Registration Dossier

Administrative data

Description of key information

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 NOAEL of 50 mg/kg bw/day was reported in rats treated with H-L3 (Harlan Laboratories Ltd., 2010). Effects observed at 200 mg/kg bw/day were considered by the authors of this CSR to be either adaptive and non-adverse, or specific to male rats and therefore not relevant for human hazard assessment. The overall NOAEL relevant for humans was 200 mg/kg bw/day.

Read-across inhalation data are available for the related substance octamethyltrisiloxane (L3, CAS 107-51-7). Whole body exposure of SD rats for 90 days resulted in liver and kidney changes (Harlan Laboratories Ltd., 2011). In the liver hepatocyte hypertrophy and accumulation of hepatic brown pigment in the bile ducts with associated periportal inflammation and bile duct proliferation were noted. In the kidneys tubular hypertrophy was noted and associated with minor changes in salt balance and plasma protein levels. Hyaline droplets noted represented alpha-2u-globulin accumulation. Based on the pigment accumulation noted in the liver at 3200 ppm the NOAEC (inhalation) was considered to be 400 ppm (3870 mg/m3).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
Reason / purpose:
reference to same study
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
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
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
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
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.
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
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.
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
gastrointestinal tract
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Hsd:Sprague DawleySD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: not stated
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: Males 263.6 - 308.7g (+/-8%) Females 181.2 - 219.8g (+/-10%)
- Fasting period before study: No
- Housing: Makrolon type-4 cages, 5/sex/cage
- Diet: Kliba Nafag 3433 ad libitum
- Water: local supply ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 27 January to 26 May 2009
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel sealed chambers
- Method of holding animals in test chamber: stainless steel wire cage units
- Source and rate of air: compressed air at 40L/min
- System of generating vapours: compressed air supplied into glass flasks containing Octamethyltrisiloxane through a metal nebulization tube. Flasks maintained at 60-80 degrees C.
- Temperature, humidity, pressure in air chamber: oxygen concentration maintained above 19% whenever possible. Temperature 19-25 degrees C, humidity 30-70%
- Air flow rate: 380L/min
- Method of particle size determination: n/a


TEST ATMOSPHERE
- Brief description of analytical method used: on-line GC
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal - once daily weighing test item reservoir before and after each exposure
Chemical - measured 4 times per hour by on-line GC according to following conditions:
Column: DB-1 (30m x 0.53mm x 1.5um)
Injector: 225 degrees C
Oven: 100 degrees C for 0.1 min, then 50 degrees C/min, to 150 degrees C for 0 min
Detector: FID, 300 degrees C
Duration of treatment / exposure:
90 days followed by 28 day recovery period for subgroup of Control and high dose animals
Frequency of treatment:
6 hours daily
Dose / conc.:
95 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
Dose / conc.:
3 200 ppm (nominal)
No. of animals per sex per dose:
Control and high dose - 20
Low and intermediate dose - 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: not stated
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during treatment, weekly during recovery

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and day 86
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 92 or 93, recovery period day 29
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 92 or 93, recovery period day 29
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: day 92 or 93, recovery day 29
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
ORGAN WEIGHT: Yes (see table 4)
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)
Other examinations:
Immunohistochemistry of kidney sections for alpha-2u globulin staining
Statistics:
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 Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test was applied to the ophthalmoscopic and macroscopic findings.
Statistical evaluation of microscopic findings was performed using the one-sided exact Fisher test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: all animals survived and no treatment-related clinical signs recorded.

BODY WEIGHT AND WEIGHT GAIN: no effect of treatment.

FOOD CONSUMPTION: no effect of treatment.

OPHTHALMOSCOPIC EXAMINATION: no treatment-related findings

HAEMATOLOGY: no effect of treatment

CLINICAL CHEMISTRY: A slight increase in cholesterol levels were observed in males (+26.5%) and females (+10.1%) at 3200 ppm and females at 400 ppm (+8.0%) compared to controls at the end of treatment. This was not apparent at the end of the recovery period.

Marginally increased sodium (between +3.2 and +3.8%) and chloride (between +1.2 and +1.8%) values were seen in females at all concentrations. Additionally, sodium was also increased in males at 3200 ppm (+ 1.6%) and marginally increased calcium values were noted in males (+3.6%) and females (+2.4%) at 3200 ppm. This was not apparent at the end of recovery.

Increased total protein and globulin values, which resulted in a decreased albumin to globulin ratio, were observed for males at 3200 ppm (+6.4% for protein and + 13.8% for globulin) and females at 400 (+4.4% for protein and +10.1% for globulin) or 3200 ppm (+5.4% for protein and +14.7% for globulin).This was not apparent at the end of recovery.

URINALYSIS: no effect of treatment.

ORGAN WEIGHTS: Increased absolute and relative liver weights were recorded at the end of the treatment period for males at 400 (+11.1% absolute/+12.1% body weight ratio) or 3200 ppm (+22.1% absolute/+24.6% body weight ratio) and for females at 3200 ppm (+26.5% absolute/+26.6% body weight ratio). The differences to Controls was less marked but still apparent at the end of the recovery period in males but not females.

Increased kidney weights were observed for males exposed to 3200 ppm (+6.7% absolute/+8.5% body weight ratio). This was not apparent at the end of the recovery period.

GROSS PATHOLOGY: no effect of treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Centrilobular hepatocellular hypertrophy was observed in the liver of four males at 400 ppm (minimal in severity) and nine males and nine females at 3200 ppm (minimal to slight in severity).

Brown pigmented crystalline material at a minimal to moderate degree was noted in all ten males at 3200 ppm. The material was characterized by accumulations/concretions of brown pigment in small and medium bile ducts and macrophages in the portal triads. Under polarized light these concretions were birefringent appearing bright red with a dark Maltese cross-like patterns in many globules. Periportal chronic inflammation was noted in all ten males and one female at 3200 ppm, and was also noted in one control male and female each, one female at 95 ppm, and one male and female each at 400 ppm. Bile duct proliferation was noted in one female each at 95 and 400 ppm, and nine males at 3200 ppm.

Brown pigment crystals were noted in seven males previously exposed to 3200 ppm at the end of the recovery period with a lower mean severity than at the end of the treatment period. Periportal chronic inflammation was noted at the end of recovery in four control males and in nine males and two females previously exposed to 3200 ppm, and bile duct proliferation was noted in eight males and two females of the same group.

Minimal to slight proximal tubular hypertrophy was noted in the kidneys of nine males and ten females at 3200 ppm. Minimal to slight hyaline droplets were observed in one male at 95 ppm, three males at 400 ppm and all males at 3200 ppm. This was not observed at the end of the recovery period.

IMMUNOHISTOCHEMISTRY:
Immunohistochemical staining of kidney sections for alpha-2u-globulin revealed a dose-related increase in staining in male rats at all doses terminated at the end of the dosing period. This was observed primarily as an increase in mean staining score of the globular staining patterns. There was evidence of incomplete recovery in alpha-2u-globulin accumulation in the Recovery Group males at 3200 ppml. There was no evidence of alpha-2u-globulin accumulation in the kidneys of female rats.
Dose descriptor:
NOAEC
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on accumulation of brown pigment and associated periportal chronic inflammation and bile duct proliferation noted at 3200 ppm
Critical effects observed:
not specified

Table 1 - Exposure conditions

Group

Temperature (oC)

Relative Humidity (%)

Oxygen concentration (%)

1

23.1±0.2 (n=91)

49.2± 1.5 (n=91)

20.3± 0.0 (n=91)

2

22.8± 0.2 (n=91)

37.0± 1.3 (n=91)

20.6± 0.0 (n=91)

3

23.8± 0.1 (n=91)

36.3± 1.0 (n=91)

20.3± 0.0 (n=91)

4

22.9± 0.4 (n=91)

39.9± 1.8 (n=91)

19.4± 0.3 (n=91)

Table 2 - Test Atmosphere Concentrations

Group

Achieved Test Atmosphere Concentration (ppm)

Target Test Atmosphere Concentration (ppm)

Test Atmosphere Concentration Relative to Target (%)

2

95.0±0.2
(n=91, CV=0.2%)

95

100.0%± 0.2%
(n=91)

3

400±1
(n=91, CV=0.1%)

400

100.1%±0.1%
(n=91)

4

3201±3
(n=91, CV=0.1%)

3200

100.0%±0.1%
(n=91)

Conclusions:
Whole body exposure of SD rats to octamethyltrisiloxane at 95, 400 or 3200 ppm for 90 days resulted in liver and kidney changes. In the liver hepatocyte hypertrophy and accumulation of brown pigment crystals in the bile ducts with associated periportal inflammation and bile duct proliferation were noted. In the kidneys tubular hypertrophy was noted and associated with minor changes in salt balance and plasma protein levels. Hyaline droplets noted represented alpha-2u-globulin accumulation.

Based on the pigment accumulation noted in the liver at 3200 ppm the No-Observed-Adverse-Effect-Level was considered to be 400 ppm.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
3 870 mg/m³
Study duration:
subchronic
Species:
rat
System:
gastrointestinal tract
Organ:
liver

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Hsd:Sprague DawleySD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: not stated
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: Males 263.6 - 308.7g (+/-8%) Females 181.2 - 219.8g (+/-10%)
- Fasting period before study: No
- Housing: Makrolon type-4 cages, 5/sex/cage
- Diet: Kliba Nafag 3433 ad libitum
- Water: local supply ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 27 January to 26 May 2009
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel sealed chambers
- Method of holding animals in test chamber: stainless steel wire cage units
- Source and rate of air: compressed air at 40L/min
- System of generating vapours: compressed air supplied into glass flasks containing Octamethyltrisiloxane through a metal nebulization tube. Flasks maintained at 60-80 degrees C.
- Temperature, humidity, pressure in air chamber: oxygen concentration maintained above 19% whenever possible. Temperature 19-25 degrees C, humidity 30-70%
- Air flow rate: 380L/min
- Method of particle size determination: n/a


TEST ATMOSPHERE
- Brief description of analytical method used: on-line GC
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal - once daily weighing test item reservoir before and after each exposure
Chemical - measured 4 times per hour by on-line GC according to following conditions:
Column: DB-1 (30m x 0.53mm x 1.5um)
Injector: 225 degrees C
Oven: 100 degrees C for 0.1 min, then 50 degrees C/min, to 150 degrees C for 0 min
Detector: FID, 300 degrees C
Duration of treatment / exposure:
90 days followed by 28 day recovery period for subgroup of Control and high dose animals
Frequency of treatment:
6 hours daily
Dose / conc.:
95 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
Dose / conc.:
3 200 ppm (nominal)
No. of animals per sex per dose:
Control and high dose - 20
Low and intermediate dose - 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: not stated
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during treatment, weekly during recovery

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and day 86
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 92 or 93, recovery period day 29
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 92 or 93, recovery period day 29
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: day 92 or 93, recovery day 29
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
ORGAN WEIGHT: Yes (see table 4)
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)
Other examinations:
Immunohistochemistry of kidney sections for alpha-2u globulin staining
Statistics:
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 Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test was applied to the ophthalmoscopic and macroscopic findings.
Statistical evaluation of microscopic findings was performed using the one-sided exact Fisher test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: all animals survived and no treatment-related clinical signs recorded.

BODY WEIGHT AND WEIGHT GAIN: no effect of treatment.

FOOD CONSUMPTION: no effect of treatment.

OPHTHALMOSCOPIC EXAMINATION: no treatment-related findings

HAEMATOLOGY: no effect of treatment

CLINICAL CHEMISTRY: A slight increase in cholesterol levels were observed in males (+26.5%) and females (+10.1%) at 3200 ppm and females at 400 ppm (+8.0%) compared to controls at the end of treatment. This was not apparent at the end of the recovery period.

Marginally increased sodium (between +3.2 and +3.8%) and chloride (between +1.2 and +1.8%) values were seen in females at all concentrations. Additionally, sodium was also increased in males at 3200 ppm (+ 1.6%) and marginally increased calcium values were noted in males (+3.6%) and females (+2.4%) at 3200 ppm. This was not apparent at the end of recovery.

Increased total protein and globulin values, which resulted in a decreased albumin to globulin ratio, were observed for males at 3200 ppm (+6.4% for protein and + 13.8% for globulin) and females at 400 (+4.4% for protein and +10.1% for globulin) or 3200 ppm (+5.4% for protein and +14.7% for globulin).This was not apparent at the end of recovery.

URINALYSIS: no effect of treatment.

ORGAN WEIGHTS: Increased absolute and relative liver weights were recorded at the end of the treatment period for males at 400 (+11.1% absolute/+12.1% body weight ratio) or 3200 ppm (+22.1% absolute/+24.6% body weight ratio) and for females at 3200 ppm (+26.5% absolute/+26.6% body weight ratio). The differences to Controls was less marked but still apparent at the end of the recovery period in males but not females.

Increased kidney weights were observed for males exposed to 3200 ppm (+6.7% absolute/+8.5% body weight ratio). This was not apparent at the end of the recovery period.

GROSS PATHOLOGY: no effect of treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Centrilobular hepatocellular hypertrophy was observed in the liver of four males at 400 ppm (minimal in severity) and nine males and nine females at 3200 ppm (minimal to slight in severity).

Brown pigmented crystalline material at a minimal to moderate degree was noted in all ten males at 3200 ppm. The material was characterized by accumulations/concretions of brown pigment in small and medium bile ducts and macrophages in the portal triads. Under polarized light these concretions were birefringent appearing bright red with a dark Maltese cross-like patterns in many globules. Periportal chronic inflammation was noted in all ten males and one female at 3200 ppm, and was also noted in one control male and female each, one female at 95 ppm, and one male and female each at 400 ppm. Bile duct proliferation was noted in one female each at 95 and 400 ppm, and nine males at 3200 ppm.

Brown pigment crystals were noted in seven males previously exposed to 3200 ppm at the end of the recovery period with a lower mean severity than at the end of the treatment period. Periportal chronic inflammation was noted at the end of recovery in four control males and in nine males and two females previously exposed to 3200 ppm, and bile duct proliferation was noted in eight males and two females of the same group.

Minimal to slight proximal tubular hypertrophy was noted in the kidneys of nine males and ten females at 3200 ppm. Minimal to slight hyaline droplets were observed in one male at 95 ppm, three males at 400 ppm and all males at 3200 ppm. This was not observed at the end of the recovery period.

IMMUNOHISTOCHEMISTRY:
Immunohistochemical staining of kidney sections for alpha-2u-globulin revealed a dose-related increase in staining in male rats at all doses terminated at the end of the dosing period. This was observed primarily as an increase in mean staining score of the globular staining patterns. There was evidence of incomplete recovery in alpha-2u-globulin accumulation in the Recovery Group males at 3200 ppml. There was no evidence of alpha-2u-globulin accumulation in the kidneys of female rats.
Dose descriptor:
NOAEC
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on accumulation of brown pigment and associated periportal chronic inflammation and bile duct proliferation noted at 3200 ppm
Critical effects observed:
not specified

Table 1 - Exposure conditions

Group

Temperature (oC)

Relative Humidity (%)

Oxygen concentration (%)

1

23.1±0.2 (n=91)

49.2± 1.5 (n=91)

20.3± 0.0 (n=91)

2

22.8± 0.2 (n=91)

37.0± 1.3 (n=91)

20.6± 0.0 (n=91)

3

23.8± 0.1 (n=91)

36.3± 1.0 (n=91)

20.3± 0.0 (n=91)

4

22.9± 0.4 (n=91)

39.9± 1.8 (n=91)

19.4± 0.3 (n=91)

Table 2 - Test Atmosphere Concentrations

Group

Achieved Test Atmosphere Concentration (ppm)

Target Test Atmosphere Concentration (ppm)

Test Atmosphere Concentration Relative to Target (%)

2

95.0±0.2
(n=91, CV=0.2%)

95

100.0%± 0.2%
(n=91)

3

400±1
(n=91, CV=0.1%)

400

100.1%±0.1%
(n=91)

4

3201±3
(n=91, CV=0.1%)

3200

100.0%±0.1%
(n=91)

Conclusions:
Whole body exposure of SD rats to octamethyltrisiloxane at 95, 400 or 3200 ppm for 90 days resulted in liver and kidney changes. In the liver hepatocyte hypertrophy and accumulation of brown pigment crystals in the bile ducts with associated periportal inflammation and bile duct proliferation were noted. In the kidneys tubular hypertrophy was noted and associated with minor changes in salt balance and plasma protein levels. Hyaline droplets noted represented alpha-2u-globulin accumulation.

Based on the pigment accumulation noted in the liver at 3200 ppm the No-Observed-Adverse-Effect-Level was considered to be 400 ppm.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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 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 NOAEL of 50 mg/kg bw/day was reported in rats treated with H-L3 (Harlan Laboratories Ltd., 2010). Males given 800 mg/kg bw/day demonstrated significantly reduced food consumption during the second week of the study.

It is of note that decreased food consumption was also observed in some dams in the reproduction/developmental toxicity screen, and that pups of these maternal animals developed secondary non-specific effects, such as no milk in stomachs, suggesting that the dams were weak and unable to feed their offspring.

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 pigment accumulation with portal bile ducts and associated with bile duct hyperplasia. 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.

A number of microscopic findings were also observed 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, which the study author considered to be adaptive changes. From these results, a no-observed-adverse-effect level (NOAEL) of 200 mg/kg bw/day was assigned based on hepatic brown pigment accumulation in males.

In an OECD Guideline 407 study (Harlan Laboratories Ltd., 2010), performed to GLP, the potential oral toxicity of the structurally-related substance octamethyltrisiloxane was evaluated in Sprague Dawley rats. Animals were exposed to target concentrations of 0, 5, 25, 250 and 1000 mg/kg bw/day by oral gavage for 28 days. Based on the findings in the liver (brown pigment accumulation accompanied by periportal chronic inflammation and bile duct proliferation), it was concluded that males were more affected than the females by treatment with octamethyltrisiloxane (L3). 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. In the absence of clear reversibility of microscopic liver findings, the NOAEL of the test item was considered to be 25 mg/kg/day in the males and 250 mg/kg/day in the females.

Other effects on the liver (liver enlargement, altered clinical chemistry and liver cell hypertrophy) are not necessarily directly related to the hepatic brown pigment findings. The current interpretation, which is consistent with findings with other siloxanes, is that the liver enlargement is an adaptive effect that should not be considered as adverse. There is currently no evidence, from studies with L3 or other substances, that an increase in liver weight has the potential to impact on other organs.

Whole body exposure of SD rats (via inhalation) to the structurally-related substance octamethyltrisiloxane (L3) at 95, 400 or 3200 ppm for 90 days resulted in liver and kidney changes (Harlan Laboratories Ltd., 2011). In the liver hepatocyte hypertrophy and accumulation of brown pigment in the bile ducts with associated periportal inflammation and bile duct proliferation were noted. In the kidneys tubular hypertrophy was noted and associated with minor changes in salt balance and plasma protein levels. Hyaline droplets noted represented alpha-2u-globulin accumulation. Based on the secondary effects of brown pigment accumulation and bile duct proliferation noted in the liver at 3200 ppm the NOAEC was considered to be 400 ppm (3870 mg/m3).

In an OECD Guideline 422 study (Dow Corning Corporation, 2008) performed to GLP, the potential inhalation toxicity of the structurally-related substance octamethyltrisiloxane was evaluated in Sprague-Dawley rats. Animals were exposed to target concentrations of 0, 836 (806 ppm analytical concentration), 1651 (1623 ppm analytical concentration) or 3135 ppm (3146 ppm analytical concentration) for 6 hours/day for up to 29 days in the toxicity test and up to 42 days in the reproductive/ developmental screening test. After 29 days, there were no overt toxic effects. Increased liver weights were observed in female rats exposed to 806 ppm and above and in male rats exposed to 3146 ppm. Histopathological examination revealed effects that were attributed to octamethyltrisiloxane exposure in the liver, kidney and thyroid gland. Effects (centrilobular hypertrophy) in the liver were evident in females at all dose levels and in males in the highest exposure group. In males, hepatic brown pigment accumulation was observed in the mid- and high exposure groups and protein droplet nephropathy at 806 ppm and above. Thyroid gland follicular hypertrophy was evident in both sexes at 3146 ppm and this was considered to be secondary to the hepatic centrilobular hypertrophy. Actual exposure concentrations were 806, 1623 and 3146 ppm. The NOAEC for general systemic toxicity was <806 ppm octamethyltrisiloxane based on protein droplet nephropathy and hepatic brown pigment accumulation.

Justification for classification or non-classification

Based on the results of the available repeated dose toxicity studies 1,1,1,3,5,5,5-heptamethyltrisiloxane is not classified for adverse effects following repeated exposures according to Regulation EC (No) 1272/2008.