Variability in the Uterotrophic Response Assay (An in Vivo Estrogenic Response Assay) in Untreated Control and Positive Control (Des-Dp, 2.5 äG/Kg, Bid) Wistar and Sprague-Dawley Rats

Abstract

Inclusion of biological outlier values was found to bias the results of rat uterotrophic assays towards false negatives, i.e., not identify uterotrophic effects in treated populations. The present investigation was conducted to identify the background variability in the rat uterotrophic assay and to evaluate the need to exclude biological outlier values in untreated control groups. The Styrene Steering Committee (SSC) of the European Chemical Industry Council (CEFIC) co-sponsored this work with Argus Research Laboratories (Argus). The rat uterotrophic response assay originally was used as a pharmacology screen to identify estrogenic agents. Classically, 5 to 10 immature female rats (18 to 22 days of age) are administered an agent for three or four days. At sacrifice on the following day (21 to 26 days of age), the uterus is removed, weighed and a uterine weight/terminal body weight ratio calculated. This in vivo assay has been adapted for use in identifying the potential estrogenicity of chemicals, generally using 10 immature female rats per group, more closely controlling the ages, and adding one or more positive control groups to demonstrate sensitivity and response of the test system. Statistically significant increases in the positive control group means for absolute and relative uterine weights, as compared with the untreated (or vehicle-treated) means, is generally interpreted as identifying a sensitive test system. The untreated (and/or vehicle-treated) control group is then compared with the various test groups, and statistically significant increases in the mean absolute and relative uterine weights are identified as evidence of estrogenicity of the agent. Although not fully described previously, the inherent biological variability existing in both untreated and treated animals, can confound interpretation of the data, especially when numbers are relatively small. Our laboratories have identified that under controlled GLP-compliant conditions, some Wistar rats [randomly assigned (weight-ordered) to groups of ten at 22 +/- 1 days of age, and sacrificed when 26 +/- 1 days of age] in untreated control groups have high relative uterine weights that skew data distributions such that statistically significant differences are not present between untreated control and positive control groups. Based on these observations, further evaluations of untreated control and positive control (DES-DP, 2.5 micrograms/kg, b.i.d.) populations of three rat strains [Wistar--Chbb:THOM-SPF, Wistar--Crl:(WI)BR and Sprague-Dawley--Crl:CD(SD)IBS BR VAF/Plus "International Genetic Standard"] were made to define when such normal findings should be considered biological outliers, and whether outlier values should be excluded from analyses. Our data indicate that body weight is not always predictive of uterine weight, that relative uterine weight outlier values occur in each of these rat strains, and that statistically significant differences exist between groups of untreated control animals when outlier values are included in analyses....

The immature mouse is a suitable model for detection of estrogenicity in the uterotropic bioassay.

“…As reported by other laboratories (13,36), there was a greater variation in body weights in all of the treatment groups in rats compared to mice. Also, rats showed a general decrease in body weight after 17β-estradiol treatment, and mice showed an increase with similar treatment.…”

“…Although much attention has been focused on identifying protocol variables and reproducibility between laboratories, direct comparison of uterine responses between rodent species has been rarely addressed. Rats are commonly used in toxicologic testing, and many studies have been performed in this species (8)(9)(10)(11)(12)(13). Because of our experience with the CD-1 mouse and our previously published data on the uterotropic responses in this strain (14)(15)(16)(17)(18), we compared uterine responses in this mouse strain with responses in the Sprague-Dawley rat.…”

The OECD program to validate the rat uterotrophic bioassay. Phase 2: coded single-dose studies.

“…The animals used would have been born on the same day, meaning that the animals were likely from a limited number of litters. In fact, some investigators have taken the precaution to also randomize their groups by litter (Christian et al 1998). As the litter of origin for each individual was not recorded, this possibility cannot be assessed here.…”

The OECD program to validate the rat uterotrophic bioassay to screen compounds for in vivo estrogenic responses: phase 1.

“…Limiting the weight variability was thought essential to limit the chances of older animals being inadvertently included in the study. Older animals could enter puberty, leading to an increase in control uterine weight and thereby adding to the variability of the results (19,22). For the adult OVX animals, ovariectomy occurred at 6 weeks of age or later, with a minimum period of 1 week after surgery before administration of the reference compounds.…”