Registration Dossier

Administrative data

Description of key information

In the key sub-chronic 90-day oral repeated dose toxicity study with 2,4,6,8,10-pentamethylcyclopentasiloxane, conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for systemic toxicity was concluded to be 150 mg/kg bw/day based on test-item related adverse effects in the urinary system at 400 and 1000 mg/kg bw/day. The findings in this study indicated that the target of toxicity was the urinary system and test-item administration resulted in the early deaths of four high dose group animals. There were a few findings in the urinary tract of some low dose (150 mg/kg bw/day) males but these were minor and there was no calculi-formation and, consequently, they were considered non-adverse. The urinary system findings generally showed some recovery, despite calculi still being present to a similar extent to that observed at the end of the treatment period. There was also an effect on the liver that was considered to represent an adaptive response to treatment that led to a secondary, rodent-specific effect on the thyroid glands as a consequence of disruption of the thyroid hormonal control mechanism (Covance Laboratories Limited, 2020a).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 39 to 52 days old
- Weight at study initiation: 178 to 242 g (males); 131 to 198 g (females)
- Fasting period before study: no
- Housing: Three or four of the same sex (main study and recovery), unless reduced by mortality or isolation in polycarbonate body cages with a stainless-steel mesh lid, changed at appropriate intervals.
- 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: not specified

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.

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

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil. The vehicle was chosen based on the test item characteristics and in consultation with the study sponsor.
- Concentration in vehicle: 0, 37.5, 100, 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no. (if required): MKCK6411
- Purity: not applicable
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 (Covance Study Number HC89KK). 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 (90 days) followed by 4 weeks of no treatment for recovery groups (control and high dose group)
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control group
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
low dose group (LD)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
middle dose group (MD)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
high dose group (HD)
No. of animals per sex per dose:
Test groups: 10 male and 10 female animals per dose
Recovery control and high dose groups: 5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels selected (0, 150, 400 and 1000 mg/kg bw/day) were based on the pathology results from the preliminary toxicity study (Covance Laboratories Limited, 2020b).
In a previous acute oral toxicity test 2,4,6,8,10-pentamethylcyclopentasiloxane 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 signs noted were ruffled fur in 2/5 animals which resolved by study Day 4. The LD50 was greater than 2000 mg/kg bw in female rats.
In the preliminary toxicity study (Covance Laboratories Limited, 2020b), dose levels of 0, 250 and 750 mg/kg bw/day administered for 21 days and 1000 mg/kg bw/day for 14 days did not show any evidence of toxicity during the in-life phase. However, changes in the kidneys and urinary bladder were noticed at necropsy and therefore, pathology was performed. This identified slight ulceration with associated inflammatory, proliferative changes and oedema in the urinary bladder of one female given 1000 mg/kg bw/day but there were no findings in the kidneys.

- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: yes (overnight)
- Rationale for selecting satellite groups: to evaluate recovery from effects
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): random
Positive control:
Not used
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. During the acclimatization and recovery periods, observations of the animals and their cages were recorded at least once per day. Signs associated with dosing were checked prior to dosing, immediately after dosing, on completion of group dosing, 1-2 hours after dosing, as late as possible in the working day.
- Cage side observations checked included: viability check, signs of ill health

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

BODY WEIGHT: Yes
- Time schedule for examinations: one week before treatment commenced (Week -1), 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.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretreatment and on study week 13
- Dose groups that were examined: All main study and recovery study animals during pretreatment and all main study and recovery study animals of Groups 1 and 4 during week 13 examination.

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

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 and recovery Week 4
- Animals fasted: Yes (overnight)
- How many animals: all main study animals and all recovery study animals
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 and recovery Week 4
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (overnight)
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: performed (before dosing) and during Week 12 of treatment
- Dose groups that were examined: on the first five main study animals and all recovery phase animals in Groups 1 and 4 and all main study animals from Groups 2 and 3
- Battery of functions tested: sensory activity / grip strength / motor activity

ESTROUS CYCLES: Yes
- Time schedule for examinations: Daily smears were taken for 4 days before scheduled termination at the end of either the treatment or recovery period. The last smear was taken on the morning of necropsy.
- How many animals: all females

OTHER:
THYROID HORMONE ANALYSIS: Yes
- Time schedule for examinations: on Week 14 (at necropsy) and on Week 5 (at necropsy)
- How many animals: all main study animals and all recovery study animals
- Parameters checked included: triiodothyronine (T3); thyroxine (T4); thyroid stimulating hormone (TSH)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. The following data types were analysed at each timepoint separately: body weight, using gains over appropriate study periods; grip strength and motor activity; hematology; blood chemistry; urinalysis; organ weights, absolute and adjusted for terminal body weight T3, T4, TSH. The following comparisons were performed: Group 1 vs 2, 3 and 4. The following statistical tests was used for grip strength, motor activity, body weight, organ weight and clinical pathology data: A parametric analysis if Bartlett's test was not significant at the 1% level. The F1 approximate test was applied. 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 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 was applied. If the H1 test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant Steel's test was performed. For two groups, comparisons were made by Wilcoxon rank sum tests. For grip strength, motor activity and clinical pathology data, if 75% of the data were the same value Fisher’s exact tests were performed. Treatment groups were compared by pairwise comparisons against the control. 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. Significant differences expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings in the animals that died or were sacrificed during the study period included:
- Recovery phase Animal No.49, which was found dead had abnormal staining of the perigenital area and the ventral surface, hunched posture and decreased activity.
- High dose animal No.148 (assigned to the recovery phase), which was euthanised for welfare reasons on day 88, showed decreased activity, distension of the abdomen, swollen vaginal area, piloerection and yellow staining at the perigenital area.
- High dose animal No.108, which was found dead on study day 78, did not display significant signs ante mortem.
- Low dose animal No.112, was euthanised on day 21 for welfare reasons due to general poor clinical condition, where the animal displayed breathing difficulties (gasping).
- High dose animal No.149 (assigned to recovery phase) was found dead without displaying clinical signs ante mortem.
- Female No. 105 (1000 mg/kg bw/day) was euthanised for welfare reasons in Week 13 after displaying decreased activity, shallow breathing and abnormal yellow staining at the perigenital area.
The death of the low dose female was clearly accidental and not related to treatment whilst for three of the four high dose females, and also of the high dose male, death was attributed to the treatment-related findings in the urinary system. The cause of death of the other high dose female was undetermined.

Overall clinical observations from all the animals included:
Chin rubbing and occasional salivation occurred at the time of dose administration in males and females receiving 400 or 1000 mg/kg bw/day. Salivation tended to be more persistent on a few days in some of the animals receiving 1000 mg/kg bw/day, and was also transiently persistent in one male receiving 400 mg/kg bw/day, as this was at the observations performed approximately 1 to 2 hours post dose. Paddling of the fore-paws at the time of dose administration was also evident on some days in a few females receiving 400 mg/kg bw/day and in a few males and females receiving 1000 mg/kg bw/day. Each of these signs are commonly observed in studies where the test material is administered by gavage and, as such, are considered of no toxicological importance.
Mortality:
mortality observed, treatment-related
Description (incidence):
A total of six animals died during study; three were sacrificed for welfare reasons and three were found dead. Four of these deaths were considered test article-related and two were considered unrelated to the test article. Test item-related mortality included one male (Animal No.49) and three females (Animals Nos. 105, 148 and 108) administered 1000 mg/kg/ bw/day. On Day 12 of the recovery phase Animal No.49 was found dead. On Day 88 of treatment Animal No.105 was euthanized for welfare reasons. On Day 88 of treatment Animal No.148 (assigned to the recovery phase) was euthanised for welfare reasons. On Day 78 of treatment Animal No.108 was found dead. The non-test article related deaths included two females (Animals No.112 and 149). On Day 21 of treatment Animal No.112 administered 150 mg/kg bw/day was euthanised for welfare reasons. On Day 14 of treatment, Animal No.149 administered 1000 mg/kg bw/day (assigned to the recovery phase) was found dead. All other animals survived to their scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The administration of the test item to males had no effect on their body weights during the first six weeks of treatment but between Week 6 and 13 there was a reduction of weight gain at 400 and 1000 mg/kg bw/day, the extent of which was dose related, though statistical significance was attained only at the highest dose. Between Week 6 and 13 the weight gains at 400 and 1000 mg/kg bw/day were approximately 14 and 25% lower than that of the control males during the same period. This resulted in overall (Week 0-13) weight gains being approximately 6 and 10% lower than the control males. During the four-week recovery period, males that previously received 1000 mg/kg bw/day gained weight but the rate of weight gain was lower than the control, indicating that no significant recovery had occurred after treatment ceased.
There were no effects of treatment upon the body weights of males receiving 150 mg/kg bw/day or at any dose in females.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The amount of food consumed by females receiving 1000 mg/kg bw/day was higher than that of controls in the majority of weeks, but this reflected a trend that was present before treatment commenced and, consequently, it was not attributed to treatment.
Food efficiency:
not examined
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 neutrophil and lymphocyte counts in males receiving 400 mg/kg bw/day (37% and 10%) and in males (54% and 27%) and females (72% and 21%) receiving 1000 mg/kg bw/day, the extent of which in males was statistically significant and dose-related. Monocyte and large unstained cell counts were also statistically significantly higher in males (100% and 55%) and females (71% and 125%) receiving 1000 mg/kg bw/day. In addition, three males receiving 1000 mg/kg bw/day (Nos. 1, 3 and 9; 120%, 230% and 690%) had high eosinophil count. As a consequence of these findings, the total leucocyte counts of males receiving 400 mg/kg bw/day (16%) and in males and females receiving 1000 mg/kg bw/day (35% and 28%) were higher than controls. The majority of the individual values were, however, within the background range (Males: 7.02x10^9 to 18.75x10^9/L for total leucocyte count, 0.71x10^9 to 2.83x10^9/L for neutrophils, 5.09x10^9 to 16.23x10^9/L for lymphocytes, 0.04x10^9 to 0.24x10^9/L for eosinophils, 0.12x10^9 to 0.59x10^9/L for monocytes and 0.02x10^9 to 0.27x10^9/L for large unstained cells (n=98). Females: 3.95x10^9 to 12.76x10^9/L for total leucocyte count, 0.33x10^9 to 2.41x10^9/L for neutrophils, 2.96x10^9 to 11.49x10^9/L for lymphocytes, 0.07x10^9 to 0.34x10^9/L for monocytes and 0.02x10^9 to 0.16x10^9/L for large unstained cells (n=97)). By the end of the four-week recovery period there was no evidence of recovery in respect of the high eosinophil counts in males and of the high monocyte counts in females, but all other leucocyte findings showed evidence that at least partial recovery had occurred.
Platelet counts were higher than controls in females receiving 400 mg/kg bw/day and in males and females receiving 1000 mg/kg bw/day but the prothrombin and activated partial thromboplastin times of these animals were unaffected. Platelet counts of females that previously received 1000 mg/kg bw/day showed full recovery but there was no evidence that any recovery had occurred in the males.
All other differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the slightly high eosinophil count in males receiving 1000 mg/kg bw/day as this was due to high values in two animals. They also included the trend towards low reticulocyte count in males since there was no consequential effect on erythrocyte count.
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 blood urea nitrogen concentration in males receiving 1000 mg/kg bw/day (47%). Five of the males (Nos. 3, 5, 7, 8 and 9; 42%-119%) had blood urea nitrogen concentrations that were notably above the range reported in the control males and in one animal (No. 3) its value exceeded the background range (2.37 to 9.04 mmol/L for urea; n=89) and was associated with a high plasma creatinine concentration that also exceeded the background range (20 to 47 μmol/L; n=99). High plasma blood urea nitrogen (56%) and creatinine (100%) concentration was also reported in one female (No. 102) receiving 1000 mg/kg bw/day, though only its creatinine concentration was 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)). By the end of the four week recovery period the plasma urea concentrations of males previously given 1000 mg/kg bw/day were still higher than controls (22%), though partial recovery had occurred, with the other findings showing full recovery.
Plasma glucose concentrations were slightly low in males receiving 1000 mg/kg bw/day (12%) but this showed full recovery. There was no similar finding in females.
There was a statistically significant dose-related increase of total cholesterol concentration in females given 400 or 1000 mg/kg bw/day (31% and 35%) but all values were within the background range (1.02 to 3.45 mmol/L (n=100)) and this was due to increased high (29% and 32%) and low density (38% and 81%) lipoprotein concentration. The total cholesterol and high and low density lipoprotein concentrations in all male dose groups also tended to be slightly higher than controls but there was no dose-response, no statistical significance and, consequently, this was less clearly attributable to treatment. Although not statistically significant, there was a small reduction of triglyceride concentration in males receiving 400 or 1000 mg/kg bw/day (24% and 36%); females were unaffected. The findings in females showed no evidence of recovery at the end of the four week recovery period but the slight trends in males showed full recovery.
Plasma phosphorus concentrations were higher than controls at all doses and in both sexes, though only in males was the extent of the increase dose-related (males: 4%, 7% and 18%; females: 8%, 12% and 12%, 150, 400 and 1000 mg/kg bw/day respectively), and only males given 1000 mg/kg bw/day were statistically significant. The majority of individual plasma phosphorus concentrations were within range of historical control range (1.48 to 2.19 mmol/L in males (n=49) and 1.01 to 1.99 mmol/L in females (n=50)). This finding showed full recovery.
There was a dose-related reduction of the albumin to globulin ratio in females given 400 or 1000 mg/kg bw/day (21% and 23%) which, in the absence of any alteration of plasma albumin concentration, was attributed to an increase in the globulin fractions of these animals. Though this particular finding showed full recovery, the total protein concentrations of previously treated 1000 mg/kg bw/day females were markedly higher than the controls (22%), due to clear increases of both the albumin and globulin fractions. Males were unaffected in this respect, though there were minor increases of total protein and albumin concentrations at all dose levels that were considered of no toxicological significance.
All other differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the lower than control bile acid concentrations in treated males since the difference was due to very high values in two control males that raised the group mean value for the control group. Other differences also included the minimally high plasma alanine amino-transferase activities in males receiving 1000 mg/kg bw/day (19%) since all values were within the background range (24 to 98 U/L (n=99)), the low alkaline phosphatase activities in males (25% and 12%) and females (27% and 31%) receiving 400 or 1000 mg/kg bw/day and low aspartate amino-transferase in females receiving 400 or 1000 mg/kg bw/day (21% and 23%) since reduced activities are of no toxicological significance.
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 (males: 150% and 233%; females: 114% and 171%) and low urinary specific gravity (males: 2% and 3%; females: 1% and 1%), resulting in the urine of these animals being of a paler appearance than that of controls, in males and females receiving 400 or 1000 mg/kg bw/day, the extent of which was statistically significant and dose-related. There was also a small increase of urinary volume in males (29%) and females (43%) receiving 150 mg/kg bw/day which associated with a small decrease of specific gravity in females (0.5%), although these findings were not statistically significant. Blood was detected in the urine of males receiving 400 or 1000 mg/kg bw/day but was not as frequently observed in females, where only one animal receiving 400 mg/kg bw/day was affected. Total urinary protein output was increased at 400 and 1000 mg/kg bw/day in males (532% and 271%) and females (209% and 422%) which was statistically significant, and there was a slightly increased glucose output in females at these doses (43% and 54%); however, this finding was not statistically significant. In males the majority of animals at these doses had high protein outputs but this was particularly marked in three males (Nos. 38, 39 and 40; 481%, 342% and 689%) receiving 400 mg/kg bw/day and in one male (No. 3; 138%) receiving 1000 mg/kg/day where protein output was >25 mg (control range was 1.28 to 3.24 mg), and also in one female (No. 109; 575%) receiving 1000 mg/kg bw/day (13.99 mg; control range 0.23 to 1.35 mg).
All of these findings showed evidence that significant recovery had occurred during the four-week recovery period.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
All differences in sensory reactivity, grip strength and motor activity from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation. Forelimb grip strength was low, compared to controls, at all doses in females but there was no dose-response and no similar trend was evident in males.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test article-related body weight-adjusted mean organ weight changes after 13 weeks of treatment occurred for kidney, liver, thyroid and thymus weights.
In the kidneys and liver, statistically significantly increased mean body weight-adjusted organ weights in both sexes administered 400 or 1000 mg/kg bw/day and in females administered 150 mg/kg bw/day were noted. The increased kidney weights correlated microscopically with tubular basophilia, presence of calculi, pelvic granuloma, pelvic urothelium hyperplasia, inflammatory infiltrate in males and females administered 1000 mg/kg bw/day and in males administered 400 mg/kg bw/day, with females displaying a lower incidence and severity of the lesions. The increased mean body weight-adjusted liver weights correlated microscopically with centrilobular hypertrophy (minimal) in males and females administered 400 or 1000 mg/kg bw/day and persisted at the recovery sacrifice in females.
Statistically significantly increased mean body weight-adjusted thyroid weights were observed in males treated with 150, 400 or 1000 mg/kg bw/day (the extent of which was broadly dose-related), which correlated microscopically with follicular cell hypertrophy (minimal) in males administered 150, 400 or 1000 mg/kg bw/day. Statistically significantly decreased mean body weight-adjusted thymus weights were observed in females treated with 150, 400 or 1000 mg/kg/ bw/day, which correlated microscopically with decreased cellularity in these animals.
At the recovery sacrifice, statistically significant higher adjusted mean liver weights were noted in females administered 1000 mg/kg bw/day which correlated microscopically with centrilobular hypertrophy (minimal). The extent of the difference from controls was less than that at the end of the treatment period, signifying that some recovery had occurred. There were no histopathological changes to account for statistically significantly higher adjusted thyroid weights in females administered 1000 mg/kg bw/day.
All other treatment-related organ weight findings showed full recovery. All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body or brain weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross pathological findings in the animals that died or were sacrificed during the study period included:
- Macroscopic findings of recovery phase Animal No.49 comprised of bilaterally enlarged kidneys with a unilaterally dilated pelvis, distended and thickened urinary bladder with abnormal contents and bilaterally distended ureters; all of which correlated with microscopic findings, which were considered to have contributed to the death of the animal.
- Macroscopic findings of high dose animal No.148 (assigned to the recovery phase), which was euthanised for welfare reasons on day 88, included bilaterally distended ureters, distension of the urinary bladder and distension and thickening of the distal vagina forming a firm swelling. The kidneys appeared pale and enlarged and had dilated pelvises.
- Macroscopic findings of high dose animal, No.108, which was found dead on study day 78, indicated the presence of bilaterally distended ureters, distension of the urinary bladder and the ureters, urinary bladder and vagina had abnormal contents (dark thick fluid). The kidneys appeared pale and enlarged and had a dilated pelvis.
- Macroscopic findings of animal No.112, which was euthanised on day 21, indicated the presence of a perforation of the lower oesophagus and pale material in the thoracic cavity partially enveloping the lungs and oesophagus.
- Macroscopic findings of high dose animal No.149 (assigned to the recovery phase) indicated a unilaterally dilated pelvis and a diverticulum in the jejunum.
- Macropathology of female No. 105 (1000 mg/kg bw/day) which was euthanised for welfare reasons in Week 13 examination showed the animal had bilaterally distended ureters with abnormal contents, distended urinary bladder and distension of the vagina by a pale calculus. The kidneys appeared pale and enlarged. The findings in the urinary system were considered the likely cause of the condition of this animal and, consequently, it was likely due to treatment.
Overall macroscopic observations from all the animals included:
- Test article-related macroscopic observations were noted in the kidneys, urinary bladder, ureters and in the liver at the terminal sacrifice.
In the kidneys, enlargement, dilated pelvis, abnormal colour and granular appearance were observed. The urinary bladder contained calculi in all males administered 400 or 1000 mg/kg bw/day and the wall was thickened in all males treated with 400 mg/kg bw/day and nine males treated with 1000 mg/kg bw/day. One male administered 400 mg/kg bw/day also presented distension of the urinary bladder. In the urinary bladder of females administered 400 or 1000 mg/kg bw/day, treatment-related findings (presence of calculi/wall thickness) were observed in single animals. In the ureters of males administered 400 or 1000 mg/kg bw/day, test item-related findings (presence of calculi/wall thickness/distension) were variably noted at the terminal sacrifice, whilst in females, one administered 1000 mg/kg bw/day had distension of the ureters.
In the liver, enlargement was noted in males and females administered 400 or 1000 mg/kg bw/day.
At the recovery sacrifice, test article-related macroscopic findings were still present in the kidneys and in the urinary bladder, but the findings in the ureters showed full recovery.
Dilated pelvis and raised areas were noted in a single male administered 1000 mg/kg bw/day, this suggested that partial recovery had occurred.
Thickened wall of the urinary bladder was observed in one female, and calculi in two males and females previously administered 1000 mg/kg bw/day.
All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague Dawley rats of this age, therefore, they were considered not test article related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology findings in the animals that died or were sacrificed during the study period included:
- Microscopic findings of recovery phase Animal No.49 comprised of nephropathy, papillary necrosis, hyperplasia of the urothelium, distended urinary bladder and the ureters, which were considered to have contributed to the death of the animal.
- Microscopic findings of high dose animal No.148 (assigned to the recovery phase), which was euthanised for welfare reasons on day 88, included findings in the urinary tract which correlated with the macroscopic observation (nephropathy in the kidneys, dilated urinary bladder). Histologically, calculi were also observed in the peritoneal cavity with concurrent peritonitis. The findings in the urinary system were considered to be the cause of peritonitis, and consequently were considered to have contributed to the death of the animal.
- Microscopic findings of high dose animal No.108, which was found dead on study day 78, in the urinary tract correlated with the macroscopic observation in these tissues (nephropathy, hemorrhage in the urinary bladder) and were considered to have contributed to the death of the animal.
- Microscopic findings of animal No.112, which was euthanised on day 21, were mainly in the oesophagus and correlated with the macroscopic observation in the tissue (perforation of the oesophagus) and indicated that the death of this animal was due to a dose administration error.
- Histological findings in high dose animal No.149 (assigned to the recovery phase) correlated with the macroscopic observations (diverticulum in the jejunum). The likely cause of death was the finding in the jejunum.
Overall microscopic observations from all the animals included:
At the terminal sacrifice, test article -related microscopic findings were noted in the kidneys, urinary bladder, ureters, liver, thyroids of males and females and in the mammary gland and the thymus of females.
A number of test article-related changes were noted in the urinary tract. The most significant finding was the presence of calculi in the urinary pelvis, causing hyperplasia of the urothelium with inflammatory infiltrate, which were noted in the majority of males administered 400 or 1000 mg/kg bw/day and, to a lesser extent and incidence, in females administered 1000 mg/kg bw/day. The presence of the calculi resulted in pelvic dilatation and gave rise to the development of the granulomatous inflammation in the pelvic area and in some cases, led to the development of nephropathy, consisting of the combination of tubular basophilia, inflammatory infiltrate, dilated tubules and interstitial fibrosis. The findings in the kidneys correlated with observed blood in the urine of males administered 400 or 1000 mg/kg bw/day and with higher total urinary protein output in males and females administered 400 or 1000 mg/kg bw/day than in controls. The changes in the kidneys were considered adverse. Presence of calculi in the urinary bladder resulted in hyperplasia of the urothelium, which was observed in all males administered 400 or 1000 mg/kg bw/day (slight to moderate) and in a majority of females administered 400 or 1000 mg/kg bw/day (minimal to moderate). It was accompanied by inflammatory infiltrate, haemorrhage, dilatation and inflammation. These changes were considered adverse.
Hyperplasia of the urothelium was observed in the ureters of two males and one female administered 1000 mg/kg bw/day and three males administered 400 mg/kg bw/day, sometimes accompanied by dilatation. These changes were considered non-adverse.
After four-weeks of recovery, test article-related microscopic findings were still evident in the kidneys, urinary bladder and livers of males and females previously administered 1000 mg/kg bw/day and in the mammary glands of females previously administered 1000 mg/kg bw/day.
Though the incidence of calculi in the kidneys was similar to that reported after 13 weeks of treatment in males, the incidence and extent of other treatment-related findings in the kidneys was less than that after 13 weeks, indicating that partial recovery had occurred. The incidence and extent of the findings in the urinary bladder had also reduced, compared to that at the end of the treatment period, indicating that partial recovery had occurred. The incidence and extent of the hepatocellular hypertrophy in both sexes was similar to that reported at the end of the treatment period, suggesting that there had been no recovery after treatment had been withdrawn for four weeks. At the recovery sacrifice, no changes were observed in the thyroids of males or females and in the thymuses of females previously administered 1000 mg/kg bw/day, signifying that full recovery had occurred.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Serum triiodothyronine (T3) and thyroxine (T4) concentrations were slightly low, when compared to controls, in females receiving 1000 mg/kg bw/day, with males at this dose also showing a small reduction of T4 concentration. These findings showed full recovery.
There was a clear and dose-related increase, compared to controls, of serum thyroid stimulating hormone levels in both sexes, the extent of which was greater in females than in males. These findings were not evident at the end of the recovery period since animals previously given 1000 mg/kg bw/day had lower serum TSH concentrations than the control.
Details on results:
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
There was no effect of treatment on estrous cycle at the end of the treatment period or the recovery phase. All females showed evidence of oestrus, demonstrating normal cycling.
Key result
Dose descriptor:
NOAEL
Effect level:
150 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
mortality
organ weights and organ / body weight ratios
urinalysis
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
ureter
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

The mean concentrations were within the applied limits of -15%/+10% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis.

Table 1: Summary of thyroid hormone findings (pg/mL)

Sex

Male

Male

Male

Male

Female

Female

Female

Female

Group

1

2

3

4

1

2

3

4

Level (ppm)

0

150

400

1000

0

150

400

1000

Triiodothyronine (T3)

 

 

 

 

 

 

 

 

Week 14

689

784

711

701

909

1110

956

694

Week R5

566

-

-

627

951

-

-

1230

Thyroxine (T4)

 

 

 

Week 14

43300

48600

40400

37600

36200

43600

37900

24800

Week R5

49500

-

-

48400

38000

-

-

48400

Thyroid stimulating hormone

 

 

 

 

 

 

 

 

Week 14

2070

2840

4320

6000

505

993

1730

2910

Week R5

1550

-

-

339

1390

-

-

873

Table 2: Summary of urinary appearance and blood in the urine

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

150

400

1000

0

150

400

1000

Urinary appearance

Week 13

  Pale yellow

  Pale brown

  Medium yellow

  Dark yellow

  Dark brown

  Dark red

No. of animals

Week R4

  Pale yellow

  Medium yellow

  Dark yellow

No. of animals

 

 

0

0

1

9

0

0

10

 

1

3

1

5

 

 

1

0

3

6

0

0

10

 

-

-

-

0

 

 

5

2

2

0

0

1

10

 

-

-

-

0

 

 

9

0

0

0

1

0

10

 

3

1

0

4

 

 

2

0

4

4

0

0

10

 

4

1

0

5

 

 

2

0

7

0

0

0

9

 

-

-

-

0

 

 

7

0

3

0

0

0

10

 

-

-

-

0

 

 

7

0

1

0

0

0

8

 

1

0

2

3

Urinary blood

Week 13

  Negative

  Trace

  1+

  2+

  3+

No. of animals

Week R4

  Negative

  Trace

No. of animals

 

 

10

0

0

0

0

10

 

5

0

5

 

 

9

1

0

0

0

10

 

-

-

0

 

 

2

1

0

3

4

10

 

-

-

0

 

 

6

1

1

0

2

10

 

1

3

4

 

 

10

0

0

0

0

10

 

5

0

5

 

 

9

0

0

0

0

9

 

-

-

0

 

 

9

0

0

1

0

10

 

-

-

0

 

 

8

0

0

0

0

8

 

3

0

3

Table 3: Test Article-Related Effects on Organ Weights after 13 Weeks of Treatment

Males

Males

Males

Males

Females

Females

Females

Females

Dose Level (mg/kg bw/day)

0

150

400

1000

0

150

400

1000

Kidney

 

 

 

 

 

 

 

 

Absolute Weight (g)

3.244

108

119

120

1.780

112

  113

  123

Body Weight Adjusted Mean (g)

3.258

102

  115**

  123*

1.798

 110*

  112**

  121**

 

 

 

 

 

 

 

 

 

Liver

 

 

 

 

 

 

 

 

Absolute Weight (g)

17.847

107

  115

  122

9.707

116

  130

  169

Body Weight Adjusted Mean (g)

18.081

101

  113**

  125**

10.020

109*

  125**

 159**

 

 

 

 

 

 

 

 

 

Thyroids and Parathyroids

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.019

126

  142

  142

0.019

105

100

121

Body Weight Adjusted Mean (g)

0.010  

 126*

  142**

  147**

 0.02

100

95

115

 

 

 

 

 

 

 

 

 

Thymus

 

 

 

 

 

 

 

 

Absolute Weight (g)

0.273

93

99

86

0.256

91

89

83

Body Weight Adjusted Mean (g)

0.274

92

98

86

0.277

 80*

 81*

  73**

*= p≤0.05; ** = p≤0.01 statistically significant difference (adjusted mean) compared with respective control mean value.

Note: Values for absolute weight and body weight adjusted organ weights for treated groups are expressed as a percentage of the control mean value.

Table 4: Test Article-Related Effects on Organ Weights after 4 Weeks of Recovery

Males

Males

Males

Males

Females

Females

Females

Females

Dose Level (mg/kg bw/day)

0

 

1000

 

 

0

 

1000

Liver

 

 

 

 

 

 

 

 

Absolute Weight (g)

22.517

 

88

 

 

11.284

 

  122

Body Weight Adjusted Mean (g)

21.298

 

99

 

 

11.413

 

  118**

 

 

 

 

 

 

 

 

 

*= p≤0.05; ** = p≤0.01 statistically significant difference (adjusted mean) compared with respective control mean value.

Note: Values for absolute weight and body weight adjusted organ weights for treated groups are expressed as a percentage of the control mean value.

Table 5: 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

150

400

1000

0

150

400

1000

Kidneys

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

10

9

10

8

Enlarged

0

0

1

6

0

0

0

0

Dilated Pelvis

0

1

0

2

0

0

1

1

Abnormal Color

0

0

1

1

0

0

0

0

Granular

0

0

0

1

0

0

0

0

 

 

 

 

 

 

 

 

 

Urinary bladder

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

10

9

10

8

Contained Calculus (i)

0

0

10

10

0

0

1

0

Thickened

0

0

10

9

0

0

0

1

Distended/Dark area(s)

0

0

1

0

0

0

0

0

Ureters

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

10

9

10

8

Contained Calculus (i)

0

0

1

1

0

0

0

0

Thickened

0

0

3

2

0

0

0

0

Distended

0

0

1

0

0

0

0

1

Liver

 

 

 

 

 

 

 

 

Number examined

10

10

10

10

10

9

10

8

Enlarged

0

0

1

3

0

0

3

6

Table 6: Incidence of Test Article-Related Macroscopic Findings After 4 Weeks of Recovery

Males

Males

Males

Males

Females

Females

Females

Females

Dose Level (mg/kg bw/day)

0

 

1000

 

0

 

1000

 

Kidney

 

 

 

 

 

 

 

 

Number Examined

5

 

4

 

5

 

3

 

Dilated pelvis

0

 

1

 

0

 

0

 

Raised areas

0

 

1

 

0

 

0

 

 

 

 

 

 

 

 

 

 

Urinary bladder

 

 

 

 

 

 

 

 

Number Examined

5

 

4

 

5

 

3

 

Calculi, present

0

 

2

 

0

 

2

 

Thickened

0

 

0

 

0

 

1

 

Table 7: 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

150

400

1000

0

150

400

1000

 

 

 

 

 

 

 

 

 

Kidney

 

 

 

 

 

 

 

 

Number Examined

10

10

10

10

10

9

10

8

 Nephropathy

 

 

 

 

 

 

 

 

Minimal

0

0

1

0

0

0

0

0

Moderate

0

0

0

3

0

0

0

0

Severe

0

0

0

0

0

0

0

1

Granuloma, Pelvic

 

 

 

 

 

 

 

 

Minimal

0

0

1

2

0

0

0

0

Slight

0

0

2

1

0

0

0

0

Moderate

0

0

0

1

0

0

0

0

Basophilia, Tubular

 

 

 

 

 

 

 

 

Minimal

0

1

1

5

0

0

0

3

Slight

0

0

0

2

0

0

0

0

Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Minimal

0

0

3

4

0

0

3

3

Slight

0

0

2

2

0

0

0

0

Moderate

0

0

0

1

0

0

0

0

Calculi

 

 

 

 

 

 

 

 

Present

0

0

8

8

0

0

1

0

Dilatation, Pelvic

 

 

 

 

 

 

 

 

Minimal

0

0

1

0

0

0

1

0

Slight

0

1

2

1

0

0

0

0

Moderate

0

0

0

1

0

0

0

0

Marked

0

0

0

2

0

0

0

0

Severe

0

0

0

0

0

0

0

1

Infiltrate, Inflammatory Cell

 

 

 

 

 

 

 

 

Minimal

0

1

2

2

0

0

0

1

Slight

0

0

0

2

0

0

1

1

Inflammation

 

 

 

 

 

 

 

 

Minimal

0

1

0

0

0

0

0

0

Slight

0

0

0

1

0

0

0

1

Cast(s), Hyaline

 

 

 

 

 

 

 

 

Minimal

0

4

1

2

0

0

1

1

Slight

0

1

0

1

0

1

0

0

Ulceration

 

 

 

 

 

 

 

 

Moderate

0

0

0

1

0

0

0

0

Dilatation, Tubular

 

 

 

 

 

 

 

 

Minimal

0

1

0

1

0

0

0

0

Hyperplasia, Epithelium

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

0

1

1

Slight

0

1

1

0

0

0

0

0

 Necrosis, Papillary

 

 

 

 

 

 

 

 

Slight

0

0

1

0

0

0

0

0

 

 

 

 

 

 

 

 

 

Urinary Bladder

 

 

 

 

 

 

 

 

Numbers Examined

10

10

10

10

10

9

10

8

Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Minimal

0

0

1

0

0

0

4

6

Slight

0

0

9

8

0

0

3

1

Moderate

0

0

0

2

0

0

0

0

Infiltrate, Inflammatory Cells

 

 

 

 

 

 

 

 

Minimal

1

0

5

6

0

0

1

1

Slight

0

0

1

1

0

0

0

0

Hemorrhage

 

 

 

 

 

 

 

 

Minimal

0

0

2

1

0

0

0

0

Slight

0

0

1

0

0

0

0

0

Calculi

 

 

 

 

 

 

 

 

Present

0

0

3

6

0

0

0

2

Edema

 

 

 

 

 

 

 

 

Minimal

0

0

1

2

0

0

0

0

Inflammation

 

 

 

 

 

 

 

 

Slight

0

0

0

0

0

0

0

2

Marked

0

0

1

0

0

0

0

0

Dilatation

 

 

 

 

 

 

 

 

Moderate

0

0

1

0

0

0

0

0

Ulceration

 

 

 

 

 

 

 

 

Slight

0

0

0

0

0

0

0

1

 

 

 

 

 

 

 

 

 

Ureters

 

 

 

 

 

 

 

 

Numbers Examined

0

0

3

2

0

0

0

1

Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Slight

0

0

3

2

0

0

0

1

Dilatation

 

 

 

 

 

 

 

 

Slight

0

0

1

0

0

0

0

0

Moderate

0

0

0

0

0

0

0

1

Infiltrate, Inflammatory Cells

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

0

0

1

 

 

 

 

 

 

 

 

 

Liver

 

 

 

 

 

 

 

 

Numbers Examined

10

10

10

10

10

9

10

8

Hypertrophy, Centrilobular

 

 

 

 

 

 

 

 

Minimal

0

3

6

7

0

0

7

7

 

 

 

 

 

 

 

 

 

Thyroids

 

 

 

 

 

 

 

 

Number examined

10

10

10

10

10

9

10

8

Hypertrophy, Follicular Cell

 

 

 

 

 

 

 

 

Minimal

1

3

8

10

0

0

5

5

Mammary

 

 

 

 

 

 

 

 

Numbers Examined

10

0

0

10

10

9

10

8

Hyperplasia, Lobuloalveolar

 

 

 

 

 

 

 

 

Minimal

0

 

 

0

3

2

3

3

Slight

0

 

 

0

0

1

2

3

 

 

 

 

 

 

 

 

 

Thymus

 

 

 

 

 

 

 

 

Numbers Examined

10

0

0

10

10

9

10

8

Cellularity Decreased, General

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

2

2

2

Table 8: 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

 

1000

 

0

 

1000

Kidney

 

 

 

 

 

 

 

 

Number Examined

 

5

 

4

 

5

 

3

 Nephropathy

 

 

 

 

 

 

 

 

Slight

 

0

 

1

 

0

 

0

Moderate

 

0

 

1

 

0

 

0

Granuloma, Pelvic

 

 

 

 

 

 

 

 

                                                                   Slight

 

0

 

1

 

0

 

0

 

 

 

 

 

 

 

 

 

 Basophilia, Tubular

 

 

 

 

 

 

 

 

Minimal

 

0

 

0

 

0

 

1

Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Minimal

 

0

 

2

 

0

 

0

Slight

 

0

 

2

 

0

 

0

Dilatation, Pelvic

 

 

 

 

 

 

 

 

Minimal

 

0

 

0

 

2

 

1

Slight

 

0

 

1

 

0

 

0

Moderate

 

0

 

1

 

0

 

0

Inflammation

 

 

 

 

 

 

 

 

Slight

 

0

 

1

 

0

 

0

Calculi

 

 

 

 

 

 

 

 

Present

 

0

 

3

 

0

 

0

Cast, Hyaline

 

 

 

 

 

 

 

 

Minimal

 

0

 

1

 

0

 

1

Infiltrate, Inflammatory Cells

 

 

 

 

 

 

 

 

Minimal

 

1

 

1

 

0

 

0

Hyperplasia, Epithelium

 

 

 

 

 

 

 

 

Slight

 

0

 

1

 

0

 

0

 

 

 

 

 

 

 

 

 

Dose Level (mg/kg bw/day)

 

0

 

1000

 

0

 

1000

Urinary Bladder

 

 

 

 

 

 

 

 

Number examined

 

5

 

4

 

5

 

3

Hyperplasia, Urothelium

 

 

 

 

 

 

 

 

Minimal

 

0

 

2

 

0

 

1

Slight

 

0

 

2

 

0

 

2

Dilatation

 

 

 

 

 

 

 

 

Minimal

 

0

 

1

 

0

 

0

 

 

 

 

 

 

 

 

 

Liver

 

 

 

 

 

 

 

 

Number examined

 

5

 

4

 

5

 

3

Hypertrophy, Centrilobular

 

 

 

 

 

 

 

 

Minimal

 

0

 

3

 

0

 

3

 

 

 

 

 

 

 

 

 

Mammary

 

 

 

 

 

 

 

 

Number examined

 

0

 

0

 

5

 

3

Hyperplasia, Lobuloalveolar

 

 

 

 

 

 

 

 

Minimal

 

0

 

0

 

2

 

1

Slight

 

0

 

0

 

0

 

1

Conclusions:
In the sub-chronic 90-day oral repeated dose toxicity study with 2,4,6,8,10-pentamethylcyclopentasiloxane, conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for systemic toxicity was concluded to be 150 mg/kg bw/day based on test-item adverse effects in the urinary system at 400 and 1000 mg/kg bw/day. The findings in this study indicated that the target of toxicity was the urinary system and test-item administration resulted in the early deaths of four high dose group animals. There were a few findings in the urinary tract of some low dose (150 mg/kg bw/day) males but these were minor and there was no calculi-formation and, consequently, they were considered non-adverse. The urinary system findings generally showed some recovery, despite calculi still being present to a similar extent to that observed at the end of the treatment period. There was also an effect on the liver that was considered to represent an adaptive response to treatment that led to a secondary, rodent-specific effect on the thyroid glands as a consequence of disruption of the thyroid hormonal control mechanism.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
urinary
Organ:
bladder
kidney
ureter

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the sub-chronic 90-day oral repeated dose toxicity study with 2,4,6,8,10-pentamethylcyclopentasiloxane, conducted according to OECD Test Guideline 408 and in compliance with GLP, male and female Sprague-Dawley rats were administered orally by gavage doses of 0, 150, 400 and 1000 mg/kg bw/day in corn oil for 13 weeks. The control animals received the vehicle dried and deacidified corn oil (Covance Laboratories Limited, 2020a). Recovery from any effects in the control and high dose animals was evaluated during a 4-week recovery period. The dose levels selected in the study were based on the pathology results from the preliminary toxicity study (Covance Laboratories Limited, 2020b), in which dose levels of 0, 250 and 750 mg/kg bw/day were administered for 21 days and 1000 mg/kg bw/day for 14 days and did not show any evidence of toxicity during the in-life phase. Changes in the kidneys and urinary bladder were noted at necropsy and slight ulceration with associated inflammatory, proliferative changes and oedema in the urinary bladder of one female given 1000 mg/kg bw/day were observed at histopathology but there were no other findings in the kidneys.

During the main sub-chronic study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examination, estrous cycle, haematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone, organ weight, macropathology and histopathology investigations were undertaken.

The administration of 2,4,6,8,10-pentamethylcyclopentasiloxane to rats did not result in any toxicologically significant clinical signs. Chin rubbing, salivation and paddling of the forepaws was observed occasionally in males and females receiving 400 or 1000 mg/kg bw/day; each of these signs are commonly observed in studies where the test material is administered by gavage and are considered of no toxicological importance. The appearance and behaviour of the animals were unaffected by the treatment. However, four treatment-related deaths occurred during the study and recovery period; treatment-related lesions in the urinary system were considered to have caused the death of these animals (three main high dose group female animals and one high dose recovery male animal). A further high dose female died during the treatment period but its death could not be attributed conclusively to treatment. A low dose female was euthanised as a consequence of a dose administration error.

Sensory reactivity responses, grip strength and motor activity were unaffected by treatment. Estrus cycles were unaffected by treatment.

Body weight gain was unaffected up to study Week 6 in males but during the reminder of the treatment period was low at 400 and 1000 mg/kg bw/day, which was considered to be dose-related. This effect occurred in the absence of any treatment-related change in food consumption. During the 4-week recovery period, males that previously received 1000 mg/kg bw/day gained weight but the rate of weight gain was lower than that of control group, indicating that there was no significant recovery after treatment ceased.

There were no treatment-related ophthalmoscopic findings.

The serum thyroxine (T4) concentrations of males and females given 1000 mg/kg bw/day were lower than those of the controls and there was a small reduction of serum triiodothyronine (T3) concentrations in the males. The reduction of T4 lead to a consequential dose-related increase of serum thyroid stimulating hormone (TSH) levels in both sexes, particularly in females. These findings showed full recovery.

The haematological examination in Week 13 indicated high neutrophil and lymphocyte counts in males receiving 400 mg/kg bw/day and in both sexes receiving 1000 mg/kg bw/day, and high monocyte and large unstained cells in males and females receiving 1000 mg/kg bw/day;  three of these males also had high eosinophil count. These findings resulted in high total leucocyte counts in males receiving 400 mg/kg bw/day and in males and females receiving 1000 mg/kg bw/day. Platelet counts were high in females receiving 400 mg/kg bw/day and in both sexes receiving 1000 mg/kg bw/day but there was no effect on prothrombin or activated partial thromboplastin time. Eosinophil and platelet counts in males and monocyte counts in females showed no evidence of recovery but at least partial recovery had occurred for all other treatment-related haematological findings.

Biochemical changes in the blood plasma that were attributed to treatment consisted of high blood urea nitrogen in males receiving 1000 mg/kg bw/day where 5/10 males had blood urea nitrogen concentrations that were notably above the reported range in the control males and one had an associated high creatinine concentration; slightly low glucose concentrations in males receiving 1000 mg/kg bw/day; a dose-related increase of total cholesterol concentration in females, due to increased high and low density lipoprotein concentrations; slightly low triglyceride concentrations in males receiving 400 or 1000 mg/kg bw/day; high phosphorus concentrations at all dose levels and in both sexes; and a dose-related reduction of the albumin to globulin ratio in females given 400 or 1000 mg/kg bw/day attributed to an increase in the globulin fraction. At the end of the recovery period the increased total cholesterol and high and low density lipoproteins concentrations persisted in females previously dosed at 1000 mg/kg bw/day but all other treatment-related findings showed at least partial recovery.

The analysis of urine samples obtained in Week 13 indicated high volume and low urinary specific gravity in males and females receiving 400 or 1000 mg/kg bw/day and there was also a small increase of urinary volume in males and females receiving 150 mg/kg bw/day which was associated with a small decrease of specific gravity in females. Blood was detected in the urine of males receiving 400 or 1000 mg/kg bw/day but this was not observed as frequently in females. High total urinary protein output occurred in males and females receiving 400 or 1000 mg/kg bw/day, with slightly high glucose output occurring in females at these doses. All of these findings showed evidence that significant recovery had occurred during the 4-week recovery period.

After 13 weeks of treatment there were increases of liver and kidney weights in females given 150 mg/kg bw/day and in males and females given 400 or 1000 mg/kg bw/day. There was also an increase of thyroid gland weight at all doses in males and a reduction of thymus weight in males and females given 1000 mg/kg bw/day. With the exception of the increased liver weights in females, where partial recovery had occurred, these findings showed full recovery.

Macroscopic findings in the urinary system that were attributed to treatment after 13 weeks consisted of enlarged kidneys, dilated pelvis and abnormal colour and granular appearance of the kidneys at 1000 mg/kg bw/day in males, with one female at this dose also having a dilated pelvis; the presence of calculi in the urinary bladder in all males given 400 or 1000 mg/kg bw/day, with the urinary bladder wall being thickened in males given 400 or 1000 mg/kg bw/day and distension of the urinary bladder in one male given 400 mg/kg bw/day; in the urinary bladder of females given 400 or 1000 mg/kg bw/day there were calculi and thickening of the wall in single animals; the presence of calculi/wall thickness/distension were variably reported in the ureters in one to three animals given 400 or 1000 mg/kg bw/day. In addition, enlargement of the liver occurred in males and females given 400 or 1000 mg/kg bw/day. After four weeks of recovery test article-related macroscopic findings were still present in the kidneys and urinary bladder but the findings in the ureters and liver showed full recovery.

Histopathological findings after 13 weeks that were attributed to treatment occurred in the kidneys [presence of calculi in the renal pelvis, causing hyperplasia of the urothelium with inflammatory infiltrate in the majority of males given 400 or 1000 mg/kg bw/day and, to a lesser extent, in females given 1000 mg/kg bw/day; the calculi caused pelvic dilatation and the development of granulomatous inflammation in the pelvic area and, in some cases, nephropathy (consisting of a combination of tubular basophilia, inflammatory infiltrate, dilated tubules and interstitial fibrosis)]; urinary bladder (presence of calculi causing hyperplasia of the urothelium in animals, particularly males, given 400 or 1000 mg/kg bw/day and which was accompanied by inflammatory infiltrate, haemorrhage, dilatation and inflammation); ureters (hyperplasia of the urothelium in three males given 400 mg/kg bw/day and in two males and one female given 1000 mg/kg bw/day that was occasionally accompanied by dilatation); liver (centrilobular hypertrophy in the majority of animals given 400 or 1000 mg/kg bw/day); thyroid glands (follicular cell hypertrophy in animals given 400 or 1000 mg/kg bw/day); mammary glands (Lobulo-alveolar hyperplasia of the female mammary gland in females given 400 or 1000 mg/kg bw/day) and thymus (decreased cellularity at all doses in females). By the end of the 4-week recovery period there was no evidence of recovery in respect of the findings in the liver, and there was partial recovery of findings in the urinary system (despite the incidence of calculi in the kidneys being similar to that at the end of the treatment period); all other findings showed full recovery.

It is concluded that oral administration of 2,4,6,8,10-pentamethylcyclopentasiloxane for 13 weeks to Sprague-Dawley rats caused a range of adverse findings at 400 and 1000 mg/kg bw/day that included reduced weight gain during the latter part of the treatment period in males, and the findings in this study indicated that the target of toxicity of the test material was the urinary system, which resulted in the early deaths of four animals (one male and three females) in the high dose group. There were a few findings in the urinary tract of some low dose males but these were minor and there was no calculi-formation and, consequently, they were considered non-adverse. The urinary system findings generally showed some recovery, despite calculi still being present to a similar extent to that observed at the end of the treatment period. There was also an effect on the liver that was considered to represent an adaptive response to treatment that led to a secondary, rodent-specific effect on the thyroid glands as a consequence of disruption of the thyroid hormonal control mechanism. There were no adverse findings in the low dose animals and, consequently, the no observed-adverse-effect level (NOAEL) in this study was considered to be 150 mg/kg bw/day.

In the preliminary dose range-finding study, not conducted according to a guideline or in compliance with GLP, 2,4,6,8,10 pentamethylcyclopentasiloxane (HD5; CAS RN 6166-86-5) in corn oil was administered orally (gavage) to male and female rats at doses of 0, 250 and 750 mg/kg bw/day for 21 days and, in the absence of any effect of treatment, a fourth group was given 1000 mg/kg bw/day for 14 days, with treatment starting one week after the other groups (Covance Laboratories Limited, 2020b). During the study, clinical condition, body weight, food consumption, water consumption, organ weight and macropathology investigations were undertaken. Limited histopathological examination of the kidneys and urinary bladder in females was also conducted. There were no unscheduled mortality or overt toxicity during the study. There were no toxicologically significant clinical signs and no effect on food intake or body weight.

One female given 1000 mg/kg bw/day had macroscopic findings in the urinary bladder (ulceration with associated inflammatory and proliferative changes) and the urethra (distension) and histopathologically there was slight ulceration with associated inflammatory, proliferative changes and edema in the urinary bladder. All other animals at this dose were unaffected. A dose-related increase in liver weight occurred in mid and high dose females but the cause of this finding was not established as there was no histopathological examination of the liver. The findings from this study indicated that doses up to 1000 mg/kg bw/day were well tolerated and that doses up to 1000 mg/kg bw/day were therefore considered suitable for administration in the main 90-day oral repeated dose toxicity study in rats.

Data for the structural analogue, 2,4,6,8-tetramethylcyclotetrasiloxane (CAS 2370-88-9), have been included to support the read-across test proposal for an extended one-generation reproduction toxicity study. See Annex 4 for justification of read-across.

In the 90-day oral repeated dose toxicity study with 2,4,6,8-tetramethylcyclotetrasiloxane (HD4), 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 a 4-week recovery period (Covance Laboratories Limited, 2020).

In the combined repeated dose toxicity study with the reproduction / developmental toxicity screening test with 2,4,6,8-tetramethylcyclotetrasiloxane (HD4), conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEC for systemic toxicity following inhalation exposure in male and female rats was 100 ppm (equivalent to 984 mg/m3 based on MW of 240.5094 for 2,4,6,8-tetramethylcyclotetrasiloxane) based on bladder stones at 3000/2000 and 1000 ppm and histopathological findings in the urinary tract at 3000/2000 and 1000 ppm (Harlan Laboratories, 2012).

Justification for classification or non-classification

Based on the available repeated dose toxicity data for the registered substance, 2,4,6,8,10-pentamethylcyclopentasiloxane does not require classification for specific target organ toxicity following repeated exposures according to Regulation (EC) No 1272/2008.