Evaluation of multigenerational effects of 2-ethylhexyl 4-hydroxybenzoate in Japanese medaka.

The Japanese medaka (Oryzias latipes) extended one-generation reproduction test (MEOGRT) (Test Guideline 890.2200) is a Tier 2 test within the Endocrine Disruptor Screening Program of the US Environmental Protection Agency (US EPA). A modified MEOGRT was used to evaluate multigenerational effects of 2-ethylhexyl 4-hydroxybenzoate (2-EHHB) under flow-through conditions starting with adults (parent generation, F0) through a 3-week reproductive phase of the second generation (F2). Fish were exposed to one of five 2-EHHB test concentrations or a dechlorinated tap water control. Fecundity was affected at the lowest exposure (5.32 µg/L) and greater sensitivity occurred in the F1 and F2 generations. Percent fertility was also diminished from no effect level observed in the F0 generation to 101 and 48.8 µg/L in the F1 and F2 generations, respectively. Growth indices were decreased for F0 adult females and F1 subadults and adults at 48.8 µg/L 2-EHHB. Histopathologic examination of gonads, liver, kidney, and thyroid yielded possible delayed reproductive tract development in F1 subadult males, masculinization of the renal phenotype in F1 adult females (renal tubular eosinophilia) and reduced hepatic energy storage (liver glycogen vacuoles) in F1 (11.3 and 48.8 µg/L) and F2 (48.8 and 101 µg/L) males and females, respectively. Endocrine-related findings included a decrease in anal fin papillae in F2 adult males at 101 µg/L. Results of this study demonstrate effects on growth, development, and reproduction that may be mediated by endocrine (weak estrogenic) and nonendocrine mechanisms. Duration of the MEOGRT should not be routinely extended beyond the OCSPP 890 guideline study design.

Amphibian Metamorphosis Assay: Investigation of the potential effects of five chemicals on the hypothalamic-pituitary thyroid axis of Xenopus laevis.

2-Ethylhexyl 4-hydroxybenzoate (2-EHHB), 4-tert-octylphenol (4-OP), 4-nonylphenol-branched (4-NP), benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) were evaluated using a 21-day Amphibian Metamorphosis Assay (AMA). Xenopus laevis larvae were exposed nominally to each chemical at 3.6, 10.9, 33.0, and 100 µg/L, except 4-NP concentrations were 1.8, 5.5, 16.5 and 50 µg/L. Endpoints included mortality, developmental stage, hind limb length (HLL), snout-vent length (SVL), body weight (BW), and thyroid histopathology. BBP and 4-OP accelerated development compared to controls at the mean measured concentration of 3.5 and 39.8 µg/L, respectively. An increase in developmental stage frequency distribution was observed for 4-OP at 39.8 and 103 µg/L, BBP at all concentrations and DBP at 143 µg/L. Normalized HLL was increased on study day (SD) 21 for all tested substances except 4-NP. Histopathology revealed accelerated development and mild thyroid follicular cell hypertrophy at all BBP concentrations, but moderate severity at 105 µg/L. Increased BW occurred for all chemicals except 4-OP. Increased SVL was observed for 4-NP, BBP and DBP on SD 21. There was insufficient evidence that 4-NP and 2-EHHB affected the hypothalamic-pituitary thyroid axis, however, BBP, DBP and 4-OP showed potential effects on amphibian metamorphosis and thyroid activity, albeit through different lines of evidence.

Evaluation and comparison of in vitro intrinsic clearance rates measured using cryopreserved hepatocytes from humans, rats, and rainbow trout.

In vitro and in silico methods that can reduce the need for animal testing are being used with increasing frequency to assess chemical risks to human health and the environment. The rate of hepatic biotransformation is an important species-specific parameter for determining bioaccumulation potential and extrapolating in vitro bioactivity to in vivo effects. One approach to estimating hepatic biotransformation is to employ in vitro systems derived from liver tissue to measure chemical (substrate) depletion over time which can then be translated to a rate of intrinsic clearance (CLint). In the present study, cryopreserved hepatocytes from humans, rats, and rainbow trout were used to measure CLint values for 54 industrial and pesticidal chemicals at starting test concentrations of 0.1 and 1 µM. A data evaluation framework that emphasizes the behavior of Heat-Treated Controls (HTC) was developed to identify datasets suitable for rate reporting. Measured or estimated ("greater than" or "less than") CLint values were determined for 124 of 226 (55 %) species-chemical-substrate concentration datasets with acceptable analytical chemistry. A large percentage of tested chemicals exhibited low HTC recovery values, indicating a substantial abiotic loss of test chemical over time. An evaluation of KOW values for individual chemicals suggested that in vitro test performance declined with increasing chemical hydrophobicity, although differences in testing devices for mammals and fish also likely played a role. The current findings emphasize the value of negative controls as part of a rigorous approach to data quality assessment for in vitro substrate depletion studies. Changes in current testing protocols can be expected to result in the collection of higher quality data. However, poorly soluble chemicals are likely to remain a challenge for CLint determination.

Monitoring Bacillus thuringiensis-susceptibility in insect pests that occur in large geographies: how to get the best information when two countries are involved.

The adoption of cotton producing insecticidal proteins of Bacillus thuringiensis, commonly referred to as Bt cotton, around the world has proven to be beneficial for growers and the environment. The effectiveness of this important genetically-modified crop can be jeopardized by the development of resistance to Bt cotton by pests it is meant to control, with the possibility that this phenomenon could develop in one country and spread to another by means of insect migration. To preserve the effectiveness of this agricultural biotechnology, regulatory agencies have developed plans to mitigate the development of resistance, and research institutions constantly monitor for shifts in Bt-susceptibility in important pests. If Bt-resistance is detected, this finding needs to be corroborated by an independent laboratory according to current regulatory requirements; a process that presents numerous challenges. We investigated the biological activity of Bt-incorporated diet on Helicoverpa virescens L. after it was stored for several days at different temperatures. Diet stored up to nine days at different temperatures (-14 to 27 degrees C) produced the same biological effect on H. virescens as freshly-prepared diet. Elevating the temperature of Bt stock solution to 76 degrees C as compared to 26 degrees C yielded significantly higher reading of apparent Cry1Ac concentration from MVP II, but not enough to elicit a significant biological response when these stock solutions were incorporated into insect artificial diet. These findings are important particularly when the confirmation of resistance is done at a distant location, such as Mexico, or when diet is shared between laboratories, and must be stored for later use, as in the case of international collaboration.

Sustainability of insect resistance management strategies for transgenic Bt corn.

Increasing interest in the responsible management of technology in the industrial and agricultural sectors of the economy has been met thorough the development of broadly applicable tools to assess the "sustainability" of new technologies. An arena ripe for application of such analysis is the deployment of transgenic crops. The new transgenic pesticidal or plant-incorporated protectant (PIP) crops have seen widespread application in the United States based on the features of higher yield, lower applications of insecticides, and control of mycotoxin content. However, open rejection of these new crops in Europe and in other countries has been a surprising message and has limited their worldwide acceptance. The US Environmental Protection Agency's (USEPA) Office of Pesticide Programs (OPP) has worked on the development and analysis of insect resistance management (IRM) strategies and has mandated specific IRM requirements for Bacillus thuringiensis (Bt) crops since 1995 under the Food, Fungicide, Insecticide, and Rodenticide Act. Improvement of data quality and sustainability of IRM strategies have been targeted in an ongoing partnership between the USEPA Office of Research and Development and the Office of Pesticide Programs that will further enhance the agency's ability to develop sustainable insect resistance management strategies for transgenic field corn (Bt corn) producing B. thuringiensis (Bt) insecticidal proteins.