AD-A112 982 LETTERMAN ARMY INST OF RESEARCH PRESIDIO OF SAN FRANC-ETC F/$ 6/20THE MUTAGENIC POTENTIAL OF: CHLOROQUINE PHOSPHATE USP.tU)MAR 82 L J SAUERS. J T FRUIN

UNCLASSIFIED LAIR-117

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N . INSTITUTE REPORT NO. 117

THE MUTAGENIC POTENTIAL OF: chloroquine phosphate USP

LEONARD J. SAUERS BA, SP5and

JOHN T. FRUIN, DVM, PhD, COL VC

TOXICOLOGY GROUP,DIVISION OF RESEARCH SUPPORT

DTIC

APR5

A

LJ MARCH 1982 Toxicology Series 27

L

LETTERMAN ARMY INSTITUTE OF RESEARCHPRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

THE MUTAGENIC POTENTIAL OF 2 ,4-DIAMINO-6-(2'-NAPTiYL-SYLFONYL) QUINAZOLINE

L. J. Sauers and J. T. Fruin Toxicology Series: 28

Reproduction of this document in whole or in part is prohibited except with the permission of the Commander,Letternan Army Institute of Research, Presidio of San Prancisco, California 94129. However, the DefenseTechnical Information Center is authorized to reproduce the document for United States Government purposes.

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Citation of trade names in this report does not constitute an official endorsement or approval of the use ofsuch items.

In conducting the research described in this report, the investigation adhered to the "Guide for the Care andUse of Laboratory Animals," as promulgated by the Committee on Revision of the Guide for LaboratoryAnimal Facilities and Care, Institute of Laboratory Animal Resources, National Research Council.

This material has been reviewed by Letterman Arm), Institute ofResearch and there is no objection to its presentation and/orpublication. The opinions or assertions contained herein are theprivate views of the author(s) and are not to be construed asofficial or as reflecting the views of the Department of the Armyor the Department of Defense. (AR 360-5)

...a 17 Feb 1982

... . ..~trL .....d....da........

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REPORT DOCUJMEN4TATION PAGE BEFORE COM4PLETING FORM1. 11191ORT NMBNER 12. GOVy ACCESSION No. 3. RECIPIENT'S CATALOG NUMBERLAIR Institute Report No. 117 A/ ) A v-4. TITLE (dad SubEl.) S YEO EOT&PRO OEEThe Mutagenic Potential of Chloroquine FINALPhosphate US? October___-_November _______

6. PERFORMING ORG. REPORT NUMBER

1. AU THOR(q) S. CONTRACT OR GRANT NUMSER()

Leonard J. Smuers, BA, SP5John T. Fruin, DYM, PhD, COL, VC

t. PERFORMING ORGANIZATION NAMIE AND ADDRESS 10. PROGRAM ELEMENT. PROJECT. TASKAREA & WORK UN IT NUMBERS

US Army Medical. Research and Development CommoandFort Detrick, MD 21201 & Tox Grp, Div of Rsch Spt, Project 3SI6273LA8758DLAIR, Presidio of San Francisco, CA 94129 WV 302. APC TLO7It. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATEUS Army Medical Research and Development Conmand March 1982Fort Detrick 13. NUMBER OF PAGESFrederick, MD 21701 ______________14. MONITORING AGENCY NAME A AOORE~g'f d~lierent htow Controlling Offi.) 15. SECURITY CLASS. (of this report)

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THIS DOCUMENT HAS BEEN APPROVED FOR PUBLIC RELEASE AND SALE: ITS DISTRIBUTIONIS UNLIMITED

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I6. SUPPLEMENTARY NOTES

I0. KEY BORDS (Contnu 4w rnverse side It necesary Wed identIfy by block numnber)

Mutagenicity, Toxicology, Ames Assay, Chloroquine Phosphate USP

*z mutagenic potential of chioroquine phosphate USP CWR1522 )was assessed byUsing the Ames Salmonella/Mammalian Microsome Mutagenicity Assay. Testerstrains TA 98, TA 100, TA 1535,_XAJU57 and TA 1538 were exposed to dosesranging from I mg/plate toZOmg/p~m late. It was determined that thetested substance did not have mutagenic poten~gi--

*Code Number of Compound

, D W3 umvior I Nov e5 s OLRTE$CU140 OF TwoS PAGE (NwSf ta o& eomatIdlWNAW

ABSTRACT

The mutagenic potential of Chioroquine Phosphate USP (WR154'I wasassessed by using the Ames Salmonella/Mammalian tMicrosome MutagenicityAssay. Tester strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 wereexposed to doses ranging from 1 mg/plate to 3.2 x 10- mg/plate. It wasdetermined that the test substance did not have mutagenic potential.

£Code number for compound.

I'Opy)SPr 105

PREFACE

TYPE REPORT: Ames Assay GLP Study Report

TESTING FACILITY: US Army Medical Research and Development CommandFort Detrick, Frederick, MD 21201Letterman Army Institute of ResearchPresidio of San Francisco, CA 94129

SPONSOR: US Army Medical Research and Development CommandDivision of Experimental TherapeuticsWalter Reed Army Institute of Research, Bldg 40Washington, D.C. 20012

PROJECT/WORK UNIT/APC: 3S162734A875BD, Medical Systems in ChemicalDefense, WU 302 Good Laboratory Training,APC TL07

GLP STUDY NUMBER: 81031

STUDY DIRECTOR: COL John T. Fruin, DVM, PhD, VC, Diplomate ofAmerican College of Veterinary Preventive Medicine

PRINCIPAL INVESTIGATOR: SP5 Leonard J. Sauers, BA

REPORT AND DATA MANAGEMENT: A copy of the final report, study protocol,and retired SOPs will be retained in theLAIR Archive. Test compounds wereprovided by the sponsor. Chemical,

analytical, purity, etc. data areavailable in Appendix A.

TEST SUBSTANCE: Chloroquine Phosphate USP

Vehicle - DMSO

INCLUSIVE STUDY DATES: October - November 1981

OBJECTIVE: To determine the mutagenic potential of the above compoundusing the Ames Assay. Tester strains TA 98, TA 100,TA 1535, TA 1537 and TA 1538 were used. The plateincorporation method was followed. The test substancewas dissolved in DMSO and this diluent was checked forsterility.

lii

ACKNOWLEDGMENTS

The authors wish to thank John Dacey, SP4 Lawrence Mullen, BS andSP4 Thomas Kellner, BA for their assistance in performing theresearch.

iv

FISignatures of Principal Scientists involved

in the Study

We, the undersigned, believe the study described in this report to be

scientifically sound and the results and interpretation to be valid. The

study was conducted to comply to the best of our ability with the Good

Laboratory Practice Regulations outlined by the Food and Drug

Administration.

LNARD J. S7 3, BA/DATE &(ONT. FRUIN, DVM, PhD/DATECOL, VC

Principal Inv stigator Study Director

¥ .1

DEPARTMENT OF THE ARMYLETTERMAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

REPLY TOATTEN EION OF:

SGRD-ULZ-QA 23 November 1981

MEMORANDUM FOR RECORD

SUBJECT: Report of GLP Compliance

I hereby certify that in relation to LAIR GLP study 81031 thefollowing inspections were made:

5 October 19817 October 19819 October 1981

14 October 198122 October 1981

The report and raw data for this study were audited on 23 November1981.

Inspection findings were reported to the Study Director on 15October 1981. These inspections will also be included in theDecember 1981 report to management.

JOHN C. JOHNSONCPT, MSQuality Assurance Officer

V-vi

TABLE OF CONTENTS

Abstract .....................................................1

Preface ....................................................iii

Acknowledgments .............................................iv

Signatures of Principal Scientists ...........................v

Report of Quality Assurance Unit ............................vi

Table of Contents ..........................................vii

BODY OF REPORT

INTRODUCTION

Rationale for Using the Ames Assay ................... 1

Description of Test, Rationale for strain selection..1

Description of Strains, History, Methods, and Data ...?2

METHODS

Rationale for Dosage Levels and Response Tabulations 3

Test Format ..........................................3

Statistical Analysis .................................i4

Chemical Analysis ....................................14

RESULTS AND DISCUSSION ...................................

CONCLUSION ...............................................5

RECOMMENDATION ...........................................5

REFERENCES ...............................................6

APPENDICES

Appendix A (report on chemical analysis) ................. 7

Appendix B (Tables 1 through 10) ........................21

Appendix C (LAIR SOP-OP-STX-1) .......................... 33

DISTRIBUTION LIST ...........................................36

Vii

THE HUTAGENIC POTENTIAL OF: chloroquine phosphate USPToxicology Series 27--Sauers and Fruin

This test substance is a candidate anti-malarial drug tested bythe Ames Assay at the request of the Walter Reed Army Institute ofResearch. The plate incorporation method was followed and thechemical was dissolved in DMS0. The DMSO was checked for sterility.

Rationale for using the Ames Assay

The Ames Salmonella/Mammalian Microsome Mutagenicity Test is oneof a standard bank of tests used by our laboratory for the assessmertof the mutagenic potential of a test substance. It is a short-termscreening assay for the prediction of potential mutagenic agents inmammals. It is inexpensive when compared to in vivo tests, yet ishighly predictive and reliable in its ability to detect mutagenicactivity and therefore carcinogenic probability (1). It relies onbasic genetic principles and allows for the incorporation of amammalian microsome enzyme system to increase sensitivity throughenzymatically altering the test substance into a potentiallymetabolite. It has proven highly effective in assessing human risk(1).

Description of Test (Rationale for the selection of strains)

The test was developed by Bruce Ames, Ph.D. from the Universityof California-Berkeley. The test involves the use of severaldifferent genetically altered strains of Salmonella typhimurium, eachwith a specific mutation in the histidine operon (2). The testsubstance demonstrates mutagenic potential if it is able to revert themutation in the bacterial histidine operon back to the wild type andthus reestablish prototrophic growth within the test strain. Thisreversion also can occur spontaneously due to a random mutationalevent. If, after adding a test substance, the number of revertants issignificantly greater than the spontaneous reversion rate, then thetest substance physically altered the locus involved in the operon'smutation and is able to induce point mutations and genetic damage (2).

In order to increase the sensitivity of the test system, twoother mutations in the Salmonella are used (2). To insure a higherprobability of uptake of test substance, the genome for thelipopolysacchride layer (LP) is mutated and allows larger molecules toenter the bacteria. Each strain has another induced mutation whichcauses loss of excision repair mechanisms. Since many chemicals arenot by themselves mutagenic but have to be activated by an enzymatic

6!1

I!

process, a mammalian microsome system is incorporated. Thesemicrosomal enzymes are obtained from livers of rats induced withAroclor 1254; the enzymes allow for the expression of the metabolitesin the mammalian system. This activated rat liver microsomal enzymehomogenate is termed S-9.

Description of Strains (History of the strains used method tomonitor the integrity of the organisms, and data pertaining tocurrent and historical control and spontaneous reversion rates)

The test consists of using five different strains of Salmonellatyphimurium that are unable to grow in absence of histidine because ofa specific mutation in the histidine operon. This histidinerequirement is verified by attempting to grow the tester strains onminimal glucose agar (MGA) plates, both with and without histidine.The dependence on this amino acid is shown when growth occurs only inits presence. The plasmids in strains TA 98 and TA 100 contain anampicillin resistant R factor. Strains deficient in this plasmiddemonstrate a zone of inhibition around an ampicillin impregnateddisc. The alteration of the LP layer allows uptake by the Salmonellaof larger molecules. If a crystal violet impregnated disc is placedonto a plate containing any one of the bacterial strains, a zone ofgrowth inhibition will occcur because the LP layer is altered. Theabsence of excision repair mechanisms can be determined by usingultraviolet (UV) light. These mechanisms function primarily byrepairing photodimers between pyrimidine bases; exposure of bacteriato UV light will activate the formation of these dimers and cause celllethality, since excision of these photodimers can not be made. Thegenetic mutation resulting in UV sensitivity also induces a dependenceby the Salmonella to biotin. Therefore, this vitamin must be added.In order to prove that the bacteria are responsive to the mutationprocess, positive controls are run with known mutagens. If afterexposure to the positive control substance, a larger number ofrevertants are obtained, then the bacteria is adequately responsive.Sterility controls are performed to determine the presence ofcontamination. Sterility of the test compound is also confirmed ineach first dilution. Verification of the tester strains occursspontaneously with the running of each assay. The value of thespontaneous reversion rate is obtained using the same inoculum ofbacteria that is used in the assay (3).

Strains were obtained directly from Dr. Ames, University ofCalifornia-Berkeley, propagated and then maintained at -80 C in ourlaboratory. Before any substance was tested, quality controls wererun on the bacterial strains to establish the validity of theirspecial features and also to determine the spontaneous reversion rate(2). Records are maintained of all the data to determine ifdeviations from the set trends have occurred.

F2

We compare the spontaneous reversion values with our ownhistorical values and these cited by Ames, et al (2). Our conclusionsare based on the spontaneous reversion rate compared to theexperimentally induced rate of mutation. When operating effectively,these strains detect substances that cause base pair mutations (TA1535, TA 100) and frameshift mutations (TA 1537, TA 1538 and TA 98)(2).

METHODS (3)

Rationale for Dosage Levels and Dose Response Tabulations

To insure readable and reliable results, a sublethalconcentration of the test substance had to be determined. Thistoxicity level was found by using MGA glates. various concentrationsof the substance, and approximately 10 cells of TA 100 per plate,unless otherwise specified. Top agar containing trace amounts ofhistidine and biotin were placed on MGA plates. TA 100 is usedbecause it is the most sensitive strain. Strain verification wasconfirmed on the bacteria, along with a determination of thespontaneous reversion rate. After incubation, the growth was observedon the plates. (The auxotrophic Salmonella will replicate a few timesand potentially express a mutation. When the histidine and biotinsupplies are exhausted, only those bacteria that reverted to theprototrophic phenotype will continue to reproduce and formmacrocolonies; the remainder of the bacteria comprises the backgroundlawn. The minimum toxic level is defined as the lowest serialdilution at which decreased macrocolony formation, below that of thespontaneous revertant rate, and an observable reduction in the densityof the background lawn occurs.) A maximum dose f 1 mgplate is usedwhen no toxicity is observed. The densities were recorded as normalslight, and no growth.

Test Format

After we have validated our bacterial strains and determined theoptimal dosage of the test substance, we began the Ames Assay. In t~eactual experiment, 0.1 ml of the particular strain of Salmonella (10cells) and the specific dilutions of the test substance are added to 2ml of molten top agar, which contained trace amounts of histidine andbiotin. Since survival is better from cultures which have just passedthe log phase, the Salmonella strains are used 16 hours (maximum)after initial inculation into nutrient broth. The dose of the testsubstance spanned a 1000-fold, decreasing from the minimum toxic levelby a dilution factor of 5. All the substances were tested with and

3

without S-9 microsome fraction. The S-9 mixture which was previouslytitered at an optimal strength was added to the molten top agar.After all the ingredients were added, the top agar was vortexed, thenoverlayered on minimum glucose agar plates. These plates contained 2%glucose and Vogel Bonner "E" Concentrate (4). The water used in thismedium and all reagents came from a polymetric system. Plates wereincubated, upside down in the dark at 37 C for 48 hours. Plates wereprepared in triplicate and the average revertant counts were recorded.The corresponding number of revertants obtained was compared to thenumber of spontaneous revertants; the conclusions were recordedstatistially. A correlated dose response is considered necessary todeclare a substance as a mutagen. Commoner (5), in his report,"Reliablilty of Bacterial Mutagenesis Techniques to DistinguishCarcinogenic and Non-Carcinogenic Chemical," and McCann et al (1) intheir paper, "Detection of Carcinogens as Mutagen: Assay of over 300Chemicals," have concurred on the test's ability to detect mutagenicpotential.

Statistical Analysis

Quantitative evaluation was ascertained by the Ames method. Heassumes that a compound which causes twice the reversion rateexperimentally as spontaneously, is mutagenic.

Chemical Analysis

Our information on the chemcial analysis was obtained from Chunand Lam (Appendix A). WR1544 * 2H3PO4 (Chloroquine Phosphate USP) LotAG, Bottle Number AJ20618.

RESULTS AND DISCUSSION

Throughout this report Chloroquine phosphate will be referred toby its respective code number WR1544.

On I October 1981, the Toxicity Level Determination was performedon the test substance. For this experiment, all sterility, strainverification, and negative controls were normal (Table 1). Notoxicity was observed at the highest dose of 1 mg/plate (Table 2).

Two Ames Assays were run to determine conclusively the mutagenicactivity of WR1544. The initial assay performed on 6 October 1981showed normal results to all strain verification and sterilitycontrols (Table 3). An unexpected response was observed for thespontaneous reversion rates taken at the end of the assay. Anunexpected response by TA 100 to positive control chemical dimethylbenzanthracene (DMBA) was also observed (Table 4). In response toWR1544, there were several incidences of no growth and abnormally lowreversion counts associated with TA 98, and TA 1538 (Table 5). Due tothese irregular results, a second assay was run on 22 October 1981.

4

In the second experiment, all sterility and strain verificationcontrols were normal (Table 6). The spontaneous reversion rate andall positive controls were normal except the response of TA 1538 toDIMBA (Table 7). A few scattered incidences of a more than doubling ofthe spontaneous reversion rate were seen in response to the testchemical. These were seen for activated TA 1537 at the 0.2 and 0.04mg/plate dose and nonactivated at the 0.2 mg/plate dose. No doseresponse was seen (Table 8).

CONCLUSION

The Ames Test is able to detect frameshift and basepair mutagenicpotential. Our results show no evidence of such potential.Therefore, on the basis of the Ames test, Chloroquine phosphate, bothin the presence and absence of metabolic activation, is not mutagenicat the levels tested.

RECOMMENDATION

None

5

REFERENCES

1. McCANN, J., E. CHOI, E. YAMASAKI, and B. N. AMES. Detection ofcarcinogens as mutagens in the Salmonella/microsome test: Assayof 300 chemicals. Proc Nat Acad Sci, USA 72:5135-5139, 1975

2. AMES, B. N., J. McCANN and E. YAMASAKI. Methods for detectioncarcinogens and mutagens with Salmonella/mammalian microsome

mutagenicity test. Mutation Res 31: 347-364, 1975

3. LAIR SOP OP-STX-1, Ames Salmonella/mammalian microsomemutagenicity test, 1 March 1981

4. VOGEL, H. J. and D. M. BONNER. Acetylornithinase of E. coli:Partial purification and same properties, J Biol Chem, 218:97-106, 1956

5. COMMONER, B. Reliability of the bacterial mutagenesis techniquesto distinguish carcinogenic and non-carcinogenic chemicals. EPA600/1 76-022, 1976

6

I

Report on Chemical Analysis

APPENDIX A

7

II

'I

I

~: I

I

t

I.

APPENDIX A

Assay of 7-Chloro-4-{ [4-(diethylamino)-l-methylbutyl]-amino}quinoline diphosphate,Chloroquine Phosphate, WR-1544, BG58689

Report No. 35721 June 1979

Contract No. DADA 17-73-C-3171SRI International Project No. 2772

Report A cept d

Byflatse

RECEIVED JAi 1 5 1980

ForHeadquarters, U.S. Army MedicalResearch and Development CommandOffice of the Surgeon General

Washington, D. C. 20314

9

APPENDIX A, continued

Preface

This report was prepared at SRI International, 333 RavenswoodAvenue, Menlo Park, California, 94025, under U.S. Department of theArmy, Contract No. DADA 17-73-C-3171, SRI International ProjectNo. 2772, "Analysis of Organic Chemical Products, InvestigationalNew Drugs and Composition Thereof." This project was supported bythe Division of Experimental Therapeutics, Walter Reed Army MedicalCenter, Walter Reed Army Institute of Research (WRAIR), U.S. ArmyMedical Research and Development Command. Melvin Heiffer, Ph.D.,of the Pharmacology Department is the project monitor.

This work was conducted in the Life Sciences Division byMrs. Jane Chiu, chemical technician and Mr. Allen Benitez,chemist, between 1-14 June 1979.

J nChiu, Chemical Technician

XAllen Benitez, Chemist : /

Peter Lim, Principal Investigator

-a

APPENDIX A, continuedAssay of 7-Chloro-4-([4-(diethylamino)-l-methylbutyl]-amino)quinoline diphosphate,Chloroquine Phosphate, WR-1544, BG58689

Report No. 357

CH3

CH- (CH 2 ) 3N(C 2H5) 2*2H 3 PO,

NH4

00C1 *H 2 6 ClN3.2H 3PO, 515.87

Objective

The objective of this investigation was to confirm the identity and to

establish the purity of the WR-1544, BG58689 sample.

Summarv

On the basis of comparative spectral and chromatographic evidence, theprincipal component in the sample has been verified as chloroquine phosphate.

Based on the USP assay, the sample is 93.9% pure; however, the carbon con-tent of the sample is only 93.3% of the calculated. The sample also contains3.2-3.8% water. Taking into consideration the water (3.5%) and calculating samplepurity on a dried basis, the respective percentages become 97.3 and 96.7. TheU.S. Pharmacopeia XIX requires a dried purity percentage of 98-102.

In addition, the sample contains a water-insoluble, and apparently non-carbonaceous substance (,v 2.9 to 3.5% by difference). Thin-layer chromato-graphic data show four very minor to trace impurities; one is uv-absorbing andthree are fluorescent. The USP chloroquine phosphate reference standard alsoshows four very minor to trace impurities. three of these appeared chromaro-graphically alike to those found in the subject sample.

11

APPENDIX A, continued

Introduction

The sample was received on 21 May 1979 and is covered by a letter fromDr. Melvin H. Heiffer dated 9 May 1979. Experimentals are recorded in Note-book No. 2979.

Exverimentals

A. Tests according to the U.S. Pharmacopeia XIX, page 82.

Descrintion. The sample is odorless, white, and granular.

PH_ (1% solution). 4.9; USP Chloroquine phosphate, 4.8. USP XIX requirespH \, 4.5.

Melting Range (Mel-temp apparatus, 1°C/min, capillary applied at 1839C).

189-209 (dec., corrected)

187-191 (dec.. - -7t ed, USP Chloroquine Phosphate Reference Standard)'

Solubility. Ex,, small portion, the sample is soluble in water.The water-inscli, _-,ion did not dissolve by increasing the water volumeor by heating.

Identificatioo. fhA ultraviolet absorption spectrum (Fig. 1) of a 1 in25,000 solution of dilute hydrochloric acid (1 in 1000) exhibits maximaand minima at t.h: same wavelengths as that of a similar solution of USPChloroquine Phc-,,hate Reference Standard, comcomitantly measured. TheA3& 3/A32, ratio is 1.09, which is exactly. the same for the USP ChloroquinePhosphate Reference Standard. USP XIX gives a ratio range of 1.00-1.15.

Loss on Dr'in% (100 = C, I mm Hg, 5 hours).

3.3% from a 306.3-mg sample

Assay. The sample (100 mg) was dissolved in 5 ml of water with the aid ofgentle sonicarion, and the "solution" was centrifuged to allow the insol-ubles to settle. The supernatant was diluted (1 to 500) with dilutehydrochloric acid (1 in 1000). The diluted solution was assayed by ultra-violet measurements at 343 and 329 rnm.

The results obtained were equated to a reference value, analogouslyderived from USP Chloroquine Phosphate Reference Standard. The reference

'Merck 8th ed. reports chloroquine phosphate to be dimorphic; one form melts193-195", the other melts 215-218*.

12

APPENDIX A, continued

standard purity was corrected to 99.9% because of an impurity (, 0.1%')found by tic. There appear to be three more very minor impurities present,but their quantities could not be estimated because of their fluorescentcharacteristics. The sample purity reported for each wavelength is theaverage of four separate determinations.

XSample Purity

343 93.9, s = 0.9

329 93.8, s = 0.9

Average 93.9, s - 0.9

B. Analyses Not Required by USP XIX

An infrared absorption spectrum (Fig. 2), recorded as a Nujol mull,compares very well with the spectrum of USP Chloroquine Phosphate ReferenceStandard (Fig. 2A).

Proton-magnetic-resonance spectrum (Fig. 3), which was determined asa D20 solution, is qualitatively and quantitatively consistent with thestructure of chloroquine phosphate.

Elemental Analysis%C %H %Cl %N %P

Calcd. for CsH26CIN 3o2H3PO,. 41.91 6.25 6.87 8.15 12.01

Found 39.12 6.06 6.30 7.63 11.01

Percentage of found/calcd. 93.3 97.0 91.7 93.6 91.7

Karl Fischer Water Determination

3.2% from a 71.17-mg sample Average 3.5%

3.8% from a 21.71-mg sampleJ

Thin-Laver Chromatographv

Adsorbent. Commercially prepared SiO 2eGF, Analtech, Inc.(0.025 x 20 x 20 cm)

Quantities spotted. 100, 200, and 500 Ug (100 Vg/pl H20, supernatantfrom centrifuged solution)

Reference samples. USP Chloroquine Phosphate Reference StandardAmmonium phosphate (Baker, lot 8648)

aThis estimation assumes comparable tlc responses from chloroquine and this

impurity.

13

ii

APPENDIX A, continued

Detection. (a) uv (254 and 365 nm)

(b) Iodine vapor

(c) Spray in sequence - 1% ammonium molybdate and1% stannous chloride in 10% HCl

Solvent system (solvent front travelled 13 cm)

MeOH/conc. NHOH, 58% (25/1, v/v)

Results. The sample solutions were prepared, quickly spotted undersubdued light, and the plates were developed in darkened tanks in orderto eliminate or reduce photodecomposition.5

The subject sample was resolved into the following spots: Rfs 0.00(ammonium phosphate; an impurity is indicated by 365 nm light ana byiodine vapor), 0.07 (very minor, detected by 365 nm light on a damp plate;spot disappears when plate dries), 0.27 ( 0.2%), 0.47 (major, WR-1544),and 0.57 (very minor, detected by 365 nm light on a damp plate; spotdisappears when plate dries).

The chromatogram of the USP Chloroquine Phosphate Reference Standarddisplayed the same spots at Rf 0.00 (less intense under 365 nm light thanfor subject sample; does not respond to iodine vapor), 0.27 ("' 0.1%'),0.47 (major, WR-1544), and 0.57. A spot at Rf 0.17 (detected under 365 nmlight on a damp plate; spot disappears when plat2 dries) was novel tothe reference sample.

Isolation of Water-Insoluble Material from Sample

The water-insoluble material that remained after the dissolution of thechloroquine phosphate was collected, washed with water and dried. Its infra-red spectrum (Fig. 4) indicates an inorganic material.

Conclusion

The subject sample is chloroquine phosphate of 93 ± 1% purity.

It also contains 3.5 ± 0.3% water, four very minor to trace unknown impuri-ties indicated by thin-layer chromatographic data, and a water-insoluble,apparently noncarbonaceous material estimated at 3.5% (by difference).

3If these precautions were not exercised, artifacts, appearing as additionalminor components on the chromatogram, would be found.

4Estimation based on visual comparisons acainst known quantities of USP Chloro-quine Phosphate Reference Standard under 254 nm light.

14

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- 115

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APPENDIX A, continued

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19

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1:

LIST OF TABLES

Date Page

Table 1 Strain Verification for Toxicity Level

Determination I Oct 81 22

Table 2 Toxicity Level Determination 1 Oct 81 23

Table 3 Strain Verification Control 6 Oct 81 24

Table 4 Quality and Positive Controls 6 Oct 81 25

Table 5 Salmonella/microsome Assay Worksheet 6 Oct 81 26

Table 6 Strain Verification Control 22 Oct 81 28

Table 7 Quality and Positive Controls 22 Oct 81 29

Table 8 Salmonella/microsome Assay Worksheet 22 Oct 81 30

APPENDIX B

21

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LAIR SOP-OP-STX-1 APPENDIX C

33

LAIR SOP-OP-STX-1 (1 May 1981)

available from LAIR Archives

35

OFFICIAL DISTRIBUTION LIST

CommnderDirectorUS Army Mcdicjl Research and D~evelopment Command Walter Reed Army Institute of ResearchATTN: Sr.RD-RiS/i~rs. Madigan Washington DC 20012Fort Detrick, Frederick.Nil) 21701

Defense Technical Information Center CommanderATTN: DTIC-DDA (12 copies) US Army Medical Research institute of InfectiousCameron Station DiseasesAlexandria VA 22314 Fort Detrick, Frederick MD 21701

Director of Defense Research and Engineering CommanderATTN; Assistant Director, Environmental and US Army Research Institute of Environmental

Life Sciences MedicineWashington DC 20301 Natick MA 01760

The Surgeon General CommanderATTN: DASG-TLO US Army Institute of Surgical ResearchWVashinaton DC 20314 Brooke Army Medical Center

Fort Sam Houston TX 78234

IIQ DA (DASG-ZX..)WASH DC 203 10

Superintendent CommanderAcademv of Health Scicnces US Army Institute of Dental ResearchATTN: AIIS-COM Washington DC 20012Fort Sam Houston TX 78234

Assistant Dean CommanderInstitate and Rcsc.7rch Support US Army Medical IBioenginecringUniformed Services Llniversity- of Hecalth Sciences Research and Development Laboratory6917 Arlington Road Fort Detrick, Frederick AID 21701Bethesda Ml) 20014

Commander CommanderUS Army Environmental Ilxsien e Agency US Army Aeromedical Research LaboratoryAberdeen Proving Ground MD 21070 Fort Rucker AL 36362

US Army Research Office CommanderATTN: Chemical and Bliolo,,ical Sciences Division US Army Biomedical LaboratoryP.O. Pox 1221 Aberdeen Proving GroundResearch Triangle Park NC 27709 Edgewood Arsenal AID 21010Biological Scienccs D~ivision CommanderOffice of Naval Research -Naval Medical Research InstituteArlington VA 22217 National Naval Medical Center

Bethesda AID 20014Director of Life Sciences CommanderUSAF Office of Scientific Research (AFSC) USAF School of Aerospace MedicineBolling AFB Aerospace Medical DivisionWashington DC 20332 Brooks Air Force Base TX 78235

36

I ATE