Document JrYQ8gw7kpnG9xOEEm2vL5rL2

AR226-2985 a* CCR Cytotest Cell Research GmbH & Co. KG CCR PROJECT 326417 SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY WITH Study Completion Date: February 17,1993 REPORT Does not contain TSCA CBl C o m * TM sa"mzd CCR In den L eppsteinsw iesen 19 D-6101 Rodorf F.R.G. T elephone: 0 61 54 - 80 7-0 Telefax 0 61 54 - 8 33 99 COPY OF GLP CERTIFICATE W . HESSISCHES MINISTERIUM FR UMWELT, ENERGIE UND BUNDESANGELEGENHEITEN GLP-Bescheinigung Bescheinigung Hiennit wird besttigt, da die Prfungseinrichtung(en) C y t o t e s t C a ll R e s e a r c h GmbH & Co KG In d e n L e p p s t e i n s w i e s i n 19 in S jO L R a d o f f ___ ` (O,AnchntQ . der RCC H o ld in g V e rw a ltu n g GmbH_________ pomi) am 0 3 . 0 8 ., 0 4 . 0 8 ., 0 5 .0 8 .- und ,0 6 .0 8 .9 2 P&IURl) * von der fr die berwachung stndigen Behrde Ober . . die Schaltung der Grundstze der Guten Laborpraxis inspiziert worden ist (sind}. . / Es wird hiennit besttigt, da folgende Prfungen in dieser Prfeinrichtung nach den Grundstzen der Guten Laborpraxis durchgefhrt werden. . Certificate It is hereby certified that the test !aciity(ies) C y t o t e s t C e l l R e s e a rc h GmbH & Co KG In d e riT e p p ^ ----------- in 6 101 Ro d o r f . (location,tainai ~~ RCC H o ld in g V e rw a ltu n g GmbH {campen/nun) o n 0---3--.-0--8--.-,----0- 4 . 0-8---.-, 0 5 .0 8 . a n d 0 6 .0 8 .9 2 (data) "" was (ware) inspected by the competent authority regar ding compliance with the Principles of Good Laboratory Practica. 1 It is hereby certified that studies in Ihis test facility are conducted in compliance with the Principles of G~-ri Laboratory Practice. - T oxikologische E ig en sch aft T o x ico lo g ical,p ro p e rties Im Auftrag . (0 r.-H eck er) ' W iesbaden, den st5r page 2 of 35 not contain TSCACB1 Sanitized. Does Compaq CONTENTS COPY OF GIiP CERTIFICATE PAGE 2 :PREFACE General Project Staff Schedule Project StaffSignatures Quality Assurance Guidelines Archiving 4 4 4 -4 5 5 5 5 STATEMENT OF COMPLIANCE 6 QUALITY ASSURANCEUNIT Statement 7 7 SUMMARY ~ Conclusion 8 8 OBJECTIVE Aims of the Study Reasons for the Study 9 9 9 MATERIALS ANDMETHODS The Test Article The Controls The Test System Mammalian Microsomal Fraction S9 Mix Pre-Experiment forToxicity Dose Selection Experimental Performance Data Recording Evaluation of Results 10 10 11 - 12 14 15 15 16 1617 BIOMETRY 18 RESULTS ' Pre-Experiment for Toxicity Tables of Results Experiment I Tables of Results Experiment II Summary of Results 19 19 20 26 32 CONCLUSIONS ' 33 REFERENCES . 34 DISTRIBUTION OF THE REPORT 35 st5r page 3 of 35 j0t contai.inTSCA CBl Sanitized. Doss* C om paq PREFACE GENERL Sponsor: Monitoring: Testing Facility CCR Project No.: Test Article: Title ISEGA Forschungs- u. Untersuchungs Gesellschaft mbH Zeppelins t r . 3-5 D-8750 Aschaffenburg Heike Krmer CCR CYTOTEST CELL RESEARCH GMBH & CO. KG D-6101 Rodorf, F.R.G. 326417 I Salmonella typhimurium Revenge Mutatioi^ |tAssay with, PROJECT STAFF Management: Dr. H.- E. Knoell Study Director: Dr. Albrecht Poth Quality Assurance Unit: Dr. Ch. Helmrich SCHEDULE Date of Protocol: t November 10, 1992 Date of 1st Amendment to Protocol: January 18, 1993 Start of Pre-Experiment: November 27, 1992 End of Pre-Experiment: December 04, 1992 Start of Experiment I: December 15, 1992 End of Experiment I: December 18, 1992 Start of Experiment II: January 05, 1993 End of Experiment .11: January 15, 1993 Date of Draft: January 18, 1993 Date of Report : February 17, 1993 st5r page 4 of 35 Sanded. CoinpanV contain TSCA Cm PROJECT STAFF SIGNATURES Study Director: D r . Albrecht Poth Management: ............. >h td Date: February 17, 1993 QUALITY ASSURANCE The study was performed in compliance with: Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, A n l a g e 1 ("Annex 1"), dated March 14, 1990 (B G B L . I s 521). "OECD Principles of Good Laboratory Practice", Paris, 1981 GUIDELINES This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: First Addendum to OECD Guidelines for Testing of Chemicals, , Section 4, No. 471, "Salmonella typhimurium, Reverse Mutation Assay", adopted Ma y 26, 1983 and EEC Directive 79/831, Annex V, B 14. ARCHIVING C C R, D-6101 Rodorf/F.R.G. will archive the following data for 30 years: Raw data, protocol, and copy of report. The following sample will be archived for at least 12 years: Sample of test article. No raw data or material relating to the study will be discarded without the sponsor's prior consent. st5r page 5 of 35 Company Sanitized. D oes not contain TSCA CBI STATEMENT OF COMPLIANCE Project Number: 326417 Test Article Study Director: Dr. Albrecht Poth Title Salmonella typhimurium Reverse Mutation Assay with To the best-of m y knowledge and belief, this study performed in the testing facility of CCR was conducted in compliance with Good Laboratory Practice Regulations: Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, Anlage 1 ("Annex 1"), dated March 14, 1990 "OECD Principles of Good Laboratory Practice, Paris, 1981 There were no circumstances that may have affected the quality or integrity of the study. ' Study Director CCR Dr. Albrecht Poth st5r page 6 of 35 Sanitized. Does not contain TSCA GBt Compaq QUALITY ASSURANCE UNIT C C R , Cytotest Cell Research GmbH & Co. KG, In den Leppsteinswiesen 19, D-6101 Rodorf, F.R.G. STATEMENT Project Number Test Article : Study Director: Title 326417 Dr. Albrecht Poth Salmonella typhimurium Reverse Mutation Assay with This report was audited by the Quality Assurance Unit and the study and/or testing facility were inspected on the following dates. Dates of QAU Inspections/ Audits November 11, 1992 December 15, 1992 January 21, 1993 f Dates of Reports to the Study Director and to Management November 11, 1992 December 15, 1992 January 21, 1993 Head of Quality Assurance Unit . Dr. Ch. Helmrich st5r Date: -/S/ ' f J J page 7 of 35 Company Sanitized. Does not contain TSCA CBS SUMMARY T?Ms study was performed to investigate the potential of m f t o induce gene mutations according to the plate incoi^o^tlon' .test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, T A 1537, t a 98 TA 100, and T A 102. ' The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration including the controls, was tested in triplicate. The test arti cle was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 ug/plate No relevant" toxic effects occurred in the test groups with and without metabolic activation in experiment I and II in all strains used. The plates incubated with the test article showed normal back ground growth up to 5000.0 |ig/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with of metabolic activation (S9 mix) . There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. CONCLUSION In conclusion, it can be stated that during the described mutage nicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, is considered to be non-mutagenic in this Salmonella typhTmuriim reverse mutation assay. st5r page 8 of 35 M W S a n ifeed . B o no. contain TSCA CBI OBJECTIVE AIMS OF THE STUDY ^he experiments were performed to assess the potential of the t? .induce ?ene mutations by means of two independent Salmonella typhimurium reverse mutation assays. Experiment I xra performed as a plate incorporation assay. As a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay. as REASONS FOR THE STUDY t h ^ = r S t - WidT?lyu US-ed assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the abilitj of an a g t to interact with DNA and produce mutations. y ro However, the bacteria most commonly used in these assays do not possess the enzyme systems which, in mammals, are known to con vert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system ij added m form of mammalian microsome enzyme activation mixture. In spite of great differences between bacterial and eucaryotic cells with respect to structure and function there is an associa tion between mutagenicity in bacteria and carcinogenicity in mammals described in literature (7,8). y Reverse mutation assays determine the frequency at which an acrent abolishes or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains, or a single strain with multiple ^markers are necessary to overcome the effects of mutacen specificity. The reversions of bacteria from growth-dependence on a particular amino acid to growth in the absence of that amino acid (reversion from auxothrophy to prototrophy) is the most widely used marker. ' 1 osr The Salmonella typhimurium histidine (his) reversion system measures his -- > his reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 15'?c; fa 100, TA 102) and frameshift (TA 1537, TA 98) mutations. ' According to the direct plate incorporation and the ore- incubation method the bacteria were exposed to the test article with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies were counted. ` To establish a dose response effect five dose levels with ade- fnnnSn sPacec* intervals were tested. The maximum dose level was 5000.0 hg/plate, unless limited by toxicity or solubility of the test article. J To validate the test, reference mutagens were tested in narallel to the test article. * st5r page 9 of 35 Company Sanitized. Does not contain TSCA CBI MATERIALS AND METHODS THE TEST ARTTrT.E; The test article and the information concerning the, test article were provided by the sponsor. Name: Batch N o .: Aggregate State at R T : Colour: Analysis: Purity: Stability: liquid Storage: 4C Expiration Date: not indicated by the sponsor On the day of the experiment, the test article ZONYL RP 18 was dissolved in Ethanol. The solvent was chosen because of its solubility properties and its relative nontoxicity for the bacte ria. st5r page 10 of 35 Sanitized Does not contain t s c a c b i Company Test: Report CCR Rrojecr j^t>4X / THE CONTROLS The Negative Controls Concurrent untreated and solvent controls were performed. The Positive Control Substances Without metabolic activation Strains; Name: Supplier: Catalogue N o .: Purity: Dissolved in: C o n c e n t r a t i o n :. Strains: Name: Supplier: Catalogue No. Purity: Dissolved in: Concentration TA 1535, TA 100 sodium azide, NaN3 SERVA, D-6900 Heidelberg, 30175 at least 99 % aqua dest. 10 ng/plate F.R.G. TA 1537, TA 98 4-nitro-o-phenylene-diamine, 4-NOPD SIGMA, D-8024 Deisenhofen, F.R.G. N 9504 > 99.9 % DMSO 50 ng/plate Strain: Name: Supplier: Catalogue N o .: Purity: Dissolved in: Concentration: TA 102 methyl methane sulfonate, MMS MERCK-SCHUCHARDT, D-8011 Hohenbrunn, F.R.G. 820775 > 99.0 % aqua dest. 1 .0, pi/plate With metabolic activation Strains: Name: Supplier: Catalogue N o .: Purity: Dissolved in: Concentration: TA 1535, TA 1537, TA 98, TA 100, TA 102 2-aminoanthracene, 2-AA SIGMA, D-8024 Deisenhofen, F.R.G. A 1381 97.5 % .DMSO 2.5 pg/plate The stability of the positive control substances in solution was unknown but a mutagenic response in the expected range is sufficient evidence of biological stability. st5r page 11 of 35 contain TSCAGSl XiQl Compaq Sanitized CoeE Test Report CCR Project 326417 THE TEST SYSTEM Characterisation of the Salmonella tvphxmnrlum Strains The strains are derived from S . typhimurium strain LT2 and 'due to a mutation in the histidine locus are histidine depen dent. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which en ables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an exci sion repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". .In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the_ampicillin resistance marker. The strain TA 102 does not contain the uvrB"~mutation. Additionally TA 102 contains the multicopy plasmid pAQl, which carries the hisG428 mutation and a tetracyclin resistance gene. TA 102 contains the ochre mutation in hisG gene. In summary, the mutations of the TA strains used in this study can be described as follows: Salmonella typhimurium TA 1537: his C 3076; rfa" ; uvrB" ; : frame shift mutations TA 98: his D 3052; rfa" ; uvrB" ;R-factor: "" TA 1535*: his G 46; rfa" ; uvrB" ; :base-pair substitutions TA 100: his G 46; rfa" ; uvrB" ;R-factor: " " TA 102: his G 428; rfa" ; uvrB+ ; R-factor: " " Regular checking of the properties of the strains with regard to membrane permeability, ampicillin- and tetracyclin-resistance as well as normal spontaneous mutation rates is performed in the laboratory of C C R according to Ames et a l . (1). In this wa y it was ensured that the experimental conditions set down by Ames were fulfilled. . The bacterial strains were obtained from Dr. Heinz Trger, Knoll AG, D-6700 Ludwigshafen, F.R.G. st5r page 12 of 35 Company Sanitized-Ees1:iOt CO fitain TSCA CBl r e s e t ie y u r u t-CK t r u j a u i ; j z o t i / Storage The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen. Precultures From the thawed ampoules of the strains 0.5 ml bacterial suspen sion was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre: 8 g Merck Nutrient Broth 5 g NaCl The bacterial 'culture was incubated in a shaking water bath for 10 hours at 37 C. Selective Agar 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilisations were performed at 121 C in an autoclave. Overlay Agar s The overlay agar contains per litre: 6.0 g Merck Agar Agar 6.0 g NaCl 10.5 m g L-histidine x HC1 x H 2O 12.2 mg biotin Sterilisations were performed at 121 C in an autoclave. st5r page 13 of 35 not contain TSCA Cl C om paq Sanitized. 0 es Test Report CCK Project: J2b41 / MAMMALIAN MICROSOMAL FRACTION S9 MIX S9 (Preparation by C C R\ The S9 liver microsomal fraction was obtained from the liver of 8 - 1 2 weeks old male Wistar rats, strain WU (SAVO-Ivanovas, med. Versuchstierzuchten GmbH, D-7964 Kisslegg, F.R.G.; weight approx. ISO - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor- 1254 (Antechnika, D-7500 Karlsruhe, F.R.G.) in olive oil 5 days previously. After cervical dislocation the livers of the animals were re moved, washed in 150 mM KC1 and homogenised. The homogenate, diluted 1+3 in KC1 was centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -70 C. Small ntimbers of the ampoules are kept at -20 C for only several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-8000 Miinchen: Bio-Rad protein assay, Catalogue 500 000 6 (6 ). The protein concentration in the S9 preparation was 31.6 mg/ml (lot 060792). S9 Mix Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 15% v/v. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl2 33 m M KC1 5 mM glucose-6-phosphate 5 mM NADP in 100 m M sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according'to Ames et al.{2). st5r page 14 of 35 *contain TSCACSi !RVSamfeed.Does^ XfcU R t i ^ U X L n u j t L l J ^ O " X / PRE-EXPERIMENT FOR TOXICITY To evaluate the toxicity of the test article a prestudy was performed with strains TA 98 and T A 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test). Toxicity of the test article ma y be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures. DOSE SELECTION According to the results of the pre-experiment the concentrations applied in the main experiments were chosen. The maximum concentration was 5000.0 pg/plate. The concentration range included two logarithmic decades. In this study six ade quately spaced concentrations were tested. Two independent exper iments were performed. As the results of the pre-experiment are in accordance with the criteria described above, these data are reported as a part of the main experiment I. st5r page 15 of 35 not "contain TSCA CB1 Company Sanitized. Does Test: Keporr c c k . project: / EXPERIMENT^!, PERFORMANCE For each strain and dose level, including the controls, a of three plates were used. ;The following materials were mixed in a test tube and poured onto the selective agar plates: 100 pil 500 nl 100 Hi 2000 nl Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control), S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation), Bacteria suspension (cf. test system, pre-culture of the strains), Overlay agar In the pre-incubation assay 100 Hi test solution, 500 h 1 S9 miy / S9 mix substitution buffer and 100 h ! bacteria suspension were mixed in a test tube and incubated at 37C for '60 minutes. After pre-incubation 2.0 ml overlay agar was added to each t u b e . The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 G in the dark. DATA RECORDING The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BI0SYS GMbH, D-6367 Karben; F.R.G.). The counter was connected to an IBM AT compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard dviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). If precipitation of the test article preclud ed automatic counting the revertant colonies were counted by hand. ` st5r page 16 of 35 Test Report CCR Project J2b41/ EVALUATION OF RESULTS The generally accepted conditions for the evaluation of the results are: - corresponding background growth on both negative control and : test plates - normal range of spontaneous reversion rates. * These values refer to the negative control without metabolic activation (+) The range of strain TA 102 was determined from our historical control datas Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical proce dure can be recommended for analysis of data from the bacterial assays at this time (5 ). A test article is considered as positive if either a dose related and reproducible increase in the number of revertants or a sig nificant and reproducible increase for at least one test concen tration is induced. A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and repro ducible positive response at any one of the test points is con sidered non-mutagenic in this system. A significant response is described as follows: A test article is considered as mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains T A 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate (4 ). Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not. ' st5r page 17 of 35 not contain TSCACB! Company Sanitized. Does Test Keport CUK project: J2fa41/ BIOMETRY ;No appropriate statistical method is available (5). st5r page 18 of 35 Company San: tized. Doss not contain TSCA CBS xesu Keporc cuit jrrujcL. **x/ RESULTS PRE-EXPERIMENT FOR TOXICITY To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100. The results are given in the following table: ' test groups concentration per plate ng Negative control Solvent control 4-NOPD Sodium azide * 2-aminoanthracene -- - 50.0 10.0 2.5 3.3 10.0 33.3 100.0 333.3 1000.0 2500.0 5000.0 revertants TA 98 --+ 37 30 2542 / / 22 34 25 34 33 25 28 30 42 40 / / 575 33 23 35 43 39 24 32 25 per plate TA 100 -- +* 76 104 82 106 // 925 / / 1119 73 108 72 98 76 84 72 85 64 88 50 84 50 76 57 64 * - - without S9 mix; + with S9 mix / *not performed The plates with the test article showed normal background growth up to 5000.0 p.g/plate in strain TA 98 and TA 100, respectively. According to the dose selection criteria, the test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 ng/plate st5r page 19 of 35 not contain TOGA CBl Sanitized. Dos Com pany Test Report CCR Project 326417 TABLES OF RESULTS EXPERIMENT I PLATE INCORPORATION TEST st5r page 20 of 35 company no. oo-- TSCACB. Test article S9 mix from : rat liver (Batch R 060792) .Test strain : TA 1535 Dose pg/plate Plate 123 without S9 mix Negative control Solvent control 33.3 100.0 -' 333.3 1000.0 2500.0 5000.0 Positive control Sodium azide (IQpg/plate) 14 15 13 20 15 15 19 22 23 18 16 14 11 13 17 17 14 13 17 7 15 12 15 19 1142 1178 1135 with S9 mix Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 14 15 19 21 22 27 26 21 21 27 26 22 22 26 20 19 21 19 21 30 28 24 21 21 Positive control 2-Aminoanthracene (2.5pg/plate) + enhancement fa c to r = 135 153 190 Z revertants/concentr. te s t a rtic le Z revertants/solvent control to Revertants/plate mean 's .d. factor* 14 1.0 17 2.9 1.0 21 2 . 1 1.3 16 2 . 0 1.0 14 3.1 15 0.9 13 5.3 15 3.5 0.9 1152 23.1 69.1 15 2 . 6 23 3.2 1.0 23 2.9 1.0 25 2 . 6 1 . 1 23 3.1 1.0 20 1 .2 0 .8 26 4.7 1 . 1 22 1.7 0.9 159 28.0 CO CO CD CO Oo str5 21 of 35 ' Company San-feed. Does not contain TSCA CBl Test article: S9 mix from : rat liver (Batch R 060792) Test strain : TA 1537 Dose pg/plate without S9 mix Negative control Solvent control 33.3 100.0 _- 333.3 1000.0 2500.0 5000.0 Positive control 4-Nitro-ophenylene-diamine (50pg/plate) Plate 123 8 10 48 7 '8 68 46 54 87 94 5 7 7 11 6 6 5 5 307 311 350 with S9 mix Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 12 14 13 10 8 15 6 .8 Positive control 2-Aminoanthracene (2.5|j.g/plate) 115 15 11 19 13 12 11 14 11 110 11 12 8 13 12 11 6 10 93 + enhancement fa c to r X revertants/concentr. te s t a rtic le X revertants/solvent control Revertants/plate mean s .d. factor* 8 2.5 6 2 . 1 1.0 7 0.6 1.2 8 2.5 1.3 5 1.2 0.8 5 1.0 0 . 8 7 1.5 1 . 1 6 2 . 6 0.9 323 23.8 50.9 13 2 . 1 12 1.5 1.0 13 5.5 1 . 1 12 1.7 1.0 11 2.3 0.9 12 2.3 1.0 9 4.6 0.7 10 1.5 0 . 8 106 11.5 8.6 str5 22 of 35 Company Sanitized. Does not contain TSCA CBi Test article S9 mix from : rat liver (Batch R 060792) Test strain : TA 98 Dose pg/plate Plate 123 without S9 wi'y Negative control 42 35 35 Solvent control 27 33 31 33.3 28 24 24 100:0 -- 31 35 36 333.3 24 37 37 1000.0 32 26 17 2500.0 19 26 39 5000.0 . 28 28 33 Positive control 4-nitro-o- phenylene-diamine (50 pi/plate) 2464 2501 2660 Revertants/plate mean s.d. factor* 37 30 . 25 34 33 25 28 30 4.0 3.1 2.3 2.6 7.5 7.5 10.1 2.9 1.0 0.8 1.1 1.1 0.8 0.9 1.0 2542 104.1 .83.8 with S9 miy Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 38 47 29 43 42 25 30 29 Positive control 2-Aminoanthracene . 520 (2.5pg/plate) 44 34 30 40 41 25 34 23 454 43 40 45 45 33 21 31 24 751 42 3.2 40 6.5 1.0 35 9.0 0.9 43 2.5 1.1 39 4.9 1.0 24 2.6 0.6 32 2.1 0.8 25 3.2 0.6 575 156.0 14.3 enhancement fa c to r Z revertants/concentr. te s t a rtic le Z revertants/solvent control str5 23 of 35 Com pany Sanitised. D oes not contain TSCACBfe Test article: S9 mix from : rat liver (Batch R 060792) Test strain : TA 100 Dose pg/plate without S9 mix Negative control Solvent control 33.3 100". o- _- 333.3 1000.0 2500.0 5000.0 Positive control Sodium azide (10pg/plate) Plate 1 23 78 82 68 80 76 89 63 86 80 63 79 75 55 66 70 50 50 51 38 53 58 45 63 64 946 942 886 Revertants/plate mean s .d . factor* 76 7.2 82 6.7 76 11.9 72 8.3 64 7.8 50 0.6 50 10.4 57 10.7 1.0 0.9 0.9 0.8 0.6 0.6 0.7 925 33.5 11.3 with S9 mix Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 103 111 8,9 114 90 82 94 89 96 71 93 102 77 80 56 67 Positive control 2-Aminoanthracene (2.5pg/plate) 1356 1083 99 116 81 73 98 58 71 68 919 104 6.1 106 15.0 84 4.9 85 11.0 88 15.0 84 23.2 76 4.6 64 6.7 1.0 0.8 0.8 0.8 0.8 0.7 0.6 1119 220.8 10.5 + enhancement fa c to r Z revertants/concentr. te s t a rtic le Z revertants/solvent control str5 24 of 35 Company Sanitized. Does not contain TSCA CBI Test article S9 mix from : rat liver (Batch R 060792) Test strain : TA 102 Dose Mg/plate Plate 12 3 without S9 mix Negative control Solvent control 33.3 100.0 -' 333.3 1000.0 2500.0 5000.0 Positive control methyl methan sulfonat (1.0|il/plate) 200 207 208 206 225 211 181 178 154 179 244 199 109 179 188 171 151 190 237 228 212 99 201 209 827 1107 1010 with S9 mix Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 280 253 252 318 364 204 284 214 Positive control 2-Aminoanthracene (2.5|ig/plate) 752 296 318 312 262 291 289 272 284 861 243 357 343 324 292 251 289 295 864 enhancement fa c to r t revertants/concentr. te s t a rtic le Z revertants/solvent control Revertants/plate mean s.d. factor-*- 205 214 171 207 159 171 226 203 4.4 9.8 14.8 33.3 43.2 19.5 12.7 5.3 1.0 0.8 1.0 0.7 0.8 1.1 0.9 981 142.2 4.6 273 309 302 301 316 248 282 264 27.2 52.5 46.3 34.2 41.9 . 42.6 8.7 43.9 1.0 1.0 1.0 1.0 0.8 0.9 0.9 826 63.8 2.7 str5 -- S B * * Does no!obtain ^ C A CEI TABLES OF RESULTS EXPERIMENT II PRE-INCUBATION TEST st5r page 26 of 35 not contain TSCACB Company Sanitized. Does Test article: S9 mix from : rat liver (Batch R 060792) Test strain : TA 1535 Dose )ig/plate Plate 1 23 without S9 mix Negative control Solvent control 33.3 100.0 -' 333.3 1000.0 2500.0 5000.0 Positive control Sodium azide (IQng/plate) 19 20 22 21 18 21 23 18 17 15 13 24 17 14 23 10 12 12 13 9 15 16 16 17 1044 1096 1091 with S9 mix Negative control Solvent control 33.3 100.0 3^3.3 1000.0 2500.0 5000.0 Positive control 2-Aminoanthracene (2.5ng/plate) 24 17 13 3 24 27 24 26 18 21 24 25 31 26 31 28 28 29 28 27 30 22 20 17 163 187 167 revertants/concentr. te s t a rtic le + enhancement fa c to r revertants/solvent control CO to Revertants/plate mean 's .d. factor+ 20 1.5 20 1.7 1.0 19 1.0 17 5.9 0.9 18 4.6 0.9 11 1.2 0.6 12 3.1 0.6 16 0.6 0.8 1077 28.7 53.9 18 5.6 27 3.5 1.0 23 4.2 0.8 23 2.1 0.9 29 2.9 1.1 28 0.6 1.0 28 1.5 1.0 20 2.5 0.7 172 12.9 6.3 str5 27 of 35 C o m p a n y Sasiitte**' D o e s noi contain T5CACBI Test article: S9 mix from : rat liver (Batch R 060792) Test strain : TA 1537 Dose ng/plate Plate 123 without S9 mi ir Negative control Solvent control 33.3 100.0 333.3 -* 1000.0 2500.0 5000.0 Positive control 4-Nitro-ophenylene-diamine (50pg/plate) with S9 mix 4 4 7: 8 7 .9 9 6 11 8 9 12 6 11 9 11 9 6 13 7 9 11 8 8 212 245 176 Negative control Solvent control 33.3 100.0 . 333.3 1000.0 2500.0 5000.0 7 13 10 13 14 13 12 15 13 14 13 9 11 18 13 12 12 7 10 8 10 11 11 9 Positive control 2-Aminoanthracene 113 94 91 (2.5ng/plate) Reversants/plate mean s .d. factors 5 1.7 8 1.0 1.0 9 2.5 1.1 10 2.1 1.2 9 2.5 1.1 9 2.5 1.1 10 3.1 1.2 9 1.7 1.1 211 34.5 26.4 10 3.0 13 0.6 1.0 13 1.5 1.0 12 2.6 0.9 14 3.6 1.1 10 2.9 0.8 9 1.2 0.7 10 1.2 0.8 99 11.9 7.5 enhancement fa c t o r I revertants/concentr. te s t a rtic le X revertants/solvent control str5 28 of 35 not contain tscacbr Company Sanitized. Does Test article S9 mix from : rat liver (Batch R 060792) Test strain : TA 98 Dose ng/plate Plate 123 without S9 mix Negative control Solvent control 33.3 100.0 333.3 -* 1000.0 2500.0 5000.0 22 30 25 23 27 26 18 29 31 19 23 25 19 18 20 20 25 20 17 19 19 17 13 26 Positive control 4-nitro-ophenylen-diamine (50 pg/plate) with S9 mix 1856 1874 1940 Negative control 42 39 33 Solvent control 39 45 43 33.3 43 37 45 100.0 39 44 42 333.3 33 46 45 1000.0 38 41 33 2500.0 * 37 41 33 5000.0 45 33 44 Positive control 2-Aminoanthracene 448 352 335 (2.5ng/plate) + enhancement fa c to r l revertants/concentr. te s t a rtic le l revertants/solvent control Revertants/plate mean s.d. factor+ 26 4.0 25 2.1 1.0 26 7.0 1.0 22 3.1 0.9 19 1.0 0.8 22 2.9 0.9 18 1.2 0.7 19 6.7 0.7 1890 44.2 74.6 38 4.6 42 3.1 1.0 42 4.2 ^ 1.0 42 2.5 1.0 41 7.2 1.0 37 4.0 0.9 37 4.0 0.9 41 6.7 1.0 378 60.9 8.9 s tr5 29 of 35 .,,M d.D oas Company Sa, TSCk OBI Test article S9 mix from : rat liver (Batch R 060792) Test strain : TA 100 Dose pg/plate Plate 123 without S9 mix Negative control Solvent control 33.3 100.0 333.3 ~' 1000.0 2500.0 5000.0 Positive control Sodium azide (lOpg/plate) 82 95 .81 79 93 86 89 99 79 79 86 91 72 87 76 82 86 78 70 89 78 98 86 78 1133 1214 1196 Revertants/plate mean s.d. factor+ 86 7.8 86 7.0 89 10.0 85 6.0 78 7.8 82 4.0 79 9.5 87 10.1 1.0 1.0 1.0 0.9 1.0 0.9 1.0 1181 42.5 13.7 with S9 m i x Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 106 109 84 103 87 78 92 . 78 91 97 92 98 87 75 88 84 90 89 80 97 105 74 84 78 Positive control 2-Aminoanthracene (2.5pg/plate) + enhancement fa c to r 1190 845 1089 l revertants/concentr. te s t a rtic le X revertants/solvent control 96 9.0 98 10.1 85 6.1 99 3.2 93 10.4 76 2.1 88 4.0 80 3.5 1.0 0.9 1.0 0.9 0.8 0.9 0.8 1041 177.4 10.6 str5 30 o f C3o5mpany S,,am-uj:zzeed".Does not contain TSCA CBl Test article S9 mix from : rat liver (Batch R 060792) Test strain : TA 102 Dose fig/plate Plate 12 3 without S9 mix Negative control Solvent control 33.3 100.0 333.3 ~* 1000.0 2500.0 5000.0 Positive control methyl methan sulfonat (1.0 jil/plate) 151 158 178 188 198 188 190 159 171 175 166 175 177 179 156 167 998 1034. 179 167 169 176 187 189 178 189 897 with S9 mix Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 Positive control 2-Aminoanthracene (2.5fig/plate) 260 197 233 217 194 224 240 237 219 218 210 219 201 199 216 256 270 245 233 245 206 213 230 174 811 924 702 enhancement fa c to r revertants/concentr. te s t a rtic le revertants/solvent control Revertants/plate mean s.d. factor+ 167 14.4 167 8.5 171 6.2 180 7.2 187 10.5 185 5.5 175 17.2 172 15.5 1.0 1.0 1.1 1.1 1.1 1.0 1.0 976 71.0 5.9 250 220 225 227 200 212 229 222 27.0 24.1 13.3 15.6 6.0 12.5 12.1 42.9 1.0 1.0 1.0 0.9 1.0 1.0 1.0 812 111.0 3.7 str5 31 of 35 Company Sanitized. Does not co"fe!n TSC CEP SUMMARY OF RESULTS Test article: S9 mix from : rat liver (Batch R 060792) without S9 mix Revertants/plate mean from three plates Dose TA 1535 TA 1537 TA 98 TA 100 TA 102 p-g/plate I / II I ! II I / II I / II I / II Neg. contr. 14 Solv. contr. 17 33.3 21 .100.0 16 333.3 14 100070 . r 15 2500.0 ` 13 5000.0 15 20 20 19 17 18 11 12 16 a5 68 79 8 10 59 59 7 10 69 37 26 30 25 25 26 34 22 33 19 25 22 28 18 30 19 76 86 82 86 76 89 72 85 64 78 50 82 50 79 57 87 205 167 214 167 171 171 207 180 159 187 171 185 226 175 203 172 Positive controls Sodium azide 1152 1077 (lOfig/plate) 4-Nitro-ophenylene-diamine (50p.g/plate) methyl methan sulfonat (1.0|xL/plate) 323 211 2542 1890 925 1181 981 976 with S9 mix Revertants/plate mean from three plates Dose TA 1535 TA 1537 p,g/plate I / II I / II TA 98 I'/ II TA 100 I / II TA 102 I / II Neg. contr. Solv. contr. 33.3 100.0 333.3 1000.0 2500.0 5000.0 16 18 23 27 23 23 25 23 23 29 20 28 26 28 22 20 13 10 12 13 13 13 12 12 11 14 12 10 99 10 10 42 38 40 42 35 42 43 42 39 41 24 37 32 37 25 41 104 96 106 98 84 85 85 99 88 93 84 76 76 88 64 80 273 250 309 220 302 225 301 227 316 200 248 212 282 229 264 222 Positive control 2-Amino- 159 172 106 99 575 378 1119 1041 826 812 anthracene (2.5(xg/plate) str5 32 of 35 Company Sanitized. Does not contain TSCA CBI CONCLUSIONS The test a-rticle (was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, T A 98, TA 100, and TA 102. ' The assay was perfcanned in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test arti cle was tested at the following concentrations: 33.3? 100.0; 333.3; 1000.0? 2500.0 and 5000.0 ng/plate No relevant toxic effects occurred in the test groups with and. without metabolic activation in experiment I and II in all strains used. The plates incubated with the test article showed normal back ground growth up to 5000.0 ng/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of ' "' ter strains were observed following treatment with at any dose level, either in the presence or absence ______ activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance. . Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies. In conclusion, it can be stated that during the described mutage nicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. st5r page 33 of 35 Company Sanitized. D o es not contain TSCA CB1 REFERENCES 1. Ames, B.N., W.E. Durston, E. Yamasaki, and F.D. Lee (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection Proc. Natl. Acad. Sci. (USA) 70, 2281-2285 2. Ames, B.N., J. McCann, and E. Yamasaki (1977) Methods for detecting carcinogens and mutagens Salmonella/mammalian microsome mutagenicity test In: B.J. Kilbey et al. (Eds.) "Handbook of Mutagenicity Test Procedures" Elsevier, Amsterdam, 1-17 with the 3. Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K . , Nestmann, E. and Zeiger, E. (1987) Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity Mutation Res. 189, 83-91 4. Hollstein, M . , J. McCann, F.A. Angelosanto and W.W. Nichols (1979) Short-term tests for carcinogens and mutagens Mutation Res. 65, 133-226 * 5. Kier, L . E . , D.J. Brusick, A.E. Auletta, E.S. Von Halle, M.M. Brown, V.F. Simmon, V. Dunkel, J. McCann, K. Mortelmans, M. Privai, T.K. Rao and V. Ray (1986) The Salmonella typhimurium/mammalian microsomal assay A report of the U.S. Environmental Protection Agency Gene-Tox Program Mutation Res. 138, 69-240 6. Lowry, Q.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent J. Biol. chem. 193,"265-275 ' 7. McCann, J. and B.N. Ames (1976) Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 Chemicals: Discussion. Proc. Natl. Acad. Sci. (USA) 73, 950-954 8. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 Chemicals. Proc. Natl. Acad. Sci. (USA) 72, 5135-5139 9. de Serres F.J. and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay Mutation Res. 64, 159-165 st5r page 34 of 35 C om pany Sanitized. Does not contain TSCA CBS DISTRIBUTION OF THE REPORT Sponsor Study Director 2x (original, copy) lx (copy) st5r page 35 of 35 Company Sanitized. D oes not contain TSC CSf