TERATOLOGY 43~159-185 (1991)

Activity Profiles of Developmental Toxicity: Design Considerations and Pilot Implementation ROBERT J. KAVLOCK, JACQUELINE A. GREENE, GARY L. KIMMEL, RICHARD E. MORRISSEY, ELIZABETH OWENS, JOHN M. ROGERS, THOMAS W. SADLER, H. FRANK STACK, MICHAEL D. WATERS, AND FRANK WELSCH US Environmental Protection Agency (R.J.K., J.M.R., M.D.W.),Burroughs Wellcome Co. (J.A.G.),Environmental Health Research and Testing, Inc. (H.F.S.),and Chemical Industry Institute of Toxicology (F.W.),Research Triangle Park, and University of North Carolina, Chapel Hill (T.W.S.), North Carolina; US Environmental Protection Agency, Washington, District of Columbia (G.L.K.);Merck, Sharp and Dohme Research Laboratories, West Point, Pennsylvania (R.E.M.); The Human Genome & Toxicology Group, Oak Ridge National Laboratory, Oak Ridge, Tennessee (E.0.)

ABSTRACT The available literature was searched for quantitative test results from both in vitro and in vivo assays for developmental toxicity for five model compounds: cyclophosphamide, methotrexate, hydroxyurea, caffeine, and ethylenethiourea. These compounds were chosen on the basis of their extensive utilization in a variety of assay systems for developmental toxicity as evidenced by their representation in the ETIC database (each generally has 100-500 citations encompassing multiple test systems). Nine cellular-based assays, six assays using whole embryos in culture, as well as Segment I1 and abbreviated exposure tests for mammalian test species are included in the database. For each assay, the critical endpoints were identified, each of which was then provided a three-letter code, and the criteria for extraction of quantitative information were established. The extracted information was placed into a computerized reference file and subsequently plotted such that the qualitative (positivehegative) and quantitative (e.g., IC,,, highest ineffective dose (HID), lowest effective dose (LED)) results across all test systems could be displayed. The information contained in these profiles can be used to compare qualitative and quantitative results across multiple assay systems, to identify data gaps in the literature, to evaluate the concordance of the assays, to calculate relative potencies, and to examine structure-activity relationships. Recent developments in the field of developmental toxicology have resulted in the proliferation of assays designed to detect agents which may be hazardous to the fetus. Increasing amounts of data from in vitro and other short-term assays have created the need for a peer-compiled and reviewed database of quantitative test results. Such a database would help identify data gaps in the literature, provide a coherent basis for reporting assay results, and maintain a repository of test results. In addition, it would provide the framework to establish the sensitivity, specificity, and accuracy of assays used for hazard identification, and to select assays for monitoring mechanisms relevant 0 1991 WILEY-LISS, INC.

to the developmental toxicity of particular classes of chemicals. To accomplish this task, we adapted an approach developed by Garrett et al. ('84) which used bar graphs to describe the data from short-term tests of genetic toxicity. For a particular chemical the data are displayed along the x-axis, with each assay being designated by a three-letter code, and a logaReceived July 3, 1990; accepted September 25, 1990. DISCLAIMER The information in this document has been funded wholly or in part by the US Environmental Protection Agency. It has been subject to the Agency's peer and administrative review, and it has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

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rithmic dose/concentration scale is shown on the y-axis. The condensed format of the data presentation facilitates the rapid visualization of the types of biological effects induced by a particular chemical and provides a mechanism for quick comparison of effects among different chemicals. This technique has now been incorporated into the data summaries for chemicals evaluated in the IARC Monographs Program on the Evaluation of the Carcinogenic Risk of Chemicals to Humans (IARC, ’87, ’88, ’89a,b, ’90; Waters et al., ’88a). The procedure we followed in modifying the genetic activity profiles for the purposes of developmental toxicity began in November of 1987 when a committee consisting of the authors and representatives of the environmental Teratogen Information Center (ETIC) was convened. The charge to the committee was to review all published test results for incorporation into the database, identify the endpoint(s) that should be included in it, establish the minimum criteria for acceptance of a test result, and to review the information in the database for accuracy. The primary extractions of the information were performed by the staff of ETIC. The results of the extractions for five pilot chemicals and the experience gained by the committee serve as the basis for this communication. METHODS

Assay selection and criteria A total of eight cellular-based assays, six assays using embryos in culture, as well as Segment I1 and abbreviated exposure tests from the mouse, rat, rabbit, miscellaneous laboratory species (hamster, guinea pig, cat, dog, etc.), and non-human primates were considered for inclusion into the database. For each assay, the salient endpoints were identified, a unique three-letter code was assigned, and data extraction criteria were determined. A complete listing of the assays, endpoints, codes, quantitative variables, and prototype references is provided in Table 1. The conventions used to assign the code followed two basic principles. The “common” designation of the assay was used if available, such as MOT for the mouse ovarian tumor assay or HPM for the human palatal mesenchyme assay. If this was not the case, the code was constructed as “Species, Stage, Endpoint” or “Species, Endpoint,

Duration.” Thus, the first letter designates the species (V, virus; D, Drosophilia; Y, Hydra; C, aves; A, amphibians; F, fish; G, hamster; M, mouse; R, rat; L, rabbit; X, misc. or multiple; P, primate; H, human). The second letter generally represents the particular endpoint (i.e., M, maternal toxicity; D, embryonic death; G, growth retardation; V, visceral anomalies; S, skeletal anomalies, F, functional effects) or culture condition (E, embryo in vitro; u, micromass culture systems). For in vivo mammalian assays, the last character distinguishes durations of exposures (2 for a “Segment II” study or X for an abbreviated exposure study). For assays based on whole embryos in vitro, the last letter defines the endpoint of the assay (e.g., G, growth retardation; M, malformation; D, death). Thus, MEG indicates mouse embryos in vitro, growth retardation; and LV2 designates rabbit, visceral malformations, Segment I1 exposure. Preliminary extraction of the information was conducted by the staff of EMTIC/ORNL according to criteria established for each assay by a committee member. Each member was responsible for several of the assays. Where possible, assignments were on the basis of prior expertise in that assay. Only data contained in publications present in the ETIC database were considered for inclusion. Data contained in abstracts were not extracted unless verified by contacting the senior author. For in vitro assays, ICs0s (concentration causing 50% inhibition) or EC,,s (concentration producing half-maximal response) were generally extracted for positive results, and the highest test concentration was extracted for negative results. For in vivo assays, the LED (Lowest Effective Dose) was extracted for positive results, while for negative results the HID (Highest Ineffective Dose) was used. Because of the desire t o extract the lowest effective concentration or dose, studies which utilized a single-dose level or concentration were generally excluded from the database, the exception being where a test protocol is routinely conducted at a singledose level. The author’s interpretation of the effective dose was utilized unless no analyses or interpretations were stated. Algorithms were developed to extract information for the latter cases. For example, in mammalian developmental toxicity assays the criteria applied were: 1)the study must have included dose-response information, 2)

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PROFILES OF DEVELOPMENTAL TOXICITY TABLE 1 . Assays, endpoints, test codes, extracted variables, and prototype paper (where applicable) used in the orofiles Gateszorv Cellular assays Pox virus expression Mouse ovarian tumor Palatal mesenchyme Drosophila cell culture

EndDoint

Code

Virion release Cell attachment Cytotoxicity Differentiation

VIR MOT HPM DMY DNT

50% inhibition of viral plaques LEDIHID for attachment inhibition LEDIHID for growth inhibition Myotube reduction a t 1%adult L D s Neurotube reduction a t 1% adult L D s

Neuroblastoma Micromass culture

Differentiation Differentiation/ proliferation

MNC XUL XuN

LEDIHID for induction of differentiation ICS~IHIDfor inhibition of limb cells IC5,IHID for inhibition of neuroblasts

Chick retina assay

Differentiation/ proliferation

CRA CRM CRH CRD

LEDIHID for aggregation LEDIHID for macromolecular synthesis LEDIHID for histapathology LEDIHID for biochemical alteration

Daston and Yonker (’87)

Mammalian organ culture

Differentiation

xoc

LEDIHID for palate, limb buds, lungs, etc

NIA

Embryogenesis

YAT YRT

LED for adult tulip regenerations LED for regenerating dissolution

Johnson (‘82)

Drosopbila larva culture Frog embryo culture

Embryogenesis Embryogenesis

DEC

LEDIHID for anomalies in emerging adults LED(IC5,)IHID for embryonic mortality LED(IC50)IHID for embryonic malformations

Schuler et al. (‘85) Sabourin et al. (‘85)

Fish embryo culture

Embryogenesis

Chick embryo culture

Embryogenesis

AED AEM FED FEM CED CEM

Rodent Embryo Culture

Embryogenesis

Embryos in vitro Hydra attentuata

Embryogenesis

Mammals in vivo Mouse

Rat

Rabbit

Extraction

Prototme reference Keller and Smith (’82) Braun e t al. (’82) Pratt and Willis (‘85) Bournias-Vardiabasis et al. (’83) Mummery et al. (‘84) Flint and Orton (‘84)

LED(IC50)/HID for embryonic mortality LED(IC50)IHID for embryonic malformations

NIA

LED(ICSO)/HIDfor embryonic mortality LED(ICSO)/HIDfor embryonic malformations

NIA

MED MEG MEM RED REG REM

Mouse, LED(ED50)IHID for embryonic mortality Mouse, LED(ED,,)MID for growth retardation Mouse, LED(EDSO)MIDfor anomalies Rat, LED(EDSO)/HIDfor embryonic mortality Rat, LED(ED5o)IHID for growth retardation Rat, LED(ED5oYHID for anomalies

NIA

MCK MM2 MG2 MD2 MV2 MS2 MF2

Chernoff and Kavlock (‘82) NIA

MMX MGX MDX MVX MSX MFX RCK RM2 RG2 RD2 RV2 Rs2 RF2 RMX RGX RDX RVX RSX RFX LM2 LG2 LD2 LV2 LS2 LF2

LEDIHID, postnatal growthIviability LEDIHID, maternal toxicity, Segment I1 LEDIHID, fetal growth effect, Segment I1 LEDIHID, embryonic death, Segment I1 LEDIHID, visceral anomalies, Segment I1 LEDIHID, skeletal anomalies, Segment I1 LEDIHID, functional effects, Segment I1 LEDIHID, maternal toxicity,

Activity profiles of developmental toxicity: design considerations and pilot implementation.

The available literature was searched for quantitative test results from both in vitro and in vivo assays for developmental toxicity for five model co...
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