Document MGan18oZe6j07qMwL7kLqmQnM

AR226-2760 FOR OU POUT USE ONLY cc; I. E. du Pent da Nemoucs and Company Kaakell Laboratory for Toxicology and Industrial Medicine Elkton Road, Newark, DE 19711 HASKELL LABORATORY REPORT NO. 1072-60 (see. page 4 for composition) Submitted by; Polymer ProductB yai.ningcon Worka Haskel Other l No .: Coda: w_13, A. -J 7& fBU.3J Uaca of Initiation: Dates of Completion; 11/lU/oO 12/^/aU MUTAGEMICI.TY P.VALUATIQa IN SALMONELLA/TYPH1MURIUM INTRODUCTION The purpose of this test is to detenaine whether the test sample causes nutationa in Salmonella typhiiauriiga strains TA 1535, TA 1537, TA 98, and TA lOOo These strains cannot produce hiafcidine because o mutations in the genes coding for enzymes involved in tha synthesis of this essential amino acid. Individual bacteria acquiring a specific nutation in these genea regain tbe ability to synthesize histldine* These revercanca form visible colonies in agar. A comparison of the number of revertants observed in the presence and absence of test sample indicates whether the sample induces mutations in these strains of bacteria. METHODS Plate Incorputdtion Assay - Solids and Nonvolatile Liquids The assay uas performed in the presence and the absence of a rat-liver homogenate activation system similar to the method described by Aaies, et al. (Mutation Res. 31^347-36/t, 1975). Treatments without activation were conducted by adding 0.1 ml -of the solvent or a solution oCgthe test. sample and 0.1 ml of an overnight culture containing approximately 10 bacteria to 2 pi of cop agar (U.6X agar, U.6X MaCl. O.U5 mM L-histidiae, O.U5 mM blotin). These components were mixed and poured on the surface of a plate containing 20 ml of Uavts minimal agar. Treatments with activation were conducted by add^g U.b ail of S-y mix to Che bacteria/test sample/top agar and pourics the mixture onto a minimal agar plate. The S-^ mix contained per ml: U.3 ml of S-y diluted with 0.15 H KCL (1.6 mg oi. irotein), 8 micromoles MgCl, 33 lalcromoles KCi, 5 micromoles Eiucose-6-phosphate, 4 micromoies NADP and tuu micromoles sodium phosphate (from a 0.2 M stock sodium phosphate solution at pH P.^). Company SanHbed. Doe. not conte-n TSC* CB1 The S-9 was the 9,000 x g supernatant of liver homogenace (I gm wet liver;3 ml 0.15 M KC1). The livers were obtained from 8 to 9 week old male Charles River CD rats given 500 mg of Aroclor 1254/kg five days before aacrlflce. The revercact colonies were counted after the places were Incubated at 37 "C for ^ti houra. Positive (known xnutagens) and negative (solvent) controls were Included In all assays. The cytotoxicity of the test sample In the presence and absence of an activation system, as measured In strain TA 1535, was the basis for selecting concentrations for the mutagenesis experiments The protocol used to determine the cycotoxicity was Identical Co the mutagenesis protocol except that 10 rather than 10 bacteria were used per pi-ata and excess histidine was present. Concentrations of test sample that were noncoxic and, if possible, sUphtly toxic .were selected for the mutageneais asb^y. The concentrations of test sample per ml of treatment medium per plate were calculated with the following assumptions; liquids - the solvent and test sample volumes were additive; solids - the addition of test sample did not change the volume of solvent* Spot Test in a Closed System The plate incorporation assay may not be the best method to test liquid samples that vaporize appreciably when this protocol is followed. A spot sesc in a close.4 system is performed on all nonaqueous liquids to aid in identifying samples with volatile mutagen.^ components. These samples are tested by differenC protocols. ,, Experiments without activation were performed by adding 10 bacteria (in 0.1 t&l) to 2.0 nil of standard mutsgenesis top agsr, sixing iiiiiae.iiai.ely, and pouring on a Davis minimal agar place. Experiments with activation were performed In the same aannt-r except that 0.5 ml of S-9 mix was also added before mixing. All components used for this assay were identical to the ones used in the plate incorporation assay. A sterile disk saturated with test compound was placed in the center of one of two replicate places* The place with the disk was inverted and the other place was stacked on top of it. Both plates were sealed in a plastic bag and incubated for 4a hours at 37C. Use of Data: 1) A Plate Incorporation Assay - Solids and Nonvolar.ile Liquids is performed if the spot test is negative. 2) If the spot test is positive, a limited Place Incorporation Assay - Solids and nonvolatile Liquids is performed with only the strains and conditions in which mutagenic activity was suggested in the spot. test. A complete assay ;s performed if mutagenic activity is observed in the limited assay. 3) If the limited assay is negative, the test sample is assayed according to the plate assay protocols for testing gases and volatile liquids. CoBgiaaySanlffzetf. Dow not conlain TSCA CBI Statistical Analyota^ The data points (number of revertante/plata, x) were transformed prior to " analysis using the power tranefonaation y " x . Two analyaes were performed. In the first analysis the response observed at each concentration waa compared to the control by a ftest of significance: to detenaine which dose levels produced significant increases in outaiion frequency. In the second analysis the significance of the -iose-responae relati nahip was tested. Linear, quadratic, and higher order dose-reBponBe effects were tested in an F-teat of significance. In addition, an anaiyaia was conducted to determine whether the dose-response relationship u&e, different in different trials. The following guidelines are used co claasify a.test sample as a mutageo oc nonmutagen in the asaay. A test sample is classified as a nonmucagen when: A. The probability ia greater than 0.05 that the numbers of revertant8 at each of the teat sample concentrations studied are not greater than the number of revertants in the solvent control. &HD B. The probability is greater thar 0.05 that there ia not a positive correlation between the numbers of revertants and increasing 0 concentrations of the te&t sample. A test sample: is classified as a mutagen when: A. The probability ia less than O.Ol chat the numbers o ravertants at one or more of the test sample concentrations studied are not greater than the number of revertant8 in the solvent control. AMD B. The probability ia less than 0.01 that there Is not a positive .-urrelatton between the number of revertanta and increasing concentrations of the teac sample. Test samples that cannot be classified by the above criteria are dealt with on a case~by~ca8e basis. A detailed description of the statistical methods is available (J. D. Irr and R. D, Snee. 1979. Statis:icat Evaluation of Mutageniclty in the CUO/UGPRT System. In Banbury Report 2-Mananalian Cell Mucagenesisi The^ Maturation of Teat Systema (ed. A. W. Hale, J. P. O'Neill, and V. K. rfcElheny), p. 263. Cold Spring Harbor Laboratory, New York. c Company Sanftlzed. Doe. nol conlam TiCA CBl ABBREVIATIONS AMD DEF1NITIOMS Positive Controls: 2AA, 2-antnoanthracene (DMSO)*; 9AAc, 9-aninoacridine (DMSO)*; MNKG, H-Tnethyl-N'-nicro-H-nitrosoguaoldine (DMSO)*; 2KF, 2-nitrofluorene (DM30)*. *aolvent (DMSO - dimethylsulfoxide). Solvent for Test Chemicals echanol. Solvent was present where the concentration oC test chemical is listed as "0"* Symbols; (T) Toxiclty indicated by decreased density of the background lawn R E S U L T S T -- -- f t HT tha initial cytolaxiclcy experiment with strain TA 1535,fja8 tested ac concentrations up to 10,000 micrograms/plate. No tonicity was observed. Higher concentrations were noe tested in the inutagenesis experiments. A spot tese in a closed system was performed and no indication of III mutagenic activity was observed. The mutagenesis data are listed in Tables I, II, TA 1535, TA 1537, TA 98 and TA 100, respactively. and IV for strains Statistical analysis of the revertant frequencies obtained in the prasance of test chemical showed no significant increasea over control frequencies and no significant positive linear dose responses in all strains in either the presence or absence of the activation system. Therefore, the rest chemical was not mufcageBic CONCLUS1QMS CUD^3 tested in Salmonella typhimurium straine TA 1535, TA i;>l7, TA 9o and tA^ulU in the presence and absence of the activation system according to the protocol described in METHODS. This teat sample was not mucagenic for these strains of bacteria. Report Approved by: Q-^Q^jL^ '^(g^^LZ^ Aprn L. Hforst Biologist . ,. by; ^/^/^/ f 'David Manager, rk-.-tApPi . Kra-hn ^^Genetic Toxicology Division Study Director Fepurc No. 1072-dU Date Issued: January i, lyol Company Sanitized. Does not contain TSCA CBI ilNNG 2694, 3019 2243, 3094 ^0 HTT" ACTIVAT10K Compound 13,783 -- 1 i Concentration (^1 plate) 0 100 500 1000 5000 10,000 10 2AA Reverta. '.s Per Trial 1 73, 37 44, 45 44, 54 46, 45 46, 54 38, 56 510, 567 Plate^ (Duplicate Plates) Trial 2 35, 29 41, 22 27, 27 35, 28 31, 32 25, 40 798, 747 C - 5 - Company Sanded. Does not TABLE II. nMuIliTA&ur'6Bt'MLTUr' AAP^TJT.lUVTlT.iVi IN SALMONELLA TYPHIML'RIUM STRAIN TA 1537 WITHOUT ACTIVATION Compound 13,783 ,, n 9AAc Concentration ^S,.UuBK; I/JoAltaI t.eC ) / 0 100 500 1000 5000 10,000 50 Revertants Per Trial 1 17, 7 10. 7 .2. 4 7. 5 8., 11 15. 11 , Plate (Duplicate Plates) Trial 2 ^,i0, 13 8 10, ' 5. 7 6. 7 5, 7 1081, 944 1283, 1591 WITH ACTIVATION Compound l^,7tfj * > : 2AA Concentration OUK/plate) 0 1UO 500 1000 3000 10,000 10 Revercants Per Trial 1 11. 4 4 > q' 12 , ? 16 , 11 , 9 11 . 8 Plate (Duplicate Plates) Trial 2 n, 6 , 8' B . i ^, 6 , 8,, 5 9,, 12 9,, 12 440, 720 583, 821 H-13,783 ^----------] 6 - ----------^,^-------,-nTSC.OB, I I I TABLE TYPHIMURIUM STRAIN TA 98 MUTACENIC ACTIVITY IN SALMONELLA WITHOUT ACTIVATION Compound Concentration Qug/plate) Revertanta Per Plate fn..nlicate Plategj TrialJ, Trial 13,783 24,- 26 25 2ai6, 2647 2895, 220 2NF c WITH ACTIVmOM CoiSit!i0und 13.7'83 I i i r i M Co ncent-racion Oiig/place) 0 100 500 1000 5000 10,000 10 2AA Revertants Per Plate (Duplicate Plates) 2 Trial 1 Trial 46, 43 48, 35 36. 51 48, 37 30. 43 .42, 63 51, 42 53, 50 44, 43 41. 39 48, 37 45, 44 3619, 3648 1838. 2395 C -7 Company SanUU.-.O."-""""^06' TABLE IV MUTAGEMIC ACTIVITY IH SALMONELLA TYPUIMURIUM STRAIN TA 100 WITHOUT ACTIVATlOti Compound 13,73 t if MNNG Concentration Cug/plate) 0 100 500 1000 5000 10,000 4 Revertants Per Plate Trial 1 121, 116 146. 161 103, 121 115, 156 115, 100 120, 121 2493, 2564 (duplicate Plates) Trial 2 114, 101, as, 85, 92, 49 (T), 103 PO 100 117 90 86 (T) 3982, 3501 WITH ACTIVATION Compound 13.7B3 * ii Concentracion (jug/plate) 0 100 5UO 1000 3UUU 10,000 2AA Revercants Per Place Trial 1 135, l5tf 144, 147 i4a, 171 132, 157 14U, 155 143, 168 3125, 316a (Duplicate. Plates) Trial 2 163 154 147 168' 156 152 156 149 12y 132 144 131 2491. 2565 Coffipaa?'