Document 8V4MO34EyKMOpDdODR9GRRxzK

I /4RX&-01W IN VITRO MICROBIOLOGICAL MUTAGENICITY ASSAYS OF 3M COMPANY'S COMPOUND T-3727 F inal Report March 1985 By! j z . _________ K atoleenO kam oto.M icrobiologist M icrobial G enetics Department and 1=3 Eduard S. R iccio , A s s is ta n t D ire c to r M icrobial G enetics Departm ent Prepared fo r; 3M Company M edical Departm ent G en eral O f f ic e s , 3M C e n te r S t. P a u l, MN 55144 A tte n tio n : D a lla s D. Zimmerman Toxicology S p e c ia lis t SRI P r o j e c t LSO-3145 A" rprp-r'oved by: il Oh 1rs-cz<*'Z> X ris tie n E. M ortelmans, Study D ire c to r M icrobial G enerics Department B. R eid , D ir e c to r foxicology Laboratory \ j j (y .. W. A. S k in n e r, V ice P r e s id e n t L ife Sciences D ivision 3(43135R)3a2v6e-n6s2w0o0od ATvWe.X: 9M10e-n3l7o3P-2a0r4k,6CA T9e4l0e2x5: 334-486 OOi.331 SUMMARY SRI I n t e r n a t i o n a l exam ined 3M Company's Compound T-3727 f o r m uta g e n ic a c t i v i t y in th e s ta n d a rd Ames S a lm o n e lla /m icrosome a s s a y w ith s t r a i n s TA1535, TA1537, TA1538, TA98, and TA100 o f th e b a c te riu m Salmo n e l la typhim urium . The assa y was perform ed in th e p re se n c e and ab sen ce of a r a t- liv e r m etabolic a c tiv a tio n system . A ll te s ts were performed in com plian ce w ith th e U n ited S ta t e s Food and Drug A d m in is tr a tio n Good Labo rato ry P ractice Standards. Compound T-3727 was r e p r o d u c ib ly n o n m u tag en ic when t e s t e d a c c o rd in g to these procedures. ii 001*132 CONTENTS SUMMARY............................................................................................... QUALITY ASSURANCE STATEMENT.................................................. INTRODUCTION.................................................................................... MATERIALS ........................................................................................ METHODS ......................................................................................... RESULTS AND DISCUSSION.............................................................. TABLES T ab le 1 .................................................................................... T ab le 2.................................................................................... ii Iv 1 3 5 10 11 12 ill 001^33 QUALITY ASSURANCE UNIT Final Report Statem ent SRI I n te r n a tio n a l a s s u r e s th e q u a lit y and i n t e g r i t y o f t h i s s tu d y , I n - V itr o M ic r o b io lo g ic a l M u ta g en ic ity A ssays o f Compound T -3 7 2 7 , f o r th e 3M Company. The stu d y was in s p e c t e d on March 1 2 , 1985 d u rin g th e c o lo n y c o u n tin g p h a se . The f in d in g s o f th e Q u a lity A ssurance U nit in sp ectio n were reported a t the tim e o f the in sp ectio n to the Study D ir e c t o r . SRI management was inform ed o f t h e i n s p e c t io n r e s u l t s on March 1 2 , 1 9 85. A d a ta a u d it was perform ed on March 2 5 , 19 8 5 . The Study D ir e c to r and SRI management were inform ed o f th e a u d it r e s u l t s on March 2 5 , 1985. The f i n a l r e p o r t was a u d ite d and review ed on March 2 5 , 1 9 85. The r e s u lt s o f th e f in a l r ep o rt rev iew were communicated to th e Study D ir e c t o r and SRI management on March 2 5 , 1 9 8 5 . The f in a l rep o rt a c c u r a te ly d e sc r ib e s th e methods and standard o p era tin g p roced u res and r e f l e c t s th e raw d a ta o f th e s tu d y . Any d e v ia t io n s from the approved protocol and standard op eratin g procedures were iv 001234 INTRODUCTION SRI I n t e r n a t i o n a l exam ined 3M Company's Compound T-3727 f o r muta g e n i c ity in th e s ta n d a r d Ames S a lm o n e lla /microsome a s s a y w ith s t r a i n s TA1535, TA1537, TA1538, TA98, and TA100 o f th e b a c te riu m S alm o n e lla typhim urlum . An A ro c lo r 1 2 5 4 -s tim u la te d , r a t - l i v e r hom ogenate m e ta b o lic a c t iv a t io n system was in c lu d e d in th e a ssa y p ro ced u re to p ro v id e m e ta b o lic s te p s t h a t th e m icroorganism s e i t h e r a r e in c a p a b le of c o n d u c tin g o r do n o t carry out under the assay conditions. The a s s a y p ro c e d u re w ith S_. typhim urlum h as p ro v en to be 80 to 90% r e l i a b l e i n d e t e c ti n g c a rc in o g e n s a s m u tag en s, and i t has a b o u t th e same r e l i a b i l i t y in id e n tify in g chem icals th a t a re n o t carc in o g en ic . However, b eca u se th e a s s a y system s do n o t alw ays p ro v id e 100% c o r r e l a t i o n w ith carcinogenicity in v estig atio n s in anim als, n eith er a p o sitiv e nor a n eg ativ e response conclusively proves th a t a chem ical is carcinogenic o r n o n carc in o g en lc to man. E v alu atio n o f experim ental r e s u lts from the Salm onella assay c o n s is ts o f com paring th e number of h is ti d in e - i n d e p e n d e n t c o lo n ie s on th e t r e a t e d a g a r p la te s w ith the number observed on the c o n tro l p la te s . Because a l l th e p la te d Salm onella in d ic a to r organism s undergo a few c e l l d iv isio n s in the presence of the te s t chem ical, the te s t is sem iq u an titativ e in n atu re . The p la te t e s t procedure does n o t p erm it q u a n tita tiv e d eterm in atio n o f the number of c e lls surviving the chem ical treatm en t. I t is the dem onstration of a m utagenic dose-response re la tio n s h ip th a t is im portant in e s ta b lis h ing m utagenicity. The te s t chem icals a re assayed a t se v e ra l dose le v e ls w ith in a non to x ic dose range--w ith the ex cep tio n of the h ig h e st dose le v e l, which sometimes e x h ib its to x ic ity . T o x ic ity is evidenced by s e v e ra l phenomena: c le a rin g o f the background b a c t e r ia l lawn grow th, form atio n o f p in p o in t 1 001235 c o lo n ie s c o n s is tin g of su rv iv in g c e l l s , and a d ecrease in the number of re v e rta n t co lo n ies below th e spontaneous background. A chem ical is considered a mutagen in the Salm onella assay i f i t e l i c i t s a re p ro d u cib le, d o se -re la te d in crease in th e number of h is tid in e re v e rta n ts per p la te in one o r more te s te r s tr a in s . The a s s a y s w ith Compound T-3727 were begun on 20 F eb ru ary 1985 and t e s t i n g was com pleted on 27 F e b ru a ry 1985. C opies o f th e f i n a l r e p o r t w i l l be k e p t in o u r f i l e s (B u ild in g M, Room 213) and in S R I's R eco rds C e n te r. The raw d a ta w i l l be r e t a i n e d in B u ild in g 205, Room 13, f o r one year a f te r th e la b o ra to ry notebook has been f i l l e d and th en w ill be sto re d i n S R I's R ecords C e n te r. A ll t h a t rem ain s o f Compound T-3727 w i l l be k e p t fo r s i x months i n o u r ch em ical s to r a g e room (B u ild in g M, Room 217) and th e n r e tu r n e d to 3M Company. 2 001236 MATERIALS Test A rticle - Name: T -3727 - P a te R e c e iv e d : 19 F eb ru ary 1985 - D e s c r ip tio n : L ig h t-am b e r waxy s o l i d - S to ra g e C o n d itio n s : S to re d a t room te m p e ra tu re in a secondary co ntainer - S p e c ia l T e s tin g C o n d itio n s : None - S ta b ility : Assured by Sponsor In d ic a to r Organisms - Species: - S trains: - Source: S alm o n ella typhim urium LT2 TA1535, TA1537, TA1538, TA98, and TA100 f o r S_. typhim urium D r. Bruce Ames, U n iv e rs ity o f C a lif o r n ia , B erkeley M etabolic A ctivation A ro c lo r 1 2 5 4 -in d u ced , r a t l i v e r S--9; SRI B atch F -4 ; ~ 26.5 mg/ml p r o te in N egative (S olvent) C ontrol M aterial A ceto n e, CAS No. 67 -6 4 -1 D ate Opened: 14 December 1984 E x p ir a tio n D a te : 14 December 1985 M a n u fa c tu re r: American S c i e n t i f i c P ro d u c ts , McGraw P a r k , IL P u r i t y : 99.7% L o t N o.: KTEA 3 001237  Positive Control Chemicals 9 -A m in o a c rid in e , CAS No. 9 0 -4 5 -9 M a n u fa c tu re r: P f a l t z and B a u e r, S tam fo rd , CT 2-A n th ram in e, CAS No. 613 -1 3 -8 M a n u fa c tu re r: Sigma C hem ical C o ., S t . L o u is , MO 2 - N itr o f lu o r e n e , CAS No. 6 0 7 -5 7 -8 M a n u fa c tu re r: A ld ric h C hem ical C o ., M ilw aukee, WI Sodium A zid e, CAS No. 26628-22-8 M a n u fa c tu re r: D ifco L a b o r a to r ie s , D e t r o i t , MI Counters Used - New B runsw ick S c i e n t i f i c B loT ran II A utom ated Colony C o u n te r, Model 0 .1 1 , SRI No. 00 30 61 26 0 0 - New B runsw ick S c i e n t i f i c B actro n ic Colony C o u n te r, Model C110, SRI No. 0012 3108 00 4 METHODS S alm onella typhimurlum S tra in s TA1535, TA1537, TA1538, TA98, and TA100 The S alm o n e lla typhim urlum s t r a i n s u sed a t SRI a r e a l l h i s t i d i n e a u x o tro p h s by v i r t u e o f m u ta tio n s In th e h i s t i d i n e o p ero n . When th e s e h is ti d in e - d e p e n d e n t c e l l s a r e grown on m inim al medium a g a r p l a t e s con ta in in g a tra c e of h is tid in e , only those c e lls th a t re v e rt to h is tid in e ind ep en d en ce ( h is '*') a r e a b le to form c o l o n ie s . The sm all amount o f h i s tid in e allow s a l l th e p la te d b a c te ria to undergo a few d iv is io n s ; in many c a s e s , t h i s grow th i s e s s e n t i a l f o r m u ta g e n e sis to o c c u r . The h i s 4, re v e rta n ts a re e a s ily v is ib le as co lo n ies a g a in st the s lig h t background grow th. The spontaneous m utation frequency o f each s t r a i n i s r e la tiv e ly c o n s ta n t , b u t when a mutagen i s added to th e a g a r , th e m u ta tio n fre q u e n c y Is In creased , u su ally In a d o se -re la te d manner. We o b ta in e d o u r S_. typhim urlum s t r a i n s from D r. B ruce Ames o f th e U niversity of C alifo rn ia a t B erkeley. In ad d itio n to having m utations in the h is tid in e operon, a l l the in d ic a to r s tra in s have a m utation ( r f a ) th a t lead s to a d efec tiv e lip o polysaccharide coat; they a lso have a d e le tio n th a t covers genes Involved in the sy n th esis o f the vitam in b io tin (bio ) and in th e r e p a i r o f u l t r a v i o l e t (u v )-in d u c e d DMA damage (u v rB ). The r f a m u ta tio n makes th e s t r a i n s more p erm eab le to many la r g e m o le c u le s, th e re b y in c re a s in g th e m utagenic e f f e c t of th e se m o lecu les. The uvrB m utatio n ren d ers the b a c te r ia unable to u se th e a c c u ra te e x c isio n r e p a ir mechanism to remove c e r t a i n c h e m ic a lly o r p h y s ic a lly Induced tyNA l e s i o n s and th e re b y enhances th e s t r a i n s ' s e n s i t i v i t y to some m u tag en ic a g e n t s . S t r a i n TA1535 i s r e v e r te d to h i s 4 by many m utagens t h a t c a u se b a s e - p a i r s u b s t i t u t i o n s . S t r a i n TA100 i s d e riv e d from TA1535 by th e in t r o d u c ti o n o f th e r e s i s t a n c e t r a n s f e r f a c t o r , p la sm id pKMIOl. T h is p la sm id i s b e lie v e d to c a u se an in c r e a s e in e r r o r - p r o n e DMA r e p a i r t h a t le a d s to many more m u ta tio n s f o r 5 001239  a g iv e n dose o f moat m utagens. In a d d i t i o n , p la sm id pKMIOl c o n f e r s r e s i s tance to the a n t ib io t ic a m p lc illln , which is a convenient marker to d e te c t the presence of the plasm id in the c e l l. The presen ce of th is plasm id a l s o makes s t r a i n TA100 s e n s i t i v e to some f r a m e s h i f t m utagens [ e . g . , ICR191, benzo(a)pyrene, a fla to x in B^, and 7 ,1 2 -d im eth y lb en z(a)an th racen e]. S t r a i n s TA1537 and TA1538 a r e r e v e r te d by many f r a m e s h i f t m u tag en s. S t r a i n TA98 i s d e riv e d from TA1538 by th e a d d i t i o n o f th e p la sm id pKMIOl, w hich makes I t more s e n s iti v e to some m utagenic a g e n ts . A ll in d ic a to r s tra in s are k e p t frozen in n u trie n t b ro th supplem ented 9 w ith 10% s t e r i l e g ly c e r o l a t -80C i n 1-ml a l i q u o t s c o n ta in in g a b o u t 10 c e l l s . New f r o z e n s to c k c u l tu r e s a r e made e v e ry th r e e months from s i n g l e colony is o la te s th a t have been checked fo r th e ir genotypic c h a ra c te ris tic s (h is , r f a , uvrB, bio) and fo r the presence of the plasm id. For each e x p erim en t, th e fro z e n 1-ml c e l l c u ltu r e s a r e allo w ed to thaw a t room te m p e ra tu re b e fo re in o c u la ti o n in 50 ml o f g lu c o se m inim al l i q u i d medium supplem ented w ith an excess of b io tin and h is tid in e . The c u ltu re s a re grown a t 37C, unshaken f o r 4 h o u r s , then g e n tly shaken (100 rpm) f o r 11 to 14 h o u rs. A ll s t r a i n s a r e g e n e tic a lly an aly zed w henever ex p erim en ts are performed. A ro clo r 1254-Stim ulated M etabolic A c tiv a tio n System Some c a r c in o g e n ic ch e m ic a ls ( e . g . , o f th e a ro m a tic am ine ty p e o r th e p o ly cy clic hydrocarbon type) are in a c tiv e unless they are m etabolized to a c tiv e form s. In anim als and man, an enzyme system in th e l i v e r o r o th e r organs ( e .g ., lung o r kidney) is capable o f m etabolizing a la rg e number o f th e s e ch em ica ls to c a rc in o g e n s . Some o f th e s e in te r m e d ia te m e ta b o lite s a r e v e ry p o te n t m utagens in th e S. typhlm urlum t e s t . Ames has d e s c rib e d th e l i v e r m e ta b o lic a c t i v a t i o n sy stem t h a t we u s e . In b r i e f , a d u l t male Sprague-Dawley r a t s (200 to 250 g) a re g iv e n a s in g le 500-mg/kg i n t r a p e r i to n e al in je c tio n of A roclor 1254 (a m ixture of p o ly c h lo rin ated b ip h e n y ls). This treatm en t enhances the sy n th e sis of enzymes involved in the m etab o lic conversion of chem icals. Four days a f te r the in je c tio n , the anim als' food 6 001240 \ I s removed b u t d rin k in g w a te r i s p ro v id e d ad l i b i t u m . On th e f i f t h day, the r a ts a re k il le d and the liv e r homogenate is prepared as fo llo w s. The li v e r s a re removed a s e p tic a lly and p laced in a prew eighed, s t e r i l e g la s s b eak e r. The organ w eight is d eterm in ed , and a l l subsequent o p e ra tio n s a re conducted in an ic e b a th . The liv e r s a re washed w ith an e q u a l volume o f c o ld , s t e r i l e 0 .1 5 M KC1, minced w ith s t e r i l e s u r g i c a l s c i s s o r s in th r e e volum es o f 0 .1 5 M KC1 (3 m l/g o f w et o r g a n ) , and homo g en ized w ith a P o tte r-E lv e h je m a p p a ra tu s . The homogenate i s c e n trifu g e d f o r 10 m in u tes a t 9000 x and th e s u p e r n a ta n t, r e f e r r e d to a s th e S-9 fra c tio n , is quickly frozen on dry ic e and sto red a t -80C. The m etabolic a c tiv a tio n m ixture fo r each experim ent c o n s is ts o f, f o r 50 ml: 5.0 ml of S-9 fra c tio n 1 .0 ml o f MgCl2 (0 .4 M) and KC1 (1 .6 5 M) 0 .2 5 m l o f g lu c o s e -6 -p h o s p h a te (1 M) 2 .0 ml o f NADP (0 .1 M) 25 .0 ml o f sodium p h o sp h ate b u f f e r (0 .2 M, pH 7 .4 ) 16.75 ml of s t e r i l e H20 . The amount of S-9 f r a c tio n d e liv e re d to each p la te is 50 p i. P la te In co rp o ratio n Assay P rio r to te s tin g , the te s t a r t i c l e is s e r ia lly d ilu te d from an i n i t i a l s to c k . In some c a s e s , a p re lim in a ry ex p erim en t i s conducted to f in d a s u ita b le dose range fo r te s tin g . The a r t i c l e is u su a lly te ste d over a minimum o f s i x dose l e v e l s , th e h ig h e s t n o n to x ic dose l e v e l b e in g 10 mg/ p la te u n less s o lu b ility , m utag en icity , o r to x ic ity d ic ta te s a lower upper l i m i t . Vhen e x tr a c ts a re made, v a rio u s u n d ilu te d a liq u o ts a re te s te d , u s u a ll y o v er a dose ran g e o f 5 to 100 o r 200 p l / p l a t e . When l i q u i d s a r e te s te d , o cca sio n ally the sample is n o t d ilu te d and v ario u s a liq u o ts a re u sed . A ll assay s a re rep ea ted a t l e a s t once on a s e p a ra te day. The p la te in c o rp o ra tio n assay i s perform ed in th e fo llo w in g way. To a s t e r i l e 13 x 100-mm t e s t tu b e p la c e d in a 43C h e a tin g b lo c k we ad d : 7 G0*!41  (1 ) 2.00 ml o f 0.6Z a g a r c o n ta in in g 0.6Z NaCI, 0 .0 5 mH b i o t i n , and 0 .0 5 mM h i s t i d i n e (2 ) 0 .0 5 ml o f i n d i c a t o r o rg an ism s (a b o u t 10** b a c t e r i a ) (3) 0.05 ml of a so lu tio n of the te s t a r tic le (4) 0.50 ml of m etab o lic a c tiv a tio n m ixture ( i f a p p ro p ria te ). This m ixture is s tir r e d gently and then poured on p la te s co n tain in g about 25 ml o f m inim al g lu c o se a g a r . A f te r th e top a g a r h a s s e t , th e p l a t e s a r e In c u b a te d f o r 48 h o u rs a t 37C. The number o f h is"*' r e v e r t a n t c o lo n ie s i s co u n ted u s in g a B ioT ran I I au to m ated co lo n y c o u n te r when p o s s i b l e . Vhen accurate counts cannot be obtained ( e .g ., because of p re c ip ita te ), the p la te s are counted manually using an e le c tr ic probe colony cou n ter. Concurrent s te r i lit y , negative (so lv e n t), and p o sitiv e co n tro ls are run w ith every experim ent. S te r ility co n tro ls Include p la tin g out sepa r a t e l y s te p s (3 ) and ( 4 ) . For n e g a tiv e c o n t r o l s , we u se s te p s ( 1 ) , ( 2 ) , ( 4 ) , and 0.05 ml of th e so lv e n t used f o r th e t e s t a r t i c l e , i f a p p ro p ri a t e . F or p o s i t i v e c o n t r o l s , we t e s t ea c h b a c t e r i a l c u l t u r e u s in g th e ste p s (1 ), (2 ), (3 ), and (4) w ith the follow ing mutagens: Sodium a z id e f o r th e b a s e - p a i r s u b s t i t u t i o n m u tan ts TA1535 and TA100 9 -A m in o ac rld in e f o r th e f r a m e s h i f t m u tan t TA1537 2 - N itr o f lu o r e n e f o r th e f r a m e s h i f t m u tan ts TA1538 and TA98 2-Anthramine fo r a l l te s te r s tr a i n s , in the presence o f m etabolic a c tiv a tio n . S ta tis tic a l A nalysis No s t a t i s t i c a l a n a ly s is i s p erfo rm e d . R e s u lts a r e a t a b u la ti o n o f th e number of c o lo n ie s ap p earin g on th e p la te s . C riteria for Interpretation P o s itiv e . A t e s t a r t i c l e i s co n sid e re d a mutagen when i t produces a rep ro d u cib le, d o se -re la te d in c re a se in the number of re v e rta n ts in one o r more s t r a i n s . T his in c re a se must o ccu r f o r a t l e a s t th re e dose le v e ls . 8 001242 N eg attv e. A t e s t a r t i c l e i s co n sid ere d a nonmutagen when no doser e l a t e d in c r e a s e in th e number o f r e v e r t a n t s i s o b se rv e d i n a t l e a s t two in d e p e n d e n t e x p e rim e n ts. The maximum dose l e v e l t e s t e d f o r n o n to x ic compounds i s 10 m g /p la te (u n le s s d i c t a t e d o th e rw is e by s o l u b i l i t y p ro b lem s). For to x ic compounds, only the h ig h e s t dose le v e l te s te d should show ev id en ce o f t o x i c i t y . I n c o n c lu s iv e . When a t e s t a r t i c l e c a n n o t be i d e n t i f i e d c l e a r l y as a mutagen o r nonmutagen in the stan d ard p la te assay , the r e s u lts a re c la ssifie d as inconclusive. R eferen ces Ames, B. N ., E. G. G urney, J . A. M i l l e r , and H. B a r ts c h . C arcin o g en s as fram e sh ift mutagens: M etabolites and d e riv a tiv e s o f 2-acetylam inoflu o re n e and o th e r aro m atic amine carcin o g en s. P ro c. N a tl. Acad. S ci. USA j t t , 3128-3132 (1 9 7 2 ). Ames, B. N ., W. E. D u rsto n , E. Y am asaki, and F. D. L ee. C arcin o g en s a r e mutagens: A sim ple t e s t system combining liv e r homogenates fo r a c tiv a tio n and b a c t e r i a f o r d e t e c t i o n . P ro c . N a tl . Acad. S c i. USA 70, 2281-2285 (1973). Ames, B. N ., J . McCann, and E. Y am asaki. Methods f o r d e t e c ti n g c a r c in o gens and mutagens w ith the S alm o n ella/mammallan-mlcrosome m u tag en ic ity t e s t . M utat. Res. 31, 347-364 (1975). McCann, J . , E . C hoi, E. Y am asaki, and B. N. Ames. D e te c tio n o f c a r c in o gens as m utagens in th e S a lm o n e lla /mlcrosome t e s t : A ssay o f 300 chem i c a l s . P ro c . N a tl. Acad. S c i. USA 7 2 , 5135-5139 (1 9 7 5 ). McCann, J . , and B. N. Ames. D e te c tio n o f c a rc in o g e n s a s m utagens in th e S alm o n ella/mlcrosome t e s t : Assay o f 300 chem icals: D iscu ssio n . P roc. N a tl. Acad. S c i. USA 73, 950-954 (1 9 7 6 ). M ortelm ans, K. E ., and B.A.D. S to c k e r. S eg reg atio n o f the m utator p ro p e r ty o f p la sm id R46 from i t s u l t r a v i o l e t - p r o t e c t i n g p r o p e r ty . Mol. Gen. G enet. 167, 317-327 (1979). 9 001243 RESULTS AND DISCUSSION 3M Company's Compound T-3727 was sc re e n e d f o r m u tag en ic a c t i v i t y In th e Ames S a lm o n e lla /m lcrosom e In v i t r o m u ta g e n ic ity a s s a y u s in g th e f iv e s ta n d a r d s t r a i n s o f S alm o n ella typhlm urlum : TA1535, TA1537, TA1538, TA98, and TA100. The a ssa y s were perform ed in d u p lic a te , b o th in the p resen c e and absence of a r a t- liv e r m etabolic a c tiv a tio n system . Three p la te s p er dose le v e l w ere te s te d . Acetone was used as th e s o lv e n t. The m ic ro b ia l m u ta g e n ic ity te s tin g of th is sample was perform ed on 20 and 27 F e b ru a ry 1985. Dose l e v e l s ra n g in g from 10 to 5000 p g /p la te were u sed f o r b o th a s s a y s (T ab les 1 and 2 ) . No d o s e - r e la te d in c r e a s e s in the number o f h istid in e -in d e p e n d e n t re v e rta n ts were observed in e ith e r a s s a y . A b la c k p r e c i p ita te was n o ted a t 5000 p g /p la te ; th e r e f o r e , th ese p la te s were hand-counted. We co n clu d e t h a t Compound T-3727 was r e p r o d u c ib ly nonm utagenic when te ste d according to these procedures. 10 C-OJ.^44 Table 1 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-3727 E xperim ent D ate: 20 F eb ru ary 1985 Compound Compound M e ta b o lic Added A ctivation per P late TA1535 H istidine R evertants per P late TA1537 TA1538 TA98 N egative Control Ace tone - + 50 p i 20 22 32 3 10 3 17 18 17 18 26 32 50 17 8 8 11 7 6 28 25 21 41 38 33 P o sitiv e Controls Sodium A zide 9-A m in o acrid ine 2-Ni tro flu o re n e 2-A nthram ine IH--* - + -- + 1 Mg 50 5 1 1 2.5 2.5 690 661 752 37 37 27 405 347 338 520 662 787 1440 1244 1552 942 895 1144 23 34 27 37 36 34 182 177 164 187 160 240 8 11 6 87 89 95 Compound T-3727 -- -- -- -- -- 10 pg 25 15 25 10 7 5 19 19 17 30 19 26 50 30 21 26 9 6 5 14 13 17 29 23 38 100 28 32 20 886 8 16 10 22 26 26 500 23 21 24 8 7 8 14 10 12 23 13 23 1000 28 16 29 5 7 7 13 16 17 29 17 19 5000* 25 23 17 6 12 6 11 12 7 23 24 22 + 10 13 15 18 15 9 9 21 25 22 53 28 31 + 50 10 15 12 5 10 8 12 24 17 37 44 35 + 100 16 16 12 6 13 13 25 27 23 25 29 23 + 500 5 11 9 5 15 9 25 24 22 28 24 27 + 1000 12 10 6 11 19 4 24 20 21 41 33 36 + 5000* 12 17 8 5 9 11 21 17 19 31 20 43 TA100 125 124 129 130 129 149 589 574 567 150 136 151 437 463 428 117 125 116 111 145 115 109 122 96 137 122 108 123 127 111 109 127 122 106 153 116 111 124 118 120 130 115 112 136 99 141 118 126 146 120 125 ?v P re c ip ita te d a t th is dose le v e l; hand-counted. cn Table 2 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-3727 E xperim ent D ate: 27 F eb ru ary 1985 Compound_____ Compound M e ta b o lic A d d e d ____ _____________________ H is tid in e R ever ta n t s p e r P l a t e __________________ A ctivation per P late TA1535 TA1537 TA1538 T98 TAIOO' Negative Control Ace tone -- + 50 p i 50 10 22 19 8 15 7 P o sitiv e Controls Sodium Azide 9-Am inoacridine 2-Ni tro flu o re n e 2-A nthram lne - -- -- -- + -- + 1 Hg 50 5 1 1 2.5 2.5 464 492 477 24 21 20 215 212 220 Compound T-37 27 -- -- -- " 10 pg 50 100 500 1000 5000* 21 26 16 20 24 17 23 14 22 20 13 17 23 25 20 19 13 22 + 10 11 10 8 + 50 10 8 8 + 100 10 15 17 + 500 12 11 14 c+ 1000 687 cH* 5000* 10 7 11 ----------------------------------------P re c ip ita te d a t th is dose le v e l: hand-counted. 7 6 10 16 6 8 8 11 11 19 21 18 8 18 19 18 26 26 111 293 346 1253 1240 1102 783 689 737 8 15 22 40 21 22 193 158 151 141 168 158 8 10 6 68 72 68 3 3 7 16 16 20 23 19 20 8 5 7 18 14 9 28 19 20 6 3 8 11 9 12 21 19 15 4 9 8 18 11 13 32 26 19 74 6 7 16 10 30 12 20 7 4 9 10 8 4 . 30 23 21 16 12 20 11 8 6 9 10 11 6 12 9 7 12 13 11 9 9 11 11 11 32 24 27 15 15 9 26 34 19 17 21 12 26 27 18 16 9 15 24 25 21 16 20 12 18 16 26 20 13 17 32 26 36 98 92 90 103 91 89 500 549 544 128 125 129 382 378 334 122 97 96 97 120 101 115 84 88 85 103 93 108 98 94 82 92 109 138 113 128 130 79 132 114 94 113 115 103 108 124 114 114 122 114 127