Chlorotrioctylstannane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 December 2002 to 17 April 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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chlorotrioctylstannane

EC Number:

219-969-8

EC Name:

Chlorotrioctylstannane

Cas Number:

2587-76-0

Molecular formula:

C24H51ClSn

IUPAC Name:

chlorotrioctylstannane

Test material form:

liquid

Specific details on test material used for the study:

- Appearance: Colourless liquid
- Storage conditions of the test material: <-18 °C, in the dark
- Expiry date: 31 July 2004

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: (Crl:(WI)WU BR)
- Age at study initiation: 10 to 11 weeks old
- Mean weight at study initiation: Males: 261.7 g; females: 180.3 g. The weight variation of the animals used did not exceed 20 % of the mean weight for each sex.
- Housing: During the premating period the animals were housed in groups of 4/sex in cages of 48 x 37.5 x 21 cm with sterilised dust free saw shavings as bedding material and environmental enrichment (shreds of paper). For mating, one male and one female were housed together in cages of 42.5 x 26.6 x 15 cm. Mated females were housed individually in the same type of cages.
- Diet: Commercial rodent diet ad libitum
- Water: ad libitum, drinking water (tap-water) supplied in polypropylene bottles with a rubber stopper and a stainless steel nipple.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: Relative humidity was between 30 to 70 %
- Air changes: The room was ventilated with about 10 air changes per hour
- Photoperiod: Lighting was artificial with a sequence of 12 hours of light and 12 hours of darkness

IN-LIFE DATES
- From: 19 February 2003
- To: 17 April 2003

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- The test material was incorporated in the basal diet by mixing in a mechanical blender. The test material was weighed in a steel tray (160 g) and the tray rinsed with the diet. The weighed test material was mixed with approximately 3 kg of diet in a small Stephan mixer for 2 minutes. Thereafter, approximately 3 kg of weighed food was used to rinse the small Stephan mixer. Mixing was continued in the Lödige mixer for 2 minutes. This was then used to prepare the diet for the dose groups.
- The experimental diets were prepared once shortly before the start of the studies; they were stored in a freezer (<-18 °C) until use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

TEST MATERIAL ANALYSIS IN THE DIET
Before the start of the study, the analytical method was validated in the matrix under examination in a separate study.
In the batch of diets prepared for the main study, the achieved concentration was assessed as well as the stability upon storage at < -18 °C for the duration of the main study (50 days).

PRINCIPLE OF THE ANALYTICAL METHOD
From each diet sample, 2.0 g was transferred into a 50 mL Greiner tube. An aliquot of the internal standard solution in methanol was added. Subsequently methanol, acetate buffer solution (pH 4.5), 20 % aqueous sodium tetraethylborate (NaBEt4) solution and hexane (with naphthalene as internal standard) were added to each sample and this mixture was shaken and heated to 60 °C. During this step, the test material and some impurities were converted into the corresponding ethylated derivatives, which were extracted into the hexane layer. Prior to GC-MS analysis, the hexane layer was washed with 2 mol/L HCl in order to remove (most of) the ethylboron compounds that interfere with the GC-MS analysis. The concentration in feed was determined by GC-MS analysis of the hexane extracts.

ANALYSES
The homogeneous distribution, stability and achieved concentration of the test material in rat feed was analysed in the batch of diets prepared for the dose-range finding study. The same diet preparation protocol was used in the main study.
Directly after mixing of each diet for the dose-range finding study, samples for homogeneity/stability experiments were taken from the mixer. Firstly, five homogeneity samples (about 50 g each) were taken in the order: top centre, middle centre, bottom centre, left centre, right centre. Secondly, five samples (of about 50 g each) for examination of the stability were taken from the top centre part of the mixer. The samples taken for the homogeneity experiments were also used for determination of the content.

In addition, analyses to determine the content (achieved concentration) of the test material in the batch of diet used in the main study were conducted. Stability of the test material in the diet was also tested after the end of the main study after ca. 50 days (17 April 2003).
Diet samples for the determination of content of the diets used in this study were taken immediately after preparation of the diets and stored at ca. -18 °C pending analysis.

CRITERIA FOR HOMOGENEITY, STABILITY AND CONTENT OF THE TEST MATERIAL IN THE DIET
- Homogeneity: For each group a one way analysis of variance (ANOVA) was performed using the sample location (1-5) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the various locations in the sample). The test material was considered homogeneously distributed in the diets if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the sample means was less than or equal to 15 %.

- Stability: For each group a one way analysis of variance (ANOVA) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the difference between the results on the first day and the last day is significant). 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 study samples used for the assessment of the homogeneity were considered to represent the achieved concentration. The content of the test material in diet was considered to be `close to intended' if the mean measured concentration was between 80 and 120 % of the intended concentration.

CHROMATOGRAPHY
The concentration of the test material in the extracts was determined using GC-MS. For calculation of the amount of test material in diet samples, the peak area of an internal standard was used. Quantitation occurred using the calibration graphs constructed from the calibration solutions (Q-value (ratio peak area test material / peak area internal standard) vs. concentration).
The following conditions were used:
- Column: fused silica HP5 MS, 30 m, 0.25 mm ID, 0.25 µm film
- Pre-column: fused silica HP5 MS, 2.5 m, 0.25 mm ID, 0.25 µm film
- Column temperature: After 3 min at 45 °C the temperature was increased to 80 °C at a rate of 5 °C/min; then the temperature was increased to 260 °C at a rate of 15 °C/min; followed by 15 min at 260 °C
- Carrier: Helium; 1.5 mL/min constant flow
- Injection volume: 1 µL
- Injection temperature: Started at 60 °C, then increased to 300 °C at a rate of 14.5 °C/s; followed by 5 min at 300 °C
- Injection method: Splitless
- Ionisation: Electron impact, 70 eV
- Mass range: 60 - 600 amu

RESULTS
The test material was considered to be homogeneously distributed in all diets. Furthermore, the test material was considered to be stable in the diets upon storage at room temperature for 7 days and upon storage at < -18 °C for 50 days. The content of the test material was considered to be close to intended for all diets.
In view of these conclusions regarding the analysis of the diets, the nominal levels of the test material were used to calculate the test material intake.

Duration of treatment / exposure:

Males were treated for 30 days. Females were dosed for a 2 week pre-mating period, throughout mating and gestation and up to postnatal day 4 or 5.

Frequency of treatment:

Continuous in the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

4 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Doses were selected on the basis of a 14 day range-finding study in which the rats were dosed at 0, 300, 1000, 5000 and 10 000 mg/kg bw. Administration of the test material via the diet up to a level of 1000 mg/kg diet for a period of 14 days was well tolerated by the rats. At 5000 (high-mid-dose) and 10 000 (high-dose) mg/kg diet effects on body weight and food intake were observed. This effect was probably due to the palatability of the diet. Furthermore, at these dose levels, the absolute and relative thymus weight was statistically significantly decreased.
- Rationale for animal assignment: Shortly before the start of the studies, the animals (males and females separately) were allocated to the various treatment groups by computer randomisation and proportionally to body weight.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity starting from the beginning of the study. 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were conducted outside the home cage prior to the first exposure and then once weekly in all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female rats were recorded on day -2 (randomisation) and on days 0 (first day of dosing), 7 and 13 of the pre-mating period. Males were weighed weekly during the mating period until sacrifice. Females were weighed during mating (day 0, 7 and 13) and mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 1 and 4 of lactation. All animals were weighed on the day of sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption of male rats was measured weekly (days 0-7, 7-13 and 21-28), except during the mating period.
- Food consumption of female rats was measured weekly during the pre-mating period (days 0-7, 7-13). Food consumption of mated females was recorded weekly during gestation (gestation days (GD) 0-7, 7-14 and 14-21) and once during lactation (postnatal days (PN) 1-4).

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to the end of the premating period (12 March 2003) blood was taken by means of orbital puncture. K2-EDTA was used as anticoagulant.
- Anaesthetic used for blood collection: Yes, under CO2/O2 anaesthesia
- Animals fasted: Yes, fasted overnight
- How many animals: 5 rats/sex/group
- The following parameters were evaluated: Haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: Prior to the end of the premating period (12 March 2003) blood was taken by means of orbital puncture. Blood was collected in heparinised plastic tubes and plasma was prepared by centrifugation.
- Anaesthetic used for blood collection: Yes, under CO2/O2 anaesthesia
- Animals fasted: Yes, fasted overnight
- How many animals: 5 rats/sex/group
- The following measurements were made in the plasma collected at necropsy: Fasting glucose, alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio of albumin to globulin, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium, sodium, potassium, chloride and inorganic phosphate.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Spontaneous motor activity measurements and Functional Observational Battery (FOB) were performed in 5 males/ group prior to the end of dosing and in 5 females/ group prior the end of lactation (PN 4) randomly selected from each group.
- Dose groups that were examined: All dose groups
- Battery of functions tested: lacrimation, salivation, pupil response to light, palpebral closure, piloerection, defecation, urination, gait, mobility, forelimb and hindlimb grip strength, landing foot splay, righting reflex, response to tail pinch, click, touch and approach of a visual object, clonic and tonic movements, ease of removal, handling reactivity, arousal, vocalisations, rearing, motor activity and body temperature.

OTHER:
- Parturition and litter evaluation
- Litter size, sexes and pup weight

Sacrifice and pathology:

SACRIFICE
All male and female parent rats were sacrificed by decapitation after CO2/O2 anaesthesia, except 5 animals/sex/group which were sacrificed by exsanguination from the abdominal aorta under CO2/O2 anaesthesia. All sacrificed animals were examined grossly for pathological changes.

GROSS PATHOLOGY
Samples of the following tissues and organs of all parental animals were preserved in a neutral aqueous phosphate-buffered 4 % solution of formaldehyde (except for the testes which were preserved in Bouin's fixative): ovaries, uterus (after counting the implantation sites; if necessary the implantation sites were made visible according to Salewski), testes, epididymides, seminal vesicles, prostate and organs and tissues showing macroscopic abnormalities.

In addition for 5 animals/sex/group, randomly selected from each group, the following organs were preserved: adrenals, axillary lymph node, bone marrow (femur), brain, caecum, coagulation glands, colon, duodenum, eyes, heart, ileum, jejunum, lungs, kidneys, liver, mammary gland (females only), mesenteric lymph node, parathyroids, Peyer's patches, pituitary, rectum, sciatic nerve, spinal cord, spleen, stomach, thymus, thyroids, trachea, and urinary bladder.

HISTOLOGY
Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 µm, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin.
Microscopic examination was performed on the collected organs of all rats of the control and high-dose groups. Examination was extended to the mesenteric lymph nodes of the male and female rats of the low- and mid-dose groups and to the thymus of the male and female rats of the low and mid-dose groups because of the effect in the high-dose group for this tissue/organ.
In addition, reproductive organs of males that failed to sire (mated female which was not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined.

OTHER
A necropsy was performed on stillborn pups and pups that died during the study; macroscopic abnormalities were recorded. Pups were examined externally for gross abnormalities and killed by hypothermia at <-18 °C on PN 4 or shortly thereafter.

Statistics:

- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- Fisher's exact probability test used to evaluate the number of mated and pregnant females and females with live pups.
- Number of implantation sites, live and dead pups were evaluated by Kruskal- Wallis nonparametric analysis of variance followed by the MannWhitney U-test.
- Red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights: one-way ANOVA followed by Dunnett's multiple comparison tests (treatment period).
- Reticulocytes and relative differential white blood cell counts: Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests.
- Histopathological changes: Fisher's exact probability test.
- Parameters assessed during functional observations were measured on different measurement scales. Continuous measures were analysed by one-way analysis of variance at each test time point, followed by post-hoc group comparisons in case of a significant result. Rank order data were analysed by Kruskal-Wallis analysis of variance at each test time point, followed by planned multiple comparisons between dose groups in case of a significant result. Categorical data were analysed by Pearson chi-square analysis.
- Motor activity data were analysed using one-way analysis of variance at each test time point, followed by post-hoc group comparisons in case of a significant result. All tests were two-sided. A level of probability of <0.05 (p< 0.05) was considered significant.
Statistical evaluations on variables associated with pups were considered on a litter basis. Additional evaluations on a pup basis were performed to attempt to identify any specific dose-related effect that may have occurred.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs were observed in the males from the start of the study until sacrifice.
Females showed no clinical signs during the premating period and gestation period apart from 1 animal of the mid-dose group (GD 21)) and 1 animal of the high-dose group (from GD 7 onwards) which were sparsely haired. During the lactation period 2 control animals (PN 4 and PN 1-4), 2 animals of the low-dose group (PN 4 and PN 4-5), 1 animal of the mid-dose group (PN 0-4) and 3 animals of the high-dose group (PN 3, PN 1 and 2 and PN 0-4) were sparsely haired. In addition, one animal in the high dose group showed a pale appearance on PN 1.

Mortality:

no mortality observed

Description (incidence):

No mortalities were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight of the male animals of the high-dose group was statistically significantly decreased from day 7 until sacrifice on day 30. Mean body weight change of the male animals of the high-dose group was statistically significantly decreased between days 0-7, 7-13 and 13-21.
During the premating period body weight of the female animals of the high-dose group was statistically significantly decreased on day 7 and 13 of the study; body weight change was statistically significantly decreased from day 0-7. During the gestation period body weight of the female animals of the high-dose group was statistically significantly decreased on GD 7, 14 and 21; body weight change was statistically significantly decreased from GD 0-7 and 7-14. During the lactation period body weight of the female animals of the high-dose group was statistically significantly decreased on PN 1 and 4; body weight change was decreased (not significantly) from PN 1-4.
Body weight and body weight change of the male and female animals of the low- and mid-dose groups were comparable to the control group during the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption expressed as g/animal/day or as g/kg body weight/day of the male animals of the high-dose group was statistically significantly decreased from days 0-7 and days 7-13 or from days 0-7, respectively. In the male animals of the high-dose group, the food consumption from days 21-28 could not be measured because some males were still caged with the females for mating. Mean food consumption expressed as g/kg body weight/day of the male animals of the low-dose group was statistically significantly increased from days 7-13; this observation was considered to be a fortuitous finding.
During the premating period, mean food consumption expressed as g/animal/day or as g/kg body weight/day of the female animals of the high-dose group was statistically significantly decreased from days 0-7.
During the gestation period, mean food consumption expressed as g/animal/day or as g/kg body weight/day of the female animals of the high-dose group was statistically significantly decreased from GD 0-7, 7-14 and 14-21. In females of the mid-dose group mean food consumption was expressed as g/animal/day was statistically significantly decreased from GD 7-14.
During the lactation period, mean food consumption expressed as g/animal/day or as g/kg body weight/day of the female animals of the high-dose group was statistically significantly decreased from PN 1-4.
During the study, no statistically significant decrease in food consumption was observed in the male animals of the low- and mid-dose groups when compared to the control group.
Apart from the statistically significant decrease in the females of the mid-dose group from GD 7-14, no statistically significant decrease in food consumption was observed during the gestation and lactation period in the females of the low- and mid-dose groups when compared to the control group.

The test material intake of the male animals of the low-, mid-, and high-dose groups, respectively, was:
- Premating period
Day 0-7: 21, 51 and 240 mg/kg body weight/day
Day 7-13: 20, 49 and 248 mg/kg body weight/day
- After mating
Days 21-28: 18, 45 and * mg/kg body weight/day
*food consumption could not be measured because the males were still caged with the females (second week of the mating period).
The test material intake of the female animals of the low-, mid-and high-dose groups, respectively, was:
- Premating period
Day 0-7: 19, 45 and 200 mg/kg body weight/day
Day 7-13: 19, 48 and 227 mg/kg body weight/day
- Gestation period
GD 0-7: 22, 53 and 234 mg/kg body weight/day
GD 7-14: 22, 50 and 210 mg/kg body weight/day
GD 14-21: 17, 41 and 170 mg/kg body weight/day
- Lactation period
PN 1-4: 26, 62 and 242 mg/kg body weight/day

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In the male animals of the low- and high-dose group haemoglobin was statistically significantly increased and in the male animals of the low-dose group the packed cell volume was statistically significantly increased as compared to the control animals. These findings were not dose-related and therefore not considered to be a treatment-related finding. The decrease in white blood cells seen in the high-dose group and the decrease in absolute number of monocytes in the mid- and high-dose groups were considered to be treatment-related findings.
In the female animals the statistically significant differences between the control and the test material treated animals were limited to an increase in mean corpuscular haemoglobin concentration in the low-, mid- and high-dose groups. Since no dose-relation was found for this finding, and because the measured main red blood cell variables (RBC, PVC) showed no significant changes, the increase in mean corpuscular haemoglobin was not considered to be toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The increase of aspartate aminotransferase, gamma glutamyl transferase, and the increase of cholesterol (total), triglycerides and phospholipids as observed in the males of the low-dose group were not confirmed in the mid- and high-dose group. Therefore these findings were not considered to be toxicologically relevant. Furthermore, in the male animals of the high-dose group a statistically significant increase in albumin/globulin ratio and a statistically significant decrease in phospholipids were observed.
In the female animals the following statistically significant findings were observed: decrease in total protein in the high-dose group, decrease in albumin in the low-and high-dose group and an increase in albumin/globulin ratio in the high-dose group. In addition, a decrease in albumin was detected in the mid-dose group which did not achieve statistical significance. A decrease in calcium was observed in the high-dose group.
These findings in the clinical chemistry were not accompanied by any histopathological alterations and therefore are considered of little if any toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In high-dose females, neurobehavioural testing at the end of the study on PN 4 revealed decreases in fore- and hind-limb grip strength and body temperature. In view of the fact that treatment-related signs of systemic toxicity were observed in these females, the decreases in grip strength were considered to be a corroborative finding to their reduced growth (more than 20 % less than that of control animals), rather than an indication of a neurotoxic potential of the test material. The decrease in body temperature might also be a reflection of their impaired health status as a result of test material-induced systemic toxicity. No changes indicative of neurotoxic potential of the test material were observed in the neurobehavioural observations and motor activity assessment in males when tested up to 4 weeks of treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the males of the high-dose group, terminal body weight was statistically significantly decreased and the absolute and relative thymus weights of the male animals of this group were statistically significantly decreased. In addition, thymus weight (absolute and relative) of the male animals was, not statistically significantly, decreased in the male animals of the mid-dose group; absolute (0.463 control versus 0.343 g mid-dose) and relative (1.364 control versus 1.007 g/kg body weight mid-dose).
In the females of the high-dose group, terminal body weight was statistically significantly decreased. The absolute weight of the adrenals of the female animals of the high-dose group was statistically significantly decreased; no effect was observed for the relative adrenal weight in this group. Absolute brain weight of the female animals of the high-dose group was comparable amongst the group. The relative brain weight (g/kg body weight) of the female animals of the high-dose group was increased; this increase was due to the decrease in body weight.
The absolute and relative thymus weight of the female animals of this group was statistically significantly decreased. In addition, thymus weight (absolute and relative) of the female animals of the mid-dose group was, not statistically significantly, decreased; absolute (0.125 control versus 0.088 g mid-dose) and relative (0.612 control versus 0.418 g/kg body weight mid-dose).
No other effects on organ weights (absolute and relative) were observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic observations reported are common findings in rats of this strain and age and occurred only incidentally. The decreased size of the thymus was not reported as a gross lesion as decreases in thymus weights were considered to reflect more accurately the diminished size of the thymus in individual animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Examination of the thymus revealed lymphoid depletion in 5/5 high-dose males and 4/4 high-dose females (thymus of 1 female of the high-dose was lost during processing). Lymphoid depletion was characterised by a decrease in the size of the thymic lobules because of an extensive loss of cortical and medullary small lymphocytes. Consequently the distinction between the cortical and medullary areas was blurred. In severe cases the cortex was very small, or partially absent. The remaining lymphoid cells visible in the cortical areas were mainly lymphoblasts. Lymphoblastic cells, reticular epithelial cells had increased, and/or greater numbers of these cells were visible because of disappearance of small lymphocytes and collapse of thymic stroma. Two mid-dose females showed lymphoid depletion in the thymus. In one female control animal thymic lymphoid depletion accompanied by tingible body macrophages was noted, which was interpreted as a case of accidental stress-related involution of the thymus. This animal, in addition, had stomach ulceration. One mid-dose and two low-dose females showed involution of the thymus. The thymic lobules in these animals were decreased in size, but exhibited normal histology. This phenomenon may be interpreted as pregnancy/lactation involution.
The mesenteric lymph nodes showed very slight to slight paracortical lymphoid depletion in 3 males and 3 females of the high-dose group and 1 mid-dose female. In the mesenteric lymph nodes of high-dose animals the presence of clusters of swollen yellowish macrophages in the medullary cords, and occasionally in the paracortical areas, was conspicuous. The medullary cords had been enlarged by these macrophage accumulations. Several low-and mid-dose animals had very slight to slight macrophage accumulations. In addition, 2 mid-dose females had moderate macrophage accumulations.
Administration of the test material caused treatment-related lymphoid depletion in the thymus in the mid- and high-dose group, paracortical depletion and the presence of clusters of swollen yellowish macrophages in the mesenteric lymph nodes, pointing to elimination by macrophages of unwanted/foreign material, most probably derived from intestinal absorption, for no other organs showed similar macrophages. These macrophages were also seen in the mesenteric lymph nodes of low-dose animals. The toxicological significance of this finding is not clear.
Examination of the reproductive organs did not reveal a treatment-related effect. All other changes observed represent common findings in rats of this strain and age and/or occurred in one or a few animals only or at comparable incidences in the treated group(s) and control.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant effects on reproduction and litter data were observed in the high-dose group: prolonged pre-coital time, decreased number of females with liveborn pups, increased number of females with stillborn pups, increased number of females with only stillborn pups, decreased gestation index, increased post-implantation loss, decreased number of pups delivered, decreased number of liveborn pups, increased number of stillborn pups, increased pup mortality postnatal day (PN) 4, decreased pup weight PN 1 and 4 and increased number of runts PN 1 and 4. In the mid-dose group the only effect on reproduction and litter data was a statistically significant decrease in the number of liveborn pups/litter. No effect on reproduction and litter data was observed in the low-dose group.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Effect level:

20 - 21 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

3

Key result

Dose descriptor:

NOAEL

Effect level:

17 - 26 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day (nominal)

System:

cardiovascular

Organ:

blood

thymus

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1: Mean Body Weights (g)

		Dose Group (mg/kg)
Sex	Day	Control	300	750	4000
Male	0	262.20 d	260.68	262.06	261.68
	7	290.08 d	291.26	287.37	278.36*
	13	310.52 d	312.62	306.25	292.50**
	21	320.54 d	324.55	317.42	294.76***
	28	335.50 d	340.11	331.70	305.12***
Female	0	179.76 d	178.88	181.81	180.64
	7	188.70 d	187.97	187.68	178.31*
	13	193.67 d	191.50	191.52	181.56*

d = ANOVA & Dunnett test

* = p<0.05

** = p<0.01

*** = p<0.001

 

Table 2: Mean Body Weight Change (g)

		Dose Group (mg/kg)
Sex	Day	Control	300	750	4000
Male	0 to 7	27.88 d	30.57	25.31	16.68***
	7 to 13	20.43 d	21.37	18.88	14.14**
	13 to 21	10.02 d	11.93	11.17	2.26**
	21 to 28	14.96 d	15.56	14.28	10.36
Female	0 to 7	8.94 d	9.09	5.87	- 2.33***
	7 to 13	4.97 d	3.53	3.84	3.25

d = ANOVA & Dunnett test

** = p<0.01

*** = p<0.001

 

Table 3: Mean Maternal Body Weight and Mean Body Weight Change during Gestation and Lactation (g)

Parameter	Time (Day)	Dose Group (mg/kg)
		Control	300	750	4000
Mean maternal body weight during gestation	0	191.31 d	186.81	190.30	181.30
	7	212.32 d	205.13	209.15	189.48***
	14	236.28 d	226.86	228.44	195.22***
	21	264.38 d	256.43	252.50	213.09***
Mean maternal body weight change during gestation	0 to 7	21.02 d	18.32	18.85	8.18***
	7 to 14	23.96 d	21.73	19.29	5.74***
	14 to 21	27.29 d	29.57	24.73	17.87
Mean maternal body weight during lactation	1	191.63 d	185.10	183.68	160.74**
	4	212.41 d	202.76	201.27	177.40**
Mean maternal body weight change during lactation	1 to 4	19.90 d	17.66	17.99	11.63

d = ANOVA & Dunnett test

** = p<0.01

*** = p<0.001

Table 4: Mean Terminal Body Weight, Mean Thymus Weight and Mean Thymus Weight Relative to Terminal Body Weight

Sex	Parameter	Dose Group (mg/kg)
		Control	300	750	4000
Male	Terminal body weight (g)	342.9 d	348.9	340.7	315.1**
	Absolute thymus weight (g)	0.463 d	0.453	0.343	0.129***
	Relative thymus weight (g/kg bw)	1.364 d	1.332	1.007	0.399***
Female	Terminal body weight (g)	210.4 d	201.5	201.4	164.2***
	Absolute thymus weight (g)	0.125 d	0.120	0.088	0.055**
	Relative thymus weight (g/kg bw)	0.612 d	0.585	0.418	0.324**

d = ANOVA & Dunnett test

** = p<0.01

*** = p<0.001 

Applicant's summary and conclusion

Conclusions:

The NOAEL for general toxicity was established on the low-dose level of 300 mg/kg diet, which is equivalent to 20 - 21 mg/kg body weight/day for the male animals and 17 - 26 mg/kg body weight/day for the female animals.

Executive summary:

The repeated dose toxicity of the test material was investigated in a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test conducted in accordance with the standardised guideline OECD 422 under GLP conditions.

Following a dose range-finding study, 12 Wistar rats per dose level were fed diets containing 0, 300, 750 and 4000 mg test material/kg diet for up to 30 days (males) or for a 2 week premating period, followed by mating and gestation and up to postnatal day 4 or 5 (females).

The following evaluations were conducted: cage side observations, detailed clinical observations, body weight, food consumption and compound intake, haematology, clinical chemistry and neurobehavioural examination. Reproductive parameters were also evaluated.

Male animals were sacrificed on day 30. Female animals with a litter were sacrificed on PN 4 or 5. Not pregnant animals between GD 23-27.

The test material was considered to be homogeneously distributed in all diets. Furthermore, it was considered to be stable in the diets upon storage at room temperature for 7 days and at < -18 °C for 50 days. The content of the test material was considered to be close to intended for all diets.

During the study no mortalities or treatment-related clinical signs were observed.

Mean body weight and body weight change in male and female animals of the high-dose group were decreased during the entire study; at the majority of the days and periods this effect was statistically significant. Body weight and body weight change of the male and female animals of the low-and mid-dose groups were comparable to the control group during the study.

Food consumption was statistically significantly decreased in the male and female animals of the high-dose group during almost the entire study. No treatment-related effects on food consumption were observed in the male and female animals of the low- and mid-dose groups.

In male animals tested after up to 4 weeks of treatment, no changes indicative of neurotoxic potential of the test material were observed during the neurobehavioural observations and motor activity assessment.

Decreases in fore- and hind-limb grip strength in female animals of the high-dose group were considered to be a corroborative finding to their reduced growth, rather than an indication of a neurotoxic potential of the test material. Decreased body temperature of the females of the high-dose group might also be a reflection of their impaired health status as a result of test material-induced systemic toxicity.

At the end of the premating period a decrease in white blood cells in the male animals of the high-dose group and a decrease in absolute number of monocytes in the male animals of the mid- and high-dose group were observed; these findings were considered to be treatment-related. In the female animals no treatment-related findings on red and white blood cell parameters were observed.

At the end of the premating period in the male animals of the high-dose group a statistically significant increase in albumin/globulin ratio and a statistically significant decrease in phospholipids were observed. In the female animals a statistically significant decrease in total protein in the high-dose group, a statistically significant decrease in albumin in the low- and high-dose group and an increase in albumin/globulin ratio in the high-dose group were observed. In addition, a decrease in albumin was detected in the mid-dose group which did not achieve statistical significance. A decrease in calcium in the female animals of the high-dose group was measured. These findings in the clinical chemistry were not accompanied by any histopathological alterations and therefore considered of little if any toxicological significance.

In the animals of high-dose group terminal body weight at necropsy was decreased. The absolute and relative weights of the thymus were statistically significantly decreased in male and female animals of the high-dose group. In addition, animals of the mid-dose group showed a (not statistically significant) decrease in absolute and relative thymus weight by approximately 25 and 30 % in the male and female animals, respectively. In females of the high-dose group a statistically significant decrease in absolute adrenal weight and a statistically significant increase in relative brain weight were observed.

No other effects on organ weights of the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus were observed. At necropsy no treatment-related macroscopic findings of parental animals were observed.

Administration of the test material caused treatment-related lymphoid depletion in the thymus in the mid- and high-dose group, paracortical depletion and the presence of clusters of swollen yellowish macrophages in the mesenteric lymph nodes. However, macrophage accumulations in the mesenteric lymph nodes occurred also in low-dose animals. The toxicological significance of this finding is not clear. Examination of the reproductive organs did not reveal a treatment-related effect.

The following statistically significant effects on reproduction and litter data were observed in the high-dose group: prolonged pre-coital time, decreased number of females with liveborn pups, increased number of females with stillborn pups, increased number of females with only stillborn pups, decreased gestation index, increased post-implantation loss, decreased number of pups delivered, decreased number of liveborn pups, increased number of stillborn pups, increased pup mortality postnatal day (PN) 4, decreased pup weight PN 1 and 4 and increased number of runts PN 1 and 4. In the mid-dose group the only effect on reproduction and litter data was a statistically significant decrease in the number of livebom pups/litter. No effect on reproduction and litter data was observed in the low-dose group.

In conclusion, based on the observed effects in the mid-dose group, decrease in monocytes in the plasma, thymus weight, microscopic findings in the thymus and decreased number of liveborn pups/litter, the No Observed Adverse Effect Level (NOAEL) for general and reproductive toxicity was established on the low-dose level of 300 mg/kg diet, which is equivalent to 20 - 21 mg/kg body weight/day for the male animals and 17 - 26 mg/kg body weight/day for the female animals.