Document 2DX4JD0ZomVBqZ0rxyEk7Q3L

AR226-3103 jm m ^ -- r J Huntingdon lili ' : I CONFIDENTIAL DPT 435/984104 irW TO^.lnrPff il , IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES '' Sponsor DuPont Speciality Chemicals, Jackson Laboratory, Chambers Works, Deepwater, N J08023, USA. Research L aboratory Huntingdon Life Sciences Ltd., P.O. Box 2, Huntingdon, Cambridgeshire, PE18 6ES, > ENGLAND. *. Report issued 27 October 1998 Pace 1 o f 27 Cosmssnv CONTENTS COMPLIANCE W ITH GOOD LABORATORY PRACTICE STANDARDS QUALITY ASSURANCE STATEMENT.......................................................... SU M M A RY ......................................................................................................... INTRODUCTION................................................................................................ TEST SUBSTANCE........................................................................................... EXPERIMENTAL PROCEDURE................................................................... ASSESSMENT OF RESULTS.......................................................................... MAINTENANCE OF RECORDS.................................................................... RESU LTS................. ........................................................................................... CONCLUSION................................................................................................... RFERENCES.................................................................................................... DPT 435/984104 Page 3 -4 5 6 7 8 12 12 14 15 16 TABLES . 1. Summary o f R esults............................ 2. I&itotic index data - first test.............. 3. M etaphase analysis data - first te s t.... 4. M itotic index data - second test.......... 5. M etaphase analysis data - second test APPENDIX Historical control d ata..... Company Santtfzad. Doss not contain TSCA CB DPT 435/984104 COM PLIANCE W ITH GOOD LABORATORY PRA CTICE STANDARDS The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid. The United Kingdom Good Laboratory Practice Regulations 1997, Statutory Instrum ent No. 654. EC Council Directive, 87/18 EEC o f 18 December 1986, (No. L15/29). OECD Principles o f Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98) 17. V_ _ o j & W f c - ..................... ...... Leslie C ^ u k 'B.Sc.(H ons.), CJBioi. M .I.Biol., Study Director, Department o f Genetic Toxicology, Huntingdon Life Sciences Ltd. ..... D ate A t* 3 : Company Sanitized. Does not contain TSCA CBS QUALITY ASSURANCE STATEM ENT DPT 435/984104 The following have been inspected or audited in relation to this study Study Phases Inspected P ro to c o l D ate of Inspection 20 August 1998 D ate of R eporting 20 August 1998 Process Based Inspections Cell culture preparation Test m aterial preparation D osing H arvesting cells Slide preparation Slide reading M edia change Record keeping R eport } } } }11 June 1998 } } } } 5 October 1998 12 June 1998 9 October 1998 Protocol: An audit of the protocol for this study was conducted and reported to the Study D irector and Company M anagement as indicated above. Process based inspections: A t or about the tim e this study was in progress inspections and audits o f routine and repetitive procedures employed on this type o f study were carried out. These were conducted and reported to appropriate Company M anagement as indicated above R eport A udit: This report has been audited by the Quality Assurance Departm ent. This audit was conducted and reported to the Study D irector and Company M anagement as indicated above. The m ethods, procedures and observations were found to be accurately described and the reported results to reflect the raw data. Kevin"p . de-Salis, B.A. (H ons.), C.Biol., M .I.Biol., Dip R.Q.A., Quality A ssurance Unit H ead, ' Departm ent O f Quality A ssurance, Huntingdon L ife Sciences Ltd. D ate :4 : Company Sanitized. Boss not enn*n nn SUMMARY DPT 435/984104 A study was performed to assess the ability human lymphocytes cultured in vitro. induce chromosomal aberrations in Hum an lymphocytes, in whole blood culture, were stimulated to divide by addition o f phytohaemagghitinin, and exposed to the test substance both in the presence and absence o f S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. Tw o hours before the end o f 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 o ^ H B H ^ t o cultured human lymphocytes, the m itotic index was for all cultures treated with the test substance and the solvent control. On the basis o f these Hata the following concentrations were selected for metaphase analysis: F irst test W ithout S9 mix - 3 hours treatm ent, 18 hours recovery: W ith S9 mix - 3 hours treatm ent, 18 hours recovery: 312.5, 625 and 1250 pg/ml. 156.3, 312.5 and 625 pg/ml. Second test W ithout S9 mix - 3 hour treatm ent, 18 hours recovery: W ith S9 mix - 3 hours treatm ent, 18 hours recovery: 312.5.450 and 550 pg/ml. 312.5.450 and 625 pg/ml. In the absence o f S9 n u x , f l H H M c a u s e d a statistically significant m crease (P<0.001) m the proportion o f metaphase figures co n tain tf| chromosomal aberrations, a t the two highest dose levels analysed when compared w ith the solvent control, in the first test. In the second test a statistically c-gnifirant increase (P<0.01) in the proportion o f metaphase figures containing chromosomal aberrations was observed a t the highest dose level analysed. In the presence o f S9 n t i x |M H B 0 % a u s e d a statistically significant increase (P < 0.001) in the proportion ofm etaphase figures containing chromosomal aberrations, a t the highest dose level analysed when compared with the solvent control, in the first test. In the second test, statistically significant increases were seen at the intermediate (P O .001) and highest (P<0.01) dose levels analysed. A quantitative analysis for polyploidy was made in cultures treated with the negative control and highest dose level. Statistically significant increases in the proportion o f polyploid cells were seen m both tests in the absence and presence o f S9 mix. All positive control compounds caused large, statistically significant increases in the proportion o f aberrant cells, demonstrating the sensitivity o f the test system and the efficacy o f the S9 mix. > It is concluded shown evidence o f clastogenic and polyploidy-inducing activity in both the presence and absence o fS 9 mix in this in vitro cytogenetic test system. 5 . Company Sanitized. Doss not contain Tsrta INTRODUCTION DPT 435/984104 This report describes a study designed to assess the ability o aberrations in human lymphocytes cultured m vitro. to cause chromosomal The study w as conducted in compliance with the foHowmg^guideline. ~ f ("1997) Genetic Toxicology: In Vitro Human lymphocytes hat* ^ ^ T s o Dt U" " y ^ results in a high mitotic yield (Nowell 1960). ^ phytohaemagglutinin (PHA, to foe culture winch In this study, blood taken from healthy the test substance. The cultures were incubated in the Pres metanhase using the m itotic inhibitor, Colcerrud . Following treatm ent the ceUs w ere am ; Chromosomes in these m etaphase cells w e exarnined for the presence o f chromosome .g ^ ^ grst cell division after initiation of aberrations. The best estim ate o f the a e , during subsequent cell divisions. In this laboratory treatm ent since certain types { blood cuiture is approximately 15 hours. On this 1 the cell cycle tim e for hum an lymphocytes m wnoie basis, the cells were examined after 21 hours. The study was performed on two separate occasions. tIn,, u^tv, toctc, a three hour treatm ent was used in both the presence and the absence o f S9 mix. ,, Aberrations w ere scored according to th e c o f the. ISCN (1985). Traditionally gaps have aberrations Some gaps, however, have been been excluded from the quantation Bodycote 1970, Satya-Prakash, H su and Pathak shown to be real discontinuities m D N A ^ both ^ ^ without gaps has 1981). In this study the total num ber o f cells containing been calculated. Many substances do n ot exert a mutagenic e systems f t * are no, found in until thev have been m etabolised by enzyme ^ cultures and test substance were O l i v e r traction (S9 m ix) prepared from ^ o w n te biduce a high ievei o t enzymtc activity (M aron and Ames 1983. Natarajan al. 1976). ' f , Furtimrdon L ift Sciences Management on.7 July 1998, by foe Sponsor r i 7P" a n T C s ^ o t o n .9 A u ,, s t 1998. The experimental phase o f foe "study was conducted between 19 August'and 21 September 1998. :6 : Company Sanitized. Dons not contain TSC C S Identity: Chemical name: Intended use: Appearance: Storage conditions: Lot number: Expiry: Purity: D ate received: TEST SUBSTANCE DPT 435/984104 Room tem perature 2 years from date o f receipt 23 June 1998 : 7 : Orees not contain TSCA CBI e x p e r im e n t a l p r o c e d u r e DPT 435/984104 CULTURE OF LYMPHOCYTES Human blood was collected aseptically from ------- _ -* ,, , n tissue culture medium (Life Technologies) supplemented with 10% foetal c a lf serum (Life Technologies), 2 0 1.U./ml peniciUin/20 pg/ml streptomycin (Imperial) and 2 mM glutam ine (Im perial). Aliquots (0.4 ml blood : 4.5 ml medium : 0.1 ml phytohaemagglutinin (Life Technologies)) o f die cell suspension were placed in sterile universal containers and incubated at 37C for approximately 48 hours. The cultures were gently shaken once daily to resuspend die cells. PO SITIV E CONTROLS In th e absence of S9 m ix Identity: S u p p lie r Appearance: Batch num ber Solvent: Final concentration: In th e presence of S9 mix Identity: S u p p lie r Appearance: Batch number: ` Solvent: Final concentration: Mitomycin C BDH Blue powder . B37741 Sterile deionised w ater 0.4 and 0.8 pg/ml, one dose level o f which was selected for the metaphase analysis C yclophospham ide Sigma Chemical Co Ltd W hite powder 87H0207 Sterile deionised w ater 20 and 30 pg/ml, one dose level o f which was selected for the metaphase analysis PREPARATION O F S9 FRACTIO N Species: Sex: Strain: Source: Age: W eight: Diet: Rat M ale Sprague-Dawley derived . Harlan Olac Ltd 7 - 8 weeks <300 g SDS R+M no. 1 (modified) nuts * : 8 : Company Sanifize'dL D sss not contain TSCA CB1 DPT 435/984104 S9 ,, a s prepared from a group o f err. 20 animals. M ixed fhncnon omdase systems m dm rat Ovenrwere s t a m S by Aroelor 1254, admmisteted as a single mtmpentoneal mjeebon m A m dns od i t a dosage o f 500 mg/kg bodyweight. On tbe fifth day after mjectton, H t a n g an overmght starvation, the rats were killed and their livers aseptically removed. The following steps were carried out a t 0 - 4C under aseptic conditions The livers were placed m 0J.5 M K Q (3^S*KC1: 1 gliver)'before being transferred to anhom ogeniser. the homogenates were centrifuged a t 9000 g for 10 minutes. H ie supernatort fiaction J was dispensed into aliquots and stored at -80C or below until required^ The efficacy o f each to c h S9 fic tio n w as tested in a bacterial mutation assay with ffie mutegemc precursors 7 ,12-dimethylbenzanthracene and 2-aminoanthracene. The stenhty was also checked. PREPARATION O F S9 M IX S9 mix contained- S9 traction (10% v/v), MgCl2 (8 mM), KC1 (33 mM), sodium orthophosphate buffer S T d (100 mM ), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filtersterilised before use. SELECTIO N O F SOLVENT Prior to commencing te s tm th e .s o lu bility o f the test s d g a n c e in solvents compatible<witti theitest return was: assessed f l ^ f l B M w a s provided a s f p f tic tiv e mgredient in w ater, i.e. 250 mg/ml. 2 garance was cleared on warming (37C) to a clear solution. On dosing'at 1% v/v i n t o l ^ S S u e c f f i - e medium, giving a final concentration o f 2500 pg/ml, a S p f e c ip h l and a colour change to yellow was observed which remained after tw o hours incubation a t 37C Further dilutions to 125, 62.5 and 31.3 pg/m l were made and on dosing a t 1/o v/v in aqueous tissue culture medium, gave final concentrations o f 1250, 625 and 313 ^ 1 T ^ s r" uI^ in decreasing levels o f precinitate, which were also present after two hours incubation a t 37 C. Concentrations with high ionic strength and osmolality m ay cause chromosomal aberrations (GaUway T a l 1987). Therefore! concentrations greater than 5000 pg/m l or 10 mM are not used m f t * system In this case, the highest final concentration used for subsequent testing was 1250 pg/ml, in order to ensure that several dose levels would be in the insoluble range. TREATM ENT O F CELLS W ITH TEST SUBSTANCE - FIR ST TEST so ill aliauots o f f t H f lf l ^ f l f t w e r e added to one set o f duplicate ^ " r i a L ^ ' .S, i 9 ^ ^ ^ 5 6 . 3 , 32.5, 625 and 1250 * W ater, the solvent control, in 50 pi aliquots, was added to two cultures. Mitomycin C, at final concentrations o f 0.4 and 0.8 pg/ml, was added to duplicate cultures. : 9 : C a n n ed . D&es not contain TSCA CBI DPT 435/984104 Immediately before treatm ent o f the second set o f cultures, 1 ml o f medium was removed from each culture and discarded. This was replaced with 1 ml o f S9 mix, followed by 50 pi aliquots o f the various Hilntinng giving the same series o f final concentrations as above. W ater (50 pi) was adffeH to tw o cultures. Cyclophosphamide was added to duplicate cultures at final concentrations o f 20 and 30 pg/ml. Three hours after dosing, the cultures were centrifuged at 500 g for 10 minutes and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours. HARVESTING AND FIX A TIO N Two hours before the cells were harvested, mitotic activity was arrested by addition o f Colcemid (Sigma) to each culture a t a final concentration o f 0.1 pg/ml. A fter 2 hours incubation, each cell suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes a t 500 g. The cell pellets were treated with 4 ml o f a hypotonic solution (0.075M KC1 prewarmed a t 37 C). A fter a 10 minute period o f hypotonic incubation at 37C, 4 ml o f ice-cold fresh fixative (3 parts m ethanol: 1 part glar-^l acetic acid) was added with gentle agitation. The cultures were then centrifuged at 200 g fo r 10 minntftg the supernatant removed, and the cell pellets resuspended in 4 ml fixative. The fixative was replaced further times until it became colourless. SLID E PREPARATION The pellets were resuspended, then centrifuged at 200 g for 10 minutes and finally resuspended in a small volume o f fresh fixative. A few drops o f 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 w ater (pH 6.8). After rinsing in buffered w ater the slides were left to air-dry and then mounted in DPX. M IC R O SC O PIC EXAM INATION The prepared slides were examined by light microscopy using a low power objective. The proportion o f m itotic cells p e r'l 000 cells in each culture was recorded except for positive control treated cultures. From these results the dose level causing a decrease in m itotic index o f a t least 50% o f the solvent contfb! 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 o f each positive control compound selected for analysis was the lowest concentration dosed unless a preliminary scan o f metaphase figures indicated an insufficient level o f aberrant cells. The selected slides were then coded. M etaphase cells were identified using a low power objective and examined at a magnification o f xlOOO using an oil immersion objective. One hundred metaphase figures were examined, where possible, from each culture. Chromosome aberrations were scored according to the classification o f the ISCN (1985). Only cells w ith 44 - 48 chromosomes were .m DPT 435/984104 analysed. Polyploid and endoreduplicaled cells were noted when seen. The vernier readings o f all aberrant metaphase figures were recorded. The incidence o f polyploid metaphase cells, out o f 500 m etaphase cells, where possible, was determined quantitatively for negative control cultures and cultures treated with the highest dose level o f the test substance used in the analysis for chromosomal aberrations. The number o f aberrant metaphase cells in each treatm ent group was compared with the solvent control value using Fisher's test (Fisher 1973). SECOND TEST Cultures were initiated and maintained as previously described. In this second test a repeat o f the first test was carried out. Concentrations as follows: W ithout S9 mix: 156.3, 312.5,450, 550, 625, 750, 1000 and 1250 pg/ml. W ith S9 mix: 78.1, 156.3, 312.5,450, 550, 625, 750 and 1250 pg/ml. D uplicate cultures were used for each treatm ent and two cultures were treated with the solvent control. Positive control cultures were treated as in the first test. Three hours after dosing, all cultures were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours. All cultures were treated with Colcemid, at a final concentration o f 0.1 pg/ml, two hours before the end o f the incubation period. They were then harvested, fixed and the slides prepared as previously described. The slides were then examined microscopically as previously described. STA B ILITY AND FO RM U LA TIO N ANALYSIS The stability o f the test substance and o f the test substance in the solvent were not determined as part of this study. Analysis o f achieved concentration was not performed as part o f this study. s : 11 : S' r ASSESSMENT OF RESULTS DPT 435/984104 An assay is considered to be acceptable if the negative and positive control values lie w ithin the current historical control range. The test substance is considered to cause a positiveT esponseifT h^follow in^ ccraditions^eT net: Statistically significant increases (P < 0 .0 1 ) in the frequency o f metaphases with aberrant chromosomes (excluding gaps) are observed at one or m ore test concentration. The increases exceed the negative control range o f this laboratory, taken at the 95% confidence lim it. The increases are reproducible between replicate cultures. The increases are not associated with large changes in osm olality o f the treatm ent medium or extrem e toxicity. Evidence o f a dose-relationship is considered to support the conclusion. A negative response is claimed if no statistically significant increases in the num ber o f 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 o r a negative response are not m et. M AINTENANCE O F RECORDS All experim ental data arising from the study (including documentary raw data, specim ens, records and other m aterials; collectively defined as the " m aterials" ) will rem ain the property o f the Sponsor. if . Huntingdon L ife Sciences shall retain the m aterials in its archive for a period o f five years from the date o f issue o f the final report. A fter such tim e, the Sponsor w ill be contacted and their advice sought on the return, disposal o r further retention of the materials.- If requested, Huntingdon Life Sciences w ill continue to retain1the m aterials, subject to a reasonable fee being agreed with the Sponsor. Huntingdon Life Sciences shall also retain a copy o f the final report in its archive at Huntirigdoh indefinitely. Company Sanitized. Does not contain TSC CBS RESULTS DPT 435/984104 FIRST TEST Toxicity data M itotic indices o f cultured human lymphocytes treated Wll .re shown in Table 2. In the absence o f S9 m i x f l ^ B f l n C a u s e d a reduction in the mitotic index to 43% o f the solvent control value a t 1250 pg7ml. This concentration was selected as the highest dose level for the metaphase analysis. Treatm ent concentrations o f 625 and 312.5 pg/ml were chosen as the intermediate and low est concentrations for m etaphase analysis. hi the presence o f S9 mix, no live cells were seen in cultures treated with the highest dose level, 1250 pg/m l. f lH H f |j[ |H ^ a u s e d a reduction in the m itotic index to 42% o f the solvent control value at 625 pg/ml This concentration was selected as the highest dose level for the m etaphase analysis. Treatm ent concentrations o f 312.5 and 156.3 pg/ml were chosen as the interm ediate and lowest concentrations for metaphase analysis. The quantitative analysis for polyploidy showed statistically significant increases in the number o f polyploid metaphase figures, when compared to the solvent control, in both tests. The concentrations o f the positive control compounds selected for analysis were mitomycin C at 0.8 pg/m l and cyclophosphamide a t 30 pg/ml. M etaphase analysis The effects of in the chromosomes o f cultured human lymphocytes are shown in Table 3 and summarised in Table 1. hi die absence o f S9 m ix f l( P B H ( |p a u s e d statistically significant increases in the proportion o f cells w ith chromosomal aBerrations (P 0 .0 0 1 ) a t the highest two dose levels analysed, 625 and ` 1250 pg/m l. These increases were to 5.5 and 7.5% aberrant cells, respectively, w hich lie well above the upper 95% limit o f the historical control range (1.5%). In the presence o f59 mix, treatm ent w itl0 B B B M B ^ e s u Ite d in a statistically significant increase in the proportion o f metaphase figures with chromosomal aberrations (PO.O Ol) a t the highest dose level analysed, 625 pg/ml. This increase was to 9.0% aberrant cells (excluding gaps), w hich lies well above the upper 95% limit o f the historical control range (1.5%). , Both positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P<0.001), in the proportion o f aberrant cells. This dem onstrated the efficacy o f the S9 m ix and the sensitivity o f the test system. 13 Company Sanitized. Does not contain TSCA C3 DPT 435/984104 SECOND TEST Toxicity d ata M itotic indices o f cultured human lymphocytes treated s^own T able 4. In the absence o f S9 m i x A M B M i caused a reduction in the mitotic index to 43% o f the solvent P r a g u e at 550 p ^ ^ ^ K n S a t i o n was selected a sth e h ip e s t dose level for th e m ^ a p h a ^ analysis. Treatment Concentrations o f 450 and 312.5 pg/ml were chosen as the intermediate and lowest concentrations for metaphase analysis. Tn the nresence o f S9 m i x f i f l M B i W > s e d a reduction in the mitotic index to 49% o f the solvent control3value a t 625 p g / m l T ^ S m S r a t io n was selected as the highest dose level for the metaphase analysis. Treatm ent concentrations o f 450 and 312.5 pg/ml were chosen as the intermediate and lowest concentrations for metaphase analysis. H e quantitative analysis for polyploidy stow ed statistically significant: increases in the number of polyploid m etaphase figures, when compared to the solvent control, in both tests. The concentrations o f the positive control compounds selected for analysis were mitomycin C at 0.8 pg/ml and cyclophosphamide a t 30 pg/ml. M etaphase analysis The effects o ^ | P f l 0D the chromosomes o f cultured human lymphocytes are shown in Table 5 and summarised in Table 1. In the absence o f S9 m i x ( |H M f c a u s e d a statistically significant increase in the p ro p o rtio n ^ cells w ith chromosomal aberrations (P<0.01) at the highest dose level analysed 550 pg/mL This increase w as to 6.5% aberrant cells, which lies well above the upper 95 / limit o f the historical contra range (1.5% ). i +u rtf on m;x treatm ent w i t h f i B S M i l e s u I t e d in statistically significant increases in p r o p o r t io n o f m etaphase figures with chromosomal aberrations (P<0.001 and P<0-01) at fce two highest dose levels analysed, 450 and 625 pg/ml, respectively. These increases were to 7.0 and 5.5/o S n t cells, respectively, which he well above the upper 95% limit o f the histoncal control range (1.5% ). , Both positive 'control compounds, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P<0.001) in the proportion o f aberrant cells. : 14: C O N C L U S IO N DPT 435/984104 It is r-nnrlnHftH t h a t d |M H 0 0 p i a s shown evidence o f clastogenic and polyploidy-inducing activity in both the presence tm cffii^ib^nee o f S9 mix in this in vitro cytogenetic test system. : 15: 0omPa>U'San|!i2ed..Do. R,, ,. REFERENCES DPT 435/984104 EVANS, H .J., and O'RIORDAN, M .L., (1975) Human peripheral blood lymphocytes for the analysis o f chromosome aberrations in mutagen tests. M utation Research, 31, 135. FISHER, R.A . (1973) The Exact: Treatment o f 2 x 2 Table in: Statistical Methods fo r Research Workers. Hafher Publishing Company, New York. GALLOWAY, S.M ., DEASY, D.A., BEAN, C .L., KRAGNAK, A.R., ARMSTRONG, M .J. and BRADLEY, M.Q. (1987) Effects o f high osmotic strength on chromosome aberrations, sister chromatid exchanges and DNA strand breaks, and the relation to toxicity. M utation Research, 189, 15. HEDDLE, J.A. and BODYCOTE, D.J. (1970) On the formation o f 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 AM ES, B.N . (1983). Revised m ethods for the Salmonella mutagenicity test. Mutation Research, 113, 173. NATARAJAN, A.T., TATES, A.D., van BUUL, P.P.W ., MELTERS, M. and de VOGEL, N. (1976) Cytogenetic effects o f mutagens/carcinogens after activation in a microsomal system in vitro. Mutation Research, 37, 83. NOW ELL, P.C. (1960) Phytohaemagglutinin: an initiator o f mitosis in cultures o f 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 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 : 16 : Company Sanitized. Does not contain TSCA CBi '""'N DPT 435/984104 TABLEI Sum mary o f Results Test 1 Exposure S9 period mix (hours) 3- (pg/ml) 0 (Water) 312.5 625 1250 0.8 (Mitomycin C) -C Excluding gaps . ..Cells wiihnherrations _ __Relative-- Including gaps Mitotic Individual values (%) Mean (%) Individual values (%) Mean (%) Index (%) 00 00 47 87 10 10 0 .0 0 .0 7 5*** 2 0 .0 *** 0 0 4 8 10 0 0 7 7 10 0 .0 0 .0 5 5*** 7 5 *** 2 0 .0 *** 95 64 43 - 3+ 0 (Water) 00 0 .0 00 0 .0 - 156.3 03 1.5 1 3 2 .0 88 312.5 01 0.5 1 2 1.5 72 625 6 12 8 12 1 0 .0 *** 42 30 (Cyclophosphamide) 1 1 9 2 0 .0 *** 1 2 10 2 2 .0 *** - Test 2 Exposure S9 period mix (hours) 3 ^ (pg/ml) 0 (Water) 312.5 450 550 0.8 (Mitomycin C) Cells with aberrations Excluding gaps Individual values (%) Mean (%) 11 01 53 85 12 12 1 .0 0.5 4.0 6.5** 24.0*** Cells with aberrations Including gaps Individual values (%) Mean (%) 11 01 54 85 12 12 1 .0 0.5 4.5 6.5** 24.0*** Relative Mitotic Index (%) 84 55 43 - 3+ 0 (Water) * - 312.5 11 13 1 .0 2 .0 11 13 1 .0 2 .0 85 * 450 68 7.0*** 6 8 7.0*** 67 625 6 5 ' 5.5** 6 5 5.5** 49 Y 30 (Cyclophosphamide) 14 17 15.5*** 15 17 16.0*** - *** pcO.OOl ** P<0.01 Otherwise P>0.01 : 17 : Comnanv Sanitized. Does not contain TSCA CBl DPT 435/984104 TABLE 2 M itotic index data - first test W ithout S9 mix, 3 hours treatm ent and 18 hours recovery Concentration of 0 (Water) Mitotic index- - -----Relative-mitotic index Incidence %Mean <%) 124/1000 152/1000 13.8 100 Polyploidy________ Incidence 0/500 1/500 % Mean 0.1 9.8 19.5 39.1 78.1 156.3 312.5 625 1250 147/1000 165/1000 169/1000 175/1000 159/1000 155/1000 154/1000 137/1000 118/1000 114/1000 139/1000 1 2 2 /1 0 0 0 87/1000 90/1000 6 8 /1 0 0 0 52/1000 15.6 17.2 15.7 14.6 1 1 .6 13.1 8.9 6 .0 113 125 114 106 84 95 64 43 7/205 7/221 3.3 *** Precipitate in cultures treated with 312.5, 625 and 1250 pg/m l on dosing Precipitate in cultures treated with 625 and 1250 pg/ml a t end o f treatm ent (3 hours) *** PO.OOl ,i Otherwise P>0.01 ? 18 Company Sanitized. Does noi contain TSCA CBI DPT 435/984104 I TABLE 2 M itotic index data - first test (continued) W ith S9 m ix, 3 hours treatm ent and 18 hours recovery Concentration/^ (^ ^ K ^ (ug/ml) 0 (Water) Mitotic index Incidence 148/1000 156/1000 %Mean 15.2 Relative -mitotie-index- (%) 100 Polyploidy Incidence 0/500 2/500 % % Mean 0.2 9.8 167/1000 16.3 107 158/1000 19.5 1 1 1 /1 0 0 0 13.7 90 162/1000 39.1 150/1000 15.5 102 160/1000 78.1 155/1000 16.2 107 168/1000 156.3 127/1000 140/1000 13.4 88 312.5 625 1 2 0 /1 0 0 0 98/1000 57/1000 70/1000 10.9 6.4 72 42 11/438 21/500 3.4 *** 1250 0 /0 0 /0 - ** Precipitate in cultures treated with 625 and 1250 pg/ml on dosing *** PcO.OOl Otherwise *P>0.01 : I I : 19 : Company Sanitized. Poes not contain TSCA CBs DPT 435/984104 TABLE 3 M etaphase analysis data - first test Without S9 mix, 3 hours treatment and 18 hours recovery Concentration of (/tg/ml) No. cells Aberrations No. o f aberrant cells Relative examined Chromatid Chromosome Others Gaps Exc. Mean Inc. Mean Mitotic type type gaps % gaps % index ctb cte csb cse ctg csg %; 0 (Water) . 100 100 0 0 .0 0 0 .0 00 100 312.5 625 1250 100 100 71 0 0 1 0 7 1 3 100 100 10 12 3 1 2 0 0 .0 0 0 .0 00 4 5.5 4 5.5 7 *** 7 *** 8 7.5 8 7.5 77 95 64 43 0 .8 Mitomycin C 50 14 2 2 50 2 1 2 1 1 0 2 0 .0 10 1 0 2 0 .0 *** 10 - 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 10 aberrations, pulverised cells and pulverised chromosomes : 20 : Company Sanitized. Does not contain Tsr'A DPT 435/984104 Concentration of Gtg/ml) 0 (Water) TABLE 3 M etaphase analysis data - first test (continued) With S9 mix, 3 hours treatment and 18 hours recovery No. cells Aberrations examined Chromatid Chromosome Others type type Gaps No of aberrant cells Relative Exc. Mean Inc. "Mean "Mt&c gaPs % gaps % index ctb cte csb cse ctg csg 100 100 0 0 .0 0 0 0 .0 0 100 156.3 312.5 625 100 100 100 100 100 100 12 1 21 17 1 4 9 1.5l 0 1 2 .0 88 33 1 0 0.5 1 1.5 72 11 2 3 6 9.0 8 1 0 . 0 42 1 1 2 SM:* 1 2 *9|i* 30 Cyclophosphamide 50 9 3 2 50 4 4 7 -1 11 2 0 .0 1 2 2 2 .0 1 9 *** 1 0 ctb Chromatidbreak 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 10 aberrations, pulverised cells and pulverised chromosomes ? : 21 : Company Sanitized. Does not contain TSCA CBS DPT 435/984104 TABLE4 M itotic index data - second test Without S9 mix, 3 hours treatment and 18 hours recovery 0 (Water) Mitoticindex - ---- Relative mitotic index Incidence %Mean (%) 183/1000 18.7 1 0 0 190/1000 Polyploidy----------- : Incidence 0/500 0/500 % Mean 0 .0 156.3 312.5 450 550 625 750 1000 1250 173/1000 141/1000 170/1000 146/1000 1 0 0 /1 0 0 0 103/1000 70/1000 90/1000 81/1000 58/1000 61/1000 57/1000 37/1000 39/1000 2 0 /1 0 0 0 16/1000 15.7 15.8 1 0 .2 8 .0 7.0 5.9 3.8 1 .8 84 84 55 43 37 32 20 10 15/500 17/500 3.2 #*# Precipitate in cultures treated from 312.5 to 1250 pg/ml on dosing *** * P<0.001 Otherwise P>0.01 , l : 22 : Company Sanitised. Does not contain TSCA CSS DPT 435/984104 TABLE 4 M itotic index data - second test (continued) With S9 mix, 3 hours treatment and 18 hours recovery ^ o r g itr a o n a ^ (ttg/ml) 0 (Water) Mitotic index Incidence 127/1000 108/1000 %Mean 1 1 .8 Relative mitotic index (%) 100 Polyploidy Incidence 0/500 2/500 %Mean 0.2 78.1 156.3 312.5 450 550 625 750 1250 1 0 1 /1 0 0 0 109/1000 10.5 89 117/1000 1 1 1 /1 0 0 0 11.4 97 98/1000 1 0 1 /1 0 0 0 1 0 .0 85 84/1000 73/1000 7.9 67 61/1000 65/1000 57/1000 59/1000 6.3 5.8 53 49 15/500 3.6 21/500 *** 47/1000 28/1000 3.8 32 35/1000 33/1000 3.4 29 Precipitate in cultures treated with 450 and 1250 pg/ml on dosing *** P<0.001 Otherwise P>0.01 : 23 : Company Sanitized, Doss not contain TSCA CBS DPT 435/984104 TABLE5 M etaphase analysis data - second test W ithout S9 mix, 3 hours treatment and 18 hours recovery Concentration of Cm m m \ (/ig/ml) No. cells Aberradons No. o f absrrant cells Relative examined Chromatid Chromosome Others Gaps Exc. Mean Inc. Mean Mitotic type type gaps % gaps % index ctb cte csb cse ctg csg % -. 0 (Water) 100 100 1 2 1 1 .0 1 1 .0 11 100 312.5 450 550 100 100 100 100 100 100 1 14 7 72 22 1 1 1 1 0 0.5 0 0.5 11 5 4.0 5 4.5 34 8 6.5 8 6.5 " 5 ** 5 84 55 43 0 .8 Mitomycin C 50 19 3 2 50 1 0 8 1 1 1 2 24.0 1 2 24.0 *|**j*|^ *** 12 12 ctb Chromatid break csb Chromosome break ctg Chromatid gap ** / Otherwise '* P<0.001 P<0.01 P>0.01 cte cse csg others Chromatid exchange Chromosome exchange Chromosome gap C ells with greater than 10 aberrations, pulverised cells and pulverised chromosomes * '? : 24 : Company Sanitized. D ecs not contain TSC Ci I DPT 435/984104 TABLE 5 M etaphase analysis data - second test (continued) With S9 mix, 3 hours treatment and 18 hours recoveiy Concentration of Og/ml) 0 (Water) No. cells -- Aberrations------- - No. ofaberrant cells-- -Relative examined Chromatid Chromosome Others Gaps Exc. Mean Inc. Mean Mitotic type type ctb cte csb cse gaps % gaps % ctg csg index % 100 100 1 1 1 1.0 1 1.0 1 0 0 11 312.5 450 625 100 100 1 4 100 100 100 100 8 10 4 6 2 2 30 Cyclophosphamide 1 0 0 12 3 3 100 11 2 6 1 1 1 1 2 .0 3 1 2 .0 3 6 7.0 6 7.0 8 *** 8 *** 6 5.5 6 5.5 5 ** 5 ** 85 67 49 14 15.5 15 16.0 17 sMe* 17 *** _ ctb Chromatid break csb Chromosome break ctg Chromatid gap *** ** Otherwise PcO.OOl P<0.01 P>0.01 cte cse csg others Chromatid exchange Chromosome exchange Chromosome gap Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes : 25 : Company Sanitized. D oss not contain TSCA CBP DPT 435/984104 A P P E N D IX H istorical control data A berrant cells seen in human lymphocytes in whole blood culture, treated w ith solvent controls from February 1995 until M ay 1998. Total no. of cells Without gaps No. of %mean aberrant cells Range of means (%) min max Upper 95% confidence limit (%) - S9 80780 477 0.59 0 3.68 1.5 + S9 75600 429 0.57 0 3 1.5 Total no. of cells With gaps No. of % mean aberrant cells Range of means (%) min max Upper 95% confidence limit (%) -S9 80780 609 0.75 0 4.47 1.76 + S9 75600 542 0.72 0 3 2 Polyploid cells seen in human lymphocytes in whole blood culture, treated w ith solvent controls from February 1995 until May 1998. * Total no. No. of %mean Range of means of cells polyploid (%) Upper 95% confidence cells limit min max (%) - S9 33391 37 0 . 1 1 0 0.5 0.5 + S9 30433 6 6 0 . 2 2 0 0.7 0.63 : 26 : Company Sanitized Does no! contain TSCA CBS DPT 435/984104 A P P E N D IX H istorical control d ata (continued) A berrant cells seen in human lymphocytes in whole blood culture, treated with positive controls from February 1995 until M ay 1998. Total no. of cells No. of aberrant cells % mean Without gaps Range of means (%) min max - S9 29876 4663 15.61 8 48 Lower 95% confidence limit 8.75 Upper 95% confidence limit (%) 31 + S9 27064 4184 15.46 9 42 10 27.95 Total no. of cells No. of aberrant cells % mean With gaps Range of means (%) min max Lower 95% confidence limit - S9 29876 4851 16.24 8.5 51.5 9.25 Upper 95% confidence limit (%) 32 + S9 27064 4342 16.04 9 42 10 29 not contain TSGA CB 27 : Company Sanitized. Does