Document XzoZQ6RkYqemExerLMLoQGbzG

CCR Cytotest Cell R esearch GmbH & Co. KG AR226-2983 CCR PROJECT 326417 SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY WITH Study Completion Date: February 17,1993 REPORT CCR In den Leppsteinswiesen 19 D-6101 Rodorf F.R.G. Telephone: 0 61 5 4 - 8 0 7-0 - Telefax 0 61 5 4 - 8 33 9 9 ' Company Sanitized. Does not contain TSCA CBI COPY OF GLP CERTIFICATE HESSISCHES MINISTERIUM FR UMWELT, ENERGIE UND BUNDESANGELEGENHEITEN G L P-B escheinigu ng B e sch ein ig u n g Hiermit wird besttigt, da die Prfungseinrichtung(en) C ytotest Cell Research GmbH & Co KG In den Leppsteinswiesen 19 " in 6101 Rodorf (On, Aracfwm) . riar RCC Holding Verwaltung GmbH (Finna) a m ..^ J L -'.JL4 -0L.* 05-08-- und.0 6 .0 8 .9 2 (Datum) von der fr die berwachung zustndigen Behrde Ober . . die Einhaltung der Grundstze der Guten Laborpraxis inspiziert worden ist (sind). E s wird hiermit besttigt, da folgende Prfungen in dieser Prfeinrichtung nach den Grundstzen der Guten Laborpraxis durchgefhrt werden. . Toxikologische Eigenschaften. r^JtUSTE Certificate It is hereby certified that the test fadiity(ies) C ytotest Cell Research GmbH & Co KG In d e n T e p p stiT i^ in 6101 Rodorf . poeuion, tddms) n, RCC Holding Verwaltung GmbH ' (companyrana) 0 3 .0 8 .,. 0 4 ,0 8 ., 0 5 .0 8 . and 0 6 .0 8 .9 2 0 0 ............ ...................... .......................... - r r i n I, , . '(tBl . " wa3 (were) inspected by the competent authority regar ding compliance with the Principles of Good Laboratory Practice. It is hereby certified that studies in this test facility are conducted in compliance with the Principles of Laboratory Practice. Toxicological, properties Im Auftrag . CPs. J(zt.2ujr (D r.-Hecker) ' Wiesbaden, den *2 $ st5r page 2 of 35 Company Sanitized. Does not contain TSCA CBI CONTENTS PAGE COPY OF GLPCERTIFICATE 2 .`PREFACE General Project Staff Schedule Project Staff Signatures Quality Assurance Guidelines Archiving 4 4 4 4 5 5 5 5 STATEMENT OF COMPLIANCE 6 QUALITY ASSURANCE UNIT Statement 7 7 SUMMARY Conclusion 8 8 OBJECTIVE Aims of the Study Reasons for the Study 9 9 9 MATERIALS AND METHODS The Test Article The Controls The Test System Mammalian Microsomal Fraction S9 Mix Pre-Experiment for Toxicity 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 Company Sanitized. Does not contasn sSCA CBS PREFACE GENERAI, Sponsor: Monitoring: Testing Facility: CCR Project No.: Test' Article: Title: ISEGA Forschungs- u. Untersuchungs Gesellschaft mbH Zeppelinstr. 3-5 D-8750 Aschaffenburg Heike Krmer CCR CYTOTEST CELL RESEARCH GMBH & CO. KG D-6101 Rodorf, F.R.G. 326417 Salmonella typhimurium Reverse Mutatior^Assay with PROJECT STAFF Management: Dr. H.- E. Knoell Study Director: Dr. Albrecht Poth Quality Assurance Unit: Dr. Ch. Helmrich SCHEDULE Date of Protocol: 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 It December 15, 1992 End of Experiment I: December 18, 1992 Start of Experiment II: January 05, 1993 End of Experiment II: January 15, 1993 Date of Draft: January 18, 1993 Date of Report : February 17, 1993 st5r page 4 of 35 Company Sanitized. Does not contain TSCA CBI PROJECT STAFF SIGNATURES Study Director: D r . Albrecht Poth & e .d Date: February 17, 1993 Management: QUALITY ASSURANCE The study was performed in compliance with: Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, Anlage 1 ("Annex 1"), dated March 14, 1990 (BGBL. 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 May 26, 1983 and EEC Directive 79/831, Annex V, B 14. ARCHIVING C C R, D-61Q1 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 Saniite!. Does not contain TSCACB1 Company STATEMENT OF COMPLIANCE Project Number: 326417 Test Article Study Director: Dr. Albrecht Poth Title Salmonella typhimurium Reverse Mutation Assay with To the best-of my 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 Company Sanitized.Dogs notconta',TMTSCA CBI 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 I 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 Dates of Reports to the StudyDirector and to Management November 11, 1992 December 15, 1992 January 21, 1993 / November 11, 1992 December 15, 1992 January 21, 1993 Head of Quality Assurance Unit D r . Ch. Helmrich Date: st5r page 7 of 35 Company Sanitized. uooJ cu,iia'n 1 U CSt SUMMARY This study was performed to investigate the potential of V p H t o induce gene mutations according to the plate incorporation .`test (experiment I) and the pre-incubation test (experiment II) . using the Salmonella typhimurium strains TA 1535, TA 1537, TA. 98, TA 100, and TA 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 jj.g/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 pg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of 3t h e f i v e t e s t e r strains were observed following treatment with B a t any dose level, either in the presence or absence 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. T h e r e f o r e i s considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay. st5r page 8 of 35 Company Sanitized. D oes not contain TSCA CBS OBJECTIVE AIMS OF THE STUDY The experiments were performed to assess the potential of the test article to induce gene mutations by means of two independent Salmonella typhimurium reverse mutation assays. Experiment I was performed as a plate incorporation assay. As a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay. REASONS FOR THE STOP? The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations. However, the b'acteria 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 is added in 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). Reverse mutation assays determine the frequency at which an agent 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 mutagen 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. ' The Salmonella typhimurium histidine (his) reversion system measures his- -- > his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100, TA 102) and frameshift (TA 1537, TA 98) mutations. According to the direct plate ` incorporation and the pre 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 quately spaced intervals were tested. The maximum dose level was 5000.0 pg/plate, unless limited by toxicity or solubility of the test article. To validate the test, reference mutagens were tested in parallel to the test article. st5r page 9 of 35 Company Sanitized. Does not contain TSCA CB1 MATERIALS AND METHODS THE TEST ARTICLE The test article and the information concerning the.test article were provided by the sponsor. Name: Batch N o . : Aggregate State at RT: Colour: Analysis : Purity: Stability: liquid Storage : 4C Expiration Date: not indicated by the sponsor &On the day of the experiment, the test a r t i c l e i m ^ ^ H 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 Company Sanitized. Does not contain TSC CBl Test Report CCK project Ab>ixi 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: Concentration: TA 1535, TA 100 sodium azide, NaN SERVA, D-6900 Heidelberg, F.R.G. 30175 at least 99 % aqua dest. 10 ng/plate Strains: Name: Supplier: Catalogue N o .: Purity: Dissolved in: Concentration: 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, jil/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 ng/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 Test Keport CUR Project J^b4/ THE TEST SYSTEM Characterisation of the Salmonella typhimurium 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 al. (1). In this way 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. Does not contain TSCA CBI it iti U ite p v jj. L. t-i-U J tili L. J ^ D i i 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 The overlay agar contains per litre: 6.0 g Merck AgarAgar 6.0 g NaCl 10.5 mg L-histidine x HC1 x H2O 12.2 mg biotin Sterilisations were performed at 121 C in an autoclave. st5r page 13 of 35 Company Sanitized. D oss not contain TSC CBI T e s t tceport cuk p r o je c t; jzb^tx / 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. 150 - 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 numbers 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 Mnchen: 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 mM KC1 5 mM glucose-6-phosphate 5 mM NADP in 100 mM 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 SanWMd. Boss Company 0 ,1 xeb L . i t e p u i ' L ouxx x x u j t s c t / PRE-EXPERIMEHT FOR TOXICITY To evaluate the toxicity of the test article a prestudy was performed with strains TA 98 and TA 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 may 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 abo-fre, these data are reported as a part of the main experiment I. st5r page 15 of 35 TSCACompany Sanitized. Coes not contain CB aoou. u c p u x u r x u je u L JZD^X / EXPERIMENTAL PERFORMANCE For each strain and dose level, including the controls, a minimum of three plates were used. ;The following materials were mixed in a test tube and poured onto the selective agar plates: 100 pi 500 pi 100 pi 2000 ul 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 pi test solution, 500 pi S9 mix / S9 mix substitution buffer and 100 pi 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 tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 Q in the dark. DATA RECORDING The colonies were counted using the ADTOCOUNT (Artek Systems Corporation, BIOSYS 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 deviations 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 Sanitized. Does not contain T SC ftC B i Compaq u IVUJ^UX. u uvU r J.UJCUt. J^u*x / 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 TA 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 Com pany Sanitized. Doe-3not contain TSC A C B i xeL xuspyxu l u i rxrujecT* BIOMETRY :No appropriate statistical method is available (5). / st5r page 18 of 35 Company Sanitized. D o ss not contain TSCA CBS xca u X UUU JCJ-WJCV-. u t 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 pg/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; 353.3; 1000.0; 2500.0 and 5000.0 pg/plate st5r page 19 of 35 Company Sanitized. Does not contain TSCA CBi Test Keport c u k jeroject nii/ TABLES OF RESULTS EXPERIMENT I PLATE INCORPORATION TEST st5r page 20 of 35 Company Sanitized,Does notcontain TSCA CBl Test article S9 mix from : rat liver (Batch R 060792) Test strain : TA 1535 Dose pg/plate Plate 12 3 Revertants/plate mean 's .d . factor+ 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) 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 14 1.0 17 2.9 1.0 21 2.1 1.3 16 2.0 1.0 14 3.1 0.8 15 2.1 0.9 13 5.3 15 3.5 0.9 1152 23.1 69.1 00 o with S9 mix Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 Positive control 2-Aminoanthracene (2.5pg/plate) + enhancement factor 14 15 2/1 22 26 21 27 26 22 26 19 21 21 30 24 21 19 27 21 22 20 19 28 21 135 153 190 I revertants/concentr. test article X revertants/solvent control 16 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 6.8 str5 21 of 35 C om pany S a n i t i z e d . D oss not contain TSCA CB Test article: S9 mix from : rat liver (Batch R 060792) Test strain : TA 1537 Dose ng/plate Plate 123 without S9 mix Negative control Solvent control 33.3 1-00. a _- 333.3 1000.0 2500.0 5000.0 Positive control 4-Nitro-ophenylene-diamine (50pg/plate) 8 10 5 487 787 6 8 11 466 546 875 945 307 311 350 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|ig/plate) 115 15 11 19 13 12 11 14 11 110 11 12 8 13 12 11 6 10 93 + enhancement factor I revertants/concentr. test article I revertants/solvent control Revertants/plate mean s .d. factors- 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 Sani llzed. D oes not contain TSC A CBl Test article: S9 mix from : rat liver (Batch R 060792) Test strain : TA 98 Dose ng/plate Plate 12 3 without S9 mix Negative control Solvent control 33.3 100.0 -* 333.3 1000.0 2500.0 5000.0 42 35 35 27 33 31 28 24 24 31 35 36 24 37 37 32 26 17 19 26 39 28 28 33 Positive control 4-nitro-ophenylene-diamine (50 fil/plate) 2464 2501 2660 with S9 mix Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 Positive control 2-Aminoanthracene (2.5ng/plate) 38 / 47 29 43 42 25 30 29 520 44 34 30 40 41 26 34 23 454 43 40 45 45 33 21 31 24 751 + enhancement factor X revertants/concentr. test article X revertants/solvent control Revertants/plate mean 's.d. factor* 37 4.0 30 3.1 25 2.3 34 2.6 33 7.5 25 7.5 28 10.1 30 2.9 1.0 0.8 1.1 1.1 0.8 0.9 1.0 2542 104.1 .83.8 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 str5 23 of 35 TSCA CBS , Does not con`am Compaq Sanitize I! I ; Test article S9 mix from : rat liver (Batch R 060792) Test strain : TA 100 Dose ng/plate Plate 123 Revertants/plate mean s .d. factor* without S9 mix Negative control Solvent control 33.3 l-00`.0 _- 333.3 1000.0 2500.0 5000.0 Positive control Sodium azide (10{ig/plate) 78 82 68 80 76 89 63 86 80 63 79 75 55 65 70 50 50 51 38 53 58 45 63 64 946 942 886 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 8 114 90 82 94 89 96 71 93 102 77 80 56 67 Positive control 2-Aminoanthracene (2.5ug/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 revertants/concentr. test article revertants/solvent control str5 24 of 35 ^ i i z o d . D o e s not conrtnTSCA ' o n ^ y wiU Test article S9 mix from : rat liver (Batch R 060792) Test strain : TA 102 Dose jig/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.Opl/plate) 200 207 208 206 225 211 181 178 154 179 244 199 109 179 188 171 151 190 237 228 212 199 201 209 827 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 (2.5jig/plate) 752 296 318 312 262 291 289 272 284 861 243 357 343 324 292 251 289 295 864 enhancement factor l revertants/concentr. test article revertants/solvent control Revertants/plate mean s.d. factor+ 205 4.4 214 9.8 171 14.8 207 33.3 159 43.2 171 19.5 226 12.7 203 5.3 1.0 0.8 1.0 0.7 0.8 1.1 0.9 981 142.2 4.6 273 27.2 309 52.5 302 46.3 301 34.2 316 41.9 248 42.6 282 8.7 264 43.9 1.0 1.0 1.0 1.0 0.8 0.9 0.9 826 63.8 2.7 str5 25 of 35 Company Sanitized, o o e # t\0'! contain TSCA CB8 TABLES OF RESULTS EXPERIMENT II PRE-INCUBATION TEST st5r page 26 of 35 Com pany Sanitized. D oes not contain TSCA COI Test article: S9 mix from : rat liver (Batch R 060792) Test strain : TA 1535 Dose ng/plate Plate 123 without 59 mix Negative control Solvent control 33.3 100'. 0 -' 333.3 1000.0 2500.0 5000.0 Positive control Sodium azide (10jj.g/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 24 17 13 31 24 27 24 26 18 21 24 25 31 26 31 28 28 29 28 27 30 22 20 17 Positive control 2-Aminoanthracene 163 187 167 (2 .5ng/plate) + enhancement factor I revertants/concentr. test article t revertants/solvent control Revertants/plate mean 's .d. factort 20 1.5 20 1.7 1.0 19 3.2 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 .tized. e s ` Compaq Sar*` un TSC CB'l Test article S9 mix from : rat liver (Batch R 060792) Test strain : TA 1537 Dose ng/plate Plate 123 without S9 TTiiyr Negative control Solvent control 33.3 100.0 333.3 "* 1000.0 2500.0 5000.0 Positive control 4-Nitro-ophenylene-diamine (50(ig/plate) with S9 mix 447 879 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 (2.5jxg/plate) Revertants/plate mean s .d . factor+ 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 factor revertants/concentr. test article revertants/solvent control s tr 5 28 of 35 rtfized. Doss n!0! contain TSCACB! Test article S9 mix from : rat liver (Batch R 060792) Test strain : TA 98 Dose pg/plate Plate 123 without S9 mix Negative control 22 30 25 Solvent control 23 27 26 33.3 . 18 29 31 100.0 19 23 25 333.3 -" 19 18 20 1000.0 20 25 20 2500.0 17 19 19 5000.0 17 13 26 Positive control 4-nitro-ophenylene-diamine (50 fig/plate) with S9 mix 1856 1874 1940 Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 42 39 33 39 45 43 43 37 45 39 44 42 33 46 45 38 41 33 37 41 33 45 33 44 Positive control 2-Aminoanthracene 448 352 335 (2.5fig/plate) + enhancement factor X revertants/concentr. test article X 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 str5 29 of 35 not contain TSCA CB1 Company SanlCzacL Boas Test article:, S9 mix from : rat liver (Batch R 060792) Test strain : TA 100 Dose jig/plate Plate 12 3 Revertants/plate mean s .d. factor* without S9 mix Negative control Solvent control 33.3 100.0 333.3 ~ 1000.0 2500.0 5000.0 Positive control Sodium azide (10p.g/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 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 mix 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 90 97 89 92 80 98 97 87 105 75 74 88 84 84 78 Positive control 2-Aminoanthracene (2.5ng/plate) enhancement factor 1190 845 1089 t revertants/concentr. test article I revertants/solvent control 96 9.0 98 10.1 85 6.1 99 3.2 93 10.4 75 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 of 35 Uzed.Coesnotc-o-.'*tain Com paq Sa'* 3! Test article: S9 mix from : rat liver (Batch R 060792) Test strain : TA 102 Dose pg/plate 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 pl/plate) with S9 mix Negative control Solvent control 33.3 100.0 333.3 1000.0 2500.0 5000.0 Positive control 2-Aminoanthracene (2.5|ig/plate) Plate 123 151 171 179 158 175 167 178 166 169 188 175 176 198 177 187 188 179 189 190 156 178 159 167 189 998 1034 897 260 219 270 197 218 245 233 210 233 217 219 245 194 201 206 224 199 213 240 216 230 237 256 174 811 924 702 + enhancement factor revertants/concentr. test article revertants/solvent control Revertants/plate mean s.d. factort 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 27.0 220 24.1 225 13.3 227 15.6 200 6.0 212 12.5 229 12.1 222 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 Garaged. notcontain TSCA CBl 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 1000.0 15 2500.0 ~ 13 5000.0 15 20 20 19 17 18 11 12 16 85 68 79 8 10 59 5 9' 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 (50|xg/plate) methyl methan sulfonat (l.Opl/plate) 323 211 2542 1890 925 1181 981 976 with S9 mix Revertants/plate mean from three plates Dose TA 1535 ixg/plate I / II TA 1537 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 (2.5fig/plate) s tr5 32 of 35 .-.^tnT SC A C B l CONCLUSIONS The test article 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, TA 98, TA 100, and TA 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 pg/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 jxg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of '' _ ' ':er strains were observed following treatment with .t any dose level, either in the presence or absence or m e i a D o u c 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 lompanS Sam:z2 "iialn TSCA CB 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, O.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 Sanitized. Boss noi contain TS*iACBI Company DISTRIBUTION OF THE REPORT Sponsor Study Director 2x (original, copy) lx (copy) & S' st5r page 35 of 35 Company Sanitized. Dose net contain TSCA