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In vitro Alternative Methods: A Growing Trend in Regulatory Safety Evaluationqrcode

Aug. 15, 2018

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Aug. 15, 2018

Co-authored by Rahul Date, Ph.D., Head – R & D section; Rajendra Nagne, Ph.D., Head - Mutagenicity section; Manish Patel, Ph.D., Head – Toxicology
Pesticides have curbed and controlled plant diseases, and helped increase crop yields as well, but have adverse effects too - thereby necessitating various precise toxicological tests and a constant risk assessment because of their (prolonged) use. Most of these methods are based on animal studies, but the traditional animal based toxicological studies need to be replaced with reliable alternative methods for pesticide evaluation, considering animal welfare and ethics requirement (1).
JRF has been monitoring the regulatory developments and upgrades in the guidelines by keeping ourselves abreast of the latest developments in this area. We have been offering GLP in vitro alternative tests to our global clients since 2013.
JRF is fully committed to the principles of 3Rs in animal testing. A few of these alternative methods serve to "replace" certain animal tests; help to "reduce" the number of animals needed in a test; or to "refine" an animal testing procedure to reduce pain and suffering. JRF offers these studies in accordance with the various global regulatory guidelines. In vitro test methods use (reconstructed) tissues, whole cells, or parts of cells.
The recent advances in cell-based research include the development of twodimensional and three-dimensional cell (co)-cultures, which very closely mimic the tissues in the human body (1-8).

Alternative (in-vitro) study list
1. Kinetics of dermal penetration of a product in human skin
This test quantitatively evaluates the potential absorption and penetration kinetics of the test formulation. The relevant test guideline is OECD 428. This test has received a major impetus in the recent past, after a ban on the usage of animals for test, in compliance with OECD 427.
JRF offers product development support by screening your product/s under development. We also conduct studies using dermatomed human, as well as rat skin. The test item could be radioactive or cold compound. The radioactive compound based dermal penetration is quantified using DPM data generated during the kinetic study using the liquid scintillation analyzer.
The cold compounds are analyzed through sophisticated LCMS/MS for the quantitative presence of the active ingredient.
2. Skin corrosion and irritation studies
These studies have replaced the conventional rodent skin irritation tests.  
Skin corrosion OECD TG 431
These studies are conducted using "Reconstructed human epidermis" (RhE); comprising non-transformed, human-derived epidermal kerat inocytes, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.
Skin corrosion is evaluated using the RhE tissue to determine if the exposure causes a potential irreversible damage to the skin, manifested as visible necrosis through the epidermis. Cell viability is measured using vital dye such as MTT, which discriminates between live and dead cells
Skin irritation: OECD 439
Skin irritations are a result of the responses in the form of erythema and oedema.  
This test determines the events leading to the dying keratinocytes releasing mediators, which begin the inflammatory cascade, which is triggered in the cells in the dermis, particularly the stromal, and endothelial cells. This test is also conducted using reconstructed human epithelium tissue. It is the dilation and increased permeability of the endothelial cells that produce the observed erythema and oedema. The release of cellular contents as a result of the adverse effects leading to skin irritation in this model is measured by enzymatic conversion of the vital dye MTT [3-(4,5- Di methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue] into a blue formazan salt that is quantitatively measured after extraction from tissues in a spectrophotometrydriven assay, based on the OD of the clear supernatant. Irritant products are identified by their ability to decrease cell viability below defined threshold levels, i.e., ≤ 50%.
3. Skin sensitization
As a product development support, JRF offers three tiered in vitro skin sensitization tests, based on “AOP-Adverse outcome pathway” principle. Such tests will eliminate the formulations, which may lead to a possible sensitization response. 
The AOP response is typically assessed. Kindly refer to the pictogram (Fig. 1) below for skin sensitization (9);
Fig. 1 Skin sensitization response
This pictogram classifies the sensitization response driven by the following events:
1. Molecular initiating event (MIE), focused on peptide reactivity 
2. Cellular response with two important key events, namely, keratinocyte and 
3. Dendritic cell response.
These tests are in vitro tests.  They replace a “Local Lymph Node Assay” and organism response determined by Guinea Pig Maximization tests for compounds identified as sensitizers.
However, if the compounds are found to be non-sensitizers, the LLNA needs to be conducted after seeking the regulator’s permission.
Let us review the in vitro sensitization regime for current relevance.
A. Direct Peptide Reactivation Assay (DPRA) & Photo-DPRA (OECD TG 442C):
These assays evaluate the potential sensitization, as a result of the depletion of the specific synthetic hepta-peptides of lysine and cysteine. Haptenization, which is the covalent binding of lowmolecular weight substances (haptens) to proteins in the skin, is considered a prominent mechanism through which the test substances could become antigenic. Therefore, information from peptide reactivity assays such as the DPR Assay, is considered relevant for the assessment of the skin sensitization potential of the product. 
We can modify this assay to make it a Photo-DPR Assay as well. 
Limitations: Can’t be used for mixtures, natural compounds (where the molar ratio with the hepta-peptides cannot be tested), and compounds with low solubility, aldehydes, compounds facilitating cysteine dimers, among others.)

B. KeratinoSens™ Assay (OECD TG 442D):
This test is based on keratinocyte activation — an immortalized adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter, fused with an ARE element from a gene that is known to be up-regulated by contact sensitizers. The Luciferase signal is measured using a Chemiluminescence measuring device. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well-established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.
• Highest final conclusion should be 2,000 uM. One can work with 1,000 uM, but a negative result cannot be conclusive. 
• Can’t be used for compounds that are hydrophobic or have LogP above 6. 
• Highly cytotoxic substances cannot be tested.

C. Human Cell Line Activation Test (h-CLAT) (OECD 442E)
The h-CLAT method, an in vitro assay, quantifies the changes of cell surface marker expression (i.e., CD86 and CD54) on a human monocytic leukemia cell line, THP-1, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP - 1 activation and may mimic Dendritic cell (DC) activation, which plays a critical role in T-cell priming. The changes of surface marker expression are measured by flow cytometry, following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether up-regulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control are calculated and used in the predict ion model to support the discrimination between sensitizers and non-sensitizers.
• Negative results with test chemicals with a Log Kow greater than 3.5 should not be considered. However, positive results obtained with test chemicals with a Log Kow greater than 3.5 could still be used to support the identification of the test chemical as a skin sensitizer.
• Compounds interfering with fluorescence spectrum overlapping that of FITC/PI cannot be tested.
JRF is committed towards reduction in animal use, and we continue to validate newer in vitro assays. JRF was one of the first laboratories in the region to thoroughly validate these in vitro assays and can offer a comprehensive list of in vitro assays for various industries.
1. https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC4206742/ 
2. Griffith, L. G. & Swartz, M. A. Capturing complex 3D tissue physiology in vitro. Nature Reviews Molecular Cell Biology 7, 211–224 (2006). 
3. Van Grunsven, L. A. 3D in vitro models of liver fibrosis. Advanced Drug Delivery Reviews 121, 133–146 (2017). 
4. Hu, T. et al. Xenobiotic metabolism gene expression in the EpiDerm™ in vitro 3D human epidermis model compared to human skin. Toxicol. Vitr. 24, 1450–1463 (2010). 
5. Huang, S., Wiszniewski, L., Constant, S. & Roggen, E. Potential of in vitro reconstituted 3D human airway epithelia (MucilAir™) to assess respiratory sensitizers. Toxicol. Vitr. 27, 1151–1156 (2013). 
6. LaPlaca, M. C., Vernekar, V. N., Shoemaker, J. T. & Cullen, D. K. ThreeDimensional Neuronal Cultures. Methods Bioeng. 3d Tissue Eng. 187–204 (2010). 
7. Lelièvre, S. A., Kwok, T. & Chittiboyina, S. Architecture in 3D cell culture: An essential feature for in vitro toxicology. Toxicol. Vitr. 45, 287–295 (2017). 
8. Mueller, D., Krämer, L., Hoffmann, E., Klein, S. & Noor, F. 3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies. Toxicol. Vitr. 28, 104–112 (2014). 
9. Skin sensitization adverse outcome pathway no 40, AOPWiki Covalent Protein binding leading to Skin Sensitization https://aopwiki.org/aops/40

The authors would like to acknowledge Dr. Abhay Deshpande, CEO - JRF Global for his valuable inputs and suggestions.

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