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toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
other: OECD Guideline No. 440 (2007)
not specified
according to guideline
other: US EPA Guideline No. 890.1600 (2009)
not specified
GLP compliance:
Type of method:
in vivo

Test material

Test animals

Details on test animals or test system and environmental conditions:
Species and Sex
Immature female rats

Strain and Justification
Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.

Supplier and Location
Charles River Laboratories, Inc. (CRL) (Portage, Michigan)

Age at Study Start
Immature female rats, along with lactating dams, arrived in the lab on postnatal day (PND) 10 or 11. Pups were weaned on PND 18 and dosed starting on PND 19. The immature and ovariectomized animal models were shown to have comparable sensitivity and reproducibility during the uterotrophic validation program; however, the ovariectomized animal was reported to have increased specificity relative to the immature model (OPPTS 890.1600). The immature model responded to agents affecting the hypothalamic-pituitary-gonadal (HPG) axis rather than agents that act only at the estrogen receptor (OPPTS 890.1600). Despite these minor differences in specificity, the immature model was selected, because: 1) Via the oral route of exposure, the immature model was more sensitive to increases in uterine weight with estrogenic compounds than the ovariectomized model (Laws et al., 2000); 2) Some alternate activities (e.g., aromatizable androgens) that can result in a positive uterotrophic response in the immature model were a concern for potential endocrine activity; thus, using the immature model made the uterotrophic assay more inclusive; and 3) The immature model was more consistent with the animal use policies of this laboratory.

Physical and Acclimation
During the acclimation period, each animal was evaluated by a laboratory veterinarian to determine the general health status and acceptability for study purposes upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International). Prior to randomization, the animals were group-housed with a dam in solid bottom cages with 7089 Teklad Diamond Soft paper pulp bedding (low phytoestrogen content; Harlan Laboratories, Inc., Indianapolis, Indiana) in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Animals were acclimated to the laboratory for at least seven days prior to the start of the studies on PND 18 (March, 24, 2011, April 28, 2011, and April 29, 2011, respectively).

Rat offspring were weaned on PND 18. After assignment to study, weanlings were group-housed (2-3 per cage) in solid bottom cages with paper pulp bedding. Cages contained a feed crock and a pressure-activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room. Records of the environmental conditions are included in the study file.
Temperature: 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
Humidity: 40-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Randomization and Identification
Before administration of the test material, the animals were weaned on PND 18 and stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Study personnel controlled for litter of origin, to the extent possible. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders on PND 18 (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Feed and Water
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. Specifications for the diet can be found in the study file. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by the supplier to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. Results of the feed and water analyses indicated no contaminants that would interfere with the conduct of the study or interpretation of the results.

Laws, S. C., Carey, S. A., Ferrell, J. M., Bodman, G. J., and Cooper, R. L. (2000). Estrogenic activity of octylphenol, nonylphenol, bisphenol A and methoxychlor in rats. Toxicol. Sci. 54, 154-167.

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on exposure:
Route, Method of Administration, Frequency, Duration and Justification
Oral gavage was the preferred route of exposure according to OECD Guideline 440. While the USEPA has proposed subcutaneous dosing, oral dosing was an option in the Endocrine Disruptor Screening Program uterotrophic test guideline (OPPTS 890.1600). The relevant route for potential human exposure to the test substance is oral.

In replicate 1, animals (6 immature female rats/dose group) were treated once a day for three days by oral gavage with 0 (vehicle control, corn oil), 1, 10, or 100 mg/kg bw/day the test substance or EE (10 μg/kg bw/day) starting on PND 19. This dosing scenario was consistent with the test guidelines and the uterotrophic validation studies (e.g., Kanno et al., 2001). In replicates 2 and 3 the study design was the same except that the sample size was increased to 10 rats/dose to improve data precision.

Kanno, J., Onyon, L., Haseman, J., Fenner-Crisp, P., Ashby, J., and Owens, W. (2001). The OECD program to validate the rat uterotrophic bioassay to screen compounds fo in vivo estrogenic responses: Phase I. Environ. Health Perspect. 109, 785-794.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Concentration verification of all dose suspensions and homogeneity of the low- and high-dose suspensions were established for each replicate prior to starting the study using gas chromatography/flame ionization detection (GC/FID).

of all dosing suspensions from mixes prior to dosing revealed mean concentrations ranging from 95.2 to 99.8% of targeted concentrations. Analyses of aliquots for all dose suspensions indicated that the test material was homogeneously distributed with a percent relative standard deviation (%RSD) of ≤ 4.2%.
Duration of treatment / exposure:
Groups of six immature Crl:CD(SD) rats/group were administered daily doses of the test substance by gavage at dose levels of 0 (vehicle control, corn oil), 1, 10, or 100 mg/kg bw/day beginning on postnatal day (PND) 19 through PND 21.
Frequency of treatment:
Duration of test:
Postnatal day 19 through 21.
Doses / concentrations
Doses / Concentrations:
0, 1, 10 or 100 mg/kg
nominal conc.
No. of animals per sex per dose:
6 female rats/group
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of six immature Crl:CD(SD) rats/group were administered daily doses of the test substance by gavage at dose levels of 0 (vehicle control, corn oil), 1, 10, or 100 mg/kg bw/day beginning on postnatal day (PND) 19 through PND 21. A positive control group was included with six rats exposed to 17α-ethynyl estradiol (EE) by gavage at 10 μg/kg bw/day. Previous work has demonstrated that corn oil vehicle given at 4 ml/kg does not significantly alter uterine weight from baseline values (Marty and Brooks, 2010, 2011, In progress; Marty et al., 2011). On TD 4, PND 22 animals were examined for vaginal patency and body weights recorded. Animals were anesthetized, euthanized, and the uteri were excised and weighed before and after blotting. Following analysis of the data, two replicates uterotrophic assays were conducted using 10 animals/dose group to investigate the reproducibility of the uterotrophic response at the mid-dose that was seen in the original study. Gavage dosing for replicate 1 began on March 25, 2011 and the rats were necropsied on March, 29, 2011. Gavage dosing for replicate 2 began on April 29, 2011 and the rats were necropsied on May 02, 2011. Gavage dosing for replicate 3 began on April 30, 2011 and the rats were necropsied on May 03, 2011.

Dose Preparation
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 were prepared daily for three days of dosing since stability of the test substance in corn oil had not been determined.

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.

Prior to randomization, cage-side examinations were conducted on dams and their litters, at least twice daily. These examinations were conducted as described above.

Body Weights/Body Weight Gains
All rats were weighed on the day of randomization (TD -1/PND 18), the three days of dosing (TD 1, 2 and 3/PND 19-21) and the day of necropsy (TD 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 was a potential indicator of an estrogenic effect.

Anatomic Pathology
Body Weights
Terminal, non-fasted, body weights were recorded for all animals on TD 4.

On TD 4, animals were anesthetized via CO2 inhalation from a compressed gas cylinder. After the 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 avoiding loss of luminal
fluid from the uterine horn. The vagina was removed from the uterus just below the cervix so that the cervix remains with the uterine body and was included in the uterine weight. An imbibed (wet) uterine weight was collected to the nearest 0.0001 g with all fluid retained in the uterus. If a leak of fluid was noted, this observation was recorded and the weight 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 to remove luminal fluid, then re-weighed to the nearest 0.0001 g for a blotted weight. Uterine handling was sufficiently rapid to avoid desiccation of the tissues. Wet and blotted uterine weight-to-terminal body weight ratios were calculated and are maintained in the study file. Uteri were preserved in 10% neutral phosphate-buffered formalin for potential future evaluation.

Histological examination of the uterine tissue was not conducted.
Replicate 1 body weights and gains were analyzed by a parametric (Steele and Torrie, 1960) analysis of variance (ANOVA). Replicates 2 and 3 body weight and body weight gains were first evaluated by Bartlett’s test (alpha = 0.01; Winer 1971) for equality of variances. Based upon the outcome of Bartlett’s test, either a parametric (Steele and Torrie, 1960) or nonparameteic (Hollander and Wolfe, 1973) ANOVA was performed. For all 3 replicates, if the ANOVA was significant at alpha = 0.05, a Dunnett’s test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni’s correction (Miller, 1966) was performed. Both the Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control to keep the experiment-wise error rate at 0.05. Both were reported at the experiment wise alpha level. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969) and were only excluded from analysis for documented, scientifically sound reasons.

For uterine weights from all replicates (blotted and wet), a Bartlett’s test was conducted to check for homogeneity of variance. Based on the outcome of the Bartlett's test (Winer, 1971; alpha=0.01), the following variables were transformed as indicated prior to the analysis.
• Log of Wet Uterine Weight for Vehicle Control versus Positive Control
• Log of Blotted Uterine Weight for Vehicle Control versus Positive Control

An analysis of covariance (ANCOVA) was performed on uterine weights 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. test substance-treated groups
• Vehicle (corn oil)-treated controls vs. EE-treated positive control group

(continued below)

Results and discussion

Effect levels

Dose descriptor:
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no effects up to and including the highest dose level

Observed effects

All animals survived until scheduled termination.

Cage-Side Observations
There were no treatment-related cage-side observations noted during the study period (data maintained in study file).

Body Weights/Body Weight Gains
There were no significant treatment-related differences in body weights or body weight gains in any study replicate when comparing the test substance-treated rats to the vehicle control rats. There were no significant differences in the body weights or body weight gains between the replicate 1 EE positive control group and the vehicle control group. Similarly, there were no significant differences in body weights between the EE positive control groups and their respective vehicle control groups in replicates 2 and 3. However, mean body weight gain in EE treated rats in replicates 2 and 3 were significantly decreased by 12.2% over the entire study interval (TD 1-4) or 14.5% over TD 1-3, respectively. These decreases in body weight gain with EE treatment were not seen in the EE proficiency study conducted in this laboratory (Marty and Brooks, In progress); however, in the uterotrophic validation study (Kanno et al., 2001), laboratories reported terminal body weights that ranged from 5.7% lower than controls to 13% greater than controls with EE treatment, indicating some variability in the responsiveness of this measurement. A decrease in body weight gain would not be unexpected with EE treatment based on previous reports on the effects of estradiol on body weight (e.g., Biegel et al., 1998).

Vaginal Opening
Precocious vaginal opening was not observed in any rats in the control or treated groups on the day of termination (PND 22). One rat in replicate 1 (#2971) and one rat in replicate 3 (#3920) that were treated with the positive control (EE) had precocious vaginal opening as noted in the study file. The presenece of premature vaginal opening in an EE-treated rat is consistent with an estrogenic response.

Anatomic Pathology
Uterine Weights
All blotted uterine weights in the vehicle control group were <0.040 g, meeting the guideline criterion for acceptable control uterine weights. Furthermore, the mean blotted uterine weight for the vehicle control groups was 0.03-0.04% of terminal body weight, which was deemed acceptable to yield sufficient assay sensitivity and was consistent with the requirements (<0.09%) of the test guideline (OPPTS 890.1600). Wet and blotted uterine weights in the vehicle control group (0.0210 g and 0.0195 g, respectively) were slightly lower than the previous historical control data (HCD) range (0.0222-0.0279 g for wet weights and 0.0200-0.0271 g for blotted weights; Table 2), but control wet and blotted uterine weights in replicates 2 and 3 were within this laboratory’s HCD range. Lower uterine weights in control animals would not be expected to lower assay sensitivity (OECD, 2003). Together, these data suggest that there was sufficient assay sensitivity.

For the test substance, there were small (23-24%), but significant increases in wet and blotted uterine weights at 10 mg/kg bw/day when compared to control uterine weights in replicate 1 (Table 1). Both wet and blotted uterine weights at 10 mg/kg bw/day were within the HCD range for this laboratory (see HCD values in paragraph above). Uterine weights were not statistically different from the control group at 100 mg/kg bw/day, the highest dose level tested. Furthermore, the control animals had atypically low uterine weights (i.e., outside HCD range), which likely contributed to the statistical significance of the mid-dose value. Thus, the marginal increase in uterine weights at 10 mg/kg/day, the atypically low control values, and the absence of a dose-response relationship suggested that the findings at 10 mg/kg/bw/day could be a “false positive” result and supported the decision to repeat the uterotrophic assay with the test substance.

With the uterotrophic assay, previous studies have shown that “false positive” results can occur. The OECD’s “Detailed Background Review of the Uterotrophic Bioassay” (OECD, 2003) describes scenarios for “false positive” uterotrophic assays. “False positive” assays typically include weak uterotrophic responses (i.e., 15-40% increases in uterine weight relative to controls); in the current study, uterine weights were increased by 23-24%. These low magnitude differences can be seen within control data sets due to group-to-group variability in the mean uterine weights: “The difference between the highest and the lowest value data sets for the vehicle controls would yield an apparent difference of 15-40% over the low (control) values” (OECD, 2003). This statement of intergroup variability is supported by the HCD data in this laboratory, where blotted uterine weights in controls range from 0.0195 g (current study) to 0.0271 g (Table 1).

In replicates 2 and 3, there were no significant effects on wet or blotted uterine weights at any dose of the test substance (ANOVA, alpha = 0.05). Both replicates 2 and 3 used larger sample sizes (10/group) to improve data precision. Thus, the increases in wet and blotted uterine weight at 10 mg/kg bw/day in replicate 1 were not reproducible and were deemed incidental and unrelated to treatment.

The coefficients of variation (CVs) for the absolute uterine weights for all three replicates averaged over dose groups (i.e., vehicle and test substance-treated) ranged from 10.5-15.3% for wet weights and 10.7-15.4% for blotted weights. These results were similar to CVs reported in the OECD validation study (Kanno et al., 2001). In the OECD validation (Kanno et al., 2001), six animals per group was sufficient for detecting a 25-35% increase in uterine weight with reasonable power if the CV remained low (i.e., in the general range of 10.0-15.0%). Thus, these data also support that the assay was sufficiently sensitive to detect an estrogenic response if one was present.

The mean absolute uterine weights (wet and blotted) were significantly increased for all three replicates for the EE positive control animals; EE-induced increases in uterine weight ranged from 695-1086% for wet weights and 407-553% for blotted weights relative to the vehicle control group. Compared to our laboratory proficiency study at 10 μg/kg bw/day EE (Marty and Brooks, in progress), the EE-induced uterine weight increases for all three replicates in the current study were similar in magnitude for both imbibed weights (0.2100-0.2490 g compared with 0.2064 g in the proficiency study), and blotted weights (0.1234-0.1273 g compared with 0.1264 g in the proficiency study). This same relationship was seen when comparing these EE results with those reported in the validation study using oral exposures in immature rats (Kanno et al., 2001). In the Kanno publication, the wet and blotted uterine weights were increased by up to 754 and 400%, respectively at 10 μg/kg bw/day EE, which is similar to the EE-induced increases in uterine weights reported in the current study. Thus, these data, coupled with the baseline data in the vehicle control group, indicated that the assay was performing as expected and would detect treatmentrelated increases in uterine weight if these increases were present.

Any other information on results incl. tables

Table 1. Absolute Wet or Absolute Blotted Uterine Weights (g)

 Dose Level (mg/kg/day)  Replicate  0  1  10  100
 Absolute Wet Uterine Weight  1  0.0210  0.0217  0.0261*  0.0224
 Absolute Blotted Uterine Weight  1  0.0195  0.0191  0.0239*  0.0190
 Absolute Wet Uterine Weight  2  0.0248  0.0252  0.0249  0.0240
 Absolute Blotted Uterine Weight  2  0.0233  0.0235  0.0231  0.0220
 Absolute Wet Uterine Weight  3  0.0264  0.0271  0.0303  0.0265
 Absolute Blotted Uterine Weight  3  0.0246  0.0256  0.0287  0.0246

*Statistically Different from Control Mean by Dunnett’s Test, Alpha = 0.05.

Table 2. Historical Control Data for Absolute Wet or Absolute Imbibed Uterine Weights (g)

 Study #(year)  1(2010) 2(2011)  3(2011)  4(2011)  5(2012)   6(2012)  7(2012) 8(2011)   9(2012)
 Absolute Wet Uterine Weight  0.0256  0.025  0.024  0.0234  0.0271  0.0236  0.0222  0.0276  0.0279
 Absolute BlottedUterine Weight  0.0231  0.0231  0.0227  0.0209  0.0254  0.0209  0.0200  0.0248  0.0271

Bolded values represent the lowest and highest values

Applicant's summary and conclusion

Overall, under the conditions of this study, the test substance was judged to be negative for estrogenicity in immature female rats at doses ≤ 100 mg/kg bw/day, the highest dose level in the uterotrophic study.
Executive summary:

To provide information on the potential for estrogenicity following short term exposure, groups of six immature female Crl:CD(SD) rats were administered the test substance, by gavage at dose levels of 0 (vehicle control), 1, 10, or 100 mg/kg bw/day beginning on postnatal day (PND) 19. Dose levels were selected based on potential human exposures with up to a 400,000X factor to allow for a large margin of exposure. A positive control groups 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 TD 4, PND 22 animals were examined for precocious vaginal opening, weighed, euthanized, and the uteri were excised and weighed before and after blotting. Based on the outcome of the first study, two replicate studies were conducted using the same study design, but included ten animals/group to improve data precision. There were no animal mortalities or treatment-related cage-side observations in the study. There were no treatment-related effects on body weights or body weight gains at any dose of the test substance. In the first study, there was a significant increase in wet and blotted uterine weights at 10 mg/kg bw/day, but not at other dose levels. Due to the minimal increase in uterine weights, the atypically low control uterine weights, and the absence of a dose-response relationship, the assay was repeated twice to verify this result. In the second and third replicates, which included larger sample sizes, there were no significant increases in wet or blotted uterine weights at any dose level of the test substance; thus, the result from the first study was not reproducible and, therefore, deemed to be unrelated to treatment. Neither the control nor treated animals exhibited precocious vaginal opening in any study replicate. Immature rats treated with the positive control compound, EE (10 μg/kg bw/day), showed significant decreases in body weight gain in two of three replicates, as well as significant increases in mean uterine wet and blotted weights in all three replicates. One EE-treated animal in each of replicates 1 and 3 had vaginal opening at termination, an effect consistent with an estrogenic response. Uterine weights from vehicle-treated control animals met the performance criteria outlined in the applicable test guidelines, indicating acceptable assay sensitivity.

Overall, under the conditions of this study, the test substance was judged to be negative for estrogenicity in immature female rats at doses ≤ 100 mg/kg bw/day, the highest dose level in the uterotrophic study.