Recommendations for harmonization of data collection and analysis of developmental neurotoxicity endpoints in regulatory guideline studies: Proceedings of workshops presented at Society of Toxicology and joint Teratology Society and Neurobehavioral Teratology Society meetings

Abstract

The potential for developmental neurotoxicity (DNT) of environmental chemicals may be evaluated using specific test guidelines from the US Environmental Protection Agency or the Organisation for Economic Cooperation and Development (OECD). These guidelines generate neurobehavioral, neuropathological, and morphometric data that are evaluated by regulatory agencies globally. Data from these DNT guideline studies, or the more recent OECD extended one-generation reproductive toxicity guideline, play a pivotal role in children's health risk assessment in different world areas. Data from the same study may be interpreted differently by regulatory authorities in different countries resulting in inconsistent evaluations that may lead to inconsistencies in risk assessment decisions internationally, resulting in regional differences in public health protection or in commercial trade barriers. These issues of data interpretation and reporting are also relevant to juvenile and pre-postnatal studies conducted more routinely for pharmaceuticals and veterinary medicines. There is a need for development of recommendations geared toward the operational needs of the regulatory scientific reviewers who apply these studies in risk assessments, as well as the scientists who generate DNT data sets. The workshops summarized here draw upon the experience of the authors representing government, industry, contract research organizations, and academia to discuss the scientific issues that have emerged from diverse regulatory evaluations. Although various regulatory bodies have different risk management decisions and labeling requirements that are difficult to harmonize, the workshops provided an opportunity to work toward more harmonized scientific approaches for evaluating DNT data within the context of different regulatory frameworks. Five speakers and their coauthors with neurotoxicology, neuropathology, and regulatory toxicology expertise discussed issues of variability, data reporting and analysis, and expectations in DNT data that are encountered by regulatory authorities. In addition, principles for harmonized evaluation of data were suggested using guideline DNT data as case studies.

Keywords: Behavior; Developmental neurotoxicity testing; Extended one-generation reproductive toxicity; Learning and memory; Morphometry; Neuropathology.

Fig. 1.

Diagram of EPA Developmental Neurotoxicity Study—OCSPP 870.6300 (adapted from Raffaele et al., 2010). Detailed clinical observations and body weights are performed periodically in the F1 generation throughout the study, before weaning and after treatment is discontinued, until around postnatal day (PND) 70 (PND 0 is defined as the day of delivery). Neurobehavioral tests for the F1 generation include automated measures of activity, auditory startle habituation, and measures of cognition (US EPA, 1998a). The numbers in the table including the total time needed to conduct motor activity in the last row are from an actual study from Dr. Sheets’ lab. The last row in bold is the approximate time it takes to run all the motor activity sessions. As illustrated in this example, some labs add extra pups distributed across groups to meet guideline requirements for 20 offspring/dose group (1 pup/sex/litter from 20 litters/dose group) in case of mortalities, logistical errors and equipment malfunction. The US EPA and OECD DNT study design requirements result in a particularly high workload at ~PND 21 (US EPA, 1998a; OECD, 2007). In cases where the 80–100 females are mated over a period of 3–5 days (the duration of the estrous cycle in rats), this may result in litters born over a similar period. Using this study design, having a range of delivery days helps to reduce certain aspects of the workload at specific days of age. On the other hand, this factor increases the logistical complexity to manage the daily workload, because the various study endpoint assessments overlap. PND 21 is a particularly busy day, with the need to measure body weight, obtain detailed clinical observations, and evaluate motor activity, auditory startle, and cognition for F1 animals from 80 to 100 litters. One consequence of the test guideline requirements is that animals must be tested over a long test day and across different days, increasing variability due to circadian rhythms and other environmental factors that can vary despite best attempts to control them.

Fig. 2.

Representative intrasession motor activity (mean ± SEM) for control male Wistar rats at PNDs 13, 17, 21, and 60 from a guideline DNT study from Dr. Sheet’s lab.

Fig. 3.

Theoretical data (mean) illustrating test session motor activity data patterns that reflect normal, as well as altered, habituation. Normal habituation in adult rats is shown in Vehicle Control and Treatment A. The specific temporal activity pattern in Control animals for a study will be determined by a range of factors, including the test equipment, test conditions, age, species and strain, etc. Regardless, habituation in control animals will be reflected by a decrease in activity measures over the course of the test session. While Treatment A shows decreased ambulatory activity relative to Control animals, Treatment A animals exhibit habituation rates comparable to Control (note parallel lines). In contrast, Treatment D exhibits an enhanced rate of habituation compared to all other groups, as illustrated by the more rapid decrease in ambulatory counts in the second half of the test session. Treatment B and C animals show reduced habituation, as indicated by the reduced rate of ambulation decrease in the second half of the test session. Note that Treatment C and Control will have comparable total session ambulation counts despite session activity patterns that are not comparable, illustrating the potential for total session data to provide misleading information. In the absence of time block data, it would not be possible to determine that Treatment C alters habituation. Similarly, while total session activity is comparable between Control and Treatment C and D, using only total session activity would mask the fact that Treatment C and D exhibit opposite alterations in habituation pattern. Note that Treatment A through D refer to different arbitrary treatments to illustrate issues to consider.

Fig. 4.

Frequency histograms for PND 13 (top), PND 17 and PND 21 (bottom) motor activity data of control animals from the laboratory of Dr. Moser. Data were collected in the figure-8 chamber and include data for males and females combined. X-axis values are photocell counts. Each chart within each age contains data for 10-min time blocks for a total of 30 min of data. The X-axis indicates the number of photocell counts, and the Y-axis indicates the frequency of counts (number of animals). For PND 13 animals, there is a high proportion of animals with little or no detected movement (frequency of 0 counts), especially after the first time block. The data set is highly skewed at PND 13. In contrast, at PND 17 and 21, there are few animals with no movement, and data are normally distributed (n’s = 43).

Fig. 5.

Frequency histograms for PND 13 (top, n = 6), PND 17 (middle, n = 20) and PND 21 (bottom, n = 20) motor activity data from Charles River Laboratories. Data were collected using Coulbourn Instruments Tru Scan system, and values are ambulatory counts from combined male and female control animals. Each chart within each age contains data for 10-min time blocks for a total of 30 min of data. The X-axis indicates the ambulatory counts and the Y-axis indicates the frequency of counts (number of animals). For PND 13 animals, there is a high proportion of animals with no movement (frequency of 0 counts), and the data set is highly skewed. Unlike the figure-8 chamber data, PND 17 activity data are also highly skewed, with a high proportion of animals with little movement.

Fig. 6.

Frequency histograms for PND 17 (top, n = 104) and 35 (bottom, n = 80) motor activity data of control animals (males and females combined) from Dr. Bowers’ laboratory. Data were collected using Med Associates activity system, and values are ambulatory counts from control animals from multiple studies. Each chart within each age contains data for 10-min time blocks, for a total of 30 min of data. The X-axis indicates the number of ambulatory counts, and the Y-axis indicates the frequency of counts (number of animals). For PND 17 animals, the data distribution is skewed at 10 min but approaches a normal distribution at later time points. At PND 35, there is a low frequency of 0 counts, and the data set is normally distributed at most time blocks. The increase in proportion of animals exhibiting little movement (increase in proportion of 0 counts) at 30 min reflects the onset of the expected reduction in activity associated with habituation at this age.

Fig. 7.

Latency data (mean ± SEM) for chemical X over eight days of training, four trials per day. Each day is the average of four trials. Inset shows individual trial data from day 1 only (circled).

Fig. 8.

Trials-to-criterion data for chemical X. Average data expressed as mean ± SEM. Individual data range from 4 (minimum possible) to 12 (maximum number of trials).