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T'lXICCII.IICV AMI AIM'1,1 III I'll \HM ACOI.OCY 5, 7f)0-7 7 1 ( ] %.I) The Injection of Chemicals into the Yolk Sac of Fertile Eggs prior to Incubation as a Toxicity Test JoSKPII McLAUGIII.IN, JK., JkAN-Pikrre MANIAC, M. JACQUELINE ^ EUKKTT, MARY K. MUTCH LKK, AM) O. CiAHTII I'lTZlIUGH Division of / harniaeoloyy, Pood and Drug ,1 dniiidsl ration, Deportment of Health, Pdueolion, and Welfare, Washington 2d, D. C. Rereived January 24, lu62 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 he 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 he a promising method in that it may he carried out much more economically im terms of money and space than would he possible with larger animals. Hundreds of chicken embryos may he observed in a minimum of space, and over a comparatively short period of lime. 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 Held except in a fragmentary wav 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. The earliest work that we have found in the literature was by Fere (189.1). During ten years after this date he published about sixty-seven papers; a review article (Fere, 1899) contains a summary of many of his studies. FIis 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. 700 * DEPOSITION L exhibit wr TOXICITY STUDIES USING CHICK EM1SKYOS 761 Since 1900 other articles have appeared in the literature, but most of them concern the effects of one or two chemicals on a small number of embrvos. One of the most informative papers is that of Ridgway and Karnofsky (1952) in which they report experiments on compounds con taining fifty-five of the elements, mainly the metallic ones, generally in jected at either the fourth or eighth day of embryonic development to study toxicity and 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 toxicity of various chemicals have been reported by Kemper (1962), Platt cl al. (1962), and Goertller ( 1962), Finally, the classic books of Romanoff and Romanoff (1949) and Romanoff (1960) contain a wealth of information on the avian embryo, . Preliminary reports of this investigation have been presented by Marline (1962) and McLaughlin and Mutchler ( 1962), LXI'KRl M F.NTAL The fertility and hatchabilitv of eggs and the livability of chicks are dependent cm 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 hatchabilitv of our supply of White Leghorn eggs1 under conditions existing in our laboratories. I he data accumulated -during two vears for 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 eggj. Eggs to be injected are first candled in order to dis card those that arc 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: 2c/o cracked, 4(7 with improperly calcined shells, 1.5^ with a tremulous air cell, lr/c with the air cell in the wrong place, and 0.5(77 with blood clots. After the elimination of such defective eggs, the hatch of control eggs averages 95(7. A further restriction is based on the weight of the eggs: all those weighing less than 52 g or more than 65 g are rejected. After 1 Tnislow F.res, ChoMcrlown. Maryland. 762 J. MC I.AUCHI.IN, JK., KT AT.. candling, (lie eggs 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 (his information, 20 or more eggs are injected with the appropriate amount of the chemical. If the chemical proves to he nontoxic, the experiment is repeated 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 (he data obtained initially and 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 of injection. The injections of pure chemicals, chemical solu tions. or 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 an Isolator box- with a sterile atmosphere created by using formaldehyde v apors (produced by mixing 2 g of potassium permanganate and SO ml of 37'jl formalin). During the period of a rear, more than fifteen hundred noniniected eggs were exposed to formaldehyde vapors; no toxic effect was noticed, After exposure to these vapors for .to minutes, the eggs are ready for injection. The large end of the egg is wiped with a sterile gauze pad moistened with a 70',i alcohol solution, and a hole is drilled in the shell in the center of the surface over the air cell (Fig. 1). ('are must be taken not to damage the shell membrane with the point of the drill-'1; this is to avoid, if possible, contact 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 slicks to the air cell and it is possible to damage it with the needle, this movement will allow the disc to float free in the egg. - Kewaunee Siii'iilillc Company. Adrian. Michigan. ;l Burgess Yilirocraiters Inc., (irayslake, Illinois. TOXICITY STCDII'S USING CIITCK KMHUYOS 763 The needle (hypodermic, 1 inch long, either no. 22 or no. 27, depending on the viscosity of the liquid to be injected) is inserted horizontally through the air cell into the yolk (Fig. 1). ( are must be taken in withdrawing the needle to avoid damogiiig the vitelline membrane since such damage could cause the yolk to spread out in the albumen. If the end of the needle has some y olk on it, the injection is not satisfactory. The needle should he 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 travs 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 3SC and a relative humidity of 60( i . The eggs are candled on the fifth day 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' (' until they hatch. FVAI.l'ATIO.V 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 1 11iimidairc Incubator Co.. New Madison, Ohio: model no. 50, rapacity T50 eggs. v Blower MnmifarTuring Co., (.Hiinrv. Illinois. WATER 764 J. MC LAIT.HUX, JR., FT AL. all llit' embryos arc killed during the first 20 hours of incubation (before the (\vo-somi(e stage); (2) the chemical is toxic lull allows a number of embryos (o develop only up to a certain point, and some possibly even to hatch; (J) (he chemical has lidle effect on the hatch; and (4) the chem ical has no effect on (he hatch or on the posthatch development of the chick ("no effect" level). If (he chemical appears to be highly toxic and all the embryos are killed, the experiment is repealed 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 chicks 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 chanties, and condition of the internal organs (liver, kidneys, heart. >1011 bladder, and spleen). It is advisable in all cases to observe (he chicks for a period of a few weeks in order to detect any delayed effects. Since as much as 50ri of the yolk remains a( (he time of hatchin.Li and 'is absorbed during the first 7 days (hereafter, effects of a toxic chemical may first be observed at this time. There mat' he weight retardation, death during the first week, or the appearance of nerve dama.ee occurring as la(e 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 eggs that fail to Iiaiih. and from a slud\ of lite -appearance and development of the chicks (hat do hatch. However, several other factors must also be considered in this evaluation: these are specific gravity, solubility, coagulat ing effect. pH. and (he ionic concentration of the chemical tested. If (lu1 chemical has a high specific gravity, there is the possibility of its settling nut in the bottom of the egg and thereby giving a value of ap parently low (oxicity. 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 are somewhat minimized. In (be case of insolul.de chemicals (hat are injected as suspensions, it is also necessary to consider particle size in the evaluation of toxicity data. In order (o have a toxic effect, the chemical must come in contact with the embryo either directly or indirectly through the bloodstream, Chemicals such as the lower aliphatic alcohols have a coagulating effect on the pro toxicity stuhf.s using chick kmiikyos 765 tein, and this coagulation may decrease the availability of the chemical as well as some yolk nutrients, and thereby alter the response of the embryo. If the pH is highly acidic or basic, a pH effect differing from the (rue loxic effect of the chemical may be obtained due to interference with (he normal acid-base equilibrium in the egg, The ionic concentration is also important for a similar reason. The ob servation of increasing toxicity with increasing concentration of a chemical should he 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 toxicity because it destroys, alters, or combines with essential nutrients such as vitamins and minerals. F.Yl'FR l.MFXTAF DATA Twenty-five thousand eggs have been used in our laboratory during the past three years to test more than 100 chemicals with the following Chemical Water (hailed) Propylene elycol Torn oil Peanut oil Sodium chloride I lexlriisc TABI.F. 1 XoXTOXIC CniMlrAI.S Solution injected Concentration Quantity (ml) -- Undiluted ' Undiluted Undiluted O.')r/o in water .VOT in waler 5.05) in waler 10.0/7 in waler 5.01 r in water 0.05 0.05 0.050.05 0.05 0.05 0.10 0.05 0.05 Per rent hatch 05 95 90 90 90 70 50 50 90 results: (1) nontoxic chemicals injected at an appropriate level allowed the embryo to develop and to hatch as did the controls: (2) toxic chemicals produced effects at dose levels which may be compared to those produc ing effects in feeding experiments using animals: and (5) this technique often provided toxicologic information which had not been shown by con ventional methods. Tabic 1 lists the results obtained with some chemicals in common use in food and shows the dosages used and the percentages of hatched chicks. ' TAKER 2 rH K M IC .M t W IT H \ H lC lt O k d KR OF T o X IC IT Y 7 66 J. MC I.AlGHUN, JR., ET AL, ri JZ <A C c X r6t JQaZ. o "d JO D ~ <-* c(J c-- tc/ o *. .2 rdt a o> -crt (g/, 5a jr *? t/. o E >-at-4t E^S ri u. rt ' Cm Oo oo r--) O Oe-l O 9 ooo o o y,, u IV u ou. Jr I iJ o jC r* H H c rJ E ca o *cUo "c E Ea ^ f/j a (Chlorinated polyplu-nyls) U n d ilu te d 0 01 -,, . 3 Konk deform ity (.short upper beak) ; -- ------------------------------ -- --------- -a______________________________ edema: g ro w th retardation TOXICITY STUDIES USING CHICK EMBRYO'S 767 These (lit(a, supplemented by an examination of the nonviable eggs and an autopsy of the chicks that hatched, have not indicated ;inv hazard from their use in food. However, it must he pointed out that any chemical, added at a sufficiently high concentration, may have some toxic effect. Since our data and those reported in the literature indicated safely, this phase of the study was not carried beyond a preliminary examination to .ascertain that nontoxicity 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 our results with several chemicals which have been shown to be hi,ably toxic to animals. To each case there is not only the low per centage of hatch at a low level of the chemical tested, but there are also congenital abnormalities and other responses that raised extremely serious questions as to the safety to the 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 de Tranciscis and Bocalatle (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 Jo_ 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). Tavanagh (1954) reported that triorlhocrcsyl phosphate (TOCT), a well-known plasticizer for nonfood use, causes paralysis when fed to adult chickens. AVe observed this paralysis in some chicks which hatched from eggs injected with 10 mg of the undiluted chemical, The compound /o/Z-diaminodiphenylmelhane has been reported bv 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 90rL of the chicks had a short mandible and leg damage consisting of a severe bending of 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, no embryos developed to more than the 5-dav stage. Dibutyl-tin-dilaurate showed no hatch at a level of 10 mg per egg. 'The majority of the embryos, which did not live more than 15 days at this TABI.E 3 \ 'm M U M S \V I II \N Iv x i U I IUATI-. O wim o s T u ic it v 768 eCj & J. MC LAUGH UN, JK., LT AL. e tc c In CC r. x: 0 r. O O O O O C 1 - O' i - - i -1- s. o oO o O OO O c/^ o n IO ir, o roc ca =3 ir. ' ~i ~r, -f >r, o >'. O rr. c poop p -- o -- O odooooood o O ir. --O OO -- ", or. o-- >pr, oir, o o o o od o o o 1o/. O-- ior, o-- d oo dddd o O' c c ,2 o V. I CJ _c x: O TOXICITY STUDIES USING CIIICK EMBRYOS 769 level, had a short and or Hexed mandihle: in addition, some embryos showed subcutaneous edema. Aroclor 1242 gave no hatch at a level of 23 mg per egg. At a level of 10 mi; j>er egg, one chick hatched out of 20 injected eggs, but died 2 clays 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 he 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. DISCISSION 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 safety of food additive chemicals and drugs, and which could be used to screen new products and eventually to correlate their toxicity 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 harrier, 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 failure of conventional testing methods to produce this effect in animals, emphasize the urgent necessity for new methods ol analysis, l'reliminary work that we have done in this area has given satis factory results (Yerrett 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 770 J. MC LAL'CHUX, JU., V.T AL. the potentiation of a few pesticides have been very encotira,"in" (Marline and Mutchler, l(,r>3). SUMMARY An waliiation of toxicity liy injection of the chemical into the yolk sac ctf fertile eggs prior lo incuhalion cave 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 arelate, selenium, Irinrlliocresyl phosphate, />,/>'-dianiinodiphenylmelhane, thiourea, Aroclnr 12-42, and dihulyl-tin-dihturale showed a Inch order of toxicity and or leralngcnic effects at certain levels. Acetone, methanol, ethanol, it-hulanol, dielhvlene glycol, ethylene glycol, iso propanol, di-2-elhyIhe.x-yl phlhalate. hydrochloric arid, carhon tetrachloride, ethyl arelate. malalhion. heplachlor. and styrene showed an intermediate order of toxicity. RF.FKRKNCKS C wanAoii, ,1. 11. (1954). The toxic effects of Iri-orlhn-cresyl phosphate on the nervous system: an experimental study in hens. J. S rural. Xcurosury,. I'sychiat. 17, ldd-171. ni. Ft;\N'r>sns, 1'.. and Hocai.aiti:. F. (1002). Lead acetate and development of the chick cmhryo. .Xatnrr 193, OS't-OUO. Tfatf:, C. (ISO.!). Note stir rinlluence, sur I'incuhation de 1'oeuf dc poulc, d'injections prealahles dans I'alhumen. de solutions de sel. de glucose, dc glycerine. Campt. Raul. Sac. ISiol., 45. SA1. I'lnf. C. (1X99). Teratogenic experimentale et pathologic generate. Cinqnantenaire dr la Sorirtt' dr Piolnyir, t'al. iuldhirr, pp. 300-309. Goi'.httu.u, K. (1902). I ter 'leratolngischc Grundversuch' am hchruteten Hiihnchen- keim seine. Miiglichkciten nnd Grenren.-A'/in. Warlikehr. 40, sou ir.iMiii'itui R, V., and JI.am it.To.v. II. I., (1931). A series of normal stages in the development of tlie chick cmhryo. J. Morphol. 88. 49-92. Karnoisky, ]). A., and Itimirw, L. 1'. (1952). Production of injury lo the central nervous system of the thick emlirio liy lead salts. J. Phanntirol. lixpll. Thrrap. 104, 1 70-180. Kimi'IR, lr. IKK,2). Thalidomid uml Fnlwicklung von Iliihneremliryonen. .1 rznei- ntittel-Forseh. 12. 040. Mel,.ut,m is. J.. Jit., and Men ill ir. M. K. ( 1902). Toxirily of some chemicals measured by injection into chicken eggs, Federation Pror. 21. 450. Al iRi iAe, J. 1'. (|oo2). Injection of chemicals into chicken eggs as a toxicity lest. Federation /'roe. 21. 450. M Mti.ne. J. I'., and M I'Ti iii.i it, M. K, (1903). Use of the chick cmhryo lcchnit|uc for delecting potentiating effects of rhcniicals. Federation Pror. 22, 188. I'l.vir, H. S,, Stiwarc, R. J. G,, and Gem, S. R. (1902). The chick cmhryo as a lest organism for toxic substances in food. Pror. Sntr. Site. (Fugl. Sent.) 21, XXX. KincwAV, ].. I'., and Karnoisky. It. A. (1952). The effects of metals on the chick cmhryo: toxicity and production of abnormalities in development. Ann. AM'. A end. Sel. 65. 205-215. TOXICITY STGDIKS USING CHICK. I.MIIKVOS 771 KoMAnoit, A. L. (1990). The Avian F.nrhryo: Strnrtnral anti Functional Develop ment, 1st ed. Macmillan, Xcav York. Ro.MA.vorr, A. I-., and Komanoit, A. J. (1949). The Avian F.kr. Wiley, Xew Y'ork. Yi.hri.tt, M. J,, and McLauciii.iv, J,, Jr. ( 1903). Use of the chick cmhryo tech nique in the evaluation of (he toxicity of drugs. Federation Prof. 22. 1.3S. Yt'snoK, W. It. (1950). The relationship of thyroid activity to the growth and the cytochrome content of the chick embryos and their organs. Inaugural dissertation, Cornell Univ., Ithaca, Xew York. 7,vt,ni rrzac, S. (1951). Cirrhosis-provoking action of insoluble diamiiio-diphenyl compounds on the rat liver. Conipt. Rend. Soc. liitd. 145. 136-138. WATER_PCB-SD0000039102