Registration Dossier

Administrative data

Description of key information

The key 90-day repeated dose inhalation toxicity study in rats with dodecamethylcyclohexasiloxane (D6) was conducted according to OECD Test Guideline 413 and in compliance with GLP (Dow Corning Corporation, 2013). The NOAEC was concluded to be 1 ppm (18.2 mg/m³), the lowest concentration tested, based on hyperplasia and inflammation in the nasal tissues at 10 (182 mg/m3) and 30 ppm (546 mg/m3), indicative of a mucosal irritation and therefore local effects. The systemic NOAEC was concluded to be greater than 30 ppm (546 mg/m³) based on the absence of adverse effects in either sex up to the highest dose tested. The inhaled local LOAEC is considered to be the mid-dose, 182 mg/m³, and this is used as the starting point for calculation of the local Derived No Effect Level.

The key repeated dose oral toxicity study with dodecamethylcyclohexasiloxane (D6) (Dow Corning Corporation, 2005) was a 4-week rat combined repeated dose toxicity study with the reproductive/developmental toxicity study screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP. A NOAEL of at least 1000 mg/kg bw/day, the highest dose tested, was reported in the study. The observed liver effects (increased absolute and/or relative liver weight in all treated groups and periportal lipidosis at all doses in females) were described as “of minimal toxicologic significance” and the thyroid effects (follicular cell hypertrophy, incidence possibly treatment-related in both sexes) were referred to as secondary and adaptive to the liver changes.

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:
key study
Study period:
from 2005-04-26 to 2005-10-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Labs, Raleigh, NC, USA
- Age at study initiation: 9 wk
- Weight at study initiation: 173-254 (f); 285-386 (m)
- Housing: 1/suspended wire mesh cage
- Diet: standard diet ad libitum
- Water: drinking water ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67.7-72.5 deg F (approximately 20 degrees celsius)
- Humidity (%): 35-59
- Air changes (per hr): 15.6
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From 2005-05-12 for 28 or 29 days in the toxicity portion of this study.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

Prepared for the whole study. Analysed (GC) 4 times to ensure homogeneity, stability and concentration.

VEHICLE
Corn oil
- Lot/batch no.: 015K0115
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC analysis on 4 occasions to ensure homogeneity, stability and concentration.
Duration of treatment / exposure:
males 28d
toxicity females 29d
reproductive females 46d
Frequency of treatment:
daily, 7 days/wk
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
330 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 males
10 toxicity and 10 reproductive females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): weight stratified randomization
- Rationale for selecting satellite groups: none
- Post-exposure recovery period in satellite groups: none
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: yes
- How many animals: all toxicity groups

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes / No / No data
- How many animals: all toxicity groups

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: FOB at start and completion
- Dose groups that were examined: males and toxicity group females
- Battery of functions tested: cage-side observations, hand-held observations, open field observations, categorical observations, hind and forelimb grip strength, landing foot splay, motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
all toxicity groups
HISTOPATHOLOGY: Yes
Microscopic examination of a relatively large range of tissues of all control and hi-dose group animals, and the lungs and thyroid of both sexes and liver of females at the low and mid doses.
ORGAN WEIGHTS
weights of a relatively large range of organs determined
Other examinations:
Examination for reproductive parameters (reported in the relevant IUCLID section).
Statistics:
ANOVA (analysis of variance): body weights, organ weight, haematology, clinical chemistry etc
ANCOVA (analysis of covariance): FOB
Cochran-Armitage: microscopic findings
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:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Prothrombin time prolonged in males at >=300 mg/kg bw/day with no clinical indication of clotting abnormalities.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased absolute and/or relative liver weight in both sexes at all doses (>=100 mg/kg bw/day; statistically significant), with a modest dose-relation in females. It was said that only the relative liver weight in high-dose females exceeded historical control values.

Relative absolute and/or adrenal weights in females were increased at all doses (>=100 mg/kg bw/day; statistically significant) without any dose relationship.

Kidney weights were increased in both sexes at all doses, but only for the low and mid dose groups was the change statistically significant. There was no dose relationship.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination identified a dose related increase in periportal lipidosis in the liver of females (controls: 4/10 minimal severity; treated groups 100, 330, 1000 mg/kg bw/day: 10/10, 10/10, 9/10 minimal to moderate severity). There was no other evidence of hepatotoxicity and the report considers this effect to be of “minimal toxicologic significance”. A subsequent re-evaluation of these data (TSCA 8(e), 2009) note that in the light of a dose-response in liver weight in females in the presence of hepatic histopathological changes in this sex, “it cannot be ruled out that the findings may be test article related”. In the context of this view, the LOAEL would be 100 mg/kg bw/day (in females).

Lung effects were considered an artefact due to the treatment route (further justification not given in study report, but presumably due to accidental dosing into the trachea).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination identified a dose related increase in periportal lipidosis in the liver of females (controls: 4/10 minimal severity; treated groups 100, 330, 1000 mg/kg bw/day: 10/10, 10/10, 9/10 minimal to moderate severity). There was no other evidence of hepatotoxicity and the report considers this effect to be of “minimal toxicologic significance”. A subsequent re-evaluation of these data (TSCA 8(e), 2009) note that in the light of a dose-response in liver weight in females in the presence of hepatic histopathological changes in this sex, “it cannot be ruled out that the findings may be test article related”. In the context of this view, the LOAEL would be 100 mg/kg bw/day (in females).

Lung effects were considered an artefact due to the treatment route (further justification not given in study report, but presumably due to accidental dosing into the trachea).

The incidence of animals with follicular cell hypertrophy in the thyroid appeared possibly related to treatment (incidence in groups 0, 100, 330, 1000 mg/kg bw/day: 0, 5, 2, 6 in males and 1, 2, 1, 5 in females). Severity was not affected by dose in either sex. This effect was considered to be secondary and adaptive, and typical of a xenobiotic which induces hepatic microsomal enzymes with increased degradation of thyroxin and triiodothyronine as a side effect.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effect observed.
Critical effects observed:
no
Conclusions:
In the oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with dodecamethylcyclohexasiloxane (D6), conducted according to OECD Test Guideline 422 and in compliance with GLP (Dow Corning Corporation, 2005), the NOAEL for systemic toxicity was concluded to be at least 1000 mg/kg bw/day, the highest dose tested. The observed liver effects (increased absolute and/or relative liver weight in all treated groups and periportal lipidosis at all doses in females) were described as “of minimal toxicologic significance” and the thyroid effects (follicular cell hypertrophy, incidence possibly treatment-related in both sexes) were referred to as secondary and adaptive to the liver changes.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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
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
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: The highest concentration tested was based on the saturated vapour concentration. The top dose was selected to avoid aerosol formation and a mixture of vapour/aerosol test atmosphere.
- Post-exposure recovery period in satellite groups: 28 days
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.
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
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
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.

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, non-staining, 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.

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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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.

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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
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
Critical effects observed:
not specified

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

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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
546 mg/m³
Study duration:
subchronic
Species:
rat
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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
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
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: The highest concentration tested was based on the saturated vapour concentration. The top dose was selected to avoid aerosol formation and a mixture of vapour/aerosol test atmosphere.
- Post-exposure recovery period in satellite groups: 28 days
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.
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
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
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.

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, non-staining, 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.

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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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.

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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
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
Critical effects observed:
not specified

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

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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
182 mg/m³
Study duration:
subchronic
Species:
rat

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 key 90-day repeated dose inhalation toxicity study with dodecamethylcyclohexasiloxane (D6), conducted according to OECD Test Guideline 413 and in compliance with GLP (Dow Corning Corporation, 2013), the test article, D6, was administered daily, 6 hours/day for 90 days at concentrations of 0, 1, 10 or 30 ppm (equivalent to 18.2, 182 or 546 mg/m3). The highest concentration tested was based on the saturated vapour concentration. The top dose was selected to avoid aerosol formation and a mixture of vapour/aerosol test atmosphere. At necropsy, test item-related findings were noted in the nasal tissues of both sexes and the liver of females at ≥ 10 ppm and the lungs of females at 30 ppm. Increased incidence and severity of minimal to mild subacute inflammation, mucous cell hyperplasia and epithelial cell hyperplasia of the squamous (30 ppm males only), respiratory and/or transitional epithelium were observed in the rostral-most section (level 1) of the nasal cavity. At level 2 only an increased incidence of mucous cell hyperplasia was noted. These observations were considered to be adverse and consistent with a mucosal irritant. The incidence and severity of the nasal findings did not resolve after a 28-day recovery period, with the exception of the mucous cell hyperplasia at level 2 in females. A higher incidence of alveolar macrophage accumulation at 30 ppm and higher incidences of minimal and mild periportal hepatocellular vacuolation at ≥10 ppm were both noted in females at the end of dosing at necropsy; the incidence of these changes was not statistically significant and they were not defined as adverse. At the end of the recovery period the extent of accumulation of alveolar macrophages in the 30 ppm group was comparable to controls and the incidence of periportal hepatocellular vacuolation was only marginally higher than controls suggesting near complete recovery. There were no other findings considered to be attributable to treatment. Based on the hyperplasia and inflammation in the nasal tissues at 10 and 30 ppm, considered to be local effects, the No-Observed-Adverse-Effect-Concentration (NOAEC) for local effects was concluded to be 1 ppm. The inhaled local LOAEC is considered to be the mid-dose, 182 mg/m3, and this is used as the starting point for calculation of the local Derived No Effect Level (refer to Section 5.11 for further information). Although not explicitly stated, the inhaled systemic NOAEC is considered to be 30 ppm (546 mg/m3) in this study as the only changes stated to be considered adverse were the findings in the nasal cavity and no adverse systemic effects were reported.

In the key oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening test with dodecamethylcyclohexasiloxane (D6), conducted according to OECD Test Guideline 422 and in compliance with GLP (Dow Corning Corporation, 2005), the test article, D6, was administered in corn oil daily, seven days a week by oral gavage to 10 rats/sex/group at 0, 100, 330 or 1000 mg/kg bw/day for up to 45 consecutive days. All female rats in each of the treatment groups expressed hepatic lipidosis with the exception of one animal in the high dose group. The severity among dose groups was not statistically different and was not dose-responsive. Thyroid effects (follicular cell hypertrophy, incidence possibly treatment-related in both sexes) were also noted and referred to as secondary and adaptive to the liver changes.

After reviewing additional oral data on D6 (administered without a vehicle) and conducting a literature review of periportal lipidosis, it was concluded that the periportal lipidosis observed in this study was more likely an exacerbation of the diffuse fatty changes of the liver seen following oral gavage administration of corn oil or an interaction between the test substance and the corn oil vehicle.  

Key evidence to support the argument that the periportal lipidosis is attributed to the corn oil vehicle comes from an older study, where the test material is administered neat. The 28-day repeated dose oral toxicity study with neat D6 (Dow Corning Corporation, 1990) did not identify any histological effects, including periportal lipidosis at a dose of 1500 mg/kg bw/day. In 2010, a review of the slides from this older 1990 study was conducted to specifically look for the periportal lipidosis and other liver effects. The review of the slides in 2010 did not find any evidence of periportal lipidosis (or other histological findings) at the dose of 1500 mg/kg bw/day for D6 when it was administered neat via oral gavage. 

In addition, well-founded and compelling reasons not to interpret observations of hepatic lipidoses in rodents as treatment-related adverse effects when the test substance is administered by corn oil gavage were found during a review of the literature (Condie et al., 1986; Bull et al., 1986; Lowe et al., 2009; Marty et al., 2007). Several studies provide evidence that the use of corn oil (CO) as the vehicle for gavage administration of test substances may increase the incidence and severity of fatty infiltrates in liver and may alter (either exacerbate or reduce depending on species and conditions) hepato- and nephro-toxicity in rats and mice. Taken together, these studies suggest caution in interpreting fatty changes in the livers of rodents administered test substances by gavage in corn oil vehicle. In particular, hyperlipidosis that occurs without a clear dose response may be due to interactions between the test substance and the corn oil vehicle rather than to the test substance alone. 

There was also an NTP study with corn oil that demonstrated significant increases in diffuse fatty changes of the liver following oral gavage administration. This study was designed to evaluate the effects of various concentrations of various oils (safflower oil, corn oil, and tricaprylin) on the incidence and pattern of neoplasms in the F344/N rat. To evaluate corn oil as well as two other gavage vehicles for potential toxicity, corn oil, safflower oil, and tricaprylin were administered by gavage to male F344/N rats for 2 years. This NTP report focused on the influence of corn oil as a gavage vehicle on the incidence and severity of pancreatic neoplasms, but additionally provides data on non-neoplastic changes. In a comparison of untreated controls, saline, and 2.5 ml/kg, 5 ml/kg, and 10 ml/kg corn oil in this study, NTP noted significant increases in “diffuse fatty changes” in the liver of rats receiving corn oil. The incidences for each of these groups, respectively, are: 3/50 (6%), 2/50 (4%), 19/50 (38%), 27/50 (54%), and 33/50 (66%). NTP noted the potential confounding effects of corn oil gavage in the interpretation of bioassay results.

Therefore, based on the additional oral data on neat D6 and the findings of the literature review of periportal lipidosis, it can be concluded that the periportal lipidosis observed in this study was more likely an exacerbation of the diffuse fatty changes of the liver seen following oral gavage administration of corn oil, or an interaction between the test substance and the corn oil vehicle, and not an inherent toxicity of the test material.

The combined repeated dose toxicity study with the reproductive/developmental toxicity study screening test is selected as the key oral study, as it is the most reliable and most recent study available for repeated dose toxicity by this exposure route.


Justification for classification or non-classification

Based on the available sub-acute and sub-chronic toxicity studies for D6, there is no evidence that classification for Specific Target Organ Toxicity (STOT) is required following repeated oral or inhalation exposure according to Regulation (EC) No 1272/2008. No data are available for the dermal route.