Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2012 to July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Dodecamethylcyclohexasiloxane (D6)
- Stability under test conditions: stable for duration of study
- Storage condition of test material: room temerature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (MI, US)
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 189-225g (males) 148-184g (females)
- Fasting period before study: no
- Housing: individually in wire mesh cages
- Diet: Lab Diet 5002 (PMI Nutrition International) ad libitum
- Water: local supply ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 22.5
- Humidity (%): 33 - 75
- Air changes (per hr): 10 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 18 September 2012 to 15 January 2013

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 litre stainless steel and glass TSE-system inhalation chambers
- Method of holding animals in test chamber: stainless steel exposure caging
- Source and rate of air: compressed air
- System of generating particulates/aerosols: heated stainless steel J-tube
- Temperature, humidity in air chamber: 22+/- 3 degrees C and 50+/-20%
- Air change rate: approx 12 (10-15) per hour


TEST ATMOSPHERE
- Brief description of analytical method used: GC/FID
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test atmosphere was conducted by gas chromatography with flame ionization detection at least once per hour during exposure.
Duration of treatment / exposure:
At least 13 weeks
Frequency of treatment:
6 hours/day, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
18.2 mg/m³ air (nominal)
Remarks:
1ppm
Dose / conc.:
182 mg/m³ air (nominal)
Remarks:
10 ppm
Dose / conc.:
546 mg/m³ air (nominal)
Remarks:
30ppm
No. of animals per sex per dose:
20 control and high dose, 10 low and intermediate dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: not stated
- Post-exposure recovery period in satellite groups: 28 days

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: twice weekly for first 4 weeks, weekly thereafter

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before dosing and Day 87
- Dose groups that were examined: all (before dosing) Control and high dose (Day 87)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of scheduled necropsy at end of dosing or recovery period
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled necropsy at end of dosing or recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No. 2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 3)
Statistics:
Data analysis were carried out using SAS 9.1.3 in the Provantis™ 8.2 system for body weight, changes in body weight, organ weight, organ to body weight ratios, food consumption, hematology, clinical chemistry and prothrombin times. These data were analyzed using a one-way Analysis of Variance (ANOVA) (Gad and Chengelis, 1998). If data do not satisfy the requirements of normality of the residuals [Shapiro-Wilks Test (Shapiro and Wilk, 1965)] and homogeneity of variance (Bartlett’s) a log or rank transformation was performed. If the ANOVA is significant (p>0.05), pair-wise comparisons of the exposed groups to control were made using the Dunnett’s test (Dunnett, 1955, 1964).

ENVIRON Health Sciences Institute conducted statistical analysis on clinical observations and histopathology data using SAS 9.3 and the analyses included in a contributing scientist report. Data from the Provantis™ 8.2 system was exported to Excel and verified data was sent electronically to the contributing scientist.
Clinical observations data was analyzed using Mixed Modeling repeated measures. Mixed models represent the fixed effect of treatments and the random effect of variability from animal to animal and observation to observation (within animal).

Microscopic findings were analyzed using a Cochran-Armitage trend test to indicate an increasing incidence trend regardless of grade. A Fisher’s Exact test was used to indicate increased incidence (non-grade specific) over the control. In addition, for those microscopic findings with one or more incidences in the control group, the graded animal only was compared using a Kruskal-Wallis test (Kruskal, 1952; Kruskal and Wallis, 1952) to determine if there was an increase in severity grade between the control group and the treated groups. Pair-wise comparisons of the graded-only animals were conducted using a Wilcoxon test if the overall Kruskal-Wallis test was significant.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Test article-related microscopic observations in the main study animals were noted in the liver in females at ≥10 ppm, in the lung in females at 30 ppm, in nasal level 1 in both sexes at ≥10 ppm, and in nasal level 2 in females at ≥10 ppm. After a 28-day non-treatment period, test article-related microscopic observations were present at 30 ppm in the nasal level 1 in both sexes, nasal level 2 in males, and in the liver in females. The incidences and severities of the nasal findings in the recovery animals indicate little or incomplete recovery while findings in the female liver were suggestive of near complete recovery. No test article-related microscopic observations were present in the lung of females indicating recovery after the 28-day non-treatment period.

Main Study
Increased incidences and severities of subacute inflammation and hyperplasia of the various epithelia were observed in the nasal cavity in nasal level 1 and increased mucous cell hyperplasia was observed in nasal level 2 (females only). Subacute inflammation was characterized by mixed inflammatory cell infiltrates (neutrophils and mononuclear cells) within the submucosa and sometimes within the mucosa. Inflammation was most common on the ventral portion of the septum, and on the turbinates and lateral wall. There was an increased incidence and severity (minimal to mild) of subacute inflammation in nasal level 1 in males and females at ≥10 ppm.

The mucosa of nasal level 1 is comprised of squamous, transitional and respiratory epithelium in varying amounts depending on the precise level of the section. Hyperplasia of the various epithelia was observed as a thickened epithelium with irregularly arranged cell layers, increased nuclear density and sometimes an increase in subepithelial glands; it was frequently present in the same areas as subacute inflammation. Epithelial hyperplasia of nasal level 1 was observed primarily in the following areas: squamous cell hyperplasia (males only) - along the lower aspect of the nasal septum; transitional epithelial hyperplasia - on the turbinates and the lateral wall; respiratory epithelial hyperplasia - along the nasal septum, ventral meatus and medial sides of the turbinates. Hyperplasia of mucous (goblet) cells was observed primarily along the nasal septum but also occasionally extended onto the turbinate mucosa, lateral walls and ventral meatus. Mucous cell hyperplasia was characterized by increased mucin within the goblet cells, increased mucous cells within transitional epithelium, and frequently an increased number of mucinous subepithelial glands in respiratory epithelium. In nasal level 1 at ≥10 ppm, test article-related increased incidences and severities of respiratory epithelial hyperplasia and transitional epithelial hyperplasia were noted. There was also an increased incidence and severity of mucous cell hyperplasia in nasal level 1 in males at 30 ppm and in females at ≥10 ppm. An increase in incidence and severity of squamous cell hyperplasia in nasal level 1 was also observed in males at 30 ppm only.

At nasal level 2, most of the mucosa is composed of respiratory and olfactory epithelium. The only test article-related finding in nasal level 2 was mucous cell hyperplasia at ≥10 ppm in females.

These findings in the nasal level 1 and 2 are considered test article-related and adverse. There were no test article-related microscopic observations in nasal sections 3, 4 and 5.

A modestly higher incidence of periportal hepatocellular vacuolation was observed in females at 10 and 30 ppm and considered to be attributable to treatment. The vacuoles were small, nonstaining, intracytoplasmic vacuoles that morphologically were consistent with lipid.

An increased incidence of minimal alveolar macrophages in the lung was noted in 30 ppm females and was characterised by the presence of small clumps of foamy macrophages within alveoli, often next to bronchi or in a subpleural location.

Recovery necropsy
In nasal level 1, increased incidences and/or severities of subacute inflammation, respiratory epithelial hyperplasia, transitional cell hyperplasia and mucous cell hyperplasia were observed in both sexes. A minimally increased incidence of squamous cell hyperplasia was noted in males. The incidences and severities in the 30 ppm group were similar to the incidences and severities observed at the primary necropsy, suggesting that there was little or incomplete recovery from these effects after the 28 day non-exposure period.

In nasal level 2, there was a minimal increase in the incidence of mucous cell hyperplasia in males only. An increase in mucous cell hyperplasia was not identified in males at the primary necropsy; however, this increase at the recovery necropsy was likely test article-related because of the presence of other types of epithelial hyperplasia in test article-exposed males at this necropsy.

A slightly increased incidence of periportal vacuolation (1/10 control vs 3/10 30 ppm) in the female rats was observed at 30 ppm. The incidence and severity at 30 ppm were less than that observed in the same group at the primary necropsy, suggesting near complete recovery from this effect.

There were no other test article-related microscopic observations noted in the other organs/tissues at the primary or recovery necropsies.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
1 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Hyperplasia in the nasal tissue (mucous cell, epithelial cell, respiratory epithelium and transitional epithelium) at 10 and 30 ppm
Dose descriptor:
LOAEC
Effect level:
10 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Hyperplasia in the nasal tissue (mucous cell, epithelial cell, respiratory epithelium and transitional epithelium) at 10 and 30 ppm

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The daily average actual concentrations +/- SD for all exposure days were as follows:

Control - BLQ

Group 2 - 1.0 +/- 0.02 ppm

Group 3 - 10 +/- 0.5 ppm

Group 4 - 30 +/- 1.4 ppm

Applicant's summary and conclusion

Conclusions:
Inhaled administration of D6 to Sprague-Dawley rats at 0, 1, 10 or 30 ppm for 90-days resulted in test article-related microscopic finding in nasal tissues, liver (females only) and lung (females only) at 10 and/or 30 ppm. The nasal tissue findings included increased incidence and severity of inflammation and hyperplasia consistent with a mucosal irritant and were considered adverse; they did not resolve after a 28-day recovery period. An increased incidence of minimal alveolar macrophages in the lung and mild periportal hepatocellular vacuolation in the liver of females did resolve after a 28-day recovery period. Based on the hyperplasia and inflammation in the nasal tissues at 10 and 30 ppm the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 1 ppm.