Use of the Benchmark Dose Approach in Toxicology Risk Assessment
Toxicity studies aim to spot and understand the adverse effects caused by a substance, focusing on how these effects change with different doses. While sometimes data is obtained from human studies usually animal assays are essential for risk assessment. The goal of dose-response curves in risk assessment is to identify critical endpoints that could impact human health, looking closely at how the severity of these effects fluctuates with the dose, usually by visual inspection and with conventional statistical tools. This dose, the Reference Point (RP), will then be used to establish a safe dose for human health, where no adverse effects will be observed.
No-Observed-Adverse-Dose-Effect-Level (NOAEL) and Benchmark Dose (BMD) are the most commonly used statistical approaches to determine the Reference Point (RP).
Benchmark Dose vs. No-Observed-Adverse-Effect-Level in Toxicology Risk Assessment
No-Observed-Adverse-Effect-Level
The NOAEL is the highest dose at which there is not an observed statistically significant increase in the frequency or severity of adverse effects between the exposed population and its appropriate control. The next higher dose showing a statistically significant adverse effect is the Lowest-Observed-Adverse-Effect level (LOAEL).
This statistical analysis is highly dependent on the dose range selection and the statistical power, implying the size of the population. When every dose in a study shows a statistically significant effect, the lowest dose (LOAEL) might be chosen as the Reference Point (RP). However, it is possible that even lower doses could be harmful. Conversely, if no dose shows a significant effect, the highest dose tested gets labeled as the NOAEL, implying it is the safest, highest dose without observed adverse effects.
Determining a NOAEL is not just statistics; it also involves expert insight. Decision-makers bring their perspectives, so different experts might come to different conclusions about where the NOAEL lies in the same dose-range finding curve.
Regarding regulatory acceptance, NOAEL has traditionally been used and well-understood. Still, agencies are moving towards BMD due to its advantages in terms of statistical rigor and the ability to use all the available data.
Benchmark Dose
The BMD is defined as the dose or concentration of a substance associated with a specific change (the benchmark response or BMR) in the adverse effect compared to the background level of that effect, estimated from the fitted dose-response curve. The BMR is usually set as a small increase over the background level. The BMDL is the BMD’s lower confidence bound and this value is normally used as the RP, as determined by Hsieh et al. in an interesting paper about the application of BMD analysis on Zebrafish data.
The BMD method is generally considered more sensitive and precise because it is based on the entire dose-response curve and uses all the available data points, allowing a more robust extrapolation outside the observed data range based on a mathematical model. While statistically robust, it can be complex and requires careful model selection and benchmark response level selection.
From the regulatory perspective, the Scientific Committee of the European Food Safety Authority (EFSA) confirmed in 2022 that the BMD approach is a scientifically more advanced method compared to the NOEL approach for deriving an RP, and appropriate for quantitative risk assessment.
Application of BMD Analysis in New Alternative Model
Zebrafish has emerged as a promising model for toxicity testing. It can be used as a High-Content Screening (HCS) method due to its time and cost-effectiveness, small size, transparency, ease of maintenance, and high fecundity.
Regarding developmental toxicity and neurotoxicity assays, usually the LOAEL strategy has been followed. As explained before, this value may or may not be derived using statistical tests; if so, these tests can be applied to different toxicity endpoints. This accentuates the need for more harmonization in data analysis as makes it difficult to compare and interpret results between laboratories.
The BMD approach utilizes a user-defined benchmark response (BMR) relative to the background response to derive a BMD, enabling an extrapolation of the results from alternative models to humans, such as zebrafish. This extrapolation is highly important because it allows us to set a reference, bringing preclinical data obtained from alternative models closer to defining the concentrations used in human clinical trials. It is also an objective comparison across laboratories that can be used for standardization and validation of the alternative assays across labs, to gain confidence in them.
Expanding the application of the BMD approach to Zebrafish data can enhance the use of this NAM in the chemical risk assessment framework. Biobide works toward that direction in collaboration with organizations such as the Organization for Economic Cooperation and Development (OECD) and the National Institutes of Health (NIH/NIEHS).
Attempts to establish a unified data analysis strategy for Zebrafish developmental toxicity and neurotoxicity assays by adopting the benchmark dose analysis have already been made. The results from BMDs have been compared with a traditionally used LOAEL approach, and a high active compound concordance has been found. The referenced study has found that the BMD is more sensitive than LOAEL. Using standardized toxicity endpoints and an appropriate BMR allows for comparing results across different Zebrafish datasets, and extrapolation from Zebrafish to humans.
Determination of Developmental Toxicity in Zebrafish
The necessary data to evaluate the sensitivity of BMD and LOAEL approaches is obtained from different alternative methods, such as the developmental toxicity assays performed in Zebrafish.
Biobide has developed several toxicity assays, including a developmental toxicity assessment. Biobide's Zebrafish Teratotox Assay has been validated as a qualified alternative assay for its regulatory use following the ICH S5(R3) Guideline. Zebrafish is the first New Alternative Model (NAM) to be qualified for developmental toxicity and reprotoxicity based on this guideline.
The study of the potential induction of developmental defects is designed in two phases, a first phase for the Dose Range Finding (DRF) and the second phase for the definitive developmental toxicity assay. Specific endpoints, such as craniofacial malformations or deformed body shape, are analyzed at 2 days post-fertilization (dpf) and 4 or 5 dpf in this second phase. Based on LC50 and EC50 values, a Teratogenic Index (TI) is calculated, and a TI threshold is established to classify compounds tested based on their teratogenic potential.
Merging zebrafish toxicity tests with the BMD approach holds remarkable potential for shaping a safer future. This innovative combination enhances our understanding of substances’ toxicity and refines the ability to establish safe exposure levels for humans. By combining the potential of zebrafish as a NAM with the statistical robustness of the BMD method, we pave the way for more accurate, reliable, and human-relevant toxicological risk assessments.
Sources
EFSA Scientific Committee, More SJ, Bampidis V, Benford D, Bragard C, Halldorsson TI, et al. Guidance on the use of the benchmark dose approach in risk assessment. EFS2 [Internet]. [published 2022 Oct]; [cited 2024 Feb 1]; Available from: https://data.europa.eu/doi/10.2903/j.efsa.2022.7584
Hsieh JH, Ryan K, Sedykh A, Lin JA, Shapiro AJ, Parham F, et al. Application of Benchmark Concentration (BMC) Analysis on Zebrafish Data: A New Perspective for Quantifying Toxicity in Alternative Animal Models. Toxicological Sciences. 2019;167(1):92-104.
Weiner AMJ, Irijalba I, Gallego MP, Ibarburu I, Sainz L, Goñi-de-Cerio F, Quevedo C, Muriana A. Validation of a zebrafish developmental defects assay as a qualified alternative test for its regulatory use following the ICH S5(R3) guideline. Reprod Toxicol. 2024;123:108513.
