Document 1yM1rwLpY67yerxp0v2nbGRMX

4 . ... . i. V ' /' /t THE "CHICK-EDEMA FACTOR" AND ITS RELATION TO THE FATTY ACID INDUSTRY . .J , / -/ y{j^A /.. -v - T" >>' ' _ - (f, f ' .,. 7 <2.--------- ! >., r /j^t Ac c '* 3 , / / V <3* __ / From th <4mJc o( I R. DURLAND Ur NOV q /'6Juu* <:Ui. JS ; FfrS'<*9->-r *-- F+SEj o -^ a. y, By Thomas McGuine Wilson-Martin Division of Wilson & Co. Philadeiphia, Penna. MUNS 037634 When tha 85th Congress in September 1958 passed and sent to the President for his signature an amendment to the Federal Food, Drug and Cosmetic Act regulating chemical additives to food, hardly an eyebrow was lifted among fatty acid producers, because of the long historical recognition of these materials as safe. This feeling of security was further enhanced with the issuance of the tentative GRAS (Gennerally Recognized As Safe) list in the Federal Register on December 9, 1958. Oleic and linoleic acids, glyceryl monostearate, magnesium stearate and mono and diglycerides, except lauric, were listed. As stearic acid La a component of glyceryl monostearate and magnesium stearate, and is often found in mono and diglycerides, it was felt that stearic acid was included by implication and the failure to list it separately was simply an oversight. This was found to be the case when a query was made to the FDA (Food and Drug Administration), for the reply received March 16, 1959 by the Fatty Acid Producers' Council (a national organization representing most of the fatty acid pro duction in the United States) said in part: "We concur in the opinion that stearic acid is generally recognized by appropriately qualified experts as being safe for food use and that it should not be subject to clearance provisions of tha Food Ad ditive Amendment for those uses which are customary and usual". Here things stood until a reading of the revised GRAS list issued November 20, 1959, indicated that instead of correcting the stearic acid oversight, now oleic and linoleic acids were also omitted, although strangely, glyceryl monostearate remained. A revision of the section dealing with emulsifying agents listed a group of mono and diglycerides requiring their production from edible fats and oils by glycerolysis. The glycerolysis limitation was the only processing requirement shown by the entire GRAS list and the raw material source statement "of edible fats or oils", also was the only such requirement on the list. The Fatty Acid Council speedily appointed a committee to meet with the FDA HONS 037635 2 to establish the background Cor these changes. It didn't take long to find out that the PDA were now concerned about U.S.P. Oleic Acid because of certain samples they had picked up in a plant manufacturing esters Cor Cood use and Cound them to aCCect chicks, with symptoms indicating a similarity to the chick edema disease oC 1957.12* 3,^,5,6,9 ^at outbreak had been traced to a fatty acid still residue product high in unsaponifiables and this finding relative to low unsaponifiable material was un expected. These oleic acids were found to abnormally affect rat9 and monkeys, as did a food grade glyceryl monooleate having 0,22% unsaponifiable, while a glyceryl trioleate was even more active. This finding was also reported later in a paper in the J.A.O.C.S. 7 and the Federation ProceedingsA0 of the A.O.A.C. by Dr. Ames and his group at Distillation Products Industries, who had this effect called to their at tention by F. W. Hill of Cornell University. He had observed abnormal efCects with a Cood grade glyceryl monooleate. The group at Distillation Products Industries found that conversion of these oleics to glyceryl esters carried over the C.E.F. (chick-edema factor) in proportion to its presence in the fatty acids. Hence, the attempt of the FDA to prevent this possibility in the production of monoglycerides by spelling out the "glycerolysis" limitation. It was felt by the FDA, based on the evidence at the time, that the C.E.F. was somehow related to fatty acid distillation procedures. Cooperative work between the FDA, some association members and feed manu facturers trying to track down the cause of the 19S7 edema outbreak, indicated that split fatty acid stock was satisfactory but distillation of the split acids showed some C.E.F. in the distillates, with even more in the still residues. Hence the working theory at the time was that distillation somehow caused the appearance of the C.E.F. One group In the FDA felt the effect was thermal in origin, while others felt it might be contamination from something such as boiler treating com pounds used in the production of the sparging 9team for fatty acid distillation. It was because of the association of the 1957 edema outbreak with low HONS 037636 -3- grade inedible tallows that the PDA now spelled oat the edible requirement for mono glycerides even though Section 402 of the Act covered this matter. While no evidence had appeared that stearic acid had the C.E.F., Lt was omitted from the GRAS classification because of the possibility that it could be pro duced from oleic acid by low pressure hydrogenation, which was known to not affect the C.E.F.^ In other words, the responsibility was placed on the manufacturers to define a grade of stearic acid that was safe for food use, rather than to exempt the commercial product generally under the GRAS clause. This situation caused considerable concern among fatty acid and emulsifier producers because it appeared to strike at the root of two of their key processes, namely, distillation and esterification. The concern was accentuated by the short time interval allowed to get in under the March 6, 1960 enforcement deadline by petitioning for a time extension. The fatty acid industry set in motion a very intensive search for chick edema positive materials. Screening tests utilizing hydrocarbon diene content, a cholestadiene test, and feeding chicks, were tried. Cooperative work with samples of C.E.F. positive commercial oleic acids kindly furnished by Distillation Products Industries rapidly established the worthlessness of the cholestadiene and hydrocarbon diene tests, leaving only chick feeding tests to fall back on, which incidentally is where we stand today. Steps were also taken to set in motion the requirements for obteining e time extension of the enforcement. In the spring of 1960 while the fatty acid industry was searching its supplies and processes to find C.E.F. positive material, the Food and Drug inspectors picked up quantities of both acids and derivatives in the field, from various pro ducers, for their own evaluation. MQNS 037637 The upshot of all this activity was that everyone came up with negative results. No C.E.F. appeared anywhere, not even in first, second, or third still -4- bottoms produced from oleic or cottonseed acids, much less in the distillates, or in stearic acids. This threw cold water on the heat and distillation theories for the C.E.F. formation. At about this time, the Association became acquainted with the concentration and isolation work on the C.E.F. being done by a group of workers at Merck 8t Co. and Procter it Gamble. Merck had assigned the men who worked on the isolation of vitamins Bg and to this task, and, in conjection with a quantitative bioassay procedure developed by Ur. Walther Ott of their nutrition labs, were following the isolation step by 9tep from large quantities of the tallow held over from the 1957 in outbreak. ` Dr. Norman Brink and his group at Merck shortly reported the isolation of what they felt was the purified C.E.F. They had obtained 1 mg from approximately 1,000 pounds of starting edema positive tallow.11 Help was asked of the Association members in supplying more concentrated sources of such edema positive fat for further work. Shortly thereafter, Merck definitely found halogen in the pure C.E.F., due to work by Dr. Trenner and his group. Within a few more weeks it was established that the halogen was chlorine, present to the extent of about 47% in a molecule, having from 10-12 carbon atoms, free of aromaticity, but showing evidence of a diene structure. It sublimed at about 225C but it was felt that this figure might be as high as 260OC. A molecular weight of approximately 380 was indicated. Thi9 agreed well with the figure previously reported by Dr. Ames based on molecular distillation work.^ The high biological activity of 1.6 million times that found in the crude fat, which readily killed chickens, rated this as a potent toxin. In the chick diet. positive response. HONS 037638 With the first finding of chlorine, and before the molecular level was established, the possibility that chlorine dioxide bleaching of fats might be the cause of the C.E.F. wa9 checked by submitting some tallow, treated in this manner, -5- and also after prolonged heating subsequent to bleaching to Dr, Ott, who found these to be negative by hi9 bioasaay procedure. The positive finding of a highly chlori nated molecule, in the pure C.E.F,, was also confirmed by workers at Procter & Gamble and the FDA, who separately isolated thi9 factor about this time. A comparison of the infra-red curves obtained by Merck and the FDA showed structural differences, and among these was the indication of aromaticity in the FDA sample. Dr. Neil Arttnan will report on the isolation work of the C.E.F. done by Procter and Gamble at the A.O.A.C. convention held in Washington concurrent with the A.O.C.S. convention here. As a result of these findings, the Merck workers were convinced that they were dealing with an unnatural material resulting from contamination and decided to discontinue any further work. Before doing so, they tested a number of chlorinated cyclodiene pesticides, as did a number of the members of our Association, the FDA, and Procter and Gamble. Also checked were chlorinated naphthalenes which had been implicated in toxic effects on cattle some years before. The workers at the FDA leaned toward the chlorinated naphthalene theory for the C.E.F., based on their data. The pesticide theory for the C.E.F. seemed the most plausible interpreteri.on of the Merck results. To check these possibilities, the following pesticides and lubricants were tested and, although found toxic to varying degrees in chicks, in no case did they 9how positive edema results. They were: Aldrin, Dleldrin, Emir in, DDT, Chlordane, Hepthachlor, Hexachlorophene, tetrachlorotetrahydronaphthalene, hexa- chlorocyclohexane, pentachlorophenol, tetrachlorotoluene and hexachloronaphthalene. The Association also sponsored small scale rendering tests at the American Maat Institute using tallow having Aldrin, Dieldrin, Endrin and Heptachlor added at a level designed to give 10 PPM of contaminant in the rendered fat. There were chick fed and all found to be negative. Fly tests on the rendered fat9 run by a member company showed 100% mortality in each case, indicating the presence of the insecticides. Prior fly tests by the same company on known C.E.F. positive tallow MOWS 037639 6 and oleic acids had shown no insecticidal activity. Another company ran the gamut of available insecticides in testing for thermal breakdown products, by heating them at a 1% level in fatty acids to over 200C for extended periods of time. In no case did a positive edema test result on feeding the acids to chicks. While this work was proceeding, the FDA issued a time extension covering food grade stearic, oleic and related straight chain acids, and also the mono and diglycerides from them, free of the chick edema factor and from edible fats and oils. The glycerolysis limitation had been dropped. The ice cream standards Lssuing a few months later had this limitation, which the Commissioner later rectified, on his own initiative, after we pointed out the discrepancy. In September of 1960 another outbreak identified by a number of independent laboratories as "Chick Edema Disease" occurred in some brooder houses in Georgia. The C.E.F. positive feed concentrate was traced to the production of a single day. Unfortunately, neither the fat supplier nor the feed manufacturer retained fat samples, so it has not been possible to unequivocally prove that contaminated fat, which in this case was *1 tallow, was the cause. The various other feed components were checked by feeding and found not at fault. Our laboratory received samples of the C.E.F. positive feed and extracted the Cat. This was tested Cor chlorinated material by a member laboratory using a Dohrman instrument and also using the Mills pesticide procedure. A finding of ap proximately 50 PPM of Chlordane was noted. That this fat contained the C.E.F. was established by feeding tests indicating a moderate level of toxicity. Other non toxic feeds taken from production about the same time showed a few PPM of Chlordane, BHC and Lindane and were found nontoxic on checking the extracted Cat. The fat supplier was positive that the particular shipment of *1 tallow contained no fatty acid residues of any kind. MON5 03 7640 Approximately 12,000,000 chickens were fed this toxic supplement utilizing - 7- 4,000 gal. of #1 tallow. Condemnations ran about 20% of the flocks with 00 - 90% condemnations at times. The affected chickens showed large accumulations of peri cardial fluid with associated liver damage. Separate tests absolved crotolaria seed and pathogenic organisms as alternate possibilities. The nature of this outbreak and other information to date makes it seem probable that fats entering fatty acid plants at times contain the C.E.F. in mar ginal quantities, which become concentrated by various processing steps. Since the findings of a chlorinated pesticide in C.E.F, positive fat work done by the research group has resulted in a finding of Chlordane in a specially concentrated edema toxic fatty acid which one of our members has prepared from some toxic residue material. Recent industry evidence substantiates the earlier findings of the FDA that vegetable fatty acids also contained the C.E.F. at times. This eliminates the necessity of postulating the animal body as a metabolic intermediate in the production of the C.E.F. from some chlorinated precuaor. In an article to appear shortly in the Journal of Poultry Science, Dr. B. L. O'Dell et al., of the University of Missouri, will report on an occurrenc of chick edema indistinguishable from that of edema positive fats, which he traced to a chlorinated biphenyl compound incorporated into an epoxy paint and mixed with sand. 15 He had coated the bottom of his galvanized chick batteries with this com position, and the chicks pecking on the sand picked up the toxic factor. The chlorinated biphenyl material responsible for this edema toxicity was found to be Aroclor 1242. Dr. O'Dell reports his belief that the active ingredient is a minor constituent in the Aroclor 1242. HONS 037641 The product is substantially biphenyl, with 42 weight percent of chlorine. Among uses for this material suggested by the manufacturer is one as a vapor sup pressant for insecticides, with from 5-25 weight percent of the chlorinated biphenyl increasing the effective kill life of Lindane by a factor of 10. 1 quote the follow ing from the manufacturer's bulletin on Aroclors. "The most pronounced effect for (<r>K ) / Increasing the kill-life of the insecticide is obtained with Lindane, Chlordane and BHC".16 Is this recommendation a concidence, or is it related to our finding chlorinated pesticides associated with edema toxic fat? The chlorine content is of the order of magnitude of the purified C.E.F. Our Association is currently trying to track down these leads. -_____ _ In addition to these problems concerned with the nature of the C.E.F., the industry has been confronted with the onerous task of feeding chickens for three week intervals as a quality control check on finished food grade acids. You can imagine the scheduling and storage problems entailed. We are trying to work out an acceptable diet and criterion for deciding what constitutes a positive finding of "edema" by collaborative work with the FDA. Origi nally, chick feeding tests were run using a laboratory procedure given the Fatty Acid Producers* Council by the FDA. It was known as the "Friedman" test. This test used visual scoring to determine an edema positive chicken. A modification requiring the presence of a definite fluid level was an Improvement. Numerous workers were not satisfied with this test for various reasons, so a collaborative study was made using a diet having higher salt levels designed to in crease the sensitivity. The diet also was nutritionally better. It issued early in 1961 as a tentative A.O.A.C. procedure.17 This test again called for visual scoring, but in work with our Association, the FDA picked a level of 0.2 ml of heart fluid on a single chick in 10 as a positive response. Using the procedure, a number of laboratories begain finding what appeared to be false positives on control fats. This would be indicated by a single chick showing a level of heart fluid of over 0.2 ml while the balance of the test group would have very low levels. On rechecking, the positive finding could not be sub stantiated. This posed the question whether the "positive" chick was abnormally HONS 037t><*l - 9- sensitive to the C.E.F. or to the high level of salt in the diet. An interesting discussion of sodium chloride levels and their relation to chick hydropericardium was given by Dr. J. C. Alexander at the Poultry Association meeting held in August of this year. After another collaborative survey during 1961 using essentially the same diet, the FDA have proposed a statistical value for deciding what constitutes a positive response. This is now being reviewed by our industry. The Fatty Acid Producers' Council currently have before the FDA a petition for a regulation covering fatty acids and derivatives therefrom. It is expected that the regulation will not issue until problems concerned with the chick bioaasay procedure are cleared up* The petition includes a reservation on the requirement for running any type of test for the C.E.F. It would seem from the evidence available to date that the presence of the C.E.F. in food grade fatty acids is an unusual and rare occurence, and our in dustry should not be required to test for this factor as a routine matter any more than we should be required to test for thousands of other toxicants which might ac cidentally get in. This should be a matter for careful production and is already a responsibility spelled out in Section 402 of the Act applying to adulterated foods. The problem should be looked at from a broader frame of reference than only the fatty acid industry. 10/30/61 HONS 037643 - 10 - REFERENCES 1. Effect of Certain Composition and Source Variables on Wholesomeness of Fat for Feed by H. D. Hathaway, C. M. Barnett and R. L. Patterson, FeedstuEfa. January 10, 1959, 2. Symposium reported in J.A.O.A.C. (Journal of the Assn, of Off, Agric. Chem.) pp. 120 148, February 1959, held October 14, 1958. 3. Ibid -- Characterization of a Type of Unidentified Compound Producing Edema in Chicks by W. B. Brew & J. D. Dore, J. H. Benedict, S. C. Potter and Endre Sipos, 4. Ibid -- Studies of the Chicken Edema Disease Factor by L. Friedman, D. Firestone, W. Horwitz, D. Banes, M, Anstead and G. Shue. 5. Ibid -- Some Chemical Characteristics of the Chicken Edema Disease Factor by J. C. Wooton and J. C. Alexander. 6. Current Status of the Toxic Principle Causing the Chick Edema Syndrome by George C, Potter, W. B. Brew, R. L. Patterson and Endre Sipos, J.A.Q.C.S., 36, 214-217 (May 1959). 7. The Occurrence of the Chick Pericardial Edema Factor in Some Oleic Acids & Products Derived Therefrom by Stanley R. Ames, William J. Swanson, Marion Ludwig and George Brokaw, J.A.Q.C.S., 37, 10-11, April 1960. 8. Studies on a Factor Causing Pericardial Edema in Chicks and Its Occurrence in Soma Oleic Acids, Federation Proc., 19, 323 (1960). 9. The Examination of Fats and Fatty Acids for Toxic Substances, David Firestone, et al., J,A.Q.C.S,, 38, 418-422 (1961). 10. Studies of the Chick Edema Factor II, Isolation of a Toxic Substance by A, Yartzoff, David Firestone, et al., J.A.Q.C.S., 38, 60-62 (1961), 11. The Isolation and Characterization of the Chick Edema Factor, by R. E. Harman, G. E. Davis, W. H. Ott, N. G. Brink, F. A. Kuehl Jr., J. Am. Chem. 3oc.t 82, 2078 - 9 (1960). 12. A chick Assay Procedure for the Edema - Producing Factor in Toxic Fat, W. H. Ott, et al., J. Poultry Science, 40, 1016-1022 (1961) HONS 037644 i 11 13. The Food and Drug Laws as Viewed by Regulatory Agencies, J. K. Kirk, J.A.O.C.S.. 37, 533-5 (I960). 14. Bioassay Method Cor the Detection of the Chick Edema Factor, J.A.Q.C.S., 44, 147-7 (1961). 15. Hydropericardium and Ascites in Chicks Fed a Chlorinated Hydrocarbon by E. L. McCune, J. E. Savage and B. L. O'Dell, University of Mo.; Columbus, Mo. Prepublication report, 16. Aroclor Compounds - Monsanto Chemical Company Bulletin 2-800-0560-53, p. 17. See also Aroclor Plasticizers Bull, Pl-306. 17. Bioassay Method for the Detection of the Chick Edema Factor, J.A.O.A.C., 44, 146-147 (1961). 18. The Significance of Sodium Chloride in Diets for the Hydropericardium Assay by J. C, Alexander, 50th Annual Meeting, Poultry Science Assn., August 1961 HONS 037645