Tris(1-chloro-2-propyl) phosphate (TCPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are organic flame retardants detected in the environment and biota for which avian toxicological data are limited. In this study, domestic chicken eggs were injected with TCPP or TDCPP (maximum dose = 51,600 and 45,000ng/g egg, respectively) to determine dose-dependent effects on pipping success, development, hepatic messenger RNA (mRNA) expression levels of genes associated with xenobiotic metabolism and the thyroid hormone (TH) pathway, and TH levels following 20-22 days of incubation. Neither compound reduced pipping success; however, TCPP significantly delayed pipping at 9240 and 51,600ng/g and reduced tarsus length at 51,600ng/g. TDCPP exposure resulted in significant decreases in head plus bill length, embryo mass, and gallbladder size at 45,000ng/g and reduced plasma free T4 levels at 7640ng/g. Type I deiodinase, liver fatty acid-binding protein, and cytochrome P450 (CYP) 3A37 mRNA levels were significantly induced by TCPP, whereas TDCPP induced CYP3A37 and CYP2H1. Chemical analysis of egg contents at incubation days 0, 5, 11, 18, and 19 revealed that > 92% of the injected TCPP or TDCPP concentration was detectable up to day 5; however, < 1% was detected by day 19. The observed phenotypic responses to TCPP and TDCPP exposure may be associated with disruption of the TH axis, which is critical for normal growth and development in birds. The effects of TDCPP on the gallbladder indicate that the disturbance of lipid metabolism is a likely mechanism of toxicity.

Risk assessors are challenged with the task of providing data for an increasing number of priority chemicals. High-throughput toxicity screening methods--which permit rapid determination of toxic, molecular, and/or biochemical effects of a wide range of chemicals--are essential to help meet this demand. The avian embryonic hepatocyte in vitro screening method has been utilized in the authors' laboratory to assess the effects of a wide range of environmental contaminants on cytotoxicity and mRNA expression of genes associated with xenobiotic metabolism, the thyroid hormone pathway, lipid metabolism, and growth. Sixteen structurally variable organic flame retardants (OFRs)--including tetrabromoethylcyclohexane (TBECH), tris(2-butoxyethyl) phosphate (TBEP), tricresyl phosphate (TCP), and tris(1,3-dichloro-2-propyl) phosphate (TDCPP)--were screened using the in vitro method in the present study. Hepatocytes from 2 avian species, chicken and herring gull, were prepared, and species differences in hepatocyte viability were observed for several OFRs. For example, TCP was not cytotoxic in chicken hepatocytes up to the highest concentration tested (300 µM), whereas the median lethal concentration (LC50) was 31.2 µM in herring gull hepatocytes. Effects on mRNA expression in chicken embryonic hepatocytes were determined using a 3 × 32 custom-made Avian ToxChip polymerse chain reaction array and were variable among OFRs; TCP, TDCPP, and tris(2,3-dibromopropyl) isocyanurate showed the most significant alterations among the target genes assessed. Overall, this rapid screening method helped prioritize OFRs for further assessment. For example, OFRs that elicited significant effects on cytoxicity or mRNA expression represent prime candidates for egg injection studies that determine adverse effects on the whole animal but are more costly in terms of time, money, and embryo utilization.

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and tris(1-chloropropyl) phosphate (TCPP) belong to a group of chemicals collectively known as triester organophosphate flame retardants (OPFRs). OPFRs are used in a wide range of consumer products and have been detected in biota, including free-living avian species; however, data on toxicological and molecular effects of exposure are limited. An in vitro screening approach was used to compare concentration-dependent effects of TDCPP and TCPP on cytotoxicity and messenger RNA (mRNA) expression in cultured hepatocytes and neuronal cells derived from embryonic chickens. TDCPP was toxic to hepatocytes (LC₅₀ = 60.3 ± 45.8μM) and neuronal cells (LC₅₀ = 28.7 ± 19.1μM), whereas TCPP did not affect viability in either cell type up to the highest concentration administered, 300μM. Real-time reverse transcription-PCR revealed alterations in mRNA abundance of genes associated with phase I and II metabolism, the thyroid hormone (TH) pathway, lipid regulation, and growth in hepatocytes. None of the transcripts measured in neuronal cells (D2, D3, RC3, and Oct-1) varied in response to TDCPP or TCPP exposure. Exposure to ≥ 10μM TDCPP and TCPP resulted in significant upregulation of CYP2H1 (4- to 8-fold), CYP3A37 (13- to 127-fold), and UGT1A9 (3.5- to 7-fold) mRNA levels. Transthyretin was significantly downregulated more than twofold by TCPP at 100μM; however, TDCPP did not alter its expression. Liver fatty acid-binding protein, TH-responsive spot 14-α, and insulin-like growth factor-1 were all downregulated (up to 10-fold) in hepatocytes exposed to ≥ 0.01μM TDCPP and TCPP. Taken together, our results indicate that genes associated with xenobiotic metabolism, the TH pathway, lipid regulation, and growth are vulnerable to TDCPP and TCPP administration in cultured avian hepatocytes. The mRNA expression data were similar to those from a previous study with hexabromocyclododecane.

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