Document JB0KK6VV5aNrNe6J0nk827LB

r.'lRt December, 1971 PCH KAH/SMA: E.K WHEELER, St. Louis tiXSp CC : W.B. Pni*f.o9i'C<!, Si. I.oujr, . W.H. Hp'fcfia/cJ, St. I.ouir,./ H.A. /Voorfeii, Ruabon P.J.ft. Marsh, Brussels Heretfjth translation of a report given us by Bayer at a recent 1*01} meeting. On the basis of this work and some longer term feeding trials which they have not yet reported in detail, Bayer maintain that biphenyls containing up to four chlorine atoms are , metabolised, whereas those containing five or more chlorines are not. Consequently they plan to restrict~1he sale of the higher "chlorinated biphenyls to controllable applications. It would seem that they have more informatioh thari-is' contained in this particular report and we expect to learn some of it at a meeting to be held in Leverkusen on Jonuary^ljtli l^Tjr* We should be pleased to have comments on the Bayer, work and details of any relevant Monsanto work which could be discussed with Bayer and/or other European producers on the 19th"Jahuaryv ' ~ R.A. BAXTER HONS 098377 Fnrbrnfnbri ken Raycr AG. Institute of Toxicology Report No.: 2933* Wuppertal - liberielrt,2%H. D71, Studies of the metabolism of tri- end hexachlorodiphenyl in the rot. lly: V)bent A. A Kimmerle, G, Introduction. In order to clarify the question whether end to whnt extent on accumulation of tri chi orodi phenj'l (TC1>) and of hrxachlorodi phenyl (liCD) occurred in the organism, investigations by residue were carried out in certain tissue* of organs and in feta of rots, after n single ornl admini*trntion. We also investigated the segregation of unchanged TCD in the faeces and, in order to obtain an indication about the degradation in the organiss, the metabolites which were present in the urine. Method. Compound *. Mixtures of isomers of purr TCD end NCR were used for investigations. Animal ran te ri 1 . Male and female SPP rats, CF^ stock. Appa rn ton. Rllliler homogni*er Gas chromatogrnnh 5790 0, a Hewlett-Packard product with K.C, detector (Ni65) Rotary vacuum evaporator Solvents. Rutrol 9 n-hexnne (llvasol) MOWS 098378 a, p, c:h] oro j orm ii.ii, benzene ft.p, cone* ammonia (25/) lOxpc riroen to 1 orrimgfnifnt, A. T..1K ], Invpwti on for the detection of TCP in the tissue, following n nine,Ip oral ndmini strntion. Using a pharyngeal probe, 5 rats each obtained per dose one single ornl dose of 2 r>0, 12'>, 10 end 1 mg of TCD/ltg in Lutrol 9 (l,0 ml solution/kg). They were killed 96 hours nfter the Blurt of the experiment. Koch do**e was matched by 2 controls, . In nnimnls of the 250 end 125 wg/kg dose, the hexene extracts of fnt, liver, kidney, spleen, heort, stomach and the content of stomach were subjected to gas chromatogrnphic investigation ; in animals of the 10 mg and 1 mg/kg dose, only the fat extracts were investigated by gas chromatogrnphy, a) Method of extraction. Kor the extraction of TOD, organR and adipose tissues were homogenised in M Dllhler" with 7*0 ml ( fnt and liver) or 5*0 ml of hexane (kidney, spleen, heart, stomach). After 10 minutes centrifugetion at 4000 rpm, the supernatant solution was siphoned off and was again extracted with 1 x 7*0 ml and 3 x 10,0 ml of hexone ( or with 1 x 4,0 and 2 x 5,0 ml). The extracts were combined in a 50 ml or 25 ml mensuring flask (extract* with small content of nctive substance were concentrated to 10,0 ml in a rotary vacuum evaporator ), HONS 093379 -3- b) (iae chroma togranhy. This vfi governed by the following conditions: Column: steel, l/fi inch, 6 ft, 60 - 80 ine^h, 10/C UCCW 9B2 on Chromnsorb W (AW - DMCS) Injection block temperature 265C Detector tempersture 290C Column lompemture 200C Chart speed 0,5 inch/min. 1,0 - 2,0 ^il of the extracts were injected into the gas chrorantograph. For comparison, 1,0 jil of a standard wob subsequently injected; the standard was so selected that the peak areas of the atnndard and analysis solution were of on approximately identical magnitude. The evaluation was carried out by calculating the product from the height and width at half of maximum intensity of the peaks. The peak area of the sample used for analysis was compared with that of the standard. The range of detection extended over k - 10 ng of TCD/^il, 2. l)e tormina ti on of TCI) separated out in the faeces unchanged, after a single oral administration. From 10 male rats, 5 received 250 mg of TOD/kg each and 5 were given 125 mg of TOD/kg each in l.utrol 9 using a pharyngeal probe. Each test included 2 control animals. The faeces of animals placed on metabolism cages was collected during 72 hours, a) Meihad of extrac Cion, The total amount of faeces from each animal separated out daily was weighed, frozen at - 15C and, before processing, dried for 2k hours in a vac cum desiccator over phosphorus pentoxide. Subsequently, homogenisation was carried out in a IHihler apparatus with 15 ml of HONS 098380 hcxnur, i' was centrifuged for 4 minutes at 4300 rpra, the aupernnlont solution who siphoned off and the residue was again extracted with 4 x 20 ml of hexane. The extracts were transferred into a 100 ml measuring flask and the TCI) concentration was determined by gnu chromatography ( conditions given enrlirr ). If the concentration of the active subrtance vns slight, the extracts were concentrated to 50 or 25 ml in a rotray vacuum evaporator. 3 Investigation of TCP decomposition products in urea, 10 female rata were given TCD by means of a pharyngeal probe in a dose of 1000 mg/kg and were subsequently placed, together with 5 control animals, over metabolism cages. The urea wos collected during 72 hours and it was frozen up to the time of processing ( -15C). a) Method_of extraction. In order to be also able to identify the decomposition products bound on glucuronic acid, half of the collected urea (24, 48 and 72 hour urea) from the treated animals and from the controls was adjusted to pH 4,5 (N CJi^COOIl), it was mixed with ^ -glucuronidase ( 3000 Fishman units/ml) and incubated for 18 hours at 37C, Subsequently, the enzymatically *plit and the untreated urea samples were adjusted to pH 9,0 ( 20J* sodium carbonate solution) and shaken out four-times with the three-fold amount of hexane (centrifugation for 10 minutes St 4000 rnra ). The hexane phases were combined and concentrated in a rotary vacuum evaporator at 45C. The residue wos absorbed in a small amount of hexane (l/lOO of the original amount of urea). mons 098381 b) Thin-1 :'\<`T chroma t ogra phy. The \irr(i extracts vrrc separated on silica gel G with chi oroform/bcnzrne/ ammonia ( 127:22 tl) ss eluent ( 2 x in the same direction of motion ) , l'or rendering the individual fractions visible, spraying was carried out with Nodamin B -solution, Folin'a and Gibb's reagent (Rodnmin B: 0,05'*' in ab.s, ethanol { Gibb's reagent: solution ofVI i rh 1 oroqui none chlorimidc in 06'/ ethanol; Folin's reagent: cone, solution (Merck) to dilute with water in a ratio of 1:3)# For the isolation of the TCJ) decompose lion products, the hexnne extracts of the 24 and 48 hour ureas trentod with fb -glucuronidase were implied onto a large number of plates (layer thickness 0,5 cm). For the localisation of the metabolites, on completion of the test, the border strips of chromatograms were sprayed with reagent solution. The silica gel layer at the height of individual metabolites was scraped out and was transferred to centrifugal tubes (100 ml). By repeated washing of the silica gel with hexane ( 5 x HO ml, centrifugation for 10 minutes at 4000 rpm), it was ponsible to dissolve out the still strongly contaminated metabolites. The extracts wore concentrated in the rotary evaporator to 0,5 - 1,0 ml, the concentrated solutions were again chromatographed ( 2 x in the same direction of motion ), the fractions were once more eluted with hexane from the silica gel and subjected to investigation by gas chromatography. 11. Inves- tiyv' ti ons J_ai\ the detection of HCB in the adipose tissue, afu-v a finale ora) adnini tration, MONS 098302 -6- by using n nh'iryjigct*2 probe, 3 rH? each were given orally 1,9 mg of llCp/kg in J.utrol 9 (l,0 ml of solution/kg). They we re killed 96 hours lifter the begin of the experiment. Two untreated onim.ilK were used ss controls. n) Me thod of extrsetion. The extraction of the Adipose tissue was carried out uncording to the method given for TCD. h) flop rhromntogrnphy. The following conditions were valid for the gns chromstogrnphic investigation Column: steel, l/8 inch, 6 ft, 100 Chrnniosorb W , (AW-DMCS) Injection block temperature Dr to c tor tr wipers ture Column tempersture Clinrt speed 120 mesh, 10 SE 30, 265C 290C 233C 0,5 inch/min. It was possible to identify h - 10 ng/ul. He `ml ts A, TCD. 1, The gns chromatogrnw of the pure TCI) mixture of isomers used for our tests shoved t lenst 8 peaks of which 3 could be identified by comparison with the corresponding pure substances. These were the following compounds : 2 , k , h 1 -tri rhlorodi nhmyl ( 2, h , 9 *-TCD), 3 , 9 , A 1 - tri chi orodi phenyl ( 3*9,9' -TCD) and 2,3,21-tri chiorodiphenyl ( 2,5,2'-TCD), see Fig. 1. )n the tis:'we of ruts, from nil the isom-rs, only 2,9,9'-TCl) could !. ONS 098383 i don 1 i fi od OG lioiir? oft^r the adrnini s trn ii on of 25<> 121, 10 and 1 n.g of Ti:i)/kg ; fee Figures 2 and 3 In the adipose tissue, 8 - lV,* of 2,4,4'-TCP were accumulated, i it the other organs only about. 1'* (rein ted to that contained in the TCI> mixture of isomer* ndministcred), Bee Tables 1 - 3 2. In the fnecea, up to 72 hours after the end of the test, altogether nbout 10J* of unchanged TCI) mixture of isomers could be identified. The main amount was separated out after 48 hours. In addition to the TO) spectrum, gns chromatograms of the treated nnimnls shoved a number of further peaks which were absent in the controls and which could not be identified; see Fig, 4. It is possible that we deal with decomposition products of TCI) separated out via the bile. 3, Unchanged TCI) was not found in urine. After break-down with ^-glucuronidase, the thin-layer chromatographic separation gave a greater number of decomposition product? which showed a phenol-tyne reaction. 6 main metabolites were found, see Fig. 5. It was possible to separate to a large extent metabolite 6 by repeated thin-layer chromatography from the Accompanying substances and it only showed 1 peak in the gns chromatogram, see Fig. 6. H. 11 C I) . (in chroma togrn phie analytic, showed that, even st a concentration of I, 4 mg of UC)>/lig, the spectrum of the total mixture of iFODiera coul< be identified in the fat extract, sec Fig. 7. The accumulation in (.he adipose tissue amounted to about 15/. An exact evaluation wr. * not possibie. HONS 098384 -8- Pi o^n . In tin* orgnni sr.i, the isomeric trichiorodinhenyIs be come hydroxylated to a certain percentage and ore, bound on glucuronic acid, separated out in urine. Introduction of OH-groups into the molecule token predominantly place in p- and o-positions. If these nre portly occupied, op for instance in 2,ft,V-TCD, hydroxylation and,consequently, separation con only occur to o very limtcd extent, if nt nil. This in pssibly tlx* rejiBon for the Accumulation of this particular isomer in the adipose nnd organ tissue. The concentration of the TCI) mixture of isomers separated out unchanged in the faeces vas much snnller then expected. On the other hand, peaks occurring additionally in the gnu chromatograms indicated decomposition products of TCD. These may possibly separate out via the bile. Hut there is also the possibility of decomoosition by intestinal bacteria. In contrast to TCP, the total spectrum of isomers could be identified in the adipose tissue, after the administration of IICI). Unfortunately, no pure isomers vere available, so as to determine hov high was the percentage of the p and o-ch1orinoted compounds in the mixture of isomers* Summary. ], The single oral administration of 2C>0, 125* 10 nnd 1 mg of TCD mixture of isomers/kg caused in rats an accumulation in the tissue by only one of the isomeric co.inound* identified as 2, h, h *-TCP. In the adipose tissue, 8 - 15 /* in other organs about 1% of 2,4,V-?CP MOMS 098385 "7" rou 1 (I be i ilen l.i f i ed. In the faeces, up to 72 hours after orn] administration, unchanged TCD voa sopnrated out. Heaide the ppoctrura of the mixture of isomers , the gas chromatogrnms of the faeces extracts of treated animals showed some additional peaks which could not be identified. In urine, unchanged TCD wns not identified. The separation in thinloyer chromatography gave, after breok-down with 0 -glucuronidase, a number of metabolites with phenolic 011-groups. 2, In contrast to the results obtained with TCD, the entire spectrum of isomers wns identified in the fat extracts, after the single administration of l,k rag/kg of tyCD. The accumulation in the adipose tissue amounted to about 15/b. Signatures of the two authors figure 1 Gas chromatograms of a. 2, kt4'-TCD, 4.2 ng/^1 b. 3,4 * 4*-TCD, 4,0 nc/;il c. 2,5,2'-TCD, 2.2 n6/fi1 d. TCD mixture of isomers, n,9 ns/^il MOMS 098386 Piguro__2. Gbb chromatogram of a. TCD mixture of isomers, 6,08 ng/ul b. 2, ^ ' -TCD (pure), 2,28 ng/ul c. fat extract TCI) rat (10 nig TCD/kg) d. fat extract (control rat ). Figure 1. Gna chromatogram of a. 2,*ifV-TCD (pure) 1, h ng/pl b. fat extract TCD rat (l,0 rag/kg) f Jv A b MOMS 09038? -10- Tr b 1 c 1: Detection of 2, h t V-tri <.h 1 orocJ i plicny 1 (2, , 4'in thr (ulipOM' tiv.Kur* of the rnt (<?' ) niter a single ornl ndmi hi * 1 n< ti on of 2^0, 125. 10 nnH 1 mg of irichloror'iphenyl (TOD,mixture of i somem )/kg (wcnn values from 5 onimnl each) Dote TOD m(j/kG V_ tfiSd/dl .s V TCD mg cUu- , 2,1,4' -l'CD mg 250 125 10 1 92,0 44 ,5 5,31. 0,33 >0,66 10,83 1,16 0,11 2,4,1' -TCD mg 2,53 2,15 0,107 0,011 8,2 14,5 9,2 10,0 fijsurc 5 figure >i. Thin-layer chromatogren of urine (in* rlu om.'togj-.'inj o P extract* obtained after break-,<w. vith /3~glucuronidnse (nil ')) n. pure V(;l>trii x turn of i sonor s , 23, H'>6 "n/pi h* fneeop extract TO)) rnt ()25 mg/k/) c. faeces extract control r;>t Klv.ent:, liloro form/brii/wie/, ,n , i (ta:>: i ) Spray; ki bl>' t reason t. u( . ci,\o:n,)in.. 'i>iri \iini 'i h . .4 lion - HONS 098388 -n- Identification of 2,4,'-trichinrodiphenyl (2,4, V-TCD) in the ,,"i""Se ti5BU<' f th' rnt <'> *ilr ornl od.i nl. t,, ti of 1 . no mr TCP (dnaUr* f/i***i ) / kg Rat i. StSielltoG &U- TCD 2,4,4'-TCD mg rag 6 7. 0 9 10 95,00 96,25 93,75 80,75 06,25 31,67 32,08 31,25 29,50 28,75 2 . 125 mg TCI) 4 fidiprst ti&Aut in g 3,550 11,963 5,730 10,516 6,507 ) / kg 2,4,4 '-TCD in .& mg % 1,94 4,50 3,23 1J5 1,23 , 6,1 14,0 10,3 5,9 4,2 u. T 0 .7) 2.4. <3. ,-*P0T) mg mg V 1 42,5 2 45,6 3 42,5 4 44,9 5 46,9 14,17 15,21 14,17 14,96 15,63 7,69% 8,928 'BN 309 <<,9,003 < t- 6'395 3. 10 mg TCP ( *'*%** / kg Rut No. S C D 2,4,4'-TCD mg ^ rag 11 12 13 14 15 3,45<< , ' 3,40 3,,0% 3-760 '<"3,10 1,15 1,13 1,00 1,20 1,33 4 1 mg TCI) ( -nUkl^rtlf /so-nutJ ftcUp ttt insUP in g 7,4 11,9 11,4 16,5 0,5 Rat K. 16 17 18 19 20 QbfrtuH tUits TCD 2,4,4 '-TC3> mg mg 0,34 0,33 0,32 0,34 0,31 0,11 0,11 0,11 0,11 0,10 Mi/Mifi-t* tdin g 13,4 11,9 14,5 12,6 9,9 2,4,4'-TCD in (/. fit mg * 1,75 2,10 3,1'5 1,75 2,00 12,4 13,8 22,2 11,7 12,0 Red MTl.uJdJ/to'' & 2,4,4'-TCD inib i*f mg i ' 0,1066 0,1011 . . 0,0873 0,1379 0,1011 9,3 9,0 8,7 11,5 7,6 2,4,4 '-l'CD Jr: <4 /* mg 0,0145 0,0121 0,0114 0,00/5 0,0056 13,2 11,0 10,4 7,2 9,6 MONS 090339 -12- Fig. 6. Gas chromatogram of n* Metabolite 6,after three-fold thin* layer chromatography b. Metabolite 6 after the 1. thinlayer chromatography c. Spectrum of the pure TCD mixture of isomers. Figure 7. V Goa rhromi:togrom of a. Fat extract HC1) rat (lf4 mg/kg) b. 1ICI) mixture of isomers (nurr), 6tG* ng^/pl HUNS 098390 v:ci), o- 1 .wr: )/k; 1 i ;>he-,y i rer.n r<- r.-ts ~'ch (o" ), kill cl ')C hour? *fter the W*in oT the 'ND'riucnt n " cAg 250 92,0 1 25 44.4S 2- . TCD mg mg 50,66 2,55 ***** / *.**1 SPLA* tups rrortKM r.' * Eg =g % TT.Q 6,2 0,171 0,6 C,025 0,07` 0,006 / S mg * mg * "g 1| ,<**' !! o.ooijjo.oo:!?,?( 0,02 0,0115 0,037 0,024 0,0S 1 4.85 2,15. 14,5 0,09 0,6 0,013 0,1 0,01 0.0059 0,03 0,014 0,09 0,005 j0,"C>' j?,2 +------- 1 ML 1, 4 ~ *" ^ 1*r. W ''-/ h.J/wt/JLyt `l'!a*M)/h '. ^ HU /,, saJ< W r-*y . ^ * A*,' A" T6CB60 SNOW i