Document Ed2KvJRQeGDVeOODyqRN3gbdR

ff /} * 2 2 i-O Y $ ? y n d u A b tic d B I O - T E S T XcdxM GJ/yujeA', S n c . 1810 F R O N T A G E R O A D N O RTH BRO O K. ILLINOIS 60062 ORIGINAL REPORT TO 3M COMPANY REVERSE MUTATION STUDIES WITH T -1485 IN FIV E SALM ONELLA STRAINS AND ONE SACCHAROMYCES STRAIN P .O . NO. L P - 003397 -40 0 AUGUST 10. 1976 IBT NO. 8540-09238 CS> 030 --OpT -- ---a(mo CO -- ' pa H? TO ro si1\?Of. CB! 9ri <Atai B I O - T E S T JaJ3>uit&u&6', 3nc. 1810 F R O N T A G E R O A D N O RTH BRO O K, ILLINOIS 60062 A u g u s t 10, 1976 D r. James E. Long M anager, Toxicology Services 3M C om pany 3M C e n te r St. P au l, M innesota 55101 Dear D r. Long: Re: IBT No. 8540-09238 - R ev erse Mutation Studies with T-1485 in Five Salmonella Strains and One Saccharomyces Strain - P.O . No. LP-003397-400 We a r e s u b m ittin g h e r e w i t h o u r l a b o r a t o r y r e p o r t p r e p a r e d connection with the above s tu d y . Very truly yours, JCC: bp J . C . Calandra President 4L V'S KduiifaL I O - T b i JtaboAGi&iiei, REPO RT TO 3M COMPANY REVERSE MUTATION STUDIES WITH T -1485 IN FIV E SALM ONELLA STRAINS AND ONE SACCHAROMYCES STRAIN P . O. NO. L P-0033 97-400 AUGUST 10, 1976 IBT NO. 8540-09238 I. Introduction A sam ple identified as T-1485 was receiv ed from 3M Company for the purpose of conducting re v e rs e m utation stu d ie s. The te s t m a te ria l was evaluated for genetic activity in m icro b ial assay s w ith and without the addition of m am m alian m etabolic activation p re p a ra tio n s. This re p o rt p re se n ts the re s u lts of the investigation. <6 V / 6 JSriditi& ual B ! O - T 5 I lab&xcUiyu&i, 9*tc, II. Sum m ary The te st m aterial, T-1485, was exam ined for m utagenic activity in a s e rie s of in v itro m icro b ial assay s using Salm onella and Sac ch ar om yces indicator organism s. The test m a te ria l was tested directly and in the p r e sence of liv e r m ic ro so m a l enzym e p re p a ra tio n s fro m A ro c lo r-in d u c e d r a ts . D o s e l e v e l s of 0. 25, 0. 5, 5, a n d 50 jug of T - 1 4 8 5 p e r p l a t e w e r e u s e d in both the nonactivation and the activation te sts. Some physiological effect w as o b s e rv e d a t the h ig h e st dose le v e l (50 p g /p la te ). The lo w e r dose levels w ere below toxic levels. The resu lts obtained with the positive control m aterials d em o n strate that the te st system s are functional with known m utagens. T h e r e s u lts of the te s t s c o n d u c te d w ith T -1 4 8 5 , both in th e a b s e n c e an d in th e p re s e n c e of th e r a t liv e r a c tiv a tio n s y s te m , w e re a ll n e g a tiv e . It w as concluded th at T -1485 did not exhibit genetic a ctiv ity in any of the in v itro a ssa y s em ployed in this in v estig atio n . Respectfully subm itted, INDUSTRIAL B IO -TEST LABORATORIES, INC. R eport p rep ared by: G e ra ld L . K e n n e d y ,( jr . , B. S. Section Head, Toxicology R eport approved by: trm # Ht7 y n u /u a i D I U * i C O i jx i& y u il& u & t, y * tc . 3 III. P ro c e d u re A. Indicator M icroorganism s The following stra in s of indicator m icro o rg an ism s w ere used in the investigation: 1. Y e ast S train : S a c c h a ro m y c e s c e r e v is ia e , s tr a in D4 2. B acteria S tra in s: Salm onella typhim urium , strain s T A -1535 T A - 1537 TA-1538 TA-98 T A - 100 B. P re p a ra tio n of L iv er H om ogenate and 9, 000 x g Cell F ra c tio n T h e t i s s u e h o m o g e n a t e a n d 9, 000 x_g_ c e l l f r a c t i o n w e r e p r e p a r e d fro m the liv e rs of adult m ale S p rag u e-D aw ley r a ts . The a n im a ls w e re tr e a te d with 500 m g /k g of A ro c lo r 1254 five days b e fo re k ill. A su fficien t n u m b er of an im als to provide the n e c e s s a ry quantities of liv e r w e re killed by cran ial blow, decapitated, and exsanguinated. The liv er was im m ediately d issected fro m each anim al using asep tic techniques and placed in ic e-co ld 0. 25 M s u c r o s e b u ffe re d w ith T r is a t a pH of 7. 4. Upon c o lle c tio n of th e d e sire d quantity of liv e rs , they w e re w ashed tw ice with fre s h buffered su cro se and com pletely hom ogenized with a m o to r-d riv e n hom ogenizing unit at 4C. The liver hom ogenate obtained fro m this step was centrifuged f o r 20 m in u te s at 9, 000 x in a r e f r i g e r a t e d c e n tr if u g e . T h e s u p e r n a t a n t fro m the centrifuged sam p le w as re ta in e d and fro z e n at -80 C. S am ples from this preparation w ere used for the activation tests. !}i*Atidi'iial B 1O - T 5 T J^aamdcyUei, nc. 4 C. Reaction M ixture The following reactio n m ixture was em ployed in the activation tests : Component Final C oncentration/m l 1. T P N (so d iu m sa lt) 2. Iso citric acid 3. T ris b u ffer, pH 7. 4 4. M gCl2 5. L iv er h om ogenate fra c tio n equivalent to 25 m g of w et tissue 6 mmoles 35 p m oles 28 ixmoles 2 pinole s D. P late T est (O verlay M ethod) A p p ro x im a te ly 10^ c e lls fro m a log p h a se c u ltu re of e ac h in d ic a to r s tra in w e re added to te s t tu b es containing 2. 0 m l of m o lten a g a r su p p lem en ted w ith biotin and a tra c e of h istid in e . F o r n o n activ atio n te s ts , the 4 dose lev els of the te st m a te ria l w ere added to the contents of the a p p ro p riate tubes and p o u re d ov er the s u rfa c e s of s e le c tiv e a g a r p la te s . In a c tiv a tio n te s ts th e 9, 000 x _ t i s s u e s u p e r n a t a n t a n d r e q u i r e d c o f a c t o r s ( r e a c t i o n m i x t u r e ) w e r e added to the overlay tubes. F o u r dose lev els of the te s t chem ical w ere added to the ap p ro p riate tu b es, w hich w ere then m ixed and the contents poured over the su rface of a m in im a l a g a r (selective m edium ) plate and a llo w ed to so lid ify . The p la te s w e r e in c u b a te d fo r 48 to 72 h o u rs a t 3 7 C, and sco red for the num ber of colonies grow ing on each plate. P o sitiv e and solvent controls w ere run with each assay . 0 *//? jKdM&al I U - I S 1 Ja&iez&yU&,, /ne. 5 E. Positive Control Chem icals Positive controls using both directly active positive chem icals and those that require m etabolic activation w ere run with each assay . The following chem icals w ere u sed for positive controls in the n o n activ a tion and activation te sts: Test Chem ical* Solvent P ro b a b le M utagenic Specificity N onactiva tion M ethylnit r os oguani din e (MNNG) 2 -N itro flu o rene (NF) Q u in a c rin e m u s ta r d (QM) W ater or Saline Dim ethylsulfoxide*** W ater or Saline BPS** FS** FS** A ctiv atio n 2 -A nthr am ine (ANTH) 2 -A cetylam inofluorene (AAF) 8 -A m inoquinoline (AMQ) D im ethylnitr os am ine (DMNA) Dim ethylsulfoxide*** Dim ethylsulfoxide*** Dim ethylsulfoxide*** Saline BPS** FS** FS** BPS** * C oncentrations given in R esults Section ** B PS = B a s e -p a ir su b stitu tio n FS = Fram eshift *** P rev io u sly shown to be nonm utagenic jrbV JV H + vi+ u*' u I w " i hm rnJ t UmCUJ<yU2<yii&&, J r tC . 6 IV. R esults A sum m ary of the plate te st resu lts is p resen ted in Table I. The te st m a te ria l was evaluated at a s e rie s of dose levels such th at the h ig h est le v e l (50 p g /p la te ) s h o w e d s o m e p h y s io lo g ic a l e f f e c t. T h e lo w e r d o se levels (0.25, 0 .5 , and 5 pg/plate) w ere below toxic levels. The re su lts obtained with the positive control m aterials dem o n strate th at the te st system s are functional with known m utagens. The resu lts of the te sts conducted with T-1485 w ere all negative. TAB1.It; i T K S T M A T L I U A L : 'J1- 148 4 liuvwrac M utation Studios in F ive Salm onella Strain s end unc S iceh a n u n y c u j S train Test <it Non.tctivulion Sulvent Conlrul (UMSO) Positive Control T-l T -1I T-lli 'l' -lV Activation Solvent Control (UMSO) Positive Control r-i T-ll T in T-JV Tebl Materi al T -1-185 T -l485 1-1485 T - 1-185 _ * v 1-1485 T - 1485 1'- 1485 T - l-185 S u m m a ry of P la te T est ltesiills Dose Level (lll`./1*1 1:) SjKMj 1: N um ber of R everlanls per Plate T i s mu: St r a i n : T A - 1 5 15 T A - 1537 T A - 1 5 3 8 T A - 9 8 T A - 1 0 0 D4* 0. 5 0. 5 5 SO _ * * '( (>.5 0. 5 5 50 -- --- -- -- Rat L i v e r Rut L i v e r Hal L i v e r Rat L i v e r Rat L i v e r Rat L i v e r 45 >11)5 77 78 66 75 54 185 u. l.J. 30 27 7 5 9 5 4 38 0 30 38 4 37 3 O A 0 A 11 >103 5 10 10 14 0 >10* 1) 1 1 0 -12 1 13 66 42 >103 155 46 132 55 46 145 60 46 108 45 48 104 40 36 124 520 21 150 27 146 28 149 25 133 37 44 36 34 35 32 T ry * cmivai'lantd pci1 plata TA- 1535 TA-1537 TA- 1538 T A-98 TA - 100 m MNNG C.'M NK M!' MNNG MNNi; 10 pt/plate 10 pi/plate 100 pg/plate 100 jig/plate 10 pi /plate 10 pi /pin e TA-15 5 T A -1537 TA- 1533 T A -98 TA -100 n1 ANTI 1 AMU AAT A.A!' AN111 DMNA 100 pg/plulc 100 jig/plate 100 pg/plate 100 jig/pi ate 100 jig/plate 100 pinoles/plal e `-erwpycrpzj- 5 oo O -i m -J