Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study conducted under GLP. The method of analysis involved derivatization. This method only measures the amount of the alkyltin moiety, MMT, present and does not identify the other ligands attached to the tin. Currently there is no analytical method available that can quantify the actual named substance, i.e., the entire organotin compound with its associated chloride ligand. All measured MMT was fully attributed to the named substance, MMTC.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
On each day of the analysis of study samples, QC samples were freshly prepared; Validation of the anlaytical method for test material in the diet at the low dose of the main study was performed, because the dose wasn't covered by the pilot study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Trichloromethylstannane
- Molecular formula (if other than submission substance): CH3Cl3Sn
- Molecular weight (if other than submission substance): 240.8 g/mol
- Smiles notation (if other than submission substance): CL[Sn](CL)(CL)C
- Structural formula attached as image file (if other than submission substance): see Fig.
- Physical state: liquid
- Analytical purity: 88%
- Composition of test material, percentage of components:
Alkyl group distribution % (mass/mass)
Monomethyltin trichloride 82.85
Dimethyltin dichloride 9.29
Trimethyltin chloride 0.02
Tin tetrachloride 4.68
Me2ClSnCH2SnCl3 0.59
MeCl2SnCH2SnCl3 1.78
Cl3SnCH2SnCl3 0.80
- Lot/batch No.: 82420
- Expiration date of the lot/batch: December 1, 2003
- Storage condition of test material: <-18 degrees C, in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 7 weeks old; satellite group- 13-14 weeks old
- Weight at study initiation: 134.9-178.5 g in males and 113.9 -138.8 in females; 178.8 - 213.5 g in females satellite group
- Fasting period before study: no
- Housing: under conventional conditions in one room in macrolon cages with sterilized wood shavings and environmental enrichment 2 (dose-range finder) or 5 rats per cage (13-week study) separated by sex. During the premating period females of the satellite groups were housed 3 or 4 per group per cage. During the gestation and lacation period the females were housed individually.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8, 13 and 13 days for the dose-range finder, 13-week and satellite groups


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 degrees C
- Humidity (%): at least 30% and not exceeding 70%, other than during room cleaning
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark


IN-LIFE DATES: dose range finder From: January 30, 2003 To: February 13, 2003
13-week study From: April 1, 2003 To: July 1, 2003- males; July 2, 2003- males; June 26 to July 14, 2003 for satellite groups

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): at the start of the study and every six weeks thereafter
- Mixing appropriate amounts with (Type of food): commercial rodent diet (rat and mouse No 3 Breeding Diet Services, Witham, England)
- Storage temperature of food: stored in a freezer


VEHICLE- not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From each diet sample, 2.0 g was transferred into a 50 mL Greiner tube. An aliquot of the internal standard solution (monoheptyltin trichloride, diheptyltin dichloride, tripropyltin chloride and tetrapropyltin in methanol) was added. Subsequently methanol, acetate buffer solution (pH 4.5), 20% aqueous NaBEt4 solution (STEB solution) and hexane (with naphthalene as internal standard) were added to each sample and this mixture will be shaken and heated to 60 degrees C. During this step, the organotin chlorides were converted into the corresponding ethylated tetraorganotin derivatives, which were extracted into the hexane layer. Prior to GC-MS analysis, the hexane layer wa washed with 2 mol/l HCl in order to remove (most of) the ethylboron compounds that interfere with the GC-MS analysis. The concentration of each test substance in feed was determined by GC-MS analysis of the hexane extracts.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 150, 750 mg trichloromethylstannane/kg diet
Basis:
nominal in diet
No. of animals per sex per dose:
10 plus 10 females for the satellite groups
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: no
- Dose selection rationale: based on dose-range finder
- Rationale for animal assignment (if not random): computer randomization
- Rationale for selecting satellite groups: to provide additional data on possible reproductive and developmental effects of the test substance
- Post-exposure recovery period in satellite groups: none
- Section schedule rationale (if not random): allowed to give birth
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (in the morning) and on working days also once in the afternoon.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily (in the morning) and on workin days also once in the afternoon.


BODY WEIGHT: Yes
- Time schedule for examinations: once during the acclimatization period, once at initiation  of the study prior to introduction of feed.  Thereafter body weights were  recorded once weekly.  Furthermore, all animals were weighed on the day  of necropsy in order to determine their correct organ to body weight  ratios.



FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Food consumption: measured per cage over weekly periods by weighing the  feeders (in g/animal/day).  Data of male rats was not  measured in weeks 10 (all animals) and 11 (some animals), because this  was hampered by the mating procedure (male rats of the 13-week study were  used to mate with female rats from the satellite group).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes the intake of substance per kg/bw/day was  calculated from the nominal dietary concentration of the substance, the  food consumption and the mean body weight in the period for which the  intake of the substance is calculated.


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


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: provided ad libitum, the amount consumed was not  measured


OPHTHALMOSCOPIC EXAMINATION: Yes   Eye examinations were carried out using an ophthalmoscope after  induction of mydriasis by a solution of atropine sulphate
- Time schedule for examinations: observations made prior to the start of  the treatment in all animals and towards the end of the treatment period  in all surviving animals of the control and high dose (750 mg/kg) groups. 
- Dose groups that were examined: prior to the start of  the treatment in all animals and towards the end of the treatment period  in all surviving animals of the control and high dose (750 mg/kg) groups. 


HAEMATOLOGY: Yes  
- Time schedule for collection of blood: at necropsy at the end of treatment, after overnight  fasting, blood samples were taken from the abdominal aorta of all  surviving animals. 
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes overnight
- How many animals: all surviving animals
- Determinations carried out included: haemoglobin  (Hb), packed cell volume (PCV), red blood cell count (RBC),  reticulocytes, total white blood cell count, differential white blood  cell count, prothrombine time, thrombocyte count.  The mean corpuscular  volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular  haemoglobin concentration (MCHC) were calculated.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:at necropsy at the end of treatment, after overnight  fasting, blood samples were taken from the abdominal aorta of all  surviving animals
- Animals fasted: Yes overnight
- How many animals: all surviving animals
- Measurements made included alkaline phosphatase  activity (ALP), aspartate aminotransferase activity (ASAT), alanine  aminotransferase activity (ALAT), gamma glutamyl transferase activity  (GGT), total protein, albumin, albumin to globulin ratio (A/G), urea,  creatinine, total bile acid, bilirubin (total and direct), cholesterol  (total), triglycerides, phospholipids, Ca, Na, K, Cl, inorganic  phosphate, fasting glucose


URINALYSIS: Yes  all animals 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 and urine  was collected.  The concentrating ability of the kidneys was investigated  by measuring the volume and density of the individual samples.  
- Time schedule for collection of urine: Shortly before the end of the  treatment (days 86-87)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes 24 hours
- The  determinations carried out with the urine collected in the renal  concentration test included appearance, glucose, pH, occult blood,  ketones, protein, bilirubin, urobilinogen, microscopy of the sediment.


NEUROBEHAVIOURAL EXAMINATION: Yes .
- Time schedule for examinations: prior to the first exposure and then once weekly up to and including week 12.
- Dose groups that were examined: all surviving animals
- Battery of functions tested: arena testing, functional observational  battery (FOB) and motor activity assessment


Sacrifice and pathology:
GROSS PATHOLOGY: Yes All animals of the 13-week study and all adult animals of the satellite group were subjected to a complete gross necropsy. organs weighed included adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroid (with  parathyroids), uterus. 
HISTOPATHOLOGY: Yes performed on  tissues and organs of all animals of the control and high dose group and  included adrenals, aorta, brain (brain stem, cerebrum, cerebellum),  caecum, colon, epididymides, eyes, GALT (gut associated lymphoid tissue,  including Peyer's patches), heart, kidneys, liver, lungs, mammary gland  (females), mesenteric lymph nodes, nerve-peripheral (sciatic),  oesophagus, ovaries, pancreas, parathyroid, pituitary, prostate, rectum,  skin (flank), small intestine (duodenum, ileum, jejunum), spinal cord (at  3 levels), spleen, sternum with bone marrow, stomach (glandular and non-glandular), sublingual salivary glands, submaxillary salivary glands,  testes, thymus, thyroid, trachea/bronchi, urinary bladder, uterus (with cervix) and all gross lesions. Lungs, liver, kidneys and gross lesions were examined microscopically in all intermediate dose groups.  Since treatment related  changes were found in the thymus and brain of males and females of the  high dose group, histopathology on these organs was extended to the animals  of the intermediate dose groups.
Statistics:
STATISTICAL METHODS: - See other information on material and methods section

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY No mortality occurred
No treatment related findings were observed

BODY WEIGHT AND WEIGHT GAIN Similar among the groups in males and females throughout the study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) Similar among the groups in males throughout the study. Food consumption was slightly higher (ca. 8%) in females of the 750 mg/kg group. This difference was statistically significant during the last three weeks of the study.


FOOD EFFICIENCY Similar among the groups in males and females throughout the study. An occasional significant difference was seen.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) N/A


OPHTHALMOSCOPIC EXAMINATION No treatment related ocular changes were observed.

HAEMATOLOGY RBC, Hb and PVC were statistically significantly increased in females and MCV and MCH were statistically significantly increased in males of the 750 mg/kg group. Thrombocytes (females) and prothrombine time (males and females) were statistically significantly decreased in the 750 mg/kg group. Absolute and relative numbers of eosinophils were significantly decreased in females of the 750 mg/kg group. Haematology parameters were similar among the control, 30 and 150 mg/kg groups, with the exception of a statistically significantly lower number of neutrophils in males of the 30 mg/kg groups, which was considered a chance finding.

CLINICAL CHEMISTRY At the end of the treatment period the following statistically significant differences (relative to the control group) were observed
ALP: increased in males of the 750 mg/kg group and decreased in females of the 30 mg/kg group;
ASAT: increased in males and females of the 750 mg/kg group;
Albumin: increased in males of the 750 mg/kg group;
Albumin/globulin ratio: decreased in females of the 750 mg/kg group;
Urea: increased in males of the 750 mg/kg group;
Creatinin: increased in males of the 750 mg/kg group;
Total bilirubin: decreased in females of the 750 mg/kg group;
Cholesterol: increased in males of the 750 mg/kg group;
Phospholipids: increased in males of the 750 mg/kg group;
Chloride: increased in males of the 750 mg/kg group;
Potassium: decreased in males of the 750 mg/kg group.



URINALYSIS Urinary pH and urinary crystals were statistically significantly increased in males and females of the 750 mg/kg group. Other semi-quantitative and microscopic urinary observations were similar among the groups. Urinary volume was statistically significantly increased and urinary density was statistically significantly decreased in males and females of the 750 mg/kg group.

NEUROBEHAVIOUR In animals of the 750 mg/kg group, some statistically significant effects were observed during neurobehavioural testing at the end of the study in week 13. In males, increases in forelimb gripstrength, landing footsplay and body temperature were measured, and a marginal effect was shown on click response. Hyperactivity was clearly observed in both males and females. The changes were considered related to treatment and toxicologically relevant.

ORGAN WEIGHTS The following organ weights were statistically significantly increased in the 750 mg/kg group:
• Absolute (males and females) and relative (males) adrenal weights;
• Absolute and relative kidney weights (males and females).
The following organ weights were statistically significantly decreased in the 750 mg/kg group:
• Absolute and relative thymus weights (males and females);
• Absolute and relative brain weights (females);
• Absolute and relative spleen weight (males);
• Absolute and relative epidydimidal weights.

GROSS PATHOLOGY No treatment related changes were observed.


HISTOPATHOLOGY: NON-NEOPLASTIC At microscopic examination, treatment related histopathological changes were observed in the thymus and the brain. Six males of the 750 mg/kg group showed a decreased cortex/medulla ratio in the thymus. This change was also present in three females. Although the incidence in the females of the 750 mg/kg group was not statistically significantly higher than that in the controls, it was considered related to treatment because the thymic change was similar to that in the males. The decreased cortex/medulla ratio was characterized by a smaller cortex while the general architecture of the thymus was not changed. Decreased cortex/medulla ratio was not observed in any of the animals of the control, 30 or 150 mg/kg groups. The treatment related histopathological changes in the brain consisted of loss of perikarya of neuronal cells in specific areas of the brain. All females and all but one male showed loss of perikarya in the pyramidal layer of the Hippocampus CA1/2. In addition, four males of the 750 mg/kg group demonstrated loss of perikarya in the piriform cortex, which was also considered related to treatment. The histopathological changes observed in the brain were not observed in any of the animals of the control, 30 or 150 mg/kg groups.


HISTOPATHOLOGY: NEOPLASTIC (if applicable) N/A


HISTORICAL CONTROL DATA (if applicable) N/A

Effect levels

Dose descriptor:
NOAEL
Effect level:
150 mg/kg diet
Sex:
male/female
Basis for effect level:
other: overall effects: Based on the changes in neurobehaviral parameters, hematology, clinical chemistry, urinalysis and organ weights and the associated histopathological findings in the thymus and brain in animals of the 750 mg/kg group.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

NOAEL: Based on the changes in neurobehavioural parameters, haematology,  clinical chemistry, urinalysis and organ weights and the associated histopathological findings in thymus and brain in animals of the 750  mg/kg group, the NOAEL in the sub-chronic toxicity study was placed at 150 mg trichloromethylstannane/kg diet (equivalent to 9.8 mg trichloromethylstannane/kg bw/day in males and  10.2 mg trichloromethylstannane/kg bw/day in females).

Dietary doses of 50, 250, 750, and 1500 mg trichloromethylstannane/kg  feed (ppm) were administered for 14 days.  No treatment-related clinical  signs were observed.  The body weights were sporadically decreased in  males of the 750 mg/kg group and throughout at 1500 mg/kg.  Body weights  were not significantly different among females.  Food consumption was  significantly decreased in males of the 750 and 1500 mg/kg groups on day  7 and 14.  Food consumption was significantly increased in females of the  50 and 250 mg/kg groups (day 7) and significantly decreased in females of  the 750 mg/kg group (days 7 and 14) and the 1500 mg/kg group (day 7).   Food conversion efficiency was significantly decreased in males of the  1500 mg/kg group (days 7 and 14).  Food conversion efficiency was not  significantly different among females.  The absolute weights of the  testes were significantly decreased in the males of the 50 and 1500 mg/kg  groups.  Absolute spleen weights, relative kidney weights and absolute  and relative liver weights were significantly decreased in the males of  the 1500 mg/kg group.  Absolute weights of the ovaries were significantly  increased in the females of the 250 mg/kg group and decreased in the  females of the 1500 mg/kg group.  Absolute and relative spleen weights  were significantly decreased in females of the 750 and 1500 mg/kg groups.   Macroscopic examination at necropsy did not reveal any treatment related  changes.
Dietary exposure of trichloromethylstannane up to 1500 mg/kg for 14 days  was tolerated; however, the body weight and food consumption decreases  were deemed palatability effects at 750 and 1500 mg/kg.  The low food  intake, low food efficiency, and organ weight effects at these doses were suggestive of a toxic response threshold.  Doses for the main and satellite studies were chosen as 30, 150, and 750 mg/kg in the diet.

Applicant's summary and conclusion

Conclusions:
The NOAEL was 150 mg/kg diet
Executive summary:

The toxicity of trichloromethylstannane in Wistar rats was examined using continuous administration via the diet for 13 consecutive weeks (OECD Test Guideline 408). In satellite groups of female rats a reproduction/developmental screening test (OECD Test Guideline 421) was performed to provide initial data on possible reproductive and developmental effects of trichloromethylstannane. The main study used four groups of 10 rats/sex (13-week study) and the satellite study used four groups of 10 female rats (reproduction/developmental screening study). For both studies, the control group was kept on control diet and three test groups received experimental diets containing 30,150 and 750 mg/kg [ppm] of the test substance. The dose levels used in both studies were based on the results of a preceding dose range finding study.

 

Clinical observations, growth, food consumption, food conversion efficiency, neurobehavioural testing, ophthalmoscopy, haematology, clinical chemistry, renal concentration test, urinalysis, organ weights and gross examination at necropsy, microscopic examination of various organs and tissues and assessment of various reproductive and developmental parameters were used as criteria for detecting the effects of treatment.

 

Results

Main study (OECD Test Guideline 408)

 

The calculated doses for the groups receiving 30, 150, or 750 mg/kg trichloromethylstannane in the diet were 1.9,9.8, and 49.7 mg/kg body weight/day in males and 2.1, 10.2, and 53.6 mg/kg body weight/day in females.

 

No treatment-related changes were observed in clinical signs, body weight, food conversion efficiency and ophthalmoscopy.

 

A number of haematological changes (statistically significant increases in RBC, Hb, PCV, MCV and MCH and decreases in thrombocytes, prothrombin time and eosinophilic leucocytes) observed in the 750 mg/kg group, were considered treatment-related and toxicologically relevant.

 

A number of clinical chemistry parameters (statistically significant increases in ALP, ASAT, albumin, urea, creatinin, cholesterol, phospholipids and chloride and a decreases in potassium) observed in the 750 mg/kg group, were considered treatment-related and toxicologically relevant.

 

The changes in urinary volume and density and in urinary pH and crystals observed in the 750 mg/kg group, were considered treatment-related and toxicologically relevant.

 

The changes in organ weights (increases in adrenal and kidney weights and decreases in thymus, brain, spleen and epidydimidal weights) observed in the 750 mg/kg group were considered treatment-related and toxicologically relevant.

 

 

The decrease in thymus weights was accompanied by a decrease in cortex/medulla ratio in the thymus of males and females, which was considered treatment-related and toxicologically relevant.

 

The treatment-related changes in neurobehavioural paran1eters (increased forelimb gripstrength, increased landing footsplay, increased body temperature and increased hyperactivity) are indicative of a neurotoxic potential of trichloromethylstannane. The neurobehavioural changes were corroborated by the treatment-related histopathological changes in the brain of most animals of the 750 mg/kg group. The observed histopathological lesion (loss of perikarya in the pyramidal layer of the Hippocampus CAl/2) was located in a functional domain that is generally consistent with the observed neurobehavioural changes and provides further evidence of the neurotoxic potential of the test substance at the 750 mg/kg level.

 

 

Conclusions

Main studv (OECD Test Guideline 408)

Based on the changes in neurobehavioural parameters, haematology, clinical chemistry, urinalysis, organ weights and the associated histopathological findings in thymus and the brain in animals of the 750 mg/kg group, the No Observed Adverse Effect Level (NOAEL) in the sub-chronic toxicity study was placed at 150 mg Trichloromethylstannane/kg diet. This level was equivalent to 9.8 mg/ kg body weight/day in males and 10.2 mg/kg body weight/day for females. 

 

Based on the effects on body weight and food consumption in the 750 mg/kg group, 150 mg

Trichloromethylstannanel kg diet (equivalent to 6.2-11.7 mg/kg body weight/day) can be considered as a NOAEL for maternal toxicity.

 

Qverall conclusion:

The NOAEL for general sub-chronic toxicity and maternal toxicity, fertility and developmental effects was placed at 150 mg trichloromethylstannane per kg diet.