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AR226-2865 K E. I. du Pont de Nemours and-Company Haskell Laboratory for Toxicology and Industrial Medicine HASKELL LABORATORY REPORT NO. Qfi|-T7 Material Tested: Material Submitted by: Seaford Textile Fibers Department, Haskell No. Other Code: Sample Ready for Testing: 8-3-77 Materials and Methods: The ability of the test sample 1o revert five strains of S a lm o n ella typ h im triw ti from histidine dependence to histidine independence is determined. Strains TA I535 and TA I00 are used to detect base-pair substitution mutations, whereas TA 1537, TA 1538, and TA 98 are used to detect frameshlft mutations.1 The assay Is performed In the presence and the absence of a rat-liver homogenate activation system. In the absence of an activation system, 0.1 ml of a solution of the test sample and approximately 108 bacteria In 0.1 ml are added to 2 ml of top agar (0.6? agar, 0.6? NaCI, 0.05 mM L-hlstldlne, 0.05 mM biotin). These components are mixed and poured on the surface of a plate containing 20 ml of Davis minimal, agar.. To treat In the presence of an activation system, 0.5 ml of S 'S 1254mix Is added to the bajcterIa-test sample-top agar mixture. S*9 Is the 9,000 x g supernatant of liver homogenate from reis given 500 mg of Aroclor /kg five days before sacrifice. The S9 mix contains per ml: 0.3 ml of S*9, 8 mM MgCl2 33 mM'KCI, 5 mM glucose-6-phosphate, 4 mM NADP and 100 mM sodium phosphate (pH 7.4). The Sr9 mix Is added to the bacteria, test sample and top agar. These components are mixed and Immediately poured over the minimal agar plate. The revertant colonies are counted after the plates are incubated at 37 for 48 hr.1 Positive (known mutagens) and negative (solvent) controls are Included in each assay. In assays In which an active, i*. system Is present, an additional negative control (l.e., no F 9 mix) Is Included. ^-Ames, McCann'and Yamasaki, Mutation Res. 3J_: 347-364,. 1975. Company Sanitized. Does nol contain TSCA C8I P. -2- The cytotoxicity of the test sample In the presence and absence of an activation svc+^m TA 1535, is the basis for selecting concentrations to be used In the mutaqenesis measured in strain determine the cytotoxicity Is Identical to the mutagenesis protocol except that l O ^r J- lT r^ h Th S1'01'0001 U5ed +o per plate and a nonllmitlng concentration of histidine is present. Concentrations of t + ^ han 10 bi3Cterii) are used if possible, slightly toxic are selected for the mutagenesis assay. f +es+ samp,e that are nontoxic anc. The data is handled as follows: Data from replicate plates within a ^ of these values from different experiments is determined. The hlqhest averaoe Vexpressed as a multiple of the control v a ' s for the sensitive st?ain(s) w L n ^ t are averaged. The average rev<frtan+s +ha+ is obtained Is numbers of revertants'observed before activity plateaus or decrease at Wh'B - a +es+ samplR 5s active, the a-erage +*linear regression analysis. The slope of the line thus o M ^ n e d u h concentra+ions tested are ubmiited to tof test sample. P 1,06 thUS pb,,i,neti ls to determine the number of revertan ./nmole or Ug ___________________________________ ____________ lc5+y experiment with TA 1535,nontoxic and slightly toxic u:-ortrati,* + tNo evidence of t o x I c I t ^ r a ^ ! o ! ^ t r i i ^ l T ^ h e ep r e L ^ r o f a h +a ^ W a t i o ^ s i S Pai+ +hV " u+a9enesIs experiment. Trial I. highest concentration tested (10,000 pg/plate). An additional h ^ I h ^ n ^ ?,th cy++ox,c,+Y experiment at the activated part of the mutagenesis experiment! Trial T A 2" <,S'0 O p i a t e ) was included in the tratlon was In the range approaching toxicity In an'at+im+ + cy|+oxic,+y experiment demonstrated that this concen- -- - m w -? sr the J h e mutagenesis data are'listed In Tables I and 11 for Trial' I and Tables 111 and IV for Trial 2. Summary; * ------- frequency Is lass t W n ! t W - . t L itTiSl ^ ' "^ . S a ^ r ^ ' ^ a ^ e d . I Notebook No. "MES :DFK:ms E - 15630. P- 13, 21, 71, Report No. 961-77 Date: November 23. 1977 Maureen E. SippeI Molecular Biologist Approved by :----- U f a A * ' David F. Krahn Chief, Molecular -Biology Section Company Sanitized. Does not contain T SCA CB < TABU 1 Trial I H2 O 11,479 -S*9* 2AA Compound Added Wg/plate 2000 II 2000 It 4000 6000 II II 8000 n 10000 it 15000 it 5 ti 10 ft 100 h TA 1535 19 15 18 24 27 23 21 21 386 H2 O 11,479 2AA * s Distilled water (solvent control) = 2-Aminoanthracene (positive control) = Test plate without S-9 and activators = Average of two plates Histidine4- Revertants Per Plate** TA '7 TA 1538 T a 98 " 8 19 40 9 16 19 15 20 30 16 20 25 14 14 26 13 17 25 13 17 32 15 15 26 2225 2897 320 TA 100 |42 144 165 143 157 158 135 163 2146 Company Sanitized. Does not contain T S CA CBi TABLE II MUTAGENIC ACTIVITY OF STRAINS TA I535T 1537, Ti oo n - , ---------- J in salmonella TTPBJMJRIUM 1538, TA 98 AND TA 100 WITHOUT METABOLIC ACTIVATION Trial I H20 11,479 MNNG 9AAc 2NF Compound Added Mg/plate 400 " 800 ' 1200 " 1600 " 2000 2" 50 " 25 " TA 1535 23 20 18 22 22 22 3269 H istid in e + Revertants Per Plate* IA 1537 TA 1538 " TA 98 12 12 33 8 11 . 12 10 9 * 1544 14 13 12 9 9 1453 18 25 21 21 23 2916 T A .I0 0 153 157 154 134 . 144 142 2695 H2O 11,479 MNNG 9AAc 2NF Distilled water (solvent control) 1 c N-Methyl-N'-nltro-N-nItrosoguanIdIne (posittye controI) = 9-ArnInoacrIdI ne (pos111ve controI) = 2-Nitrofluorene (positive control) = Average of two plates . Company Sanitized. Does not contain TSC A CBi TABLE 111 T S g l T a . TA .537, TA 1536. TA Trial 2 Compound Added *r 1 H20 11,479 -S-9* 2A/V 24 ' 4000 2000 . 4000 6000 8000 10000 15000 5 Mg/plate II (I ft ir it 11 ii . 13 15 - . 17 21 15 23 16 , 10 ir 100 VI 116 * 1rHiIs1tJljdl ine+ Revertants IA 1538 Per Plate** TA 98 13 37 39 12 10 6 10 8 .6 4 261 39 37 31 28 39 31 35 1250 24 36 35 34 46 38 40 1166 H2 O 11,479 2AA # * = D i s t i lle d water (solvent co n tro l) 2-AmI noanthracene (positive control) Test plate without S*9 and activatorsAverage of two plates TA 100 162 146 130 130 115 22 129 163 1517 Company Sanitised. Does not contain TSCA CB # TABLE IV MUTAGENIC ACTIVITY OF STRAINS TA 15357 - - 1537, TA l538 TA 98 AND TA " T o Q ^ l T H a i ^ ^ T A B O ^ ^ C T t v A T i o N Trial 2 h2o 11,479 MNNG 9AAc 2NF Compound Added_______ _______ 800 1600 2400 3200 4000 2 50 25 p g/p 1ate vr it it it tv it it ft j a 1535 29 37 37 34 29 30 2352 H !stld tn e + TA 1337 Revertants TA 1538 Per Plate* TA 98 14 23 19 12 21 28 13 24 24 12 16 26 9 20 26 5 22 . 22 522 1332 1614 h2o 11,479 MNNG 9AAc 2NF # P*s^M^^^eiMsolve|v^on+rol N-Me+hyI-N' -nItro-N-nItrosoguanIdIne (p o sitive control) 9-Amlnoacrldtne (p o sitive control) . 2-Nltrofluorene (positive control) Average of two p la te s . TA 100 127 148 155 123 95 66 1086 Company Sanitized. Does not contain T S C A CBI