Rethinking chronic toxicity and carcinogenicity assessment for agrochemicals project (ReCAAP): A reporting framework to support a weight of evidence safety assessment without long-term rodent bioassays.

Rodent cancer bioassays have been long-required studies for regulatory assessment of human cancer hazard and risk. These studies use hundreds of animals, are resource intensive, and certain aspects of these studies have limited human relevance. The past 10 years have seen an exponential growth of new technologies with the potential to effectively evaluate human cancer hazard and risk while reducing, refining, or replacing animal use. To streamline and facilitate uptake of new technologies, a workgroup comprised of scientists from government, academia, non-governmental organizations, and industry stakeholders developed a framework for waiver rationales of rodent cancer bioassays for consideration in agrochemical safety assessment. The workgroup used an iterative approach, incorporating regulatory agency feedback, and identifying critical information to be considered in a risk assessment-based weight of evidence determination of the need for rodent cancer bioassays. The reporting framework described herein was developed to support a chronic toxicity and carcinogenicity study waiver rationale, which includes information on use pattern(s), exposure scenario(s), pesticidal mode-of-action, physicochemical properties, metabolism, toxicokinetics, toxicological data including mechanistic data, and chemical read-across from similar registered pesticides. The framework could also be applied to endpoints other than chronic toxicity and carcinogenicity, and for chemicals other than agrochemicals.

Reducing the need for animal testing while increasing efficiency in a pesticide regulatory setting: Lessons from the EPA Office of Pesticide Programs' Hazard and Science Policy Council.

As part of EPA's commitment to reducing animal testing, the Office of Pesticide Programs (OPP) created the Hazard and Science Policy Council (HASPOC). This group considers requests for waiving animal study requirements for human health risk assessments and makes recommendations based on a weight-of-the-evidence approach. Since its inception in 2012, the HASPOC has evaluated over one thousand requests to waive animal studies required by default for pesticide evaluation. Here, the number of studies waived, and the types of studies represented were analyzed to determine the impact of the HASPOC decisions in terms of animal and monetary savings. Overall, the waiving of studies by HASPOC resulted in over 200 thousand animals saved. There were also savings of over $300 million in study costs and over $6 million in study review costs as well as less time spent in study processing and review by EPA staff. Thus, the HASPOC has built significant efficiencies into the risk assessment process while continuing to protect human health.

Evaluation of high-throughput genotoxicity assays used in profiling the US EPA ToxCast chemicals.

Three high-throughput screening (HTS) genotoxicity assays-GreenScreen HC GADD45a-GFP (Gentronix Ltd.), CellCiphr p53 (Cellumen Inc.) and CellSensor p53RE-bla (Invitrogen Corp.)-were used to analyze the collection of 320 predominantly pesticide active compounds being tested in Phase I of US. Environmental Protection Agency's ToxCast research project. Between 9% and 12% of compounds were positive for genotoxicity in the assays. However, results of the varied tests only partially overlapped, suggesting a strategy of combining data from a battery of assays. The HTS results were compared to mutagenicity (Ames) and animal tumorigenicity data. Overall, the HTS assays demonstrated low sensitivity for rodent tumorigens, likely due to: screening at a low concentration, coverage of selected genotoxic mechanisms, lack of metabolic activation and difficulty detecting non-genotoxic carcinogens. Conversely, HTS results demonstrated high specificity, >88%. Overall concordance of the HTS assays with tumorigenicity data was low, around 50% for all tumorigens, but increased to 74-78% (vs. 60% for Ames) for those compounds producing tumors in rodents at multiple sites and, thus, more likely genotoxic carcinogens. The aim of the present study was to evaluate the utility of HTS assays to identify potential genotoxicity hazard in the larger context of the ToxCast project, to aid prioritization of environmentally relevant chemicals for further testing and assessment of carcinogenicity risk to humans.

Effect of p53 genotype on gene expression profiles in murine liver.

The tumor suppressor protein p53 is a key regulatory element in the cell and is regarded as the "guardian of the genome". Much of the present knowledge of p53 function has come from studies of transgenic mice in which the p53 gene has undergone a targeted deletion. In order to provide additional insight into the impact on the cellular regulatory networks associated with the loss of this gene, microarray technology was utilized to assess gene expression in tissues from both the p53(-/-) and p53(+/-) mice. Six male mice from each genotype (p53(+/+), p53(+/-), and p53(-/-)) were humanely killed and the tissues processed for microarray analysis. The initial studies have been performed in the liver for which the Dunnett test revealed 1406 genes to be differentially expressed between p53(+/+) and p53(+/-) or between p53(+/+) and p53(-/-) at the level of p < or = 0.05. Both genes with increased expression and decreased expression were identified in p53(+/-) and in p53(-/-) mice. Most notable in the gene list derived from the p53(+/-) mice was the significant reduction in p53 mRNA. In the p53(-/-) mice, not only was there reduced expression of the p53 genes on the array, but genes associated with DNA repair, apoptosis, and cell proliferation were differentially expressed, as expected. However, altered expression was noted for many genes in the Cdc42-GTPase pathways that influence cell proliferation. This may indicate that alternate pathways are brought into play in the unperturbed liver when loss or reduction in p53 levels occurs.

Alterations in gene expression profiles and the DNA-damage response in ionizing radiation-exposed TK6 cells.

Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis.

An evaluation of the mode of action framework for mutagenic carcinogens case study II: chromium (VI).

In response to the 2005 revised U.S Environmental Protection Agency's (EPA) Cancer Guidelines, a strategy is being developed to include all mutagenicity and other genotoxicity data with additional information to determine whether the initiating step in carcinogenesis is through a mutagenic mode of action (MOA). This information is necessary to decide if age-dependent adjustment factors (ADAFs) should be applied to the risk assessment. Chromium (VI) [Cr (VI)], a carcinogen in animals and humans via inhalation, was reassessed by the National Toxicology Program (NTP) in 2-year drinking water studies in rodents. From these data, NTP concluded that the results with Cr (VI) showed clear evidence of carcinogenicity in male and female mice and rats. Cr (VI) is also mutagenic, in numerous in vitro assays, in animals (mice and rats) and in humans. Accordingly, Cr (VI) was processed through the MOA framework; postulated key steps in tumor formation were interaction of DNA with Cr (VI) and reduction to Cr (III), mutagenesis, cell proliferation, and tumor formation. Within the timeframe and tumorigenic dose range for early events, genetic changes in mice (single/double-stranded DNA breaks) commence within 24 hr. Mechanistic evidence was also found for oxidative damage and DNA adduct formation contributing to the tumor response. The weight of evidence supports the plausibility that Cr (VI) may act through a mutagenic MOA. Therefore, the Cancer Guidelines recommend a linear extrapolation for the oral risk assessment. Cr (VI) also induces germ cell mutagenicity and causes DNA deletions in developing embryos; thus, it is recommended that the ADAFs be applied.

[Sentinel node biopsy in vulvar cancer]. / La technique du ganglion sentinelle dans le cancer de la vulve : état des lieux.

In vulvar cancer, lymph node status is a major prognostic factor. Currently, the reference regarding nodal exploration is the groin lymphadenectomy responsible for a significant morbidity. The sentinel node technique in breast cancer has become a standard of care. This technique has been studied for fifteen years in vulvar cancer, on small numbers because of its low incidence. There is not yet consensus about its use in practice. This article is a focus on this technology, its feasibility and the benefits of sentinel node detection applied to vulvar cancer.

[Hysteroscopic injection in sentinel node biopsy for endometrial cancer increases the frequency of para-aortic drainage: Is it necessary to reconsider the role of para-aortic lymphadenectomy?]. / L'injection hystéroscopique dans la biopsie du ganglion sentinelle pour cancer de l'endomètre augmente la fréquence du drainage para-aortique: faut-il reconsidérer la place de la lymphadénectomie para-aortique?

In cancer research, regional lymph node status is a major prognostic factor and a decision criterion for adjuvant therapy. The sentinel node procedure, which has emerged to reduce morbidity of extensive lymphadenectomy, remains a major step in the surgical management of various cancers. In endometrial cancer, the sentinel node biopsy is still at the stage of feasibility. The main problem of the diffusion of the sentinel node biopsy in endometrial cancer is the absence of injection site consensus. The different injection sites (myometrial, pericervical and hysteroscopy) allow to identify para-aortic lymph node drainage. In this article, we review the interest of hysteroscopic injection in endometrial cancer. This injection site could contribute to select the patients potentially being able to profit from a lombo-aortic lymphadenectomy.