Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18.03.2009 to 03.08.2010
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Screening Toxicity testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V.
- Age at study initiation: Approximately seven weeks.
- Weight at study initiation: Males: 222-239 g; Females: 163-188 g.
- Fasting period before study: No data
- Housing: Groups of five in Makrolon type-4 cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Eight 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: 19.03.2009 To: 07.05.2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
dried and deacidified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared weekly. Decamethyltetrasiloxane (L4) was weighed into a glass beaker on a balance. Thereafter the remaining vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature. Homogeneity of the test substance 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:
After experimental start, samples of the control group as well as three samples of about 2g of each concentration were taken prior to dosing for analysis of homogeneity, concentration and stability. Samples of about 2g of each concentration were taken during week 3 after commencement of dosing to confirm homogeneity and concentration. Analysis was determined by gas chromatography coupled to a flame ionisation detector and quantified with the area under the peak.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily (seven days/week)
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Five (additional five animals in control and 1000 mg/kg bw/day groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on dose range-finding study.
- Rationale for selecting satellite groups: To investigate reversibility of any adverse effects observed.
- Post-exposure recovery period in satellite groups: yes, 14 days.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for viability and mortality were recorded twice daily. The animals were observed for clinical signs once before commencement of administration as well as twice daily on days 1 to 3, once daily on days 4 to 28 (treatment period), and once daily during days 1 to 14 (recovery period).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during the acclimatization, treatment and recovery periods and before necropsy.

FOOD CONSUMPTION: Food consumption was recorded once during the acclimatization period and weekly thereafter.

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 AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 weeks in main and recovery groups. After 6 weeks in recovery group.
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes, for 18 hours
- How many animals: All animals
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 weeks in main and recovery groups. After 6 weeks in recovery group.
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table No.1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During 18 hours fasting period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.
- Dose groups that were examined: All
- Battery of functions tested: appearance, behaviour, respiration, reflexes, motor activity.

Functional Observation Battery (FOB): During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.

Vaginal smear for estrus stage: A vaginal smear was taken from all females during the last week of treatment and the stage of estrus was evaluated.
Sacrifice and pathology:
Sacrifice after 4 weeks for the main group. Sacrifice after 6 weeks for the recovery group.

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)
Statistics:
The following statistical methods were used to analyse body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings: 1) 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.2) 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. 3) Fisher's exact-test.

Results and discussion

Results of examinations

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:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no deaths and no general clinical signs associated with the test substance. In females, slight chromodacryorrhea was evident in one female during weeks 1, 2 and 3 of treatment.

BODY WEIGHT AND WEIGHT GAIN: No treatment-related changes in body weights or body weight gains were noted after the treatment period or at the end of the recovery period. Marginally higher body weight noted in males at 1000 mg/kg bw/day were not significantly higher than those of the controls and considered to be within the range of typical variation. On Day 8 of treatment, the mean body weight gain of the males treated with 1000 mg/kg bw/day was significantly elevated (p<0.05) when compared with the controls.
FOOD CONSUMPTION: No effects.

FOOD EFFICIENCY: No effects.

HAEMATOLOGY: After four weeks of treatment, no test substance-related differences were noted in males or females. At 1000 mg/kg bw/day, significantly lower hemoglobin and hematocrit were noted in males only. The respective platelet counts in males and females were significantly elevated when compared with the controls, yet remained distinctly lower than the mean platelet count of the historical control values. Therefore, these differences were considered to be incidental. The mean activated thromboplastin time in females was significantly lower when compared with controls, but this difference was not evident in males.

At 250 mg/kg bw/day, the red cell count, hemoglobin and hematocrit vlaues of males were all significantly lower than those of the controls, whereas females were unaffected. The red cell distribution width was significantly lower in males but this finding was not dose related and therefore considered to be unrelated to the test item. The mean absolute reticulocyte count was lower in females and in males but only in females did the difference attain statistical significance. The mean relative number of 'large unstained cells' was significantly lower in females when compared with controls.

At 25 mg/kg bw/day significant reductions in the men relative basophil and the mean absolute monocyte counts in males and females, respectively, were significantly lower when compared with their respective control values. These differences were not dose-related.

By the end of the two week recovery period, none of the minor differences noted in the 1000 mg/kg bw/day group were considered to be late effects of the treatment. In males and females, the mean cell volume was significantly lower than the respective controls. In females, the mean cell hemoglobin was also significantly reduced. There was a significant reduction in the mean number of high fluorescence reticulocytes of females but not in males.

CLINICAL CHEMISTRY: After four weeks of treatment, a number of statistically significant and toxicologically relevant changes were noted. Significantly elevated blood glucose levels were noted in males and females treated with 25 mg/kg bw/day (p<0.05), males treated with 250 mg/kg bw/day (p<0.01), and in males and females treated with 1000 mg/kg bw/day (both p<0.01). Significantly reduced mean total bilirubin levels were noted in both sexes at 25 mg/kg bw/day (p<0.05), both sexes at 250 mg/kg bw/day (p<0.01) and both sexes at 1000 mg/kg bw/day (p<0.01).

The mean urea level was significantly elevated in males treated with 1000 mg/kg bw/day (p<0.05). The mean cholesterol level was significantly elevated in females treated with 1000 mg/kg bw/day (p<0.01) when compared with controls. Males at this dose level were unaffected.

Aspartate aminotransferase activity was significantly lower in females at 25 mg/kg bw/day (p<0.01), both sexes at 250 mg/kg bw/day (p<0.05 in males and p<0.01 females), and in females at 1000 mg/kg bw/day (p<0.01) when compared with controls. Alkaline phosphatase activity was also significantly reduced in females at 250 mg/kg bw/day (p<0.05) and 1000 mg/kg bw/day (p<0.01).

Lactate dehydrogenase activity was significantly lower in males and females treated with 1000 mg/kg bw/day (both p<0.05). Lower creatine kinase levels were statistically significant in females treated with 25 mg/kg bw/day (p<0.05) and 1000 mg/kg bw/day (p<0.01). Reductions of these parameters are generally not associated with systemic toxicity.

Females treated with 1000 mg/kg bw/day also showed a dose-related, statistically significant increase in phospholipid levels (p<0.01) when compared with controls. Increased gamma glutamyltransferase activity was significantly elevated (p<0.01) in females treated with 1000 mg/kg bw/day, and as all but one female showed elevated values for this parameter, it was considered to be test substance-related.

There were also changes in electrolyte and transport proteins. At 1000 mg/kg bw/day, potassium and chloride were significantly elevated (both p<0.01) and phosphorus was reduced (p<0.01) in males. In females, sodium was significantly elevated (p<0.05) and phosphorus was reduced (p<0.01). The pattern of change in albumin and globulin levels were generally similar in both sexes. At 250 and 1000 mg/kg bw/day, albumin values were significantly reduced in males (p<0.01 at both dose levels) and females (p<0.05 and p<0.01, respectively). However, only the resulting changes in total proteins of females attained statistical significance (p<0.05 and p<0.01, respectively), and the resulting albumin/globulin ratios of both sexes were significantly reduced (p<0.01).

After the recovery period, phosphorus levels remained significantly lower (p<0.05) in males previously treated with 1000 mg/kg bw/day. In females, significantly reduced total bilirubin (p<0.05) and significantly reduced bile acids (p<0.05) were noted, although the latter difference was due to a markedly higher control value rather than a reduction in the value recorded in the females that were treated. The mean aspartate aminotransferase activity of the females was significantly reduced (p<0.05) when compared with the controls, and the calcium level was also significantly reduced (p<0.01) when compared with the controls. The different values were either unchanged after the end of the treatment period, due to outlying values or contrary to changes generally not associated with systemic toxicity.

URINALYSIS: No adverse findings.

ORGAN WEIGHTS: After four weeks treatment, test substance-related changes included significantly elevated mean absolute liver weights, mean liver-to-body weight ratios and mean liver-to-brain weight ratios in males and females treated with 250 mg/kg bw/day and 1000 mg/kg bw/day (p<0.05 or p<0.01). At 250 mg/kg bw/day, the differences in mean absolute liver weights were 32.3% and 32.8%, respectively, for males and females. At 1000 mg/kg bw/day, the differences were 23.3% and 60.7% for males and females, respectively.

Although these changes were considered to be largely adaptive in nature, they were not completely reversible after the recovery period in the remaining treated females. Significantly elevated mean absolute liver weights, mean liver-to-body weight ratios and mean liver-to-brain weight ratios (all p<0.01) persisted after two weeks recovery when compared with the controls.

After two weeks recovery, males had significantly elevated mean absolute and relative thyroid weights (all p<0.05) that were not evident after the treatment period. However, in the absence of microscopic findings these were considered not be late effects. In females, a significantly elevated mean kidney-to-brain weight ratio was noted that was not observed after the treatment period.

No difference in organ weights were noted at 25 mg/kg bw/day after the end of the treatment period or in the remaining animals after the recovery period.

GROSS PATHOLOGY: Treatment-related macroscopic findings included accentuated lobular pattern on the liver in both males and females treated with 250 and 1000 mg/kg bw/day at the end of the treatment period. All other findings were within the range of normal background lesions that may be recorded in animals of this strain and age.

HISTOPATHOLOGY: In the liver, minimal to slight brown pigment accumulation in the intrahepatic bile duct in males treated with 250 and 1000 mg/kg bw/day was observed. These pigments were brownish in haematoxylin and eosin stain and negative for bile pigment in the Hall stain. Bile duct proliferation as well as periportal chronic inflammation at minimal to slight severity was recorded in males treated with 1000 mg/kg bw/day.

Hepatocellular hypertrophy was recorded at minimal severity in three males treated with 1000 mg/kg bw/dat at the end of the treatment period. Incidence and severity of periportal fatty change in the liver was increased (the severity was dose-related, but not statistically significant) in females treated with 25, 250 and 1000 mg/kg bw/day.

In the thyroid, follicular cell hypertrophy at minimal severity was recorded in a single male treated with 1000 mg/kg bw/day.

The remaining findings were within the range of normal background lesions.

IMMUNOHISTOCHEMICAL STAINING: Findings in the kidney sections subjected to immunohistochemical staining for alpha-2u-globulin indicated a test substance-related accumulation of alpha-2u-globulin in male rats from the 1000 mg/kg bw/day group compared to rats from the concurrent control groups.

THYROID HORMONE ANALYSIS: In the absence of any morphological findings that would support a possible effect upon T3, T4 and TSH, these analyses were omitted.

FOB: There were no significant test substance-related changes.

LOCOMOTOR ACTIVITY: Slight increased values were noted during the early stages of the locomotor activity in males and females treated with 1000 mg/kg bw/day when compared with the controls, and was considered to be a slight test substance-related effect. Significant elevated mean locomotor activity was noted in males treated with 1000 mg/kg bw/day from 10-20 minutes (p<0.01), from 20-30 minutes (p<0.05) and from 0-60 minutes (p<0.01). The remaining measurement intervals of these males and the males treated with 25 mg/kg bw/day and 250 mg/kg bw/day compared favourably with those of the control males. The mean locomotor activity of the females treated with 1000 mg/kg bw/day was significantly elevated during 0-10 minutes (p<0.05), when compared with the control females.

VAGINAL SMEAR FOR ESTRUS STAGE: The number of estrus cycles did not show test substance-related differences.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
other: brown pigment accumulation was considered to be an adverse finding, due to secondary periportal chronic inflammation and bile duct proliferation

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
bile duct
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 1 Hepatic findings at terminal sacrifice

Findings/incidence/mean severity grade  Group 1    (0 mg/kg bw/d)  Group 2    (25 mg/kg bw/d)  Group 3   (250 mg/kg bw/d)  Group 4   (1000 mg/kg bw/d)
   5 M  5 F  5 M  5 F  5 M  5 F  5 M  5 F
Brown pigment accumulation #  -  -  -  1/1.0  -  5/1.8*  -
 Bile duct proliferation #  -  -  -  -  -  -  5/1.4*  -
 Periportal chronic inflammation #  -  -  -  -  -  -  5/1.4*  -
 Hepatocellular hypertrophy #  -  -  -  -  -  -  3/1.0  -
 Fatty change: perilobular  -  3/1.0  -  5/1.2  5/1.8  1/1.0  5/2.8

*: p<0.01 by one-sided exact Fischer Test

#: p<0.01 by Armitage/Cochran Trend Test

Table 2 Hepatic findings at the end of the recovery period

 Findings/incidence/mean severity grade  Group 1     Group 4   
   5 M  5 F  5 M  5 F
Brown pigment accumulation  -  -  5/1.4*  -
 Bile duct proliferation  -  -  2/1.0  -
 Periportal chronic inflammation  -  -  5/1.2*  -
 Hepatocellular hypertrophy  -  -  -
 Fatty change: perilobular  -  2/1.0  -  4/1.5

*: p<0.01 by One-sided Exact Fischer Test

#: p<0.01 by Armitage/Cochran Trend Test

Applicant's summary and conclusion

Conclusions:
In a 28-day oral gavage study conducted to OECD 407 and to GLP (reliability score 1) the NOAEL for decamethyltetrasiloxane was 25 mg/kg bw/day based on significantly elevated mean absolute liver weights, mean liver-to-body weight ratios and mean liver-to-brain weight ratios in males and females treated with 250 mg/kg bw/day and 1000 mg/kg bw/day (p<0.05 or p<0.01). In addition, brown pigment accumulation, observed after four weeks of treatment in five males at 1000 mg/kg bw/day and one male at 250 mg/kg bw/day, was considered to be an adverse finding, due to secondary periportal chronic inflammation and bile duct proliferation in five males at 1000 mg/kg bw/day. In females at 1000, 250 and 25 mg/kg bw/day, periportal fatty change was not accompanied by degeneration or inflammation and was considered to be non-adverse. After the recovery period, the severity of perilobular fatty change was reduced in the females previously treated at 1000 mg/kg bw/day.