Document O5pQ7b7qbayZLJM0kE4D9R6w

Aftt2j6-02H? IN VITRO MICROBIOLOGICAL MUTAGENICITY ASSAYS OF 3M COMPANY'S COMPOUND T-3752 F in al Report June 1985 K athleen Okamoto, M ic ro b io lo g ist M icro b ial G enetics Department and ___________________ Edward S. R ic c io , 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 Department G e n e ra l 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 : Janine R. Gleason Toxicology S p e c ia lis t (SE! SRI P r o je c t LSC-3145 Approved by: Q k cX i'T U X .u O K ris tie n E. M ortelm ans, Study D ire c to r M ic ro b ia l Genet^ireq D epartm ent rn B. R eid, D ire c to r foxicology L aboratory W. A. S k in n e r, V ice P r e s id e n t L ife Sciences D ivision 1343135R1 3a2v6e-n6s2w0o0od ATvWe.X: 9M10e-n3l7o3P-2a0r4k,6CA T9e4l0e2x5: 334-486 001211 SUMMARY SRI I n t e r n a t i o n a l exam ined 3M Com pany's Compound T-3752 f o r m u tag en ic a c t i v i t y i n th e s ta n d a r d Ames S a lm o n e lla /m icrosom e 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 a lm o n e lla typh im u rlu m . Compound T-3752 was a l s o sc re e n e d f o r reco m b in p g en ic a c t i v i t y i n th e y e a s t Saccharom yces c e r e v i s i a e D3 assay * B oth a s s a y s w ere performed in the presence and absence of a r a t- l iv e r m etabolic a c tiv a tio n system . A ll te s ts were perform ed in com pliance w ith th e U nited S ta te s Food and Drug A d m in is tr a tio n (FDA) Good L a b o ra to ry P r a c t i c e S ta n d a r d s . Compound T-3752 was r e p r o d u c ib ly nonm utagenic and n o n reco m b in o g en ic when t e s t e d a c c o rd in g to th e s e p ro c e d u r e s . ii G 02 CONTENTS SUMMARY................................................................................................. QUALITY ASSURANCE STATEMENT................................................... INTRODUCTION....................................................... MATERIALS............................................................................................ METHODS................................................................................................. RESULTS AND DISCUSSION.................................. TABLES T a b le 1...................................................................................... T ab le 2 ..................................................................................... T a b le 3 ................................................................................ T a b le 4 ....................................................... ii iv 1 3 5 12 13 14 15 16 ill 001213 QUALITY ASSURANCE UNIT Final Report Statem ent SRI I n t e r n a t io 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 , In V it r o M ic r o b io lo g ic a l M u ta g en ecity A ssa y s Of Compound T -3 7 5 2 , f o r th e 3M Company. I n s p e c tio n s and a u d its were perform ed during d i f f e r e n t p hases o f th e s tu d y , and t h e S tudy D ir e c t o r and SRI management w ere n o tifie d of the fin d in gs o f the Q uality Assurance U n it. T h is 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 e r a tin g p r o c e d u r e s o f th e s tu d y . Any d e v ia t io n s from th e approved p r o to c o l were made w ith th e proper a u t h o r iz a t io n and d o cu m en ta tio n . Any d e v i a t i o n s from sta n d a rd o p e r a tin g p r o c e d u r e s were documented. i Manager o f R egulatory A frai rs and- Q u a lity A ssurance 'if-. '. g__ iv 00214 INTRODUCTION SRI I n t e r n a t i o n a l exam ined 3M Company's Compound T-3752 f o r muta g e n i c i t y in th e s ta n d a rd Ames S a lm o n e lla /m icrosom e 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 a lm o n e lla typhlm u riu m . Compound T-3752 was a l s o t e s t e d f o r reco m b in o g en ic a c t i v i t y i n th e y e a s t Saccharom yces c e r e v i s i a e D3 a s s a y . An A ro c lo r 1 254s tim u la te d , r a t - l i v e r homogenate m e ta b o lic a c tiv a tio n system was in c lu d e d in the assay procedures to provide m etabolic step s th a t the m icroorganism s e i t h e r a re in c a p a b le o f co n d u ctin g o r do n o t c a r r y o u t under th e a s s a y c o n d itio n s. The a s s a y p ro c e d u re w ith JS. typh lm u riu m h a s p roven to b e 80 t o 90% r e li a b le in d e te c tin g carc in o g en s as m utagens, and i t has about 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 c a rc in o g e n ic . The assay p ro c e d u re w ith Si. c e r e v i s i a e i s a b o u t 60% r e l i a b l e i n d e t e c t i n g c a r c in o gens as ag en ts th a t In crease m ito tic reco m b in atio n . However, because th e a s s a y sy stem 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 c a r c i n o g e n i c i t y in v e stig a tio n s in anim als, n e ith e r a p o sitiv e nor a negative response co n clu siv ely proves th a t a chem ical is carcinogenic or noncarcinogenic to man. E v alu atio n o f experim en tal r e s u lts from th e Salm onella assay c o n s is ts o f com paring th e number o f h is tid in e -in d e p e n d e n t c o lo n ie s on th e tr e a te d agar p la te s w ith th e number observed on th e c o n tro l p la te s . Because a l l th e p la te d S alm onella in d ic a to r organism s undergo a few c e l l d iv is io n s in the presence o f 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 not perm it q u a n tita tiv e d e te rm in a tio n o f th e number o f c e lls su rv iv in g the chem ical tre a tm e n t. I t i s th e d em o n stratio n of a m utagenic dose-response re la tio n s h ip th a t i s im portant in e s ta b lis h in g m utagenicity. 1 The te s t chem icals a re assayed a t s e v e ra l dose le v e ls w ith in a non to x ic d o se ra n g e ----w ith th e e x c e p tio n o f th e h ig h e s t d o se l e v e l , w hich sometimes e x h ib its to x i c it y . T o x ic ity i s evidenced by s e v e ra l phenomena: c le a rin g of th e background b a c te r ia l lawn grow th, form ation of p in p o in t co lo n ies c o n s is tin g of su rv iv in g c e l ls , and a d ecrease in th e number of re v e rta n t co lo n ies below th e spontaneous background. A chem ical i s co n sid ered a mutagen in th e Salm onella assay i f i t e l i c i t s a re p ro d u c ib le , d o se -re la te d in c re a se in the number of h is tid in e r e v e r ta n ts per p la te in one or more t e s t e r s t r a i n s . The y e a s t S accharom yces c e r e v i s i a e D3 i s a e u k a r y o tic m icro o rg an ism capable of d etectin g m ito tic recom bination, as expressed through a m utation le a d in g to a d e fe c tiv e enzyme in th e a d e n in e -m e ta b o liz in g pathw ay, r e s u l t ing in a red-pigm ented colony. In th is assay, the yeast c e lls are exposed to s e v e ra l c o n c e n tra tio n s of the te s t chem ical, u s u a lly ranging from a c o n c e n tr a ti o n t h a t r e s u l t s in no k i l l i n g to one t h a t c a u s e s 50% k i l l i n g . Any c o n c e n tr a tio n t h a t in d u c e s 90% k i l l i n g i s c o n s id e re d t o x i c . When th e number of g e n e tic a lly a lte r e d co lo n ies per m i l l i l i t e r (y ie ld ) and th e r a tio of a lte re d c o lo n ie s to su rv iv o rs (freq u en cy ) from th e tr e a te d c e lls a re u n e q u iv o c a lly l a r g e r th a n th o s e o f th e s o l v e n t - t r e a t e d c o n t r o l s , we con clu d e th a t th e ex p o su re o f th e c e l l s to th e compound in d u ces m ito tic recom b in a tio n . I f th is event is d o s e -re la te d , th e o b se rv a tio n i s termed a po sitiv e response. The a s s a y s w ith Compound T-3752 w ere begun on 24 May 1985 and t e s t i n g was com pleted on 14 June 1985. C opies of th e f i n a l r e p o r t w ill be k e p t in o u r f i l e s ( B u ild in g M, Room 213) and i n S R I's R eco rd s 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 1 3 , f o r one y e a r a f t e r th e la b o r a to ry notebook has been f il le d and then w ill be sto red in SRI's Records C e n te r . A ll t h a t rem ain s o f Compound T-3752 w i l l be k e p t f o r s i x m onths in o u r c h em ica l 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 MATERIALS Test A rticle - Name: T-3752 - D ate R ece iv ed : 15 May 1985 - D e s c r ip tio n : Amber 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 container - S p e c ia l T e s tin g C o n d itio n s : None " S ta b ility : A ssured by Sponsor In d ic a to r Organisms - Species: - S trains: - Source: S alm on ella typhim urium LT2; Saccharom yces c e r e v is la e TA1535, TA1537, TA1538i TA98, and TA100 f o r S_. tyP^lsuTC'iurci D3 f o r S_. c e r e v i s l a e D r. B ruce Ames, U n iv e r s ity o f C a l i f o r n i a , B e rk e le y , f o r th e S a lm o n e lla ; D r. F . K. Zimmermann, W. Germany, f o r th e Saccharomyces M etabolic A ctivation A ro c lo r 1 2 5 4 -in d u c e d , r a t l i v e r S--9; SRI B atch F -5 ; ~ 31.5 mg/ml p ro te in Animal S u p p lie r: Simonsen L a b o ra to rie s , G ilro y , C a lifo rn ia N egative (S o lv en t/D ilu en t) C ontrol M aterial D im e th y ls u lfo x id e (DMSO), CAS No. 6 7-68-5 D ate Opened: 3 and 8 May and 3 Ju n e 1985 E x p i r a tio n D a te : 3 and 8 May and 3 Ju n e 1986, r e s p e c t i v e l y M a n u fa c tu re r: A m erican S c i e n t i f i c P r o d u c ts , McGraw P a rk , IL P u r i t y : 0.12% ~H 20 ( f o r a l l ) L ot N o.: 4948 KVLX ( f o r a l l ) 3 001217  P o s itiv e C ontrol Chem icals 9 -A m in o a c rid in e , CAS No. 9 0-45-9 M a n u factu rers P f a l t z and B au er, S tam fo rd , CT 2 -A n th ram in e, CAS No. 6 13-13-8 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 07-57-8 M a n u fa c tu re r: A ld ric h C hem ical C o ., M ilw aukee, WI Sodium a z i d 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 1 , 2 : 3 ,4 -D ie p o x y b u ta n e , CAS No. 1464-53-5 M a n u fa c tu re r: P f a l t z and B au er, S tam fo rd , CT S te r ig m a to c y s tin , CAS No. 10048-13-2 M a n u fa c tu re r: A ld ric h C hem ical C o ., M ilw aukee, WI C ounters Used - New B runsw ick S c i e n t i f i c B loT ran II Automated C olony C o u n te r, Model C H I , SRI N os. 0030 6126 0 0 , 0030 0151 0 0 , and 0012 3318 00 - 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 Nos. 0030 1471 0 0 , 0012 3108 0 0 , and 0013 0788 00 4 00 8 METHODS Salm onella typhlm urlum S tra in s TA1535, TA153), TA1538. TA98. and TA100 The S a lm o n e lla typhlm 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 e ro n . When th e s e h is tid 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 tain 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 independence (h is + ) a re ab le to form c o lo n ie s . The sm all amount o f h is tid in e allow s a l l th e p la te d b a c te r ia 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 ti a l f o r m utagenesis to o c c u r. The h is + re v e rta n ts are e a s ily v is ib le as colonies ag ain st the s lig h t background grow th. The spontaneous m u tatio n frequency o f each s tr a i n i s r e la tiv e ly c o n s ta n t, b u t when a m utagen i s added to th e a g a r, th e m u ta tio n freq u e n c y is in c re ased , u su a lly in a d o se -re la te d manner. We o b ta in e d o u r S_. ty p h im u ritm s t r a i n s from D r. B ruce Ames o f th e U n iv ersity o f C a lifo rn ia a t B erkeley. In a d d itio n to having m utations in the h is tid in e operon, a ll the in d ic a to r s tra in s have a m utation (rfa ) th a t leads to a d efectiv e lipopolysaccharide co at; they also have a d e le tio n th a t covers genes involved in th e sy n th e sis of th e vitam in b io tin (b io ) 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 DNA damage ( u v rB ). The r f a m u ta tio n makes th e s t r a i n s more perm eable to many la r g e m o le c u le s, th e re b y in c re a sin g th e m utagenic e f f e c t o f th e se m o lecu les. The uvrB m u tatio n re n d e rs th e b a c te r ia unable to use 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 i c a l l y in d u c ed DNA l e s i o n s and th e re b y enhan ces 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 ts . S t r a i n TA1535 i s re v e r te d to h is + by many m utagens t h a t cau se b a s e - p a ir s u b s t i t u t i o n s . S t r a i n TA100 i s d e r iv e d from TA1535 by th e i n t r o d u c t i 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 pKMlOl. T h is p la sm id i s b e lie v e d to c a u s e an I n c r e a s e in e r r o r - p r o n e DNA 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 a g iv e n d o se o f m ost m u tag en s. I n a d d i t i o n , p la sm id pKMlOl c o n f e r s r e s i s tance to th e a n tib io tic a m p lc lllin , which i s a con v en ien t marker to d e te c t 5 001219 th e p resen ce o f th e plasm id in the c e l l . The p resen ce o f t h i s 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 if t m utagens [ e . g . , ICR191, b en zo (a )p y re n e, a f la to x in B^, and 7 , 1 2 -d im e th y lb e n z (a )a n th ra c e n e ]. S tr 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 ta g e n s. S t r a i n TA98 i s d e r iv 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 i t i v e to some m utagenic a g e n ts . A ll in d ic a to r s tr a in s a re kept 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 - 8 0 C in 1-m l a l i q u o t s c o n ta in in g ab o u t 10 c e l l s . New f r o z e n s to c k c u l t u r e s a r e made e v e ry t h r e e m onths from s in g le 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 i s , r f a , uvrB , b io ) and fo r the p resen ce o f th e p lasm id . For each ex p erim en t, th e fro zen 1-m l c e l l c u ltu r e s a re allow ed to thaw a t room te m p e ra tu re b e f o r e i n o c u l a t i o n in 50 ml o f g lu c o s e m in im al l i q u i d medium supplem ented w ith an excess o f b io tin and h is ti d in e . The c u ltu r e s a re grown a t 37C, un sh ak en f o r 4 h o u r s , th e n g e n t ly sh ak en (100 rpm) f o r 11 to 14 h o u rs. A ll s tr a in s a re g e n e tic a lly analyzed whenever experim ents are perform ed. A ro clo r 1 254-S tim ulated M etabolic A c tiv a tio n System Some c a rc in o g e n ic ch em icals ( e . g . , o f th e aro m a tic amine type o r th e p o ly cy clic hydrocarbon type) are in a c tiv e u n less th ey 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 or kidney) i s capable o f m etab o lizin g a la rg e number o f th e s e c h e m ic a ls t o c a r c 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 li te s a r e v e r y p o te n t m utagens in th e S . ty phim urium t e s t . Ames h as d e s c r ib 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 m ale Sprague-D aw ley r a t s (200 to 250 g) a re g iv en a s in g le 500-mg/kg i n t r a p e r i to n e a l in je c tio n o f A roclor 1254 (a m ix tu re o f p o ly c h lo rin a te d b ip h e n y ls ). T his tre a tm e n t enhances th e s y n th e s is o f enzymes involved in th e m etab o lic co n v ersio n o f ch em icals. Four days a f te r th e in je c tio n , th e an im als' food i s removed b u t d r in 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 d a y , th e r a t s a re k ille d and the li v e r homogenate is prep ared as fo llo w s. 6 001220 The l i v e r s a r e removed a s e p ti c a l ly 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 i s 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 li v e r s a re washed w ith an eq u al volume o f c o ld , s t e r i l e 0.15 M KCl, minced w ith s t e r i l e s u r g ic a l s c i s s o r s i n th r e e volum es o f 0 .1 5 M KCl (3 m l/g o f w et o r g a n ) , and homogenized w ith a P o tte r-E lv e h je m a p p a ra tu s . The homogenate i s cen trifu g e d fo r 10 m inutes a t 9000 x and th e s u p e rn a ta n t, r e f e r r e d to as th e S-9 f r a c tio n , i s q u ic k ly fro z e n on d ry ic e and sto re d a t - 8 0 C. The m etab o lic a c tiv a tio n m ix tu re fo r each experim ent c o n s is ts o f , f o r 50 m l: 5 .0 ml o f S-9 f r a c tio n 1 .0 ml o f MgCl2 ( 0 .4 M) and KCl (1 .6 5 M) 0 .2 5 ml 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) 2 5 .0 ml o f sodium p h o sp h ate b u f f e r ( 0 .2 M, pH 7 .4 ) 16 .7 5 ml o f s t e r i l e H20 . The amount o f S-9 f r a c t i o n d e l iv e r e d to each p l a t e i s 50 |i l . P la te In co rp o ra tio n Assay P r io r to te s tin g , th e te s t a r t i c l e is s e r i a l l y d ilu te d from an in i t i a l sto ck . G enerally, a p relim in ary experim ent i s conducted to fin d a s u ita b le dose ran g e fo r t e s t i n g . The a r t i c l e i s u s u a lly te s te d over a minimum o f s i x d o se l e v e l s , th e h ig h e s t n o n to x ic d o se l e v e l b e in g 10 mg/ p la te u n le ss s o lu b ility , m u tag en icity , o r to x ic ity d ic ta te s a low er upper l i m i t . When e x t r a c t s a r e made, v a r io u s u n d ilu te d a l i q u o t s a r e t e s t e d , u s u a l l y o v e r a d o se ra n 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 c c a sio n a lly th e sample i s not d ilu te d and v a rio u s a liq u o ts a re used. A ll assays are repeated a t le a s t once on a separate day. The p la te in c o rp o ra tio n a ssa y 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 4 3 C h e a tin g b lo c k we add: 7 001221 (1) 2 .0 0 ml o f 0.6% a g a r c o n ta in in g 0.6% N aC I, 0 .0 5 mM 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 o f a s o lu tio n o f th e t e s t a r t i c l e (4) 0.50 ml of m etabolic a c tiv a tio n m ixture ( i f a p p ro p ria te ). This m ixture i s s t i r r e d g e n tly and then poured on p la te s c o n ta in in g about 25 ml of m inim al g lu c o se a g a r. A fte r th e top ag ar has s e t , th e p la te s a re in c u b a te d f o r 48 h o u rs a t 3 7 C. The number o f h is + r e v e r t a n t c o lo n ie s i s c o u n te d 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 . When accurate counts cannot be obtained ( e .g ., because of p re c ip ita te ), the p la te s a r e counted m anually u sin g an e l e c t r i c probe co lo n y c o u n te r. C oncurrent s t e r i l i t y , n eg ativ e (s o lv e n t/d ilu e n t) , and p o s itiv e con tr o ls are run w ith every experim ent. S te r ility co n tro ls Include p la tin g o u t s e p a r a t e l y s t e p s (3 ) and ( 4 ) . For n e g a tiv e c o n t r o l s , we use s t e p s ( 1 ) , ( 2 ) , ( 4 ) , and 0.0 5 ml of th e s o lv e n t/d ilu e n t used fo r th e te s t a r t i c l e , i f a p p r o p r i a t e . For p o s i t i v e c o n t r o l s , we t e s t eac h b a c t e r i a l c u ltu re using step s (1 ), (2 ), (3 ), and (4 ) w ith th e follow ing m utagens: 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 ta n ts TA1535 and TA100 9 -A m in o a c rid in e f o r th e f r a m e s h if t m u ta n 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 ta n ts TA1538 and TA98 2-Anthram ine fo r a l l te s te r s tr a in s , in the presence of m etabolic a c tiv a tio n . We r o u t i n e l y ch ec k f o r t r u e r e v e r t a n t ( h l s + ) c o lo n ie s by r e p l i c a p la tin g from th e p aren t to m inim al g lu co se agar p la te s supplem ented w ith b io tin . C riteria for Interpretation P o s i t i v e . A t e s t a r t i c l e i s c o 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 crease in th e number o f re v e rta n ts in one or more s tr a i n s . T his In crease should occur fo r a t le a s t th re e dose le v e ls . N e g a tiv e . A t e s t a r t i c l e i s c o n s id e re d a nonmutagen when no d o s e r 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 in 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 d o se l e v e l t e s t e d f o r n o n to x ic 8 Q01ZZZ 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 th e s p o n so r o r by s o lu b ility problem s). For to x ic compounds, only the h ig h e st dose le v e l t e s t e d should show e v id e n c e 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 a ssa y , th e r e s u lt s a re c la ssifie d as inconclusive. Saccharom yces c e r e v i s i a e D3 The y e a s t c e r e v i s i a e D3 i s a d i p l o i d m icro o rg an ism h e te ro z y g o u s fo r a m u ta tio n le a d in g to a d e f e c tiv e enyzme in th e a d e n in e -m e ta b o liz in g p ath w ay . When grown on medium c o n ta in in g a d e n in e , c e l l s homozygous f o r th is m utation produce a red pigm ent. These homozygous m utants can be g e n e ra te d from th e h e te ro z y g o te s by m ito tic reco m b in atio n . The frequen cy o f t h i s re c o m b in a tio n a l ev e n t may be In c re a s e d by in c u b a tin g th e o rg an ism s w ith v a rio u s c a rc in o g e n ic o r recom binogenic a g e n ts . The recom binogenic a c t i v i t y o f a compound o r i t s m e ta b o lite i s d eterm in ed from th e number o f red-pigm ented co lo n ies appearing on te s t p la te s . A c u l t u r e o f S_. c e r e v i s i a e i s s to r e d a t 4C . F or ea c h e x p e rim e n t, b r o t h c o n ta in in g 0 .0 5 Z MgSO^, 0.15% KH2PO^, 0.45% (N H ^ jS O ^ 0.35% p e p to n e , 0.5% y e a s t e x t r a c t , and 2% d e x tr o s e i s in o c u la te d w ith a lo o p f u l o f th e s to c k c u ltu r e and in cu b ated o v e rn ig h t a t 30C, w ith sh ak in g . The in v itr o y east m ito tic recom bination assa y in suspension i s con d u cted as fo llo w s . The o v e rn ig h t c u l tu r e i s c e n trifu g e d and the c e l l s a r e re su sp e n d e d a t a c o n c e n tr a tio n o f 1 0 8 c e l l s / m l i n 67 mM p h o sp h a te b u f f e r (pH 7 . 4 ) . To a s t e r i l e t e s t tu b e a r e ad d ed : 1.0 ml o f th e resuspended c u ltu re 0 .5 ml of e ith e r the m etabolic a c tiv a tio n m ixture o r b u ffe r 0.2 ml o f th e te s t chem ical 0 .3 ml of b u ffe r. S e v e r a l d o s e s o f th e t e s t c h e m ic a l a r e t e s t e d (up to 5% w /v o r v /v ) in each ex p erim en t, and a p p ro p ria te s o lv e n t/d llu e n t c o n tro ls are in c lu d e d . 1 ,2 :3 ,4-D iepoxybutane w ithout m etabolic a c tiv a tio n and ste rlg m a to c y stin w ith a c tiv a tio n are used as p o s itiv e c o n tro ls . 9 001223 The s u s p e n s io n m ix tu r e i s in c u b a te d a t 30#C f o r 4 h o u rs on a r o l l e r drum. The sam ple i s th e n d il u te d s e r i a l l y in s t e r i l e p h y s io lo g ic s a l i n e , and 0 .2 ml o f th e 10" and 10" 3 d ilu tio n s i s sp read on p la te s c o n ta in in g th e same i n g r e d i e n t s a s th e b r o th p lu s 2.0% a g a r ; f i v e p l a t e s a re s p r e a d w ith th e 10" 3 d i l u t i o n and th r e e p la te s a r e sp read w ith th e 10" 5 d il u tio n . The p la te s are in cu b ated fo r 3 days a t 3 0 C, follow ed by a t l e a s t 1 day a t 4C to en h an ce th e dev elo p m en t o f th e re d p igm ent i n d i c a t i v e o f ad e n in e -d e fic ie n t hom ozygosity. P la te s co n tain in g the 10" 3 d ilu tio n are scanned w ith a d is s e c tin g m icroscope a t 10x m a g n ific a tio n , and th e number o f m ito tic recom binants (red c o lo n ie s or red s e c to rs ) i s re c o rd e d . The su rv iv in g f r a c tio n o f organism s i s determ ined from the t o t a l number o f co lo n ies appearing on th e p la te s o f the 10" ^ d ilu tio n . C riteria for In terp retatio n P o s itiv e . A p o s itiv e response in th is assay i s in d ic a te d by a dose- r e la te d in c re a s e o f more th an th re e fo ld in th e a b s o lu te number o f m ito tic recom binants per m i l l i l i t e r and in th e r e la tiv e number o f m ito tic recom binants per 105 su rv iv o rs. N e g a tiv e . When no r e p r o d u c ib le reco m b in o g en ic a c t i v i t y i s o b ta in e d in any of the assays perform ed, the te s t r e s u lts are considered to be n e g a tiv e . I n c o n c l u s i v e . When a t e s t a r t i c l e c a n n o t b e i d e n t i f i e d c l e a r l y a s causing a p o sitiv e or a negative response, th e re s u lts are c la s s ifie d as in c o n c lu s iv e . S ta tis tic a l A nalysis No s t a t i s t i c a l a n a l y s i s i s p erfo rm ed f o r any o f th e a s s a y s . The r e s u lt s o f th e p la te in c o rp o ra tio n assay are a ta b u la tio n o f th e number o f c o lo n ie s a p p e a rin g on th e p l a t e s . The r e s u l t s o f th e S, c e r e v i s l a e D3 assay are ta b u late d a f te r c a lc u la tin g th e number o f m ito tic recom binants p e r 1 0 5 s u r v iv o r s . A ll c a l c u la tio n s a r e e x p re sse d w ith two s i g n i f i c a n t d ig its. 10 001.324 R eferen ces Ames, 8 . N ., E. G. G urney, J . A. M i l l e r , and H. B a r ts c h . C arc in o g e n s as fram e sh ift m utagens: M etab o lites and d e riv a tiv e s of 2-acety lam in o flu o re n e and o th e r arom atic amine carcinogens* P ro c. N a tl. Acad. S c i. USA 69 , 3128-3132 (1 9 7 2 ). Ames, B. N ., W. E. D u rs to n , E. Y am asaki, and F. D. L e e . C a rc in o g e n s a r e m utagens: A sim ple te s t system com bining 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 r o c . N a t l . A cad. S c i . USA 7 0 , 2281--2285 (1973) . Ames, B. N ., F . D. L ee , and W. E. D u rs to n . An im proved b a c t e r i a l t e s t system fo r th e d e te c tio n and c l a s s i f i c a t io n o f m utagens and c a rc in o g e n s. P ro c . N a tl . A cad. S c i . USA 70 , 782-786 (1 9 7 3 ). Ames, B. N ., J . McCann, and E. Y am asaki. Methods f o r d e t e c t i n g c a r c in o gens and m utagens w ith th e S alm o n ella/mammalian-microsome m u ta g e n ic ity t e s t . M utat. Res. 31, 347-364 (1975). B ru s ic k , D. J . , and V. W. M ayer. New d ev elo p m en ts i n m u ta g e n ic ity s c re e n ing techniques w ith y e a st. E nviron. H ealth P e rsp e c t. 83-86 (1973). K ie r , L. D ., E. Y am asaki, and B. N. Ames. D e te c tio n o f m u tag en ic a c t i v i t y i n c i g a r e t t e smoke c o n d e n s a te s . P ro c . N a tl. A cad. S c i. USA 71, 4159-4163 (1974) . McCann, J . , E. C h o i, E. Y am asaki, and B. N. Ames. D e te c tio n o f c a r c in o gens a s m utagens in th e S alm o n ella/mlcrosome t e s t : Assay of 300 chem i c a l s . P r o c . N a tl . A cad. S c i. USA j[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 Salm onel1a / mlcrosome t e s t : A ssay o f 300 ch em ica ls: D is c u s sio n . P ro c . N a tl. Acad. S c i. USA 73, 950-954 (1 9 7 6 ). M o rtelm an s, K. E . , and B.A.D. S to c k e r . S e g r e g a tio n o f th e m u ta to r p r o 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 . M ol. Gen. G enet. 167, 317-327 (1979). Zimmermann, F. K ., and R. S ch w aier. I n d u c tio n o f m ito tic gene c o n v e rs io n w ith n itro u s a c id , l-m e th y l-3 -n itro -l-n itro s o g u a n id in e and o th e r a lk y la t in g ag en ts in Saccharomyces c e r e v is ia e . Mol. Gen. G enet. 100, 63-76 (1967). 11 001*325 RESULTS AND DISCUSSION 3M Com pany's Compound T-3752 was s c 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 icrosom 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 i v e s ta n d a rd s t r a i n s o f S alm o n ella typhim urium : TA1535, TA1537, TA1538, TA98, and TA100. The a ssa y s were perform ed in d u p lic a te , u sin g th re e p la te s per dose, both in the presence and absence of a r a t - l i v e r m etabolic a c tiv a tio n sy ste m . DMSO was u sed a s th e s o l v e n t . The m ic ro b ia l m u ta g e n ic ity te s t i n g o f t h i s sam ple was perform ed tw ice a t dose l e v e l s ra n g in g from 10 to 5000 y g /p la te . (T a b le s 1 and 2 ) . No in c re a se s in th e number of re v e rta n t co lo n ie s over th e spontaneous back ground were observed under any o f th e assay co n d itio n s used. Background lawn th in n in g was o b se rv e d i n th e f i r s t a s s a y o n ly w ith s t r a i n TA1537 on a l l o f th e p la te s a t 5000 p g /p la te and on two o f th e th r e e p la te s a t 1000 p g /p la te w ith o u t a c tiv a tio n . A li g h t p r e c i p ita te was noted a t 5000 p g /p la te ; th e re fo re , th e se p la te s were hand-counted. The r e s u lts are presented in Tables 1 and 2. Compound T-3752 was a l s o s c re e n e d f o r reco m b in o g en ic a c t i v i t y i n th e y e a s t Saccharom yces c e r e v l s l a e D3 a s s a y f o r m i t o t i c r e c o m b in a tio n . The assay s w ere perform ed tw ice on s e p a ra te d ay s, b oth in th e p resen ce and a b sen ce o f a r a t - l i v e r m e ta b o lic a c t i v a t i o n sy ste m . DMSO was u sed a s th e s o lv e n t. S in ce no to x i c it y was seen in th e ra n g e -fin d in g a ssa y , th e f i r s t ex p erim en t was perform ed a t dose le v e ls o f 0 .0 5 , 0 .1 , 0 .5 , 1.0 and 5.0% ( w /v ) . No in c r e a s e s in th e number o f m i t o t i c re c o m b in a n ts w ere o b s e rv e d . The c o n firm a to ry assay was perform ed under c o n d itio n s id e n tic a l to th o s e used in th e I n i t i a l a s s a y . A gain, no recom binogenic re sp o n se was o b se rv e d . The r e s u l t s o f th e s e two ex p erim en ts a re p re s e n te d in T ables 3 and 4. In c o n c lu s io n , Compound T-3752 was r e p r o d u c ib ly n o n m utagenic and nonrecom binogenic when te s te d a c c o rd in g to th e p ro c e d u re s o u tlin e d in t h i s rep o rt. 12 001226 Table 1 Compound______ N egative C ontrol DMSO P o sitiv e C ontrols Sodium a z id e 9-A m inoacridine 2-N itrofluorene 2-Anthramine ' Compound T-3752 M etabolic A ctivation - + - + - + -- -- + + + + + + IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-3752 Experiment Date: 28 May 1985 Compound Added per P late ______________________ H i s t i d i n e R e v e rta n ts p e r P l a t e TA1535 TA1537 TA1538 TA98_____ TA100* 50 pi 50 19 22 27 6 8 16 6 3 73 9 10 22 29 15 23 17 39 19 26 28 117 104 108 31 35 17 130 122 135 1 Mg 50 5 1 1 2.5 2.5 372 280 386 175 165 170 1686 1329 1284 18 33 28 230 214 808 28 18 17 13 5 11 158 177 61 31 33 25 10 K 50 100 500 1000 5000* 17 16 24 10 3 8 24 18 17 10 9 15 5 5 6 9 20 28 15 14 22 5 7 6 14 15 20 12 11 21 4 6 7 23 17 27 9 15 17 8+ 2* 13 14 15 21 14 11 18 5* 7* 8* 15 24 15 10 50 100 500 1000 5000* 13 7 17 13 8 5 26 34 33 12 8 3 13 12 7 22 30 31 14 5 13 6 5 6 35 18 36 10 3 7 4 9 6 14 31 18 13 13 8 9 11 8 28 23 22 8 6 9 7* 5 + 9 + 28 35 27 818 883 848 19 31 21 97 109 355 20 14 19 14 18 20 13 21 27 17 22 30 19 19 12 21 17 16 41 30 32 39 33 28 31 27 34 36 33 29 32 24 29 43 39 50 342 321 346 148 112 135 230 280 330 148 122 143 106 117 82 122 137 105 143 114 142 140 151 154 106 89 93 98 121 101 87 117 138 109 78 85 125 126 86 126 132 96 93 97 124 E x p erim en t perform ed on 14 Ju n e 1985. ^Background lawn th in n in g . '^P recip itated a t th is dose le v e l; hand-counted. Table 2 IN VITRO ASSAYS WITH SALMONELLA TYPHIMORIUM COMPOUND T-3752 Experiment Date: 6 June 1985 Compound M etabolic A ctivation Compound Added per P late TA1535 H istid in e R evertants per P late TA1537 TA1538* TA98*_______ TA100 N egative C ontrol DMSO + 50 pi 50 31 37 27 18 14 13 3 10 3 998 16 15 18 20 17 23 160 129 123 30 24 23 38 24 18 136 114 122 P o sitiv e C ontrols Sodium a z id e 9-Am inoacrldine 2-N itrofluorene 2-A nthram ine I--1 - - - + - + 1 50 5 1 1 2.5 2.5 562 586 621 600 666 636 456 307 390 1111 1017 842 675 452 502 24 18 20 23 17 34 173 174 127 202 160 256 109 108 194 306 336 311 33 30 38 13 13 12 214 182 177 51 59 49 Compound T-3752 - 10 pg 42 24 34 9 10 12 21 17 11 15 24 13 117 137 136 - 50 32 33 36 3 10 11 14 17 17 24 30 20 153 179 127 - 100 33 27 45 13 9 4 15 13 8 28 38 26 148 140 150 - 500 35 42 33 6 9 8 19 11 17 23 18 14 140 152 130 -- 1000 31 33 31 6 8 10 7 13 12 10 25 14 143 126 122 - 50001 39 24 31 11 7 8 12 16 15 12 14 17 133 103 130 + 10 16 11 10 13 7 12 18 16 19 25 20 29 174 108 136 + 50 10 13 17 9 7 13 30 17 32 22 18 29 140 108 136 + 100 10 11 20 12 3 17 23 24 36 22 24 23 153 128 146 o + 500 17 16 12 9 7 6 31 32 17 24 27 20 147 138 121 o + 1000 12 15 9 10 12 11 22 22 18 38 29 20 130 128 147 H* + 5000+ 18 18 18 15 11 18 37 35 33 28 17 28 150 101 130 fo fO oo E x p erim en t perform ed on 14 Ju n e 1985. ^ P rec ip itated a t th is dose le v el; hand-counted. Table 3 IN VITRO ASSAYS WITH SACCHAROMYCES CERE VIS IAE D3 COMPOUND T-3752 Experiment Date: 24 May 1985 Compound M etabolic A ctivation Negative C ontrol EMSO + P o sitiv e C ontrols 1 ,2 :3 ,4-Diepoxybutane Sterigm atocystin - + Compound T-3752 -- - - + + + + + Percent C oncentration (w /v) 0.025 0.005 0.005 0.05 0.1 0.5 1.0 5.0 0.05 0.1 0 .5 1.0 5.0 S u rv iv in g C ells per ml (x 10" 7) 7 .3 7.2 7.1 6.8 7.4 7.2 7.1 6.9 7.1 7.6 7.8 6.4 7.2 6.0 8.0 S u rv iv o rs (%) 100 100 97 93 100 99 97 95 97 100 100 89 100 83 100 M itotic R ecom binants per ml (x 10" 3) 4.5 7.5 1331 2 303 9 6 7 11 5 7 9 8 6 7 M itotic R ecom binants p e r 10 ^ S u rv iv o rs 6.2 10 1900 2.9 410 13 8.5 10 15 6.6 9.0 14 11 10 8.8 *A11 c a l c u l a t i o n s a r e e x p re s s e d to two s i g n i f i c a n t f ig u r e s * Table 4 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 COMPOUND T-3752 Experiment Date: 7 June 1985' ON oo H* fv CJ o Compound M etabolic A ctivation Negative C ontrol DMSO + P o sitiv e C ontrols 1 ,2 :3 ,4-Diepoxybutane Sterigm atocy stin - - + Compound T-3752 -- -- + + + + + Percent C oncentration (w /v ) 0.025 0.005 0.005 0.05 0 .1 0.5 1.0 5.0 0.05 0.1 0.5 1.0 5.0 Surviving C ells per ml (x 10" 7) 7.3 6.9 7.3 7.3 7.3 7.5 7.0 6.9 6.9 7.5 7.2 7.6 7.1 7.3 7.7 S u rv iv o rs (Z) 100 100 100 100 100 100 96 95 95 100 100 100 100 100 100 M itotic R ecom binants per ml ( x 10" 3) 8 10 1512 8 434 8 9 13 6 6 6 7 9 11 11 M itotic R ecom binants per 105 ^ S u rv iv o rs 11 14 2100 11 590 11 13 19 8.7 8.0 8.3 9.2 13 15 14 *A11 c a l c u l a t i o n s a r e e x p re s s e d to two s i g n i f i c a n t f i g u r e s .