ToxSci Advance Access originally published online on January 8, 2008
Toxicological Sciences 2008 102(2):392-412; doi:10.1093/toxsci/kfn002
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Two-Generation Reproductive Toxicity Evaluation of Dietary 17β-Estradiol (E2; CAS No. 50-28-2) in CD-1 (Swiss) Mice
* Health Sciences Unit, RTI International, Research Triangle Park, North Carolina 27709
Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina 27709
GE Plastics, Pittsfield, Massachusetts 01201
Toxicology/Regulatory Services, Inc., Charlottesville, Virginia 22911
¶ Bayer MaterialScience, Pittsburgh, Pennsylvania 15205
|| Bayer Healthcare AG, Wuppertal, Germany D-42096
||| The Dow Chemical Co., Midland, Michigan 48674
|||| American Chemistry Council, Arlington, Virginia
1 To whom correspondence should be addressed at RTI International, 3040 Cornwallis Road, P.O. Box 12194, Hermann Laboratory Bldg., Research Triangle Park, NC 27709-2194. Fax: (919) 541-5956. E-mail: rwt{at}rti.org.
Received September 17, 2007; accepted December 27, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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No information exists on reproductive/developmental effects in mice exposed to dietary 17β-estradiol (E2) over multiple generations. Therefore, under OECD Test Guideline 416 with enhancements, CD-1 mice (F0 generation, 25 mice/sex/group) were exposed to dietary E2 at 0, 0.001, 0.005, 0.05, 0.15, or 0.5 ppm (
0, 0.2, 1, 10, 30, or 100 µg E2/kg body weight/day) for 8 weeks prebreed, 2 weeks mating, 3 weeks gestation, and 3 weeks lactation. At weaning, selected F1 offspring (F1 parents; 25/sex/group) and extra retained F1 males (one per litter) were exposed to the same dietary concentrations and durations as the F0 generation; study termination occurred at F2 weaning; F1/F2 weanlings (up to three per sex per litter) were necropsied with organs weighed. At 0.5 ppm, effects were increased F1/F2 perinatal loss, prolonged F0/F1 gestational length, reduced numbers of F2 (but not F1) litters/group, reduced F1/F2 litter sizes, accelerated vaginal patency (VP) and delayed preputial separation (PPS), increased uterus + cervix + vagina weights (UCVW) in F0/F1 adults and F1/F2 weanlings, and decreased testes and epididymides weights (TEW) in F1/F2 weanlings. At 0.15 ppm, effects were increased UCVW in F0/F1 adults and F1/F2 weanlings, accelerated VP, delayed PPS, and reduced TEW in F1/F2 weanlings. At 0.05 ppm, UCVW were increased in F1/F2 weanlings, and PPS was delayed only in extra retained F1 males. There were no biologically significant or treatment-related effects on F0/F1 parental body weights, feed consumption, or clinical observations, or on F0/F1 estrous cyclicity, F0/F1 andrology, or F1/F2 anogenital distance at any dose. The no observable effect level was 0.005 ppm E2 (1 µg/kg/day). Therefore, the mouse model is sensitive to E2 by oral administration, with effects on reproductive development at doses of 10- 100 µg/kg/day. Key Words: 17β-estradiol; CD-1 mice; 2-generation reproductive toxicity study; OECD 416; acquisition of puberty; estrous cyclicity; andrology.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Basic research investigating endocrine-active chemicals (predominantly xenoestrogens), using new and unique study designs, parameters, and routes of exposure, is being performed in mice. The results from these studies have caused scientific, regulatory, and societal interest and concern. Proper evaluation of effects from endocrine-active substances for risk assessment requires a validated and robust study design with multiple dose groups, a robust number of animals/group, administration of test material by a relevant route of administration, with exposures during sensitive life stages over generations, and evaluating a number of specific and apical systemic, reproductive, and developmental parameters. Therefore, this study was designed to meet or exceed OECD Testing Guideline 416 (OECD, 2001
) under OECD Good Laboratory Practice Principles (OECD, 1998, 2002), to evaluate the systemic, reproductive, and/or developmental effects of exposure to an endogenous estrogen (E2) administered in the feed to CD-1 (Swiss) mice over two offspring generations.
A one-generation study of dietary E2 in CD-1 (Swiss) mice, previously performed in this laboratory (Tyl et al., in press), provided initial information on the appropriate dose range of interest (doses at which viable offspring were produced), to provide data on the appropriate and sensitive parental and offspring parameters. That study employed E2 dietary concentrations of 0, 0.005, 0.05, 0.5, 2.5, 5, 10, and 50 ppm, equivalent to 0, 1, 10, 100, 500, 1000, 2000, and 10,000 µg/kg/day. Dietary E2 concentrations of 2.5 ppm (intake of 500 µg/kg/day) and higher were incompatible with successful pregnancies in the CD-1 mouse. At 0.5 ppm, F0 females exhibited increased uterine weights; for F1 offspring, the stillbirth index was increased, the live birth index was reduced, and litter size was reduced; F1 females exhibited swollen vaginal area detected as a clinical observation, as well as increased uterine weights at weaning and pubertal necropsies; acquisition of vaginal patency (VP) was accelerated (by 8.1 days), and acquisition of preputial separation (PPS) was delayed (by 8.2 days); at pubertal necropsy, 20% of F1 males had uni- or bilateral undescended testes (Tyl et al., in press). Therefore, the current two-generation reproductive toxicity study design (OECD, 2001) expanded the dietary E2 range used in the one-generation study which resulted in offspring at dietary concentrations of 
0.5 ppm, to E2 dietary concentrations of 0, 0.001, 0.005, 0.05, 0.15, and 0.5 ppm, equivalent to 0, 0.2, 1.0, 10, 30, and 100 µg/kg body weight/day.
The objectives of this study were to: (1) evaluate the potential of E2, administered in the feed to CD-1 (Swiss) mice, to produce possible alterations in parental fertility, maternal pregnancy and lactation, and systemic and reproductive growth and development of the offspring for two offspring generations, 1 litter/generation (i.e., to establish the responsiveness of this mouse strain to dietary E2); (2) develop a baseline (historical positive and negative databases) by which to characterize the parameters in CD-1 mice and to help judge xenoestrogen effects in mice, including the use of extra retained F1 males (one per litter) to address biological variability; and (3) identify sensitive, relevant, and appropriate endpoints (and any inappropriate and unnecessary endpoints) for subsequent testing of xenoestrogens in the CD-1 mouse model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Test Substance and Dose Formulations
The test substance, E2 (CAS No. 50-28-2, Batch No. 021K1267, Supplier Product No. E8875), was received from Sigma-Aldrich (St Louis, MO); purity 99.0% (with purity confirmed at Research Triangle Institute [RTI], pre- and post-study). The vehicle was Purina Certified Ground Rodent Diet, No. 5002 (PMI Feeds, Inc., St Louis, MO). The diets were formulated using a stock solution of E2 in acetone (0.0200 mg E2/ml acetone), diluted with acetone to the appropriate concentrations, and then mixed with feed to make the premixes. The premixes were air dried under a hood in stainless steel pans using stainless steel "rakes" to ensure dryness and to break up any clumps. The premixes were then mixed with undosed feed to produce the specified dietary concentrations in the required quantities. The vehicle control group feed also had an acetone premix. The homogeneity and stability of the dosed feed preparations were assessed at concentrations of 0.001, 0.005, and 50 ppm. Homogeneity was confirmed and stability confirmed at room temperature for at least 9 days and frozen (
–20°C) up to 61 days. The dosed feed was formulated, based on the stability data, at least monthly and stored frozen. The feed in the feed jars was changed at least every 7 days. Analyses of the formulated diets were conducted by liquid chromatography/mass spectrometry on all dietary concentrations from the first four formulation dates and for every fourth formulation date thereafter. Standards for acceptable accuracy of mixing were the mean of the analyzed samples were within ± 20% of nominal, and the % RSD (relative standard deviation) for triplicate analyses did not exceed 15%. Only formulations satisfying the acceptability standards were administered to the animals. The analytical range for all dietary concentrations used, for all formulation dates scheduled for analyses, was 89.1–120% of nominal (with % RSDs from 0.6 to 13%), with an estimated limit of detection (LOD) of 0.00023 ppm. The control diets did not contain detectable E2 (< LOD).
Animals and Husbandry
One hundred sixty-five (165) virgin female mice and the same number of virgin male mice (Caesarean-originated VAF Crl:CD-1 (ICR)BR outbred albino; known as the Charles River CD-1 mouse) were received from Charles River Breeding Laboratories, Inc. (Raleigh, NC). There were 300 animals (150 males and 150 females) assigned to the study at the initiation of the treatment period, 25/sex/group in six groups. Mouse antibody screening Level 1 (BioReliance Corp., Rockville, MD) and clinical observations confirmed the animals were suitable for use on this study. The males and females were 5 weeks of age (35 days old) on the scheduled animal receipt date (weights upon arrival: 22–24 g for males, 19–21 g for females). Exposures began when the F0 males and females were 6 weeks old (males 22.17–31.61 g, females 20.58–26.12 g).
F0 animals, prior to initiation of exposure, and F1 offspring selected to be parents of the F2 generation or to be retained as extra F1 males, were identified by uniquely numbered ear tags (National Band and Tag Co., Newport, KY). F1 pups, prior to selection at weaning, were not uniquely identified, except for F1 females selected for parents of the next (F2) generation in each litter, because examination for VP began on postnatal day (pnd) 18 prior to weaning.
All necropsies or humane termination of adult moribund animals were performed after terminal anesthesia with CO2. Culled F1 and F2 offspring were sacrificed on pnd 4 by decapitation. Any F1 or F2 offspring that became moribund during lactation were terminated by decapitation (pnd 0–4) or by CO2 asphyxiation (pnd 4–21).
The animals were individually housed upon arrival, during the acclimation period, and upon the initiation of the treatment periods; by mating pairs (one male:one female) during the mating period; individually as plug-positive females from gestational day (gd) 0 until birth of their litters; individual dams with their litters throughout the lactational period until weaning (pnd 21); and individually as selected F1 postweanlings, in solid-bottom polypropylene cages (5'' x 11.5'' x 7'') with stainless steel wire bar lids (Laboratory Products, Rochelle Park, NJ). Sani-Chip was used as cage bedding (P. J. Murphy Forest Products, Inc., Montville, NJ). Males and females were housed in the same animal room to prevent the females from becoming anestrous (Whitten, 1956).
Temperature and relative humidity (RH) in the ARF animal rooms were continuously monitored, controlled, and recorded using an automatic system (Siebe Environmental Controls/Barber-Colman Company, Loves Park, IL). The target environmental ranges were 66–77°F (19–25°C) for temperature and 30–70% RH, with a 12-h light cycle per day (NRC, 1996). There were no excursions in RH or temperature that affected the design, conduct, results, or conclusions of this study. At all times, the animals were handled, cared for, and used in compliance with the NRC Guide (NRC, 1996) and under an RTI Institutional Animal Care and Use Committee protocol.
The formulated diets were available ad libitum in glass mouse feed jars with stainless steel snap-on or screw-on caps and stainless steel wire-mesh inserts. Contaminant and nutrient levels of the feed were provided by the supplier. Contaminant levels were below certified levels, and nutrient levels were at or slightly above certified levels. In addition, each of the four lots of feed used was analyzed by the supplier for the concentration of the phytoestrogens genistein (mean ± SEM: 149 ± 36 ppm), daidzein (142 ± 19 ppm), and glycitein (41 ± 4 ppm). The feed was stored at 60–70°F, and the period of use did not exceed 6 months from the milling date.
Water was available ad libitum in glass water bottles with Teflon-lined, plastic screw caps and stainless steel sipper tubes. Contaminant levels of the water were measured at regular intervals (per EPA specifications). Contaminant levels were below the maximal levels established for potable water.
Study Design
A graphic representation of the study design is presented in Figure 1. The study began with 25 males/group and 25 females/group (the F0 generation) to yield at least 20 pregnant females/group at or near term, and six groups. Animals were assigned to groups by means of randomization stratified by body weight, such that the mean body weights by sex of all groups were homogeneous at treatment initiation.
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The target dietary concentrations, from 0, 0.001, 0.005, 0.05, 0.15, and 0.5 ppm (equivalent to
0, 0.2, 1.0, 10, 30, and 100 µg/kg body weight/day, respectively), provided E2 intakes from 0.20 (male) and 0.18 (female) µg/kg/day at 1 ppb to 100 (male) and 90 (female) µg/kg/day at 0.5 ppm, to encompass the range for effect and no effect levels of E2 in parental and offspring mice (see Table 1 for study organization, target dietary concentrations, target calculated intakes, and actual intakes for male and female parental F0 mice).
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Experimental Evaluations
Parental Animals (F0 and F1)
Observations for mortality were made twice daily (A.M. and P.M.). Clinical examinations were conducted and recorded at least once daily throughout the course of the study. The body weights of the F0 and F1 parental male mice were recorded initially and weekly through mating. The body weights of the F0 and F1 parental female mice were recorded in the same manner until confirmation of mating. During gestation, females were weighed on gd 0, 7, 14, and 17. Dams producing litters were weighed on pnd 0, 4, 7, 14, and 21. Body weight gains were computed for all intervals for all periods.
Feed consumption measurements were recorded for all F0 and F1 parental animals every 7 days, each time the feed was changed, throughout the prebreed exposure period. Feed consumption collection periods corresponded with the collection of the animals’ weekly body weight data and were employed to calculate intake on a µg or mg E2/kg of body weight ratio. During pregnancy, feed consumption was recorded for gd 0–7, 7–14, and 14–17. During lactation, maternal feed consumption was measured for pnd 0–4, 4–7, 7–14, and 14–21, although maternal feed consumption after pnd 14 was confounded by the contribution from the pups, because pups were self-feeding by this time. Feed was changed at least weekly throughout the study. Feed consumption was not measured during the period of cohabitation because two adult animals (one male and one female) were in the same cage, but consumption was visually monitored and the feed was added/changed as necessary.
Estrous cyclicity and normality were evaluated by daily vaginal smears for all F0 and F1 parental females during the last 3 weeks of the prebreed exposure period.
Animals of the F0 generation were 6 weeks of age at the commencement of treatment. They were administered dosed feed at their respective formulations for 8 weeks prior to mating (i.e., until they were 14 weeks of age). The animals were then mated on the basis of one male to one female, selected randomly within each dose group for a period of 14 days. Females were examined daily during the cohabitation period for the presence of copulation plug in the vaginal tract. The observation of a copulation plug in the vaginal tract was considered evidence of successful mating; the date a vaginal plug was observed was designated gd 0. Once the vaginal plug was observed, the male and female from that mating pair were individually housed.
Selected animals of the F1 generation were administered dosed feed at their respective formulations ad libitum 7 days/week for at least 8 weeks before mating. Developmental landmarks were evaluated as described below. The F1 animals were 11 to 13 weeks of age at the initiation of the mating period. The F1 animals were mated as described above for the F0 animals, including, but not limited to, pairing one male:one female and cohabitation for 14 days or until a vaginal copulation plug was observed, whichever came first (with no change in pairing). Male and female siblings were not used as breeding pairs.
Beginning on gd 17, each plug-positive F0 or F1 female was observed twice daily (A.M. and P.M., approximately 9:00 A.M. and 3:00 P.M.) for evidence of delivery. The day of birth was designated as pnd 0. The dams were allowed to rear their young to pnd 21. If a dam with a litter died or was euthanized moribund, her litter was euthanized (by methods appropriate for their age) at her termination. Any female that did not show evidence of successful mating after 14 days of cohabitation continued on the original weekly weighing schedule and was monitored for evidence of pregnancy and delivery. Maternal and offspring information during lactation was collected for females that delivered a live litter, regardless of whether they had a confirmed gd 0.
Progeny (F1 and F2)
All live pups were counted, sexed, and examined as soon as possible on the day of birth (designated pnd 0) to determine the number of viable and stillborn members of each litter. Thereafter, litters were evaluated for survival on pnd 4, 7, and 14 and at weaning on pnd 21. All live F1 and F2 pups were individually counted, sexed, weighed, and examined grossly at pnd 0, 4, 7, 14, and 21. The body weights and sexes were recorded on an individual basis, but the pups were not uniquely identified at this stage. All pups were examined for physical abnormalities at birth and throughout the prewean and postwean periods. Any pups that were stillborn, died, or were euthanized moribund during lactation were necropsied, when possible, to investigate the cause of death and for any malformations or variations, especially of the reproductive system.
At birth (pnd 0), all live F1 and F2 offspring had Anogenital distance (AGD) measured by an eyepiece diopter (accurate to 0.01 mm) attached to a stereomicroscope and a microscope stage grid, and individual body weights were recorded. At pnd 21, all necropsied F1 and F2 offspring (up to three per sex per litter) also had AGD measured by the same method, and individual body weights were recorded.
On pnd 4, the size of each F1 and F2 litter was adjusted by eliminating extra pups by random selection within sex from litters with more than 10 pups to yield 10 pups, with as nearly as possible five males and five females per litter. Natural litters with 10 or fewer pups were not standardized. All culled pups were sacrificed by decapitation and examined for visceral alterations, especially those of the reproductive system. Pups with gross lesions were retained in buffered neutral 10% formalin (BNF); normal pups were discarded.
Beginning on pnd 18, each F1 female pup selected for the parental generation (by randomization procedures stratified by body weight) to produce F2 litters (see below) was observed for VP, because exposure to dietary E2 at 0.5 ppm resulted in accelerated VP acquisition beginning prior to weaning in a previous one-generation study (Tyl et al., in press). Observations for VP continued until every selected female had this response. The date, age, and body weight were recorded for each female on the day of acquisition, and body weight was recorded on pnd 21. The F1 male pups were selected at weaning on pnd 21 (by randomization procedures stratified by body weight) to be parents of the F2 generation. Then, one additional F1 male pup/litter was randomly selected to be retained, with exposures continuing for 3 months, and necropsied when the F1 parental males were necropsied (at approximately 13 weeks of age). A total of 25 F1 offspring/sex/group were selected as parents. During the prebreed exposure period, each F1 male was observed for cleavage of the balanopreputial gland (PPS), beginning on pnd 22. Separation was recorded daily for each animal until all males had this response. The date, age, and body weight was recorded for each male on the day of acquisition, and body weight was recorded on pnd 30.
In addition at weaning (pnd 21), up to three F1 and F2 pups/sex/litter were randomly selected and subjected to a gross necropsy, with organs weighed and retained in fixative. Organs were not weighed for any pups that died. The status of the F1 weanling testes was evaluated at necropsy after euthanasia by abdominal incision and localization of the testes low in the abdominal cavity at the inguinal ring (undescended) or in the scrotal sacs (descended).
Gross Necropsy and Organ Weights
Euthanasia was by CO2 asphyxiation. Sacrifice of the parental F0 and F1 males occurred after the completion of the gestation period of their F1 and F2 litters, respectively. Sacrifice of the parental F0 and F1 females occurred after the weaning of their F1 and F2 litters, respectively. In addition, the retained F1 males (one per litter) were sacrificed at the same time as the F1 parental males. After euthanasia of the F0 and F1 females, prior to necropsy, a vaginal smear was taken from each female, stained (with Toluidine blue), and examined microscopically to determine the stage of estrus at demise. All F0 and F1 parental animals in all groups, as well as the retained F1 males (one per litter), were subjected to a complete gross necropsy. In addition, the uterus of each parental F0 and F1 female was examined, and the number of nidation scars (implantation sites) was documented. Selected tissues were weighed and retained as outlined below. All gross lesions and the tissues listed below were retained in BNF, except for one testis per male which was fixed in Bouin's fixative for 24 hours and then retained in 70% ethanol, and the other testis/male which was frozen at –20°C.
The organs weighed and retained from F0 and F1 parental animals and retained F1 adult males are presented in Table 2. Organ weights are reported as absolute and relative to terminal body weight.
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A complete gross necropsy and retention of tissues was conducted for any parental animal that appeared moribund (and was humanely sacrificed) or that died during the study. Organ weights were not recorded, and histopathology was not performed on tissues from animals found dead on study.
Histopathology.
Embedment of fixed tissues for examination was in paraffin. Tissue slides were stained with hematoxylin and eosin, except for male testes slides, which were stained with Periodic Acid Schiff and hematoxylin. Full histopathology was performed on all of the retained organs listed in Table 2 from high-dose and control F0/F1 parental animals (not from retained F1 males).
In addition, reproductive organs of the animals from low and all intermediate doses (0.001 through 0.15 ppm) suspected of reduced fertility (e.g., those that failed to mate, conceive, sire, or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility, or morphology were affected) were subjected to histopathological evaluation.
Testis.
Testicular histopathological examination was conducted to identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells, or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis included the caput, corpus, and cauda (Russell et al., 1990).
Ovary.
Ovaries from 10 randomly selected F0 and F1 adult females from the control group and the 10 females from the high-dose group used for histopathology were examined for enumeration of primordial follicles. Five ovarian sections were taken at least 100 µm apart from the inner third of each ovary. Examination included enumeration of the total number of primordial follicles from these 10 sections per female for comparison with numbers in control ovaries. Examination also confirmed the presence or absence of small, growing, and antral follicles and corpora lutea in comparison with control ovaries (Heindel et al., 1989).
Mammary glands.
The most anterior (axillary) pair of mammary glands in F0 and F1 adult females was retained in BNF (adhered to file cards to keep the preparation flat). The retained mammary glands from 10 randomly selected F1 adult females/group in all E2-exposed groups and mammary glands from all F1 control females were subjected to histopathologic examination. The mammary glands from the F0 adult females were not evaluated histopathologically.
Andrology.
At the time of F0 and F1 parental male sacrifice, one testis from each male was frozen at –20°C for subsequent enumeration of testicular homogenization-resistant spermatid heads and calculation of daily sperm production (DSP) and efficiency of DSP (LeBlond and Clermont, 1952; Robb et al., 1978). In addition, one cauda epididymis was immediately removed, weighed, and seminal fluid from the cauda assessed for sperm number, motility, and morphology. Sperm motility (motile and progressively motile) was assessed immediately after necropsy in all F0 and F1 adult males. Caudal sperm number (from homogenized cauda frozen at the time of necropsy) was recorded and morphology (at least 500 sperm per male, if possible) from sperm slides made at necropsy was evaluated. Testicular spermatid head counts, DSP, and efficiency of DSP were evaluated from frozen testes from high-dose and control F0 and F1 parental males. No treatment-related effects were observed in the high-dose samples, so these parameters were not examined in other groups. Motility (and progressive motility) and number of cauda epididymal sperm and testicular homogenization-resistant spermatid head counts were assessed in F0 and F1 parental males using an HTM-IVOS Automated Sperm Analysis System (Version 12.1 c, Hamilton-Thorne Research, Beverly, MA). Sperm morphology was evaluated manually. No andrology was performed on retained F1 males.
Nonpregnant females.
The fixed (BNF) uterus from any parental F0/F1 female failing to produce a litter was stained with potassium ferricyanide for confirmation of pregnancy status. This staining procedure did not interfere with subsequent histopathologic evaluation.
Gross Necropsy and Organ Weights (F1 and F2 Pups)
Up to three F1 and F2 pups/sex/litter were randomly selected for euthanasia by CO2 asphyxiation for gross necropsy examination on pnd 21, with the organs presented in Table 2 weighed and retained. Organ weights were reported as absolute and relative to terminal body weight. Gross lesions also were retained in fixative (BNF or Bouin's for testes). The retained organs from the F1/F2 weanlings were not subjected to histopathologic examination.
Statistical Analyses
The unit of comparison was the F0 and F1 male, the F0 and F1 female, the pregnant F0 and F1 female, the retained F1 male, or the F1 and F2 litter (from birth to weaning). All data collected from F1/F2 pups from birth to weaning were analyzed as litter data. Once the F1 pups were weaned, eartagged, in their own cages and directly exposed to the dosed feed, they were treated as individual, independent units. Quantitative continuous data (e.g., parental and pup body weights, organ weights, feed consumption, day of acquisition of VP and PPS, etc.) were compared among the five treatment groups and the vehicle control group using either parametric analysis of variance (ANOVA) under the standard assumptions or robust regression methods (Huber, 1967; Royall, 1986; Zeger and Liang, 1986), which do not assume homogeneity of variance or normality. The homogeneity of variance assumption was examined via Levene's Test (Levene, 1960). If Levene's Test indicated lack of homogeneity of variance (p < 0.05), robust regression methods were used to test all treatment effects. These methods use variance estimators that make no assumptions regarding homogeneity of variance or normality of the data, and they were used to test for overall treatment group differences (via Wald chi-square test) and individual t-tests for exposed versus control group comparisons when the overall treatment effect was significant (REGRESS procedure of SUDAAN Release 8.0; Research Triangle Institute, 2001).
If Levene's Test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied for comparing the treatment groups. The GLM procedure in SAS 8.0 was used to evaluate the overall effect of treatment and, when a significant treatment effect was present, to compare data from each exposed group with the control data via Dunnett's test (Dunnett, 1955, 1964). For the litter-derived percentage data (e.g., periodic pup survival indices), the ANOVA was weighted according to litter size. A one-tailed test (i.e., Dunnett's test) was used for all pairwise comparisons to the vehicle control group, except that a two-tailed test was used for parental and pup body weight and organ weight parameters, feed consumption, percent males per litter, AGD, VP, and PPS (GLM procedure of SAS Release 8.0; SAS Institute, Inc., 2000).
Frequency data, such as reproductive indices (e.g., mating and fertility indices), were not transformed. All indices were analyzed by chi-square test for Independence for differences among treatment groups (Snedecor and Cochran, 1967). When Chi-Square revealed significant (p < 0.05) differences among groups, then a Fisher's Exact Probability Test, with appropriate adjustments for multiple comparisons, was used for pairwise comparisons between each treatment group and the control group (SAS Institute, Inc., 2000).
Acquisition of developmental landmarks (e.g., VP and PPS), as well as AGD, was also analyzed by analysis of covariance (ANCOVA; in addition to ANOVA analysis or robust regression analysis) using body weight at acquisition or measurement as the covariate. In addition, age at acquisition of puberty (VP, PPS) was also analyzed, with the individual body weights on pnd 21 for females and on pnd 30 for males as the covariate.
Correlated data (e.g., body and organ weights at necropsy of pups on pnd 21, with more than one pup per sex per litter) were analyzed using general estimating equations (GEE) techniques (Zeger and Liang, 1986) in the SUDAAN software package (Research Triangle Institute, 2001). GEE techniques were used to evaluate the overall effect of treatment and pairwise comparisons of exposed group values to the control group values. Student's t-test was used to analyze quantitative continuous data collected for parameters from only two groups (e.g., ovarian primordial follicle counts and selected andrological parameters) (SAS Institute, Inc., 2000).
A test for statistical outliers (SAS Institute, Inc., 1999) was performed on parental body weights, feed consumption (in g/day), and F0 and F1 adult, F1 and F2 weanling, and F1 retained male organ weights. When examination of pertinent study data did not provide a plausible, biologically-sound reason for inclusion of the data flagged as "outlier," the data were excluded from summarization and analysis and were designated as outliers. The criterion for statistical significance was p 0.05. If the overall ANOVA or chi-square p value was significant, then the appropriate pairwise comparisons were made, and those pairwise comparisons that were statistically significant are presented in the summary tables. If the overall ANOVA or chi-square p value were not significant, then pairwise comparisons were not made. All values in the text, specified as "reduced" or "increased" relative to the control group value, were statistically significantly different unless otherwise indicated.
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RESULTS |
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Parental Systemic Parameters
Fate and Body Weights
There were no treatment-related deaths for F0 or F1 males. There were no statistically or biologically significant effects on F0 or F1 parental male body weights at any dietary concentration throughout the 8-week prebreed period or the 2-week mating period (Fig. 2 for F0 males; the data for F1 males were equivalent, data not shown). There was a slight reduction in body weights for the extra retained F1 males at 0.5 ppm for study days (sd) 0 and 7 which fully resolved beginning on sd 14 to their termination; these differences were transient and did not occur in the parental F1 males; the significance, if any, is unknown.
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There were no treatment-related deaths for F0 or F1 females. F0 and F1 female body weights were unaffected across all groups during the 8-week prebreed period. Body weights were reduced at 0.5 ppm at gd 17 for F0 females and at gd 14 and 17 for F1 females, with all earlier gestational time points equivalent across all groups for both generations. Lactational body weights were equivalent across all groups for both generations except for reductions on pnd 7 for F0 and F1 females at 0.5 ppm (Fig. 3 for F0 females; the data for F1 females were essentially the same as for the F0 females, data not shown).
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There were no treatment-related clinical observations for F0 or F1 males or females in any group at any time (data not shown).
Feed Consumption and E2 Intake
F0 and F1 male and female feed consumption was equivalent across all dose groups by sex for all intervals (data not shown). F0, F1, and extra retained F1 male and F0 and F1 female E2 intake (µg/kg/day) exhibited the appropriate increases across groups (i.e., intake was five times greater at 0.005 ppm than at 0.001 ppm, etc.) and the anticipated decreases within groups over time during the prebreed/postwean exposure periods, because body weights were increasing and feed consumption was similar over time. Figure 4 presents the E2 intake for the F0 males; intake for the F1 generation was essentially the same as for the F0 males. Total (designated "maternal") feed consumption and therefore total E2 intake were increased during the latter part of the lactational period for F0 and F1 dams, almost exclusively because the F1 and F2 pups began to self feed on or about pnd 14, and 30–40% of maternal feed consumption (and therefore E2 intake) during the last week of lactation is considered to be the result of pup consumption. There is no literature for the timing of the onset of mouse pup self-feeding but it was observed in the present study and in rats by Tyl et al. (2002) and was measured in rats by Hanley and Watanabe (1985). Figure 5 presents the total E2 intake for the F0 females; intake for the F1 females was the same as for the F0 females.
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Adult Male Organ Weights and Andrology
The organ weights from F0 and F1 parental males and retained F1 males are presented in Table 3 (absolute and relative, as % of sacrifice weight, organ weights are included in Supplementary Data Table 1). Statistically significant differences at 0.5 ppm in the relative weights of the paired kidneys (in F1 parental and retained males), of the pituitary (in F0 parental males and F1 retained males), and in absolute and relative thyroid weights in the F1 parental (but not F0 or retained F1) males were not considered treatment related based on lack of consistency across and within (e.g., parental vs. retained F1 males) generations, and the small magnitude of changes.
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Andrologic parameters in F0 and F1 adult parental males are presented in Table 4. There were no effects on any parameters in either generation across all groups for percent motile and percent progressively motile sperm, and in either generation at 0 or 0.5 ppm for epididymal sperm concentration, testicular homogenization-resistant spermatid head counts, DSP per testis, efficiency of DSP per gram testis, or percent abnormal sperm.
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Adult Female Organ Weights
Selected systemic and reproductive organ weights, ovarian primordial follicle counts, and stage of estrus at scheduled necropsy are presented in Table 5 for F0 and F1 adult parental females (absolute and relative, as % of sacrifice weight, organ weights are included in Supplementary Data Table 2). In adult females, an increase in absolute and/or relative UCV weight was observed at 0.15 and 0.5 ppm in F0 adult females and at 0.05, 0.15, and 0.5 ppm in F1 adult females. Occasional statistically significant differences in other organ weights were not considered treatment related based on lack of consistency across generations, small magnitude of changes, and/or lack of dose response.
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There was an apparent, slight increase in the percentage of F0 and F1 adult females at 0.5 ppm in estrus at scheduled necropsy, but this was not statistically or biologically significant. There was no effect on prebreed estrous cycle length in any group in either generation (Table 5).
Adult Histopathology
Ovarian primordial follicle counts/female were equivalent at 0 and 0.5 ppm for both F0 and F1 adult parental females, 10/group (Table 5). Histopathology of all the retained organs for F0 and F1 parental males and females at 0.5 ppm exhibited no treatment-related changes (relative to the control organs) in incidence or severity of findings at any dose in either generation (data not shown).
Gestational and Lactational Parameters
Treatment-related parental and offspring gestational and lactational effects (Table 6), were observed only at 0.5 ppm that included: prolonged gestational length by 0.7 days in F0 and 0.6 days in F1 females; reduced gestational index [defined as (no. females with live litters/no. pregnant females) x 100] in F1 females; reduced live birth index and increased stillbirth index (neither one statistically significant), increased percent postimplantation loss in F1 and F2 litters, reduced number of F2 (but not F1) litters/group (not statistically significant), and reduced numbers of total and live pups/litter on pnd 0 (precull) and 21 (postcull) in F1 and F2 litters (collectively termed "perinatal loss"). Reduced numbers of implantation sites per litter were observed in F1 dams at 0.5 ppm; a slight but statistically significant reduction in implantation sites in F1 dams at 0.001 ppm was also observed but was not considered treatment related because a similar finding was not observed in F0 dams at this dose, and there was no effect on this parameter in F1 dams at 0.005, 0.05, or 0.15 ppm (Table 6). There were no effects on any post birth survival index throughout lactation in any group for F1 or F2 offspring. Pup body weights/litter (sexes separately or combined) were equivalent across all groups throughout lactation for F1 and F2 pups except for an increase at 0.5 ppm on pnd 0 and 4 for F1 and F2 pups and on pnd 21 for F2 pups (Table 6). The increased body weights at 0.5 ppm for both F1 and F2 pups were most likely due to the reduced litter size and/or the prolonged gestational length at this dose in both offspring generations. F1 and F2 pup AGD at birth was equivalent across all groups for both sexes (Table 6). F1 female AGD on pnd 21 at weaning was equivalent across all groups. F1 male absolute AGD on pnd 21 was reduced at 0.001, 0.005, and 0.15 ppm (but not at 0.05 or 0.5 ppm), and adjusted AGD was reduced at all dietary concentrations with no dose–response pattern. For F2 male pups, absolute and adjusted AGD on pnd 21 were unaffected. For F2 female pups, AGD was increased at 0.005, 0.05, 0.15 ppm (absolute and adjusted), and at 0.5 ppm (absolute but not adjusted) with no dose–response pattern (Table 7).
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F1 and F2 Weanling Males
Selected F1 and F2 weanling systemic and reproductive organ weights and parameters are presented in Table 7 for males (absolute and relative, as % of sacrifice weight, organ weights are included in Supplementary Data Table 3). Decreased absolute testes and epididymides weights (TEW) were observed in F1 and F2 weanlings at 0.15 and 0.5 ppm. In addition, apparent increases in undescended testes (defined here as testes near the inguinal canal and not in the scrotum) were observed at weaning at 0.15 ppm in F1 and F2 weanlings and at 0.5 ppm in F2 weanlings at necropsy. One confounder is that the testes of young mice are easily displaced through the open inguinal canal; therefore, the toxicological significance of this finding is uncertain (see discussion). Statistically significant differences in thyroid and pituitary weights were observed but were not considered to be treatment related due to lack of consistency between generations and highly variable responses across doses (Table 7).
F1 and F2 Weanling Females
Selected F1 and F2 systemic and reproductive organ weights and parameters are presented in Table 8 for weanling females (absolute and relative, as % of sacrifice weight, organ weights are included in Supplementary Data Table 4). Enlarged/fluid filled uterus (uni- or bilateral) and enlarged, open, swollen, and/or thickened vagina were observed in a dose-related manner at necropsy of the F1 and F2 females at 0.15 and 0.5 ppm. Increased absolute and relative uterus (plus cervix and vagina; UCV) weight and the uterus visibly enlarged and the vagina enlarged, open, and thickened were observed in F1 and F2 weanlings at 0.05, 0.15, and 0.5 ppm. Increased ovarian weight was also observed in F1 (absolute and relative) and F2 females (relative only) at 0.5 ppm. Relative thyroid weights were significantly reduced for F1 females at 0.5 ppm, and absolute and relative thyroid weights were significantly reduced at 0.15 and 0.5 ppm in F2 females and associated with large differences in F1 versus F2 control thyroid weights. For paired adrenal glands, a statistically significant increase was reported only at 0.001 ppm only for relative weight and only in F2 weanlings. The weight changes reported in ovary, thyroid, and adrenals were not considered to be biologically significant or treatment related due to lack of consistency between generations and for adrenals, the inconsistent dose–response pattern (Table 8).
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F1 Postwean Parameters
Acquisition of puberty in F1 females (VP) was accelerated at 0.15 ppm by 6.8 days and at 0.5 ppm by 7.3 days for the age in days at acquisition (Fig. 6) but not when adjusted for body weight at acquisition (with comparison at same physiological state regardless of chronological age). VP was accelerated relative to body weight on pnd 21 (with comparison at the same chronological age regardless of physiological state) by 6.3 days at 0.15 ppm and by 6.1 days at 0.5 ppm. In the ANCOVA model, F1 female body weight on pnd 21 and at acquisition and dose were all statistically significant covariates. Acquisition of puberty in F1 males selected to be parents of the F2 generation (PPS) was delayed at 0.15 and 0.5 ppm for age in days at acquisition (by 3.9 and 8.6 days, respectively; Fig. 6), delayed at 0.5 ppm when adjusted for body weight at acquisition (by 4.4 days) and delayed at 0.15 and 0.5 ppm when adjusted by body weight on pnd 30 (by 3.2 and 7.1 days, respectively). In the ANCOVA model, body weights on pnd 30 and at acquisition of PPS for F1 males selected for mating, and dose, were all statistically significant. Acquisition of puberty in the extra retained F1 males was delayed at 0.05, 0.15, and 0.5 ppm for age of acquisition (by 1.7, 3.3, and 9.4 days, respectively; Fig. 6) or when adjusted by body weight on pnd 30 (by 1.9, 2.8, and 8.1 days) and delayed at 0.15 and 0.5 ppm when adjusted by body weight at acquisition (by 2.8 and 8.2 days, respectively). Age at acquisition of puberty is adjusted for body weight at acquisition (so all animals are at the same physiological state) regardless of age, and at an arbitrary age selected during the period of acquisition (i.e., pnd 21 for females and pnd 30 for males; so they are the same chronological age regardless of physiological state) to correct for the confounding of body weight, because smaller animals acquire puberty later (so delays could be due to, and therefore secondary to, systemic toxicity). In the ANCOVA model, F1 extra male body weight on pnd 30 and dose were statistically significant covariates; body weight at acquisition was not a statistically significant covariate.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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A one-generation study (Tyl et al., in press
) identified critical endpoint parameters for evaluation of estrogenic effects in mice and established dietary doses for the present study. The present study examined these effects of estrogen using the regulatory guideline two-generation protocol (OECD, 2001), enhanced, in mice. The results at 0.5 ppm included increased perinatal F1/F2 pup losses, reduced litter sizes in F1/F2 offspring and associated increased body weights during lactation, accelerated puberty in F1 females (also seen at 0.15 ppm), and delayed puberty in F1 males at 0.5 (and 0.15) ppm. F0 and F1 females also exhibited reduced gestational weights at 0.5 ppm, which were viewed as secondary to (and due to) the increased F1/F2 embryofetal loss. The weanling and adult reproductive effects at 0.5 ppm were considered unlikely to be due to the embryofetal loss and subsequent maternal gestational body weight reduction because the postnatal survival and growth were unaffected. There was also increased UCV weight at 0.15 and 0.5 ppm in F0/F1 adults and in F1/F2 weanling females (also at 0.05 and 0.15 ppm) and reduced paired TEW in F1/F2 male weanlings at 0.5 (and 0.15) ppm, but there were no effects on testes or epididymides weights in either the F0 or F1 adult males. The above findings were all consistent with the results of the one-generation study (Tyl et al., in press). A slight (0.6–0.7 day), prolonged gestational length in F0/F1 females, was also observed at 0.5 ppm in this study; a similar prolongation (0.3 days) was also observed in the one-generation study at the same dose. The toxicological relevance and significance, if any, of this prolongation of 7–17 hours is unknown. Reduced gestational index (percentage of pregnant females which delivered live litters) was observed in the F1 females in this study, but not in the F0 females in either this two-generation study or the one-generation study. Effects on gestational index were also not observed in F0 or F1 females in the E2 0.5 ppm group (positive control) of a subsequent two-generation study in mice (Tyl et al., unpublished data). Therefore, this effect was not considered treatment related. In the present study, the F0 and F1 adult animals exhibited similar sensitivity to dietary E2. The F1/F2 weanling animals exhibited different responses than did the adults and appeared more sensitive than the adults; these weanling effects were transient and not observed in adults. F1 and F2 weanling responses were comparable.
There are some statistically significant differences in this study (and in all other toxicity studies) that are not considered to be related to E2 exposure, but are Type I errors that are inherent in these statistical comparisons. With a fiducial limit of 95% (p 0.05), one in twenty statistically significant findings are in fact due to chance (Type I error). Therefore, statistically significant differences observed in isolated parameters with inconsistencies between generations, with inconsistent patterns of dose response, and/or small magnitude of changes without concomitant biologic effects (e.g., no histopathologic correlation) are not considered to represent effects of E2 exposure. Weanling organ weights exhibited the greatest number of inconsistent dose–response patterns. Periweanling animals are in a dynamic growth phase and therefore, even when all weanlings are necropsied at exactly the same age in days as done in this study, there are variations in organ weights within as well as between groups. These sources of variability as well as Type I errors are considered responsible for the inconsistent dose–response patterns occasionally observed.
Results of the present study and the previous one-generation study in mice provide for direct comparisons of the responses of E2 in reproductive toxicity studies in mice versus rats (Biegel et al., 1998a,b; Cook et al., 1998; Tyl et al., 2006, in press). Table 9 summarizes the key findings of the two species at the highest dietary concentrations that produced viable offspring (2.5 ppm in rats, 0.5 ppm in mice). E2 intakes at these concentrations were approximately equivalent: 0.12 mg/kg/day in mice, 0.17 - 0.2 mg/kg/day in rats. Responses observed in both mice and rats included; decreased adult female body weight during in-life, decreased number of litters/group, decreased number of live pups/litter on pnd 0, decreased total number of pups/litter on pnd 0 and accelerated VP and delayed PPS. There were no effects of E2 in either species for AGD. There were also responses observed only in CD-1 mice, only in CD(SD) rats, and effects on parameters evaluated for only one species or the other (Table 9).
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These comparisons indicate that, although there are differences in responsiveness of some endpoints, overall, the ability to detect effects of an orally administered estrogen in rats and mice is comparable at approximately equivalent E2 intakes.
Other known estrogenic substances that have been evaluated in rodent reproductive/developmental toxicity study designs by the oral route of exposure include the synthetic estrogen, ethinyl estradiol (EE; Guo et al., 2005), the phytoestrogen, genistein (Kyselova et al., 2004; Rozman et al., 2006), and the synthetic nonsteroidal estrogen, diethylstilbestrol (DES; Kyselova et al., 2004). Similarities in the results reported for orally administered E2 and other orally administered estrogenic compounds include infertility at high doses (DES), decreased litter size (genistein and DES), accelerated VP (EE, genistein, and DES), decreased testes (genistein and DES) and epididymides (DES) weight. Differences include increased UCV weight and delayed PPS observed for E2, but not for the other estrogenic compounds, offspring body weight reduction (EE, genistein, and DES, but not E2), presence of effects on andrological parameters and AGD (genistein and DES, but not E2), decreased prostate, seminal vesicle, and ovary weight observed with genistein and DES (but not E2) and altered histopathology of male mammary glands observed with EE and DES (but not E2). Differences observed among previous studies evaluating chemicals with estrogen-like activity and this study with E2 are readily explained because of differences in study designs, endpoints examined or other mechanisms of action (e.g., antiandrogenic activity of DES). The present study was conducted primarily to define the effects of orally administered estrogen in a definitive study design and therefore identified the robust responses to an estrogen. Standard regulatory guidelines for reproductive toxicity studies require measurement of age at VP as a measurement of the onset of puberty. Some investigators have suggested that VP may not be reliable marker of puberty in mice because estrous cyclicity sometimes does not begin until later (days or weeks), whereas in rats VP signals the onset of estrous cycling (Clark, 1999). In the present study (and in the rat E2 studies), accelerated VP was clearly present following exogenous estrogen exposure, suggesting that this is a robust marker of the peripubertal E2 surge in both mice and rats. The relationship, if any, of accelerated VP on the adverse reproductive outcomes in rodents at high E2 doses is not known.
No E2-related effects were observed in AGD in the published E2 mouse or rat multigeneration studies. Measurement of AGD in F2 offspring on pnd 0 is a triggered endpoint in standard guideline multigeneration studies when treatment-related effects on sex ratio or sexual maturation are observed in F1 offspring. For this study however, AGD on pnd 0 and 21 (absolute and adjusted for body weight) for F1 and F2 pups was added to the study design to ascertain if there were AGD effects on pnd 0, whether they were transient or permanent, and present in both offspring generations. Based on the lack of consistent effects on AGD on pnd 0 or 21 in either F1 or F2 generations in this study and the lack of effects seen in previous multigeneration studies on weanling AGD; this measurement at weaning in multigeneration studies is not considered useful. It is clear that AGD on pnd 0 or 21 is also not an indicator of estrogenicity in rodents, consistent with the evidence that AGD is driven by androgen (5
-dihydrotestosterone; DHT) secretion rather than by estrogens (Imperato-McGinley et al., 1992).
There were no effects on the presence or duration of estrous cyclicity or ovarian primordial follicle counts in adult F0/F1 female mice, and no treatment-related changes in incidence or severity of any histopathologic findings in F0/F1 parental animals (including no histopathological effects on F1 axillary mammary glands). As in the previous one-generation E2 study (Tyl et al., in press), there were no effects of E2 on nipple/areolae retention in F1 and F2 male mice or adult prostate weight. In contrast, vom Saal et al. (1997) reported prostate weight changes in adult offspring from CF-1 mouse dams (six to eight per group) subcutaneously implanted with various levels of E2 in silastic tubing from gd 13–19. Adult offspring prostate weight changes showed a non-monotonic response related to different increased serum E2 concentrations as measured in fetuses. Calculated doses (assuming complete release of E2 from the silastic tubing at equivalent daily levels across the exposure period and a 30 gram maternal mouse) ranged from approximately 120–1430 µg/kg body weight/day which exceeded the highest dose used in the present study and the doses that allowed for viable offspring in the one-generation study (Tyl et al., in press). Major differences (including castration and T-implantation of the adult male offspring, group size, route of administration, mouse strain, study design, housing, timing and duration of exposure, doses, age of animals at termination, etc.) between the vom Saal et al. study and the present multi-generation study preclude direct comparisons. However, the robustness of the present study, along with the results of the one-generation study, strongly support the conclusion that prostate weight as well as other parameters noted above are not affected by orally administered estrogen in mice.
Based on observations made in the pilot study with estradiol (Tyl et al., in press), the location of the testes at necropsy of the male weanlings was noted in the present study. These observations revealed an apparent increase in the incidence of undescended testes at 0.15 ppm in F1 and F2 weanlings and at 0.5 ppm in F2 weanlings. The concern therefore arose as to whether these findings represented a true, permanent cryptorchid state, a transient delay in testis descent, or a spurious finding related to physical displacement of the testes from the scrotum.
Testicular descent is regulated by the development and subsequent tightening of the gubernacular cords, which anchor the posterior portion of the testes to the scrota (Hutson et al., 1997). The molecular mechanism for the development and function of the gubernacular ligaments is dependent on testosterone, DHT and Insl 3 (insulin-like 3), all produced by the Leydig cells of the testis (Gilbert, 1997; Kubota et al., 2002). Impairment of the gubernaculum has been associated with reduced fetal Insl3 gene expression (Shono et al., 2005; Wilson et al., 2004), and mice mutant for the Insl3 gene have irreversible cryptorchidism (Nef and Parada, 1999).
Testis descent occurs in two phases: in utero intra-abdominal descent and perinatal inguino-scrotal descent (Hutson, 1985). These phases of testes descent occur sequentially such that during the intra-abdominal phase, the testes move from their initial position at the genital ridge near the kidney to the lower abdomen and the subsequent phase of inguino-scrotal descent involves additional maturation of the gubernaculum to pull the testis through the inguinal canal into the scrotum (Hutson, 1985). Interestingly, exposure to some exogenous estrogens (e.g., DES by subcutaneous injection) causes a disruption in the first phase of testis descent, potentially due to a failure in the development of the gubernaculum (Emmen et al., 2000; Hutson et al., 1997). Based on these data, it is feasible to speculate that exposure to E2 in this study could potentially lead to disruption of the first phase of testis descent; however, it was noted that the undescended testes observed in this study were present in the lower abdomen near the inguinal canal Additional histopathological evaluation of target tissues in weanlings would be necessary to determine if these observations actually represent a delay in testis descent. Another important observation was that no adult F1 males from any group had descended testes at necropsy.
Disruption of normal male reproductive development through exposure to anti-androgenic agents (e.g. DES, flutamide, vinclozolin) typically presents with a host of phenotypic effects including, but not limited to: retained nipples and areolas, hypospadias, decreased anogenital distance, reduced androgen-responsive organ weights (e.g., ventral prostate, seminal vesicles), and permanent cryptorchidism. When comparing the incidence of these effects with respect to dose, a profile of disruption of male reproductive development becomes apparent where each end point has a different relative sensitivity (Gray et al., 2001).
Taken together, the observation of an increased incidence of undescended testes in weanlings in this study is considered of uncertain toxicological significance because: (1) all testes were located low in the abdominal cavity near the inguinal canal and not near the kidneys, which would be expected from disruption of the in utero phase of testis descent; (2) there were no F1 males in any group with undescended testes at adult necropsy (the F2 generation was terminated at weaning); (3) this observation was made in the absence of other markers of endocrine-mediated activity (e.g., hypospadias, retained nipples, decreased anogenital distance) that are typically found in males at doses lower than those that cause permanent cryptorchidism; (4) adult F1 male reproductive functions (andrology and male reproductive indices) were equivalent across all groups; and (5) there were some F1 and F2 male weanlings in all other groups (including a few in the control group) that were observed with undescended testes.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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In conclusion, this study demonstrates the dose-related effects of 17β-estradiol in mice following the OECD guideline for a reproductive toxicity study and provides valuable insight for future evaluations of potential estrogenic effects in this species following exposure to other chemicals. Based on the results of this study and the literature, there is no evidence that either species is more or less sensitive to the overall E2 effects; therefore, either rodent species is a suitable model for detection of reproductive/developmental effects from estrogenic compounds. The results of the study are summarized as follows:
At 0.001 ppm (0.2 µg/kg/day) and 0.005 ppm (1 µg/kg/day), there were no treatment-related effects on any parameter for parental or offspring animals.
At 0.05 ppm (10 µg/kg/day), increased weights of the UCV in F1/F2 weanling females, and delayed PPS in extra retained F1 males were observed.
At 0.15 ppm (30 µg/kg/day), weight of the UCV was increased for F0 (absolute) and F1 (relative to body weight) parental females, and VP was accelerated. For F1/F2 weanling males, paired TEW were reduced, and PPS was delayed in parental and retained F1 males.
At 0.5 ppm (100 µg/kg/day), there was significant systemic and reproductive toxicity in parental animals (observed at lower doses), including reduced gestational index in F1 (but not in F0) females, developmental toxicity in F1 and F2 offspring (reduced litter sizes), and accelerated VP and delayed PPS.
There were no effects at any concentration on prostate weight, andrologic parameters, estrous cyclicity, ovarian primordial follicle counts, or histopathologic findings.
Therefore: the no observable effect level was 0.005 ppm (1 µg/kg/day) and the lowest observable effect level was 0.05 ppm (10 µg/kg/day).
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Polycarbonate/BPA Global Group, Arlington, VA.
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ACKNOWLEDGMENTS |
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We wish to thank Mr M. D. Crews, Ms A. B. Goodman, Mr J. E. Gray, Ms R. T. Krebs, Ms N. M. Kuney, Mr C. G. Leach, Ms L. L. McDonald, Ms A. J. Parham, Ms L. B. Pelletier, Ms M. L. Reith, Mr W. P. Ross, Ms K. D. Vick, Mr T. W. Wiley, and Ms V. I. Wilson of the RTI Laboratory Animal Sciences Group; Mr D. L. Hubbard, Mr R. A. Price, and Mr J. E. Larson of RTI's Materials Handling Facility; Ms. T. Uenoyama and Mr M. S. Gardner of RTI's Analytical Chemistry Group; Ms D. J. Smith and Ms D. A. Drissel, Managers, Ms C. D. Keller, Ms C. A. Ingalls, Ms M. M. Oh, Ms M. D. Phillips, Ms E. D. Shinuald, Ms D. D. Rowe, Ms J. E. Jones, Ms S. C. Wade, and Ms P. Hall of RTI's Quality Assurance Unit; and Ms C. A. Winkie, and Ms D. B. Bynum, RTI LST administrative staff.
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