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SRPTT-7.f -A a a s - 1*200 MIN 314/022208 n u Anilin* 0a 0 ^ 0 Z -O m ro ro -~c?mo 2"DTg** < O pri oo 5*, o jr P E R F L U 0R 00C T A N E SU L F 0N Y L FLUORIDE (POSF) BACTERIAL REVERSE M UTATION TEST 223 \ S "^b *-*-*-1 Sponsor 3M Center 3M Corporate Toxicology Building 220-2E-02 St Paul MN 55133-3220 USA Research Laboratory Huntingdon Life Sciences Ltd. Woolley Road Alconbuiy Huntingdon Cambridgeshire PE28 4HS ENGLAND Report issued 23 October 2002 Page 1 o f29 080002  ' 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 S. typhimurium TA100: test 1 (range-finding).... ......... 20 4. Results obtained with S. typhimurium TA100: test 2, with pre-incubation ............................ 21 5. Results obtained with S. typhimurium TA 1535: test 1 (range-finding)................................... 22 6. Results obtained with S. typhimurium TA 1535: test 2, with pre-incubation ......................... 23 7. Results obtained with S. typhimurium TA1537: test 1 (range-finding)................................... 24 8. Results obtained with/?, typhimurium TA 1537: test2, with pre-incubation ......................... 25 9. Results obtained with E. co/z WP2wvM/pKM101 (CM891): test 1 (range-finding).............. 26 10. Results obtained with E. coli WP2zzvrA/pKM101 (CM891): test 2, with pre-incubation 27 APPENDICES 1. Historical control data.......................................................................................... ...................... 28 2. Eye Research Centre GLP Compliance Statement 2001 .......................................................... 29 oooop3 ' 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 of 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 of the test sample was the Sponsor's responsibility. tL Kenneth Ma;,, Study Director, Huntingdon Life Sciences Ltd. ..... Date :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 15 January 2002 31 October 2001 24 October 2001 4 Februaiy 2002 Date of Reporting 16 October 2001 15 January 2002 31 October 2001 24 October 2001 5 Februaiy 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. R eport 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 o f this study to reflect the raw data. \ ................ Angela M a n n in g s , B.Sc., M.Sc., Ph.D., M.R.Q.A., Group Manager, Department of Quality Assurance, Huntingdon Life Sciences Ltd. Date c=>2sOC2><-- 000005 :4 : RESPONSIBLE PERSONNEL Kenneth May, B.Sc., C.Biol., M.LBiol. Study Director Elizabeth Farrall, B.Sc. Scientist MIN 314/022208 000006 :5: . MIN 314/022208 SUMMARY In this in vitro assessment of 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 WP2ww*A/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. 000007 :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 o f 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 o f Director General, Agricultural Production Bureau. NohSan No. 4200. Joint Directives o f J EPA, J MHW and J MITT. (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, Marn 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 o f 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 o f Escherichia coli that are incapable o f synthesising the amino acid, tryptophan, which is required for growth. The strains used cany additional mutations that render them more sensitive to mutagens. The S. typhimurium strains have a defective cell coat, which allows greater permeability o f 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. 000008 : 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. 000009 :8: Identity: Appearance: Storage conditions: Lot number: Expiry date: Purity: Specific gravity: Date received: TEST SUBSTANCE MIN 314/022208 Perfluorooctanesulfonyl fluoride (POSF) Clear liquid Room temperature 040227 Sponsor's responsibility; assumed stable for duration o f study >95.5% ca 1.8 14 June 2001 000010 :9 : EXPERIMENTAL PROCEDURE MIN 314/022208 BACTERIAL STRAINS The following strains were used: S. typhinwriwn TA1535: contains a histidine missense mutation (hisG46) but is also deficient in a DNA repair system (wvrB) 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 ffameshift mutagens. S. typhimicrium 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. typhimicrium TA1537: bears a histidine ffameshift mutation (hisC3076). Like TA1535, it is defective in a DNA repair system and lipopolysaccharide coat. It is sensitive to agents causing ffameshift mutations involving insertion or deletion o f a single base-pair. S. typhimurium TA98: contains another histidine ffameshift mutation (AM)3052). 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 ffameshift mutations), but not by simple alkylating agents causing base-pair substitutions. E. coli WP2wvrA/pKM 101 : (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. typhimicrium were obtained from the National Collection of Type Cultures, London, England. The strain o f 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 o f 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 o f plates of nutrient agar and counting the resultant colonies. 09011 : 10: MIN 314/022208 POSITIVE CONTROLS In the absence of S9 mix 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: Identity: CAS No.: Supplier: Lot number: Purity: Appearance: Solvent: Concentration: In 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-Furyl)-3-(5-nitro-2-fuiyl) 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 WP2zrvrA/pKM101 (CM891) 2-Ammoanthracene 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 WP2wvrA/pKM 101 (CM891) 000012 : 11 : 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 cor 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 o f S9 fraction was tested for sterility and efficacy. Date of preparation: 17 July 2001 (test 1); 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 o f 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. 000013 : 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 of 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 of 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 of 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 o f the test substance would be detected by a substantial-reduction in revertant colony counts or by the absence o f 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. : 13 : MIN 314/022208 ASSESSMENT OF RESULTS A ccep tan ce 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 o f 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 of 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 of 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 o f 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 of 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 o f the study. 000015 : 14 : MIN 314/022208 MAINTENANCE OF RECORDS 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 of the final report in its archive indefinitely. 000016 : 15 : MIN 314/022208 RESULTS 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 of 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 o f 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 o f the background lawn o f 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 o f 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. 90917 : 16: MIN 314/022208 REFERENCES AMES, B.N., McCANN, J. and YAMASAKI, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonellalmaxxm&Visa. 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.H.L. (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 o f 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. M utation Res. 113, 173-215. 000018 : 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 (ig/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) + + + + + + + + -f - 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 000019 : 18: TABLE2 Results obtained with S. typhimurium TA98: 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 2-Nitrofluorene None; 10^ 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 00 0 000 000 00 00 00 (5000 pg/plate) + 43 48 39 (2500 pg/plate) 4- 42 36 36 (1750 pg/plate) + 42 42 36 (1250 pg/plate) + 36 43 30 (700 pg/plate) + 51 42 46 + 53 44 49 (5000 pg/plate) - 21 39 34 (2500 pg/plate) - 34 38 23 (1750 pg/plate) - 37 35 35 (1250 pg/plate) - 29 31, 29 (700 pg/plate) - 32 38 23 - 38 39 31 (5 pg/plate) + 449 550 567 (1 pg/plate) - 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 000020 : 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^ dilution of 21 overnight culture, plated on nutrient agar S9 mix + present - absent + (5000 pg/plate) - Revertant colony counts* and means AB / 00 00 00 C Mean sd 0 00 0 00 0 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/piate) (1250 pg/plate) (700 pg/plate) (350 pg/plate) (175 pg/plate) (5 pg/plate) (0.5 pg/plate) + + + + + + + + + - 125 169 137 144 23 148 130 152 143 12 153 164 154 .157 6 159 141 165 155 12 154 131 114 133 20 159 145 131 145 14 143 159 128 143 16 148 140 131 140 9 119 131 122 124 6 125 132 88 115 24 125 121 139 128 9 88 118 124 110 19 138 97 136 124 23 119 118 125 121 4 122 122 109 118 8 112 110 141 121 17 573 507 568 549 37 360 453 471 428 60 - 168 160 173 167 7 * Except plate nos. 1,2 and 21 (total colony counts) sd Standard deviation 000021 : 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"6 dilution of 17 overnight culture, plated on nutrient agar (5000 pg/plate) S9 mix + present - absent + - 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) (0.5 pg/plate) + + + + + + - 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 000022 : 21 : MIN 314/022208 TABLES Results obtained with S. typhimurium TA1535: 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 Sodium azide None; 10-6dilution 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 (ig/plate) (5000 pg/plate) (2500 pg/plate) (1750 pg/plate) (1250 pg/plate) (700 pg/plate) (350 pg/plate) (175 pg/plate) (2 pg/plate) (0.5 pg/plate) + + + + + + + + - - 14 21 16 17 4 20 26 16 21 5 29 22 12 21 9 14 26 20 20 6 22 23 19 21 2 17 23 20 20 3 14 20 19 18 3 31 28 19 26 6 21 15 22 19 4 15 19. 14 16 3 20 14 24 19 5 17 15 21 18 3 19 16 24 20 4 16 15 19 17 2 21 20 20 20 1 22 20 21 21 1 282 324 247 . 284 39 231 252 251 245 12 - 184 165 175 175 10 * Except plate nos. 1, 2 and 21 (total colony counts) sd Standard deviation 000023 : 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"6 dilution of 17 overnight culture, plated on nutrient agar (5000 pg/plate) S9 mix + present ~ absent + -' - 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) (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 090024 : 23 : TABLE 7 Results obtained with S. typhimurium TA1537: 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 9-Aminoacridine None; 10"* dilution of 21 overnight culture, plated on nutrient agar (5000 pg/plate) S9 mix + present - absent + - Revenant 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) (5 pg/plate) (30 pg/plate) + + + + + + + + + - 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 tSO025 : 24 : TABLE 8 Results obtained with S. typhimurum 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"6dilution of 17 overnight culture, plated on nutrient agar S9 mix + present - absent + - (5000 gg/plate) - Revertant colony counts* and means A B C Mean sd 000 00 0 00 0 00 00 00 (5000 gg/plate) (2500 gg/plate) (1750 gg/plate) (1250 gg/plate) (700 gg/plate) (5000 gg/plate) (2500 gg/plate) (1750 gg/plate) (1250 gg/plate) (700 gg/plate) (5 gg/plate) (30 gg/plate) + + + + + + - 16 22 22 16 24. 29 22 28 23 26 20 19 98 10 14 89 10 17 13 9 16 10 315 281 269 276 23 20 20 15 14 26 13 8 14 12 13 15 238 317 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 000026 : 25 : MIN 314/022208 TABLE 9 Results obtained with E. coli WP2HwA/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 S9 mix + present - absent 4* (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) (10 pg/plate) (0.05 pg/plate) 444444* 444- - Revertant colony counts* and means A B C Mean sd 0 00 000 000 00 00 00 107 131 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-Fuiyl)-3-(5-nitro-2-furyl) acrylamide 000027 : 26 : TABLE 10 Results obtained with E. coli WP2wvrA/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-21 None; IO'6 dilution of 17 overnight culture, plated on nutrient agar (5000 pg/plate) S9 mix + present - absent + - Revenant colony counts* and means A B C Mean sd 0 0 0 00 0 0 0 00 0 0 0 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) (10 pg/plate) (0.05 pg/plate) + + -f + + + - - 125 136 154 122 126 159 132 145 184 143 137 144 147 148 121 139 144 131 m 131 115 110 153 152 501 465 1041 899 122 128 7 165 147 22 145 . M3 17 172 150 20 166 164 21 165 149 15 128 141 11 155 138 17 145 140 8 139 134 5 135 120 13 140 148 7 583 516 60 957 966 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-furyI) acrylamide 000028 : 27 : 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 controls Strain S9 mix M inim um M axim um M ean No. of values Standard deviation Upper 99% limit Lower 99% lim it TA100 -+ TA1535 -+ WP2uvrA/pKM101 (C M 891) -+ TA98 -+ 78 81 12 10 79 73 23 29 149 165 33 29 167 200 48 54 109 114 18 19 121 134 38 41 309 313 307 311 214 218 311 315 16 22 3 3 17 23 4 5 151 168 33 29 170 204 49 55 76 78 12 10 76 69 22 28 TA1537 - +. 77 23 34 12 13 308 312 35 23 35 76 Positive controls Strain S9 mix Minimum Maximum Mean No. of values Standard deviation TA 100 +- (a) (d) (h) 212 237 263 860 1327 1350 405 564 561 346 441 808 120 175 189 TA1535 +- (b ) (d ) (i) 53 112 63 781 1130 1145 205 419 229 341 434 796 139 196 114 WP2vr A/pKM 101 (CM891) -' + (c) (e) (j) 294 244 188 2312 1533 1704 1331 679 659 112 415 549 488 183 250 TA98 TA1537 -+-+ (f) (fa) ( g ) ( h ) 123 123 39 67 993 1031 1933 543 320 517 350 246 788 810 569 810 115 165 337 80 (a) ENNG 3 pg (b) ENNG 5 pg (c) ENNG 2 pg (d) Sodium azide 0.5pg (e) AF-2 0.05 pg " (f) 2-Nitrofluorene 1 pg (g) 9-Aminoacridme 30 pg (h) Benzo[a]pyrene 5 pg (i) 2-Aminoanthracene 2 pg (j) 2-Aminoanthracene 10 pg Ml : 28 : MIN 314/022208 APPENDIX 2 Eye Research C entre GLP Compliance Statement 2001 i A * EVV THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNTTED KINGDOM GOOD LABORATORY PRACTICE SX4.TEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 8&320 EEC LABORATORY TEST TYPE Huntingdon Life Sciences Eye Research Centre Eye Suffolk IP23 7PX Analytical Chemistry Clinical Chemistry Ecosystems Environmental Fate Environmental Toxicity Mutagenicity Phys/Chem Testing Toxicology DATE OF INSPECTION 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. At the time of the inspection no deviations were found of sufficient magnitude to affect the validity of non-clinical studies performed at these facilities. rp Dr. Roger G. Alexander Head, UK GLP Monitoring Authority 0004)3$ : 29 : Enquiry number: 23923D pertt-7m,i Huntingdon Life Sciences ' - [ ' i L b l . 2- PROTOCOL PERFLUOROOCTANESULFONYL FLUORIDE (POSF) BACTERIAL REVERSE MUTATION TEST Sponsor 3M, Center 3M Corporate Toxicology Building 220-2E-02 St Paul MN 55133-3220 USA Total number of pages: 12 Research Laboratory Huntingdon Life Sciences Ltd. Woolley Road Alconbury Huntingdon Cambridgeshire PE28 4HS ENGLAND mm2 i. Page / Enquiry number: 23923D Huntingdon Life Sciences PROTOCOL APPROVAL PERFLUOROOCTANESULFONYL FLUORIDE (POSF) BACTERIAL REVERSE MUTATION TEST Management Huntingdon Life Sciences Ltd. 3M Center Sponsor Date Please sign both copies o f this page, retain one fo r your records and return one to the Study D irector a t H untingdon Life Sciences. Study D irector approval o f the protocol is given on the study details page o f the protocol once such details have been established and agiAeed. The com pletedpage w ill be issuedprior to the start o f the study. 000032 Enquiry number: 23923 D Huntingdon Life Sciences PROTOCOL APPROVAL - p i L i l Z. PERFLUOROOCTANESULFONYL FLUORIDE (POSF) BACTERIAL REVERSE MUTATION TEST Huntingdon Life Sciences Ltd. ~7 Date "i ^ * 3M Center Sponsor 3' / Date 7 / ' Please sign both copies o f this page, retain one fo r your records and return one to the Study D irector at H untingdon Life Sciences. Study D irector approval o f the protocol is given on the study details page o f the protocol once such details have been established and agreed. The com pletedpage w ill be issuedprior to the start o f the study. f'- w 000033 Enquiry number: 23923D STUDY DETAILS PAGE Huntingdon Life Sciences Study number: Study title: Test substance Identity: Batch number: Expiry: Appearance: Storage conditions: Purity/Assay: Specific Gravity: Solvent: Stability of test substance formulation: Analysis of achieved concentration: Sponsor's Monitoring Scientist: Head, Department of Genetic Toxicology: Study Director: Person acting in the temporaiy absence o f the Study Director: Location o f study: Proposed study dates Experimental start: Experimental completion: Draft report: MING 14 Bacterial reverse mutation test Perfluorooctanesulfonyl Fluoride (POSF) 040227 Sponsor's responsibility; assumed stable for duration o f study Clear liquid Room temperature >95.5% ca 1.8 To be determined Not assessed in this study N ot assessed in this study John Butenhoff Dr M. Gillian Clare Mr Kenneth May Miss Elizabeth Farrall Dept, of Genetic Toxicology, Huntingdon Life Sciences Ltd., Eye, Suffolk, IP23 7PX, England 20 November 2001 5 December 2001 4 January 2002 STUDY DIRECTOR APPROVAL OF PROTOCOL 2^0 1 Date Enquiry number: 23923D H u n tin g d o n Life Sciences CONTENTS PROTO COL A PPRO V A L........................... L INTRODU CTIO N................................. 2. EXPERIM ENTAL PR O CED U R E..... 3. ASSESSMENT OF RESULTS ........... 4. REPORTIN G............ ............... ............. 5. MAINTENANCE OF RECORDS...... 6. GOOD LABORATORY PRACTICE 7. QUALITY ASSURAN CE................... 8. HEALTH & SA FETY .......... .............. . 9. REFER EN C ES.............................. ....... Page ..... ii 2 3 .6 .7 .7 .8 .8 .8 .9 HMISo Enquiry number: 23923D 1. INTRODUCTION Huntingdon Life Sciences The object o f this study is to assess the mutagenic potential of the test substance in a bacterial system. This procedure complies with the following guidelines: OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology: Bacterial Reverse M utation 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 JEPA, JM HW and JM ITI (31 October 1997). KANPOAN No. 287 E ISE IN o. 127 KIKYOKU No. 2 (31 October 1997). JM HW Genotoxicity Testing Guideline, PAB Notification No. 1604 (1 Novem ber 1999). Official Notice of J MOL (8 February 1999). The method described is also designed to comply with ICH (1995 & 1997), and follows the recommendations of 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. A technique based on similar principles has also been described by Green (1984). This system employs mutant strains of Escherichia coli which are incapable of synthesising the amino acid tryptophan required for growth. 000036 Enquiry number: 23923D Huntingdon Life Sciences The strains used carry additional mutations which render them more sensitive to mutagens. The S. typhimurium strains have a defective cell coat which allows greater perm eability o f test substances into the cell. All the strains are deficient in normal DNA repair processes. In addition three of them possess a plasmid (pKM IOl) which introduces an error-prone repair process, resulting in increased sensitivity to some mutagens. 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 m ix) prepared from rats previously treated with a substance (Aroclor 1254) known to induce a high level of enzyme activity. 2. EXPERIMENTAL PROCEDURE Bacterial strains The following strains will be used:- S. typhimurium T A 1535 hisG 46 i f a wvrB S. Typhimurium TA1537 hisC3076 lfa i/vrB S. typhimurium TA98 hisD3052 rfa wvrB pKM IOl S. typhimurium TA100 /iisG46 rfa uvrB pKM IOl E. coli WP2 trp uvrA pK M IO l (CM891) 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 M arine Bacteria. Aberdeen, Scotland. Batches of the strains are obtained from master stocks held in liquid nitrogen. The test batches are aliquots of nutrient broth cultures and are stored at -80C. Dimethyl sulphoxide (DM SO) is added to the cultures at 8% v/v as a cryopreservative. Each batch of frozen strain is tested, where applicable, for cell m em brane permeability {rfa m utation), sensitivity to UV light and the pKMIOl plasmid which confers resistance to ampicillin. The responses of the strains to a series of diagnostic mutagens are also assessed. For use in tests an aliquot o f frozen culture will be added to 25 ml of nutrient broth and incubated, with shaking, at 37C for 10 hours. These cultures provide approximately 109 cells per ml which will be measured by dilution plating. Positive controls In the absence of S9 mix Identity: CAS No.: Solvent: Concentration: Sodium azide 26628-22-8 DMSO 0.5 pg/plate for strains TA1535 and TA100 'DOW S? Enquiry number: 23923D Identity: CAS No.: Solvent: Concentration: Identity: CAS No.: Solvent: Concentration: Identity: CAS No.: Solvent: Concentration: 9-Aminoacridine 90-45-9 DMSO 30 pg/plate for strain TA1537 Huntingdon Life Sciences 2-Nitrofluorene 607-57-8 DMSO 1 jig/plate for strain TA98 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) 3688-53-7 DMSO 0.05 jig/plate for strain CM891 In the presence o f S9 mix Identity: CAS No.: Solvent: Concentration: 2-Aminoanthracene 613-13-8 DMSO 2 pg/plate for strain TA1535 10 pg/plate for strain CM891 Identity: CAS No.: Solvent: Concentration: Benzo[a]pyrene 50-32-8 ' DMSO 5 pg/plate for strains TA1537, TA98 and TA100 Preparation of S9 fraction Species: Sex: Strain: Age: Weight: Rat M ale Sprague-Dawley derived 7-8 weeks <300 g S9 fraction will be prepared from a group, of usually ca 10 animals. Mixed function oxidase systems in the rat liver will be stimulated by Aroclor 1254, administered in an appropriate vehicle as a single intraperitoneal injection at a dosage of 500 mg/kg body weight. On the fifth day after injection, following an overnight starvation, the rats will be killed and their livers aseptically removed. The following steps will be carried out at 0-4C under aseptic conditions. The livers will be placed in 0.15 M KC1 (3 ml KC1 : 1 g liver) before being transferred to a homogeniser. Following preparation, the hom ogenate will be centrifuged at 9000 g for 10 minutes. The supernatant fraction (S9 fraction) will be dispensed into aliquots and stored at -80C or below until required. All batches of S9 fraction will be tested for sterility and efficacy. P reparation of S9 mix S9 mix contains: S9 fraction (10% v/v), M gCF (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 will be filter-sterilised before use. 000038 Enquiry number: 23923D Huntingdon Life Sciences Selection of solvent/vehicle A suitable solvent will be selected. If solubility cannot be achieved in a suitable solvent, then a vehicle will be chosen in which a suspension o f test substance can be prepared. The identity of the solvent or vehicle will be documented. Mutation test procedure First test The test substance will be added to cultures of the five tester strains usually at a minimum of seven concentrations. The highest concentration will usually be 50 mg/ml of the test substance in the chosen solvent/vehicle, which will provide a final concentration of 5000 pg/plate. This is the standard limit dose recommended in the regulatory guidelines this assay follows. Normally the concentrations will be separated by ca half-logio intervals. The negative control will be the chosen solvent/vehicle. The appropriate positive control compounds will also be included. Aliquots of 0.1 ml of the test solution/suspension will be placed in airtight glass vessels; this volume may be adjusted if necessary for reasons of test substance solubility or solvent/vehicle toxicity. 0.5 ml S9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) will be added, followed by 0.1 ml of a 10 hour bacterial culture. The mixtures will be incubated at 37C for 30 m inutes with shaking before addition of 2 ml of agar containing histidine (0.5 mM) and tryptophan (0.5 mM). The mixtures will be thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 ml minimal agar. Each Petri dish will be individually labelled with a unique code corresponding to a sheet, identifying the contents of the-dish. Three Petri dishes will be prepared for each dose level. If a solvent/vehicle is used for which there are no historical data to demonstrate that it causes no deleterious or mutagenic effects then a set of untreated control plates will also be prepared containing only bacterial culture and S9 mix or phosphate buffer. Plates will also be prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and phosphate buffer. All plates will be incubated at 37C for 48-72 hours. After this period the appearance of the background bacterial lawn will be examined and revertant colonies counted using an automated colony counter. Any toxic effects of the test substance will 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 used in the second test will be the same as that used in the first. If toxic effects are observed a lower concentration may be chosen. It should be ensured that if a lower concentration is chosen, signs of bacterial inhibition will be present at the top concentration. Ideally a minimum of three non-toxic concentrations should be obtained. If this is not achieved then the first test may be repeated using a more appropriate dose range. If precipitate is 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. This should be taken into account when selecting the dose levels for the second assay and may necessitate the repetition of the first test if this requirement has not been met. 000033 Enquiry number: 23923D Huntingdon Life Sciences Second test If a clear positive response is obtained in the first test, the second test will be an exact repeat of the first. However, a minimum of only five concentrations of test substance will be used. If a negative or equivocal response is obtained a variation on the above procedure may be used. Variations include (but are not restricted to) different S9 concentrations or narrowing the dose range. If the required number of non-toxic dose levels is not obtained, the second test may be repeated using a more appropriate dose range. It may also be repeated to confirm a positive response. Additional testing may be performed if no clear response is obtained. The exact design of any additional test will be at the discretion of the Study Director. 3. 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 of mean revertant colony numbers over the negative control. Analysis The mean number of 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 of at least twice the concurrent solvent/vehicle controls, with some evidence o f a positive doserelationship (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. N o statistical analysis will be performed. If exposure to a test substance does not produces 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 of 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 D unnetf 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. 000040 Enquiry number: 23923D 4. REPORTING H u n tin g d o n Life Scien ces The report will contain details of the test substance, methodology, results and interpretation of the data. Tabulated results will show individual plate counts, mean revertant colony counts and their standard deviation. W here appropriate, graphs will also be included. Good Laboratory Practice and Quality Assurance statements will be included. In the absence of ongoing communications, Huntingdon Life Sciences reserves the right to finalise, sign and issue the final report from this study six months after issue o f the draft. In such an event, all materials will be transferred to the archive. Any subsequent requests for modifications, corrections or additions to the final report will be the subject of a formal report amendment (or new study, as appropriate) and will be subject to additional cost. Upon study completion, two types of report are issued: Draft report: Following QA audit, for review by the Sponsor Final report: After approval by the Sponsor Reports will be supplied on A4 paper and the following num ber of copies will be supplied: Draft report: 1 unbound (double sided) Final report: 1 bound (double sided with original signatures) ... 1 unbound (single sided) Any additions or corrections to an authorised final report will be documented as a formal amendment to the protocol. 5. MAINTENANCE OF RECORDS 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 of the final report in its archive indefinitely. 000041 Enquiry number: 23923D Huntingdon Life Sciences 6. GOOD LABORATORY PRACTICE The study will be conducted in compliance with the principles of Good Laboratory Practice Standards as set forth in: The UK Good Laboratory Practice Regulations 1999 (Statutory Instrument No 3106). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98) 17. EC Commission Directive 1999/11/EC of 8 March 1999 (Official Journal No L 77/8). 7. QUALITY ASSURANCE The following will be inspected or audited in relation to this study. Protocol Audit : Study protocol. Process based inspections : Routine and repetitive procedures will be inspected on representative studies, not necessarily on this study. Report Audit : The draft report and study data will be audited before issue of the draft report to the Sponsor. QA findings will be reported to the Study Director and Company Management promptly on completion of each action, except for process based inspections, which will be reported to appropriate Company Management only. 8. HEALTH & SAFETY In order for Huntingdon Life Sciences to comply with the Health and Safety at Work etc. Act 1974, and the Control of Substances Hazardous to Health Regulations 1994, it is a condition of undertaking the study that the Sponsor shall provide Huntingdon Life Sciences with all information available to it regarding known or potential hazards associated with the handling and use of any substance supplied by the Sponsor to Huntingdon Life Sciences. The Sponsor shall also comply with all current legislation and regulations concerning shipment of substances by road, rail, sea or air. Such information in the form of a completed Huntingdon L ife Sciences test substance data sheet must be received by Safety Management Services at Huntingdon Life Sciences before the test substance can be handled in the laboratory. At the discretion o f Safety M anagement Services at Huntingdon Life Sciences, other documentation containing the equivalent information may be acceptable. ;i t; 000042 Enquiry number: 23923D 9. REFERENCES Huntingdon Life Sciences AMES, B.N., McCANN, J. and YAMASAKI, E. (1975) Methods for detecting carcinogens and mutagens' with the Sa/raone/ia/mammalian microsome mutagenicity test. M utation Research, 31,347-364. GATEHOUSE, D.G., ROWLAND, I.R., WILCOX, P,, CALLANDER, R.D. and FORSTER, R. (1990) Bacteria] mutation assays in: KIRKLAND, D.J. (Ed.). UKEMS Sub-committee on Guidelines fo r Mutagenicity Testing. Report. Part I revised. Basic M utagenicity Tests: UKEMS Recommended Procedures, pp. 13-61. Cambridge University Press, Cambridge. GREEN, M.H.L. (1984) Mutagen testing using trp+ reversion in Escherichia coli in: KILBEY, B.J., LEGATOR, M., NICHOLS, W. and RAMEL, C. (Eds.). Handbook o f M utagenicity 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., M IDDLETON, B,, M ITCHELL, 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 Guidelines fo r Mutagenicity Testing. Report. Part 111. Statistical Evaluation o f Mutagenicity Test Data, pp.26-65. Cam bridge University Press, Cambridge. MARON, D.M. and AMES, B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Research, 113, 173-215. 000043