Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA OPPTS 870.3550 (Reproduction/ Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
WIL Research Laboratories, LLC, Ashland, US
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): EEA-NH4
- Physical state: colorless, clear liquid
- Analytical purity: 30%
- Lot/batch No.: 100628
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: Males: 330 - 407 g; Females: 213 - 261 g
- Housing: individually in clean, stainless steel wire-mesh cages suspended above cage-board
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (e.g. ad libitum): municipal water
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 50% ± 20%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
sterile for injection
Details on exposure:
The vehicle was dispensed approximately weekly for administration to the control group and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group. The vehicle was mixed throughout preparation, sampling, and dose administration procedures and was stored refrigerated.
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH)
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For assessment of 10-day stability and aliquot resuspension homogeneity, aliquots approximately equivalent in volume to a daily dispensation aliquot, were transferred from the pre-initiation batch into the same type of storage container that was used for the daily aliquots for dosing. After refrigerated storage for 10-days, each aliquot was resuspended for a minimum of 30 minutes and samples were withdrawn from the top and bottom strata for assessment of 10-day stability and aliquot resuspension homogeneity. Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group). Samples collected from the first and fourth weeks of dose administration were analyzed to verify concentration; all other samples were stored frozen (approximately -20°C) for possible analysis. All analyses were conducted by the Analytical Chemistry Department at WIL Research using a validated high performance liquid chromatography tandem mass spectrometry method in the negative electrospray ionization mode.
Duration of treatment / exposure:
The males were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses.
The females were dosed during study day 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-51 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 40-52 doses.
Frequency of treatment:
once daily
Details on study schedule:
Males and females were approximately 11 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating and were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-51 doses; females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 40-52 doses.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 25 and 100 mg/kg bw/day (the dosing formulations were adjusted for purity (30%))
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Dosage levels were selected based on the results of the available 28-day repeated dose oral toxicity study.
The selected route of administration for this study was oral (gavage) because this is the potential route of exposure for humans. Historically, this route had been used extensively for studies of this nature.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked were moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: physical examinations were recorded weekly and on the day of necropsy, signs of toxicity were recorded daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded weekly throughout the study and on the day of euthanasia. Individual female body weights were recorded weekly until evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible, for dose calculation purposes only), 1, and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Estrous cyclicity (parental animals):
not determined as this study is a screening study
Sperm parameters (parental animals):
not determined as this study is a screening study
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION:
Intact offspring that died were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). The carcass of each pup was then discarded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following completion of the mating period.
- Maternal animals: All surviving animals on lactation day 4. Females that failed to deliver were euthanized on post-mating day 25 or post-cohabitation day 25. Females with total litter loss were euthanized on the same day of litter loss.

GROSS NECROPSY
- Gross necropsy consisted of examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY
The following tissues and organs were placed in 10% neutral-buffered formalin (except as noted):
Coagulating glands, Mammary gland (females only), Ovaries and oviducts (2), Pituitary gland, Prostate gland, Seminal vesicles (2), Testes with epididymides (Bouin’s solution) (2) and vasdeferens, Uterus with cervix and vagina, All gross lesions.

ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies:
Brain, Epididymides, Kidneys, Liver, Ovaries with oviducts, Pituitary gland, Testes.
Postmortem examinations (offspring):
SACRIFICE
On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital. Gross lesions and malformations (if any) observed at the detailed physical examinations on PND 4 were preserved in the appropriate fixative for possible future examination.
Statistics:
Each mean was presented with the standard deviation, standard error, and the number of animals used to calculate the mean. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999).
Histopathological findings were analyzed by the Fisher’s Exact Test (Steel, 1980). Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites and corpora lutea, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, and pre-coital intervals were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Reproductive indices:
Reproductive indices determined were:
Male and Female Mating Index (%), Male Fertility Index (%), Male Copulation Index (%), Female Fertility Index (%), Female Conception Index (%)
Offspring viability indices:
Litter parameters determined were:
Mean Live Litter Size, Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-selection) (% Per Litter), Postnatal Survival for All Other Intervals (% Per Litter)

Results and discussion

Results: P0 (first parental animals)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
5 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
effects on fertility
Effect level:
100 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
5 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion