AMRL-TR-76-78

MUTAGEN AND ONCOGEN STUDY ON 1,1-DIMETHYLHYDRAZINE FINAL REPORT

LITTON BIONETICS, INC.5516 NICHOLSON LANEKENSINGTON, MARYLAND 20795

DECEMBER 1976

Approved for public release; distribution unlimited

AEROSPACE MEDICAL RESEARCH LABORATORYAEROSPACE MEDICAL DIVISIONAIR FORCE SYSTEMS COMMANDWRIGHT-PATTERSON AIR FORCE BASE, OHIO 45433

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TECHNICAL REVIEW AND APPROVALAMRL-TR-76-78

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FOR THE COMMANDER

VERNON L. CARTER, JR., COLONEL, USAF, VCDeputy DirectorToxic Hazards DivisionAerospace Medical Research Laboratory

AIR FORC[ - 17 JANUARY 77 - 100

SECURITY CLASSIFICATION OF THIS PAGE (When Data Entered)

READ INSTRUCTIONSREPORT DOCUMENTATION PAGE BEFORE COMPLETING FORM1. REPORT NUMBER 12, GOVT ACCESSION NO. 3. RECiPIENT'S CATALOG NUMBER

AMRL-TR- 76-784. TITLE (and Subtitle) S. TYPE OF REPORT & PERIOD COVERED

MUTAGEN AND ONCOGEN STUDY ON 1,I-DIMETHYLWDRAZI E FINAL6. PERFORMING ORG. REPORT NUMBER

7. AUTHOR(s) 8. CONTRACT OR GRANT NUMBER(s)

David Brusick, Ph.D., Dale W. Matheson, Ph.D. F33615-76-C-0515

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

Litton Bionetics, Incorporated AREA & WORK UNIT NUMBERS

5516 Nicholson Lane 61102F, 2312, 2312V1,Kensington, MD 20795 2312V115

"I1. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATEAerospace Medical Research Laboratory DECEMBER 1976Aerospace Medical Division, Air Force Sys Cmd 13. NUMBER OF PAGESWright-Patterson Air Force Base, Ohio 45433 27

"14. MONITORING AGENCY NAME & ADDRESS(if different from Controlling Office) 15. SECURITY CLASS. (of this report)

UnclassifiedISa. DECL ASSI FICATION/.DOWNGRADING

SCHEDULE

16. DISTRIBUTION STATEMENT (of this Report)

Approved for public release; distribution unlimited.

17. DISTRIBUTION STATEMENT (of the abstract entered In Block 20, if different from Report)

18. SUPPLEMENTARY NOTES

19. KEY WORDS (Continue on reverse aide if necessary and identify by block number)1,l-Dimethylhydrazine

MutagenesisDNA RepairMicrosome Activation

20. ABSTRACT (Continue on reverse aide If necessary and identify by block number)

A three tier test was organized into a matrix of assays employing microbialcells, mammalian cells in culture, and in vivo experiments in jice.1,1-dimethylhydrazine exhibited genetic activity in several in vitro assays.The responses were consistently obtained under conditions of microsomal enzymactivation suggesting the formation of an active metabolite. The dominantlethal test results were negative.

DD I JAN73 1473 EDITION OF I NOV 65 IS OBSOLETESECURITY CLASSIFICATION OF THIS PAGE (When Data Entered)

PREFACE

This research was initiated by the Toxicology Branch, Toxic HazardsDivision, Aerospace Medical Research Laboratory. Experiments wereperformed under Contract F33615-76-C-0515 by Litton Bionetics, Inc.,5516 Nicholson Lane, Kensington, Maryland 20795.

The experiments were conducted by David Brusick, Ph.D., and Dale W.Matheson, Ph.D., of Litton Bionetics, Inc., Kensington, Maryland 20795.Kenneth C. Back, Ph.D., was contract monitor for the Aerospace MedicalResearch Laboratory.

1. INTRODUCTION

The detection and subsequent confirmation of mutagenic substancescapable of producing germ cell mutations requires a multifacetedtesting program. The components of such a program should be ableto detect both point mutations and chromosomal aberrations sincethese two classes of genetic alterations represent the types oftransmissible mutations that are of concern to man. The testsincluded in a mutagenicity evaluation program for chemicals shouldnot only be sensitive and reproducible, but also relevant to normalexposure and pharmacological conditions encountered in the environ-ment. These latter two conditions are often difficult to achievesince good human model systems are lacking. It may be argued thatif a single toxicologic end point, e.g., mutation, can be demon-"strated in several different test species, then application of theresponse to a wide range of species, including man, can be made.Therefore, a mutagenicity evaluation program should contain aseries of assays covering many phylogenetic levels.

We feel that the program conducted in this study offered as many ofthe essential test criteria as possible for an accurate evaluationof l,l-dimethylhydrazine for genetic activity. Selected tests fromTiers I, II, and III were organized into a matrix of assays employingmicrobial cells, mammalian cells in culture, and in vivo experimentsin rats and mice.

Tests utilizing these organisms measured point mutations (forwardand reverse), chromosomal aberrations, and mitotic recombinationalevents induced by acute and subchronic exposure to the test sub-stance.

Figure 1 illustrates the composition of the test program preparedfor the genetic evaluation of the test substance. A briefsummary of each of the assays is listed as follows:

A. In Vitro Microbial Assays

In these assays, the test substance was evaluated for muta-genic and recombinogenic activity in strains of Salmonella andSaccharomyces, respectively. Metabolic activation of thecompound was obtained by combining hepatic microsomes withthe test system. Nonactivation and activation semiquantita-tive plate tests were conducted.

B. In Vitro Mutation Assay in Mammalian Cells

In this assay, the mutations were measured in cultured mousecells (L5178Y). Both direct and in vitro activation assayswere performed. The specific event detected by these cellswas forward mutation at the thymidine kinase (TK+/- - TK-/-)locus, which is an autosomal recessive trait. The combined invitro tests from A and B gave a very sensitive measurement ofthe test substance's ability to induce point mutations andmitotic recombination.

TEST SUBSTANCEITOXICITY TESTING

I I i i i II I I

IN VITRO FORWARD MUTATION INDUCTION DOMINANTPLATE-7A9ýYS TESTS USING OF UNSCHEDULED LETHAL

WITH SALMONELLA MOUSE LYMPHOMA DNA SYNTHESIS ASSAYS INTYPHiMU-RUR- L5178Y CELLS IN WI-38 MICE AND

CELLS RATS

DETECTION OF POINT MUTATIONS DETECTION OF DETECTION OFINDUCED CHROMOSOME

DNA REPAIR ABERRATIONS

EVALUATION FOR GENERAL ACTIVITY

FIGURE 1

COMPOSITION OF THE GENETIC EVALUATION PROGRAM

2

C. Unscheduled DNA Synthesis

A second component of the in vitro mammalian cell assay systemutilized the human diploid WI-38 strain of cells. This cellstrain obtained from human embryonic lung was used to measuretest chemical-induced DNA repair in cells not undergoingscheduled (S phase) DNA synthesis.

Normal DNA synthesis occurs in the S phase of the cell cyclewith little or no synthesis occurring in any of the otherphases (GO, G1, G2, or M). The detection of significant DNAsynthesis during these stages (UDS) is indicative of thestimulation of repair enzyme systems. Exposure of WI-38 cellsto various forms of radiation or to chemicals known to bemutagenic or carcinogenic has resulted in the stimulation ofUDS (1).

The detection of UDS in WI-38 cells involved exposure of thecells to the test chemical followed by the addition of tritiatedthymidine ( 3H-TdR) to the culture. If DNA damage has beeninduced, the 3H-TdR will be incorporated during the repair ofthe DNA. This incorporation can be detected by scintillationcounting.

D. Dominant Lethal Assay

This assay was designed to determine the ability of a compoundto induce genetic damage to the germ cells of treated male miceand rats leading to fetal wastage. Chromosome aberrations,including breaks, rearrangements, and deletions, are believedto produce the dominant lethality. The male mice were exposedto several dose levels of the test compound for five days andthen sequentially mated to two virgin untreated females eachweek over the period of spermatogenesis. At mid-pregnancy thefemales were killed and scored with respect to the number ofliving and dead implants as well as to the level of fertility.These results were then compared to data from control animals.This assay was designed to detect compounds capable of inducingchromosome alterations.

E. Background

Hydrazines and methylhydrazines react with pyrimidine bases,especially at high pH, breaking the pyrimidine ring and causingthe removal of the base from DNA (2). Based on this type ofreaction with DNA, the potential for hydrazine and/or deriva-tives to exhibit mutagenic, teratogenic, and carcinogenicactivity might be expected.

3

Some information on the mutagenic and carcinogenic propertiesof hydrazine and some of its derivatives has been published.Hydrazine in relatively high doses causes leukemia, reticulumcell sarcoma, and lung adenomas in mice (3). Hydrazine ismutagenic in T4 phage (4), Salmonella typhimurium (5) andDrosophila melanogaster (6), but was not active in a dominantlethal assay (7). Symetrical dimethylhydrazine induced mitoticgene conversion (8).

Because the test agent is structurally related to chemicalsthat are established mutagens and carcinogens, and because ofthe excellent correlation between mutagenicity and carcino-genicity, mutagenesis studies might provide insight intopotential toxicologic problems associated with the testagent.

2. MATERIALS

A. In Vitro Microbial Assays

The test chemical was examined in a series of microbialassays employing histidine-requiring mutants of Salmonellatyphimurium. The assays were conducted so that the compoundwas tested directly and in the presence of a mouse livermicrosome activation system.

The compound was evaluated at a minimum of four dose levelsunder both test conditions with the highest dose level showingsome evidence of toxicity. In addition to these tests, spottests (5) were conducted with the Salmonella mutants plusadditional strains of bacteria; S. typhimurium strain G-46 andE. coli strain WP2 uvrA- (11).

1. Preparation of Tissue Homogenates and 9,000 x a Cell Fractions

Male mice (sufficient to provide the necessary quantitiesof tissues) were killed by cranial blow, decapitated, andbled. Organs were immediately dissected from the animalusing aseptic techniques and placed in ice-cold 0.25 Msucrose buffered with Tris buffer at a pH of 7.4. Uponcollection of the desired quantity of organs, they werewashed twice with fresh buffered sucrose and completelyhomogenized with a motor-drive homogenizing unit at 4C.The whole organ homogenate obtained from this step wascentrifuged for 20 minutes at 9,000 x 9 in a refrigeratedcentrifuge. The supernatant from the centrifuged samplewas retained and frozen at -80C. Samples from thesepreparations were used for the activation studies.

2. Reaction Mixture

The following reaction mixture was employed in the activa-tion tests:

4

Component Final Concentration/ml

TPN (sodium salt) 6 pMIsocitric acid 35 1MTris buffer, pH 7.4 28 IjMMgCI 2 2 vIMHomogenate fraction equivalent to 25 mg

of wet tissue

3. Solvent and Control Compounds

Preparation and dilution of test compounds were done indimethylsulfoxide (DMSO). Positive control compoundswere included as reference points and to ensure that theassay was functioning with known mutagens. Direct actingmutagens were employed in nonactivation assays and mutagensrequiring microsomal activation were used in activationassays. The compounds and the concentrations employedare provided in the data tables.

4. Bacteria Cultures

Overnight cultures of Salmonella typhimurium G-46, TA-1535,TA-1537, TA-1538, TA-98, and TA-1O0 were employed alongwith E. coli strain WP2 uvrA- and Saccharomyces cerevisiaestrain D4. All cultures were monitored regularly forstability of markers and contamination.

B. In Vitro Mutation A in Mammalian Cells

The test chemical was tested for mutagenic activity in aforward mutation assay employing cultured mouse cells (L5178Y).The cell line is heterozygous for the Thymidine Kinase (TK+/-)gene and the assay detects homozygous TK-/- mutant clones.The compounds were tested directly and in the presence of amouse liver microsome activation system.

1. Preparation of Tissue Homogenates and 9,000 x a CellFraction: The activation system employed in this assaywas the same as described for the Microbial Assays.

2. Reaction Mixture: The same reaction mixture as describedfor the Microbial Assays was used for these studies.

3. Solvent and Control Compounds: Preparation of stockchemicals was done in DMSO. All dilutions of test chemicalswere made in F1 op culture medium. Positive controlmutagens active directly and requiring microsome activationwere employed with all tests.

5

4. Cells and Media: TK BUdR-sensitive L5178Y mouselymphoma cells were used in this assay. Growth mediumfor this line consists of Fischer's mouse leukemia mediumsupplemented with 10% horse serum and sodium pyruvate(Flop). Cloning medium consists of Fischer's medium plus20% gorse serum and agar (0.37%). Selective medium forTK-/- cells was prepared by adding BUdR to the cloningmedium.

C. Unscheduled DNA Synthesis (UDS) Assay

Nondividing WI-38 cells were exposed to three concentrationsof the test compound and 3H-thymidine. Treatment was directand under conditions of microsome activation. The amount of3H-thymidine incorporated into the DNA was measured by scin-tillation counting.

1. Preparation of Mouse Liver Microsomes: A 9,000 xsupernatant of mouse liver was prepared as described inthe Microbial Assays. This supernatant was then centri-fuged at 105,000 x 9 for 60 minutes and the pelletedmicrosomes resuspended in 0.25 M sucrose. This microsomepreparation was added to the reaction mixture in place ofthe 9,000 x 9 cell fraction.

2. Reaction Mixture: The reaction mixture was the same asused in the Microbial Assays except purified microsomesreplaced the 9,000 x 9 supernatant.

3. Solvent and Control Compounds: Any stock solutions ofchemicals were prepared and diluted in DMSO. Positivecontrol chemicals that act directly and require microsomeactivation were employed.

4. Cells and Media: Human diploid embryonic lung cells (WI-38) were obtained from Flow Laboratories and used inthese assays. The growth medium (GM) employed was Eagle'sminimal essential medium (EMEM) supplemented with 10%fetal calf serum. Step-down medium (SM) was amino aciddepleted to reduce cell division, and hydroxyurea medium(HUM) was the medium used to inhibit S phase growth. Allmedia were based on EMEM.

D. Dominant Lethal Assay (DLA)

The test chemical was tested in a standard subchronic mouseDLA. All animals were dosed by intraperitoneal injectionsover five consecutive days, rested for two days, and mated.

6

1. Animals: Seven- to eight-week-old male random bred mice(ICR, Flow) were used for treatment. Female mice of thesame strain, age, and weight were used for the matings.

2. Animal Husbandry: Male mice were housed five to a cagewhile being dosed with the compound, and then housedseparately with two females for mating. All animals wereoffered a 4% fat diet and water ad libitum. Water wasacidified according to approved laboratory animal healthstandards.

Animals were identified by ear punch. Sanitary cages andbedding were used and changed two times per week at whichtimes water containers were cleaned, sanitized and filled.Cages were repositioned on racks once a week, and theracks repositioned within rooms monthly. Personnelhandling animals or working with animal facilities wearhead and face masks as well as suitable garments.Individuals with respiratory or other overt infectionsare excluded from the animal facility.

3. Positive and Negative Control Chemicals: Triethyl-enemelamine (TEM) was administered intraperitoneally (IP)at a level of 0.3 mg/kg in 0.85% saline as a positivecontrol. Negative control animals received an IP injec-tion of the corn oil or water solvents.

E. Test Chemicals

The test sample was obtained from the United States AirForce. The test compound l,l-dimethylhydrazine (UDMH) was aclear liquid (less than 100 ml) in an amber bottle.

3. METHODS

A. In Vitro Microbial Assays

Overnight cultures of S. typhimurium TA-1535, TA-1537, TA-1538,TA-98, TA-IO0, E. coli WP2 uvrA-, and S. cerevisiae D4 weregrown in complete broth. Approximately 108 cells from aculture were added to test tubes containing 2.0 ml of moltenagar supplemented with biotin and a trace amount of histidine.Four dose levels of the test chemical were added to the appro-priate tubes and the contents poured over selective medium.In activation tests 0.5 ml aliquots of the reaction mixturecontaining the microsomes were added to the tubes containingcells and chemical just prior to pouring onto the selectivemedium. After the overlays solidified, the plates were placedin a 37C incubator for 48 to 72 hours. The plates were thenscored for the number of colonies growing in the agar overlay.Positive and solvent controls using both direct-acting muta-gens and promutagens that required metabolic activation wererun with each assay. Supplementary spot tests were alsoconducted according to the methods described by Ames et al.(12).

7

The data are presented in Table 1. Concentrations of the test

and positive control chemicals are given in the data tables.

B. In Vitro Mutation Assay in Mammalian Cells

1. Toxicity

The solubility, toxicity, and doses for the test chemicalwere determined prior to screening. The effect of thechemical on the survival of the indicator cells wasdetermined by exposing the cells to a wide range ofchemical concentrations in complete growth medium.Toxicity was measured as loss in growth potential of thecells induced by a five-hour exposure to the chemical.Four doses were selected from the range of concentrationby using the highest dose that showed no loss in growthpotential as the penultimate dose and by bracketing thiswith one higher dose and two lower doses. Toxicityproduced by chemical treatment was monitored during theexperiment.

2. Test

a. Nonactivation assay

The procedure used is a modification of that reportedby Clive and Spector (13). Prior to e ch treatment,cells were cleansed of spontaneous TK- by growingthem in a medium containing thymidine, hypoxanthine,methotrexate, and glycine (THMG). This mediumpermits the survival of only those cells thatproduce the enzyme thymidine kinase, and can thereforeutilize the exogenous thymidine from the medium.The test compound was added to the cleansed cells ingrowth medium at the predetermined doses for fivehours. The mutagenized cells were washed, fed, andallowed to express in growth medium for t~ree days.At the end of this expression period, TKImutants were detected by cloning the cells in theselection medium for ten days. Surviving cellpopulation were determined by plating diluted ali-quots in nonselective growth medium.

b. Activation Assay

The activation assay differs from the nonactivationassay in the following manner only. Two and fivetenths ml of the reaction mixture was added to 10 mlof growth medium. The desired number of cleansedcells was added to this mixture, and the flask wasincubated on a rotary shaker for five hours. Theincubation period was terminated by washing thecells twice with growth medium. The washed mutagen-ized cells were then allowed to express for threedays and were cloned as indicated for the nonacti-vated cells.

8

c. Data Analysis

A mutation frequency for each test dose was deter-mined by dividing the number of mutants/mil by thenumber of surviving cells/ml (adjusted to "10 4) asindicated by plating efficiency. These data arepresented in Table 2. Concentrations of the test andpositive control chemicals are given in the datatables.

C. Unscheduled DNA Synthesis

1. Cell Preparation

Normal human diploid WI-38 cells were seeded at 5 x I05cells in a 100 mm tissue culture dish and grown to conflu-ency in GM. Once reaching confluency, the cells wereswitched to SM for 5 days. The contact inhibition imposedby confluency and the use of SM held the cells in anonproliferating state.

2. Treatment

On the day of treatment, cells held in G1 phase wereplaced in HUM. After 30 minutes this medium was replacedby 5 ml of HUM containing the control or test chemicaland 1.0 pCi of 3HTdR. Each treatment was at three con-centrations. Exposure was terminated by washing thecells twice in cold BSS containing an excess of coldthymidine.

3. DNA Extraction and Measurement of 3HTdR Incorporation

Treated plates were frozen at -20C until processed.After thawing, the cells on the 100 mm plate were coveredwith 2.5% SDS in 1 x SSC and scraped from the dish with arubber policeman. The cells were washed and precipitatedfrom the SDS by three changes of 95% ethanol and centri-fuged at 10,000 x 9. Additional lipid components wereremoved by extraction in ethanol ether at 70C. Thispellet was washed in 70% ethanol, further incubated at70C in O.3N NaOH, and the DNA extracted in 50 pl IN PCAat 70C. The DNA was separated into two 25 1il aliquots.One of these was dissolved in 10 ml of hydromix scintil-lation cocktail (Yorktown Co.) and counted in a Becki;ianLiquid Scintillation spectrometer. The second aliquotwas added to 275 pl of IN PCA and read at 260 nm in aGilford spectrophotometer. The values were corrected forlight scatter and converted to Vg of DNA. Followin;gliquid scintillation counting, the data were combinedwith the DNA extraction values and expressed as Disintegra-,tion Per Minute Per pg DNA (DPM/pg DNA).

9

4. Activation Assays

The activation tests were conducted according to themethods described above except that 0.62 ml of a purifiedmicrosome preparation (100,000 x 9 pellet) was added tothe test mixture.

5. Dosage Determinations

Doses were determined from preliminary toxicity tests inwhich cells were seeded in 16 mm wells (Linbro plate).A wide range of concentrations was tested in the wells,and toxicity was monitored visually by altered cell mor-phology and adhesion. The three doses used in theexperiments were selected.

The results of these tests are given in Table 3. Theconcentrations of test and control compounds are given inthe data tables.

D. Dominant Lethal Assay

The dominant lethal assay is designed to assess the ability ofthe test compound or its metabolic products to reach thetestes of treated male animals and induce genetic activity inthe developing gametes during spermatogenesis.

l,l-dimethylhydrazine was administered to male mice weighing

30 ± 2.5 gms.

1. Stock Solutions

The compound was prepared daily from stock solutions.l,l-dimethylhydrazine was dissolved in distilled water.

2. Compound Administration

Dosages were determined from LD50 data supplied by thecontract monitor with a high dose of 1/10 the LD50, anintermediate dose at 1/3 the high level, and a low doseof 1/10 the high level. Compound was injected intra-peritoneally into each animal daily for five days. Alldosages and routes of administration were determined inconsultation with Dr. Kenneth Back of the United StatesAir Force.

10

Calculated dosages are as follows:

1,1l-dimethylhydrazine

Mice

LD50 125 mg/kgHigh 1/10 LD50 12.5 mg/kgInt. 1/30 LD50 4.2 mg/kgLow 1/100 LD50 1.25 mg/kg

3. Animal Husbandry

Ten male mice were housed five animals to a cage duringthe five days of dosing. After two days of rest, eachmale was caged with two virgin females from Monday throughFriday. This sequence was repeated weekly with two newfemales each week for eight weeks. Fourteen days fromthe midweek in which they were caged with the males,females were sacrificed, dissected, and the number ofdead, living, and total embryos in the uterus recorded onthe standard forms. These data were statistically analyzedfor indications of dominant lethality, and compared withcontrol data for significance.

4. Data

The results of the Dominant Lethal Assay are given in Tables4-9.

4. RESULTS

The results of the genetic studies are presented in the followingseries of tables:

11

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12

KEY

MOUSE LYMPHOMA ASSAY TABLE

COLUMN

A, B, C, D Day = Expression day cell counts (x 106)

E AGS = Represents cell population growth during expression.The value is obtained by subtracting the Day 1 countsfrom the terminal day counts.

F %GS = Percent suspension growth is obtained by expressingthe AGS values for treated cells as a percent of theAGS for the negative controls E treated x 100

E control

G MC = Mutant counts. The total number of colonies countedin the BUdR plates.

H VC = Viable counts. The total number of colonies countedin the VC plates.

I %CE = Cloning efficiency VC counts in treated culturesVC counts in control cultures x 100

J GF = Growth factor Percent suspension growth (column F) xPercent clonal growth (column I)

100

MF(x 10-4) = Mutation frequency MC counts (column C u column x 10-

13

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14

TABLE 3

MEASUREMENT OF UDS IN WI-38 CELLS TREATED WITH UDMH

Concentration Activity a Percent

Test (pl/ml) DNA(pg) DPM Index of Control

Nonactivation

Solvent Control 9.02 76 8.4 -Positive Control MNNG (lOig/ml) 1.76 86 48.9 582UDMH 0.1 9.26 84 7.7 92

0.5 4.45 54 12.1 1441.0 7.94 71 10.5 125

Activation

Solvent Control 13.64 78 5.7 -Positive Control 2-AAF (30g/ml) 2.45 60 24.5 430UDMH 0.1 6.54 78 11.9 209

0.5 8.88 120 13.5 2371.0 10.88 115 10.6 186

aActivity Index = DPM/pg DNA (DPM = Disintegrations/minute)

bpercent of Control = Activity Index Treated x 100Activity Index Control

15

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21. 2 00

5. INTERPRETATIONS AND CONCLUSIONS

l,l-dimethylhydrazine (UDMH) was evaluated for its genetic activityand ability to stimulate DNA repair using a battery of in vitro andin vivo assays.

A. Microbial Assays (Table 1)

There was no clear indication of any mutagenic activity by UDMHfor the microbial strains shown in the results section. Concen-trations sufficient to produce clear toxicity were used. Marginalresponses were noted in activation tests with Salmonella TA-98 andpossibly TA-1538 but neither of these responses was sufficientlystrong to be considered significant in and of themselves.

B. Mouse Lymphoma' Assay (Table 2)

The results of this assay show a moderately strong, dose-relatedmutagenic response to UDMH. This was especially true in themicrosome activation tests. In these tests, there was afifteen (15) fold increase in mutation frequency at 0.1 pl/ml.Concurrently, the absolute increase in numbers of TK-/- mutantswas between 2 and 3 fold greater than the solvent control. Thusselection of pre-existing mutants can be ruled out.

C. UDS A in WI-38 Cells (Table 3)

In these tests, there was no response in nonactivation tests.The data from activation studies indicate a possible effect(a greater than 200% increase in the Activity Index). However,the effect was dose related only over the two lower dose levelsand dropped off at 1.0 pIl/ml. Cellular toxicity at the high dosemight explain the response in this situation.

D. Dominant Lethal Assay in Mice (Tables 4-9)

No significant trends indicating the induction of dominant lethalitywere observed in this study. A decrease in reproductive performancewas noted at weeks 1 and 5, but this was not compound relatedsince it was observed in the controls as well. The reason forthis reduced fertility at week 5 was not determined. Reducedfertility during the first week of mating is normal.

E. Conclusions

The accumulated data from the test battery suggest that UDMH ismetabolically activated in vitro (liver microsomes) to an inter-mediate that possesses genetic activity. The forward mutationinduction of TK-/- mutants was the only strong response obtained,

22

but supported data from certain microbial tests (TA-98) and theDNA repair test in WI-38 cells were consistent in established atrend of activity although not sufficient to indicate activity ifexamined as an isolated study. The negative results from thedominant lethal assay are consistent with the mutagenic activityof hydrazine while it is also mutagenic in microbial tests but notactive in the dominant lethal assay. The chemical reactivity of theactive molecule may be responsible for the lack of response in thistest since other methylating agents (MNNG) and nonfunctionalalkylating agents (ý-propiolactone) are also negative in dominantlethal tests but mutagenic in a wide range of in vitro systems.Based on correlation data collected on the in vitro test systems,it can be concluded that UDMH exhibits biological activitiesconsistent with most known mutagens and carcinogens.

6. REFERENCES

1. Stich and Laishes, Pathobiology Annual, Vol. 3, p. 341, 1973.

2. Freese, Chemical Mutagens, Vol. 1, A. Hollaender (ed.), p. 1,Plenum Press, New York, 1971.

3. Juhasz, Potential Carcinogenic Hazards from Drugs, U.I.C.C.Monograph, Vol. 7, R. Truhart (ed.), p. 180, Springer-Verlag,Berlin, 1967.

4. Freese et al., Proc. Nat. Acad. Sci. (USA), 47:845, 1961.

5. Ames, Chemical Mutagens, Vol. 1, A. Hollaender (ed.), p. 267,Plenum Press, New York, 1971.

6. Auerbach, Science, 158:1141, 1967.

7. Epstein and Shafner, Nature, 219:385 1968.

8. Zimmermann and Schwaier, Naturwiss., 54:251, 1967.

9. Miller and Miller, Cancer Res., 25:1292, 1965.

10. Ames et al., Proc. Nat. Acad. Sci. (USA), 70:2281, 1973.

11. Green and Muriel, Mutation Res., 38:3, 1976.

12. Ames et al., Mutation Res., 31:347, 1975.

13. Clive and Spector, Mutation Res., 31:17, 1975.

23SU. S. GOVELRNMENT PRINTING OFFICE: 1976 - 757-001/151