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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 December 2008 to 22 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 287-335 g: Females: 179-211 g.
- Fasting period before study: no data
- Housing: individually in Makrolon type-3 cages (except in pairing period)
- Diet (e.g. ad libitum): standard diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 December 2008 to 7 February 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
dried and deacidified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. The test substance was weighed into a glass beaker on a tared precision balance and approximately 100% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (7 days). During the last week of the treatment, samples were taken from the middle only to confirm concentration. The samples were analysed by GC coupled to an FI detector following an analytical procedure developed at Harlan Laboratories.
Duration of treatment / exposure:
Males: at least 28 days (including 14 days prior to pairing and pairing periods)
Females: approximately 49 days (14 days prior to pairing, then through pairing and gestation, and up until offspring reach 4 days post-partum)
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose range-finding study
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: Yes, once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported, if present.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Males: Weekly during pre-pairing and after pairing periods; Females: Pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14-21 postcoitum, and days 1-4 postpartum. No food consumption was recorded during the pairing period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 postpartum.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 18 hours
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 postpartum.
- Animals fasted: Yes, 18 hours
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, at one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 postpartum) relevant parameters were evaluated with five P generation males and five P generation females from each group. This functional observation battery (FOB) assessment was conducted following the daily dose administration. Animals were observed for the following:
a) Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behaviour (e.g. circling, stereotypy) and posture as well as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes.
Sacrifice and pathology:
Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 postpartum.
GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2). For non-reproductive organs/tissues 5/10 animals examined
Statistics:
The following statistical methods were used to analyse food consumption, body weights, functional observational battery, locomotor activity and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnetttest when the data could not be assumed to follow a normal distribution.
• Fisher’s exact-test was applied if the variables could not be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no deaths or clinical signs of toxicity. In group 4, all dams were noted to push their head through the bedding material after administration starting between day 3 and 8 of the gestation period until the end of the study. One dam was noted to have ruffled fur on days 4 and 5 of the lactation period. Salivation before oral gavage administration occurred during the second week of treatment and on two occasions during the pairing period in one male in group 4 and occasionally during the after-pairing period in one male in group 3 and in three males in group 4. During the gestation and lactation periods, salivation was noted before oral gavage administration in five and one dams respectively. These observations were considered to be a sign of discomfort and not considered to be toxic effects.
Mortality:
no mortality observed
Description (incidence):
There were no deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: In group 4, mean body weight gain was statistically significantly reduced starting on day 10 of the pre-pairing period and on day 4 of the after pairing period onwards. This reduction led to a statistically significant decrease of the mean body weight on day 14 of the pre-pairing period and on day 3 (-5.6% compared to the control) of the after pairing period onwards. This was considered to be a test substance-related effect. In group 3, mean body weight gain was statistically significantly decreased between day 8 and 14 of the pre-pairing period (+7.3% versus +9.0%). This reduction led to a statistically significant decrease of mean body weight starting on day 11 of the pre-pairing period until the end of the study. This was likely due to the treatment with the test substance. In group 2, mean body weight and mean body weight gain were not considered to be affected by the treatment with the test substance.

Females: Mean body weight and mean body weight gain were not affected by the treatment with the test substance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In group 4, mean food consumption was statistically significantly reduced between days 8 and 14 of the pre-pairing period (-12.9% compared to that of control group). This was considered to be a test substance related effect. In groups 2 and 3, mean food consumption was not considered to be affected by the treatment with the test substance. The statistically significantly lower mean food consumption observed in group 2 on days 8/9 of the after pairing period (-10.6% compared to the control) was considered to be incidental since there was no dose-dependency.

Females: Mean food consumption was not affected by the treatment with the test substance for the whole duration of the treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Males: In group 4, platelet count was statistically significantly higher (+15.9% compared to the control group). Since the other coagulation parameters were not affected, this was not considered to be an adverse effect. In group 3, the statistically significantly higher prothrombin time (+5.0% compared to the control) was not considered to be a test substance-related effect since there was no dose-dependency.

Females: The assessment of the hematology data did not reveal any test substance-related effects in females. In group 3, the statistically significantly lower counts of erythrocytes, concentration of hemoglobin and hematocrit did not follow a dose dependent pattern and were therefore considered
to be incidental.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males: In group 4, the concentration of cholesterol was statistically significantly increased (+52.8% compared to the control group). The concentration of total bilirubin was statistically significantly reduced (-51.7% compared to the control group). These changes were considered to be test substance-related. The statistically significantly lower concentration of glucose in group 4 (-26.5% compared to the control) and of total bilirubin in group 3 (-43.2% compared to the control) were within the range of the historical control data and therefore considered to reflect the range of normal biological variation.

Females: The assessment of the clinical biochemistry data did not reveal any test substance-related effects in females. In group 4, the statistically significantly lower concentration of creatinine (-23.3% compared to the control) and total blirubin (-39.2% compared to the control) were within the range of the historical control data. The statistically significantly higher concentration of cholesterol in groups 3 and 4 (+26.0 and +55.0% compared to the control, respectively) was also within the tolerance limit of the historical control data. In group 3, the statistically significantly higher concentration of triglycerides (+110.8%) was not dose dependent.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
None of the parameters under investigation during the functional observational battery gave an indication of a test substance-related effect. The incidence of observations noted such as salivation, rearing number, and spontaneous vocalization when the rat was removed from the cage did not give any indication of test substance-related effect since there was no dose-dependency. The statistically significantly lower mean grip strength of hind paws in males in groups 2 and 4 (-29.9% and -26.3%, respectively) was within the range of the historical control data. The statistically significantly lower grip strength of fore paws in females in group 3 (-38.8%) was considered to be incidental since there was no dose-dependency. Body temperature in males and females was similar in all groups. Locomotor activity was assessed quantitatively in terms of low beam counts in the activity monitor did not give any indication of test substance-related effects.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males: In group 4, absolute and relative weights of liver and kidney were statistically significantly increased (absolute weights increased by 18.7% and relative weights by about 26.0%). This was considered to be a test substance-related effect. The statistically significantly lower absolute weight of thymus and relative weight of heart were within the range of the historical control data.

Females: In group 3 and 4, absolute and relative weight of liver was statistically significantly and dose dependently increased (absolute weight increased by 12.2% and 27.5% and relative weights by 18.1% and 40.3%, respectively). Taking into consideration the histopathology findings, this was considered to be a test substance related effect. The statistically significantly lower absolute weight of adrenals in group 4 was within the range of the historical control data.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In group 4, the enlargement of the size of kidneys observed in two males and the pelvic dilation that occurred in one male correlated at the microscopic level with renal tubular lesions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 200 and 800 mg/kg bw/day, during the histopathology examination an increase in focal or multifocal tubular degeneration/regeneration and of hyaline droplets/granules were observed in kidney of males but not in females. These tubular lesions were accompanied by an increase in pelvic dilation. Furthermore, at 800 mg/kg bw/day minimal to moderately increased hepatic pigment accumulation within the bile ducts associated with minimal to moderate reactive bile duct hyperplasia was observed in males.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the hepatic brown pigment accumulation noted at 800 mg/kg bw/day in males.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In an oral repeated dose toxicity study (combined with a reproduction/developmental toxicity screening test), performed to GLP and conducted according to OECD Guideline 422, a no-observed-adverse-effect level (NOAEL) of 200 mg/kg bw/day was reported in rats treated with 1,1,1,3,5,5,5-heptamethyltrisiloxane based on hepatic brown pigment accumulation in male rats.

Under polarised light some pigment accumulations show birefringence, but this finding was not consistent in size or between animals. Pigment accumulation was considered to be an adverse finding due to observed secondary periportal chronic inflammation and bile duct proliferation. The weight effects in the liver are considered to be adaptive effects, and therefore not adverse.

The effects in the male kidney consisting of protein droplet nephropathy and tubular degeneration are considered to be species specific as they represent alpha-2u-globulin accumulation, although immunohistochemistry to definitively identify it was not performed. However, in a 28-day repeat dose study on another substance (1,3-diethenyl-1,1,3,3-tetramethyldisiloxane, CAS 2627-95-4) similar kidney findings were recorded and immunohistochemistry was performed and confirmed the presence of alpha-2u-globulin (WIL, 2011). Therefore, as the kidney findings in both studies were similarly described it is reasonable to assume the presence of alpha-2u-globulin in this study, so the kidney findings are therefore not included in NOAEL considerations.
Executive summary:

In an oral repeated dose toxicity study (combined with a reproduction/developmental toxicity screening test), performed to GLP and conducted according to OECD Guideline 422, 1,1,1,3,5,5,5-heptamethyltrisiloxane was assessed for its toxicity in Wistar rats.

 

Four groups of ten male and ten female rats were treated by oral gavage with the test material (in corn oil) at 0, 50, 200 or 800 mg/kg bw/day. Following two weeks of treatment, animals were allowed to mate for up to two weeks. In total, males were treated for at least 28 days (including the pre-mating period of 14 days), while females were treated for approximately 49 days (a pre-mating period of 14 days, a mating period of up to 14 days, throughout gestation and up until 4 days post-partum). Animals were observed throughout the study for any overt signs of toxicity or morbidity, until sacrifice and gross necropsy. Changes in body weight, food consumption, and clinical chemistry and haematology were recorded.

 

All animals survived until scheduled necropsy and there were no clinical signs of toxicity (signs of discomfort such as increased salivation were noted but were not considered to be toxic effects).

 

Males given 800 mg/kg bw/day demonstrated significantly reduced food consumption during the second week of the study. Significant decreases in body weight and body weight gain were noted in males given at least 200 mg/kg bw/day. Levels of cholesterol were significantly increased, and total bilirubin significantly decreased in males given 800 mg/kg bw/day. In females given at least 200 mg/kg bw/day, the absolute and relative weight of the liver was significantly increased in a dose-dependent manner, while males given 800 mg/kg bw/day also demonstrated significant increases in liver, and kidney, weight. Adverse, apparently substance-related microscopic findings were reported in the kidneys and livers of males given at least 200 mg/kg bw/day and 800 mg/kg bw/day respectively, and included protein droplet nephropathy, kidney tubular degeneration/regeneration, and hepatic brown pigment accumulation. A number of microscopic “adaptive changes” were also seen in the spleen and thyroid gland of females given at least 200 mg/kg bw/day and in males given 800 mg/kg bw/day.

 

From these results, a no-observed-adverse-effect level (NOAEL) of 200 mg/kg bw/day was assigned to males and females for general toxicity, based on the microscopic findings in the liver (hepatic brown pigment accumulation) of males.