AD A 4555 THE MUTAGENIC P0ENTIAL 0FF )-234-TETRAHYORO) -6-ME THY -2-METHY IOXO..U)LE ERM AN ARMY INS 0 O R ESEAR CH PR E SI00 OF SAN

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MICROCOPY RESOLUTION TEST CHARTNATIONAL BUREAU OF STANDARDS-I963-A

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IA 124555

INSTITUTE REPORT NO. 136

THE %lUTAGENIC POTENTIAL OF:

(E)-1,2,34-tradwd-6-metyl--12nwMhyl-1 oxo-2-buiyl)quinolkiw (CHR 5)

LEONARD J. SAUERS, BA, SP.5andJOHN T. FRUIN, DVM, PhD, COL VC

TOXICOLOGY GROUP,

DIVISION OF RESEARCH SUPPORT

DTICNOVEMBER 7962N

LETTERMAN ARMY INSTITUTE OF RESEARCH 118'.. PRESIDIO OF SAN FRANCISCO. CALIFORNIA 94129 E

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Toxicology Series 37 -- Sauers and Fruin

Reproduction of this document in whole or in pant is prohibited except with Ihe kewmio o( theC. mmander, Lenerma Army institute of Research, Presidio of San Francui o, CaWfomia 94129.However, the De sm Technical Infonmation Center is authorized to reproduce the document forUnited Staes Government purposes.

Destroy this repot when it is no lonet needed. Do not return it to the originator.

Otationo( trade names in this report does not constitute an official endorsement or approval of theuse of h itenu

This material has been reviewed by Letterman Army Instituteof Research and there is no objection to it pre tation an/or publication. The opinions or amrtions contained hereinae the private views of the authot(s) and anre not to be con-strued a official or as reflecting the views of the Departmentof the Anmy or the Department of Defense. (AR 3 5)

Thi document has been approved for public -oels and sale; its distribution is umlkmitd

UNCLASSIFIEDSECtUITY CLASSIFICATION OF THIS PAGE Men Date Rfnteu.

REPORT DOCUMENTATION PAGE READ Wrn c 70MBEFORtE COMPIL IIG FORM

1. =PONT NUMBER 2. GOVT ACCESSION NO S. RECIPIENT*S CATALOG NUMBER

JLIR Institute Report No. 136 Lp_ ___4___2_!_____

4. TITLE (ME "d10) S. TYPE OF REPORT & PERIOD COVERED

The Mutagenic Potential of (E)-l,2,3,4-tetrahydro- Final6-methyl-l-(2-methyl-l-oxo-2-butenyl) quinoline 21 April - 21 May 1982

(CHR 5) 6. PERFORMING ORO. REPORT WUNDER

7. AUTN!I 0) S. CONTRACT OR GRANT NUMUER(S)

LeonaJ'rd J. Sauers, BA, SP5John T. Fruin, DVM, PhD, COL VC9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT. PROJECT. TASK

AREA & WORK UNIT NUMBERS

US Army Medical Research and Development Command

Fort Detrick, MD 21201 & Tox Grp, Div of Rsch Spt, Project 3M162779A871LAIR. Presidio of San Francisco. CA 9412911. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE

US Army Medical Research and Development Command November 1982

Fort Detrick IS.NUMBER OF PAGES

S 1701294MONITOING AGENCY NAME & ADDRESS(fi EdUtimt hrom Controfiil Offles) IS. SECURITY CLASS. (of this mpolf)

UNCLASSIFIED

ISO. DECLASSIFICATION/DOWNGA4ADINGSCHEDULE

1. DiSTRIUUTION STATEMENT (of this Ropef)

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

17. DISTRIBUTION STATEMENT (of mho oabgat ontomd In Block 20, If t ront hoe Reoprt)

11. SJPPLEIIiENTARY NOTES

19. KEY WORDS (Cmtha amm rere~e aide it necesely and lietify by Meek easer)

Mutagenicity, Toxicology, Ames Assay, (E)-1,2,3.4-tetrahydro-6-methyl-1-(2-methyl-l-oxo-2-butenyl) quinoline

20 ASITlAcr ( m m @60; N nemuuem mI IE l - 3g.by Mk N moe )

4he mutagenic potential of (E)-1,2,3,4-tetrahydro-6-methyl-1-(2-methyl-1-oxo-2-butenyl) quinoline (CHR 5) was assesed by using the Ames salmonella/Mawalian Microsome Mutagenicity Assay. Tester strains TA 98, TA 100, TA 1535,

TA-37 and TA 1538 were exposed to doses ranging from mg/plate to 3.2 x I0 mg/plate. It was determined that the test substanoe)did not have mutagenicpotential. E-- - .1

D W7 M f mow F I v i sD LeT a UNCLASSIFIED

SECITY CLASIFICATION OF THIS PAGE (Nim Data it.re0

|

ABSTRACT

The mutagenic potential of (E)-1,2,3.4-tetrahydro-6-methyl-1-(2-methyl-l-oxo-2-butenyl) quinoline (CHR 5) was assessed by using theAmes Salmonella/Mammalian Microsome Mutagenicity Assay. Tester

strains TA 98, TA 100, 1TA 1535, TA 1537 and T1 1538 were exposed todoses ranging from 10- mg/plate to 3.2 x 10- mg/plate. It wasdetermined that the test substance did not have mutagenic potential.

Accession For

NTIS GRA&IDTIC TAB

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UnannouncedJustification

0ByDistribution/

Availability Codes

Avail and/orist Special

COOV

PREFACE

TYPE REPORT: Ames Assay GLP Study Report

TESTING FACILITY: Letterman Army Institute of ResearchPresidio of San Francisco, CA 94129

SPONSOR: Same as above

PROJECT: 3M162779A871, Development of Repellents Against MedicallyImportant Arthropods, WU 201, APC TLO1

GLP STUDY NUMBER: 82012

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

PRINCIPAL INVESTIGATOR: SP5 Leonard J. Sauers, BA

RAW DATA AND DATA MANAGEMENT: A copy of the final report, retiredSOPs, raw data, and chemical, analytical, stability, andpurity data of the test compound will be retained in theLAIR Archives.

TEST SUBSTANCE: (E)-1,2,3,4-tetrahydro-6-methyl-1-(2-methyl-1-

oxo-2-butenyl) quinoline (CHR 5)

INCLUSIVE STUDY DATES: 21 April - 21 May 1982

OBJECTIVE: To determine the mutagenic potential of the abovecompound using the Ames Assay. Tester strains TA 98,TA 100, TA 1535, TA 1537 and TA 1538 were used. Theplate incorporation method was followed. The testsubstance was dissolved in ethanol and this diluentwas checked for sterility.

|IH

71NL.

ACKNOWLEDGMENTS

Trhe authors Wish to thank PFC Paul lMauk, 8S; Carolyn Lewis, MS;and John Dacey for their assistance in performing the research.

iv

Signatures of Principal Scientists involvedin 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.

-fiftw - -- - -/ VOW PL NADIJOHN T. FRUIN, DVM., PhD/DATE

COL, VCPrincipal Investigator Study Director

. V

DEPARTMENT OF THE ARMYLETTERMAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

'REPLY TO

ATTENTION OF:

SGRD-ULZ-QA 9 Aug 82

MEMORANDUM FOR RECORD

SUBJECT: Report of GLP CompI lance

I hereby certify that In relation to LAIR GLP study #82012 the followingInspections were made:

21 Apr 8223 Apr 82

The report and raw data for this study were audited on 5 Aug 82.

Routine inspections with no adverse findings are reported quarterly, thus theseInspections are also included in the 7 July 82 report to management and the StudyDirector.

N .JO SONCPT, MSQuality Assurance Officer

Vi

II

S t

TABLE OF CONTENTS

Abstract... ........................................................................... £

Preface.......................................................................

Aocnowledgiments ................................................ 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...................................... 4

Chemical Analysis......................................... 4

RESULTS ...................................................... 4

DISCUSSION........................................... o........ 4

CONCLUSIONS .................................................. 4

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

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

APPENDICES

Appendix A (Chemical Analysis).................................... 7

Appendix B (Tables 1 through 5) ................................. 13

DISTRIBUTION LIST . . .. . .. .. . ... . .. . . ................................... .. .21

vii

THE MUTAGENIC POTENTIAL OF: (E)-1,2,3,4-ttrahydro-6-ethyl-l-(2-rmthyl-l-oxo-2-butenyl)quineiw (CHR 5) - Sus and Fruin

Rationale for using the Ames Assay

The Ames Salmonella/Mamalian Microsome Mutagenicity Test is oneof a standard bank of tests used by our laboratory for the assessmentof the mutagenic potential of a test substance. It is a short-termscreening assay, which we use for the prediction of potentialmutagenic agents in mammals. It is inexpensive when compared to invivo tests, yet is highly predictive and reliable in its ability todetect mutagenic activity and therefore carcinogenic probability (1).It relies on basic genetic principles and allows for the incorporationof a mammalian microsomal enzyme system to Increase sensitivitythrough enzymatically altering the test substance into an activemetabolite. 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, two othermutations 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 enzymaticprocess, 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 mialan system. This activated rat liver microsomal enzymehomogenate is termed S-9.

. ... _ m . .. _ I I_ I

__ __ _ __ _ __ _ __ _ __ _ __ _ _ _

Description of Strains (History of the strains used, method tomonitor the integrity of the organisms, and data p-rtaining 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 %P 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 occur because the LP layer is altered. Theabsence of excision repair mechanisms can be determined by usingultraviolet (UY) 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 largar number ofrevertants are obtained, then the bacteria are 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 by 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. These records are keptin the archives of the Quality Assurance Unit.

In this series of tests for the detection of mutagenic potentialof different agents, we compare the spontaneous reversion values withour own historical values and these cited by Ames et al (2). Ourconclusions are based on the spontaneous reversion rate compared tothe experimentally induced rate of mutation. When operating

i2

effectively, these strains detect substances that cause base pairmutations (TA 1535, TA 100) and frameshift mutations (TA 1537, TA1538, and TA 98).

METHODS (3)

Rationale for Dosage Levels and Dose Response Tabulations

To insure readable and reliable results, a sublethal concentrationof the test substance had to be determined. This toxicity level wasfound by using MGA piates, various concentrations of the substance,and approximately 10 cells of TA 100 per plate, unless otherwisespecified. Top agar containing trace amounts of histidine and biotinwere placed on MGA plates. TA 100 is used because it is the mostsensitive strain. Strain verification was confirmed on the bacteria,along with a determination of the spontaneous reversion rate. Afterincubation, the growth was observed on the plates. (The auxotrophicSalmonella will replicate a few times and potentially express amutation. When the histidine and biotin supplies are exhausted, onlythose bacteria that reverted to the prototrophic phenotype willcontinue to reproduce and form macrocolonies; the remainder of thebacteria comprises the background lawn. The minimum toxic level isdefined as the lowest serial dilution at which decreased macrocolonyformation, below that of the spontaneous revertant rate, and anobservable reduction in the density of the background lawn occurs). Amaximum dose of 1 mg/plate is used when no toxicity is observed. Thedensities were recorded as normal, slight, and no growth.

Test Format

After we 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 inoculation into nutrient broth. The dose of the testsubstance spanned a 10uO-fold, decreasing from the minimum toxic levelby a dilution factor of 5. All the substances were tested with andwithout S-9 microsome fraction. The optimal titer of the S-9 wasdetermined by the supplier, and 0.5 ml was added to the molten topagar. After all the ingredients were added, the top agar was mixed,then overlaid on minimum glucose agar plates. These plates contained2% glucose and Vogel Bonner "E" Concentrate (4). The water used inthis medium and all reagents came from a polymetric system. Plateswere incubated, upside down in the dark at 37 C for 48 hours. Plateswere prepared in triplicate and the average revertant counts wererecorded. The corresponding number of revertants obtained wascompared to the number of spontaneous revertants; the conclusions wererecorded statistically. A correlated dose response is considered

3

necessary to declare a substance as a mutagen. Commoner (5), in hisreport, "Reliablilty of Bacterial Mutagenesis Techniques toDistinguish Carcinogenic and Non-Carcinogenic Chemical," and McCann etal (1) in their paper. "Detection of Carcinogens as Mutagen in theSalmonella/Mamallian Microsome Mutagenicity ' Assay of over 300Chemicals," have concurred on the test's abil,.y to detect mutagenicpotential.

Statistical Analysis

Quantitative evaluation was ascertained by the method of Ames (2).He assumed that a compound which causes twice the spontaneousreversion rate and a correlated dose response is mutagenic

Chemical Analysis

Our information on the chemical analysis of CHR5 was obtained fromStarks Associates (Appendix A).

RESULTS AND DISCUSSION

Throughout this report,(E)-1,2,3,4-tetrahydro-6-methyl-]-(2-methyl-]-oxo-2-butenyl) quinolinewill be referred to by its respective code name, CHR 5.

On 21 April 1982, the toxicity level determination was performedon the test compound. All sterility, strain verification and negativecontrols were normal (Table 1). toxic response Was observed at thehighest dose used; therefore, 10 mg/plate was designated as theinitial dose for the assay (Table 2).

On 23 April 1982, the Ames Assay was performed on CHR 5. Allstrain verification and sterility controls were normal for thisexperiment (Table 3). Expected results were obtained for all positiveand negative controls (Table 4). The bacterialjtrains were exposedto doses ranging from 10 mg/plate to 3.2 x 10 mg/plate of testsubstance. In no case was a dose response or a doubling of thespontaneous reversion rate observed (Table 5).

CHR 5 was tested previously (LAIR Institute Report 109). At thattime, a solution of an unknown concentration was assayed. The Amestest was repeated due to the new lot and controlled concentration.

CONCLUSION

Based on the Ames Assay, CHR 5 is not mutagenic at the levelstested.

4

RECOMMENIDATION

We recommend that candidate insect repellent CHR 5 be tested

further with other toxicological assays if efficacy tests show this

comWound to be a promising repellent.

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

3. LAIR SOP OP-STX-1, Ames Salmonella/mammalian microsomemutagenicity test, 15 February 1982

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

Chemical Analysis APPENDIX A

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DATA WENTT FOE COMPOUNDS

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LIST OF TABLES

Date Page

Table 1 Strain Verification for Toxicity Level

Determination 21 Apr 82 15

Table 2 Toxicity Level Determination 21 Apr 82 16

Table 3 Strain Verification Control 23 Apr 82 17

Table 4 Quality and Positive Controls 23 Apr 82 18

Table 5 Salmonella/microsome Assay Worksheet 23 Apr 82 19

APPENDIX B

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OFFICIAL DISTRIBUTION LIST

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21

i

DATE,

FILMED

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