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Evaluation and comparison of in vitro intrinsic hepatic clearance rates measured using cryopreserved hepatocytes from humans, rats, and rainbow trout
Citation:
Black, S., J. Nichols, K. Fay, S. Matten, AND S. Lynn. Evaluation and comparison of in vitro intrinsic hepatic clearance rates measured using cryopreserved hepatocytes from humans, rats, and rainbow trout. TOXICOLOGY. Elsevier Science Ltd, New York, NY, 457:152819, (2021). https://doi.org/10.1016/j.tox.2021.152819
Impact/Purpose:
In vitro and in silico methods that can reduce the need for animal testing are being used with increasing frequency to assess chemical risks to human health and the environment. The rate of hepatic biotransformation is an important species-specific parameter for determining bioaccumulation potential and extrapolating in vitro bioactivity to in vivo effects. One approach to estimating hepatic biotransformation is to employ in vitro systems derived from liver tissue to measure chemical (substrate) depletion over time which can then be translated to a rate of intrinsic clearance (CLint). In the present study, isolated cryopreserved hepatocytes from humans, rats, and rainbow trout were used to measure CLint values for 54 industrial and pesticidal chemicals. A data evaluation framework that emphasizes the behavior of heat-treated controls (HTC) was developed to identify datasets suitable for rate reporting. Measured or estimated (“faster than” or “slower than”) CLint values were determined for 124 of 228 (54%) species-chemical-test concentration datasets with acceptable analytical chemistry. A large percentage of tested chemicals exhibited low HTC recovery values, indicating a substantial abiotic loss of test chemical over time. An evaluation of KOW values and Henry’s law constants for individual chemicals suggested that in vitro test performance declined with increasing chemical hydrophobicity and volatility, although differences in testing platforms for mammals and fish also likely played a role. The current findings emphasize the value of negative controls as part of a rigorous approach to data quality assessment for in vitro substrate depletion studies. Changes in current testing protocols can be expected to result in the collection of higher quality data. However, chemicals with high volatility and poor solubility are likely to remain a challenge for CLint determination.
Description:
Computational models that describe chemical uptake and elimination by fish and humans are being used in increasing frequency to assess risks associated with environmental and workplace contaminants. An important input to such models is the rate of hepatic biotransformation, as this activity may substantially determine the extent to which a chemical accumulates over time. Unlike most inputs to these models, the rate of hepatic biotransformation cannot be predicted with acceptable accuracy from a compound's structure or properties. Alternative methods are needed, therefore, to obtain this information. One such approach involves the measurement of biotransformation using an in vitro system derived from liver tissue followed by extrapolation of this information to the intact animal. To date, this in vitro-in vivo extrapolation (IVIVE) approach has been used to predict risks to humans associated with nearly 60% of the ToxCast Phase I and Phase II libraries. Similar methods have been employed to assess the bioaccumulation potential of more than 100 environmental contaminants. Ultimately, the success of these IVIVE methods depends on the quality of information produced by the in vitro test systems. It is of interest, therefore, to develop a rational basis for assessing the chemical domain of applicability for specific in vitro testing platforms. In the presented work, in vitro assays employing liver cells from humans, rats, and rainbow trout were used to measure the rate of biotransformation for 56 industrial and pesticidal chemicals. Data acceptable for rate reporting were obtained for approximately half of the tested species and chemical combinations. In a substantial number of cases, however, unacceptable chemical losses from inactivated negative controls made it impossible to obtain a high quality rate prediction. Test failures were often associated with chemicals exhibiting high hydrophobicity (log Kow > 6) and/or volatility (log Henry's constant > 1 x 10-6 atm m3/mole), particularly in the rat and human assays, although other factors appeared to play a role. These findings underscore the need to incorporate negative controls into the assays and use the derived information to assess data quality. Although simple improvements to the assays may result in acceptable data for a substantial percentage of chemicals, chemicals that exhibit a high degree of hydrophobicity or volatility are likely to present continuing challenges. The framework for data evaluation presented in this work represents the first known effort to systematically evaluate data generated by these widely used in vitro assays. Future use of this framework should lead to improved risk assessments by helping to identify those chemicals for current methods are well-suited and those for which alternative methods
URLs/Downloads:
DOI: Evaluation and comparison of in vitro intrinsic hepatic clearance rates measured using cryopreserved hepatocytes from humans, rats, and rainbow trout