ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 28, 25-32 (1993) Calculation of the EC50 and Its Confidence Interval When Subtoxic Stimulus Is Present P. H. VAN EWUK* AND J. A. HOEKSTRAT *Solvay Duphar BV. P.O. Box 900, 1380 DA Weesp, The Netherlands: and National Insta of Public Health and Environmental Protection, P.O, Box: 1. 3720 BA Bilthoven. The Netherlands Received January 30, 1992 In many experiments in toxicology and ecotoxicology with continuous response, like growth, 4 stimulus is found for low doses. while higher doses are toxic. This subtoxie stimulus is called hormesis. The standard logistic model cannot be used when hormesis occurs. A model is given ‘which can deseribe this phenomenon, The model has the ECS0 as one of ts parameters. This has the advantage that the ECS0 and its confidence interval can be determined directly by nonlinear regression. Also, the tests forthe presence of hormesisang lack of fit are deseribed. «1995 acateme INTRODUCTION In ecotoxicology and toxicology many experiments are done to establish the dose response relationship for a certain compound and organism. The results of these ex- periments are often analyzed by a logistic model and summarized in the form of the EC5O. the concentration or dose which gives a 50% effect. The logistic model can be applied to dichotomous data such as survival or death and continuous data. eg ‘weight or biomass. The authors restricted the study to the continuous case. For continuous responses a stimulus for low doses of otherwise toxic compounds is found in many of these experiments (Stebbing. 1982). This stimulus is called hor- esis. If hormesis occurs, the standard logistic model does not fit. Common practice is then to use the model anyway or drop part of the data. A better solution was proposed by Brain and Cousens (1989) who extended the logistic model. This extension naturally implements hormesis in the logistic model. However, nonlinear regression with their model does not give the ECS0 explicitly. Therefore, the researchers propose a reparameterization of this model, which will give the EC50 and its confidence interval directly. and describe how to test for hormesis. A MODEL FOR HORMESIS, Extension of the Logistie Model ‘Often dose-response toxicity data follow a sigmoidal curve. This curve can be de- scribed by the logistic model. The general form of the logistic model is -—* ee PETE eh OP TE px or a) where xy = ECSO = exp(—g). 2s (0147-6513/93 $5.00, Copyright © 983 Acad Pr ne Ac of wpedacton am fr sored26 VAN EWUK AND HOEKSTRA ‘This can be written as D I Tear —blog x + 3). In model (1) k stands for the value of at x = O and g = —log(EC50). The parameter Kis estimated from the complete set of data, not just from the control, as are the other parameters. If 6 > 0, the response is monotonically decreasing and the x-axis an asymptote. as curve 1 in Fig. 1. The parameter —h stands for the slope of the line on logit-log-scale. On the original scale (1° vs x), b relates to the slope of the tangential line in the point of inflection, where x = EC30: [dv/dx].-2em = —b*k/4xo). For the data in Table | and Fig. | the logistic model obviously shows a lack of fit If this model is fitted to the data anyway, the control response will probably be over- estimated and the EC50 estimate can be misleading. To allow for hormesis, Brain and Cousens (1989) generalize model (1) into KU +f) T+ ex? @) This model can describe an increase for low levels of x, cf. Fig. I, curve 3. In the AIDS-literature, model (2) is known as the linear-logistic model, ef. Isham (1988, 1989). There, however, generally it is assumed that b < 0, which results in a curve with an exponential shape for low values of x and a straight line for high values. In model (2) the intuitive meaning of the parameters is lost, apart from the inter- pretation of the parameter k, which still stands for the response at x = 0. The EC50 (x9) should be calculated from the equation oon om) a} tN Fic, 1, Fitted curves by nonlinear regression for case study isobutylalcohol in nutrient solution. Curve 1 standard logistic model fited to all available data; curve 2, standard logistic model fitted to data, after exclusion of concentrations with mean response > control response: curve 3, linear logistic model (5) fitted toall available dataSUBTOXIC STIMULUS AND EC50-ESTIMATION 27 ex§ — Yo — 1 = 0. ) This equation has no explicit solution for xo. So, the estimation of the ECS0 involves two steps: first, find the parameter estimates for model (2 second, solve Eq. (3) numerically The construction of a confidence interval for the ECS0 also needs additional caleu- lations. ECSO as a Parameter in the Model Since it is very cumbersome to find the EC30 and its confidence interval from model (2), the model is rewritten so that the ECSO is included as a parameter. Then, no extra step is needed and an approximate confidence interval can be constructed from the estimated standard error which is in general supplied by nonlinear regression procedures. First, Eq. (3) is solved for g, leading to cen Bt | a d Substitute (4) in (2) kat fi A ey ©) T+ Qf + Delx/xa)" ‘The parameter k stands for the response at x = 0; f stands for hormesis: if > 0 the curve shows an increase for low doses. The parameter xo is the ECSO. The parameter ‘bi loses its simple interpretation. Numerically it is better to replace Xp by = log(p). This means one has to replace Xp in formula (5) with e*. The Fit of the Model Because of the restriction toa continuous response, nonlinear regression procedures will fit model (5) to the data, e.g. PROC NONLIN from SAS (SAS Institute Inc... TABLE | RESULTS OF AN EXPERIMENT WITH ISORUTYLALCOHOL IN, NUTRIENT SOLUTION (HULZEROS cf al., IN PREPARATION) Shoot wei Replication Concentration 1 2 Mean 0 1.126 0.833 0.980 0.32 1.096 1.106 L101 t 1.163 1.336 1.250 32 0.985 0.754 0.870 10 0.716 0.683, 0.700 32 0.560 0.488 0.524 100 0.375 0.344 0.36028 VAN EWUK AND HOEKSTRA 1990) and FITNONLINEAR from GENSTAT (Payne ef a/., 1987). Some extra input is needed, however. Most packages require initial estimates or a grid over which starting values for the parameters are searched. The following procedure was adopted to find initial estimates. ‘The parameter k can be easily estimated by the mean of the control responses. The parameter b is found by linear regression of log(./(max(’) ~ »)) on log(x). Only data with response lower than the mean response (k) for the control are included. The slope of this regression line is an estimate of —b. The ratio -intercept/slope from the same regression can be used as initial estimate for u, A more robust and therefore more reliable estimate for wis = ¥ wy*log(concentration,/5 with w; = |) — 0.5ek| 1 Ifthe response lies near k/2. than w, will be great, while concentrations with responses far from k/2 will hardly contribute to the estimation. While fitting a nonlinear model the derivatives of the function to the parameters have to be calculated. This can be done numerically, but it is better to supply these derivatives in formula, The derivatives can be found in the Appendix in SAS code. Tests for Hormesis and for Lack of Fit Since hormesis is described by only one parameter, the test for hormesis is formed by the ratio of the extra explanation of the linear logistic model and the residual variation, Formesin = (SSEtogise ~ SSEnmiogisc)/* where SSEvgsue = residual sum of squares for logistic model, SSEjinogiv: ~ residual sum of squares for linear logistic model, 4” = estimate of residual variation. €.2., SSEvnipsie/ residual degrees of freedom. This statistic follows approximately a FU. df) distribution, where df stands for the degrees of freedom of 6” When a design is replicated, ie.. more than one result per dose, itis possible to test for lack of fit for the total model. The procedure is comparable to the test for hormesis. Foot in which SSE = residual sum of squares for the current model, SS,.9 ~ sum of squares between replications, dfe = residual degrees of freedom for current model, dies = degrees of freedom between replications, 4° ~ estimate of residual variation, 2. SSsep/dhep. This Figrshould be compared with the Fldfe ~ dep» Up) distribution. A CASE STUDY: ISOBUTYLALCOHOL IN NUTRIENT SOLUTION Table | lists the results of an experiment in which isobutylalcohol was dissolved in nutrient solution in which lettuce plants were grown. The biomass of the shoot was determined after 21 days. The standard logistic model was fitted to these data (fit 1) and to the data, after exclusion of concentrations with mean response greater than the mean control response (fit 2). The third fit included all data and used the linear logistic model (5),SUBTOXIC STIMULUS AND ECS0-ESTIMATION 29 TABLE 2 RFSULTS FOR [sont 'YLALCOHOL EXPERIMENT: PARAMETER ESTIMATES, Data Model k Nv ! 1 All data Standard 110 29 Logistic «0.93, 1.29) (12, 66) 2 < control Standard 0.99 41 Logistic (0.82, 1.16) (0241.14) (17,97) 3 Allddata Linear 097 1228) 3s Wn Logistic (0.79. 1.14) (17.140) (13.97) 3, 4.6) Initial estimate 0.98 0.24 68 ° aa Note. Model (1) was used for ft and 2, Mode! (5) for fit 3. Far fit 2 concentrations with mean response ereater than mean control response were excluded. For each parameter the estimate is given and the 95% confidence interval (in parentheses). The parameter & stands for the intial response (x), forthe slope. ‘for the ECS0. and /for hormesis “Robust estimate w ~ Slog (concentration,yDu. with w, = jy OSek| Table 2 gives the parameter estimates of various fits. The parameter for control weight (k) is high for the standard logistic model. This lack of fit is illustrated in Fig. 1. The ECS0 estimate from the second fit (logistic model, part of the data excluded) is 43% higher than for the standard logistic model, while the linear logistic model (tit 3) gives an intermediate result. 23% higher. Table 3 presents the sums of squares and statistics derived from these. For all fits a calculation was made for a lack of fit test. based on the variation between replicates («2,,). For fit 1, the logistic model. it is found that: TABLE 3 RESULTS FOR ISORUTYL ALCOHOL EXPERIMENT: SUMS OF SQUARES P value Fit Data Model SSE dle MSE lof 1 All data Standard 0.242 n 0.0220 0.035 Logistic 2 < Control Standard 0.076 7 0.0109 0.26 Logistic 3 All data Line: 0.125 lo 0.0125 0.18 Logistic Between replicates (dig) All data 0.065 7 0.0092 = Control 0.045 5 0.0089 ‘Note: Model (1) was used for fit I and 2, model () for fit 3, For ft 2 concentrations with mean response ereater than mean control response were excluded. SSE stands for the residual sum of squares (error. dle forthe residual degrees of Freedom, MSF. the residual mean square isthe ratio ofthe two. The last columa, (P value, of) gives the result of the lack of ft test30 VAN EWUK AND HOEKSTRA iat = {(SSE ~ SSp)(dfe ~ dep) }/ 7p = {(0.242 — 0.065)/(11 — 7)}/0.0092 = 4.81 Since this is greater than the 5% critical value for a F(4, 7) statistic, 4.12, the lack of fit is significant. The P value is 0.035. For fit 2, the MSE is much lower than that for model (1) and the lack of fit test not significant. This is misleading, however, since the doses with mean response greater than the control response were excluded in the calculation of the MSE and the lack of fittest. For these doses Fig. | shows a clear lack of fit. Fit 3, the linear logistic model (5). gives no significant lack of fit (P = 0.18). Hence, the linear logistic model describes the data satisfactorily. A test can be made for the presence of hormesis: Prormess = (SSEjgitic ~ SSEiintegss)/ MSE ig Thus, the hormesis is significant (P = 0.012), (0.242 — 0.125)/0.0125 = 9.38 DISCUSSION The linear logistic model can be implemented into a nonlinear regression procedure. The author reparameterization looks a bit more complicated but has the advantage of a straightforward estimation of the ECSO. If there is no hormesis, the corresponding parameter fin model (5) equals 0. As described in the case study it is simple to test for hormesis. When model (5) was fitted to 72 datasets (see Van Ewijk and Hoekstra, in preparation), it was found, that fcan be negative, even significantly. Then f describes the asymmetry in the model and indicates the inappropriateness of the standard logistic model. AAs uswal in nonlinear regression, numerical problems can arise if the data do not contain enough information to estimate all parameters. Of course, the extra parameter Fenlarges this risk, especially in those cases where no hormesis exists. APPENDIX: SAS-CODE FOR THE FIT OF THE LINEAR LOGISTIC MODEL TITLE * SAS-code for fit of linear logistic model * DATA horm; INFILE ‘final76.dat’ FIRSTOBS=2; INPUT dos resp; IF dos >0.01 THEN Idos=LOG(dos) ; ELSE Idos=LOG(0.01); “the first part of the file finds initial estimates PROC MEANS NWAY DATA=horm; calculation of mean for control VAR resp; CLASS dos, OUTPUT OUT=hormmn MEAN=respmn ; DATA contrmn; SET hormmn:SUBTOXIC STIMULUS AND ECS0-ESTIMATION 31 IF dos=0: k=respmn: *k = mean of control; DATA hormcomb: MERGE horm hormmn ; IF -N-=1 THEN SET contrmn; logitr=LOG(resp/(k-resp) ): CALL SYMPUTCinik’.k) : *initial estimate for k. PROC REG DATA=hormcomb OUTEST=estlin: ‘linear regression to find: MODEL logitr=Idos: *initial estimate for b and xo: RUN; DATA -NULL- : SET estlin: CALL SYMPUTCinib’ -ldosy. CALL SYMPUT(inix0".EXP(-INTERCEP/Idos)); LET inif=0 ; *now the initial estimates are known, nonlinear regression can be performed: PROC NLIN DAT! orm : PARMS k=&inik x0=&inixO b=Sinib f=&init ; initial estimates IF dos > 0.0001 THEN DO; * specification of derivatives xx0b=(dos/x0)**b + fxO=241*x041 denom=1+ fx0* xx0b y= k*(L + f#dos) / denom : DER.k = y/k ; DER.b = ~y / denom * fx0 * LOG(dos/x0) * xxOb : DER.x0 = y /denom * xx0b * (~2*f + fx0 * b/x0) 5 DER.f = k* dos / denom ~ y/denom * 2 * x0 * xx0b + END: ELSE DO; *derivatives for control need denom=1 *special care DER.f= 0 DER.x0=0 ; END; MODEL resp RUN; “fit of the model REFERENCES, BRAIN P., AND COUSENS. R. (1989). An equation to describe dose responses where there is stimulation of growth at low doses, Heed Res, 29, 93-96. HULZEROS. E. M.. ADEMA. D. M. M.. DIRVEN-VAN BREEMEN, E. M., HENZEN, L., BAARSELMAN. R.. AND VAN GESTEL. C. A. M, Phytotoricity studies with Lactuca Sativa in soil and nutrient solution. 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