Document NG5kQ45rKZ5r8Lok9N3RDKY7Q

I MEMORANDUM DEPARTMENT Ol HEALTH, EDUCATION, a\D wr.Ll Akl V PUBLIC HEALTH SERVICE FOOD AND drug admlsis i ration j to ? William Papageorge Manager, Product Acceptability Date: July 21, 1976 from : chief, Epidemiology Unit SUBJECT: Final Report of the Subcommittee on the Health Effects of Polychlorinated Biphenyls and Polybrominated Biphenyls. A copy of the above report is enclosed for your information. Prank Cordle, Ph.D., M.P.H. Chief, Epidemiology Unit, HFF-108 Bureau of Foods . Food and Drug Administration Washington, DC 20204 l i i. -, .. HONS 201593 r- /C*'**'/' r Evidence o f Tctriichlorodibenzofuran (TCDF) in Aroclor 1254", and the Urine of Rat* Following Dietary Exposure to Aroclor 1254"* by Aucjm CuHi.rr', Vialyn W. Ruasc1. Ralph W. Jkhhihcs', EllSh C. Villahm**', and Kf.hatf. D. KiMbAiinc*"** Unviron'urniai Protrction Agtitty Chtmlltt Tosicoiotj Uioniory 4T10 Ru(ontHithuf Chnmbltt, C. 30341 ' INTRODUCTION 1 Recent reports and pest findings indicate that impurities found In some technical products ney result In disease following exposure, Chlorecne, X-dleesee and the chick edema factor are claselc examples of disease resulting from exposure to chlorlneted compounds, contaminants In the same, or both1. Chlorinated dibenzodloxlns (CDD) and chlorinated dlbenaofurans (CDF) have been the most Implicated as contaminants of polychlorinated hlphenyla (PCB). VOS et^ si, accounted for differences in the toxicltles of three commercially available PCB preparations, namely: Ptienoclor DPG, French; Clophen A60, German; and Aroclor 1260*. l>.. by the presence of two polar compounds In the third fractions (25% Et^O In hsxans) of ths French and German products, neawly tetra and pentachlorodibenzofurans. Chlorinated dlbenzofurane, including the tetrachlorodibenaofuran as well ee pentachloronaphthelene were also Identified In e Japanese PCB (Kanechlor WOO)3. Neither was found In the U, S. product, Aroclor 1260*. . This paper reports the details of the mass spectral findings of a tstrachloro compound with a molscular wslght of 304 in the urine of rate following prolonged exposure to Aroclor 1254*. It alao reports evidence In support of tbs presence of a similar compound in Aroclor 1254* itself. Environmental Protection Agency, Research Triangle Park, M,C, 27711. ?U,S. Consumer Product Safety Comailsalon, 1330 Heat Peachtree Street, N-H., Atlanta, Georgia 30309. Environmental Protection Agency, Region IV, 1421 Peachtree Street, N.E., Atlanta, Georgia 30309. > "The Coca-Cola Export Corporation, P.0, Drawer 1734, Atlanta, Georgia 30301, , ^Center for Disease Control, Toxicology Branch, 1S00 Clifton Road, N.E.,-Atlanta, Georgia 30333. * Portions of this paper were presented at the 163rd National ACS.Meeting, Division of Pesticide Chemistry, April 10., 1972, Boston, Hess. ' e* Correspondence end reprint requesta; Dr, R. D. Kimbrough, . *4 lerimiittUl CwimIhiIh # Tttltiliff. 44, imbf ****>Vt * Yirt I*** IW ,. M0NS 201594 i EXPERIMENTAL In the first study sixteen-hour urine samples were collected frost sight male Sherman strain rats. Seven had been on e dietary level of 100 ppm Aroclor 1254* (between 13-S mgAg/day) for times varying from k-Sfl days. One had bsen on a dietary level of 500 ppm Aroclor 1254R (about 25 mgAg/day) for 252 days. These rats were started on the experimental diet when they were about US days old. The total Aroclor consumption of all rats over the entire period of exposure up to the time the urine was collected was 3,0 grams. Urine averaged 11 mis per rat and was extracted separately with hexane at e pH of about 7 and eluted from micro eillca gel columns with a ljl mixture of benzene:hexane according to the method of Curley nt al. , Samples were combined prior to mats spectral analysis. ' In a second study, seven-day urine samples were collected from four female rats that had been fed Aroclor 125U^, 100 ppm (7.5 mgAg/ day) for eight months. The pooled urine sample, MU0 mis - pH 6.6, was extracted six times with 50 mla of diethyl ether-hexane (3:1). Each 50 mis of extract waa centrifuged and the aupornatee combined. The extrset was evaporated to 15 mis and partitioned with acetonitrile es described by Mills , Prior to mass spectral analysis the sample was methylated using a procedure similar to that of Stanley. Aroclor 125NR, 1,7 grams, was dissolved in 300 mle of hexanb. The chromatography column had an 1,0. of approximately 3<i ami and waa filled with 180 grams of PR grade activated florisil. The column was pre-washed with hexsne and tha Aroclor standard was added in 300 mis hexane. The fractions and volumes collected are listed in Table I. Analysis of each fraction using Coulson Conductometry revealed a general pattern indicative of Aroclor 125<R. TABLE 1 Elution of Aroclor 125^ from Florisil Elute ' Vol. (mis) Fraction Hexane 5% Et-0 in Hexane 25% LtjO in Hexane 50% EtjO in Hexane 50% EtjO in Hexane 1200 1200 200 mis each 500 mla 500 mla I II III, IV, IX X RESULTS AMD DISCUSSION The mssa spectrum resulting from tha analysis of authenlc 2,3,7,6 tetrachlorodibensofuran (TCDF) la shown In figure 1. The molecular ion K*/e 336 with a chlorine isotopic cluster Indicative of S la the pantachlorodlbensofuran (PCDF) obtained aa an impurity during tha ayntfnsia of TCDF. The low intensity Ion at N/e 275 la the fragment resulting from tha loaa of 63 maaa units from PCDF. The molecular 154 ion at K+/e 304, TCDF, contains the base peak. The fragmentogrsphy of TCDF ia characterized by the loss of 63 maaa units (C0C1) to yield the fragment at MVe 241 and subsequent loss of 70 mass units (2C1) to yield MVe 171. Doubly charged ions were observed at MtJ/e 152 and M+2/e 120.5 with isotopic clusters synonomous with those of their singly charged counterparts at MVe 304 end MVe 241 respectively. The characteristic lose of C0C1 hes been observed in the fragmentation of similarly structured compounds, namely the chlorinated dioxins end higher chlorinated dibenzofurans Curley et al.7. The urine spectrum from the first study (Fig 2A) shows the presence of pentechlorobiphenyl (MVe 324) and the fragment resulting from the loss of 70 mass units to yield HVe 254. Low intensity lone at Kf/e 288 and MVe 290 were obeerved. The molecular ion at H+/c 304 contains an isotopic cluster indicative of 4 Cl with one major fragment at H*/e 235 indicative of the loss of 2 Cl. Observation of the spectrum below MVe 230 revealed that it was attributed entirely to PCB'e. Fraction V from the Aroclor 1254 florieil elution (Fig 2H) contains the hexachlorobiphenyl (H+/e 358), pentechlorobiphenyl (MVe 324) in addition to tri end tetrachlorobiphenyl fragments (H+/o 288 and MVe 253) resulting from the lose of 2 Cl from 358 end 324 respectively. Also present in the spectrum is the moleculer ion . MVe 304 with an iaotopic cluster indicative of 4 Cl. Thia ion compares favorably with TCDF. The low intensity of the ion et 304 precluded observance of any fragmentation. Figure 2C ahows the spectrum produced by urine that had been methylated. The spectrum indicatea the presence of a monomethoxy derivative of pentechlorobiphenyl (M+/e 354) with major fragment ions indicative of: (1) lose of methyl end carbon monoxide (M+-43) to yield KVe 311 and (2) subsequent loss of methyl and carbon monoxide end two chlorines (M+-113) to yield M+/e 241. Thia spectrum also contains a molecular ion with an isotopic cluster indicative of four chlorinea at MVe 304. Mesa spectral evidence doea not establish unequivocally the presence of TCDF. However, it can be said that the molecular ion, 304, is not the result of fragmentation of a higher molecular weight component of the iaomeric commercial Aroclor 1254 mixture. There is e noticsabla difference in the relative intensities of the ' molecular ions, MVe 304, in the urine and standard Aroclor 1254*. The amount of Aroclor represented in Fraction V is 1.7 grams, w])ile the urine represents 3.0 grams consumed over sn extended period. The urine sample enrichment in the analyzer tube was low necessitating a greatly amplified normalized spectrum. Hutxinger et el.'9* have reported the presence of hydroxylatsd biphanyle in urine resulting from metabolism in addition to the preaene* of oxygen derivatives resulting from Irradiation of Aroclor 1254*'fllma( spectre In both instances show the presence of MVe 306 with am isotopic pattsrn indicative of four chlorines. This author end co-workers have observed a molecular ion matching the 306 apaclee reported by Mutzinger, however its fragmentation hae been characterized by the predominance of the 1S5 HONS 201596 % A1ISN31NI FIGURE 1 3 SO a IL. fiu. 120 190 MO 190 T 200 Z h || 1------ 220 240 M+/e 260 280 Figure 1 ' Mass Spectrue resulting fro* the direct probe analysis, at 30C and 70 eV, of authenicated 2,3,7,8 tetrachlorodibenzofuran; see Figure 2A for other conditions. MOMS 201597 Figure 2A Haas spectrum resulting from GC-HS analysis of SB oils of rat urine. LKB 9000 GC-HS, mass marker io.3 mans unit. GC column temperature, 209*Cj flash heater 235C; glass coiled column, 6'xl/M" 1.5 OV-17/1,95 QF-1 on 60/80 mesh chromosorb "W" H.P., A.V,, DMCS; carrier gas (He) 30 pel and 45 cc/min.; separator, 320*Ci source 290*C-, energy, 70 V; accelerating voltage, 3.5 KVi trap current, SO uAi box current, 50 uA; leak current, 8 uA. PFK. 1ST HONS 20159a fragment resulting from the loss of 70 mass units. The molecular iun n+/e 30<i was also present in methylated urine obtainod from 4 Tumslc rats that had consumed Aroclor 1254* for eight months (rig 2C)- This finding Indicates thst s 304 tetrachloro component is present in the urine snd et s trace level in the analyzed commercial PCB preparation. A trace level of the 304 tetrachloro component wee also present in the analyzed commercial PCB preparation. One would have to consider the possibility of such a compound existing in the PCB preparation from a review of the purification process. This procsss consists of distillation of the crude materiel et reduced pressures (about 50 mm Kg) snd slsveted temperatures (150*C-300*C) in the presence of a few tenths of 1% of lime or sodium hydroxide, Pspsgeorge10, Hubbard1^-, snd could lead to hydroxylstion snd tha subsequent loss of HC1 could leed to s dlbenzofuran derivative. ACKNOWLEDGMENT The authors are indeed grateful to Dr's. A. Poland, J.J. Wade end A.S. Knnds, School of Medicine snd Department of Chemistry, University of Rochester, Rochester, New York for a gift of the authenticated 2,3,7,8 tetrachlorodibcnzofuran. REFERENCES ` KIMBROUGH, R.D. , Arch. Environ. Hlth., 25, 125 (1972). VOS, J.G., KOEMAN, J.H., VAN PER MAAS, H.L., TEN NOEVER DC BRAUN, M.C., snd DE VOS, R. H., Td. Coamet. Toxicol., B, 625 (1970). " ROACH, J.A.G. snd POKERANTZ, I.H., Bull. Environ. Contamin. Toxicol., 12, 338 (1974). CURLEY, A., BURSE, V.W., GRIM, M.E., JENNINGS, R.W., and UNDER, R.E., Environmental Res., 4_, 481 (1971). HILLS, P.A., J. Ass. Off. Agri. Chem.. 42, 734 (1959). STANLEY, C.W., J. Agr. food Cham., 14, 321 (1965). 1 CURLEY, A., JENNINGS, R.W., BURSE, V.W., VILLANUEVA, E.C., Pesticido Chemictry, M_, 71 (1974). . HVrZINGEK, O., NASH, D.N., SAFE, 6,, DEFREITAS, A.8.H., - HORSTKOH, R.J., WILDISH, D.J., snd ZITKO, V., Science, 178, 313 ' (1972). . HUTZINGER, 0.. SAIT, S., end ZITKO, V., Environmental Hlth. Farapaotivee, No. 1, 15 (1972). . Personal Coeeeunication. W, B. PAPAGEORGE, June 22, 1973. HUBBARD, H.L. , Encyclopedia of Chemical Technology, , 289 (1965). 156 MOMS 201599