Document 8X47ydxaZLyE1vdxbe0QGwQy

Ar s s Q> _ \ 5 & 0 SRPTT-7^1 MIN 314/022208 PERFLUOROOCTANESULFONYL FLUORIDE (POSF) -p ' BACTERIAL REVERSE MUTATION TEST Sponsor 3M Center 3M Corporate Toxicology Building 220-2E-02 St Paul MN 55133-3220 USA Research Laboratory Huntingdon Life Sciences Ltd. Woolley Road Alconbury Huntingdon Cambridgeshire PE28 4HS ENGLAND Report issued 23 October 2002 Paee 1 o f29 001570 MIN 314/022208 CONTENTS Page COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS............................... 3 QUALITY ASSURANCE STATEMENT........................................................................................ 4 RESPONSIBLE PERSONNEL......................................................................................................... 5 SUMMARY ....................................................................................................................................... 6 INTRODUCTION.............................................................................................................................. 7 TEST SUBSTANCE .......................................................................................................................... 9 EXPERIMENTAL PROCEDURE................................................................................................... 10 ASSESSMENT OF RESULTS ........................................................................................................ 14 DEVIATIONS FROM PROTOCOL................................................................................................ 14 MAINTENANCE OF RECORDS ................................................................................................... 15 RESULTS.......................................................................................................................................... 16 CONCLUSION ................................................................................................................................. 16 REFERENCES.................................................................................................................................. 17 TABLES 1. Results obtained with S. typhimurium TA98: test 1 (range-finding) ..................................... 18 2. Results obtained with S. typhimurium TA98: test 2, with pre-incubation ............................. 19 3. Results obtained with 5. typhimurium TA100: test 1 (range-finding).................................... 20 4. Results obtained with S. typhimurium TA 100: test 2, with pre-incubation ........................... 21 5. Results obtained with S. typhimurium TA1535: test 1 (range-finding)............................... 22 6. Results obtained with S.typhimurium TA1535: test 2, with pre-incubation .......................... 23 7. Results obtained with S. typhimurium TA1537: test 1 (range-finding)............................... 24 8. Results obtained with S.typhimurium TA1537: test 2, with pre-incubation .......................... 25 9. Results obtained with E. coli WP2vrA/pKM101 (CM891): test 1 (range-finding)............... 26 10. Results obtained with E. coli WP2wvrA/pKM101 (CM891): test 2, with pre-incubation.... 27 APPENDICES 1. Historical control data............................................................................................................... 28 2. Eye Research Centre GLP Compliance Statement 2001 ......................................................... 29 01571 :2: MIN 314/022208 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS The study described in this report was conducted in compliance with the following Good Laboratory Practice Standards, with the exceptions stated below, and I consider the data generated to be valid. The UK Good Laboratory Practice Regulations 1999 (Statutory Instrument No. 3106). EC Commission Directive 1999/11/EC o f 8 March 1999 (Official Journal No. L 77/8). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. In line with normal practice in this type of short-term study, the protocol did not require analysis of the dose form. The expiry date o f the test sample was the Sponsor's responsibility. Study Director, Huntingdon Life Sciences Ltd. Date 001572 :3 : MIN 314/022208 QUALITY ASSURANCE STATEMENT The following inspections and audits have been carried out in relation to this study: Study Phase Protocol Audit Process Based Inspections S9 Preparation Formulation and Treatment Plate Scoring Report Audit Date of Inspection 16 October 2001 IS January 2002 31 October 2001 24 October 2001 4 February 2002 Date of Reporting 16 October 2001 IS January 2002 31 October 2001 24 October 2001 5 February 2002 Protocol Audit: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Process Based Inspections: At or about the time this study was in progress inspections o f routine and repetitive procedures employed on this type of study were carried out. These were conducted and reported to appropriate Company Management as indicated above. Report Audit: This report has been audited by the Quality Assurance Department This audit was conducted and reported to the Study Director and Company Management as indicated above. The methods, procedures and observations were found to be accurately described and the reported results of this study to reflect the raw data. Angela M/dennings, B.Sc., M.Sc., Ph.D., M.R.Q.A., Group Manager, Department of Quality Assurance, Huntingdon Life Sciences Ltd. =03 Date == 2 O O < 0 001573 :4 : RESPONSIBLE PERSONNEL Kenneth May, B.Sc., C.Biol., M.I.Biol. Study Director Elizabeth Farrall, B.Sc. Scientist MIN 314/022208 001574 :5: MIN 314/022208 SUMMARY In this in vitro assessment o f the mutagenic potential of Perfluorooctanesulfonyl fluoride (POSF), histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2wvrA/pKM101 (CM891), were exposed to the test substance. Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). Both tests involved a pre-incubation stage in airtight vessels. Concentrations o f Perfluorooctanesulfonyl fluoride (POSF) up to 5000 pg/plate were tested in the mutation tests. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any concentration of Perfluorooctanesulfonyl fluoride (POSF) in either mutation test The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that, under the test conditions employed, Perfluorooctanesulfonyl fluoride (POSF) showed no evidence of mutagenic activity in this bacterial system. 001575 :6 : MIN 314/022208 INTRODUCTION This report describes a study designed to assess the mutagenic potential of Perfluorooctanesulfonyl fluoride (POSF) in a bacterial system. The study was conducted in compliance with the following guidelines: OECD Guidelines for the Testing of Chemicals. (1997) Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471. EC Commission Directive 2000/32/EC Annex 4D-B. 13/14. Mutagenicity - Reverse mutation test in bacteria. No. L 136/57. US EPA (1998) Health Effects Test Guidelines. OPPTS 870.5100 Bacterial reverse mutation test. EPA 712-C-98-247. Japan Ministry of Agriculture, Forestry and Fisheries. (1985) Notification of Director General, Agricultural Production Bureau. NohSan No. 4200. Joint Directives of J EPA, J MHW and J MITI. (31 October 1997) Kanpoan No. 287, Eisei No. 127 and Kikyoku No. 2 (31 October 1997). JMHW Genotoxicity Testing Guideline, PAB Notification No. 1604 (1 November 1999). Official Notice of J MOL. (8 February 1999). The method described was also designed to comply with ICH (1995 & 1997), and followed the recommendations o f the United Kingdom Environmental Mutagen Society (Gatehouse et al 1990). The in vitro technique described by Ames and his co-workers (Ames, McCann and Yamasaki 1975, Maron and Ames 1983) enables the mutagenic effect of a test substance to be determined by exposing specially selected strains of Salmonella typhimurium to the test substance. Normally S. typhimurium is capable of synthesising the essential amino acid, histidine, but the mutant strains used in this test are incapable of this function. When these strains are exposed to a mutagen, reverse mutation to the original histidine independent form takes place in a proportion of the population. These are referred to as revertants, and are readily detected by their ability to grow and form colonies on a histidine deficient medium (supplemented with biotin, since these strains are also incapable of biotin synthesis). A technique based on similar principles has also been described by Green (1984). This system employs mutant strains of Escherichia coli that are incapable of synthesising the amino acid, tryptophan, which is required for growth. The strains used carry additional mutations that render them more sensitive to mutagens. The 5. typhimurium strains have a defective cell coat, which allows greater permeability of test substances into the cell. All the strains are deficient in normal DNA repair processes. In addition, three of them possess a plasmid (pKMIOl), which introduces an error-prone repair process, resulting in increased sensitivity to some mutagens. 001576 :7: MIN 314/022208 Many substances do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell. Therefore, the bacteria and test substance are incubated in both the absence and presence of a supplemented liver fraction (S9 mix) prepared from rats previously treated with a substance (Aroclor 1254) known to induce a high level of enzyme activity. The protocol was approved by Huntingdon Life Sciences Management on 18 July 2001, by the Sponsor on 31 July 2001 and by the Study Director on 15 October 2001. The study was conducted at Huntingdon Life Sciences Ltd., Eye Research Centre, Eye, Suffolk, IP23 7PX, England. Experimental start date: 21 November 2001. Experimental completion date: 31 January 2002. 001577 Identity: Appearance: Storage conditions: Lot number: Expiry date: Purity: Specific gravity: Date received: MIN 314/022208 TEST SUBSTANCE Perfluorooctanesulfonyl fluoride (POSF) Clear liquid Room temperature 040227 Sponsor's responsibility; assumed stable for duration of study >95.5% ca 1.8 14 June 2001 001578 O EXPERIMENTAL PROCEDURE MIN 314/022208 BACTERIAL STRAINS The following strains were used: S. typhimuriim TA1535: contains a histidine missense mutation (hisG46) but is also deficient in a DNA repair system (uvrB) and has a defective lipopolysaccharide coat on the cell wall (rfa mutation). It is reverted by many agents causing base-pair substitutions, but is not sensitive to frameshift mutagens. S. typhimurium TA100: is the same as TA1535 but contains a resistance transfer factor conferring ampicillin resistance and increasing sensitivity to some mutagens (plasmid pKMIOl). In addition to base-pair substitutions, it is also able to detect certain frameshift mutagens. S. typhimurium TA1537: bears a histidine frameshift mutation (AisC3076). Like TA1535, it is defective in a DNA repair system and lipopolysaccharide coat. It is sensitive to agents causing frameshift mutations involving insertion or deletion o f a single base-pair. S. typhimurium TA98: contains another histidine frameshift mutation (AD3052). Again it has a defective DNA repair system and lipopolysaccharide coat but also contains the pKMIOl plasmid. It is reverted by agents causing deletion of two adjacent base-pairs (double frameshift mutations), but not by simple alkylating agents causing base-pair substitutions. E. coli WP2wvrA/pKM101 : (CM891) contains an ochre mutation. It is reverted by many agents causing A-T base-pair substitutions at the trpE locus or by G-C base-pair substitutions in transfer RNA loci elsewhere in the chromosome. It is also deficient in a DNA repair system (uvrA), and is more readily reverted by certain mutagens than its parent strain WP2. It also contains the pKMIOl plasmid. The strains of S. typhimurium were obtained from the National Collection of Type Cultures, London, England. The strain of E. coli was obtained from the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland. Batches of the strains were obtained from master stocks held in liquid nitrogen. The test batches were aliquots of nutrient broth cultures and were stored at -80C. Dimethyl sulphoxide (DMSO) was added to the cultures at 8% v/v as a ciyopreservative. Each batch of frozen strain was tested, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light and the pKMIOl plasmid, which confers resistance to ampicillin. The responses of the strains to a series of diagnostic mutagens were also assessed. For use in tests, an aliquot of frozen culture was added to 25 ml of nutrient broth and incubated, with shaking, at 37C for 10 hours. These cultures were intended to provide approximately 109 cells per ml, which were measured by spreading aliquots (0.1 ml) of a 10'6dilution of the overnight cultures on the surface of plates o f nutrient agar and counting the resultant colonies. 001579 : 10 MIN 314/022208 POSITIVE CONTROLS In the absence o f S9 mix Identity: CAS No.: Supplier: Lot number: Purity: Appearance: SolventConcentration: Identity: CAS No.: Supplier Lot number Purity: Appearance: Solvent: Concentration: Identity: CAS No.: Supplier: Lot number: Purity: Appearance: Solvent: Concentration: Identity: CAS No.: Supplier: Lot number: Purity: Appearance: Solvent: Concentration: the presence of S9 mix Identity: CAS No.: Supplier: Lot number: Purity: Appearance: Solvent: Concentration: Sodium azide 26628-22-8 Sigma Chemical 77H0079 min. 99.5% White powder DMSO (Aldrich, A.C.S. spectrophotometric grade) 0.5 pg/plate for strains TA1535 and TA100 9-Aminoacridine 90-45-9 Sigma Chemical 106F-06681 >97% Yellow powder DMSO (Aldrich, A.C.S. spectrophotometric grade) 30 pg/plate for strain TA1537 2-Nitrofluorene 607-57-8 Aldrich Chemical Company 80501-24227 98% Beige powder DMSO (Aldrich, A.C.S. spectrophotometric grade) 1 pg/plate for strain TA98 2-(2-FuryI)-3-(5-nitro-2-furyl) acrylamide (AF-2) 3688-53-7 Wako Pure Chemical Industries Ltd. PAE 1151 98-102% Red powder DMSO (Aldrich, A.C.S. spectrophotometric grade) 0.05 pg/plate for strain WP2mrA/pKMl 01 (CM891) 2-Aminoanthracene 613-13-8 Aldrich Chemical Company 52234-024 96% Green powder DMSO (Aldrich, A.C.S. spectrophotometric grade) 2 pg/plate for strain TA1535 10 pg/plate for strain WP2wrA/pKM101 (CM891) : 11 : 001580 MIN 314/022208 Identity: CAS No.: Supplier: Lot number Purity: Appearance: Solvent: Concentration: Benzo[a]pyrene 50-32-8 Aldrich Chemical Company 07778-105 98% Yellow powder DMSO (Aldrich, A.C.S. spectrophotometric grade) 5 pg/plate for strains TA1537, TA98 and TA100 PREPARATION OF S9 FRACTION Species: Sex: Strain: Source: Weight: Rat Male Sprague-Dawley derived Charles River UK Ltd. <300 g S9 fraction was prepared from a group of ca 10 animals according to the method described by Ames, McCann and Yamasaki (1975). Mixed function oxidase systems in the rat livers were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in com oil at a dosage of 500 mg/kg body weight. On the fifth day after injection, following overnight fasting, the rats were killed by cervical dislocation and their livers aseptically removed. The following steps were carried out at 0-4C under aseptic conditions. The livers were placed in 0.15 M KC1 (3 ml KC1 : 1 g liver) before being transferred to a Potter-Elvehjem homogeniser. Following preparation, the homogenate was centrifuged at 9000 g for 10 minutes. The supernatant fraction (S9 fraction) was dispensed into aliquots and stored at -80C or below. Each batch of S9 fraction was tested for sterility and efficacy. Date of preparation: 17 July 2001 (test I); 15 January 2002 (test 2) PREPARATION OF S9 MIX The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KC1 (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM). All the cofactors were filter-sterilised before use. FORMULATION OF TEST SUBSTANCE The solubility of the test substance was assessed in dimethyl sulphoxide (DMSO), ethanol, acetone and hexane. It was insoluble in DMSO, ethanol and acetone, and was known to be insoluble in water. Although soluble in hexane at 50 mg/ml, the volume of hexane required to administer the test substance at this concentration was found to be too toxic towards the test system. It was, therefore, decided to administer the test substance by direct addition without the use of a solvent All concentrations cited in this report are expressed in terms of the Perfluorooctanesulfonyl fluoride (POSF) sample as received. 001581 : 12: MIN 314/022208 MUTATION TEST PROCEDURE First test (range-finding) The test substance was added to cultures of the five tester strains at seven concentrations. The highest concentration o f test substance tested was 5000 pg/plate (obtained by addition of 2.8 pi of the test substance). This is the standard limit concentration recommended in the regulatory guidelines this assay follows. The other concentrations were 2500 pg/plate (1.4 pi), 1750 pg/plate (1.0 pi), 1250 pg/plate (0.7 pi), 700 pg/plate (0.4 pi), 350 pg/plate (0.2 pi) and 175 pg/plate (0.1 pi). Untreated controls and the appropriate positive controls were also included. Following the addition of the above aliquots of the test substance (or 0.1 ml o f positive control solution) to airtight glass vessels, 0.5 ml S9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) was added, followed by 0.1 ml o f a 10 hour bacterial culture. The mixtures were incubated at 37C for 30 minutes with shaking before addition of 2 ml o f agar containing histidine (0.5 mM) and tryptophan (0.5 mM). The mixtures were thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 ml minimal agar. Each Petri dish was individually labelled with a unique code corresponding to a sheet, identifying the contents of the dish. Three Petri dishes were used for each concentration. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at 37C for ca 72 hours. After this period the appearance of the background bacterial lawn was examined and revertant colonies counted using a Domino automated colony counter. Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration normally used in the second test would be the same as that used in the first If toxic effects were observed a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at the top concentration. Ideally a minimum of three non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating dose levels should be obtained, unless otherwise justified by the Study Director. Second test The second test was an exact repeat of the first test, except that only five concentrations were used. 5000 pg/plate was again chosen as the top concentration. STABILITY AND FORMULATION ANALYSIS The stability of the test substance and the stability and homogeneity of the test substance in the test system were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study. 001582 : 13 : MIN 314/022208 ASSESSMENT OF RESULTS Acceptance For a test to be considered valid the mean of the solvent/vehicle control revertant colony numbers for each strain should lie within the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range will be maintained as a rolling record over a maximum of five years. Also, the positive control compounds must cause at least a doubling o f mean revertant colony numbers over the negative control. Analysis . The mean number o f revertant colonies for all treatment groups will be compared with those obtained for the solvent/vehicle control groups. Evaluation If exposure to a test substance produces an increase in revertant colony numbers o f at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-relationship (increased revertant colony counts at concentrations below that at which the maximal increase is obtained), in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be considered to show evidence of mutagenic activity in this test system. No statistical analysis will be performed. If exposure to a test substance does not produce an increase in revertant colony numbers in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed. If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after the additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance o f any increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al (1989) and will usually be analysis of variance followed by Dunnett's test. Biological significance should always be considered along with statistical significance. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained. DEVIATIONS FROM PROTOCOL Although the protocol indicated that a solvent or vehicle would be employed, it was not possible to obtain a solution or suspension of the test substance that would be compatible with the test system. The test substance was, therefore, added directly to the test system. Since this procedure complies with the test guidelines that this study follows, this deviation does not impact on the integrity of the study. 001583 : 14 : MAINTENANCE OF RECORDS MIN 314/022208 All raw data, samples and specimens (if appropriate) arising from the performance o f this study will remain the property of the Sponsor. Types of sample and specimen which are unsuitable, by reason of instability, for long term retention and archiving may be disposed of after the periods stated in Huntingdon Life Sciences Standard Operating Procedures. All other samples and specimens and all raw data will be retained by Huntingdon Life Sciences in its archive for a period of five years from the date on which the Study Director signs the final report. After such time, the Sponsor will be contacted and his advice sought on the return, disposal or further retention of the materials. If requested, Huntingdon Life Sciences will continue to retain the materials subject to a reasonable fee being agreed with the Sponsor. Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and a copy o f the final report in its archive indefinitely. 001584 RESULTS MIN 314/022208 The results obtained with Perfluorooctanesulfonyl fluoride (POSF) and positive control compounds are presented in Tables 1 to 10. The mean values quoted have been corrected to the nearest whole number. The absence o f colonies on sterility check plates confirmed the absence o f microbial contamination. The total colony counts on nutrient agar plates (see Tables) confirmed the viability and high cell density of the cultures of the individual organisms. The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix. FIRST TEST (RANGE-FINDING) No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Perfluorooctanesulfonyl fluoride (POSF) at any concentration in either the presence or absence of S9 mix. No visible thinning of the background lawn of non-revertant cells was obtained following exposure to Perfluorooctanesulfonyl fluoride (POSF). A maximum exposure concentration of 5000 pg/plate was, therefore, selected for use in the second test. SECOND TEST No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Perfluorooctanesulfonyl fluoride (POSF) at any concentration in either the presence or absence of S9 mix. No visible thinning of the background lawn of non-revertant cells was obtained following exposure to Perfluorooctanesulfonyl fluoride (POSF). CONCLUSION It is concluded that, under the test conditions employed, Perfluorooctanesulfonyl fluoride (POSF) showed no evidence of mutagenic activity in this bacterial system. 001585 : 16 : MIN 314/022208 REFERENCES AMES, B.N., McCANN, J. and YAMASAKI, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella!mammalian microsome mutagenicity test. Mutation Res. 31, 347-364. GATEHOUSE, D.G., ROWLAND, I.R., WILCOX, P., CALLANDER, R.D. and FORSTER, R. (1990) Bacterial mutation assays in: KIRKLAND, D.J. (Ed.). UKEMS Sub-committee on Guidelines fo r Mutagenicity Testing. Report. Part I revised. Basic Mutagenicity Tests: UKEMS Recommended Procedures, pp. 13-61. Cambridge University Press, Cambridge. GREEN, M.HX. (1984) Mutagen testing using trp+ reversion in Escherichia coli in KILBEY, B.J., LEGATOR, M., NICHOLS, W. and RAMEL, C. (Eds.). Handbook o f Mutagenicity Test Procedures. Second edition, pp.161-187. Elsevier Science Publishers BV, Amsterdam. ICH (1995) Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests. ICH (1997) Genotoxicity: A Standard Battery of Genotoxicity Testing of Pharmaceuticals. MAHON, G.A.T., GREEN, M.H.L., MIDDLETON, B., MITCHELL, I.de G., ROBINSON, W.D. and TWEATS, D J. (1989) Analysis of data from microbial colony assays in: KIRKLAND, D J. (Ed.). UKEMS Sub-committee on Guidelinesfo r Mutagenicity Testing. Report. Part III Statistical Evaluation o fMutagenicity Test Data, pp.26-65. Cambridge University Press, Cambridge. MARON, D.M. and AMES, B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res. 113, 173-215. 001586 : 17 : TABLE 1 Results obtained with S. typhimurium TA98: test 1 (range-finding) MIN 314/022208 Plate No. Addition 1+ None; S9 mix sterility check 1- None; buffer sterility check 2 POSF; sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 POSF 9 POSF 10 Untreated 11 POSF 12 POSF 13 POSF 14 POSF 15 POSF 16 POSF 17 POSF 18 Untreated 19 Benzo[a]pyrene 20 2-Nitrofluorene None; 10"6 dilution of 21 overnight culture, plated on nutrient agar S9 mix + present - absent + - (5000 pg/plate) - Revertant colony counts* and means A B C Mean sd 000 000 000 00 00 00 (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (350 pg/plate) (175 pg/plate) (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (350 pg/plate) (175 pg/plate) (5 pg/plate) (1 pg/plate) + + + + + + + + + - 46 44 56 45 41 42 48 45 50 43 51 50 48 50 48 61 35 52 56 55 55 41 36 51 41 37 37 37 38 35 43 31 41 39 46 49 34 39 43 37 42 41 44 36 21 30 30 38 566 546 542 234 205 209 49 6 43 2 48 3 48 4 49 1 49 13 55 1 43 8 38 2 37 2 38 6 45 5 39 5 40 3 34 12 33 5 551 13 216 16 - 133 128 129 130 3 * Except plate nos. 1,2 and 21 (total colony counts) sd Standard deviation 001587 : 18: TABLE 2 Results obtained with S. typhimurium TA98: test 2 MIN 314/022208 Plate Mo. Addition 1+ None; S9 mix sterility check 1 - None; buffer sterility check 2 POSF; sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 Untreated 9 POSF 10 POSF 11 POSF 12 POSF 13 POSF 14 Untreated 15 Benzo[a]pyrene 16 2-Nitrofluorene None; 10"6 dilution of 17 overnight culture, plated on nutrient agar S9mix + present - absent + (5000 pg/plate) - (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (5 pg/plate) (1 pg/plate) + + + + + * + - Revenant colony counts* and means A B C Mean sd 000 000 000 00 00 00 43 48 39 42 36 36 42 42 36 36 43 30 51 42 46 53 44 49 21 39 34 34 38 23 37 35 35 29 31 29 32 38 23 38 39 31 449 550 567 504 467 443 43 5 38 3 40 3 36 7 46 5 49 5 31 9 32 8 36 1 30 1 31 8 36 4 522 64 471 31 106 96 117 106 11 * Except plate nos. 1, 2 and 17 (total colony counts) sd Standard deviation 001588 : 19 : TABLE 3 Results obtained with S. typhimurium TA100: test 1 (range-finding) MIN 314/022208 Plate No. Addition 1+ None; S9 mix sterility check 1 - None; buffer sterility check 2 POSF; sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 POSF 9 POSF 10 Untreated 11 POSF 12 POSF 13 POSF 14 POSF 15 POSF 16 POSF 17 POSF 18 Untreated 19 Benzo[a]pyrene 20 Sodium azide None; 10"6dilution of 21 overnight culture, plated on nutrient agar S9 mix + present - absent + (5000 pg/plate) - (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (350 pg/plate) (175 pg/plate) (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (350 pg/plate) (175 pg/plate) (5 pg/plate) (0.5 pg/plate) + + + + + + + + + - - Revertmt colony counts* and means A B C Mean sd 00 0 00 0 000 00 00 00 125 169 137 148 130 152 153 164 154 159 141 165 154 131 114 159 145 131 143 159 128 148 140 131 119 131 122 125 132 88 125 121 139 88 118 124 138 97 136 119 118 125 122 122 109 112 110 141 573 507 568 360 453 471 144 23 143 12 157 6 155 12 133 20 145 14 143 16 140 9 124 6 115 24 128 9 110 19 124 23 121 4 118 8 121 17 549 37 428 60 168 160 173 167 7 * Except plate nos. 1, 2 and 21 (total colony counts) sd Standard deviation 001589 : 20: TABLE 4 Results obtained with S. typhimurium TA100: test 2 MIN 314/022208 Plate No. Addition 1+ None; S9 mix sterility check 1 - None; buffer sterility check 2 POSF; sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 Untreated 9 POSF 10 POSF 11 POSF 12 POSF 13 POSF 14 Untreated 15 Benzo[a]pyrene 16 Sodium azide None; 10"6dilution of 17 overnight culture, plated on nutrient agar S9 mix + present -absent (5000 pg/plate) (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (5 pg/plate) (0.5 pg/plate) - + + + + + + + - - Revenant colony counts* and means A B C Mean sd 000 000 000 00 00 0 "0 131 145 119 148 132 148 122 154 139 147 132 166 165 125 143 131 145 131 111 118 123 126 137 121 135 128 147 119 128 124 150 124 125 143 123 122 752 706 705 528 501 549 132 13 143 9 138 16 148 17 144 20 136 8 117 6 128 8 137 10 124 5 133 15 129 12 721 27 526 24 109 110 111 110 1 * Except plate nos. 1, 2 and 17 (total colony counts) sd Standard deviation 001590 : 21 : TABLE 5 Results obtained with S. typhimurium TA1535: test 1 (range-finding) MIN 314/022208 Plate No. Addition 1+ None; S9 mix sterility check 1 - None; buffer sterility check 2 POSF; sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 POSF 9 POSF 10 Untreated 11 POSF 12 POSF 13 POSF 14 POSF 15 POSF 16 POSF 17 POSF 18 Untreated 19 2-Aminoanthracene 20 Sodium azide None; 10-6dilution of 21 overnight culture, plated on nutrient agar (5000 |ig/plate) S9 mix + present - absent + - Revertant colony counts* and means A B C Mean sd 000 000 000 00 00 00 (5000 ug/plate) + 14 21 16 (2500 fig/plate) 4* 20 26 16 (1750 ug/plate) + 29 22 12 (1250 ug/plate) + 14 26 20 (700 pg/plate) 22 23 19 (350 ug/plate) + 17 23 20 (175 ug/plate) + 14 20 19 + 31 28 19 (5000 ug/plate) - 21 15 22 (2500 ug/plate) - 15 19 14 (1750 ug/plate) - 20 14 24 (1250 ug/plate) - 17 15 21 (700 ug/plate) - 19 16 24 (350 ug/plate) - 16 15 19 (175 ug/plate) - 21 20 20 - 22 20 21 (2 ug/plate) + 282 324 247 (0.5 ug/plate) - 231 252 251 17 4 21 5 21 9 20 6 21 2 20 3 18 3 26 6 19 4 16 3 19 5 18 3 20 4 17 2 20 I 21 1 284 39 245 12 - 184 165 175 175 10 * Except plate nos. 1,2 and 21 (total colony counts) sd Standard deviation 001591 : 22 : TABLE 6 Results obtained with S. typhimurium TA1535: test 2 MIN 314/022208 Plate No. Addition 1+ None; S9 mix sterility check 1 - None; buffer sterility check 2 POSF; sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 Untreated 9 POSF 10 POSF 11 POSF 12 POSF 13 POSF 14 Untreated 15 2-Aminoanthracene 16 Sodium azide None; 10"6dilution of 17 overnight culture, plated on nutrient agar S9 mix + present - absent + (5000 pg/plate) - Revertant colony counts* and means A B C Mean sd 000 000 000 00 00 00 (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 |ig/plate) (700 pg/plate) (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (2 pg/plate) (0.5 pg/plate) + + + + + + + - 20 13 13 19 15 13 12 13 13 20 22 14 21 14 19 22 19 19 17 14 16 15 27 16 14 16 22 21 14 16 22 16 27 27 21 19 145 137 121 253 237 288 15 4 16 3 13 1 19 4 18 4 20 2 16 2 19 7 17 4 17 4 22 6 22 4 134 12 259 26 - 129 129 161 140 18 * Except plate nos. 1,2 and 17 (total colony counts) sd Standard deviation 001592 : 23 : TABLE 7 Results obtained with S. typhimurium TA1S37: test 1 (range-finding) MIN 314/022208 Plate No. Addition 1+ None; S9 mix sterility check I - None; buffer sterility check 2 POSF; sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 POSF 9 POSF 10 Untreated 11 POSF 12 POSF 13 POSF 14 POSF 15 POSF 16 POSF 17 POSF 18 Untreated 19 Benzo[a]pyrene 20 9-Aminoacridine None; 10-6dilution of 21 overnight culture, plated on nutrient agar S9 mix + present -absent + (5000 pg/plate) - (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (350 pg/plate) (175 pg/plate) (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (350 pg/plate) (175 pg/plate) (5 pg/plate) (30 pg/plate) + + + + + + + - - Revertant colony counts* and means A B C Mean sd 000 000 000 00 00 00 23 22 15 26 22 16 23 27 21 21 27 19 21 22 26 26 21 24 17 24 24 22 22 26 19 15 10 15 10 13 17 17 10 17 19 10 16 16 12 13 9 15 13 13 10 16 15 13 355 394 360 489 457 453 20 4 21 5 24 3 22 4 23 3 24 3 22 4 23 2 15 5 13 3 15 4 15 5 15 2 12 3 12 2 15 2 370 21 466 20 103 139 107 116 20 * Except plate nos. 1,2 and 21 (total colony counts) sd Standard deviation 001593 74- TABLE 8 Results obtained with S. typhimurium TA1537: test 2 MIN 314/022208 Plate No. Addition 1+ None; S9 mix sterility check 1 - None; buffer sterility check 2 POSF; sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 Untreated 9 POSF 10 POSF 11 POSF 12 POSF 13 POSF 14 Untreated 15 Benzo[a]pyrene 16 9-Aminoacridine None; 10"6 dilution of 17 overnight culture, plated on nutrient agar S9 mix + present absent + (5000 pg/plate) - Revertant colony counts* and means A B C Mean sd 000 000 000 00 00 00 (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (5 pg/plate) (30 pg/plate) + + + + + + + - 16 22 23 22 16 20 24 29 20 22 28 15 23 26 14 20 19 26 9 8 13 10 14 8 8 9 14 10 17 12 13 9 13 16 10 15 315 281 238 269 276 317 20 4 19 3 24 5 22 7 21 6 22 4 10 3 11 3 10 3 13 4 12 2 14 3 278 39 287 26 - 124 129 124 126 3 Except plate nos. 1,2 and 17 (total colony counts) sd Standard deviation 001594 : 25 : MIN 314/022208 TABLE 9 Results obtained with E. coli WP2vrA/pKM101 (CM891): test 1 (range-finding) Plate No. Addition 1+ None; S9 mix sterility check 1 - None; buffer sterility check 2 POSF; sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 POSF 9 POSF 10 Untreated 11 POSF 12 POSF 13 POSF 14 POSF 15 POSF 16 POSF 17 POSF 18 Untreated 19 2-Aminoanthracene 20 AF-2T None; 10"6 dilution of 21 overnight culture, plated on nutrient agar S9mix + present -absent + - (5000 pg/plate) - Revertant colony counts* and means A B C Mean sd 0 00 0 00 000 00 00 00 (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (350 pg/plate) (175 pg/plate) (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (350 pg/plate) (175 pg/plate) (10 pg/plate) (0.05 pg/plate) + + + + + + + + - 4- 107 131 87 119 136 96 126 122 116 123 96 119 122 144 123 122 138 119 117 121 119 123 121 133 101 112 99 111 82 89 132 118 116 85 138 85 110 86 110 95 74 84 81 111 73 88 124 123 320 423 405 559 529 531 108 22 117 20 121 5 113 15 130 12 126 10 119 2 126 6 104 7 94 15 122 9 103 31 102 14 84 11 88 20 112 21 383 55 540 17 - 162 188 217 189 28 * Except plate nos. 1,2 and 21 (total colony counts) sd Standard deviation t 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide 001535 : 26: TABLE 10 Results obtained with & coll WP2vrA/pKMIOl (CM891): test 2 MIN 314/022208 Plate No. Addition 1+ None; S9 mix sterility check 1- None; buffer sterility check 2 POSF; sterility check 3 POSF 4 POSF 5 POSF 6 POSF 7 POSF 8 Untreated 9 POSF 10 POSF 11 POSF 12 POSF 13 POSF 14 Untreated 15 2-Aminoanthracene 16 AF-2t None; 10"6dilution of 17 overnight culture, plated on nutrient agar S9 mix + present -absent + (5000 pg/plate) - Revertant colony counts* and means AB C Mean sd 00 0 00 0 00 0 00 00 00 (5000 pg/plate) + 125 136 122 128 7 (2500 pg/plate) + 154 122 165 147 22 (1750 pg/plate) + 126 159 145 143 17 (1250 pg/plate) + 132 145 172 150 20 (700 pg/plate) + 184 143 166 164 21 + 137 144 165 149 15 (5000 pg/plate) - 147 148 128 141 11 (2500 pg/plate) - 121 139 155 138 17 (1750 pg/plate) - 144 131 145 140 8 (1250 pg/plate) - 131 131 139 134 5 (700 pg/plate) - 115 110 135 120 13 - 153 152 140 148 7 (10 pg/plate) + 501 465 583 516 60 (0.05 pg/plate) - 1041 899 957 966 71 - 190 175 189 185 8 Except plate nos. 1, 2 and 17 (total colony counts) sd Standard deviation t 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide 001596 -.21 MIN 314/022208 APPENDIX 1 Historical control data Presented below are the historical control data from the period 1 April 1997 to 30 September 2001. U ntreated con trob Strain S9 mix TA100 -+ TA1535 -+ WP2vrA/pKM10l (CM891) -+ TA98 -+ TA1537 -+ M inim um 78 81 12 10 79 73 23 29 7 7 M axim um 149 165 33 29 167 200 48 54 23 34 Mean 109 114 18 19 121 134 38 41 12 13 No. o f values 309 313 307 311 214 218 311 315 308 312 Standard deviation 16 22 3 3 17 23 4 5 35 Upper 99% limit 151 168 33 29 170 204 49 55 23 35 Lower 99% limit 76 78 12 10 76 69 22 28 7 6 Positive controb Strain S9 mix Minimum Maximum Mean No. of values Standard deviation TA100 + (a) (d) (h) 212 237 263 860 1327 1350 405 564 561 346 441 808 :120 175 189 TA1535 +- ( b ) ( d ) CO 53 112 63 781 1130 1145 205 419 229 341 434 796 139 196 114 W P 2iw A/pKM 101 (CM891) +- - (c ) <e) (j) 294 244 188 2312 1533 1704 1331 679 659 112 415 549 488 183 250 TA98 -+ (f) (h) 123 123 993 1031 320 517 788 810 115 165 TA1537 - (g ) 39 1933 350 569 337 + (h ) 67 543 246 810 80 (a) ENNG 3 fig (b) ENNG 5 fig (c) ENNG 2 fig (d) Sodium azide O.Sfig (e) AF-2 0.05 fig (f) 2-Nitrofluorene 1 fig (g) 9-Aminoacridine 30 fig (h) Benzo[a]pyrene 5 fig (i) 2-Aminoanthracene 2 fig (j) 2-Aminoanthracene 10 fig 0015^7 : 28 : MIN 314/022208 APPENDIX 2 Eye Research Centre GLP Compliance Statement 2001 THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE STATEMENT O F COMPLIANCE IN ACCORDANCE W ITH DIRECTIVE 8&320 EEC LABORATORY TEST TYPE Huntingdon Life Sciences E ye R esearch C en tre Eye Suffolk JP237PX Analytical Chemistry Clinical Chemistry E cosystem s Environmental Fate Environmental Toxicity M utagenicity Phys/Chem Testing Toxicology DATE O F INSPECTIO N 29thJanuary 2001 A general inspection for compliance with the Principles of Good Laboratory Practice was carried out at the above laboratory as part of UK GLP Compliance Programme. A t the time of the inspection no deviations were found of sufficient magnitude to affect the validity of non-clinical studies performed at these facilities. Dr. Roger G. Alexander Head. UK GLP Monitoring Authority U0l5b>8 : 29 :