AD-AI65 756 NUTAGENIC POTENTIAL OF P-DITHIANE(U) LETTEEMfi RNY 1/1INST OF RESEARCH PRESIDIO OF SAN FRANCISCO CAS K SRNO ET AL. AUG 05 LRIR-2S?

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MICROCOP RESOUTION TEST CHART

an INSTITUTE REPORT NO. 207 LEC

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( MUTAGENIC POTENTIAL OF p-DITHIANE

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STEVEN K. SANO, BA, SP5 ._ .S

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DON W. KORTE JR, PhD, MAJ MSC

TOXICOLOGY GROUP o,,

DIVISION OF RESEARCH SUPPORT

IP,

AUGUST 1955 Toxicology Sries 95

GLP Study 94031

LETTERMAN ARMY INSTITUTE OF RESEARCHPRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

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Nutagenic potential of p-dithiane (Toxicology Series 95)--Sano and Korte

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Ot~ t,- IA///a-Zt 3 Aug '85(Signatim and dae)

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This document has been approved for public release and sale, its distribution is unlimited.

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Mutagenic Potential of p-Dithiane Final24 Sep - 12 Oct 1984

S. PERFORMING ORG. REPORT NUMBeER

7. AUTHOR(&) 8. CONTRACT OR GRANT NUMBER(@)

Steven K. Sano, BA SF4Don W. Korte, Jr, PhD, MAJ, MS

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT. PROJECT, TASK

Toxicology Group, Division of Research Support ARAA'WR UI*UBR

Letterman Army Institute of Research 41~627WA75

Prsdoof San Francisco, CA 94129-6800 WU 308, APC TL05

US Army Medical Research and Development Command August 1985Fort Detrick, MD 21701-5012 13. NUMBER OFPAGES

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10. SUPPLEMENTARY NOTES

19. KEY WORDS (Continue an reverse ad* it necessary and Identity by block nr.bor)

Mitaenicity, enetic Toxicology Ames Assay, p-Dithlane

20. AGSTRACr (Conotse star1

eee. IfrCnec.ewy and Idenltfy byp bloc-k r,.mb.r-

A ---.*he mlltagenic potent'l4l of p-dithianf- was assecssed by using the AmesSa'-monel laMaramal tan 4,4crosome MutagelfIcitv Assay. Testor strains TA98, T.\h)O,TN1535, TA110~7, and TA1t 38 were exposed, Lo doses ranging. from 5 mg/plate to0.17116 mg/plate. The Ltqt :onpound was niot nutagentc kiader conditions of thisassay.

DO 1473 EDpTloA OF I Nov 6%I.S -01 tIUCA I['~.8 ~t~ Sa0P1

ABSTRACT

The mutagenic potential of p-dithiane was assessed by using theAmes Salmonella/Mammalian Microsome Mutagenicity Assay. Tester strainsTA98, TA100, TA1535, TA1537, and TA1538 were exposed to doses rangingfrom 5 mg/plate to 0.0016 mg/plate. The test compound was notmutagenic under conditions of this assay.

Key Words: Mutagenicity, Genetic Toxicology, Ames Assay,

p-DithianeH

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PREFACE

TYPE REPORT: Ames Assay 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 CommandUS Army Medical Bioengineering Research andDevelopment LaboratoryFort Detrick, MD 21701-5010

WORK UNIT: 3516277A875 Medical Defense Against ChemicalAgents Projects; WU 308; APC TLO5 , .-

GLP STUDY NUMBER: 84031 ,

STUDY DIRECTOR: MW Don W. Korte Jr, PhD

PRINCIPAL INVESTIGATOR: SP4 Steven K. Sano, BA

REPORT AND DATA MANAGEMENT: A copy of the final report, study protocols,raw data, retired SOPs, and an aliquot ofthe test compound will be retained in theLAIR Archives.

TEST SUBSTANCE: p-Dithiane (TA039)

INCLUSIVE STUDY DATES: 24 September - 12 October 1984

OBJECTIVE: The objective of this study was to determine the mutagenicpotential of p-dithiane (Batch Number 3030TH, LAIR Code TA039)by using the Ames Salmonella/Mammalian MicrosomeMutagenicity Assay.

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ACKNOWLEDGMENTS

The authors wish to thank SP6 James Justus, BA; SP4 Paul Mauk, BA;PFC James Martin; and Mr. John Dacey, for their assistance inperforming the research.

IV.

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SIGNATURES OF PRINCIPAL SCIENTISTS AND MANAGERS INVOLVED IN THE STUDY

We, the undersigned, declare that GLP study number 84031 wasperformed under our supervision, according to the procedures decribedherein, and that this report is an accurate record of the resultsobtained.

'A4

DON W. KORTE, JR Ph. 9 DATEJIAJw MSC d

Study Director

STEVEN K. SANO, B.A. / DATESP4, USA

Principal Investigator

CONRAD WHEELER, Ph.D. / DATEDAC

Analytical Chemist

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DEPARTMENT OF THE ARMYLETTERMAN ARMY INSTITLJTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

SCRD-ULZ-QA 18 August 1985

MLMO~RANDUM FOR RECORD

SUBJECT: Report of GLP Compliance

1. I hereby certify that in relation to LAIR GLP Study 84031 the following

NA I inspections were made:

10 October 1984

12 October 1984

2. The report and raw data for this study were audited on 10 may 1984.I 3. Routine inspections with no adverse findings are reported quately,r? thus these inspections are also included in the 21 January 1985 report to

Management and the Study Director.

GARY D UTCHER

Quality Assurance Unit

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TABLE OF CONTENTS

Abstract ........................................................i $i

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

Acnwegmns................................................ iv

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

Report of Quality Assurance Unit ................................Vi

*,Table of Contents ............................................. Vii

BODY OF REPORT

INTRODUCTION

objective of the Study .................................. 1

METHODS

Test Compound ...........................................1Test Solvent ............................................ 2Chemical Preparation ....................................2Test Strains ............................................2Test Format .............................................2

RESULTS ..................................................... 4DISCSSIO......................................I

DISCUSSION ................................................. 11

COCLUSDION..............................................1

RECOERENDAEION...............................................12

APPEENIX...................................................13

DISTRIBUTION LIST .............................................. 18

166t

vii

Sano-- I

Mutagenic Potential of: p-Dithiane (TA039)--Sano and Korte

he Ames Salmonella/Mammalian Microsome Mutagenicity Assay is ashort-term screening assay that utilizes histidine auxotrophic mutantstrains of Salmonella typhimurium to detect those compounds which arepotentially mutagenic in mammals. A mammalian microsomal enzyme systemis incorporated in the assay to increase sensitivity by simulating invivo metabolic activation of the test compound. The Ames assay is an

inexpensive yet highly predictive and reliable assa)y for detecting' mutagenc activity and thus carcinogenic potential 41.

- Objective of the Study

The objective of this study was to determine the mutagenicpotential of p-dithiane (Batch Number 3030TH, LAIR Code TA039) by usingthe Ames Salmonella/Mammalian Microsome Mutagenicity Assay.

METHODS

Test Compound

Chemical name: p-Dithiane

Chemical Abstract Service Registry No.: 51330-42-8

Structural formula:

.5..

Empirical formula: C4H5 S2

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Sano--2

Storage: Ten grams of p-dithiane (Batch Number 3030TH) werereceived from Aldrich Chemical Company, Inc (Milwaukee, WI) on 22August 1984 and assigned the LAIR Code number TA039. The test compoundwas stored in a dessicator at room temperature (21'C) until use.

Chemical Properties/Analysis: Data characterizing the chemicalcomposition and purity of the test material were obtained from AldrichChemical Co, Inc and confirmed by Infrared Spectrometer performed by

the Toxicology Group, LAIR (Presidio of San Fiancisco, CA) (AppendixA).

Test Solvent

The test compound and the positive control chemicals were dissolvedin grade I dimethyl sulfoxide (Lot Number I0OF-0269) obtained fromSigma Chemical Co (St. Louis, MO).

Chemical Preparation

p-Dithiane was stored in a dessicator at room temperature (21 0C)until used. On the day before dosing, 300 mg of the test compound wasmeasured into a sterile vial and again stored at room temperature. Onthe day of dosing, the 300 mg sample was dissolved in a 6 ml volume ofgrade I dimethyl sulfoxide (Lot Number I00F-0269) to achieve a 5% (w/v)solution. Aliquots of this solution were used to dose the test plates.The dosing procedure was completed within 20 minutes of dissolving thetest compound.

Test Strains

Salmonella strains TA98, TA100, TA1535, TA1537, and TA153R,obtained directly from Dr. Bruce Ames, University of California,Berkeley, were used. These strains were maintained in our laboratoryat -800C. Quality controls were run concurrently with the testsubstance to establish the validity of their special features and todetermine the spontaneous reversion rate. Descriptions of the strains,their genetic markers, and the methods for strain validation are givenin the LAIR SOP, OP-STX-l (2).

Test Format

p-Dithiane was evaluated for mutagenic potential according of Ames

et al (3). A detailed description of the to the methods methodology isgiven in LAIR SOP, OP-STX-1 (2).

Toxicity Tests

Toxicity tests were conducted to determine a sublethalconcentration of the test substance. This toxicity level was found by

4................................................

..............................

Sano--3

using minimal glucose agar (NGA) plates, concentrations of p-dithianeranging from 1.6 x 10

- 3 mg/plate to 5 mg/plate, and approximately 108

cells of TA100 per plate. Top agar containing trace amounts ofhistidine and biotin were placed on the plates. Strain verificationwas confirmed on the bacteria, along with a determination of thespontaneous reversion rate. After incubation, the growth on the plateswas observed. Since none of the plates showed decreased macrocolony

formAtion (below the level of the spontaneous reversion plates) or anobservable reduction in the density of the background lawn, a maximum -L

"limit" dose of 5 mg per plate was used in the mutagenicity assay.

Mutagenicity Assay

The test substance was evaluated over a 1000-fold range ofconcentrations, decreasing from the minimum toxic level (the maximum or

ltmht dose) by a dilution factor of 5 both with and without 0.5 ml ofthe 9-9 microsome fraction. The S-9 was purchased from Litton .-

Bionettcs (Kensington, MD). The optimal titer of this S-9, asdetermined by Litton Bionetics, was 0.75 mg protein/plate. After all 1-%-

the ingredients were added, the top agar was mixed, then overlaid onMGA plates. These plates contained 2% glucose and Vogel Bonner "E"Concentrate (4). The water used in this medium and in all reagentscame from a Polymetric model 200-3 Water Purifier (Sunnyvale, CA).Plates were incubated upside down in the dark, at 37*C for 48 hours.Plates were prepared in triplicate and the average revertant countswere recorded. The average number of revertants at each dose level was *1compared to the average number of spontaneous revertants (negativecontrol). The spontaneous reversion rate (with and without S-9) wasmonitored by averaging the counts from two determinations runsimultaneously with the test compound assay. The spontaneous reversionrate was determined by inoculating one set of plates before and one setafter the test compound assay plates so that any change in spontaneousreversion rate during the dosing procedure would be detected. This

spontaneous reversion rate was also compared with historical values forthis laboratory and those cited in Ames et al (3). Concurrent c1sterility and strain verification controls were run. All reagents,test compounds, and media were checked for sterility by plating samplesof each on MGA media and incubating them at 370C with the test plates.The Salmonella strains were verified by a standard battery of tests.The following tests were run to determine if:

- Lipopolysaccharide layer (LP) alteration causes growth inhibitionin the presence of crystal violet. "

- An ampicillin-resistant R factor has allowed growth in strainsTA98 and TAIO in the presence of ampicillin impregnated disks. V.q

- Absence of excision repair n&.chanism has inhibited growth in the

presence of ultraviolet ligl .

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Four known mutagens were tested as positive controls to confirm theresponsiveness of the strains to the mutation process. Thesecompounds, benzo [a] pyrene, 2-aminofluorene, 2-aminoanthracene and N-methyl-n-'nitro-n-nitrosoguanidine, were obtained from Sigma ChemicalCo (St. Louis, MO). The test compound and mutagens were handled duringthis study in accordance with the standards published in NIH Guidelinesfor the Laboratory Use of Chemical Carcinogens (DHHS Publication No.(NIH) 81-2385, May 1981).

Data Interpretation

According to Brusick (5), a compound is considered mutagenic if thefollowing criteria are met:

1. For strain TA98 and TAOO, a positive dose response (correlateddose response) over three dose concentrations is achieved with atleast the highest dose yielding a revertant colony count greaterthan or equal to twice the spontaneous revertant colony count forthe strain. A strong correlated dose response in strain TA100without a doubling of the individual colony count may also beconsidered positive.

2. For strains TA1535, TA1537, and TA1538, a correlated doseresponse over three concentrations is achieved with at least onedose yielding a revertant colony count three times thespontaneous colony count for the strain.

RESULTS

On 3 October 1984, the toxicity level determination was performedon p-dithiane (Table 1). For this experiment all sterility, strainverification, positive and negative controls were normal (Table 2). Notoxicity was observed after exposure of the tester strain (TA100) tothe highest dose used (5 mg/plate).

Normal results were obtained for all sterility, strainverification, and negative controls during the Ames Assay performedduring the 3-day period 10 to 12 October 1984 (Tables 3-4). p-Dithianedid not induce any appreciable increase in the revertant colony counts

* relative to those of the negative control cultures (Table 5).

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DISCUSSION

Certain test criteria must be satisfied before an Ames assay can beconsidered a valid assessment of a compound's mutagenic potential.First, the special features of the Ames strains must be verified.These features include demonstration of ampicillin resistance, LP layeralterations, and DNA excision repair deficiencies. Second, theSalmonella strains must be responsive to the mutagenic process byexposing the strains to known mutagens. Third, the optimalconcentration of the test compound must be determined by treating TAIOOwith a broad range of doses and observing the potential toxic effectson macrocolony and microcolony formation. If these tests are performed

and expected data are obtained, then the results of Ames assay can beconsidered valid.

After validation of bacterial strains and selection of optimalsublethal doses, p-dithiane was evaluated in the Ames assay. Criteriafor a positive response are a correlated dose-response relationship forthe positive strains and a two-fold (strains TA98 or TAIO0) or three-fold (strains TA1535, TA1537, or TA1538) increase in revertant colonycounts relative to the respective negative control counts (5).

p-Dithidne did not induce the requisite dose-response relationship orthe increase in revertant colony counts necessary for a positiveresponse. Thus, the results of this assay indicate that p-dithiane is 6

not mutagenic when evaluated in the Ames assay.

CONCLUSION

p-Dithiane, both with and without metabolic activation, is not

mutagenic in the Ames assay as conducted in this study.

RECOMMENDATION

p-Dithiane should be tested in other genetic toxicity assays inaccordance with the Toxic Substance Control Act. S

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Sano--12

RE FERENCES

1. McCann JE, Choi E, Yamasaki E, Amses BN. Detection of carcinogensas mutagens in the Salmonella/microsome test: Assay of 300chemicals. Proc Nat Acad Sci, USA 1975;72:5135-5139.

2. Ames Salmonella/Mammalian Microsome Mutagenicity Assay. LAIR

Standard Operating Procedure OP-STX-l, Letterman Army Institute of

Research, Presidio of San Francisco, California, 15 November 1983.

3. Aimes TIN, McCann J, Yanasaki E. Methods for detection ofcarcinogens and mutagens with Salmonella/Mammalian microsome

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

4.Vogel HJ, Bonner DM. Acetylornithinase of E. coli: Partialpurification and some properties. J Biol Chem 1956;218:97-106.

5. Brusick D. Genetic Toxicology. In: Hayes AW, ed. Principles andMethods of Toxicology. New York: Raven Press, 1982: 223-272.

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CHEMICAL DATA

Chemical name: 1,4-Dithiane

Chemical Abstracts Service Registry No.: 505-29-3

Chemical structure:

Molecular formula: CHgS,

Molecular weight: 120.24

Physical state: White crystals

Melting point: I1-120C (data supplied by source)

Source: Aldrich Chemical Co.Milwaukee, WI

Lot number: 3030TH

Analytical data: Compound was described as 97% pure by source.Analysis provided by sponsor demonstrated a purity

of 99.92%.* NR and IR analyses were performed afterrec eipt of the compound: .NNR (80 MHz, d6-DMSO): 6 ,

2.82 (Singlet, 8 H, -Ch,-).t IR (KBr): 2945, 2905, ',

1410. 1280, 1270, 1150, q05, and 90 -1 t...R andIR data were Identical to published standard IR;

' and

NMR spectra.

Stability: No decomposition of 1,4-dithiane was detected by TR after

66 hx in 0uSO.'

*Rosencrance AB. IMemorandum for Dr. Reddy]. SUBJECT: Results from the

the chemical analysis of three compounds slated for toxicity testing(4 july 9!) Frederirc, Maryland: USA"18RD1. .-

4 ¢ heelir, CR. litrocellulose-Nttro~uiidtae Projects. l.aboratorvNotebo. 4-u5-010.2, p

4. Letterman Army Institute wc Research.

Presidio of San Francisco, CA.

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M1 A 1x.,, ,.. AIriI f u, , i C l ....I to.. l' .I I 1 1, so ,xxt c k'.

.;adtItr Research la.boritorv, lac.. '-tdtL -,tanxdard spectra.Phlladelphli: The S.,tler Research !.0,,ratorv, Inc., l'-2: InfraredSpectroram "7752.

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aldrich chemical compang. Inc.

ANALYTICAL DATA

Dale June 18, 1984

Our: D21770-0 Para-dithiane, 971.

Batch No.: 303 0TH

Analylical Results:

Appearance Off white crystals

m.p. 111-113 deg. C b.p.

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a" spectal Deis:

I.R. Conforms to structure and standard as illustrated onpage 160 B of Edition III, of "The Aldrich Libraryof Infrared Spectra".

U.V.N-,,R.-,:

% A Ntar .

a)a Assay: .

V.P C.

% Totraticn 99.9%,S-Content

Other 0fs OLKB/kb Anna Napiorkowski, Manager

Quality Control/Quality Assurance

APPENDIX A (cont.) " "

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T..ilty Testin

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analysis:Other

% of' Total Formula Cnound ihlte

c 7 .8 C 5 NS Benzothlazcl0.61 Cal..NS 2-Met~y * eit~a~ ~c~0.25 C 1 1'i11 Aniline 3 or0.12 C, EO,0 2 Dipheny d inul r'.':.-0.11 C7 jdn (.Hos 9enz.jor e

0.03 CO 14JS tethyltezrzth~jzcl y I n nt!

1.06 C14i 8.32 1,14-Dithinne

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OFFICIAL DISTRIBUTION LIST

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