~w138a IMPERIAL TOBACCO LIMITED RES~RtH LABOR4TORY ~- ~ MOKIRWI IHE MV~A~NICITY BY THE AMES TEST 13F AIO~~L ~F~ LWI UT nlulxlll ~_VLn~HI[S FROH_RIDdj~i~iR?Kl~iC~S~- Research laooratoy Report No, 162 BATCo document for legal Services : Health Canada 20 May 1949 CII, BUS. L'?HaRr R.n.T. ~O. :il:., Ct~~THnmrT0N lit R/1,JrlkkL:%L LM ~IPqll ,,: ~iu "hQ ~~··e 10, b bi A!: Inilijl O~ LlwrP Uorators. P,O, Rox 6500, ktnal, lur, I~ v0u Vil;l ~: ~~~i·: lyl: nr~on~ g ~wpill(n RI:~(~ll, ~t~q I:fl~· nqinIn lulnnl.~ IFlil Zl~i:lnv (H IHI I~lt CI:(llr.nTIOY I1EC5 1I1! i" Date Ly 22, 1981, DSiinlnS WIC( LrCPII. nv[N II II RSOEO. PtJP ~c~e;FP~Gt~ TO ~et~ "~i ~c~ `3t( B,A,T. $. L,C5. 6 wA --·· ·;· ··--f·~· 1 H(RuiUI RnPrt Io· 162 ~1 i .On Il,l~~~'~slsbi;r,8njhe ihli C~ndenEliel fm Ridomii-irel tld Flue·Cund Tabaeco Cigaretter. j Ceoy Ilo~, 9, ,; G"I~AQ! i ji,6 P c; BATCo document for Legal Senrices : Health canada 20 May 1999 Research Ldborator~ Report No, 162 COPY No. IMPERIAL TOBACCO LIMITED RESEARCH LABORATORY MDNTAEtlL THE nUTAGENICIr( BY IHE AMES TEST OF AIOOM11 ANO CONDENSATES mOM RIOOMI1-fREA:ED FIVE-CURED TOBACCO C[GARETTES Project No, r-7708 Author: M.H, Biiimoria Issued By: R,S, Wade Gate issued: May 13, 1981 DISTRIBUfTON: Capy No. 1, library 1A, Dr, P.J, lunn 2, Mr, S,H, Fibb 19. Mr. M,F, Scherba~ 3. Mr, R,S, Wade 20~ Or. G.W, Boswail 4. Mr. 5,M, CandIish 21, Mr. L. Bouchard 5, Or. T,A, Smith 22, Mr. H. Roubieek 6. Mr, E, Rittershaus 23. Miss R,R. Smith 7. Mr, F, Seehofer 24. Mr. B,J. Stirling 8. Or, C.3,P, Oe Sigueira 25, Mr, R,i, Rice 9, Or, L.C, F, BtackmanJ 26~ Mr,A,Schaffer 10, Mr. A,L, Heard 27, Mr. E,P. Gage 11. Mr. A,L, Heard 28, Mr. P. Leblond 12. Or, i,W, Hughes 29. Mr. W, :ennyson 13, Or. R.A, Sanford 30, Mr. ~.J, Ross ·1J. Dr, R.I1. Sanford 31, Mr, C~d~ Brown 15. Mr. R,G. Nicholls 32, Mr, C, Harten 16. Mr, R.G. Nieholls II, Dr. M, H. 9i 1 i mo ri a BATCo document for Legal Services : Health Canada 20 May 1999 ~URURI severe ~ae b Ya mould toe oa!PR~C oortior oi at h~~l mnKco crop In 1979 le~ to he use oi ~do~l on~~~t~e ~dre Ontario ~bacco crop in 1980 wnen It was found eo ~e very cffec~ve. In both these yeaa rhe ~1~ R~elr~ Station produced Ridomil treated tobacco on an exw~ment~ ~~~s to r:udy the ef~c~ven~l of Me hn~c~e under On;ario nrawing c~n~Cons. Since n3thinp w~ tn~n about he muh~~d~ of ~id~nil or the effect of Ridomii treatment of tobacco on the biological activity of cigarette smoke, it was decided to examine both Ridomil as well a QC~cn~l dp~tn Ar ~~ th, 109 and nl H~~. ~aP( t3aaccos in the ~er m~~ae~~ty system. Ridomil was not mulaponic in the Amel system either with or without be 31~a ol a rat liver ectivation gna. ~r relui:s d le examination of:he smoke condensater suggest that Rfdomil, if apolied *o~~~ ~ ~e ·sbrln~l iostructions, ma mt b~ap rhede rdn$ d 9~1 03~9~~ ~Lp~ dPbat n~HI mnC ~ mP~i if ~ese are not strictly adhered ~~ B~Co document for legal Services : Health Canada 20 May 1999 INTROOUG;ION in 1979 a considerable portion of the Cntario flue·clred tcaaeco croo was severely daPged by blue mould infection. At tnat time the Delhi Resear:h Station evaluated several fungicider, including Ridomil, for Luture usS in con:rclling tf~ir infection of tobacco in Canada, in 19BO, Ridornii was used on almost the entire Ontario tobacco croo and no blue mould damage Was observed. Rjdbmil, a Ciba·6eigy product had been found to be ery efiectire in blue ;pouid control in other countries (i). Since nothing was known about the lutagenicity of Ridomil Or of the e'fect of iiidoPil treat~?t of BbvcP on tbe biological activity of cigarette snake, it was dPci~ed to enaAine hotil Ridbli. as tell as ciilarel:rs rrdde wjtt Ridonil-trea:el ~Obdcco 1~ tke Ames lutagenicity test, These tooaccos were produced experimentally in 1979 ar,d 19ari by the Oelii Rerelr:1 Station. This report presentS these results. BATCo document for Legal Services : Health Canada 20 May 1999 Ridomii The cona~ercia: saale of "Af danil" /:iba-Geigy) used rlr in ~ct a formulation consis:inc],i 1 par; ai the iyitair hngiridl b~laxyl C~. 12,6-direthylphenyl )·N·(me~houyacetyl) dl anine methyl elter~ and 3 parts ( wettable powder usee to ~Mke it dirperiable i, rater, It wo3ld have been rmre appropriate to :ert the active Ingredient and wettable Powder Separa:eiy 11,,!1 as in ccmbind tian, but Ry were not available. The Ridomil Has tested at levllr of 3, id, 100, 510 Ind 191146, cg per ~iatc, with and without :he Iprague-lawley rat liver activati0e ryl:em i21, ~l~i~Ep. In 1979 the tobaccos were Produced by the Deihi Research Station at two dj fferent JOCatiOng 13j, Each appiicati6n Ridomil per acre. An consisted of ],,~ (,.i,) of aPPropriate control (no appiication of RidamilJ was produced at each rite, ~e locati on i, iaoni; has applied 3 tires, while at location 2, tobaccos were produced w;rh four and five aopiicatronr of One experiment by the Ames test, Since no difference war obtained between the ?xa cO'ltnli,:h. reieif~ iron:he ta lo:l of tobacco are discussed together, In 1380 the control and treated Research Station at separate loeati tdCacCI1E were produced b? tse Oelhi ons On the station and replicated plots employed !4,j)l This war considered necessary to prevent accidental con· taminati6n of the control tPbae:J by Bidonil, in the experientdl nlots, Ri domi i war aPplied at two level s, vi2., four applicati~ni at ihe n3rmdl rate 3 02, (l,i.j ler acre, an? four applicati O"' at twice this rate, and at tj~ ~arettes Cigarettes were prodllced fr:m these Montreal, eapl~yinl a medium ef~iciencr t0tdccas ]y inoerial Tobacco Ltd., deliver about 10 r~ tlr;er Cigarette. 'ilter with ventilated :ilpinP to Leaf and mate analysis were also B BA~Co document for Legal Services : Health Canada 20 May 1999 carried out by this company. ?inoice condensates Smke condensates for mutagenicity testing were produced using Ihe ~inrich Bcrgu~aldt capillary ress machine, weighed and dissolved in dimethylrlllfDxide. The condensate solution was diluted appro3ridtely to glve100 ug, 1SO ug,200 uqand400 ug)erplate, fovrreclications at the above dose levels, each in triplicate, were undertaken, ~ rat liver activation system was used te activate the conlensa;er. ~utaqenic studies airtidlne-reauir:ng Salmonella lyphimvriurn tester strains, viz,, id·1535, TA·100, iA·1537, Tb·1538 and n.gg, kindly provided ~y Professor 3.N, kes ,Here used fur detenining whether or not the con~nerciai fungicide lidomil was mutagenic in this system, strain irl·1535 is reverted by bare lair atagens, while iA·100, a ~lasrid·containing Strain derived i*on TA·1535, is generally nare sensitive to mutagens, Strains:A-1537, i~·153R and TA·PS (siasmid-containicg strain derived from T~-1538) detect fra~esaiit n!utaoens, Snx,ke condenrate is a franeshift mutalen jS,lj, a?d conleoueP!lv, only ;:rain TA-98 Has Ised t3 compare the nutagenicitier of cmtrol Ind Aidonii· treated tobaccos. :he aicrobiologieal Iracedurer and 3reoaraejon of the Ssragle·0dwiey rat liver pbrt-mitochoodrial supe'natant jS-o), iave been dde~uatei? described by ker et al. (2), and their proceC~nr were close;? adhered to, Itatistical analysis of data Condensate mutagenicities were calculated in three ways, In the first orocedure:he ~vtagenici:ier were calculated using regression analysis of lag:ransforned data. the regression lines yere checked for laraileli~a by an analysis of variance JANOVG) test (8i. The intercepti, using a csnmon Ilsoe, were employed ta express Putagenicitier. in the second omcedu~, reqrer~ibn analysis of untransfon~d data was undertaken, and slopes were used to express mutagenicitier, In the third procedure, the m3taaeni[itiei Hete expressed as the number of Hirt revertantlr per ug of condensate at each a BATCo document for Legal Services : Health Canada 20 May 1999 Of the dose levels, Mean and standard deviations ::,~Li, ,,,, ,,,,i,,,,, x;n!i. 1, 4,,Y:~,I:1::I::,4:,""~( detenniOe rhether the differences be~xeen means were Significant or not. For all three ~ethods of lutageni city ealculatiO"I the Student-NeM~n.iIeul IMI PerCO~I ib detenine whi oh of the iiiierencer in "utllenicitier ~en signifiont. Oifferenees with p,0.0~ were eons i dered si Inificant. BATCo document for Legal Services : Health Canada 20 May 1999 RESULTS Ridbnil was not mutagenic in the Ames 1,590 ug per plate system at n, 10, ln0, 501 and as will be evident from iable I (first CO1WR1 unaer elCn strain), ~ Positive reaction would have shown a dose·aF0nre IDldti0nShi3. i.e,, anj ~n~nlring Iwier of iacterial re'iertants of the responsie strain WOUld have jeen recorded with increasi"9 concentration of Ridomil, addition Oi a rat liver meeabolic activation system failed to convert Ridomi i to int2nnedia:e[r) mu!aiien:e toward these strains (recond col umn under each ban*ial strain), but it did ccnvert 3,1·benlo~Jpyrene and 2-asino- an:aracene (Positive cbntnlsj to inteodiatejil ,,t,l,,ic towards resoonrive strains, #(1 evidence dr the toxicity of aidoril tcwardr tnere rtrainr rar evi:enced either with ar without the 'at liver Inefaoolic aetivatiOn system. Since Ridamil ii Ipplied to toaa~ce ie~,,,~ it is eorrible the latter C3UI: Possess eo2ymes caoabie of tranrfonning Uir funpi~ide t0,,tagenic metajol ites, Thus, bmans handiino thp tnh~r~.... ~,, to R;domil. hilt Ilr· ,. ... ' ~~~- IVVYLI·U IUUI(I oe exposed not Only '' '"* V1'U lo metaboi i teS prodaced by the :Jbac:o plant. There. fore, it would rave been useful to inclule a metabolic activation lyftem prepared from toaaceo leaves, 'long iri:n tae Idal;ar! ii,,, IYI~EI, to invertigate tne Passibility Of :nOIiO.Sli01; 01R'dPmil t0 intermediltellj mutagenic in the Ames test, but time did not ~enrit it. The nega:ie rerl;Iti obtained in tnii ?alterili nutageniiity test I;a~le ii suggest thil: the cnancl of illdomjl '3~W;r are retate indeed. cluling mutageniric!rcinapenic da~age in However in order to be more certain that there is "O ;lalard involved in the use of Ridomil 00 tobacco, testing of concennlei prepared from ci~~r(:!pl ~dl fr0m dii0li:tnlted tolc:0, wdI Uodertlipi. a :,, ,,,,,,,ia ~i .niul rmlvtl: ~s~or!i, with smol(e c0"denlate, as well "S the oroductj of 'I'dcail io·ned ~Y the DOfSi~ie Etaboi i sm by plant enzymes. In "xlsinatiol of the 1379 gidomil-~reated i" Fi3ure i and Table 2. ii no significant difrere":e between control and tobacco to chi[il three aopiic3tions of Ridomil ere ~de, four and tolacc~s Hith signific~"tly higi~er ,utagenicfiYe I(id0mil aDPlicatidrl gaveactiviti However, the higher dlthauoh statistically iicnifi:ant,'ere malier;han those "hic~ have been observed between light ant dark grades of ilue-cu·ed tobaccos n the :regllent apol icayionr,: tile aqueaus iunlicidai v BA~Co document for Legal Services : Health Canada 20 May 1999 dispersion medi~n itself, result in chenical chances which are reflected in these srall increases in m~tagenicitles. I: should also be pointed out that the plots on whicn tile tobaccos were grown were not rellicaied, and that Ridomil Has applied so ;ate in the ieaion ;hat 131 Di the toiaccor were harvested ~eilre ail the apPlicationr uere completed. R(domii residue values (see Table 3) received from Ciba· Geigy for these cilaretter dio not, nowever, reflect the Increasing numbers of appji~atilnE of aidomil. Significlar differences in sugar and nicotine contents of the tobaccos and to a lesser degree in the tar and nicotine deliveries for the four and five applications were also observed, As a result 3f there various anomalies, it was decided to repeal the mutagenicity Study using the 1SRO sampies wi;ere resljcated ~lotr were en~lo~ed and Ridomil was applied ~i;owi~g :le supplier's ,ecomneldation for conmerciai crops ane also at ~o tires the recsmrended rate. The residues resultin? from these treatnentr are repo~ted In ~hle i. ihe analyser, in taii case, Here ionductel in the ind'vijual primings after curing as weil as on a composite sample of pnmrngs, The effect of the double rate of Ridomil is evident in all but the las: Friri~S and ior sore unexplained reason in the com?osite blend. The nctageniciiy studies carried out using cigarettes mace from the 1930 croo are plpsenteC (n Figure 2 and Table 5. From the table it will be seen that aPP;iCdliln :i the Fungicide Ridomil to tobacco 19Bn crop) at either the normal rate or double the nomai rate dic not result in any difference in lutagenic activity 'me the control tobacco. Only Players Check 23, used as a continuity control gave results ligniHcantly different (hiSherj than all i above mentioned cigarettes, Mutagenicities calculated by all three procedural enabled identical Conclusions to be drawn, This contrasts with the increase in autageniclty seen in 1979, Euggesting that the supplier's recoii~nenddtions for the appiicaticr of Pido~it should be followed. The negative results obtained in the 1998 tobacco stuly, along with the oaservat:on ~iat Ricomi; ~y itieii ii not m~tapenic, iu?gesi that this !unoiciCe, if applied dccordin5 to the manufacturer' S instructions, doer not increase mutagenic activity of smoke condensate. BATCo document for Legal Services : Health Canada 20 May 1999 REFERENCES i, ~8, lucar, SdF~c, n0 (1980) 147. 2. 3,~ der, ~. McCann Ind i. rl~~kI, Mutation Res,, 31 (1975) 367. I GS. Boswal:, I~r~~ ~~ao Ltd., ~ ~~d 6 ~,~ l97p. I S.L Q!d, Report presented a! the P~,, ~~18 $Ikg~p MeetI ng, 10 Nov, 1900, Sl'mcoe, Ont. L Ct Bndl, Br Caaad it ~I ;Oth e~ia oi tha ~~~iPnl gedgll B~D·l~r ef ~LC, 1~~ RL )980, London, U.L L ~~~n, i ~MY~ ~d&~ k~, ~~~ ~~ k~. ~I,, ~LL, n (1974) 4159, ~~~Bili~~a~~~~i~ed ~~enat~ns~ b rl Bn#b~ ~~ir~~l Ba~ ad k~~~op in ekna end ~~nee~n~ 3, ~lcl i 50~, Ne Yo~, 1960, ~ L~~ Steel ICJ~. T~~,hl*l~a Pdhla~nJalS tatistics, McG~M Hill Beck Co., New Ycrk, 1960. 10, M,H, SIlimeria, Imoe~al iobacco Ltd., mem3 dated 11 Jan. 1980. 11, G,W, Boswali, Im~erial To~acco l:d,, mem dated 20 Feb, 1~81, BA~Co document for legal Services : Health Canada 20 ray 1999 ~-i. AM15 MUTAGENICITy OF SMOKE CcNDENSAS~S FROM RIDOMlliREAtEn~ ~LTES 97 eo f E f p: Fl vl X u O d u Z 111 r CONTROLS 13 b 3 APPLICATIONS ~4 3 dS " O ioa ~200 3W 4W J SMOKE CONDENSATE_ rE PER PLATE N S; 3 iv BATCo document for legal Services : Health Canada 20 May 1999 RCUR~- MES MUTAGENIClfY OF SMOKE CONDENSAiES fRoM a~DoMI1·TREATED r -- --- rlvlll\L I 1 Lj ~980) 400t w I Av/ x CONTROL i I a NaRMAL~RATE ) a DOUBLE'R47E I /~ ~ CHECK 23 '00 2bo 300 ~D s SMOKE CONDENSATE- ~G PER PlrllE C BATCo document for legal Services : Health Canada 20 May 1999 PwnO~ a oo·h? · IL llka\5Yl * \5 C *9*rl a ". cn I b oi ·~*cl a r * ~C~·~ h ~* · r o·Drr o * I e**, O + r L ul ' ' I I ·~ i l~i I I I ; cmi:i4! orr~o · I p , 1·,· · o i 1 n 3 It n ci]l I I I c cl I f L 1 i i qL i LY-rO · I r*Yrr * * 1 rb I 4 n+HO~ 1 n UE rrrrr I IO Ill i 2! 1 O 4 "' *E ,,,,, i n·nr~ 1, O P Y r Y I I 51r E i F1 I I 1 ' . I *I I I I I I C OL 5 f CRh·· I i I U 5 i i 1 · -----· -~-- I --~ i I PP ap·19 1 1 D i:= V i -- ,,.,. I, 4 r - -- - · d Ii e 1 10 ~ I rU ~ * ttl j ·aa,a lu 15 iur· ·8: F J e u ~u ii D U Lr '1 ' ; -~ " f5 1 B E EL. ii rp L~~U· i I C Le ,I n h u :a , 5 i! t Ir :I Ou i I a a n · rv O ;v E 5 " ;I; n L :f * -L I -I c e~. c i I I I 1 u I- i I I I c Eli II I I 1 r -D n 0* eLI I I I r gr * rl ·O~c* n c u· ii i' / I O '" o- j I I . -......;-. . 1 ·I·~ + L C * I a h , U C 5 1 ,F O- d~ppg oi r I~ h rL rOO 8 eo PC] 5 5 C 5;1 pr re O- 5 n; a BATCo d6cllent for ~0~ S,rvices : HIJtC Canada 20 ray 1899 1 ~r u a c* E H FIY -L~ · L~ * nr r ~u I Idr u~W I* a L~ H w I- I n ~t '~n Orc(wI(u -· 41 u-l CCU ~n H uOl Im z a c~E L'1 -Er h n Eb H E in i, I I I W B I . ,, Olt Rm c· h r ~tL 10 n~ Y U-"Lu CO dC O ii cu~h * i : n3 ·9 D r rcr c~ ~· 'O" A dPLOI rl CD a C iaae ~r rO a - r·, ICI E =ol~~· ru u* n d ftLCUr I ul I I r r U/ r I nn Y· a Ca *h · r OV hL NEC r ·n u c ML Hn * a ,, i -i~__ I d 1 i n v u E~ c I i P: C * Y -I * I ~ "- aC Cm n c rl' I I ~ i I u N (F C UI ~L h~ P O ~ U : : LL c'd ~ i u L oc h i ~1 ~I p :El i fi,, 1_ -.-·--· ,,LoU- ~3 , w Epr I I i n u 5 J * uc e vl E s- ii P Ir C~ eI O W 1 I Y- ~C 6- 1 Uc V L= U ·D 0- P O IY ha hD UC- : '" dd cp o e I E EI u~ a 4~ O c~ r· r -~* u ct v * L ; f E C *a ; ; i n U B O Vvl 1 I ~N *E E L H* an D I P ·e ~D P u I I- I NN '' * Y ,Le uH I r IV VI~ U =': 0~C e· w nl * Pe L -I H h L *- J ~_ N L-~ I IV Ur a h PC · ,,, aa Arn a t;; dd dd o; i I E·u OP OLr UU i I~ --C--- ""' 1PO II- n r * C C E P P vwE * O I I·I j u uuu u :E sC i, cZ i ·r Lr Lr 1 ,r ·I : ,r ~, la ·s s di E II D i ~ n0 U 1J 01 ~6 'V u~ ,a I $: a~ 4 a ~~ *e ,,S3Do j: : 4 "* " L~ BATCo document for Legal Servlcea : Heath Clnsdt 20 hfay 1999 TABLE 3 Ridomil residues on flue-cured tobacco (1979 crop)" Code Ho, and Ridomil residue · Oescri pti on P919 cigarette location 1 617- Control 0.95 680 · 3 applications 3,20 of Ridonil Location 2 239 · Control 0.40 476 - 4 applications 1 1,90 of Ridomil 282 · j applications 1 2.10 of Ridomil k Residue data obtained from a Ciba-Geigy Canada 1;j, report(S]~ BATCo document for Legal Services : Health Canada 20 May 1999 Table 4 Riiolil residues an fiue·cured tobacco (1980 croajt Oay between i - - aoelicatian and Code no, and descriot~i~ harvest 7 4 applications at P applications at normal rate double the rate 6.2 11.11 1,2 samoles ap3ear i 1- 36 mixed) 3.9 to have b9en : O 7 reversed Reridve data cbbineo irorn an :mplrill Tcaarco Ltd., menn (il), BAfCo document for Legal Services : Health Canada 20 May 1999 II e n u Fi r Y ·* n rlu u *n n c** r~WI I~ 1* · Ilr 3 YHW IY D nr I cc a ZI 1 OJC~ln rl uul lo urlY CCU * L ,.u X t E ill U~I C~E -jy EC~ -IY h *·n ,r, t ill L 'I- r OPC 10 · d -Lelld Nll Y n re I Uc,,Y dd o d u -EEI~L 1 y~u E* 40 u 1~0r net t UIL·I O" 1 rr (n ii tr 12nu:l: L 1 u I- c ,, Pa a · g: vrm h N I u ldn D L IIrl;IPr N O I * L * O? e e a --I : I r n Eu Pt O re n D O U ·u h h L hi 03 Q 1 ,e hm ~ ~ e o O? L~ L. 00 D I 03 euI C Uu Ir O unJ L c CEI OYL P1 E ,Ec O u 0 O' ~UI * 0- f ··IV r I I u- * D O Uu ~~ ~ D I L LZ P 1 h L OC I uO ·h · C * a· H C* ,.,,,. a D Ic W 1 ~"" "L U c O- D 1 PS P ,r * H* O D L L· 11 L L 0 mi P o Y Il L e Irr*O ·C ·I, rn r re r r U *C 0 eP i ,. Pc n u -' rl % D L; r- rn u .U I· · h C1 0 C; c. I·1L· ;,= '' dd d d L- LU h PO L-l UY 1Uc gr, ~IC n I U 5 00 P ·- D P* ~ V c r C~ u rLn r II·oo U* UI Ile 1 --lrT* *~ S EE Or~'Y U I I .O L··l * u ·r 01 r 1 O P J YE .u L ~~ Ir U 0 p~ r a *r r3 U~LC3 H IU IIEls: UU~ L D ·* * tz 01 ~*. ·*· C U ~·ln~r ~n r N Q N BATCo document for LIJ Olnker : Health Canada 20 ral 1911 G iv BATCo document for Legal Services : Health Canada 20 May 1999