-
Michael G. MacNaughton, Lt Col, USAF, BSC
AMRL-TR-76-80 Deputy Director, Toxic Hazards Divisio
/1 p~5 47 7
MUTAGEN AND ONCOGEN STUDY OF METHYLHYDRAZINEFINAL REPORT
LITTON BIONETICS, INC.5516 NICHOLSON LANEKENSINGTON, MARYLAND
20795
DECEMBER 1976
Approved for public release; distribution unlimited
O13 o01
AEROSPACE MEDICAL RESEARCH LABORATORYAEROSPACE MEDICAL
DIVISIONAIR FORCE SYSTEMS COMMANDWRIGHT-PATTERSON AIR FORCE BASE,
OHIO 45433 STINFO CO"PY
-
NOTICES
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TECHNICAL REVIEW AND APPROVAL
AMRL-TR-76-80
This report has been reviewed by the Information Office (01) and
is releasable to the National Technical InformationService (NTIS).
At NTIS, it will be available to the general public, including
foreign nations.
This technical report has been reviewed and is approved for
publication.
FOR THE COMMANDER
VERNON L. CARTER, JR., COLONEL, USAF,.VCDeputy DirectorToxic
Hazards DivisionAerospace Medical Research Laboratory
AIR FORCE - 17 JANUARAY 77 - 100
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SECURITY CLASSIFICATION OF THIS PAGE (then Date Entered)
READ INSTRUCTIONSREPORT DOCUMENTATION PAGE BEFORE COMPLETING
FORMI. REPORT NUMBER 2. GOVT ACCESSION NO. 3. RECIPIENT'S CATALOG
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AM•RL-TR-7 6-80 0
4. TITLE (and Subtitle) 5. TYPE OF REPORT & PERIOD
COVERED
MUTAGEN AND ONCOGEN STUDY OF METHYLHYDRAZINE FINAL
6. 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, TASKAREA & WORK UNIT NUMBERSLitton Bionetics,
Incorporated 61102F, 2312, 2312V1,
5516 Nicholson Lane 2312V15
Kensington, MD 20795
11. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATEAerospace
Medical Research Laboratory DECEKBER 1976Aerospace Medical
Division, Air Force Sys Cmd 13. NUMBER OF PAGESWright-Patterson Air
Force Base, Ohio 45433 38
14. MONITORING AGENCY NAME & ADDRESS(If different from
Controlling Office) IS. SECURITY CLASS. (of this report)
Unclassified
15a. DECL ASSI FICATION/,DOWN GRADINGSCHEDULE
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 side if necessary and
identify by block number)
Methylhydraz ineMutagenesisDNA RepairMicrosome Activation
20. ABSTRACT (Continue on reverse side 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 rats and mice.Methylhydrazine (NH) was mutagenic for
Salmonella typhimurium TA-1535 insuspension tests. Results from all
other assays conducted as part of thisevaluation were considered to
be negative.
DD JARM 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.
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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.
LBI feels that the program conducted in this study offered as
many ofthe essential test criteria as possible for an accurate
evaluationof methylhydrazine (MH) for genetic activity. Selected
tests from TiersI, 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 brief
summaryof 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.
1
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TEST SUBSTANCE
ITOXICITY TESTING
S~II .. . .. . ... .I .. .. . .. ..... I . ... .
IN VITRO FORWARD MUTATION INDUCTION DOMINANTPLATE"A-- YS TESTS
USING OF UNSCHEDULED LETHAL
WITH SALMONELLA MOUSE LYMPHOMA DNA SYNTHESIS ASSAYS INTY P H
-RUiRMR-- 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
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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. Male mice and rats wereexposed to several dose
levels of the test compound for fivedays and then sequentially
mated to two virgin untreated femaleseach week over the period of
spermatogenesis. At mid-pregnancythe females were killed and scored
with respect to the numberof living and dead implants as well as to
the level of fertility.These results were then compared to data
from control animals.
E. Background
Hydrazines and MH react with pyrimidine bases, especially athigh
pH, breaking the pyrimidine ring and causing the removalof the base
from DNA (2). Based on this type of reaction withDNA, the potential
for hydrazine and/or derivatives to exhibitmutagenic, teratogenic,
and carcinogenic activity might beexpected.
Some information on the mutagenic and carcinogenic propertiesof
hydrazine and some of its derivatives has been published.
3
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Hydrazine in relatively high doses causes leukemia,
reticulumcell sarcoma, and lung adenomas in mice (3). Hydrazine
ismutagenic in T4 phage (4), S. typhimurium (5) and
Drosophilamelanogaster (D. melanogaster) (6), but was not active in
adominant lethal assay 7). Symetrical dimethylhydrazineinduced
mitotic gene 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 S. typhimurium.The assays
were conducted so that the compound was testeddirectly and in the
presence of a mouse liver microsome activa-tion 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 WP2uvrA"
(11).
1. Preparation of Tissue Homogenates and 9,000 x g 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.
4
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2. Reaction Mixture
The following reaction mixture was employed in the activa-tion
tests:
Component Final Concentration/ml
TPN (sodium salt) 6 1MIsocitric acid 35 pMTris buffer, pH 7.4 28
]MMgCI 2 2 pMHomogenate 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 S. typhimurium G-46, TA-1535, TA-1537,
TA-1538, TA-98, and TA-1O0 were employed along withE. coli strain
WP2uvrA and S. cerevisiae strain D4. Allcultures were monitored
regularly for stability of markersand contamination.
B. In Vitro Mutation Assay 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 R
CellFractions: 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 testchemicals were made in Flop
culture medium. Positivecontrol mutagens active directly and
requiring microsomeactivation were employed with all tests.
5
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4. Cells and Media: TK+/- BUdR-sensitive L5178Y mouselymphoma
cells were used in this assay. Growth medium(GM) for this line
consists of Fischer's mouse leukemiamedium supplemented with 10%
horse serum and sodiumpyruvate (Flo ). Cloning medium consists of
Fischer'smedium plus 2B% horse serum and agar (0.37%).
Selectivemedium for TK-/- cells was prepared by adding BUdR to
thecloning medium.
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 x g supernatant of mouse liver was prepared asdescribed
in the Microbial Assays. This supernatant wasthen centrifuged at
105,000 x g for 60 minutes and thepelleted microsomes resuspended
in 0.25 M sucrose. Thismicrosome preparation was added to the
reaction mixturein place of the 9,000 x 9 cell fraction.
2. Reaction Mixture
The reaction mixture was the same as used in the Micro-bial
Assays except purified microsomes replaced the 9,000x 9
supernatant.
3. Solvent and Control Compounds
Any stock solutions of chemicals were prepared and dilutedin
DMSO. Positive control chemicals that act directlyand require
microsome activation were employed.
4. Cells and Media
Human diploid embryonic lung cells (WI-38) were obtainedfrom
Flow Laboratories and used in these assays. The GMemployed was
Eagle's minimal essential medium (EMEM)supplemented with 10% fetal
calf serum (FCS). Step-downmedium (SM) was amino acid depleted to
reduce cell division,and hydroxyurea medium (HUM) was the medium
used toinhibit S phase growth. All media were based on EMEM.
6
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D. Dominant Lethal Assay (DLA)
The test chemical was tested in mouse and rat DLA. Allanimals
were dosed by intraperitoneal (IP) injections overfive consecutive
days, rested for two days, and mated.
1. Animals
a. Mice: Seven- to eight-week-old male random bredmice (ICR,
Flow) were used for treatment. Femalemice of the same strain, age,
and weight were usedfor the matings.
b. Rats: Ten- to twelve-week-old Sprague-Dawley malerats from a
random bred closed colony (Flow) wereused for treatment. Females of
the same strain,age, and weight were used for the matings.
2. Animal Husbandry
Each species was housed in separate rooms of our
animalfacility.
Male mice were housed five to a cage while being dosedwith the
compound, and then housed separately with twofemales for
mating.
All animals were offered a 4% fat diet and water adlibitum.
Water was acidified according to approvedlaboratory animal health
standards.
Animals were identified by ear punch. Sanitary cages andbedding
were used and changed two times per week at whichtimes water
containers were cleaned, sanitized, andfilled. Cages were
repositioned on racks once a week,and the racks 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
Triethylenemelamine (TEM) was administered IP at a levelof 0.3
mg/kg in 0.85% saline as a positive control.Negative control
animals received an IP injection of thecorn oil or water
solvents.
E. Test Chemicals
The test sample was obtained from the United States AirForce. MH
was a clear liquid (less than 100 ml) in an amberbottle.
7
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3. METHODS
A. In Vitro Microbial Assays
Overnight cultures of S. typhimurium TA-1535, TA-1537,
TA-1538,TA-98, TA-lO0, E. coli WP2 uvrA-, and S. cerevisiae D4
weregrown in complete broth. ApproximateTy 10- 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).
The data are presented in Table 1. Concentrations of the testand
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 GM. Toxicity
wasmeasured as loss in growth potential of the cells inducedby a
five-hour exposure to the chemical. Four doses wereselected from
the range of concentrations by using thehighest dose that showed no
loss in growth potential asthe penultimate dose and by bracketing
this with onehigher dose and two lower doses. Toxicity produced
bychemical treatment was monitored during the experiment.
8
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2. Test
a. Nonactivation assay
The procedure used was a modification of that reported byClive
and Spector (13). Prior to each treatment, cellswere cleansed of
spontaneous TK-/- by growing them in amedium containing thymidine,
hypoxanthine, methotrexate,and glycine (THMG). This medium permits
the survival ofonly those cells that produce the enzyme thymidine
kinase,and can therefore utilize the exogenous thymidine from
themedium. The test compound was added to the cleansed cellsin GM
at the predetermined doses for five hours. Themutagenized cells
were washed, fed, and allowed to expressin GM for three days. At
the end of this expressionperiod, TK-/- mutants were detected by
cloning the cells in theselection medium for ten days. Surviving
cell popula-tions were determined by plating diluted aliquots
innonselective GM.
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 GM. The desired number of cleansed
cells wasadded to this mixture, and the flask was incubatedon a
rotary shaker for five hours. The incubationperiod was terminated
by washing the cells twicewith GM. The washed mutagenized cells
were thenallowed to express for three days and were cloned
asindicated for the nonactivated cells.
c. Data Analysis
A mutation frequency for each test dose was deter-mined by
dividing the number of mutants/ml by thenumber of surviving
cells/ml (adjusted to 10-4) asindicated by plating efficiency.
These data arepresented in Table 2. Concentrations of the testand
positive 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 105cells in
a 100 mm tissue culture dish and grown to con-fluency in GM. Once
reaching confluency, the cells wereswitched to SM for five days.
The contact inhibitionimposed by confluency and the use of SM held
the cells ina nonproliferating state.
9
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2. Treatment
On the day of treatment, cells held in G, 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 3 HTdR. 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 3 HTdR 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 g. Additional lipid components
wereremoved by extraction in ethanol ether at 70C. Thispellet was
washed in 70% ethanol, further incubated at70C in 0.3N NaOH, and
the DNA extracted in 50 pl IN PCAat 70C. The DNA was separated into
two 25 ul aliquots.One of these was dissolved in 10 ml of hydromix
scintil-lation cocktail (Yorktown Co.) and counted in a
Beckmanliquid 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 pg of DNA. Followingliquid scintillation counting, the
data were combinedwith the DNA extraction values and expressed as
disintegra-tion per minute per pg DNA (DPM/pg DNA).
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 g 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.
10
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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.MH was administered to male mice
weighing 30 ± 2.5 gm. In
addition, MH was also dosed to male rats weighing 325 ± 25
gm.
1. Stock Solutions
The compound was prepared daily from stock solutions. MHwas
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 IP intoeach animal daily for five days. All dosages
and routesof administration were determined in consultation withDr.
Kenneth Back of the United States Air Force.
Calculated dosages are as follows:
Methylhydrazine
Mice
LD50 26.0 mg/kgHigh 1/10 LD50 2.6 mg/kgInt. 1/30 LD50 0.86
mg/kgLow 1/100 LD50 0.26 mg/kg
Rats
LD50 21.5 mg/kgHigh 1/10 LD50 2.15 mg/kgInt. 1/30 LD50 0.72
mg/kgLow 1/100 LD50 0.215 mg/kg
3. Animal Husbandry
a. Mice: Ten male mice were housed five animals to acage during
the five days of dosing. After two daysof rest, each male was caged
with two virgin femalesfrom Monday through Friday. This sequence
wasrepeated weekly with two new females each week foreight weeks.
Fourteen days from the midweek in
11
-
which they were caged with the males, females werekilled,
dissected, and the number of dead, living,and total embryos in the
uterus recorded on standardforms. These data were statistically
analyzed forindications of dominant lethality, and compared
withcontrol data for significance.
b. Rats: The protocol for the experiment using ratsdiffered from
that of the mice only in the sequenceof mating lasting seven weeks
and in that corporalutea were counted and recorded. These scores
wereused to determine evidence of compound-inducedpreimplantation
losses.
4. Data
The results of the Dominant Lethal Assay are given inTables 4 to
17.
4. RESULTS
The results of the genetic studies are presented in the
followingseries of tables:
12
-
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13
-
TABLE 1A
Results From Suspension Tests of MH Using
S. typhimurium Strain TA-1535a
Compound Population Mutant MutationTest Concentration/ml Counts
Counts Freq. (xlO- 8 )
Activation
Solvent Control (a) - 2698* 227 8.4(b) - 1369** 86 6.3
Positive Control DMN 100 vmoles 1264 4798 379.6MH 1 Pl 1043**
332 31.8MH 5 Ill 1958* 9855 503.3
"**Identifies treated group with appropriate solvent
control.
aThe suspension assay was conducted using the same mouse liver
activation
system described for the plate assays shown in Table I. The
protocol waschanged such that rather than add all test components
to semisolid overlayagar, they were suspended in saline and
incubated 60 minutes at 370C on arotary shaker. After incubation
samples were removed and assayed for thenumbers of surviving cells
and numbers of revertants. Mutation frequencieswere calculated for
each test. Dimethylnitrosamine (DMN) was used as thepositive
control compound.
14
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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.
VC counts in treated culturesI %CE = Cloning efficiency VC
counts in control cultures x 100
J GF = Growth factor Percent suspension growth (column F)
xPercent clonal growth (column I)
100
K MF(x 10-4) = Mutation frequency MC counts column G) X 10-4VC
counts (column Hx
15
-
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16
-
TABLE 3
MEASUREMENT OF UDS IN WI-38 CELLS TREATED WITH MH
Concentration Activitya Percent
Test (Ial/ml) DNA(pg) DPM Index of Controlb
Nonactivation
Solvent Control - 9.02 76 8.4 -Positive Control MNNG (lOig/ml)
1.76 86 48.9 582MH 0.1 9.64 102 10.6 126
0.5 16.88 83 4.9 581.0 9.25 102 10.4 124
Activation
Solvent Control - 13.64 78 5.7 -Positive Control 2AAF (301ig/ml)
2.45 60 24.5 430MH 0.1 22.56 145 6.4 112
0.5 14.85 79 5.3 931.0 Sample Lost
aActivity Index = DPM/pg DNA (DPM = Disintegrations/minute)
bpercent of Control Activity Index Treated X 100Activity Index
Control
17
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5. INTERPRETATIONS AND CONCLUSIONS
MH was evaluated for its genetic activity and ability to
stimulateDNA repair using a battery of in vitro and in vivo
assays.
A. Microbial Assays (Table 1)
There were no clear indications of mutagenic activity by MH
inany of the microbial assays reported in Table 1. The toxicityof
MH for bacteria and yeast was high and concentrations of 10pl/plate
were consistently too toxic to use.
Because hydrazines have certain properties similar to
nitrosamines,this compound was also examined for mutagenicity in a
suspensionassay. Dimethylnitrosamine was found to be inactive in
thestandard plate assay, but the chemical is highly mutagenicwhen
tested in a suspension procedure. The results for MHunder the same
test conditions were also found to be positive(Table ]A). Mutagenic
activity was observed at MH concentrationsof I pl and 5 pl/ml after
a 60-minute incubation with a mouseliver activation system.
B. Mouse Lymphoma Assays (Table 2)
The data from these tests were clearly negative.
C. UDS Assay in WI-38 Cells (Table 3)
The data from these tests were clearly negative. The samplecells
at the high dose level (activation assay) were lost bybreakage in
the centrifuge. However, there was no indicationof a trend or any
activity in an equivalent dose in nonactiva-tion tests.
D. Dominant Lethal Assays
Dominant lethality, which is indicated by a high percentage
ofdead implants to total implants, was not demonstrated by thedata
from either mice or rats administered MH. In comparison,the
positive control compound TEM demonstrated a clear domin-ant lethal
effect in mice during weeks 1 through 3 and in ratsduring weeks 1
through 5.
1. Mice (Tables 4-9)
Implant data for week 5 were not statistically analyzeddue to
the low number of pregnant females in the low andintermediate
dosage groups. The reason for the lowfertility in these animals is
unknown, but does notappear to be compound related since the
negative controlgroup also only contained one pregnant animal.
Implantdata from the other weeks did not reveal any of the
dose-related trends indicative of compound-induced
geneticactivity.
32
-
2. Rats (Tables 10-17)
Increased lethality compared to negative controls significantat
p < 0.01 was indicated for week 7 dosages. The ratiosof dead to
total implants for these animals fall within therange of variation
encountered for all levels of tests through-out the testing period;
arid, therefore, the statistical signi-ficance is associated with
the week 7 negative control animalsnot having any dead implants. In
addition, no dose-related trends were observed. In light of these
observationsand since the ratios are considerably less than those
forweeks 1 through 5 of the positive controls, we did notconsider
the data as indicating biologically significantactivity for MH in
rats.
E. Conclusions
MH was mutagenic in activation, microbial reversion tests ifthe
tests were conducted as suspension tests and not if conductedas
standard plate tests. This differential activity is similarto the
type of results obtained with dimethyl- and diethylnitro-samines.
Except for the mutagenicity of MH in tests with S.typhimurium
TA-1535, there were no indications of genetic -activity for MH in
any of the other tests conducted as part ofthis evaluation.
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.
33
-
10. Ames et al., Proc. Nat. Acad. Sci. (USA), 70:2281, 1973.
II. 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.
34SU S. GOVERNMENT PRINTING OFrICE: 1976 - 757-001/153