C-

Institute Report No. 292

O Mutagenic Potential of Diethyleneglycol Dinitratein the Ames Salmonella/Mammalian

Microsome Mutagenicity Test

Steven K. Smo, BA, SGTend

Don W. Korb, Jr, PD, MAI, MSC

GENETIC TOXICOLOGY BRANCHDIVISION OF TOXICOLOGY D I

S ELECTE-DEC j19080

September1988 Toxicology Serles: 147

LETTERMAN ARMY INSTITUTE OF RESEARCHPRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

b PaSm4 88 12 -'-i)ft . .. ........... a d*.I

-- " " "' m mat iW~li H

UNCLASSIFIEDS1CaRITY CLASSIFICATION OF THIS PAGE

Form A~mrovedl

REPORT DOCUMENTATION PAGE OA4 No. 070"188

Ia. REPORT SECURITY CLASSIFICATION lb. RESTRICTIVE MARKINGSUNCLASSIFIED

2&. SECURITY CLASSIFICATION AUTHORITY 3. DISTRIBUrION/AVAILABILITY OF REPORTApproved for public release; distribution

2b. DECLASSIFICATION /DOWNGRADING SC1EDULE is unlimited.

4. PERFORMING ORGANIZATION REPORT NUMBER(S) S. MONITORING ORGANIZATION REPORT NUMBER(S)

Institute Report No. 292

G. NAME OF PERFORMING ORGANIZATION 6b. OFFICE SYMBOL 7a. NAME OF MONITQ.ING-OR.GANIZATIQNGenetic Toxicology Branch ()k ) US " omedical Research and DevelopmentDivision of Toxicology SGRD-ULE-T Laboratory

6. ADDRESS (Cit, State, and ZIP Coe) 7b. ADDRESS (City, State, and ZIP Code)Letterman Army Institute of Research Ft. DetrickPresidio of San Francisco, CA 94129-6800 Frederick, MD 21701-5010

Ba. NAME OF FUNDING /SPONSORING Sb. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATION US AM Medical (fw ap able)

Research & Development CommandSc. ADDRESS (City, State, and ZIP Code) 10. SOURCE OF FUNDING NUMBERS

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Frederick, MD 21701-5012 ELEMENT NO. NO. NO. 0 SSION NO.

62720A 835 AB DA 30391311. TITLE (Icid* SKVfudt Gaifcation) MNtagenic Potential of Diethyleneglycol Dinitrate in the Ames

Salmonella/Mammalian Microsome Mutagenicity Test

12. PERSONAL AUTHOR(S)Steven K. Sano and Don W. Korte Jr.

13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT (Year MoDth,DiY) 15. PAGE COUNT

Institute FROM 8/19/8S TO8/30/8S September 198 1816. SUPPLEMENTARY NOTATION

Toxicology Series 147

17. COSATI CODES 1S. SUBJECT TERMS (Continue on revere if necenary and identify by block number)FIELD GROUP SU -IMutagnicity/ Diethyleneglycol Dinitrate

Gene ic Toxcology DEGCIrAmes Test Propellant. ,, -

I APSTRACT (Continue on reverse if necenary and identify by block number)

"-The mutagenic potential of diethyleneglycol dinitrate (DEGDN) was assessed by using theAmes Salmonella/Mammalian Microsome Mutagenicity Test. Tester strains TA97, TA98, TAI00,and TA102 were exposed to doses ranging from 5 1l/plate to 0.0016 ji/plate. The testcompound was not mutagenic under conditions of thitest .,

20. DISTRIBUTION I AVAILABILITY OF ABSTRACT 21. ABSTRACT SECURITY CLASSIFICATIONI[UNCLASSIFIEDIUNLIMITED C SAME AS RPT. [3 DTIC USERS UNCLASSIFIED

. AME OF RESPONSIBLE INDIVIDUAL 22b TELEPHONE (Include Area Code) 22c. OFFICE SYMBOLEdwin S. Beatrice. COL. MC 415-561-3600 SGRD-UL-Z

DO Form 1473. JUN 86 Previous a obsoete. SECURITY CLASSIFICATION OF THIS PAGEUNCLASSIFIED

ABSTRACT

The mutagenic potential of diethyleneglycol dinitrate(DEGDN) was assessed by using the Ames Salmonella/MammalianMicrosome Mutagenicity Test. Tester strains TA97, TA98,TAlO0, and TA102 were exposed to doses ranging from5 p.1/plate to 0.0016 p.1/plate. The test compound was notmutagenic under conditions of this test.

Key Words: Mutagenicity, Genetic Toxicology, Ames Test,Diethyleneglycol Dinitrate, DEGDN, Propellant

Aecn~on For

N T 1S 0 RL cIDIC T.I'i3

UnanrnouncedJustification-

Distribution/ rAvaila bility Codes

ALaiicdorDist Special

PRZFACE

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 CommandUS Army Biomedical Research and Development LaboratoryFort Detrick, Frederick, MD 21701-5012Project Officer: Gunda Reddy, PhD

PROJECT/WORK UNIT/APC: #3E162720A835/180/TLBO

GLP STUDY NUMBER: 85014

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

PRINCIPAL INVESTIGATOR: SGT Steven K. Sano, BA

REPORT AND DATA MANAGEMENT: A copy of the final report,study protocol, retired stabilityand purity data on the testcompound, tissues, and an aliquot ofthe test compound will be retainedin the LAIR Archives.

TEST SUBSTANCE: Diethyleneglycol dinitrate (DEGDN)

INCLUSIVE STUDY DATES: 19 Aug - 30 Aug 85

OBJECTIVE: The objective of this study was to determine themutagenic potential of diethyleneglycol dinitrate (LAIR CodeTP047) by using the Ames Salmonella/Mammalian MicrosomeMutagenicity Test.

iii

ACKNOWLEDGMENTS

CPT John W. Harbell, PhD, MSC; SGT Lillie D. Witcher,BS; SP4 John R.G. Ryabik, BS; Mr. John Dacey; and Ms. JoanneWong provided research assistance.

iv

SIGNATURES OF PRINCIPAL SCIENTISTS INVOLVED IN THESTUDY

We, the undersigned, declare that GLP study number 85014was performed under our supervision, according to theprocedures described herein, and that this report is anaccurate record of the results obtained.

DON W. KORTE JR, Pt)// DATE CONRAD WHEELER, PhD/ DATEMAJ, MSC DACStudy Director Analytical chemist

STEVEN K. SAN% BA / DATESGT, USAPrincipal Investigator

v

DEPARTMENT OF THE ARMY

LETTERMAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO, CALIFORNIA 941296800RFPLY TO

A T TENTION OF:

SGRD-ULZ-OA (70-1n) 15 September 1988

MEMORANDUM FOR RECORD

SUBJECT: GLP Compliance for GLP Study 85014

1. This is to certify that in relation to LAIR GLP Study85014, the following inspections were made:

16 August 1985 - Protocol Review27 August 1985 - Plate Incorporation

2. The institute report entitled "Mutagenic Potential ofDiethyleneglycol Dinitrate in the Ames Salmonella/MammalianMicrosome Mutagenicity Test, "Toxicology Series 147, wasaudited on 20 July 1988.

C ROLQM LWSChief, 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

BODY OF THE REPORT

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

Objective of the Study ............................ 2

MATERIALS AND METHODS .................................. 2

Test Compound ..................................... 2Test Solvent ...................................... 2Chemical Preparation .............................. 2Test Strains ...................................... 3Test Format ....................................... 3Data Interpretation ............................... 5Deviations/Changes ................................ 5Storage of Raw Data and Final Report .............. 5

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

DISCUSSION ............................................. 9

CONCLUSION ............................................. 9

REFERENCES ............................................ 10

APPENDIX ................................................... 11

Appendix A. Chemical Data ............................ 12Appendix B. Individual Plate Scores .................. 15

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

vii

Mutagenic Potential of Diethyleneglycol Dinitrate inthe Ames Salmonella/Xammalian Microsome MutagenicityTest--Sano and Korte

INTRODUCTION

The Department of Defense is considering the use ofdiethyleneglycol dinitrate (DEGDN), triethyleneglycoldinitrate (TEGDN), or trimethylolethane trinitrate (TMETN) asa replacement for nitroglycerin in munition formulations. A"health effects" review conducted for the US Army BiomedicalResearch and Development Laboratory (USABRDL) identifiednumerous gaps in the toxicology database of these compounds(1). Consequently, USABRDL has tasked the Division ofToxicology, LAIR, to conduct an initial evaluation of thehealth effects of DEGDN, TMETN, TEGDN, and two DEGDN-basedpropellants, JA-2 and DIGL-RP. This initial evaluationincludes the Ames mutagenicity test, acute oral toxicitytests in rats and mice, acute dermal toxicity tests inrabbits, dermal and ocular irritation studies in rabbits, anddermal sensitization studies in guinea pigs. This reportcontains the results of a study that assessed the mutagenicpotential of DEGDN in the Ames Salmonella/Mammalian MicrosomeMutagenicity Test.

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(2).

This evaluation of DEGDN utilizes a revision of the AmesSalmonella/Mammalian Microsome Mutagenicity Test (3). Twonew tester strains, a frame-shift strain (TA97) and a straincarrying an ochre mutation on a multicopy plasmid (TAl02),are added to the standard tester set. TA97 replaces TA1537,TA1535 and TA1538 which are removed from the recommended set.TA98 and TA100 are retained.

Sano and Korte--2

Objective of the Study

The objective of this study was to determine themutagenic potential of diethyleneglycol dinitrate (LAIR CodeTP047) by using the revised Ames Salmonella/MammalianMicrosome Mutagenicity Test.

MATZRIALS AND METHODS

Test Compound

Chemical name: Diethyleneglycol dinitrate

Code number: LAIR Code No. TP047

Physical state: Liquid

Source: Hercules IncorporatedWilmington, Delaware

Storage: Diethyleneglycol dinitrate was received fromRadford Army Ammunition Plant (Radford, VA) and assigned theLAIR Code number TP047. The test compound was stored at roomtemperature (210C) until used.

Chemical Properties/Analysis: Data provided by HerculesInc., characterizing the chemical composition and purity ofthe test material, are presented in Appendix A along withconfirmatory analysis of the test material performed by theDivision of Toxicology, LAIR (Presidio of San Francisco, CA).

Test Solvent

The positive control chemicals and the test compoundwere dissolved in grade I dimethyl sulfoxide (lot 113F-0450)obtained from Sigma Chemical Co. (St. Louis, MO).

Chemical Preparation

Diethyleneglycol dinitrate was stored at roomtemperature (21 C) until used. On the day of dosing, 300 giof the test compound was measured into a sterile vial anddissolved in 5.7 ml of grade I dimethyl sulfoxide to achievea 5% (w/v) solution. Aliquots of this solution were used todose the test plates.

Sano and Korte--3

Test Strains

Salmonella strains TA97, TA98, TA100, and TA102,obtained directly from Dr. Bruce Ames, University ofCalifornia, Berkeley, were used. These strains weremaintained in our laboratory at -800C. Quality control testswere run concurrently with the test substance to establishthe validity of their special features and to determine thespontaneous reversion rate. Descriptions of the strains,their genetic markers, and the methods for strain validationare given in the LAIR SOP, OP-STX-I (4).

TePt Frmat

Diethyleneglycol dinitrate was evaluated for mutagenicpotential according to a revised Ames method (3). A detaileddescription of the methodology is given in LAIR SOP, OP-STX-I(4).

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 diethyleneglycol dinitrate ranging from 1.6x 10-3 pl/plate to 5 /plate, and approximately 108 cells ofTA100 per plate. Top agar containing trace amounts ofhistidine and biotin was placed on the plates. Strainverification was confirmed on the bacteria, along with adetermination of the spontaneous reversion rate. Afterincubation, the growth on the plates was observed. Sincenone of the plates showed a decrease in the number ofmacrocolonies (below the number in the spontaneous reversionpltes) or an observable reduction in the density of thebackground lawn, a maximum "limit" dose of 5 gi per plate wasused in the mutagenicity test.

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 was purchased from Microbiological Associates Inc.(Bethesda, MD). The optimal titer of this S-9, as determinedby Microbiological Associates Inc., was 0.75 mgprotein/plate. After all the ingredients were added, the topagar was mixed, then overlaid on MGA plates. These platescontained 2% glucose and Vogel Bonner "E" Concentrate (5).The water used in this medium and in all reagents came from a

Sano and Korte--4

Technic Model 301 Reverse Osmosis Pre-Treatment Water System(Seattle, WA), LAIR SOP, OP-STX-94 (6). Plates wereincubated upside down in the dark at 37°C for 72 hr. Plateswere prepared in triplicate and the average revertant countswere recorded. The average number of revertants at each doselevel was compared to the average number of spontaneousrevertants (negative control). The spontaneous reversionrate (with and without S-9) was monitored by averaging thecounts from two determinations run simultaneously with thetest compound. The spontaneous reversion rate was determinedby inoculating one set of plates before and one set after thetest compound plates so that any change in spontaneousreversion rate during the dosing procedure would be detected.This spontaneous reversion rate was also compared withhistorical values for this laboratory and those cited inMaron and Ames (3). Sterility and strain verificationcontrols were run concurrentiy. All reagents, testcompounds, and media were checked for sterility by platingsamples of each on MGA media and incubating them at 370C withthe test plates. The Salmonella strains were verified by astandard battery of tests. The integrity of the differentSalmonella strains used in the assay was verified by thefollowing standard tests:

-Lack of growth (inhibition) in the presence of crystalviolet which indicates that the prerequisite alterationof the lipopolysaccharide layer of the cell wall ispresent.

-Growth in the presence of ampicillin-impregnated diskswhich indicates the presence of an ampicillin-resistantR Factor.

-Lack of growth (inhibition) following exposure toultraviolet light which indicates the absence of the DNAexcision-repair mechanism (for all strains exceptTAI02).

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(a]pyrene, 2-aminofluorene, 2-aminoanthraceneand 4-nitroquinoline-n-oxide, were obtained from SigmaChemical Co. (St. Louis, MO). The test compound and mutagenswere 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).

, ! II I

Sano and Korte--5

Data Interpretation

According to Brusick (7), 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 TAI00. A strong correlateddose response in strain TAI00 without a doubling of theindividual colony count may also be considered positive.

Maron and Ames (3) consider a compound mutagenic intester strains TA97 and TA102 if a correlated dose responseover three concentrations is achieved with the highest doseyielding a revertant colony count greater than or equal totwice the spontaneous colony count.

Deviations/Changes

A 72-hr rather than a 48-hr incubation period was used.According to Maron (personal communication, 1985), theadditional 24-hr growth enables all of the revertant colonies,especially TA102, to be detected with the colony counter.

Storage of 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 LAIR archives.

RESULTS

On 23 August 1985, the toxicity of diethyleneglycoldinitrate was determined (Table 1). For this experiment allsterility, strain verification, and negative controls werenormal (Table 1). No toxicity was observed after exposure ofthe tester strain (TA100) to the highest dose used (5g/plate).

Normal results were obtained for all sterility andstrain verification tests during the Ames Test performed on27-30 August 1985 (Table 2). Diethyleneglycol dinitrate didnot induce any appreciable increase in the revertant colonycounts relative to those of the negative control cultures(Table 3).

A copy of the raw data is included in Appendix B.

Sano and Korte--6

TABLZ 1: TOXICITY DETERMINATION rOR DZGDN

GLP STUDY NUMBER 85014 23 Aug 1985 PERFORMED BY SANO/WONG

TOXICITY DETERMINATION REVERTANT PLATE COUNT (TA100)

CONCENTRATION OF TEST COMPOUND MEAN (1SD) BACKGROUND LAWN*

START RUN NEGATIVE CONTROL 102 (14.0) NL5.0 gl/plate 72 (15.4) NL1.0 gi/plate 77 (5.1) NL0.2 g1/plate 75 (1.0) NL0.04 /plate 75 (5.5) NL0.008 gi/plate 80 (9.9) NL0.0016 il/plate 73 (8.7) NLEND RUN NEGATIVE CONTROL 96 (8.1) NL

STRAIN VERIFICATION FOR TOXICITY DETERMINATION (TA100)

HISTIDINE REQUIREMENT NG*AMPICILLIN RESISTANCE GUV NGCRYSTAL VIOLET

SENSITIVITY (ZONE SIZE) NG (12mm)STERILITY CONTROL NG

STERILITY CONTROL FOR TOXICITY DETERMINATION

MATERIAL TESTED OBSERVATION*

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

TARL 2: STRAIN VERIFICATION AND STERILITY TESTINGFOR THE MUTAGENICITY DETERMINATION

or DEGDN (TP047)

GLP STUDY NUMBER 85014 12 SEP 1985 PERFORMED BY SANO/WONG

STRAIN VERIFICATION

OBSERVATIONS*STRAINS TA97 TA98 TA 100 TA102

HISTIDINE REQUIREMENT NG NG NG NGAMPICILLIN RESISTANCE G G G GUV REPAIR NG NG NG GCRYSTAL VIOLET

SENSITIVITY NG NG NG NG(ZONE SIZE) (13mm) (10mm) (9mm) (10mm)

STERILITY CONTROL 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) NG

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

Sano and Korte--8

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S~no and Korte--9

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 TAl02).Second, the Salmonella strains must be susceptible tomutation by known mutagens. Third, the optimal concentrationof the test compound must be determined by treating TA100with 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 the Ames test can be considered valid.

After validation of bacterial strains and selection ofoptimal sublethal doses, diethyleneglycol dinitrate wasevaluated in the Ames test. Criteria for a positive responseare a correlated dose-response relationship and a twofoldincrease in revertant colony counts relative to therespective negative control counts (3,4,7). Diethyleneglycoldinitrate did not induce the requisite dose-responserelationship or the increase in revertant colony countsnecessary for a positive response. Thus, the results of thistest indicate that diethyleneglycol dinitrate is notmutagenic when evaluated in the Ames test.

CONCLUSION

Diethyleneglycol dinitrate was evaluated for mutagenicpotential in the Ames Test, both in the presence and absenceof metabolic activation, and did not induce a positivemutagenic response under conditions of this study.

PH -.. ... . .II I I . ...... . .... ..... .... ... .. -

Sano and Korte--10

REFZRENCES

1. Holleman JW, Ross RH, Carroll JW. Problem definitionstudy on the health effects of diethyleneglycoldinitrate, triethyleneglycol dinitrate, andtrimethylolethane trinitrate and their respectivecombustion products. Frederick, Maryland: US ArmyMedical Bioengineering Research and DevelopmentLaboratory, 1983, DTIC No. ADA 127846.

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

3. Maron DM, Ames BN. Revised methods for the SalmonellaMutagenicity Test. Mutation Res 1983;113:173-215.

4. Ames Salmonella/Mammalian Microsome Mutagenesis Test.LAIR Standard Operating Procedure OP-STX-1, Presidio ofSan Francisco, California: Letterman Army Institute ofResearch, 15 November 1983.

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

6. Operation of the Technic Model 301 Reverse Osmosis Pre-Treatment Water System and the Corning Model MP-1 GlassStill. LAIR Standard Operating Procedure OP-STX-94,Presidio of San Francisco, California: Letterman ArmyInstitute of Research, 29 July 1985.

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

Sano and Korte--1.

Appendix A. Chemical Data.............................. 12Appendix B. Individual Plate Scores................... 15

Sano and Korte--12

Appendix A: CHEMICAL DATA

Chemical name: Ethanol, 2,2'-oxybisdinitrate

Alternate chemical name: Diethyleneglycol dinitrate (DEGDN)

Chemical Abstracts Service Regiscry No.: 693-21-0

LAIR Code No.: TP047

Chemical structure:

02N-O-CH2CH2-O-CH2CH2-O-N02

Molecular formula: C4H8N207

Molecular weight: 196

Physical state: Pale yellow liquid

Density (g/cm3): 1.381

Analytical data: Refer to the attached data sheet, ARRCOMForm 213R. The compound chromatographedas a single peak (retention time 5.4 min)by HPLC analysis under the followingconditions: column, Brownlee RP-18 (4.6x 250 mm); solvent system, 30% water, 70%acetonitrile; flow rate, 0.9 ml/min;detection wavelength, 205 nm.2 NMR (300MHz, CD3CN): 3.75 8 (complex multiplet,4H,-C12-O-Cji2-), 4.61 complex

1 Holleman JW, Ross RH, Carroll JW. Problem definition studyon the health effects of diethyleneglycol dinitrate,triethyleneglycol dinitrate, and trimethylolethane trinitrateand their respective combustion products. Frederick,Maryland; US Army Medical Bioengineering Research andDevelopment Laboratory, 1983; DTIC No. ADA127846, p. 17.

2 Wheeler CR. Toxicity Testing of Propellants. LaboratoryNotebook #85-12-023, p. 31. Letterman Army Institute ofResearch, Presidio of San Francisco, California.

Sano and Korte--13

Appendix A (cont.): CHEMICAL DATA

multiplet, 4H,-C 2ONO2).3 Additional singletsignals of approximately equal intensity wereobserved at 2.08 d, and were due to sampleimpurities. Integration of all signals in thespectrum demonstrated that the samplecontained 96.6% DEGDN. The impurities werenot identified. IR(KBr): 2896, 1632, 1429,1390, 1373,1279, 1139, 1032, 909, 857, 758,707, 655, 572 cm-

1 .4

Stability: The DEGDN was shipped containing 18% acetone(a desensitizer) and arrived at LAIR on 12December 1984. The acetone was removed byrotary evaporation prior to studies with thepropellant. Analysis of the compound one yearafter it was received gave the resultsdescribed above.

Source: Radford Army Ammunition Plant, Radford, Virginia(prime contractor: Hercules Inc., Wilmington,Delaware).

Lot No.: RAD84MO01S214

3 Ibid. pp. 44-48.

4 Ibid. pp. 49-50.

Sano and Korte--14

Appendix A (cont): CHEMICAL DATA

DESCRIPTION SHEET FOR EXPLOSIVES, CHEMICALS, ETC h1AP.* 7- joAR 138 15 Of

ro. poms DATE

December 5. 1984MATIrnALDiethylene GlycolDinitrace (DECDN)

MANJFACTURER )fECULgS NCOUO,"5.D ONTRACT 0o.

RADFORD -A ANIIZTION PLANT DAAO-77-C-4007S*CiTI$ZN A - 01SCRIl I N- q F ,OS ...

PlfOM SMIR 21 'THRU NUMBER TOTAL NO. LOTS TOTAL NET AMOUNT ACCEPTED

RADS4HooIszl4 z - 1PLACE MANUFACTUIE0 SO ICATIONAN A NOM NT/D MAI" NO.RADFORD ARMY AUNTION PLANT, RADFORD, VIGINIA DOD-D-6O01S

SE ON B - DISCRIPTION OF MATERIAL.

Requirements Limit Results

82.2"C Potassium Iodide 10 minutes minimum 12Starch Paper Heat Test (KI)

Nitrogen, Z 14.10 minimum 14.15

Water. Z Info Only 0.43

Acidity None None

Alkalinity None None

B l*Am$ DEGDN is desensitized ith 5o more of acetone tor a total wei nt ot 5 lbs. annpacked in a DOT 60 S gallon drum with a DOT 2S liner. overpacked in a DOT-6J 30 galloncapacity drum with vermiculite as a cushioning agent around the 5 gallon drum and contain,in the 30 gallon drum. Requested by shipping Order A.MCCOM and COR letter SMCRA dated4ovember 28, 1984 (DOT Exemption 5704).

SE ~ON C - CER[TIFACATIONISAMD

P LS CONDUC ? f rKH ABOVL AT IrAL COMPLIES WITH ALL SPECIFICATION

HErCULSS INCORPORATED 0EoUIEEMENTS ANO IS CERTIP[O TIP UE ANa CON ICT.

HERCULES INCORPORATED 12-5-84 z, .

rN 1Ov I OkS46t13O LOTS AlE MER1Y ACCEPTED

FOB THE COMMANOER

AIRCON Fora 213-A, 10 Aug 77 SEQUENlCE No. -;4

Sano and Korte--15

Appendix B: INDIVIDUAL PLATE SCORES

TOXICITY DETERMINATION WITH TA100

COMPOUND DOSE/plate PLATE 1 PLATE 2 PLATE 3

NEGATIVE CONTROL 116 88 102

(Start Run)

TP047 5.0 gi 62 65 90

TP047 1.0 gI 78 81 71

TP047 0.2 p. 74 75 76

TP047 0.04 pi 75 70 81

TP047 0.008 pi 73 75 91

TP047 0.0016 pi 71 66 83

NEGATIVE CONTROL 101 87 101(End Run)

I I

Sano and Korte--16

Appendix B (cont.): INDIVIDUAL PLATE SCORES

MUTAGENICITY TESTS WITHOUT S-9

COMPOUND DOSE/plate TA97 TA98 TA100 TA102

NEG CONTROL 49 25 91 190(start run) 51 31 113 183

66 30 108 180

NEG CONTROL 65 21 97 176(END RUN) 54 32 116 192

54 24 123 188

NQNO* 2.0 pg 732 731615 630102 710

TP047 5.0 gi 89 29 131 15386 26 108 20779 38 125 199

TP047 1.0 l 54 25 98 17865 21 102 19241 20 97 179

TP047 0.2 pi 78 19 90 18470 31 101 17363 32 77 207

TP047 0.04 gI 50 36 104 17242 35 98 17541 33 105 187

TP047 0.008 gil 62 27 92 21063 23 81 19668 30 97 185

TP047 0.0016 gl 78 37 107 17357 29 76 18369 13 103 175

* 4-nitroquinoline-n-oxide

Sano and Korte--17

Appendix B (cont.): INDIVIDUAL PLATE SCORES

MUTAGENICITY TESTS WITH S-9

COMPOUND DOSE/plate TA97 TA98 TA100 TA102

NEG CONTROL 65 34 89 262(Start Run) 45 38 83 271

70 33 90 252

NEG CONTROL 97 26 86 273(End Run) 85 38 108 266

80 37 1 246

2-aminofluorene 2.0 gg 327 1347 530 358362 1095 557 392368 1028 553 412

benzo(a)pyrene 2.0 gg 500 492532 485553 436

2-aminoanthracene 2.0 gg 1186 1198924 1144

1474 1055

TP047 5.0 gl 86 18 120 23869 30 118 19383 36 106 159

TP047 1.0 gl 80 32 89 26056 37 81 28266 27 94 234

TP047 0.2 gl 72 20 81 24587 21 86 26481 22 83 233

TP047 0.04 il 60 32 91 25448 30 98 23853 22 68 227

TP047 0.008 .l 70 28 107 23361 28 94 25397 lost 97 266

TP047 0.0016 gi 84 30 80 23670 21 69 21772 22 96 250

Sano and Korte--18

Distribution List

Commander CommandantUS Army Biomedical Research and Academy of Health Sciences

Development Laboratory (27) United States ArmyATTN: SGRD-UBZ-C ATTN: Chief, EnvironmentalFort Detrick, Frederick, MD 21701-5010 Quality Branch

Preventive Medicine DivisionDefense Technical Information Cente: (HSHA-IPM)

(DTIC) (2) Fort Sam Houston, TX 78234ATTN: DTIC-DLACameron Station Commander US Army MaterielAlexandria, VA 22304-6145 Command

ATTN: AMSCGUS Army Medical Research and 5001 Eisenhower Avenue

Development Command (2) Alexandria, VA 22333ATTN: SGRD-RMI-SFort Detrick, Frederick, MD 21701-5012 Commander

US Army Environmental HygieneCommandant AgencyAcademy of Health Sciences, US Army ATTN: Librarian, HSDH-AD-LATTN: AHS-CDM Aberdeen Proving Ground. MD 21010Fort Sam Houston, TX 78234

DeanChief School of MedicineUSAEHA Regional Division, West Uniformed Services University of theFitzsimmons AMC Health SciencesAurora, CO 80045 4301 Jones Bridge Road

Bethesda, MD 20014ChiefUSAEHA Regional Division, North CommanderFort George G. Meade, MD 20755 US Army Materiel Command

ATTIN: AMCEN-AChief 5001 Fisenhower AvenueUSAEHA Regional Division, South Alexandria, VA 22333Bldg. 180Fort McPherson, GA 30330 HQDA

ATTN: DASG-PSP-ECommander Falls Church, VA 22041-3258USA Health Services CommandATTN: HSPA-P HQDAFort Sam Houston, TX 78234 ATTN: DAEN-RDM

20 Massachusetts, NWWashington, D.C. 20314

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