Scientific and Regulatory Policy Committee Best Practices: Recommended ("Best") Practices for Informed (Non-blinded) Versus Masked (Blinded) Microscopic Evaluation in Animal Toxicity Studies.

This article describes the Society of Toxicologic Pathology's (STP) five recommended ("best") practices for appropriate use of informed (non-blinded) versus masked (blinded) microscopic evaluation in animal toxicity studies intended for regulatory review. (1) Informed microscopic evaluation is the default approach for animal toxicity studies. (2) Masked microscopic evaluation has merit for confirming preliminary diagnoses for target organs and/or defining thresholds ("no observed adverse effect level" and similar values) identified during an initial informed evaluation, addressing focused hypotheses, or satisfying guidance or requests from regulatory agencies. (3) If used as the approach for an animal toxicity study to investigate a specific research question, masking of the initial microscopic evaluation should be limited to withholding only information about the group (control or test article-treated) and dose equivalents. (4) The decision regarding whether or not to perform a masked microscopic evaluation is best made by a toxicologic pathologist with relevant experience. (5) Pathology peer review, performed to verify the microscopic diagnoses and interpretations by the study pathologist, should use an informed evaluation approach. The STP maintains that implementing these five best practices has and will continue to consistently deliver robust microscopic data with high sensitivity for animal toxicity studies intended for regulatory review. Consequently, when conducting animal toxicity studies, the advantages of informed microscopic evaluation for maximizing sensitivity outweigh the perceived advantages of minimizing bias through masked microscopic examination.

Novel Bacterial Topoisomerase Inhibitor Gepotidacin Demonstrates Absence of Fluoroquinolone-Like Arthropathy in Juvenile Rats.

Fluoroquinolone use in children is limited due to its potential toxicity and negative effects on skeletal development, but the actual effects/risks of fluoroquinolones on bone growth and the mechanisms behind fluoroquinolone-driven arthropathy remain unknown. Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibiotic with a unique mechanism of action that is not anticipated to have the same risks to bone growth as those of fluoroquinolones. Gepotidacin is in phase III clinical development for uncomplicated urinary tract infections (ClinicalTrials.gov identifiers NCT04020341 and NCT04187144) and urogenital gonorrhea (ClinicalTrials.gov identifier NCT04010539) in adults and adolescents ≥12 years of age. To inform arthropathy and other potential toxicity risks of gepotidacin in pediatric studies, this nonclinical study assessed oral gepotidacin toxicity in juvenile rats from postnatal day (PND) 4 to PND 32/35 (approximately equivalent to human ages from newborn to 11 years), using both in-life assessments (tolerability, toxicity, and toxicokinetics) and terminal assessments (necropsy with macroscopic and microscopic skeletal femoral head and/or stifle joint examinations). Gepotidacin doses of ≤300 mg/kg of body weight/day were well tolerated from PND 4 to PND 21, and higher doses of ≤1,250 mg/kg/day were well tolerated from PND 22 when the dose levels were escalated to maintain systemic exposure levels up to PND 35, with no observed treatment-related clinical signs, effects on mean body weight gain, or macroscopic findings on articular surfaces. A dose of 1,000 mg/kg/day was not tolerated during the dosing period from PND 4 to 21, with effects on body weight gain, fecal consistency, and body condition. Microscopic effects on articular surfaces were evaluated after 32 days of gepotidacin treatment at the highest tolerated dose. After 32 days of treatment with the highest tolerated gepotidacin dose of 300/1,250 mg/kg/day (systemic concentrations [area under the curve {AUC} values] of 93.7 µg · h/mL [males] and 121 µg · h/mL [females]), no skeletal effects on articular surfaces of the femoral head or stifle joint were observed. The absence of treatment-related clinical signs and arthropathy in juvenile rats provides evidence to support the potential future use of gepotidacin in children.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Dog.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.asp) is a joint initiative of the societies of toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in most tissues and organs from the dog used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions, lesions induced by exposure to test materials, and relevant infectious and parasitic lesions. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Opinion on Current Use of Non-Blinded Versus Blinded Histopathologic Evaluation in Animal Toxicity Studies.

The Society of Toxicologic Pathology (STP) explored current institutional practices for selecting between non-blinded versus blinded histopathologic evaluation during Good Laboratory Practice (GLP)-compliant, regulatory-type animal toxicity studies using a multi-question survey and STP-wide discussion (held at the 2019 STP annual meeting). Survey responses were received from 107 individuals representing 83 institutions that collectively employ 589 toxicologic pathologists. Most responses came from industry (N = 46, mainly biopharmaceutical or contract research organizations) and consultants (N = 24). For GLP-compliant animal toxicity studies, histopathologic evaluation usually involves initial (primary) non-blinded analysis, with post hoc informal blinded re-examination at the study pathologist's discretion to confirm subtle findings or establish thresholds. Initial blinded histopathologic evaluation sometimes is chosen by study pathologists to test formal hypotheses and/or by sponsors to address non-pathologist expectations about histopathology data objectivity. Current practice is that a blinded histopathologic evaluation is documented only if formal blinding (ie, using slides with coded labels) is employed, using simple statements without detailed methodology in the study protocol (or an amendment) and/or pathology report. Blinding is not an appropriate strategy for the initial histopathologic evaluation performed during pathology peer reviews of GLP-compliant animal toxicity studies. [Box: see text].

Scientific Regulatory Policy Committee Points to Consider*: Nuisance Factors, Block Effects, and Batch Effects in Nonclinical Safety Assessment Studies.

Detection of test article-related effects and the determination of the adversity of those changes are the primary goals of nonclinical safety assessment studies for drugs and chemicals in development. During these studies, variables that are not of primary interest to investigators may change and influence data interpretation. These variables, often referred to as "nuisance factors," may influence other groups of data and result in "block or batch effects" that complicate data interpretation. Definitions of the terms "nuisance factors," "block effects," and "batch effects," as they apply to nonclinical safety assessment studies, are reviewed. Multiple case examples of block and batch effects in safety assessment studies are provided, and the challenges these bring to pathology data interpretation are discussed. Methods to mitigate the occurrence of block and batch effects in safety assessment studies, including statistical blocking and utilization of study designs that minimize potential confounding variables, incorporation of adequate randomization, and use of an appropriate number of animals or repeated measurement of specific parameters for increased precision, are reviewed. [Box: see text].

An Integrated Multi-Omics Analysis Defines Key Pathway Alterations in a Diet-Induced Obesity Mouse Model.

Obesity is a multifactorial disease with many complications and related diseases and has become a global epidemic. To thoroughly understand the impact of obesity on whole organism homeostasis, it is helpful to utilize a systems biological approach combining gene expression and metabolomics across tissues and biofluids together with metagenomics of gut microbial diversity. Here, we present a multi-omics study on liver, muscle, adipose tissue, urine, plasma, and feces on mice fed a high-fat diet (HFD). Gene expression analyses showed alterations in genes related to lipid and energy metabolism and inflammation in liver and adipose tissue. The integration of metabolomics data across tissues and biofluids identified major differences in liver TCA cycle, where malate, succinate and oxaloacetate were found to be increased in HFD mice. This finding was supported by gene expression analysis of TCA-related enzymes in liver, where expression of malate dehydrogenase was found to be decreased. Investigations of the microbiome showed enrichment of Lachnospiraceae, Ruminococcaceae, Streptococcaceae and Lactobacillaceae in the HFD group. Our findings help elucidate how the whole organism metabolome and transcriptome are integrated and regulated during obesity.

Alisertib plus induction chemotherapy in previously untreated patients with high-risk, acute myeloid leukaemia: a single-arm, phase 2 trial.

BACKGROUND: Increased aurora A kinase (AAK) expression occurs in acute myeloid leukaemia; AAK inhibition is a promising therapeutic target in this disease. We therefore aimed to assess the activity of alisertib combined with 7 + 3 induction chemotherapy in previously untreated patients with high-risk acute myeloid leukaemia. METHODS: We did a single-arm, phase 2 trial of patients recruited from the Dana-Farber/Harvard Cancer Center in the USA. Eligible patients had previously untreated acute myeloid leukaemia, an Eastern Cooperative Oncology Group performance status of 0-2, and were at high risk of disease as defined by the presence of an adverse-risk karyotype, the presence of secondary acute myeloid leukaemia arising from previous myelodysplastic syndrome or myeloproliferative neoplasm, the presence of therapy-related acute myeloid leukaemia, or being 65 years or older. Enrolled patients received 7 + 3 induction chemotherapy of continuous infusion of cytarabine (100 mg/m2 per day on days 1-7) and intravenous bolus of idarubicin (12 mg/m2 per day on days 1-3). Oral alisertib (30 mg) was given twice per day on days 8-15. Patients could receive up to four consolidation cycles with cytarabine and alisertib, and alisertib maintenance for 12 months. The primary endpoint was a composite including the proportion of patients achieving complete remission and those with a complete remission with incomplete neutrophil or platelet count recovery. Analyses were per-protocol. This study is registered with Clinicaltrials.gov, number NCT02560025, and has completed enrolment. FINDINGS: Between Dec 31, 2015, and Aug 1, 2017, we enrolled a total of 39 eligible patients. 19 (49%) of 39 patients had secondary acute myeloid leukaemia and three (8%) had therapy-related acute myeloid leukaemia. At mid-induction, 33 (85%) of 39 patients showed marrow aplasia, six (15%) received re-induction. The median follow-up was 13·7 months (IQR 12·7-14·4). Composite remission was 64% (two-stage 95% CI 48-79), with 20 (51%) of 39 patients achieving complete remission and five (13%) achieving complete remission with incomplete neutrophil or platelet count recovery. The most common grade 3 or 4 adverse events included febrile neutropenia (16 [41%] of 39), neutropenia (12 [31%]), thrombocytopenia (13 [33%]), anaemia (11 [28%]), anorexia (nine [23%]), and oral mucositis (four [10%]). No treatment-related deaths were observed. INTERPRETATION: These results suggest that alisertib combined with induction chemotherapy is active and safe in previously untreated patients with high-risk acute myeloid leukaemia. This study met criteria to move forward to a future randomised trial. FUNDING: Millennium Pharmaceuticals.

A phase 1 study of the antibody-drug conjugate brentuximab vedotin with re-induction chemotherapy in patients with CD30-expressing relapsed/refractory acute myeloid leukemia.

BACKGROUND: Outcomes for patients with relapsed/refractory acute myeloid leukemia (R/R AML) remain poor. Novel therapies specifically targeting AML are of high interest. Brentuximab vedotin (BV) is an antibody-drug conjugate that is specific for human CD30. In this phase 1 dose escalation study, the authors evaluated the safety of BV combined with mitoxantrone, etoposide, and cytarabine (MEC) re-induction chemotherapy for patients with CD30-expressing R/R AML. METHODS: Using a standard dose escalation design, the authors evaluated 3 dose levels of BV (0.9 mg/kg, 1.2 mg/kg, and 1.8 mg/kg) administered once on day 1 followed by MEC on days 3 through 7. RESULTS: There were no dose-limiting toxicities noted and the maximum tolerated dose was not reached. The recommended phase 2 dose of BV was determined to be 1.8 mg/kg when combined with MEC. The side effect profile was similar to that expected from MEC chemotherapy alone, with the most common grade ≥3 toxicities being febrile neutropenia, thrombocytopenia, and anemia (toxicities were graded using the National Cancer Institute Common Terminology Criteria for Adverse Events [version 4.0]). Among the 22 patients enrolled on the trial, the composite response rate was 36%, with a composite response rate of 42% noted among those who received the highest dose of BV. The median overall survival was 9.5 months, with a median disease-free survival of 6.8 months observed among responders. Approximately 55% of patients were able to proceed with either allogeneic hematopoietic stem cell transplantation or donor lymphocyte infusion. CONCLUSIONS: The combination of BV with MEC was found to be safe in patients with CD30-expressing R/R AML and warrants further study comparing this combination with the use of MEC alone in this population (ClinicalTrials.gov identifier NCT01830777). LAY SUMMARY: The outcomes for patients with relapsed/refractory acute myeloid leukemia (R/R AML) are exceptionally poor. New and emerging treatment combinations are actively being studied in an effort to improve outcomes. The authors examined the combination of brentuximab vedotin, an antibody product that recognizes a marker called CD30, with mitoxantrone, etoposide, and cytarabine (MEC), a common chemotherapy regimen, in patients with R/R AML that expressed the CD30 marker. The authors found that the combination was safe and well tolerated. Future studies comparing this new combination with the use of MEC alone can help to inform its effectiveness for this patient population.

Nutrient composition and safety evaluation of simulated isobutanol distillers dried grains with solubles and associated fermentation metabolites when fed to male Ross 708 broiler chickens (Gallus domesticus).

Saccharomyces cerevisiae genetically engineered to enhance butanol production will be used in a manufacturing process similar to that of fuel ethanol production, including co-production of distillers products for animal feed. A poultry feeding trial was conducted with simulated isobutanol-derived dried distillers grains with solubles (bDDGS), comprising non-fermentable corn solids and heat-inactivated Butamax modified yeast (BMY), to determine potential health effects. Simulated dried distillers grains were produced in 2 variants: bDDGS containing 10% (B10) or 50% (B50) BMY. The BMY concentrations were selected based on a conservative estimate from ethanol-derived distillers grains (eDDGS) approximating 2.5 and 12-fold margins of exposure. The B10 and B50 DDGS were evaluated in a 42-day feeding trial using male Ross 708 broiler chickens fed diets containing eDDGS, B50 DDGS, or B10 DDGS without or with isobutanol, 2,3-butanediol, and isobutyric acid metabolites each at target concentrations of 2 (B10-2), 5 (B10-5), or 10 (B10-10) times the anticipated specification limit in the commercial product. Diets were fed (n = 50 broilers/treatment) in 3 phases: starter phase with 8% DDGS and grower and finisher phases each with 15% DDGS. No statistically significant differences or diet-related effects on mortality, clinical pathology, or organ weights, and no microscopic observations associated with consumption of diets containing B10, B50, or B10 supplemented with metabolites at any targeted exposure level were observed. A lower (P < 0.05) mean absolute bursa of Fabricius weight in the B10-10 group compared to the B10 group was considered to be within the range of biological variability. A non-significant trend toward lower weight, gains, and feed intake, and higher feed:gain ratio was observed in the B10-10 group, and was considered a non-adverse palatability effect of consuming high concentrations of metabolites. These results demonstrate that consumption of phase diets containing simulated DDGS from a novel isobutanol production process was well-tolerated.

A GNPTAB nonsense variant is associated with feline mucolipidosis II (I-cell disease).

BACKGROUND: Mucolipidosis II (ML II; I-cell disease) is caused by a deficiency of N-acetylglucosamine-1-phosphotransferase (GNPTAB; EC 2.7.8.17), which leads to a failure to internalize acid hydrolases into lysosomes for proper catabolism of various substances. This is an autosomal recessive lysosomal storage disease and causes severe progressive neuropathy and oculoskeletal dysfunction in humans (OMIM 252500). A naturally occurring disease model has been reported in juvenile domestic cats (OMIA 001248-9685) with clinical signs similar to human patients. We investigated the molecular genetic basis of ML II in a colony of affected cats by sequencing the coding and regulatory regions of GNPTAB from affected and clinically healthy related and unrelated domestic cats and compared the sequences to the published feline genome sequence (NCBI-RefSeq accession no. XM_003989173.4, Gene ID: 101100231). RESULTS: All affected cats were homozygous for a single base substitution (c.2644C > T) in exon 13 of GNPTAB. This variant results in a premature stop codon (p.Gln882*) which predicts severe truncation and complete dysfunction of the GNPTAB enzyme. About 140 GNPTAB variants have been described in human ML II patients, with 41.3% nonsense/missense mutations, nine occurring in the same gene region as in this feline model. Restriction fragment length polymorphism and allelic discrimination real-time polymerase chain reaction assays accurately differentiated between clear, asymptomatic carriers and homozygous affected cats. CONCLUSION: Molecular genetic characterization advances this large animal model of ML II for use to further define the pathophysiology of the disease and evaluate novel therapeutic approaches for this fatal lysosomal storage disease in humans.

Cabozantinib is well tolerated in acute myeloid leukemia and effectively inhibits the resistance-conferring FLT3/tyrosine kinase domain/F691 mutation.

BACKGROUND: Cabozantinib, a tyrosine kinase inhibitor of FMS-like tyrosine kinase 3 (FLT3), MET, AXL, vascular endothelial growth factor receptor, and KIT, is approved for use in multiple malignancies. We assessed the safety and tolerability of cabozantinib in AML, given up-regulation of multiple relevant pathways. METHODS: Adults were eligible if they were 18 years old or older with relapsed/refractory AML or if they were 70 years old or older with newly diagnosed AML but were ineligible for conventional therapy. Cabozantinib was administered in 28-day cycles, and dose escalation occurred via cohorts. A pharmacodynamic evaluation of serial plasma samples via a plasma inhibitory assay (PIA) was used to assess FLT3-inhibitory activity in FLT3-mutant cell lines. RESULTS: Among 18 patients enrolled, 5 were found to harbor FLT3/ITD mutations. Sixteen patients (89%) had relapsed/refractory AML, and most were treated with 2 or more lines of prior treatment. No dose-limiting toxicities (DLTs) were detected at the first dose level (40 mg daily), but 2 patients experienced DLTs at the next level (60 mg daily). The remaining patients were then dosed at 40 mg daily, the maximum tolerated dose (MTD). Additional grade 2 or higher toxicities, possibly/probably related to cabozantinib, included fatigue, nausea, transaminitis, and electrolyte imbalance. No patients had a marrow response according to formal criteria, but 4 had peripheral blast reductions; 2 of these 4 patients transiently cleared circulating blasts. One patient experienced a reduction in marrow blasts, and 1 had stable disease. The FLT3-inhibitory activity of plasma samples, as assessed with the PIA, revealed potent and sustained inhibition in FLT3/ITD and, notably, F691 tyrosine kinase domain (TKD)-mutant cells. CONCLUSIONS: Cabozantinib is well tolerated in AML patients at an MTD of 40 mg daily and is a potent inhibitor of FLT3/ITD- and F691 TKD-altered tyrosine kinases. Cancer 2018;124:306-14. © 2017 American Cancer Society.

Safety evaluation of HOWARU® Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM®, Lactobacillus paracasei Lpc-37®, Bifidobacterium animalis subsp. lactis strains Bl-04®, and Bi-07®, and their combination (HOWARU® Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 1012 CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 1012 CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics.

Development of dietary soluble fibres by enzymatic synthesis and assessment of their digestibility in in vitro, animal and randomised clinical trial models.

The aim was to develop novel fibres by enzymatic synthesis, to determine their total dietary fibre by AOAC method 2009.01 and to estimate their potential digestibility and assess their digestibility in vivo using glycaemic and insulinaemic responses as markers in mice and randomised clinical trial models. We found that fibre candidates to which α-(1,2) branching was added were resistant to digestion in the mouse model, depending on the amount of branching. These results show that in vivo models are needed to reliably assess the digestibility of α-glycosidic-linked oligomeric dietary fibre candidates, possibly due to absence of brush border α-glucosidase activity in the current in vitro assessment. α-(1,3)-linked and α-(1,6)-linked glucose oligomers were completely digested in humans and mice. In conclusion, it is possible to develop dietary soluble fibres by enzymatic synthesis. Adding α-(1,2) branching increases their resistance to digestion in vivo and can thus improve their suitability as potential fibre candidates. Clinical Trial Registry: ClinicalTrials.gov, NCT02701270.

Safety evaluation of AB-LIFE(®) (Lactobacillus plantarum CECT 7527, 7528 and 7529): Antibiotic resistance and 90-day repeated-dose study in rats.

AB-LIFE(®) is a probiotic product consisting of equal parts of three strains of Lactobacillus plantarum (CECT 7527, 7528, and 7529) blended with inert excipients. Whole genome sequencing was performed on each of the three strains. Antibiotic resistance was evaluated by genomic mining for resistance genes, and assessment for transferability. No risk of transfer potential was identified for any antibiotic resistance genes in the three strains. AB-LIFE(®) was evaluated for potential subchronic oral toxicity in rats, with dosages of 300 and 1000 mg/kg BW/day (equivalent to 5.55 × 10(10) and 1.85 × 10(11) CFU/kg BW/day). Survival of the three test strains through the gastrointestinal tract was supported by fecal analysis. No adverse effects were identified with respect to in-life parameters, clinical or anatomic pathology, translocation, or fecal chemical analyses. The no-observed-adverse-effect level (NOAEL) for AB-LIFE(®) in male and female rats was 1000 mg/kg BW/day (1.85 × 10(11) CFU of AB-LIFE(®)/kg BW/day), the highest dose level evaluated. These results, in conjunction with a previous acute toxicity study in rats, support the conclusion that AB-LIFE(®) is safe for human consumption.

Oral repeated-dose systemic and reproductive toxicity of 6:2 fluorotelomer alcohol in mice.

6:2 fluorotelomer alcohol (6:2 FTOH) was evaluated for potential systemic repeated-dose and reproductive toxicity in mice. 6:2 FTOH was administered by oral gavage to CD-1 mice as a suspension in 0.5% aqueous methylcellulose with 0.1% Tween-80 at dosages of 1, 5, 25, or 100 mg/kg/day. The no-observed-adverse-effect level (NOAEL) for systemic toxicity was 25 mg/kg/day (males) and 5 mg/kg/day (females), based on effects at higher doses on mortality, clinical observations, body weight, nutritional parameters, hematology (red and white blood cell), clinical chemistry (liver-related), liver weights, and histopathology (liver, teeth, reproductive tract, and mammary gland). However, 6:2 FTOH was not a selective reproductive toxicant. The NOAEL for reproductive toxicity was >100 mg/kg/day; no effects on reproductive outcome were observed at any dosage. The NOAEL for viability and growth of the offspring was 25 mg/kg/day, based on clinical signs of delayed maturation in pups, and reductions in pup survival and pup body weight during lactation at 100 mg/kg/day. While the severity of the effects was generally greater in mice than previously reported in CD rats, the overall NOAELs were identical in both species, 5 mg/kg/day for systemic toxicity and 25 mg/kg/day for offspring viability/growth. 6:2 FTOH was not a selective reproductive toxicant in either species; no effects on reproductive outcome occurred at any dose level, and any effects observed in offspring occurred at dose levels that induced mortality and severe toxicity in maternal animals.

Comparative in vivo and in vitro analysis of possible estrogenic effects of perfluorooctanoic acid.

Previous studies suggested that perfluorooctanoate (PFOA) could activate the estrogen receptor (ER). The present study examined the hypothesis that PFOA can activate ER using an in vivo uterotrophic assay in CD-1 mice and an in vitro reporter assay. Pre-pubertal female CD-1 mice fed an estrogen-free diet from postnatal day (PND)14 through weaning on PND18 were administered 0, 0.005, 0.01, 0.02, 0.05, 0.1, or 1mg/kg PFOA or 17ß-estradiol (E2, 0.5mg/kg) from PND18-20. In contrast to E2, PFOA caused no changes in the relative uterine weight, the expression of ER target genes, or the morphology of the uterus/cervix and/or vagina on PND21. Treatment of a stable human cell line containing an ER-dependent luciferase reporter construct with a broad concentration range of PFOA caused no change in ER-dependent luciferase activity; whereas E2 caused a marked increase of ER-dependent luciferase activity. These data indicate that PFOA does not activate mouse or human ER.

Pathway-based toxicity: history, current approaches and liver fibrosis and steatosis as prototypes.

The Human Toxicology Project Consortium (HTPC) was created to accelerate implementation of the science and policies required to achieve a pathway-based foundation for toxicology as articulated in the 2007 National Research Council report, Toxicity Testing in the 21st Century: a Vision and a Strategy. The HTPC held a workshop, "Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action," in January, 2013, in Baltimore, MD, to further the science of pathway-based approaches to liver toxicity. This review was initiated as a thought-starter for this workshop and has since been updated to include insights from the workshop and other activities occurring in 2013. The report of the workshop has been published elsewhere in this journal (Willett et al., 2014).

Building shared experience to advance practical application of pathway-based toxicology: liver toxicity mode-of-action.

A workshop sponsored by the Human Toxicology Project Consortium (HTPC), "Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action" brought together experts from a wide range of perspectives to inform the process of pathway development and to advance two prototype pathways initially developed by the European Commission Joint Research Center (JRC): liver-specific fibrosis and steatosis. The first half of the workshop focused on the theory and practice of pathway development; the second on liver disease and the two prototype pathways. Participants agreed pathway development is extremely useful for organizing information and found that focusing the theoretical discussion on a specific AOP is extremely helpful. In addition, it is important to include several perspectives during pathway development, including information specialists, pathologists, human health and environmental risk assessors, and chemical and product manufacturers, to ensure the biology is well captured and end use is considered.

Pathology review of proliferative lesions of the exocrine pancreas in two chronic feeding studies in rats with ammonium perfluorooctanoate.

Two chronic dietary studies, conducted years apart, with ammonium perfluorooctanoate (APFO) in Sprague Dawley rats have been previously reported. Although both included male 300 ppm dietary dose groups, only the later study, conducted in 1990-1992 by Biegel et al., reported an increase in proliferative lesions (hyperplasia and adenoma) of the acinar pancreas. An assessment of the significance of the differences between both studies requires careful consideration of: the diagnostic criteria for proliferative acinar cell lesions of the rat pancreas (for example, the diagnosis of pancreatic acinar cell hyperplasia versus adenoma is based on the two-dimensional size of the lesion rather than distinct morphological differences); the basis for those criteria in light of their relevance to biological behavior; and the potential diagnostic variability between individual pathologists for difficult-to-classify lesions. A pathology peer review of male exocrine pancreatic tissues from the earlier study, conducted in 1981-1983 by Butenhoff et al., was undertaken. This review identified an increase in acinar cell hyperplasia but not adenoma or carcinoma in the earlier study. Both studies observed a proliferative response in the acinar pancreas which was more pronounced in the study by Biegel et al. Definitive reasons for the greater incidence of proliferative lesions in the later study were not identified, but some possible explanations are presented herein. The relevance of this finding to human risk assessment, in the face of differences in the biological behavior of human and rat pancreatic proliferative lesions and the proposed mechanism of formation of these lesions, are questionable.