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NOTOX Safety& Environmental Research LV. REPORT EVALUATION OF THE MUTAGENIC ACTIVITY OF T-5874 IN AN IN VITRO MAMMALIAN CELL GENE MUTATION TEST WITH L5178Y MOUSE LYMPHOMA CELLS (WITH INDEPENDENT REPEAT) NOTOX Project 115921 NOTOX Substance 38187 page 1 of 22 - C-,EIVED -9 1994 TOAICULOGY Hambakenwtrte-tWeg 1 P.O. 8ox 3476, S203 DL 's-H*'rlgtyl%pw:wpm The Netherlands Tel--07'4- 41 9S 7S fole 67-4 Al AS 43 REPORT EVALUATION OF THE MUTAGENIC ACTIVITY OF T-5874 IN AN IN VITRO MAMMALIAN CELL GENE MUTATION TEST WITH L5178Y MOUSE LYMPHOMA CELLS (WITH INDEPENDENT REPEAT) NOTOX Project 115921 NOTOX Substance 38187 page 1 of 22 - RECEIVI-=-D N 0 @,'-9 1994 TOAIC;OLOGY T -5874 STATEMENT OF GLP COMPLIANCE NOTOX Project 115921 NOTOX B.V., Is-Hertogenbosch, The Netherlands The study described in this report was conducted in compliance with the most recent edition of: The OECD Principles of Good Laboratory Practice which are essentially in conformity with: The United States Food and Drug Administration. Title 21 Code of Federal Regulations Part 58. The United States Environmental Protection Agency (FIFRA). Title 40 Code of Federal Regulations Part 160. The United States Environmental Protection Agency (TSCA). Title 40 Code of Federal Regulations Part 792. With the exception that the stability of the test substance in the vehicle was unknown Study Director Ing. E.J. van de Waart Date: page 2 T-5874 QUALITY ASSURANCE STATEMENT NOTOX Project 115921 NOTOX B.v., Is-Hertogenbosch, The Netherlands. study procedures were subject to periodic inspections and general non study specific processes were also inspected at periodic intervals. This report was audited by the NOTOX Quality Assurance Unit and the methods and results accurately reflect the raw data. DATES OF QAU INSPECTIONS/ AUDITS January 17, 1994 February 10, 1994 February 23, 1994 March 28, 1994 REPORTING DATES January 17, 1994 February 14, 1994 February 23, 1994 March 28, 1994 Quality Assurance Manager C.J. Mitchell B.Sc. ..J,4v@@. j@@iv Date: L, -0-iLt page 3 - T-5874 REPORT APPROVAL STUDY DIRECTOR: NOTOX Project 115921 Ing. E.J. van de Waart Date MANAGEMENT: Dr. I.C. Enninga Technical Director ...................... Date 22Cc>> IoOtt@++ I'1cCl3I4C934 page 4 - T-5874 PREFACE Sponsor Study Monitor Testing Facility Study Director Technical Coordinator Study Plan NOTOX Project 115921 3M Belgium Chemical EBC Canadastraat 11 B-2070 ZWIJNDRECHT Belgium Mr. R.H. Cox NOTOX B.V. Hambakenwetering 3 5231 DD Is-Hertogenbosch The Netherlands Ing. E.J. van de Waart C.M. Verspeek Start : January 18, 1994 Completed : February 14, 1994 TEST SUBSTANCE Identification Description Batch Purity Specific Gravity Instructionsfor test substance storage Stability under storage conditions Expiry date Stable for at least 4 hours in vehicle T-5874 Cream solid 2334 100% 1.7 At room temperature in the dark Stable January 01, 1996 Water no Dimethylsulphoxide: not indicated VEHICLE The test substance was dissolved in dimethylsulphoxideof spectroscopic quality (Merck). Test substance concentrations were prepared directly prior to use. The final concentrationof the solvent in the culture medium amounted to 0.8% (v/v). page 5 - T-5874 NOTOX Project 115921 GUIDELINES The study procedures described in this report were based on the following guidelines: Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 476: ugenetic Toxicology: In Vitro Mammalian Cell Gene Mutation Testsu, (adopted April 4, 1984). European Economic Community (EEC), Directive 87/302/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; "Other Effects-Mutagenicity: In Vitro Mammalian Cell Gene Mutation Test". EEC Publication no. L133 (adopted May 30, 1988). ARCHIVING NOTOX B.V. will archive the following data for at least 10 years: protocol, report, test article reference sample, all specimens and raw data. OBJECTIVE Purpose of the study The objective of this study was to evaluate the test substance for its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells. The assay was conducted in the absence and presence of a metabolic system (S9-mix). The TK mutational system detected base pair mutations, frame shift mutations and small deletions. Justification for selection of the test system L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions. Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). TK deficient cells can not incorporate the analogue into its phosphorylated derivative (nucleotide); the nucleotides needed for cellular metabolism are obtained solely from de novo synthesis. In the presence of TK, TFT is converted into nucleotides, which are lethal to the cells. Thus, cells which will survive in culture medium containing TFT are mutated, either spontaneously or by the action of the test substance, giving rise to a TK-deficient phenotype. A test article which induces a positive response in this assay is presumed to be a potential mammalian cell mutagen. page 6 - cn CD 0 Cl) C:) 0 C+ C:) U3 < CD co CT) c7L r+ co CD OL r) C+ C= 3 Cl) a Cl) C3- c: C-) r*) co rn riC= ;o (D rTi 0 7-) OL C-+, 0 :3 0) U) r)C-) OL (D C CL -I - 0 - 1: C+ -o rt,a c: a) -.. -1 to 0 CD =3 -J 0 CD to ic =T a a -M CD 0 CL C:L =r -. 0 c :E E3 23 W CL) w in 0 (/)0 r+. c =3 " oc (n U3 a)-o -. a) :3 - C/) (1)co C+ --ba CD 0 (1)C3L 0 CT a ro-0 @ M (D -t Cl) cl CL o o tn cn- c CD -. W to w r+ m r) rt, :1> CO -. C(3 < 0) a Cl) -1 m C3L a = CD 0 C CL r-r9 -. c :r_ E3 =r -. 0 (=I0 0 -- =r :3 --b (A C) 0 0 0 Cf) c+ m 0) 0 C3L =) CL :3 0 CD -t 01" CL r+l rt, 0 ::r 0 CO 0) a 0-0 co 8pp,-@ mm =r C+ a co cr co - a) 0 CL a --ICL 10 -n --I r+l co (+ 0m CC3 0 c: r+ CT = 0) o -- SO a co ll) U) C: C3L t@)-0 -11 -rl l@- 0 C:) a0 CD 0 CL E3 @. -0 C: =-) CD r+l :C CD -o L" a @ 4r+ 0 0 X co =r cz =.3 0 r_ co r_ rt- U3 0) =) C+ a @ :5 r+l -") a OL Cl)& m CL =F -0 1< -7 co :iE E3 CO M :3 ". -A. U, @. C+ CL -0 0 Co 0 r+l ciD Mr -- C: :3 E3 CD C:) 0 11) 0 =) :3 =T CL (n CA 0 =r (n CD Co -0 CD '0 0 C3. rt.tn x a m OL) a a --3 :3 --b ---c r+l 0 23 ::r CO Cr 0 IE -0 n c o m n a) t-n -t 9 --)CD t,) 0 0 -t)-0 CD -4) c -1 CD CD CT 0 CO n n 00 CD rt, X a) C+ E3 C).CD -0 [37 Q) (1)-0 =3 0 CL @ 0 :E 0 tn CD C am @. C+ Ct) rt.--jCl 77 91)D CD M =r r+ CD rt- c ri--o 0) 0 r%) -1 0 r+,W c: 1< 0m -li=r @ a r+- CT 0 c c-+-o a a =r CD r-)9 C: =r :r (D M -. -) 0 cl 0 O@ CD a = -. c- X C-) (1) rric m co OL) a -u a 0 4 0 rn C+ C3. (D En IE x 0 @. @ . @. (/) C)Lin c: rt-C: CD -0 33 LI) =r c)-0 :3 ::r- 0 to " (n m 3E c: C+ -- -) C-) CD c+.0) o c+ m =) m 1< a@ -M (1)0 MM 0 C+ X 0C 0 =) C U) rt-Cl) E3 n gk) Cl) =@3. :CIOE CO 0 C') n Cl m to CD -i :3 tn =r ct co 0 0 77 r) r+.C -3 c+ CD c (1) " CD tn -)0 V) 0 a) c 0 0 m :3 0) (D 0 C+ =r =T CD CD 0 (n 7;z, m CD u0 CD -C")I) " (A a 2E 4< CD 0 -) 0 CD -o @ En 91) rt, (/0) E3 -3 OL) CD c3L 0 0 :3 ti) CL 0 =3 =) rtI 0 CC3 m @o 0 C') w r- r- in 0 M 0 (n C)C=) rt, M -3 0 CT :3 %0 11) =3 -i c 0 m co co (-Im, oo tn " - -. H Cl)(+ DC, -4 0) I E3 C+ t@a c0 m 0 :)Cl) %D o ri. c: 91)1< cn riCO -.-D 0 @%< fl) CD :3 r) rt.CL U) -I C:L - 0 CD CD 0 0 a) a cn 0 =3 -.0 c+ =r :3 CD c@ cr < C 1< m a) -0 a) cl L< :T c+ c cn co U) CL co 0 33 [k) @.'O 0 -) -0 C') -4)r) 9 &m m 91) $I) =r o m -t r+ < 09 M CD 0 o o m E3 -o -- M-0 (D -4 -1 -7 =r C3L C 0) @X - -% rt-0 (D C+ -3 - 0) :r a =) 0) C+ -- cn Li %D CO 0) - C(3 0 (A CO CD r)co -n -q 0 --4:) :3 X C tn -. co ::rM C+ c: tn cn cn r+l 12) =) r+l ci3 -0. @.4 0 0 cn %D -0 -4 m #,n ;a & 0 - Ct) C: -J. C#) -3 -b CD cl0 Cf) co cn 0 CD M -1 & 0 ;a =1 =r -t 12) r- co m u %.n -3 (+ @. @- x --j=r 0) @j 12) 0) m rt OD cn :3 CL 0 tn rrir+-;t m =r CD C-) CD 0 0 (M CD r) 0N m c m :3 c CL -c CD rt-ci m co Ef) c r+ co co rn > 0 rn T-5874 NOTOX Project 115921 Environmental conditions All incubations were carried out in a humid atmosphere (80-95%) containing 5% C02 in air in the dark at 370C. The temperature and C02percentage were monitored during the experiment. REFERENCE SUBSTANCES Neqative control: The vehicle of the test article. Positive controls: Without metabolic activation (-S9-mix)@.* Ethylmethanesulphonate (E S; CAS no. 62-50-0; purity 98%; Janssen Chimica) (2 mM) was used. EMS causes direct alkylation of DNA. With metabolic activation +S9-m Dimethyinitrosamine (DMN; CAS-no. 62-75-9, purity 99%, Janssen Chimica) (0.5 mM) was used. DMN had to be activated by microsomal enzymes present in the S9-mix, resulting in a methyldiazonium ion which could react with cellular DNA. Solvents for Reference Substances Hank's balancee salt solution without calcium and magnesium. Solutions of reference substances were prepared immediately before use. METABOLIC ACTIVATION SYSTEM Preparation of S9-homo ena e Rat liver microsomal enzymes were routinely prepared from adult male Wistar or Sprague Dawley rats, which were obtained from BRL, Switzerland. The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP'S. The rats were injected intraperitoneally with a solution (20, % w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (DOC) sterile 0.1 k sodium phosphate buffer (pH 7.4) containing 0.1 mm Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-1960C). page 8 T-5874 NOTOX Project 115921 Ames, B.N., Mc Cann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Res., 31, 347-364. preparation of S9-mix S9-mix was prepared immediately before use and kept on ice during the test. S9-mix contained per ml: 1.02 mg MgC12.6H20; 2.46 mg KCI; 1.7 mg glucose-6phosphate; 3.4 mg NADP; 4 umol HEPES and 0.5 ml S9. The above solutions were mixed and filter (0.22 pm)-sterilized (apart from the S9-fraction,. which was added after filter-sterilization of the S9-mix components). Metabolic activation was achieved by adding 0.2 ml liver S9-mix to each ml of cell suspension. EXPERIMENTAL PROCEDURE Selection of Dose Levels/Cytotoxicity Test Prior to the actual mutagenicity test cytotoxicity data were obtained by treating 6 x 106 cells, suspended in 6 ml of F-10 medium buffered with 20 mM HEPES, in the absence of serum in a sterile 30 ml centrifuge tube with a range of test substance concentrations in approximately half log steps, both in the absence and presence of S9-mix. The centrifuge tubes were rotated for 3 h on a roller mixer at 370C. After the exposure, the cells were separated from the treatment solutions by 3 centrifugation steps (115 g, 8 min), each followed by removal of the supernatant and resuspension of the cells, twice in Hank's balanced salt solution and finally in F-10 medium. The cells in the final suspension were counted in an "Improved Neubauer" haemocytometer. Relative cytotaxicity, expressed as the reduction after approximately 24 h and 48 h growth compared to nontreated control cells, was used to determine a suitable concentration range (4 doses) of the test substance to be applied to cultures prepared for mutagenicity testing. In case the test substance was not toxic and/or difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium. In general concentrationslexceeding 5 mg/ml were not tested. Cleansing Prior to mutagenicity (and cytotoxicitx) testing, the cells were grown for 1 day in culture medium containing 10- M hypoxanthine, 2 x 10,7 M aminopterin and 1.6 x 10-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on medium containing hypoxanthine and thymidine only. After this period cells were returned to normal medium at least for 1 day before starting the experiment. - page 9 - T-5874 NOTOX Project 115921 Mutagenicity test* The test substance was tested both with and without S9-mix in two independent experiments. 6 x 106 cells (106/ml), or 12 x 106 cells (106/ml) for test substance concentrations expected to be strongly toxic, for -each selected dose cells were exposed for 3 h to the test substance in HEPESbuffered Ham's F-10 medium without serum. For this purpose the cell suspensions were placed in 30 ml centrifuge tubes on a roller mixer at 370C. Solvent and positive controls were included. After exposure, cells were washed twice with HBSS, counted and seeded in F-10 culture medium for expression of the mutant phenotype. The cultures were subcultured at least every other day in order to maintain log phase growth. The expression period for TFT-resistant mutants was 3 days. Immediately after exposure to the test substance 3 x 200 cells of each dose were plated into P90 petri dishes containing 15 ml cloning medium to determine cell survival (cloning efficiency). The cell survival was counted after 10-14 days with the Artek colony counter or the naked eye. Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown, M.M.M., 1979, Validation and characterization of the L5178Y/TK Mouse lymphoma mutagen assay system, Mutation Res., 59, 61-108. Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. and Salsburg, D.S., 1980, Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation, Mutation Res., 72, 447-474. Jotz, i@.-and Mitchell, A.D., 1981, Effects of 20 coded chemicals on the forward mutation frequency at the thymidine kinase locus in L5178Y mouse lymphoma cells. In: Evaluation of short-term tests of carcinogens. F.J. de Serres and J. Ashby (Eds.), Elsevier-North Holland. Van der Hoeven, J.C.M., Bruggeman, I.M. and Debets, F.M.H., 1984, Genotoxicity of quercetin in cultured mammalian cells, Mutation Res., 136, 9-21. Clive, D., Caspary, W., Kirby, P.E., Krehl, R., Moore, M., Mayo, J. and Oberly, T.J., 1987, Guide for performing the mouse lymphoma assay for mammalian cell mutagenicity, Mutation Res., 189, 143-156. Mutant selection After the expression period a total number of 1.5 x 106 cells were plated in ten P90 petri dishes, each containing 15 ml selective medium (TFTselection). TK-deficient mutants formed microcolonies in 10-17 days, which were counted with the naked eye. Cloning efficiencies at the time of mutant selection were determined as described above. The mutant frequency was expressed as the number of mutants per 105 surviving cells. page 10 T-5874 NOTOX Project 115921 ACCEPTABILITY OF ASSAY A mutation assay was considered acceptable if it met the following criteria: a) The absolute cloning efficiency of the solvent controls was > 50%. b) At least three of the four doses of the test substance had an acceptable number of surviving cells (106) analysed for expression of the TK mutation. c) The spontaneous mutant frequency in the untreated or solvent control was < 5 per 105 clonable cells. d) The positive controls (ethylmethanesulfonateand dimethylnitrosamine) induced significant (at least 2-fold) increasesin the mutant frequencies. DATA EVALUATION AND STATISTICAL PROCEDURES No formal hypothesis testing was done. A test substance was considered positive (mutagenic) in the mutation assay if: a) It induced at least a 2-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner; and b) The results were reproduciblein an independently repeated test. A test substance was considered negative (not mutagenic) in the mutation assay if: a) None of the tested concentrations showed a mutant frequency at least twice that of the solvent control. b) The results were confirmed in an independently repeated test. The preceding criteria were not absolute and other modifying factors might enter into the final evaluationdecision. - page 11 - T-5874 NOTOX Project 115921 RESULTS CYTOTOXICITY TEST/DOSAGE SELECTION Preliminary solubility tests indicated that T-5874 was soluble in DMSO at 120 mg/ml but precipitated in the exposition medium at concentrations of 333 vg/ml and upwards. Table 1 shows the results of the preliminary cytotoxicity test with T-5874 in the presence and absence of a metabolic activation system (S9-mix). In the absence of S9-mix T-5874 inhibited the growth of the lymphoma cells in suspension at a test substance concentration of 100 vg/ml by 58% and at 333 jig/mlby 81%. In the presence of S9-mix the test substance inhibited the growth of the lymphoma cells in suspension at 333 ug/ml by 44%. MUTAGENICITY TEST Based on the results of the cytotoxicity test (Table 1), the following dose range was selected for mutagenicity testing: Without S9-mix: 10 to 333 ug/ml culture medium With S9-mix : 10 to 333 pg/ml culture medium EMS (2 mM) was used as a positive control in the test without metabolic activation, whereas DMN, (0.5 mM) served as a positive control for the assay with metabolic activation. Tables 2 and 3 show the percentages of cell survival and the mutant frequencies for various concentrations of T-5874. Individual colony counts of cloning and selective plates, and cell counts during subculturing are listed in Tables 4-9 of the appendix. In the absence of S9-mix the test substance induced a slight (2.3-fold) increase in the mutant frequency in experiment 1. In experiment 2 a slight, less than 2-fold increase in the mutant frequency was observed. However, the increase in the mutant frequency was not reproducible (not 2fold in the second experiment) and not dose related. Therefore, the test substance is considered not mutagenic in the absence of S9-mix. In the presence of S9-mix the test substance induced no significant increase in the mutant frequency in both independent experiments. The spontaneous mutant frequencies in the solvent-treated control cultures were within our historical control data range (2.0 + 1.2 in the absence of S9-mix and 1.4 + 0.8 in the presence of S9-mix; indicated are means + S.D. for n=40 and 41 respectively). Mutant frequencies induced by positive control chemicals were increased by 5- to 7-fold for EMS and by 9- to 10-fold for DMN. It was therefore concluded that the test conditions were optimal and that the metabolic activation system (S9-mix) functioned properly. CONCLUSION In conclusion, T-5874 is not mutagenic in the TK mutation test system under the experimental conditions described in this report. T-5874 NOTOX Project 115921 TABLE 1 PRELIMINARY CELLS CYTOTOXICITY DETERMINATION IN L5178Y MOUSE LYMPHOMA Dose (pg/ml) Cells/ml (x 105) After After After 0h 24 h 48 h Suspension growth Total % of control Without metabolic activation (-s9-mix) Solvent control 5.9 6.3 4.5 65.3 100 1 6.2 5.1 4.7 58.1 89 3.3 6.1 4.9 5.4 63.0 96 10 4.8 4.2 4.4 34.7 53 33 4.9 4.8 5.1 46.9 72 100 5.2 3.1 4.4 27.7 42 3333 5.5 1.01 3.7 12.7 19 ------------------------------------------------------------------------- Solvent control 6.5 1 6.4 3.3 6.4 10 6.0 33 6.3 100 6.2 333 3 5.5 With metabolic activation (+S9 mix) 6.0 6.4 97.5 100 6.0 6.7 100.5 103 6.6 6.1 100.7 103 5.9 6.1 84.4 87 7.4 5.9 107.4 110 6.2 5.7 85.6 88 5.1 5.0 54.8 56 Solvent control = DMSO Subculture = 1.6 x 105 cells/ml No subculture 2 Total growth = Cell count after 0 h x (24 h cells/ml) x (48 h cells/ml) cells subcultured cells-subcultured directly after after 24 h treatment 3 Test substance precipitated slightly in the exposition medium page 13 T-5874 NOTOX Project 115921 TABLE 2 CYTOTOXIC AND MUTAGENIC RESPONSE OF T-5874 IN THE MOUSE LYMPHOMA L5178Y TEST SYSTEM EXPERIMENT 1 DOSE (ug/ml) C.E. AT DAY 0 (% OF CONTROL) C.E. AT DAY 3 (ABSOLUTE %) MEAN NO. OF MUTANTS PER PLATE MUTATION FREQUENCY x 105. Without metabolic activation (-S9-mix) Solvent control 100 60 10 102 61 33 103 60 100 90 62 3331 70 77 2.1 2.3 2.4 2.6 3.4 3.8 4.8 5.2 1.8 1.6 2 mM EMS 129 60 14.2 15.9 -------------------------------------------------------------------------- With metabolic activation (+S9-mix) Solvent control 100 58 10 83 57 33 94 59 100 92 59 3331 61 83 0.5 mM DMN 30 38 1.8 1.3 0.9 0.5 2.6 11.3 2.1 1.5 1.0 0.6 2.1 19.8 C.E. = Cloning Efficiency Solvent control = DMSO EMS = Ethylmethanesulphonate DMN = Dimethylnitrosamine Test substance precipitated slightly in the exposition medium page 14 T-5874 NOTOX Project 115921 TABLE 3 CYTOTOXIC AND MUTAGENIC RESPONSE OF T-5874 IN THE MOUSE LYMPHOMA L5178Y TEST SYSTEM EXPERIMENT 2 DOSE (ug/ml) C.E. AT DAY 0 (% OF CONTROL) C.E. AT DAY 3 (ABSOLUTE %) MEAN NO. OF MUTANTS PER PLATE MUTATION FREQUENCY x 105 Without metabolic activation (-S9-mix) Solvent control 100 85 10 95 98 33 106 99 100 59 94 3331. 65 97 4.2 3.3 5.8 4.0 3.7 2.5 6.1 4.3 5.2 3.6 2 mM EMS 94 91 23.2 17.1 -------------------------------------------------------------------------- With metabolic activation (+S9-mix) Solvent control 100 103 10 82 78 33 92 100 100 96 73 3331 91 101 0.5 mM DMN 41 46 3.4 3.2 2.4 1.3 3.9 14.9 2.2 2.8 1.6 1.2 2.6 21.6 C.E. = Cloning Efficiency Solvent control = OMSO EMS = Ethylmethanesulphonate DMN = Dimethylnitrosamine Test substance precipitated slightly in the exposition medium page 15 - T-5874 NOTOX Project 115921 APPENDIX Individual Colonv Counts and cell Counts during Expression Period FORMULAS AND CALCULATIONS FOR L517BY MOUSE LYMPHOMA TEST SYSTEM. Mutant frequency per 105 survivors Total number of mutant 100 x colonies on selective plates cloning efficieit-cy- number of seeded cells Cloning efficiency Averaqe No. of colonies on cloninq plates- X 100% 200 L517BY MOUSE LYMPHOMA TEST SYSTEM - Cell counts during expression period - Cloning efficiency immediately after exposure - Mutation experiments, individual colony counts Tables 4-6 Tables 7-9 Experiment 1 Experiment 2 Abbreviations used: OMN, dimethylnitrosamine EMS, ethylmethanesulphonate Solvent control: Dimethylsulphoxide page 16 T-5874 NOTOX Project 115921 TABLE 4 L5178Y MOUSE LYMPHOMA TEST SYSTEM CELL COUNTS AND SUBCULTURE DATA Experiment 1 DAY 0 DAY 2 DAY 3 itotal amount ofitotal amount ofl I Subculture dose Icells before@ icelis after I 1 x 106 Ice1ll 3)lc ml (ug/mi)ltreatment x 1061treatment x 1061%1)ltotal amount 1 Subculture countl 6 I x 10 ICell x 1051% 2)lTotal amount 4)lc/ml count x 105 1% 2) DMSO 6 10 6 33 6 100 6 333 12 EMS 6 DMSO 6 10 6 33 6 100 6 333 6 DMN 6 3.0 2.3 1.9 2.5 5.8 2.6 3.8 3.5 4.2 3.9 3.0 3.5 Without Metabolic Activation (-s9-mix) 50 2.8 38 2.2 32 1.8 42 2.4 48 5.7 43 2.4 5.0 100 4.8 96 4.3 86 3.5 76 2.7 54 4.4 With Metabolic Activation (+S9-mix) 63 3.0 58 3.0 70 3.0 65 3.0 50 2.8 58 3.0 6.0 100 6.1 102 6.9 115 6.4 107 4.4 73 3.1 52 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 5.3 100 5.5 104 5.3 100 5.4 102 4.6 87 5.7 108 5.9 100 5.2 88 5.5 93 5.4 92 4.9 83 3.2 54 (1) cells after treatment x 100% cells before treatment (2) cell count x 100% cell count of control (3) (4) cell density cell density 0.4 x 105 ctml 1.6 x 105 ciml page 17 T-5874 NOTOX Project 115921 TABLE 5 L517BY MOUSE LYMPHOMA TEST SYSTEM - CLONING EFFICIENCY DAY 0 EXPERIMENT 1 Dose (pg/mi) No. of colonies/ cloning plate 1 2 3 Mean No. of colonies/plate Cloning efficiency absolute relative (% of control) Without S9-mix Solvent control 109 105 108 107 54 100 10 ill 113 104 109 55 102 33 106 112 112 110 55 103 100 91 101 97 96 48 90 333 98 68 60 75 38 70 EMS 137 146 131 138 69 129 ------------------------------------------------------------------------ With S9-mix Solvent control 168 174 163 168 10 139 135 146 140 33 162 150 163 158 100 169 159 138 155 333 101 102 105 103 OMN 59 53 39 50 84 100 70 83 79 94 78 92 52 61 25 30 page 18 T-5874 NOTOX Project 115921 TABLE 6 L517BY MOUSE LYMPHOMA TEST SYSTEM EFFICIENCY SELECTION DATA AND CLONING EXPERIMENT 1 Dose pg/ml INumber of colonies/SelectionplatelTotaliNo. of coloniesIMeaniMF I -----------------------------I-N-o-.--I/cloningplate I I 1 1 2 3 4 5 6 7 8 9 101 11 2 3 1 1 Without Metabolic Activation (-S9-mix) Solvent I control 1 0 3 3 4 3 0 2 2 10 44 44 02 2 1 33 4 2 55 03 5 4 100 58 44 45 3 6 333 53 00 01 2 1 EMS 12 17 15 9 22 19 11 16 1 3 21 3 0 24 3 3 34 6 3 48 2 4 18 9 12 142 120 123 116 114 122 128 127 117 114 138 119 113 148 145 169 105 119 134 With Metabolic Activation (+S9-mix) Solvent I control 1 1 0 1 3 2 4 1 3 2 1 18 10 1 0 1 2 2 1 0 2 2 1 2 13 33 01 11 00 30 03 9 100 01 00 00 20 02 5 333 1 1 3 5 1 1 3 1 5 5 26 OMN 20 13 11 10 10 7 12 11 11 8 113 118 117 114 110 115 117 114 119 117 112 117 125 165 183 151 75 73 81 MF Mutant frequency per 105 survivors 120 2.31 121 2.61 119 3.81 123 5.21 154 1.61 119 15.91 .1 I I I I I 116 2.11 114 1.51 117 1.01 118 0.61 166 2.11 76 19.81 1 page 19 - T-5874 NOTOX Project 115921 TABLE 7 L5178Y MOUSE LYMPHOMA TEST SYSTEM - CELL COUNTS AND SUBCULTURE DATA Experiment 2 DAY 0 DAY 2 itotal amount ofITotal amount ofl I Subculture I dose icells before icells after 6 I I x 10 ICell (ug/ml)ltreatment x 1061treatment x 106 1%1)lTotal amount3)lc/m, I I Subculture I countl I x 106 icall x 1051%2)lTotal amount4)lciml DAY 3 count x 105 I I 1%2) Without Metabolic Activation (-S9-mix) DMSO 6 10 6 33 6 100 6 333 12 ENS 6 4.1 68 3.0 4.2 100 4.3 72 3.0 3.0 71 3.9 65 3.0 3.2 76 4.3 72 3.0 2.1 50 9.0 75 6.0 2.0 48 4.0 67 3.0 4.4 105 With Metabolic Activation (+S9-mix) omso 6 10 6 33 6 100 6 333 6 OMN 6 3.7 62 3.0 5.4 100 4.4 73 3.0 5.1 94 3.9 65 3.0 5.4 100 3.8 63 3.0 5.9 109 3.5 58 3.0 4.4 al 4.0 67 3.0 2.0 37 (1) cells after treatment x 100% cells before treatment (2) cell count x 100% cell count of control (3) (4) cell density cell density 0.4 x 105 c/ml 1.6 x 105 c/ml 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 6.0 100 6.1 102 4.8 so 6.0 100 5.8 97 6.0 100 6.3 100 5.2 83 6.2 98 4.5 71 5.9 94 3.7 59 page 20 T-5874 NOTOX Project 115921 TABLE 8 L5178Y MOUSE LYMPHOMA TEST SYSTEM - CLONING EFFICIENCY DAY 0 EXPERIMENT 2 Dose (ug/ml) No. of colonies/ cloning plate 1 2 3 Mean No. of colonies/plate Cloning efficiency absolute relative (% of control) Without S9-mix Solvent control 147 147 141 145 73 100 10 138 134 138 137 69 95 33 155 147 158 153 77 106 100 84 85 INF 85 333 97 92 92 94 43 59 47 65 EMS 133 135 143 137 69 94 -------------------------------------------------------------------------- With S9-mix Solvent control 168 149 189 169 10 130 147 INF 139 33 167 152 149 156 100 161 165 162 163 333 166 139 156 154 DMN 75 63 INF 69 85 100 70 82 78 92 82 96 77 91 35 41 INF Plate infected with fungi page 21 T-5874 NOTOX Project 115921 TABLE 9 L517BY MOUSE LYMPHOMA TEST SYSTEM - SELECTION DATA AND CLONING EFFICIENCY EXPERIMENT 2 Dose jig / ml Solvent control 10 33 100 333 EMS INumber of colonies/Selection platelTotallNo. of coloniesIMeanIMF I I ------------------------------I--N-o-. I/cloning plate I I I 1 1 2 3 4 5 6 7 8 9 101 11 2 31 1 1 1 I Without-Metabolic Activation (-S9-mix) I I I I 0 4 4 3 5 2 8 5 6 5 42 6 3 4 8 5 7 5 7 6 7 58 2 7 4 1 2 3 8 5 3 2 37 10 5 4 5 5 7 9 4 7 5 61 5 6 9 4 4 6 6 3 6 3 52 24 27 26 24 16 15 28 25 23 24 232 150 187 172 199 202 183 189 198 203 189 182 190 204 195 182 180 180 184 I 170 3.31 195 4.01 197 2.51 187 4.31 194 3.61 181 17.11 1 1 With Metabolic Activation (+S9-mix) Solvent I control 1 3 3 3 4 4 2 4 3 4 4 34 10 3 3 4 8 1 5 4 2 2 0 32 33 4 0 1 1 3 4 -6 3 2 0 24 100 1 0 2 0 1 6 l 0 2 0 13 333 3 6 7 5 2 1 4 3 5 3 39 DMN 12 15 12 18 17 16 18 13 12 16 149 201 226 190 144 151 171 201 204 194 161 140 138 180 223 201 77 105 95 206 2.21 155 2.81 200 1.61 146 1.21 201 2.61 92 21.61 1 MF = Mutant frequency per 105 survivors page 22