Octamethyltrisiloxane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 December 2009 - 20 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Octamethyltrisiloxane

EC Number:

203-497-4

EC Name:

Octamethyltrisiloxane

Cas Number:

107-51-7

Molecular formula:

C8H24O2Si3

IUPAC Name:

2,2,4,4,6,6-hexamethyl-3,5-dioxa-2,4,6-trisilaheptane

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Netherlands
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: Males 196-220g, females 152-183g
- Fasting period before study: no
- Housing: In groups of 5, in Makrolon type-4 cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: corn oil (dried and deacidified)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared daily. The test material was weighed into a glass beaker, and the vehicle was added to give the appropriate final concentration of the test item in the dose formulation. Each dose level was prepared individually. The mixtures were stirred using a magnetic stirrer and used at room temperature. The homogeneity of the rest item in the vehicle was maintained during daily administration period using a magnetic stirrer.

VEHICLE
- Lot/batch no. (if required): 049103168

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed using a GC method. After experimental start and during week 3, duplicate samples of the control group and three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hour; due to organizational reasons the samples were transferred to the analytical department ca. 4.5 hours after preparation). The samples were delivered to the analytical department and stored at -20C +/-5 until analysis.

Duration of treatment / exposure:

Treatment: 28 days
Recovery: 14 days

Frequency of treatment:

Daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

The groups comprised of 5 animals per sex which were terminated after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14 day treatment-free recovery period after which they were terminated.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous range finding toxicity study (C53644)

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on days 1-3, once daily on days 4-28 and once daily during days 1-14 of the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimatization, treatment and recovery periods and before necropsy.


HAEMATOLOGY: Yes
Complete blood cell count including: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin and haemoglobin concentration, haemoglobin concentration distribution width, reticulocyte count, reticulocyte maturity index, total leukocyte count, differential leukocyte count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells, platelet count, methaemoglobin, Heinz bodies, prothrombin/thromboplastin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 weeks, and 6 weeks (recovery)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined. Glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, bile acids, gamma-glutamyl-transferase, creatinine kinase, sodium, potassium, chloride, calcium, phosphorus (inorganic), total protein, albumin, globulin and albumin/globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: After 4 weeks, and 6 weeks (recovery)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined. Urine volume, specific gravity, colour, appearance, pH, nitrite, protein, glucose, ketones, urobilinogen, erythrocytes, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER: Weekly behavioural observations were carried out for the animals in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before initial dosing and once weekly (weeks 1 to 3) during treatment period. Vaginal smears were taken over four days from all females in Week 4, and the stage of estrus was evaluated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes (see table 1)

Other examinations:

Organ weights were recorded for adrenal glands, brain (including section of medulla/pons, cerebral and cerebella cortex), epididymides, heart including auricles, kidneys, liver, ovaries, pituitary gland, seminal vesicles and prostate gland (including coagulating glands), spleen, testes, thymus, thyroid, uterus. Relative organ weights were calculated on the basis of body weight and brain weight. Terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios and organ to brain weight ratios were determined.

Statistics:

The following statistical methods were used to analyse body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios and macroscopic findings.
• 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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived until scheduled necropsy.
No test-item related findings were noted and no abnormalities were recorded at any dose level.
In three male animals at 1000 mg/kg/day clinical signs were noted during the treatment phase: animal No. 28 had hunched posture and slight visible body weight loss from day 20 onwards as well as slightly ruffled fur at day 14 and again from day 22 to the end of observation time were observed. Slight dyspnea was recorded at days 13 and 14 and again from day 19 to 25. Slight salivation was observed from day 19 to 22. In animal 35, slight to moderate hair loss was observed in the right axillary region from day 10 to the end of observation. These findings are considered to be typical background findings.

BODY WEIGHT AND WEIGHT GAIN
A test item-related reduction in the body weight was recorded in males at 1000 mg/kg/day when compared to the control animals.
The mean body weight in these males was significantly lower on day 8 (-9%), day 15 (-11%), day 22 (-14%) and day 28 (-11%) of the treatment phase (all p<0.01) as well as day 1 (-15%), day 8 (-11%; both p<0.01) and day 14 (-9%; p<0.05) of the recovery period compared to the control group. In addition, the mean body weight gain was significantly reduced in the same animals on days 8, 15, 22 and 28 of treatment (p<0.01), but was increased compared to the control animals during recovery (day 8; p<0.05). This was considered to be a compensatory reaction.
Increased mean body weight gain compared to control was noted during recovery in females at 1000 mg/kg/day (day 14; p<0.01).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No test item-related difference in the mean daily food consumption was noted at any dose level. Changes in mean food consumption were noted in male and female animals at 1000 mg/kg/day. The food consumption was increased in these animals during treatment days 22 - 28 and during
the recovery period when compared to the control groups.


HAEMATOLOGY
After the four-week treatment phase, test item related and statistically significant changes were noted in males at 1000 mg/kg/day. These included increased number of red blood cells as well as decreased mean corpuscular volume and mean cell hemoglobin (with normal mean cell
hemoglobin concentration). The decrease in MCV and MCH persisted at the end of the recovery period. Although the red blood cell count was not significantly different from control at the end of the recovery, it was still much higher at this time point compared to the control animals and even to
the data at the end of the treatment phase. Therefore, it is considered to be relevant although not statistically significant. However, the lack of statistical significance was due to a single outlier and the smaller sample size. Mean corpuscular hemoglobin level was below the control levels in the female animals at 1000 mg/kg/day. The reticulocyte maturity index showed a higher fraction of mature (low fluorescence reticulocytes) and reduced fraction of immature (middle fluorescence and high fluorescence reticulocytes) cells only in males. In the differential white blood cell count, the relative and
absolute number of eosinophils was below the control level. Significant changes of these parameters were no longer evident after the 2-week recovery period. Other significant changes (see below) noted in the hematology parameters in male and female animals are within the historical control data for animals of this strain, sex and age, based on outliers or due to statistical method and, therefore, considered not to be test item-related.
Within the normal range of variability were reduced relative lymphocyte count (p<0.05), increased number of neutrophils (p<0.05) and platelets (p<0.01), increased hemoglobin distribution width (p<0.01) in male animals at 1000 mg/kg/day as well as decreased hemoglobin and hematocrit (both p<0.05) in males at 250 mg/kg/day. In females, red blood cells (p<0.05) and hematocrit (p<0.01) were reduced at 5 mg/kg/day and reduced red cell distribution width 1000 mg/kg/day, but also within the range of historical data. The mean relative methemoglobin level was the same in males at 250 mg/kg/day and 1000 mg/kg/day, but is mentioned significantly different the latter group due to the rounding difference during calculation. This parameter was also within the historical data range. The mean thromboplastin time was significantly lower in males at 250 mg/kg/day and at 1000 mg/kg/day and in females at 25 mg/kg/day and 1000 mg/kg/day (but not at 250 mg/kg/day) when compared with the control animals. As the control values exceed the range of historical data, the statistical differences were considered to be artefacts.

CLINICAL CHEMISTRY
After four weeks of treatment, several test item-related effects were noted in the clinical biochemistry parameters.
Test item-related changes in liver-associated enzymes noted in males at 1000 mg/kg/day included significantly increased mean activities of aspartate aminotransferase (p<0.05), alanine aminotransferase (p<0.05) and gamma glutamyl transferase (p<0.01). Females of the same dose
level were unaffected. Total cholesterol and phospholipid levels were significantly elevated (p<0.01) in males at 250 mg/kg/day and in both sexes at 1000 mg/kg/day. Triglyceride level was increased (p<0.01) only in females at 1000 mg/kg/day and this difference persisted until the end of the recovery period (p<0.05).
Mean total bilirubin level was increased (p<0.01) in males at 250 mg/kg/day and 1000 mg/kg/day. In females, a significant increase was noted only at 250 mg/kg/day (p<0.01). As no effect was noted in females at 1000 mg/kg/day, this was considered to be not test item related.
An increase in glucose level was noted in males and females at 5 mg/kg/day, males at 25 mg/kg/day and females at 250 and 1000 mg/kg/day when compared to the controls but was within the range of historical data for animals of this strain, sex and age and considered to be not
related to treatment. Significant changes in protein parameters noted in males and females at 250 mg/kg/day and/or 1000 mg/kg/day included increased total protein and globulin, and decreased albumin and albumin/globulin ratio. However, with exception of higher globulin and lower albumin/globulin ratio in males at 1000 mg/kg/day, all changes were within the historical data range and considered not to be test item-related.
Significantly elevated sodium and chloride serum levels were evident in males at 1000 mg/kg/day (p<0.01) after four weeks of treatment. An increase in serum potassium was noted in males at 25 mg/kg/day (p<0.05), 250 mg/kg/day (p<0.05) and 1000 mg/kg/day (p<0.01). In females at 1000 mg/kg/day, mean serum potassium level was also significantly increased after four weeks of treatment but did not persist after the recovery period. Phosphorous level was lower than control level in males at 250 mg/kg/day (p<0.05) as well as males and females at 1000 mg/kg/day (p<0.05 and p<0.01, resp.). However, the values in females were still within the historical data.
Further significant changes in females included increased sodium and chloride at 25 mg/kg/day and decreased calcium at 5 mg/kg/day, all within the range of the historical data and considered not to be test item-related. After two weeks of recovery, several significant differences were noted: significantly higher potassium persisted in males (p<0.05) when compared with the control values. Although the triglyceride level remained significantly higher in females (p<0.05) when compared with the control value, it was markedly lower than at the end of the treatment period and most likely
caused by a low control value. Decreased bile acid (p<0.01), decreased mean activity of alanine aminotransferase (p<0.01) and decreased mean activity of alkaline phosphatase (p<0.05) in males as well as decreased albumin levels in females (p<0.05) were noted. These differences were either within the range of the historical control data, or considered to be of no toxicological relevance.


URINALYSIS
Mean urine volume was significantly increased in males at 1000 mg/kg/day (p<0.01) and the relative density was decreased in the same animals after four weeks of treatment when compared to the control group. However, these values are within the range of historical data and do not
appear in females of the same dose group. Nevertheless, this effect was considered to be toxicologically relevant (but not adverse) due to the combination with changes in serum electrolyte levels (see Clinical Biochemistry) and microscopic changes. The significant decrease in protein noted in males at 1000 mg/kg/day (p<0.05) was in the range of historical data. The ketones were also significantly decreased at 1000 mg/kg/day (p<0.01) with control levels above the historical data range. The urine pH in females at 1000 mg/kg/day was significantly different from control. Although the pH values appear identical in females at 25 and 1000 mg/kg/day, the statistical significance resulted from a rounding difference and different numbers of animals (n = 5 in group 2 versus n = 10 in group 4). In all cases, the exact raw data values were used for statistical analysis, but are rounded for printing. Therefore, the statistical significance of this parameter was considered to be an artefact.

NEUROBEHAVIOUR
No test-item related differences compared to the control groups were recorded in the mean forelimb or hindlimb grip strength values of male and female animals at any dose levels. The mean grip strength was significantly reduced (p<0.05) in females at 5 mg/kg/day. As there
were no significant differences in the higher dose groups compared to the control, this finding is considered to be incidental.

No test item-related effects on the locomotor activity were observed.
When compared to the control group, significantly reduced locomotor activity was recorded in males at 1000 mg/kg/day at 20 - 30 minutes (p<0.05), 30 - 40 minutes (p<0.01) and 40 - 50 minutes (p<0.05). In females of this dose level, significantly increased locomotor activity was
noted at 0-10 minutes (p<0.05) and 40-50 minutes (p<0.05). These differences are considered to be within the normal range of variability.

ORGAN WEIGHTS
Test item-related increases in mean absolute and relative liver weights were recorded in male animals at 25 mg/kg/day, 250 mg/kg/day and/or 1000 mg/kg/day and in females at 250 mg/kg/day and 1000 mg/kg/day after four weeks of treatment. Mean absolute liver weights were significantly elevated in males at 250 and 1000 mg/kg/day (+56% for both groups) and in females at 1000 mg/kg/day (+37%; all p<0.01). The mean liver/body weight ratio was significantly increased in males at 25 mg/kg/day (+12%; p<0.05), 250 mg/kg/day (+52%; p<0.01) and 1000 mg/kg/day (+78%; p<0.01) and in females at 250 mg/kg/day (+12%; p<0.05) and 1000 mg/kg/day (+45%; p<0.01). Furthermore, the mean liver/brain weight ratio was significantly higher in males at 250 mg/kg/day (+57%; p<0.01) and 1000 mg/kg/day (+62%; p<0.01), and in females at 1000 mg/kg/day (44%; p<0.01).
Reduced mean absolute weights of the heart (-16%) and pituitary gland (-18%) were noted in males at 1000 mg/kg/day (p<0.05). Elevated mean kidney/body weight ratio (p<0.01) was recorded in male animals at 1000 mg/kg/day. Significantly reduced mean heart/brain weight ratio (p<0.05) was noted in males at 1000 mg/kg/day. Significantly decreased mean absolute brain weights were recorded in female animals at
250 mg/kg/day (-4%; p<0.05) and at 1000 mg/kg/day (-4%; p<0.01). After two weeks of recovery, significantly lower mean absolute brain weights (-5%; p<0.01), lower combined absolute mean weights of the seminal vesicles/prostate glands (-15%; p<0.01) and lower weight of epididymides (-8%; p<0.05) were noted in males at 1000 mg/kg/day. Elevated mean testes/body weight ratios (p<0.05) and mean testes/brain weight ratios were
recorded (p<0.05) in these animals compared to controls and mean seminal vesicles and prostate/brain weight ratio were decreased (p<0.05). Elevated mean liver/body weight and liver/brain weight ratios (+23%; p<0.01 and +15%; p<0.05, respectively) were recorded in males at 1000 mg/kg/day. In the corresponding females, a significantly higher mean liver/body weight ratio (+12%; p<0.01) was also noted.

GROSS PATHOLOGY
Test item-related enlargement and dark or black brown discoloration of the liver were noted in males at 250 mg/kg/day (100% of treated animals, both p<0.01) and at 1000 mg/kg/day (80 - 100%, p<0.01 or p<0.05, respectively) and in females at 1000 mg/kg/day (40%) at necropsy
after four weeks of treatment. Additional findings noted in individual males or females were considered to be not related to treatment: dark red foci in the mucosa of the stomach (one control male), unilateral renal pelvis dilation (one male at 5 mg/kg), red foci or reddish discoloration of the thymus (one control male and one control female, one male at 250 mg/kg/day, one male at 1000 mg/kg/day, one female at 5 mg/kg/day), dark red nodules in epididymal adipose tissue (one male at 250 mg/kg/day), dilation of the uterus (one female at 25 mg/kg/day and one female at 250 mg/kg/day).
After two weeks of recovery, black brown discoloration of the liver was still present in all males at 1000 mg/kg/day (p<0.01), and enlarged / discoloured lymph nodes were recorded in one of these males. No findings were noted in the females at necropsy after the 2-week recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC
LIVER:
Hepatic brown l pigment accumulation in the intra-hepatic bile ducts was recorded at minimal to moderate severity in animals of both sexes at 1000 mg/kg/day and in males at 250 mg/kg/day after the treatment period. Brown pigment accumulation was recorded in Kupffer cell as well as in
hepatocytes in males at 250 mg/kg/day and 1000 mg/kg/day at the end of the four week treatment period. Under polarised light some pigment accumulations show birefringence, but this finding was not consistent in size or between animals. Periportal chronic inflammation and bile duct proliferation at minimal to slight severity was recorded in both sexes at 1000 mg/kg/day and in males at 250 mg/kg/day at the end of the treatment period. Hepatocellular hypertrophy (centrilobular) was recorded at minimal severity in both sexes at 1000 mg/kg/day and in males at 250 mg/kg/day after four weeks of treatment. With the exception of hepatocellular hypertrophy in both sexes and bile duct proliferation in females, these findings persisted in animals at 1000 mg/kg/day after the 14 day recovery period.

KIDNEY:
Hyaline droplets at minimal to slight severity were recorded in males at 25 mg/kg/day, 250 mg/kg/day and 1000 mg/kg/day at the end of the treatment period. The hyaline droplets were not evident in animals after 14 day recovery period. Immunohistochemistry for a-2u globulin (globular staining) showed a similar dose effect as seen with hyaline droplets indicating that the hyaline droplets are not relevant for humans.

THYROID GLANDS:
Follicular cell hypertrophy at minimal severity was recorded in two males and one female at 1000 mg/kg/day at the end of 28-day treatment period.
The follicular cell hypertrophy was not evident in animals after the 14-day recovery period.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: Based on the findings in the liver (brown pigment accumulation accompanied by periportal chronic inflammation and bile duct proliferation), and the absence of clear reversibility.

2

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: lack of reversibility of macroscopic liver findings.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL of 25 mg/kg/day in the males and 250 mg/kg/day in the females was determined in a 28 day oral gavage study in rat, conducted according to current OECD guideline and in compliance with GLP.