Afl-A203 827

tIn.;titiite Report No. 322

Mutagenic Potential of2-(E)-HydroxyiminomethyI-l-methoxymethy1-3-

methylimidazolium Chloride in the AmesSal1ionell/a/Mammalian Microsome Mutagenicity Test

Steven K. Sano, BA, SGT, USAand

Don W. Korte, Jr., PhD, MAI, MSC

GENETIC TOXICOLOGY BRANCH O% LET

DIVISION OF TOXICOLOGY JAN 2 6198q

November 1988 Toxicology Series: 122

A! ,w for publIc roI.'wcvm;

LETTERMAN ARMY INSTITUTE OF RESEARCHPRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

Mutiagenic Potential of 2-(E)-Hydroxyiminometiyl-l.rnethoxymethyl.3.methylimidazoliumChloride in the Ames Salmonella/Mammalian Microsome Mutagenicity Test (ToxicologySeries 122)--Sano and Korte

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11. TITE IcueScurfty Classification) (U) Mutagenic Pot ial of 2-(E)-Hydroky-iminomethyl-l-methoxymethyl-3-methylimidazoli Chloride in the Ames

qa~nn~ja/ammlia Mirosme Muta nincitv est12. PERSONAL. AUTHOR(S)

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FIELI GROUP ISUB-GROUP Mutagenicity, Genetic Toxicology, Ames Test,2- (E) -Hydroxyiminomethyl-1-methoxymethyl-3- ~

-meth limidazoliukchloridei Oxime. lrA'dctzl c/r, Owl -i19~ ABST(Continue on reverse if necessary and identify by block number)

The mutagenic potential of 2-(E)-HYDROXYIMINOMETHYL-1-METHOXYMETHYL-3-METHYLIMIDAZOLIUN CHLORIDE was assessed by using the Ames SalmonellalMarmalianMicrosome Mutagenicity Test. Tester Strains TA98, TA100, TA1535, TA1537, andTA1538 were exposed to doses ranging from 5.0 mg/plate to 0.0016 mg/plate.The test compound was not mutagenic under conditions of this test.

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ABSTRACT

The mutagenic potential of 2-(E)-HYDROXYIMINOMETHYL-1-METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE was assessed byusing the Ames Salmonella/Mammalian Microsome MutagenicityTest. Tester strains TA98, TAI00, TA1535, TA1537, and TA1538were exposed to doses ranging from 5.0 mg/plate to 0.0016mg/plate. The test compound was not mutagenic underconditions of this test.

Key Words: Mutagenicity, Genetic Toxicology, Ames Test, 2-(E)-HYDROXYIMINOMETHYL-1-METHOXYMETHYL-3-METHYLIMIDAZOLIUMCHLORIDE, Oxime

Accession ForNTIS GRA&IDTIC TAB 5Unannounced 0Justification

ByDistribution/Availability Codes

Avlfli aud/or

r 3t 1 ,-;pecial

i

PREFACE

TYPE REPORT: Ames Test GLP Study Report

TESTING FACILITY:

US Army Medical Research and Development CommandLetterman Army Institute of ResearchPresidio of San Francisco, CA 94129-6800

SPONSOR:

US Army Medical Research and Development CommandWalter Reed Army Institute of ResearchWashington, D.C. 20307-5100Project Officer: H.A. Musallam

PROJECT/WORK UNIT/APC: 3M162734A875/308/TLEO

GLP STUDY NUMBER: 85007

STUDY DIRECTOR: MAJ Don W. Korte, Jr., PhD, MSC

PRINCIPAL INVESTIGATOR: Steven K. Sano, BA, SGT, USA

REPORT AND DATA MANAGEMENT:

A copy of the final report, study protocol, retiredSOPs, stability and purity data on the test compound,and an aliquot of the test compound will be retained inthe LAIR Archives.

TEST SUBSTANCE: 2-(E)-HYDROXYIMINOMETHYL-I-METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE

INCLUSIVE STUDY DATES: 25 February 1985 - 22 March 1985

OBJECTIVE:

The objective of this study was to determine themutagenic potential of 2-(E)-HYDROXYIMINOMETHYL-I-METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE (LAIR CodeTP50) by using the Ames Salmonella/Mammalian MicrosomeMutagenicity Test.

iii

ACKNOWLEDGMENTS

MAJ John W. Harbell, PhD, MSC, and Mr. John Daceyprovided scientific guidance and research assistance.

iv

SIGNATURES OF PRINCIPAL SCIENTISTS INVOLVED IN THESTUDY

We, the undersigned, declare that GLP Study 85007 wasperformed under our supervision, according to the proceduresdescribed herein, and that this report is an accurate recordof the results obtained.

A 1/W A- 3DON W. KORTE JR, P I DATEMAJ, MSCStudy Director

STEVEN K. SANV BA / DATESGT, USAPrincipal Investigator

CONRAD WHEELER, PhD/ DATEDACAnalytical chemist

v

DEPARTMENT OF THE ARMY

LETTERMAN ARMY INSTITUTE OF RESEARCHPRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129-800

REPLY TO

ATTENTION OF:

SGRD-ULZ-QA 3 December 198R

MEMORANDUM FOR RECORD

SUBJECTz GLP Statement of Compliance

1. This is to certify that the protocol for GLP Study 85007 w~sreviewed on 21 February 1985.

2. The institute report entitled "Mutagenic Potential of 2-(E)-Eydroxyiminomethyl-l -methoxymethyl-3-methylimidazolium Chloridein the Ames Salmonella/Mammalian Microsome Mutagenicity Test,"Toxicology Series 122, was audited on 14 November 1988.

CAROLY M. LEWIS, MSDiplomate, American Board of ToxicologyChief, Quality Assurance

vi

TABLE OF CONTENTS

Abstract .................................................. i

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

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

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

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

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

INTRODUCTION .............................................. 1

Objective of the Study ............................... 1

MATERIALS AND METHODS ..................................... 1

Test Compound.........................................1Test Solvent. ......................................... 2Chemical Preparation ................................. 2Test Strains ......................................... 2Test Format ......................................... 2Data Interpretation .................................. 4Devibtions from the Protocol/SOP ..................... 4Storage of the Raw Data and Final Report ............. 4

RESULTS ................................................... 5

DISCUSSION ................................................ 5

CONCLUSION ............................................... 10

REFERENCES ............................................... 11

APPENDICES ............................................... 12

Appendix A: Chemical Data .......................... 13Appendix B: Individual Plate Scores ................ 15

OFFICIAL DISTRIBUTION LIST ............................... 20

vii

Mutagenic Potential of 2-(E) -BYDROXYIMINOMETBYL-l-MZTNOXYMETHYL-3-METHYLIKIDAZOLIUM CHLORIDE in the AmesSalmonella/Mammalian Microsome Mutagenicity Test--Sanoand Korte

INTRODUCTION

2-(E)-HYDROXYIMINOMETHYL-1-METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE was synthesized for a UnitedStates Army Medical Research and Development Command programcharged with developing more effective oximes for treatmentof nerve agent poisoning. The Ames Test is one of a seriesof tests in which these compounds will be evaluated todetermine their relative potential for further development.

The Ames Salmonella/Mammalian Microsome MutagenicityTest is a short-term screening test that utilizes histidineauxotrophic mutant strains of Salmonella typhimurium todetect compounds that are potentially mutagenic in mammals.A mammalian microsomal enzyme system is incorporated in thetest to increase sensitivity by simulating in vivo metabolicactivation of the test compound. The Ames Test is aninexpensive yet highly predictive and reliable test fordetecting mutagenic activity and thus carcinogenic potential(1).

Objective of the Study

The objective of this study was to determine themutagenic potential of 2-(E)-HYDROXYIMINOMETHYL-l-METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE (LAIR Code TP50)by using the revised Ames Salmonella/Mammalian MicrosomeMutagenicity Test.

MATERIALS AND METHODS

Test Compound

Chemical Name: 2-(E)-HYDROXYIMINOMETHYL-I-METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE

LAIR Code Number: TP50

Physical State: Colorless prisms

Sano and Korte--2

Source: SRI International, Menlo Park, CA

Storage: 2-(E)-HYDROXYIMINOMETHYL--METHOXYE THYL-3-METHYLIMIDAZOLIUM CHLORIDE was received from SRIInternational, 333 Ravenswood Ave., Menlo Park, CA 94025 andassigned the LAIR Code number TP50. The test compound wasstored in a desiccator at 50C until used.

Chemical Properties/Analysis; Data provided by SRIInternational characterizing the chemical composition andpurity of the test material are presented in Appendix A alongwith confirmatory analysis of the test material performed bythe Division of Toxicology, LAIR (Presidio of San Francisco,CA).

Test Solvent

The positive control chemicals were dissolved in gradediriethyl sulfoxide (lot 113F-0450) obtained from SigmaChemical Co. (St. Louis, MO). The test compound wasdissolved in sterile deionized water obtained from aPolymetric model 200-3 Water Purifier (Sunnyvale, CA).

Chemical Preparation

On the day of dosing, 300 mg of the test compound wasmeasured into a sterile vial and dissolved in 6 ml of steriledeionized water to achieve a 5% (w/v) solution. Aliquots ofthis solution were used to dose the test plates.

Test Strains

Salmonella strains TA98, TAI00, TA1535, TA1537, and1'A1538 obtained directly from Dr. Bruce Ames, University ofCalifornia, Berkeley, were used. These strains weremaintained in our laboratory in liquid nitrogen. Qualitycontrol tests were run concurrently with the test substanceto establish the validity of their special features and todetermine the spontaneous reversion rate. Descriptions ofthe strains, their genetic markers, and the methods forstrain validation are given in the LAIR SOP, OP-STX-I (2).

Test Format

2-(E)-HYDROXYIMINOMETHYL-1-METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE was evaluated for mutagenicpotential according to the methods of Ames et al (3). Adetailed description of the methodology is given in LAIR SOP,OP-STX-l (2).

Sano and Korte--3

Toxicity Tests:

Toxicity tests were conducted to determine a sublethalconcentration of the test substance. This toxicity level wasfound by using minimal glucose agar (MGA) plates,concentrations of 2-(E)-HYDROXYIMINOMETHYL-1-METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE ranging from 1.6 x 10- 3 mg/platet-j 5 mg/plate, and approximately 108 cells of TAI00 perplate. Top agar containing trace amounts of histidine andbiotin was placed on the plates. Strain verification wasconfirmed on the bacteria, along with a determination of thespontaneous reversion rate. After incubation, the growth onthe plates was observed. Since none of the plates showed adecreased number of macrocolonies (below the spontaneousrate) or an observable reduction in the density of thebackground lawn, the highest dose selected for themutagenicity test was 5.0 mg/plate.

Mutagenicity Test:

The test substance was evaluated over a 1000-fold rangeof concentrations, decreasing from the minimum toxic level(the maximum or limit dose) by a dilution factor of 5, bothwith and without 0.5 ml of the S-9 microsome fraction. TheS-9 (batch R-315) was purchased from Litton Bionetics(Kensington, MD). The optimal titer of this S-9, asdetermined by Litton Bionetics, was 0.75 mg protein/plate.After all the ingredients were added, the top agar was mixed,then overlaid on MGA plates. These plates contained 2%glucose and Vogel Bonner "E" concentrate (4). Plates wereincubated upside down in the dark at 370C for 48 hours.Plates were prepared in triplicate, and the average revertantcounts were recorded. The average number of revertants ateach dose level was compared to the average number ofspontaneous revertants (negative control). The spontaneousreversion rate (with and without S-9) was monitored byaveraging the counts from two determinations runsimultaneously with the test compound. The spontaneousreversion rate was determined by inoculating one set ofplates before and one set after the test compound plates sothat any change in spontaneous reversion rate during thedosing procedure would be detected. This spontaneousreversion rate was also compared with historical values forthis laboratory and those cited in Ames et al (3). Sterilityand strain verification controls were run concurrently. Allreagents, test compounds, and media were checked forsterility by plating samples of each on MGA media andincubating them at 370C with the test plates. The integrity

Sano and Korte--4

of the different Salmonella strains used in the assay wasverified by the following standard tests:

-Lack of growth (inhibition) in the presence of crystal violetwhich indicates that the prerequisite alteration of thelipopolysaccharide layer (LP) of the cell wall is present.

-Growth in the presence of ampicillin-impregnated diskswhich indicates the presence of an ampicillin-resistant RFactor in the TA98 and TA100 strains.

-Lack of growth (inhibition) following exposure toultraviolet light which indicates the absence of the DNAexcision-repair mechanism.

Four known mutagens were tested as positive controls toconfirm the responsiveness of the strains to the mutationprocess. Each strain must be tested with at least onepositive control but may be tested with several. Thesecompounds (benzo[alpyrene, 2-aminofluorene, 2-aminoanthracene,and N-methyl-N'-nitro-N-nitrosoguanidine) were obtained fromSigma Chemical Co. (St. Louis, MO). The test compound andmutagens were handled during this study in accordance with thestandards published in NIH Guidelines for the Laboratory Useof Chemical Carcinogens (DHHS Publication No. (NIH) 81-2385,May 1981).

Data Interpretation

According to Brusick (5), a compound is consideredmutagenic if a positive dose response (correlated doseresponse) over three dose concentrations is achieved with atleast the highest dose yielding a revertant colony countgreater than or equal to twice the spontaneous colony countfor the tester strains TA98 and TA100, or three times thespontaneous colony count for strains TA1535, TA1537, andTA1538. A strong correlated dose response in strain TA100without a doubling of the individual colony count may also beconsidered positive.

Deviations from the Protocol/SOP

There were no deviations from the protocol or standardoperating procedures.

Storage of the Raw Data and Final Report

A copy of the final report, study protocols, raw data,SOPs, and an aliquot of the test compound will be retained inthe L:IR Archives.

Sano and Korte--5

RESULTS

On 8 March 1985, the toxicity of 2-(E)-HYDROXYIMINOMETHYL-1-METHOXYMETHYL-3-METHYLIMIDAZOLIUMCHLORIDE was determined (Table 1). For this experiment allsterility, strain verification and negative controls werenormal (Table 1). Exposure of the tester strain (TA100) tothe highest dose showed neither a decrease in the number ofmacrocolonies nor an observable reduction in the density ofthe background lawn. Therefore, the highest dose selectedfor the mutagenicity test was 5.0 mg/plate. Normal resultswere obtained for all sterility and strain verification testsduring the Ames Test performed on 11-14 March 1985 (Table 2).2-(E)-HYDROXYIMINOMETHYL-1-METHOXYMETHYL-3-METHYLIMIDAZOLIUMCHLORIDE did not induce any appreciable increase in therevertant colony counts relative to those of the negativecontrol cultures (Table 3). A tabular presentation of theraw data is included in Appendix B.

DISCUSSION

Certain test criteria must be satisfied before an AmesTest can be considered a valid assessment of a compound'smutagenic potential. First, the special features of the Amesstrains must be verified. These features includedemonstration of ampicillin resistance, alterations in the LPlayer, and deficiency in DNA excision-repair (except TA102).Second, the Salmonella strains must be susceptible tomutation by known mutagens. Third, the optimal concentrationof the test compound must be determined by treating TAI00with a broad range of doses and observing the potential toxiceffects on formation of macrocolonies and microcolonies. Ifthese tests are performed and expected data are obtained,then the results of an Ames Test can be considered valid.

After validation of bacterial strains and selection ofoptimal sublethal doses, 2-(E)-HYDROXYIMINOMETHYL-l-METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE was evaluated inthe Ames Test. Criteria for a positive response include botha correlated dose response over three dose concentrations,and a revertant colony count at least two times (TA98, TAI00)or three times (TA1535, TA1537, TA1538) the spontaneousrevertant colony count (5). 2-(E)-HYDROXYIMINOMETHYL-I-METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE did not induce therequisite dose-response relationship or the increase inrevertant colony counts necessary for a positive response.Thus, the results of this test indicate that 2-(E)-HYDROXYIMINOMETHYL-1-METHOXYMETHYL-3-METHYLIMIDAZOLIUMCHLORIDE is not mutagenic when evaluated in the Ames Test.

Sano and Korte--6

TABLE 1: TOXICITY LEVEL DETERMINATION FOR TP50

GLP STUDY NUMBER 85007

TOXICITY DETERMINATION REVERTANT PLATE COUNT (TAI00)

CONCENTRATION EAN +D ACKGROUND LAWN*

5.0 mg/plate 108 31.6 NL1.0 mg/plate 112 12.8 NL0.2 mg/plate 122 5.9 NL0.04 mg/plate 121 13.2 NL0.008 mg/plate 127 14.0 NL0.0016 mg/plate 122 8.5 NL

STRAIN VERIFICATION FOR TOXICITY DETERMINATION

TA10O*

HISTIDINE REQUIREMENT NGAMPICILLIN RESISTANCE GUV NGCRYSTAL VIOLET SENSITIVITY NGSTERILITY CONTROL NG

STERILITY CONTROL FOR TOXICITY DETERMINATION

MATERIAL TESTED BSF I*MINIMAL GLUCOSE AGAR PLATES NGTOP AGAR NGDILUENT WATER NGNUTRIENT BROTH NGTEST COMPOUND (HIGHEST DOSE) NG

*NL=Normal Lawn, G=Growth, NG=No Growth

Sano and Korte--7

TABLE 2: STRAIN VERIFICATION AND STERILITY TESTINGFOR THE MUTAGENICITY DETERMINATION OF TP50

GLP STUDY NUMBER 85007

STRAIN VERIFICATION

OBSERVATIONS*

HISTIDINE AMPICILLIN UV CRYSTAL STERILITYSTRAIN REOUTREMENT RES.TA±NCE REPAIR WOTLT CONTROL

TA98 NG G NG NG NGTAI00 NG G NG NG NGTA1535 NG NG NG NG NGTA1537 NG NG NG NG NGTA1538 NG NG NG NG NG

STERILITY CONTROL FOR MUTAGENICITY DETERMINATION

MATERIAL TESTED OBSERVATION*

MINIMAL GLUCOSE AGAR PLATES NGTOP AGAR NGDILUENT WATER NGNUTRIENT BROTH NGTEST COMPOUND (HIGHEST DOSE) NGS-9 NG

*G = Growth, NG = No Growth

Sano and Korte--8

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CONCLUSION

2-(E)-HYDROXYIMINOMETHYL--METHOXYMETHYL-3-METHYLIMIDAZOLIUM CHLORIDE was evaluated for mutagenic

potential in the Ames Test, in both the presence and the

absence of metabolic activation, and did not induce a

positive mutagenic response under conditions of this study.

Sano and Korte--li

REFERENCES

1. McCann J, Choi E, Yamasaki E, Ames BN. Detection ofcarcinogens as mutagens in the Salmonella/MammalianMicrosome Mutagenicity Test: Test of 300 chemicals.Proc Nat Acad Sci, USA 1975;72:5135-5139.

2. Ames Salmonella/Mammalian Microsome Mutagenesis Test.LAIR Standard Operating Procedure OP-STX-1, Presidio ofSan Francisco, California: Letterman Army Institute ofResearch, 29 August 1986.

3. Ames BN, McCann J, Yamasaki E. Methods for detection ofcarcinogens and mutagens with Salmonella/MammalianMicrosome Mutagenicity Test. Mutat Res 1975;31:347-364.

4. Vogel HJ, Bonner DM. Acetylornithinase of E. coli:Partial purification and some properties. J Biol Chem1956;218:97-106.

5. Brusick D. Genetic toxicology. In: Hayes AW, ed.Principles and methods of toxicology. New York: RavenPress, 1982:223-272.

Sano and Korte--12

APPENDICES

APPENDIX A: Chemical Data....................................13

APPENDIX B: Individual Plate Scores......................... 15

Sano and Korte--13

APPENDIX A: Chemical Data

Chemical Name: 2-((hydroxyimino)methyl)-1-methoxymethyl)-3-methyl-lH-imidazolium chloride

Alternate Chemical Names: 2[(Hydroxyimino)methyl]l-1-(methoxymethyl)-3-methylimidazolium chloride,2-(E)-Hydroxyiminomethyl-l-methoxymethyl-3-methylimidazolium chloride

Chemical Abstracts Service Registry No: 91900-09-3

LAIR Code Number: TP50

Chemical Structure:

/CH3

N

ICH20CH 3

Molecular Formula: C7H12C1N302

Molecular Weight: 205.5

Source: Clifford D. Bedford, PhDSRI International, Physical Sciences DivisionMenlo Park, CA

SRI Reference Number: BHH-0001

Sano and Korte--14

APPENDIX A (cont.): Chemical Data

Analytical Data: Data supplied by SRI International includedmelting point, elemental analysis, and NMR and IR spectra.1IMelting point: 168-172"C (dec) . Elemental analysiscalculated for C7H12C1N302: C, 40.88; H, 5.88; N, 20.43; Cl,17.24. Found: C, 41.00; H, 6.01; N, 20.67; Cl, 17.02. 1HNMR (60 MHz, d6-DMSO) 8 3.37 (s, 3H, OCH3), 4.03 (s, 3H,NCH3), 5.77 (s, 2H, NCH20), 8.08 (d, 1H, J=1.5 Hz, aryl),8.17 (d, 1H, J=l.5 Hz, aryl), 8.62 (s, 1H, CHN), 13.45 (s,1H, NO) . IR (KBr) 2700 (broad), 1590, 1525, 1420, 1395,1320, 1300, 1270, 1240, 1205, 1120, 1060, 1005, 925, 875, 790cm- I . The IR spectrum obtained upon receipt of the compoundconfirmed the identity of the material.

2

1 Bedford CD. Notebook reference 5337-15. Stanford Researchinternational, Physical Sciences Division, Menlo Park, CA.

2 Wheeler CR. Nitrocellulose-Nitroguanidine Projects.Laboratory Notebook #84-05-010.3, p18. Letterman Armyinstitute of Research, Presidio of San Francisco, CA.

Sano and Korte--15

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OFFICIAL DISTRIBUTION LIST

Commander CommanderUS Army Medical Research US Army Medical Bioengineering Research

& Development Command and Development LaboratoryATTN: SGRD-RMS/Mrs. Madigan ATTN: SGRD-UBG-MFort Detrick, MD 21701-5012 Fort Detrick, Bldg 568

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ATTN: LibraryOffice of Under Secretary of Defense Fort Detrick, Bldg 568

Research and Engineering Frederick. MD 21701-5010ATT'N: R&AT (E&LS). Room 3D129The Pentagon CommanderWashington, DC 20301-3080 US Army Research Institute

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