Trend in Regulatory Safety Evaluation: An In vitro Alternative Approach

In an interview with AgroPages, Dr. Rajendra Nagane, Ph.D., Head -Mutagenicity and In vitro studies at JRF Global, a leading Indian CRO company, talks about JRF’s efforts towards the in vitro alternative studies in the global agrochemical and specialty chemicals industry.  

The regulators are moving away from the animal testing, what are the initiatives at JRF to adopt it?

JRF keeps abreast of the latest development in the guidelines, and continuously monitors the regulatory development updates across the globe. Since 2013, GLP In vitro alternative tests is offered to the global clients. The principles of 3Rs of animal testing is completely adopted at JRF. The alternative methods serve to "reduce" the number of animals required for the testing test; aids to "replace" some animal tests; or "refine" an animal testing procedure to reduce pain and suffering. These studies are offered in accordance with the various global regulatory guidelines. The In vitro test methods use (reconstructed) tissues, whole cells, or parts of cells.

Can the Skin irritant or Skin corrosion be predicted?

Initial prediction on Skin irritation and corrosion can be predicted based on the pH and the Physico chemical properties of the compound. Additionally, the QSAR tools are available to get some information on the behaviour of the compounds. 

How is Skin Corrosion evaluated?

In vitro skin corrosion testing is conducted using reconstructed human skin tissues (RhE) to determine if the exposure causes irreversible damage to the skin, manifested as visible necrosis through the epidermis. Cell viability is measured using vital dye MTT, and this discriminates between live and dead cells.

Can you explain the phenomenon of in vitro skin irritation?

Skin irritations are a result of the responses in the form of erythema and oedema. This test establishes the outcome leading to the damaged keratinocytes releasing intermediaries, which begin the inflammatory response. Dilation increases the permeability of the endothelial cells, which causes the formation of erythema and oedema. This test is also conducted using reconstructed human epithelium tissue. The release of cellular contents because 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. Optical density is quantitatively measured after extraction from tissues in a multi-mode plate reader. Irritant products are identified by their ability to decrease cell viability below-defined threshold levels, i.e., ≤ 50%.

What is top-down or bottom-up procedure in skin irritation study?

The bottom-up procedure can be performed when it is expected the test item is non-irritant and non-corrosive.  Skin irritation study (OECD 439) is conducted first, and if results show a non-irritant response; then no further testing is required. In the case of the Top-down approach, skin corrosion (OECD 431) is performed primarily, and if results show skin corrosive response, then no further test is required. In general, the top-down approach is used when we expect the test item to have a corrosive/irritant response. In the case of the bottom-up approach, if the skin irritation shows clearly negative response, then no further testing is required. The priority of the test can be decided based on the physicochemical data available. The animal study is required only if we do not get a clear indication.  

When is the skin corrosion not required to be conducted?

As per ECHA guidance, the Skin corrosion study may not be required to be conducted if the test item is:

1. Strong acid (pH ≤ 2.0) or base (pH ≥ 11.5) and the available information indicates that it should be classified as skin corrosive (Category 1)  

2. Spontaneously flammable in the air or contact with water or moisture at room temperature 
   

3. It is classified as acutely toxic by the dermal route (Category 1). 
   

4. An acute toxicity study by the dermal route does not indicate skin irritation up to the limit dose level (2000 mg/kg body weight). 
   

What are the offerings of JRF for in vitro skin sensitisation study?

The battery of three-tiered in vitro skin sensitization tests is offered at JRF based on “AOP-Adverse outcome pathway” principle. These batteries of tests classify the formulations, as a possible sensitizer or non-sensitizer response. The following key events drive the sensitization response:

1. Peptide reactivity using DPRA assay: The test item is covalently bound to the skin protein when it comes in contact with the skin protein. If the test item binds with the peptide the free peptide will be less, and the depletion in the free peptide due to Cys and Lys adducts is measured. Peptide depletion over 6.38% indicates sensitiser.

2. Keratinocyte response: It is based on Luciferase Reporter Gene Test Method is measured. A luciferase reporter gene is used to assess Keratinocyte activation. An increase ≥ 1.5-fold indicates sensitiser (Cell viability - ≥ 70%).
   

3. Dendritic cell response: Core expression of CD54 and CD86 cell marker is measured. Overexpression of CD54 and CD86 are measured to evaluate the sensitisation potential of the test item. 
   

What are the limitations of the DPRA technique?

The limitation of DPRA technique is that it may not provide conclusive results for low solubility compounds, complex mixtures, natural compounds whose molar ratio with the hepta-peptides cannot be tested and compounds aiding cysteine dimers. 

How is an in vitro h-CLAT method used to differentiate between the sensitiser and non-sensitiser compound? 

The h-CLAT method is an in vitro assay which quantifies the overexpression of cell surface markers i.e., CD86 and CD54 on THP-1 cells. Following exposure cells with test item for 24 hours cells are processed. These surface markers of monocytic THP - 1 activation mimic Dendritic cell (DC) activation. These cell surface marker expressions are quantified by flow cytometry, following cell staining with fluorescence-tagged antibodies. Simultaneous cytotoxicity evaluation is carried out to measure whether up-regulation of surface marker expression occurs at cytotoxic concentrations. The RFI values of surface markers are compared to the control and it is used in the prediction model to categorize between sensitizers and non-sensitizers.

What is an alternative to the LLNA assay?

In vitro battery of tests is used as a replacement for in vivo a “Local Lymph Node Assay”. The in vitro studies are widely useful to determine if the compound is a sensitizer or non-sensitizer.  LLNA needs to be performed only if the compound is not classified as sensitizer or non-sensitizers based on in vitro study results. In vivo testing is generally conducted after seeking permission from the regulators. 

When is the In vivo testing required?

In vivo testing is required only if:

1. In chemico in vitro tests available are not applicable for the test substance; or the results obtained from such methods are not adequate for classification and risk assessment.

2. Testing In-conclusive: If the test substance has low solubility or logP > 7, testing of metal compounds, testing for mixtures/UVCB, less than 2000 μM (Keratinosens assay).
   

How are the radioactive and non-radioactive compounds analysed in an in vitro dermal absorption study?

The test item can be C₁₄ radiolabelled or non-radio labelled for this study. The radioactivity associated with the absorbed material is measured by liquid scintillation analyser during the study based on dermal penetration. The non-radiolabelled compounds are analysed through sophisticated HPLC or LCMS/MS for the quantitative presence of the active.

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This article was initially published in AgroPages ‘Annual Review 2020’ magazine. Download it to read more articles.