Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD 440 (Uterotrophic Assay in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: USEPA OPPTS 890.1600 (Uterotrophic Assay)
Deviations:
no
GLP compliance:
no
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: XU-18838.00 - solid
Details on test material:
- Name of test material (as cited in study report): XU-18838.00
- Molecular formula (if other than submission substance): C37H44O2
- Molecular weight (if other than submission substance): 520.8
- Physical state: off white solid
- Lot/batch No.: 085385-090

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Portage, Michigan)
- Age at study initiation: Postnatal day 19
- Weight at study initiation:
- Fasting period before study: no
- Housing: group-housed (2-3 per cage) in solid bottom cages with paper pulp bedding
- Diet (e.g. ad libitum): Animals were provided Teklad Diet #2016 (Harlan Laboratories, Inc., Indianapolis,
Indiana), a low phytoestrogen rodent diet (total genistein equivalents < 325 μg/g diet) in
meal form ad libitum.
- Water (e.g. ad libitum): municipal water was provided ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

IN-LIFE DATES: From: December 6, 2011 To: December 9, 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was administered in corn oil, such that a dose volume of 4 ml/kg body
weight yielded the targeted dose. Dose volumes were adjusted using the most current
body weight. Dose suspensions (XU-18838.00) and dose solutions (XU-18844.00) were
prepared daily for the three days of dosing because the stability of XU-18838.00 and XU-
18844.00 in corn oil has not been determined.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil (Sigma-Aldrich, St. Louis, Missouri) was used as a vehicle for the test articles.
Vehicle information, supplied by the manufacturer, is included in the study file. Corn oil
was selected as the vehicle due to the solubility properties of the test materials and
because it has been shown in previous studies that corn oil does not exert any
estrogenicity or toxicity in immature female rats.
- Concentration in vehicle: 250, 500, or
1000 mg/kg bw/day
- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration verification of the first dose suspension (XU-18838.00) and dose
solution (XU-18844.00) preparations was established concurrent with the start of the
study using gas chromatography/mass spectrometry detection (GC/MS).
Analyses of all dosing suspensions of XU-18838.00 from the initial mix revealed mean
concentrations ranging from 89.1 to 96.5% of targeted concentrations.
Duration of treatment / exposure:
3 days
Frequency of treatment:
daily
Duration of test:
4 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250, 500 or 100 mkd
Basis:
nominal conc.
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
Six immature Crl:CD(SD) rats/group were administered daily doses of XU-18844.00 or
XU-18838.00 at dose levels of 0, 250, 500, or 1000 mg/kg bw/day in corn oil for three
days by gavage beginning on PND 19. A positive control group included six rats
exposed to 17α-ethynyl estradiol (EE) at 10 μg/kg bw/day.
On test
day 4 (PND 22), animals were examined for vaginal patency and body weights recorded.
Animals then were anesthetized, euthanized, and the uteri was excised and weighed
before and after blotting.
Statistics:
Body weights and gains were analyzed by a forced parametric test. For uterine weights
(blotted and wet), a Bartlett’s test was conducted to check for homogeneity of variance.
Based on the outcome of the Bartlett's test (alpha=0.01), the following
variables were transformed as indicated prior to the analysis.
• Log of Wet Uterine Weights for Vehicle Control vs. Positive Control
An analysis of covariance (ANCOVA) was performed with dose in the model and
terminal body weight as the covariate. The interaction term (Dose x Terminal body
weight) was not included in the model. Data from the following groups were statistically
compared:
• Vehicle (corn oil)-treated controls vs. XU-18838.00 animals
• Vehicle (corn oil)-treated controls vs. XU-18844.00 animals
• Vehicle (corn oil)-treated controls vs. EE-treated positive control group
If the dose effect was significant at alpha = 0.05, one-sided (upper) least square means
(Searle, 1971) with Dunnett’s correction were used to determine
differences (experiment wise alpha = 0.05) since the least square means were adjusted for
the covariate.

Results and discussion

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no indication of estrogenicity in immature female rats at doses up to 1000 mg/kg/day XU-18838.00

Applicant's summary and conclusion

Conclusions:
Based on these uterotrophic assay results, there was no indication of estrogenicity in immature
female rats at doses <1000 mg/kg/day XU-18838.00 when compared to the vehicle
controls.
Executive summary:

To provide information on the potential for estrogenicity following short term exposure to XU-18838.00 , groups of six immature female Crl:CD(SD) rats were administered vehicle control or XU-18838.00 by gavage at dose levels of 250, 500, or 1000 mg/kg bw/day beginning on postnatal day (PND) 19. A positive control group of six rats was exposed to 17α-ethynyl estradiol (EE) by gavage at 10 μg/kg bw/day. Rats in all groups were dosed once daily for three days. On test day 4 (PND 22), animals were examined for precocious vaginal opening, weighed, euthanized, and the uteri were excised and weighed before and after blotting. There was no animal mortality or treatment-related clinical observations in this study. Body weight/body weight gains were not affected at 1000 mg/kg bw/day XU-18838.00. There were no treatment-related increases in mean uterine weights (wet or blotted) in immature animals treated with 1000 mg/kg bw/day XU-18838.00. The positive control compound, EE (10 μg/kg bw/day), had no significant effects on body weights or body weight gains relative to the vehicle-treated control animals; however, immature rats treated with EE showed significant increases in mean wet and blotted uterine weights. None of the animals in this study had precocious vaginal opening. Vehicle-treated control uterine weights met the performance criteria outlined in the applicable test guidelines, indicating acceptable assay sensitivity.

Based on these uterotrophic assay results, there was no indication of estrogenicity in immature female rats at doses 1000 mg/kg/day XU-18838.00 when compared to the vehicle controls.