Document RJ01mJ6QYwbDjvB4Gy4GVdE28

AR226-2996 . STUDY TITLE: CHROMOSOME ABERRATION ASSAY IN CH IN E SE HAMSTER V79 CELLS IN VITRO WITH REPORT ' DATA REQUIREMENTS: O EC D Guideline 473 E E C Directive 92/69 EPA 40 CFR Ch. 1 (7-1-86 ed. 798.5385) AUTHOR: Dr. Wolfgang Vlkner STUDY COMPLETION: July 06, 1993 PERFORMING LABORATORY: C C R - Cytotest Cell Research GmbH & Co. KG P.O. Box 1252, D-6101 Rossdorf, Germany LABORATORY PROJECT ID: C C R 326430 I SPONSOR: ISEG A Forschungs- und ! Untersuchungsgesellschaft mbH D-8750 Aschaffenburg ! Study Monitor i Heike Krmer I Page 1 of 32 RCC Group ISCACBI Sanitized. D oes not co^ta. Company COPY OF GLP CERTIFICATE i HESSISCHES MINISTERIUM . . FR UNWELT, ENERGIE UND BUNDESANGELEGENHEITEN GLP-Bescheinigung B escheinigung C ertific ate Hierm it wird b esttig t, d a d ie P rfungsalnrichtung(en) Cytotest Cell Research GmbH & Co KG It Is h e re b y certified th a t th e t e s t fa d lity (ie s) Cytotest Cell Research GnbH & Co KG In den Leppsteinswiesen 19 In den Leppsteinswiesen 19' ~ !n 6101 Rodorf ' (Ort. ArovTii) . . 6101 Rodorf . (location, ftddrcu) rinr RCC Holding Verwaltung &nbH RCC Holding Verwaltung GmbH (Fwn*) (company name) 0 3 .0 8 ., 0 4 .0 8 ., 05.08.- und 06.08.92 03.08., 0 4 .0 8 ., 05.08. and 06.08.92 e n ..................... m ` (Datum) , (dt| _ von d e r (r die b erw ach u n g zu stn d ig en B ehrde b e r die E inhaltung d e r G ru n d s tz e d a r G uten L& bcrpraxls inspiziert w orden ist (sind). * w as (were) inspected by the com petent authority reg ar ding com pliance w ith th e Principles of G o o d L aboratory Practice. E s w ird hierm it b e st tig t, d a folgende P rfungen in d ie se r Prfeinrichtung n a c h d e n G rundstzen der G u ten L aborpraxis durchgefhrt w erden. ' . It is h e re b y ce rtifie d th a t s tu d ie s in this t e s t facility a re conducted in c o m p lia n c e with th e P rin cip les of G o o d . Laboratory P ractice. ' . Toxikologische Eigenschaf Toxicological, properties Im Auftrag . ' Kpf'- jCi.tujr (Dr.'-Hecker) ' Wiesbaden, den 09. ca7922r page 2 of 32 Company Sanitized. Does not contain TSCA CBI CONTENTS COPY OF GLP CERTIFICATE PREFACE General . Project Staff ' Schedule Project Staff Signatures Quality Assurance Guidelines Archiving STATEMENT OF COMPLIANCE QUALITY ASSURANCE UNIT Statement SUMMARY .. C o n c l u s i o n OBJECTIVE Aims of the Study Reasons for the Study MATERIALS AND METHODS The Test Article The Controls The Test System Mammalian Microsomal Fraction S9 Mix Pre-Test for Toxicity Dose Selection Experimental Performance Data Recording A c c e p t ability of the Assay Evaluation of Results BIOMETRY , RESULTS Tables of Results Pre-Test for Toxicity EXPERIMENT I Number of Polyploid Cells and Mitotic Index _ Structural Chromosomal Aberrations E X P E R I M E N T II Number of Polyploid Cells and Mitotic Index _ Structural Chromosomal Aberrations DISCUSSION REFERENCES DISTRIBUTION PAGE 2 4 4 4 5 5 5 6 7 8 8 9 9 10 10 10 11 11 11 13 14 15 15 16 18 18 19 20 21 21 21 22 22 23 26 26 27 30 32 32 ca7922r Company Sanitized, page 3 of 32 not contain TSCA CB1 PREFACE GENERAL Sponsor: Study Monitor: Testing Facility: CCR Project No.: Test Article: Title: ~ ISEGA Forschungs- und Untersuchungsgesellschaft mbH D-8750 Aschaffenburg Heike Krmer CCR CYTOTEST CELL RESEARCH GMBH & CO. KG D-6101 Rodorf 326430 Chromosome Aberration Assay in Chinese Hamste_V79 Cells in vitro withf PROJECT STAFF Management: Study Director: Author: Head of Quality Assurance Unit: Technical Coordination: SCHEDULE / Dr. Hans-Emil Knoell Dr. Albrecht Heidemann Dr. Wolfgang Vlkner Dr. Christiane Helmrich Frauke Khner Date of Protocol: Start of Pre-Test: November 11, 1992 December 01, 1992 Start of Experiment I : End of E x p e r i m e n t I :' Start of E x p e r i m e n t II: End of Experiment II: March May March May 01, 1993 24, 1993 29, 1993 05, 1993 Date of Draft: M a y 26, 1993 Date of Report: July 06, 1993 ca7 922r page 4 of 32 Company;Sarsstic-cjd. unoo--' ^' !-"'-"t contain T S C A CBt  PROJECT STAFF SIGNATURES Study Director: Dr. A ltre ' " ' ' Author: Date: July 06, 1993 Dr. W o l f g a n g Vlknr Management : Date: July 06, 1993 Dr. F '~ `1 Knoell Date: 06, 1993 QUALITY ASSURANCE The study was performed in compliance with: "C h e m i k a l i e n g e s e t z ( " C h e m i c a l s Act") of t h e F e d e r a l R e p u b l i c of Germ a n y , A n l a g e 1 ("A n n e x 1"), d a t e d M a r c h 14, 1990 (BGBL. I S. 521).." "The OECD Principles of Good Laboratory Practice", Paris 1981. GUIDELINES ' This study was conducted according to the procedures indicated by the following internationally accepted guidelines and recom mendations : First Add e n d u m to the OECD Guideline for Testing of Chemicals, S e c t i o n 4, No. 473, a d o p t e d M a y 26, 1983, v i t r o _ M a m m a l i a n C y t o genetic Test" EEC D i r e c t i v e 92/69, L 383 A, A n nex V, B 10, d a t e d D e c e m b e r 29, 1992. Environmental Protection Agency, Code of Federal Regulations, Title 40, S u bpart F-G e n e t i c Toxicology, R e v i s i o n July 1, 1986, "In vitro mammalian cytogenetics" ca7922r page 5 of 32 Company Sanitized. D oss sot contain TSC CBt ARCHIVING C C R, D-6101 Rodorf will archive the following data for 30 years: raw data, protocol and copy of report. . The following specimen and samples will be archived 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. f ca79 22r page 6 of 32 Company Sanitized. D1 not contain TSCA CSI STATEMENT OF COMPLIANCE Project Number: Test Article: Study Director: Title : 326430 Dr. Albrecht Heidemann Chromosome Aberration Assay Hamster V79 Cells in vitro with Chines To the best_o my knowledge and belief, this study performed in the testing facility of CCR was conducted in compliance with the Good Laboratory. Practice Regulations : Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, A n l a g e 1 ("Annex 1"), d a t e d M a r c h 14, 1990 (BGBL. I S. 521)." "The OECD Principles of Good Laboratory Practice", Paris 1981. There were no circumstances that may have affected the quality or integrity, of the study. '- Study Director CCR Dr. Albrecht Heidemann ca7922r page 7 of 32 Company Sanitized. D oss not contain TSCA CBt QUALITY ASSURANCE UNIT C C R, C y t o t e s t Cell Research GmbH & Co KG, In d e n L e p p s t e i n s w i e s e n 19, D-6101 Rodorf STATEMENT Project Number: Test Article: Study Director: Title: _- 326430 Dr. Albrecht Heidemann C h r o m o s o m e A b e r r a t i o n A s s a y j-n C.h in sat Hamster V79 Cells in vitro withi 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 11/ 1992 March 02, 1993 April 02, 1993 April 21, 1993 June 28 ,1993 , Dates of Reports to the Study Director and to Management November 11, 1992 March 02, 1993 April 02, 1993 April 21, 1993 June 28 ,1993 Head of Quality Assurance Unit ca7922r Dr. Christiane Helmrich Date: .Wr^.- X . ...... -icl c page 8 of 32 Company Sanitized. Docs not co; :ain TSCA CBI SUMMARY rpj^g tsst was assssssd for its potsntial to induce aberrations in V7 9 cells of the Chinese hamster in vitro in two independent experiments . The c h r omosomes w e r e p r e pared 18 h and 28 h a f ter s t art of treat ment with the test article. The treatment interval was 4 h with .'metabolic activation, 18 h a nd 28 h w i t h o u t m e t a b o l i c activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations. ' The f o llowing concentrations* were e v a l u a t e d (18 h: 3 c o n c e n t r a tions; 28 h: highest concentration): Experiment I without S9 mix: 18 h: 30; 100; 300 pg/ml 28 h: 300 pg/ml with S9 mix: 18 h: 30; 100; 300 pg/ml 28 h: 300 pg/ml Experiment II without S9 mix: 18 h: 30; 100; 200 pg/ml 28 h: 200 pg/ml with S9 mix: j 18 h: 30; 100; 200 'pg/ml 28 h: 200 pg/ml The concentration range of the test article was determined in p r e - e x p e r i m e n t s . In experiment II the highest concentration was 200 pg/ml due to the limited solubility of the test article. _ The colony forming ability of V79 cells as indicator for a toxic ity response was not affected by treatment with concentrations up to 300 pg/ml of the test article (with and without S9_mix). In the absence of S9 mix, in experiment I the mitotic index was slightly reduced after treatment with the test article. However, this effect could not be confirmed in experiment II._ _ _ In the presence of S9 mix in both experiments the mitotic index was not reduced. In both independent experiments, there were no statistically relevant increases in'cells with structural aberrations after treatment with the test article. __ In both experiments, no biologically relevant increase m the rate of polyploid metaphases was found after treatment with the test article as compared to the controls. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations. CONCLUSION In conclusion, it can be stated-that in the study described and under the experimental conditions reported^ the test article did not induce structural chromosomal aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. * cannot be g i v e n accurately; see p a g e 15 ca7 922r page 9 of 32 Company Sanitized. Does nc contain TSCA CB{ OBJECTIVE AIMS OF THE STUDY This in vitro assay was performed to assess the potential of the test article to induce structural chromosomal aberrations by means of two independent chromosomal aberration experiments in the Chinese hamster cell line V79. REASONS FOR THE STUDY In vitro methods are valuable when it is desirable to accurately control the concentration and exposure time of cells to the test article under study. However, due to the limited capacity for metabolic activation of potential mutagens an exogenous metabolic activation system is necessary. This in v i t r o t est is an a s s a y for the d e t e c t i o n of structural chromosomal aberrations. These aberrations are frequently lethal to the damaged cells. However, cytogenetic damage in somatic cells is an indicator of a potential to induce more subtle^ chro mos o m a l d a m a g e that is c o m p a t i b l e w i t h cell division. Similar damage induced in germinal cells may lead to heritable cyto genetic abnormalities. Heritable cytogenetic abnormalities are known to have deleterious effects in man, e.g. induction of neoplastic events or birth defects. . The V7 9 cells were exposed to the test article both with and without exogenous metabolic activation. The cells were then harvested at sequential intervals and chromosome preparations were made. The stained preparations were examined and metaphase cells were scored for chromosomal aberrations. Chromosomal aberrations are generally evaluated in first post treatment mitoses. With the majority of chemical mutagens, in duced aberrations are of the chromatid type, but chromosome type aberrations also occur. / The time at which the aberration frequency is at the maximum va ries from agent to agent. Because different chemicals have ef fects at different parts of the cell cycle and V79 cultures are asynchronous, multiple post-treatment sample times are necessary to precisely define the response. Due to mitotic delay or meta bolic and pharmacokinetic effects the appearance of the first post-treatment mitosis can be considerably delayed. Therefore samples t a k e n at 18 h and 28 h aft e r b e g i n n i n g of t r e a t m e n t cover the intervals in which maximum aberration frequency is expected. For the assessment of clastogenic activity three concentrations were e v a l u a t e d at the central sampling time of 18 h and one con c e n t r a t i o n at 28 h. The h i g h e s t c o n c e n t r a t i o n s h o u l d e x h x b i t a cytotoxic effect, if possible. To validate the test, reference mutagens were tested in parallel to the test article. ca7922r p a g e 10 o f 32 Company Sanitized. Dc^c-10fc . " G5"2in TSCA CBI 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 .: not indicated by the sponsor Chemical name: Aggregate State at RT: Colour : Analysis: Molecular Weight not indicated by the sponsor not indicated by the sponsor Purity: Stability: Storage: Expiration Date: 35% in Isopropanol and H^O a) Pure: not indicated by the sponsor In solvent: not indicated by the sponsor 4 0C not indicated by the sponsor On the day of the experiment (immediately before treatment), the test a r t i c l e w a s d i s s o l v e d in e t h a n o l (E. M e r c k , D - 6 1 0 0 D a r m stadt; p u r i t y > 99 % ). The solvent was c hosen a c c o r d i n g to its solubility properties and its non-toxicity to the c e l l s . The final concentration of ethanol in the culture medium did not exceed 1 % v/v. ca7922r p a g e 11 o f 32 Company Sanitized. D oss not contain TSCA CBI THE CONTROLS The Negative Controls Concurrent negative (culture medium) and solvent controls (etha nol) were performed. -v- The Positive Control Substances Without metabolic activation Name: Supplier: EMS; Ethylmethanesulfonate Merck-Schuchardt, D-8000 Mnchen Catalogue n o .: 820774 (purity > 98 %) Dissolved in: utrient medium Final Concentration: 0,4 mg/ml = 3.25 mM Solution prepared on day of experiment. r The stability of the positive control substance in solution was proven by the mutagenic response in the expected range. With metabolic activation Name : Cyclophosphamide Supplier: 'SERVA, D-6900 Heidelberg C a t a l o g u e n o .: 17681 (purity > 99.5 %) . Dissolved in: nutrient medium Final C o n c entration: 0.93 jig/ml = 3.3 p M (in d e v i a t i o n to proto- cpl where 2.8 pg/ml = 10.0 pM was given) The s t a b i l i t y of C P A at r o o m tem p e r a t u r e is good. A t 20 C only 1 % of C PA is hydrolysed per day in aqueous solution. ca7922r p a g e 12 o f 32 Company Sanitized. D ocs net contain TSCA CBI . THE TEST SYSTEM Reasons for th e Choice of the C e l l L i n e V7_9 The V79 cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate (doubling time of clone V 7 9/T5 in stock cultures: 12 h, deter mined on O ctober 22, 1992) and a high plating e f f i c i e n c y of u n t r e a t e d c e l l s (as a r u l e m o r e t h a n 50 %) b o t h n e c e s s a r y for the a p p r o p r i a t e p e r f o r m a n c e of t h e study, r e c o m m e n d - t h e u s e of this cell line. The cells have a stable karyotype with a modal chromosome n u m b e r of 22. Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays with cell cultures as many chemicals only develop mutagenic potential when they are metabolized by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures w i t h liver m i c r o s o m e p r e p a r a t i o n s (S9 mix). Cell Cultures Large stocks of the V7 9 cell line (supplied by LMP, Technical University Darmstadt, D-6100 Darmstadt) were stored in liquid nitrogen in th e cell bank of C C R a l l o w i n g the r e p e a t e d use of the same cell culture batch in experiments. Before freezing, eac batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experi ments remained similar because of the reproducible characteris tics of the c e l l s . Thawed stock cultures w e r e p r o p a g a t e d at 37 C in 80 c m 2 plastic flasks (GREINER, D - 7 4 4 3 F r i c k e n h a u s e n ). A b o u t 5 x 10 c e l l s per flask w e r e s e e d e d in 15 ml of M E M (minimal^ e s s e n t i a l medium; Biochrom KG; D-1000 Berlin 46)' s u p p l e m e n t e d w i t h 10 % fetal calf serum (FCS; Biochrom). The cells were subcultured twice weekly. The cell c u l t u r e s w e r e i n c u b a t e d at 37 C in an a t m o s p h e r e with 4.5 % carbon dioxide' (95.5 % air). ca7922r page 13 of 32 Company Sanitized. Dogs not contain TSCA CBI MAMMALIAN MICROSOMAL FRACTION S9 MIX S9 {P r e p a r a t i o n b y C C R) The S9 liver microsomal fraction was obtained from the livers of 8 - 1 2 weeks old male rats, strain Wistar/WU (SAVO, m e d . Ver suchstierzuchten GmbH, D-7964 Kisslegg; weight approx. 150 - 200 g) w h i c h r e c e i v e d a s i n g l e i.p. i n j e c t i o n of 500 m g / k g b.w. Aroclor 1254 (Antechnika, D-7500 Karlsruhe) in olive oil 5 days previously. After cervical dislocation the livers of the animals were re moved, wash e d in 150 mM KC1 and homogenized. The homogenate, di l u t e d 1+ 3 w i t h KC1 was c e n t r i f u g e d twice at 9.000 g for 10 minutes (4 C). A stock of the s u p e r n a t a n t c o n t a i n i n g t h e microsomes w a s f r o z e n in a m p oules of 2 o r 3 m l a n d s t o r e d a t -70 C. Small n u m b e r s o f the ampoules w e r e k e p t at -20 C for o n l y sever al weeks before use. The protein content was determined using the analysis kit of Bio-Rad Laboratories, D-8000 Mnchen: Bio-Rad p r o t e i n assay, Catalogue No. 500 000 6. The p r o t e i n concentration in the S9 prepar a t i o n is u s u a l l y b e tween 20 and 45 mg/ml. In experiment I and II the protein concen tration was 38.5 mg/ml (Lot. No.: 071292). S9 Mix An appropriate'quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentra tion of 0.75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2 33 m M KC1 . 5 mM glucose-6-phosphate 4 mM NADP in 100 m M sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath. The S9 mix p r e p a r a t i o n was perf o r m e d a c c o r d i n g to Ames et a l . (1)* i ca7922r p a g e 14 o f 32 Company Sarnfized. Docs not contain TSCA CBf PRE-TEST FOR TOXICITY A pre-test was performed in order to determine the toxicity of the test article. The general culturing and experimental condi tions in this pre-test were the same as described below for the mutagenicity experiment. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per /concentration) after treatment with the test article was observed and compared to the controls. Toxicity of the^ test article was evidenced by a reduction in colony forming ability. Concurrent with the pre-test high density cultures (approx. 200,000 cells/slide) were treated with the test article to simu late the conditions of the main experiment. Cell number and morphology were examined qualitatively 4 h and 24 h after the start of treatment (with metabolic activation: after washing with Saline G ) . DOSE SELECTION A c c o r d i n g to -the res u l t s f r o m this p r e - t e s t 6 c o n c e n t r a t i o n s (18 h i n t e r v a l ) w e r e c h o s e n to be applied in the chromosomal aberration assay. The h i g h e s t concen t r a t i o n (300 jig/ml) used in t h e p r e - t e s t and in the main experiment I was limited by the solubility of the test article in ethanol and other appropriate solvents (for example aqua bidest. or DMSO). As no toxic effect could be observed in the pre-test (colony forming ability) the cytogenetic experiments were performed with the concentrations listed below. Experiment I without S9 mix: 18 h: 1.0; 3.0; 10.0; 30.0; 100.0; 300 ng/ml 28 h: 10.0; 30.0; 100.0; 300.0 ng/ml w i t h S9 mix: 18 h: 1.0; 3.0; 1 0 . O'; 30.0; 100.0; 300 [xg/ml 28 h: 10.0; 30.0; 100.0; 300.0 ng/ml In experiment I the stock solution (30 mg/ml) f or formulation of the highest concentration was not completely dissolved. Addition ally, a test article precipitation in the aqueous culture medium was observed. Therefore, in experiment II the concentration range was o n l y set u p to 200 |ig/ml. Experiment II without S9 mix: 18 h: 1.0? 3.0? 10.0; 30.0? 100.0; 200 ng/ml 28 h: 10.0; OL*J O 100.0; 200.0 ng/ml with S9 mix: 18 h: 1.0; 3.0; 10.0; 30.0? 100.0; 200 ng/ml 28 h: 10.0; 30.0; 100.0; 200.0 jig/ml However, 200 pg/ml were not completely dissolved too, so the concentrations used in each experiment cannot be given accu rately. The t r e a t m e n t interval was 4 h with m e t a b o l i c ac t i v a t i o n , 18 h ca7922r P a9 e 15 of ^ Company Sanitized. D ess noi contain TSCA CB! and 28 h w i t h o u t m e t a b o l i c a ctivation. Per c o n c e n t r a t i o n d u p l i cate cultures were used. . The preparations of all test groups in both experiments were assessed qualitatively for possible toxic effects (low number of remaining/surviving cells, low metaphase number, metaphase quali- In both experiments, in presence of S9 mix no toxicity could be .observed u p to the highest concentrations used (see pages 22 and 26). In a b s ence of S9 mix, in experiment I the mitotic index was slightly reduced up to the highest concentration used (300 pg/ml) in both fixation intervals, but in experiment II no-reduction of the mitotic index was found at the highest concentration used (200 jig/ml) . There f o r e , in c y t o g e n e t i c e x p e r i m e n t I, in the absence and presence of S9 mix cultures after treatment with 300.0 pig/ml (18 h and 28 h) as the h i g h e s t c o n c e n t r a t i o n were evaluated for cytogenetic damage; in experiment II, the highest concentration evaluated was 200.0 ng/ml at each fixation interval with and without S9 mix. EXPERIMENTAL PERFORMANCE Seeding of the Cultures Three or four days old exponentially growing stock cultures more than 50 % c o n f l u e n t were t r y p s i n i z e d at 37 C for a p p r o x imately 5 minutes. Then the enzymatic digestion was stopped by adding comp l e t e c u l t u r e m e d i u m .a n d a s i n g l e cell s u s p e n s i o n w a s p r e pared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA). The Ca-Mg--free salt solution was composed as follows NaCl 8000 mg KCl Glucose NaHC03 400 mg 1000 mg 350 mg (per litre). Prior to the trypsin treatment the cells were rinsed with Ca-Mg- free salt solution containing 200 mg/1 EDTA-(Ethylene diamine tetraacetic acid). ' The cells were seeded into Quadriperm dishes (Heraeus, D -- 6450 Hanau) w h i c h contained m i c roscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x 1 0 - 1 x 1 0 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS (complete medium). ca7922r p a g e 16 o f 32 Company Saniiized. Does no! contain TSCA CBf Treatment Exposure time 4 hours (with S9 mix): .In b o t h i n d e p e n d e n t e x p e r i m e n t s , a f t e r 48 h (28 h p r e p a r a t i o n interval) and 55 h (18 h p r e p a r a t i o n interval) the culture m e dium was replaced with serum-free medium containing different concen trations of the test article and 50 nl/ml S9 mix. After 4 h the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time. The "Saline G" solution is composed as follows (per litre): NaCl - ' 8000 mg KC1 400 mg Glucose 1100 mg N a 2H P 0 4 .7H20 290 mg K H 2P 0 4 150 mg pH is adjusted to 7.2 Exposure time 18 and 28 hours (without S9 mix): In both independent experiments, after 48 h (28 h preparation interval) and 55 h (18 h p r e p a r a t i o n interval) t he culture m e d i u m was replaced with complete medium (10 % F C S ) containing different concentrations of the test article without S9 mix. This medium was not changed until preparation of the cells. f' A l l c u l t u r e s w e r e i n c u b a t e d a t 37 C in a h u m i d i f i e d a t m o s p h e r e with 4.5 % C02 (95.5 % air). - Preparation of the Cultures 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 jig/ml culture m e d ium) to the cultures. 2.5 h later, the cells were treated on the slides in the chambers with hypo tonic solution (0.4 % K C l ) f or 20 m i n at 37 C. A f t e r incubation' in the hypotonic solution the cells were fixed with 3 + 1 metha nol + glacial acetic acid. Per experiment both slides per group were prepared. After fixation the cells were stained With Giemsa (E. Merck, D-6100 D a r m s t a d t ) . I ca7 922r p a g e 17 o f 32 Company Sanitized. Does not contain TSCA CBi A n a l y s i s of M e t a p h a s e Cells_ Evaluation of the cultures was performed (according to standard p r o t o c o l of t h e " A r b e i t s g r u p p e der I n d u s t r i e , C y t o g e n e t i k " (4)) using NIKON microscopes with lOOx oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disinte grations werp recorded as structural chromosomal aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides (deviation to prot o c o l w h e r e for the p o s i t i v e controls only 50 metaphases should be scored). Only metaphases with characteristic c h r o m o s o m e numbers of 22 1 were included in the analysis. To d e s c r i b e a c y t o t o x i c e f f e c t the m i t o t i c i n d e x (% cells m m i t o sis). w a s d e t e r m i n e d . In addition, the n u m b e r of p o l y p l o i d ce s was s c o r e d '(% po l y p l o i d metaphases; in the c a s e of this aneuploid cell line polyploid means a near tetraploid karyotype). DATA RECORDING The data generated were recorded in the raw data file. The re sults are presented in tabular form, including experimental groups with the test article, negative and positive controls. ACCEPTABILITY OF THE ASSAY The chromosomal aberration assay is considered acceptable if it meets the following criteria: a) The n u m b e r of abe r rations found in the- n e g a t i v e a nd/or solvent controls falls within the range of historical laboratory c o ntrol data: 0.00 % - 4.00 %. . b) The p o s i t i v e control s u b s tances s h o u l d p r o d u c e significant increases of the number of cells with structural chromosomal aberrations. ' ca7 922r p a g e 18 o f 32 Company Ssniiized. Doss s*cf c ` lain TSCA OBJ EVALUATION OF RESULTS A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points. A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly posi tive response at any one of the test points is considered non mutagenic in this system. This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together. ~ # ca7 922r page 19 of 32 Company Sanitized. Dc s not contain TSC CBf BIOMETRY A statistical evaluation of the results was not necessary to be performed. The mean aberration rates of the test groups after treatment with the test article did not exceed the control values. .. Ex c e p t i o n was the test a r t i c l e group 200 Jig/ml at f i x ation inter val 28h in experiment II. Stati s t i c a l significance at the five per cent level (p < 0.05) was evaluated for this test article group by means of the chi- square test. Evaluation was performed only for cells carrying aberrations exclusive gaps. Solvent control versus Test group 200.0 jig/ml fixation interval 28 h S9 mix - p value 0.1 > p > 0.05 ca7 922r C o m p a q Saniti: :sd. Dos p a g e 20 o f 32 ?contain TSCA OBI RESULTS TABLES OF RESULTS Table 1: Pre- t e s t for t o x i c i t y In the pre-test the toxicity of the test article was examined determining the colony forming ability of cultures treated with the test article. Colony forming ability without and with metabolic activation; . Per flask approx. 500 cells were seeded. cone, per ml negative control . ' solvent control ethanol 0.1 jxg 0.3 |ig 1.0 |xg 3.0 p.g 10.0 jig 30.0 jig 100.0 jig 300.0 jig negative control solvent control ethanol 0.1 jig 0.3 jig 1.0 jig 3.0 jig . 10.0 jig 30.0 jig 100.0 jig 300.0 jig S9 colonies counted mix flask I flask II 302 261 277 ' 251 320 _ 306 294 312 254 - 294 355 282 243 264 284 315 296 295 309 274 + 288 279 + 303 252 + 252 255 + 263 287 + 304 231 + 281 266 + 284 267 + 317 310 /' + 265 301 + 297 238 mean 328.5 271.5 260.0 257.5 302.0 310.5 295.0 303.5 281.5 284.0 283.5 277.5 253.5 275.0 267.5 273.5 275.5 313.5 283.0 267.5 relative survival % 100.0 95.8 94.8 111.2 114.4 108.7 111.8 103.7 104.6 100.0 91.4 99.1 96.4 98.6 99.3 113.0 102.0 96.4 ABBREVIATIONS The following abbreviations of structural chromosomal aberrations are used in the following tables: a = cap- ig = iso-gap; gaps are achromatic lesions of chromatid or chromosome type where no dislocation of chromosomal material is visible (independent of the size of the achromatic region). b - break; ib = iso-break; f = fragment; if = deletion* id = iso-deletion; ma = multiple aberration ( more than 4 events in one cell [excluding gaps]^ onl y ^ J ^ h a n g e s ar^ recorded additionally in these cells); ex c > n^isinte- change; cx = chromosome type exchange; cd - chromosomal disi g r a t i o n (= pulverization) ca79 22r page 21 of 32 Company Sanitized. Doss nof contain TSCA CBI V o 03 to tno oo 3 xBrs3s co3o. N(U1) a oO0) 7oCou'3yrc4*i*" -J H rO <O>/)i{PQD) O 09 -- KJ Ol-h LNOJ Experiment I Table 2: Number of Polyploid Cells and Mitotic Index experiment I; fixation intervals 18 h and 28 h; without and with metabolic activation cone, S9 fixation per ml mix interval polyploid cells* culture total mean 12 mitotic index (% )** culture abs. rei.*** 12 Negative control Solvent control ethanol Positive control EMS Test article n tl 1.0 % 0.4 mg 30.0 p,g 100.0 |ig 300.0 p.g - - X 18 h 18 h 18 h 18 h 18 h 18 h 347 3.5 20.0 22.9 21.5 100.0 134 2.0 17.1 20.9 19.0 100.0 369 4.5 10.3 17.3 13.8 64.3 909 4.5 11.4 10.2 10.8 56.8 404 2.0 14.1 14.3 14.2 74.7 437 3.5 13.1 12.4 12.8 67.1 Negative control Solvent control ethanol Positive control CPA Test article 1.0 % 0.93 p,g 30.0 p,g 100.0 p,g' 300.0 p,g + + + + + + 18 h 18 h 18 h 18 h 18 h 18 h 325 2.5 16.8 16.4 16.6 100.0 5 5 10 5.0 11.1 16.0 13.6 100.0 325 2.5 14.3 12.5 13.4 80.7 123 1.5 14.7 16.5 15.6 115.1 213 1.5 17.0 16.1 16.6 122.1 459 4.5 15.1 18.0 16.6 122.1 Solvent control ethanol 1.0 % _ Test article 300.0 pg 28 h 28 h 527 3.5 13.5 14.9 14.2 100.0 112 1.0 10.6 10.5 10.6 74.3 Solvent control ethanol 1.0 % + Test article 300.0 p.g + 28 h 28 h 022 1.0 20.4 17.2 18.8 100.0 404 2.0 17.3 20.7 19.0 101.1 * The number of polyploid cells was determined in 100 cells per culture of each test group. ** The mitotic index was determined in 1000 cells per culture of each test group. *** For the positive control groups, the relative values of the mitotic index are related to the negative controls; for the test article treatment groups the values are related to the solvent controls ompany Sanffizecf. D oss nof c o n fa i T SC A CBf npj LhDJ KJ h O 'a p iQID tLoO ol-h LO to T a b l e 3: S t r u c t u r a l c h r o m o s o m a l a b e r r a t i o n s e x p e r i m e n t I; f i x a t i o n i n t e r v a l 18 h; w i t h o u t m e t a b o l i c a c t i v a t i o n Negative control Solvent control ethanol Positive control EMS Test article II ll cone. S9 per ml mix cells aberrant cells (%mean) scored incl. excl. exchan gaps gaps ges types of aberrations found -gaps- g ig -chromatid typefa f d ex - cult. 1 100 cult. 2 100 -total- 200 5.0 2.0 0 . 0 30 50 80 0 10 i 00 2 10 0 0 0 1 . 0 % - cult. 1 100 cult. 2 100 -total- 200'" 5.5 3.0 0.5 30 30 G0 2 10 110 320 1 0 1 0.4 mg - cult. 1 100 cult. 2 100 -total- 200 13.5 11.5 7.0 30 40 70 500 6 310 8 8 1 0 14 30.0 p,g cult. 1 100 cult. 2 100 -total- 200 7.5 2.5 0 . 0 31 70 10 1 2 10 0 10 220 0 0 0 100.0 g,g cult. 1 100 cult. 2 100 -total- 200 9.0 3.0 0 . 0 30 10 0 13 0 300 110 410 0 0 0 300.0 p,g - cult. 1 100 cult. 2 100 -total- 200 8.0 2.5 0 . 0 50 70 12 0 1 10 3 10 420 0 0 0 -chromosome typeib if id cx 000 0 020 0 0 20 0 0000 0000 0000 0 o' 0 1 000 1 000 2 100 0 0000 100 0 0000 0 10 0 0 10 0 000 0 000 0 000 0 -otherma cd 00 00 00 00 00 00 00 10 10 00 00 00 00 00 00 00 00 00 For abbreviations see RESULTS/ABBREVIATONS V ,/ !.. ieoq -pazsM^ES ueduioc o D-iJ to to h t'i> ul'* o U Xp)j rT H iQro CoO to >o TO o Hi to NJ T a b l e 4: S t r u c t u r a l c h r o m o s o m a l a b e r r a t i o n s e x p e r i m e n t I; f i x a t i o n i n t e r v a l 18 h; w i t h m e t a b o l i c a c t i v a t i o n Negative control Solvent control ethanol Positive control CPA Test article H U cone. S9 per ml mix + 1.0 %. + 0.93 mg + 30.0 (xg + 100.0 ng + 300.0 p,g + cells aberrant cells (%mean) scored incl. excl. exchan- gaps gaps ges cult. 1 100 cult. 2 100 -total- 200 6.5 4.0 0.0 cult. 1 100 cult. 2 100 -total- 200 ,, 8.0 2.5 0.5 cult. 1 100 cult. 2 100 -total- 200 22.0 18.0 8.0 cult. 1 100 cult. 2 100 -total- 200 2.0 0.0 0.0 cult. 1 100 cult. 2 100 -total- 200 3.0 1.5 0.0 cult. 1 100 cult. 2 100 -total- 200 3.5 2.0 0.0 types of aberrations found -gapsg ig -chromatid type- fa f d ex 1 30 2 10 0 20 3' 0 0 0 50 5 10 0 50 60 11 0 000 2 10 2 10 0 0 0 51 6 3 18 12 0 690 9 17 1 12 12 1 17 20 20 40 000 000 000 0 0 0 20 20 40 0 20 0 10 030 0 0 0 10 30 40 03 10 13 0 0 0 0 0 0 -chromosome typelb if id cx 0000 0 20 0 0200 0000 000 1 000 1 110 030 14 0 000 000 000 0 2 2 0 0 0 0000 0000 0000 0 00 0 0000 00Q0 -otherma cd 00 00 00 10 00 10 10 00 10 00 00 00 00 00 00 00 00 00 For abbreviations see RESULTS/ABBREVIATONS Company Op-jJ KJ HK J to \3 H(C9U ao<?0*) n c fp o'pXf' M. i(QD H CD UO>> KU>l oO 03 M i U> KJ T a b l e 5: S t r u c t u r a l c h r o m o s o m a l a b e r r a t i o n s e x p e r i m e n t I; f i x a t i o n i n t e r v a l 28 h; w i t h o u t a n d w i t h m e t a b o l i c a c t i v a t i o n Solvent control ethanol Test article Solvent control ethanol Test article cone. S9 per ml mix cells aberrant cells (%mean) scored incl. excl. exchan- gaps gaps ges 1.0 % - cult. 1 100 cult. 2 100 -total- 200 5.5 2.0 0.5 300.0 ng - cult. 1 100 cult. 2 100 -total- 200' 3.5 1.5 0.0 types of aberrations found -gaps- -chromatid type- g ig b1 f d ex 30 31 61 1. 0 0 110 2 10 0 1 1 10 40 50 0 10 200 2 10 0 0 0 1.0 % + 300.0 jig + cult. 1 100 cult. 2 100 -total- 200 cult. 1 100 cult. 2 100 -total- 200 3.0 1.0 0.0 2.0 0.5 0.0 11 30 41 20 10 30 10 0 0 10 110 030 000 030 0 0 0 0 0 0 -chromosome typeib if id cx 0000 00 00 0000 0000 0000 0000 0000 000 0 0000 0000 000 0 000 0 -otherma cd 00 00 00 00 00 00 00 00 00 00 00 00 For abbreviations see RESULTS/ABBREVIATONS Company n p> UNDi Nhi to 5.30 NO ao<w:0i oo Z0i, 3 "tin "i0aQ) to Experiment II Table 6: Number of Polyploid Cells and Mitotic Index experiment II; fixation intervals 18 h and 28 h; without and with metabolic activation cone. S9 fixation per ml mix interval polyploid cells* culture total riiean 12 mitotic index (%)** culture abs. rei.*** 12 Negative control Solvent control ethanol Positive control EMS Test article 1.0 % 0.4 mg 30.0 ng 100.0 pg 200.0 |ig Negative control Solvent control ethanol Positive control CPA Test article 1.0 % 0.93 (xg 30.0 |xg 100.0 p.g 200.0 |xg + + + + + + Solvent control ethanol 1.0 % Test article 200.0 p,g Solvent control ethanol Test article 1.0 % 200.0 juj 18 18 18 18 18 18 18 18 18 18 18 18 28 h 28 h 28 h 28 h 246 123 4 6 10 022 0 33 4 15 3 14 156 437 10 1 3 14 022 2 13 224 011 325 3.0 1.5 5.0 1.0 1.5 2.5 2.0 3.0 3.5 0.5 2.0 1.0 1.5 2.0 0.5 2.5 18.3 13.2 8.1 11.6 9.4 9.8 13.0 13.1 7.7 9.4 13.3 16.5 16.8 14.5 13.5 13.7 17.5 13.7 11.2 12.9 8.7 15.4 17.1 15.9 9.8 10.6 15.1 7.0 12.3 14.3 14.2 15.8 15.7 13.2 7.9 10.5 11.4 13.2 14.0 13.7 11.1 14.6 17.3 14.8 10.2 11.1 13.3 15.0 100.0 100.0 50.5 79.8 86.3 100.0 100.0 100.0 79.3 106.2 126.3 108.0 100.0 108.3 100.0 112.8 The number of polyploid cells was determined in 100 cells per culture of each test group. * iFnLr mTihlc Pn lfC, -Ulln-IkXcwonat!rodlf ermined groups, 1000 cells per culture of each test group the relative values of the mitotic index are controls, for the test article treatment groups the values are related to the related solvent to the negative controls ompany bsninzeu. Duoo-s>s contain T SC A CBI Pn) -j VO to tho U vD(DQDJ to O t-h LNOJ T a b l e 7: S t r u c t u r a l c h r o m o s o m a l a b e r r a t i o n s e x p e r i m e n t II; f i x a t i o n i n t e r v a l 18 h; w i t h o u t m e t a b o l i c a c t i v a t i o n Negative control Solvent control ethanol Positive control EMS Test article II II cone. S9 per ml mix cells aberrant cells (%mean) scored incl. excl. exchan- gaps gaps ges - ' cult. 1 100 cult. 2 100 -total- 200 4.0 1.5 0.5 1 . 0 %- - cult. 1 100 cult. 2 100 -total- 2 0 0 ' 5.0 2.0 0 . 0 0.4 mg - cult. 1 100 cult. 2 100 -total- 200 13.5 10.5 5.5 30.0 pg - cult. 1 100 cult. 2 100 -total- 200 3.0 1 . 0 0.0 100.0 pg 200.0 pg - cult. 1 100 cult. 2 100 -total- 200 cult. 1 100 cult. 2 100 -total- 200 6.5 0.5 0 . 0 4.5 2.0 0 . 0 types of aberrations found -gaps- g ig -chromatid typeb f d ex 50 J 110 0 00 50 O' 0 11 0 0 0 0 20 40 60 010 10 0 110 0 0 0 61 20 81 330 7 340 6 6 7 0 13 10 30 40 61 60 12 1 30 20 50 000 200 20 0 000 100 1 00 2 00 010 21 0 0 0 0 0 0 0 0 0 0 -chromosome type- 1b if id cx 0 -0 o 1 0000 000 1 0 1o o 0100 0200 -otherma cd 0o 01 01 Qo oo 00 o O' o o 000 1 000 1 oo 00 00 0000 0000 0000 0000 0000 0000 1000 0000 1000 00 00 00 00 00 00 00 00 00 For abbreviations see RESULTS/ABBREVIATONS Ix o & ttvoj IO H o o3 o Q5 in m N (D a a O ort* o s' --1 w t) CD o > (B o KJ C0 CO O Hi U> NJ T a b l e 8: S t r u c t u r a l c h r o m o s o m a l a b e r r a t i o n s experiment II; fixation interval 18 h; w i t h m e t a b o l i c activation Negative control Solvent control ethanol Positive control CPA Test article II II cone. S9 per ml mix + 1.0 %- + 0.93 mg + 30.0 pg + 100.0 pg + 200.0 pg + cells aberrant cells (%mean) scored incl. excl. exchan- gaps gaps ges cult. 1 100 cult. 2 100 -total- 200 4.0 1.5 0.0 cult. 1 100 cult. 2 100 -total- 200' 4.0 2.5 0.5 cult. 1 100 cult. 2 100 -total- 200 24.0 21.0 10.5 cult. 1 100 cult. 2 100 -total- 200 4.0 2.0 0.5 cult. 1 100 cult. 2 100 -total- 200 4.5 2.5 0.0 cult. 1 100 cult. 2 100 -total- 200 2.0 0.5 0.0 types of aberrations found -gaps- -chromatid type- g ig b f d ex 10 40 50 310 000 3 10 0 0 0 40 00 40 0 20 0 10 030 1 0 1 60 61 12 1 5 10 0 11 3 13 0 12 B 23 0 23 20 20 40 0 20 0 10 030 0 0 0 10 50 60 030 020 050 0 0 0 20 10 30 0 10 0 000 0 0100 For abbreviations see RESULTS/ABBREVIATONS -chromosome typeib if id cx 0000 0000 0000 0000 0 10 0 0 10 0 0 10 0 050 0 0600 0000 000 1 0001 0000 0000 0000 0000 0000 0000 -otherma cd 00 00 00 00 00 00 00 00 00 00 10 10 00 00 00 00 0 '0 00 o T a b l e 9: S t r u c t u r a l c h r o m o s o m a l a b e r r a t i o n s 3 experiment II; fixation intervai 28 h; w i t h o u t an d wi th me ta b o l i c activation N) Solvent control ethanol Test article cone. S9 Per m1 mix cells aberrant cells (%mean) scored incl. excl. exchangaps gaps ges 1-0 % - cult. 1 100 cult. 2 100 -total- 200 2.5 0.5 0.0 200.0 pg - cult. 1 100 cult. 2 100 -total- 200v 3.5 2.5 0.5 types of aberrations found -gaps- g g -chromatid typefa f d ex 40 10 50 0, 0 00 00 0 0 0 0 0 o 00 20 20 02 10 12 0 0 0 0 0 0 -chromosome typeib if id cx 10 00 0000 10 00 0 10 0 000 1 0 10 1 -otherma cd 00 00 00 00 10 10 Solvent control ethanol Test article 1.0 % + 200.0 pg + cult. 1 100 cult. 2 100 -total- 200 cult. 1 100 cult. 2 100 -total- 200 5.5. 1.0 0.0 3.0 1.0 0.0 60 40 10 0 30 20 50 000 000 000 10 00 10 0 0 0 0 0 0 0 0 0 0400 000 0 0400 000 0 10 00 10 00 00 00 00 00 00 00 For abbreviations see RESULTS/ABBREVIATONS Company Sanitized. Dcas not contain T S C A C BI 'O 0) 0) to UD O hh LO to DISCUSSION The test a r t i c l e was assessed for its potential to induce structural^chroraosomal aberrations in V7 9 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. The chromosomes w e r e prepared 18 h a nd 28 h a f t e r start of treat ment with the test article. The treatment intervals were 4 h (with S9 mix) and 18h and 28 h (without S9 m i x ) . In each experimental group two parallel cultures were set u p . Per culture 100 metaphases were scored for structural chromosomal aberrations. The f o l l o w i n g c o n c e n t r a t i o n s were e v a l u a t e d (18 h: 3 c o n c e n t r a tions; 28 hr highest evaluable concentration): Experiment I without S9 mix: 18 h: 30.0; 100.0; 300 jig/ml 28 h: 300.0 p g / m l with S9 mix: 18 h: 30.0; 100.0; 300 ng/ml 28 h: 300.0 jig/ml Experiment II without S9 mix: 18 h: 30.0; 100.0; 200 jig/ml 28 h: 200.0 pg/ml with S9 mix: 18 h: 30.0; 100.0; 200 pg/ml 28 h: 200.0 pg/ml The c o n c e n t r a t i o n range u s e d in this s t udy (300 p.g/ml as highest concentration) was limited by the solubility of the test article in ethanol and other appropriate solvents. In the pre-test on toxicity (colony forming ability) in the absence and presence of S9 mix after treatment with concentra tions up to 300.0 pg/ml (with and without S9 mix) the colony forming ability was not reduced. In the a b sence of S9 mix, in experiment I the mitotic indices were reduced after treatment with the highest concentration used (300 jig/ml) at b o t h f i x a t i o n i n t e r v a l s w h e r e a s in e x p e r i m e n t II (highest concentration 200 jig/ml) no reduction of the mitotic index was o b s e r v e d .'These effects are considered being biologi cally irrelevant since there was no dose relation in the test groups used. In the p r e s e n c e of S9 mix, in both experiments the mito t i c in dices were not reduced after treatment with the test article at both fixation intervals. ca7922r page 30 of 32 Company Sanitized. D ees not contain TSCA CBi In both experiments, in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberra tions (excl. gaps). The aberration rates of the cells after treat m e n t w i t h t h e test article (exp.I: 0.0 % - 3.0 %; exp. II: 0.5 % - 2.5 %) w e r e in the range of the solvent control v a l u e s (exp. I: 1.0 % - 3.0; exp. II: 0.5 % - 2.5 %) and w i t h i n o u r h i s t o r i c a l control range: 0.0 % - 4.0 %. Also, the number of cells carrying exchanges was not increased as c o m p a r e d t o th e solvent'c o n t r o l s . Tables 2 and 6 show the occurrence of polyploid metaphases. In both experiments, no increase in the rate of polyploid metaphases (maximum 4.5 %) as c o m pared to the rates of the c o n t r o l s (maximum 5.0 %) w e r e f o u n d after t r e a t m e n t w i t h the test article. In b o t h e x p e r i m e n t s , EMS (0.4 m g/ml) and C P A (0.93 |j.g/ml) were used as positive controls and showed distinct increases in cells with structura'l chromosomal aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article ZONYL RP 18 did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. ca7922r p a g e 31 of 32 Company Sanitized. Dees not contain TSCA CBF REFERENCES 1. B.N. Ames, J. McCann, a nd E. Y a m asaki (1977) _ Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. ' B.J. Kilbey et a l . (Eds.) "Handbook of Mutagenicity Test Procedures", Elsevier, Amsterdam, 1-17 . 2. M.O. B r a d l e y , B. Bhuy a n , M.C. Francis, R. L a n g e n b a c h , A. P e t e r s o n and E. H u b e r m a n (1981) Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of the literature. A report of the gene-tox program. M u t a t i o n Research 87, 81-142 3. R.J. Preston, W. Au, M.A. Bender, J.G. Brewen, A.V. Carrano, J.H. H e d d l e , A . F . M c F e e , S. W o l f f and J.S. W a s s o m (1981) Mammalian in vivo and in vitro cytogenetic assays.. A report of the U.S. EPA's gene-tox program. M u t a t i o n Rese a r c h 87, 143-188 4. E n g e l h a r d t G . , (1987) "Arbeitsgruppe der Industrie, Cytogenetik" Standard-Protokoll zur cytogenetischen Auswertung von Mitose- und Meiosechromosomen bei der Routineuntersuchung. 5. S.H.H. S w i e r e n g a , J.A. H e d d l e , E.A. Sigal, J . P.W. Gilman, R.L. Brillinger, G.R. Douglas and E.R. Nestmann (1991) Recommended protocols based on the a survey of current practice in genotoxicity testing laboratories, IV: Chromoso me aberration and sister chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocyte cultures. M u tation Research 246, 301-322 6. D.J. K i r k l a n d (1992) _ Chromosomal aberration tests In vitro: problems with proto col design and interpretation of results. M u t a g e n e s i s 2, 95-106 7. C.L. Bean, M. J. A r m s t r o n g a nd S.M. G a l l o w a y (1992) Effect of sampling time on chromosome aberration yield for 7 chemicals in Chinese hamster ovary cells. Mutation Research 265, 31-44 8. M. J. Ar m s t r o n g , C.L. B e a n and S.M. G a l l o w a y (1992) _ A quantitative assessment of the cytotoxicity associated with chromosomal aberration detection in Chinese hamster ovary cells. ' Mutation Research 265, 45-60 DISTRIBUTION OF THE REPORT Sponsor Study Director 2x (lx original, lx copy) lx (copy) ca7 922r page 32 of 32 Company Sanitized. D oss not contain TSCA CBf RCC Research & Consulting C o m p a n y Ltd Postfach C H - 4 4 5 2 Itingen . Switzerland Phone: 41/61 975 11 11 Fax: 41/61 971 52 84 RCC Research & Consulting C o m p a n y Ltd 1, route de Troinex CH-1227 Carouge Switzerland Phone: 41/22 342 66 70 Fax: 41/22 342 66 43 RCC Umweltchemie G m b H & Co. KG In den Leppstelnswiesen 19 D -- 6101 Rossdorf Federal Republic of Germany Phone: 49/6154 80 70 Fax: 49/6154 8 33 99 RCC Umweltchemie A G Postfach r H - 4 4 5 2 Itingen s i tzerland Phone: 41/61 975 11 11 Fax: 41/61 971 52 66 l i : b^l . Biological Research Laboratories Ltd Wolferstrasse 4 C H - 4 4 1 4 Fullinsdorf . " Switzerland Phone: 41/61 901 42 42 Fax: 41/61 901 25 65 CCR Cytotest Cell Research G m b H & Co. K G in den Leppsteinswiesen 19 D-6101 Rossdorf Federal Republic of Ge rmany, Phone: 49/6154 80 70 Fax: 49/6154 8 33 99 RCC Registration & Consulting C o m p a n y A G Landstrasse 33 C H - 4 4 5 2 Itingen Switzerland Phone: 41/61 971 50 00 Fax: 41/61 971 50 72 RCC N O T O X . Research & Consulting C o m p a n y B.V. .P.O.Box 3476 NL-5203 DL 's-Hertogenbosch The Netherlands Phone: 31/73 41 95 75 Fax: 31/73 41 85 43 RCC (UK) Ltd Research & Consulting Company Ltd Willow Court, Netherwood Road Hereford HR2 6JU United Kingdom Phone: 44/432 35 40 52 Fax: 44/432 35 23 40 RCC Group -,-uPV A^ CcHqpany Sanitized. D oss not contain TSCA CBi