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Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13.10.2003 to 30.04.2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Laboratory Animal Services, Switzerland
- Age at study initiation: (P) x 6-8 wks
- Weight at study initiation: (P) Males: 144-184 g; Females: 162-190 g
- Fasting period before study: None
- Housing: During the pre-pairing period, males and females were housed individually. During the pairing period, the rats were housed two females/one male in Makrolon pairing cages. After positive mating or at the end of the pairing period, the males and females were housed individually again; males until necropsy and the females for the birth and rearing of young. On the day of weaning, the dam was separated from its litter.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): 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: 20.10.2003 To: 30.04.2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was administered in dried corn oil. Mixtures of the test substance in the vehicle (weight:volume) at the appropriate concentrations were freshly prepared once per week using a magnetic stirrer.
Details on mating procedure:
- M/F ratio per cage: 1:2
- Length of cohabitation: 21 days maximum
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 21 days of unsuccessful pairing replacement of first male by another male
- Further matings after two unsuccessful attempts: no
- 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:
Verification analyses of the actual test substance concentrations, stability during administration (2 hours) and over seven days, and homogeneity in the prepared mixtures were performed on the prepared mixtures on three days (one during pre-pairing, one during gestation and one during the lactation period). On the day of preparation, samples of each dose concentration were taken before dosing. Samples from before dosing were taken from the top, middle and bottom of the container. Later, samples were taken from the middle of the container for verification of stability at room temperature for two hours and for seven days. Analysis was performed using gas chromatography.
Duration of treatment / exposure:
Exposure period: Males: Exposed for a 70-day pre-pairing period, during pairing and until the last litter reached day 7 post-partum. Females: Exposed during pairing and until the last litter reached day 7 postpartum. Premating exposure period (males): 70 days. Premating exposure period (females): 14 days . Duration of test: until the last litter reached day 7 postpartum.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 male and 24 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in conjunction with the sponsor, based on the results of preceding prenatal developmental and repeated dose toxicity studies.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined at least twice daily for mortality and signs of a reaction to treatment and/or symptoms of ill health.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from start of treatment until necropsy. Organs weighed were: pituitary gland, liver, kidneys, testes, prostate, seminal vesicles with coagulating glands, epididymides, ovaries and uterus with cervix and oviducts.

FOOD CONSUMPTION:
- Food consumption for females was recorded weekly from start of treatment to delivery (except mating period). During lactation, food consumption was recorded on days 1, 7 and 14 post-partum. Since pups begin to consume material feed on or about lactation day 14, food consumption was not recorded after this day. For males, food consumption was recorded weekly from treatment start until necropsy, except during mating.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
Daily determination of the estrous cycle stage beginning at pairing start until evidence of positive mating.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testes weight, epididymis weight, prostate weight, epididymides weight, histopathology of testes, prostate, seminal vesicles and epididymides.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and examined macroscopically.

PARAMETERS EXAMINED
The following parameters were examined in offspring on day 21 post-partum: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross external and internal abnormalities, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: yes, they were autopsied and/or preserved in fixative for possible further examination.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals when the last litter was at least seven days old.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was directed to the reproductive organs. Implantation sites were counted for all dams (uteri placed in solution of ammonium sulphide).

HISTOPATHOLOGY / ORGAN WEIGHTS
- All adults, including those that died before scheduled sacrifice, or were killed in a moribund condition. The tissues histopathologically examined in all animals of the control and high dose groups were as follows: pituitary glands, liver, kidneys, testes, prostate, seminal vesicles, epididymides, ovaries, uterus, cervix and vagina. In addition, the following organs were histopathologically examined in all groups: ovaries, uterus, cervix and vagina in non-pregnant positively mated females (with day 0 post coitum); tested, epididymides, seminal vesicles with coagulating glands and prostate in males that failed to mate.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected at standardisation were killed and examined macroscopically.
- After weaning at post-partum day 21, all pups were sacrificed and examined internally and externally for abnormalities.
Statistics:
The following statistical methods were used to analyse body weights, food consumption, reproduction and pup data:
- means and standard deviations.
- if the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
- The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

For pup data, the litter was the appropriate unit for statistical comparison.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs that were attributable to treatment with the test item were noted. All females survived until scheduled necropsy. At 1000 mg/kg bw/d, starting during early/mid gestation, all females displayed signs of discomfort after dosing (pushing head through bedding). This behaviour was noted as long as the females were dosed (i.e., one day prior to scheduled necropsy).
Mortality:
no mortality observed
Description (incidence):
All males survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/d, mean body weight gain of males during the pre-pairing period was slightly decreased, resulting in a slightly lower mean body weight at the end of the pre-pairing period (375 g compared with 409 g in the vehicle control).  Although statistical significance was only reached on single days, this reduction was considered to be test item related.  During the pairing and after pairing period, lower absolute body weights at 1000 mg/kg bw/d persisted, while body weight gain was similar to that of the vehicle control. Body weight development of females was not affected by treatment with the test item.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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):
In the liver of males, the severity of glycogen deposition was slightly increased at 1000 mg/kg bw/d. This finding was considered in relation with the nutritional state of the animals and of no adverse character. Kidneys A slightly increased severity of tubular hyaline change occurred in males of Group 4 (1000 mg/kg bw/d) mainly in the outer cortex. The grade was 2.5 versus 2.0 in the controls.  Probably this change reflects an increased accumulation of alpha-2-microglobulin which is a male rat specific phenomenon of no toxicological relevance for humans. 
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):
For both generations, the fertility rate was high resulting in at least 23 litters per group for evaluation of reproduction data.  At all dosages, there were no treatment related effects on mean or median pre-coital time, fertility indices, mean duration of gestation and number of implantations, post-implantation loss, pup survival or litter size from birth through to weaning.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Decreased body weight gain of males, and increased absolute and relative liver and kidney weights in P males and females. Histopathology revealed effects on livers and kidneys of males.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive effects
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No test-item related findings or clinical signs were noted at first litter check on day 0 postpartum or during the lactation period. 
Mortality / viability:
no mortality observed
Description (incidence and severity):
No effect on pup survival.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Pup weights at birth and during lactation were unaffected by treatment with the test item.  
Food consumption and compound intake (if feeding study):
not examined
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test-item related findings were noted at macroscopic examination of pups.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No treatment-related effects on sex ratios were noted.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no effects on any reproductive parameters.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

In a good quality one-generation reproductive toxicity study (reliability score 1) conducted to OECD Test Guideline 415 and GLP, the parental and reproductive NOAELs for [3-(2,3-epoxypropoxy)propyl]trimethoxysilane were = 1000 mg/kg bw/d, in rats. Treatment with the test substance resulted in signs of discomfort after dosing (noted for P females from early /mid gestation onwards), decreased body weight gain of males, and increased absolute and relative liver and kidney weights in P males and females. Histopathology revealed effects on livers and kidneys of males.

Please see attached document for result tables.

Applicant's summary and conclusion

Conclusions:
In a good quality one-generation reproductive toxicity study (reliability score 1) conducted to OECD Test Guideline 415 and GLP, the parental and reproductive NOAELs for [3-(2,3-epoxypropoxy)propyl]trimethoxysilane were 500 and = 1000 mg/kg bw/d, in rats. Treatment with the test substance resulted in signs of discomfort after dosing (noted for P females from early /mid gestation onwards), decreased body weight gain of males, and increased absolute and relative liver and kidney weights in P males and females. Histopathology revealed effects on livers and kidneys of males. Based on these data a NOAEL for parental animals was established at 500 mg/kg bw/d. A NOAEL for reproductive effects was established at 1000 mg/kg bw/d.