Biochemical, Genetic, and Molecular Interactions in Development - part 10 doc

Fetal Skeletal Risk Assessment Issues 387 Prior to 1998, regulatory testing guidelines for developmental toxicity assessments specified expo- sure from gestational day (GD) 6 (time of implantation of conceptuses into the uterine lining) to the end of major organogenesis (closure of the secondary palate), GD 15 for rodents, and GD 18 (4) or 19 (3) for rabbits. Current International Conference on Harmonization guidelines (20) have retained this duration of exposure. Recent EPA (11,21), FDA (6), and OECD (12) testing guidelines specify exposure beginning after implantation is complete (or on the day of insemination; see below) and continuing until the day before scheduled sacrifice at term. This corresponds to GD 6 (or 0) through 19 (or 20) for rats, GD 6 (or 0) through 17 (or 18) for mice, and GD 6 (or 0) through 28 (or 29) for rabbits. The day vaginal sperm or a copulation plug is found (rodent) or mating is observed (rabbit) is designated GD 0. The rationale for starting exposures after implantation is complete is based on two possible con- founding scenarios: • If the initial (parent) test material is teratogenic and the metabolite(s) is not, and if metabolism is induced by exposure to the parent compound, then exposure beginning earlier than implantation (with concomitant induction of enzymes and enhanced metabolism) will result in the conceptuses being exposed to less of the teratogenic moiety, and the study may be falsely negative. • If the test chemical and/or metabolite(s) interfere with implantation, then exposure prior to implantation will result in few or no conceptuses available for examination at term. However, there are situations when initiation of exposure should begin before uterine implantation. These include the following: •For exposure regimens that are anticipated to result in slow systemic absorption (e.g., topical application, subcutaneous injection or insertion of an osmotic mini-pump for continuous infusion, or dosing via feed or water), steady-state or maximal blood levels may not be attained until the very end (or beyond) of major organogenesis if exposures begin on GD 6 or 7. • For materials that are known to have cumulative toxicity (due to build-up of chemical and/or insult) after repeated exposures, exposure should begin on GD 0 (or earlier) so the conceptuses are developing in a fully affected dam. Fig. 2. New risk assessment/risk management paradigm (2). 388 Tyl et al. • For materials that are known to deplete essential components, such as vitamins, minerals, cofactors, etc., exposures should begin early enough so that the dam is in a depleted state by the start of organogenesis (or by GD 0). • For materials that are innocuous as the parent chemical but that are metabolized to teratogenic forms, expo- sures should begin early enough so that postimplantation conceptuses are exposed to maximal levels of the teratogenic metabolites. • For test materials that are known not to interfere with implantation, so exposure can encompass the entire gestational period and offspring will be available for examination. The previous specification for cessation of exposure prior to term allowed for a postexposure recovery period for both the dam and the fetuses, and an assessment could be made regarding whether the observed maternal effects (body weights, clinical observations) are transient or permanent. How- ever, the fetal evaluations take place only at term, and there is no commonly employed way to detect early adverse effects on the conceptus that resolve (are repaired, or compensated for, or result in in utero demise) earlier in gestation. What was observed at term was the net result of the original insult and any repair or compensation that occurred subsequently in the dam and conceptus after a post- exposure period. Thus, no detailed necropsy information on the dam was obtained during the expo- sure period (unless satellite females were used), and there was no way to distinguish effects during Table 1 Regulated Materials Requiring Risk Assessment 1. U.S. FDA (Food and Drug Administration) a. Food additives (preservatives, flavorings, dyes, etc.) b. Vitamins, not “natural products” (biologically active) c. Food substitutes (e.g., fat substitutes, artificial sweeteners) d. Pharmaceuticals (biologically active) e. Veterinary pharmaceuticals (biologically active) f. Food use pesticides (biologically active) g. Food use chemicals (e.g., chemical wrapping, artificial sausage skins, etc.) h. Medical devices NOTE: Attention to mechanism, human focus: disease state, clinical trials (IND [investigational new drug], NDA [new drug application], FDA approval) 2. U.S. EPA (Environmental Protection Agency) a. U.S. FIFRA (Federal Insecticide, Fungicide and Rodenticide Act) i. Pesticides (biologically active) ii. Food use pesticides (biologically active) iii. Biological pesticides (e.g., BT—Bacillus thuringiensis) iv. Biochemical pesticides (biologically active) v. Engineered plants (e.g., transgenic plants make pesticidal chemicals or are otherwise resistant to pests; GMOs [genetically modified organisms]) NOTE: Attention to nontarget species, environmental migration, contamination, transformation, bioaccumulation, etc. (registration and labeling, reregistration, data “call-ins”) b. U.S. TSCA (Toxic Substances Control Act) i. Commodity chemicals (use based on physio-chemical properties) —Paints, paint thinners, inks, plasticizers, plastics, medical supplies, PVC piping, solvents, carpets, car parts, chemical intermediates, etc. ii. Options the Agency uses to obtain/require animal testing —Test rules —Negotiated test agreements —Voluntary submissions/programs (e.g., HPV [High Production Volume] initiative) NOTE: Minimal information required for PMN (premanufacturing notice), SNUR (significant new use registration); EPA has 90 d to respond to submission Fetal Skeletal Risk Assessment Issues 389 exposure from those occurring afterward. Most germane to this chapter, the fetal skeleton primarily develops during the fetal period, so the previous maternal exposure period ended prior to fetogenesis, and the fetal skeleton developed after maternal exposure had ended. The new developmental toxicity testing guidelines require exposure until term, which includes the postembryonic fetogenesis period. The rationale for continuing exposure until term includes the following: • Maternal exposure until term is a better model for human exposure than exposure only during a portion of gestation with abrupt cessation at the end of embryogenesis. • Maternal responses at term with continued exposure (e.g., changes in organ weights, hematology, clinical chemistry, histopathology) can be better interpreted in terms of causality; there is no confounding mater- nal postexposure period for compensatory changes to occur. • Many systems continue to develop in the fetal period, both in terms of increases in cell size and number, and of differentiation of specialized cells, tissues, organs, and systems (e.g., skeletal, central nervous, pul- monary, renal, gastrointestinal, etc.). The effects on these processes occurring in the presence of continued maternal exposure will be manifested at term (for most of the systems) and will not be confounded by com- pensatory processes that may occur in a postexposure period. • The male fetal reproductive system is established and differentiates internally and externally in utero, beginning on GD 13–14 in rodents, so effects may occur from continued exposure to possible endocrine- active or reproductively toxic compounds by other mechanisms and may be detected. However, at term, only the testes and epididymides can be reasonably assessed, and most effects are not discernible until weaning, puberty, or adulthood (such as morphological and/or functional effects on accessory sex organs, adult testicular spermatogenesis, and epididymal sperm transit, etc.). Recently in the authors’ laboratory, a comparison was made of parameters of maternal and devel- opmental effects in control CD ® (Sprague–Dawley) rat dams dosed by oral gavage from GD 6 through 15 vs CD ® rat dams dosed from GD 6 through 19 (22). Although the GD 6 through 15 dosing was employed for earlier studies (1992–1997) and the GD 6 through 19 dosing was predominantly used for more recent studies (1996–1998), there was an overlap in time between studies using the different dosing durations. The authors concluded that the longer dosing regimen with no recovery period resulted in signifi- cant depression of maternal body weight and weight gain end points, as well as a reduction in fetal body weights. Presumably, the stress of continued handling and dosing was responsible for these differences. The three vehicles used (methylcellulose, corn oil, and water) were equally represented in both data sets. The concern was whether dosing with a potentially toxic test material would result in even fur- ther reductions because of a synergistic effect of the longer dosing period and the toxicity of the test material. The unexpected decrease in the number of implants and live fetuses may be the result of the differences in times of performance of the two groups of studies. In the early 1990s, Charles River Laboratories selected all offspring from larger litters (i.e., rather than a set number per litter, regard- less of litter size) as breeders, so that the average litter size rose. In the latter 1990s, a more balanced selection program was instituted [the CD ® (SD) “international gold standard”] to halt and reverse the increasing litter sizes and to minimize spontaneous differences in CD ® rats in the different Charles River breeding facilities. The decreased incidence of hydronephrosis (a common malformation), as well as enlarged lateral ventricles of the brain and of rudimentary ribs on Lumbar I (both variations), may represent genetic drift in this strain over the years evaluated. The relative developmental delay of the fetal skeleton, evidenced by the increased incidences of dumbbell cartilage and bipartite ossification centers in the thoracic centra, was likely because of decreased fetal body weights at term in the litters under the longer dosing regimen (i.e., the fetuses are delayed in late gestational development but are appropriate in the development of their systems, especially the skeletal system, for their size; ref. 23). Any embryofetal adverse outcome must be interpreted in the context of maternal toxicity. This is especially true of the development of the skeletal system because fetal well being (e.g., body weight) is absolutely dependent on maternal well-being. Therefore, the maternal animals should be evaluated in-life for at least clinical signs of toxicity, body weights, body weight gains, and feed and/or water con- sumption (as g/d and g/kg body weight/d). At maternal necropsy, body and organ weights are essen- 390 Tyl et al. tial, including gravid uterine weight, so gestational/treatment period weight gain, minus the contribu- tion of the gravid uterus, can be calculated as a measure of maternal toxicity separate from any embryo- fetal toxicity (e.g., reduced numbers of fetuses/litter and/or reduced fetal body weights/litter will result in reduced gravid uterine weight which, in turn, will cause reduced maternal body weight in the intact female) as well as at least a gross evaluation of organs. ADVERSE IN UTERO EMBRYOFETAL OUTCOMES There are four general categories of adverse in utero embryofetal outcomes: 1. Prenatal death, including preimplantation loss (the difference between the number of eggs ovulated and the number of conceptuses implanted) and postimplantation loss (the difference before the number of con- ceptuses implanted and the number of live fetuses at term; nonlive implantations include resorptions, indic- ative of early postimplantation demise, and dead fetuses, indicative of late postimplantation demise). 2. Fetal malformations and variations, including individual alterations; alterations by system; pooled exter- nal, visceral, and skeletal alterations; and all alterations. Usually, the alterations are separated into malfor- mations and variations for summarization and analysis. See below for a discussion of the definitions and characteristics of malformations and variations. 3. Developmental delays or growth retardation, including reduced body weights and reduced ossification, especially in those areas which ossified late, which are frequently associated with immaturity or delayed development (reduced fetal body weight) caused by toxicity. For example, typical skeletal delays include reduced ossification in fore- and hindpaw bony structures, such as metacarpals, metatarsals (hand, foot), and phalanges (fingers, toes); carpals and tarsals (wrist and ankle), are usually not ossified in terms of rodents or rabbits; caudal vertebrae, pubis (but usually not ilium or ischium), skull plates, sternebrae (especi- ally 5 [last to ossify], 6, 2, or 4 in that order), and cervical centra (especially 1; last to ossify; see the section titled Term Rodent Skeletal Components and Historical Control Incidences of Fetal Skeletal Malformations and Variations in this chapter). In addition, other evidence of delays includes enlarged renal pelvis in rats (24), reduced size of organs (e.g., liver or lung lobes, etc.) or other structures (e.g., long bones of the limbs), and dilated lateral ventricles (without tissue compression) of the cerebrum. Developmental delays or growth retardation also includes delayed organ development (reduced size and/or level of differentiation). 4. Functional deficits, which are not assessed in a developmental toxicity study design. They can be assessed in postnatal offspring exposed prenatally, which is done in other study designs. CLASSIFICATION OF FETAL MALFORMATIONS AND VARIATIONS In the test animal and human literature on malformations, there is no case to date where a teratogenic agent causes a new, never-before-seen malformation. What is detected is an increase in the incidence of the malformation(s) above that seen in the general population in humans and that seen in historical and concurrent control groups for animal studies. The current view is that teratogenic agents act on sus- ceptible genetic loci and/or on susceptible developmental events. Therefore, the response seen is influ- enced by the genetic background and will vary by species, strain, stock/colony, or race (in the case of humans) and individual (the last is more variant in, and therefore more relevant to, genetically hetero- geneous populations than in and to inbred strains). The outcome is also influenced by timing and dur- ation of exposure. There is genetic predisposition to certain malformations that characterizes specific species, strains, races, and individuals. Historical control data are indispensable (along with concurrent controls) to determine the designation and occurrence of the present finding(s) in the context of the background “noise” of the population on test or at risk. The general considerations for designation of a finding as a malformation, a variation, or a delay are imprecise, may vary from study to study and teratologist to teratologist, are relatively arbitrary, and are not necessarily generally accepted. However, the following are the general classification criteria for skeletal findings currently used in the authors’ laboratory (refs. 25 and 26; see also Figs. 7–15 in this chapter). Fetal Skeletal Risk Assessment Issues 391 Malformations • Incompatible with or severely detrimental to postnatal survival (e.g., exencephaly, anencephaly, spina bifida, cleft palate, ectopia cordis, gastroschisis, missing limbs [amelia]) • Involves replication, reduction (if extreme), or absence of essential structure(s) or organs (e.g., missing, extra, or small limbs, digits, ribs, other skeletal components) •Abnormal fusion of skeletal components (long bones of appendages, digits, ribs, etc.), dichiria (double hand), sympodia (fusion of legs), craniostenosis (abnormal/accelerated closure of anterior/posterior fontanelles and suture margins of skull plates), syndactyly (fused digits), vertebral scoliosis, cleft or “lobster claw” hand • Skeletal components unossified in an abnormal pattern (e.g., cleft sternum) • Result from partial or complete failure to migrate, close, or fuse (e.g., cleft palate, cleft lip, facial clefts, forked ribs, open neural tube, exencephaly, anencephaly, spina bifida, cranioraschisis) • May include syndromes of otherwise minor anomalies •Exhibit a concentration- or dose-dependent increased incidence (and/or severity) across dose groups, with a quantitative and/or qualitative change across dose groups (e.g., meningocele A meningomyelocele A menin- goencephalocele A exencephaly; foreshortened face A facial cleft A facial atresia; short tail A no tail A anal atresia; short rib A missing rib; brachydactyly (short digits) A oligodactyly (absence of some digits) A adactyly (absence of all digits); missing distal limb bones (hemimelia) A missing distal and some prox- imal limb bones A amelia (missing limbs) • Rare in concurrent and historical control fetuses • Cervical ribs Transitional Findings These may be upgraded to “malformation” or downgraded to “variation” status, depending on sever- ity and/or frequency of occurrence. • Nonlethal and generally not detrimental to postnatal survival • Generally irreversible • Frequently may involve reduction or absence of nonessential structures (e.g., innominate or brachiocepha- lic artery, as long as the subclavian artery to the arm and common carotid artery to the head are still present) • Frequently may involve reduction in number or size (if extreme) of nonessential structures or may involve their absence • Exhibit a dose-dependent increased incidence Variations • Nonlethal and not detrimental to postnatal survival • Generally reversible or transitory, such as wavy rib, extra rib (especially rudimentary) • May occur with a high frequency and/or may not exhibit a dose-related increased incidence (e.g., extra ribs on Lumbar I in mouse, rat, and rabbit fetuses) • Detectable change (if not extreme) in size of specific structures (subjective); e.g., shortened long bones of forelimb (humerus, radius, ulna, and/or hind limb femur, tibia, fibula), reduced renal papilla, small/large/ accessory spleen, short rib (XIII in rodents, XII in rabbits), etc. Please note that there is less variability in the development of the fetal skeleton the closer to term it is evaluated (i.e., if the term sacrifice is on GD 21 for rats, GD 18 for mice, and GD 31–32 for rabbits, rather than on GD 20 for rats, GD 17 for mice, and GD 28–30 for rabbits), but the risk of delivery before scheduled sacrifice is much greater. In addition, the sacrifice order must be random or selection of one from each dose group in rotation. If the dams and their fetuses are necropsied by group, there will be uncorrectable confounding. If done in the order of high, mid, low, and control groups, then there will be a “dose”-related reduction in fetal body weight and skeletal ossification due to the tim- ing of sacrifice (independent of any treatment-related effects), since late gestation, even a few hours close to term, is the time of rapid growth and development, especially of the fetal ossified skeleton (27). Skeletal defects may be primary (intrinsic to the cells and tissues that will form the skeletal com- ponents) or they may be secondary to a defect in another system that impacts on the development of associated skeletal components. Examples of the first situation are cleft sternum, cleft or “lobster 392 Tyl et al. claw” hand, duplication or loss of limb components (if not caused by amniotic band syndrome), etc. Examples of the second situation are exencephaly (when the overgrowth of the brain is considered primary, causing subsequent failure of the skull plates to form), spina bifida (where the initial failure of the neural folds to fuse into a closed neural tube subsequently prevents normal formation of the associated vertebrae), etc. The skeletal system is formed of two types of bones: (1) the flat, plate-like bones of the face, cranial vault, and scapulae are dermal elements that develop directly in a connective tissue membrane (i.e., “membranous bone”); and (2) the deeper, three-dimensional bones go through a membranous phase, then form as cartilaginous anlagen, and then finally the cartilaginous structures are replaced by bone- forming cells (osteoblasts and osteocytes) and extracellular bony matrix (i.e., cartilage replacement bone). The genesis of the rat skeleton has been previously described (28–30). With the new regulatory requirement for identifying and evaluating both cartilaginous and bony components of the fetal skeleton, knowledge of the status and nomenclature of both types of compo- nents at term is essential (excellent sources are ref. 31 for the rat, mouse, and rabbit; refs. 32 and 33 for the rabbit; ref. 34 for the mouse, etc.). The designation of skeletal malformations and variations, especially in the vertebral column, are now primarily based on the status of the cartilaginous components. If the cartilage is abnormal or variant, then the ossified bone that develops will follow the cartilagi- nous form and be abnormal variant. Examples of fetal malformations at term under this classification include fused cartilage:lumbar centrum; unilateral cartilage, unilateral ossification center:lumbar cen- trum; bipartite cartilage, dumbbell ossification center:lumbar centrum; split sternal cartilage; fused rib cartilage; rib cartilage attached to sternum; discontinuous rib cartilage; bipartite cartilage, normal ossi- fication center/dumbbell ossification center/unossified ossification center:thoracic centrum, and so on. In contrast, if the cartilage is normal, then unossified ossification centers in thoracic centra are designated as variations. If the cartilage is dumbbell, then unossified dumbbell or bipartite ossifica- tion sites in the thoracic and lumbar centra are also designated as variations. If cartilage is present for other bones (such as the pubis), even though there is incomplete ossification, the findings are desig- nated as variations. TERM RODENT SKELETAL COMPONENTS AND HISTORICAL CONTROL INCIDENCES OF FETAL SKELETAL MALFORMATIONS AND VARIATIONS The fetal skeletal components, which ossify last during late gestation, are the most sensitive to insult and are, therefore, usually the most affected by (and associated with) maternal and other embryo- fetal toxicity (33,35). These skeletal regions include the fetal skull (the GD 20 rat skull is illustrated in Fig. 3), the ribs and sternum (the GD 20 rat clavicle, ribs, and sternum are illustrated in Fig. 4), and the distal appendages (the rat GD 20 fore- and hind-paws are illustrated in Fig. 5). The complete fetal rat skeleton on GD 20 is presented in Fig. 6. Note the large anterior and posterior fontanelles in the GD 20 skull (Fig. 3), the predominantly cartilaginous rib cage in the GD 20 thorax (Fig. 4), and the pattern of ossification of units of the fore- and hind paws (Fig. 5). The bones of the wrist (carpals) and ankle (tarsals) are not yet ossified in the GD 20 rat. The bones of the hand (metacarpals) and foot (metatarsals) are ossified to a variable extent, with the forepaw usually ahead of the hindpaw in ossi- fication. In Fig. 5, four of the five metacarpals and three of the five metatarsals are ossified. The pha- langeal bones are also ossified to a variable extent in the GD 20 rat. There are ossification sites in the proximal phalanges of digits II and III of the forepaw, and no ossification sites yet in any phalanges of the digits in the hindpaw. The alizarin red S stained tips of the digits are the nails (not bones). A beau- tiful and detailed colored atlas of the double-stained fetal skeletons of Jcl:ICR mice on GD 18, Wistar rats on GD 21, and Kbl:JW rabbits on GD 28 is strongly recommended for the reader’s review (31). Historical control fetal incidences of malformations and variations have been published periodi- cally to aid in the interpretation of findings in a particular study and to illustrate the background inci- dence of effects of interest in rats (36–41), in mice (37,38,42), and in rabbits (38,40,42–45). Historical Fetal Skeletal Risk Assessment Issues 393 control fetal skeletal malformations and variations from the authors’ laboratory are presented to illu- strate the changing incidences over time within specific stocks and strains of rats, mice, and rabbits. These changing incidences are caused by founder effects, genetic drift, and spontaneous fluctuations (causes unknown) and are presented to make the point that concurrent and historical control data are absolutely essential to place the fetal skeletal findings in treatment groups in a given study in the proper context and to provide the background for deciding whether the effects are biologically and toxicologically significant (whether or not they are statistically significant). This information will Fig. 3. Rat gestational day 20 cranium. A, Dorsal view; B, ventral view. Ossified bone is clear, cartilaginous bone is black (modified from ref. 31 with permission). 394 Tyl et al. Fig. 4. Rat gestational day 20 rib cage. A, Lateral view; B, ventral view. Ossified bone is clear, cartilaginous bone is black (modified from ref. 31 with permission). Fig. 5. Rat gestational day 20 appendages. A, Forepaw, dorsal view; B, hindpaw, dorsal view. Ossified bone is clear, cartilaginous bone is black (modified from ref. 31 with permission). Fetal Skeletal Risk Assessment Issues 395 also inform subsequent risk assessment procedures and decisions (see below) based on the most sen- sitive, biologically relevant end points in the animal model. The historical control data from the authors’ laboratory are based on three CD-1 ® (Swiss) mouse studies with 70 dams and 841 fetuses evaluated on GD 17 (date of copulation plug = GD 0), 28 CD ® (Sprague–Dawley) rat studies with 652 dams and 10,033 fetuses evaluated on GD 20 (date of vaginal sperm = GD 0), and 11 New Zealand White rabbit studies with 224 does and 1800 fetuses evaluated on GD 30 (date of maternal breeding = GD 0). The rats and mice were all obtained from Charles River Laboratories (Raleigh, NC; Portage, MI; and New York facilities). The rabbits were all obtained from Covance Laboratories (Denver, PA; previously known as Hazleton Research Products [HRP], Inc.). Figures 7 through 9 provide data on the fetal incidence of all skeletal malformations and varia- tions over time in rats (Fig. 7), mice (Fig. 8), and rabbits (Fig. 9). In general, the incidence of skeletal malformations is always lower than the incidence of skeletal variations, and the peaks and troughs of malformations do not mirror the peaks and troughs of variations (and vice versa). The incidences of both parameters vary greatly over time, with no apparent trends. Pooling the control incidences across Fig. 6. Rat gestational day 20 axial and appendicular skeleton. Ossified bone is clear, cartilaginous bone is black (from refs. 25 and 26 with permission). 396 Tyl et al. Fig. 7. Historical incidence of total fetal rat skeletal malformations and variations on gestational day 20 (over 28 studies). Fig. 8. Historical incidence of total fetal mouse skeletal malformations and variations on gestational day 17 (over three studies). [...]... heredity, 351 molecular genetics, 351 I IFN- , see InterferonIGF-I, see Insulin-like growth factor-I IL-1, see Interleukin-1 IL-4, see Interleukin-4 IL-6, see Interleukin-6 IL-11, see Interleukin-11 Indian hedgehog, cartilage growth regulation overview, 229, 230 cartilage mechanotransduction mediation, 94, 95 chondrocyte differentiation signaling, 44–46 long bone development orchestration with retinoic acid,... morphogenetic proteins, 123 Hedgehog, 124 Wnt, 123 HoxD genes, HOXD13 mutation in skeletal disease, 24 site-specific recombination studies in mice, overview, 101 103 prospects, 111 regulatory sequences, Hoxd10, 105 , 107 Hoxd11, 105 , 107 Hoxd12, 106 , 107 Hoxd13, 107 targeted deletions, Hoxd11, 107 , 108 , 110 Hoxd13, 108 targeted inversions, 110 trans-allelic targeted meiotic recombination, 110, 111 Index Hox... 314, 315 interactions, 319 Interferon- (IFN- ), osteoclast differentiation role, 203, 204 Interleukin-1 (IL-1), osteoclast differentiation role, 202 Interleukin-4 (IL-4), osteoclast differentiation role, 202 Interleukin-6 (IL-6), osteoclast differentiation role, 202, 203 Interleukin-11 (IL-11), osteoclast differentiation role, 203 Ipriflavone, osteoclast differentiation, 207 L Larsen syndrome, candidate... Permitting Alizarin Red Staining of Skeleton and Histological Study of Viscera Supplement to Teratology Workshop Manual, pp 163–173 14 Inouye, M (1976) Differential staining of cartilage and bone in fetal mouse skeleton by Alcian blue and alizarin red S Congenit Anomal 16, 171–173 15 Kimmel, C A and Trammel, C A (1981) A rapid procedure for routine double staining of cartilage and bone in fetal and adult... with increasing dose? For example, exposure to dietary boric acid during gestation in rats and mice (but not rabbits) results in a dose-related decrease in the incidence of extra ribs on Lumbar I (associated with dose-related decreases in fetal body weights; refs 62 and 65) If the incidence of skeletal variations goes down with increasing dose, but the incidence of skeletal malformations goes up with increasing... 86; and triploid fetuses, ref 87) ENDPOINTS Both hazard- and risk-based studies examine endpoints Hazard-based research is important to identify new hazardous test materials; explore, identify, and define new endpoints; evaluate mechanism(s); and to enhance risk-based evaluations (Table 2) For rigorous risk-based studies, endpoints used (Table 3) must exhibit the following: • Reproducibility: within... 159–162, 166–169 mutation in skeletal disease, 24 parathyroid hormone-related peptide signaling loop disruption by fibroblast growth factor 421 receptor-3 activating mutations, 355, 356 retinoic acid effects on expression, 166, 167 Insulin-like growth factor-I (IGF-I), hind-limb suspension model effects, 266 osteogenic protein-1 synergism in osteoblast differentiation, 173, 179, 180 vitamin D, effects on osteoblast... retinoid signaling, growth plate chondrocyte signaling, 154, 155 long bone development orchestration with Indian hedgehog, 159–162, 166–169 molecular mechanisms, 153, 154 overview, 149, 150, 162–165 retinoic acid receptor signaling and establishment of chondrogenic template in developing limb, 150–152 Wnt signaling in mesenchymal condensation and chondrogenesis, 8 10 Chordin, bone morphogenetic protein... morphogenetic protein binding and regulation, 121 Wnt binding and regulation, 122 Cervical supernumary ribs (CSNRs), chemical agents in induction in rodents, methanol induction, 378, 379, 381 types of agents, 375 humans, 375, 381, 407 c-fos, osteoclast differentiation role, 198, 199 Chondoma, candidate gene loci, 28 Chondrocalcinosis, candidate gene loci, 28 Chondrocyte, Cbfa1 expression in differentiation,... One striking exception is the skeletal response in fetal CD-1® mice and rats (but not NZW rabbits; ref 62) to gestational exposure to dietary boric acid (63–70) With increasing dietary concentrations in ppm (and therefore increasing dose in mg/kg/d) in the presence of dose-related decreases in fetal 404 Tyl et al Table 2 Studies to Identify Hazard In vitro/ex vivo scenarios with limited end points • . malformations and variations have been published periodi- cally to aid in the interpretation of findings in a particular study and to illustrate the background inci- dence of effects of interest in rats. system is established and differentiates internally and externally in utero, beginning on GD 13–14 in rodents, so effects may occur from continued exposure to possible endocrine- active or reproductively. (63–70). With increasing dietary concentrations in ppm (and therefore increasing dose in mg/kg/d) in the presence of dose-related decreases in fetal 404 Tyl et al. body weight, the incidence of