The US Environmental Protection Agency is increasingly employing new approach methods (NAMs), including in vitro plasma binding and hepatocyte clearance experiments to collect chemical-species specific data. This paper presents data from plasma binding experiments using rapid equilibrium dialysis (RED) devices and plasma from humans, rats, and rainbow trout with a 4-h incubation time. A total of 54 chemicals, utilizing two concentrations, were tested across the three species resulting in 238 chemical-species specific datasets. Mass balance controls for chemical plasma stability and dialysis system recovery were used to evaluate the datasets and almost 40 % of the datasets (92/238 datasets) produced quantitative measurements. Cross-species comparisons and evaluations of the impact of physicochemical properties on chemical-assay performance were also evaluated. Comparisons of human-rat plasma binding revealed rat plasma generally demonstrated higher fup values for chemicals than human. While fup values in trout plasma were frequently lower than rat or human plasma. A comparison with literature data was performed and correlations between plasma binding, expressed as fraction unbound in plasma (fup), and log Kow across all three species indicate that the strongest relationship occurs at log Kow values between 1.5 and 4. The obtained datasets exhibited a wide range of behaviors, emphasizing the need for a robust approach to data quality assessment. The broader analysis of fup values indicates that chemicals with log Kow > 4.5 will be highly bound (fup ≤ 0.0001), difficult to measure, and have low reproducibility across laboratories, suggesting that use of different methods may be needed across different physicochemical properties.

The United States Environmental Protection Agency has required an unprecedented insect resistance management (IRM) program for Bt cotton. The specific IRM strategies and requirements for Bt cotton (Bollgard(tm) cotton) in the United States have been developed by a coalition of stakeholders including EPA, USDA, academic scientists, public interest groups, Monsanto Company, and grower organizations.There are seven basic IRM requirements: structured refuge, resistance monitoring, remedial action plan, compliance assurance, grower education, grower agreements, and annual reports. The mandatory refuge requirements for Bollgard cotton are: (1) 5% external, unsprayed refuge option, (2) 20% external, sprayed refuge option, (3) 5% embedded refuge option, or (4) a community refuge option that may utilize the 5% external, unsprayed refuge option and/or the 20% external, sprayed refuge option. Each refuge option also has specific deployment requirements. The current registration for Cry1Ac plant-incorporated protectant as expressed in cotton (known as Bollgard cotton) expires September 30, 2006, except for the 5% external, unsprayed structured refuge option, which will expire September 30, 2004. Additional research data will be integral to strengthening the existing IRM plans for Bollgard cotton and promoting greater sustainability.

Microbial Bacillus thuringiensis (Bt) insecticides have been used for over 40 years. In the United States, Bt formulations are primarily applied to control lepidopteran pests on fruit and vegetable crops, to control gypsy moth in forests and to control dipteran pests (mosquitoes and blackflies) that bite humans. A highly selective insecticide with activity conferred primarily by insecticidal crystal proteins (ICPs), Bt is generally not harmful to humans, non-target wildlife or beneficial arthropods. Its selectivity and unique mode of action make it an important alternative to conventional chemical insecticides, and many integrated pest management (IPM) programs for particular fruit and vegetable crops as well as certified organic production include the use of Bt. Agricultural commercialization and adoption of plant-incorporated Bt presents new opportunities to expand the use of Bt ICPs for agricultural pest control, but also raises concerns about the potential for accelerated development of pest resistance to Bt. The relative risks and benefits of microbial and plant-incorporated Bt products are introduced.

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.

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.

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.

Regulatory risk assessments are an important part of the introduction of insect-resistant genetically modified (GM) crops (e.g., Bacillus thuringiensis [Bt] crops) into the environment to ensure the safe use of such products. In doing so, the regulatory assessment process can be clearly beneficial to integrated pest management (IPM) programs. In general, the regulatory framework for insect-resistant GM crops includes an assessment of the following: effects of the insecticidal trait on non-target organisms, other potential adverse environmental impacts, evolution of resistance to target pests, and environmental and agronomic benefits of the insecticidal trait. Each country’s regulatory system is dependent on the overall environmental risk management goals, relevant and available risk information, scientific capacity, and the available financial resources. A number of regulatory activities can help to ensure that new products such as Bt crops fit well within IPM programs: (1) evaluation of the environmental safety of new products, and their ability to enhance IPM; (2) encouragement of the adoption of new technologies with improved environmental safety profiles; (3) adoption of an expedited regulatory review system; and (4) encouragement and appropriate oversight of sustainable use of such products. Governmental regulation of insect-resistant GM crops can also hinder IPM programs by creating significant barriers to the adoption of such technologies. Such barriers include: (1) absence of functioning regulatory systems in many developing countries; (2) meeting the obligations and understanding the various interpretations of international treaties, e.g., Cartagena Protocol on Biosafety; (3) lack of public sector research to generate data supporting the safety of these crops; and (4) regulatory costs involved in the development and commercialization of novel products for small market sectors. Ways in which regulatory data requirements can be globally harmonized need to be considered to decrease the regulatory barriers for insect-resistant GM crops and comparable technologies. International organizations can play a key role in rationalizing regulatory systems; however, public sector research will also be needed to make sure that the risk assessment process is scientifically sound and transparent.

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 °C) produced the same biological effect on H. virescens as freshly-prepared diet. Elevating the temperature of Bt stock solution to 76 °C as compared to 26 °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.

This chapter discusses the role that economic analysis plays in pesticide regulation for plant-incorporated protectants and compares that to how economic analysis is used in conventional pesticide regulatory decisions. The goal is to provide a description, for research economists, of what makes economic research on agricultural biotechnology relevant to regulatory decision makers. It is our hope that in providing this perspective, economists will be able to develop a stronger sense of what types of research questions and approaches could actually inform policy. This enhanced understanding would serve the interests of those researchers seeking to make a policy contribution and could provide useful, independent analysis to help policymakers in making regulatory decisions.

Sharlene Matten from the US-Environmental Protection Agency (EPA) Office of Pesticide Programs, (on detail to CAST as Science Policy Fellow from February-June 2003) reports on a recent symposium whose goal was to provide a collective framework in which proactive resistance management could be developed.

Sharlene Matten from the U.S. Environmental Protection Agency (EPA) Office of Pesticide Programs, Biopesticides and Pollution Prevention Division (7511C) in Washington D.C. discusses the US system of voluntary resistance management labeling guidance.

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.

ABSTRACT The uncB gene codes for the a subunit of the F o proton channel sector of the Escherichia coli F 1 F o ATPase. Control of expression of uncB appears to be exerted at some step after translational initiation. Sequence analysis by the perceptron matrices (G. D. Stormo, T. D. Schneider, L. Gold, and A. Ehrenfeucht, Nucleic Acids Res. 10:2997–3011, 1982) identified a potential ribosome binding site within the uncB reading frame preceding a five-codon reading frame which is shifted one base relative to the uncB reading frame. Elimination of this binding site by mutagenesis resulted in a four- to fivefold increase in expression of an uncB ′-′ lacZ fusion gene containing most of uncB . Primer extension inhibition (toeprint) analysis to measure ribosome binding demonstrated that ribosomes could form an initiation complex at this alternative start site. Two fusions of lacZ to the alternative reading frame demonstrated that this site is recognized by ribosomes in vivo. The results suggest that expression of uncB is reduced by translational frameshifting and/or a translational false start at this site within the uncB reading frame.