Neurotoxicity test validation, positive controls and proficiency: Are chemicals necessary?

Abstract

The USEPA neurotoxicity guidelines require the use of positive control data in support of toxicology studies submitted to the Agency and emphasize the use of chemicals to accomplish this requirement. These guidelines, though, propose a number of different rationales for the use of chemicals as positive control agents. We re-evaluated the potential roles of positive control data in addressing three questions: 1) what does the test measure? 2) is the performing laboratory proficient in the use of the test? 3) do the complementary data submitted in support of neurotoxicity studies conducted with the test material provide enough context for the interpretation of the biological significance of an effect? While, for most types of guideline neurotoxicity tests, the use of test chemicals has been emphasized for positive control testing, the use of non-chemical procedures (i.e., systematic manipulation of the experimental parameters of a test, which poses less risk of adverse effects to the test animals) should be strongly considered as a potential alternative.

Introduction

The United States Environmental Protection Agency (USEPA; the Agency) neurotoxicity guidelines typically require the use of chemicals to “detect chemically induced increases and decreases in [motor] activity” [19], to evaluate “effects in neonatal animals perinatally exposed to chemicals” [20], to “provide evidence that the experimental procedures are sensitive to substances known to affect operant behavior” [21], to “provide evidence that the experimental procedures are sensitive to substances or procedures known to affect peripheral nerve function” [22], or to “demonstrate the capability of the laboratory performing the testing to conduct the procedure” [23]. These represent a spectrum of guideline studies; some are required for registration, others are required if results from other studies suggest a significant potential for neurotoxicity. A summary of the endpoints for each of these studies is presented in Table 1.

The USEPA addresses the nature of the positive control chemicals and indicates that “Permanently injurious substances need not be used for the behavioral tests.” [19], [21]. Acrylamide and trimethyl tin are suggested for nervous system pathology [19]. In the “Peripheral nerve function” [22] and the “Neurophysiology: sensory evoked potentials” [23] guidelines, the USEPA states that the positive control data do not have to include chemicals. The Agency recommends “procedures” and states that: “Temperature change could be used as a positive control procedure …” [22] or “ … it should be demonstrated that the mean of an amplitude-sensitive dependent measure increases monotonically as a function of stimulus intensity …” [23].

Regarding the USEPA developmental neurotoxicity guidelines mentioned above [20], the Agency has reported that some positive control data packages submitted to the Agency were poor and lacked adequate information [5]. The main deficiencies encountered within the context of the USEPA developmental neurotoxicity guideline included absence of positive control data, positive control data without effects, inappropriate control chemical, inappropriate dose and/or route of administration, lack of statistical analysis and use of one sex.

Several reasons have been given in the guidelines by the Agency for requesting positive control data, but these reasons are neither uniform nor fully developed. The stated reasons are to address the “sensitivity” of the test, its “reliability”, the “competence” of the laboratory, the “ability to detect increases and decreases” and/or the “significance” of effects seen. Further, the Agency offers little guidance in selecting specific chemicals (or procedural manipulations) to be used as positive controls for specific tests, leaving important details (dose, route, timing, etc.) up to the registrant. Though this lack of prescription allows some freedom in how validation studies are conducted, it also increases the chances of inappropriate selections and decisions, which may result in wasted use of laboratory resources and animals, and invalid positive control studies.

Further evaluation of the guideline statements, the Agency's specific comments to registrants over the years, as well as some reflection on scientific test requirements have led us to rephrase the questions that should be asked about a neurotoxicology test, as follows:

• “What does the test measure?”

• “Does the laboratory have the competence to perform the test?”

• “Do the complementary data submitted in support of neurotoxicity studies conducted with the test material provide enough context for the interpretation of the biological significance of an effect?”

The purpose of this commentary is to provide a conceptual framework for the evaluation of what needs to be performed before a test (old or new) can be satisfactorily used within the context of the guidelines.