MB Research Labs - 10th World Congress Posters

In Vitro and Alternative Toxicology

MB Research Labs is honored to present the following posters at the 10th World Congress - Alternatives and Animal Use in the Life Sciences in Seattle, Washington, August 20th - 24th, 2017. As technology and advanced techniques improve, replacements for live animal studies are becoming more and more common place. At MB Research, we are dedicated to the development and use of in vitro and alternative toxicology testing methods. For more information about our presented posters, please click on the title.

MB Research Laboratories, Spinnerstown, PA

Micheal Carathers, Bennett Varsho, Puneet Vij, and George DeGeorge.

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To explore the applicability domain of the h-CLAT beyond pure chemicals and into mixtures, we analyzed THP-1 cell expression of CD86 and CD54 cell surface markers from an expanded set of OECD proficiency chemicals and several mixtures. The h-CLAT correctly predicted 14 of 15 of these chemicals, an Accuracy of 93.7%. A variety of products from the petroleum, agrochemical, food, cosmetic and chemical industries were obtained via retail outlets and evaluated, including: PPD and non-PPD based hair dyes, propolis extract, fuel additives, pesticides, and adhesives. The h-CLAT correctly predicted the sensitization properties of these mixtures with an accuracy of 91%. Thus, we show the feasibility of the h-CLAT to determine the sensitization potential for both pure chemicals and mixtures.


1MB Research Laboratories, Spinnerstown, PA, 2CellSystems® Biotechnologie Vertrieb GmbH, Troisdorf, Germany

Lisa Pratt1, Matthew J. Troese1, Dirk Weisensee2, Oliver Engelking2, George L. DeGeorge1.

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Reconstructed human epidermis has been shown to release IL-18 in response to dermal sensitizers. The exposure concentrations resulting in a greater than a threshold positive response (SI ≥ 2.0) correlates with their potency in the In Vitro Sensitization Assay (IVSA). In our experiments, NBB and DNCB were strong inducers of IL-18 secretion (EC2.0 = 0.028% and 0.03%). Isoeugenol (IE) and Cinnamaldehyde (CA) were moderate sensitizers, while Resorcinol and HCA (EC2.0 = 22%) were weak sensitizers. Sensitizer potency ranked as follows: NBB > DNCB > PPD, IE ≈ CA > RES > HCA, with NBB, DNCB and PPD classified as strong, IE and CA as moderate, and RES and HCA classified as weak sensitizers. Of the total of 20 chemicals tested, 7 were irritants, 2 were non-sensitizers and only Chlorobenzene was incorrectly predicted as a weak sensitizer. In summary, measuring IL-18 release from RHE allows for highly accurate and sensitive identification of dermal sensitizers.


MB Research Laboratories, Spinnerstown, PA

Matthew J. Troese, Lisa Pratt, George L. DeGeorge.

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We co-cultured a reconstructed human epidermal tissue (RHE) with human plasmacytoid Dendritic Cells (pDCs) for use as a dermal sensitization assay. RHE tissues were placed at the air-liquid interface above a media suspension of pDC. The tissues are topically dosed with test materials. After 4 hours of incubation, the RHE tissues and pDC were separately cultured for an additional 20 hours. Media was analyzed for IL-18 by ELISA, and pDC were analyzed for changes in CD86 expression. A positive response from the RHE tissues was defined as a 2-fold increase in IL-18 secretion, and for pDCs, a 1.5 fold increase in CD86 expression. Increases in both secretion of IL-18 and expression of CD86 were detected after exposure to dermal sensitizers. A prediction model was developed where a chemical is defined as a sensitizer if either a positive result occurs in either the IL-18 or in CD86. From three individual experiments, we obtained an Accuracy of 100%, 83%, and 83%.


1MB Research Laboratories, Spinnerstown, PA, 2Cell Systems® Biotechnologie Vertrieb GmbH, Troisdorf, Germany

Matthew Troese1, Bennett Varsho1, Dirk Weisensee2 and George DeGeorge1.

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Skin releases IL-18 in response to dermal sensitizers. Using a 3D skin model (epiCS®) in the In Vitro Sensitization Assay (IVSA), we measured IL-18 secretion as a biomarker of sensitization. We are able to achieve 90% accuracy when testing 20 chemicals. Analysis of the data revealed that test chemical concentrations that induced a 2-fold increase in IL-18 secretion (Stimulation Index; SI-2) was proportional to the potency of the sensitizer. In the epiCS® IVSA test system tissues can be topically dosed, like skin, with a wide variety of substances, so we obtained sensitizing and non-sensitizing mixtures from commercial sources; including hair dyes, caulking, adhesives, antimicrobial fuel additives and propolis (dietary supplement). A positive response (SI≥2) was detected for all the known sensitizing mixtures. In summary, the IVSA was able to correctly distinguish pure sensitizing chemicals and mixtures from non-sensitizing materials with high Accuracy and Sensitivity.


MB Research Laboratories, Spinnerstown, PA

Bennett Varsho, Micheal Carathers, Debra Hall, and George DeGeorge.

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In the mid-1990s, regulators moved from allowing the use of strong positive control agents in animal studies for dermal sensitization to requiring the use of weak to moderate sensitizers. This shift arose from the desire to prove that the performing laboratories could identify weak to moderate sensitizers under their testing conditions, reducing concerns regarding false negatives. Twenty years later, replacement agents (including MBT and HCA) are in widespread use and demonstrate the test methods’ capacity to identify weak to moderate sensitizers. In our laboratory, the Guinea Pig Maximization Test (GPMT) returns 30-45% positive responders (PR) with MBT, while the Buehler Method returns 20-65% PR with HCA. In the LLNA, 25% HCA returns 40-100% PR using both the flow cytometric method and the more recent BrdU-ELISA method. Factors potentially affecting positive response rates, including induction and/or challenge concentrations, vehicle effects, are compared and reported in detail.


MB Research Laboratories, Spinnerstown, PA

Micheal Carathers, Puneet Vij, Bennett Varsho, Edwin Delacruz, and George DeGeorge.

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The classification of severe ocular irritation as distinct from corrosion is determined by assessing reversibility of damage to the corneal epithelium. Here, we demonstrate a dual ex vivo assay (Bovine Corneal Opacity and Permeability (BCOP) and Porcine Corneal Opacity Reversibility Assay (PorCORA)) for distinguishing moderately/severely irritating from corrosive substances. Chemicals with known EPA classifications were first evaluated using BCOP assay. Test substances with an In Vitro Irritation Score (IVIS) ≥ 20 were considered moderately irritating to corrosive and were further assayed in the PorCORA. In PorCORA, substances causing stain retention persisting passed Day 21 were deemed corrosive and if cleared by Day 21, classified as severe irritant. Of the 21 chemicals, 6 of 6 Category I chemicals induced irreversible damage, while ocular damage caused by 15 of 15 Category II and III chemicals completely reversed by Day 21 in PorCORA.


1 SeCAM, Magliaso, Switzerland, 2 International Association for Soaps, Detergents and Maintenance Products, Brussels, Belgium, 3 MB Research Labs, Spinnerstown (PA), U.S.A., 4 Henkel AG & Co. KGaA, Duesseldorf, Germany, 5 Unilever Safety and Environmental Assurance Centre, Bedford, U.K., 6 The Procter & Gamble Company, Egham, U.K.

Chantra Eskes1, Caroline Bertein2*, Micheal Carathers3*, George DeGeorge3, Martina Hermann4, Penny Jones5, Pauline McNamee6, Bennett Varsho3

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Currently adopted OECD test methods for eye hazard do not allow identification of UN GHS Cat. 2 chemicals. A key reason is that these assays may not cover all relevant mechanisms of ocular damage. In particular, test methods adopted to identify UN GHS Cat. 1 were not designed to predict chemicals classified in vivo due to tissue effects persisting 21 days after exposure. In contrast, PorCORA was designed to specifically address (ir)reversibility of corneal damage in ex vivo porcine corneas cultured for 21 days. Detergent and cleaning products having a balanced distribution of Cat. 1/Cat. 2/No Cat. based on existing in vivo data were tested using PorCORA with an extended washing procedure. All 5 in vivo Cat.1 products based on persistence of effects, as well as 9 of 12 in vivo non-Cat. 1 products were correctly identified by the assay. PorCORA appears therefore as a promising assay for further investigation e.g. within Defined Approaches for eye hazard identification.


In Vitro Toxicology Testing Services

Cytotoxicity

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In Vitro Toxicology

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MB Research Labs specializes in predictive, 3D Tissue and cell-based in vitro toxicity assays using primary cells from many different tissues and organs. We utilize many proprietary GLP assays to allow for use in nearly all levels of drug and product development from early stage screening to pre-clinical in vivo toxicity studies.

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Toxicology Test Methods - In Vitro and Alternative


In Vitro and Alternative Ocular Irritation Assays


MatTek EpiOcular™, SkinEthic corneocyte models

  • These models mimic characteristics of the epithelium of the eye
  • Unique properties of 3D models allow test articles to be applied topically, allowing testing of solids, organics, and insoluble materials

  • Uses fertilized chicken eggs
  • INVITTOX PROTOCOL Number 47

  • Uses excised bovine corneas normally discarded
  • Measures two endpoints; 1) changes in opacity, reflecting protein denaturation and corneal injury, and 2) fluorescein permeability reflecting damage to corneal epithelium
  • Optional histological endpoint is available (H & E staining)
  • Measurement of pro-inflammatory mediators in liberated corneal cells (by flow cytometry or ELISA)

  • Uses excised porcine cornea normally discarded
  • Corneas maintained in culture for up to 21 Days
  • Measures "Days to Recover" after exposure to possible Irritants
  • Allows discrimination between Severe Irritant and Ocular Corrosive

  • Uses fertilized chicken eggs on embryonic day 10 or 14 of development
  • Measures hemodynamic effects, injury, and anti-angiogenic effects to the membrane microvasculature
  • Especially useful for alcohol-containing formulations

  • Uses tiered testing strategy of alternative ocular irritation assays (CAMVA, BCOP, PorCORA, PorFocal) to determine regulatory classification of a test material without the use of live animals.
  • Currently Under Development

MB Research uses normal human primary epidermal keratinocytes or fibroblasts (or mouse keratinocytes and fibroblasts)
  • Cells are representative of the eye and skin epithelium
  • Human cells are derived from neonatal, adolescent, or aged donors
  • Cytokine expression or release (LDH, PGE-2, interleukins) can be measured
  • Low serum or serum free protocol options are available

  • Using primary human cell or mouse cell lines in serum-free or low serum media
  • Rate of proliferation or apoptosis is measured by vital dye uptake (MTT or neutral red) or by BrdU incorporation and flow cytometry

  • OptiSafe™ In Vitro Eye Irritation Test
  • An in vitro test method in which a test substance is applied to a semi-permeable membrane. Damage to macromolecules in the membrane is measured to assess the test substance’s potential to cause eye irritation.
  • Can be used to determine the irritation potential of cosmetics, creams, and a wide variety of consumer products. Results are presented as GHS, EPA classifications, an ocular irritation score and class

In Vitro Dermal Irritation Assays

The 3D models available at MB Research (MatTek EpiDerm™, SkinEthic systems and CellSystems EpiCS®) allow the application of the test material directly to the "skin" surface and permit the assessment of dermal toxicity via a wide variety of parameters.

  • Cytotoxicity via MTT, neutral red viability assay, or flow cytometry
  • Histological examination (H & E) of the epidermal layers, including the stratum corneum
  • Measurement of inflammatory mediator and lytokine release (PGE-2, nitric oxide, TNFa, IL1a, and other interleukins)
  • Dissolution of the tissue equivalent into a single cell suspension, with subsequent flow cytometric analysis allows evaluation of gene expression and peroxide production (antioxidant status of cells)

In Vitro Dermal Corrosivity Assays

  • An accepted alternative to the Draize rabbit corrosivity test that recently has been validated by ICCVAM for use for DOT, FHSA, CPSC, EPA and FDA testing
  • Allows Packing Group assignment of test materials
  • Can be used to compare the relative corrosivity of materials within Packing Groups for product development purposes. See www.corrositex.com for more information.

  • Validated by ECVAM and ICCVAM as an in vitro alternative corrosivity test based on human skin cells
  • Allows testing of material not compatible with Corrositex™


In Vitro Phototoxicity Assays

  • With 3T3 mouse fibroblast cells via the EC/COLIPA validated protocol. See www.3t3nru.com for more information.
  • Using the MatTek EpiDerm™ protocol currently under review by ECVAM
  • Human keratinocytes or fibroblasts


Cytotoxicity Assays

The 3D models available at MB Research (MatTek EpiDerm™, SkinEthic systems and CellSystems EpiCS®) allow the application of the test material directly to the "skin" surface and permit the assessment of dermal toxicity via a wide variety of parameters.

  • Cytotoxicity via MTT, neutral red viability assay, or flow cytometry
  • Histological examination (H & E) of the epidermal layers, including the stratum corneum
  • Measurement of inflammatory mediator and lytokine release (PGE-2, nitric oxide, TNFa, IL1a, and other interleukins)
  • Dissolution of the tissue equivalent into a single cell suspension, with subsequent flow cytometric analysis allows evaluation of gene expression and peroxide production (antioxidant status of cells)

MB Research also performs other in vitro and alternative assays. Please refer to our Capabilities List or feel free to contact a Client Services Representative for assistance.

In Vitro Toxicology Links:

Solving the In Vitro Toxicology Puzzle

MB Research Labs works closely with other leaders in the field of in vitro toxicology and is dedicated to advancing techniques that reduce the use of live animals in Toxicological Research. Below are several related sources that are helpful in developing and validating novel in vitro toxicology testing methods.