Registration Dossier

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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the key combined repeated dose toxicity study with the reproduction / developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP (Dow Corning Corporation, 2002), the NOAEC for reproductive toxicity was concluded to be 500 ppm (4.55 mg/L) hexamethylcyclotrisiloxane (D3; CAS 541-05-9; EC No. 208-765-4) based on decreased implantation sites and litter size at 2500 ppm dose group.

The registrants propose to conduct an extended one-generation reproductive toxicity study, conducted according to OECD Test Guideline 443 and in compliance with GLP, with the registered substance, hexamethylcyclotrisiloxane (CAS 541-05-9), after approval by ECHA.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-03-25 to 2002-11-07
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:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada, 188 LaSalle, St. Constant, Canada
- Age at study initiation: >=8 wk
- Weight at study initiation: 187-238 g (f); 250-313 g (m)
- Housing: 1/suspended wire cage
- Diet: standard diet ad libitum
- Water: drinking water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 2002-03-25 To: 2002-11-07
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 450 l steel and glass Rochester-style inhalation chambers with stainless steel exposure caging.
- Method of holding animals in test chamber: individual stainless steel caged compartments
- Method of conditioning air: air passed through series of purification filters
- System of generating vapour/aerosols: Test substance placed in warming chamber (75 deg C). The liquid TS was then metered into a heated metal J-tube for vapourization.
- Air change rate: at 10 exchanges of chamber volume per h

TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: automatic sampling from chamber

Acclimatization for 3 h on 2 days
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: continuously until evidence of copulation
- Proof of pregnancy: vaginal plug or sperm in vaginal smear (day 0)

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 ionisation detection (GC/FID) and each chamber was evaluated at least once per hour during the exposure period. For each day of exposure the mean test atmosphere D3 concentration (actual) as determined from the GC/FID analysis, was compared with the theoretical concentration (nominal), derived from the estimated chamber air flow rate and test substance consumption, as a quality control measure to evaluate exposure system performance.
Duration of treatment / exposure:
Exposure period: 28 days for males and 46 days for females.
Premating exposure period (males): 2 weeks.
Premating exposure period (females): 2 weeks.
Mated femaes were exposed up to Day 19 of gestation.
Dams were not exposed during the lactation period.
Frequency of treatment:
6 hours/day; 7 days/week
Details on study schedule:
In this screening study only the parental animals (F0) were exposed, and an F1 generation produced. No further matings were scheduled to produce an F2 generation.
Remarks:
Doses / Concentrations:
100, 500, and 2500 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
99, 512 and 2492 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10 per reproductive group and toxicity group
Control animals:
other: yes, control group was exposed to filtered air
Details on study design:
- Dose selection rationale: based on the results of a previous 90-day inhalation study (DCC, 2001)
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily or twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

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: Yes
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (see Table 1)
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Ketamine, HCL/Xylazine
- How many animals: all toxicity groups

CLINICAL CHEMISTRY: Yes (see Table 1)
- Time schedule for collection of blood: at sacrifice
- How many animals: all toxicity groups

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to exposure and during wk 4
- Dose groups that were examined: all toxicity groups
- Battery of functions tested: FOB and motor activity examinations. These included: cage-side, hand-held, open field and categorical observations, rectal temperature, hindlimb/forelimb grip strength and landing foot splay. Motor activity was also determined using a Cage Rack Activity System.

Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Parameters examined in all F0 males: testes and epididymis weight.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, litter weight gain and physical abnormalities.

GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities for pups found dead and pups sacrificed on Day 4 post-partum.
Postmortem examinations (parental animals):
Toxicity group males and females: necropsy and microscopic examination of tissues in Table 2.

Reproductive/developmental phase females were not subjected to macroscopic examination at necropsy, however the number of corpora lutea and implantation sites was recorded. No microscopic evaluation was conducted on organs and tissues of reproductive/developmental phase females.
.
Postmortem examinations (offspring):
Pups found dead and pups sacrificed on day four postpartum were examined for external gross abnormalities only. No pup tissues were collected and the carcasses were discarded.
 
Statistics:
Statistical methods:'s test and Kolmogorov-Smirnov test. Parametric data was tested using one-way Analysis of Variance (ANOVA) followed by Dunnett's Test (if significant); nonparametric data was tested by Kruskal-Wallis Test followed by Wilcoxon. Categorical data and histomorphology findings were evaluated with Fisher's Exact Test. Statistically significant probabilities are reported at either the p<0.05 or p<0.01 levels. 
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Observations recorded during the weekly detailed physical examinations were not different from those recorded during the daily clinical examinations. The clinical observations that were indicative of an adverse effect of the treatment were minimal and involved an increased incidence of anogenital staining and brown staining on the head. Anogenital staining was observed in 30% of males, 40% of toxicity group females and 100% of reproductive group females in the 2500 ppm groups. A single occurrence was observed in one reproductive group female in the 100 ppm group. Staining on head was not observed in the control group animals and was noted in two male rats, one in the 100 ppm group and another in the 2500 ppm group. In contrast, the incidence of staining on the head in females was increased in the 2500 ppm group. 90% of the toxicity group females and 100% of the reproductive group females in the 2500 ppm groups exhibited this finding at least once during the in-life phase of the study. There were no other clinical effects of toxicity.
Mortality:
no mortality observed
Description (incidence):
All adult animals survived to their scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weights for 100, 500, and 2500 ppm-exposed males were not statistically different from the control group at any time; however, group mean body weight for males in the 2500 ppm exposure group was consistently lower than any other group after day 1. The difference in mean body weight between the control and 2500 ppm exposure group grew larger with time and resulted in a decrease of 6% relative to control on day 29. Similarly, group mean body weight gain was consistently lower for males in the 2500 ppm exposure group relative to controls for each time period. The decreases were statistically significant at week 1 (35% decrease) and for total weight gain (39% decrease).
Group mean body weights for 100, 500 and 2500 ppm-exposed toxicity group females were consistently lower than the control group. However, the decreases (approximately 6% relative to control) reached statistical significance only for the 2500 ppm exposure group on day 8 and 30. Group mean body weight gain was not statistically different in test substance-exposed females relative to control. However, the mean value for total body weight gain was 25% lower for the 2500 ppm exposure group relative to control. These data suggest that exposure to 2500 ppm results in a slight decrease in body weight/body weight gain.
Group mean body weight for 500 and 2500 ppm -exposed reproductive group females were consistently lower (≤ 8%) than the control group. However, the decreases were not statistically significant except for the 2500 ppm exposure group on gestation day 20 (8% lower relative to Control). Similarly, group mean body weight gain for the exposed reproductive group females in the 500 and 2500 ppm exposure groups were typically lower (30%), though not statistically significant, than controls prior to week 3 of gestation. Females in the 2500 ppm exposure group demonstrated a statistically significant 21% decrease in body weight gain at gestation week 3. These changes in body weight and body weight gain are consistent with those observed for the toxicity group females (pre-mating) and with the effects on litter size (gestation and post-partum periods).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean weekly food consumption values were generally lower than control for male and toxicity group females exposed to the test substance. Statistically significant decreases (<15% relative to control values) were demonstrated for 2500 ppm group males (weeks 1 and 2), 2500 ppm group females (weeks 1, 2, and 3), and 500 ppm group females (weeks 2 and 3). There were no statistically significant differences in mean weekly food consumption values for the exposed reproductive group females relative to control. However, values were generally lower than control (2-12%). The one exception being a greater consumption of food (21%) during the parturition period (gestation day 20 - postpartum day 0/1) that likely represents differences in parturition (litter size and care).
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematology measures for the exposed animals were similar to controls. Differences were slight and the mean values for each group were within published ranges for animals of this strain, age and sex. Statistically significant differences were demonstrated for hemoglobin concentration in males (5% decrease at 2500 ppm) and females (4% increase at 100 ppm) and for platelet content in males (17% increase at 2500 ppm). In males, the modest elevation of platelets may represent a secondary thrombocytosis in response to the slight decrease in hemoglobin. The slight decrease in hemoglobin at 2500 ppm males may be related to the observed decrease in food consumption, decrease in body weight and/or effects on the kidney. The slight increase in hemoglobin for females in the 100 ppm exposure group is not considered treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry measures for test substance-exposed rats were generally similar to those for control groups. Exceptions include a statistically significant decrease in alkaline phosphatase activity,
decreased sodium concentration, and an increase in urea nitrogen in the 2500 ppm group males. Statistically significant changes in the exposed females included decreased calcium and glucose concentrations (500 and 2500 ppm females) and increased cholesterol (2500 ppm females).
In males, the 28% increase in urea nitrogen and 2% decrease in sodium in the 2500 ppm groups correlate with the observed kidney histopathology. The 27% decrease in alkaline phosphatase activity observed for males in the 2500 ppm group is consistent with a reduction in food consumption; decreases in this parameter are not indicative of organ toxicity.
In females, the 3-4% decrease in calcium in the 500 and 2500 ppm exposure groups was not considered treatment-related based on the magnitude of the changes and that the values were within the normal range for this strain and age of rat. The 18-19% decrease in serum glucose levels for females in the 500 and 2500 ppm exposure groups represent moderate changes that may be related to the general health condition of the animals (decreased food consumption, slight decrease in body weight). Similarly, the 30% increase in blood cholesterol may be related to the test substance-induced liver effects.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related changes noted in unusual body movements, abnormal behavior, posture or resistance to removal.
Palpebral closure, lacrimation, pupil size, and reactivity, salivation, muscle tone, and extensor thrust response were unaffected by treatment. There was a statistically significant change in the "reactivity to handling" categorical observation for 2500 ppm toxicity group females. This change reflects a greater proportion of rats in this exposure group exhibiting "minimal struggling" as compared to
"moderate struggling with little or no vocalization" for the control group.
No differences were apparent between the control and treated groups in open field observations (level of ambulatory activity including rearing, responsiveness to sharp noise, touch, tail pinch, gait evaluation, quantity of urine and fecal pellets voided).
No differences were apparent between the control and treated groups for skin or hair coat, respiration, muscle movements, eyes, urine or feces, soiling, posture, or general abnormalities.
There were no treatment-related changes noted for rectal temperature, hindlimb grip strength, forelimb grip strength or landing foot splay.
Habituation, the characteristic of decreasing motor activity with time in the observation cage, was analyzed with the data from males and females combined and with the sexes separate. When combined there was no statistical difference between the exposed and control rats. When analyzed separately, there was no statistical difference between the exposed female rats and the controls. However, analysis of the data for males showed a statistically significant difference in habituation in the 500 ppm exposure group relative to control. Because this effect was not dose-responsive (i.e. only present in the 500 ppm exposure group and not in the 100 and 2500 ppm exposure groups), was not present in the exposed females, and was not detected in the combined data set, it was not considered treatment-related.
The effect of exposure on motor activity (i.e. total number of locomotor counts per session) was evaluated statistically as a sexes-combined data set and then again with the sexes separated. The combined evaluation demonstrated reduced motor activity at the 2500 ppm exposure level relative to control. When analyzed separately, a statistically significant decrease in motor activity was demonstrated for 2500 ppm exposure group males and not females. Motor activity for males exposed to 2500 ppm test substance was markedly less than that for the other exposure groups. Similarly, the total motor activity counts for 2500 ppm exposed females were lower than that of the other exposure groups however the difference (baseline verses treated) was not as great as that for
the males.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histomorphologic effects attributable to treatment were observed in the liver of rats of both sexes, and in the kidneys and seminal vesicles of males. Other findings were of an incidence and character common to rats of this age and stock and not attributable to treatment.
In the liver there was centrilobular hepatocellular hypertrophy in all rats of both sexes exposed to 2500 ppm test substance. The change was uniformly graded as very slight in males and slight in females. The liver weight increases at the highest exposure in both sexes correlate with this finding. This is considered an adaptive change related to induction of cellular processes for increased xenobiotic metabolism/excretion. Treatment-related histomorphologic effects on the liver were not detected at lower exposure levels in either sex. The changes in liver were considered to be an adaptive response to the type of class of the test substance as a cyclic siloxane.
In the kidney, changes characterized as protein droplet nephropathy (PDN) were observed in male rats only. The severity and incidence were exposure related; all males exposed to 2500 ppm had slight to moderate PDN and there was a modest increase in the incidence and severity of tubular degeneration/regeneration (observed as small foci of basophilic tubules). At the 500 ppm exposure level, 9 of the 10 rats had very slight PDN with no increase in basophilic tubules. At 100 ppm, 1 of the 10 rats had very slight PDN changes, a finding that could be due to biologic variation since protein droplets are normally visible in the kidneys of most control male rats. PDN was characterized by an increase in the portion of renal cortical tubules (P2 segment) with visible eosinophilic droplets, often with angular droplet shapes suggesting crystallized material. With this increase in droplets there were increases in renal tubular cell necrosis, as indicated by nuclear pyknosis and karyorrhexis. Grade one, very slight, indicated a subtle increase in these findings over the background change in control males. Grades two and three, slight and moderate, were associated with several fold increases in the portion of tubules with visible droplets, angular droplet shapes, and cortical tubular cell degeneration. These changes correlated with a modest increase (6%, not statistically significant) in kidney weight as well as a 28% increase in serum urea nitrogen and a very small decrease in serum sodium in the high exposure group males. These histomorphologic and clinical chemistry kidney findings are consistent with a nephropathy, affecting only male rats, caused by a2u-globulin retention in renal cortical tubules and accumulation in the renal cortex. This mechanism is specific to male rats and not associated with human risk.
The seminal vesicles were slightly atrophied in 4 of 10 males in the 2500 ppm exposure group. The change was characterized by decreased fluid in the gland lumen and decreased cell height and epithelial folding of the luminal epithelium. This finding correlated well with the noted decrease in organ weight.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
With regard to sexual function and fertility the treatment had no effect on evidence of mating or gestation length. All mated females presented with positive evidence of mating on or before day seven of pairing and all but one of the reproductive phase females became pregnant. The exception was a female from the 2500 ppm exposure group (Animal D0033). The uterus from this female was negative for implantation sites. Fertility in males was not affected by the statistically significant decrease in absolute weight of the epididymides and decreased absolute/relative weight of the seminal vesicles with slight atrophy of seminal vesicles in four of the ten males at 2500 ppm; these four males successfully impregnated the females with which they were mated. The epididymides, testes and prostate from the exposed rats were histologically normal.
Exposure to the test substance had no effects on gestation length, pup sex ratio, pup weight, pup viability, and corpora lutea counts (PND 4). Statistically significant decreases were noted for litter size [number of pups per litter on PND 0 (33% decrease relative to control) and 4 (41% decrease relative to control)], litter weight (27% decrease relative to control on PND 0 and 4), and number of uterine implantation sites (33% decrease relative to control) in the 2500 ppm exposure group only.
Decreased litter size is considered responsible for lower mean body weight, body weight gain, and food consumption observed during gestation for this exposure group.
One reproductive group female (Animal D0035) in the 2500 ppm exposure group was observed squealing, shaking and with "bulging" eyes for approximately 10 minutes during the exposure period on study day 31. This same rat was observed to have an abnormal posture during the exposure period on study day 33. Physical and/or behavioral abnormalities were not observed at the end of the exposure period during return to the housing caging. No other rats in the exposure chamber exhibited these findings. This rat had a normal gestation period (22 days) and gave birth to 14 pups, none of which survived to PND 4. The loss of this litter represents the only complete litter loss for this exposure group. The only other rat on study to experience a complete litter loss was from the 100 ppm exposure group. The significance of this relationship, abnormal behaviors observed during exposure and litter loss, is not known.
Key result
Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
reproductive performance
Critical effects observed:
yes
Lowest effective dose / conc.:
2 500 ppm
System:
male reproductive system
Organ:
seminal vesicle
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced litter weights on postnatal days 0 and 4 (highest dose group only) due to reduced litter size. Pup weight was unaffected.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased litter size at 2500 ppm
Critical effects observed:
no
Reproductive effects observed:
yes
Lowest effective dose / conc.:
2 500 ppm (analytical)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
In the key combined repeated dose toxicity study with the reproduction / developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP (Dow Corning Corporation, 2002), the NOAEC for reproductive toxicity was concluded to be 500 ppm (4.55 mg/L) based on decreased implantation sites and litter size at 2500 ppm dose group.
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
4 550 mg/m³
Study duration:
subacute
Species:
rat
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the key combined repeated dose toxicity study with the reproduction / developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP (Dow Corning Corporation, 2002), Sprague-Dawley male and female rats were exposed to hexamethylcyclotrisiloxane vapor via whole body inhalation at concentrations of 0, 100, 500 or 2500 ppm (approximately 0.61, 4.5 or 22.8 mg/L). The animals were exposed 6 hours per day, 7 days per week for up to 39 days. Females in each exposure level were divided into a toxicity group (10 animals/group) and a reproductive group (10 animals/group). A single group of males (10 animals/group) was used for both the toxicity and reproductive phases of the study. Males and toxicity group females were treated for 28 and 29 days, respectively. Reproductive group females were treated for 14 days prior to the mating period, during the mating period and then up to and including gestation day 19.

A mating phase was initiated after the first two weeks of exposure by pairing reproductive group females with males of the same treatment group until positive evidence of mating was obtained. Reproductive and developmental parameters evaluated included evidence of mating, duration of gestation, mean litter size, mean live litter size, mean litter weight, mean ratio live births/litter size, corpora lutea count, and the number of uterine implantation sites. Dams and pups were euthanized on PND 4 and examined for external gross lesions.

In general, whole-body inhalation exposure of male and female Sprague-Dawley rats to hexamethylcyclotrisiloxane vapour at concentrations of up to 2500 ppm was well-tolerated, as suggested by minimal effects on body weight and food consumption, the lack of significant clinical signs of toxicity, and minimal effects on clinical pathology parameters. Increased liver weight and centrilobular hepatocellular hypertrophy were demonstrated in both sexes exposed to 2500 ppm. These effects are considered to be an adaptive response characteristic of this class of cyclic siloxanes. No liver effects were identified at lower exposure levels.

In males exposed to 2500 ppm, there were statistically significant decreases in the absolute weight of epididymides (10 %) and in the absolute (30 %) and relative (27 %) weight of the seminal vesicles. Slight atrophy of the seminal vesicles was identified microscopically in four of the 10 males in the 2500 ppm exposure group. The epididymides, testes, and prostate from exposed rats were histologically normal.

With regard to sexual function and fertility the treatment had no effect on evidence of mating or gestation length. All mated females presented with positive evidence of mating on or before day seven of pairing and all but one of the reproductive phase females became pregnant. Fertility in males was not affected by the statistically significant decrease in absolute weight of the epididymides and decreased absolute/relative weight of the seminal vesicles with slight atrophy of seminal vesicles in four of the ten males at 2500 ppm; these four males successfully impregnated the females with which they were mated. The epididymides, testes and prostate from the exposed rats were histologically normal.

Negative effects on implantation sites and litter size parameters were observed for female rats exposed to 2500 ppm. There was a 33 % decrease in litter size and in the number of implantation sites. Gestation length, pup sex ratio, pup weight and viability, and corpora lutea counts were unaffected. No effects were observed at lower exposure concentrations.

The NOAEC for reproductive toxicity was concluded to be 500 ppm (4.55 mg/L) based on decreased implantation sites and litter size at 2500 ppm dose group.

Data for the final hydrolysis product, dimethylsilanediol (DMSD)

Data for the final hydrolysis product, dimethylsilanediol (DMSD), have been included in the dossier to allow risk characterisation for exposure of humans via the environment.

In the combined repeated dose toxicity study with the reproduction / developmental toxicity screening test with dimethylsilanediol, conducted according to OECD Test Guideline 422 and in compliance with GLP, dimethylsilanediol was administered by oral gavage in corn oil for 28 (toxicity group females) or 29 (males) days to 10 rats/sex/group (exception, female 50 mg/kg group where N=9) at 0, 50, 250 or 500 mg/kg bw/day (Dow Corning Corporation, 2009). A single group of males was used for both the toxicity and reproductive phases of the study. Reproductive group females were treated (10 rats/dose group) for 14 days prior to the mating period, during the mating period and through post-partum day 3. Clinical observations were performed daily immediately following exposure. Body weight measurements were performed weekly. All animals received a detailed physical examination once before the first dose (to allow for within-subject comparisons), and weekly thereafter. Additional body weights on reproductive females were obtained on gestational days (GD) 0, 7, 14, and 20, within 24 hours of parturition, and on post-partum day four. Individual food consumption was recorded at least weekly, except during the cohabitation period. Functional observational battery (FOB) and motor activity evaluations were performed on males and toxicity group females once prior to initiation of exposures and during the fourth week of exposure. Blood samples for haematology and serum chemistry evaluations were collected at the scheduled necropsy from males and toxicity group females. Complete necropsies were performed on the males and the toxicity group females and selected organs were weighed. Microscopic examination was performed on protocol specified tissues on all toxicity group animals from the control and 500 mg/kg bw/day dose groups. Target tissues examined from the low- and mid-dose levels included liver, lung, prostate gland and thyroid gland from male rats and liver and lung from female rats.

Mating was initiated after the first two weeks of exposure by pairing reproductive group females with males of the same treatment group until positive evidence of mating was obtained. Reproductive and developmental parameters evaluated included evidence of mating, pregnancy, duration of gestation, mean litter size, mean live litter size, mean litter weight, and mean ratio of live births/litter size. Dams and pups were euthanized on post-partum day 4 and examined for external gross lesions. The number of corpora lutea, and the number of uterine implantation sites were determined for all reproductive group females.

Oral gavage administration of dimethylsilanediol to male and female Sprague-Dawley rats at concentrations of up to 500 mg/kg bw/day for 28 (toxicity females) or 29 (males) consecutive days was generally well tolerated. For the toxicity group males at 250 and 500 mg/kg bw/day significant soiling was observed (abdominal region and urogenital soiling). Soiling of the muzzle was a significant abnormal observation in the toxicity group females at 500 mg/kg bw/day. Both abdominal soiling and urogenital soiling were significant abnormal observations in the reproductive group females at 500 mg/kg bw/day.

There were no statistically significant differences across exposure groups in the mean body weights on any day for toxicity group females and reproductive toxicity group females. In the male group there was a significant difference across treatment groups in week 2, however, there was not a significant difference between control and any of the treatment groups for that week. With respect to body weight gain, male group 4 animals had a significant decrease in body weight gain during week 4 and in total gain from day 1 to 29. For toxicology females there was significant decrease in body weight gain during week 3. There were no statistically significant differences in body weight gain for the reproductive females in any of the treatment groups during any of the measured intervals. There were no differences in the average daily food consumption between control and treatment groups for the reproductive females group or the toxicity male and female groups for any of the measured time periods. There were no significant differences of toxicological significance between the control and treatment groups in either sex for the FOB ranked tests. There were no significant differences between either male or female treatment groups and their respective controls for the FOB continuous test and motor activity. There were no treatment-related changes associated administration on rat neurobiological function as evaluated with FOB and motor activity parameters. The significant changes that were noted in haematological parameters and prothrombin times for toxicity group males and females were within or slightly below historical control values. The significant changes that were noted in clinical chemistry parameters for toxicity group males and females were within or slightly below historical control values. The increased liver weights in toxicity males and females at 250 and 500 mg/kg bw/day correlated with the histopathologic finding of centrilobular hypertrophy. There were no other treatment-related differences in organ weights, absolute and relative for toxicity group males and females.

There were three primary effects of the test article observed in the liver, including centrilobular hypertrophy in both sexes, periportal hepatocellular vacuolation (microvascular lipidosis, females only), and brown pigment accumulation (males only) which was accompanied by chronic inflammation and bile duct hyperplasia. Centrilobular hypertrophy is considered an adaptive change. Hepatic lipidosis, unless severe, is generally considered non-adverse. Hepatic brown pigment is considered an adverse effect. Follicular cell hypertrophy was observed in the thyroid gland of mid- and high-dose male rats. This may reflect an adaptive secondary effect and adverse in the rat, but the mechanism is generally not applicable to species with significant levels of thyroid binding globulin (Capen,et al., 2002). Lung (males and females) and prostate gland were considered possible target tissues; however, further examination and inclusion of animals from the mid- and low-dose groups did not support this interpretation. There were no treatment-related effects apparent for any of the reproductive endpoints: gestation length, litter size, litter weight, ratio live births/litter size, litter sex ratio, number of implantation sites, number of corpora lutea, mating and fertility indices.

Based on the results of this study, the systemic toxicity NOAEL (No-Observed-Adverse-Effect-Level) for dimethylsilanediol in rats via oral administration in corn oil is considered to be 250 mg/kg bw/day based on hepatic brown pigment accumulation in and around the bile ducts, with associated bile duct hyperplasia and chronic inflammation at 500 mg/kg bw/day. In the absence of adverse effects on reproductive or developmental parameters in this study, a NOAEL of ≥500 mg/kg bw/day is assigned for reproductive and developmental toxicity.

Effects on developmental toxicity

Description of key information

In a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP (Dow Corning Corporation, 2002), the NOAEC for developmental toxicity was concluded to be 500 ppm (4.55 mg/L) hexamethylcyclotrisiloxane (D3; CAS 541-05-9; EC No. 208-765-4) based on decreased implantation sites and litter size at 2500 ppm dose group.

The registrants propose to conduct a prenatal developmental toxicity study in first species (rat) and second species (rabbit), conducted according to OECD Test Guideline 414 and in compliance with GLP, with the registered substance, hexamethylcyclotrisiloxane (CAS 541-05-9), after approval by ECHA.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-03-25 to 200211-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 422
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Canada, 188 LaSalle, St. Constant, Canada
- Age at study initiation: >=8 wk
- Weight at study initiation: 187-238 g (f); 250-313 g (m)
- Housing: 1/suspended wire cage
- Diet: standard diet ad libitum
- Water: drinking water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 2002-03-25 To: 2002-11-07
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 450 l steel and glass Rochester-style inhalation chambers with stainless steel exposure caging.
- Method of holding animals in test chamber: individual stainless steel caged compartments
- Method of conditioning air: air passed through series of purification filters
- System of generating particulates/aerosols: TS placed in warming chamber (75 deg C). The liquid TS was then metered into a heated metal J-tube for vapourization.
- Air change rate: at 10 exchanges of chamber volume per h

TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: automatic sampling from chamber

Acclimatization for 3 h on 2 days
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 ionisation detection (GC/FID) and each chamber was evaluated at least once per hour during the exposure period. For each day of exposure the mean test atmosphere D3 concentration (actual) as determined from the GC/FID analysis, was compared with the theoretical concentration (nominal), derived from the estimated chamber air flow rate and test substance consumption, as a quality control measure to evaluate exposure system performance.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: continuously until evidence of copulation
- Proof of pregnancy: vaginal plug or sperm in vaginal smear (day 0)
Duration of treatment / exposure:
Exposure period: 28 days for males and 46 days for females.
Premating exposure period (males): 2 weeks.
Premating exposure period (females): 2 weeks.
Mated femaes were exposed up to Day 19 of gestation.
Dams were not exposed during the lactation period.
Frequency of treatment:
6 hours/day; 7 days/week
Duration of test:
46 days
No. of animals per sex per dose:
10
Control animals:
other: yes, control group was exposed to filtered air
Details on study design:
- Dose selection rationale: based on the results of a previous 90-day inhalation study (DCC, 2001)
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily or twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

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: Yes
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (see Table 1)
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Ketamine, HCL/Xylazine
- How many animals: all toxicity groups

CLINICAL CHEMISTRY: Yes (see Table 1)
- Time schedule for collection of blood: at sacrifice
- How many animals: all toxicity groups

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to exposure and during wk 4
- Dose groups that were examined: all toxicity groups
- Battery of functions tested: FOB and motor activity examinations. These included: cage-side, hand-held, open field and categorical observations, rectal temperature, hindlimb/forelimb grip strength and landing foot splay. Motor activity was also determined using a Cage Rack Activity System.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
Examination for gross external malformations only.
Statistics:
Statistical methods: Bartlett's test and Kolmogorov-Smirnov test. Parametric data was tested using one-way Analysis of Variance (ANOVA) followed by Dunnett's Test (if significant); nonparametric data was tested by Kruskal-Wallis Test followed by Wilcoxon. Categorical data and histomorphology findings were evaluated with Fisher's
Exact Test. Statistically significant probabilities are reported at either the p<0.05 or p<0.01 levels.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Observations recorded during the weekly detailed physical examinations were not different from those recorded during the daily clinical examinations. The clinical observations that were indicative of an adverse effect of the treatment were minimal and involved an increased incidence of anogenital staining and brown staining on the head. Anogenital staining was observed in 100% of reproductive group females in the 2500 ppm groups. A single occurrence was observed in one reproductive group female in the 100 ppm group. 100% of the reproductive group females in the 2500 ppm groups exhibited staining on the head at least once during the in-life phase of the study. There were no other clinical effects of toxicity.
Mortality:
no mortality observed
Description (incidence):
All adult animals survived to their scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weight for 500 and 2500 ppm -exposed reproductive group females were consistently lower (≤ 8%) than the control group. However, the decreases were not statistically significant except for the 2500 ppm exposure group on gestation day 20 (8% lower relative to Control). Similarly, group mean body weight gain for the exposed reproductive group females in the 500 and 2500 ppm exposure groups were typically lower (S 30%), though not statistically significant, than controls prior to week 3 of gestation. Females in the 2500 ppm exposure group demonstrated a statistically significant 21% decrease in body weight gain at gestation week 3. These changes in body weight and body weight gain are consistent with those observed for the toxicity group females (pre-mating) and with the effects on litter size (gestation and post-partum periods).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no statistically significant differences in mean weekly food consumption values for the exposed reproductive group females relative to control. However, values were generally lower than control (2-12%). The one exception being a greater consumption of food (21%) during the parturition period (gestation day 20 - postpartum day 0/1) that likely represents differences in parturition (litter size and care).
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Description (incidence and severity):
Not examined for reproductive toxicity group females.
Clinical biochemistry findings:
not examined
Description (incidence and severity):
Not examined for reproductive toxicity group females.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Description (incidence and severity):
Not examined for reproductive toxicity group females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related effects were noted on ovary and uterine weights for toxicity group females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No effects seen in female reproductive organs and tissues.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No effects seen in female reproductive organs and tissues.
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases in the number of uterine implantation sites (33% decrease) when compared to the control in the 2500 ppm group.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no effects on pup viability.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no effects on gestation length.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
All of the reproductive group females presented with positive evidence of mating on or before the seventh day of pairing and all but one of the reproductive group females became pregnant. The exception was a female from the 2500 ppm exposure D (Animal D0033). The uterus from this female was negative for implantation sites.
Other effects:
no effects observed
Description (incidence and severity):
There were no effects on number of corpora lutea.
Key result
Dose descriptor:
NOAEC
Effect level:
500 ppm
Based on:
test mat.
Basis for effect level:
pre and post implantation loss
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases were noted for litter size (number of pups per litter on PND 0 and 4) and litter weight (27% decreases relative to control on PND 0 and 4) at 2500 ppm.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases were noted for litter size (number of pups per litter on PND 0 and 4) and litter weight (27% decreases relative to control on PND 0 and 4) at 2500 ppm.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in litter size and weights
Abnormalities:
no effects observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
2 500 ppm
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
In the combined repeated dose/reproductive and developmental inhalation toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP, exposure to 2500 ppm (22.8 mg/L) of the test substance was associated with decreased implantation sites and litter size at 2500 ppm dose group. Therefore, the NOAEC for reproductive and developmental toxicity was concluded to be 500 ppm (4.55 mg/L).
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
4 550 mg/m³
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test, conducted according to OECD Test Guideline 422 and in compliance with GLP (Dow Corning Corporation, 2002), Sprague-Dawley male and female rats were exposed to hexamethylcyclotrisiloxane vapor via whole body inhalation at concentrations of 0, 100, 500 or 2500 ppm (approximately 0.61, 4.5 or 22.8 mg/L). The animals were exposed 6 hours per day, 7 days per week for up to 39 days. Females in each exposure level were divided into a toxicity group (10 animals/group) and a reproductive group (10 animals/group). A single group of males (10 animals/group) was used for both the toxicity and reproductive phases of the study. Males and toxicity group females were treated for 28 and 29 days, respectively. Reproductive group females were treated for 14 days prior to the mating period, during the mating period and then up to and including gestation day 19.

A mating phase was initiated after the first two weeks of exposure by pairing reproductive group females with males of the same treatment group until positive evidence of mating was obtained. Reproductive and developmental parameters evaluated included evidence of mating, duration of gestation, mean litter size, mean live litter size, mean litter weight, mean ratio live births/litter size, corpora lutea count, and the number of uterine implantation sites. Dams and pups were euthanized on PND 4 and examined for external gross lesions.

In general, whole-body inhalation exposure of male and female Sprague-Dawley rats to hexamethylcyclotrisiloxane vapour at concentrations of up to 2500 ppm was well-tolerated, as suggested by minimal effects on body weight and food consumption, the lack of significant clinical signs of toxicity, and minimal effects on clinical pathology parameters. Increased liver weight and centrilobular hepatocellular hypertrophy were demonstrated in both sexes exposed to 2500 ppm. These effects are considered to be an adaptive response characteristic of this class of cyclic siloxanes. No liver effects were identified at lower exposure levels.

In males exposed to 2500 ppm, there were statistically significant decreases in the absolute weight of epididymides (10 %) and in the absolute (30 %) and relative (27 %) weight of the seminal vesicles. Slight atrophy of the seminal vesicles was identified microscopically in four of the 10 males in the 2500 ppm exposure group. The epididymides, testes, and prostate from exposed rats were histologically normal.

With regard to sexual function and fertility the treatment had no effect on evidence of mating or gestation length. All mated females presented with positive evidence of mating on or before day seven of pairing and all but one of the reproductive phase females became pregnant. Fertility in males was not affected by the statistically significant decrease in absolute weight of the epididymides and decreased absolute/relative weight of the seminal vesicles with slight atrophy of seminal vesicles in four of the ten males at 2500 ppm; these four males successfully impregnated the females with which they were mated. The epididymides, testes and prostate from the exposed rats were histologically normal.

Negative effects on implantation sites and litter size parameters were observed for female rats exposed to 2500 ppm. There was a 33 % decrease in litter size and in the number of implantation sites. Gestation length, pup sex ratio, pup weight and viability, and corpora lutea counts were unaffected. There were no external malformations in any of the pups. No effects were observed at lower exposure concentrations.

The NOAEC for developmental toxicity was concluded to be 500 ppm (4.55 mg/L) based on decreased implantation sites and litter size at 2500 ppm dose group.

Data for the final hydrolysis product, dimethylsilanediol (DMSD)

Data for the final hydrolysis product, dimethylsilanediol (DMSD), have been included in the dossier to allow risk characterisation for exposure of humans via the environment.

In the prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, dimethylsilanediol was administered orally to pregnant female rats during gestation days 6 to 19 at doses of 0, 250, 500 or 1000 mg/kg bw/day (WIL Research, 2013).


Administration of dimethylsilanediol to pregnant rats at doses of 0, 250, 500 or 1000 mg/kg bw/day resulted in a maternal No-Observed-Adverse-Effect-Level (NOAEL) of less than 250 mg/kg bw/day based on higher mean liver weights and corresponding microscopic findings of hepatocellular hypertrophy and periportal hepatocellular vacuolation at all doses.

A higher mean litter proportion of post implantation loss, with a corresponding lower mean litter proportion of viable foetuses, was considered attributable to treatment and adverse in the 1000 mg/kg bw/day group.

Mean foetal body weights at the three doses were 5.3%, 10.5%, and 23.7% lower, respectively, than the concurrent control group. The mean foetal weight in the 250 mg/kg bw/day group was within the historical control data range and was only 0.2 g lower than the concurrent control group mean. Furthermore, litter size can be affected by treatment so additional statistical analysis of the foetal weight data using an appropriate statistical method specific for this type of data, which takes litter size into account, demonstrated that foetal weight at 250 mg/kg bw/day was comparable with control and not statistically significant and therefore unaffected by treatment at this dose.

Foetal malformations were noted in the 500 and 1000 mg/kg bw/day groups, but not in the 250 mg/kg bw/day group. Foetal variations were recorded in all dose groups with some visceral (small thyroid gland) and skeletal variations in the 250 mg/kg bw/day group showing a dose-response relationship. However, many of the foetal variations noted in the 250 mg/kg bw/day group were either recorded at an incidence that was not statistically significant and/or within the historical control incidence. Furthermore, as many, but not all, of these variations (skeletal developmental delays) correlate with foetal body weight, and foetal body weight was not affected by treatment, then the variations noted at the low dose of 250 mg/kg bw/day are also considered not to be attributable to treatment.

It is important to note that 1) malformations are alterations in foetal morphology that are expected to be permanent and to interfere with the function or viability of the organism, and 2) variations are alterations that are transient, do not interfere with the function or viability of the organism, and are often seen in control animals. Based on the fact that variations are not considered adverse the variations recorded in the prenatal developmental toxicity study on dimethylsilanediol should not be considered as adverse findings and the lowest dose of 250 mg/kg bw/day is the NOAEL based on reduced foetal body weight and malformations at the middle and high doses.

Therefore, the NOAEL for prenatal developmental toxicity was considered to be 250 mg/kg bw/day.

In the combined repeated dose toxicity study with the reproduction / developmental toxicity screening test with dimethylsilanediol, conducted according to OECD Test Guideline 422 and in compliance with GLP, the NOAEL for general systemic toxicity was 250 mg/kg bw/day based on hepatic brown pigment accumulation in and around the bile ducts, with associated bile duct hyperplasia and chronic inflammation at 500 mg/kg bw/day, and ≥500 mg/kg bw/day for reproductive and developmental toxicity (Dow Corning Corporation, 2009).

Justification for classification or non-classification

Based on the results of the OECD 422 study, classification and labelling for reproductive toxicity is not warranted because inhalation of D3 vapour had no adverse effects on sexual function or fertility. With regards to development of the offspring, exposure to D3 had no effect on pup sex ratio, pup weight, pup viability or corpora lutea counts at any exposure concentration and no external malformations were noted in any pups. There was a negative effect on litter size parameters (decrease in litter size and litter weight due to a decrease in total number of implantation sites) at 2500 ppm (22800 mg/m3; a concentration that exceeds the NOAEC from repeated dose toxicity studies), but no adverse effects were observed at lower exposure concentrations relevant to human exposure. Therefore D3 is not classified for toxicity to reproduction under Regulation (EC) No 1272/2008.

Additional information