An expert-driven literature review of "negative" chemicals for developmental neurotoxicity (DNT) in vitro assay evaluation.

To date, approximately 200 chemicals have been tested in US Environmental Protection Agency (EPA) or Organization for Economic Co-operation and Development (OECD) developmental neurotoxicity (DNT) guideline studies, leaving thousands of chemicals without traditional animal information on DNT hazard potential. To address this data gap, a battery of in vitro DNT new approach methodologies (NAMs) has been proposed. Evaluation of the performance of this battery will increase the confidence in its use to determine DNT chemical hazards. One approach to evaluate DNT NAM performance is to use a set of chemicals to evaluate sensitivity and specificity. Since a list of chemicals with potential evidence of in vivo DNT has been established, this study aims to develop a curated list of "negative" chemicals for inclusion in a "DNT NAM evaluation set". A workflow, including a literature search followed by an expert-driven literature review, was used to systematically screen 39 chemicals for lack of DNT effect. Expert panel members evaluated the scientific robustness of relevant studies to inform chemical categorizations. Following review, the panel discussed each chemical and made categorical determinations of "Favorable", "Not Favorable", or "Indeterminate" reflecting acceptance, lack of suitability, or uncertainty given specific limitations and considerations, respectively. The panel determined that 10, 22, and 7 chemicals met the criteria for "Favorable", "Not Favorable", and "Indeterminate", for use as negatives in a DNT NAM evaluation set. Ultimately, this approach not only supports DNT NAM performance evaluation but also highlights challenges in identifying large numbers of negative DNT chemicals.

Comparison of chemical-induced changes in proliferation and apoptosis in human and mouse neuroprogenitor cells.

There is a need to develop rapid and efficient models to screen chemicals for their potential to cause developmental neurotoxicity. Use of in vitro neuronal models, including human cells, is one approach that allows for timely, cost-effective toxicity screening. The present study compares the sensitivity of human (ReN CX) and mouse (mCNS) neuroprogenitor cell lines to chemicals using a multiplex assay for proliferation and apoptosis, endpoints that are critical for neural development. Cells were exposed to 0.001-100 µM concentrations of 11 chemicals (cadmium, chlorpyrifos oxon, dexamethasone, dieldrin, ketamine, lead, maneb, methylmercury, nicotine, trans-retinoic acid, and trimethyltin) reported in the literature to affect proliferation and/or apoptosis, and 5 chemicals (dimethyl pthalate, glyphosate, omeprazole, saccharin, and d-sorbitol) with no reports of effects on either endpoint. High-content screening of markers for proliferation (BrdU incorporation) and apoptosis (activated caspase 3 and p53) was used to assess the effect of chemicals in both cell lines. Of the chemicals tested, methylmercury, cadmium, dieldrin, chlorpyrifos oxon, trans-retinoic acid, and trimethyltin decreased proliferation by at least 50% of control in either the ReN CX or mCNS cells. None of the chemicals tested activated caspase 3 or p53 in the ReN CX cells, while methylmercury, cadmium, dieldrin, chlorpyrifos oxon, trimethyltin, and glyphosate all induced at least a doubling in these apoptotic markers in the mCNS cells. Compared to control, cadmium, trans-retinoic acid, and trimethyltin decreased cell viability (ATP levels) by at least 50% in the ReN CX cells, while cadmium, dieldrin, and methylmercury decreased viability by at least 50% in the mCNS cells. Based on these results, BrdU is an appropriate marker for assessing chemical effects on proliferation, and human cells are more sensitive than mouse cells for this endpoint. By contrast, caspase 3 and p53 were altered by environmental chemicals in mouse, but not in human cells. Therefore, these markers are not appropriate to assess the ability of environmental chemicals to induce apoptosis in the ReN CX cells.