Registration Dossier

Administrative data

Description of key information

Based on the results of the key OECD 422 study, the NOAEC for 2,4,6,8-tetramethylcyclotetrasiloxane for systemic toxicity following inhaled administration in male and female rats is 100 ppm (equivalent to 984 mg/m3 based on MW of 240.5094 for 2,4,6,8-tetramethylcyclotetrasiloxane). In addition, there is an ongoing 90-day study with the registered substance, which will be included in the dataset at a later date, when study results are available.

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:
supporting study
Study period:
19.11.2003 to 19.05.2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: USEPA OPPTS 870.3650
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: No data
- Age at study initiation: Nine weeks
- Weight at study initiation: Males: 294.2-351.5; Females: 200.2-260.2 g
- Fasting period before study: None
- Housing: individually housed in suspended wire-mesh cages (pregnant rats in shoebox cages)
- Diet (e.g. ad libitum): Ad libitum (except during FOB)
- Water (e.g. ad libitum): Ad libitum (except during FOB)
- Acclimation period: Six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2-22.5
- Humidity (%): 36.0-62.0
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09.02.2004 To: 19.04.2005
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Conducted over nitrogen atmopshere. Test substance was placed into a volumetric flask and corn oil added to achieve the desired volume. The weight of the test substance added to the flask was used to calculate nominal dose solution concentrations. Dosing solutions were prepared at least once every two weeks consistent with the previously determined 15-day stability. The concentration, homogeneity and stability of the test substance in vehicle for at least 15 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: Various
- Amount of vehicle (if gavage): Up to 3 ml/kg
- Lot/batch no. (if required): 122K0131
- Purity: Considered 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of methyltrimethoxysilane (MTMS) in corn oil dosing solutions was determined prior to the beginning of the definitive study.
Duration of treatment / exposure:
Toxicity group females and males were treated for 28 and 29 days, respectively. Reproductive group females were treated for 14 days prior to the mating period, during the mating period, and then up to and including post partum day 3, for a total of up to 51 days.
Frequency of treatment:
Daily, seven days/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
corn oil
Dose / conc.:
50 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:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a range-finding study
- Rationale for animal assignment (if not random): Weight stratified randomisation process
- Rationale for selecting satellite groups: According to OECD TG 422
- Post-exposure recovery period in satellite groups: None
Observations and examinations performed and frequency:
Mortality/Morbidity: Animals were observed at least twice daily in their cages for moribundity and mortality throughout the in-life phase of the study.

Daily Observations: General clinical examinations were made at lease once a day and were conducted immediately after dosing. The examinations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system functions, motor activity and behavior patterns. Findings were recorded for individual animals. General clinical examinations were not performed on days when detailed physical examinations were performed.

Detailed Physical Examinations: All animals received a detailed physical examination once before the first dose administration (to allow for within-subject comparisons), and weekly thereafter. Examinations were made outside the home cage in a standard arena at approximately the same time each day. Observations were detailed and carefully recorded. Examinations included, but were not limited to, changes in skin, fur eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movement, stereotypies, difficult or prolonged parturition or bizarre behavior were recorded. The presence or absence of findings was recorded for individual animals.

Body weights and food consumption were recorded weekly. Additional body weights for reproductive group females were obtained on gestational day 0, 7, 14, and 20, and within 24 hours of parturition, and on postnatal day 4. Individual food consumption was determined for each group following group specific schedules.

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: On day of scheduled termination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: Males and toxicity phase females
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day of scheduled termination
- Animals fasted: Yes
- How many animals: Males and toxicity phase females
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of dosing and during the  last week of dosing. 
- Dose groups that were examined: adult males and  all toxicity group females
- Battery of functions tested: Unusual body movements, abnormal behaviour and/or posture, and resistence to removal, palperal closure, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, reactivity to handling, level of ambulatory activity including rearing, responsiveness to sharp noise, touch, tail pinch, gait evaluation and quantity of urine and fecal pellets voided, behavior, skin or hair coat, respiration, muscle movements, eyes, urine or feces, soiling, general abnormalities and posture, rectal temperature, hindlimb/forelimb grip strength and landing foot splay. Motor activity assessment involved obtaining baseline motor activity data prior to the first dose administration to assist in evaluating potential treatment-related effects by allowing each rat to act as its own control.
Sacrifice and pathology:
Males and toxicity group females were sacrificed after they had been  treated for 28 days.  Reproductive group females and pups were sacrificed  
on day 4 postpartum.  Complete necropsies were performed on the males and the toxicity group females and selected organs were weighed (adrenals, brain, heart, kidneys, liver, lungs, spleen, thymus, uterus, ovaries, testes, prostate, seminal vesicles, and epididymides). Microscopic examination was performed on protocol-specified tissues from all animals in the control and high dose group males and toxicity group females. Microscopic examination of the liver, thyroid, jejunum and duodenum was extended to males and toxicity group females in the 50 and 250 mg/kg dose groups. In addition, the adrenal gland from these middle dose groups was evaluated for the toxicity group females.
Statistics:
All data analyses were carried out using SAS version 8.2. Statistically significant probabilities were reported for /-values of <0.05, <0.02 and <0.01.

Body weight, body weight changes, organ weight, organ to body weight ratios, food consumption, hematology data, clinical chemistry and prothrombin times were analyzed using a one-way Analysis of Variance (ANOVA) if the data satisfied the requirements of normality of the residuals and homogeneity of variance as determined using the Shapiro-Wilk test for normality and Levene’s test for homogeneity of variance. If the data did not satisfy the parametric requirements, a Kruskal-Wallis test was used. If the ANOVA or Kruskal-Wallis test was significant, pair-wise comparisons of the dosed groups to control were made using the Dunnett’s Test or a Wilcoxon test, respectively.

The red blood morphology findings in the hematology data that were reported by severity grade; polychromasia, anisocytes, poikilocytosis, and ananthocytes were analyzed using a Cochran-Armitage trend test to indicate an increasing incidence trend regardless of grade with Fisher’s Exact tests used to indicate increased incidence (non-grade specific) over the control. To determine shifts in severity, analysis using the Kruskal-Wallis test was performed on the graded data only if there was at least once incidence of severity grade seen in the controls group. If the overall Kruskall-Wallis test was significant, pair wise comparisons of the graded animals between the control and treated groups was done using the Kruskal-Wallis.

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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:
not examined
Details on results:
There were two unscheduled deaths (one female each at 0 and 50 mg/kg  bw/day); both were related to dosing errors. Clinical signs included transient inactivity or salivation following dosing.  Statistically  significant decreases in body weight gain and food consumption were noted for 1000 mg/kg bw/day group males.  Increased liver weight was observed  for male and female animals at 250 and 1000 mg/kg bw/day. Exposure to  MTMS was associated with organ weight and/or histomorphological changes  in males (liver, thymus, thyroid, duodenum, jejunum)  and females (liver, thyroid, duodenum, jejunum, and adrenal gland) at  dose levels at or above 250 mg/kg bw/day. A marked increase in  prothrombin time was observed for males at 250 and 1000 mg/kg bw/day  whereas females were unaffected.  Exposure was also associated with  increased blood platelet concentration for males and females, and increased red blood cell concentration in males at 1000  mg/kg bw/day.  The NOAEL for systemic toxicity was 50 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

Toxic Response/Effects by Dose Level:

50 mg/kg bw/day Methyltrimethoxysilane:
Males: None
Females:  None

250 mg/kg bw/day Methyltrimethoxysilane:
Males: 
Increased liver weight (absolute(relative): 19%* (25%*)
Thyroid gland follicular cell hyperplasia/hypertrophy: (incidence 6*/10)
Decreased thymus weight (absolute(relative): 28%*(24%*)
Increased prothrombin time: 2-fold*

Females:
Increased liver weight (absolute(relative): 37%* (41%*)
Centrilobular hepatocellular hypertrophy (incidence 5*/10)
Periportal vacuolation (incidence 10*/10)
Thyroid gland follicular cell hyperplasia/hypertrophy:
(incidence 10*/10)

1000 mg/kg bw/day Methyltrimethoxysilane:
Males:  
Increased liver weight (absolute(relative): 33%* (47%*) 
Diffuse hepatocellular hypertrophy (incidence 10*/10)
Thyroid gland follicular cell hyperplasia/hypertrophy: (incidence 10*/10)
Mucosal lipidosis: duodenum  (incidence 4*/10)
Mucosal lipidosis: jejunum (incidence 7*/10)
Decreased thymus weight (absolute(relative): 35%*(29%*)
Ancanthocytosis (incidence 5*/10)
Increased red blood cell concentration: 16%*
Increased prothrombin time: 2-fold*
Increased serum alanine aminotransferase (48%*)
Increased blood platelet concentration: 16*

Females (Toxicity Phase):
Increased liver weight (absolute(relative)): 197%* (200%*)
Centrilobular hepatocellular hypertrophy (incidence 10*/10)
Periportal vacuolation (incidence 8*/10)
Thyroid gland follicular cell hyperplasia/hypertrophy:(incidence 10*/10)
Adrenal gland hyperplasia/hypertrophy (incidence 10*/10)
Adrenal gland apoptosis (incidence 9*/10)
Adrenal gland lymphocytic infiltration: zona reticularis:(incidence 5*/10)
Mucosal lipidosis: duodenum  (incidence 5*/10)
Mucosal lipidosis: jejunum (incidence 5*/10)

Ancanthocytosis (incidence 4*/10)

Increased blood platelet concentration: 21%*

Where "*" denotes statistically different from control (p<0.05)

Conclusions:
Exposure to methyltrimethoxysilane was associated with organ weight and/or histomorphological changes in males (liver, thymus, thyroid, duodenum, jejunum, and red blood cell) and females (liver, thyroid, duodenum, jejunum, and adrenal gland) at dose levels at or above 250 mg/kg bw/day. A marked increase in prothrombin time was observed for males at 250 and 1000 mg/kg bw/day whereas females were unaffected. Exposure was also associated with increased blood platelet concentration for males and females at 1000 mg/kg bw/day. These data support a NOAEL for the toxicity phase of the study of 50 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
RccHan:WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Male 266 to 347g Female 181 to 225g
- Fasting period before study: No
- Housing: Individually in Makrolon type-3 cages
- Diet: Pelleted standard Kliba Nafag 3433 rodent diet (Provimi Kliba SA, Switzerland) ad libitum except when animals in inhalation chambers or prior to blood sampling
- Water: Community tap water ad libitum except when animals in inhalation chambers
- Acclimation period: Minimum 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: 06 January to 01 March 2011
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel sealed chamber
- Method of holding animals in test chamber: stainless steel wire cage unit
- Source and rate of air: 40L/min
- System of generating aerosols: test material in glass flask, air pumped through flask at 40L/min
- Temperature, humidity in chamber: 22.0 to 22.3 degrees C, 37.4 to 52.4%
- Air change rate: 10-15 per hour


TEST ATMOSPHERE
- Brief description of analytical method used: weighing test item reservoir before and after each exposure (nominal). On-line GC (analytical)
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
3 times per hour via on-line GC
Duration of treatment / exposure:
28 to 49 days, 6 hours daily
Frequency of treatment:
once daily
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
3 000 ppm (nominal)
Dose / conc.:
2 000 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: Target atmosphere concentrations were selected based on 14-Days Dose Range-Finding Inhalation Toxicity Study in the Han Wistar Rat
- Post-exposure recovery period: 14 days for subset of Control and high dose groups

High dose of 3000 ppm reduced to 2000 ppm from day 12 due to early deaths of 3 females and 1 male at the higher dose.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Male - twice weekly. Female - twice weekly then days 0, 7, 14 and 20 PC and days 0 and 4 PP

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

WATER CONSUMPTION: No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters listed in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day prior to scheduled necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No. 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Male - before termination. Female - day 3 or 4 PP
- Dose groups that were examined: all
- Battery of functions tested: sensory activity/motor activity/grip strength
Sacrifice and pathology:
ORGAN WEIGHT: Yes (see table 4)
GROSS PATHOLOGY: Yes (see table 5)
HISTOPATHOLOGY: Yes (see table 5)
Statistics:
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 would 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 if the variables could be dichotomized without loss of information.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item caused early death of some animals at the high-dose level.

During the treatment with the test item at the dose level of 3000 ppm, one male and three females were found dead either after exposure in the exposure chamber. After reduction of the high-dose level to 2000 ppm on day 12, one male and two additional females were found dead; either in the exposure chamber or in the home cage. For one or two days before death, clinical signs including ruffled fur and decreased activity were observed in some animals.

No further clinical signs or observations were noted in any other animals.
Mortality:
mortality observed, treatment-related
Description (incidence):
During the treatment with the test item at the dose level of 3000 ppm, one male and three females were found dead either after exposure in the exposure chamber. After reduction of the high-dose level to 2000 ppm on day 12, one male and two additional females were found dead; either in the exposure chamber or in the home cage.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item at 3000/2000 ppm caused reduction of body weight gain during the dosing period of up to 10% compared to control in males. This reduction was statistically significant on a number of occasions. For females, similar reductions were noted during the first half of the dosing period, thereafter, body weight gain continued to be lower in the recovery subset females but was comparable to controls for the remainder in this group.

No effects on body weighs were observed at the dose levels of 1000 and 100 ppm.

Body weight gain during the recovery period was generally comparable with controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item caused a dose-dependent decrease of food consumption in males at all dose levels.

At the dose levels of 3000/2000 and 1000 ppm, decrease of food consumption was statistically significant during the dosing period. Mean food consumption during this period was reduced by about 12% to 17% at the dose level of 1000 ppm and by up to 29% at the dose level of 3000/2000 ppm (percentages refer to the respective values compared to the control group).

At the dose level of 100 ppm, decrease of food consumption was statistically significant from day 5 to 8 and not statistically significant thereafter.
Treatment with the test item caused decrease of food consumption in females at the dose level of 3000/2000 ppm during the first half of the treatment period. This reduction was up to 34% compared to control and was statistically significant on several days. Thereafter food consumption continued to be lower in the recovery subset females but was comparable to controls for the remainder in this group.

No effects on food consumption of females were observed at the dose levels of 1000 and 100 ppm. Mean food consumption at these dose levels was similar to the respective control values during the entire study period.

During the recovery period food consumption was comparable to controls for both males and females.
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):
No test item-related effects on haematology parameters were observed in males or females at any dose level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item at the dose levels of 1000 and 3000/2000 ppm caused a dose dependent, statistically significant increase of urea concentration in males. This change was not noted at the end of the recovery period. Biochemical blood parameters at all dose levels were similar to the respective control values for females.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No findings which were considered to be test item-related were noted in males or females at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the high-dose level in males, terminal body weight at the end of the dosing period was decreased by 9.6% compared to control, this reduction was statistically significant.

In males at the dose level of 3000/2000 ppm, statistically significantly higher by 15% (percentage refer to the value compared to the control group) kidney to body weight ratio was noted. Kidney weight relative to the brain weight was also statistically significantly higher than the respective control values. Absolute kidney weight at this dose level was slightly, not statistically significantly higher than the control value. Although increase of kidney weights was only moderate, this change was considered to be test item-related as it was also observed in the recovery groups in both sexes.

At the dose levels of 3000/2000 and 1000 ppm in males, a dose-dependent and statistically significant reduction of absolute weights of brain was noted. No changes of brain weights were observed in females at any dose level. No further findings which may have indicated a specific effect of the test item on the central nervous system were noted as well as no histopathological changes of the brain tissue were observed in males up to the highest dose level. For this reason the toxicological relevance of this finding remained equivocal. However, because of the dose dependency and statistical significance of this effect, its relation to the treatment could not be excluded
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment of males with the test item at the dose level 3000/2000 and 1000 ppm caused an increased incidence of changes in urinary tract noted during macroscopic examination at scheduled termination at the end of the dosing or recovery periods as well as decedent animals. At the dose level of 3000/2000 ppm, the following findings were considered to be test item related: pelvic dilatation, stones in urinary bladder and thickened mucosa/wall of urinary bladder. At the dose level of 1000 ppm, the following findings were considered to be test item-related: stones in urinary bladder and pelvic dilation.

Treatment of females with the test item at the dose levels of 1000 and 3000/2000 ppm caused an increased incidence of changes in urinary tract found during macroscopic examination of females at scheduled termination at the end of the dosing or recovery periods, as well as decedent females. At the dose level of 3000/2000 ppm, the following findings were considered to be test item related: pelvic dilatation, stones in urinary bladder, enlarged kidneys, dilatation of ureter, hemorrhagic contents or hemorrhagic watery fluid, thickened or discoloured mucosa/wall of urinary bladder and urinary bladder distended with urine or dilated. In addition, dark red foci in the vagina and/or thickened mucosa of the vagina were noted. These changes were possibly secondary to the inflammatory changes and lesions in the urethra (identified within histopathological examinations) and therefore were secondary effects to the treatment with the test item.

At the dose level of 1000 ppm, urinary bladder containing stones was found in one female. This finding was considered to be caused by the treatment with the test item.

The following findings were observed only in decedent animals: advanced autolysis, dark red discoloration of the lung, crateriform retractions in the stomach, Peyers patches not visible, enlarged adrenal glands, dark red discoloration of internal organs (including thymus, lungs, ovaries, adrenal glands and mandibular lymph nodes), reddish discoloration of bronchial lymph nodes and watery clear fluid in the abdominal cavity. These findings were considered to possibly arise peri-/post-mortem.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were observed mainly in the urinary organs in both sexes, both in the survivors and decedents, at the dose levels of 1000 and 3000/2000 ppm. The thyroid gland (surviving males) and heart (decedent females) were also considered to be the main target organs.

In the kidney, the following findings were noted in both sexes at the dose levels of 1000 and 3000/2000 ppm or only at the dose level of 3000/2000 ppm: granular deposits in the pelvis and in the tubules, pyelitis (with foreign-body granuloma), urothelial hyperplasia and ulcer/erosion, tubular simple dilation, increased mitoses in the tubular epithelium, pelvic dilation, tubular basophilia, hyaline droplets and mononuclear cell foci. Specific to decedent animals, tubular necrosis/degeneration and slight papillary necrosis, pelvic dilation, tubular basophilia and tubular simple dilation were mainly observed. Granular deposits in the pelvis, pyelitis (with foreign-body granuloma) and urothelial hyperplasia and ulcer/erosion were observed with a low frequency. Fibrosis, inflammatory cell infiltration and mononuclear cell foci were also observed.

In the ureter, the following findings were observed at the dose level of 3000/2000 ppm: granular deposits in the lumen (in males), inflammatory cell infiltration (in males) and luminal dilation (in males and females). Luminal dilation and inflammatory cell infiltration in the surrounding soft tissue were observed in decedent animals.

In the urinary bladder, the following findings were observed at the dose levels of 1000 and 3000/2000 ppm in both sexes: urothelial hyperplasia and increased inflammatory cell infiltration (with foreign-body granuloma) and at the dose level of 3000/2000 in both sexes: granular deposits in the cavity or submucosa, hemorrhage, ulcer and edema (only in males).

In the urethra, marked injury was observed specifically to the decedent females. Granular deposits, necrosis involving the mucosa, submucosa and surrounding tissues, inflammatory cell infiltration, luminal dilation and urothelial hyperplasia were observed in the urethra of all decedent females.
Treatment-related findings in the heart were specific to the decedents. Myocardial necrosis/degeneration, inflammatory cell infiltration, fibrosis, myocardial mineralization and hemorrhage were observed.

Urinary obstruction was indicated in most of the decedent animals by the marked luminal distention of the urethra, distention with urine of the urinary bladder (macroscopically), luminal distention of the ureter, pelvic dilation and tubular simple dilation of the kidney, and was probably caused by the existence of the calculi or the injury including necrosis and inflammation at the lower urinary tract (urethra). Urinary obstruction might have caused uremia and subsequent death. The remarkable heart lesions observed in this study might have caused heart failure and thereby also been the direct cause of death. The heart lesions observed in the decedents can be induced by uremia.

At the end of the recovery period, similar kidney, urinary bladder and urethral findings were still noted and myocardial necrosis/degeneration, inflammatory cell infiltration and fibrosis was noted in one male.

In the thyroid gland, amorphous materials in the colloid were observed in males of all treatment groups with dose-dependency in its severity and incidence. No further indicators of thyroid injury (necrosis, apoptosis, fibrosis, other degenerative changes, etc.), this change was considered to have been caused by metabolic change in the thyroid and was not noted at the end of the recovery period.

At the dose level of 3000/2000 ppm, treatment-related findings in the liver, hepatocellular hypertrophy and increased mitoses were found in decedents. Hepatocellular hypertrophy was macroscopically correlated with enlargement of the organ. The significance of these changes remains unknown.
Testicular tubular degeneration found in two males at each dose level of 100 and 1000 ppm and in four males at the dose level of 3000/2000 ppm was in most cases unilateral and within the range of historical control data. However, because this effect was noted in both decedent males in the recovery group, it cannot be excluded to be the secondary treatment effect. The marked renal lesions may have caused inanition making effects on the testes of these decedents.

Alveolar edema in the lung and congestion in the several organs were observed only in the decedent animals. Although these can be agonal or post mortem change, the possibility of the secondary changes associated with the heart disorder or uremia cannot be excluded.

Cortical hypertrophy of the adrenal gland (macroscopically correlated with enlargement of the organ), atrophy of the spleen (macroscopically correlated with reduction in size of the organ) and atrophy of the thymus and ulcer in the forestomach (macroscopically correlated with mucosal crateriform retractions) were observed only in the decedent animals. No other related findings were observed in the same organs and therefore, these changes were considered to be the secondary effect caused by stress.

In the vagina, mucification with mucus plug was observed in the decedent females. This change may have been caused by the stimulation by the marked inflammatory changes occurred in the urethra or associated with the possible inanition caused by the renal lesions in these animals.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Bladder stones and histopathological findings in urinary tract at 3000/2000 and 1000 ppm, plus reversible reduction of food consumption and reversible pathology change in thyroid gland in males at 100 ppm
Remarks on result:
other: equivalent to 984 mg/m3 based on MW of 240.5094 for 2,4,6,8-tetramethylcyclotetrasiloxane
Critical effects observed:
not specified

Inhalation technical data:

Test atmosphere concentrations

Group

Achieved Test Atmosphere Concentration (ppm)

Target Test Atmosphere Concentration (ppm)

Test Atmosphere Concentration Relative to Target (%)

2

100 ± 1

(n=37, CV=0.5%)

100

99.9

3

1000 ± 3

(n=40, CV=0.3%)

1000

100.0

4 and 6

From 06 to 17-Jan-2011

2987 ± 55

(n=11, CV=1.8%)

3000*

100.0

From 18-Jan to 14-Feb-2011

2000 ± 9

(n=29, CV=0.4%)

2000*

99.6

* The dose level of 3000 ppm was administered from day 1 to 11 of the treatment period. The dose level of 2000 ppm was administered starting from day 12 onwards.

Exposure conditions

Group

Temperature [°C]

Relative humidity [%]

Oxygen concentration [%]

1 and 5

22.1 ± 0.1 (n=40)

51.3 ± 1.1 (n=40)

20.3 ± 0.0 (n=40)

2

22.1 ± 0.1 (n=37)

39.5 ± 1.2 (n=37)

20.2 ± 0.0 (n=37)

3

22.4 ± 0.1 (n=40)

38.8 ± 1.4 (n=40)

20.1 ± 0.0 (n=40)

4 and 6

22.2 ± 0.1 (n=40)

42.2 ± 1.9 (n=40)

20.0 ± 0.1 (n=40)

Conclusions:
Based on bladder stones at 3000/2000 and 1000 ppm and histopathological findings in the urinary tract at 3000/2000 and 1000 ppm and reversible reduction of food consumption and reversible pathology change observed in the thyroid gland in males at the dose level of 100 ppm, a NOAEC (no observed adverse effect concentration) for general toxicity in males and females was considered to be 100 ppm (equivalent to 984 mg/m3 based on MW of 240.5094 for 2,4,6,8-tetramethylcyclotetrasiloxane).
Executive summary:

In a well-conducted, GLP compliant, OECD 422 study (reliability 1) treatment with the test item at the dose level of 3000 ppm caused early death of three females and one male. After reduction of the high-dose level to 2000 ppm, two further females and a male were found dead. Uremia resulting from the impairment of the urinary tract was indicated as a cause of the early deaths. The early deaths of animals might be due to the initial higher exposure to the test item at the concentration of 3000 ppm. All remaining animals survived the scheduled study period.

Clinical laboratory investigations, necropsy and histopathology results supply evidence that the urinary tract was a target organ for the 2,4,6,8-tetramethylcyclotetrasiloxane. During macroscopic and microscopic examinations stones/granular deposits in the urinary tract, injury, inflammation and dilation of urethra, urinary bladder, ureter or kidney were found in males and females which died before scheduled termination but also in most of the survivors at the dose level of 3000/2000 ppm and some males and females at the dose level of 1000 ppm. Changes in the urinary tract were still observed in both sexes at the high-dose level after the recovery period. In addition, in males and some females at the dose level of 3000/2000 ppm, higher kidney/body weight ratio was noted. Increased blood concentration of urea was recorded in some males at the high-dose level. This indicated that uremia was probably an important consequence of the urinary tract dysfunction. Uremia was postulated to be a direct cause of the pre-term deaths or, alternatively, to cause heart failure and consequent deaths of males and females at the high-dose level. Impairment of the urinary tract observed in males and females at the dose levels of 3000/2000 and 1000 ppm was considered to be adverse.

In addition to urinary tract, thyroid gland was also considered to be a target organ for the test item-related toxicity. During histopathological examination, amorphous materials in the colloid of the thyroid gland were observed in males of all treatment groups with dose dependency in its severity and incidence. This was considered to have been caused by a metabolic change in the thyroid gland. The similar change was observed in recovery animals but showing no difference in its incidence and severity between the control and high dose groups. No further indicators of thyroid injury were observed and therefore the effect on thyroid gland was considered not to be adverse.

Treatment with 2,4,6,8-tetramethylcyclotetrasiloxane caused a reduction of food consumption in males at all dose levels and in females at the dose level of 3000/2000 ppm. Body weight gains and body weights were reduced at the dose level of 3000/2000 ppm in both sexes. Reduction of food consumption, body weight gain and body weights, were reversible. During the recovery period, values of food consumption and body weight gain at the high-dose level were similar to the respective control values in both sexes. Although body weights during the recovery period remained slightly lower in males and females (in females, statistically significantly on day 8 of the recovery period), the differences were only minor and tended to decrease. For these reasons, effects on food consumption, body weight gain and body weights were considered not to be adverse.

At the terminal examinations, at the dose levels of 3000/2000 and 1000 ppm in males, a dose dependent and statistically significant reduction of absolute weights of brain was noted. No changes of brain weights were observed in females at any dose level. Reduction of brain weights is not expected to be a result of systemic toxicity even if body weights are reduced. Within this study, no behavioral or functional dysfunctions which indicated a specific effect of the test item on the central nervous system as well as no histopathological changes of the brain tissue were observed in males up to the highest dose level. However, because of the dose dependency and statistical significance of this effect, its relation to the treatment could not be excluded even if a toxicological relevance of this effect remained equivocal.

Based on bladder stones at 3000/2000 and 1000 ppm and histopathological findings in the urinary tract at 3000/2000 and 1000 ppm and reversible reduction of food consumption and reversible pathology change observed in the thyroid gland in males at the dose level of 100 ppm, a NOAEC (no observed adverse effect concentration) for general toxicity in males and females was considered to be 100 ppm (equivalent to 984 mg/m3 based on MW of 240.5094 for 2,4,6,8-tetramethylcyclotetrasiloxane).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
984 mg/m³
Study duration:
subacute
Species:
rat
System:
urinary
Organ:
bladder
kidney

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
RccHan:WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Male 266 to 347g Female 181 to 225g
- Fasting period before study: No
- Housing: Individually in Makrolon type-3 cages
- Diet: Pelleted standard Kliba Nafag 3433 rodent diet (Provimi Kliba SA, Switzerland) ad libitum except when animals in inhalation chambers or prior to blood sampling
- Water: Community tap water ad libitum except when animals in inhalation chambers
- Acclimation period: Minimum 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: 06 January to 01 March 2011
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel sealed chamber
- Method of holding animals in test chamber: stainless steel wire cage unit
- Source and rate of air: 40L/min
- System of generating aerosols: test material in glass flask, air pumped through flask at 40L/min
- Temperature, humidity in chamber: 22.0 to 22.3 degrees C, 37.4 to 52.4%
- Air change rate: 10-15 per hour


TEST ATMOSPHERE
- Brief description of analytical method used: weighing test item reservoir before and after each exposure (nominal). On-line GC (analytical)
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
3 times per hour via on-line GC
Duration of treatment / exposure:
28 to 49 days, 6 hours daily
Frequency of treatment:
once daily
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
3 000 ppm (nominal)
Dose / conc.:
2 000 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: Target atmosphere concentrations were selected based on 14-Days Dose Range-Finding Inhalation Toxicity Study in the Han Wistar Rat
- Post-exposure recovery period: 14 days for subset of Control and high dose groups

High dose of 3000 ppm reduced to 2000 ppm from day 12 due to early deaths of 3 females and 1 male at the higher dose.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Male - twice weekly. Female - twice weekly then days 0, 7, 14 and 20 PC and days 0 and 4 PP

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

WATER CONSUMPTION: No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters listed in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day prior to scheduled necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No. 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Male - before termination. Female - day 3 or 4 PP
- Dose groups that were examined: all
- Battery of functions tested: sensory activity/motor activity/grip strength
Sacrifice and pathology:
ORGAN WEIGHT: Yes (see table 4)
GROSS PATHOLOGY: Yes (see table 5)
HISTOPATHOLOGY: Yes (see table 5)
Statistics:
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 would 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 if the variables could be dichotomized without loss of information.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item caused early death of some animals at the high-dose level.

During the treatment with the test item at the dose level of 3000 ppm, one male and three females were found dead either after exposure in the exposure chamber. After reduction of the high-dose level to 2000 ppm on day 12, one male and two additional females were found dead; either in the exposure chamber or in the home cage. For one or two days before death, clinical signs including ruffled fur and decreased activity were observed in some animals.

No further clinical signs or observations were noted in any other animals.
Mortality:
mortality observed, treatment-related
Description (incidence):
During the treatment with the test item at the dose level of 3000 ppm, one male and three females were found dead either after exposure in the exposure chamber. After reduction of the high-dose level to 2000 ppm on day 12, one male and two additional females were found dead; either in the exposure chamber or in the home cage.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item at 3000/2000 ppm caused reduction of body weight gain during the dosing period of up to 10% compared to control in males. This reduction was statistically significant on a number of occasions. For females, similar reductions were noted during the first half of the dosing period, thereafter, body weight gain continued to be lower in the recovery subset females but was comparable to controls for the remainder in this group.

No effects on body weighs were observed at the dose levels of 1000 and 100 ppm.

Body weight gain during the recovery period was generally comparable with controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item caused a dose-dependent decrease of food consumption in males at all dose levels.

At the dose levels of 3000/2000 and 1000 ppm, decrease of food consumption was statistically significant during the dosing period. Mean food consumption during this period was reduced by about 12% to 17% at the dose level of 1000 ppm and by up to 29% at the dose level of 3000/2000 ppm (percentages refer to the respective values compared to the control group).

At the dose level of 100 ppm, decrease of food consumption was statistically significant from day 5 to 8 and not statistically significant thereafter.
Treatment with the test item caused decrease of food consumption in females at the dose level of 3000/2000 ppm during the first half of the treatment period. This reduction was up to 34% compared to control and was statistically significant on several days. Thereafter food consumption continued to be lower in the recovery subset females but was comparable to controls for the remainder in this group.

No effects on food consumption of females were observed at the dose levels of 1000 and 100 ppm. Mean food consumption at these dose levels was similar to the respective control values during the entire study period.

During the recovery period food consumption was comparable to controls for both males and females.
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):
No test item-related effects on haematology parameters were observed in males or females at any dose level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test item at the dose levels of 1000 and 3000/2000 ppm caused a dose dependent, statistically significant increase of urea concentration in males. This change was not noted at the end of the recovery period. Biochemical blood parameters at all dose levels were similar to the respective control values for females.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No findings which were considered to be test item-related were noted in males or females at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the high-dose level in males, terminal body weight at the end of the dosing period was decreased by 9.6% compared to control, this reduction was statistically significant.

In males at the dose level of 3000/2000 ppm, statistically significantly higher by 15% (percentage refer to the value compared to the control group) kidney to body weight ratio was noted. Kidney weight relative to the brain weight was also statistically significantly higher than the respective control values. Absolute kidney weight at this dose level was slightly, not statistically significantly higher than the control value. Although increase of kidney weights was only moderate, this change was considered to be test item-related as it was also observed in the recovery groups in both sexes.

At the dose levels of 3000/2000 and 1000 ppm in males, a dose-dependent and statistically significant reduction of absolute weights of brain was noted. No changes of brain weights were observed in females at any dose level. No further findings which may have indicated a specific effect of the test item on the central nervous system were noted as well as no histopathological changes of the brain tissue were observed in males up to the highest dose level. For this reason the toxicological relevance of this finding remained equivocal. However, because of the dose dependency and statistical significance of this effect, its relation to the treatment could not be excluded
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment of males with the test item at the dose level 3000/2000 and 1000 ppm caused an increased incidence of changes in urinary tract noted during macroscopic examination at scheduled termination at the end of the dosing or recovery periods as well as decedent animals. At the dose level of 3000/2000 ppm, the following findings were considered to be test item related: pelvic dilatation, stones in urinary bladder and thickened mucosa/wall of urinary bladder. At the dose level of 1000 ppm, the following findings were considered to be test item-related: stones in urinary bladder and pelvic dilation.

Treatment of females with the test item at the dose levels of 1000 and 3000/2000 ppm caused an increased incidence of changes in urinary tract found during macroscopic examination of females at scheduled termination at the end of the dosing or recovery periods, as well as decedent females. At the dose level of 3000/2000 ppm, the following findings were considered to be test item related: pelvic dilatation, stones in urinary bladder, enlarged kidneys, dilatation of ureter, hemorrhagic contents or hemorrhagic watery fluid, thickened or discoloured mucosa/wall of urinary bladder and urinary bladder distended with urine or dilated. In addition, dark red foci in the vagina and/or thickened mucosa of the vagina were noted. These changes were possibly secondary to the inflammatory changes and lesions in the urethra (identified within histopathological examinations) and therefore were secondary effects to the treatment with the test item.

At the dose level of 1000 ppm, urinary bladder containing stones was found in one female. This finding was considered to be caused by the treatment with the test item.

The following findings were observed only in decedent animals: advanced autolysis, dark red discoloration of the lung, crateriform retractions in the stomach, Peyers patches not visible, enlarged adrenal glands, dark red discoloration of internal organs (including thymus, lungs, ovaries, adrenal glands and mandibular lymph nodes), reddish discoloration of bronchial lymph nodes and watery clear fluid in the abdominal cavity. These findings were considered to possibly arise peri-/post-mortem.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were observed mainly in the urinary organs in both sexes, both in the survivors and decedents, at the dose levels of 1000 and 3000/2000 ppm. The thyroid gland (surviving males) and heart (decedent females) were also considered to be the main target organs.

In the kidney, the following findings were noted in both sexes at the dose levels of 1000 and 3000/2000 ppm or only at the dose level of 3000/2000 ppm: granular deposits in the pelvis and in the tubules, pyelitis (with foreign-body granuloma), urothelial hyperplasia and ulcer/erosion, tubular simple dilation, increased mitoses in the tubular epithelium, pelvic dilation, tubular basophilia, hyaline droplets and mononuclear cell foci. Specific to decedent animals, tubular necrosis/degeneration and slight papillary necrosis, pelvic dilation, tubular basophilia and tubular simple dilation were mainly observed. Granular deposits in the pelvis, pyelitis (with foreign-body granuloma) and urothelial hyperplasia and ulcer/erosion were observed with a low frequency. Fibrosis, inflammatory cell infiltration and mononuclear cell foci were also observed.

In the ureter, the following findings were observed at the dose level of 3000/2000 ppm: granular deposits in the lumen (in males), inflammatory cell infiltration (in males) and luminal dilation (in males and females). Luminal dilation and inflammatory cell infiltration in the surrounding soft tissue were observed in decedent animals.

In the urinary bladder, the following findings were observed at the dose levels of 1000 and 3000/2000 ppm in both sexes: urothelial hyperplasia and increased inflammatory cell infiltration (with foreign-body granuloma) and at the dose level of 3000/2000 in both sexes: granular deposits in the cavity or submucosa, hemorrhage, ulcer and edema (only in males).

In the urethra, marked injury was observed specifically to the decedent females. Granular deposits, necrosis involving the mucosa, submucosa and surrounding tissues, inflammatory cell infiltration, luminal dilation and urothelial hyperplasia were observed in the urethra of all decedent females.
Treatment-related findings in the heart were specific to the decedents. Myocardial necrosis/degeneration, inflammatory cell infiltration, fibrosis, myocardial mineralization and hemorrhage were observed.

Urinary obstruction was indicated in most of the decedent animals by the marked luminal distention of the urethra, distention with urine of the urinary bladder (macroscopically), luminal distention of the ureter, pelvic dilation and tubular simple dilation of the kidney, and was probably caused by the existence of the calculi or the injury including necrosis and inflammation at the lower urinary tract (urethra). Urinary obstruction might have caused uremia and subsequent death. The remarkable heart lesions observed in this study might have caused heart failure and thereby also been the direct cause of death. The heart lesions observed in the decedents can be induced by uremia.

At the end of the recovery period, similar kidney, urinary bladder and urethral findings were still noted and myocardial necrosis/degeneration, inflammatory cell infiltration and fibrosis was noted in one male.

In the thyroid gland, amorphous materials in the colloid were observed in males of all treatment groups with dose-dependency in its severity and incidence. No further indicators of thyroid injury (necrosis, apoptosis, fibrosis, other degenerative changes, etc.), this change was considered to have been caused by metabolic change in the thyroid and was not noted at the end of the recovery period.

At the dose level of 3000/2000 ppm, treatment-related findings in the liver, hepatocellular hypertrophy and increased mitoses were found in decedents. Hepatocellular hypertrophy was macroscopically correlated with enlargement of the organ. The significance of these changes remains unknown.
Testicular tubular degeneration found in two males at each dose level of 100 and 1000 ppm and in four males at the dose level of 3000/2000 ppm was in most cases unilateral and within the range of historical control data. However, because this effect was noted in both decedent males in the recovery group, it cannot be excluded to be the secondary treatment effect. The marked renal lesions may have caused inanition making effects on the testes of these decedents.

Alveolar edema in the lung and congestion in the several organs were observed only in the decedent animals. Although these can be agonal or post mortem change, the possibility of the secondary changes associated with the heart disorder or uremia cannot be excluded.

Cortical hypertrophy of the adrenal gland (macroscopically correlated with enlargement of the organ), atrophy of the spleen (macroscopically correlated with reduction in size of the organ) and atrophy of the thymus and ulcer in the forestomach (macroscopically correlated with mucosal crateriform retractions) were observed only in the decedent animals. No other related findings were observed in the same organs and therefore, these changes were considered to be the secondary effect caused by stress.

In the vagina, mucification with mucus plug was observed in the decedent females. This change may have been caused by the stimulation by the marked inflammatory changes occurred in the urethra or associated with the possible inanition caused by the renal lesions in these animals.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Bladder stones and histopathological findings in urinary tract at 3000/2000 and 1000 ppm, plus reversible reduction of food consumption and reversible pathology change in thyroid gland in males at 100 ppm
Remarks on result:
other: equivalent to 984 mg/m3 based on MW of 240.5094 for 2,4,6,8-tetramethylcyclotetrasiloxane
Critical effects observed:
not specified

Inhalation technical data:

Test atmosphere concentrations

Group

Achieved Test Atmosphere Concentration (ppm)

Target Test Atmosphere Concentration (ppm)

Test Atmosphere Concentration Relative to Target (%)

2

100 ± 1

(n=37, CV=0.5%)

100

99.9

3

1000 ± 3

(n=40, CV=0.3%)

1000

100.0

4 and 6

From 06 to 17-Jan-2011

2987 ± 55

(n=11, CV=1.8%)

3000*

100.0

From 18-Jan to 14-Feb-2011

2000 ± 9

(n=29, CV=0.4%)

2000*

99.6

* The dose level of 3000 ppm was administered from day 1 to 11 of the treatment period. The dose level of 2000 ppm was administered starting from day 12 onwards.

Exposure conditions

Group

Temperature [°C]

Relative humidity [%]

Oxygen concentration [%]

1 and 5

22.1 ± 0.1 (n=40)

51.3 ± 1.1 (n=40)

20.3 ± 0.0 (n=40)

2

22.1 ± 0.1 (n=37)

39.5 ± 1.2 (n=37)

20.2 ± 0.0 (n=37)

3

22.4 ± 0.1 (n=40)

38.8 ± 1.4 (n=40)

20.1 ± 0.0 (n=40)

4 and 6

22.2 ± 0.1 (n=40)

42.2 ± 1.9 (n=40)

20.0 ± 0.1 (n=40)

Conclusions:
Based on bladder stones at 3000/2000 and 1000 ppm and histopathological findings in the urinary tract at 3000/2000 and 1000 ppm and reversible reduction of food consumption and reversible pathology change observed in the thyroid gland in males at the dose level of 100 ppm, a NOAEC (no observed adverse effect concentration) for general toxicity in males and females was considered to be 100 ppm (equivalent to 984 mg/m3 based on MW of 240.5094 for 2,4,6,8-tetramethylcyclotetrasiloxane).
Executive summary:

In a well-conducted, GLP compliant, OECD 422 study (reliability 1) treatment with the test item at the dose level of 3000 ppm caused early death of three females and one male. After reduction of the high-dose level to 2000 ppm, two further females and a male were found dead. Uremia resulting from the impairment of the urinary tract was indicated as a cause of the early deaths. The early deaths of animals might be due to the initial higher exposure to the test item at the concentration of 3000 ppm. All remaining animals survived the scheduled study period.

Clinical laboratory investigations, necropsy and histopathology results supply evidence that the urinary tract was a target organ for the 2,4,6,8-tetramethylcyclotetrasiloxane. During macroscopic and microscopic examinations stones/granular deposits in the urinary tract, injury, inflammation and dilation of urethra, urinary bladder, ureter or kidney were found in males and females which died before scheduled termination but also in most of the survivors at the dose level of 3000/2000 ppm and some males and females at the dose level of 1000 ppm. Changes in the urinary tract were still observed in both sexes at the high-dose level after the recovery period. In addition, in males and some females at the dose level of 3000/2000 ppm, higher kidney/body weight ratio was noted. Increased blood concentration of urea was recorded in some males at the high-dose level. This indicated that uremia was probably an important consequence of the urinary tract dysfunction. Uremia was postulated to be a direct cause of the pre-term deaths or, alternatively, to cause heart failure and consequent deaths of males and females at the high-dose level. Impairment of the urinary tract observed in males and females at the dose levels of 3000/2000 and 1000 ppm was considered to be adverse.

In addition to urinary tract, thyroid gland was also considered to be a target organ for the test item-related toxicity. During histopathological examination, amorphous materials in the colloid of the thyroid gland were observed in males of all treatment groups with dose dependency in its severity and incidence. This was considered to have been caused by a metabolic change in the thyroid gland. The similar change was observed in recovery animals but showing no difference in its incidence and severity between the control and high dose groups. No further indicators of thyroid injury were observed and therefore the effect on thyroid gland was considered not to be adverse.

Treatment with 2,4,6,8-tetramethylcyclotetrasiloxane caused a reduction of food consumption in males at all dose levels and in females at the dose level of 3000/2000 ppm. Body weight gains and body weights were reduced at the dose level of 3000/2000 ppm in both sexes. Reduction of food consumption, body weight gain and body weights, were reversible. During the recovery period, values of food consumption and body weight gain at the high-dose level were similar to the respective control values in both sexes. Although body weights during the recovery period remained slightly lower in males and females (in females, statistically significantly on day 8 of the recovery period), the differences were only minor and tended to decrease. For these reasons, effects on food consumption, body weight gain and body weights were considered not to be adverse.

At the terminal examinations, at the dose levels of 3000/2000 and 1000 ppm in males, a dose dependent and statistically significant reduction of absolute weights of brain was noted. No changes of brain weights were observed in females at any dose level. Reduction of brain weights is not expected to be a result of systemic toxicity even if body weights are reduced. Within this study, no behavioral or functional dysfunctions which indicated a specific effect of the test item on the central nervous system as well as no histopathological changes of the brain tissue were observed in males up to the highest dose level. However, because of the dose dependency and statistical significance of this effect, its relation to the treatment could not be excluded even if a toxicological relevance of this effect remained equivocal.

Based on bladder stones at 3000/2000 and 1000 ppm and histopathological findings in the urinary tract at 3000/2000 and 1000 ppm and reversible reduction of food consumption and reversible pathology change observed in the thyroid gland in males at the dose level of 100 ppm, a NOAEC (no observed adverse effect concentration) for general toxicity in males and females was considered to be 100 ppm (equivalent to 984 mg/m3 based on MW of 240.5094 for 2,4,6,8-tetramethylcyclotetrasiloxane).

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 the 14-day range finder inhalation study, RCC Han rats (5 animals/sex/concentration) were exposed to 2, 4, 6, 8-tetramethylcyclotetrasiloxane at concentrations of 0, 100, 1000 and 4000 ppm for 14 consecutive days. There were no statistically significant differences in body weights and % of body weight gain. None of the macroscopic findings in males and females were statistically significant when compared to control values. One male animal in the high dose group was killed in extremis and there was one female spontaneous death in the high dose group. There were no statistically significant differences in absolute organ weights for males. The liver weight/body weight ratio (14% increased) and the kidney weight/brain weight ratios (18%) were statistically increased in males of the high dose group. In females, liver weight was statistically increased (33%) in the high dose group. Also in females, the liver weight/ body weight (37%) and liver weight/brain weight ratios were statistically increased (31%).

In the key 28-day inhalation study (Harlan, 2012) conducted to OECD 422 and in compliance with GLP, inhalation of 2, 4, 6, 8 -tetramethylcyclotetrasiloxane was not tolerated at the high dose concentration of 3000 ppm and resulted in the early death of three females and one male. After reduction of the high-dose level to 2000 ppm, two further females and a male were found dead.

Clinical laboratory investigations, necropsy and histopathology results all indicate that the urinary tract was a target organ for 2,4,6,8-tetramethylcyclotetrasiloxane. During macroscopic and microscopic examinations, stones/granular deposits in the urinary tract, injury, inflammation and dilation of urethra, urinary bladder, ureter or kidney were found in males and females which died before scheduled termination but also in most of the survivors at the dose level of 3000/2000 ppm and some males and females at the dose level of 1000 ppm. Changes in the urinary tract were still observed in both sexes at the high-dose level after the recovery period. In addition, in males and some females at the dose level of 3000/2000 ppm, higher kidney/body weight ratio was noted and increased blood concentration of urea was recorded in some males at the high-dose level. Uremia was considered to be a direct cause of the pre-term deaths or, alternatively, to cause heart failure and consequent deaths of males and females at the high-dose level. Impairment of the urinary tract observed in males and females at the dose levels of 3000/2000 and 1000 ppm was considered to be adverse.

In addition, the thyroid gland was also considered to be a target organ for the test item-related toxicity. Amorphous material in the colloid of the thyroid gland were observed in males of all treatment groups with dose dependency in its severity and incidence and was considered to have been caused by a metabolic change in the thyroid gland although this was considered not to be adverse.

Treatment with 2,4,6,8-tetramethylcyclotetrasiloxane also resulted in reductions in food consumption in males at all dose levels and in females at the dose level of 3000/2000 ppm and body weight gains and body weights at 3000/2000 ppm in both sexes.

Based on the results of this study the NOAEC for 2,4,6,8-tetramethylcyclotetrasiloxane for systemic toxicity following inhaled administration in male and female rats is 100 ppm (equivalent to 984 mg/m3 based on MW of 240.5094 for 2,4,6,8-tetramethylcyclotetrasiloxane).

There are no repeat dose toxicity studies for 2,4,6,8-tetramethylcyclotetrasiloxane for the oral or dermal route of exposure.

A 28 day repeat dose study is available for the read-across substance trimethoxy(methyl)silane. This substance hydrolyses rapidly to methylsilanetriol, which is also the proposed final hydrolysis product of 2,4,6,8-tetramethylcyclotetrasiloxane. Exposure to trimethoxy(methyl)silane was associated with organ weight and/or histomorphological changes in males (liver, thymus, thyroid, duodenum, jejunum, and red blood cell) and females (liver, thyroid, duodenum, jejunum, and adrenal gland) at dose levels at or above 250 mg/kg bw/day.  A marked increase in prothrombin time was observed for males at 250 and 1000 mg/kg bw/day whereas females were unaffected. Exposure was also associated with increased blood platelet concentration for males and females at 1000 mg/kg bw/day. These data support a NOAEL for the toxicity phase of the study of 50 mg/kg bw/day. This study is used as the starting point for the calculation of an oral DNEL for general population. The hydrolysis product is relevant as exposure is via the environment, where hydrolysis would have occurred. The current DNEL is an interim for risk characterisation and will be updated when the results of the oral repeated dose 90-day study with the registered substance are available.



Justification for classification or non-classification

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