Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here, we evaluated transcriptomic dose responses using RNA-sequencing in paired FFPE and frozen (FROZ) samples from 2 archival studies in mice, one <2 years old and the other >20 years old. Experimental treatments included 3 different doses of di(2-ethylhexyl)phthalate or dichloroacetic acid for the recently archived and older studies, respectively. Total RNA was ribo-depleted and sequenced using the Illumina HiSeq platform. In the recently archived study, FFPE samples had 35% lower total counts compared to FROZ samples but high concordance in fold-change values of differentially expressed genes (DEGs) (r =0.99), highly enriched pathways (90% overlap with FROZ), and benchmark dose estimates for preselected target genes (<5% difference vs FROZ). In contrast, older FFPE samples had markedly lower total counts (3% of FROZ) and poor concordance in global DEGs and pathways. However, counts from FFPE and FROZ samples still positively correlated (r = 0.84 across all transcripts) and showed comparable dose responses for more highly expressed target genes. These findings highlight potential applications and issues in using RNA-sequencing data from FFPE samples. Recently archived FFPE samples were highly similar to FROZ samples in sequencing quality metrics, DEG profiles, and dose-response parameters, while further methods development is needed for older lower-quality FFPE samples. This work should help advance the use of archival resources in chemical safety and translational science.

Brown (2017): Evolution of chemical-specific adjustment factors (CSAF) based on recent international experience; increasing utility and facilitating regulatory acceptance, Critical Reviews in Toxicology, DOI: 10.1080DOI: 10. /10408444.2017 The application of chemical-specific toxicokinetic or toxicodynamic data to address interspecies differences and human variability in the quantification of hazard has potential to reduce uncertainty and better characterize variability compared with the use of traditional default or categorically-based uncertainty factors. The present review summarizes the state-of-the-science since the introduction of the World Health Organization/International Programme on Chemical Safety (WHO/IPCS) guidance on chemical-specific adjustment factors (CSAF) in 2005 and the availability of recent applicable guidance including the WHO/IPCS guidance on physiologically-based pharmacokinetic (PBPK) modeling in 2010 as well as the U.S. EPA guidance on data-derived extrapolation factors in 2014. A summary of lessons learned from an analysis of more than 100 case studies from global regulators or published literature illustrates the utility and evolution of CSAF in regulatory decisions. Challenges in CSAF development related to the adequacy of, or confidence in, the supporting data, including verification or validation of PBPK models. The analysis also identified issues related to adequacy of CSAF documentation, such as inconsistent terminology and often limited and/or inconsistent reporting, of both supporting data and/or risk assessment context. Based on this analysis, recommendations for standardized terminology, documentation and relevant interdisciplinary research and engagement are included to facilitate the continuing evolution of CSAF development and guidance.ARTICLE HISTORY

1,2-Cyclohexanedicarboxylic acid, 1,2-diisononylester (DINCH), a polyvinyl chloride plasticizer, has food, beverage, and medical device applications that may result in general population exposure. Although no apparent toxicity information in humans was identified, there is a substantial data set in lab animals to serve as the basis of hazard identification for DINCH. Target tissues associated with repeated dietary DINCH exposure in lab animals included liver, kidney, and thyroid and mammary glands. In contrast to some phthalate ester plasticizers, DINCH did not show evidence of hepatic peroxisomal proliferation, testicular toxicity, or liver tumors in rats. Liver and thyroid effects associated with DINCH exposure were attributed to compensatory thyroid stimulation secondary to prolonged metabolic enzyme induction. The toxicological significance of mammary fibroadenomas in female rats is unclear, given that this common benign and spontaneously occurring tumor type is unique to rats. The weight of evidence suggests DINCH is not genotoxic and the proposed mode of action (MOA) for thyroid gland lesions was considered to have a threshold. No adverse reproductive effects were seen in a two-generation study. An oral reference dose (RfD) of 0.7 mg/kg-d was derived from a human equivalent BMDL₁₀ of 21 mg/kg-d for thyroid hypertrophy/hyperplasia seen in adult F₁ rats also exposed in utero. The total uncertainty factor of 30x was comprised of intraspecies (10×) and database (3×) factors. An interspecies extrapolation factor was not applied since rodents are more sensitive than humans with respect to the proposed indirect MOA for thyroid gland lesions.

Polyethylene terephthalate, a copolymer of terephthalic acid (TPA) or dimethyl terephthalate (DMT) with ethylene glycol, has food, beverage, and drinking water contact applications. Di-2-ethylhexyl terephthalate (DEHT) is a plasticizer in food and drinking water contact materials. Oral reference doses (RfDs) and total allowable concentrations (TACs) in drinking water were derived for TPA, DMT, and DEHT. Category RfD and TAC levels were also established for nine C(1)-C(8) terephthalate esters. The mode of action of TPA, and of DMT, which is metabolized to TPA, involves urinary acidosis, altered electrolyte elimination and hypercalciuria, urinary supersaturation with calcium terephthalate or calcium hydrogen terephthalate, and crystallization into bladder calculi. Weanling rats were more sensitive to calculus formation than dams. Calculi-induced irritation led to bladder hyperplasia and tumors in rats fed 1000 mg/kg-day TPA. The lack of effects at 142 mg/kg-day supports a threshold for urine saturation with calcium terephthalate, a key event for calculus formation. Chronic dietary DMT exposure in rodents caused kidney inflammation, but not calculi. Chronic dietary DEHT exposure caused general toxicity unrelated to calculi, although urine pH was reduced suggesting the TPA metabolite was biologically-active, but of insufficient concentration to induce calculi. Respective oral reference doses of 0.5, 0.5, and 0.2 mg/kg-day and total allowable drinking water concentrations of 3, 3, and 1 mg/L were derived for TPA, DMT, and DEHT. An oral RfD of 0.2 mg/kg-day for the terephthalate category chemicals corresponded to a drinking water TAC of 1 mg/L.

Low-dose extrapolation and dose-related transitions are paramount in the ongoing debate regarding the quantification of cancer risks for nongenotoxic carcinogens. Phenobarbital (PB) is a prototypical nongenotoxic carcinogen that activates the constitutive androstane receptor (CAR) resulting in rodent liver tumors. In this study, male and female CD-1 mice administered dietary PB at 0, 0.15, 1.5, 15, 75, or 150 mg/kg-day for 2 or 7 days to characterize multiple apical and molecular endpoints below, at (∼75 mg/kg-day), and above the carcinogenic dose level of PB and examine these responses using benchmark dose modeling. Linear toxicokinetics were observed for all doses. Increased liver weight, hepatocellular hypertrophy, and mitotic figures were seen at 75 and 150 mg/kg-day. CAR activation, based on Cyp2b qPCR and pentoxyresorufin dealkylase activity, occurred at doses ≥ 1.5 mg/kg-day. The no-observable transcriptional effect level for global gene expression was 15 mg/kg-day. At 2 days, several xenobiotic metabolism and cell protective pathways were activated at lower doses and to a greater degree in females. However, hepatocellular proliferation, quantified by bromodeoxyuridine immunohistochemistry, was the most sensitive indicator of PB exposure with female mice more sensitive than males, contrary to sex-specific differences in sensitivity to hepatocarcinogenesis. Taken together, the identification of low-dose cellular and molecular transitions in the subtumorigenic dose range aids the understanding of early key events in CAR-mediated hepatocarcinogenesis.