Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 February 2020 to 25 September 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4,6,8-tetramethylcyclotetrasiloxane
EC Number:
219-137-4
EC Name:
2,4,6,8-tetramethylcyclotetrasiloxane
Cas Number:
2370-88-9
Molecular formula:
C4H16O4Si4
IUPAC Name:
2,4,6,8-tetramethyl-1,3,5,7,2,4,6,8-tetroxatetrasilocane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 35 to 41 days
- Weight at study initiation: Males: 131 to 204 g; Females: 106 to 153 g
- Fasting period before study: no
- Housing: Three or four of the same sex (main study and recovery), unless reduced by mortality or isolation.
- Diet (e.g. ad libitum): Teklad 2014C Diet ad libitum
- Water (e.g. ad libitum): Potable water from the public supply ad libitum
- Acclimation period: 7 days before commencement of treatment

DETAILS OF FOOD AND WATER QUALITY: No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 hours light: 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
dried and deacidified corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The formulations were prepared in ascending order of concentration and prepared in a glove box under nitrogen. Dose formulations were prepared weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 12.5, 37.5, 150 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of formulations during storage were confirmed as part of another study and samples for homogeneity and stability analysis were not performed during the study. Samples of each formulation prepared for administration in weeks 1 and 12 of treatment were analysed for achieved concentration of the test item.
Duration of treatment / exposure:
13 weeks followed by a 4-week recovery period
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control group
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
low dose group (LD)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
middle dose group (MD)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
high dose group (HD)
No. of animals per sex per dose:
Main groups: 10 males and 10 females per group
Recovery groups: 5 males and 5 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous acute oral toxicity test the test substance did not show any evidence of toxicity at the dose level of 2000 mg/kg bw after single oral administration to five female rats, observed over a period of 14 days (Harlan Laboratories, 2010). All animals survived until the end of the study period. The only clinical sign noted was slightly ruffled fur in 1/5 animals which resolved by study Day 4. The LD50 was greater than 2000 mg/kg bw in female rats.
The doses used in this study (0, 50, 150 and 600 mg/kg bw/day) were selected based on the pathology findings in a preliminary toxicity study (Covance, 2020) in which dose levels of 0, 250 and 750 mg/kg bw/day were administered for 21 days and a dose level of 1000 mg/kg bw/day was administered for 14 days. There was no evidence of toxicity during the in-life phase. There were, however, findings in the kidneys (tubular basophilia with pelvic dilatation and accumulation of hyaline droplets in males given 750 or 1000 mg/kg bw/day, erosion of the urothelium in individual males at these doses and urothelial hyperplasia and foreign material in the renal pelvis in a few males given 750 mg/kg bw/day) and urinary bladder (ulceration with associated inflammation, edema and urothelial hyperplasia at 750 and 1000 mg/kg bw/day, foreign material in the lumen in one male given 750 mg/kg bw/day and urothelial vacuolation in one male given 1000 mg/kg bw/day). These findings indicated that the highest dose in the 90-day study should be below 750 mg/kg bw/day. Therefore, the highest dose in the main 90-day study, was chosen with the aim to induce toxicity but no severe suffering or death.

- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Rationale for selecting satellite groups: To assess recovery or persistance of effects. Recovery groups were included in the control and high dose animals.
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): random
Positive control:
not used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for evidence of ill-health or reaction to treatment; signs associated with dosing were checked daily during the first week of treatment and twice weekly during Weeks 2 to 13; A viability check was performed near the start and end of each working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

FOOD CONSUMPTION:
- Time schedule for examinations: The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No effect was observed and consequently, quantitative measurements were not performed

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretreatment and on week 13
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, recovery week 4
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13, recovery week 4
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: week 13, recovery week 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: Sensory reactivity, grip strength, motor activity assessments

IMMUNOLOGY: No

OTHER: Oestrous cycles
- Time schedule for examinations: Daily smears were taken for 4 days before scheduled termination at the end of either the treatment or recovery period
- Dose groups that were examined: all female animals

THYROID HORMONE ANALYSIS
- Time schedule for examinations: week 13 and week 14 at necropsy, recovery week 4 and recovery week 5 at necropsy
- Dose groups that were examined: all animals
- Anaesthetic used for blood collection: Yes (isoflurane)
- Parameters analysed: triiodothyronine (T3); thyroxine (T4); thyroid stimulating hormone (TSH)


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)

HISTOPATHOLOGY: Yes (see table 4)
Statistics:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead. Where there were only two groups, comparisons were made using t-tests. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed. Where there were only two groups, comparisons were made using Wilcoxon rank sum tests. For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, Fisher’s exact tests were performed. For organ weight data, analysis of covariance was performed using terminal body weight as covariate, unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights. Significant differences between the groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The test substance caused piloerection and hunched posture during the first week of treatment.
Piloerection was observed at all doses and in both sexes, with the incidence of affected animals and frequency of the finding being dose-related. Piloerection occurred most commonly on Day 2 and was evident throughout the day in most cases. In some animals, particularly in those receiving 600 mg/kg bw/day, this was observed initially at the end of Day 1 and persisted through to Day 3 or 4. There was also transient piloerection on Day 77 in a high dose male but this isolated occurrence was not attributed to treatment. Two high dose females (Nos. 149 and 150) displayed hunched posture, with this being first observed 1 to 2 hours post-dose or at the end of the working day on Day 1 and persisting through Day 2, but was not evident from Day 3. A low dose male showed similar hunched posture but as this was not observed at higher doses in males it is of uncertain relationship to treatment.
Transient chin rubbing, salivation and paddling of forepaws occurred at the time of dose administration at 150 and 600 mg/kg bw/day in males and females. Paddling of the forepaws also occurred in a control male and chin rubbing occurred in a control female. These are commonly observed signs in studies where the test material has been administered by gavage and, as such, are considered of no toxicological importance.
Mortality:
no mortality observed
Description (incidence):
There were no treatment-related deaths. Two deaths occurred during the treatment period, neither of which was attributed to treatment.
Control female No. 143 was euthanised for welfare reasons on Day 3 due to general poor clinical condition where the animal displayed breathing difficulties (gasping and laboured breathing), piloerection and dull eyes. The macroscopic examination revealed a perforated oesophagus and adhesions of the lungs and bronchi that indicated that its condition was a consequence of a dose administration error.
Male No. 39 (150 mg/kg bw/day) was found dead in Week 10 but there were no significant signs ante mortem. The cause of death was not determined at the macroscopic or histopathological examination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The administration of the test substance to males had no effect on their body weights during the first four weeks of treatment but between Week 4 and 13 there was a reduction of weight gain at 150 and 600 mg/kg bw/day, the extent of which was dose related. Between Week 4 and 13 the weight gains at 150 and 600 mg/kg bw/day were approximately 13 and 18% lower than that of the control males during the same period, but only the difference at 600 mg/kg bw/day attained statistical significance. This resulted in overall (Week 0-13) weight gains being approximately 3 and 8% lower than the control males.
In females there was no effect of treatment on their body weights during the first eight weeks but between Week 8 and 13 there was a reduction of weight gain at 150 and 600 mg/kg bw/day, the extent of which was dose related. Between Week 8 and 13 the weight gains at 150 and 600 mg/kg bw/day were approximately 44 and 51% lower than that of the control females during the same period. Since this effect occurred late in the study, the overall body weights were not significantly different from the control females.
During the four-week recovery period the animals that previously received 600 mg/kg bw/day gained weight but the rate of weight gain was lower than the control, particularly in females, indicating that there was no significant recovery after treatment ceased.
There was no effect of treatment upon the body weights of males and females receiving 50 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption during either the treatment or recovery period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related ophthalmoscopic findings.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematological examination in Week 13 indicated, when compared to controls, high lymphocyte, eosinophil, monocyte and large unstained cell count in males receiving 600 mg/kg bw/day, resulting in a consequential increase of the total leucocyte counts in these animals. The majority of individual values were, however, within the background range. The haematological examination in Week 13 indicated, when compared to controls, high lymphocyte, eosinophil, monocyte and large unstained cell count in males receiving 600 mg/kg bw/day, resulting in a consequential increase of the total leucocyte counts in these animals. The majority of individual values were, however, within the background range.
Platelet counts were slightly higher than controls in females receiving 600 mg/kg bw/day, though the majority of individual values were within the background range and this finding persisted to the end of the recovery period. These animals showed a small prolongation of prothrombin time and a slight shortening of activated partial thromboplastin time, though most individual values were within background ranges,which were no longer apparent at the end of the recovery period.
There were a few statistically significantly differences from controls reported for some of the erythrocyte indices (low mean cell volume and red cell distribution width in males receiving 600 mg/kg bw/day and low mean cell haemoglobin and mean cell volume in females receiving 600 mg/kg bw/day). With the exception of the red cell distribution width in the high dose males, where five values were below the background range, the majority of values were within the background range. There was, however, no effect upon any other erythrocyte index, particularly haemoglobin concentration, and, in consequence, these findings were considered of no toxicological significance, despite still being evident at the end of the recovery period.
All other differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma in Week 13 indicated, when compared to controls, high urea/blood urea nitrogen concentration in males receiving 600 mg/kg bw/day. Six of the males (Nos. 4, 5, 6, 7, 9 and 10) had blood urea nitrogen concentrations that were notably above the range reported in the control males, three of which (Nos. 4, 5 and 6) had values that exceeded the background range (2.37 to 9.04 mmol/L for urea; n=89), but their plasma creatinine concentrations were similar to controls. There was full recovery by the end of the recovery period. Females were unaffected at the end of the treatment period but at the end of the recovery period one previously treated female (No. 146) showed a marked increase of both urea/blood urea nitrogen and creatinine concentration (18.2 mmol/L and 85 μmol/L, respectively) that was well above the background range (3.99 to 9.85 mmol/L for urea (n=90) and 30 to 59 μmol/L for creatinine (n=100)).
There was a small increase of total cholesterol concentration in males and females receiving 600 mg/kg bw/day but the majority of values were within the background range (0.90 to 2.83 mmol/L for males (n=99) and 1.01 to 3.45 mmol/L for females (n=100)). This was due to increased high and low density lipoprotein concentration. These findings showed full recovery. There was no associated effect on plasma triglyceride concentrations.
There was a reduction of the albumin to globulin ratio in males and females receiving 600 mg/kg bw/day but the majority of values were within the background range (1.09 to 1.57 for males (n=99) and 1.25 to 1.87 for females (n=100)). In the absence of any alteration of plasma albumin concentration, this was attributed to a small increase in the globulin fractions of these animals. This finding showed full recovery in males but remained low in females where there was no evidence that any recovery had occurred and, in these animals, there was a reduction of albumin concentration.
All other differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The analysis of urine samples obtained in Week 13 indicated, when compared to controls, high volume and low urinary specific gravity in males and females receiving 600 mg/kg bw/day and blood was detected in the urine of three males and two females at this dose. An increase in total urinary protein and, to a lesser extent, glucose and a significant decrease in creatinine was observed in males and females receiving 600 mg/kg bw/day. The protein output in one of these males (No. 9) was severely increased compared to controls (50.93 mg; control range was 1.44 to 3.76 mg).
By the end of the four-week recovery period there were two animals (one male and one female) that showed marked effects, but the urinary composition for the remaining animals, that had previously received 600 mg/kg bw/day, was similar to that of the controls. Male No. 48 had a moderately high urinary volume and low specific gravity, leading to pale yellow colouration of the urine, and this animal also had slightly high protein output (7.800 mg output; control range was 2.926 to 5.146 mg). Female No. 146 had a more marked increase of urinary volume and decreased specific gravity, also leading to pale yellow colouration of the urine, and this animal also had severely high protein output (35.910 mg output; control range was 0.648 to 1.260 mg). The creatinine concentrations remained low for males that previously received 600 mg/kg bw/day. This indicated that these animals had not recovered after treatment was withdrawn. The effect on glucose output in both sexes showed full recovery.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity responses and grip strength were unaffected by treatment. Motor activity was unaffected by treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was an increase in body weight adjusted means noted in the adrenal glands, kidneys and liver of males administered 600 mg/kg bw/day and the liver at 150 mg/kg bw/day. There was an increase in the absolute weight of the liver of females administered 150 or 600 mg/kg bw/day. There was an increase in body weight adjusted kidney weight in females administered 600 mg/kg bw/day. An increase in adrenal gland weight in females was not statistically significant and no associated histopathological changes were apparent.
After the 4-week off treatment recovery period these differences were not present indicating recovery had taken place. A statistically significant weight increase in the body weight adjusted thyroid and parathyroid weight was present in animals previously administered 600 mg/kg bw/day.
Since there were no findings in the blood plasma that were indicative of hepatotoxicity, such as increased alkaline phosphatase or alanine and aspartate amino-transferase activity and there were no treatment-related histopathological findings in the liver as well as full reversibility of the effect, the liver enlargement was considered to be due to liver enzyme induction and to be an adaprive response to treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The liver was enlarged in females administered 600 mg/kg bw/day and the kidneys were enlarged in a few females administered 600 mg/kg bw/day. The urinary bladder contained calculi and/or was thickened in the majority of males and females administered 600 mg/kg bw/day and contained calculi in two males administered 150 mg/kg bw/day. After 4 weeks off-treatment the urinary bladder changes persisted in males and females previously administered 600 mg/kg bw/day. No macroscopic changes were present in the liver and kidney after the recovery period.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were present in the thyroids, kidneys and urinary bladder of males and females. In males changes were also present in the adrenal and pituitary glands.
Follicular cell hypertrophy was present in males and females given 600 mg/kg bw/day. The presence in one male given 50 mg/kg bw/day and one male given 150 mg/kg bw/day was not considered to be significant. Thyroid follicular cell hypertrophy was considered secondary to mixed function oxidase induction in the liver that was suggested by the enlargement and increased liver weights of these animals. The underlying mechanism of the effect on the thyroid gland is considered to be increased hepatic clearance of thyroid hormones. This results in increased stimulation of a normally functioning thyroid gland, leading to follicular cell hypertrophy.
In the kidney the incidence of basophilic tubules and tubular dilatation was increased in males given 150 or 600 mg/kg bw/day and in females given 600 mg/kg bw/day. Pyelitis, urothelial hyperplasia, giant cell reaction and eosinophilic material/debris were present in the renal pelvis of males and females given 600 mg/kg bw/day. Eosinophilic material in the renal pelvis was also present in one male given 50 mg/kg bw/day and one male 150 mg/kg bw/day, without any other evidence of pathology this was considered unlikely to be of any toxicological significance.
In the urinary bladder urothelial hyperplasia, ulceration or erosion and inflammatory change were present in males and females administered 600 mg/kg bw/day. Eosinophilic material/ debris was also present in the lumen, mainly in males. Urothelial hyperplasia was present in males and females given 150 mg/kg bw/day, with inflammation and ulceration/erosion also present in occasional males given 150 mg/kg bw/day. Hyperplasia of the urothelium of the urethra (within the prostate) was present in males given 150 or 600 mg/kg bw/day.
Vacuolation in the pituitary pars distalis was present only in males given 600 mg/kg bw/day. Increased hepatic degradation of thyroid hormones may have caused the vacuolation in the pars distalis of the pituitary gland in males.
In the adrenal gland of males there was an increase in vacuolation in the cortex at all doses. This showed no dose-related incidence, and whilst there was a minor increase in weight in males given 600 mg/kg bw/day, this did not correlate with the incidence seen in other groups. This is a commonly observed finding in male rats and did also occur in one control recovery male, but it was absent in recovery males previously given 600 mg/kg bw/day. It was therefore, concluded that this represented an uneven background incidence, rather than being related to the test substance administration.
After the 4-week off treatment recovery period the follicular cell hyperplasia persisted in the thyroids, although it showed a minor decrease in incidence and occurred only at a minimal level indicating partial recovery. In the kidneys the tubular dilation in both sexes and the pyelitis in females showed full recovery but there was no clear evidence that the other kidney changes seen after 13 weeks of treatment had recovered. In the urinary bladder, the eosinophilic material/debris and the ulceration/erosion was no longer present, the submucosal inflammation had partially recovered but there was no evidence of recovery from the other finding in males and females, nor was there any evidence of recovery from the findings in the urethra of males. The vacuolation in the pituitary gland was absent after the recovery period.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Serum triiodothyronine (T3) concentrations were considered to have been unaffected by treatment. The higher than control concentrations after 13 weeks for males given 50 or 150 mg/kg bw/day was not attributed to treatment since there was no similar finding at 600 mg/kg bw/day.
The serum thyroxine (T4) concentrations of males given 600 mg/kg bw/day were statistically significantly lower than those of the controls and there was a trend towards low T4 concentration in females at this dose. T4 concentrations at 50 and 150 mg/kg bw/day tended to be higher than those of the controls, particularly in females where the differences were statistically significant. By the end of the recovery period the serum T4 concentrations in animals previously given 600 mg/kg bw/day tended to be slightly higher than controls, indicating that recovery had occurred.
There was a clear increase of serum thyroid stimulating hormone levels after 13 weeks in females given 150 mg/kg bw/day and in males and females given 600 mg/kg bw/day. These findings were not evident at the end of the recovery period in animals previously given 600 mg/kg bw/day signifying that full recovery had occurred.
The reduction of thyroxine levels and increase of thyroid stimulating hormone were considered secondary to mixed function oxidase induction in the liver that was suggested by the liver enlargement.

There was no effect of treatment on oestrous cycle at the end of the treatment period. All females showed evidence of oestrus, demonstrating normal cycling.
Details on results:
There was a reduction of weight gain from Week 4 in males and from Week 8 in females at 150 and 600 mg/kg bw/day, the extent of which was dose-related, but this occurred in the absence of any reduction in food consumption or associated clinical signs. This was indicative of a non-specific toxic response to treatment and the possibility that it occurred as a consequence of the toxic responses in the urinary system.
There was an increase in leucocyte numbers in the peripheral blood of males and females given 600 mg/kg bw/day which were attributed to the inflammatory changes in the kidneys and bladder. There was an increase of platelet count in the high dose animals was a possible adaptive response to the blood loss that caused the haematuria. Platelet numbers were still raised at the end of the recovery period, but the increased leucocyte counts had shown recovery.
There was high blood urea nitrogen concentration in the majority of males given 600 mg/kg bw/day as a consequence of the effect on the kidneys.
There were high volume, low urinary specific gravity and increased glucose output at 600 mg/kg bw/day.
There was a dose-related increase in kidney weights at 150 or 600 mg/kg bw/day.
There were calculi in the urinary bladder, leading to thickening, in two males given 150 mg/kg bw/day and in the majority of males and females given 600 mg/kg bw/day.
There were tubular basophilia and dilatation that occurred at 150 mg/kg bw/day in males and at 600 mg/kg bw/day in both sexes. At 600 mg/kg bw/day there was also clear evidence of tubular damage (pyelitis, urothelial hyperplasia, giant cell reaction and eosinophilic material/debris in the renal pelvis, the presence of eosinophilic material/debris in the lumen of the urinary bladder in males and haematuria and proteinuria in both sexes) and hyperplastic change in the urothelium, the cause of which was likely to be the calculi that were detected in the urinary bladder. In the urinary bladder, urothelial hyperplasia, ulceration or erosion and inflammatory change were present in males and females administered 150 or 600 mg/kg bw/day and there was also hyperplasia of the urothelium of the urethra (within the prostate) in males given 150 or 600 mg/kg bw/day. Following recovery, calculi were still evident in the urinary bladder of the majority of previously treated animals at 600 mg/kg bw/day and, as a consequence, the damage caused by these calculi was also still evident, particularly in one male and one female.
There were several findings in this study that indicate a reversible effect on the liver. After 13 weeks of treatment there was an increase of liver weight at 150 and 600 mg/kg bw/day in both sexes and at macroscopic examination the livers of females given 600 mg/kg bw/day were considered enlarged. There were, however, no findings in the blood plasma that were indicative of hepatotoxicity, such as increased alkaline phosphatase or alanine and aspartate amino-transferase activity. There were findings that suggested a non-adverse effect on hepatic function (high total cholesterol concentration, due to increased high and low density lipoprotein concentrations, in males and females receiving 600 mg/kg bw/day, and increased globulin fraction in males and females receiving 600 mg/kg bw/day). Despite the increase in liver weight, there were no treatment-related histopathological findings. This indicated that there may have been a low degree of hepatocellular enlargement due to adaptive enzyme induction, but at a level that was not evident during the histopathological examination.
Thyroid follicular cell hypertrophy was present after 13 weeks in males and females given 600 mg/kg bw/day and this was considered secondary to mixed function oxidase induction in the liver that was suggested by the enlargement and increased liver weights of these animals, though no hepatocellular hypertrophy was reported. The underlying mechanism of the effect on the thyroid gland is considered to be increased hepatic clearance of thyroid hormones where thyroxine is de-iodinated more rapidly, leading to the reduced thyroxine levels that occurred at 600 mg/kg bw/day in both sexes, but particularly males. This then leads to a disruption of the normal feedback control of the thyroid gland and an increase of thyroid stimulating hormone release from the pituitary gland, and in this study there was an increase of serum TSH levels in both sexes at 600 mg/kg bw/day. This results in increased stimulation of a normally functioning thyroid gland, leading to follicular cell hypertrophy though, unusually in this study, thyroid weights were not increased after 13 weeks of treatment, though there was a slight increase of thyroid weight at the end of the recovery period in males. There was also an increase of TSH at 150 mg/kg bw/day in females but this occurred in the absence of any reduction of thyroxine concentrations and did not cause any histopathological change in the thyroid glands. Increased hepatic degradation of thyroid hormones may also have caused the vacuolation in the pars distalis of the pituitary gland in males.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
urinalysis

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

The mean concentrations in formulations prepared for administration in week 1 and 12 were in the range -12.7 to +12.8% of the intended concentration. There were generally within the applied limits of -15%/+10% of the nominal concentration, with the single exception of the low dose (Group 2; 12.5 mg/mL) formulation in Week 1 where the relative mean error was slightly above applied limits (+12.8%). Overall, these results demonstrated accurate formulation. The difference from mean remained within 3%, confirming the precision of analysis.

Table 5: Thyroid hormone analysis

Group

1M

2M

3M

4M

1F

2F

3F

4F

Treatment

Control

Low dose

Mid dose

High dose

Control

Low dose

Mid dose

High dose

Level (ppm)

0

50

150

600

0

50

150

600

Triiodothyronine (T3)

 

 

 

 

 

 

 

 

Week 14

654

741

791*

558

1000

994

1160

850

Week R5

552

-

-

568

1000

-

-

1110

Thyroxine (T4)

 

 

Week 14

42200

49000

51600

34000*

39300

48400**

49500**

34500

Week R5

38300

-

-

43800

35600

-

-

44900

Thyroid stimulating hormone

 

 

 

 

 

 

 

 

Week 14

535

1280

979

1990***

521

462

1060*

1030*

Week R5

1300

-

-

1670

1100

-

-

1030

Significant when compared to Group 1: * - p<0.05; ** - p<0.01; *** p<0.001.

Table 6: Summary of urinary appearance and blood in the urine

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

50

150

600

0

50

150

600

Urinary appearance

Week 13

  Pale yellow

  Medium yellow

  Dark yellow

  Pale brown

No. of animals

Week R4

  Pale yellow

  Medium yellow

  Dark yellow

No. of animals

 

 

3

4

3

0

10

 

0

4

1

5

 

 

1

5

4

0

10

 

-

-

-

0

 

 

1

6

2

0

9

 

-

-

-

0

 

 

 

6

1

1

1

9

 

1

4

0

5

 

 

0

10

0

0

10

 

0

4

0

4

 

 

2

8

0

0

10

 

-

-

-

0

 

 

2

8

0

0

10

 

-

-

-

0

 

 

7

3

0

0

10

 

1

4

0

5

Urinary blood

Week 13

  Negative

  Trace

  1+

  2+

  3+

No. of animals

Week R4

Negative

  Trace

  1+

  2+

  3+

No. of animals

 

 

10

0

0

0

0

10

 

5

0

0

0

0

5

 

 

10

0

0

0

0

10

 

-

-

-

-

-

0

 

 

9

0

0

0

0

9

 

-

-

-

-

-

0

 

 

4

2

0

1

2

9

 

2

0

1

0

2

5

 

 

10

0

0

0

0

10

 

4

0

0

0

0

4

 

 

10

0

0

0

0

10

 

-

-

-

-

-

0

 

 

10

0

0

0

0

10

 

-

-

-

-

-

0

 

 

8

0

1

0

1

10

 

2

0

0

1

2

5

Table 7: Test Article-Related Effects in Organ Weights After 13 weeks of Treatment

Males

Males

Males

Males

Females

Females

Females

Females

Dose Level (mg/kg bw/day)

0

50

150

600

0

50

150

600

Adrenal Gland

 

 

 

 

 

 

 

 

Body Weight adjusted Weight (g)

0.053

0.050

0.052

0.062**

 

 

 

 

Kidneys

 

 

 

 

 

 

 

 

Body Weight adjusted Weight (g)

3.146

3.046

3.167

3.583**

1.832

1.898

1.853

2.080*

Liver

 

 

 

 

 

 

 

 

Absolute Weight (g)

15.968

16.777

17.850

18.366

9.234

9.551

9.953*

13.342**

Body Weight adjusted Weight (g)

16.051

16.131

17.699*

19.066**

 

 

 

 

Statistically significant difference (absolute or relative) compared with respective control mean value * p ≤ 0.05; ** p ≤ 0.01

Table 8: Test Article-Related Effects in Organ Weights – After 4 weeks of Recovery

Males

 

Females

 

Dose Level (mg/kg bw/day)

0

600

0

600

Thyroids and Parathyroids

 

 

 

 

Absolute Weight (g)

0.020

0.026*

 

 

* = Statistically significant difference (absolute or relative) compared with respective control mean value * p  ≤  0.05; ** p ≤ 0.01.

 

Table 9: Incidence of Test Article-Related Macroscopic Findings After 13 weeks of Treatment

Males

Males

Males

Males

Females

Females

Females

Females

Dose Level (mg/kg bw/day)

0

50

150

600

0

50

150

600

Liver

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

10

10

10

10

 Enlarged

0

0

0

0

0

0

0

8

 

 

 

 

 

 

 

 

 

Kidneys

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

10

10

10

10

 Enlarged

0

0

0

0

0

1

0

3

 

 

 

 

 

 

 

 

 

Urinary Bladder

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

10

10

10

10

 Contained calculus(i)

0

0

2

9

0

0

0

7

 Thickened

0

0

0

10

0

0

0

8

 

Table 10: Incidence of Test Article-Related Macroscopic Findings After 4 weeks of Recovery

Males

Males

Females

Females

Dose Level (mg/kg bw/day)

0

600

0

600

Urinary Bladder

 

 

 

 

Number Examined

5

5

4

5

 Contained calculus(i)

0

5

0

3

 Thickened

0

5

0

4

Table 11: Incidence and Severity of Test Article-Related Microscopic Findings After 13 weeks of Treatment

Males

Males

Males

Males

Females

Females

Females

Females

Dose Level (mg/kg bw/day)

0

50

150

600

0

50

150

600

Thyroid Glands

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

10

10

10

10

 Hypertrophy, Follicular Cell

 

 

 

 

 

 

 

 

Minimal

0

1

1

7

0

0

0

5

Slight

0

0

0

2

0

0

0

3

 

 

 

 

 

 

 

 

 

Kidneys

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

10

10

10

10

 Basophilia, Tubular, Focal/Diffuse

 

 

 

 

 

 

 

 

Minimal

2

1

5

3

0

0

1

1

Slight

0

0

1

6

0

0

0

2

Moderate

0

0

0

1

0

0

0

0

 Dilatation, Tubular, Focal/Diffuse

 

 

 

 

 

 

 

 

Minimal

1

2

1

4

0

0

0

4

Slight

0

0

2

4

0

0

0

2

Moderate

0

0

0

1

0

0

0

0

 Eosinophilic Material/Debris, Pelvis

 

 

 

 

 

 

 

 

Minimal

0

1

1

3

0

0

0

2

Slight

0

0

0

4

0

0

0

1

Moderate

0

0

0

1

0

0

0

0

 Giant Cell Reaction, Pelvis

 

 

 

 

 

 

 

 

Slight

0

0

0

2

0

0

0

1

Moderate

0

0

0

2

0

0

0

0

 Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Minimal

0

0

0

5

0

0

1

7

Slight

0

0

0

4

0

0

0

2

Moderate

0

0

0

1

0

0

0

0

 Pyelitis

 

 

 

 

 

 

 

 

Minimal

0

1

0

3

0

0

1

2

Slight

0

0

0

1

0

0

0

1

Moderate

0

0

0

1

0

0

0

0

 

 

 

 

 

 

 

 

 

Urinary Bladder

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

10

10

10

10

 Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Minimal

0

0

2

0

0

0

3

2

Slight

0

0

2

1

0

0

0

4

Moderate

0

0

2

8

0

0

0

3

 Inflammation, Submucosal

 

 

 

 

 

 

 

 

Minimal

0

0

1

1

0

0

0

1

Slight

0

0

1

5

0

0

0

3

Moderate

0

0

0

1

0

0

0

0

 Ulceration/erosion

 

 

 

 

 

 

 

 

Minimal

0

0

1

0

0

0

0

1

Slight

0

0

0

1

0

0

0

1

Moderate

0

0

0

3

0

0

0

0

 Eosinophilic Material/Debris, Lumen

 

 

 

 

 

 

 

 

Minimal

0

0

0

1

0

0

0

0

Slight

0

0

0

3

0

0

0

0

Moderate

0

0

0

1

0

0

0

1

 Giant Cell Reaction

 

 

 

 

 

 

 

 

Slight

0

0

0

1

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Adrenal Glands

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

 

 

 

 

 Vacuolation, Cortical

 

 

 

 

 

 

 

 

Slight

0

6

5

4

 

 

 

 

 

 

 

 

 

 

 

 

 

Pituitary

 

 

 

 

 

 

 

 

Number Examined

10

10

9

10

 

 

 

 

 Vacuolation, Pars Distalis

 

 

 

 

 

 

 

 

Minimal

0

0

0

5

 

 

 

 

Slight

0

0

0

3

 

 

 

 

 

 

 

 

 

 

 

 

 

Urethra (within prostate)

 

 

 

 

 

 

 

 

Number Examined

0

6

3

7

 

 

 

 

 Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Minimal

0

0

1

1

 

 

 

 

Slight

0

0

0

0

 

 

 

 

Moderate

0

0

2

6

 

 

 

 

 

Table 12: Incidence and Severity of Test Article-Related Microscopic Findings after 4 weeks of Recovery

 

Males

Males

Males

Males

Females

Females

Females

Females

Dose Level (mg/kg bw/day)

0

50

150

600

0

50

150

600

Thyroids

 

 

 

 

 

 

 

 

Number Examined

5

 

 

5

0

 

 

0

 Hypertrophy, Follicular Cell

 

 

 

 

 

 

 

 

Minimal

0

 

 

5

0

 

 

2

 

 

 

 

 

 

 

 

 

Kidneys

 

 

 

 

 

 

 

 

Number Examined

5

 

 

5

5

 

 

5

 Basophilia, Tubular, Focal/Diffuse

 

 

 

 

 

 

 

 

Minimal

0

 

 

3

0

 

 

1

Slight

0

 

 

1

0

 

 

0

 Eosinophilic Material/Debris, Pelvis

 

 

 

 

 

 

 

 

Minimal

0

 

 

2

0

 

 

1

Slight

0

 

 

2

0

 

 

0

 Giant Cell Reaction, Pelvis

 

 

 

 

 

 

 

 

Slight

0

 

 

0

0

 

 

1

Moderate

0

 

 

3

0

 

 

0

 Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Slight

0

 

 

3

0

 

 

0

Moderate

0

 

 

1

0

 

 

1

 Pyelitis

 

 

 

 

 

 

 

 

Minimal

0

 

 

3

0

 

 

0

Slight

0

 

 

1

0

 

 

0

 

 

 

 

 

 

 

 

 

Urinary Bladder

 

 

 

 

 

 

 

 

Number Examined

0

 

 

5

0

 

 

4

 Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Slight

0

 

 

1

0

 

 

2

Moderate

0

 

 

4

0

 

 

1

Marked

0

 

 

0

0

 

 

1

 Inflammation, Submucosal

 

 

 

 

 

 

 

 

Minimal

0

 

 

0

0

 

 

0

Slight

0

 

 

1

0

 

 

1

Moderate

0

 

 

0

0

 

 

1

 Ulceration/erosion

 

 

 

 

 

 

 

 

Slight

0

 

 

0

0

 

 

2

Giant Cell Reaction

 

 

 

 

 

 

 

 

Slight

0

 

 

1

0

 

 

0

Urethra

 

 

 

 

 

 

 

 

Number examined

0

 

 

2

 

 

 

 

 Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Slight

0

 

 

2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
In the 90-day oral repeated dose toxicity study with the registered substance, conducted according to OECD Test Guideline 408 and in compliance with GLP, the concluded NOAEL for systemic effects was 50 mg/kg bw/day (the lowest dose tested) due to effects in the urinary system in male and female rats at 150 and 600 mg/kg bw/day some of which were persistent after the 4-week recovery period.