 15 ppm diet with the appropriate amount of RM3 diet. The controls received the unsupplemented RM3 diet. Directly after preparation the diets were stored in a freezer (<-18°C) until use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The diets were extracted with a methanol/water solution. The organic tin chlorides were ethylated with sodium tetraethyl borate (NaBEt4). The methyl ethyl tin compounds were subsequently extracted with hexane. The concentrations of MMTTC and DMDTC were determined using a Carlo Erba/Fisons QMD-1000 GC/MS under the following conditions:

Column: fused silica 60 m x 0.32 mm; 0.25 µm DB-1
Column temperature: After 3 minutes at 50°C at a rate of 5°/min. to 140°C; then at a rate of 10°/min. to 260°C; 10 minutes at 260°C
Carrier: Helium (70 kPa)
Injection volume: 1 µl
Injection temperature: 250°C
Injection method: splitless
Ionisation: Electron Impact, 70 eV
Mass range: 100 - 250 amu
Mass fragments used for quantification:
DMTDC low concentrations: m/z = 179
DMTDC high concentrations: m/z = 193
MMTTC low concentrations: m/z = 193
MMTTC high concentrations: m/z = 205
TMTMC: m/z = 165
Internal standard (naphthalene) m/z = 102

Homogeneous distribution of the test substance in rat feed was demonstrated by using the same diet preparation protocol and concentrations that were used in the present study. Moreover, the stability of the test substance in rat feed under simulated experimental conditions (7 days at room temperature in an open container) and after 5 weeks in a freezer was demonstrated.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Dose / conc.:

1 ppm

Remarks:

(nominal in diet)

Dose / conc.:

6 ppm

Remarks:

(nominal in diet)

Dose / conc.:

15 ppm

Remarks:

(nominal in diet)

Dose / conc.:

200 ppm

Remarks:

(nominal in diet)

No. of animals per sex per dose:

10 per sex per dose group (main study)
6 per sex per dose group (satellite groups)

Control animals:

yes, plain diet

Details on study design:

The study comprised five main groups of 10 males and 10 females each and five satellite groups (neuropathology study) of 6 males and 6 females each. One control group receiving unsupplemented basal diet and four test groups receiving diets containing different levels of the test substance for 13 weeks. These groups are intended to provide information on the subchronic oral toxicity of the test substance and to establish a no-observed-adverse-effect level (NOAEL).

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimise loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded. Any animal showing signs of severe debility or intoxication, particularly if death appeared imminent, was killed by exsanguination or by whole body perfusion fixation (satellite animals used for neuropathology), under ether anaesthesia to prevent loss of tissues by cannibalism or autolytic degeneration.

BODY WEIGHT:
The body weight of each animal was recorded twice during the acclimatization period, at initiation of treatment and once weekly thereafter. In addition, the animals were weighed on the day of scheduled necropsy (terminal body weight, for calculation of the organ to body weight ratios).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured per cage over weekly periods by weighing the feeders. The results were expressed in g per animal per day.
The intake of the test substance per kg body weight per day was calculated from the nominal concentration of the test substance in the diet, the food consumption and the body weight (mean of the body weights measured at the start and at the end of each week).

FOOD EFFICIENCY:
The efficiency of food utilization was calculated and expressed in g weight gain per g food consumed.

WATER CONSUMPTION:
Water intake was measured per cage by weighing the bottles. The results were expressed in g per animal per day. The consumption was measured daily during four days in week 1, 6 and 12 for rats of the main groups only.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic observations were made prior to the start of the study in all rats of the main groups and towards the end of the treatment period in all rats of the main and satellite control group and the main and satellite 15 ppm dose-group. Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

HAEMATOLOGY:
At necropsy at the end of treatment, blood samples were taken from the abdominal aorta of all surviving rats of the main groups whilst under ether anaesthesia. K2-EDTA was used as anticoagulant.

The following parameters were examined: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time and thrombocyte count.

The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY:
Clinical chemistry determinations were made on all surviving rats of the main groups. Fasting glucose was determined shortly before the end of the treatment period (on nominal day 88) in blood collected from the tip of the tail after deprivation of food for 16 hours. The collection of fasting blood was combined with the collection of urine (see next section). At necropsy at the end of treatment, blood was collected from the abdominal aorta in heparinized plastic tubes and plasma was prepared by centrifugation.

The following measurements were made in the plasma: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT) , alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, albumin/globulin ratio, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl) and inorganic phosphate.

URINALYSIS:
In the urine samples collected from the rats in the renal concentration test, the following determinations were carried out in individual samples: appearance, glucose, pH, occult blood, ketones, protein, bilirubin, urobilinogen and microscopy of the sediment.

RENAL CONCENTRATION TEST:
On nominal days 87 and 88, all surviving rats of the main groups were deprived of water for 24 hours and of food during the last 16 hours of this period. During the last 16 hours of deprivation, the rats were kept in metabolism cages (one rat per cage) and urine was collected. The concentrating ability of the kidneys was investigated by measuring the urinary volume and density of the individual samples.

NEUROBEHAVIOURAL EXAMINATION:
Before the start of the study and in weeks 1, 4, 8 and 13, spontaneous motor activity measurements and Functional Observational Battery (FOB) tests were performed in four animals of both sexes of each main group (two animals with the lowest identification numbers from each cage) and in all animals of each satellite group.

Sacrifice and pathology:

GROSS PATHOLOGY:
Based on the severe clinical signs and mortality observed in the high-dose (200 ppm) group, it was decided to sacrifice the main and satellite high-dose groups after approximately one month of treatment. The surviving animals of the main and satellite high-dose groups were considered moribund and were killed by exsanguination from the abdominal aorta under ether anaesthesia or by whole body perfusion fixation, under ether anaesthesia, respectively.

After completion of the 13 week treatment period, all surviving animals of the main groups were killed in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under ether anaesthesia and then examined macroscopically for pathological changes. A thorough autopsy was also performed on all animals that died intercurrently or that had to be killed because they were moribund.

The following organs of all surviving animals of the main groups were weighed: adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid (with parathyroids), liver and uterus.

Samples of the following tissues and organs of all animals of the main groups were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin): adrenals, aorta, * axillary lymph nodes, brain (brain stem, cerebrum and cerebellum), caecum, colon, epididymides, * eyes, * exorbital lachrymal glands, * femur with joint, heart, kidneys, liver, lungs, mammary glands (females), *mandibular (cervical) lymph nodes, mesenteric lymph nodes, nerve-peripheral (sciatic nerve), oesophagus, ovaries, pancreas, parathyroids, parotid salivary glands, pituitary, prostate, rectum, * seminal vesicles with *coagulating glands, * skeletal muscle (thigh), skin (flank), small intestines (duodenum, ileum, jejunum), spinal cord (three levels), spleen, sternum with bone marrow, stomach (glandular, non-glandular), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, trachea/bronchii, urinary bladder, uterus (with cervix), * vagina and all gross lesions.

* The tissues marked with an asterisk were preserved but not processed for histopathological examination, unless histopathological examination was considered necessary on the basis of the results of gross observations.

HISTOPATHOLOGY:
The tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Histopathological examination was performed on all tissues and organs listed above of all animals of the main control group and the main high-dose (200 ppm) group and of all animals that died during the study. Based on the clinical findings in the high-dose animals, the premature termination of the high-dose group and after consultation with the sponsor, it was decided to extend the histopathology to the main 15 ppm dose-group. Gross lesions were examined microscopically in all rats of all main groups.

Other examinations:

NEUROPATHOLOGY:
The animals of the satellite high-dose (200 ppm) group were sacrificed on 7, 8 and 9 July 1999 by whole body perfusion fixation, under ether anaesthesia. Based on the neurotoxic findings in the high-dose animals, the premature termination of the high-dose group and after consultation with the sponsor, it was decided to extend the neuropathology to the satellite 15 ppm dose-group.

The surviving animals of the satellite groups were killed on 13 to 17 September 1999. They were subjected to whole body perfusion fixation, under ether anaesthesia, with buffered glutaraldehyde/formaldehyde mixture (pH 7.2 - 7.4; 12 minutes; pressure: 130 mm Hg), preceded by saline solution (approximately 30 seconds; pressure: 130 mm Hg). This was done in such a sequence that the average time of killing was approximately the same for each group. After perfusion, relevant tissues were taken from the skull (the brain), the spinal column (spinal cord with roots and spinal ganglia) and the left hind limb (the sciatic nerve, the tibial nerve, the sural nerve and the plantar nerve). These tissues were stored overnight in the fixative. The remaining tissues and the intact right hind limb were also stored in the fixative. Subsequently, the tissue were rinsed in phosphate-buffer (pH 7.2-7.4) for a period of at least 48 hours at 2-10°C.

For embedding in paraffin, the following tissues from the satellite control and the 15 and 200 ppm groups were further dissected:
— transversal slabs from the cerebrum:
— telencephalon
— diencephalon
— transition zone diencephalon/mesencephalon
— transversal slab from mesencephalon (pons cerebri)
— transversal slab from cerebellum and medulla oblongata
— transversal slab from medulla oblongata

These tissues were dehydrated in ethanol and embedded in paraffin. Sections (2-5 µm thick) were cut from these blocks and sections were stained with either haematoxylin and eosin (H&E). Sections were examined by light microscopy.

For embedding in epoxy resin, the following tissues from the satellite control and the 15 and 200 ppm groups were further dissected:
— transversal slabs from spinal cord:
— the cervical spinal cord
— the thoracic spinal cord
— the lumbar spinal cord
— the dorsal spinal nerve root at the level of L4
— the ventral spinal nerve root at the level of L4
— the spinal ganglion at the level of L4
— Gasserian ganglion

— transversal slabs from nerve tissue from left hind limb:
— the sciatic nerve
— the tibial nerve
— the sural nerve
— the plantar nerve

These tissues were post-fixed with osmiumtetroxide and were dehydrated in a series of increasing ethanol solutions, followed by propylene oxide before embedding in epoxy resin.

Semi-thin sections were cut from these blocks. The semi-thin sections were stained with toluidine blue and were examined light-microscopically. All epoxy-resin embedded tissues of the perfusion-fixed animals of the satellite control group and the satellite 15 and 200 ppm groups were examined.

The fixed tissues obtained from the animals from the satellite 200 ppm dose group were stored at 2-10°C in monthly-refreshed phosphate-buffer (pH 7.2- 7.4; containing 0.02% sodium azide) prior to processing for microscopy. The fixed tissues obtained from the animals from the satellite 1 and 6 ppm dose groups were stored at 2-10°C in monthly-refreshed phosphate-buffer (pH 7.2-7.4; containing 0.02% sodium azide; awaiting possible processing for neuropathology) and were archived in buffered formalin after completion of the study. During storage, the fixed tissues in phosphate-buffer were visually inspected weekly for contamination.

Statistics:

The statistical procedures used in the evaluation of data were as follows:

- body weight: one-way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests;
- water intake: one-way analysis of variance (Anova) followed by least significant difference tests;
- food consumption/efficiency, red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, urinary volume and density, and organ weights: one-way Anova followed by Dunnett's multiple comparison tests;
- relative differential white blood cell counts, and semi-quantitative urinary determinations: Kruskal-Wallis non-parametric Anova followed by Mann-Whitney U-tests;
- histopathological changes: Fisher's exact probability test.

Probability values of p<0.05 were considered significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Three females of the high-dose (200 ppm) group died towards the end of the first month of treatment. Almost simultaneously, most of the females and a number of males of the high-dose group started to show severe neurological signs, including tremors and convulsions. Based on the death of three females and the severe nature of the clinical signs, all animals of the high-dose group were considered moribund and killed for humane reasons.

The daily observations did not reveal treatment-related clinical signs among the animals of the other groups. The few signs that were observed in these animals are common findings in Wistar rats and were randomly distributed among the remaining groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three females of the high-dose (200 ppm) group died towards the end of the first month of treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Generally, from day 7 to day 28 the body weight of the animals of the high-dose (200 ppm) group was lower than that of the controls, except on day 14 for male rats. On days 7 and 28 the difference reached the level of statistical significance for male rats.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was statistically significantly decreased in high-dose (200 ppm) animals on day 7 (males and females) and increased on day 28 (females). Food consumption was similar in the other groups throughout the study.
Due to the well-known decrease in food consumption per kg body weight with increasing age of rats, the intake of test substance per kg body weight decreased in the course of the study. The mean intakes of MMTTC/DMTDC in the groups receiving 1, 6, 15 or 200 mg per kg diet were 0.06, 0.39, 0.98 and 16.81 mg/kg bw/day in males and 0.07, 0.41, 1.02 and 17.31 mg/kg bw/day in females, respectively.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food conversion efficiency values tended to be decreased in high-dose animals on days 21 (males) and 28 (males and females). This decrease was statistically significant in high-dose males on day 21. Food conversion efficiency values were similar in the other groups throughout the study.

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

no effects observed

Description (incidence and severity):

Water consumption was generally similar among the groups throughout the study. The water consumption was statistically significantly decreased in females dosed with 6 ppm or with 200 ppm on day 1 only.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmoscopic examination of rats of the 15 ppm dose-group and the controls in week 13 of the study did not reveal any treatment-related ocular changes.

Haematological findings:

no effects observed

Description (incidence and severity):

No statistically significant changes in red blood cell and coagulation variables or total and differential white blood cell counts were observed.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry values showed statistically significantly increased alanine amino transferase and aspartate amino transferase activities in males of the 1 ppm dose-group. In the absence of similar changes at higher dose -levels, the increases at the 1 ppm level are considered fortuitous findings.
No other statistically significant changes in clinical chemistry values were observed between treated rats and controls.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinary density was statistically significantly increased in females of the 6 ppm dose-group. In the absence of a similar change at the next higher dose-level, this is considered a fortuitous finding. No other statistically significant changes were observed in urinary volume or density, semi-quantitative urinary observations or microscopy of the sediment.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In the first week of administration of the test substance, no effects of treatment were observed. In week 4 of treatment, increased activity was observed in females of the high-dose (200 ppm) group, together with increased rearing in the open field. Also convulsive activity was observed in a number of females from this group. These effects were not observed in males at this test time point. In males, increased landing footsplay was measured in the 200 ppm group. These effects were considered treatment-related and toxicologically relevant. Shortly after week 4, the high-dose (200 ppm) animals were excluded from the study, so no behavioural observations were recorded at later test time points for this group.
In weeks 8 and 13, no toxicologically relevant effects were observed.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Some high-dose animals demonstrated gross skin changes, which, at least in part, were probably related to treatment with the test material. Apart from that, no treatment-related gross changes were observed at autopsy. No treatment-related changes were observed in absolute or relative organ weights

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs:
One animal of the high-dose (200 ppm) subgroup for neuropathology was found dead on day 27. The other animals of this high-dose subgroup were killed moribund on day 31. Convulsions, blepharospasm and tremors were observed in these animals prior to whole body perfusion fixation. One animal showed hypothermia and four animals showed a hunched posture.
Inspection of the remaining high-dose animals, the control animals and animals of the 15 ppm subgroup, assigned for neuropathological examination did not reveal treatment-related changes prior to fixation.

Macroscopic observations:
No treatment-related gross changes were observed at necropsy.

Microscopic observations:
Microscopic examination of the cerebrum of the rats of the high dose (200 ppm) group showed pronounced neuronal death in a number of areas. This effect was most pronounced in female rats. Predominantly affected in both sexes were the hippocampal pyramidal neurons in the CA2/CA1 area and the CA4 neurons, embraced by the polymorphous layer of the dentate gyrus. Strikingly, neuronal death was not observed in the hippocampal CA3 area. The dying neurons typically showed karyorhexis and karyolysis and intense eosinophilic cytoplasm. Little or no glial reaction was observed. Occasionally, the surrounding neuropil showed very slight edema. Also the granular neurons of the dentate gyrus were locally affected (mainly in female rats), although the extent of damage was far less compared to the CA 1/2 and CA4 pyramidal cells. In addition, neuronal death was observed in the (pre- / para-)subiculum. Other predominantly lesioned areas in the cerebrum were the piriform and entorhinal cortices and the amygdala. The adjacent perirhinal cortex showed dying neurons as well.
In the fore-brain (telencephalon) the tenia tecta and adjacent area of the olfactory nuclei were affected, as was the frontal part of the piriform cortex.
In general, the morphological status of the neocortex was good, particularly in the male rats. In female rats, the results of the 4 brain levels, taken together, indicated that incidentally a single or just a few dying neurons were observed in the orbital cortex (one animal), the insular, retrosplenial and temporal cortices (five animals), the parietal cortex (three animals), the occipital cortex (two animals) and frontal cortex (one animal). These dying neurons, which showed a pathological appearance as described for those in the predominantly lesioned areas, were located mainly in those parts of these cortical regions neighbouring one of the severely affected predilection areas. In the male rats single cell necrosis of in the neocortex was observed only in the cingulate cortex of one animal and in the orbital, cingulate and parietal cortices of another animal.
One animal showed a slight dilatation of the lateral ventricle, a phenomenon which was observed to a lesser extent in another animal.
Microscopic examination of other protocol organs for neuropathology revealed neuronal micro-vacuolization in both the spinal ganglion (L4) and Gasserian ganglion in the control and in both dose groups. Neuronal microvacuolization may be induced by processes like hypoxia or ischemia but also by certain toxins. In.this study, neuronal micro-vacuolization was observed in the lumbal spinal ganglion in 4/5 females and 6/6 males of the high-dose (200 ppm) group, in 6/6 females and 6/6 males of the 15 ppm group, in 6/6 females and 6/6 males of the control group, and in the Gasserian ganglion in 2/4 females and 5/6 males of the high-dose group, in 4/6 females and 3/6 males of the 15 ppm group, in 4/5 females and 4/6 males of the control group. In short, no dose-response relationship was found and, therefore, the observed neuronal micro-vacuolization was not considered to be treatment-related.
Incidentally, degenerating axons (1 to 3) were observed in peripheral nerves, and dorsal and ventral roots of animals of the high-dose (200 ppm) group, but in animals of the other groups as well. This incidental axonal degeneration was not considered treatment-related, because the finding was within the range of the normal background pathology for rats of this strain and age.
Also in the spinal cord (3 levels) degenerating axons were present. In addition, small numbers of apparent 'swollen axons' (i.e. the axon is retracted from the relatively thinned and widened myelin sheet), were observed in animals of all groups. These swollen axons were sparsely distributed over the white matter with minor preference for the ventral part of the spinal cord. In the lumbal spinal cord their number was slightly higher in the high-dose (200 ppm) group (in 4/5 females and 4/6 males of the 200 ppm group, in 1/6 females and 0/6 males of the 15 ppm group, in 1/6 females and 2/6 males of the control group). If at all treatment-related, these effects were very mild.
Summarizing, the results of the neuropathology showed treatment-related neuronal death in a number of predilection areas in the tel-and mesencephalon. Predominantly lesioned areas in the mesencephalon were the hippocampal region in broader sense (i.e. including subiculum and dentate gyrus), the piriform, entorhinal and perirhinal cortices and amygdala. In the telencephalon, neuronal death was present in the olfactory nuclei and tenia tecta. Neuronal death was observed in both sexes but was most pronounced in female rats and was, for both sexes, restricted to animals of the high-dose (200 ppm) group.
In the spinal cord, incidental swollen axons were observed in the white matter and their number was slightly increased in animals of the high-dose (200 ppm) group. This effect, if at all treatment related, was very mild. No treatment -related effects were observed in the peripheral nerves, spinal roots and spinal ganglia.
It was concluded that treatment of rats with 200 ppm MMTTC/DMTDC (30/70) induced marked neural lesions in a number of areas in the cerebrum, whereas no neuropathology was found in the 15 ppm MMTTC/DMTDC (30/70) group and in the control group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At microscopic examination treatment-related histopathological changes were observed in the brain, the kidneys and the thymus of the animals treated with 200 ppm of the test substance. The histopathological changes in the brain comprised cell death, especially in the Ammon's horn, the dentate gyrus and the piriform cortex, and submeningeal oedema. In females, the extensiveness of the cell death was slightly higher than in males. The kidneys demonstrated treatment-related tubular dilatation in the cortical area. The tubules involved were the last parts of the distal tubules and the first part of the collecting tubules. In general, the tubular dilatation was not accompanied by degenerative or inflammatory changes. In one control and some treated females very slight to slight nephrocalcinosis in the kidneys was found. Nephrocalcinosis is a common finding in female rats. The distribution of 1 in the control group, 0 in the 1 ppm group, 4 in the 15 ppm group and 4 in the 200 ppm group was not considered to be an indication of a relationship with treatment but a fortuitous finding. Two control and six 15 ppm females showed minimal mineralisation in the ovaries. The difference in incidence in these groups was not statistically significant. Mineralisation in ovaries of 200 ppm females was not observed. However, those animals were exposed to the test substance for only one month. Speculations with respect to a possible incidence of this change after 3 months exposure cannot be founded. Therefore, the mineralisation in the ovaries was not considered to be treatment-related. In females treated with 200 ppm an increased incidence of corticomedullary haemorrhage in the thymus was observed. Most of the high-dose females also showed cortical lymphoid depletion in the thymus. This was not considered a direct toxic effect of the test substance, but an indirect effect caused by stress, due to the severe effects of the test material. All females and males treated with 200 ppm showed decreased accumulation of brown pigment in the spleen. Accumulation of brown pigment is a normal phenomenon in rats, which gradually increases with age. The 200 ppm animals were killed at an early stage of the study, at which they had not yet accumulated pigment in the spleen. Therefore, the decreased splenic pigment was not an effect of the treatment, but was the consequence of the difference in age between these animals and those of the other treatment groups and the controls, that were killed two months later. The aforementioned treatment-related histopathological changes were not observed in dose-group treated with 15 ppm. It was concluded that, based on the microscopic examination, 15 ppm was the no observed adverse effect level under the given conditions. Therefore, the animals of the lower dose groups were not examined microscopically.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

15 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Neurological effects.

Remarks on result:

other: 0.98 mg/kg bw/day (actual dose received)

Dose descriptor:

NOAEL

Effect level:

15 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Neurological effects

Remarks on result:

other: 1.02 mg/kg bw/day (actual dose received)

Critical effects observed:

not specified

The achieved concentrations in the present study were close to the nominal levels and were all in accordance with the criteria for content of the test substance (the acceptable maximum mean concentration).

Conclusions:

On the basis of the neurotoxic effects observed in the high-dose group the No-Observed-Adverse-Effect Level in the present study was placed at 15 ppm. This level was equivalent to 0.98 (males) and 1.02 (females) mg of MMTTC/DMTDC (30/70) per kg body weight per day.

Executive summary:

The objective of this study was to examine the possible sub-chronic oral toxicity of the test substance MMTTC/DMTDC (30/70). The test substance was administered to main groups of 10 male and 10 female Wistar rats and satellite groups (for neuropathology) of 6 male and 6 female Wistar rats at constant concentrations of 0, 1, 6 or 15 ppm in the diet for 13 consecutive weeks or at a constant concentration of 200 ppm for approximately one month.

After about one month of treatment, neurotoxic effects of the test substance became evident at the highest dose-level. Three high-dose females died and a large number of high-dose animals of both sexes started to develop signs of neurotoxicity, including tremors and convulsions. On account hereof, all animals of the main and satellite high-dose groups were sacrificed after approximately one month of treatment. High-dose (200 ppm) males and females showed slight growth retardation, which was associated with decreased food intake during the first week and with slightly lower food conversion efficiency in weeks three and four. Body weight, food consumption or food conversion efficiency were not affected by the test substance at the lower dose levels. Neurobehavioural and motor activity testing showed clear treatment-related neurotoxic effects in animals of the 200 ppm group in week 4 of the study. In week 1 of the study these effects were not yet observed, which is in accordance with the onset of the neurological clinical signs in the 200 ppm group after about 4 weeks. No toxicologically relevant effects were observed in any of the other dose-groups, at any of the other time-points investigated. Ophthalmoscopy, haematology, clinical chemistry, urinalysis or absolute and relative organ weights did not reveal any treatment-related changes after dietary exposure to concentrations up to 15 ppm for 13 weeks. These parameters were not measured for animals of the 200 ppm group, because of the premature termination of this group. Microscopic examinations (general pathology) demonstrated clear treatment-related changes in the kidneys, thymus and brain of animals of the 200 ppm group. The effect on the thymus was not considered a direct toxic effect, but an effect caused by stress. The effects on the kidneys and brain were considered toxicologically relevant. None of the effects was present in the next lower dose-group treated with 15 ppm. Microscopic examinations (neuropathology) demonstrated treatment-related pathological changes in several parts of the brain (neuronal death) of animals of the 200 ppm group. No significant treatment-related changes were observed in the peripheral nervous system. The effects occurred in both sexes, but were most pronounced in female rats.

On the basis of the neurotoxic effects observed in the high-dose group the No-Observed-Adverse-Effect Level in the present study was placed at 15 ppm. This level was equivalent to 0.98 (males) and 1.02 (females) mg of MMTTC/DMTDC (30/70) per kg body weight per day.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across to structurally similar substance dimethyltin dichloride (DMTC) (EC Number 212-039-2, CAS Number 753-73-1), see attached justification.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

neuropathology

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

neuropathology

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.62 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Two reliable 90-day studies were available on the read-across substance for this endpoint. Both identified neurotoxicity as the key effect. In addition, two shorter high quality studies were also available as supporting information. The overall quality of the database was therefore considered to be high.

System:

nervous system

Organ:

brain

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated Dose Toxicity Oral: Read-across to DMTC (Dimethyltin dichloride CAS No. 753-73-1)

Two reliable 90-day studies were available to address this endpoint. Beyrouty (1997a) is a well reported 90 day study (oral: drinking water) performed to GLP. In Beyrouty (1997a), rats were treated for up to 13 weeks with a methyltin chloride mixture containing 90% dimethyltin dichloride at concentrations of 25, 75 or 200 ppm. The study resulted in several deaths, reduced body weight, decreased food and water intake, blood biochemical changes, behavioural effects such as aggressiveness and neuropathological lesions at 200 ppm (equivalent to 15.5 and 19.4 mg/kg/day for males and females, respectively). For the 75 ppm group (equivalent to 5.2 and 6.7 mg/kg/day for males and females, respectively), one male died, body weights were reduced (males only), food and water intake were decreased, and behavioural changes (such as reduced motor activity for females) and neuropathological lesions were noted. At 25 ppm (equivalent to 1.6 and 2.2 mg/kg/day for males and females, respectively), no mortality occurred and treatment-related findings were limited to reduced food (males only) and water intake and neuropathological lesions. The no observed effect level was considered to be below 25 ppm.

The other reliable 90-day study selected was Appel (2000). This study gives a NOAEL of 15 ppm (equivalent to 0.98 mg/kg bw/day for male rats and 1.02 mg/kg day/bw for female rats). After about one month of treatment, neurotoxic effects of the test substance became evident at the highest dose-level of 200 ppm. Three high-dose females died and a large number of high-dose animals of both sexes started to develop signs of neurotoxicity, including tremors and convulsions. On account hereof, all animals of the main and satellite high-dose groups were sacrificed after approximately one month of treatment. High-dose (200 ppm) males and females showed slight growth retardation, which was associated with decreased food intake during the first week and with slightly lower food conversion efficiency in weeks three and four. Body weight, food consumption or food conversion efficiency were not affected by the test substance at the lower dose levels. Neurobehavioural and motor activity testing showed clear treatment-related neurotoxic effects in animals of the 200 ppm group in week 4 of the study. In week 1 of the study these effects were not yet observed, which is in accordance with the onset of the neurological clinical signs in the 200 ppm group after about 4 weeks. No toxicologically relevant effects were observed in any of the other dose-groups, at any of the other time-points investigated. Ophthalmoscopy, haematology, clinical chemistry, urinalysis or absolute and relative organ weights did not reveal any treatment-related changes after dietary exposure to concentrations up to 15 ppm for 13 weeks. These parameters were not measured for animals of the 200 ppm group, because of the premature termination of this group. Microscopic examinations (general pathology) demonstrated clear treatment-related changes in the kidneys, thymus and brain of animals of the 200 ppm group. The effect on the thymus was not considered a direct toxic effect, but an effect caused by stress. The effects on the kidneys and brain were considered toxicologically relevant. None of the effects was present in the next lower dose-group treated with 15 ppm. Microscopic examinations (neuropathology) demonstrated treatment-related pathological changes in several parts of the brain (neuronal death) of animals of the 200 ppm group. No significant treatment-related changes were observed in the peripheral nervous system. The effects occurred in both sexes, but were most pronounced in female rats.

On the basis of the neurotoxic effects observed in the high-dose group the No-Observed-Adverse-Effect Level in the present study was placed at 15 ppm. This level was equivalent to 0.98 (males) and 1.02 (females) mg of MMTTC/DMTDC (33.5/66.5) per kg body weight per day.

Justification for classification or non-classification

In accordance Annex VI of Regulation (EC) No. 1272/2008 (CLP), the substance meets the following criteria for classification for repeat-dose toxicity:

STOT Rep. Exp 1. with the association hazard phrase H372 'Causes damage to the nervous system and the immune system following prolonged or repeated exposure.