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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 December to 22 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” Health Effects Test Guideline OPPTS 870.3650
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd.
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 287-335 g: Females: 179-211 g.
- Fasting period before study: no data
- Housing: individually in Makrolon type-3 cages (except in pairing period)
- Diet (e.g. ad libitum): standard diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 December 2008 To: 7 February 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
dried and deacidified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. The test substance was weighed into a glass beaker on a tared precision balance and approximately 100% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: nightly until evidence of copulation was observed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (7 days). During the last week of the treatment, samples were taken from the middle only to confirm concentration. The samples were analysed by GC coupled to an FI detector following an analytical procedure developed at Harlan Laboratories.
Duration of treatment / exposure:
Males: at least 28 days
Females: approximately 49 days
Frequency of treatment:
Daily
Details on study schedule:
Animals were treated for two weeks, then moved to mating cages for up to two weeks, until evidence of mating was observed. Males were treated for a total of 28 days (including 14 days prior to pairing), and females for approximately 49 days (14 days prior to pairing, through the pairing and gestation periods, up until day 4 postpartum)
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose range-finding study
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally, females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes, once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported, if present.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Males: Weekly during pre-pairing and after pairing periods; Females: Pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum. No food consumption was recorded during the pairing period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 postpartum.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 18 hours
- Parameters checked in table 1 (Section 7.5.1) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 postpartum.
- Animals fasted: Yes, 18 hours
- Parameters checked in table 1 (Section 7.5.1) were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: functional observation battery (FOB) and locomotor activity
Oestrous cyclicity (parental animals):
Not applicable
Sperm parameters (parental animals):
Parameters examined in P males: testis weight, 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, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals when no longer needed for the assessment of reproductive effects
- Maternal animals: all surviving animals on day 5 postpartum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera
- Number of implantation sites and corpora lutea recorded for all dams with litters. Uteri visualised for possible haemorrhagic areas of implantation sites (with ammonium sulphide solution)

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 2 (Section 7.5.1) were prepared for microscopic examination and weighed
Postmortem examinations (offspring):
SACRIFICE
- Pups were sacrificed on day 4 postpartum

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examination [no further details on exact parameters checked]

HISTOPATHOLOGY / ORGAN WEIGHTS
- Not performed on pups
Statistics:
The following statistical methods were used to analyse food consumption, body weights, functional observational battery, locomotor activity and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett test when the data could not be assumed to follow a normal distribution.
• Fisher’s exact-test was applied if the variables could not be dichotomized without loss of information.
Reproductive indices:
Fertility and gestation indices, mean precoital time, conception rate, duration of gestation, corpora lutea, implantation rate, post-implantation loss and pup sex ratio.
Offspring viability indices:
Viability index, birth index, mean litter size and post-natal loss

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
See Section 7.5.1 for details on systemic effects.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
(Data for control, low-, mid- and high-dose groups, respectively)
Fertility index: 100% in all groups
Gestation index: 100% in all groups
Mean precoital time (mean): 2.3, 2.5, 2.9, 2.8
Conception rate: 100% in all groups
Duration of gestation (days): 21.2, 21.5, 21.6, 21.6
Corpora lutea count (mean per dam): 14.7, 15.2, 15.3, 16.1
Implantation rate (mean number of implantations per litter): 13.8, 13.8, 14.8, 14.2
Post-implantation loss (per dam): 1.0, 1.2, 0.4, 2.0

Details on results (P0)

Although post-implantation loss in group 4 was significantly higher than in the control group, the loss as a percentage of total implantations and mean post-implantation loss was within the range of historical control data. A statistically significantly reduced birth index in group 4 was not considered to be an adverse effect of treatment as mean litter size was similar to the control group.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse reproductive effects observed at any tested concentration

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At first litter check, in group 4 no milk in the stomach was observed in 13 pups in three litters. This was considered to be treatment-related. One pup was noted to have the right hind leg cannibalized and autolysis was observed in two pups which were found dead. One pup in group 3 had abdomen blue swollen and one pup in group 2 had tail thread-like. These findings were considered to be incidental.

During lactation, in group 4 twelve pups in one litter and six pups in another were noted to have cold skin at touch on day 2 postpartum or at last litter check. This was considered to be related to the treatment with the test substance. Autolysis was observed in all pups in one litter (same dam lost its whole litter on day 3 postpartum). The other observations noted were no milk in the stomach in one pup and in six pups in another litter, right hind leg cannibalized in one pup were considered to be incidental.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There was increased post-natal pup loss in the highest dose group (see Table in Section 7.8.2)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean pup weights on day 1 postpartum were unaffected by treatment with the test item. During the lactation period (to day 4 postpartum), in group 4 body weight development was reduced although not statistically significant (+22.0% versus 37.3% in the control).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At macroscopic examination: In group 4, no milk in the stomach was noted in seven pups in one litter and in six pups in another litter and in all pups in a further litter which were found dead between days 2 and 3 postpartum. Autolysis was observed in two pups in one litter. No macroscopical findings were noted in groups 1, 2 and 3.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on post-natal pup loss and reduced body weight gain.

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In an oral reproductive toxicity screening study, performed to GLP and conducted according to OECD Guideline 422, the no-observed-adverse-effect level (NOAEL) for toxicity to reproduction with 1,1,1,3,5,5,5-heptamethyltrisiloxane in rats was at least 800 mg/kg bw/day, the highest dose tested.
Executive summary:

In an oral reproductive toxicity screening study (combined with a repeated dose toxicity study and developmental toxicity screening test),

performed to GLP and conducted according to OECD Guideline 422, 1,1,1,3,5,5,5-heptamethyltrisiloxane was assessed for its ability to induce reproductive toxicity in Wistar rats.

Four groups of ten male and ten female rats were treated by oral gavage with the test substance (in corn oil) at 0, 50, 200 or 800 mg/kg bw/day. Following two weeks of treatment, animals were allowed to mate for up to two weeks. In total, males were treated for at least 28 days (including the pre-mating period of 14 days), while females were treated for approximately 49 days (a pre-mating period of 14 days, a mating period of up to 14 days, throughout gestation and up until 4 days post-partum). Animals were observed throughout the study for any overt signs of toxicity or morbidity, until sacrifice and gross necropsy. A number of reproductive parameters were measured, including fertility and gestation indices, mean precoital time, conception rate, duration of gestation, corpora lutea, implantation rate, post-implantation loss and pup sex ratio.

 

All animals survived until scheduled necropsy. Fertility index, gestation index and conception rate were 100% in all groups and mean precoital time and duration of gestation were unaffected by treatment. There were no adverse effects of treatment with 1,1,1,3,5,5,5-heptamethyltrisiloxane on fertility, and a no-observed-adverse-effect-level (NOAEL) of at least 800 mg/kg bw/day (the highest dose tested) was determined for toxicity to reproduction on the basis of this screening test.