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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 6 May 1999 to 3 Sept 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Hexamethylcyclotrisiloxane
EC Number:
208-765-4
EC Name:
Hexamethylcyclotrisiloxane
Cas Number:
541-05-9
Molecular formula:
C6H18O3Si3
IUPAC Name:
hexamethyl-1,3,5,2,4,6-trioxatrisilinane

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: not stated
- Age at study initiation: 9 wk
- Weight at study initiation: 118-141 g (m); 93-121 g (f)
- Housing: suspended wire mesh cages, individually housed
- Diet: standard diet ad libitum
- Water: drinking water ad libitum (except during exposure)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-25
- Humidity (%): 29-72
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h

IN-LIFE DATES: From: 1999-06-06 To: 1999-09-03

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Method of holding animals in test chamber: exposure tube
- Source and rate of air: conditioned, filtered compressed air
- Air flow rate: 19-31 lpm

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography
- Samples taken from breathing zone: taken from 5 locations in the exposure chamber 1/hr

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
target concentrations: 0, 15, 150, 600, 2500 ppm
measured concentrations: 0, 14.9, 150, 601, 2519 ppm
Duration of treatment / exposure:
90 days, 6 hr/day, 5 days/wk
Frequency of treatment:
daily (excluding weekends)
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (analytical)
Dose / conc.:
14.9 ppm (analytical)
Dose / conc.:
150 ppm (analytical)
Dose / conc.:
601 ppm (analytical)
Dose / conc.:
2 519 ppm (analytical)
No. of animals per sex per dose:
10 for the 15, 150 and 600 ppm groups
20 for the 2500 ppm groups
25 for controls
Details on study design:
Post-exposure recovery period in satellite groups: 4-wk (0, 2500 ppm)
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2x/day, 1x/day at weekends

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

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: Yes
- Time schedule for examinations: start and completion of study
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: See Table 1

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes
- How many animals: all
- Parameters examined: See Table 1

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 2)
HISTOPATHOLOGY: Yes (see Table 2)
ORGAN WEIGHTS: Yes (see Table 2)
Other examinations:
None.
Statistics:
Mean and standard deviations were calculated for all measured parameters. Group means were compared by ANOVA. Where significant effects were identified a further comparison was made with the control group using Dunnett’s test. The level of statistical significance was p<=0.05 in all cases.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related clinical signs were limited to the 600 and 2500 ppm groups for both sexes. There was an exposure-dependent increased incidence of wet ventral abdominal and dorsal rump fur in both of these groups. In addition, the incidence of yellow discoloured inguinal fur, red material around the mouth and rough hair coat were increased in the 2500 ppm group. These signs were primary observed immediately following daily exposure completion. No other test substance-related clinical signs were observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
No deaths occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was statistically significantly reduced in the 2500 ppm males during the first three weeks of exposure. No other test substance-related effects on body weight were observed. The reduced body weight gain in the first three weeks of the study in males at 2500 ppm, resulted in statistically significantly lower group mean body weight gain in 2500 ppm male group relative to the control group throughout the treatment period. There were no statistically significant or remarkable differences in female body weight for any of the dose levels.
During the recovery period, mean body weight in the 2500 ppm males remained statistically significantly lower than the control group; however, body weight gain upon cessation of treatment was increased significantly. Mean body weight and body weight gain for the 2500 ppm females during recovery were similar to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by the test substance exposure.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No effects observed.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin were statistically significantly decreased and platelets were statistically significantly increased for the 2500 ppm males at the end of the 13-week exposure period. Erythrocytes were also decreased for the 2500 ppm males but the change was not statistically significant. Also, exposure dependent and statistically significant decreases in erythrocytes, haemoglobin and haematocrit were present for the 600 ppm males. In addition, the incidence of several atypical cells (anisocytes, poikilocytes, microcytes and hypochromic) was increased for the 2500 ppm males and the incidence of microcytes was somewhat increased for the 2500 ppm females at the end of exposure. Also, for the 2500 ppm females at the end of the treatment period, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration were statistically decreased, but the differences from control were slight and there were no effects on erythrocytes, haemoglobin or haematocrit. The only other statistically significant value at the end of the exposure period was an increased percentage of mature neutrophils for the 2500 ppm females. The increase was not large and the total white blood cell count was not increased. Therefore, these differences from the control group were not attributed to the treatment.
At recovery evaluation, haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin remained slightly decreased and platelets remained slightly increased in the 2500 ppm males. The differences from the control group were statistically significant, but the magnitude of the differences was less than at the end of the exposure period. The incidence of several atypical cells (anisocytes, microcytes and hypochromic) remained elevated for the 2500 ppm males; no differences from control in red cell morphology were present for the 2500 ppm females. Also, in the 2500 ppm males, the erythrocyte count and the percentage of nucleated red blood cells were increased statistically significantly at the recovery evaluation. Mean corpuscular volume and mean corpuscular haemoglobin were statistically significantly decreased at the recovery evaluation in the 2500 ppm females, but, as at the end of exposure there were no effects on erythrocytes, haemoglobin or haematocrit, and the differences were not large (<3%). Statistically significantly elevated platelets were seen for the 2500 ppm females, but the differences were not large and no effect on platelets count was seen at the end of the exposure period. The only other statistically significantly different values at the recovery evaluation were decreased leukocytes, lymphocytes and mean corpuscular haemoglobin concentration for the 2500 ppm males. However, the differences were not large and the values were within the normal limits for these parameters. None of these changes were considered to be associated with exposure to the test substance.

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the 13-week clinical chemistry evaluation, albumin, globulin and total protein (TP) and gamma glutamyl transpeptidase (GGT) were statistically significantly increased and chloride was statistically significantly decreased for both sexes in the 2500 ppm group. Potassium and calcium were statistically significantly increased for the 2500 ppm males and triglycerides were statistically significantly increased for the 2500 ppm females. Glucose was statistically significantly decreased for the 600 and 2500 ppm males. Exposure-related, but not statistically significant decreases in alkaline phosphatase (AP) were present in all male groups and in the 150, 600 and 2500 ppm females.
Other statistically significantly differences at the end of the 13-week exposure period included decreased sodium in the 2500 ppm males, decreased alanine aminotransferase (ALT) in the 150 and 600 ppm females, decreased aspartate aminotransferase (AST) in the 600 ppm males and the 150 and 2500 ppm females and increased cholesterol in the 15 and 150 ppm females. These differences were of slight magnitude and were not exposure-dependent and were therefore not considered to be associated with exposure to the test substance.
At the recovery evaluation, AP remained statistically significantly decreased for the 2500 ppm males, but less so than at 13 weeks, and the 2500 ppm female AP level was comparable to the control. All other clinical chemistry changes observed at 13 weeks had returned to levels comparable to the control at the recovery evaluation; therefore, the effects seen at 13 weeks were reversible upon cessation of exposure. There were no other test substance-related changes in clinical chemistry.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weight was increased in all treated groups on an absolute, relative to body weight and relative to brain weight basis at the end of the exposure period. Although the differences were not statistically significant, an exposure-dependent response was observed. The increases for the 15 ppm females were slight. In the 2500 ppm group, thymus weight was decreased in both sexes and adrenal gland weight was increased in 2500 ppm females only, when evaluated on an absolute, relative to body weight and relative to brain weight basis; not all of these differences were statistically significant.
At the recovery necropsy, liver weight was comparable to control for the 2500 ppm males; although significantly higher when evaluated relative to body weight, the percent difference was 6.4%, which directly parallels the final body weight that was 6.3% lower than control group. Recovery liver weight for the 2500 ppm females was still elevated approximately 10% above the control value; this increase was statistically significant only when evaluated relative to body weight, and the difference was much lower than that observed at the 13-week necropsy (approximately 70%). Adrenal weight for the 2500 ppm females was also still somewhat increased, but the difference was not statistically significant. And, as for the liver, the magnitude of the increase was much less than that seen at the 13-week necropsy, i.e. approximately 14% at recovery versus approximately 35% at the end of exposure. No effects were apparent on the thymus weight. All effects seen on organ weights at the end of exposure exhibited substantial partial to full recovery and were therefore reversible upon cessation of exposure. There were no other test substance-related changes or differences in organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test-substance related gross necropsy findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the end of the exposure period, diffuse hepatocytic hypertrophy was observed in all 600 and 2500 ppm animals and in 7/10 and 5/10 150 ppm males and females, respectively. The severity of this finding was exposure dependent. Hepatocytic hypertrophy in the centrilobular region was observed in a single 600 ppm male, 9/10 150 ppm males and 6/10 15 ppm males. The severity of this finding was exposure-dependent at the 15 and 150 ppm exposure levels, and minimal for all six 15 ppm rats in which it was seen. Centrilobular hypertrophy was not specifically noted for any females. Diffuse hypertrophy was characterised by uniformly increased size of hepatocytes. Centrilobular hypertrophy was diagnosed when only hepatocytes in the centrilobular to midzonal regions were affected, or when the severity in the centrilobular region was markedly different from the periportal region. Thus, the regional distribution of hepatocytic hypertrophy appeared to be exposure dependent. No hepatocytic hypertrophy was evident in the 2500 ppm males or females at the recovery necropsy. Thus, this change was fully reversible within 28 days following cessation of exposure.
Adrenal cortical cell vacuolation was seen for essentially all rats on the study, including the controls, at both the end of exposure and recovery. The severity of this finding was higher in the 600 and 2500 ppm groups (both sexes) at the end of exposure, but similar between the control and 2500 ppm groups at recovery. This change was fully reversible at the highest exposure level given (2500 ppm) within 28 days after cessation of exposure.

Histopathological findings: neoplastic:
no effects observed
Details on results:
The test substance induced effects at all doses but up to 150 ppm these were considered transitory, adaptive/metabolic, not clearly adverse or giving no evidence of organ toxicity.

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
600 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
organ weights and organ / body weight ratios
Remarks on result:
other: equivalent to 5459 mg/m3

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 500 ppm (analytical)
System:
other: effects on body weight gain, heamatology and clinical chemistry parameters, and liver / body weight ratio
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
In the 90-day inhalation repeated dose toxicity study, conducted according to OECD Test Guideline 413 and in compliance with GLP, the NOAEC for systemic toxicity, concluded by the study authors, was 150 ppm (1400 mg/m3) for male and female rats based on clinical signs, body weight changes, effects on blood and clinical biochemistry parameters, organ weights and histopathology at 600 ppm and higher. The test substance-related effects at concentrations up to 150 ppm were considered transitory, adaptive/metabolic, not clearly adverse or giving no evidence of organ toxicity. The reviewer of the study, however, concluded that the true NOAEC for systemic toxicity in male and female rats is at least 600 ppm (equivalent to 5459 mg/m3) based on not fully reversible statistically significant changes in body weights, haematology and blood chemistry parameters seen in 2500 ppm recovery male rats as well as not fully reversible statistically significantly increased liver / body weight ratio seen in recovery males and females from 2500 ppm group.