Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

The embryonic zebrafish is a useful vertebrate model for assessing the effects of substances on growth and development. However, cross-laboratory developmental toxicity outcomes can vary and reported developmental defects in zebrafish may not be directly comparable between laboratories. To address these limitations for gaining broader adoption of the zebrafish model for toxicological screening, we established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) program to investigate how experimental protocol differences can influence chemical-mediated effects on developmental toxicity (i.e., mortality and the incidence of altered phenotypes). As part of SEAZIT, three laboratories were provided a common and blinded dataset (42 substances) to evaluate substance-mediated effects on developmental toxicity in the embryonic zebrafish model. To facilitate cross-laboratory comparisons, all the raw experimental data were collected, stored in a relational database, and analyzed with a uniform data analysis pipeline. Due to variances in laboratory-specific terminology for altered phenotypes, we utilized ontology terms available from the Ontology Lookup Service (OLS) for Zebrafish Phenotype to enable additional cross-laboratory comparisons. In this manuscript, we utilized data from the first phase of screening (dose range finding, DRF) to highlight the methodology associated with the development of the database and data analysis pipeline, as well as zebrafish phenotype ontology mapping.

Tris(chloropropyl) phosphate (TCPP) is a member of organophosphate flame retardants used commonly as a replacement for polybrominated diphenyl ethers in consumer and commercial products. Flame retardants have been shown to modulate immune function in vivo and in vitro and there is evidence that at least some related compounds such as organophosphate pesticides can cause developmental immunotoxicity. Developmental immunotoxicology studies were conducted by administering 0, 2500, 5000, or 10,000 ppm TCPP in feed to pregnant Hsd:Sprague Dawley SD rats from gestation day 6 through weaning on postnatal day 28. Feed exposure to TCPP was continued in the F1 offspring until terminal euthanasia at ∼16 to 21 weeks of age when assessments for developmental immunotoxicity were conducted. Innate, humoral, and cell-mediated immune function were assessed in the F1 adults. The antibody-forming cells (AFCs) response to sheep red blood cells was reduced in male and female F1 rats in the 10,000 ppm treatment group but coincided with reduced bodyweights. The AFC response was also significantly reduced in male rats exposed to 5000 ppm where only moderate effects on bodyweights occurred. TCPP exposure affected baseline T-cell proliferation without stimulation; however, the relevance of this change for immunotoxicity risk is unknown. TCPP exposure did not affect cytotoxic T-lymphocyte activity. Only minor and inconsistent treatment-related effects on hematology, innate NK cell function, and immune cell population distributions in the spleen were observed. Taken together, these data indicate that TCPP has the potential to impact humoral immune responses following developmental exposure.