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Photocytotoxicity Test Method for Fluorescence Imaging Products

Catalog of Regulatory Science Tools to Help Assess New Medical Devices 

This regulatory science tool (RST) is a method to evaluate the photochemical damage potential of products that include an optical imaging system and fluorescent contrast agent.

Technical Description

Described is a test method for evaluating the safety of fluorescence imaging products comprised of an optical imaging system and fluorescence contrast agent. This tool may be used alone or in conjunction with testing for generation of reactive molecular species using chemical assays [1]. It is based on the in vitro 3T3 neutral red uptake (3T3-NRU) assay described in the Organization for Economic Co-Operation and Development Test Guideline 432 (OECD TG432) [2,3], but implements an alternative protocol to accommodate fluorescence imaging products. When evaluated for a specific fluorophore concentration and light dose, this tool enables assessment of cytophototoxic potential. 

In brief, this modified version of the 3T3-NRU assay approach [4] involves adding the candidate fluorophore to cells at a clinically-relevant concentration and performing optical exposure at clinically-relevant irradiance levels and exposure times [5]. The assay includes a chromophore that stains viable cells; and can be quantified by absorbance measurements. Changes in cell viability with fluorophore concentration and irradiation can be assessed to identify suitable parameters for the product. For further information on this method and error levels, see Vig et al. [5]. 

Given this tool’s ability to evaluate cytotoxicity in novel and established fluorophores [5], it may have relevance to fluorescence imaging products with a range of applications, including vascular imaging and intra-operative visualization of tumors and tumor margins.

Intended Purpose 

This assay-based test method is intended for evaluating the cytophototoxic potential of contrast-enhanced fluorescence imaging products. Results can provide insight into the potential for cellular damage at specific levels of agent concentration and optical radiation, and thus facilitate identification of parameter combinations that may reduce photocytotoxic risk. Additionally, this tool may be used to provide objective, quantitative comparisons of cytophototoxic potential between different imaging products.

Testing

Additional information on testing is provided in Vig et al. [5], which describes bioeffects testing for several established and novel fluorescence agents.  For example, when evaluated at clinically relevant concentrations, IRDye800 showed a Mean Photo Effect (MPE) of 0.005 or less at radiant exposure levels up to 6 J/cm2, whereas the MPE for Rose Bengal at radiant exposure of 0.2 J/cm2 was 0.344. Such large differences are expected given that IRDye800 is well known as an in vivo imaging dye whereas Rose Bengal is more commonly used for photodynamic therapy. Results from Vig et al. demonstrated the tool’s ability to generate reproducible cytophototoxicity findings across six fluorophores, consistent with findings from prior in vitro and in vivo studies.

Limitations

Applicability to specific patient populations, clinical scenarios, or tissue types has not been systematically evaluated. Measurements with this method [5] indicate limited interference from products that overlap spectrally with the chromophore component of the 3T3-NRU assay, yet this robustness may not extend to devices with high optical illumination levels or fluorescence agents with very high yields.

Supporting Documentation

  1. Gaitan B, Frankle L, Vig S, Oskoui E, Adwan M, Chen Y, Elespuru R, Huang HC, Pfefer TJ. Quantifying the photochemical damage potential of contrast-enhanced fluorescence imaging products: singlet oxygen production. Photochem Photobiol. 2022;98(4):736-747. https://doi.org/10.1111/php.13638 
  2. OECD. Test No. 432: In Vitro 3T3 NRU Phototoxicity Test, OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing; 2019. https://doi.org/10.1787/9789264071162-en 
  3. Prieto Peraita M, Griesinger C, Amcoff S, Whelan M. EURL ECVAM Recommendation on the 3T3 Neutral Red Uptake Cytotoxicity Assay for Acute Oral Toxicity Testing. Publications Office of the European Union; 2013 (JRC79556).
  4. Ceridono M, Tellner P, Bauer D, et al. The 3T3 neutral red uptake phototoxicity test: practical experience and implications for phototoxicity testing—the report of an ECVAM-EFPIA workshop. Regul Toxicol Pharmacol. 2012;63(3):480-488.  https://doi.org/10.1016/j.yrtph.2012.06.001 
  5. Vig S, Gaitan B, Frankle L, Chen Y, Elespuru R, Pfefer TJ, Huang HC. Test method for evaluating the photocytotoxic potential of fluorescence imaging products. Photochem Photobiol. 2023; 100(6):1561-1578. https://doi.org/10.1111/php.13836 

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