Hexamethylcyclotrisiloxane

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylcyclotrisiloxane

EC Number:

208-765-4

EC Name:

Hexamethylcyclotrisiloxane

Cas Number:

541-05-9

Molecular formula:

C6H18O3Si3

IUPAC Name:

hexamethyl-1,3,5,2,4,6-trioxatrisilinane

Test material form:

solid

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100, 500, and 2500 ppm
Basis:
nominal conc.

Doses / concentrations 2

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)

Examinations

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. 

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

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

Applicant's summary and conclusion

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.