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:v.\li_uUK<\ AM) AIM'ULD m arvacoi.ogy 5, 760-771 (196.1) The Injection of Chemicals into the Yolk Sac of Fertile Eggs prior to Incubation as a Toxicity Test josr.i'H McLaughlin, Jr., Jean-Pierre Maruac, M. Jacqueline Yerrett, Mary K. Mutchler, and 0. Garth Fitzhucji Division of Pharmacology, Food and Drug Administration, Department of Health, Education, and Welfare, Washington 2S, D. C. Received January 24, 1963 The increasingly large number of food additive chemicals introduced into the market each year has necessitated the development of rapid and reliable methods for the evaluation of their toxicity. Toxicologic studies of all these chemicals by the usual methods using animals are very diffi cult, and such studies sometimes give inconclusive results. The toxicity of some chemicals, and especially of food additives, may be determined by injection of the chemical into the yolk sac of fertile eggs prior to incubation and subsequent observation of the effects of the chem ical on the embryonic development of the chick. This appears to be a promising method in that it may be carried out much more economically m terms of money and space than would be possible with larger animals. Hundreds of chicken embryos may be observed in h minimum of spnee, and over a comparatively short period of time. The feasibility of using such large numbers is valuable also in the statistical evaluation of toxicity data. ' A review of the literature shows how little work has been done in this field except in a fragmentary way on isolated cases. Most of the reports refer to injections of chemicals made after the fourth or eighth day of incubation and examination of the embryos killed before they hatch. M.e earliest work that we have found in the literature was by Fr< (1393). During ten years after this date he published about sixty-seven papers; a review article (Fer, 1899) contains a summary of many of his studies. His work consisted mainly of injection before incubation, but the eggs were usually opened on the third day of incubation. His interest was mainly in the teratogenic effect of chemicals. 760 ..us using cirrcK emqkyos 761 Since 19C0 other a; tides have appeared in the literature, but most of them concern the effects of one or two chemicals on a small number of embryos, One of the most informative papers is that of Ridgway and Kjrnof.'hy (1952) in which they report experiments on compounds con taining fifty-live of the elements, mainly the metallic ones, generally in jected at either the fourth or eighth day of embryonic development to study toxicity ami teratogenic effects. Hamburger and Hamilton (1951) have described an elegant method for determining the stages of develop ment of the chick, embryo. More recent investigations employing the chick embryo to study the tuxicity of various chemicals have been reported by Kemper (1902), Platt a at. ,.962), and Goertiler (1962). Finally, the classic books of Romanoff and Romanoff (1949) and Romanoff (1960) contain a wealth of information on the avian embryo. Preliminary tepotts uf ibis investigation have been presented by MarJiac ( 1962) and McLaughlin and Mutchicr (1962). EXPERIMENTAL The fertility and hatchability of eggs and the livability of chicks are dependent on a complex interrelationship of ecological factors, among which are the genetic background and the age of the mated birds, the nutri tional status and general management of the flock, and seasonal variations. In view of this the initial phase of our work, which started in 1959, was devoted to a study of the hatchability of our supply of White Leghorn eggs1 under conditions existing in our laboratories. The data accumulated during two years Mr control eggs showed that the hatchability of these eggs was, in fact, consistent, reproducible, and very high. The possibility of a seasonal variation occurring in responses to compounds introduced into the eggs was also examined by repeated testing of several chemicals at all seasons of the year. No'important variation was detected. Selection of eggs. Eggs to be injected are first candled in order to dis card those that are defective and to outline with a pencil the exact location of the air cell. In our laboratory 9 of 5000 eggs had to be discarded: 2% cracked, 4% with improperly calcified shells, 1.59b with a tremulous air cell, 1% with the air cell in the wrong place, and 0.5% with blood clots. After the elimination of such defective eggs, the hatch of control eggs averages 95%. A further restriction is based on the weight of the eggs: all those weighing less than 52 g or more than 63 g arc rejected. After 1 TruiMtv Egfcs, Chrstcrlown, Maryland. 7 62 J. MC LAUCIIUN, JK., KT AI,, candling, the e^gs arc randomized in order to avoid series of infertile eggs in any one experiment. The initial experiment with a given chemical is for range-finding and is performed at two or more concentrations of the chemical with 10 eggs per level. On the basis of this information, 20 or more eggs are injected with the appropriate amount of the chemical. It the chemical proves to be nontoxic, the experiment is repented with the minimum number of eggs that will give a reliable and reproducible value for the hatchability. In the case of a toxic chemical, additional eggs are injected to determine the specific effects of the chemical. The total number of eggs used for a chemical depends upon the data obtained initially ar.d upon the kind of information desired. Hence, data for some chemicals are based on less than one hundred eggs, whereas data for others are based on several hundred eggs. Technique oj injection. The injections of pure chemicals, chemical solu* lions, cr suspensions are made at volumes up to 0.10 ml. When necessary, dilutions are made with solvents such as water, propylene glycol, corn oil, peanut oil, or other nontoxic solvents. In order to avoid contamination, the injections are carried out in ail Isolator Box- with a sterile atmosphere created by using formaldehyde vapors (produced by mixing 2 g of potassium permanganate and 50 ml of 37r/o formalin). During the period of a year, more than fifteen hundred noninjecled eggs were exposed to formaldehyde vapors; no toxic effect was noticed. After exposure to these vapors for 30 minutes, the eggs are ready for injection. The large end of the egg is wiped with a sterile gauze pnd moistened with a 709c alcohol solution, and a hole is drilled in the shell in the center of the surface over the air cell (Fig. 1). Care must be taken not to damage the shell membrane with the point of the drill*; this is to avoid, if possible, contaci of the air with the egg membrane. Fine particles of shell are re moved with an aspirator to prevent the needle from carrying them into the yolk. Immediately before the injection, each egg is shaken with a quick twist of the wrist. Since the germinal disc occasionally sticks to the air cell and it is possible to damage it with the needle, this movement will allow the disc to lloa. free in the egg, a Kewaunee Scientific Company, Adrian, Michigan. s Burgess Yibrocratters Inc., Grayslake, Illinois. TOXICITY STUDIES USIXC CHICK KMDRYOS 763 The needle (hypodermic, 1 inch long, either no. 22 or no. 27, depending on ihe viscosity of the liquid to be injected) is inserted horizontally through the air cell into the yolk (Fig. I). Care must be taken in withdrawing the needle to avoid damaging the vitelline membrane since such damage could cause the yolk to spread out in the albumen. If the end of the needle has some yolk on it, (he injection is not satisfactory, The needle should be wiped with a sterile gauze pad between each injection. As soon as the egg has been injected the hole in the shell is covered with a small piece of Scotch tape, care being taken not to cover the entire air cell. GERMINAL DISC Incubation and hatching. The injected eggs are put into the incubator trays with the large end up; the trays are placed in the incubator,4 which automatically rotates .hourly and is maintained at an optimum temperature of 38C and a relative humidity of 607c. The eggs are candled on the fifth clay of incubation and every day thereafter. Clear eggs and dead embryos are removed for examination. On the seventeenth day of incubation the fertile eggs are transferred to the hatcher' and kept at a temperature of 37*0 until they hatch. EVALUATION OF DATA The injection of the chemical into the egg may produce one of four possible results; (1) the chemical is highly toxic at the level injected, and * Humid.iirc Incubator Co , XVw MaUXon, Ohio, mortal no. SO, capacity 450 eggs. 1 IJroucr ManuIacl Jim^ Co., uginc>. Illinois. J. MC LAUCIIUN, JR., KT AL. ill the embryos are killed during the first 20 hours of incubation (befoie the two-somite stage); (2) the chemical is toxic but allows a number of embryos to develop only up to a certain point, and some possibly even to hatch, (3) the* chemical has little effect on the hatch; and (4) the chem ical lias no effect on the hatch or on the posthatch development of the chick ("no effect" level). If the chemical appears to be highly toxic and all the embryos are killed, the ex)>eriment is repeated with smaller doses of the chemical until some hatch is obtained. If the chemical is toxic, but allows the embryos to de velop for a longer period of time, dead embryos are examined pathologically and the ducks that do hatch are examined for eye damage, color of the feathers, weight, length of the legs, form of the beak and of the rump, hematologic changes, and condition of the internal organs (liver, kidneys, heart, guil bladder, and spleen). It is advisable in all cases to observe the chicks for a period of a few weeks in order to detect any delayed effects. Since as much as J0% of the yolk remains at the time of hatching and is absorbed during the first 7 days thereafter, effects of a toxic chemical may first be observed at this time. There may be weight retardation, death during the first week, or the appearance of nerve damage occurring as late as 2-6 weeks after hatching. The toxicity of a chemical is evaluated mainly from the percentage of hatch at varying dosages of the chemical as compared to noninjected (control) eggs, from a study of the embryonic development of the egjts that fail (o hatch, and from a study of the appearance and development of the chicks that do hatch. However, several other factors must also be considered in this evaluation: these are specific gravity, solubility, coagulat ing effect, pH, and the ionic concentration of the chemical tested. If the chemical has a high specific gravity, there is the possibility of its settling out in the bottom of the egg and thereby giving a value of ap parently low toxicity. The solubility is quite important since the availability of the chemical for utilization in the chick embryo is partially dependent on Its solubility in the egg. However, since egg yolk is an emulsion, solubility problems arr somewhat minimized. In the case of insoluble chemicals that are injected as suspensions, it is also necessary to consider particle size in the evaluation of toxL-ty data. In order to have a toxic effect, the chemical must come in contact with the embryo cither directly or indirectly through the bloodstream. Chemical* such as the lower aliphatic alcohols have a coagulating effect on the pro* si ti h IV. Ih \v, Pt, Co Pet Sot Oty rest the pro ing ofti ven 1 in ft HONS 071935 TOXICITY STUDIES USING CHICK EMBRYOS 765 tcin, and 'his coagulation may decrease the availability of the chemical as well as some yolk mu.icnts, and thereby alter the response of the embryo. If the pH is highly acidic.or basic, a pH effect differing from the true to.vh. effect of the chemical may be obtained due to interference with the normal acid-base equilibrium in the egg. The ionic concentration is also important foe a similar reason. The ob servation of increasing toxicity with increasing concentration of a chemical should bo interpreted cautiously, since highly concentrated solutions may upset the physical equilibrium of the yolk by causing osmotic effects. Finally, the introduction of a chemical into the yolk may cause a special type of tuxicity because it destroys, alters, or combines with essential nutrients such as vitamins and minerals. EXPERIMENTAL DATA Twenty-five thousand eggs have been used in our laboratory during the [last three years to test more than 100 chemicals with the following Chemical Water (boiled) Pr.,)yJenc idycol Cora oil Peanut oil Sodium chloiido Dextrose TABLE 1 Nontoxic Chemicals . Solution injected Concentration Quantity (ml) -- Undiluted Undiluted Undiluted 0.9% in water 5.0% in water 5.07o in water 10.0% in water 5 0% in water 0.05 0.05 0.05 0.05 0.05 0.05 0.10 0.05 0.05 Per cent hatch 95 95 90 90 90 70 30 70 90 results: (l) nontoxic chemicals injected at an appropriate level allowed the embryo so develop and to hatch as did the controls; (2) toxic chemicals produced effects at do.xc levels which may be compared to those produc ing effects in feeding experiments using animals; and (3) this technique often provided toxicologic information which had not been shown by con ventional methods. Table l lists the results obtained with some chemicals in common use in food and shows the dosages used and the percentages of hatched chicks. \ MW&m rvvs&Soj. 071936 **:>> v\gj o r'*'.A\-V; V : /' \`,>KV*'5iv s'" ,,',is3 ."'`^-4 1 i"ii f* & fi kn> M if .. C1?'^ 1 $i O j I*-,.3 - *0 ` " i Mercuric chloride Triorthocrcsyl phosphate 10% in water 5% in water 005 OjOS ^P'-Diarainodiphenylmethane Sodium selenite Oihulyl-tin-dilaurate Aroclor 1242 (chlorinate*! pulyphenyis) 10% in ethanol 0.2% in water 0.1% in rater 0.1% in rater Undiluted Undiluted Undiluted 005 005 005 002 001 0.025 0.01 All embryos died at the beginning of incubation Some embryos lived up to 34 days Hatched on the 28th day of incuba tion; growth retardation Most of the developing embryos died on the 15th day of incubation Hatched chicks showed growth re tardation and developed paralysis 2~6 weeks after hatching Leg damage; beak deformity (short mandible} 80 0 Beak deformity (short upper beak); edema; growth retardation TOXICITY iLUUIUS USING CHICK KMDRYOS These data, supplemented by an examination of the nonviable eggs and an autopsy of the clucks that hatched, have noi indicated any ha2ard from their use in food. However, it must be pointed out that any chemical, added at a sulnciently high concentration, may have some toxic effect. Since uur data and those reported in the literature indicated safety, this phase of the study was not carried beyond a preliminary examination to a.ccemiin ib.jt nomoxicily also was shown for these chemicals by the chick embryo technique. This is of theoretical as well as practical importance for the evaluation of any new technique to be used in toxicologic studies. Table 2 lists uu: results with several chemicals which have been shown to be highly toxic to animals. In each case there is not only the low per centage of hutch at a low level of the chemical tested, but there are also congenital abnormalities and other responses that raised extremely serious questions as to ihe safety to Uic consumers of any food contaminated with these chemicals. Lead acetate resulted in no hatch at a level of 1 mg per egg. Autopsy of the dead embryos showed extensive brain damage, as has been reported by dc Francises and 3ocalattc (1962) and by Karnofsky and Ridgway (1952). * Mercuric chloride showed no hatch even at a level of 0.5 mg per egg. Thiourea is a known carcinogen with a basic effect on the thyroid gland. The hatch time was delayed with increasing amounts of this chemical. At a level of 5 mg per egg no chicks hatched and the embryo required 35 days to develop to the stage normally attained at 20 days. At 2.5 mg per egg some chicks hatched, but most of them had to be helped out of the shell. This effect has been reported also by Yushok (1950). Cavanagh (1954) reported that triorthocresyl phosphate (TOCP), a well-known plasticizer ior nonfood use, causes paralysis when fed to adult chickens. We observed this paralysis in some chicks which hatched from eggs injected with 10 mg of the undiluted chemical. The compound />,/-diarninodipheylmethane has been reported by Zylberszac (1951) to cause cirrhosis of the liver in rats. We found this chemi cal to be extremely teratogenic at a level of 5 mg per egg; more than of the chicks had a short mandible and leg damage consisting of a severe bending uf the tibia and a general shortening of the bones of the leg. Sodium selenite proved to be highly toxic. At a level of 0.1 mg per egg, :to embryos developed to more than the 5-day stage. DibutyUin-dilaurate showed no hatch at a level of 10 mg per egg. The majority of the embryos, which did not live more than IS days at this r^Ks-if it|is 'W'' 's-v i" Yw*1 HONS 071938 Chemical Acetone n-Bulanol Carbon tetrachloride Dicthylenc glycol Ethyl acetate Ethyl alcohol Ethylene glycol Di-2-elhylhcxyI phthalatc Hcptachlor Hydrochloric acid Isopropanol Malathion Methanol Styrene TABLE 3 CurM1CALS WITH AH IfCTIRM F.DIATf OiUrtR or Toxicity Solution injected Concentration Quantity (ml) Per cent hatch Undiluted Undiluted Undiluted Undiluted Undiluted Undiluted Undiluted Undiluted 1% in propylene glycol 4% in water Undiluted Undiluted Undiluted i% in *Q% ethanol 0 05 0.01 0 02* 0.03* 0.04 005 0.10 0.05 0.10 O.OS 0.10 0.05 0.10 0.30 0.05 0.10 0.05 0.0S 0.10 0.05 0.05 0.10 0.05 0.10 0.0$ 0.10 005 70 95 70 20 0 40 0 SO 55 35 15 95 60 0 SO 65 95 75 65 SO 35 15 60 | 90 65 95 Remarks * At these levels: corneal opacity, cataract, cleft palate, nerve and kidney damage u, * > a m r Short legs; bleaching cffeet on chick down f E f. '; -' ' -' { TONICITY STUDIES USING CHICK EMBRYOS 769 level, had a short a:ul/or Hexed mandible; in addition, some embryos showed subcutaneous edema. Aroclor 1242 gave no hatch at a level of 25 mg per egg. At a level of 10 mg per egg, one chick hatched out of 20 injected eggs, but died 2 days later, Some embryos, which were examined after they died, showed beak deformities (often a short upper beak), edema, and growth retardation. Table 3 lists preliminary data on the toxicity of some chemicals which have been shown to be toxic in some degree to animals, and which may be found in some processed foods. Included in this group are various solvents, plasticizers, and insecticides. Some of the chemicals on this list require further study, including observation of the chicks until they reach ma turity, before they may be classified as to low or high toxicity. DISCUSSION The injection of chemicals into the yolk of fertile eggs prior to incuba tion is a method which can be advantageously used as an element in the evaluation of the safely of food additive chemicals and drugs, and which could be used to screen new products and eventually to correlate their tuxicily with that of similar products already tested. if one considers that a chemical injected into the yolk may be com pared to a substance which has the power to cross the placental barrier, this technique, in addition to being an embryonic feeding study, assumes further importance in that it is also a reproduction study. The unfortunate experiences recently suffered with chemicals that have teratogenic effects in humans, and the Lilurc of conventional testing methods to produce this effect in animals, emphasize the urgent necessity for new methods of analysis. Preliminary work that we have done in this area has given satis factory results (Yerreu and McLaughlin, 1963). Since this represents a system in which the chemical is in direct contact with the embryo throughout development, it is more than likely that any toxic or teratogenic effects would be readily observed. However, there is always the possibility that the chicken will not be a species susceptible to a particular compound, just as it has been shown that the other commonly used species of animals do not respond to all chemicals in a similar manner. It is also possible for the chicken to be more sensitive to a chemical than other species. Finally, this technique may be applied also to the study of the syner gistic effects of chemicals. Results of experiments in our laboratory on .4 "'.wjwwiw -I'.wiy* /j-ingj yfl fair W^.V.'V 770 J. MC LAUGI1UN, JK., ET AL. the potentiation of a few pesticides have been very encouraging (Marliac and Mutchler, 1963). SUMMARY An evaluation of toxicity by injection of the chemical into the yolk sac of fertile egtts prior to incubation gave the following results: Water, propylene glycol, corn oil, peanut oil, isotonic saline solution, and isotonic glucose solution showed no toxicity or a very low order of toxicity. Mercuric chloride, lead acetate, selenium, triorthocrcsyl phosphate, p,p'-d!amino* diphcuylmcthane, thiourea, Aroelor 1242, and dibutyl-tin-dilaurate showed a high order of toxicity and/or teratogenic effects at certain lovels. Acetone, methanol, ethanol, n-butanoi, dicthyJene glycol, ethylene glycol, bo* propanol, di-2-cthylhcxyl phthaiitc, hydrochloric add, carbon tetrachloride, ethyl acetate, malaihion, heptachlor, and styrene showed an intermediate order of toxicity. REFERENCES Cavanacu, J. B. (1954). The toxic effects of tri-orlbo-crcsyl phosphate on the nervous system: an experimental study in hens. J. Neurol, S'eurosnrg. Ptyckiai IT, 163-172. oc Frakciscis, P., and Bocalatte, F. (1962). Lead acetate and development of the chick embryo. Nature 193, 989*990. ' Fx, C. (1893). Note sur I'inftuence, sur 1'incubaljon de 1'oeuf de poule, dlnjectiwt# prcalables dans I'albumcn, de solutions de set, de glucose, de gtycMne. Cornel. PmJ, Soc. Biol., 46, 831. Frst, C. (1899). Teratogenic expirimenule et pathologic gfalrale. CinqHontenoirt de la SociMl de Biologit, Vol. jubilaire, pp. 360*369. Courttle*, K. (1962). Der Ucrntologische Grundversuch'am bebruteten HShnchen* keim seine Mbglichkeiten und Crcnzcn. JCIIh. Woeheckr. 40, 809. Hamdurckr, V., and Hamilton, H. L. (1951). A series of normal stages In the development of the chick embryo. J, Morpkol. 88, 49*92. Kaakofsky, D. A., and Rjduway, U. P. (1952). Production of injury to the central nervous system of the chick embryo by lead salts. J. Pharmacol. Exptl. Tktrap. 104, 176-186. Kr.MrrR, F. (1962). Thalidomtd und Entwicklung von Hiahnertmbryonen. Annri* mitiel-Forsch, 12, 640. McLauculut, J,, Jr., and Mutchler, M. K. (1962). Toxicity of some chemicals measured l>>- injection into cbicken eggs. Federation Proe. 21, 450. Marlmc, J. P. (1962). Injection of chemicals into chicken eggs at a toxicity test. Federation Proc. 21, 450. Marliac, J. P., and Mutchler, M. K. (1963). Use of the chick embryo technique lor detecting potentiating effects of chemicals. Federation Proc. 22, JS8. Platt, B. S, Stewart, R. J. C., and Gupta, S. R. (1962). The chkk embryo as a test organism for toxic substances in-food. Proc. Nvtr. Soc. {Engl. Scot.) 21, XXX. Riocway, L. P., and Karsotsky, D. A. (1952). The effects of metals on the chick embryo: toxicity and production of abnormalities In development. Ann. AM'. Acad. Sci. 55, 203-215. TOXICITY STUDIES USING CHICK EMBRYOS 771 KoMAXufY. A. L. (1960). The Avian Embryo: Structural and Functional Develop ment, 7it cd. Macmillan, Xcw York. Ro.MA.Norr, A. L., and Romanoff, A. J. (1949). The Avian Egg. Wiley, New York. Ytafir.TT. M. J , and McLauchlin, J, J*. (196J). Use of the chick embryo tech nique in Ihc evaluation of the toxicity of drugs. Federation Proc. 32, 118. YtsitoK, W. D (i960). 'Hie relationship ni thyroid activity to the growth lad the cytochrome cc.ucnt of the chick embryos and their organs. Inaugural dissertation, Cornell Unis., Itliaui, New York. ZviHKHStAt', S. (19511 ' rrhosis-prosoking action of insoluble diamino-diphenvl compounds on tire rat liver. Compt. Pcd. Soc. Biol. 146, 116-118. HONS OH 9+i