**11. Discussion**

450 Toxicity and Drug Testing

expected value for the response variable is 0.1287 g/210L with an empirical 95% confidence interval of 0.1215 to 0.1360g/210L, determined from the distribution of results in figure 9. The sampling/method component was also correctly identified as having the largest

Fig. 9. A distribution of 10,000 Monte Carlo simulated measurement results

Several measurements performed in forensic toxicology are qualitative in nature. These measurements typically take the form of a binary response (i.e., pass/fail, yes/no, over/under, present/absent, etc.). They are classification in nature where materials are assigned to discrete groups based on measurement results. Diagnostic tests are one important example of qualitative analyses. Their qualitative results are important indicators of whether some specified threshold has been exceeded or not and are important for the determination of further confirmatory analyses. In some cases the measurement system will respond simply with binary results (green light/red light). At other times the measurement system is quantitative on a continuous scale which can be dichotomized. For example, a prearrest breath test instrument employing a fuel cell might measure the breath alcohol on a continuous concentration scale but is interpreted as being greater than or equal to 0.080 g/210L or less than 0.080 g/210L. In either case, the response is considered binary and thus qualitative. The uncertainty associated with qualitative analyses has received much less attention than that of quantitative analysis. The uncertainty in qualitative analyses is basically probabilistic in nature - that is, we are interested in the probability of being correct in our decision. We are concerned primarily with the probability of false positive and false negative results. While there are a number of statistical methods for estimating the uncertainty associated with qualitative or diagnostic test results, there is no consensus as to which is to be preferred. (EURACHEM/CITAC, 2003, Pulido, et.al., 2003, Ellison, et.al., 1998) Some methods involve the simple determination of false-positive (FP) and falsenegative (FN) fractions which in turn assess the probability of making a wrong decision. (Pepe, 2003) Other qualitative and quantitative methods employ Baye's Theorem which is argued by many as a superior approach to estimating and interpreting measurement

contribution to total uncertainty of 85%.

**10. Uncertainty in qualitative analysis** 

Several examples have been presented here for estimating measurement uncertainty in the context of forensic toxicology. By no means do these examples imply that all possible uncertainty components have been considered. These examples were intended primarily to illustrate the general approach and computations involved. Moreover, while an example may have assumed a blood alcohol context, it could just as well have been applied in the context of breath or drug analysis. While the general approach will be relevant to most methods in forensic toxicology, each laboratory will need to identify and quantify its uncertainty components unique to its protocols and instrumentation. The examples and discussion presented here have also assumed independence among the input or predictor variables. This is certainly not always a valid assumption. In some measurement contexts there will be significant correlation between input variables which must be accounted for. (GUM, EURACHEM/CITAC, Ellison, 2005) While these concepts may be new to some practicing toxicologists, the concept of measurement uncertainty should not raise concerns for the forensic sciences. The emphasis should be on their ability to quantify confidence of measurement results. They should be presented in a manner that emphasizes and demonstrates their fitness-for-purpose. Modern technology should enhance and simplify these computations as well. Spreadsheet programs can be developed which require only the entry of specific values followed by the generation of all uncertainty results. Moreover, such computations can even be incorporated into the software of aalytical instruments. Such technology, when validated, should greatly simplify the process.

Several factors are responsible for the emphasis today on reporting measurement results along with their uncertainty. These include legal, economic, liability, accrediting and technological considerations. As professional toxicologists concerned with providing measurement results of the highest possible quality, we must be prepared to make this extra effort of providing the relevant uncertainty. Since there is no consensus regarding the best approach for computing uncertainty at this time, toxicologists should be familiar with the several approaches suggested here and then select and validate the one which best suits their analytical, procedural and legal context. The literature is rich with material regarding measurement uncertainty and should be carefully reviewed by toxicologists. (Drosg, 2007, Williams, 2008, Fernandez, 2011 Ekberg,et.al., 2011) This effort wll enhance the quality and interpretability of our measurement results and help establish a foundation of "evidence based forensics". The unavoidable fact of measurement uncertainty results in the risk of making incorrect decisions. While ignoring the uncertainty increases this risk, providing the uncertainty reduces and quantifies the risk for the decision maker. This fact alone should motivate the legal community to request and forensic toxicologists to rigorously estimate and provide such estimates.

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Toxicokinetics (TK) refers to the kinetics of absorption, distribution, metabolism, and elimination (ADME) processes where both first and zero order kinetics are expected and these processes can vary over a wide range of doses. The goal of TK and pharmacokinetic studies are similar, which is to define the ADME properties of a drug candidate (Dixit & Ward, 2007). Therefore, the wide range of studies to define these ADME properties (e.g., in vitro and in vivo metabolism, animal mass balance, and distribution studies) performed in the pharmacokinetic evaluation of the drug candidate can also serve to help guide the toxicokinetic evaluation of the same drug candidate with the knowledge that first and zero order kinetics might be expected in the ADME processes at the higher doses of this drug

Now it is widely accepted that toxic effects can be better extrapolated from animals to humans when these comparisons are based on TK instead of dose alone. For example, the safety margin that is based on the ratio of the animal exposure at no observed adverse effect level (NOAEL) to human exposure at the efficacious dose is a key predictor of human safety risk. To calculate this safety margin, the animal and human exposure is determined by analyzing drug and metabolites concentrations in plasma, which is the most practical and widely accepted way of assessing this risk (Dixit & Ward, 2007). However, most safety issues are not observed in the plasma but in the organs and/or tissues. Therefore, is sampling plasma a good measure of the safety margin for the risk

Sampling plasma and extrapolating this exposure to organs or tissues assumes that 1) concentration of drug in plasma is in equilibrium with concentrations in tissues, 2) changes in plasma drug concentrations reflect changes in tissue drug concentrations over time, and 3) distribution of drug and its metabolites is not affected by polarized cells (e.g., drug transporters and enzymes) that protect a lot of these tissues. Drug transport into tissues may not be a passive process and may depend on drug transporters (Ward, 2008), thus these assumptions may result in an inaccurate assessment of target organ exposure to drug and metabolites. Even without a drug candidate being a substrate for a drug transporter, lysosomal trapping of weak bases (e.g., liver and lung) or accumulation in membranes (e.g., muscle) can occur that can give rise to preferential distribution of the drug and its metabolites (MacIntyre & Cutler, 1988). Therefore, plasma is sometimes not a good

**1. Introduction** 

candidate in the safety studies.

assessment of safety?

P.D. Ward

*USA* 

*Johnson & Johnson,* 

*Pharmaceutical Research and Development, L.L.C.,* 

Williams, A. (2008). Principles of the EURACHEM/CITAC Guide 'Use of Uncertainty Information in Compliance Assessment'. *Accred Qual Assur*, Vol.13, pp. 633-638. **19** 
