Document kDqpzqr7j1Xk34QdGmXYZ4M0b

CONFIDENTIAL AR226-3110 DPT478/992105 This report is considered by the Study Director to be the `final drift''and has bien submitted fthe Huntingdon Life Scienees.Quality Assurance Department for Audit _ _ The Sponsor is requested to review this document and commnni|aterafi.y coipients^to^fc_S^Jdrectc-,as soon as possible. When these comments have been received1andfon completion_of the~QA^audit, fhe-FINAL REPORT containing Study Directorand QA statements willbe issued.: ' ~ ~ ~ PLEASE NOTE ^ ? n_ _ __ In compliance with GLP any changs to;the final;reporhaftefiie_-de o f issue wjU hiin die fonm ofa separate, amendment to the report.: : - - . . f ^^ ^ -- - z e- Date: 19 January 1999 "" V. 1.0 MOUSE MICRONUCLEUS TEST Sponsor DuPont Specialty Chemicals, Jackson Laboratory, Chambers Works, Deepwater, NJ 08023, USA. Research Laboratory Huntingdon Life Sciences Ltd., Eye, Suffolk, IP23 7PX, ENGLAND. Report issued Page 1 of 25 Company Sanitized. Do s not contain TSC CBI v .y  DPT478/992105 CONTENTS Page COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS......................... 4 QUALITY ASSURANCE STATEMENT................................................................................. 5 SUMMARY................................................................................................................................. 6 INTRODUCTION....................................................................................................................... 7 TEST SUBSTANCE.................................................................................................................... 9 EXPERIMENTAL PROCEDURE................................. ............................................................. 10 ASSESSMENT OF RESULTS.................................................................................................... 13 MAINTENANCE OF RECORDS.................................................................. 14 RESULTS..................................................................................................................................... 15 CONCLUSION............................................................................................................................. 16 REFERENCES.................................................... 17 TABLES 1. Summary of results and statistical analysis...................................................................... 2. Results for individual animals - 24 hour sampling time.................................................. 3. Results for individual animals - 48 hour sampling time................................................... 18 19 20 :2: Company Sanitized. Does not contain TSCA CBI APPENDICES 1. Mortality data.................................................................. 2. Preliminary toxicity test - Clinical signs and mortalities 3. Micronucleus test - Clinical signs and mortalities......... . 4. Historical vehicle control values..................................... 5. Historical positive control values.'.................................. DPT478/992105 Page 21 22 23 24 25 :3: Company Saniti Do A contain TSCA CB1 DPT478/992105 COMPLIANCE WITH GOOD LABORATORY PRACTICE 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 Laboratoiy Practice Regulations 1995;(Statutory Instrument No. 654). v\ OECD Principles of Good Laboratory Practice (as revisedun 1997), ENV/MC/CHEM(98)17. " 'S\ ... J j ? EC Council Directive 87/18/EEC of 18 December 198 cial Journal No. LI 5/29). \ Christine E. Mason, B.Sc., \ : . , Study Director, \. - Huntingdon Life SciencesrLtL X \i; S V /I X' r' v V'.- % Date :4 : Company Sanitized. Does not contain TSCA C3i QUALITY ASSURANCE STATEMENT DPT478/992105 The following have been inspected or audited in relation to this study: Study Phases Inspected Protocol ' Date of Inspection Date of Reporting Process Based Inspections Report Protocol: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Process based inspections: At or about the time this study was in progress inspections and audits of other routine and repetitive procedures employed on this type of study were carried out. These were promptly reported to appropriate Company Management. 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. Helen Comb, B.Sc., Principal Auditor, Department of Quality Assurance, Huntingdon Life Sciences Ltd. . Date :5 : Company Sanitized. Decs not contain TSCA CBS SUMMARY DPT478/992105 This study was designed to assess the potential induction of micronuclei bone marrow cells of mice. Mice were treated with a single intraperitoneal administration of the test substance at dose levels of 50, 100 and 200 mg/kg bodyweight. A preliminary toxicity test had previously shown that a dose of 200 mg/kg was expected to be approximately the maximum tolerated; this level was therefore selected as an appropriate maximum'fonuse- in the micronucleus test. . ' /. \ . The test substance and negative control were administered by.intraperitoneal injection^The negative control group received the vehicle, purified water. A positive controLsgroup was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyw ei^t / - y Bone marrow smears were obtained from five male and five female,animals in the negative control, each of the test substance groups and the posifiv.e'cpntrol group 2%hours after dosing. In addition bone marrow smears were obtained from five niale and'Tiveffemale animals in the negative control and high level treatment groups 48 hours after dosing. jQnelsmearifrom each animal was examined for the presence o f micronuclei in-'2000. immatxLrbzerythrocytes. The proportion of immature erythrocytes was assessed by examinationmf at leasttlOOO erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocyteSrwas also kept. ` . ' ;V. r.^ . V Mice treated with the test suftstance..did'TToti'sho.w*'any significant increase in the frequency of micronucleated immature,erythrocytesrat either sampling time. '... There was no significant decrease in thezproportion of immature erythrocytes after treatment of the animals with tfte4est substance. Ni No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial d e ^ y s**TM the/proportion o f immature erythrocytes were observed in mice treated w i t h j B H H | ^ S andricilled'24 or 48 hours later, compared to vehicle control values (p>0.01 in each cSSe). The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes. It is concluded th.a did not show any evidence of causing chromosome damage or bone marrow cel1l1 to?xicity whei idministered by intraperitoneal injection in this in. vivo test procedure. :6 : Company Sanitized. ,c. ftot CSftt' ISC AC St INTRODUCTION DPT478/992105 The purpose of this study was to assess the potential o n H B B B f e f t o induce mutagenic effects in mice following acute intraperitoneal administration u sm ^ ^ M w S c y to g e n e tic system (Boiler and Schmid 1970, MacGregor et al 1987, Mavoumin et al 1990). The intraperitoneal route was chosen for this particular study to maximise potential absorption of the test substance. The procedures used were based on the recommendations of the following guidelines: OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, Guideline 474. EEC Annex to Directive 92/69/EEC (1992) Part B : Methods for determination of toxicity, B.12. Mutagenicity (Micronucleus test). O.J. No. L 383 A, 35,154. US EPA (1998) Health Effects Test Guidelines. OPPTS 870.5395 Mammalian erythrocyte micronucleus test. EPA 712-C-98-226. The bone marrow micronucleus test, originally developed by Matter and Schmid (1971), is a widely employed and internationally accepted short-term assay for identification of genotoxic effects (chromosome damage and aneuploidy) associated with mutagens and carcinogens (Mavoumin et al 1990). This in vivo system allows consideration of various factors including pharmacokinetics, metabolism and DNA repair which cannot be accurately modelled in an in vitro system. Young adult mice are chosen for use because of the high rate of cell division in the bone marrow, the wealth of background data on this species, and their general suitability for toxicological investigations. In mitotic cells in which chromosomal breakage has been caused by the test substance or its metabolites, acentric fragments of the chromosomes do not separate at the anaphase stage o f cell division. After telophase these fragments may not be included in the nuclei of the daughter cells and hence will form single or multiple micronuclei (Howell-Jolly bodies) in the cytoplasm of these cells. Micronuclei are seen in a wide variety of cells, but erythrocytes are chosen for examination since micronuclei are not obscured by the main nucleus and are therefore easily detected in this cell type (Boiler and Schmid 1970). A few hours after the last mitosis is completed, erythroblasts expel their nucleus. Young erythrocytes, less than 24 hours old, stain blue with Giemsa due to the presence of ribonucleic acid and are termed polychromatic (immature). This ribonucleic acid gradually disappears so that more mature erythrocytes stain orange/pink (ie they show normochromatic staining). Virtually all the chromosome damage detected in immature erythrocytes will have been caused during the recent exposure to the test substance. Mature erythrocytes may also be examined for the presence of micronuclei. No substantial increases in the incidence of micronuclei in mature erythrocytes would usually be expected 24 hours after administration of a chromosome-damaging agent; any micronucleus-like artefacts (which could otherwise possibly give a false positive result) are therefore readily distinguishable in this cell type (Schmid 1976). :7: Comp :y Sanitizerdr>. r>. Dcntsin TSCA C31 DPT478/992105 Substances which interfere with the mitotic spindle apparatus will cause non-disjunction (unequal separation of the chromosomes at anaphase resulting in aneuploidy) or lagging chromosomes at anaphase which may not be incorporated into the daughter nuclei. These lagging chromosomes are not excluded from the erythroblast with the main nucleus and hence also give rise to micronuclei. Any toxic effects of the test substance on the nucleated cells may lead either to a reduction in cell division or to cell death. These effects in turn lead to a reduction in the number o f nucleated cells and immature erythrocytes; to compensate for this, peripheral blood is shunted into the bone marrow (von Ledebur and Schmid 1973). If the proportion of immature erythrocytes is found to be significantly less than the control value, this is taken as being indicative of toxicity. A very large decrease in the proportion would be indicative o f a cytostatic effect. The bone marrow is sampled twice after treatment to allow for variations in the rate of absorption and metabolism of test substances and to allow for any delay in erythrocyte production as a result of cytostatic or cytotoxic effects (MacGregor et al 1987). The protocol was approved by Huntingdon Life Sciences Management on 8 October 1998, by the Sponsor on 21 October i998 and by the Study Director on 20 November 1998. The study was performed between 30 November 1998 and 5 January 1999 at the Department of Genetic Toxicology, Huntingdon Life Sciences, Eye, Suffolk, IP23 7PX, England. Subsequently statistical analysis was performed by the Department of Statistics, Huntingdon Life Sciences, Huntingdon, Cambridgeshire, PEI 8 6ES, England. :8: Company Saniti::zsd. Dcos not contain 7SCA C3I Identity: Chemical name: TEST SUBSTANCE DPT478/992105 Intended use: Appearance: Storage conditions: Batch number: Expiiy date: Purity: Date received: fluoromonomers Pale yellow slurry Room temperature M 2 years from date of receipt 25% 23 June 1998 The above information with regard to the physical characterisation of the test substance is the responsibility of the Sponsor. :9 : Company Sanitized. De Tita.r TSCa C d EXPERIMENTAL PROCEDURE DPT478/992105 ANIMALS i- All animals in this study were Specific Pathogen Free CD-1 outbred albino mice o f Swiss origin. Males weighed between 28 and 30 grams and females weighed between 22 and 24 gramsWn despatch from Charles River UK Limited, Margate, Kent, England J ,* On arrival the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and tail marked. Each group was kept, with the sexes separated, in plastic cages and maintained in a controlled environment, with the thermostat set at 22C and relative humidity set at 50%. The room was illuminated by artificial light for 12 hours per day. All animals were allowed free access to pelleted expanded rat and mouse No. 1 maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) and tap water ad libitum. Food and tap water are routinely analysed for quality at source. Dietary contaminants are not suspected of having any significant effect on parameters measured in this test in this laboratory at any time over the last ten years. All animals were acclimatised for a minimum of five days, examined daily and weighed prior to dosing. TEST SUBSTANCE FORMULATION AND ADMINISTRATION Suspensions of the test substance were individually prepared on the day of the test, using identical methods for each phase of the test, in purified water (prepared by reverse osmosis o f tap water). All dosages cited in this report are stated in terms of the purity of the test substance, ie 25%. Stability and homogeneity o f the test substance and of the test substance in the vehicle were not determined in this test and remain the responsibility of the Sponsor. Chemical analysis of dosing formulations for achieved concentration or stability was not performed in this study. Mitomycin C, obtained from Sigma Chemical Company, batch numbers 48H2510 and 48H2511 were used as the positive control compound. They were prepared as a solution in purified water, at a concentration of 0.6 mg/ml and pooled, just prior to administration. All animals in all groups were dosed with the standard volume of 20 ml/kg bodyweight. The test substance and negative control were dosed by intraperitoneal injection, and mitomycin C, the positive control compound, was administered orally by intragastric gavage. DATES OF DOSING Preliminary toxicity test: Micronucleus test: Group 1 - 2: 30 November 1998 Group 3 - 4: 2 December 1998 7 December 1998 : 10 : Compsny Sanitized. Does not contain TSCA C51 DPT478/992105 PRELIMINARY TOXICITY TEST The purpose of this test was to determine a suitable dose level for use in the micronucleus test. The dosages employed were used to give an approximate indication of the maximum tolerated dose, ie the highest dosage which would be expected to elicit signs of toxicity without producing extreme clinical signs or having a significant effect on survival. The experimental design is shown below: Concentration (mg/ml) Dosage (mg/kg)* Number of mice Male Female 12.5 6.25 10 11.25 250 2 2 125 2 2 200 2 2 225 2 2 * Dosage expressed in terms of purity of test substance, ie 25% Following dosing, the animals were observed regularly during the working day for a period of 48 hours and any mortalities or clinical signs of reaction during the experiment were recorded. At the end of this observation period, surviving animals were sacrificed and discarded. MICRONUCLEUS TEST From the results obtained in the preliminary toxicity study (see RESULTS), dose levels of 50, 100 and 200 mg/kg bodyweight were chosen for the micronucleus test. The experimental design is shown below: Group Treatment Concentration (mg/ml) Dose (mg/kg)* Number of mice Male Female 1 Vehicle control - - io . io * Dosage expressed in terms of purity of test substance, ie 25% : 11 : Company Sanitised. Does not contain TSCA CSi DPT478/992105 Following dosing, the animals were examined regularly and any mortalities or clinical signs of reaction were recorded. Five males and five females from the negative control, each of the test substance groups and the positive control group were sacrificed 24 hours after dosing. In addition five male and five female animals in the negative control and high level treatment groups were sacrificed 48 hours after dosing. The animals were killed by cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 ml of pre-filtered foetal calf serum using a 2 ml disposable syringe fitted with a 21 gauge needle. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner (Schmid 1976). At least three smears were made from each animal. The prepared smears were fixed in methanol (> 10 minutes). After air-drying the smears were stained for 10 minutes in 10% Giemsa (prepared by 1 : 9 dilution of Gurr's improved R66 Giemsa (BDH) with purified water). Following rinsing in purified water and differentiation in buffered purified water, the smears were air-dried and mounted with coverslips using DPX. The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic eiythrocytes per animal. Usually only one smear per animal was examined. The remaining smears were held temporarily in reserve in case o f technical problems with the first smear. Micronuclei are identified by the following criteria: Large enough to discern morphological characteristics Should possess a generally rounded shape with a clearly defined outline Should be deeply stained and similar in colour to the nuclei of other cells - not black Should lie in the same focal plane as the cell Lack internal structure, ie they are pyknotic There should be no micronucleus-like debris in the area surrounding the cell. The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 eiythrocytes. A record of the number of micronucleated mature erythrqcytes observed during assessment of this proportion was also kept as recommended by Schmid (1976). t ! i i i . `"N : 12 : C o m p ly Sarni'^od. Do izi contain TSCA C3I DPT478/992105 ASSESSMENT OF RESULTS The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. Non-parametric statistical methods were chosen for analysis of results because: They are suited to analysis o f data consisting of discrete/integer values with ties such as the incidence of micronucleated immature erythrocytes The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non parametric analysis and classical analysis of variance are very similar) 'Outliers' are frequently found in the proportion of immature erythrocytes for both control and treated animals; non-parametric analysis based on rank does not give these values an undue weighting. Unless there is a substantial difference in response between sexes (which occurs only rarely) results for the two sexes are combined to facilitate interpretation and maximise the power of statistical analysis. For incidences o f micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachusetts). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected (Agresti et al. 1990); for individual inter group comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum o f ranks test and Jonckheere's test for trend. A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (P<0.01); individual and/or group mean values should exceed the laboratory historical control range (Morrison and Ashby 1995). A negative result is indicated where individual and group mean incidences o f micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P>0.01) and where these values fall within the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response. Bone, marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature eiythrocytes (P<0.01). This decrease would normally be evident at the 48 hour sampling time; a decrease at the 24 hour sampling time is not necessarily expected because o f the relatively long transition time of eiythroid cells [late normoblast - immature erythrocyte (approximately 6 hours) -> mature erythrocyte (approximately 30 hours)]. : 13 : Company Sanitiz; oas not contain TSCA CSI DPT478/992105 MAINTENANCE OF RECORDS All specimens, raw data and study related documents generated during the course o f the study at Huntingdon Life Sciences, together with a copy of the final report, are lodged in the Huntingdon Life Sciences Archives. Such specimens and records will be retained for a minimum period of five years from the date of issue of the final report. At the end o f the five year retention period the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any item be discarded without the Sponsor's knowledge. Huntingdon Life Sciences will also retain a copy of the final report in its Archives indefinitely. : 14: Company Sanitised. C not contain TSCA CSi RESULTS Mortality data for the entire experiment are presented in Appendix 1. DPT478/992105 PRELIMINARY TOXICITY TEST The details of any toxic reaictions observed are given in Appendix 2. Results showed that a dose level of 200 mg/kg was approximately the maximum tolerated, and this level was considered to be an appropriate maximum for use in the micronucleus test. Dose levels of 50, 100 and 200 mg/kg bodyweight were chosen for use in the main test. MICRONUCLEUS TEST Table 1 gives a summary of the results of the micronucleus test and the results of statistical analysis. The results for individual animals at the 24 and 48 hour sampling times are presented in Tables 2 and 3 respectively. Appendix 4 summarises the vehicle control values for micronucleated polychromatic erythrocytes obtained in previous, unrelated experiments. Appendix 5 summarises the corresponding values obtained for the positive control in previous, unrelated experiments. Clinical signs and mortalities No mortalities were obtained in the micronucleus test. Clinical signs for animals treated with the test substance are detailed in Appendix 3. Clinical signs for the high level group were consistent with the maximum tolerated dose having effectively been achieved. No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test. Micronucleated immature erythrocyte counts (mie) The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes at either sampling time (P>0.01). Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated immature erythrocytes. Micronucleated mature erythrocytes (mme) The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time. Proportion o f immature erythrocytes (% ie/ie + me) The test substance failed to cause any significant decreases in the proportion of immature eiythrocytes (P>0.01). Mitomycin C caused a statistically significant decrease in the proportion (PO.01). : 15 : Company Sanitized. Do t contain TSCACS! CONCLUSION DPT478/992105 No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substontialdeagase in the proportion of immature erythrocytes were observed in mice treated w i t l n ^ m j M n d killed 24 or 48 hours later, compared to vehicle control values (p>0.01 in eacn"55se). It is concluded t h a t j ^ m m ^ ^ i i d not show any evidence of causing chromosome damage or bone marrow cell toxicity whenadministered by intraperitoneal injection in this in vivo test procedure. : 16: Company Sanitised. Docc r.c-t certain TSCA CSI REFERENCES DPT478/992105 AGRESTI, A., MEHTA, C.R. and PATEL, N.R. (1990) Exact inference for contingency tables with ordered categories. Journal o f the American Statistical Association, 85,453. BOLLER, K. and SCHMID, W. (1970) Chemical mutagenesis in mammals. The bone marrow of the Chinese hamster as an in vivo test system. Haematological findings after treatment with Trenimon (translation). Humangenetik, 11, 34. GIBBONS, J.D. (1985) Nonparametric Statistical Inference, 2nd edition, Marcel Dekker, New York. MacGREGOR, J.T., HEDDLE, J.A., HITE, M., MARGOLIN, B.H., RAMEL, C., SALAMONE, M.F., TICE, R.R. and WILD, D. (1987) Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes. Mutation Research, 189,103. MATTER, B. and SCHMID, W. (1971) Trenimon-induced chromosomal damage in bone marrow cells of six mammalian species, evaluated by the micronucleus test. Mutation Research, 12,417. MAVOURNIN, K.H., BLAKEY, D.H., CIMINO, M.C., SALAMONE, M.F. and HEDDLE, J.A. (1990) The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the US Environmental Protection Agency Gene-Tox Program. Mutation Research, 239,29. MORRISON, V. and ASHBY, J. (1995) High resolution rodent bone marrow micronucleus assays of 1,2-dimethylhydrazine : implication of systemic toxicity and individual responders. Mutagenesis, 10, 129. SCHMID, W. (1976) The micronucleus test for cytogenetic analysis. In: HOLLANDER, A. (ed.) Chemical Mutagens, Principles and Methodsfo r their Detection, Vol. 4, p.31. Published by Plenum Press, New York. von LEDEBUR, M. and SCHMID, W. (1973) The micronucleus test. Methodological aspects. Mutation Research, 19, 109. : 17: Company Sanitized. Do ccr.iain TSCA CSS TABLE 1 Summary of results and statistical analysis DPT478/992105 Sampling time Treatment Dose(mg/kg) % ie/(ie+me) f Incidence mie Incidence _________________ ________ _____________________ (mean) mme (total) % ie/(ie+me) Proportion of immature erythrocytes mie Number of micronucleated cells observed per 2000 immature erythrocytes examined mme Number of micronucleated cells calculated per 2000 mature erythrocytes examined Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities): *** P < 0.001 (highly significant) ** P < 0.01 (significant) otherwise P>0.01 (not significant) f Occasional apparent errors of 1% may occur due to rounding of values for presentation in the : 18: Cosnpany Ss;tif 'd. r?o :'j3i contain TSCA C81 DPT478/992105 Treatment Vehicle control Mitomycin C % ie/(ie+me) mie me mme TABLE 2 Results for individual animals - 24 hour sampling time Dose (mg/kgX. 50 100 200 12 Animal number M201 M 202 M 203 M204 M 205 F 206 F 207 F 208 F 209 F 210 M211 M 212 M 213 M214 M215 F 216 F217 F218 F 219 F 220 M 221 M 222 M 223 M 224 M 225 F 226 F 227 F 228 F 229 F 230 M 231 M 232 M 233 M 234 M235 F 236 F 237 F 238 F 239 F 240 M 241 M 242 M 243 M 244 M 245 F 246 F 247 F 248 F 249 F 250 % ie/(ie+me) 44 46 44 34 44 45 49 50 ' 43 45 40 45 44 40 48 34 41 48 46 46 45 45 42 40 45 46 45 47 44 48 43 41 46 36 42 44 43 43 40 43 34 34 39 44 33 34 50 37 35 38 Incidence mie 0 1 1 0 0 0 2 1 0 0 2 0 1 2 0 0 2 0 1 1 0 0 0 0 1 0 0 2 1 1 1 0 3 2 2 0 0 0 1 0 42 69 100 56 97 59 15 75 92 51 me 640 612 650 794 567 653 555 608 621 683 736 630 631 752 788 701 601 645 730 580 609 551 666 672 641 640 618 646 639 583 644 723 673 703 785 792 793 849 691 590 798 863 638 702 751 702 526 631 674 649___ Incidence mme 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 1 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 2 0 2 2 0 Proportion of immature ei throcytes Number of micronucleatei cells observed per 2000 immature erythrocytes Total number of mature ei throcytes examined for micronuclei Number of micronucleatei mature erythrocytes : 19 : Company Ssnitlz-sd. Docs r.zt co: n TSCAC5I DPT478/992105 % TABLE 3 Results for individual animals - 48 hour sampling time Treatment Vehicle control r~ n / n Dose (rfig/kg) 200 Animal % Incidence number ie/(ie+me) mie M 301 M 302 M303 M 304 M 305 F 306 F 307 F 308 F 309 F 310 44 41 47 38 46 43 . 36 46 43 45 2 1 0 0 0 0 0 0 0 1 M331 48 M 332 42 M333 38 M 334 43 M 335 46 F 336 37 F 337 45 F 338 45 F 339 34 . F3.4)___ ____ M_____ 0 1 0 0 0 0 0 1 0 0 me 577 611 641 692 787 632 699 543 656 623 580 648 890 743 598 693 608 627 769 693 Incidence mme 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 % ie/(ie+me) mie me mme Proportion of immature erythrocytes Number of micronucleatea cells observed per 2000 immature erythrocytes Total number of mature erythrocytes examined for micronuclei Number of micronucleatedi mature erythrocytes APPENDIX 1 Mortality data DPT478/992105 Group Treatment Dosage (mg/kg) Preliminary 1 250 Toxicity Test 2 125 3 200 4 ' 225 Micronucleus Test 1 2 50 3 100 4 200 5 Mitomycin C 12 Mortality ratio = No. of deaths No. dosed Males Females Combined 0/2 0/2 0/4 0/2 0/2 0/4 0/2 0/2 0/4 1/2 0/2 1/4 0/10 0/10 0/20 0/5 0/5 0/10 0/5 0/5 0/10 0/10 0/10 0/20 0/5 0/5 0/10 : 21 : Company Ssrutued. Dc;s ordain CA CSi DPT478/992105 APPENDIX 2 Preliminary toxicity test - Clinical signs and mortalities Treatment Dose (mg/kg) : 250 H 125 200 225 Approx, Male Female Male time after dosing (hr: min) 0: 15 U U, SR 3 :50 P, U, HP, P, U, HP, 6:40 SR SR P, U, HP, P, U, HP, SR SR 22:40 P, U, HP, HP, 1(P,U) SR 1(REPC) 30:30 P, U, HP, HP, 1(U,P) SR 1(REPC), 1(EPC) ' 46:05 P, U, HP, HP, 1(P) 0:20 2:20 SR 1OEPC1 ! i U, 1(HP): 18 :20 HP, 1(U) 26: 10 HP, 1(P,U) 41 :45 HP, 1(P) 47:55 HP, 1(P) 0:20 4:55 21 :00 22:35* 28:45 45:30 Mortalities 0/2 0/2 0/2 Female Male Female Male Female U, HP # U, 1(P) U, HP P,U P, U, HP, U, HP P, U, HP, P, U, HP SR SR P, U, HP, HP, 1(P) P, U, HP, P, U, HP, SR SR 1(EPC), 1(EPC) 1(EPC) 1(REC) 1(P, U, HP, SR UG, EC, KIE) P, U, HP, HP, 1(P) 1(P, U, HP, P, HP, SR SR EPC) 1(EPC) 1(EPC) P, HP, 1(U, HP, 1(P) 1(P, U, HP, HP, 1(P, SR EPC) SR EPC, EPC) UG1 0/2 0/2 0/2 1/2 0/2 * Observations performed on one male animal only. Type of reaction: EPC Eyes Partially Closed, EC Eyes Closed, HP Hunched Posture, KIE Killed in Extremis, P Piloerection, SR Slow Respiration, REC Right Eye Closed, REPC Right Eye Partially Closed, U Underactive, UG Unstable Gait. Clinical signs shown refer to all animals within that dose group and sex, except where x(...), x denoting the number of animals displaying the clinical sign(s) within the brackets. : 22 : Compcr '.arized. corsi: TSCA CSf APPENDIX 3 Micronucleus test - Clinical signs and mortalities DPT478/992105 Treatment Dose (mg/kg) Approx, time after dosing (hr: min) 1 :25 5:25 21 : 55 1 :05 5:05 21 :40 1: 10a 5: 10a 2 1 :45a 0:30b 4:35b 21:05b 28: 15b 4 4 : 50b 50 Male Female J 100 Male Female ----- 200 Male Female 1(P, HP, SJ) 1(P, HP, SJ) 1(HP),2(P) 1(P) - P, U, HP P, U, HP, 2(SR) P, HP U, HP, 2(SR), 3(P) P>U, HP, 3(SR), 1(EC) P, HP P, HP P, HP U, 4(HP) P, HP, 3(U) P, HP, 3(P, U, HP) 1(SR), 3(p; u, HP) 3(P, HP) 3(P, HP) 3(P, HP) Mortalities 0/5 0/5 0/5 0/5 0/10 0/10 a 24 hour sacrifice b 48 hour sacrifice NB No adverse clinical signs were noted for the vehicle or positive control groups throughout the experiment Type of reaction: EC Left Eye Closed, HP Hunched Posture, P Piloerection, SJ Swollen Jaw, SR Slow Respiration, U Underactive. Clinical signs shown refer to all animals within that dose group and sex, except where x(...), x denoting the number of animals displaying the clinical sign(s) within the brackets. 23 : Company Sanifszc ;t contsin TEGA CBI APPENDIX 4 DPT478/992105 Historical vehicle control data (August 1995 - June 1998) Percentage of micronucleated immature erythrocytes (individual animals) j O-OJ 0.6-1.0 1.1-1.5 1.6-2.0 2.1-2-5 26-3.0 3.1-3.5 3 .6 4 .0 . (.1 4 5 4.6.S.O 5.1-55 5.6-6.0 6.1-6J Frequency distribution of micronucleated immature erythrocytes per 1000 cells '- Historical vehicle control data (August 1995 - June 1998) Percentage of micronucleated immature erythrocytes (group mean values) Frequency distribution of micronucleated immature erythrocytes per 1000 cells : 24 : Company Ssr.iiizscL L 0 COiuE TSCACE! DPT478/992105 APPENDIX 5 Historical positive control data (August 1995 - June 1998) Frequency of micronucleated erythrocytes (individual animals) Frequency distribution of micronucleated immature erythrocytes per 1000 cells Historical positive control data (August 1995 - June 1998) Frequency of micronucleated immature erythrocytes (group means) U-10 11-20 21-30 31-40 41-30 31-60 61-70 71-10 11-90 Frequency distribution of micronucleated immature erythrocytes per 1000 cells : 25 : Ctwapgty Sanitized. Coe: it contain TSCACBi