ToxSci Advance Access originally published online on September 27, 2007
Toxicological Sciences 2008 101(1):91-100; doi:10.1093/toxsci/kfm253
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Quantitative Extrapolation of In Vitro Whole Embryo Culture Embryotoxicity Data to Developmental Toxicity In Vivo Using the Benchmark Dose Approach
National Institute of Public Health and the Environment (RIVM), 3720 BA Bilthoven, The Netherlands
1 To whom correspondence should be addressed at National Institute of Public Health and the Environment (RIVM), 3720 BA Bilthoven, The Netherlands. Fax: +31-3027-44446. E-mail: ah.piersma{at}rivm.nl.
Received July 3, 2007; accepted September 10, 2007
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ABSTRACT |
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If in vitro data are to be used as a basis for hazard characterization, a translation of an in vitro concentration toward an in vivo dose must be made. In this study we examined the correlation between dose descriptors from the in vitro Whole Embryo Culture (WEC) test and in vivo developmental toxicity tests. We applied the Benchmark Dose (BMD) approach to estimate equipotent in vitro concentrations (Benchmark Concentrations [BMCs]) and equipotent in vivo doses (BMDs). Using the data generated in an European Center for the Validation of Alternative Methods validation study we found that the BMCs were highly reproducible among laboratories. The three endpoints analyzed (head length, crown–rump length, and total morphological score) were strongly correlated. A clear in vitro–in vivo correlation was found between BMCs and BMDs. However, a considerable uncertainty would remain if the BMDs were estimated from the BMC using this correlation: the confidence interval of such an in vivo dose estimate would span various orders of magnitude. Differences in toxicokinetic properties among the compounds explained at least part of the scatter of the in vitro–in vivo correlation. But also heterogeneity in the design of the available in vivo studies underlies much of the scatter, and this puts a limit on validating in vitro data as predictors of in vivo data. Further analysis of the in vitro–in vivo correlation would therefore require high-quality in vivo data, generated by appropriate (and similar) study designs.
Key Words: embryotoxicity; Whole Embryo Culture; in vitro; in vivo; toxicokinetics; Benchmark Dose approach.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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A variety of in vitro and in silico methods have been and are being developed with the aim to reduce testing in experimental animals. At present, these models are mainly used for screening and categorizing compounds, although, for most endpoints, in vivo testing is still required for deriving a dose level that is used as a Point of Departure (PoD), in the hazard characterization of the compound. The translation of an in vitro concentration toward an in vivo dose (PoD) is not trivial. However, if this translation could be achieved, the in vitro models might be considered to be used as a stand alone, without the need for an additional in vivo study (provided that the in vitro test sufficiently covers the in vivo endpoints for which it is deemed predictive).
For assessing embryotoxic effects, a promising in vitro test system that seems to realistically mimic embryogenesis in vivo is the rodent postimplantation Whole Embryo Culture (WEC) system. This method is used to assess the effects of chemicals on the development of the complete embryo within its intact visceral yolk sac outside the uterus, during a critical phase in organogenesis. Although the test uses embryos, both exposure and assessment of effects occur in vitro, and for simplicity, it will be referred to as an in vitro test. Recently, within the framework of an European Center for the Validation of Alternative Methods validation study (Genschow et al., 2002
), a heterogeneous set of 20 chemicals were tested in the WEC system in four different labs. This set of compounds was composed such that it contained similar amounts of compounds considered strongly teratogenic, weakly teratogenic, or not teratogenic. The latter classification was based on existing in vivo embryotoxicity and teratogenicity data, and was established by consensus among the experts in the ECVAM validation study (Genschow et al., 2002).
The validation study yielded a large data set, which provides an excellent opportunity to study the applicability of dose–response modeling of in vitro data and to compare the outcomes with in vivo data. In this study we applied the Benchmark Dose (BMD) approach to the in vitro observations to examine the reproducibility of the WEC system, and to examine the correlation between in vitro Benchmark Concentrations (BMCs) with the PoDs derived from in vivo studies. We discuss the results in view of current proposals of changing testing procedures, in particular by reducing the number of in vivo studies.
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MATERIAL AND METHODS |
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The WEC test.
In the postimplantation, WEC rat embryos are removed from the dams at gestation day 10, and these embryos are then cultured and exposed for 48 h in vitro to various concentrations of the compound of interest. During this period, major aspects of organogenesis are realized, including heart development, closure of the neural tube, development of ear and eye, brachial bars and limb buds. Disturbance during this period may lead to general retardation of growth and development or to specific malformations in one or several organ anlagen. After culture, the morphology of the embryos is carefully assessed and a series of endpoints are scored (Brown and Fabro, 1981
). In this study we selected the evaluations for three of these endpoints: head length (HEAD), crown–rump length (CRL), and total morphological score (TMS) calculated as the sum of scores for all organ anlagen. Comparison of control embryos with exposed embryos forms the basis for conclusions regarding the embryotoxicity of tested compounds. Further details on the method can be found in Piersma et al. (2004).
Derivation of BMCs.
In the ECVAM validation study (Genschow et al., 2002), concentrations of the 20 compounds to be tested were selected based on a particular sequential scheme, where new concentrations were chosen based on findings at earlier applied concentrations. In this study, concentration–response data for each compound (n = 20), endpoint (n = 3), and laboratory (n = 4) were considered separately, resulting in 20 x 3 x 4 = 240 data sets. Each of these data sets was analyzed using the BMD approach, that is, a dose–response model was fitted to the data, and the fitted model was used to determine an estimate of the concentration associated with a particular effect size. For each of the three endpoints considered, a 5% change compared with the controls was chosen. The associated concentration is the BMC, for which
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), but it may be noted that for the purpose of establishing the relative potencies of the compounds this choice is not crucial. For instance, a 10% change in response for the BMR would have resulted in similar distances between the BMCs.
For each separate data set a model was selected according to the procedure described in Slob (2002). Figure 1 illustrates the method of deriving a BMC for a particular data set (in this example 5-fluorouracil).
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In some cases, the concentration–response data were not suitable for deriving a BMC value. In these cases, only one or two concentrations had been tested: because these concentrations did not show a clear response, testing of other concentrations was omitted.
The calculations were done using the PROAST software, a general software tool for dose–response modeling developed at RIVM, which will soon be available from the RIVM web site.
In vivo developmental toxicity studies.
For all 20 substances, a literature search for embryotoxic and teratogenic effects in vivo was performed. Literature searches were performed with the substance name together with combinations of the search terms rat, teratogen, teratogenic, teratogenicity, malformation, development, embryo, fetus. Because the in vitro WEC developmental toxicity tests were performed using rat embryos, the literature search was focused primarily on developmental toxicity studies in the rat. For 5-bromo-2'-deoxyuridine, data from mice were used in the absence of rat data. No in vivo study was found for isobutyl-ethyl valproic acid, and this compound was excluded from further analysis.
BMD derivation from in vivo data.
For the 19 remaining substances reductions in fetal weight and/or increased incidences of malformations were selected as the endpoints for estimating the (in vivo) BMDs, based on visual inspection of the data (not formal dose–response analysis). The reason for choosing these endpoints is that they represent the key effects in developmental toxicity tests, and one or both of these endpoints were affected by all those compounds that induced developmental toxicity. It is important to base the BMDs on the same endpoints for all compounds, as the resulting BMDs should reflect equipotent doses. The criterion for the BMD for each substance was defined as a 10% decrease in fetal weight and/or a 10% additional incidence of malformations. These values were chosen for practical reasons (observable changes), and are not crucial in the context of this study. As already mentioned (see derivation of BMCs), the aim is to obtain equipotent doses to establish the relative potencies of the different substances in vivo. The various studies that were often available for the same compound were evaluated as a whole, in a sort of weight of evidence approach, taking into account the quality of the study, maternal toxicity, gestation day(s) of exposure, exposure route, etc. The variation in study types and study results for the same compound was quite large, overwhelming the potential increase in precision that would result from a formal dose–response analysis. Besides that, a formal dose–response analysis was often not possible due to limited reporting of the data, or due to the very limited number of dose groups in the study design.
Correlation between in vitro and in vivo data.
Equipotent in vitro doses (the calculated BMCs) were plotted against equipotent in vivo doses (the calculated BMDs) to establish the correlation between BMCs and BMDs, and to explore to what extent an in vitro BMC could predict an in vivo BMD. A straight line was fitted to the data on a double logarithmic scale by minimizing the sum of products of the horizontal and vertical distances of the data to the line (Bokkers and Slob, 2005). The slope (b) of this fitted line represents the power of the relationship y = axbon the original scales, and indicates if the relationship between BMCs and BMDs is linear or nonlinear (on the original scales): if the slope equals 1, it is linear, if it is smaller than 1, the curve bends off at higher values.
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RESULTS |
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In Vitro BMCs
Table 1 shows the BMC values resulting from the reported concentration–response data by each of the four labs, for each of the three endpoints. The agreement between the BMCs obtained by the four laboratories was quite good, although lab 3 deviated from the general pattern for various compounds (Table 1) and showed a substantially larger variation in background values (Fig. 2) than the other three labs. The latter three labs showed a very consistent pattern (Table 1) in BMCs. The compounds were ranked with respect to the (geometric) means of the BMCs from the different labs, omitting the results from lab 3 because of its somewhat deviating results. As Table 1 shows, including lab 3 would have led to only a slightly different ranking, although for some compounds the difference would have been large (e.g., methoxyacetic acid). There is a reasonable agreement between the ranking based on the in vitro results, and the a priori assessed category of embryotoxic potency based on expert judgment using all available information (mainly in vivo results). The most deviating compounds in this comparison are diphenhydramine and acrylamide, which are in the mid-range according to the WEC test (for all three endpoints: TMS, CRL, and HEAD), but were considered nonteratogenic according to the experts (Table 1). For D-(+)-camphor, the agreement of the BMCs with the expert category was good for CRL and HEAD, but not so good for TMS. For dimethadione a good agreement was obtained for TMS, but not for HEAD.
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Some of the cells in Table 1 are empty. In these cases, no BMC value could be derived. This happened in some cases when only one concentration was tested without showing an effect, or when no dose–response was observed (see methods). If higher concentrations would have been tested for these chemicals, a BMC value might have been derived.
Figure 3 shows that a high correlation exists between the (geometric) mean BMC values for the three endpoints: CRL, HEAD, and TMS.
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In Vivo BMDs
Table 2 provides details on the animal studies that played an important role in assessing the BMD for each substance. The aim was to derive an in vivo dose for each of these compounds that would result in equally strong embryotoxic effects. To that end, the dose was established at which a 10% decrease in fetal weight and/or 10% additional incidence of malformations was reported, and that dose was considered an (rough) estimate of the in vivo BMD. For those studies where both endpoints were reported, these effects occurred at similar doses (Table 2).
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For three substances (6-aminonicotinamide, pentyl-4-yn-valproic acid, and methoxyacetic acid) developmental effects considerably larger than 10% were observed at all dose levels tested. The BMDs for these three substances were calculated by introducing in the model the dose–response data obtained in other studies for the same substance (6-aminonicotinamide) or structurally related substances (i.e., hexyl-4-yn-valproic acid for pentyl-4-yn-valproic acid; 2-methoxyethanol and di (2-methoxy-ethyl)phthalate for methoxyacetic acid), assuming that the slope of the dose–response would be equal (see Table 2).
On the other hand, for four substances, developmental effects lower than 10% were observed at all dose levels tested. For these substances, only lower bounds for the BMD could be derived (BMD > highest dose level tested; see Table 2). For another substance, acrylamide, the developmentally toxic effects observed concurred with effects in the dams, and were hence regarded as a category 1 (nonteratogenic) compound by the ECVAM expert group in the WEC validation study. However, it cannot be excluded that (possibly at higher doses) acrylamide has, in addition, a direct embryotoxic effect. Therefore, the dose at which these developmental toxic effects occurred was considered as a lower bound for the BMD for acrylamide.
Correlation In Vitro and In Vivo
Figure 4 shows the in vitro BMC values plotted against the in vivo BMD values for the 19 compounds. For some substances, the in vivo BMD is depicted by an arrow, to indicate that, theoretically, the in vivo BMD should be somewhere above the lower bound of the arrow (see previous paragraph).
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This figure shows a fairly good correlation between the BMC and the BMD, although considerable dispersion remains. In vivo BMD10s increased less than proportional with increasing in vitro BMC05s. On a double logarithmic scale a straight line can be fitted with a slope of 0.7. This slope is lower than one, which indicates that the curve would bend on the nontransformed concentration/dose scales (i.e., its slope would progressively decrease with increasing doses).
The selection of the compounds to be used in the ECVAM validation study was partly based on toxicokinetic considerations (Brown, 2002). For example, the compounds tested were themselves active agents (i.e., they did not need metabolic activation) and readily passed the placenta. Nevertheless, there could still be relevant differences in other toxicokinetic properties of the selected substances that might explain some of the scatter in the BMD to BMC correlation. Table 3 compiles two toxicokinetic parameters, absorption and half-life for all the compounds. Systemic absorption was high (40–100%) for all compounds for which we found data. Half-life ranged between 14 min and 3 h for most compounds, but some compounds had considerably longer half-life (up to 10 days). Another factor that could explain some of the scatter in the correlation between BMC and BMD is the varying dosing regimes in the in vivo studies. Thus, a single dose was administered in some studies, whereas up to 20 repeated doses were administered in others.
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To cause an equally large effect a single dose might be equal to, but generally higher than the dose in a repeated dosing scheme (unless the repeated dosing scheme missed the critical window and the single dose did not). Therefore, one may imagine that a single dose study might result in a higher BMD than a repeated dose study. Similarly, one may imagine that the BMD of a compound would have been higher had the half-life of the compound been smaller. Figure 5 shows the same plot as in Fig. 4, but now with (dashed) upwards arrows for those compounds with one (or both) of these two properties (more than 2 dosing days and/or long half-life). This figure illustrates that taking these two aspects into account could potentially improve the correlation between the in vitro BMC and the in vivo BMD. Further, it can be seen that the slope of the fitted line (on double log-scale) would move closer to unity. This implies that the relationship between BMC and BMD plotted on the nontransformed concentration/dose scales would get closer to a straight line.
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DISCUSSION |
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Our analysis of the available WEC data confirms earlier conclusions that this particular in vitro test system has a high reproducibility among laboratories (Genschow et al., 2002
; Piersma et al., 2004). One of the laboratories showed some deviating results in comparison with the other three laboratories, possibly related to a factor not systematically kept under control in this lab (e.g., temperature during incubation) as indicated by relatively large variations in the background values. The three endpoints analyzed (HEAD, CRL, and TMS) were strongly correlated. Comparison of the type of developmental effects observed in the WEC test with those observed in vivo for the same substance did not reveal concordance between endpoints (data not shown). This is probably caused by the reductionistic nature of the WEC model and/or by the difference in exposure conditions as compared to the in vivo model. For example, in vivo developmental effects such as delayed ossification, hydronephrosis, and cleft palate cannot be scored in the WEC test. In addition, the relative sensitivity of the various tissues may depend on the exposure window, which is relatively narrow in the WEC test. Thus, eye effects are more often observed in the WEC test than in in vivo studies. The ranking of embryotoxic potency resulting from these in vitro results shows a good correlation with the three categories of embryotoxicity assessed by expert judgment. However, for some of the compounds the BMCs did not completely agree with this categorization, in particular two of them (diphenhydramine and acrylamide), whereas another two agreed only partly (dimethadione and D-(+)-camphor). For instance, dimethadione was correctly categorized by the parameter TMS, but not by HEAD. It should be noted that any discrepancy is not necessarily due to a limitation of the WEC test: the characterization of in vivo embryotoxicity by expert judgment in some cases had to be based on the limited database available.
Ranking on the basis of BMC values implies ranking on effective concentrations reaching the embryo. However, the classification for embryotoxicity is not only based on the effective doses (concentrations) administered in vivo, but also on the relationship between embryotoxicity and maternal toxicity. The applied doses in the in vivo studies for substances like diphenhydramine and acrylamide might not have been high enough to achieve embryotoxic effects or might have induced maternal toxicity complicating the interpretation of developmentally toxic effects. For some studies, it is not trivial to conclude whether the developmentally toxic effects observed are a consequence of maternal toxicity. The assumption that the developmentally toxic effects are secondary to maternal toxicity when they co-occur may lead to an underestimation of the direct developmental toxic potency of a substance (e.g., acrylamide). In contrast to the in vivo studies, maternal effects do not interfere in the WEC test. Thus, comparing the effective concentration in the WEC test (extrapolated to an in vivo dose) with the dose that induces maternal toxicity in vivo could support or reject a direct developmental effect.
Results show that for the substances for which a BMD could be derived, a clear in vitro–in vivo correlation exists. However, a considerable uncertainty would remain if the BMDs were estimated from the BMC using this correlation: the confidence interval of such an estimate would span various orders of magnitude. The substances for which only a lower bound level for the BMD could be assessed were not in disagreement with the overall picture, but, of course, incorporating these values does not really help in establishing the correlation. Further, differences in dosing regimens in the in vivo study seemed to account for at least part of the scatter in the BMC–BMD correlation. These notions illustrate that evaluation of an in vitro–in vivo correlation may be strongly hampered by errors in the estimated (equipotent) BMD.
In vitro dose descriptors that we used in this study were the geometric means of the data obtained in three of the four laboratories that participated in the ECVAM validation of the WEC test. We excluded data from one laboratory (lab 3) because we aimed at exploring the possibilities of extrapolating in vitro to in vivo data by considering the highest quality data available. Of course, if a correlation as established in Fig. 4 was actually used in practice, potential errors in a single available BMC, like the ones found in lab 3, should be taken into account. Obviously, this would only add to the large prediction errors in extrapolating BMCs to BMDs.
Further analysis of in vitro–in vivo relationships that could be useful in human risk assessment strongly depends on the availability of a high-quality in vivo database. High quality is defined here as being based on similar study designs, especially with respect to route of administration, number of administrations, exposure window during gestation, etc. In addition, general quality aspects are relevant, such as purity of the test substance and animal facility standards as prescribed in Organization for Economic Co-operation and Development Test Guidelines. Admission to the numerous confidential studies that have been performed by industry under, for example, the European Pesticide Act could substantially improve the evaluation of in vitro–in vivo relationships.
We hypothesize that in vitro alternative tests might be particularly useful in the category and read-across approaches where data on relatively closely related compounds can be used to predict embryotoxicity of related compounds for which limited or no (in vivo) data are available. Indeed, within a category, effective exposure regimes are expected to be similar (assuming that chemicals within a category act on the same target). For example, if a critical window of exposure (e.g., certain gestational days) exists for a certain chemical this is likely to be the critical window for related chemicals. In vitro models such as the WEC may thus prove useful in screening and prioritizing compounds within classes for further development.
Toxicokinetic properties might have a considerable impact on the in vitro–in vivo correlation. Therefore, extrapolating a BMC to a BMD would only be valid for substances with toxicokinetic properties comparable with those of the substances used to define the correlation. As an alternative, a quantitative toxicokinetic model might be developed for each substance, and the resulting internal dose estimates could then be correlated with the in vitro concentrations. We are currently exploring this approach by developing quantitative toxicokinetic models for some of these test substances. Any improvements in quantitative toxicokinetic modeling will be of value in extrapolating in vitro to in vivo data, and research in this area should be considered as important as the development of alternative tests. The development of a structure-based toxicokinetic model (i.e., a generic model were input parameters are based on quantitative structure—property relationships) would be a useful tool, although a long way is foreseen before this can be achieved.
In conclusion, given the large remaining scatter in the correlation between in vitro BMCs and in vivo BMDs, replacing the in vivo animal study by the WEC test for the purpose of quantitative risk assessment is not yet feasible.
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FUNDING |
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Dutch Ministry of Health, Welfare and Sports (V/340720); Dutch Ministry of Housing, Spatial Planning and the Environment (M/602100); and Departament d'Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya postdoctoral fellowship to G.J.
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