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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:
24 September 2001 to 06 September 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(1996)
Deviations:
yes
Remarks:
(additionally 10 females per dose group were added to the study to determine the repeated dose toxicity to females)
Qualifier:
equivalent or similar to
Guideline:
other: USEPA Health Effects test guideline OPPTS 870.3650 (2000)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,2-ethanediamine, N-{3-(triethoxysilyl)propyl}-
- Substance type: Alkoxysilane
- Physical state: Colourless liquid
- Lot/batch No.: GD06513
- Expiration date of the lot/batch: February 2002
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature in a tightly sealed container under nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD (SD)IGBS VAF/Plus
- Source: Charles River Laboratories, Inc. , Portage, MI
- Age at study initiation: 8 weeks
- Weight at study initiation: males 253-301 g, females 181-229 g
- Fasting period before study: None
- Housing: individually in suspended wire-mesh cages elevated over Bed-O'Cobs bedding; during cohabitation the females were pared 1 to 1 with a male in a suspended wire-mesh cage; pregnant females were moved into shoebox cages containing Enviro-dri and Bed-O'Cobs combination bedding (no later than three days prior to their expected delivery date until remainder of the study)
- Diet: PMI Certified Rodent Chow #5002 (PMI Nutritional International, St. Luis, MO), ad libitum
- Watert: Municipal water, further purified by reverse osmosis, ad libitum
- Acclimation period: 5 days

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

IN-LIFE DATES: From: 01 October 2001 to: 11 April 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared daily by adding an appropriate amount of test substance to a measured amount of vehicle (dried and de-acidified) under nitrogen atmosphere. The dosing formulations were maintained under nitrogen atmosphere until the time of dosing.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was dried and de-acidified to remove residual water before use as test substance hydrolyses in contact with moisture.
- Lot/batch no.: 070K0127
- Purity: 100%
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of copulation occurred or up to 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: Until evidence of copulation occurred or up to 2 weeks
- Proof of pregnancy:vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after unsuccessful attempt: no (non-pregnant females were sacrificed 21 days post mating)
- After successful mating each pregnant female was caged in shoebox cages containing Enviro-dri and Bed-O'Cobs combination bedding (no later than three days prior to their expected delivery date until remainder of the study)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test substance in the vehicle were verified by gas chromatopgraphy with a flame ionisation detection (FID). Concentration verification was conducted on a weekly basis.
Duration of treatment / exposure:
Males: 28 days (beginning 2 weeks prior to mating)
Toxicity phase female: 29 days (beginning 2 week prior to mating)
Reproductive phase females: 39-44 days (2 weeks prior to mating, throughout mating and pregnancy until day 3 postpartum)
Frequency of treatment:
Daily (7 days/week)
Details on study schedule:
Not applicable to this screening study .
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and 20 females (10 females in reproductive group and 10 females in toxicity group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 7 day oral range-finding study

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for morbidity, moribundity and mortality. General clinical observations were made at least once per day, approximately one hour after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first dosing and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly beginning approximately one week prior to test substance administration, on the first day of dosing and just prior to scheduled necropsy.

FOOD CONSUMPTION:
Individual food consumption was recorded weekly for female animals in the toxicity group for four weeks. Individual food consumption for the male animals and the reproductive group females were recorded during the first two weeks of treatment. Food consumption was not measured during the cohabitation period. Food consumption was measured for the reproductive group females throughout gestation until terminal sacrifice.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes, ketamine HCl/Xylazine
- Animals fasted: Yes, overnight
- How many animals: All male and toxicity group females
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Animals fasted: Yes
- How many animals: All male and toxicity group females
- Parameters checked in table 1 were examined.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB): Yes
- Time schedule for examinations: Prior to the start of dosing and during the fourth week of the treatment. The assessments were conducted following the daily dose administration.
- Dose groups that were examined: All male and toxicity group females.
- Battery of functions tested: cageside observations, hand-held observations, open field observations, categorical observations, measurements/counts, motor activity.
Oestrous cyclicity (parental animals):
Apparently not investigated.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testes weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No, all animals sacrificed on day 4 postpartum.

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, runts, 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 not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after 28 days of dosing.
- Maternal animals: All surviving animals on day 3 postpartum.
- Toxicity group females: All surviving animals after 29 days of dosing.

GROSS PATHOLOGY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- Postpartum day 4

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Not conducted.
Statistics:
All data analysis was carried out using SAS. In all comparisons, the family wise error rate was held at 5% (alpha=0.05). Prior to this analysis, an exploratory analysis was carried out on all variables tested. Bartlett's test was used to check for heterogeneity of variances and a Kolmogorov-smirnov test was used to test normality of the data. Parametric data was then tested using one-way Analysis of Variance (ANOVA) followed by Dunnett's Test (if significant). Non-parametric data were tested by Kruskal Wallis Test followed by Wilcoxon (if significant). For variables that had multiple measurements across time Repeated Measurement ANOVA was used to analyse the data. Categorical data were tested for equal proportions using the Fisher's Exact Test.

Statistically significant probabilities are reported at either the p≤0.05 or p≤0.01 levels.
Reproductive indices:
Fertility
Offspring viability indices:
Survival

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
nasal sounds/squeaky vocalisation in every dose group, but not in controle animals
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY:
One male in the 125 mg/kg bw/day dose group was found dead on study day 17 due to renal disease unrelated to treatment. There were two females in the reproductive groups that died due to dosing errors. All other animals survived to scheduled sacrifice. Clear perioral soiling was slightly more common in the high dose group. In the high dose groups there were increased nasal sounds, laboured breathing and/or soft squeaky vocalisation for one male and four toxicity and five reproductive group females. At 125 mg/kg bw/day, one toxicity group and one reproductive group female had increased nasal sounds. At 25 mg/kg bw/day, one toxicity group female exhibited soft squeaky vocalisation for three days of the study. The incidence of nasal sounds/squeaky vocalisation was noted in some animals and several times in others to a maximum of 18 days during the study. These findings were not observed in control rats.

BODY WEIGHT AND WEIGHT GAIN:
Mean body weights in males and females in the 25, 125 and 500 mg/kg bw/day groups were comparable to the control group values throughout the study; no statistically significant differences were noted. Reductions (not statistically significant) in weekly body weight gain were observed in the 25 and 125 mg/kg bw/day group males during week 4. These reductions were not considered to be test substance-related since the difference did not occur in a dose-dependent manner and no reductions were noted in female body weight gains during this time period.

FOOD CONSUMPTION:
There were no statistically significant differences in food consumption in any group throughout the study period.

HAEMATOLOGY:
There were no treatment-related effects on haematology parameters. However, in the toxicity group females there was a statistically-significant increase in platelet counts compared to controls. The counts for the treated groups were within published historical control ranges, whereas controls in the present study were somewhat below those ranges. No biological/toxicological significance is attributed to treatment.

CLINICAL CHEMISTRY: There were no treatment-related effects on clinical chemistry. However, in females, there was a statistically significant decrease in the chloride value (1.9%) in the high dose group, and a slight decrease (1.4%) in sodium in the middle and high dose groups. There was no dose-response. Sodium values for all female groups, including controls, were within or slightly below published historical control ranges. Chloride values for all groups were slightly above published control ranges. There were no associated clinical or morphological findings, so the findings were not thought to be biologically or toxicologically significant.

FOB:
Cage side observations: There were no treatment-related changes noted in unusual body movements, abnormal behaviour, posture or resistance to removal.
Handling observations: Palpebral closure, lacrimation, pupil size and reactivity, salivation, muscle tone, extensor thrust response and reactivity to handling were not affected by the treatment.
Open Field Observations: No differences were apparent between the control and treated groups in the open field observations.
Categorical Observations: No differences between control and treated groups when skin or hair coat, behaviour, respiration, muscle movements, eyes, urine or faeces, soiling, posture or general abnormalities were evaluated.
Measurements/counts: There was no effect on rectal temperature, hindlimb grip strength or landing foot splay assessments.
Motor activity: There were no effects on motor activity.

ORGAN WEIGHTS:
There were no treatment-related effects on organ weights. There was no dose-response associated with a statistically-significant small increase in relative prostate weight in the 25 mg/kg bw/day group.

GROSS PATHOLOGY:
There were no findings attributable to the test substance throughout the study period.

HISTOPATHOLOGY:
There were no treatment-related findings throughout the study period.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS):
No adverse effects were detected throughout the study period.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No adverse effects were detected throughout the study period.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant adverse effects on parent animals.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING):
No adverse findings were observed throughout the study.

CLINICAL SIGNS (OFFSPRING):
No adverse findings were observed throughout the study.

BODY WEIGHT (OFFSPRING):
No adverse findings were observed throughout the study.

GROSS PATHOLOGY (OFFSPRING):
No adverse findings were observed throughout the study.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on offspring were observed.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In an oral gavage study conducted similar to OECD 422 and to GLP the NOAEL for N-(3-(trimethoxysilyl)propyl)ethylenediamine relating to repeated dose (parental systemic) effects and to reproductive toxicity was at least 500 mg/kg bw/day, as no significant adverse effects were observed up to the highest dose of 500 mg/kg bw/day tested in rats.