Document jyaYdGMa5zXNennwnV4Xwq499

Copies +o; E. I. du Pent de Nemours and-Company Haskell Laboratory for Toxicology and Industrial Medicine Material Submitted by:||UHHI^Textile Fibers Department, "'Sea ford <w Sample Ready for Testing; MUTAGENIC ACTIVITY OR Materials and Methods: The ability of the test sample 1o revert five strains of Salmonella typhuma'i dependence to histldlne Independence Is determined. Strains TA 1535 and TA 100 are used to detect ba mutations, whereas TA 1537, TA 1558, and TA 98 are used to detect frame-ishlft mutations.1 The assay Is performed In the presence and the absence of a rat-liver homogena+e activation sy o* an activation system, O.I ml of a solution of the test sample and approximately l08 bacteria In O.I of top agar (0.6? agar, 0.651 NaCI, 0.05 mM L-hls+ldlne, 0.05 mM b-lo+ln). These components are mixed surface of a plate containing 20 ml of Davis minimal agar.. To treat In the presence of an activation mi-; is added to the bacteria-test sample-top agar mixture. S-9 Is the 9,000 x g supernatant of liver given 500 mg of Aroclor J254/kg five days before sacrifice,. The S-9 mix contains per ml: 0.3 ml of 55 mM-KCI, 5 mM glucose-6-phosphate, 4 mM NADP and 100 mM sodium phosphate (pH 7.4). The S9 mix Is '>es+ sample and top agar. These components are mixed and Immediately poured over the minimal agar pl colonies are counted after the plates are Incubated at 57 for 48 lhr.1 system Posl.+ive (known mu+agens) and negative (solvent) controls are is present, an additional negative control (I.e., no p.9 mix) ^nes, McCann'and Yamasaki, Mutation Res. 5j_; 347-364.. 1975. Included In each essay. is included. In assa Comoany Sanitized. Does AR226-2865 The cyto+oxicl+y of the test sample In the presence and absence of an activation system, as me TA 1555 is the basis for selecting concentrations to be used in the mutagenesis experiment. The prot determine the cy+o+oxiclty Is Identical to the mu+cgenesis protocol except that !03 rather than lO8 ti per plate and a nonlimi+ing concentration of his+idlne Is present. Concentrations of test sample that if possible, slightly toxic are selected for the mutagenesis assay. The data is handled as follows: Data from replicate plates within a single experiment are ave of these values from different experiments Is determined. The highest average number of revertan+s t expressed as a multiple of the control va' s for the sensitive strain(s). When a test sample is act numbers of revertants'observed before activity plateaus or decreases at the various concentrations te linear regression analysis. The slope of the line thus 9b;alned Is used to determine the number of r of test sample. - Ici+y experiment with TA 1535, non+oxic and slightly toxic _Jwere chosen for the nonac+lva+ed part of the mu+agenesis No evid_en_c_e _o_f _t_o_xi_c_lt_y_w_a_s _d_em_o_n_st_ra_ted In the presence of ah activation system In the cy+o+oxicl+y e highest concentration tested (10,000 ug/plate). An additional high concentration (15,000 ug/pla+e) wa activated part of the mu+agenesis experiment. Trial 1. A subsequent cy+o+oxici+y experiment demonstra tration was in the range approaching toxicl+y. In an attempt to more closely approach the toxic range part of the test, additional higher concentrations were included In +he mu+agenesis experiment. Trial The mutagenesis data are listed In Tables 1 and II for Triiil I and Tables III and IV for Tri Summary: {HjHHBHBI||^H^IH^BHIIIHBIIHIHkas tested In Salmonella typkimurium strains TA 1538, TA9^aridl^TUU^^TnT^chemIcaTl^w^n!rragefflc for .these strains of bacteria in the presen activation system In experiments performed by the described protocol., A chemical Is classified as non reversion frequency Is less than two times.+he spontaneous frequency,, and if less than 0.02 rever+an+s ell Notebook No. E-15630, p. 13. 21, 71, "MES:DFK:ms <"- Report No. 961-77 Date: November 23, 1977 Report by: Approved by; -LS^^ Maureen E. SIppe Molecular Biolog David F. Krahn Chief, Molecular Biology Company Sanitized. Doe Trial 1 HzO 11,479 -S-9* 2AA Compound Added lig/pla+e 2000 " 2000 " 4000 " 6000 " 8000 " 10000 " 15000 " 5 " 10 " 100 " TABLi. 1 1538. TA 98 AND TA 100 WITH 1 ABOLIC ACTIVATIO TA 1555 19 15 18 24 27 23 21 21 386 His+ldlne-1- Reavertants Per Plate*4 TA 1537 TA 1538 TA 9 8 19 40 9 16 15 20 16 20 14 14 13 17 13 17 15 15 2225 320 19 30 25 26 25 32 26 2C97 HzO 11,479 2AA * *" * s Distilled xa+er (solvent control) 'fiWHUH--------^tf = 2-Aniinoan+hracene (positive con+roI) = Test plate without S'9 and ac+Ivators = Average o-f two plates ---- Company Sanitized. D TABLE I MUTAGENIC ACTIVITY OF STRAINS TA 1535. TA 1537, TA 1538, TA 98 AND TA 100 WITHOUT METABOLIC ACTIVATIO Trial \\y0 11,479 MNNG 9AAc 2NF Compound Added yg/pla+e 400 " 800 " 1200 1600 " " ... 2000 " 2 " * 50 " /7.>^i " " TA 1555 23 20 18 22 22 22 3269 His+idlne-*- Rever+an+s Per TA 1537 TA 1538------ 12 12 8 11 12 10 9 * 1544 14 13 12 9 9 1455 "2 11,479 MNNG 9AAc 2NF ts DIs+Illed water (solvent control) c N-Methyl-N'-nl+ro-N-nl+rosoguanIdtne (positive control) = 9-AmInoacrIdlne (positive control) * = 2-Ni+rofluorene (positive control) = Average of two plates Company Sanitized. Do TABLE III MUTAGENIC ACTIVITY OFifl STRAINS TA 1555, 1FA 1537, TA 1538, TA 98 flW --------ISNAU4'OSELIA TXP TA 100 WI-m METABOLIC ACTIVATION Trial 2 Compound Added H^O 11.479 -S-9* 2AA- ' ' 4000 2000 . 4000 6000 8000 10000 15000 5 10 100 iig/pla+e ii ti It It 11 11 11 It It 11 TA 1555 24 13 15 . . 17 21 15 23 16 116 HIs+Idln<3"*' Rever+an+s Per Plate TA 1537 TA 1538 TA 9 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 HzO 11,479 2AA * ** = Distilled water (solvent cor1+1-01) -f--------l = 2-Amlnoan+hrace ne (positive con+roI) = Test pla+e without S'9 and ac+lva+ors. = Average of two p1a+es ----1 Company Sanitized. TABLE IV HBHMHMMBHMBIfiN MUTACiENIC ACTIVITY OFJ SALMONELLA TYPRIMU STRAINS TA 1555,,^A^37, TA 15i38, TA 98 AND TA 100 WITHOUT METABOLIC ACTIVATION Trial 2 HzO 11,479 MNNG 9AAc 2NF Compound Added 800 1600 2400 5200 4000 2 50 25 pg/pla+e ii n i ii n ii n TA 1555 29 HIs+Idtna'*' Rever-t^nts Per Plate* TA 1557 TA 1558 TA 98 14 25 19 57 12 57 ' 15 . 54 12 29 9 50 5 2552 . 522 21 24 16 20 22 1552 28 24 26 26 . 22 1614 "2 11,479 MNNG ?AAc 2NF * i ^ M = Distilled rater (solvent c:ontrol) ^^^^IBBIHHHi^B = N-Me+hyl-N''-nI+ro-N-nt+rosoguanIdIne (positive control) = 9-AmlnoacrldIn9 (positive control) a 2-NI+rofluorene (positive control) s Average of two plates Company Sanitized. -