Toxicity testing is moving from animal-based studies to faster, more ethical in vitro approaches that focus on mechanistic toxicology. The use of bisphenol A (BPA) replacement compounds is increasing and there is limited toxicity data available for these compounds in avian species. The overall goals of this thesis were to: a) determine if avian cell lines are suitable alternatives to primary hepatocytes for chemical screening; b) generate toxicity data for five BPA replacement compounds: bis(4-hydroxyphenyl)methane (BPF), bis(3-allyl-4-hydroxyphenyl)sulfone (TGSH), 7-bis(4-hydroxyphenylthio)-3,5-dioxaheptane (DD-70), 2,2-bis(4-hydroxyphenyl)hexafluoropropane (BPAF) and 4-hydroxyphenyl 4-isoprooxyphenylsulfone (BPSIP) in three in vitro models: primary chicken embryonic hepatocytes (CEH), double-crested cormorant (DCCO) embryonic hepatocytes (DCEH) and chicken LMH cell line; and 3) prioritize two replacements for early-life stage testing (ELS). LMH cells cultured as 3D spheroids, as opposed to 2D monolayer, had enhanced mRNA expression and CYP1A activity and were therefore used for screening. Additionally, an immortalized DCCO hepatic cell line, DCH22, was established, which may be useful for future avian toxicity testing. DD-70 and BPAF were the most cytotoxic across the three in vitro models. TGSH and DD-70 altered expression of genes associated with multiple toxicity pathways, but not estrogen response, and are potential non-estrogenic replacements. BPAF, BPF and BPSIP are potential estrogenic replacements. In general, the replacements were more cytotoxic and/or transcriptionally active than BPA. There was species-specific variability in toxicity; the replacements were more transcriptionally active in CEH compared to DCEH. LMH spheroids were more sensitive to estrogenic endpoints than CEH. DD-70 and BPAF were prioritized for ELS studies based on in vitro results. All of the replacements modulated the expression of genes related to bile acid regulation in vitro and an increase in gallbladder mass was observed in chicken embryos after exposure to DD-70 or BPAF. Overall, this thesis evaluated the utility of LMH cells cultured as spheroids as an animal free alternative for chemical screening, established a DCCO cell line, and generated novel cytotoxicity and gene expression data for five BPA replacement compounds in three in vitro avian models and determined ELS toxicity of two replacement compounds.

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General toxicity evaluation and risk assessment for human exposure is essential when developing new pharmaceuticals and chemicals. Developmental toxicology is an important part of this risk assessment which consumes large resources and many laboratory animals. The prediction of developmental toxicity could potentially be assessed in vitro using embryo-derived pluripotent stem cells for lead characterization and optimization. This thesis explored the potential of short-time assays with pluripotent stem cells of embryonic origin using toxicogenomics. Three established pluripotent stem cell lines; P19 mouse embryonal carcinoma (EC) cells, R1 mouse embryonic stem (mES) cells, and SA002 human embryonic stem (hES) cells were used in the studies. Valproic acid (VPA), an antiepileptic drug which can cause the neural tube defects spina bifida in human and exencephaly in mouse, was used together with microarrays to investigate the global transcriptional response in pluripotent stem cells using short-time exposures (1.5 - 24 h). In addition to VPA, three closely related VPA analogs were tested, one of which was not teratogenic in mice. These analogs also differed in their ability to inhibit histone deacetylase (HDAC) allowing this potential mechanism of VPA teratogenicity to be investigated. The results in EC cells indicated a large number of genes to be putative VPA targets, many of which are known to be involved in neural tube morphogenesis. When compared with data generated in mouse embryos, a number of genes emerged as candidate in vitro markers of VPA-induced teratogenicity. VPA and its teratogenic HDAC inhibiting analog induced major and often overlapping deregulation of genes in mES cells and hES cells. On the other hand, the two non-HDAC inhibiting analogs (one teratogenic and one not) had only minor effects on gene expression. This indicated that HDAC inhibition is likely to be the major mechanism of gene deregulation induced by VPA. In addition, a comparison between human and mouse ES cells revealed an overlap of deregulated genes as well as species specific deregulated genes.