Document 0J3RaOB694jo0eYp9ygG9bVMR

AR226-3029 I) CCR PROJECT 509800 GENE MUTATION ASSAY IN CHINESE HAMSTER V79 CELLS IN VITRO ( V79/ HPRT) WITH REPORT Study Completion Date: October 06,1995 RCC Company Sanitized. Does not contain TSC Test Report CCR Project 509800 COPY OF GLP CERTIFICATE HESSISCHES MINISTERIUM FR UMWELT, ENERGIE, JUGEND, FAMILIE UND GESUNDHEIT GLP-Bescheinigung B eicheinigtm g C ertificate H ierm it w ird besttigt, da die P rfeinrichtungen) CCR C y to te s t Ceil R esearch GmbH & Co. KG in 6 4 3 8 0 R odorf, In den L epp stein sw iesen 19 (Ort, Anschrift) der RCC/CC R Holding Verwaltung GmbH (Firma) am 0 5 ./0 6 ./0 7 . April 1995 (D atum ) von d er fr die berwachung zustndigen Behrden ber die E inhaltung der G rundstze der G utes Laborpraxis inspiziert w orden is t (sind). Es wird hierm it besttigt, da folgende Prfungen in dieser Prfeinrichtung n ach den G rundstzen der Guten Laborpraxis durehgefhrt werden. I t is hereby certified th a t the te st facilitates) CCR C y to test Cell R esearch GmbH & Co. KG in 64380 Ro&dorf, In d en Leppsteinsw iesen 19 (location, address) o f RCC/CCR Holding V erw altungs GmbH (company name) . on 0 5 ./0 6 ./0 7 . April 1995 (date) w as (were) inspected by th e com petent au th o rity regarding com pliance w ith the Principles of Good Laboratory Practice. It is hereby certified th a t studies in this te st facility are conducted in com pliance with the Principles of Good Laboratory Practice. P rflfk a te g o ria n a e h S 19 d Aba. 3 C h e m ik a lie n g e se tz in der F assu n g vom 29.J u li 1994 (BGBl. I S. 1703), z u le tz t g e n d e rt mm 2 7 . S eptem b er 1994 (BGBl. I S. 2705) in V erb in d u n g m it d er A llgemeinen V erw altungsvorschrift zum Verfahreryder behrdlichen berwachung der Einhaltung der G rundstze der Guten L a b o rp rax is vom 21. O ktober 1990 (BAnz. 204 a vom 31.10.1990): Toxikologische Eigenschaften Toxicological properties P r fk ate g o rie gem OECD P anel o n Good L aboratory P ractice (January 1992) Prfungen auf toxikologische Eigenschaften P r fu n g en a u f m u ta g e n e E igensch aften (in vitro, in vivo] Toxicity stu d ies M utagenicity studies Im A uftrag 3^ iuJL f , (Dr. H ecker) W iesbaden, den August 1995 Company Sanitized. Do*--g not contain TSCA CB! -Page 2 of 29 - Test Report CCR Project 509800 CONTENTS COPY OF GLP CERTIFICATE PREFACE General Project Staff Schedule Project Staff Signatures Good Laboratory Practice Guidelines Archiving STATEMENT OF COMPLIANCE QUALITY ASSURANCE UNIT SUMMARY - ' Conclusion OBJECTIVE Aims of the Study Relevance of the Test System MATERIALS AND METHODS Test Article Controls Test System Mammalian Microsomal Fraction S9 Mix Pre-Test on Toxicity Dose Selection Experimental Performance Data Recording Acceptability of the Assay Evaluation of Results ' RESULTS Pre-Test on Toxicity RESULTS AND DISCUSSION REFERENCES DISTRIBUTION OF THE REPORT Annex: Tables of Results Experiment I Tables of Results: Experiment II DEVIATIONS TO THE PROTOCOL BIOMETRY 2 7 8 g g 10 10 10 11 H 12 12 13 14 14 16 18 18 ig 20 20 21 22 22 23 26 29 29 c n c n tn o i^ ^ ^ js,. not contain TSC& CBl C om paq Sanitize0*D eS -Page 3 of 29 - T est Report C C R Project 509800 PREFACE General Sponsor: Study Monitor: Testing Facility: CCR Project No.: Test Article: Title: ISEGA Forschungs- und Untersuchungsgesellschaft mbH Zeppelinstrae 3 D-63741 Aschaffenburg Dr, Derra CCR CYTOTEST CELL RESEARCH GMBH & Co. KG In den Leppsteinswiesen 19 D-64380 Rodorf, F.R.G. Gene Mutation Assay in in vitro (V79/HPRT) wit Project Staff Study Director: Management: Quality Assurance Unit Schedule Date of Protocol Start of Pre-Test: End of Pre-Test: Start of Experiments: End of Experiments: Date of Draft: Date of Final Report: Dr. Hans-Eric Wollny Markus Arenz Frauke Hermann April 13,1995 July 27, 1995 July 31, 1995 August 01, 1995 Sept. 11,1995 Sept. 13, 1995 Oct. 06, 1995 contitain TSCA CBl Company s r f W * Doe* n' -Page 4 of 29 - Test Report C C R Project 509800 Project Staff Signatures Study Director Dr. Hans-Eric Wollny Management Markus Arenz Date: October 06, 1995 Good Laboratory Practice The study was performed in compliance with: Chemikaliengesetz ("Chemicals Act") o f the Federal Republic of Germany, Anlage 1 ("Annex 1"), dated July 25, 1994 (BGBL. 1 1994 S. 1703)." "The OECD Principles of Good Laboratory Practice", Paris 1981. contain TSCA CSS Company Sanitized- Does no. -Page 5 of 29 - Test Report C C R Project 509800 Guidelines . This study followed the procedures indicated by the following internationally accepted ' guidelines and recommendations: Second Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 476, adopted April 4, 1984, "In vitro Mammalian Cell Gene Mutation Tests" EEC Directive 87/302, L 133, p. 61 - 63 Archiving C C-R, D-64380 RoBdorfiT.R.G. will archive the following data for 30 years: Raw data, protocol and copy o f report. The following sample will be archived for at least 2 years following the date on which the report is audited by the Quality Assurance Unit: sample of the test article No raw data or material relating to the study will be discarded without the sponsor s prior consent. Compaq -Page 6 of 29 - Test Report C C R Project 509800 STATEMENT OF COMPLIANCE Project Number: 509800 Test Material : Study Director: Dr. Hans-Eric Wollny Title: Gene Mutation Assay in Chinese Hamster_y79 Cells in vitro (V79/HPRT)w1 titmUx This study performed in the testing facility of C C R was conducted in compliance with Good Laboratory Practice Regulations. Chemikaiiengesetz ("Chemicals Act") o f the Federal Republic o f Germany, Anlage 1 ("Annex 1"), dated July 25, 1994 (BGBL. I 1994 S. 1703)." "The OECD Principles o f Good Laboratory Practice", Paris 1981." There were no circumstances that may have affected the quality or integrity of the study. Study Director CCR Dr. Hans-Eric Wollny Date: O cX o faO G , W f * rot contain TSCACB Sanitized. Does Company -Page 7 of 29 - Test Report CCR Project 509800 QUALITY ASSURANCE UNIT :C C R, Cytotest Cell Research GmbH & Co. KG, In den Leppsteinswiesen 19, D-64380 Rodorf, F.R.G. Statement Project Number: 509800 Test Material : Study Director: Title: ' Dr. Hans-Eric Wollny Gene Mutation Assay in in vitro (V79/HPRT) wi 79 Cells This report was audited by the Quality Assurance Unit and the conduct of this study was inspected on the following dates. Phases and Dates of QAU Inspections/ Audits Protocol Audit: Process Inspection Draft Audit: April 18, 1995 August 11, 1995 Sept. 22, 1995 Dates o f Reports to the Study Director and to Management April 18, 1995 August 11, 1995 Sept. 22, 1995 Head o f Quality Assurance Unit Frauke Hermann ........ i . . ^ . . i <k S . . ^ C ^ M . . . D ate: c w ^ Ofc Company Sanitized. Does not contain TSC CBi -Page 8 of 29 - Test Report CCR Project 509800 SUMMARY gn The study was performed to investigate the potential tations at the HPRT locus in V79 cells o f the Chinese hamster. induce gene mu The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. ' The test article was tested with the following concentrations: Experiment I: without S9 mix:1.0; 10.0*; 100.0; 200.0; 250.0* and 300.0 pg/ml with S9 mix:1.0; 10.0*; 100.0; 200.0; 250.0* and 300.0 pg/ml Experiment II: without S9 mix:1.0; 100.0; 200.0; 250.0* and 300.0 pg/ml with S9 mix:1.0; 100.0; 200.0; 250.0* and 300.0 pg/ml No relevant toxic effects occurred up to the limit of solubility and above. The highest con centration (300 pg/ml) resulted in a slight perturbation of the medium due to formation of small undisolved droplets. Survival at the lowest concentration was approximately in the range of the negative control. Up to the highest investigated concentration no relevant increase in mutant colony numbers was observed in both independent experiments. Appropriate reference mutagens were used as positive controls and showed a distinct in crease in induced mutant colonies. Conclusion In conclusion it can be stated that during the described mutagenicity test and under the ex perimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells. Therefore considered to be non-mutagenic in this HPRT assay. * not evaluated, culture not continued rscA C S i Company SanSiizedLDoes jt contain -Page 9 of 29 - Test Report C C R Project 509800 OBJECTIVE Aims of the Study This in vitro assay is performed to assess the potential o f the test article to induce gene mutations by means o f two independent HPRT experiments using the Chinese hamster cell line V79. Relevance of the Test System In vitro, methods are valuable when it is desirable to accurately control the concentration and exposure time of cells to the test article under study. However, due to the limited ca pacity for metabolic activation of potential mutagens an exogenous metabolic activation system is necessary. This in vitro test is an assay for the detection of forward gene mutations in mammalian cells. Gene mutations are considered to be an initial step in the carcinogenic process (2). The V79 cells are exposed to the test article both with and without exogenous metabolic activation. At a defined time interval after treatment the descendants o f the treated original population are monitored for the loss o f functional HPRT enzyme. HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6TG. These cells are able to proliferate in the presence o f 6TG whereas the non-mutated cells die. However, the mutant phenotype requires a period o f time before it is completely expressed. The phenotypic ex pression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expres sion period is terminated by adding 6TG to the culture medium (3). Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to deter mine the surviving cells. After a suitable period the colonies are counted. Mutant frequen cies are calculated from the number o f mutant colonies corrected for cell survival. In order to establish a concentration response effect o f the test article at least four concen tration levels are tested. These concentration levels should yield a concentration related toxic effect. The highest concentration level should induce a reduced level of survival. To demonstrate the sensitivity of the test system reference mutagens are tested in parallel to the test article. -tized. Does not contain rs c A c s i Compaq -Page ID of 29 - Test Report CCR Project 509800 MATERIALS AND METHODS Test Article The test article and the information concerning the test article were provided by the sponsor. Name: Batch No.: not indicated by the sponsor Aggregate State at RT: liquid . Colour: yellow Analysis: Purity: Stability Stability not indicated by the sponsor 35 % in Isopropanol and water pure: not indicated by the sponsor In solvent: not indicated by the sponsor Storage: 4 C Expiration Date: not indicated by the sponsor On the day o f the experiment (immediately before treatment), the test article was dissolved in Ethanol (E. MERCK, D-64293 Darmstadt; purity 99.8 %). The solvent was chosen ac cording to its solubility properties, and its non-toxicity for the cells. The final concentration of DMSO in the culture medium did not exceed 1 % v/v. ;not contain TSC GBl C o m p M SanKIzed. 0 -Pase 11 of 29- Test Report CCR Project 509800 Controls : Negative Controls Concurrent negative and solvent controls were performed. Positive Control Substances W ithout metabolic activation Name: Supplier: Catalogue no.: Dissolved in: ~ Final concentration: EMS; Ethylmethanesuifonate Merck-Schuchardt, D-85662 Mnchen, F.R.G. 820774; (Purity: > 98%) nutrient medium 0.6 mg/ml = 4.8 mM Solution prepared on day of experiment. With metabolic activation Name: Supplier: Catalogue no.: Dissolved in Final concentration: DMBA; 7,12-dimethylbenz(a)anthracene SIGMA CHEMIE GMBH, D-82041 Deisenhofen, F.R.G. D 3254; (Purity: approx. 95%) DMSO, Dimethylsulfoxide; final concentration in nutrient medium 1 % 3.85 pg/ml = 15.0 pM The stability o f both positive control substances in solution is unknown, but a mutagenic response in the expected mutation range is sufficient evidence of biological stability. The dilutions o f the stock solutions were prepared on the day of the experiment and used im mediately. Test System Reasons for the Choice of the Cell Line V79 The V79 cell line has been used successfully in in vitro experiments for many years. Espe cially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of negative control cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number o f 22 (3). Company Sanitized. D oss not contain TSC CBt -Page 12 of 29- Test Report C CR Project 509800 Cell Cultures Large stocks o f the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni cal University; D-64293 Darmstadt; F.R.G.) are stored in liquid nitrogen in the cell bank of C C R allowing the repeated use o f the same cell culture batch in experiments. Before free zing, the level of spontaneous mutants was depressed by treatment with HAT-medium as described in [3], Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters o f the experiments remain similar because o f the reproducible characteristics o f the cells. Thawed stock cultures are propagated at 37 C in 80 cm2 plastic flasks (GREINER, D72632 Frickenhausen, F.R.G.). About 5x10s cells are seeded into each flask with 15 ml of MEM (minimal essential medium; SEROMED, D -12247 Berlin, F.R.G.) supplemented with 10 % foetal calf serum (FCS; Boehringer Mannheim, 68261-Mannheim, F.R.G.). The cells are siibcultured-iwice weekly. The cell cultures are incubated at 37 C in a 4.5 % carbon dioxide atmosphere (95.5% air). For the selection of mutants the medium is supplemented with 11 pg/ml thioguanine (6TG, SIGMA GmbH, D-82041 Deisenhofen, F.R.G.). Mammalian Microsomal Fraction S9 Mix Lacking metabolic activities o f cells under in vitro conditions are a disadvantage o f assays with cell cultures as many chemicals only develop a mutagenic potential after metabolisation by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with mammalian liver microsome prepara tions (S9 mix). S9 (Preparation by C C R) The S9 liver microsomal fraction was obtained from the livers o f 8 - 12 weeks old male Wistar rats, strain Hanlbm (BRL, CH-4414 Fiillinsdorf weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Et tlingen, F.R.G.) in olive oil 5 days previously. After cervical dislocation the livers o f the animals were re-moved, washed in 150 mM KC1 and homogenised. The homogenate was diluted 1+3 in KC1 and centrifuged at 9,000 g for 10 minutes at 4 C. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80 C. Small numbers of the ampoules are kept at 20 C for up to several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 Mnchen: Bio-Rad protein assay, Catalogue 500 000 6 (6).' The protein concentration in the S9 preparation was 33.2 mg/ml (lot 220595). Company Sanitized. Does no! contain TSCA OB! -Pnge 13 of 29 - Test Report C CR Project 509800 S9 Mix . An appropriate quantity o f S9 supernatant was thawed and mixed with S9 cofactor solution ' to result in a final protein concentration o f 0.75 mg/ml in the cultures. Cofactors were ad ded to the S9 mix to reach following concentrations: 8 mM MgCl2 33 mM KC1 5 mM glucose-6-phosphate 4 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.(l). Pre-Test on Toxicity A pre-test was performed in order to determine the concentration range for the mutageni city experiments. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment. The following method was used in the pre-test: XTT-Assay: The XTT-assay is based on the cleavage o f the yellow tetrazolium salt XTT to form an or ange formazan dye by hydrogenase activity in active mitochondria. 18 - 20 h after treatment with the test article the XTT-assay was initiated by adding a mixture o f XTT-labeiling rea gent with an electron coupling reagent (PMS). After 4 h of incubation the absorption was determined at 450 nm (690 nm reference) using an ELISA reader (SLT, Labinstruments Austria, A-5082 Grodig). The viabilities of the cells are calculated as percentages o f the solvent controls and reported as tables. Company Sanitized- Does not contain TSCA CB1 -Pnpe 14 nf 7<J- Test Report CCR Project 509800 Dose Selection According to the recommendations of the guidelines (see page 6), several concentrations (usually at least four) o f the test article should be used. These should yield a concentrationrelated toxic effect. The highest concentration should produce a low level o f survival and the survival in the lowest concentration should approximate the negative control. Relatively insoluble substances should be tested up to their limit o f solubility under culture conditions. For ffeely-soluble nontoxic substances the maximum concentration should be as recommen ded in the in vitro cytogenetic assay, namely 5 mg/ml or 10 mM. If the maximum concen tration is based on cytotoxicity the cloning efficiency should be reduced to less than 50 % and/or culture growth at subcultivation should be at least 20% of the corresponding solvent control. In the pre-test o f toxicity (XTT-assay) the extinction (measured at 450/690 nm) was not significantly reduced after treatment with concentrations up to the limit o f solubility at 300 pg/ml with and without metabolic activation (see table PRE-TEST OF TOXICITY). Experiment I and II were performed with four concentrations ranging from 1.0 to 300.0 pg/ml with and without metabolic activation. Therefore, the test article was tested with the following concentrations: Experiment I: without S9mix: 1.0; 100.0; 200.0 and 300.0 pg/ml with S9mix: 1.0; 100.0; 200.0 and 300.0 pg/ml Experiment II: without S9mix: 1.0; 100.0; 200.0 and 300.0 pg/ml with S9mix: 1.0; 100.0; 200.0 and 300.0 pg/ml Company Sanitized. Does not contain TSCA CBI -Page 15 of 29 - Test Report CCR Project 509800 Experimental Performance Seeding Three days old exponentially growing stock cultures (more than 50 ^ confluent) were trypsinized at 37 C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentra tion for all subculturing steps was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Labo ratories, Detroit, USA). The Ca-Mg-free salt solution was composed as follows (per litre): NaCl KC1 Glucose NaHCO 8000 mg 400 mg 1000 mg 350 mg Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA (ethylene diamine tetraacetic acid). The cell suspension was seeded into plastic culture flasks (Greiner, D-72632 Frickenhau sen). Approximately 1.5xl06 (single culture) and 5 x l0 2 cells (in duplicate) were seeded in MEM with 10 % FCS (complete medium) for the determination o f mutation rate and toxi city, respectively (see experimental scheme). Treatment After 24 h the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 50 pl/ml S9 mix. After 4 h this medium was replaced with complete medium after two washing steps with "saline G '. The "saline G" solution was composed as follows (per litre): NaCl KC1 Glucose Na2HP04x7H20 KH2PO4 8000 mg 400 mg 1100 mg 290 mg 150 mg pH is adjusted to 7.2 Experimental Scheme: Segment a): Procedure for determination of toxicity Segment b): Procedure for determination of mutation rates Company -Page 16 of 29 - Test Report C C R Project 509800 Day 1: Subculturing o f a log-phase culture which showed an initial spontaneous mutation rate at the beginning of the experiment of 14.0 (experiment I) and 2.9 (experiment II) mutants per 10 cells. a) About 500 cells in 5 mi medium/25 cm2-plastic-flask for cloning efficiency; in duplicate per experimental point - b) lxlO6 cells in 30 ml medium/175 cm2-plastic-flask for the mutagenicity test, 1 flask per experimental point Day 2: Treatment of a) and b) experiment I Day"5: Subculturing ofb) in 175 cm2-plastic-flasks 1.5xl06 cells in 30 ml medium/175 cm2plastic-flasks experiment II Day 6: see day 5 Day 8: Fixation and staining o f colonies in a)-flasks determination of concentration-related cloning efficiency Day 9: Subculturing ofb) in five 80 cm2-plastic-flasks containing selective medium: mutant selection (about 3-5xl0s ceils/flask); subculturing ofb) in two 25 cm2-flasks for cloning efficiency (about 500 ceils/flask) Day 16: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (cloning ef ficiency). Day 18: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (mutant selection). The cultures were incubated at 37 C in a humidified atmosphere with 4.5 A>CO2. The co lonies were stained with 10 % methylene blue in 0.01 % KOH solution (E. MERCK, D64293 Darmstadt, F.R.G.). The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope (Nikon, D-40407 Dsseldorf, F.R.G.). Company Sanitized. Does not contain TSC CBI _D'w<a I 7 rvf 7Q Test Report CCR Project 509800 Data Recording The data generated were recorded in the raw data. The results are presented in tabular form, including experimental groups with the test article, negative and positive controls. Acceptability of the Assay The gene mutation assay is considered acceptable if it meets the following criteria: a) the numbers o f mutant colonies per 106 cells found in the negative and/or solvent con trols fall within the laboratory historical control data range: 0 - 4 5 mutants/10 cells. b) the positive control substances must produce a significant increase in mutant colony fre quencies. c) the cloning efficiency (absolute value) o f the negative and/or solvent controls must ex ceed 50 %. The data o f this study comply with the above mentioned criteria [a) and b) see mutation rate, tables III and VI, c) see tables II and V, factor calculated referring to the C.E. of the untreated cultures]. Sem pany Sanitized. D s^s not contain TSGA CBS tQrsf TO Test Report C C R Project 509800 Evaluation of Results A test article is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response for one o f the test points, A test article producing neither a concentration- related increase o f the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system. A significant response is described as follows: The test article is classified as mutagenic if it induces a reproducible mutation frequency that is at least three times higher than the spontaneous mutation frequency in the experiment at one or more o f the concentrations. The test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the correspon ding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0 - 45 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed. Sanfesd. Do not contain TSCACBI Company -Patte 19 of 29 - Test Report C C R Project 509800 RESULTS Pre-Test on Toxicity Without S9 mix: Blanc Neqative control Solvent control test article ~ test article test article test article test article test article test article test article concentration Mg/ml / / / 0.3 1.0 3.0 10.0 30.0 100.0 200.0 300.0 extinction (450/690 nm) % of the mean standard corresponding deviation control* 0.18 0.00 0.00 1.22 0.03 105.57 1.17 0.02 100.00 1.19 0.04 102.35 1.21 0.07 104.17 1.16 0.03 99.44 1.14 0.04 97.16 1.08 0.05 91.36 1.12 0.06 95.14 1.10 0.06 93.33 1.13 0.07 96.36 With S9 mix: Blanc Negative control Solvent control test article test article test article test article test article test article test article test article concentration pg/ml '/ / / 0.300 1.000 3.000 10.000" 30.000 100.000 200.000 300.000 -extinction (450/690 nm) % of the mean standard corresponding deviation control* 0.178 0.007 0.000 1.009 0.054 105.361 0.966 0.048 100.000 0.926 0.074 94.893. 0.914 - 0.069 93.306 0.872 0.038 87.977 0.852 0.032 85.534 0.829 0.030 82.568 0.775 0.024 75.763 0.716 0.032 68.181 0.688 0.063 64.724 * corrected with the blanc Company SanSis id. Does nui contain TSCA CBS -Pnve*.?() nf . Test Report C CR Project 509800 RESULTS AND DISCUSSION The test artici the HPRT loci_____w . . vas assessed for its potential to induce gene mutations at of the Chinese hamster. The study was performed in two independent main experiments, using identical procedures, both with and without liver microsomal activation. No relevant toxic effects occurred up to the limit o f solubility and above. In both experiments the numbers of mutant colonies per 106 cells did not exceed the values of the corresponding controls substantially and remained well within the historical range. Furthermore, there was no indication o f a concentration depend increase o f mutant colonies. In both experiments of this study (with and without S9 mix) the range of the negative con trols was from 3.0 up to 17.9 mutant colonies per 106 cells; the range o f the groups treated with the test article was from 5.7 up to 20.2 mutant colonies per 10 cells. EMS (0.6 mg/ml) and DMBA (3.85 pg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells. Company Sarr -Pasc 21 oP29 - Test Report C C R Project 509800 REFERENCES 1. B.N. Ames, J. McCann, and.E. Yamasaki Methods for detecting carcinogens and mutagens with the Salmonella/mammalian micro some mutagenicity test In: B.J. Kilbey et al. (Eds.) "Handbook of Mutagenicity Test Procedures", Elsevier, Amsterdam, 1-17, 1977 2. P. Howard-Flanders Mutagenesis in mammalian cells Mutation Research 86, 307-327, 1981 3. M.O. Bradley, B. Bhuyan, M.C. Francis, R. Langenbach,. Peterson and E. Huberman Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of the literature: A report of the gene-tox program Mutation Research 87, 81-142, 1981 4. S. Rettig Modellierung, Simulation und statistische Analyse des HGPRT-Mutagenittstests, The sis, Technical University o f Darmstadt, 1990 5. EEC Directive 92/69, L 383 A, Annex V, B 10, dated December 29, 1992 DISTRIBUTION OF THE REPORT Sponsor Study Director 2x (original, copy) lx (copy) -Page 22 ot 29 - Tesi Report CCR Project 509800 1, Annex: Tables of Results Experiment 1 Table I: Toxicity data; experiment I Compari; cone. per ml S9 number of cells per flask* mix seeded found mean l/ll I II CE%** CE*** absolute relative cells/ml cell den- at 1st sub- sity; % of cultivation control column 1 23 4 5 6 7' 8 9 10 Negative control (Untreated cells) 523 306 301 303.5 58.0 Negative control 0.00 pg - 523 236 274 255.0 48.8 100 2011333 100 Solvent control with Ethanol Positive control with EMS Test article 0.00 pg - 0.6 mg 1.00 pg - 523 214 523 189 523 253 266 201 v 245 240.0 195.0 249.0 45.9 37.3 47.6 100 2098000 76.5 103.8 1779333 2258667 100 88.5 107.7 Test article 10.00 pg - 523 203 244 223.5 42.7 93.1 2312000 110.2 Test article 100.00 pg - 523 234 214 224.0 42.8 93.3 1848667 88.1 Test article 200.00 pg - 523 197 184 190.5 36.4 79.4 1880667 89.6 Test article 250.00 pg - 523 193 183 188.0 35.9 78.3 1876667 89.5 m M Test article 300.00 pg - 523 175 204 189.5 36.2 79.0 1875333 89.4 N Negative control 0.00 pg + 523 269 214 241.5 46.2 100.0 2190000 100.0 a a Solvent control with DMSO 0.00 pg 523 252 214 233.0 44.6 100.0 2272000 100.0 o Solvent control with Ethanol 0.00 pg + 523 224 255 239.5 45.8 100.0 2468000 100.0 3 Ori* o o Positive control with DMBA 3.85 pg + 523 158 179 168.5 32.2 72.3 1542667 67.9 3**> Test article 1.00 pg + 523 200 195 197.5 37.8 82.5 2346667 95.1 3 Test article 10.00 pg + 523 186 211 198.5 38.0 82.9 2476667 100.4 --3 Test article <i> O Test article 100.00 pg + 200.00 pg + 523 207 523 241 226 216.5 223 232.0 41.4 44.4 90.4 96.9 2646000 2497333 107.2 101.2 > o 03 Test article Test article 250.00 pg + 300.00 p + 523 237 523 229 222 229.5 233 231.0 43.9 44.2 95.8 96.5 2060667 2444667 83.5 99.1 only colonies with more than 50 cells 7 days after seeding were scored CE absolute (value column 6 / value column 3 x 100 CE relative (value column 6 / value column 6 of corresponding control x 100) -Page 23 of 29 - Test Report CCR Project 509800 Table II: Mutagenicity data; experiment I (part 1: cell survival) cone. per ml S9 number of cells per flask* mix seeded found mean l/ll I II factor** cells calculated seeded cells*** survived column 1 23 4 5 6 7 8 9 Negative control Solvent control with DMSO 0.00 pg 0.00 pg - 505 296 301 298.5 513 303 353 328.0 0.59 0.64 435000 414000 57124 264702 Positive control with EMS 0.6 mg - 504 318 325 321.5 0.64 414000 264089 Test article Test article 1.00 pg 10.00 pg - 504 347 365 356.0 0.71 culture was not continued* 450000 317857 Test article T est article Test article 100.00 pg 200.00 pg 250.00 pg - 500 325 360 342.5 511 398 406 402.0 0.69 0.79 culture was not continued* 408000 402000 279480 316250 Test article Negative control Solvent control with DMSO 300.00 pg 0.00 pg + 0.00 pg + 503 380 503 381 500 336 410 395.0 409 395.0 328 332.0 0.79 0.79 0.66 426000 414000 478500 334533 325109 317724 Solvent control with Ethanol 0.00 pg 509 362 372 367.0 0.72 396000 285525 Positive control with DMBA 3.85 pg + 502 291 302 296.5 0.59 432000 255155 Test article Test article Test article 1 est article Test article Test article 1.00 pg 10.00 pg 100.00 pg 200.00 pg 250.00 pg 300.00 pg + + t* + + + 507 345 342 343.5 0.68 culture was not continued* 511 366 349 357.5 0.70 502 375 362 368.5 0.73 culture was not continued* 507 358 317 337.5 0.67 463500 420000 486000 522000 314028 293836 356755 347485 ** * ------- -- " *" ---- * ' U*M< UVUWUlftftg |TViW factor calculated (value column 6 / value column 3) CU * * * cells survived after plating in TG containing medium (value column 8 / value column 7) # concerning concentration range and toxicity four concentrations were selected to be evaluated at the end of the experiment Company Sanitized. Doss nof contain TSC CB -Page 24 of 29 Test Report CCR Project 509800 U Table III: Mutagenicity data; experiment I (part 2: mutation rates) cone. per ml S9 mix column Negative control Solvent control with DMSO Positive control with EMS Test article T est article Test article Test article Test article Test article Negative control Solvent control with DMSO Solvent control with Ethanol Positive control with DMBA Test article Test article Test article Test article Test article Test article 12 0.00 pg 0.00 pg - 0.6 mg - 1.00 pg 10.00 pg 100.00 pg 200.00 pg 250.00 pg 30.00 pg 0.00 pg + 0.00 pg 0.00 pg + 3.85 pg + 1.00 pg 10.00 pg 100.00 pg 200.00 pg 250.00 pg 300.00 pg + + + + + number of mutant colonies per flask* found after platinq in TG medium I II III IV V 34 5 6 7 67 2 5 52 4 3 mean 8 standard mutant** deviation colonies per 106 cells 9 10 3 4.6 2.1 , 17.9 3 3.4 1.1 12.8 137 118 117 151 136 131.8 57 8 4 4 5.6 culture was not continued* 63 5 6 3 4.6 3 5 6 11 3 5.6 culture was not continued* 75 4 5 9 6.0 26 3 4 4 3.8 98 1 6 4 5.6 14.3 1.8 1.5 3.3 2.0 1.5 3.2 499.1 17.6 16.5 17.7 17.9 11.7 17.6 44 5 2 5 4.0 1.2 14.0 107 5 1 7 4| 117 103 103 43 3 culture was not continued* 44 3 10 6 6 culture was not continued* 11 61 5 89 103.8 5 4.0 2 2.8 7 7.2 3 3.8 10.1 1.0 1.3 1.6 1.9 406.8 12.7 9.5 20.2 10.9 only colonies with more than 50 cells 7 days after seeding were scored value column 8 (this page) x 10* / value column 9 o f table II concerning concentration range and toxicity four concentrations were selected to be evaluated at the end of the experiment Company Sanitized. Does not contain TSCA CBI -Page 25 of 29 - Company Sanitized. Doss not contain TSCA CBI Test Report CCR Project 509800 Tables of Results: Experiment II Table I: Toxicity data; experiment II cone. per ml S9 number of cells per flask* mix seeded found mean l/ll I II CE%** CE*** absolute relative cells/ml cell den- at 1st sub- sity; % of cultivation control column 1 23 4 5 6 7 8 9 10 Negative control (Untreated cells) 552 377 402 389.5 70.6 Negative control Solvent control with Ethanol Positive control with EMS 0.0 pg 0.00 pg 0.6 mg - 552 308 552 334 552 263 304 306.0 261 297.5 229 246.0 55.4 53.9 44.6 100 100 80.4 2329333 2437333 2336667 100 100 100.3 Test article Test article Test article T est article Test article Negative control Solvent control with DMSO Solvent control with Ethanol Positive control with DMBA Test article Test article T est article T est article Test article 1.00 pg 100.00 .pg 200.00 pg 250.00 pg 300.00 pg 0.00 pg - 0.00 pg + 0.00 pg + 3.85 pg + 1.00 pg 100.00 pg 200.00 pg 250.00 pg 300.00 pg + -t* + + 552 326 552 283 552 297 552 256 552 293 501 244 501 201 501 207 501 146 501 194 501 215 501 217 501 195 501 214 310 318.0 251 267.0 281 289.0 334 295.0 291 292.0 196 220.0 197 199.0 213 210.0 152 149.0 208 201.0 200 207.5 222 219.5 219 207.0 226 220.0 57.6 48.4 52.4 53.4 52.9 43.9 39.7 41.9 29.7 40.1 41.4 43.8 41.3 43.9 106.9 89.7 97.1 99.2 98.2 100.0 100.0 2626667 2175333 1870000 2176000 2710667 2995333 3155333 100.0 3060000 74.9 95.7 98.8 104.5 98.6 104.8 2293333 2958000 2173333 1767333 2904667 3184667 107.8 89.3 76.7 89.3 111.2 100.0 100.0 100.0 72.7 96.7 71.0 57.8 94.9 104.1 only colonies with more than 50 cells 7 days after seeding were scored CE absolute (value column 6 / value column 3 x 100 CE relative (value column 6 / value column 6 o f corresponding control x 100) -Page 26 of 29 - ) ig8 V. Test Report CCR Project 509800 cone. per ml S9 number of cells per flask* mix seeded fc und mean l/ll I II factor** ceils calculated seeded survived column 1 23 4 5 6 7 Negative control Solvent control with DMSO Positive control with EMS 0.00 pg 0.00 pg 0.6 mg - 501 347 341 344.0 519 400 423 411.5 512 314 362 338.0 0.69 0.79 0.66 Test article 1.00 pg - Test article 100.00 pg - Test article 200.00 pg - Test article 250.00 pg - Test article 300.00 pg Negative control ~ 0.00 ug ...+ Solvent control with DMSO 0.00 pg Solvent control with Ethanol 0.00 pg 500 366 348 357.0 512 355 370 362.5 511 385 356 370.5 0.71 0.71 0.73 culture was not continuiid* 510 394 399 396.5 0.78 501 419 389 404.0 0.81 502 370 355 362.5 0.72 501 353 348 350.5 0.70 Positive control with DMBA 3.85 pg + 508 338 306 322.0 0.63 Test article Test article Test article Test article Test article 1.00 pg 100.00 pg 200.00 pq 250.00 pg 300.00 pq + + + + 502 364 502 381 500 AC\Q 349 356.5 385 383.0 anUOo ______*5eo.U culture was not continue`d* 504 .....3571 377] 36T1 0.71 0.76 0.79 073 *** only colon]es with more hnn *** factor calculated (value column 6 / value column 3) cells survived after plating in TG containing medium (value column 8 / value column 7) 8 390000 414000 456000 499000 390000 408000 390000 414000 385500 387000 390000 387000 414500 445000 433500 9 267784 328249 301031 356286 276123 295820 303206 333844 278374 270746 247205 0 7 A R * ir> 316242 352440 315664 concerning concentration range and toxicity four concentrations were selected to be evaluated at the end :zed. Doss not contain TSCA CBI Table III: Mutagenicity data; experiment II (part 2: mutation rates) -Page 27 of 29 - Test Report CCR Project 509800 cone. per ml S9 number of mutant colonies per flask* mean standard mutant** mix found after plating in TG medium deviation colonies I II III IV V per 106 cells column 1 23 4 5 6 7 89 10 Negative control 0.00 pg - 22 1 4 4 2.6 Solvent control with DMSO 0.00 pg - 58 1 5 4 4.6 Positive control with EMS 0.6 mg - 122 123 136 140 142 132.6 Test article 1-00 pg - 13 1 0 3 1.6 Test article 100.00 pg - 12 1 4 1 1.8 Test article 200.00 pg - 73 2 1 2 3.0 Test article 250.00 pg - culturevwas not continued* Test article 300.00 pg - 34 3 2 4 3.2 Negative control 0.00 pg + 01 1 1 2 1.0 Solvent control with DMSO 0.00 pg + 11 2 1 1 1.2 Solvent control with Ethanol 0.00 pg 22 1 2 2 1.8 Positive control with DMBA 3.85 pg + 119 106 116 130 133 120.8 Test article Test article Test article Test article Test article 1.00 pg .100.00 pg 200.00 pg 250.00 pg 300.00 pg + + + + i: 0 5 1 Sl 13 66 2 4 23 1 culture was not continued* 4| 2[ 5| 2 2 3 6 1.6 4.6 2.0 4.4 1.3 2.5 9.5 1.3 1.3 2.3 0.8 0.7 0.4 0.4 10.9 1.1 1.7 1.0 1.5 9.7 14.0 440.5 4.5 6.5 10.1 10.6 3.0 4.3 6.6 488.7 5.8 14.5 5.7 13.9 * only colonies with more than 50 cells 7 days after seeding were scored ** value column 8 (this page) x 106 / value column 9 of table II # concerning concentration range and toxicity four concentrations were selected to be evaluated at the end y Sanitized. Doss nsi contain TSC CBS -Page 28 of 29 - Test Report CCR Project 509800 DEVIATIONS TO THE PROTOCOL There were no deviations to the protocol. BIOMETRY- Since the distribution o f mutant cells does not follow known statistical models, an adequate statistical method is not available. D ie b er ein stim m u n g m it dem O r ig in a l w ir d b e s t t i g t . Company aszed. Bogs not conta-n TSCA CBS -Page 29 of 29 -