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2018 Society of Toxicology Annual Meeting

MB Research Labs - Protecting You Like One of Our Own.

Join Us on the Riverwalk at the 57th Annual Meeting of the Society of Toxicology and ToxExpo, in beautiful downtown San Antonio, Texas, March 11-15, 2018

About MB Research

MB Research Labs, a Contract Toxicology Testing organization, serves the in vivo and in vitro toxicology testing needs of both government and industry.

Complete support services including consultation, protocol development, quality assurance, analytical chemistry and archive facilities are integral components of studies performed at MB.

Our technical and support procedures are in full compliance with OECD, FDA, and EPA-OCSPP. For more information about our capabilities, please click here.

Industries Served:

MB conducts Good Laboratory Practice (GLP) compliant toxicology assays as well as low-cost screening studies for clients in the cosmetic, consumer product, chemical, biotech and pharmaceutical industries.

Scientific Poster Presentations

Mon 10:45am - 12:15pm. Session: Ocular Toxicology, Abstract 1495, Poster Board P709
Micheal Carathers, Puneet Vij, Ed Delacruz, Bennett Varsho, and George DeGeorge
  1. MB Research Laboratories, Spinnerstown, PA, United States

The Draize Rabbit Eye Test (DRET) assesses damage to a number of different ocular structures, which are scored and weighted based on toxicological importance. The structures are as follows: Cornea (CO), Conjunctiva (Conj), and Iris (IR). Corneal irritation is assessed by opacities on the cornea, Conj irritation is assessed by increased vascularization, and iridial damage is assessed by function of the iris (ability to constrict or dilate pupil) and deepening of the rugae. The heaviest weighting is on corneal damage, which is 80 points out of the total 110-point scale devised by Draize. Next are conjunctival effects, which is 20 points, and finally IR is only 10 points. Since the CO scores have the heaviest weight, and most often are the drivers of eye irritation, we developed an ex vivo corneal model to assess these effects. The PorCORA is an ex vivo ocular assay, which can distinguish between a material’s potential to cause severe (reversible) versus corrosive (irreversible) damage. We tested a total of 56 chemicals and dilutions of chemicals ranging from corrosive (GHS category 1) to non-irritating (GHS not categorized). Using Cooper Statistics, we arrived at an accuracy of 88% with a positive and negative predictivity of 91% (Category 1) and 85% (not Category 1), respectively. To determine if these Cooper Statistics could be improved, we used the in vivo drivers of classification concept based on Barroso et al., 2016. Upon re-examination of our data based on this published database and methodology, we found that eight chemicals had invalid tests (animals euthanized prior to day 21) or produced GHS classification not driven by corneal opacities. These chemicals were removed from our dataset. Without these eight chemicals, the accuracy improved to 90%. Moreover, the major change was in our sensitivity, which increased to from 80% to 87%.

Mon 3:00pm - 4:30pm. Session: Genetic Toxicity, Abstract 1522, Poster Board P740
Matthew Troese, and George DeGeorge
  1. MB Research Laboratories, Spinnerstown, PA, United States

Currently there are no regulatory approved photogenotoxicity assays. Therefore we evaluated photogenotoxicity in reconstructed human epidermis (RhE) by detection of DNA damage from double-stranded breaks by measurement of the phosphorylation of histone H2AX (γ-H2AX). Test materials were applied to the media of the 3D reconstructed tissues overnight to mimic a systemic exposure. For each treatment, separate groups of tissues were kept either in the dark or exposed to UV light irradiation for 60 minutes (6 J/cm2). Tissues were then dissociated and the cells were incubated with a viability stain, fixed, permeabilized and then stained with a fluorescently labeled anti-human γ-H2AX antibody. Cells were analyzed by flow cytometry for γ-H2AX-positive cells as a marker of DNA damage. The change in γ-H2AX levels for each material was calculated as a fold change over control γ-H2AX levels. We first tested the known photogenotoxic chemical 8-methoxypsoralen (8-MOP) and non-photogenotoxic aminotriazole which does not absorb UV or visible light. 8-MOP yielded a fold increase of 2.01 at a concentration of 0.01%, whereas aminotriazole only yielded a change of 1.10 at 1.0%. Additional known photogenotoxic chemicals were then tested. We evaluated ciprofloxacin, levofloxacin and sparfloxacin, and all were able to generate fold increases in γ-H2AX levels. Fold changes of 7.92, 3.65 and 8.04 were the largest increases detected for ciprofloxacin, levofloxacin and sparfloxacin, respectively. We concluded that a 3D skin model was a successful means by which to evaluate photogenotoxicity, by the mechanism of double-stranded DNA breaks. The data obtained from the 3D tissues correlate with the known hazards of these chemicals, which are well characterized and known to cause DNA damage. Thus it is possible that this 3D tissue assay may alleviate to some degree the number of false positives obtained with current genotoxicity or photogenotoxicity assay using suspension or monolayers cell-based assays, which would greatly improve hazard testing (and thus risk assessment) resulting in more accurate labeling of materials for human health.

Tues 1:30pm-3:00pm. Session: Autoimmunity/Hypersensitivity, Abstract 2097, Poster Board P433
Puneet Vij, Micheal Carathers, Dan Sergeyev, Bennett Varsho, and George DeGeorge
  1. MB Research Laboratories, Spinnerstown, PA, United States

Skin sensitization is one of the main hazards resulting from skin exposure to a chemical, and is an important concern. The h-CLAT was developed based on the role of Langerhans cell (LC) activation during the induction phase of skin sensitization and uses the THP-1 cell line as an LC surrogate. This in vitro skin sensitization test measures the augmentation of CD86 and CD54 expression in THP-1 cells (a human monocytic leukemia cell line) following a 24-hour exposure to a test substance. Herein we outline and address some pitfalls, issues and remediation in the h-CLAT procedures.

The reactivity check, performed two weeks after thawing a new batch of THP-1 cells, requires that the viability of cells treated with the vehicle control and those treated with lactic acid (LA) be ≥90%. Both 2,4-dinitrochlorobenzene (DNCB) and nickel sulfate (NiSO4) should produce a positive response in CD86 and CD54 expression, and LA should produce negative response. If these requirements are not met, remedial steps should be taken, possibly including thawing a new batch of cells. Proactive procedures should be made to bank frozen cell stocks at various passages. The solubility of the test substance should be evaluated and confirmed visually. An appropriate solvent (saline, culture medium, or dimethyl sulfoxide [DMSO]) should completely dissolve the test substance. If not, we present how alternative vehicles – chosen with scientific justification – can be qualified to broaden the applicability domain. A reliable CV75 should be derived from at least two independent screens; however, if there is difficulty attaining this, we present options for consideration. Eight concentrations are used for each test substance in the experiment; however, if no cytotoxicity is achievable, we present advice on how the concentration could be increased; bearing in mind the final concentration should not exceed 5000 μg/ml (saline) or 1000 μg/ml (DMSO). For flow cytometry, important details include preparation of FACS buffer and 1% globulin the day before use and storage at 4°C, as well as other considerations. High quality maintenance of the flow cytometer is essential for reliable data acquisition. In addition, historical data on the doubling time and passage number should be maintained and monitored.

Wed 9:15am to 10:45ampm. Session: Alternatives to Mammalian Models II, Abstract 3122, Poster Board P750
Bennett Varsho, Chris Kalimtzis, Dan Sergeyev, Blair Yasso, and George DeGeorge
  1. MB Research Laboratories, Spinnerstown, PA, United States

Acute toxicity categories are assigned, in part, by their acute oral systemic toxicity estimate, as determined by lethality studies in rats. Our goal was to develop a metabolically competent assay system that models human biologic responses to exogenous chemicals such that cardiovascular, nervous, endocrine, digestive and excretory systems as well as general homeostatic mechanisms are not only represented but allowed to interact naturally in situ.

We administered seven reference chemicals (i.e., strychnine, caffeine, sodium nitrite, acetylsalicylic acid, ethanol, sodium chloride and sucrose) directly into the yolk and/or the albumen of embryonated chicken eggs on either Incubation Day (ID) 5, 6, 7 or 14, and observed the eggs for 48 hours for viability. LD50 values for each experimental condition were calculated following Log-Probit methodology. Our work demonstrates that embryonated chicken eggs respond to reference chemical toxicity in a dose-responsive manner, and are able to stratify lethality responses based on the relative toxicity of agents used in our experiments. Egg LD50 values were generally similar to rat LD50 values, and a robust validation could qualify IOOTA as not only an inexpensive alternative assay to mammalian hazard identification, but could also serve as a stop-gap assay until sufficiently accurate in vitro and computational models are developed.