Document RwN3ydzBO5JY3M82v6bJ8O78

^ tfe lT M .a S L G -/\2 l SRPT T-76.I MIN 315/022356 vZ <"2Ovr?0 r ; -C O & ^53 3C*C 3 O p t o cn PERFLUOROOCTANESULFONYL FLUORIDE (POSF) T'lkC?1,3 /A VITRO MAMMALIAN CHROM OSOM E ABERRATION TEST IN HUMAN LYMPHOCYTES Sponsor 3M Center 3M Corporate Toxicology Building 220-2E-02 St Paul MN 55133-3220 USA CONTAIN NO CBI Page 1 of 31 Research Laboratory Huntingdon Life Sciences Ltd Woolley Road Alconbury. Huntingdon Cambridgeshire PE284HS ENGLAND Report issued 22 October 2002 600044 CONTENTS CONTENTS................................................................................. MENT 315/022356 Page 2 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ................. 4 QUALITY ASSURANCE STATEMENT....,................. ............................................... .......... 5 RESPONSIBLE PERSONNEL................ 6 SUMMARY............................. ........................................ ....... ................................................. 7 INTRODUCTION............................................................. ............................................,............ 8 TEST SUBSTANCE......................... 10 EXPERIMENTAL PROCEDURE...... ................................. .................................................... 11 ASSESSMENT OF RESULTS................................................................... 15 MAINTENANCE OF RECORDS............................................ 15 RESULTS........... ............................................................................................................... !....... 16 CONCLUSION............ ................................................................................. ............................ 17 REFERENCES.............. 18 MIN 315/022356 CONTENTS (continued) Page TABLES 1. Summary of Results..... ......................................................... *......................................... 2. Mitotic index data - first test................................ 3. Metaphase analysis data - first test................................................................... 4. Mitotic index data - second te st....................................... .......................................... 5. Metaphase analysis data - second test............................................................................. 19 20 22 24 26 APPENDICES 1. Historical negative control data.......................... 2. Historical positive control data...................................................... 3. Apparatus for vapour/gas phase exposure of cultivatedmammalian cells..................... 4. Eye Research Centre GLP Compliance Statement, 2001........................................ 28 29 30 31 :3 : mmm 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. 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 substance was the responsibility of the Sponsor. Ms Linda Allais,!DEA Tox., DESS Pharm. Vet., France Study Director, Department of Genetic Toxicology, Huntingdon Life Sciences Ltd. Date 006947 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 Formulation 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. Angela Jennings, B.Sc., M .Sc.,' Group Manager, Department of Quality Assurance, Huntingdon Life Sciences Ltd. I M.R.Q.A., 5: c2o2_ Date O0048 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 : summ ary MIN 315/022356 A study was performed to assess the ability of Perfluorooctanesulfonyl Fluoride (POSF) to induce chromosomal aberrations in human lymphocytes cultured in vitro. 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, 2.5 and 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 315/022356 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 In Vitro Mammalian Chromosome Aberration Test. EPA 712-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: 00051 MIN 315/022356 The protocol was approved by Huntingdon Life Sciences Management on 18 July 2001, by the Sponsor on 31 August 2001 and by the Study Director on 10 January 2002. The study was conducted at Huntingdon Life Sciences Ltd., Eye, Suffolk, EP23 7PX, England. The experimental start and completion dates of the study were 15 January 2002 and 15 March 2002, respectively. :9 : 000052 ft : Identity: CAS No.: Appearance: Storage conditions: Batch number: Expiry: Purity: Date received: TEST SUBSTANCE MEN 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: 0000 $ 3 . EXPERIMENTAL PROCEDURE MEN 315/022356 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 penicillin/20 pig/ml streptomycin and 2.0 mM glutamine. Aliquots (0.4 ml blood : 4.5 ml 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 31K2500 Sterile purified water 0.2 /tg/ml (3 hour treatment) 0.1 /xg/ml (continuous treatment) Cyclophosphamide Asta Medica Ltd White powder ON465 (Test 1) and 1H485 (Test 2) Sterile purified water 10^g/ml PREPARATION OF S9 FRACTION Species: Sex: Strain: Source: Age: Weight: Rat Male Sprague-Dawley derived Charles River UK 7 - 8 weeks <300 g : 11 : 000054 MIN 315/022356 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 com oil at a dosage of 500 mg/kg 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 KC1 (3 ml KC1: 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% v/v), MgCl2 (8 mM), KC1 (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 C, 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 /rg/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 10 ptg/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. 000055 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 37C, 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 pre-cleaned microscope slides which were then allowed to air-dry. 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 air-dry 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 xlOOO 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: 090056 MIN 315/022356 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 (Xg/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: 000057 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 (PcO.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 : 000058 RESULTS MIN 315/022356 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 (P<0.001) 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, 1 and 2% v/v atmosphere. 000059' MEM 315/022356 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% v/v. 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 (PcO.OOl) 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: 000060 REFERENCES MIN 315/022356 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 Methodsfo r 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 fo r Human Cytogenetic Nomenclature, HARNDEN, D.G. and KLINGER, H. P. (Eds). S. Karger AG, Basel. MARON, D.M. and AMES, 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., MEDERS, 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 o f the UKEMS sub-committee on guidelines fo r mutagenicity testing. Report, Part I revised p. 62. Cambridge University Press, Cambridge. : 18: 000001 MIN 315/022356 TABLE 1 Summary of Results Test 1 Exposure S9 period mix (hours) 3- Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmosphere) 0 (Air) 1.25 2.5 5 0.2 tg/rnl (Mitomycin C) Cells with aberrations Excluding gaps Cells with aberrations Including gaps Individual values (%) 11 54 30 5T a 18 a24 Mean (%) 1.0 4.5 1.5 3.0 2 1 .0 *** Individual values (%) 22 55 32 71 a24 a28 Mean (%) 2 .0 5.0 2.5 4.0 26.0*** Relative Mitotic Index (%) 100 60 72 57 - 3+ 0 (Air) 22 2 .0 4 4 4.0 100 1.25 1 3 2 . 0 1 5 3.0 92 2.5 35 4.0 3 6 4.5 80 5 11 1.0 1 2 1.5 52 10/rg/ml (Cyclophosphamide) a 2 0 23 2 2 .0 *** a 2 2 29 26.7*** - Test 2 Exposure S9 period mix (hours) 20 - Concentration o f Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmosphere) 0 (Air) 0 .8 1 2 0.1 /ig/ml (Mitomycin C) Cells with aberrations Excluding gaps Individual values (%) 20 02 21 00 97 Mean (%) 1.0 1.0 1.5 0.0 g 0* * * Cells with aberrations Including gaps Individual values (%) 20 03 31 00 97 Mean (%) 1.0 1.5 2 .0 0.0 gQ* * * Relative Mitotic Index (%) 100 69 66 61 - 3+ 0 (Air) 11 1.0 2 2 2 11 1.0 3 2 5 0 1 0.5 1 2 7.5 b0 1 0.7 b 0 2 10/tg/ml (Cyclophosphamide) a 18 a 18 18.0*** a2 0 a 2 0 *** PcO.OOl Otherwise P>0.01 a 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 2 .0 2.5 1.5 1.3 2 0 .0 *** 100 73 70 39 - : 19: 000S2 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 /1 0 0 0 92/1000 % Mean 9.0 Relative mitotic index # (%) 100 Polyploidy Incidence 1/500 1/500 % Mean 0 .2 1.25 51/1000 5.4 60 57/1000 2.5 74/1000 6.5 72 55/1000 5 57/1000 5.1 57 2/500 0 .2 45/1000 0/500 10 a a ' 20 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 : 00003 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 atmosphere) 0 (Air) Mitotic index # Incidence 97/1000 1 0 0 /1 0 0 0 % Mean 9.9 Relative mitotic index # (%) 100 Polyploidy Incidence 1/500 1/500 % Mean 0 .2 1.25 6 8 /1 0 0 0 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 Very few live cells, no metaphases present on slide No cells, no metaphases present on slide Not assessed 03 : 21 : 000064 MIN 315/022356 TABLE 3 Metaphase analysis data - first test Without S9 mix, 3 hours treatment and 17 hours recovery Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmosphere) No. cells Aberrations examined Chromatid Chromosome Others type type ctb cte csb cse Gaps ctg csg No. o f aberrant cells Relative Exc. Mean Inc. Mean Mitotic index gaps % gaps % % 0 (Air) 100 100 1 1 1 1 1 .0 2 2 .0 100 11 2 1.25 2.5 5 100 100 100 100 100 100 5 2 5 4 1 0 . 2 /rg/ml (Mitomycin C) . 50 50 8 13 2 1 2 3 5 4.5 5 5.0 60 14 5 3 1.5 3 2.5 72 20 2 2 5 3.0 7 4.0 57 11 4 9 21.0 1 2 26.0 1 1 12 *** 14 *** _ ctb Chromatid break csb Chromosome break ctg Chromatid gap *** Otherwise P<0.001 P>0.01 cte cse csg others Chromatid exchange Chromosome exchange Chromosome gap Cells with greater than 8 aberrations, pulverised cells and pulverised chromosomes : 22: 000065 MIN 315/022356 TABLE3 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. cells Aberrations examined Chromatid Chromosome Others type type ctb cte csb cse Gaps ctg csg No. o f aberrant cells Relative Exc. Mean Inc. Mean Mitotic index gaps % gaps % % 0 (Air) 100 100 2 2 3 2 2 . 0 4 4.0 100 22 4 1.25 2.5 5 100 100 100 100 100 100 1 3 5 3 1 1 1 3 1 0 /ig/ml (Cyclophosphamide) .. 50 100 71 19 2 2 4 1 2 .0 1 3.0 92 23 5 1 3 4.0 '3 4.5 80 25 6 1 1 . 0 1 1.5 52 11 2 2 1 0 2 2 . 0 11 26.7 _ 1 1 23 *** 29 *** ctb Chromatid break csb Chromosome break ctg Chromatid gap te* Otherwise P<0.001 P>0.01 cte cse csg others Chromatid exchange Chromosome exchange Chromosome gap Cells with greater than 8 aberrations, pulverised cells and pulverised chromosomes : 23 : # 000066 MIN 315/022356 TABLE4 Mitotic index data - second test Without S9 mix, 20 hours continuous treatment Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmosphere) 0 (Air) Mitotic index # Relative mitotic index # Incidence 126/1000 107/1000 % Mean 11.7 (%) 100 Polyploidy Incidence 0/500 0/500 % Mean 0.0 0 .1 104/1000 10.9 113/1000 0 .2 114/1000 10.9 103/1000 0.4 90/1000 9.4 97/1000 0 .6 105/1000 8.5 64/1000 0 .8 71/1000 8 .1 90/1000 1 72/1000 7.7 81/1000 2 65/1000 7.1 77/1000 5 14/1000 2 .2 29/1000 # Calculations have been made using rounded values 93 93 80 73 69 66 61 19 0/500 0/500 0.0 : 24 : 000067 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) (% v/v atmosphere) 0 (Air) Mitotic index # Incidence 69/1000 94/1000 % Mean 8 .2 Relative mitotic index # (%) 100 Polyploidy Incidence 0/500 1/500 % Mean 0.1 0 .6 89/1000 8 .2 100 74/1000 0 .8 72/1000 7.6 93 79/1000 1 56/1000 7.0 85 83/1000 2 60/1000 6 .0 73 59/1000 5 58/1000 5.7 70 56/1000 7.5 29/1000 3.2 39 0/71 0.0 35/1000 0/241 # Calculations have been made using rounded values 000088 MIN 315/022356 TABLE 5 Metaphase analysis data - second test Without S9 mix, 20 hours continuous treatment Concentration of Perfluorooctanesulfonyl Fluoride (POSF) (% v/v atmosphere) No. cells examined Chromatid type ctb cte Aberrations Chromosome Others type csb cse Gaps ctg csg No. o f aberrant cells Relative Exc. Mean Inc. Mean Mitotic index gaps % gaps % % 0 (Air) 100 100 1 2 2 1 .0 2 1 .0 00 100 0 .8 1 0 0 100 2 1 100 2 100 1 / 2 100 100 0 . 1 /xg/ml (Mitomycin C) . 100 100 63 1 32 2 0 1 . 0 0 1.5 69 12 3 1 2 1.5 3 2.0 66 11 0 0 .0 0 0 .0 00 61 9 8.0 9 8.0 _ 1 7 *** 7 *** ctb Chromatid break csb Chromosome break ctg Chromatid gap *** Otherwise PcO.OOl P>0.01 cte cse csg others Chromatid exchange Chromosome exchange Chromosome gap Cells with greater than 8 aberrations, pulverised cells and pulverised chromosomes : 26 : lit* MIN 315/022356 TABLES Metaphase analysis data - second test (continued) With S9 mix, 3 hours treatment and 17 hours recovery Concentration of No. cells Perfluorooctanesulfonyl examined Chromatid Fluoride (POSF) (% v/v atmosphere) type ctb cte Aberrations Chromosome Others type csb cse Gaps ctg csg No. o f aberrant cells Relative Exc. Mean Inc. Mean Mitotic index gaps % gaps % % 0 (Air) 100 100 1 1 1 1 1 .0 2 2 .0 100 11 2 2 100 1 100 1 5 100 100 1 7.5 50 100 1 1 0 /g/ml 50 8 3 (Cyclophosphamide) 50 9 1 2 2 1 1 .0 3 2.5 73 11 2 1 0 0.5 1 1.5 70 11 2 0 0.7 0 1.3 39 11 2 1 9 18.0 1 0 2 0 . 0 3 1 9 *** IQ *** _ ctb Chromatid break csb Chromosome break ctg Chromatid gap *** Otherwise PcO.OOl P>0.01 cte cse csg others Chromatid exchange Chromosome exchange Chromosome gap Cells with greater than 8 aberrations, pulverised cells and pulverised chromosomes difilli I : 27 000070 O ccu re n ce (%) APPENDIX 1 MIN 315/022356 H isto rical negative co n tro l data (January 1999 - D e ce m b e r 2001) W ithout S9 m ix Including gap s, upper 99% co n fide n ce limit = 6.0%, mean = 3.5% H Excluding gap s, upper 99% co n fid e n ce limit = 3.5%, mean = 1.11 % Frequency of aberrant m etaphases H isto rical negative co n tro l data (January 1999 - D e ce m b e r 2001) With S9 m ix Including gaps, upper 99% confidence limit = 5.0%, mean = 1.93% Excluding gaps, upper 99% confidence limit = 3.0%, mean = 0.96% O ccurrence (%) F re q u e n cy o f a b e rra n t m e ta p h a se s (%) : 28 : 000071 APPENDIX 2 MIN 315/022356 H istorical p o sitiv e co n tro l data (January 1999 - D ecem ber 2001) Without S9 mix Excluding gaps, low er 99% confidence limit = 8.5%, mean = 17.12% including gaps, low er 99% confidence limit = 10.0%, mean = 19.95% Occurrence (%) Occurrence (%) F req u e n cy of aberrant m e ta p h a se s (%) Historical positive control data (January 1999 - Decem ber 2001) With S9 m ix Excluding gaps, lower 99% confidence limit = 7.0%, mean = 14.95% Including gaps, lower 99% confidence limit = 8.5%, mean = 18.40% Frequency of aberrant metaphases : 29 : 000072 MIN 315/022356 APPENDIX 3 APPARATUS FOR VAPOUR/GAS PHASE EXPOSURE OF CULTIVATED MAMMALIAN CELLS FIGURE 1 Culture bottle (volume 160ml) <-------- Septum cap Culture medium containing mammalian cells S9 mix (volume ca 5ml) FIGURE 2 Roller apparatus : 30 : 000073 MIN 315/022356 APPENDIX 4 Eye Research Centre GLP Compliance Statement, 2001 THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 8&32C EEC LABORATORY TEST TYPE Huntingdon Life Sciences Eye Research Centre Eye Suffolk EP23 7PX Analytical Chemistry Clinical Chemistry E cosystem s Environmental Fate Environmental Toxicity M utagenicity Phys/Chem Testing Toxicology DATE OF INSPECTION 29* January 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 o f the inspection no deviations were found of sufficient magnitude to affect the validity of non-ciinical studies performed at these facilities. Dr. Roger G. Alexander Head, UK GLP Monitoring Authority : 31 : 000074 Enquiry number: 23923E pctlT-7(,M t. Huntingdon Life Sciences I _ " I (tf> k l . 3 PROTOCOL / PERFLUOROOCTANESULFONYL FLUORIDE (POSF) IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES Sponsor 3M Center 3M Corporate Toxicology Building 220-2E-02 St Paul MN 55133-3220 USA Research Laboratory Huntingdon Life Sciences Ltd Woolley Road Alconbury Huntingdon Cambridgeshire PE28 4HS ' ENGLAND 000075 Total num ber of pages: 12 Huminadim Life Sciences Lid., registered in EnglandNo: IS!5730 Page/ Enquiry number: 23923E Huntingdon Life Sciences PROTOCOL APPROVAL PERFLUOROOCTANESULFONYL FLUORIDE (POSF) IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES Management Huntingdon Life Sciences Ltd 3M Center Sponsor 51 Date 7 Please sign both copies o f this page, retain one fo r your records and return one to the Study Director at Huntingdon Life Sciences. Study Director 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 completedpage will be issuedprior to the start o f the study. Enquiry number: 23923E Huntingdon Life Sciences STUDY DETAILS PAGE Study number: MIN/315 Study title: In vitro mammalian chromosome aberration test in human lymphocytes Test substance Identity: Lot number: Expiry: Appearance: Storage conditions: Purity/Assay: Specific Gravity: Solvent: Perfluorooctanesulfonyl Fluoride (POSF) 040227 Sponsor's responsibility; assumed stable for duration of study Clear liquid Room temperature >95.5% ca 1.8 To be determined Stability of test substance formulation: Not assessed in this study Analysis of achieved concentration: Not assessed in this study Sponsor's Monitoring Scientist: John Butenhoff - Head, Department of Genetic Toxicology: Dr Gillian Clare Study Director: Ms Linda Allais Person acting in the temporary absence of the M r Lincoln Pritchard Study Director: Location of study: Dept. Genetic Toxicology, Huntingdon Life Sciences Ltd., Eye, Suffolk, IP23 7PX, England Proposed study dates: Start: Completion: Draft report: ca 14 January 2002 ca 15 M arch 2002 Within four weeks of experimental completion STUDY DIRECTOR APPROVAL OF PROTOCOL L o c i, 7 000077 Enquiry number: 23923E CONTENTS PROTOCOL A PPRO V A L........................... 1. INTRODUCTION.............................. . 2. BA CK G RO U N D ................................... 3. EXPERIM ENTAL PRO CED U RE..... 4. ASSESSM ENT OF R E SU L T S........ 5. REPORTING......................................... 6. M AINTENANCE OF RECORDS...... 7. GOOD LABORATORY PRACTICE 8. QUALITY A SSURAN CE................... 9. HEALTH AND SAFETY.................. . 10. REFEREN CES............................ . Huntingdon Life Sciences Page .....ii ....2 ....2 ....2 .... 6 ..... 7 ..... 7 7 8 8 9 000678 Enquiry number: 23923E Huntingdon Life Sciences 1. INTRODUCTION The object of this study is to assess the mutagenic potential of the test substance in an in vitro cytogenetic test system using cultured human lymphocytes. This procedure complies 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 In Vitro M ammalian Chromosome Aberration Test. EPA 712-C-98-223. 2. BACKGROUND Most chemical mutagens produce recognisable chromosomal damage expressed as structural aberrations. A well established test system using cultured human lymphocytes has frequently been used to observe these aberrations (Evans and O'Riordan 1975, Scott, Dean, Danford and Kirkland 1990). The aberrations are best observed at the metaphase stage of cell division, when the chromosomes are contracted. Division of the cells can be arrested at metaphase using the mitotic inhibitor, Colcemid, which prevents formation of the mitotic spindle. The best estimate of the aberration frequency is at the first cell division after treatment since certain types of damage will be lost before subsequent cell divisions. The cultures will be harvested after a period of time which is approximately 1.5 times the cell cycle after initiation of treatment (usually 18 - 21 hours). 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 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 enzymic activity (Maron and Ames 1983, Natarajan et al. 1976). 3. EXPERIMENTAL PROCEDURE Culture of lymphocytes Blood will be taken from healthy male donors and diluted with tissue culture medium (RPM I 1640 containing 10% foetal calf serum, heparin and antibiotics). Lymphocytes, which do not normally undergo cell division, will be stimulated to do so by the addition of the naturally occurring mitogen, phytohaemagglutinin (Evans and O 'Riordan 1975, Nowell 1960). The cultures will be prepared as 5 ml aliquots (0.4 ml blood: 4.5 ml medium: 0.1ml PHA solution) in sterile universal containers and incubated at 37C. The cultures will be occasionally shaken to resuspend the cells. 4 $ Nisi ' m m 7d Enquiry number: 23923E Huntingdon Life Sciences Positive controls In the absence of S9 mix Identity: Supplier: Appearance: Solvent: Concentration: In the presence of S9 mix Identity: Supplier: Appearance: Solvent: Concentration: Mitomycin C BDH Biochemical or other suitable supplier Blue powder W ater 0.1 - 1.6 pg/ml Cyclophosphamide Asta Medica Ltd. or other suitable supplier White powder W ater 5 - 3 0 pg/ml P rep aratio n of S9 fraction Species: Sex: Strain: Age: W eight: Rat M ale Sprague-Dawley derived 7-8 weeks <300g S9 fraction will be prepared from a group o f usually ca 10 animals. M ixed 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 bodyweight. 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 hom ogeniser. Following preparation, the homogenate will be centrifuged .at 9000 g for 10 m inutes. 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 rep aratio n of S9 mix S9 mix contains: S9 fraction (10% v/v), M gCL (8 mM), KC1 (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors will be filter-sterilised before use. 090080 Enquiry number: 23923E Huntingdon Life Sciences Culture treatment Two sets of cultures will be established, one set to be treated in the absence o f S9 m ix, the other in its presence. All lym phocyte cultures will be incubated for approxim ately 48 hours, following stimulation with PHA, before exposure to the test substance. The cultures wall be centrifuged and the cells will be resuspended in fresh culture medium (final volume 5 ml, containing S9 mix (1 ml) where appropriate). Atmospheres of the test m aterial at concentrations of 1.25, 2.5, 5, 10, 20, 40 and 70% v/v (70% v/v is the maximum practicable concentration) will be established in sealed glass bottles (160 ml internal volume) with septum caps. Air will be withdrawn from each bottle and then an appropriate volume o f test m aterial liquid will be introduced using a syringe and needle, inserted through the septum cap, to produce atmospheres at the required concentrations. After evaporation of the volatile material and equilibration of the atm ospheres at 37C, the lymphocyte cultures (5 ml, containing S9 mix where appropriate) will be injected into the glass bottles. The glass bottles will then be incubated on their sides at 37C in a roller apparatus which rotates the bottles once every eight minutes approximately. The lymphocytes coat the inside of the bottles and will be imm ersed in culture medium once every revolution and exposed directly to the treatment atmospheres for the rest of each revolution. This system allows maximum exposure to the treatment atmosphere, while maintaining good cell growth. Negative and positive control cultures will contain an atmosphere of air. Positive control cultures will be treated with Mitomycin C (in the absence of S9 mix) and cyclophosphamide (in the presence of S9 mix). At the higher concentrations there is an increased risk of artefactual increases in aberrations due to osmotic effects (Galloway et al. 1987). Change of pH in the medium, indicated by a colour change, may also cause artefactual damage (Morita, Watanabe, Takeda and Okumura 1989, Kitching, Mason and Jones 1997). Accordingly, any such colour changes will be recorded. Treatment media may be retained for osmolality and pH measurement if deemed necessary'. Duplicate cultures will be used for each treatment. S9 homogenate will be present in appropriate cultures at a final concentration of 5% v/v. All cultures will be identified using unique number/colour codes. In the absence and presence of S9 mix, cultures will be incubated for three hours in the presence of the test substance. At the end of this three hour period, the cultures will be centrifuged and the cells will be cultured in fresh medium under an atmosphere o f air in universal containers. The cultures will then be incubated until the scheduled harvest time (usually 18-21 hours). Harvesting Two hours before the cells are harvested, mitotic activity will be arrested by the addition of Colcemid at a final concentration of 0.1 pg/ml. The cells will then be harvested by centrifugation and treated with a hypotonic solution (0.075M KC1) to cause swelling. The cells will be fixed in cold m ethanokglacial acetic acid (3:1 v/v) and stored at 4 C until the slides are prepared. 1 00081 Enquiry number: 23923E Huntingdon Life Sciences Slide preparation A homogenous cell suspension will be prepared and aliquots of this cell suspension will be dropped onto pre-cleaned microscope slides and left to air-dry. The prepared slides will be stained in 10% Giemsa, left to air-dry and then mounted in DPX. All of the rem aining cell cultures will be stored at approximately 4C until the slide analysis has been completed. Microscopic examination The prepared slides will be examined by light microscopy. The incidence of mitotic cells per 1000 cells will be assessed (except for positive control treated cultures). From these results, at least three dose levels for subsequent metaphase analysis will be selected. The highest dose level will usually be that causing a depression in mitotic index of at least 50% when compared with the solvent control level or, if the test substance lacks toxicity, the highest concentration used in the test. Intermediate and low concentrations will be selected at the discretion of the Study Director. The intermediate dose level will usually show some evidence of toxicity, whereas the lower dose level will be non-toxic. When this range of toxicity occurs at test concentrations which have precipitate visible in the culture medium at the end of the treatment period, one or more of the concentrations selected for metaphase analysis will be above the solubility limit. If a clear toxicity pattern is not apparent, the study may be repeated. The dose levels used in the repeat test may differ from those used in the initial assay. The concentration of positive control compounds selected for analysis will be the lowest concentration dosed unless a preliminary scan of metaphase figures indicates an insufficient level of aberrant cells. Following mitotic index assessment but prior to metaphase analysis all slides will be coded. Metaphase figures will be examined by light microscopy and the incidence of chromosomal aberrations per 100 metaphase, spreads per culture will be scored. Nomenclature of chromosomal damage will be based on the system proposed by the ISCN (ISCN 1985). Only cells with 44-48 chromosomes will be analysed. The vernier readings of aberrant metaphase figures will be recorded. The incidence of polyploid metaphase cells will be determined for negative control cultures and cultures treated with the highest dose level of the test substance used in the analysis for chromosomal aberrations. Five hundred metaphase cells (where possible) will be analysed for polyploidy. Despite evidence showing some gaps to be real discontinuities in DNA (Heddle and Bodycote 1970, Satya-Prakash, Hsu and Pathak 1981) the exact nature of the chromatid gap is uncertain and may not be a true indicator of damage. Because of this uncertainty results will be analysed both including and excluding gaps. A gap will be defined as a non-staining region, smaller than the width of a chromatid, where there is minimal misalignment of the chromatid. Enquiry number: 23923E Huntingdon Life Sciences Second test Following the results of the first test, a second independent test will be conducted. The test treatment period for the set of cultures to be treated in the absence of S9 mix will be dependent on the results of the first test, i.e. 3 hour treatment in both the presence and absence of S9 mix. If a negative result is obtained a different treatment regime will be employed. Normally this will entail continuous treatment of cultures in the absence of S9 mix, pulse treatment will again be used for cultures in the presence of S9 mix. The need to vary treatment conditions will be evaluated on a case-by-case basis at the discretion of the Study Director. All cultures will be harvested at the same time as in the first test. The procedures used are as those described previously. The choice of test concentrations may vary from the first test e.g. a narrower range may be used on the basis of the toxicity observed in the first test. 4. ASSESSMENT OF RESULTS An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range. The numbers of aberrant and polyploid metaphase figures in each treatment group will be compared with the solvent control value using a one-tailed Fisher's test (Fisher 1973). This is.auseful test for analysing data when comparing two independent samples. It is used when the observed events fall into one or other o f two mutually exclusive classes. The test determines whether the two groups differ in the proportions with which they fall into the two classifications. The test substance will be considered to be positive if the following conditions are met: Statistically significant increases 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 will be considered to support the conclusion. A negative response will be claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies is 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. 000083 Enquiry number: 23923E Huntingdon Life Sciences 5. REPORTING The report will contain details of the test substance, methodology, results and interpretation of the data. The tabulated results of mitotic index and metaphase analysis data for each culture will be shown. Good Laboratory Practice and Quality Assurance statements will be included. 6. M AINTENANCE O F 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. 7. 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). 00$4 Enquiry number: 23923E Huntingdon Life Sciences 8. QUALITY ASSURANCE The following will be inspected or audited in relation to this study Protocol Audit Study specific 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 M anagem ent prom ptly on completion of each action, except for process based inspections which will be reported to appropriate Company Management only. 9. HEALTH AND SAFETY In order for Huntingdon Life Sciences to comply with the Health and Safety at W ork 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 Life 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 of Safety M anagement Services at Huntingdon Life Sciences, other documentation containing the equivalent information may be acceptable. 000085 Enquiry number: 23923E Huntingdon Life Sciences 10. REFERENCES 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 Treatm ent of 2 x 2 Table in: Statistical M ethods f o r 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 o f 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 fo r Human Cytogenetic Nomenclature, ed. Hamden, D.G. et al., S. Karger, Switzerland. KITCHENG, J.D., MASON, C. and JONES E. (1997) pH and clastogenicity - is it relevant to human lymphocytes? Mutagenesis, 12, 106. MARON, D.M. and AMES, B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutation Research, 113, 173. MORITA, T., W ATANABE, Y., TA KED A, K. and OKUM URA, K. (1989) Effects of pH on the in vitro chromosomal aberration test. M utation Research, 225, 55. NATARAJAN, A.T., TATES, A.D., van BUUL, P.P.W., MEHERS, M. and de VOGEL,. N. (1976) Cytogenetic effects o f 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) M etaphase chromosome aberration assays in vitro in: KIRKLAND, D.J. (Ed.) Basic M utagenicity Tests: UKEMS Recommended Procedures. Report o f the UKEMS Sub-Committee on guidelines fo r mutagenicity testing. Report. Part I revised, p. 62. Cambridge University Press, Cambridge. Study Number: M IN /315 Protocol Amendment N um ber: 1 Huntingdon Life Sciences PERFLUOROOCTANESULFONYL FLUORIDE (POSF) IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES Study Director Total num ber of pages: 4 N um ber of pages for internal distribution: 4 : Ms. Linda Allais, DEA Tox., DESS Pharm. Vet., France. The signature of the Study Director authorises the implementation of this amendment to protocol. In this amendment, deleted statements are struck through and new statements are underlined. Any changes to the study design after the date of this authorising signature will be documented in a further formal amendment. AMENDMENT APPROVAL F or Huntingdon Life Sciences Ltd F or tfie Sponsor Approved by: 2, Date: L 1 , . 2 - O r ) '~ \j Study Num ber: M IN /315 Protocol Am endment N um ber: 1 Huntingdon Life Sciences PERFLUOROOCTANESULFONYL FLUORIDE (POSF) IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES Reasons for amendments : To rectify the following paragraphs, as detailed on the Protocol: Original statement: Study Details page (page iii) Stability of test substance formulation: Not assessed in this study 5. Reporting The report will contain details of the test substance, methodology, results and interpretation of the data. The tabulated results of mitotic index and metaphase analysis data for each culture will be shown. Good Laboratory Practice and Quality Assurance statements will be included. 8. Q uality Assurance The following will be inspected or audited in relation to this study. Protocol Audit : Study specific protocol. 000083 Study Num ber: M IN /315 Protocol Amendment Number: 1 Huntingdon Life Sciences PERFLUOROOCTANESULFONYL FLUORIDE (POSF) IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES Revised statement: Study Details page (page iii) Stability and homogeneity of test substance formulation: Not assessed in this study 5. Reporting The report will contain details of the test substance, methodology, results and interpretation of the data. The tabulated results of mitotic index and metaphase analysis data for each culture will be shown. 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 of the draft. In such an event, all materials will be transferred to the archive. Anv 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 OA audit, for review bv the Sponsor Final report: After approval by the Sponsor Reports will be supplied on A4 paper and the following number of copies will be supplied: Draft report: 1 unbound (double sided) Final report: 1 bound (double sided with original signatures) 1 unbound (single sided) Anv additions or corrections to an authorised final report will be documented as a formal amendment to the report. 000GS9 Study Num ber: M IN /315 Protocol Amendment Number: 1 Huntingdon Life Sciences 8. Quality Assurance The following will be inspected or audited in relation to this study. Protocol Audit : Study specific protocol and amendments. ?.qO ON N1V1NOO 000090