Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 December 2002 to 07 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Minor deviations which not have influence on the validity of study.
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS:
- Species: Albino rats
- Strain: Wistar outbred (Crl:(WI)WU BR)
- Age at study initiation: approximately 7 weeks old males and females (13-14 weeks age of females in the satellite study)
- Weight at study initiation:  135.0 - 167.1 g (mean 150.7 g) for males;  112.6 - 148.5 g (mean 126.2 g) for females (body weights for females in the satellite groups at the start of treatment ranged from 194.2 - 229.6 g (mean 208.5 g).

- Number of animals: 
Dose-range finding study: 21 males and 21 females
Main study (13-week subchronic study) - 43 males and 43 females (4 dose groups of 10 rats/sex/dose group)
Satellite study (reproduction/developmental toxicity screening): 44 females

Upon arrival animals were put in a quarantine room and were given time to acclimate to the new surroundings. The quarantine and acclimatisation periods between arrival and experimental start date were 9, 13, and 13 days for the dose-range finding study, 13-week study, and satellite study, respectively.

The animals were housed in one room, in macrolon cages, with sterilised wood shavings as bedding material.
2 (dose-range finding study) or 5 rats (13-week study) were housed per cage, separated by sex. During the premating period, females of the satellite groups were housed 3 or 4 per dose group per cage. During gestation and lactation, the females were housed individually.

The room was ventilated with about 10 air changes per hour and was maintained at a temperature of 22 ± 3 °C at a relative humidity of 30-70 %. Lighting was artificial with 12-hour light and dark cycles.

Feed and drinking water were provided ad libitum. A commercial rodent diet was provided.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
ADMINISTRATION / EXPOSURE:
- Duration of exposure: 14 days or 13 consecutive weeks, dose-range finding study and subchronic study, respectively
- Type of exposure: via the diet (rodent diet: Rat & Mouse No.3 breeding  diet RM3)
- Doses: 0, 300, 1500 and 7500 mg MBTC/kg diet for subchronic study

SATELLITE GROUPS AND REASONS THEY WERE ADDED: Four satellite groups of  female rats were added.  The objective was to provide initial data on the  possible reproductive and developmental effects of butyltrichlorostannane  after oral administration. For the description of the method and results, see section 7.8.3 Toxicity to Reproduction, Other studies.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in the matrix under examination, viz. RM3 diet, before the start of the study. The test material and other organotin chlorides used as internal standards were converted into the corresponding ethylated tetraorganotin derivatives, which were extracted into the hexane layer. GC-MS analysis was then performed.

The homogeneity, stability, and achieved concentration (content) of the test material in RM3 rat feed was analysed in the batch of diets prepared for the dose-range finding study and the 13-week study.

From these analyses, it was concluded that the test material was homogenously distributed in all diets, the test material was stable in the diets at room temperature for 7 days and in the freezer at <-18 °C for 6 weeks, and the content of the test material was close to the nominal level for all diets.
Duration of treatment / exposure:
Dose-range finding study: 14 days
Main subchronic study: 13 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg diet
Dose / conc.:
1 500 mg/kg diet
Dose / conc.:
7 500 mg/kg diet
No. of animals per sex per dose:
Dose-range finding study: 4 animals per sex per dose
13-week study: 10 animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
ADMINISTRATION / EXPOSURE:
- Duration of exposure: 14 days or 13 consecutive weeks, dose-range finding study and subchronic study, respectively
- Type of exposure: via the diet (rodent diet: Rat & Mouse No.3 breeding diet RM3)
- Dose-range finding study: 0, 100, 300, 1000, 5000 mg/kg diet
- 13 week subchronic study doses: 0, 300, 1500 and 7500 mg/kg diet
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND FREQUENCY:
- Clinical signs: at least once daily (in the morning) and on working days also once in the afternoon.
- Mortality: at least once daily (in the morning) and on working days also at least once in the afternoon.
- Body weight: once during the acclimatisation period, once at initiation of the study prior to introduction of feed. Thereafter, body weights were recorded once weekly. In the dose range finding study, body weights were recorded twice weekly. Furthermore, all animals were weighed on the day of necropsy in order to determine their correct organ to body weight ratios. - Food consumption: measured per cage over weekly periods by weighing the feeders (in g/animal/day). The efficiency of food utilisation was calculated and expressed in g weight gain/g food consumed. Food consumption of male rats of the main study was not measured in weeks 10 (all animals) and 11 (some animals), because this measurement was hampered by the mating procedure (male rats of the 13-week study were  used to mate with the female rats from the satellite group).
- Water consumption: provided ad libitum, the amount consumed was not measured.
- Intake of test material: the intake of test material per kg/bw/day was calculated from the nominal dietary concentration of the the test material, the food consumption and the mean body weight in the period for which the intake of the test material is calculated.
- Neurobehavioural testing: arena testing, functional observational  battery (FOB) and motor activity assessment.
- Ophthalmoscopic examination: 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 7500 mg/kg groups. 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, after overnight fasting, blood samples were taken from the abdominal aorta of all  surviving animals.  Determinations carried out included: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white  blood cell count, differential white blood cell count, prothrombin time,  thrombocyte count. The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were calculated.
- Biochemistry: at necropsy at the end of treatment, after overnight fasting, blood samples were taken from the abdominal aorta of 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 and renal concentration test: Shortly before the end of the treatment (days 86-87), 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 (one rat per cage) and urine was collected. The concentrating ability of the kidneys was investigated by measuring the volume and density of the individual samples. The determinations carried out with the urine collected in the renal concentration test included: appearance of the urine, glucose, pH, occult blood, ketones, protein, bilirubin, urobilinogen, microscopy of  the sediment.
Sacrifice and pathology:
- All animals were subjected to a complete gross necropsy. In the dose-range finding study, the animals were killed after 14 days of treatment. In week 14, the animals of the 13-week study were killed on a number of successive working days so that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under anaesthesia.

ORGANS EXAMINED AT NECROPSY:
- Macroscopic: organs weighed included adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroid (with parathyroids), uterus.  
- Histopathological examination was performed on tissues and organs of all animals of the control and treatment groups 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.
Other examinations:
A 14-day dose range finding study was performed to determine the dose levels to be used in the main study. For this purpose 4 animals per sex per dose were administered the test material daily via the diet at dose levels of 0, 100, 300, 1000, 5000 mg/kg diet. In the dose range finding study, the animals were examined for clinical signs, mortality, body weights, food consumption and gross necropsy (adrenals, kidneys, liver, spleen, testes and ovaries).
Statistics:
- Test material analysis:
Homogeneity: one way analysis of variance (Anova) using the sample location (1-5) as grouping factor. The test material was considered to be homogeneously distributed in the diets if p > 0.01 and/or if the relative standard deviation (RSD) between the samples means was less than or equal to 15 %.
Stability: one way analysis of variance (Anova) using time as grouping factor. The test material was considered to be stable in the diets if p > 0.01 and/or if the mean concentration on the last day was between 80 and 120 % of the mean concentration on the first day (t =0).
Achieved concentration: for each concentration level, the mean of the concentrations, as measured in the diet samples used for the assessment of the homogeneity, was considered to represent the achieved concentration. The content of the test material in the diet was considered to be 'close to intended' if the mean measured concentration was between 80 and 120 % of the intended concentration.
- Body weight: one way analysis of covariance (covariate: body weight on  day 0) followed by Dunnett's multiple comparison tests.
- Neurobehavioral observations: parameters assessed during functional  observations were measured on different measurement scales (e.g. continuous, rank, categorical). Continuous measures were analysed by analysis of variance techniques and other parametric tests where appropriate. Rank and categorical data were analysed non-parametrically. Motor activity data were analysed using analysis of variance techniques.
- Food consumption and food efficiency: one way analysis of variance (Anova) followed by L.S.D. tests.
- Red blood cell and clotting potential variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, volume and density of the urine, organ weight: one way analysis of variance (Anova) followed by Dunnett's multiple comparison tests.
- Further details below

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related findings were observed.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights were similar among the groups in males and females throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Food consumption: Food consumption was slightly higher in males of the 7500 mg/kg group. This difference was statistically significant on days: 21, 35, 49 and 63. Furthermore, an occasional statistically significant difference was seen in the 300 and 1500 mg/kg groups. Food consumption in females was similar among the groups throughout the study. An occasional statistically significant difference was seen in all dose groups.
Overall intake of the test material for the 300, 1500 and 7500 mg/kg groups was 19, 96 and 521 mg/kg bw/day, respectively for males and 20, 101 and 533 mg/kg bw/day, respectively for females.
Analysis of the test material in diet samples revealed that the test material dose was close to the nominal level for all diets. Mean measured concentrations ranged from 93 to 109 % of nominal concentrations.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
- Food conversion efficiency: Similar among the groups in males and females throughout the study. An occasional significant difference was seen in males.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related ocular changes were observed.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The number of reticulocytes was statistically significantly increased in males of the 7500 mg/kg group. Mean corpuscular haemoglobin was statistically significantly decreased in females of the 7500 mg/kg group. Prothrombin time was statistically significantly decreased in males and females of the 7500 mg/kg group. The total numbers of white blood cells and lymphocytes were statistically significantly increased in males of the 7500 mg/kg group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males of the 7500 mg/kg group, ALP, ASAT, GGT, A/G ratio, bile acids, triglycerides, phospholipids and potassium were statistically significantly increased and sodium was statistically significantly decreased. In females of the 300 mg/kg group, ASAT was statistically significantly increased. In females of the 7500 mg/kg group, triglycerides were statistically significantly increased.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinary pH was higher in males of the 1500 and 7500 mg/kg groups. Further semi-quantitative and microscopic urinary observations were similar among the groups.
- Renal concentration test: Urinary density in males of the 1500 mg/kg group was statistically significantly lower relative to the control group. This change was not considered related to the treatment, in the absence of a dose-response relation. No other changes were observed in urinary volume and density among the groups.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test material.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute (males) and relative (males and females) liver weights were statistically significantly increased in the 7500 mg/kg group. No other statistically significant differences were observed among the groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related changes were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related changes were observed.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No treatment related changes were observed.
Other effects:
not examined
Details on results:
NOAEL: Although not accompanied by histopathological findings, the changes in haematology, clinical chemistry and liver weights are indicative of liver damage. On the basis of the increases in ASAT, GGT,  bile acids, triglycerides and relative liver weights in animals of the 7500 mg/kg group, the No Observed Adverse Effect Level (NOAEL) in the  sub-chronic toxicity study was placed at 1500 mg/kg diet. This level was equivalent to 96 mg/kg bw/day in males and 101 mg/kg bw/day for females.

OTHER: RANGE FINDING STUDY Dietary doses of 100, 300, 1000, and 5000 mg/kg feed (ppm) were administered for 14 days. No clinical abnormalities were observed. Body weights were not significantly different among the groups. Food consumption and food conversion efficiency were similar among the control and treated groups. Absolute and relative organ weights (ovaries, testes, adrenals, kidneys, spleen and liver) were not significantly different among the groups, except for the relative liver weight of the females of the 5000 mg/kg group which was slightly (ca. 8 %) but significantly increased. Macroscopic examination at necropsy did not reveal any treatment-related changes. Dietary exposure of the test materialup to 5000 mg/kg diet was well tolerated by rats for 14 days. The only possible treatment-related change was a  slight, but significant increase in liver weights of females of the 5000 mg/kg group. Dose for the main subchronic (13 -week) and satellite studies were chosen as 300, 1500, and 7500 mg/kg diet based on the dose-range finding study.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 mg/kg diet
Sex:
male/female
Basis for effect level:
other: The NOAEL of 1500 mg/kg diet was equivalent to 96 mg/kg bw/day in males and 101 mg/kg bw/day in females.
Key result
Dose descriptor:
LOAEL
Effect level:
7 500 mg/kg diet
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
other: Based on increases in liver enzymes, bile acids, triglycerides in blood, and relative liver weights in animals at the 7500 mg/kg diet group (equivalent to 521-533 mg/kg/day)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Although not accompanied by histopathological findings, the changes in haematology, clinical chemistry, and liver weights are indicative of liver damage. On the basis of increases in ASAT, GGT, bile acids, triglycerides, and relative liver weights in animals of the 7500 mg/kg diet group, the NOAEL in this subchronic toxicity study was placed at 1500 mg/kg diet. This level is equivalent to 96 mg/kg bw/day in males and 101 mg/kg bw/day in females.
Executive summary:

The toxicity of the test material was examined in Wistar rats using continuous administration in the diet for 13 consecutive weeks (OECD Test Guideline 408). In satellite groups of female rats, a reproduction/developmental screening study was also performed. The 13 -week study used four groups of 10 rats per sex per group. The control group was kept on an untreated diet and three test groups received diets containing 300, 1500, and 7500 mg/kg (ppm) of the test material in the feed. These dose levels were based on the results of a preceding dose-range finding study that used doses of 0, 100, 300, 1000, and 5000 mg/kg diet. Clinical observations, growth, food consumption, food conversion efficiency, neurobehavioral testing, ophthalmoscopy, haematology, clinical chemistry, renal concentration test, urinalysis, organ weights and gross examination at necropsy, histopathology examination were used for detection of toxicity.

The calculated intake values for the three test groups receiving 300, 1500, and 7500 mg/kg diet were 19, 96, and 521 mg/kg bw/day for males and 20, 101, and 533 mg/kg bw/day in females. A treatment-related increase in urinary pH was seen in males of 1500 and 7500 mg/kg groups, but this change was not considered toxicologically relevant. The males of the 7500 mg/kg group showed significant increases in reticulocytes, total white blood cells, lymphocytes, ALP, ASAT, GGT, bile acids, triglycerides, potassium, and absolute and relative liver weights, and decreased prothrombin time. In females of the 7500 mg/kg group, triglycerides and relative liver weights were significantly increased and prothrombin time was decreased. These changes were considered treatment-related and toxicologically relevant. No other treatment-related changes that were significant were observed in the high dose group, or the lower groups.

Although not accompanied by histopathological findings, the changes in haematology, clinical chemistry, and liver weights are indicative of liver damage. On the basis of increases in ASAT, GGT, bile acids, triglycerides, and relative liver weights in animals of the 7500 mg/kg diet group, the NOAEL in this subchronic toxicity study was placed at 1500 mg/kg diet. This level is equivalent to 96 mg/kg bw/day in males and 101 mg/kg bw/day in females.