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the Chick Embryo Model for Teratogenicity Testing

Date: Sep 1987 (est.)
Length: 8 pages
87849734-87849741
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87849734/87849741
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SCRT, SCIENTIFIC REPORT
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BENNETT,MELANIE/LAB 20 LIFE SCIENCES
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G3
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87849733/9741

Related Documents:
Named Organization
FDA, Food and Drug Administration
Who Scientific Group
Request
R1-048
Named Person
Fisher
Jelinek
Johnson
Mann
Schoenwolf
Schowing
Verrett
Wilson
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05 Jun 1998
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xte11e00

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The Chick Embryo Model for Teratogenicity Testing The chick embryo is a convenient experimental system for-embryo- logic and teratologic studies. It has been described as a classic system and is the most widely used whole-embryo system (Johnson, 1985). The chick embryo has been used to assess the toxicity and teratogenic effects of a broad variety of chemicals for more than a century by numerous investigators. The Food and Drug Administration (FDA) has utilized the injection of chemicals into developing chicken embryos to assess toxicity and to screen for teratogenic activ-ity. Beginning in 1959, the program included the study of various direct and indirect food additives, drugs, pesticides, mycotoxins, and other natural and synthetic chemicals. Later (1970) the FDA utilized this system in reviewing the group of direct food additives-that since 1958 has been classified-as "Generally Recognized As Safe" (GRAS) to determine teratogenic effects in this species. - However, in these investigations there have been many variations with respect to time and route of administration, length of development, and method of examination or analysis for abnormal occurrences. As a result, comparison of data from-different investigators is difficult and there is no one preferred protocol (Verrett et al., 1980). - Despite its extensive use in teratological research the-chick embryo has never been employed in routine testing procedures. When the-first official testing guidelines were outlined in 1966, theoretical and technical knowledge regarding the chick embryo were in a preliminary state. At that time there were widespread unfavorable attitudes toward the chick embryo model. The World Health Organization Scientific Group (1967) stated unequivocally that the method was "not recommended" because "It was too sensi- tive to a wide range of agents and affords no parallel with the anatomical and physiological relationship existing between the pregnant mammal and her conceptus," i.e., the chicken lacks a placenta and there are no similarities in either anatomical or physical characteristics. Other objections raised by WHO included pharmacokinetic dissimilarities inherent in the closed character of the avian egg with respect to injected xenobiotics and high non-specific sensitivity resulting in an unjustifiable number of false positive results (Jelinek, 1982). More recently, techniques and procedures have improved and attitudes toward the chick embryo model have been modified. The need for rapid toxicological testing of large numbers of chemicals and concern over the number of animals used in toxicity testing have made the chick embryo model more appealing. Wilson (1978) stated that the chick embryo possesses more of the essen- tial features of an optimal test system than any other in vitro
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-2- model under consideration and recommended its use as a prelim- inary teratological screen. It would serve as a means to prior- itize chemicals so that only those requiring more detailed developmental toxicity testing would subsequently be evaluated in pregnant animals. Agents causing significant disruption of development in the chick should be subjected to more rigorous specialized tests if these agents are likely to occur in appre- ciable amounts in the human environment. Agents not-causing embryotoxicity would be of less concern. The author stressed that validation would be the key factor in instituting the chick embryo model as a preliminary screen. Direct comparisons with mammals of the relative effectiveness of particular agents and doses would have to be conducted. In a review paper, Johnson (1985) acknowledges that at least two of three reasons previously advanced for not seriously consider- ing the chick embryo model are invalid. The two incorrect assumptions were based on the supposition that the chick egg could be used for NOEL (No observed effect level) determinations. According to Johnson, the determination of a whole-animal NOEL can not be the goal of a prescreen, but rather is the goal of the standard three segment evaluations using pregnant animal sur- rogates. The complexities of the intact maternal/placental/ developmental unit far exceed what can be expected of simplified systems. The third reason Johnson maintains is still relevant; - that is, the chick egg markedly overestimates the hazard poten- tial of test substances and as presently proposed, raises too many alarms. -Further, it would, perhaps, dilute attention from the relatively-fewer xenobiotics actually hazardous to embryonic development. -He points out that quantification of the relevant dosage is a major limiting factor and the high degree of respon- siveness demonstrated by chick eggs is perhaps their greatest difficulty as a prescreen; and states that "Embryonated eggs - react positively to most substances." In support of the chick embryo model, Johnson points out that Verrett et al. (1980) established that the treatment dose, route of administration, and the developmental stage at administration all markedly influence the outcome. These results were the basis of Verrett's conclusion that the chick embryo reacts in a specific manner to test agents and that the test may be useful for screen- ing large numbers of compound to identify those that need to be subjected to more rigorous evaluation. Johnson agrees that Verrett's data provide several instances of specific responses, but maintains that it is difficult to relate these responses to those of other developing systems. Johnson further comments that Jelinek (1982) presents data which demonstrate specific effects and no-effect levels correlated in a dose-related manner correctly dependent on the embryonic stage treated. In Jelinek's opinion, reasons previously stated for not
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-3- using the chick egg for teratogenicity testing are either incor- rect or overcome by recent refinements of technique. Jelinek purports that the Chicken Embryotoxicity Screening Test (CHEST) shows promise for validation in large-scale embryotoxicity testing. - CHEST employs-several well-investigated morphogenetic systems during the organogenesis period of development. CHEST utilizes the window technique and subgerminal (day 2) and intraamniotic (days 3 and 5) injections. It requires 126 embryos and 5 mg of test substance. Jelinek feels that CHEST may also be useful in testing heat-sensitive-human metabolites and differentiating between enzyme-induced and receptor-mediated effects. Johnson (1985) concurs that the CHEST method is interesting and worthy of additional study, but cautions that its applicability for prescreening and hazard prioritization remains to be pub- lished in a form allowing one to become confident of usefulness. He stated that-"such comparative attempts have been reported but whether the experimental outcomes and doses employed to achieve them have practical utility is difficult to perceive." Schowing (1985) surveyed the various technical aspects of exper- imentation related to the chick embryo as a model for teratology and reported that, although the chick embryo had been rejected as a tool for evaluating teratogenicity in man, it appeared that the chick embryos generally show a high degree of sensitivity and respond similarly to rodent embryos. His review of the litera- ture indicated that substances teratogenic in the chick are alsa teratogenic or-toxic in the rat, mouse, and rabbit. Agents which are teratogenic in various species, in particular the chick, are more likely to be teratogenic for man than agents which are teratogenic for only one species. - - Efforts to modify and improve the methodology of chicken embryo assays are ongoing. This is evidenced by the studies of Mann-et al. (1973) and Fisher and-Schoenwolf (1983) who report improve- ments in the windowing technique. Windowing eggs is a procedure required to expose embryos in ovo. A small window is cut through the shell with standard techniques and the embryo is either manipulated surgically or a particular exogenous agent is applied. The agent may be dropped directly-onto the surface of the embryo and overlying vitelline membrane, or by injecting it into-the yolk or subgerminal cavity. Other methods of test article administration include immersion of eggs in a solution of the test article or explanting the embryo for direct administration. Both Mann's group and Fisher and Schoenwolf reported that opening the egg alone (windowing) at early stages was highly teratogenic. Mann's group found-that the presence of intact shell above an introduced ai-r space was even more teratogenic than windowing. They developed a method of gently re-expanding-the intact air cell to displace the air above the embryo which resulted in normal development. Fisher and Schoenwolf found that neural tube
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-4- defects (the most common anomaly attributed to windowing) were virtually eliminated when the air space introduced over the embryo by windowing was filled with albumen or saline, and the egg (with its window sealed with tape) was rotated 180°, so that the embryo rested adjacent to an undisturbed area of the shell. In conclusion, the predominant attitude of the scientific com- munity toward the chick embryo as a model for teratogenicity testing seems to be that the system shows promise as a prelimi- nary screen used to prioritize and identify chemicals which need further teratological testing. More work needs to be done in areas such as standardization of test subjects, administration technique, method of evaluation, and validation. According to Johnson (1985), this further research will be necessary in order to develop a uniform test method, a definable means for determin- ing dosage, and a method for correlating chick embryo data to that of pregnant animals in order to determine the predictability of the system. - Note: Summary tables of the advantages and disadvantages of the_ chick embryo model as well as an outline of a procedure for an embryotoxicity screen (Wilson, 1978) are attached. /tb:2
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Advantages of Using the Chick Embryo as a Model for Teratogenicity Testin Ready availability Relatively inexpensive Short incubation time Small size Ease of accessibility to the embryo - Known embryological development Large numbers can be used with limited personnel and equipment Requires moderate skill and minimal expenditure of time Highly sensitive to a broad range of chemical and physical agents known to affect mammalian embryos Closely parallels mammalian development in many morphological and functional aspects Possesses in ovo a drug metabolizing capacity (> day 2 of incubation) - - Carries a complete set of morphogenetic systems
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Disadvantages of Using the Chick Embryo as a Model for Teratogenicity Testing High inherent variability High degree of responsiveness; overestimates the hazard potential of test substances Low reliability (when ordinary yolk sac injection is used) Lack of standardized method for compound administration Difficulty in quantifying relevant dose (some test materials and vehicles are not freely miscible in the yolk, or do not migrate upwards to the vicinity of the embryo; dose delivery variable) - Lack of standard method of evaluation Lack of standardization of experimental subjects (variability of developmental stages within the same stock) No clear correlation with results obtained with mammalian systems Lack of validation
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Procedure for Embryotoxicity Screen Using Chick Embryos a. Determine the LD dose of the test material in 30-h chick embryos by injecdRg several log dilutions of a molar solution into the subgerminal yolk, using 10 fertile eggs for each dilution. Use as small an injection volume as is feasible (about 0.05 mL) delivered with a small-gauge hypodermic needle or a finely drawn pipette through an opening-in the shell. The lowest dilution that kills all 10 embryos within 3 days can be taken as the LD100' b. Inject 60 30-h fertile incubating eggs with 1/2, 60 with 1/4, and 60 with 1/8 dilutions of the LD 0. Inject 60 eggs similarly with an equal volume of vehicl~ ~s control. The eggs may be candled before and at intervals after injection as a check on the viability of the embryo. c. Open all eggs from each dosage group and the control group on day 10 of incubation and examine surviving embryos for gross-external abnormality. If none of the original dosage groups yield survivors, use smaller dilutions of the LD until one is found in which the number of survivors appigh- mates (within 10%) that of controls. If the percentage of malformed survivors in this non-lethal dilution of the test compound does not exceed the malformation rate in controls, the test-is negative for teratogenesis. d. Using the largest 10-day non-teratogenic dose, as determined in (c) above, inject 36 additional 30-h eggs and allow the chicks to hatch. A similar number of vehicle-treated controls should also be allowed to hatch. If hatchability, gross appearance, location, and general behavior in the hatchlings do not differ significantly from those in con- trols, the test is negative for embryotoxicity. If struc- tural or functional features different from controls are observed, smaller dilutions should be used until one is found at which hatched chicks do not differ structurally or functionally from controls. e. If the test compound does not cause embryolethality, terato- genicity, or abnormality in hatchling chicks at dilutions equivalent to a reasonable multiple (e.g., 100X) above the maximal ones known or estimated to occur in the environment, the test is considered negative. Otherwise the test is positive, and consideration-should be given to testing in pregnant mammals, provided the potential value of the compound warrants, or provided it is likely to occur at more than negligible concentrations in the environment of women of reproductive age.
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REFERENCES: Fisher, M. and Schoenwolf, G. C. 1983. The Use of Early Chick Embryos in Experimental Embryology and Teratology: Improvements in Standard Procedures. Teratology 27:65. Jelinek, R. 1982. Use of Chick Embryo in Screening for Embryotoxicity. Teratogenesis, Carcinogenesis, and Mutagenesis 2:225. Johnson, E. M. 1985. A Review of-Advances in Prescreening for .Teratogenic Hazards. Prog. Drug Res. 29:121. Mann, R. A., Moore, K. L. and Persaud, T. V. N. 1973. -Limita- tions in the Use of the Early Chick Embryo as a Teratological Model. Teratology 7:22A. Schowing, J. 1985. Chick Embryos as Experimental Material for Teratogenic Investigations. Concepts Toxicol. 3:58. Verrett, M. J., Scott, W. F., Reynaldo, E. F., Alterman, E. K. and Thomas, C. A. 1980. Toxicity and Teratogenicity of.Food Additive Chemicals in the Developing Chicken Embryo. Toxicology and Applied Pharmacology 56(2): 265. Wilson, J. G. 1978. Review of In Vitro Systems with Potential for Use in Teratogenicity Screening. Journal of Environmental Pathology and Toxicology 2:149

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