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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:
No data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Combined Repeated Dose and Reproductive / Developmental Toxicity Screening Test (Precursor Protocol of GL 422)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) males: 317 - 423 g, females: 250 - 277 g
- Fasting period before study: no fasting period
- Housing: individual in stainless steel cages
- Diet: CRF-1, pelleted, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 41 - 71
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared more than once a week. Concentration of stock solution was 200 mg/ml and sotred at 4°C in a refrigerator. The stock solution was diluted with corn oil to achieve the concentration of the dosing solutions.

VEHICLE
- Lot/batch no.: V3T0416 and V4K3008
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Day 18 of gestation until day 4 of lactation: individual in plastic cage with wood chips
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration of dosing solution was verified by GC at the study initiation and at the end of the study. The range of concentration was acceptable becuase it was between 93.6 - 101.2%.
Duration of treatment / exposure:
Premating exposure period (males): 14 days prior to mating
Premating exposure period (females): 14 days prior to mating
Duration of test: males: 43 days; females: until day 6 of lactation
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
(Males)
6 males/dose
for satellite group: 6 males/dose

(Females for reproduction toxicity study)
12 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a foregoing range finding study, in which animals were orally exposed to 0, 250, 500 and 1000 mg/kg bw/day (Hashima, year not availble, 401223P). No mortality was observed in all groups. Reddish urine, decrease in food consumption and occult blood was observed in administered groups. Therefore, 62.5, 250 and 1000 were selected as the dose levels for the main study.

Post-exposure period: Yes, for a sub group of males and females for 14 days

Additional details regarding this study are provided in section 7.5.1.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day during administration period, daily during recovery period, and once before necropsy

DETAILED CLINICAL OBSERVATIONS: Yes (including FOB)
- Time schedule: once before administration, Day 7, 14, 21, 28, 35 and 41 (males)
- Time schedule: once before administration, Day 8 and 15 of administration; Day 1, 8 and 15 of gestation; Day 3 of lactation
- Time schedule: once before administration, Day 8, 15, 22, 29, 36 and 42 (females for satellite group)

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week
Males and satellite females: Day 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 during administration period, Day 1, 4, 8, 11, 14 and 15 during recovery period
Pregnant females: Day 1, 4, 8, 11, 15 and 18, Day 0, 7, 11 and 21 during gestation, Day 0, 4, 6 and 7 during lactation

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day after last administration or after recovery period
- Anaesthetic used for blood collection: Yes (Pentobarbital-Na)
- Animals fasted: Yes
- How many animals: all administered animals
- Parameters checked: RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, Platelets, Reticulocytes, PT, APTT, Fibrinogen, WBC, Differential leukocytes: Lymphocyte, Neutrophis, Eosinophis, Basophil, Monocyte

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day after last administration or after recovery period
- Animals fasted: Yes
- How many animals: all administered animals
- Parameters checked: AST, ALT, ALP, gamma-GTP, T-potein, Albumin, A/G, T-bilirubin, Urea nitrogen, Creatinine, Glucose, T-cholesterol, Triglycerides, Na, K, Cl, Ca, Inorganic-P

URINALYSIS: Yes
- Time schedule for collection of urine: on Day 2 and 37 in fasted males, Day 3 and 38 in non-fasted males, Day 2 during administration period and Day 5 of lactation in fasted females, Day 3 during administration period and Day 6 of lactation in non-fasted females, after recovery period in females
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes/No
- Parameters checked: Color, pH, Protein, Glucose, Ketone body, Bilirubin, Occult blood, Urobilinogen, Urinary sediments, Epithelial cells, Erythrocytes, Leukocytes, Casts, Crystals
Oestrous cyclicity (parental animals):
Oestrous cycle length and normality were evaluated in females by vaginal smears prior to mating, and optionally during mating, until evidence of mating was found.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
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, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after administration period or recovery period
- Maternal animals: All surviving animals after day 6 of lactation

ORGAN WEIGHT: Yes: Brain, pituitary, thyroids, thymus, heart, liver, spleen, kidney, adrenals, testes, epididymides, ovaries, uterus, lung

GROSS PATHOLOGY: Yes: heart, lung, trachea, liver, pancreas, sublingual gland, submandibular gland, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, thymus, spleen, submandibuar lymph node, mesentric lympha node, kidney, urinary bladder, testis, epididymis, seminal vesicle, prostate, ovary, uterus, pituitary, adrenal, thyroid, parathyroid, cerebrum, cerebellum, medulla oblongata, spinal code, sciatic nerve, eyeball, Harderian gland, bone (sternum or femur), and mammary gland

HISTOPATHOLOGY: Yes: heart, lung, trachea, liver, pancreas, sublingual gland, submandibular gland, esophagus, stomach, duodenum, jejunum, ileum. cecum. colon, rectum, thymus, spleen, submandibuar lymph node, mesentric lympha node, kidney, urinary bladder, urethra, ovary, uterus, pituitary, adrenal, thyroid, parathyroid, cerebrum. cerebellum. medulla oblongata, spinal code, sciatic nerve, eyeball, Harderian gland, bone (sternum or femur), bone marrow (sternum or femur), and mammary gland
Postmortem examinations (offspring):
SACRIFICE
- All F1 offspring at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
Barlett test, Dunnett test, chi-square test, Cochran-Armitage test
Reproductive indices:
- Copulation index: (number of pairs with successful copulations/number of pairs)x100
- Fertility index: (number of pregnant females/number of pairs with successful copulation)x100
- Implantation index: (number of implantation scars/number of corpora lutea)x100
- Gestation index: (number of dams having live pups/number of pregnant dams)x100
- Delivery index: (number of pups born/number of implantation scars)x100
- Birth index: (number of live pups born/ number of implantation scars)x100
Offspring viability indices:
- Sex ratio at birth: (number of male pups/number of female pups
- Live birth index: (number of live pups born/number of pups born)x100
- Viability index: (number of live pups on Day 4 of lactation/number of live pups born)x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
Two males and 1 female from the 1000 mg/kg group died on Day 16 and 17 (males) and Day 8 (female). Transient salivation, soiled hair, a decrease in locomotor activity, reddish urine, hypothermia, perioral smudges, perianal soiling, diarrhea, bradypnea, and piloerection were noted in the dying animals. Transient salivation, soiled hair and reddish urine were noted in the surviving males and females of the 1000 and 250 mg/kg groups.

BODY WEIGHT AND WEIGHT GAIN
Low body weights were noted in males on Day 4 to 42 during administration period and Day 1 and 4 during recovery period.
Females of the 1000 mg/kg group showed also decrease in body weight.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The number of estrous cases before pairing in 1000 mg/kg was less than that of the control group.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Moderate atrophy of seminiferous tubule, slight degeneration of semiferous tubule, slight vacuolization of Sertoli cell and slight retention of spermalid were observed in 1000 mg/kg. Slight decrease in sperm and slight or moderate cell debris in lumen in epididymis were observed in 1000 mg/kg.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No changes were observed in reproductive parameters.

ORGAN WEIGHTS
Body weight of males in 1000 mg/kg after administration period was increased. Decrease in absolute weight of thymus, increase in absolute weight of kidney, increase in relative weight of spleen, kidney and adrenals were observed in males of 1000 mg/kg after administration period. In same group, decrease in absolute weight of pituitary was also observed but this was not caused by the test item, since no change was detected in its relative weight. Absolute weight of brain was also increased. However, this change was not regarded as compound-related effect but caused by the difference of body weight.
No changes in body weight were detected in females after administration groups. Relative weight of thymus was decreased in females of 62.5 and 250 mg/kg. Decreased absolute weight of thymus, decreased relative weight of thymus and increased relative weight of liver were observed in females after administration period.
No changes of body weight was detected in males after recovery period. Decreased absolute weight of pituitary and increased increased relative weight of heart was observed in males in 62.5 mg/kg. However, these changes were not considered as tox effect of the test item, since there was no dose-response. Absolute weight of kidney was increased in males of 250 mg/kg but this was not caused by the test item due to lack of dose dependency. Relative weight of kidney was increased in males. Absolute weight of pituitary was also decreased. This was not caused by the test item because no change was detected in relative weight.

GROSS PATHOLOGY
See Section 7.5.1

HISTOPATHOLOGY: NON-NEOPLASTIC
See section 7.5.1

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects on reproductive performance.
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Low number of oestrous cases.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Significant histopathological changes in the urinary bladder of males and females, which were not reversed at the end of the recovery period in the higher dose groups

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed

Details on results (F1)

VIABILITY (OFFSPRING)
No changes were observed.

CLINICAL SIGNS (OFFSPRING)
No changes were observed.

BODY WEIGHT (OFFSPRING)
Litter weight on Day 4 of lactation was decreased in 1000 mg/kg. However, this change occurred accidentally since no changes were found in mean pups weight on Day 0 and 4 of lactation, male and female weight on Day 0 and 4.

GROSS PATHOLOGY (OFFSPRING)
One dead pup showed atrophy of lung in 250 mg/kg administered group. This change was not related to the test item since no dose-responsibility was found. One survival pup showed dilatation of pelvis in 1000 mg/kg administration group. This change was not regarded as test compound related effect but accidentally happened.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were noted.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Reproductive functions of male and female rats.

 

F0 (mg/kg/day)

Parameter

0

62.5

250

1000

Number of females

12

12

12

11

No. of estrous cases before pairing (14 days)

3.2±0.4

3.2±0.4

3.1±0.3

2.5±0.7*

Copulation index

100

91.7

91.7

90.9

No. of conceiving days

2.8±1.4

1.8±1.3

2.8±3.8

4.7±2.4

 

 

 

 

 

Female fertility index

100.0

100

100.0

100.0

 

Table 2: Observation of pups (F1).

 

F1 (mg/kg/day)

Parameter

0

62.5

250

1000

Length of gestation

22.2±0.4

22.5±0.7

22.5±0.5

22.6±0.5

Corpora lutea

16.7±1.8

16.3±3.5

15.8±1.5

15.6±1.3

Implantation scars

15±1.5

14.5±4.3

14.5±1.9

13.7±1.3

Implantation index (%)

90.1±6.4

87.2±17.7

91.3±7.6

88±6.8

Gestational index

100

100

100

100

Pups born

14.3±1.6

13.3±4.6

13.9±1.9

12.9±1.1

Stillbirths

0.4±0.7

0.1±0.3

0.3±0.5

0.2±0.6

Live pubs born

13.9±1.7

13.2±4.6

13.6±1.7

12.7±1.1

Sex ratio at birth

1.74±1.25

0.96±0.73

0.94±0.54

1.18±0.77

Delivery index (%)

92.9±7.7

88.7±13.5

94.7±6.3

93.1±7.3

Birth index (%)

92.8±7.7

88.6±13.5

94.6±6.3

93.0±7.3

Live birth index (%)

97.2±4.5

99.4±2.1

98.2±3.1

98.6±4.4

Live pups on day 4 of lactation

13.8±1.7

12.9±4.4

13.5±1.7

12.4±1.1

Sex ratio on day 4 of lactation

1.74±1.24

0.97±0.72

0.93±0.53

1.14±0.8

Viability index (%)

99.4±2.0

98.3±4.1

98.7±2.8

97.7±5.0

External abnormalities (%)

0

0

1.2±4.0

0

Acaudate

0

0

1.2±4.0

0

 

Table 3: General signs of pups.

 

F1 (mg/kg/day)

Parameter

0

62.5

250

1000

No. of pups

(normal/death)

 

 

 

 

day 0 of lactation

167/5

145/1

150/3

127/2

day 1 of lactation

166/1

145/0

149/1

125/2

day 2 of lactation

166/0

144/1

148/1

124/1

day 4 of lactation

166/0

143/1

148/0

124/0

day 4 of lactation

166/0

142/1

148/0

124/0

 

Table 4: Body weights of pups.

 

F1 (mg/kg/day)

Parameter

0

62.5

250

1000

Male weight

 

 

 

 

day 0 of lactation

6.7±0.6

6.6±0.8

7.0±0.7

6.8±0.7

day 4 of lactation

10.7±1.0

10.5±1.7

10.7±1.1

10.5±0.7

Female weight

 

 

 

 

day 0 of lactation

6.3±0.4

6.3±0.6

6.8±0.6

6.5±0.5

day 4 of lactation

10.1±0.9

9.8±1.2

10.6±1.0

10.3±0.6

Mean pups weight

 

 

 

 

day 0 of lactation

6.6±0.5

6.6±0.8

6.9±0.6

6.7±0.6

day 4 of lactation

10.4±0.9

10.3±1.7

10.7±1.0

10.4±0.7

Litter weight

 

 

 

 

day 0 of lactation

91.0±9.0

83.4±25.5

93.9±14.4

84.7±7.9

day 4 of lactation

143.4±11.7

126.8±35.1

142.8±18.0

128.6±10.5*

Significantly different from control group (*: p<0.05)

 

 

Applicant's summary and conclusion

Conclusions:
In a reliable study with trimethoxy(vinyl)silane, according to OECD 422 and in compliance with GLP, NOAELs for reproductive performance of parental animals were estimated to be 1000 mg/kg bw/day for males and 250 mg/kg bw/day for females. The NOAEL for offspring was 1000 mg/kg bw/day. The NOAEL for systemic toxicity of the parents was 62.5 mg/kg bw/day.