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. I' . s PERFLUOROOCTANESULFONYLFLUORIDE (POSF) T-7bb I,3 IN MTRO MAMMALIAN CHROMOSOME ABERRATION TEST M HUMAN LYMPHOCYTES Sponsor 3M Center 3M Corporate Toxicology Building 220-2E-02 St Paul MN 55133-3220 USA Page 1of 31 Research Laboratory Huntingdon Life SciencesLtd Woolley Road Alconbury Huntingdon Cambridgeshire PE28 4HS ENGLAND Report issued 22 October 2002 MIN 315/022356 CONTENTS Page CONTENTS ................................................................................................................................ 2 COMPLIANCEWITH GOOD LABORATORYPRACTICE STANDARDS....................... 4 QUALITY ASSURANCE STATEMENT................................................................................. 5 RESPONSIBLE PERSONNEL.................................................................................................. 6 SUMMARY ................................................................................................................................ 7 INTRODUCTION....................................................................................................................... 8 TEST SUBSTANCE................................................................................................................... 10 EXPERIMENTALPROCEDURE............................................................................................. 11 ASSESSMENT OF RESULTS .................................................................................................. 15 MAINTENANCE OF RECORDS ............................................................................................. 15 RESULTS .................................................................................................................................... 16 CONCLUSION ........................................................................................................................... 17 REFERENCES ............................................................................................................................ 18 :2: MIN 315/022356 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. 3 106). EC Commission Directive 1999111EC 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 substance was the responsibility of the Sponsor. Study Director, Department of Genetic Toxicology, Huntingdon Life Sciences Ltd. & .2&.... Date ...L. L 2 :4: .' QUALITY ASSURANCE STATEMENT The following have been inspected or audited in relation to this study. MIN 315/022356 Study Phase Date of Inspection Date of Reporting Protocol Audit 11 January 2002 11 January 2002 Study Based Inspection Culture treatment 17 January 2002 17 January 2002 Process Based Inspections Formu1ation Culture Establishment Harvesting and slide preparation Slide scoring S-9 preparation 14 January 2002 11 February 2002 7 February 2002 5 March 2002 15 January 2002 14 January 2002 11 February 2002 8 February 2002 5 March 2002 15 January 2002 Report Audit 15 April 2002 15 April 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. Study Based Inspection: An inspection of a phase of 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 of 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 to reflect the raw data. Group Manager, Department of Quality Assurance, Huntingdon Life Sciences Ltd. :5 -..G?.G@?2.* Date -a RESPONSIBLE PERSONNEL Linda Allais, DEA Tox., DESS Pharm. Vet., France, Study Director, Department of Genetic Toxicology. Lincoln Pritchard, B.Sc., Study Supervisor, Department of Genetic Toxicology. MIN 315/022356 :6: SUMMARY MIN 315/022356 A study was performed to assess the ability of Perfluorooctanesulfonyl Fluoride (POSF) to induce chromosomal aberrations in human lymphocytes cultured in v i m . Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid@,the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. In order to assess the toxicity of Perfluorooctanesulfonyl Fluoride (POSF) to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis: First test With and without S9 mix - 3 hours treatment, 17 hours recovery: 1.25,2.5and 5% v/v atmosphere. Second test Without S9 mix - 20 hours continuous treatment: 0.8, 1 and 2% v/v atmosphere. With S9 mix - 3 hours treatment, 17 hours recovery: 2,5 and 7.5% v/v atmosphere. In both the absence and presence of S9 mix, Perfluorooctanesulfonyl Fluoride (POSF) caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level, when compared with the solvent control, in either test. A quantitative analysis for polyploidy was made in cultures treated with the negative control and highest dose level. No statistically significant increases in the proportion of polyploid cells were seen. All positive control compounds caused large statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. It is concluded that the test substance Perfluorooctanesulfonyl Fluoride (POSF) has shown no evidence of clastogenic activity in this in vitro cytogenetic test system, under the experimental conditions described. :7: INTRODUCTION MIN 3 15/022356 This report describes a study designed to assess the ability of Perfluorooctanesulfonyl Fluoride (POSF) to cause chromosomal aberrations in human lymphocytes cultured in vitro. The study was conducted in compliance with the following guidelines: OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology: In Vitro Mammalian Chromosome Aberration Test, Guideline 473. US EPA (1998) Health Effects Test Guidelines. OPPTS 870.5375 I n Vitro Mammalian Chromosome Aberration Test. EPA 7 12-C-98-223. Human lymphocytes have been used in this type of study for a number of years (Evans and O'Riordan 1975, Scott, Dean, Danford and Kirkland 1990). They are cultured in vitro but do not divide unless stimulated to do so. This is achieved by adding phytohaemagglutinin (PHA)to the culture that results in a high mitotic yield (Nowell 1960). In this study, blood taken from healthy male non-smoking donors was pooled and diluted with tissue culture medium. The cultures were incubated in the presence of PHA before being treated with the test substance. Following treatment the cells were arrested at metaphase using the mitotic inhibitor, Colcemid@. Chromosomes in these metaphase cells were then examined for the presence of chromosome aberrations. The best estimate of the aberration frequency is at the first cell division after initiation of treatment since certain types of damage may be lost during subsequent cell divisions. In this laboratory the cell cycle time for human lymphocytes in whole blood culture is approximately 13-14 hours. The study was performed on two separate occasions. In the first test, a three hour treatment was used in both the presence and the absence of S9 mix. In the second test, a continuous treatment was used without S9 mix, and the test with S9 mix was a repeat of the first test. Aberrations were scored according to the classification of the ISCN (1985). Traditionally gaps have been excluded from the quantitation of chromosome aberrations. Some gaps, however, have been shown to be real discontinuities in DNA (Heddle and Bodycote 1970, Satya-Prakash, Hsu and Pathak 1981). In this study the total number of cells containing aberrations both with and without gaps has been calculated. Many substances do not exert a mutagenic effect until they have been metabolised by enzyme systems that are not found in cultured cells. Therefore the cultures and test substance were 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 enzymic activity (Maron and Ames 1983, Natarajan et al. 1976). :8: MIN 315/022356 The protocol was approved by Huntingdon Life Sciences Management on 18 July 2001, by the Sponsor on 3 1 August 2001 and by the Study Director on 10 January 2002. The study was conducted at Huntingdon Life Sciences Ltd., Eye, Suffolk, IP23 7PX, England. The experimental start and completion dates of the study were 15 January 2002 and 15 March 2002, respectively. :9: Identity: CAS No.: Appearance: Storage conditions: Batch number: Expiry: Purity: Date received: TEST SUBSTANCE MIN 315/022356 Perfluorooctanesulfonyl Fluoride (POSF) 307-35-7 Clear liquid Room temperature 040227 Sponsor's responsibility; assumed stable for the duration of the study >95.5 % 14 June 2001 : 10: EXPERIMENTAL PROCEDURE MIN 3 15/022356 CULTURE OF LYMPHOCYTES Human blood was collected aseptically from healthy, non-smoking male donors, pooled and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 1 unit/ml Heparin, 20 I.U./ml penicillid20 pg/ml streptomycin and 2.0 mM glutamine. Aliquots (0.4 ml blood : 4.5ml medium : 0.1 ml phytohaemagglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37C for approximately 48 hours. The cultures were gently shaken daily to resuspend the cells. POSITIVE CONTROLS In the absence of S9 mix Identity: Supplier: Appearance: Batch number: Solvent: Final concentration: In the presence of S9 mix Identity: Supplier: Appearance: Batch number: Solvent: Final concentration: Mitomycin C Sigma Chemical Co Ltd Blue powder 3 1K2500 Sterile purified water 0.2 pg/ml (3 hour treatment) 0.1 pg/ml (continuous treatment) Cyclophosphamide Asta Medica Ltd White powder ON465 (Test 1) and 1H485 (Test 2) Sterile purified water 10 P g / d PREPARATION OF S9 FRACTION Species: Sex: Strain: Source: Age: Weight: Rat Male Sprague-Dawley derived Charles River UK 7 - 8 weeks ~ 3 0 g0 11 : MIN 3 151022356 S9 fraction was prepared from a group of ca. 10 animals. Mixed function oxidase systems in the rat livers were stimulated by Aroclor 1254, administered as a single intraperitoneal injection in corn oil at a dosage of 500 mgkg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed 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 KCl (3 ml KCl : 1 g liver) before being transferred to an homogeniser. Following preparation, the homogenates were centrifuged at 9000 g for 10 minutes. The supernatant fraction (S9 fraction) was dispensed into aliquots and stored at -80C or below until required. PREPARATION OF S9 MIX S9 mix contained: S9 fraction (10% vh), MgC12 (8 mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate ( 5 mM), NADP (4 mM). All the cofactors were filter-sterilised before use. TREATMENT OF CELLS WITH TEST SUBSTANCE - FIRST TEST After approximately 48 hours, the cultures were centrifuged and the cells were resuspended in fresh culture medium. Atmospheres of Perfluorooctanesulfonyl Fluoride (POSF) were established in sealed glass bottles (160 ml internal volume) with septum caps to give final concentrations of 1.25, 2.5, 5 , 10, 20,40 and 70% v/v atmosphere. Air was withdrawn from each bottle and then an appropriate volume of Perfluorooctanesulfonyl Fluoride (POSF) was introduced using a syringe and needle, inserted through the septum cap. After evaporation of Perfluorooctanesulfonyl Fluoride (POSF) and equilibration of the atmospheres at 37 OC, the lymphocyte cultures were injected into the bottles. The glass bottles were then incubated on their sides at 37 "C in a roller apparatus (see appendix 3: Apparatus for vapour/gas phase exposure of cultivated mammalian cells), which rotates the bottles once every eight minutes approximately. The lymphocytes coat the inside of the bottles and were immersed in culture medium once every revolution and exposed directly to Perfluorooctanesulfonyl Fluoride (POSF) for the rest of the revolution. The solvent control (Air) was established in duplicate cultures and contained an atmosphere of air. Mitomycin C, at a final concentration of 0.2 pg/ml, was added to duplicate cultures and also contained an atmosphere of air. Immediately before treatment of the second set of cultures, 1 ml of medium was removed from each culture and discarded. This was replaced with 1 ml of S9 mix. The cultures were then added to the appropriate glass bottle giving the same series of final concentrations as above. The duplicate solvent control cultures were established under an atmosphere of air. Cyclophosphamide was added to duplicate cultures at a final concentration of 10pg/ml that were contained in an atmosphere of air. Three hours after dosing, the cultures were centrifuged at 500 g for 5 minutes. The cells were rinsed and resuspended in fresh medium under an atmosphere of air in universal containers. They were then incubated for a further 17 hours. : 12: MIN 315/022356 HARVESTING AND FIXATION Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid@ (Sigma) to each culture at a final concentration of 0.1 pg/ml. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with a hypotonic solution (0.075M KC1 prewarmed at 37C). After a 10 minute period of hypotonic incubation at 37"C, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced further times until it became colourless. SLIDE PREPARATION The pellets were resuspended, then centrifuged at 500 g. for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto precleaned microscope slides which were then allowed to airdry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to airdry and then mounted in DPX. The remaining cultures in fixative were stored at 4C until slide analysis was completed. MICROSCOPIC EXAMINATION The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures. From these results the dose level causing a decrease in mitotic index of approximately 50% of the solvent control value or, if there was no decrease, the maximum achievable concentration was used as the highest dose level for the metaphase analysis. The intermediate and low dose levels were also selected. The concentration of each positive control compound selected for analysis was the lowest concentration dosed unless a preliminary scan of metaphase figures indicated an insufficient level of aberrant cells. The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of xlO00 using an oil immersion objective. One hundred metaphase figures were examined, where possible, from each culture. This number was reduced in cultures showing a high level of aberrant cells. Chromosome aberrations were scored according to the classification of the ISCN (1985). Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. The vernier readings of all aberrant metaphase figures were recorded. The incidence of polyploid metaphase cells, out of 500 metaphase cells, was determined quantitatively for negative control cultures and cultures treated with the highest dose level of the test substance used in the analysis for chromosomal aberrations. The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973). 13 MIN 3 15/022356 SECOND TEST Cultures were initiated and maintained as previously described. In this second test a continuous treatment was used in the absence of S9 mix. In the presence of S9 mix, a three hour treatment was used, as in the first test. The harvest time was at 20 hours for both parts of the test. Concentrations of Perfluorooctanesulfonyl Fluoride (POSF) were as follows: Without S9 mix: 0.1,0.2,0.4,0.6,0.8,1, 2 and 5% v/v atmosphere. With S9 mix: 0.6,0.8, 1, 2,5 and 7.5% v/v atmosphere. Duplicate cultures were used for each treatment and the solvent control. Mitomycin C, at a final concentration of 0.1 pg/ml, and Cyclophosphamide, at a final concentration of 10 pg/ml, were added to duplicate cultures. Three hours after dosing, the cultures containing S9 mix were centrifuged. The cells were rinsed and resuspended in fresh medium under an atmosphere of air in universal containers. They were then incubated for a further 17 hours. Cultures treated in the absence of S9 mix were incubated for 20 hours. All cultures were treated with Colcemid@,at a final concentration of 0.1 pg/ml, two hours before the end of the incubation period. They were then harvested, fixed and the slides prepared as previously described. The slides were then examined microscopically as previously described. STABILITY, HOMOGENEITY AND FORMULATION ANALYSIS The stability and homogeneity of the test substance and of the test substance in the solvent were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study. : 14: ASSESSMENT OF RESULTS MIN 315/022356 An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range. The test substance is considered to cause a positive response if the following conditions are met: Statistically significant increases (P<O.Ol) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration. The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit. The increases are reproducible between replicate cultures. The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity. Evidence of a dose-relationship is considered to support the conclusion. A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level. A further evaluation may be carried out if the above criteria for a positive or a negative response are not met. MAINTENANCE OF RECORDS All raw data, samples and specimens (if appropriate) arising from the performance of 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. : 15: RESULTS MIN 319022356 FIRST TEST Toxicity data Mitotic indices of cultured human lymphocytes treated with Perfluorooctanesulfonyl Fluoride (POSF) are shown in Table 2. In the absence of S9 mix, Perfluorooctanesulfonyl Fluoride (POSF) caused a reduction in the mitotic index to 57% of the solvent control value at dose level of 5 % v/v atmosphere. The dose levels selected for the metaphase analysis were 1.25,2.5 and 5% v/v atmosphere. In the presence of S9 mix, Perfluorooctanesulfonyl Fluoride (POSF) caused a reduction in the mitotic index to 52% of the solvent control value at dose level of 5% v/v atmosphere. The dose levels selected for the metaphase analysis were 1.25,2.5 and 5% v/v atmosphere. The quantitative analysis for polyploidy showed no increase in the number of polyploid metaphase figures when compared to the solvent control. Metaphase analysis The effects of Perfluorooctanesulfonyl Fluoride (POSF) on the chromosomes of cultured human lymphocytes are shown in Table 3 and summarised in Table 1. In both the absence and the presence of S9 mix, Perfluorooctanesulfonyl Fluoride (POSF) caused no statistically significant increase in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent control. Both positive control compounds, Mitomycin C and Cyclophosphamide, caused large statistically significant increases (PcO.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system. SECOND TEST Toxicity data Mitotic indices of cultured human lymphocytes treated with Perfluorooctanesulfonyl Fluoride (POSF) are shown in Table 4. In the absence of S9 mix, Perfluorooctanesulfonyl Fluoride (POSF) caused a reduction in the mitotic index to 61% of the solvent control value at dose level of 2% v/v atmosphere. The dose levels selected for the metaphase analysis were 0.8, 1and 2%v/v atmosphere. : 16 MIN 3151022356 In the presence of S9 mix, Perfluorooctanesulfonyl Fluoride (POSF) caused a reduction in the mitotic index to 39% of the solvent control value at dose level of 7.5% v/v. The dose levels selected for the metaphase analysis were 2, 5 and 7.5% vlv. The quantitative analysis for polyploidy showed no increase in the number of polyploid metaphase cells when compared to the solvent control. Metaphase analysis The effects of Perfluorooctanesulfonyl Fluoride (POSF) on the chromosomes of cultured human lymphocytes are shown in Table 5 and summarised in Table 1. In both the absence and the presence of S9 mix, Perfluorooctanesulfonyl Fluoride (POSF) caused no statistically significant increase in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent control. Both positive control compounds, Mitomycin C and Cyclophosphamide, caused large, statistically significant increases (P<O.OO1) in the proportion of aberrant cells. CONCLUSION It is concluded that the test substance Perfluorooctanesulfonyl Fluoride (POSF) has shown no evidence of clastogenic activity in this in vitro cytogenetic test system, under the experimental conditions described. : 17: REFERENCES MIN 3 15/022356 EVANS, H.J. and O'RIORDAN, M.L. (1975) Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutation Research, 31, 135. FISHER, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methodsfor Research Workers. Hafner Publishing Company, New York. GALLOWAY, S.M., DEASY, D.A., BEAN, C.L., KRAGNAK, A.R., ARMSTRONG, M.J. and BRADLEY, M.O. (1987) Effects of high osmotic strength on chromosome aberrations, sister chromatid exchanges and DNA strand breaks, and the relation to toxicity. Mutation Research, 189, 15. HEDDLE, J.A. and BODYCOTE, D.J. (1970) On the formation of chromosomal aberrations. Mutation Research, 9, 117. ISCN (1985) An International System for Human Cytogenetic Nomenclature, HARNDEN, D.G. and KLINGER, H. P. (Eds). S. Karger AG, Basel. MARON, D.M. and A M E S , B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research, 113, 173. NATARAJAN, A.T., TATES, A.D., van BUUL, P.P.W., MEIJERS, M. and de VOGEL, N. (1976) Cytogenetic effects of mutagens/carcinogens after activation in a microsomal system in vitro. Mutation Research, 37, 83. NOWELL, P.C. (1960) Phytohaemagglutinin: an initiator of mitosis in cultures of normal human leukocytes. Cancer Research 20,462. SATYA-PRAKASH, K.L., HSU, T.C. and PATHAK, S. (1981) Chromatid lesions and chromatid core morphology. Cytogenetics and Cell Genetics, 30, 248. SCOTT, D., DEAN, B., DANFORD, N.D. and KIRKLAND, D.J. (1990) Metaphase chromosome aberration assays in vitro in KIRKLAND, D.J. (Ed.)Basic Mutagenicity Tests: UKEMS Recommended Procedures. Report of the UKEMS sub-committee on guidelines for mutagenicity testing. Report, Part I revised p. 62. Cambridge University Press, Cambridge. : 18: MIN 31YO22356 TABLE 1 Summary of Results Test 1 Exposure s 9 period mix (hours) Concentration of Perfluorooctanesulfon yl Fluoride (POSF) (% v/v atmosphere) 0 (Air) 1.25 2.5 5 0.2 pg/ml (Mitomycin C) Cells with aberrations Excluding gaps Cells with aberrations Including gaps Individual values (%) 1 1 54 30 5 1 "18 a24 Mean (%I 1.o 4.5 1.5 3.0 21.0*** Individual values (%) 22 55 32 71 "24 '28 Mean 2.0 5.0 2.5 4.0 26.0*** Relative Mitotic Index (%) 100 60 72 57 + 0 (Air) 22 2.0 44 4.0 100 1.25 1 3 2.0 15 3 .O 92 2.5 35 4.0 36 4.5 80 5 1 1 1.o 12 1.5 52 1Okg/ml (Cyclophosphamide) "20 23 22.0*** '22 29 26.7*** Test 2 Exposure period (hours) 20 s9 mix - Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmosphere) 0 (Air) 0.8 1 2 0.1 pg/ml (Mitomycin C) Cells with aberrations Excluding gaps Cells with aberrations Including gaps Individual values (%) 20 02 21 00 97 Mean (%) 1.o 1.o 1.5 0.0 8.0*** Individual values (%) 20 03 3 1 00 97 Mean (%) 1.o 1.5 2.0 0.0 8.0*** Relative Mitotic Index (%) 100 69 66 61 3 + 0 (Air) 2 1 1 1.o 22 2.0 100 1 1 1.o 32 2.5 73 5 01 0.5 12 1.5 70 7.5 bO 1 0.7 bO 2 1.3 39 - 1Okg/ml (Cyclophosphamide) " I 8 "18 18.0*** "20 "20 20.0*** *** Otherwise PcO.001 P20.01 ' 50 cells were analysed from these cultures due to high levels of aberrations seen b 50 cells were analysed from this culture due to insufficient metaphases present on slide : 19: MIN 315/022356 TABLE 2 Mitotic index data - first test Without S9 mix, 3 hours treatment and 17 hours recovery Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmosphere) 0 (Air) Mitotic index # Incidence 8 8/ 1000 92/ 1000 % Mean 9.0 Relative mitotic index # (%) 100 Polyploidy Incidence 1/500 1/500 '70 Mean 0.2 1.25 51/1000 5.4 60 57/ 1000 74/1000 6.5 72 55/1000 5 57/1000 5.1 57 2/500 0.2 45/ 1000 Oh00 10 a a b b 40 b b 70 b b # Calculations have been made using rounded values a Very few metaphases present on slide b No cells, no metaphases present on slide : 20 : MIN 315/022356 TABLE 2 Mitotic index data - first test (continued) With S9 mix, 3 hours treatment and 17 hours recovery Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmomhere) Mitotic index # Incidence 97/1000 1oo/1000 % Mean 9.9 Relative mitotic index # (%I 100 Polyploidy Incidence 1/ 5 00 1/500 % Mean 0.2 1.25 68/1000 9.1 92 113/1000 2.5 64/1000 7.9 80 93/1000 5 76/1000 5.1 52 0/500 0.0 26/ 1000 0/500 10 a a 20 b b 40 b b 70 b # Calculations have been made using rounded values a Very few live cells, no metaphases present on slide b No cells, no metaphases present on slide Not assessed : 21 MIN 3 15/022356 TABLE 3 Metaphase analysis data - first test Without S9 mix, 3 hours treatment and 17 hours recovery Concentration of Perfluorooc tanesulfonyl Fluoride (POSF) (% v/v atmosphere) 0 (Air) No. cell: Aberrations :xamined Chromatid Zhromosome Ithers type ctb cte type csb cse 100 1 100 1 No. of aberrant cells Gaps Exc. Mean Inc. Mean gaps % gaps % ctg csg 1 1.0 2 2.0 1 2 telative Mitotic index % 100 1.25 100 5 100 2 2.5 100 5 100 5 100 4 100 1 5 4.5 5 5.0 60 4 5 3 1.5 3 2.5 72 0 2 5 3.0 7 4.0 57 1 1 0.2 p d m l (Mitomycin C) 50 8 50 13 9 21.0 12 26.0 1 12 *** 14 *** ctb Chromatid break csb Chromosome break ctg Chromatid gap *** Otherwise P<O.Ool P10.01 cte cse csg others Chromatid exchange Chromosome exchange Chromosome gap Cells with greater than 8 aberrations, pulverised cells and pulverised chromosomes : 22 : MIN 3 1YO22356 TABLE 3 Metaphase analysis data - first test (continued) With S9 mix. 3 hours treatment and 17 hours recovery Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmosphere) No. cell: :xamined Chromatid type ctb cte Aberrations Zhromosome we csb cse 0 (Air) 100 2 100 2 Gaps ctg csg No. of aberrant cells Exc. Mean Inc. Mean gaps % gaps % Relative Mitotic index % 3 2 2.0 4 4.0 100 2 2 4 1.25 100 1 100 3 2.5 100 5 100 3 5 100 1 100 1 1 2.0 1 3.0 92 2 3 5 1 3 4.0 3 4.5 80 2 5 6 1 1.0 1 1.5 52 1 1 2 10 Pdd (Cyclophosphamide) 50 7 1 100 19 2 2 10 22.0 11 26.7 11 23 *** 29 *** ctb Chromatid break csb Chromosome break ctg Chromatid gap *** Otherwise P<O.OOl P20.01 cte cse csg others Chromatid exchange Chromosome exchange Chromosome gap Cells with greater than 8 aberrations, pulverised cells and pulverised chromosomes : 23 : MIN 3 151022356 TABLE 4 Mitotic index data - second test Without S9 mix, 20 hours continuous treatment Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmomhere) 0 (Air) Mitotic index # Incidence 12611000 107/1000 % Mean 11.7 Relative mitotic index # 100 Polyploidy Incidence 015 00 01500 % Mean 0.0 0.1 104/1000 10.9 93 113/1OOO 0.2 114/1OOO 10.9 93 1034 000 0.4 90/1000 9.4 80 97/1000 0.6 10511000 8.5 73 64/1000 0.8 71/1000 8.1 69 90/ 1000 1 72/ 1000 7.7 66 81/1000 2 6511000 7.1 61 77/1000 5 14/1000 2.2 19 29/1000 # Calculations have been made using rounded values 0/500 0.0 01500 : 24 : .. MIN 315/022356 TABLE 4 Mitotic index data - second test (continued) With S9 mix, 3 hours treatment and 17 hours recovery Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% vlv atmosphere) 0 (Air) Mitotic index # Incidence 6911000 9411000 % Mean 8.2 Relative mitotic index # (%I 100 Polyploidy Incidence 01500 11500 % Mean 0.1 0.6 8911000 8.2 100 74/1000 0.8 7211000 7.6 93 7911000 1 5611000 7.0 85 83/1000 2 60/ 1000 6.0 73 5911000 5 5811000 5.7 70 5611000 7.5 2911000 3.2 39 017 1 0.0 3511 000 0124 1 # Calculations have been made using rounded values : 25 : MIN 3 151022356 TABLE 5 Metaphase analysis data - second test Without S9 mix, 20 hours continuous treatment Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmosphere) 0 (Air) No. cell: :xaminec 100 100 Chromatid type ctb cte Aberrations Chromosomf lthers type csb cse ~ 2 Gaps ctg csg No. of aberrant cells Exc. Mear Inc. Mean gaps % gaps % 2 1 .o 2 1.o 0 0 Relative Mitotic index % 100 0.8 100 100 1 100 100 1 2 100 100 0 1 .o 0 1.5 69 1 2 3 1 2 1.5 3 2.0 66 1 1 0 0.0 0 0.0 61 0 0 0.1 pg/ml 100 (Mitomycin C) 100 3 1 2 2 9 8.0 9 8.0 1 7 *** 7 *** ctb Chromatid break csb Chromosome break ctg Chromatid gap *** Otherwise P<O.OOl P10.01 cte cse csg others Chromatid exchange Chromosome exchange Chromosome gap Cells with greater than 8 aberrations, pulverised cells and pulverised chromosomes : 26 : APPENDIX 2 MIN 3 15/022356 Historical positive control data (January 1999 - December 2001) Without S9 mix Ecluding gaps, lower 99% confidence lin-it = 8.5%, mean = 17.12% Including gaps, low er 99% confidence limit = 1O.O%, mean = 19.95% A 30al 2 5 - 0 5 20- L 2 15- 8 10- 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Frequency of aberrant metaphases (YO) Historical positive control data (January 1999 - December 2001) With S9 mix 0 Excluding gaps, lower 99% confidence lirrit = 7.0%, mean = 14.95% 0including gaps, lower 99% confidence limit = 8.5%,mean = 18.40% 40 1 - s 35 30 25 5 20 L 15 8 10 5 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Frequency of aberrant metaphases : 29 : MIN 3 15/022356 APPENDIX 3 APPARATUS FOR VAPOUIUGAS PHASE EXPOSURE OF CULTIVATED MAMMALIAN CELLS FIGURE 1 Culture bottle (volume 160ml) I.......................................................................... 01 4--- r-1 Septumcap Culture medium containing mammalian cells f S9 mix (volume ca 5ml) FIGURE 2 Roller apparatus Culture bottle Rotating bottle holder : 30 : Motorised rollers .. . APPENDIX 4 Eye Research Centre GLP Compliance Statement, 2001 MIN 315/022356 . e 4 0 4 THEDEPARTMENTOFHEALTHOFTHEGOVE~ OF THE UNITED KINGDOM SlXIEMENTOFcOMPLIANCE IN ACCORDANCE WITH DJRJKIWE88/320EEC LABORATORY TEST TYPE Huntingdon Life Science8 Eye Ruearch Centre Eye SIlilOlk IP23 7PX Analytical Cherubtry cllniepl Chemistry ECO8yyrtemr Environmental Fate Environmental Toxlcity Mutagenicity Phydchem Tmting Toricology DATE OF INSPECTION 29* January 2001 A general inspection for compliance with the Principles of Good Laboratory Practice was carded out at the above laboratory as part of UKGLP Compliance Rogramme. 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. Dr.Roger 0.Alexander Head, UK GLP MonitoringAuthority :31: