Document 1grd3KpBY0dM3eayynqNob4pE

u o n u y iu ie s i u e ii n e a e a rc n u m o ri & UO. I\la AR226-2986 CCR PROJECT 326428 . MICRONUCLEUS ASSAY IN BONE MARROW C ELLS OF THE MOUSE WITH . REPORT Study Completion Date: > February 17,1993 C C R In den Leppsteinswiesen 19 D-6101 Rodorf F.R .G . Telephone: 0 61 54 - 80 7-0 Telefax 0 61 54 - 8 33 99 Company Sanitized. D oes not contain TSCA C  % *^ ks, HESSiSCHES MINISTERIUM FORUMWELT, ENERGIE UND BUNDESANGELEGENHEITEN GLF-Bescheinfgung Bescheinigung Hiermit wird besttigt, da die Prfungsainrichtung(an) C y t o t e s t C e l l - R e s e a r c h GmbH & Co KG In d e n L e p p s t e i n s w i e s e n 19 in 6101 R o d o r f _ ' (Ort Amevn) . der RCC H o l d i n g V e r w a lt u n g GnbH (Firm *) am 0 3 - 0 8 ., 0 4 . 0 8 ., 0 5 .0 8 .- u n d .0 6 .0 8 .9 2 (Datum ) * von der fr d e berwachung zustndigen Behrde Ober . .die Einhaltung der Grundstze dar Guten Laborpraxis inspiziert worden ist (sind). Es wird hiermit besttigt, da folgende Prfungen in fieser Prfeinrichtung nach den Grundstzen d a r Guten Laborpraxis durchgefhrt werden. f . Toxikologische Eigenschaft Certificate It is hereby certified that the test facilities) Cytotest Cell Research GmbH & Co KG In den Leppstemswiesen Ti" -- in 6101 Rodorf . (location, address) of RCC Holding Verwa 1tung GmbH (com pany aanta) " m 0 3 .0 8 ., 0 4 .0 8 ., 0 5 .0 8 . a n d .0 6 .0 8 .9 2 . {<***) _ ~-- was (were) inspected by the competent authoriiy recar- d n g compfianca with the Principles of Good Laboratory Practica, 1 it is hereby certified that studies in this lest facility are conducted in compliance with the Principles of Good Laboratory Practice. Toxicological, properties Im Auftrag . * CP/. j i z t i u x (Or.-Hecker) ' Wiesbaden, den m n t e e c .re 2 s noi esula Copy of GLP-Certificate PREFACE 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 theStudy MATERIALS AND METHODS The Test Article Controls ~ The Test System Experimental Performance Data Recording Evaluatipn of Results BIOMETRY RESULTS CONCLUSIONS Pre-Experiment forToxicity Summary of Results Tables of Results distribution references . . PAGE 2 4 4 4 4 5 5 2 g ? 8 8 g 10 10 10 12 12 24 \5 28 ig 20 20 21 22 25 27 27 mnteec.re 3 Company Sanitized. Does not contain TSCA CB GNRT. 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.: Test Article: Title : PROJECT STAFF 326428 D 0 Micronuc 1eus Assay in Bone Marrow Cells of the Mouse witlffi Management: Dr. H.-E. Khoell Scientific consultant: Prof. Dr. Herbert G. Miltenburger Study Director: , Dr. Wolfgang Volkner Quality Assurance Unit: Dr. Christiane Helmrich SCHEDULE Date of Protocol: Start of Experiment: End of Experiment : Date of Draft: Date of Report : November 09, 1992 November 11, 1992 January 26, 1993 January 26, 1993 February 17, 1393 m n t e e c .re Company Sanitized. Does not contain TSCA CBi Study Director: Dr. Wolfgang Volkner Date: February 17, 1993 Management: Dr. Hans ? Knoell QUALITY ASSURANCE u IDate: Feobr ry 17, 1993 J 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. "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 79/831> 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." mnteec.re 5 Company Sanitized.D oes not contain T8C CM CCR, D-6101 Roi3dorf will archive the following data for 30 years; raw data, protocol and copy of report. 12ey f s ?Win9 SpeCimen and ">1 i U be archieved for at feast sample of test article, microscopic slides. No raw data or material relating to the study will be . without the sponsor's prior consent. iscarded m n t e e c .re Company Sanitized. Does not contain TSCA CBS - m o u vu Project Number: Test Article : 326428 Study Director: Dr. Wolfgang Volkner Title . Wcronucieus Assay in Bone Marrow Cells of the Mouse with** 1----- To the best of m y knowledge and hei,-af ,, . the tasting_fa.ciJ.ity of CCR as conducted n ' L T ' f -7 PSrfCTed ^ Laboratory Practice Regulations: compliance with Good Chemikaliengesetz ("Chemicals Act-M o -f .. Germany, Anlage 1 ("Annex 1 ) dated if ^ Federal ^ p u b l i c of 521). 1 * 1 >' dated March 14, 1990 (BGBL. i s . "0ECD * Good Laboratory Practice., Paris, l m integrity^fthe^st^y?nCeS that " 7 hm effe e d the quality or Study Director CCR Dr. Wolfgang Volkner 6 .(/; -H ) Date: A4t A lfo m n t e e c .re Company Sanitized Does not contain TSCA C0 C C R , Cytotest Cell Research GmbH s Co. KG, In den LePPSteinswiesen 19, d -6101 RoBdorf STATEMENT Project Number: 326428 Test Article : ' Study Director: Title Dr. Wolfgang Vlkner Micronucleus Assay in " Cells of tiie Mouse wit Dates of QAU Inspections/ Audits November 1 0 , 1992 November 20-, 1992 January 29, 1993 Head of Quality Assurance Unit and the were inspected on the following Dates of Reports to the Study Director and to Management November 10, 1992 November 20, 1992 January 29, 1993 D r . Christiane Helmrich Date: m n t e e c .re 8 SBpany Sanitized. D oes not contain TSCA CBt SUMMARY the bone marrow1 o T t o e ^ o u a e . ^ 1^ potential ,,, 01"3 " 0 ^ o c j t e CE) in was used a s ^ n e ^ t i v V c o n t r o l ^ h e *1 T * dedonl" d - This solvent 10 ml/kg b . w . . 24 h and 4 8 ^ a f t e l i T 3<?nlnls1:ebed orally was test article the bone narrow oells ^ere o S e o S 'V ^ 10" * * * analysis. s wers collected for micronuclei S f UP -- -luated ~-:s TM- article the ratio between p ^ l y o h r o M t ^ a ^ ^ With * 1* * throcytes (NCE) was deternnin^rt^n ^ nd normochromatic erv- the number of NCE per 1000 PCE. Sa"e Sample and r e p o r t e d ^ ThS ^ ^ p a ^ i o ^ S o ^ i - Castigated: The hi8ghesPt T rati0n lnte^ i; o S ' 4 % ^ . f 0# estimated b y ^ Z l ^ r ^ e n t to ^ s u ^ t a b l ('2i mg/fcg> was pressed slight toxic reactions'*. ^ 6 Suitable* The animals ex . ter treatment with the test a r t i H a +-u . increased as cosgiared to the corresoond>a " indicating no HCBs 1,33 not thus ^thee^ nteesgtu eanrtciyc^lfe^wha^sd oebtseecrvteed^ m i c rroonnuuoclleeii ^afftte3r^ tir*ea"tm0e--nt 1*witihn ?S S : S T T ^ s r - ~ flndi- 13 " * -- -- to he Of a biological ^ r ? r c o an " t r o T ^ S r Showe?"a S n ^ ^ " * 383d 38 micronucleus frequency. distinct increase of induced CONCLtTSTnw and under ^he'erpertaenta^TOni-t11131 dUrlng the st"d y described did not induce K c " n u o i e / T s d e t ? T e s T ^ ' the test article test with o n e marrow cells of the NM?!Un e d h j the micrnucleus Therefore o the NMRl-mouse. mi-cronucleus assay. considered to be non-mutagenic in this mnteec.re Company Sanitized. D oes not contain TSCA CDj O B JEC T IV E 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 reguired 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 chromosomes that are not included in the daughter nuclei. or 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 t e e c .re 10 Company Sanitized. D oes not contain TSCA CB| 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. / mnteec.re 11 Company Sanitized. D oes not contain TSCA CBf MATERIALS AND METHODS THE TEST ARTICLE The test article and the information concerning the test article were provided by the sponsor. Name: Batch N o . v Aggregate State at RT: Colour: liquid not indicated Purity: Analysis : Stability: Pure: not indicated In vehicle: not indicated Storage : 4 C Expiration date: not indicated On the d a y 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. mnteec.re 12 . Company Sanitized. Does not contain TSCA CBf 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 n o . 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. t mnteec.re 13 Company Sanitized. D oes not contain TSCA CB1 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 numbdr. mnteec.re 14 Conpany Sanitized. D oes not contain TSCA CBJ Husbandry The animals were kept conventionally. The experiment was conduct ed under standard laboratory conditions. Housing: Cage Type: Bedding: Feed: Water: . . Environment: single . MakroIon 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. / mnteec.re 15 Company Sanitized. D oes not contain TSCA CBI EXPERIMENTAI, 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 pos-t-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 Sanitized. Does not contain TSCA c 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 supe~matant 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 lOOx 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. mnteec.re 17 Company Sanitized. D oes not contain TSCA CBf 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 t e e c .re 18 Company Sanitized. D oes not contain TSCA CBJ BIOMETRY Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-pararaetric 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 t mnteec.re 19 Sanitized. D oes not contain TSCA C IUaiWWki I<*/ p r e -e x p e r t m e h t f o r toxicity In a pre-experiment 4 animals (2 males, 2 females ) received grally a single dose of 2000 mg/kg b.w. J| ] p H | ^ | H V P issolv'ed in aqua deionized. The volume administered was 10 ml/k^Hb.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 i n 111 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. mnteec.re 20 iifl2e& Does TSA C B f 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 ' 4a test article 2000 48 0.10 0-3 1000/ 663 0.07 0-2 1000/ 732 1.58 8-24 1000/ 923 0.03 0-1 1000/736 0.11 0-2 1000/ 666 mnteec.re 21 Sanitized. Does not contain TSCA CBl Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/NCE (HCE = normochromatic erythrocytes) scoring 24 hours after treatment Table Is vehicle animal sex test group dose micronuclei no. mg/kg in 1000 PCE b.w. per animal PCE/NCE 1 m aqua deion. 0 2 m ^ It a 3m tf tt 4m It a 5m It a 7f a a 8f a a- 9f a a 10 f n a 11 f a a sum mean percent cells with micronuclei 2 2 2 1 2 0 1 1 1 1 13 1.3 0.13 1000/ 1000/ 1000/ 1000/ 1000/ 1000/ 1000/ 1000/ 1000/ 1000/ 669 600 594 536 482 418 726 683 846 834 10000/ 6388 1000/ 639 Table II: test article animal _ nor ' 13 14 15 16 17 19 20 21 22 23 sex test group mm m dose mg/kg b.w. mi 1 200 m' m 1 a` ma a ma a f, a a fa a fa a fa a f a .a sum mean percent cells with micronuclei micronuclei in 1000 PCE per animal PCE/NCE 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 15 1.5 0.15 10000/ 7158 1000/ 716 Table III: test article animal no. 25 26 27 28 29 31 32 33 34 35 sex test group dose mg/kg 1 1m I m b-w"I 666 m a ^m a m a a fmffff . a a a a a a a a a a a a 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/ 851 1 1000/ 497 0 1000/ 602 0 1000/ 709 10 1.0 0.10 10000/ 5627 1000/ 663 mnteec.re 22 Qompgjiy Sanitized. Does not contain TSCA CBS 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 r ___( _ mg/kg a b.w. _ \m i 2000 m m m m f f f f f +i * It 0 0 It It It It It It It ft It 0 It 0 -~ ' 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 50 51 52 53 55 56 57 58 59 60 m m m m m f f f f f f CP0A n 0 tt 0 0 0 0 *0 0 300 0 0 0 0 0 0 0 0 tt 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 ICO0/ 790 not scorable 15 1000/ 693 20 1000/ 881 158 15.8 1.58 10000/ 9232 1000/ 923 mnteec.re .23 Sanitized. Does not contain TSCACfl^ Micronuclei 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 b.w. 61 62 63 64 65 67 68 69 70 71 m aqua deion. ma m m m f a n 8 n f f a n f it f ti 08 n m a a 8 8 8 8 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 anniom.al 73 74 75 76 77 79 80 81 82 83 sex test group dose mg/kg r _________J k b.w. m m m m m f f f f f I[ 8 8 8 8 8 8 8 V i r 8- 8 8 8 8 a 8 sum mean percent cells faith 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 mnteec.re 24 Sanitized. D oes not contain TSCA CBft OUIM OLUOIUIM O The test: articlel assessed in the micronucleus assay for its potential to jOrLuce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was dissolved in aqua deionized. The 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.. As determined in a pre-experiment 2000 mg/kg b.w. of the test article were tolerated by the animals. Slight toxic reactions were observed. The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs o f the corresponding negative controls, indicating t h a t U H H H | H K a d no"cytotoxic properties. At preparation interval 24 hours no substantial enhancement in the frequency of the detected micronuclei after treatment with the test article was observed. The mean micronucleus values were in the range of the corresponding negative control rate. In comparison to the corresponding negative control at prepara tion interval 48 hours, however, there was a statistically significant enhancement in-- the frequency^of the detected micronu clei after treatment with mnteec.re 25 Sanitized. Doe not contain t sc a CBfi This statistically significant effect is considered to be of minor importance: 1. 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 %. . 2. The micronucleus frequency of 0.11 % PCEs with micronu clei after treatment with 2000 mg /kg b . v M | | | B H K s within the range of the historical control data presented. 3. 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 t e e c .re 26 erapanf Sanitized. Does not contain TSCA CBS. DISTRIBUTION OF THE REPORT Sponsor: Study Director: 2x (original, copy) lx (copy) REFERENCES 1. Heddle, J.A (1973) A- rapid Jin-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, Press, New York, pp. 31-53 Plenum 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 niicronuclei 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 dimethylbenzanthra cene. 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 mnteec.re 27 ggpipa^f Ssnlfeeci Bees net contain ysc m