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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Remarks:
combined repeated dose and reproduction / developmental screening
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: 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

Administration / exposure

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:
6 hours
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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):
effects observed, treatment-related
Food efficiency:
not examined
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:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
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.

BODY WEIGHT AND WEIGHT GAIN
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
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.

HAEMATOLOGY
No test item-related effects on haematology parameters were observed in males or females at any dose level.

CLINICAL CHEMISTRY
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.

FUNCTIONAL OBSERVATIONAL BATTERY
No findings which were considered to be test item-related were noted in males or females at any dose level.

ORGAN WEIGHTS
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 PATHOLOGY
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.

HISTOPATHOLOGY: NON-NEOPLASTIC
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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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 NOAEL (no observed adverse effect level) for general toxicity in males and females was considered to be 100 ppm.
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 NOAEL (no observed adverse effect level) for general toxicity in males and females was considered to be 100 ppm.