AD-AL05 978 LETTERMAN ARMY INST OF RESEARCH PRESIDIO OF SAN FRANC--ETC

F/6 6/20THE MUTAGENIC POTENTIAL OF '4 NITROPHENYL BIS(2-THIENYL )-PWOSPII-ETCIU,

SEP 81 L J SAILERS, F R PULLIAM, J T FRUIN

UNCLASSIFIED LAIR-IONNI

IihE~hE~ENDE

INSTITUTE REPORT NO. 104

THE MUTAGENIC POTENTIAL OF: 4-nitrophenyt bis(2-thienyl) phosphinate4-nitrophenyl 2-furyl (methyl) phosphinate4-cyanophenyl bis(2-furyl) phosphinate4-nitrophenyl bis(2-furyl) phosphinate

LEONA RD J. SA UERS, BA, SP5FREDDICA R. PULLIAM, 85, SSGandJOHN T. FRUIN, D VM, PhD, L TC VC

TOXICOLOGY GROUP,OC22

DI VISION OF RESEARCH SUPPORT C2218

()S

i..SEPTEMBER 1981 Toxicology Series 17

LETTERMAN ARMY INSTITUTE OF RESEARCH PRESIDIO OF SAN FRANCISCO CALIFORNIA 94129

Toxicology Series 17

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

Destroy this report when it is no longer needed. Do not return it to the originator.

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

This material has been reviewed by Letterman Army 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)

(Signature and date)

*1|

[INCI ASSITFIDSECURITY CLASSIFICATION OF THIS PAGE (When Dlate Entered)

REPOT DCUMNTATON AGEREAD INSTRUCTIONSREPOT DCUMNTATON AGEBEFORE COMPLETING FORM

I. REPORT NUMBER 2 VTACESSION NO. 3. RECIPIENT'S CATALOG NUMBER

LAIR Jnsti tute Repgrt-Ro. 104 2GOTA6'4. TITLE ad Se-4hdit) The Mutageflic Potential of 4 nitro- s. TYPE OF REPORT & PERIOD COVEREDphenyl bis(2-thienyl)phosphinate; 4-nitrophenyl Final p- __p2-furyl(methyl)phosphinate; 4-cyanophenyl bis(2-. IJun-- Septwb"-J911furyl)phosphinate; 4-nitrophenyl bis(2-furyl) 6. P ERFORMING ORG. REPORT NUMBERphosphinatef

7. AUTNOR(qJ 0. CONTRACT OR GRANT NUNMbCR(&)

Leonard J./Sauers, BA, SP5; LFreddica RIJPulliam BS, SSG; ~* 1~~'//John T. FruinDV WPhD, LTC VC; ______________

(-. PERFGR144GRtZ ATION NAME AND ADDRESS 10. PROGRAM LLEMENI. PROiLCT. TAA(

ToxicologyGruDvofRsacSpotLetterman Army Institute of Research Project 3516772A875Presidio of San Francisco, CA 94129 WU 304

*II. CONTROLLING OFFICE NAME AND ADDRESS 4*,R PfT? DATE

U .S. Army Medical Research and Development Comman e..fIr~lFort Detrick nMKSROrbEFrederick, MD 21701 WUBRF 38 '~

14. MONITORING AGENCY NAME & ADDRESS(II different from Controlling 01f1ice) 15. SECURITY CLASS. (of this reportT

/711 A .J..l .~715sa. DECL ASSI FICATION! DOWNGRADING16. DISTRIBUTION STATEMENT (of this Report)

APPROVED FOR PUBLIC RELEASE: DISTRIBUTION UNLIMITED.

17. LI.;TrZIBUTiON STATEMENT (of tho abstract entered In Block 20, It different from Report)

IS. SUPPLEMENTARY NOTES

19. KEY WORDS (Continuo an reverse side it necessary mid identify by block n"~Mb.r)'4 Mutagenicity; Toxicology, Ames Assay, 4-nitrophenyl bis(2-thienyl )phosphinatn-4-nitrophenyl 2-furyl (methyl )phosphinate; 4-cyanophenyl bis(2-furyl )phosphinate;4-nitrophenyl bis(2-furyl )phosphinate.

ABSTRACT (Continue on reverse side If necessary and Identify by block number))VThe mutagenic potential of 4 nitrophenyl bis(2-thienyl)phosphinate (414); 4-nitrophenyl 2-furyl(methyl)phosphinate (72Y~) 4 cyanophenyl bis(2-furyl)phos-phinate (82T); 4-nitrophenyl bis(2-furyl)phosphinate (87W) was assessed byusing the Ames Salmonella/Mammalian Microsome Mutagenicity Assay. Testerstrains TA 98, TA 100, TA 15 5 T 537 and TA 1538 were exposed to dosesranging from 1 mg/plate t( mg/plate. It was determined that none ofthe tested substances had mu tagenic9 potential. *Cede-number of compound.

DDFR 1473 EDITION OI NOV 651W6SOhLETE 2ACLASSIF'IED 71-'- ..~SECURITY CLASSIFICATION OF THIS PAGE (Whenm Date Entered)

ABSTRACT

The mutagenic potential of 4 nitrophenyl bis(2-thienyl)phosphinate(41*); 4-nitrophenyl 2-furyl(methyl)phosphinate (72*); 4-cyanophenylbis(2-furyl)phosphinate (82*); 4-nitrophenyl bis(2-furyl)phosphinate(87*) was assessed by using the Ames Salmonella/Mammalian MicrosomeMutagenicity Assay. Tester strains TA 98, TA 100, TA '535, TA 1537,and 1538 were exposed to doses ranging from I mg/plate to 3.2 x 10-4mg/plate. It was determined that none of the tested substances hadmutagenic potential.

* Code number for compound.

I

rA ccassi( For - - ANTIS GRA&IDTIC TAB ElUnannounced 0Juztificatio

ByDistribution/

Availability CodesjAvail and/or

Dist Special

PREFACE

AMES ASSAY REPORT:

SUBSTANCE CODE NO.

4 nitrophenyl bis(2-thienyl)phosphinate 414-nitrophenyl 2-furyl(methyl)phosphinate 72

4-cyanophenyl bis(2-furyl)phosphinate 824-nitrophenyl bis(2-furyl)phosphinate 87

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

SPONSOR: Biomedical Laboratory, Aberdeen Proving GroundsAberdeen, MD 21005

PROJECT: Toxicity Testing of Phosphinate Compounds - 35162772A875

GLP STUDY NUMBER: 81013

STUDY DIRECTOR: LTC John T. Fruin D.V.M.,PhD.CO-PRINCIPAL INVESTIGATORS: SSG Freddica R. Pulliam, B.S.

SP5 Leonard J. Sauers, B.A.

RAW DATA: A copy of the final report, study protocol and retired SOPsuill be maintained in the LAIR archives. Thst substanceswere provided by sponsor. Chemical, analytical, stability,

purity, etc. data are available from the sponsor.

PURPOSE: To determine the mutagenic potential of the above compounds

using the Ames Assay. Tester strains TA 98, TA 100, TA1535, TA 1537, and TA 1538 were used.

iii

ACKNOWLEDGMENTS

The authors wish to thank John Dacey and SP4 Larry Mullen,

BS for their assistance in performing the research and for help

in preparation of.this report.

lv

Signatures of Principal ScientistsInvolved in the Study

We, the undersigned, believe the study, GLP number 81013, describedin this report to be scientifically sound and the results and inter-pretation to be valid. The study was conducted to comply to the bestof our ability with the Good Laboratory Practice Regulations outlined

by the Food and Drug Administration.

FEDICA R. PULLIAM, BS Date i/JOHN T. FRUIN, DVM, PhD_ fate

SSG LTC, VC

Co-Investigator Study Director

• t~b .L ,.~di~-

EONARD J. UIAURS, BA tePS

Co-Investigator

I

VI

DEPARTMENT OF THE ARMYLETTERMAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

'>J~J~C: R~ p ~lof' GLP '1ompl~i,,-ric

~r1 :r-by Ixcrtify I2u- in r(eI.tion to LkIR GLP study 310I the following

i.-.-to.,.fth no 4I' indsn- ar eportpd qunrterly, tihus this

nspEr,1,t-in is.- also jn,-Ljt'~d in the July 191report to raa-nw.'E(lnT.

Qulity Assurance Offic(-er

vi

TABLE OF CONTENTS

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

Preface ..................................................... ii

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 ..... I

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

METHODS

Rationale for Dosage Levels and Response Tabulations .... 3

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

Statistical Analysis .................................... 4

RESULTS AND DISCUSSION ..................................... 4

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

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

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

APPENDIX (Tables I through 6) ................................. 7

DISTRIBUTION LIST ........................................... 28

vii

IA

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 assessmentof 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 mutagenic

activity and therefore carcinogenic probability (1). It relies onbasic genetic principles and allows for the incorporation of amammalian microsome enzyme system to increase sensitivity through

enzymatically altering the test substance into an active metabolite.It has proven highly effective in assessing human risk (I).

Description of Test (Rationale for the selection of strains)

The test was developed by Bruce Ames, Ph.D. from the University

of California-Berkeley. The test involves the use of several differ-ent genetically altered strains of Salmonella typhimurium, each with aspecific mutation in the histidine operon (2). The test substancedemonstrates mutagenic potential if it is able to revert the mutationin the bacterial histidine operon back to the wild type and thusreestablish 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 revertantsis significantly greater than the spontaneous reversion rate, then

the test substance physically altered the locus involved in theoperon's mutation and is able to induce point mutations and geneticdamage (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 moleculesto enter the bacteria. Each strain has another induced mutationwhich causes loss of excision repair mechanisms. Since manychemicals are not by themselves mutagenic but have to be activated byan enzymatic process, a mammalian microsome system is incorporated.These microsomal enzymes are obtained from livers of rats induced

with Aroclor 1254; the enzymes allow for the expression of the

metabolites in the mammalian system. This activated rat liver

microsomal enzyme homogenate is termed S-9.

Description of Strains (History of the strains used, methods to

monitor the integrity of the organisms, and data pertaining to

current and historical controls and spontaneous reversion rates)

The test consists of using five different strains of Salmonella

typhimurium that are unable to grow in absence of histidine becavseof a specific mutation in the histidine operon. This histidinerequirement is verified by attempting to grow the tester strains on

minimal glucose agar (MGA) plates, both with and without histidine.The dependence on this amino acid is shown when growth occurs only in

4 its presence. The plasmids in strains TA 98 and TA 100 contain anampicillin resistant R factor. Strains deficient in this plasmiddemonstrate a zone of growth inhibition around an ampicillin

-1 impregnated disc. The alteration of the LP layer allows uptake bythe Salmonella of larger molecules. If a crystal violet impregnateddisc is placed onto a plate containing any one of the bacterialstrains, a zone of growth inhibition will occur because the LP layeris altered. The absence of excision repair mechanisms can bedetermined by using ultraviolet (UV) light. These mechanismsfunction primarily by repairing photodimers between pyrimidine bases;

exposure of bacteria to UV light will activate the formation of thesedimers and cause cell lethality, since excision of these photodimerscan not be made. The genetic mutation resulting in UV sensitivityalso induces a dependence by the Salmonella to biotin. Therefore,this vitamin must be added. In order to prove that the bacteria areresponsive to the mutation process, positive controls are run withknown mutagens. If after exposure to the positive control substance,a larger number of revertants are obtained, then the bacteria areadequately responsive. Sterility controls are performed to determinethe presence of contamination. Sterility of the test compound isalso confirmed in each first dilution. Verification of the testerstrains occurs spontaneously with the running of each assay. Thevalue of the spontaneous reversion rate is obtained using the sameinoculum of bacteria 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 if

deviations from the set trends have occurred.

We compared the spontaneous reversion values with our ownhistorical values and those cited by Ames et al (2). Ourconclusions are based on the spontaneous reversion rate compared tothe experimentally induced rate of mutation. When operatingeffectively, these strains detect substances that cause base pair

2

mutations (TA 1535, TA 100) and frameshift mutations (TA 1537, TA1538 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. This

toxicity level was found by using MGA pla es, various concen-trations of the substance, and approximately 10 cells of TA 100 perplate, unless otherwise specified. Top agar containing trace amountsof histidine and biotin were placed on MGA plates. TA 100 is usedbecause it is the most sensitive strain. Strain verification was

confirmed 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 fewtimes and potentially express a mutation. When the histidine andbiotin supplies are exhausted, only those bacteria that reverted tothe prototrophic phenotype will continue to reproduce and form macro-colonies; the remainder of the bacteria comprises the background lawn.The minimum toxic level is defined as the lowest serial dilution atwhich decreased macrocolony formation, below that of the spontaneousrevertant rate, and an observable reduction in the density of thebackground lawn occurs.) A maximum dose of 1 mg/plate is used when notoxicity is observed. The densities 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. Inthe actual experiment, 0.1ml of the particular strain of Salmonella(10 cells) and the specific dilutions of the test substance wereadded to 2 ml of molten top agar, which contained trace amounts ofhistidine and biotin. Since survival is better from cultures whichhave just passed the log phase, the Salmonella strains were used 16hours (maximum) after initial inoculation into nutrient broth. Thedose of the test substance spanned more than a 1000- fold, decreasing

from the minimum toxic level by a dilution factor of 5. All thesubstances were te3ted with and without S-9 microsome fraction. TheS-9 mixture which was previously titered at an optimal strength wasadded to the molten top agar. After all the ingredients were added,the top agar was vortexed, then overlayered on minimum glucose agarplates. These plates contained 2% glucose and Vogel Bonner "E'Concentrate (4). The water used in this medium and all reagents camefrom a polymetric system. Plates were incubated, upside down in thedark at 37 C for 48 hours. Plates were prepared in triplicate andthe average revertant counts were recorded. The corresponding number

3

of revertants obtained was compared to the number of spontaneous-4 revertants; the conclusions were recorded statistically. A

correlated dose response is considered necessary to declare asubstance as a mutagen. Commoner (5), in his report, "Reliablilty ofBacterial Mutagenesis Techniques to Distinguish Carcinogenic andNon-Carcinogenic Chemical," and McCann et al (1) in their paper,"Detection of Carcinogens as Mutagen: Assay of over 300 Chemicals,"have concurred on the test's ability to detect mutagenic potential.

Statistical Analysis

Quantitative evaluation was ascertained by two independent

methods. Ames et al (2) assumed that a compound which caused twicethe spontaneous reversion rate is mutagenic. Commoner (5), developedthe MUTAR Ratio, which is stated in the following equation:

MUTAR = (E - C)/CAv

Here, C is the number of spontaneous revertant colonies on controlplates obtained on the same day and with the same treatment andstrains. E is the number of revertants in response to the compound;C A is the number of spontaneous revertants on control platesca culated from historical records. The explanation of the resultsof this equationi can be determined by the method of Commoner (5).This variation determines the probability of correctly classifyingsubstances as carcinogens on the basis of their mutagenic activity.The E values were recorded by strain, with and without S-9. Valuesfor C and CAV were recorded separately.

We used the formula and logged all values for our permanent records.

RESULTS AND DISCUSSION

I Throughout this report, each of the test substances will bereferred to by the respective code number:

Substance Code No.

4 nitrophenyl bis(2-thienyl)phosphinate 414 -nitrophenyl 2-furyl(methyl)phosphinate 724 -cyanophenyl bis(2-furyl)phosphlnate 824 -nitrophenyl his(2-furyl)phosphinate 87

l4

On 1 June 1981, the Toxicity Level Determination was performed

on the 4 test chemicals. All positive, negative and sterility

controls for this experiment were normal (Table I). At the highestdose used, 1.0 mg/plate, no toxicity was observed (Table 2A-2D).

On 17 June 1981, the Ames Assay was performed using the 4 test

substances. For this experiment, all sterility and strain

verification controls were normal (Table 3). Expected responses were

observed for all negative and positive controls (Table 4).

For all the chemicals tested, there were no incidence of

mutagenicity (Table 5A-5D).

The MUTAR values listed in Tables 6A-6D were within the normallimits.

CONCLUSION

On the basis of the Ames Assay , test compounds 41, 72, 82, and87 are not mutagenic at the levels tested.

RECOMMENDATION

We recommend that organophosphinate compounds 41, 72, 82, and 87

be tested by using other toxicological testing systems if efficacy

tests show these chemicals to be promising antidotes.

REFERENCES

1. McCANN, J., E. CHOI, E. YAMASAKI, and B. N. AMES. Detection ofcarcinogens as mutagens in the Salmonella/microsome test: Assay of300 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 microsome mutagenicitytest, I March 1981

4. VOGEL, H. J. and D. H. BONNER. Acetylornithinase of E. coli: Partial>1 purification and same properties. J Biol Chem 218: 97-106, 19565. COMMONER, B. Reliability of the bacterial mutagenesis techniques to

distinguish carcinogenic and non-carcinogenic chemicals. EPA 600/176-022, 1976

I

*1

.!6

LIST OF TABLES

Code Page

Table I Strain Verification for Toxicity Level 9Determination

Tir

Table 2A Toxicity Level Determination 41 10

Table 2C Toxicity Level Determination 82 12

Table 2D Toxicity Level Determination 872 1

Table 2D Toxicity Level Determination 87 13

Table 3 Strain Verification and Sterility Controls 14

Table 4 Positive and Negative Controls 15

Table 5A Ames Assay Worksheet 41 16

Table 5B Ames Assay Worksheet 72 18

Table 5C Ames Assay Worksheet 82 20

Table 5D Ames Assay Worksheet 87 22

Table 6A Mutagenic Activity Ratio 41 24

Table 6B Mutagenic Activity Ratio 72 25

Table 6C Mutagenic Activity Ratio 82 26

Table 6D Mutagenic Activity Ratio 87 27

APPENDIX

, - , , , i II II I Il l 7

TABLE 1

STRAIN VERIFICATION FOR TOXICITY LEVEL DETERMINATION4 Salmonel la/Microsome Assay

Histidine Ampicillin uvr-B rfa Crystal Sterility ResponseStrain No. Requirements Resistance Deletion Violet Control(a

.1TA 100 NG G NG 15.46 mm NG +

TA 1537 NG NGNG 14.11 mm NG +

1TG NA G NA NA +

Diluent NA NA NA NA NG +

Positive Control - MNNG -Average -161;

TestCompound (s)

*(a 1 ~ NA NA NA NA NG +(b 2 NA NA NAN G+

(~) Z. NANANA NA NG+

32 NA NA NA NA NG +

(e A~A NA NA NA NA NA NA

G =Growth; NG =No Growth; NT = Not Tested; NA =Not Applicable;WT =Wild Type; (a) + =Expected Response; -=Unexpected Response

Spontaneous Revertants

Strain Time Average

TA 100 Beginning 146 148 155 140

TA 100 End 1122 118 1149

Test Inculated By: Sauers. Pulliam. Dacev. Mullen Date 1 June 1981

Test Read By: Sauers, Pulliam _____Date 3 June 1981

9

BIZ~D~@ PmDIA&.=ma yIjin

TABLE 2A

TOXICITY LEVEL DETERMIINATION4 Salmonel la/Microsome Assay

Substance assayed: (1) Code #41 (2) _ ___________

(3) _________ (4) ____________(5)_____________

Date: 3 June 1981 Performed by: Sauers, Pulliam, Dacey. Mullen

Substance dissolved in: (1) D14SO (2) ______(3) ______(4) ______(5) _______

Visual estimation of background lawn onNutrient Agar Plates: NG =no growth

ST =slight growthI NL -normal growthTA 100Revertant Plate Count

Test Compound BackgroundConcentration Plate #1 Plate #2 Plate #3 _ Average Lawn

1.0 mg/pl 135 105 138 126 NL

10-1 125 150 106 127 NL

10-2 121 124 117 121 NL

10-3 138 132 126 132 NL

10-4 128 118 132-- 126 NL

105134 125 152 137 11L

10-6 154 139 123 139 NL

10- 139 160 179 159 NL

10

TABLE 28

4 ~TOMM~tY LEVEL 0ETERMlNATIONSalmonel la/Microsome Assay

Substance assayed: (1) Code #72 (2) _ ___________

(3) ___________(4) _ __________(5) ____________

Date: 3 June 1981 Performed by: Pulliam, Sauers, Dacey, Mullen

Substance dissolved in: (1) DMSO (2) ______(3) ______4 ~ ~(4) _______(5) ________Visual estimation of background lawn onNutrient Agar Plates: NG = no growth

ST = slight growthNL =normal growth

TA 100-$ Revertant Plate Count

Test Compound BackgroundConcentration Plate #1 Plate #2 Plate #3 Average Lawn

1.0 mg/pl 180 194 166 180 NL

10-1 193 232 228 218 KL

10-2 206 193 181 193 NL

10-3 194 193 190 192 NL

10- 130 135 113 126 NL

10-5 125 116 89 110 NL

10-6 108 113 142 121 NL

107159 140 146 148 NL

______ _________

K TABLE 2CTOXICITY LEVEL DETERMINAT T NSalmonella/Microsome Assay

Substance assayed: (1) Code #82 (2) _ ___________

(3) ___________(4) ____________(5)_____________

Date: 3 June 1981 Performed by: Sauers, Pulliam, Dacey, Mullen

Substance dissolved in: (1) DMSO (2) ______(3) ______

(4)______ (5)________Visual estimation of background lawn onNutrient Agar Plates: NG = no growth

ST = slight growthI NL = normal growth

TA 100

ITest Compound RvratP teCutBackgroundConcentration Plate #1 Plate #2 Plate #3 Average Lawn

1.0 mg/pl 141 148 108 132 NL

10-1 108 134 154 132 NL

10-2 132 102 142 125 NL

103133 148 144 142 NL

10-4 130 143 164 146 NL

10-5 154 125 123 134 NL

10-6 136 123 109 123 NL

10-7 139 132 139 137 NL

I 12

TABLE 2D

TOXICITY LEVEL DETERMINATION

4 Salmonella/Microsome As,.ay

Substance assayed: (1) Code #87 (2) _ ___________

(3) ___________(4) ____________(5)_____________

Date: 3 June 1981 Performed by: Sauers, Pulliam, Dacey, Mullen

Substance dissolved in: (1) DMSO (2) ______(3) ______

(4) _______ (5)________Visual estimation of background lawn onNutrient Agar Plates: NG =no growth

ST = slight growth

TA 100NL = normal growth

Revertant Plate Count.1Test Compound Background

Concentration Plate #1 Plate #2 Plate #3 Average Lawn

1.0 mg/pl 125 103 121 116 NL

10-1 167 158 128 151 NL

102140 123 132 132 NL

10-3 140 158 131 143 NL

__0___4 141 119 136 132 NL

10-5 139 97 123 120 NL410-6 139 165 116 140 NL10-7 122 140 182 148 NL

13

j 0 + ++ + + )Un-0

2.- - 0. Ct -jA4 -) u0

-o~ 2n ~ .

0 1O C) LO

>4 Ci 0'J - - 1

- 14 0

2 Ei

UJ L) DCD CD C3 DD-j I I CD E- ' w - .

X 0

Zf 0 oI C.)

- o -,5 Z: r, 0. 0. 4

M 22a m 04

,414

4\ 00 k10r. CCoC~ C )

F- C\ koLf -- ..-r

00

x Q)

00

00

(c) C C

00

00

.ro In - CDL ~ (U)Q

00

zr .-

-1 C4 00 ("E' l L

0 li C-.

m~ 2l C'.) 0

z 00 -00 o-c

4o0 0 cu 0 d)0

>1 16

4-)A

4 - . -~ CM - C-k 0o .- C-

Cfl - : - C) LO 00 ,

00 - kC o

V) C) C- a) 00 C

ri t C 00- ~ C- cok

"a CJM C C - ~i. C)C

u (n. C)

m CC '--CO c c; C -L C :) C:)N

CM CZ -. -M CQ 0*1 00

-i 4-i

-o 0000 C)

"; C.iL) 0k

C~C)

zC) C) C)j L

C) C) V)

Q 0 0 j 0 Q)

-4-)

c'.J - .l-.

(\i 00* N'- U.J C 0

0 n O o u L k . r-

m " in -

- ~ c CDo * .io -- ccc-j 0 o -n

-~c Cn - -

col co o ^ZJ in-L 0- -. -r

C3,

0 ,0 d) 0 d

-4-a) (CS MU

-o Co

- C7) cl.)

18

-r 7-(NL ) >40Ci -:I r r

-~~ LO-- \

.-) -zj- r- *c n Cto C'j- rm-

-1.1!Ij L

Voo .~r- C~)j- LLn v-o at

,- to("1 0

co r- 00

Z. L.- LO - r -'to LC)~Ct

>1 >1 c '

a- CA) CLA CJN- -iC'i LA A)C-- -~ -4- -

C) to ) i- C

j ,-E~ t.0 M 0 0-0 co C

E C)NC

uE C) C) CD

WN (NJ (UJ

0 0 0

19

'- .- Z 0 C CenL LO a, u..~

- - ,-. - - - -f%. - C'j-

-4 M ,-: -2 - - I C

-A 0

E 4

C) > - L

0~~00LL)- ~ -- ~ C

0 -j'0 O

t. O

0 Qn 0) () 0.. L)

z ~ t CLr.o~ sj c

0)e

-0 0

02

ID -4 ccc

ll ~ r- O-r

C.,~~,C c- po C

-., C\.

r- ~ 00m Y~fC,7 Cc C3\1 r-CCJ

C'. C\J * C'

cn oc

0 W. 0- (

a) 4-)P -

Sfl CL-.-

CL -

C)S E; C

0U CD) ) C)

- h-- i C)L) C'

Az 41-w

0 )0 a)0 a)

C C 21

:d-C\j k 400C") C'.j - - C.

en .C-.-

I -

In; C) C O N

43 N.04 'LC .0

z~ C)Uj ~ ~ ~ ~ ~ ~ C .- 2 f - C):r 0)Co lZ-.- N- -

-:: C fl 00~ C'J co mf 3--

w~ 00-*:j - c

ION C, ~ C-N~N ---

t3 C) CCD

U--

C) 03 a)

$4-)

4-3(7) U)) (a

0z co i)0 0j CL E4C >4C >

-- , L) -4- en

4-c1 00 00E: 1 I~ -0 C)

NOME==

- - c.J ~ -C)(n in C).*0 '

r- 0 -0 -. -.

: - kz '.01.-C- - ,

C) ~~i

- - o

C40--. C) -

~ flj- ~ - - ~C) -*

oj co 00 -r-

-j P.. -C '

opC~ cdl - -- M~ r- 3 r f C

-~~~ %.0 C)'0~ ~ ) .~

L)U U .'- (ACCO ~ C0 ) 0 C) 01- -u i a)

1 a > -

W. CIL

00 C

0 E) CD CD$C C C00 00

E 0.40 0 0 0 L

CO - 23

TABLE 6A

MUTAGENIC ACTIVITY RATIO

Substance Assayed: Code #41 Dissolved in: DMSO

Study, Nu:riber: 81013 Date: 15 July 1981 By: Sauers

Concentration Strain MU~TAR MUTA Concentration Strain IMUTAR MUTA

___________(act) _ ___(actl

1.0 mg/plate TA 98 0.32 * 0.008 mg/plate TA 1535 * 0.26

0.2 mg/plate TA 98 0.32 * 0.0016 mg/pl. TA 1535 * *

0.04 mg/olate TA 98 0.24 * 0.00032 mg/pl. TA 1535 0.27 0.32

).008 mg/plate TA 98 * 0.24

).0016 mg/pl. IA 98 0.2 * 1.0 mg/plate TA 1537 *

0.00032 mg/Ql. TA 98 0.4 * 0.2 mq/plate TA 1537 *

0.04 mg/plate TA 1537 0.15 *

1.0 rin/plate TA 100 0.25 * .008 mg/plate TA 1537 0.15 *

0.2 mg/plate TA 100 0.35 0.05 0.0016 mg/Dl. TA.1537 * *

9.04 mq/plate TA 100 0.17 0.02 000032 ma/pl. TAL 5 r .4 n .5_

4.008 mg/plate TA 100 0.05 0.42 1G.0016 mogll. TA ion L2 n 1.0 molate fTA 1538a.3

-0 0032 mQ/D. TA 1 006 fi0_ 1 0.2 mg/Llate TA 15305 *

0.04 m/late TA 1538 0.27 *

]_.0 mg/plate TA 153E ) .008 ma/plat 5TA153q 0 _21

0.2 mg/plate TA 153 * * ).0016 m/pl. TA 1538 0.48 *

0.04 mn/plate TA 153 * 0.13 0.0032 mg/pl TA 153B10.37 0.07

(, ct): S- o fraction was added

i Icul.tLed '. tlue r. suIted in a negative MUTAR or zero MUTAR

24

TABLE 6B

MUTAGENIC ACTIVITY RATIO

Substance Assayed: Code #72 Dissolved in: DMSO

Study Number: 31013 Date: 15 July 1981 By: Sauers

Concentration Strain MUTAR MUTAR Concentration Strain MUTAR UTAF(act) (.ct

1.0 mg/plate TA 98 0.12 * 0.008 mg/plate TA 1535 * *

0.2 mg/plate TA 98 * * 0.0016 mg/pl. TA 1535 * 0.060.04 mg/plate TA 98 9.36 * 0.00032 mg/pl. TA 1535 * 0.26

0.008 mg/plate TA 98 * *

0.0016 mg/pl. TA 98 0.24 * 1.0 mg/plate TA 1537 0.46

0.00032 mg/pl. TA 98 0.16 * 0.2 mg/plate TA 1537 * *

10.04 mg/plate TA 1537 0.15 *

1.0 mg/plate TA 100 0.14 * 0.008 mg/plate TA 1537 0.15 *

0.2 mg/plate TA 100 0.28 * .0016 mg/pl. TA 1537 * *

0.04 mg/plate TA 100 0.08 * .00032 mg/pl. TA 1537 * *

3.008 mg/plate TA 100 0.17 *

.0016 mg/pl. TA 100 * * 1.0 m/Dlate TAJ538 * *

0.00032 ng/pl. TA 100 0.14 0.01 0.2 mg/plate TA.1538 0.4. *

0.04 mg/plate TA 1538 0.48 *

1.0 mg/plate TA 1535 * * 0.008 mq/plate TA 1538 0.27 *

0.2 mg/plate TA 1535 * * 0.0016 m/pl. TA 1538 0.27 *

P.04 mg/plate TA 1535 * 0.19 0.00032 mng/pl. TA 1538 0.32 0.07

(ict): 2-9 fraction was added

ci: cll , 1 L~d vilue resulLed in a negativc MUT\R or zero MUTAR

25

TABLE 6C

NUTAGENIC ACTIVITY RATIO

Substance Assayed: Code #82 Dissolved in: DMSO

Study Number: 81013 Date: 15 July 1981 By: Sauers

Concentration Strain NUTAR MUTA Concentration Strain NUTAR MLT(act) (act)___

1.0 mg/plate TA 98 0.12 * 0.008 mg/plate TA 1535 * 0.06

0.2 mg/plate TA 98 0.44 * 0.0016 mg/pl. TA 1535 * *

0.04 mg/plate TA 98 * * 0.00032 mg/pl. TA 1535 * 0.13

0.008 mg/platelTA 98 0.08 *

0.0016 mg/pl. TA 98 0.24 * 1.0 mg/plate TA 1537 * *

0.00032 mg/pl. TA 98 0.32 * 0.2 mg/plate TA 1537 0.31 *

S______0.04 mg/plate TA 1537 * *

1.0 mg/plate TA 100 0.07 * 0.008 mg/plate TA 1537 0.31 *

0.2 mg/plate TA 100 0.18 * 0.0016 mg/p1. TA 1537 0.15 *

0.04 mg/plate TA 100 D .00032 mg/pl. TA 1537 * *

).008 mg/plate TA 100 0.23 * _ _

O.O1 *gpl TA1 1.0 mg/plate TA 1538 0.21 0.14

0.00032 ma/1 TA~fl 10 .-7 2 mg/plate TA 1538 0.21 0.071

________.04 mg/plate TA 1538 0.27 *

1.0 mg/plate TA 153 .008 mg/plate TA 1538 0.16

0.2 mg/plate TA 1 * * ).0016 mg/pl. TA 1538 0.16

0.04 mg/plate 1~ * 2r- .00032 mg/pl. ITA 1538 10.16 *

O~ct S-9 tict ion wI. added

ri ICUIted XvIlUc resulted in a negativc NI2:\R or zero MUTAR

26

TABLE 6D

MUTACENIC ACTIVITY RATIO

Substance Assayed: Code #87 Dissolved in: DMS0

Study Number: 81013 Date: 15 July 1981 By: Sauers

Concentration Strain MUTAR MUTAR Concentration Strain MUTAR MUTA(act) (act 1

1.0 rag/plate TA 98 0.36 * 0.008 mq/Dlate TA 1535 0-.19_ 0.13

0.2 mg/plate TA 98 0.16 * 0.0016 mg/pl. TA 1535 *

0.04 mg/plate TA 98 0.16 * 0.00032 mg/pl. TA 1535 *

0.008 mg/plate TA 98 0.32 *

).0016 mg/p1. TA 98 0.12 * 1.0 mg/plate TA 1537 0.31 1

D.00032 mg/pl. TA 98 * * 0.2 mg/plate TA 1537 0.46 0.15

0.04 mg/plate TA 1537 0.31 *

1.0 mq/plate TA 1joc 0.17 * 0.008 mg/plate TA 1537 0.77 *

0.2 mg/plate TA 100 0.11 * 0.0016 mg/p1. TA 1537 *

0.04 mg/plate TA 100 0.06 * 0.00032 mc/pl. TA 1537 0.31 *

0.008 mg/plate TA 100 0.04 0.05

0.0016 mg/pl. TA 100 0.12 * 1.0 mglplate TA 1538- 05

0.00032 mg/pl. A 100 * * 0.2 mg/plate TA 1538 0.37 *

0.04 mg/plate TA 1538 0.21 *

1-. mg/olate TA*153 * * .008 mg/plate TA 1538 0.32 *

0.2 m/lat TA 153 * . 0016 mg/pl. TA 1538 0.11

,.04 mg/olate TA 15351* 0.000 3 2 mg/pl. TA 1538 * *

(act): S-9 fraction was added

cAIculated viluc resulted in a negative MIUTAIR or zero MUTAR

27

OFFICIAL DISTRIBUTION LIST

Comander DirectorUS Army Medical Research and Development Command Walter Reed Army Institute of ResearchATTN: SGRD-SI/ Mrs. Madigan Washington DC 20012Fort Detrick, Frederick MD 21701Defense Technical Information Center Commander

ATTN: 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 ResearchWashington DC 20314 Brooke Army Medical Center

Fort Sam Houston TX 78234

HQ DA (DASG-ZXA)WASI DC 20310

Superintendent CommanderAcademy of Health Sciences US Army Institute of Dental ResearchATTN: AHS-COM Washington DC 20012Fort Sam Houston TX 78234

Assistant Dean CommanderInstitute and Research Support US Army Medical BioengineeringUniformed Services University of Health Sciences Research and Development Laboratory6917 Arlington Road Fort Detrick, Frederick MD 21701Bethesda MD 20014

Commander CommanderUS Army Environmental Hygiene Agency US Army Aeromcdical Research LaboratoryAberdeen Proving Ground MD 21070 Fort Rucker AL 36362

US Army Research Office CommanderATTN: Chemical and Biological Sciences Division US Army Biomedical LaboratoryP.O. Box 1221 Aberdeen Proving GroundResearch Triangle Park NC 27709 Edgewood Arsenal MD 21010Biological Sciences Division Commander

Office of Naval Research Naval Medical Research InstituteArlington VA 22217 National Naval Medical Center

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

28