Document 70pKZ831Vd5Rw8K296nRe7bqj

9ruk(JMuLi B I O - T E S T Jltxk&iJ&ugA', Jtic. 1810 F R O N T A G E R O A D N O RTH BRO O K, ILLINOIS 60062 REPORT TO 3M COMPANY REVERSE M UTATION STUDIES WITH T-1485 IN OF INVEE SSAACLCMHOANREOLMLYAC SETSRSATIRNASINAND P . O. NO. L P -003 397 -400 AUGUST 10, 1976 IBT NO. 8540-09238 9nuA0Gl BIO - TES T laJstoJxyuA', Dnc. 1810 F R O N T A G E R O A D N ORTH BR O O K , ILU N O IS 60062 A ugust 10, 1976 DMra.naJgamere, s TEo.xiLcoolnoggy S erv ices 3M Com pany 3M C enter St. Paul, M innesota 55101 Dear D r. Long: Re: IBT No. 8540-09238 - R everse M utation Studies w ith T-1485 in Five Salmonella Strains and One Saccharom yces S train - P .O . No. LP-003397-400 _______________________ We a re su bm ittin g h erew ith o ur la b o ra to ry re p o rt p re p a re d in connection with the above stud y . Very truly yours, JCC: bp J. C. Calandra P re sid e n t jjKdudi'UoL 1 O T b I Jicuso^aiaxiai, Uric. 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-003397-400 AUGUST 10, 1976 IBT NO. 8540-09238 I. Introduction A sam ple identified as T-1485 was received from 3M Company for the purpose of conducting re v erse m utation studies. The test m aterial was evaluated for genetic activity in m icrobial assays with and w ithout the addition of m am m alian m etabolic activation p rep aratio n s. This rep o rt presents the resu lts of the investigation. Jridu/^t'uai S ! O T 5 T JL^botedaniei, 'Jnc. 2 II. Sum m ary The te st m a teria l, T-1485, w as exam ined for m utagenic activity- in a se rie s of in vitro m icrobial assays using Salm onella and S accharom yces indicator organism s. The test m aterial was tested directly and in the p re sence of liver m icrosom al enzyme preparations from A roclor-induced ra ts. D ose lev e ls of 0. 25, 0. 5, 5, and 50 pg of T-1485 p e r p la te w e re u se d in both the nonactivation and the activation tests. Some physiological effect w as o b se rv e d at the h ig h est dose lev el (50 p g /p la te). The low er dose levels w ere below toxic levels. The resu lts obtained w ith the positive control m aterials dem onstrate that the test system s are functional with known m utagens. The re su lts of the te sts conducted w ith T-1485, both in the absence and in the presence of the ra t liv er activation system , w ere all negative. It w as concluded that T-1485 did not exhibit genetic activity in any of the in v itro assay s em ployed in this investigation. R espectfully subm itted, INDUSTRIAL BIO -TEST LABORATORIES, INC. R e p o rt p r e p a r e d by: G e ra ld L . K en n eduya,L^ Jri , B .S . Section Head, Toxicology Report approved by: trm U iuutii/ueu. d i u * i : o i jlauo/uu& uei, 'Jnc. 3 III. P rocedure A. Indicator M icroorganism s The following strain s of indicator m icroorganism s w ere used in the investigation: 1. Y east Strain: Saccharom yces cerevisiae, strain D4 Z. B a cte ria S train s: S alm onella typ h im uriu m , stra in s T A -1535 T A -1537 T A -1538 TT AA --91800 B. P rep aratio n of L iver H om ogenate and 9, 000 x g Cell F raction T he tis s u e h o m o g e n a te a n d 9, 000 x_g_ c e ll fra c tio n w e re p r e p a r e d from the liv ers of adult m ale Sprague - Dawley ra ts. The anim als w ere tre ate d w ith 500 m g/kg of A roclor 1254 five days before kill. A sufficient num ber of anim als to provide the n ecessary quantities of liv er w ere killed by cranial blow, decapitated, and exsanguinated. The liv er was im m ediately d issected from each anim al using asep tic techniques and placed in ice-co ld 0. 25 M su cro se buffered w ith T ris at a pH of 7. 4. Upon collection of the desired quantity of liv e rs, they w ere v/ashed tw ice with fresh buffered sucrose and com pletely hom ogenized w ith a m o tor-driv en hom ogenizing unit at 4C. The liver hom ogenate obtained from this step was centrifuged fo r 20 m in u tes at 9, 000 x in a re fr ig e ra te d c e n trifu g e . The su p e rn a ta n t from the centrifuged sam ple was retained and frozen at -80 C. Sam ples from this preparation w ere used for the activation tests. !)r'Au^t'al B 1O - T 5 5 T J.aluyiaiyei, 9*tc. C. R e a c tio n M ix tu re The following reactio n m ixture was em ployed in the activation tests : C om ponent Final C oncentration/m l 1. TPN (sodium salt) 2. Isocitric acid 3. T ris buffer, pH 7.4 45.. ML igvCelr^ h o m o g e n a te fr a c tio n equivalent to 25 mg of w et tis sue 6 pinoles 35 pm oles 28 pm oles 2 pm oles D. P late T est (O verlay M ethod) A pproxim ately 10^ cells from a log phase culture of each in d icato r strain w ere added to test tubes containing 2. 0 m l of m olten agar supplem ented with biotin and a tra ce of histidine. F or nonactivation tests, the 4 dose levels of the te st m a teria l w ere added to the contents of the app ro p riate tubes and p ou red over the su rfaces of selectiv e ag ar p lates. In activation te s ts the 9, 000 x g_ tis s u e s u p e rn a ta n t and r e q u ir e d c o fa c to rs (re a c tio n m ix tu re ) w e r e added to the overlay tubes. Four dose levels of the test chem ical w ere added to the appropriate tubes, which w ere then m ixed and the contents poured over the surface of a m inim al agar (selective m edium ) plate and allo w ed to so lid ify . The p la te s w ere in cu b ated fo r 48 to 72 h o u rs a t 37 C, and scored for the num ber of colonies growing on each plate. P ositive and solvent controls w ere run w ith each assay. KcUidZfUal 1 O - i b 1 Jiai&vzt&ii&i, *tc. 5 E. Positive C ontrol C hem icals Positive controls using both directly active positive chem icals and those that req u ire m etabolic activation w ere run with each assay . The following chem icals w ere used for positive controls in the nonactiva tion and activation tests: Test N onactiva tion Chem ical* Solvent M ethylnitrosoguanidine W ater or Saline (MNNG) 2 -N itro flu oren e (NF) D im ethylsulfoxide*** Q uinacrin e m u sta rd (QM) W ater o r Saline P ro b a b le M utagenic S p e c ific ity BPS** FS** A ctivation 2 -A nthram ine (ANTH) 2-A cetylam inofluorene (AAF) 8-A m inoquinoline (AMQ) D im ethylnitros am ine (DMNA ) D im ethylsulfoxide*** D im ethyls ulfoxide *** D im ethyls ulfoxide#** Saline BPS** FS** FS** BPS** * C oncentrations given in R esults Section ** BPS = B ase-pair substitution FS = Fram eshift *** P reviously shown to be nonm utagenic W I w - I t. W I J-'MJCyVZityU&i., JHC. 6 IV. R esults A sum m ary of the plate test resu lts is presented in Table I. The test m aterial was evaluated at a series of dose levels such that the highest le v e l (50 p g /p la te) show ed som e p h y sio lo g ical effect. T he lo w er dose levels (0.25, 0.5, and 5 pg/plate) w ere below toxic lev els. The resu lts obtained with the positive control m aterials dem onstrate that the test system s are functional w ith known m utagens. The re su lts of the te sts conducted with T-1485 w ere all negative. Tobt Nonaclivjlion Aclivation TAMM-: l T K S T M A T L l U A L : T- I 1H < Kuv ci' yO M u t a t i o n S t u d i o s i n F i v o S a l m o n e l l a S t r a i n s a n d u m S. u d m r o m y e o s St r a i n SumiiM ry of Plate Test Reduits (iiimp Test Material Dose Level NmnOer of Roverlanls per Plate (|ii'/Til;itO Spot: i es T i s :3iu: St r a i n : T A - 1 5 P i T A - 1547 T A - 1 5 3 8 T A - 9 8 T A - 1 0 0 Solvent Control (l)M.SO) Positive Control T-J T -11 T -111 T-IV Solvent Control (DMO) P o iti vt ; Oonl r o l T-i t - ii t -m T-JV T- l-18b l - 148 b l - l 185 r i-i8s _ '1-1485 T - 1485 T-1485 t - i 185 ** 0. Zb 0. 5 5 50 *** . 5 0. b 5 5 ----- - Rat River Rat l ,i v e r Rat River Rai Li ver Rat Liver Rat Livor 1b 7 1 1 >1(0 >10 J >10^ 77 b b 7 8 9 10 00 b l 7 b t 11 12 113 4Z 46 132 40 145 46 108 18 104 54 8 zo 36 124 0 30 > 1 0 i >1U3 5Z0 60 za 0 21 150 (.Z Z-l ZI 27 146 30 Z7 ZI 28 149 Z7 Z3 zo 25 133 D4* 66 155 55 60 4S 40 37 44 30 34 35 32 * T i y ^ conve r iaut s per plate TA - 1535 T A - 15 37 TA - 1538 T A -98 TA- l00 m MN NO OM NK NK MN NC, MNN< ; 10 p l /p l a te 10 ji 1/ pl .i 1e 100 liy/pljla 00 Uli/plalc 10 p l / p l a t e 10 pl / pla e TA - 15 ib TA - 1537 T A - 15 33 T A -98 TA - 100 1)4 ANTI l AMU AAF AAL ANTI 1 PMN A 100 P t/p la te 0 0 p p*/ p l a t e 100 p g /p lale 100 py/plaLe 00 pg/pla te 100 pnioles /plal e oc VUi ot* `ysr^zrsor'sj- j_ g 03 o !71