Document yrg8DZE1edMdY3r3wbOon24b2

FROM (NAME & LOCATION) Elmer P. Wheeler, Medical Dept. DATE SUBJECT REFERENCE June 12, 1969 Report on "Human Poisoning With Kane-Chlor1' . CC: J. R. Durland - Tokyo VI. R. Richard TO Dr. Robert E. Keller Attached is a summary of the Japanese interim report. on the incident where humans were poisoned with the Japanese chlorinated biphenyl when it leaked into and contaminated rice bran oil. Briefly, the original newspaper articles were vastly exaggerated since they indicated as many as 14,000 people may have suffered poisoning. This number was reduced to 600 and the enclosed report indicated that there are 178 diagnosed cases and 111 suspected cases. The principal symptoms are related to the dermitological effects (chloracne, blackheads, rash, etc.). The clinical investigations report possible more serious effects including those on pregnant women and subsequent new births. I don't know that you want to spend much time on the reports of the clinical and the epidemilogical com mittees. You may, however, be interested in the report of the analytical investigations beginning'on page 53Included in the determinations were gas chromatography results on tissue from two or more of the patients. ' If you should make Xerox copies of the analytical portion of the report, would you make one for me? John Durland has asked that these reports be returned to him when we're through with them. EPW: ju. Enclosure osVJ229014 // STLCOPCB4053663 KJ U The First Interim Report by The Analytical Sub-Committee . (Nov. 18, 1968) Chairman: Prof. Hisao Tsukamoto Kyushu Univ. Pharmacological Dept. Members: Profs. Makizumi, Kuratsune, Nagai(Medical) Prof. Yoshimura (Pharmacological) Prof. Ueno (Technological Engineering) Prof. Inagami Assist. Profs. Yamada, Sugano(Agricultural) Prof. Takeshita(Industrial Productivity) * Prof. Yamaguchi (Kurume Medical U.) Dr. Manako (Fukuoka Pref. Sanitation Research) I. On constant^of the oil 1) Acid value (by Sugano) Analysis was done in the standard analysis method for oils and fats. .. Kanemi's rice oil owned by one ofthe oil disease patients, 0.75. The control oil considered not related with the oil disease, 0.57 - 0.70. 2) Peroxides (by Inagami and Sugano) Done in the standard methods. Pt's Kanemi oil . IodinemethoLdodin method mag/kg OD500/pmg 13.4 - 39.4 0.431 - 0.79 Control oil 1.2 - 12.1 0.044 - 0.458 DSW 229015 y' STLCOPCB4053664 V' J KJ II, Metals (Inorganic matters) 1) A rsenic i) Arsenic in oil a)' Gutzeit Method (by Yoshimura, Kuratsune, Yamaguchi) The rice oil samples of six patients and . four control oil samples were prepared in the moist method and the combustion method and analized in Gutzeit method. The results were all under 1 ppm as arsenious acid, with no significant difference between the patients' oil and the control oil, b) Xray-fluorescence analysis and radiation analysis (by Yamada) The result of the former on one patient's oil was 0+5 ppm. The result of the latter on another patient's oil was below 0.1 ppm. ii) Urine before and after BAL. BAL was given to three patients of one family and discharge of arsenic in urine before and after the . administration was measured. No significant figure was obtained in any of the cases. DSW 229016 i. STLCOPCB4053665 -55- III. Organic Chlorines 1) P C P i) Color reaction (by Inagami) Alkali extract from patients' oil sample and its acid vapor distillation fluid were placed to color reaction tests in 4-amino-antiphyrine method, and results were negative. DSW 229017 STLCOPCB4053666 V./ VJ 2) Other metals i) Chemical analysis (by Yoshimura, Pref. Sanitation Lab.) Rice oil samples used by six patients were prepared in the moist method and in the conbustion method for analysis of Pb and Ni, and the results were all negative. ii) Xray-fluorescence analysis and radiation analysis (by Yamada) The patients' oil and the control oil (Nisshoku Salad Oil) showed no marked difference in the fluouscence spectre of heavy metal elements. By radiation analysis one patient's oil . (Kanemi Rice Oil) showed the following results. Na 7 ppm Mn 0.3 ppm Ni 2 ppm or less Cu 1 ppm or less 2n 2 ppm or less Hg 1 ppm or less 3) Discussion It was concluded that, in view of the above results, the oil used by patients did not contain arsenic or any other heavy metal in quantity enough to cause the disease. sw 229018 STLCOPCB4053667 3) Carbonyl (by Sugano) In 24-dinitropheny1-hydrazine method The pt's rice oil (Total carbonyl mag/kg) 16.5 Control oil 9.6 - 11.0 4) Unsaponificated material By Prefectural Sanitary Research Lab. in the Sanitation Test Method. Two samples of pt's oil Control oil (2 samples) 3.01%, 3.51% 2.73 3.08 5) Fatty acid composition (by Nagai) Both the patients' rice oil and the control oil mostly consisted of palmitic acid, oleic acid, linoleic acid, and the gas-chro patterns were also very much alike. 6) Discussion As mentioned in the above, the difference of the oil used by the patients and the control oil was in peroxide and carbonyl values, higher in the patients' oil. - However, the patients' oil by the time of the examina tion had passed more time than the control, and these differences may be due to . natural denature of the oil. os**9019 STLCOPCB4053668 -58 ii) Gas chromatography (by Yoshimura) Patients' Kanemi Rice Oil samples and a sample of a recent lot Kanemi Rice Oil, as well as samples of two other brands were separated into acid, at each, alkali and neutral parts^ and PCP was sought at the acid part using the Shimazu GL-2C Type apparatus (detector . ECD column : 1.5% SE-30 Auakron AS 4mm x 0.75). In all samples, the peaks were close - to that25 o* f*..the 'standard PCP RT, with no significant difference between the patients' oil and others. iii) Thin layer gas chromatography(by Inagami and Yoshimura) The above i) and ii) samples were used for search of PCP in this method also. No evidence of its existence was recognized. iv) Discussion . The above is sufficient to prove that PCP cannot be considered a cause of the disease. . . 2) Heat catalyst diphenyl chloride(Kane Chlor) i) Gas chromatography (by Inagami, Makizumi, Yoshimura, & Prefectural Sanitation Lab.) a) Preparation of samples Kanemi Rice Oil samples of four patients were prepared in *saponification method or in *loAC method to get . concentrated organic chlorine contents as samples for gas-chromatography.(*The oil is heated in 15% alcoholic NaOH for 60 minutes at 100C, and the un- saponificated matter is extr^ + ed. using ethyl ether). DSW 229020 STLCOPCB4053669 -59- (** The oil solution in hexane or petroleum ether is put to an agitation extraction process with acetonitrile saturated with the same solvent, and the extract is thrown in 2% NaCl or mirabilite aqueous solution and extracted again with petrole.um ether and the extract is put through a florisil column to get the ethyl ether or the hexane content in the extract solve out). Several other brands of food oil considered not related with the disease were also gas chromatographed as control samples. b) The type of the equipment and the experiment conditions i) Shimazu GC-IC Type(Makizumi) Detector: Pulse type electron-capture detector (ECD) and hydrogen flame ionization detector (HFID) were connected for simaltaneous recording. Column: 1.5% SE-30 on Chronosorb W (60 - 80 mesh) Glass column 4mm x 2625m Column temp.: 200, Detector temp.: 200 Carrier Gas: N, (1.6kg/cm2) . J1 . DSW 229021 STLCOPCB4053670 -60- ( E C D: Sensitivity 10^, Range 0.8 or 1.6 HFID: Sensitivity 10^, Range 0.8 or 1.6 ii) Apparatus: Shimazu GC-IC-Type Gas Chromatography(Yoshimura) Detector: . Hydrogen flame ignition detector(HFID) Column: 1.5% OV-17 on Shimalite W Glass Column 4mm :x 2,625m Column temp.: 200, Detector temp.: 200 . Carrier gas: ^(SOml/min, 3.2kg/cm^) : H2 40ml/min. Sensitivity 10^, Range 3.2 . c) Conclusion The most typical chromotograms of the samples of patients' oil prepared in AoAC process, tested by ECD under conditions of the above b) i) and o are the attached that of Kane-Chlor (10"g)/|Figs. 1. and 2, in which extreme peaks are frequent and the patterns, are very similar, clearly showing that the patients' oil contains much Kane-Chlor. (The sample of Kane Chlor was obtained from Kanemi Warehouse KK Production Dept.) The control oil samples do not show such a pattern. DSW 229022 STLCOPCB4053671 -61- The Figs. 3 and 4 are another typical chromatogram of a patient's oil sample and that of Kane-Chlor ^ (10"6g) done in AoAC process, tested by HIFD under conditions of b)ii). The two figures are alike again. ii) Infra red spectrum(Yoshimura & Takeshita) The acetone extraction of the above patients' Kanemi Rice Oil sample was done and its organic chlorine part was sepa rated and purified by florisyl column chromatograph, which, in chloroform solution, was found to show almost the same . spectrum as that of Kane-Chlor, as the figures enclosed. iii) Organic chlorine contents in oil a) By chemical analysis(Yoshimura, Kuratsune, Inagami, and Prefectural Sanitation Lab.) Done in the "General Quantitative Determination Method of Organic Chlorines" of the "Sanitation Test Methods" on samples of six patients' Kanemi Rice Oil samples and control rice oil samples. The method was to dissolve the oil into benzene, wash it with water to remove init by adding organic chlorine, and then to reduce^isopy1-alcohol, metal sodium to it and the resulted NaCl was titrated. The results were, DSW 229023 STLCOPCB4053672 -62- Sample A B C D E F Control Cl content(ppm) 1,070 1,080 1,020 1,080 1,170 1,500 Trace or none b) Radiation analysis (Yamada) Three patients' Kanemi Rice Oil samples and control oil samples were analyzed using Kyoto University Atomic Pile Laboratory's pile KUR (non-breaking). Neutron ray group, 5 x 1012 n/cm2, sec; radiation time 10 minutes, 1 hour; measurements, RCL 512 and 1024; channel analyzer Ge(Li); detector 2cm2 and 22cm2. ' Sample A' B' C Control A brand " b" M Q It / Cl content(ppm) 1,170 1,105 1,300 2 8 18 DSW 229024 STLCOPCB4053673 -63- When the above A sample was washed twice with 70C ANC^Q : 4), twenty times in quantity of the sample, almost none of Na remained in the oil, while almost all Cl remained. From this, it was estimated that the chlorine in the oil was organic chlorine. c) Discussion a) From the above a) and b) results, the patients' oil was confirmed to contain 1000 - 1500 ppm (as Cl) organic chlorine . . If the composition of organic chlorine in the patients' oil will be found to be same as that of Kane-Chlor, it will be concluded that the patients oil contains 2000 - 3000 ppm Kane-Chlor as Kane-Chlor Cl content is about 48% as will be reported in the next iv). Other Kanemi Rice Oil samples than the above are still under study. r iv) Chlorine content in Kane-Chlor . . ` a) Burning in oxygen flask(Ueno) . According to Schoniger's method, the sample specimen 10 - 20 mg was capsuled and burned in an oxygen-filled and sealed flask, and the gas obtained from burning it was absorbed by the absorption fluid in the flask, consisting of 0.1 N NaOH 10 ml and 30% H2O2 2 ml, and titrated with 0.01N Hg (^3)2 , and the unused Kane-Chlor(obtained from Kanemi Warehouse KK), was found ' to contain 47.5% chlorine. / DSW 229025 STLCOPCB4053674 -64- b) Sodium biphenyl analysis j(Ueno) 20mg of sample was dissolved in toluene, added with IN sodium biphenyl solution lOgms,shaken for 30 minutes, and organic halogenated material was taken out, at which point the designated test chemical is added in excessive quantity so that the chemical*s blue-green color will remain , neutralized with nitric acid, extracted more than twice with 0.1 N HNO3, and titrated with Hg(N03)2- The chlorine content of the Kane-Chlor sample examined in this method was 48.5%. c) Reduction method, one of the Sanitation Test Methods (Yoshimura-and Pref. Sanitation Lab.) The method is same as the above iii) a). The result was 47.3%. ' d) Discussion . The above three results 47.5, 48.5 and 47.3% were almost equal with the 48% as specified in Kane-Chlor specifications. The samples used in the above three analyses were all from unused lots. v) Kane-Chlor composition(Ueno) Kane-Chlor, first, having its chlorine removed by using Raney alloy and NaOH aqueous solution, was gas-chromatographed, and the result pattern showed that two new peaks appeared in the DSW 229026 STLCOPCB4053675 -65- small RT area in addition to the pattern of the unreacted Kane-Chlor, of which, the one of large RT was identical with RT of biphenyl (Kane-Chlor material) and the other, with that of phenyl ticrohexan standard product. The Kane-Chlor raw material, that is, Yawata Chemical's biphenyl, showed a single peak at gas-chromatography, which was identical with biphenyl standard product RT. From these results, it is not considered likely that other aromatic compound than biphenyl is in the Kane-Chlor composition. IV. Search for other foreign materials Patients' oil samples were separated into acid, alkali, and neutral parts or distilled by vaporization and compared with control oil samples at each of these with efforts for seach of any agricultural chemical, food additive, etc. in methods such as thin layer gas-chromatography, ultra violet absorption spectrum and all sorts of color reactions, but no specific foreign material was found mixed in. V. Search for Kane-Chlor component contents in living tissues (Yoshimura, Makizumi, Inagami) specimen Skin bij.-cpsie-s of a few of the oil disease patients prepared at Kyushu U. Dermatological Dept, were gas-chromatographed for finding Kane-Chlor component contents in them. DSW 229027 '. STLCOPCB4053676 -66- 1) Skin fat i) Preparation of specimen Two patients' abdominal skin fat specimen, one pt's cheek skin fat specimen and three normal people's abdominal, neck and breast skin fat specimen(control) were chosen for the examination. The tissue was cut off by scissors and put through ethanol extrac- times tion three:^for 10 - 15 minutes each time at 50 - 60C. Then it was infiltrated in the same manner, using of. . ethyl ether and hexane. All/.the extracted fluid on * was placed^a hot plate for solvent removal and the residual matter was extracted with ethyl ether: petroleum ether = 1.5 : 8.5, and filtered, and the solvent was removed from the filtrate, which was dissolved in a small quantity of petroleum ether, poured ina.florisil column to dissolve the ethyl- ether and petroleum ether out, to be made ready for gas chromatograph. . . ii) Gas chromatograph apparatus and conditions a) Shimazu GC-IC Type gas chromatograph(Yoshimura) Detector: Election-Capture Detector(ECD) Other details are the same as stated in Page b) (2). T3&7* } STLCOPCB4053677 -67- b). Shimazu GC-IC Type Gas-chromatograph (Makizumi) Same as stated in Page b) (1). iii) Results The typical gas chromatogram of the above ii) a) is the attached Fig. 6(Extracts of a pt's and Kane-Chlor). Fig 7 is of a control case and Fig 8 is of another pt's, co-chromatographed with Kane-Chlor. . The Fig 6 shows several peaks which are equal-patterned as those of Fig 7 in the small RT area, but there are peaks in the large RT area also, such not being seen in Fig 7, which are very similar to Kane-Chlor components' latter half peaks, and the identification was confirmed by co-chromatography(Fig. 8). Extracts of two other patients, though there were minor ' differences of height, showed very similar patterns as of Fig. 6, while chromatograms of three control cases were very similar to Fig. 7. The same samples were analyzed bn ii) b) equipment also, whose conditions are different, and showed data to lead to the same conclusion. From the above results, it is suspected that some of the Kane-Chlor components remain in pts' fat composition for a fair length of time after the rice oil intake as some Kane-Chlor component peaks were confirmed in the pts' extract chromatograms. DSW 229029 J' STLCOPCB4053678 - 68- spec iraen 2) Other bjiopsies* ' Besides skin fat, perspiration, nails, mother!s milk, fetus, placenta, etc. were suspected to contain Kane- Chlor. Analyses are underway. VI. Conclusion 1) The constant measurements of the pts' Kanemi Rice Oil showed abnormal values in peroxides and carbonyl, but these were natural in view of the production dates of the oil. This fact does not necessarily relate with the disease as a cause. 2) Arsenic and other inorganic metals cannot be considered to have caused the disease. 3) PCP could not be the cause either. ' 4) Patients' oil was confirmed to contain much Kane-Chlor, a heat catalyst. The chlorine contents of the pts' oil samples were 1000 - 1300 ppm. 5) The chlorine content of unused Kane-Chlor obtained from Kanemi Warehouse KK was about 48%. 6) The major component of Kane-Chlor is biphenyl. DSVJ 22903 STLCOPCB4053679 -69- ( 7) The oil disease patients' skin fat gas-chromatograms show that a few of Kane-Chlor components remain there for a fair length of time after the rice oil intake. / STLCOPCB4053680