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CCR C ytotest Cell R esearch GmbH & Co. KG AR226-2984 CCR PROJECT 326428 MICRONUCLEUS ASSAY IN BONE MARROW CELLS OF THE MOUSE WITH REPORT Study Completion Date: ' February 17,1993 CCR In d en Leppsteinsw iesen 19 D-6101 Rodorf F.R.G. T elephone: 0 61 54 - 80 7-0 Telefax 0 61 54 - 8 33 99 Company Sanitized. Does not contain TSCA CBi Mg;? HESSISCHES MINISTERIUM FR UMWELT, ENERGIE UND BUNDESANGELEGENHEITEN GLP-3 escheinigung Bescheinigung Hiermit wird besttigt, da die Prfungseinrichtung(en) C ytotest Cell- Resea'rch GmbH & Co KG In den Leppsteinswiesen 19 in 6101 Rodorf_____________ '_________ (Oft. Aracrxtn) . dar J.C. Holding Verwaltung GmbH (Fama) am 0 3 .0 8 ., 0 4 .0 8 ., 0 5 .0 8 .-und.06.08.92 (Datum) von der fr die berwachung zustndigen Behrde Ober . die Einhaltung der Grundstze der Guten Laborpraxis inspiziert worden ist (sind). Es wird hiermit besttigt, da folgende Prfungen in dieser Prfeinrichtung nach den Grundstzen der Guten Laborpraxis durchgefhrt werden. Toxikologische Eigenschaft Certificate It is hereby certified that the test iatity(ies} C ytotest Cell Research GmbH & Co KG Trr*diM'*lji^stiTis^*esn Ti( r .in__61_01__R_o_d_o_rf________________ . {location,atdrsw) Of, --RC-C-H-o-ld-i-ng--V-e-rwa-lt-un-g- G,,m,,bH,,____ (company naaw| 03.08., 04.08., 05.08. and.06.08.92 was (were) inspected by the competent authority regar ding compliance with the Principles of Good Laboratory Practice. It is hereby certified that studies in this test facility are conducted in compGanca with the Principles of Good Laboratory Practice. - _ Toxicological, properties Im Aufft-ag . ` CPr". </dct U X (0r. -Hecker) ' Wiesbaden, den JH. 0 e?. m n teec.re 2 Company Sanitized. Does not contain TSG CBI Copy of GLP-Certificate Pr e f ac e General Project Staff Schedule Project Staff Signatures Quality Assurance Guidelines Archiving STATEMENT OF COMPLIANCE QUALITY ASSURANCE UNIT Statement SUMMARY Conclusion OBJECTIVE Aims of the Study Reasons for the Study MATERIALS AND METHODS The Test Article Controls The Test System Experimental Performance Data Recording Evaluation of Results BIOMETRY RESULTS Pre-Experiment for Toxicity Summary of Results Tables of Results CONCLUSIONS DISTRIBUTION REFERENCES - PAGE 2 4 4 4 4 5 5 5 6 7 8 8 9 9 10 10 10 12 12 13 14 16 18 18 19 20 20 21 22 25 27 27 m n teec.re 3 Company Sanff'red. Doss rot T3C/1. PREFACE GENERAL Sponsor: ISEGA Forschungs- und Untersuchungsgesellschaft mbH D-8750 Aschaffenburg Study Monitor: Heike Krmer Testing Facility: CCR CYTOTEST CELL RESEARCH GMBH & CO. KG D-6101 Rodorf CCR Project No.: 326428 Test Article: Title: PROJECT STAFF Micronucleus Assay in Bone Marrow Cells of the Mouse wi1 fl Management: Dr. H.-E. Knoell Scientific consultant: Prof. Dr. Herbert G. Miltenburger Study Director: , Dr. Wolfgang Volkner Quality Assurance Unit: Dr. Christiane Helmrich SCHEDULE Date of Protocol: November 09, 1992 Start of Experiment:End of Experiment: November 11, 1992 January 26, 1993 Date of Draft: January 26, 1993 Date of Report: February 17, 1993 mnteec.re 4 Company Sanitized. Does not contain TSCA CBI PROJECT STAFF SIGNATURES Study Director: Dr. Wolfgang Vdlkner Management : Date: February 17, 1993 Dr. Sans ' 11 Date: Febrary 17, 1993 QUALITY ASSURANCE * The study was performed in compliance with: Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, Anlage 1 ("Annex 1"), dated March 14, 1990 (BGBL. I S. 521). * "The OECD Principles of Good Laboratory Practice", Paris 1981. GUIDELINES , This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: First Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted May 26, 1983, "Micronucleus Test". EEC Directive 79/83T, Annex V, B 12 Environmental Protection Agency, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, Revision July 1, 1986 "In vivo mammalian bone marrow cytogenetics tests : Micronucleus assay." m n teec. re 5 Company Sanitized. Cogs not contain TSC CBI ARCHIVING CCR, D-6101 Rofldorf will archive the following data for 30 years: raw data, protocol and copy of report. The following specimen and samples will be archieved for at least 12 years: sample of test article, microscopic slides. No raw data or material relating to the study will be discarded without the sponsor's prior consent. m nteec.re 6 Company Sanitized. Does not contain TSC CBi o imi c m e n i \ j r u u iv ir u A n u c Project Number: Test Article Study Director: Title 326428 Dr. Wolfgang Vlkner Micronucleus Assay in Cells of the Mouse wit To the best of my knowledge and belief, this study performed in the testing facility of CCR was conducted in compliance with Good Laboratory Practice Regulations: Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, Anlage 1 ("Annex 1"), dated March 14, 1990 (BGBL. I s. 521). , "OECD Principles of Good Laboratory Practice", Paris, 1981 There were no circumstances that may have effected the quality or integrity of the study. Study Director ' CCR Dr. Wolfgang Vlkner Date: /fifj m n teec.re 7 Company Sanitized. Does not contain TSCA CBS WUrtLI I T A bU H A N U 't UNIT C C R, Cytotest Cell Research GmbH & Co. KG, In den Leppsteinswiesen 19, D-6101 Rodorf STATEMENT Project Number: Test Article : Study Director: Title ^ 326428 Dr. Wolfgang Vlkner Micronucleus Assay in Bone Marrow Cells of the Mouse with] This report was audited by the Quality Assurance Unit and the study and/or testing facility were inspected on the following dates. Dates of QAU Inspections/ Audits November 10, 1992 November 20, 1992 January 29, 1993 Dates of Reports to the Study Director and to Management November 10, 1992 November 20, 1992 January 29, 1993 Head of Quality Assurance Unit Dr. Christiane Helmrich Date: /rU ........ 7 Yf, '*%7J m n teec.re 8 Company Sanitized. Does not contain TSCA CBI SUMMARY * s study was performed to investigate the potential oi Ito induce micronuclei in polychromatic erythrocytes* (PCE) iri bone marrow of the mouse. The test article was dissolved in aqua deionized. This solvent was used as negative control. The volume administered orally was 10 ml/kg b . w . . 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythro cytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic ery throcytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE . The following dose levels of the test article were investigated: 24 h preparation interval: 200, 666, and 2000 mg/kg b.w.. 48 h preparation interval: 2000 mg/kg b.w. . The highest dose recommended by guideline (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals ex pressed slight toxic reactions. After treatment with the test article the number of NCEs was not increased as c o m p a r e d t ^ t h e corresponding negative controls thus indicating t h a m H j H H f f B l h a d no cytotoxic effectiveness. At preparation interval 24 hours no substantial enhancement in the frequency of the detected micronuclei after treatment with the test article was observed. In comparison to the corresponding negative control there was a statistically significant enhancement in the frequency of the detected micronuclei at preparation interval 48 hours after treatment with the test article. However, this finding is not considered to be of a biological relevance. An appropriate reference mutagen (cyclophosphamide) was used as positive control which showed a distinct increase of induced micronucleus frequency. CONCLUSION In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bonemarrow^oells of the NMRI-mouse. T h e r e f o r e ] Q H B j j ^ | ^ P | i s considered to be non-mutagenic in this micronucleus assay. m n teec.re Company S:niiizecS. Does noi contain TSC CBS U d fc O I IVfc AIMS OF THE STUDY This in vivo experiment was performed to assess the mutagenic properties of the test article by means of the micronucleus test in bone marrow cells of the mouse. REASONS FOR THE STUDY . The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of chromosomal damage. Micro nuclei arise from acentric chromosomal fragments or whole chromo somes induced by clastogens or agents affecting the spindle apparatus (1,2,3,4). Polychromatic erythrocytes (PCE) in the bone marrow of the mouse are the cell population of choice for mammalian cells in vivo. PCEs are newly formed red blood cells and are easily identifiable by their staining properties. These cells have the advantage that the micronuclei can be readily detected because the nucleus is extruded from the erythroblast after the last cell division . The first appearance of micronuclei in PCEs is at least 10-12 hours after a clastogenic exposure. This lag is due to the time required for the affected erythroblast to differentiate into a PCE. This differentiation process includes: 1. The time required for the damaged erythroblast to proceed to mitosis. 2. The mitotic delay induced by the treatment. 3. The formation of micronuclei due to acentric fragments or chromosomes that are not included in the daughter nuclei. 4. The time required for the expulsion of the main nucleus after the last mitosis to become a micronucleated PCE. This newly formed cell population persists for about 20 hours in the bone marrow of mouse. During this time micronucleated PCEs accumulate in the bone marrow as the production of micronuclei extends over a considerable period of time. The time at which the micronucleus frequency is at a maximum va ries from agent to agent (5). Due to mitotic delay or metabolic and pharmacokinetic effects the appearance of micronucleated PCEs can be considerably delayed. Therefore a single sampling time is not optimal. Results obtained with model mutagens showed that samples taken at 24 h and 48 h after treatment cover the inter vals in which maximum frequencies of micronuclei occur. m n teec.re 10 Company Sanitized. Dees not contain TSCA CBI For the initial assessment of clastogenic activity a single dose level at the maximum tolerated dose or that producing some indi cation of cytotoxicity (change in the ratio of polychromatic to normochromatic erythrocytes) and sampling at 24 h and 48 h after treatment is recommended. For verification two additional dose levels are tested at the central sampling time 24 h after treat ment to establish a dose response effect. To validate the test, a reference mutagen is tested in parallel to the test article. t m nteec.re 11 Company Sanitized. Does not contain TSCA C8f ivirt I c n iM L O MIMU IVIC I n u u THE TEST ARTICLE The test article and the information concerning the test article Were provided by the sponsor. Name: Batch N o .: Aggregate State at RT: liquid Colour: not indicated Purity: Analysis : Stability: Pure: not indicated In vehicle: not indicated Storage : 4 C Expiration date: not indicated On the day of the experiment, the test article was dissolved in aqua deionized. The solvent was chosen to its non-toxicity for the animals. All animals received a single standard volume of 10 ml/kg body weight orally. m n teec.re 12 Company Sanitized. Does not contain T5CA CBI THE CONTROLS ~ The Negative Control The vehicle of the test article was used as negative control. Name: Route and Frequency of Administration: Volume Administered: aqua deionized orally, once 10 ml/kg b.w. . The Positive Control Name: Supplier: Catalogue no. : Dissolved in: Dosing: Route and Frequency of Administration: Volume Administered: CPA; Cyclophosphamide SERVA, D-6900 Heidelberg 17681 physiological saline 30 mg/kg b.w. orally, once 10 ml/kg b.w. Solution prepared on day of administration. The stability of CPA at room temperature is good. At 20C only 1 % of CPA is hydrolysed per day in aqueous solution. / mnteec.re 13 containTSCA CBI Company Sanitized.Doe - THE TEST SYSTEM Reasons for the Choice of the Experimental Animal Species The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There .are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test (1,2,3,4,5). Strain: Source: Number of Animals: Initial Age at Start of Acclimatization: Acclimatization: Initial Body Weight at Start of Treatment: NMRI Charles River Wiga GmbH Sandhofer Weg 7, D-8741 Sulzfeld 1 84 (42 males/42 females) minimum 10 weeks minimum 5 days 20 - 40 g According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of C C R for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage numbfer. mnteec.re 14 Company Sanitized. Does net certain TSCA CB! Husbandry The animals were kept conventionally. The experiment was conduct ed under standard laboratory conditions. Housing: Cage Type: Bedding: Feed: Water: . . Environment: single Makro Ion Type I, with wire mesh top (EHRET GmbH, D-7830 Emmendingen) granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe) pelleted standard diet, ad libitum (ALTROMIN 1324, D-4937 Lage/Lippe) tap water, ad libitum (Gemeindewerke, D-6101 Rodorf) temperature 21 3C relative humidity 30-70% artificial light 6.00 a.m. - 6.00 p.m. m nteec.re 15 c o m p san ited. D o o o . TSGA CBI EXPERIMENTAL PERFORMANCE Pre-Experiment: for Toxicity A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study. Dose Selection It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test articles. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physio logical space available. Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment. Study Procedure Test Groups : Six males and six females were assigned to each test group. The animals were identified by their cage number as shown below in the table. Test group hours posi;-treatment 24 48 male/female male/female Negative control 1- 6/ 7-12 61-66/67-72 Low dose 13-18/19-24 - / - Medium dose ' 25-30/31-36 - / - High dose 37-42/43-48 73-78/79-84 Positive control 49-54/55-60 - / - mnteec.re 16 San^d-Ooesno.-- Compaq tscacbi Treatment : Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve ani mals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24 and 48 hours after treat ment . Preparation of the Animals: The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspend ed cell pellet was spread on a slide. The smear was air-dried and then stained with May-Griinwald (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mount ed with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample. Analysis of Cells: Evaluation of the slides was performed using NIKON microscopes with 10Ox oil immersion objectives. 1000 polychromatic erythro cytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and ex pressed in normochromatic erythrocytes per 1000 the PCE s . The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group. m nteec. re 17 Company Sanitized, Does nt conta* r ~CA CB! DATA RECORDING The data generated are recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups, negative and positive control. The micronucleated cells per thousand and the ratio of polychromatic to normochromatic erythrocytes are presented for each animal. EVALUATION OF RESULTS . A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychro matic erythrocytes nor a statistically significant and reproduci ble positive response at any of the test points is considered non-mutagenic in this system. This can be confirmed by means of the nonparametric Mann-Whitney test (6). However, both biological and statistical significance should be considered together. m n teec.re 18 Company Sanitized. Doss BIOMETRY Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test. Negative control versus Test group ' 200 mg/kg b.w.; 24 h 666 mg/kg b.w.; 24 h 2000 mg/kg b.w.; 24 h 2000 mg/kg b.w.; 48 h Significance - n.t* n.t* + P 0.395 0.014 + = significant; - = not significant; n.t. = not tested * = mean micronucleus frequency was not above the mean corresponding negative control value f m n teec.re 19 Company Sanitized. Oss not contain T5CA CBt RESULTS PRE-EXPERIMENT FOR TOXICITY In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w., ZONYL RP 18 dissolved in aqua deionized. The volume administered was 10 ml/kg b.w.. The treated animals expressed toxic reactions as shown below in the table: toxic reactions reduction of ' spontaneous activity eyelid closure apathy hours post-treatment male/female 1 h 6 h 24 h 48 h 2/2 2/2 2/2 2/2 0/0 1/1 1/1 1/1 0/0 0/0 1/0 1/1 In accordance to the guideline-draft "EEC Directive 79/831, Annex V, B 12" 2000 mg/kg b.w. of the test article was used as maximum dose in the micronucleus assay. m n teec.re 20 not contain TSCA wBl Company Sanitized- Do3 Summary of results :test group dose mg/kg sampling b.w. time (h) PCEs with micronuclei (Z) range PCE/NCE vehicle 0 24 0.13 0-2 1000/ 639 test article 200 24 0.15 0-3 1000/ 716 test article 666 24 test article 2000 24 cyclo phosphamide 30 24 vehicle 0 * 48 test article 2000 48 0.10 0-3 1000/ 663 0.07 0-2 1000/ 732 1.58 0.03 8-24 1000/ 923 0-1 1000/ 736 0.11 0-2 1000/ 666 m n teec.re 21 Company Sanitized, Does not contain TSC CBS micronuciei in poiycnromatic eryrnrocytes (,'Ufi) and relationsmp FCE/NCE (NCE = normochromatic erythrocytes) scoring 24 hours after treatment Table I: vehicle animal sex test group dose micronuclei no. mg/kg in 1000 PCE b.w. per animal PCE/NCE 1 m aqua deion. 0 2m n tt 3m n R 4m n R 5m tt R 7f it R 8f n H 9f 10 f 11 f n n tt R tt tt 2 1000/ 669 2 1000/ 600 2 1000/ 594 1 1000/ 536 2 1000/ 482 0 1000/ 418 1 1000/ 726 1 1000/ 683 1 1000/ 846 1 1000/ 834 sum mean percent cells with micronuclei 13 1.3 0.13 10000/ 6388 1000/ 639 Table II: test article animal . s.ex _..test....group-- rinse . micronuclei.. no. mg/kg in 1000 PCE 1 b.w. per animal PCE/NCE 13 m 1 200 14 mW " -1 15 m H R 16 m R H 17 m R H 19 f R n 20 f 21 f R tt R R 22 f 23 f n tt R R 1 1000/ 817 0 1000/ 763 1 1000/ 629 2 1000/ 679 1 1000/ 598 1 1000/ 805 3 1000/ 789 2 1000/ 545 1 1000/ 766 3 1000/ 767 sum mean percent cells with micronuclei 15 1.5 0.15 10000/ 7158 1000/ 716 Table III: test article animal no. sex test group i ** ' ~ dose mg/kg b.w. 25 26 27 m im m m m tt tl 666 R tt 28 m R a 29 m R R 31 f R R 32 f R R 33 f a a 34 f R R 35 . R H sum mean percent cells with micronuclei micronuclei in 1000 PCE per animal PCE/NCE 2 1000/ 763 3 1000/ 620 0 1000/ 675 1 1000/ 485 2 1000/ 696 1 1000/ 729 0 1000-1 85-1 1 1000/ 497 0 1000/ 602 0 1000/ 709 10 1.0 0.10 10000/ 6627 1000/ 663 m n teec.re 22 Company Sanitized. D s not M l T8CA CEI Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/NCE (NCE = normochromatic erythrocytes) scoring 24 hours after treatment Table IV: test article animal no. 37 38 39 40 41 43 44 45 46 47 sex test group dose mg/kg b.w. m 2000 m m* m m f It fn fo f0 f ' sum ,, . mean percent cells with micronuclei micronuclei in 1000 PCE per animal PCE/NCE 2 1000/ 705 1 1000/ 835 0 1000/ 584 0 1000/ 799 0 1000/ 810 0 1000/ 603 2 1000/ 674 1 1000/ 708 1 1000/ 810 0 1000/ 789 7 0.7 0.07 10000/ 7317 1000/ 732 Table V: cyclophosphamide animal no. sex test group dose mg/kg b.w. 49 m 50 m 51 m CPnA n 52 m 53 m 55 f 56 f 57 f it it n 58 59 60 f f f ," tt it 30It tt tt If tt tt tt n it it sum mean percent cells with micronuclei micronuclei in 1000 PCE per animal PCE/NCE 14 1000/ 1097 12 1000/ 983 24 1000/ 767 13 1000/ 1122 21 1000/ 989 8 1000/ 1122 14 1000/ 788 17 1000/ 790 not scorable 15 1000/ 693 20 1000/ 881 158 15.8 1.58 10000/ 9232 1000/ 923 m n teec. re 23 Compa sanitized. Doss n o te ?nisi*rt HIP 1 Micrnuclei in polychromatic erythrocytes (PCE) and relationship PCE/NCE (NCE - normochromatic erythrocytes) scoring 48 hours after treatment Table VI: vehicle animal sex test group dose no. mg/kg D.W. 61 m aqua deion. 0 62 m 63 m 64 m 65 m 67 f 68 f n 69 f 70 f 71 f sum mean .percent cells with micronuclei micronuclei in 1000 PCE per animal PCE/NCE 0 1000/ 774 1 1000/ 605 0 1000/ 758 0 1000/ 796 1 1000/ 624 0 1000/ 711 0 1000/ 742 0 1000/ 814 0 1000/ 795 1 1000/ 741 3 0.3 0.03 10000/ 7360 1000/ 736 Table VII: test article animal sex no. 73 74____ _ 75 76 77 79 80 SI 82 83 m m m m m f f f f f test group dose mg /kg b.w..... n J ;i ; sum ,, i, ' . m?a? percent cells with micronuclei micronuclei in 1000 PCE per animal PCE/NCE 1 1000/ 687 2 1000/ 567 2 1000/ 492 0 1000/ 756 1 1000/ 648 2 1000/ 733 1 1000/ 455 1 1000/ 721 1 1000/ 771 0 1000/ 827 11 1.1 0.11 10000/ 6657 1000/ 666 m n teec.re 24 Company Sanitized. Does net contain TSC Company Sanitized. Does not contain TSCA CBI This statistically significant effect is considered to be of minor importance: The corresponding actual negative control rate at prepa ration interval 48 hours was low in this study. The mean historical negative control value obtained within the last 20 experiments at preparation interval 48 hours was 0.096 %. The range varied from 0.03 % to 0.18 %. The micronucleus frequency of 0.11 % PCEs^ with micronu clei after treatment with 2000 mg /kg b.w.f| within the range of the historical control a ^ ^ p r e s ^ i t e d . The number of micronuclei per animal did not exceed 2 per 1000 PCEs. Therefore, the statistically significant response is not consid ered to be an indication for an induced mutagenic effect due to the test article. 30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase in induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the NMRI-mouse. m n teec.re - 26 Company Sanitized. Does not contain TSCA CSI U l Ilil U IlU N U h IH E R E F U H I Sponsor: 'Study Director: 2x (originai, copy) lx (copy) REFERENCES 1. Heddle, J.A (1973) A rapid In .vivo test for chromosomal damage Mutation Research, 18, 187-190 2. Schmid,W. (1976) The micronucleus test for cytogenetic analysis In: A. Hollaender (Ed.), Chemical Mutagens, Vol.4, Plenum Press, New York, pp. 31-53 . 3. Matter, B.E., and J. Grauwiler (1974) Micronuclei in mouse bone marrow cells, a simple in vivo model for the evaluation of drug induced chromosomal aberrations Mutation Res., 23, 239-249 4. Heddle, J.A. and A.V, Carrano (1977) The DNA content of micronuclei induced in mouse bone marrow by X-irradiation: evidence that micronuclei arise from acentric chromosomal fragments. Mutation Research, 44, 63 5. Salomone, M.F., J.A. Heddle, E. Stuart and M. Katz (1980) Towards an improved micronucleus test - studies on three model agents, mitomycin C, cyclophosphamide and dimethylben zanthracene. Mutation Research 74, 347 6. Krauth, J. (1971) Locally most powerful tied rank test in a Wilcoxon situation Annals of Mathematical Statistics, 42, 1949 - 1956 m n teec.re 27 Company Sanitized. Does not contain TSCA CBS