Nicola Stagg

Assessment of Possible Carcinogenicity of Oxyfluorfen to Humans Using Mode of Action Analysis of Rodent Liver Effects

Oxyfluorfen is a herbicide that is not genotoxic and produces liver toxicity in rodents, following repeated administration at high dose levels. Lifetime rodent feeding studies reported in 1977 with low-purity oxyfluorfen (85%) showed no increase in any tumor type in rats (800 ppm, high dose) and only a marginally increased incidence of hepatocellular tumors in male CD-1 mice at the highest dose (200 ppm). To evaluate the potential carcinogenicity of the currently registered oxyfluorfen (> 98% purity), we conducted a series of short-term liver mode of action (MOA) toxicology studies in male CD-1 mice administered dietary doses of 0, 40, 200, 800, and 1600 ppm for durations of 3, 7, 10, or 28 days. MOA endpoints examined included liver weight, histopathology, cell proliferation, nuclear receptor-mediated gene expression, and other peroxisome proliferator-specific endpoints and their reversibility. Minimal liver effects were observed in mice administered doses at or below 200 ppm for up to 28 days. Increased liver weight, single-cell necrosis, cell proliferation, and peroxisomal acyl-CoA oxidase (ACO) were observed at 800 ppm after 28 days, but there was no increase in peroxisomes. Expression of Cyp2b10 and Cyp4a10 transcripts, markers of constitutive androstane receptor and peroxisome proliferator activated receptor α nuclear receptor activation, respectively, were increased at 800 and 1600 ppm after 3 or 10 days. Collectively, these data along with the negative genotoxicity demonstrate that oxyfluorfen (> 98% purity) has the potential to induce mouse liver tumors through a nongenotoxic, mitogenic MOA with a clear threshold and is not predicted to be carcinogenic in humans at relevant exposure levels.

Quantitation of soybean allergens using tandem mass spectrometry.

Soybean (Glycine max) seed contain some proteins that are allergenic to humans and animals. However, the concentration of these allergens and their expression variability among germplasms is presently unknown. To address this problem, 10 allergens were quantified from 20 nongenetically modified commercial soybean varieties using parallel, label-free mass spectrometry approaches. Relative quantitation was performed by spectral counting and absolute quantitation was performed using multiple reaction monitoring (MRM) with synthetic, isotope-labeled peptides as internal standards. During relative quantitation analysis, 10 target allergens were identified, and five of these allergens showed expression levels higher than technical variation observed for bovine serum albumin (BSA) internal standard (∼11%), suggesting expression differences among the varieties. To confirm this observation, absolute quantitation of these allergens from each variety was performed using MRM. Eight of the 10 allergens were quantified for their concentration in seed and ranged from approximately 0.5 to 5.7 μg/mg of soy protein. MRM analysis reduced technical variance of BSA internal standards to approximately 7%, and confirmed differential expression for four allergens across the 20 varieties. This is the first quantitative assessment of all major soybean allergens. The results show the total quantity of allergens measured among the 20 soy varieties was mostly similar.

Heat stability, its measurement, and its lack of utility in the assessment of the potential allergenicity of novel proteins

Thermal stability has been reported as a shared characteristic among some of the major food allergens and appears to have originated from the observation that some cooked foods retain their ability to cause allergic reactions by Immunoglobulin E (IgE) binding and the subsequent cascade of events that mediate allergic reactions. Based on this observation, the thermal stability of novel food proteins, like those in transgenic crops, is considered correlative with allergenic risk and has prompted requests from some regulatory agencies for additional testing to address safety concerns. Because human testing and serum IgE screening are not feasible nor are they necessarily useful for evaluating the thermal stability of a novel food protein, a protein function assay is often used to assess the thermal stability in the context of an allergenicity risk assessment. Some regulatory authorities also require immunodetection using polyclonal IgG antibodies and gel based methods. Here we review why heat stability as measured by these functional and immunodetection assays does not correlate with allergenicity and provides no useful safety information in assessing the allergenic potential of novel food proteins.

Workshop Proceedings Challenges and Opportunities in Evaluating Protein Allergenicity Across Biotechnology Industries

A workshop entitled “Challenges and Opportunities in Evaluating Protein Allergenicity across Biotechnology Industries” was held at the 51st Annual Meeting of the Society of Toxicology (SOT) in San Francisco, California. The workshop was sponsored by the Biotechnology Specialty Section of SOT and was designed to present the science-based approaches used in biotechnology industries to evaluate and regulate protein allergenicity. A panel of experts from industry and government highlighted the allergenicity testing requirements and research in the agricultural, pharmaceutical/biopharma, and vaccine biotechnology industries and addressed challenges and opportunities for advancing the science of protein allergenicity. The main learning from the workshop was that immunoglobulin E-mediated allergenicity of biotechnology-derived products is difficult to assess without human data. The approaches currently being used to evaluate potential for allergenicity across biotechnology industries are very different and range from bioinformatics, in vitro serology, in vivo animal testing, in vitro and in vivo functional assays, and “biosimilar” assessments (ie, biotherapeutic equivalents to innovator products). The challenge remains with regard to the different or lack of regulatory requirements for allergenicity testing across industries, but the novel approaches being used with bioinformatics and biosimilars may lead to opportunities in the future to collaborate across biotechnology industries.

Autolytic Degradation of Skipjack Tuna during Heating As Affected by Initial Quality and Processing Conditions

UNLABELLED Several factors were studied as affecting protein degradation and texture of skipjack tuna muscle following ambient pressure thermal processing (precooking). These included degree of mushy tuna syndrome (MTS) evidenced in the raw meat, raw meat pH, abusive thawing/holding, and precooking temperature/time. Slurries and intact pieces from frozen skipjack tuna, either tempered for 2 h or thawed and held at 25 °C for 22 h (abusive treatment) were heated at temperatures ranging from 40 to 80 °C for up to 2 h, and also at 90 °C for 1 h, with or without prior adjustment of pH to 5 or 7 to favor cathepsin or calpain activity, respectively. Proteolysis of precooked samples was monitored by Lowry assay and SDS-PAGE; cooked texture of intact meat was measured using a Kramer shear press and by sensory profile analysis. Proteolysis maximally occurred in slurries of skipjack tuna muscle that had been abusively stored (22 h at 25 °C) and adjusted to pH 5 prior to heating at 55 °C. Intact pieces of tuna abusively thawed/held for 22 h with subsequent heating at 55 °C also evidenced the most proteolysis and were the least firm in texture. Raw fish that evidenced higher severity of MTS when raw displayed higher levels of proteolysis prior to cooking, which were further increased after cooking at 55 °C. PRACTICAL APPLICATION The kinetic data presented here can be used to optimize processing conditions for skipjack tuna canning to minimize textural degradation and optimize quality.