Registration Dossier

Administrative data

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 440, EPA 890.1600 and in accordance with the Principles of Good Laboratory Practices (GLP)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

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:
other: OECD Guideline No. 440
Deviations:
yes
Remarks:
Minor exceptions related to test material characterization being not conducted accoridng to GLP and dose analyses of the postive control were not conducted, however, these did not have any adverse impact on the study outcome
Qualifier:
according to
Guideline:
other: US EPA Guideline No. 890.1600
Deviations:
yes
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of method:
in vivo
Endpoint addressed:
other: uterotrophic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Unoxol™ Diol Diglycidyl Ether
- Molecular formula: C14H24O4
- Molecular weight: 256.3
- Analytical purity: Non-GLP characterization information lists the purity of the test material as 98.84% by gas chromatography
- Lot/batch No.: Lot # 201000773-15

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Portage, Michigan)
- Age at study initiation: Immature post natal day (PND) 11
- Housing: weanlings were group-housed (2-3 per cage)
- Diet (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
- Water: ad libitum
- Acclimation period: at least seven 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 cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material and EE were administered in a corn oil vehicle, 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 for the test material were prepared daily, just prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): recommended by variou sregulatory agencies
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Prior to the start of the study, dose preparation procedures were tested by verifying the concentration and homogeneity of UDDGE dose suspensions using gas chromatography/flame ionization detection (GC/FID). Due to the lack of stability data, the dose suspensions were prepared each day prior to use, and therefore, were not analyzed concurrently for concentration verification or homogeneity. The concentration and homogeneity of EE suspensions were not evaluated. Stability of the test material in the vehicle was not determined. Dose suspensions were prepared daily, just prior to dosing.
Duration of treatment / exposure:
from post natal day (PND) 19-21
Frequency of treatment:
daily from post natal day (PND) 19-21
Post exposure period:
not applicable
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
25 mg/kg body weight/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/kg body weight/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
500 mg/kg body weight/day
Basis:
nominal conc.
No. of animals per sex per dose:
six immature Crl:CD(SD) rats/group
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
Six immature Crl:CD(SD) rats/group were administered daily doses of UNOXOL™ diol diglycidyl ether (UDDGE) (25, 100, or 500 mg/kg bw/day) by gavage from postnatal day (PND) 19-21. A positive control group included 6 rats exposed to 17α-ethynyl estradiol (EE) at 10 μg/kg bw/day to verify assay sensitivity. In addition to a vehicle (corn oil) control group, an untreated control group was included in the study to confirm that the vehicle (corn oil) did not alter baseline uterine weights and thereby diminish the sensitivity of the assay to detect uterine
weight changes. On test day 4 (PND 22), animals were examined for vaginal patency and body weights were recorded. Animals were anesthetized, euthanized, and the uteri were excised and weighed before and after blotting to obtain imbibed and blotted uterine weights, respectively. Uterine weights from the UDDGE and EE groups were compared with uterine weights in the vehicle-treated control group to determine whether an estrogenic response was observed.

Examinations

Examinations:
Daily In-Life Observations - A cage-side examination was conducted at least twice daily. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could have been observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality and the availability of feed and water at least twice daily.

Body Weights/Body Weight Gains - All rats were weighed on the day of randomization (test day 0/PND 18), the three days of dosing (test days 1, 2 and 3/PND 19-21) and the day of necropsy (test day 4/PND 22).

Vaginal Opening - All test animals were examined for vaginal patency in the animal room prior to transport to the necropsy room on PND 22. Precocious vaginal opening is a potential indicator of an estrogenic effect.

Anatomic Pathology – Necropsy - Body Weights - Terminal, non-fasted, body weights were recorded for all animals on test day 4.

Euthanasia - Animals were anesthetized via CO2 inhalation from a compressed gas cylinder. After each animal was sufficiently anesthetized, it was euthanized by cervical dislocation immediately prior to tissue excision.

Uterine Weights - The uteri were excised and trimmed of excess fat and connective tissue. The ovaries were removed at the oviduct/uterine junction to avoid loss of luminal fluid from the uterine horn. The vagina was removed from the uterus just below the cervix so that the cervix remained with the uterine body and was included in the uterine weight. A wet uterine weight was collected to the nearest 0.1 mg with all luminal fluid retained in the uterus. If a leak of fluid was noted, this observation was recorded, and the weight was excluded from statistical analysis. The uteri were weighed in a tared weigh boat. After a wet weight was collected, each uterine horn was nicked three times, covered with a saline-moistened absorbent paper, and blotted by gently placing a 500 gram weight over the paper-covered uterus for approximately 3 seconds to remove luminal fluid, then re-weighed to the nearest 0.1 mg to determine the weight post-blotting. Uterine handling was sufficiently rapid to avoid desiccation of the tissues. Wet and blotted uterine weight-to-terminal body weight ratios were calculated. Uteri were preserved in 10% neutral phosphate-buffered formalin.

Histopathology - Histological examination of the uterine tissue was not conducted.
Positive control:
17α-ethynyl estradiol (CAS# 57-63-6), Sigma Aldrich lot# 090m1241v was used as a positive control. This positive control group included 6 rats exposed to 17α-ethynyl estradiol (EE) at 10 μg/kg bw/day to verify assay sensitivity.

Results and discussion

Details on results:
Analyses of all dosing suspensions from the initial mix revealed mean concentrations ranging from 86.7 to 88.9% of targeted concentrations. Analyses of aliquots for the low and high dose suspensions indicated that the test material was homogeneously distributed with a percent relative standard deviation (%RSD) of ≤ 4.8%.

Mortality - All animals survived until scheduled termination.

Clinical signs - There were no cage-side observations noted during the study period.

Body weight and gain - There were significant decreases in body weight gains (17-33%) on TD 1-4 in the vehicle-treated control group compared with the untreated control rats; however, the magnitude of this effect was small (2.2 g over the 4 day period) and there were no significant differences in body weights throughout the study. Body weight gains have not been significantly affected in other uterotrophic assays using corn oil vehicle; thus, this finding was considered incidental and deemed not to impact assay sensitivity. There were no significant differences in the body weights at any dose level of UDDGE when compared to the vehicle control group. On TD 4, body weight gains were decreased by 19% at 500 mg/kg bw/day; however, this result was considered equivocal due to the lack of statistical significance and small sample sizes. In the range-finding study, body weight gains were decreased 7% at this dose level. There were no effects on body weight gain at <100 mg/kg bw/day. Mean body weight and body weight gain for the EE positive control animals were similar to the vehicle control animals throughout the study.

Vagina opening - Precocious vaginal opening was not observed in any rats in the control or UDDGE treated groups on the day of termination (PND 22). Two (7592 and 7593) of the six rats treated with the positive control (EE) had precocious vaginal opening and this was considered consistent with an estrogenic response.

Anatomic pathology – Uterine weights - The comparison of the untreated and vehicle-treated control groups was designed to evaluate whether the vehicle was having an uterotrophic effect that might alter assay sensitivity. There was no significant difference in uterine weights, either pre- or post-blotting, between the untreated and vehicle control animals (ANCOVA, alpha=0.05). All uterine weights post-blotting in both the vehicle and untreated control groups were <40 mg, meeting the guideline criterion for acceptable control uterine weights. Thus, there was no effect of vehicle on baseline uterine weights and no effect on assay sensitivity. For UDDGE, there were no statistically identified treatment-related effects on absolute uterine weights, either before or after blotting (ANCOVA, alpha=0.05). Thus, there was no indication of estrogenicity at any dose level of UDDGE when compared to the vehicle controls.
The mean uterine weight after blotting for the vehicle control group was 0.04% of terminal body weight, which was deemed acceptable to yield sufficient assay sensitivity and was consistent with the requirement (<0.09%) of the test guideline (OPPTS 890.1600). The coefficients of variation (CVs) for the absolute uterine weights averaged over dose groups (i.e., vehicle and UDDGE-treated) were 10.8% for weights prior to blotting and 11.5% for weights after blotting. The mean absolute uterine weights (wet and blotted) were significantly increased for the EE positive control animals (856% and 477%, respectively) relative to the vehicle control group.

Gross pathology - In the 500 mg/kg bw/day group, four of six animals had hematocysts of the nonglandular mucosa of the stomach, an indication of irritation by UDDGE. There were no treatment-related gross observations at <100 mg/kg bw/day UDDGE

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, there was no indication of estrogenicity at any dose level of UNOXOL™ diol diglycidyl ether (UDDGE) when compared to the vehicle controls. The no-observed-effect level (NOEL) for estrogenic effects of UDDGE was 500 mg/kg bw/day, the highest dose level tested.
Executive summary:

Groups of 6 immature female Crl:CD(SD) rats were administered UNOXOL™ diol diglycidyl ether (UDDGE) by gavage at dose levels of 0 (vehicle control), 25, 100, or 500 mg/kg bw/day beginning on postnatal day (PND) 19. A positive control group of 6 rats was exposed to 17α-ethynyl estradiol (EE) daily by gavage at 10 μg/kg bw/day. An untreated control group also was included to confirm that the vehicle (corn oil) had no impact on uterine weights. 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 were no treatment-related effects on body weights at any dose of UDDGE, although body weight gains were decreased by 19% on test day 4 at 500 mg/kg bw/day. Body weight gains were not affected at <100 mg/kg bw/day UDDGE. There were no treatment-related increases in mean uterine weights (wet or blotted) in immature animals treated with < 500 mg/kg bw/day UDDGE. At necropsy, four of six high-dose UDDGE animals had hematocysts of the nonglandular stomach mucosa, which was attributed to irritation by the

test material. There were no gross lesions observed at < 100 mg/kg bw/day UDDGE. The corn oil vehicle alone did not alter uterine weights. The positive control compound, EE (10 μg/kg bw/day), had no effect on body weight and body weight gain relative to the vehicle-treated control animals; however, immature rats treated with 10 μg/kg bw/day EE showed significant increases in mean wet and blotted uterine weights. Two of the EE positive control animals exhibited precocious vaginal opening, an effect consistent with an estrogenic response.

Thus, there was no indication of estrogenicity at any dose level of UDDGE when compared to the vehicle controls. The no-observed-effect level (NOEL) for estrogenic effects of UDDGE was 500 mg/kg bw/day, the highest dose level tested.