Developmental Neurotoxicology: History and Outline of Developmental          Neurotoxicity Study Guidelines

Hiroaki Aoyama; Naofumi Takahashi; Yasufumi Shutoh; Atsuko Motomura; Kevin M. Crofton

doi:10.14252/foodsafetyfscj.2015012

Abstract

The present work provides a brief review of basic concepts in developmental neurotoxicology, as well as current representative testing guidelines for evaluating developmental neurotoxicity (DNT) of xenobiotics. Historically, DNT was initially recognized as a “functional” teratogenicity: the main concern was that prenatal and/or early postnatal exposures to chemicals during critical periods of central nervous system (CNS) development would cause later functional abnormalities of the brain. Current internationally harmonized DNT study guidelines are thus intended to predict adverse effects of test compounds on the developing CNS by observing such postnatal parameters as motor activity, startle response, and learning and memory, as well as neuropathological alterations. The reliability of current DNT study guidelines and sensitivity of testing methodologies recommended in these guidelines have been confirmed by retrospective evaluations of the many international and domestic collaborative validation studies in developed nations including Japan.

1. Introduction

Not a little concern has been growing on adverse effects of xenobiotics which would be taken through maternal and/or children’s foods and drinks on the developing nervous system of fetuses, infants and/or children¹^,²^,³^,⁴⁾. One of the most well-known examples is the neurological abnormalities caused by developmental exposures to methyl mercury. Maternal intake of methyl mercury contaminated fish in Japan led to the well-known fetal Minamata disease⁵^,⁶^,⁷^,⁸⁾. Children’s exposures to developmental neurotoxicants also occur via both inhalation and dermal exposures to environmental contaminants. Other well-known or suspected developmental neurotoxicants include ethanol, antidepressant or anticonvulsant pharmaceuticals, arsenic, lead, toluene, polychlorinated biphenyls (PCBs), organophosphate or organochlorine pesticides and a variety of industrial chemicals³^,⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵⁾. The number of chemicals known to be developmentally neurotoxic in humans is limited at present. This is likely due to the limited number of large-scale epidemiological studies on suspected developmental neurotoxicants that allow causative conclusions due to the difficulties in confounding covariates⁸^,¹²⁾.

Scientific countermeasures to protect children’s health are required to avoid or limit exposures to environmental contaminants that may harm neurological development. Such measures must include development and use of effective strategies and/or reliable guidelines to predict developmental neurotoxicity (DNT) of xenobiotics⁹^,¹⁶⁾, efficiently conducting appropriate neurotoxicity studies according to the validated strategies and/or guidelines, and conducting accurate hazard/risk assessments based on the results of scientifically reliable data. Due to the current inability to mitigate adverse neurological outcomes once manifest in children, we believe that scientific approaches must allow management of chemical exposures to levels below those associated with potential risk (eg, acceptable daily intake (ADI)).

DNT studies use standardized testing methodologies to detect behavioral and/or neuropathological abnormalities in rodents. A number of assessments of the reliability and sensitivity of the DNT guidelines have been conducted over the past few decades: available data sets generated according to the current DNT study guidelines were evaluated retrospectively based on the results of many large-scale validation exercises. The tremendous efforts in developing and standardizing DNT study guidelines are briefly summarized in a later section (Section 3). The most recent review was conducted by Makris and colleagues¹⁷⁾. These authors concluded in 2009 that the Organisation of Economic Cooperation and Development (OECD) DNT guideline “represents the best available science for assessing the potential for DNT in human health risk assessment, and data generated with this protocol are relevant and reliable for the assessment of these end points.” They also reviewed the extensive history of international validation, peer review, and evaluation of the guideline methods using a wide variety of chemicals. These authors confirmed previous reports that the DNT guidelines are capable of detecting known human developmental neurotoxicants¹⁸⁾. Multiple independent expert scientific peer reviews affirm these conclusions³^,¹⁷^,¹⁹⁾. However, the large cost and time required for DNT testing means that a large percentage of environmental contaminants remain unevaluated for their potential DNT risks. And future studies using newer advanced technologies are likely to reveal currently unknown mechanism(s) of DNT. Many stakeholders and/or consumers in Japan and other countries appear to be unaware of the details in a DNT study or its utility in protecting public health.

The following provides a brief review of: the basic concepts of DNT; a history of developing DNT test guidelines including validation efforts; and a succinct description of the current DNT test guidelines. The intent is to provide a context for the DNT risk of environmental contaminants in our food and drinking water. Readers who are interested in developmental neurotoxic effects of pharmaceuticals such as antidepressant, anticonvulsant and/or antiepileptic drugs are referred to other excellent reviews²⁰^,²¹^,²²^,²³^,²⁴⁾.

2. Basic Concept of Developmental Neurotoxicity

Experienced teratologists and/or reproductive toxicologists recognize that DNT can be perceived as a special type of delayed teratogenicity. The major concern is that central nervous system (CNS) teratogens have the potential to induce neurofunctional abnormalities such as impairment in sensory, motor, learning/ memory, emotional, or social behaviors. These are in addition to morphological abnormalities (malformations) that can be detected by gross and/or microscopic examinations at birth or after birth. A technical term, behavioral teratology, was coined more than 4 decades ago to describe this²⁵⁾. Developmental neurotoxicity is currently defined as “potential functional and morphological hazards to the nervous system which may arise in the offspring from exposure of the mother during pregnancy and lactation” according to the United States Environmental Protection Agency (U.S. EPA) DNT test guideline²⁶⁾. The concept is essentially the same in OECD DNT test guideline, although there are minor differences in wording²⁷⁾.

It has long been suspected that measurements of CNS function may be more sensitive (i.e., occur at low exposures) indicators of DNT than grossly visible malformations and/or brain morphological assessments²⁸⁾. A survey designed to evaluate the contribution of F1 neurobehavioral testing for hazard identification in pharmaceutical safety assessment studies revealed that behavioral parameters were the most sensitive in 3 studies among 113 surveyed (2.6%). However, the majority of these studies detected developmental effects on parameters other than neurobehavioral endpoints, such as body weight gains, clinical signs and physical developmental landmarks of pups, as well as sexual maturation of weanlings¹⁹⁾. It is important to note that the neurobehavioral testing conducted in these pharmaceutical safety studies did address all of the neuro-endpoints required for DNT guideline studies. A latter study of 69 DNT studies submitted to the U.S. EPA Office of Pesticide Programs (OPP) in support of pesticide registration found that 15 had been used to determine the point of departure for one or more risk assessment scenarios, and an additional 13 were determined to have the potential for use as a point of departure for future risk assessments³⁾. Neuro-endpoints affected at the lowest doses indicated that no single parameter was consistently more sensitive than another, but early postnatal assessments tended to be more sensitive than adult assessments.

Another important historical concept is that not only chemicals with toxic effects on adult CNS but also those exerting no overt neurotoxicity in adults may sometimes exert severe developmental neurotoxic effects when the developing brain of fetuses and/or infants is exposed during the critical period²⁹^,³⁰^,³¹⁾. A typical example of such chemicals is 5-bromo-2’-deoxyuridine (BrdU). Studies have shown that prenatal exposures to BrdU caused severe microscopic abnormalities in fetal CNS, which in turn induced postnatal behavioral alterations such as locomotor hyperactivity, impaired learning and memory, and lower anxiety, although the compound is known to be genotoxic but not neurotoxic in adults³²^,³³^,³⁴^,³⁵^,³⁶^,³⁷^,³⁸^,³⁹⁾. These observations support the fact that fetal and/or infantile exposures to certain chemicals during the organogenesis and/or developing periods of the CNS may later cause functional CNS defects including postnatal behavioral abnormalities regardless of their primary effect(s) in adulthoods.

Taken together, this history supports the need for developing fast and cost-efficient approaches to screen DNT, not only for the limited numbers of chemicals tested to date, or shown to be neurotoxicants in adulthoods but also tens of thousands of other chemicals of which effects on developing CNS remain unclear⁹⁾.

3. Brief History of DNT Test Guideline Development

The origin of DNT studies can date back to the early 1960s when papers describing behavioral teratogenicity of certain chemicals began to appear in scientific journals¹⁷^,¹⁹⁾. A number of papers followed that to examine potential behavioral teratogenicity of known human development neurotoxicants, including ethanol, methyl mercury, antidepressant or anticonvulsant drugs, organophosphate pesticides and others as described before⁷^,¹¹^,¹³^,¹⁵⁾. The rapid rise in numbers of behavioral teratology studies led the implementation of large-scale inter-laboratory collaborative studies in the developed nations for standardizing the methodologies for accurate hazard identifications of chemicals, for examining the validity and/or reliability of each measurement, and for developing an internationally acceptable DNT test guideline for regulatory uses¹⁷^,¹⁹⁾. Table 1 briefly summarizes the activities made by Japan, the United States, and European countries.

Table 1. History of large-scale collaborative studies for developing DNT test guidelines^a).

Date	Activities in Japan (Japanese Teratology Society)	Activities in Europe and/or North America
1978–1984		US Collaborative Behavioral Teratology Study (CBTS) for examining intra- and interlaboratory reliability and sensitivity⁴⁰^,⁴¹^,⁴²^,⁴³^,⁴⁴⁾
1984–1986	The first Behavioral Teratology (BT) collaborative study for standardizing methodologies⁴⁵^,⁴⁶^,⁴⁹^,⁵⁰⁾
1985–1988		European interlaboratory collaborative study to assess sensitivity of procedures⁵³^,⁵⁴^,⁵⁵⁾
1992–1995	The second Behavioral Teratology (BT) collaborative study for setting up a core battery⁴⁷^,⁴⁸^,⁵¹^,⁵²⁾
1995		IPCS (International Programme on Chemical Safety) interlaboratory study for evaluating test validity, reliability, and measurement validity⁵⁶^,⁵⁷^,⁵⁸^,⁵⁹^,⁶⁰^,⁶¹^,⁶²⁾
2000		ILSI workshop on DNT testing
2003	Japanese Interlaboratory Study
2004–2008		ILSI RSI DNT Working Group

a): Details of the collaborative studies can be found in the corresponding literature (reference numbers are provided on eachcolumn).

The first collaboration to standardize and validate the methodologies was the Collaborative Behavioral Teratology Study (CBTS). This was conducted to contribute to the development of the US DNT test guideline during 1978–1984. CBTS used a standardized neurodevelopment test battery including negative geotaxis, auditory startle habituation and maze-based learning and memory tests: these parameters were evaluated for their utility and repeatability with positive control substances such as methylmercury and amphetamine by participating laboratories⁴⁰^,⁴¹^,⁴²⁾. The CBTS results were compared with those with the Cincinnati Test Protocol for further evaluating the reliability and/or sensitivity of test methods applied⁴³^,⁴⁴⁾. These efforts later yielded the first version of DNT test guideline released from U.S. EPA in 1991¹⁷⁾.

Similar projects were also conducted in our country since the Japanese Teratology Society established the Behavioral Teratology Meeting (BTM) as a satellite organization of the society in 1982¹⁶^,⁴⁵^,⁴⁶⁾. The BTM led a series of Japanese collaborative studies for selecting and standardizing the reliable testing methodologies for correct identifications of behavioral teratogenicity of several known neurotoxicants during 1984–1986 and during 1992–1995¹⁶^,⁴⁵^,⁴⁶^,⁴⁷^,⁴⁸^,⁴⁹^,⁵⁰^,⁵¹^,⁵²⁾. They proposed a core battery to examine such parameters as reflexes and sensory function, activity and emotionality, and memory and learning with appropriate methodologies based on the results of first project⁵²⁾. The second collaborative studies successfully demonstrated that the proposed core battery detected developmental disorders including behavioral dysfunction caused by prenatal treatment of dams with phenytoin or retinoic acid⁴⁷^,⁴⁸⁾. Behavioral testing methodologies in the core battery included examinations of righting reflex, negative geotaxis and air righting for evaluating reflexes and sensory function, open-field test for monitoring motor activity, and Biel maze test (mandate) and shuttle box test (optional) for examining learning and memory.

Activities in European countries included inter-laboratory collaborative studies to assess sensitivity of behavioral test methods for detecting neurotoxicity of methylmercury during 1985–1988 ⁵³^,⁵⁴^,⁵⁵⁾. These studies demonstrated that behavioral tests were more sensitive than reproductive examinations, and that specific behavioral parameters evaluated by automated measures were generally more sensitive than nonspecific behavioral measures in European interlaboratory collaborative studies⁵³^,⁵⁴⁾. The International Programme on Chemical Safety (IPCS) also led a project for evaluating the test validity and reliability of adult neurotoxicity tests with such neurotoxicants as triethyltin, acrylamide, parathion, DDT, toluene, N,N’-methylene bisacrylamide and lead acetate⁵⁶^,⁵⁷^,⁵⁸^,⁵⁹^,⁶⁰^,⁶¹^,⁶²⁾. Several laboratories in the United States also participated in the project. The overall conclusion was that behavioral parameters were detectable of prototypic neurotoxic effects across the participating laboratories⁵⁶⁾ providing additional support for the behavioral measures used in DNT guidelines.

These activities repeatedly examined the sensitivity and reliability of testing methodologies for detecting behavioral abnormalities in rodents and those confirmed as effective for predicting adverse effects of neurotoxicants have been incorporated into the current DNT test guidelines. Thus, the DNT studies according to internationally accepted test guidelines are thought to be the most reliable and sensitive enough for predicting potential DNT of any chemicals¹⁷⁾.

4. Current DNT Test Guidelines

4.1 Outlines

There are two test guidelines accepted internationally for examining DNT of chemicals at present: one of them, OPPTS 870.6300, was issued by U.S. EPA in 1991 and revised in 1998²⁶⁾. The other, Test Guideline No. 426 (TG 426), was published by OECD in 2007²⁷⁾. Scientific recommendations are essentially the same between the two guidelines. Recommended parameters in F1 offspring include functional observations, motor activity, learning and memory, and startle response, which are followed by postmortem evaluations of brain weights, neuropathology and brain morphometrics²⁶^,²⁷⁾. These have been confirmed as “best practice” at present as described before¹⁷⁾. However, small differences and/or discrepancies still exist in the treatment regimen, timing of each measurement, behavioral tests required, brain fixation methods and sample sizes in each measurement. Table 2 lists these differences to help with some confusion among applicants for registration of their products and/or regulators⁶³⁾.

Table 2. Major differences in recommendations between OECD and US EPA guidelines (TG426 and OPPTS 870.6300).

	Recommendations
	TG426	OPPTS 870.6300
Treatment period	GD 6 through PND 21	GD 6 through PND 10 (option to PND21)
Direct dosing to pups	Not discussed	Should be considered
No. of animals for behavioral tests	20 males and 20 females/group	10 males and 10 females/group
Functional observations	Weekly pre-weaning, bi-weekly post-weaning	Specific days recommended
Behavioral ontology	At least 2 measures required	Not required
Motor Activity	1 to 3 preweaning days, one adolescence and one adult day	Specific days recommended; preweaning (13,17, 21) and adult (~60)
Motor and sensory function	Sensory modalities and motor functions specified; startle as example	Auditory startle specified
Animal ages at sacrifice for neuropathology	PND 22 and PND 60-70	PND 11 and PND 60 (±2)
No. of animals for pathological examination	10 males and 10 females/group	6 males and 6 females/group
Brain fixation	Immersion or perfusion at PND 22	Immersion at PND 11
Brain weight measurement	After fixation at PND 22 and before fixation at study termination	Before fixation at PND 11 and at study termination

Both guidelines recommend the use of rats as preferred test species and the use of 4 dose groups, including the control. The highest dose level should either show evidence of DNT or cause minimal maternal toxicity (eg, reduced weight gain). The lowest dose level should provide a no-observed-adverse-effect level (NOAEL) for both dams and F1 offspring. Each group should consist of 20 or more pregnant females to obtain sufficient numbers of F1 offspring (4 males and 4 females in each litter) to be examined²⁶^,²⁷⁾. As shown in Fig. 1, the test compound is recommended to be given to dams via the route of suspected human exposure from gestation day 6 through lactation day 10 (OPPTS 870.6300) or 21 (TG426). U.S. EPA currently recommends the extension of treatment period to postnatal day (PND) 21 to make it compatible with OECD protocol (internal EPA policy).

Fig. 1.

A flowchart of the DNT study according to recommendations of current test guidelines released from OECD and/or US EPA. Note that postnatal behavioral tests, as well as neuropathological examinations including brain weight measurements, are scheduled at around weaning and at termination of the study.

4.2 Postnatal Behavioral Tests

F1 pups and weanlings in each litter are allocated in one of three cohorts, in principle, for examining above mentioned parameters (functional observations, motor activity, learning and memory, and startle response) at different ages. The timing of measurements is flexible in these guidelines, more so in TG426. This may be welcomed by competent scientists and/or laboratories as it adds some flexibility in arranging limited staff, laboratory areas, and testing equipment. Figure 2 provides examples of some of the specialized testing equipment that require separate and dedicated laboratory space. Examples for arrangement of cohorts for testing are provided in TG426 in detail²⁷⁾. The followings are principle recommendations: pre- and post-weaning F1 offspring should be allocated for multiple conducts of functional observations; motor activity should be measured frequently enough (preferably three times or more) during the pre-weaning period in order to observe the onset of intersession habituation, and before termination; weanlings should be examined for learning and memory and startle response, just after weaning and as adults before study termination (Fig. 1).

Fig. 2.

Examples of apparatuses for postnatal behavioral tests, which are routinely used in our laboratory. A, a computer-assisted system for recording motor activities; B, a computer-assisted system for monitoring auditory startle reactions; C, a water M-maze; D, a computer-assisted system for monitoring passive avoidance.

Methodologies for evaluating the learning abilities and memory are also highly flexible. Representative testing methods include, but are not limited to, passive avoidance, delayed-matching-to-position, olfactory conditioning, Morris water maze, Biel or Cincinnati maze, radial arm maze, T-maze, and retention of schedule-controlled behavior. The requirement is to use these methods to assess both the acquisition (learning) and retention (memory) of behaviors. All of these testing methodologies have been confirmed to be sensitive and reliable⁶⁴^,⁶⁵^,⁶⁶^,⁶⁷^,⁶⁸^,⁶⁹^,⁷⁰^,⁷¹^,⁷²^,⁷³^,⁷⁴^,⁷⁵^,⁷⁶⁾.

The following developmental parameters, common in standard reproductive toxicity studies, are also examined in the standard DNT study: clinical signs and body weights of pups and weanlings, pre-weaning landmarks for physical development such as pinna unfolding, eye opening and incisor eruption, behavioral ontogeny such as righting reflex and negative geotaxis, and sexual maturation. The large number of test methods and extensive age-specific test requirements makes the scale of DNT study one of the most complex among the variety of reproductive and developmental toxicity tests.

4.3 Neuropathology and Brain Morphometrics

The central and peripheral nervous systems (CNS and PNS, respectively) of F1 offspring are recommended to be examined histopathologically and morphometrically²⁶^,²⁷⁾. A cohort of weanlings on PND 22 (OECD TG426) or between PND 11/PND 22 (EPA TG) from each dose group are evaluated after fixation by either immersion or perfusion. F1 young adults at study termination (PND 70) are examined for both CNS and PNS tissues after being fixed by perfusion. Perfusion fixation is preferable for proper brain fixation, yet the methodology may not be applicable for pups at PND 11–22 due to the difficulty in perfusing such small animals⁷⁷⁾. Special attention should be taken at processing of brain samples to avoid shrinkage artifacts by unnecessarily prolonged storage in fixative and/or use of inappropriate embedding materials. The guidelines recommend the use of osmium for post-fixation together with plastic (epoxy) embedding, although paraffin embedding after routine fixation is acceptable²⁶^,²⁷⁾.

Brains of pups and juveniles grow very rapidly in rats, where obvious age-dependent differences are seen in microstructures of the brains, as well as in size and weight (Figs. 3–6). The size of brains is also reported to vary widely among individuals in the same days of age during the lactation period depending on prenatal gestation lengths and/or litter sizes⁷⁸⁾. Thus, neuropathologists need to devise appropriate methodology for sectioning brain tissue with variable sizes at accurately the same plane for neuropathological examinations and morphometrics. One good method involves use of a brain matrix mold with plumb cuts for making sections in the same plane (Figs. 7 and 8). Staining methods recommended for CNS and/or PNS tissues are a myelin stain with luxol fast blue and cresyl violet (Klüver-Barrera’s stain) and a silver stain for visualizing axons (Bielschowsky’s or Bodian’s stain) for animals killed at study termination, as well as a routine hematoxylin and eosin stain for those killed at PND 22 or earlier²⁶^,²⁷⁾. Additional useful staining techniques are glial fibrillary acidic protein (GFAP) or lectin histochemistry, a fluoro-jade stain and a silver stain for characterizing particular types of neuronal alterations such as glial and microglial abnormalities, necrotic neurons and neural degeneration⁷⁹^,⁸⁰^,⁸¹^,⁸²^,⁸³⁾.

Fig. 3.

Postnatal growth and development of brains in young rats. Note age-dependent differences in size, shape and proportion of the cerebrum (red) and cerebellum (blue).

Fig. 4.

Comparison of macroscopic areas (mm²) of dorsal brain surface among young rats in various days of age. Values represent mean ± standard deviation. , males; , females. Note an age-dependent gradual increase in area of both cerebral and cerebellum hemispheres, in which the cerebellum grows more rapidlythan the cerebrum.

Fig. 5.

Progress in myelination at the cerebrum including hippocampus of young rats. Klüver-Barrera’s stain.

Fig. 6.

Age-dependent microscopic characteristics of the cerebral cortex in young rats. Note the development of axons and dendrites to form the network system. Bodian’s stain.

Fig. 7.

A brain matrix mold for cutting various sizes of brain samples at the same plane (A) and examples of planes to be cut in each sample obtained at postnatal day 22 (B) or 70 (C).

All major brain regions in representative sections should be examined microscopically for any neuropathological alterations including cellular damages such as vacuolation, degeneration and necrosis, tissue changes such as gliosis, leukocyte infiltration and cyst formation, and developmental insults²⁶^,²⁷⁾. Pathologists are requested to evaluate neuropathological lesions qualitatively, in which the region and type of each alteration should be identified with the range of severity. A variety of alterations indicative of developmental insult are also to be recorded. These include changes in the shape and size (gross and/or relative) of various brain regions, in age-matched populations of cells and axonal projections, in the degree of proliferation, migration, differentiation and apoptosis of cells, in patterns of myelination, and in particular enlargements of brain ventricles. In our laboratory, 8 sections are routinely made from each brain sample to examine such major regions as olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, tectum, tegmentum, cerebral peduncles, pons, medulla oblongata and cerebellum, in principle (Fig. 8).

Fig. 8.

A set of brain sections for our routine examination. Major regions to be examined in detail include, but are not limited to, olfactory bulbs at level 1, cerebral cortex at levels 2 through 6, corpus callosum at levels 3 and 4, striatum at level 3, hippocampus at levels 4 and 5, thalamus and hypothalamus at level 4, tectum, tegmentum and cerebral peduncles at level 5, pons at levels 6 and 7, cerebellum at level 7, and medulla oblongata at level 8. Lateral, third and fourth ventricles, as well as cerebral aqueduct, are also seen in these sections at levels 3 through 7. Hematoxylin and eosin stain.

Brain morphometric analyses, in which linear and/or areal measurements are commonly made for the specific brain regions of interest, are useful for detecting quantitative differences in volume of the specific brain regions between the control and treated groups⁸⁴^,⁸⁵^,⁸⁶^,⁸⁷^,⁸⁸⁾. The evaluations were shown to be helpful for interpreting the data on brain weights and/or morphology in a model study with methylazoxymethanol (MAM) as a positive control⁸⁹^,⁹⁰⁾. Based on these facts, U.S. EPA recommends estimating the thickness of major layers at representative locations within the neocortex, hippocampus and cerebellum²⁶⁾. Figure 9 illustrates our standard measures according to those proposed by Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology⁹¹⁾ with slight modifications. The measures include the thicknesses of both frontal and parietal cerebral cortex, corpus callosum at 2 different planes and hippocampus, the width of striatum, and the vertical height of cerebellum with a total of 3 brain planes, of which sensitivity and reliability have been confirmed in the previous study with MAM⁸⁹⁾.

Fig. 9.

Examples of brain morphometric parameters. Our parameters consist of the thickness of cerebral cortex at frontal and parietal regions (1 and 2), that of corpus callosum (3) and the width of striatum (4) at level 3, the thicknesses of hippocampus (5) and corpus callosum (6) at level 4, and the vertical height of cerebellum (7) at level 7. Klüver-Barrera’s stain.

Pathologists are clearly aware that neuropathological methods will evolve over time to incorporate new technologies for better characterizing DNT, although the methodologies recommended in the current guidelines are “best practices” at present. The current DNT study guidelines may or may not be revised in the future to include such novel methodologies as real-time measurements of molecular markers (eg, mRNA) and/or brain imaging (eg, MRI). However, many pathologists agree that “qualitative histopathology and limited morphometry of conventionally stained brain tissues from juvenile and young adult rats will remain the cornerstone for assessing the adverse impact of chemicals to the developing mammalian nervous system”⁹¹⁾.

4.4 Evaluation and Interpretation of Study Results

As is true in many other toxicological studies, interpretation of DNT study data must involve expert judgement, as well as adequate statistical and dose-dependency analyses⁶⁷^,⁹²^,⁹³^,⁹⁴^,⁹⁵⁾. The study director and collaborative scientists should discuss the biological significance of their observations: a lack of statistical significance cannot always be a concrete rationale for contradicting treatment-related adverse effects²⁷⁾. Emphasis should be placed whether neurodevelopmental effects were induced in F1 offspring at a certain dose level in the absence of maternal general toxicities or not. However, one must be clear in that evidence of maternal toxicity does not equate to a lack of DNT, but instead like all other confounds means that without additional data, one cannot state equivocally that the DNT was caused by the maternal toxicity. Since DNT studies provide only relatively crude indicators of maternal health (eg, body weight, survival, food and water consumption, clinical observations), lacking additional data, a conservative interpretation for public health protection is that evidence of offspring neurotoxicity should be considered as such, and not a necessarily a direct result of maternal toxicity. The use of a weight-of-evidence-approach is highly recommended⁹⁶⁾.

5. Future DNT Testing Strategies

Although a huge numbers of xenobiotics should be evaluated for potential developmental neurotoxicity⁴^,⁹^,¹⁰⁾, only a limited numbers of chemicals, mostly pesticides, have been tested according to the current DNT study guidelines as described above⁹^,¹⁷^,⁹⁷^,⁹⁸⁾. This is due, at least in part, to the fact that current DNT study guidelines are costly and time-consuming neurotoxic examinations, even if the methodology itself is reliable and powerful⁹^,⁹⁷⁾. It may also be true for many Japanese laboratories that the scale of DNT study is too large to complete with limited numbers of neurotoxicologists and/or relatively small areas for behavioral tests that should be done at approximately the same days of animal age. Because of these reasons, full-scale DNT studies have rarely been conducted in Japan as far as we know.

North American and European regulatory authorities and stakeholders have been making an effort to overcome the problem in DNT risk assessment, a lack of DNT hazard information in many chemicals except for some pesticides. They propose a new paradigm, an integrated testing strategy for assessing DNT, based on a consensus that a large-scale DNT study is not necessarily be routinely conducted due to high costs and the use of large numbers of animals⁹⁸⁾. The strategy consists of in silico and in vitro high-throughput systems to estimate environmental hazards to human health, simple in vivo screening assays with alternative species such as zebrafish, and application of adverse outcome pathway (AOP) concept⁹^,⁹⁷^,⁹⁸^,⁹⁹^,¹⁰⁰^,¹⁰¹⁾.

The first step in this strategy is to screen a large number of chemicals for DNT based on quantitative structure–activity relationship with structure-based in silico models, which is followed by rapid in vitro cell-free and/or cell-based screenings to narrow candidates for further study⁹^,¹⁰⁰⁾. Chemicals categorized into a high-priority group are then examined by a rapid in vivo screening assay with zebrafish for evaluating hazards on CNS and/or PNS of intact organisms¹⁰²⁾. Implementation of these screening systems is expected to be time and cost-efficient since only a limited number of chemicals will be efficiently selected for a definitive test in rats according to current DNT study guidelines.

Generated data sets for DNT may also be incorporated into databases that catalog all available toxicology information; include chemical structure, physical-chemical properties, in vitro screening results, in vivo toxicology assay data with alternatives and traditional neurotoxicity test data with mammals¹⁰³^,¹⁰⁴^,¹⁰⁵⁾. Information from each test system can be mapped to AOPs in the future: sufficient accumulation of data sets will enable us to link the toxic action of prototypic xenobiotics in intact organisms to chemical structure and properties through key events such as initiating molecular event, cellar response and organ response⁹⁹⁾. This in turn allows us to draw a new vision of toxicity testing where toxic profiles of unknown chemicals including DNT will be predicted through a series of in silico and in vitro screenings that requires less use of experimental animals.

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