The American environmental mutagen society

Mutation Research, 26 (I974) 443-461 ~!_)Elsevier Scientific Publishing Company, Amsterdam - Printed in The N e t h e r l a n d s

THE

AMERICAN

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MUTAGEN

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A b s t r a c t s of P a p e r s p r e s e n t e d a t t h e F o u r t h A n n u a l M e e t i n g , M a r c h 8 t h - I l t h ,

I974

The Washington Hilton Hotel, Washington D.C. ( U . S . A . )

CONTENTS I NAUMAN, C. H., R. VILLALOBOS-PIETRINI AND R. C. SAUTKZILIS (Upton, N.Y.), Response of a

mutable clone of Tradescantia to gaseous chemical mutagens and to ionizing radiation. 2 SPARROW, A. H., AND L. A. SCHAIREa, (Upton, N.Y.), Mutagenic response of Tradescantia to t r e a t m e n t with X-rays, EMS, DBE, ozone, SO~, N~O and sevcrul insecticides. ] I-IICKEY, H. J,, R, C. CLELL&ND, D. E. BOYCE A~n E. J. BowERs (Philadelphia, Pa.), Atmospheric sulfur dioxide, nitrogen dioxide and lead as mutagenic hazards to h u m g n health. 4 MALISZEWSKI,W. M, B., AND G. FICSOR (Kalamazoo, Mich.), Streptozotocin-induced univalents in male mice. 5 ROTH, D. (New York, N.Y.), Disparate photosensitized damage in DNA a t t w o lutitudes, 6 VIG, B. K. (Rene, Nov.), Somatic crossing over in Glycinc max (L.) Merrill : Differential response to sH-emitted fl-partlcles and ~°Co-emitted y-rays. 7 CUMmNG, R. ]3., AND G. A. SF~OA(Oak Ridge, Tenn.), Initial ethylation of D N A in sperm and testes of the mouse as a function of administered dose of ethyl methanesulfonate. 8 SEGA, G. A., AND R. B. CU~rMING(Oak Ridge, Tenn.), Ethylation pattern in mouse sperm as a function o:f developmental stage and time after exposure to ethyl methanesulfonate. 9 KOI-IN, I-re. I., AND R, W. MELVOLD (Boston, Mass.), Temporal variations in spontaneous mutation rate of the histocompatibility loci of the mouse. Io ONG, TONG-MAN (Research Triangle Park, N.C.), Mutagenicity of nitrofuran derivatives in

Neurospora 6rassa. II HOFFMANN, O. R., AND H. V. MALLING (Research Triangle Park, N.C.), Detection of 8azaguanine-resistant m u t a n t s in a Ncurosporu hcterokaryon. 12 WHONO, W:EN-ZONC,, AND H. E. BROCKMAN (Normal, ILL), Mutagenicity of I C R - I 7 o at low concentrations and high survivals in iVeurospora crassa. 13 STRAUS, D. S. (Research Triangle Park, N.C.), Detection o[ tandem gone duplications in

Escherichia ~oli. I 4 IGALI, S., AND R. C. YON BORSTEL (Edmonton, Alberta, Canada), Induction of m u t a t i o n s and lethality in Saccharomyces cerevisiae after exposure to several indazole analogs of hycanthone. 15 ZUBF.RI, 1~. I., AND G, FICSOR (Kalamazoo, Mich.), Glucose-mediated mutagm]icity in Salmo-

~zella typhimurium. 15 :FRANTZ, C. N,, A~D H. V. MALLING (Reseurch Triangle Park, N.C.), Metabolism of dimethylnitrosamine to a m u t a g e n by rodent liver microsomes. 17 WE~KES, U., F. GLETT~N AND D. BI~USlCK (Washington, D.C.), Conversion of dimethyl- and diethylnitrosamine to mutagenic metabolites by microsome fractions from liver, lung and kidney tissues of four mouse strains. 18 BRUSlCK, D., AND H. ANDREWS (Washington, D.C.), A comparison of the genetic activity of chemical mutagens in Saccharomyces cerevisiae strains D3, D4 and D 5 utilizing in vitro assays with and without hepatic enzyme activation. 19 ELLIS, JR., J. H., V, A. RAY AND H. ]~. I-IOLDEN (Groton, Corm.), Comparative studies of 6chloropurlne in host-mediated and in vitro bacterial assays. 20 HOLDEN, H. E., V. A. RAY, J. FLORIO A N D M. O. WAI-IR~;NBURG (Groton, Conn.), I n rive a n d in vitro cytogcnetic studies with 6-chloropurine. 2I L:~c,A'roa, M. S., M. STOECKEL AND T. CONNOR (Providence, R,I.), Techniques for isolating mutagenic substances from urine and blood of treated mammals using h i s t i d i n e auxotrophs of S. @phimurium as the indicator organisms. 22 CONNOR, T., IV[. STOECI{EL AND M. S. LEC-ATOI~{Providence, R.I.), Niridazole, a direct acting frameshift mntagen, not affected by microsomal enzyme preparation which c a n be detected in t h e host-mediated assay. 2 3 HOOI(, ]~. B., P. BRINSON, J. I?ECK, P. GREENWALD, N. I:~ATCHER AND O. STANECI(Y (Albany, N.Y.), A search for chromosomal breakage in individuals exposed to spray adhesives.

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24 VO~SANOE~, G., AND K. I-IIRSCHHORN (New York, N.Y.), Absence of increased chromosome aberrations due to aerosol spray adhesives. 25 WALLENSTEIN, S., J. BROBST A~D R. E. BAGDON (Hanover, N.J.), Establishment of a positive control and statistical techniques for the dominant lethal test in mice. -°6 JO~tGENSON, T. A., G. W. NEWELL, R. J. SPANGGORD, D. L. KAX"AND P. L, GRIBLINC (~Ienlo Park, Calif.), Mutagenic activity of triethylenemelamine administered orally in the diet and drinldng water. 27 ARNOLD, D. W., G. L. I(~NNEDY AND M. L. KE~LZNGER(Northbrook, Ill.), Comparison of the mutagenic effects of triethylenemelamine on rats and mice in the dominant lethaI assay. 28 MICHEL, T. M., AND M. S. LEOA¢OR (Providence, R.I.), A comparative s t u d y of mutageninduced DNA repair synthesis and cytogenetic alterations in vivo. ~9 FARRow, IV[. G. (Paoli, Pa.), Improved microculture techniques utilizing the whole blood of the dog, guinea pig, monkey, mouse or rat. 30 WEBER, E., I(. BIDWEI.I. AND IVI. S. LEaATOI~ (Providence, R.I.), An evaluation of the micronuclei test for routine cytogenetic analysis. 31 TAN, J . C. (Valparaiso, Ind.), Application of survey sampling techniques in the evaluation of mutagenlcity data in cytogenetie studies.

I NAUMAN,

C. H., I{. VILLALOBOS-PIETRINI AND R. C. SAUTKULIS, Biology Department, Brookhaven National Laboratory, Upton, N.Y. 11973 (U.S.A.).

Response of a mutable clone of Tradescantia to gaseous chemical rnutagens and to ionizing radiation Clone 6 of Tradescantia (No. OLO6),mutable for flower color, has been propagated vegetatively from a single axillary shoot following whole plant irradiation, and is thought to be a perielinal chimera. The stamen hairs of clone 6 can be scored for the same mutations as the parent clone (clone o2), and have a spontaneous pink mutation rate as much as 29 times higher than that of clone o2. The spontaneous rate for clone 6 varies from a high of 3.6 pink events/Ioo hairs when flowers are picked directly from the stock plants, to a low of 1. 9 pink events/zoo hairs when flowers are taken from cuttings which have been maintained under identical conditions for I I - I 5 days. Relative to the response of clone o2 to X-rays, the high spontaneous rate in mutable clone 6 is equal to t h a t induced by 3 o tad, while the low spontaneous rate is equal to t h a t induced by 19 rad. Colorless mutations, unlike pink mutations, exhibit a similar spontaneous frequency in both clones. X-irradiation induces in mutable clone 6 a pink mutation response which, when the spontaneous mutation rate is subtracted, is only slightly different from the rate observed for clone o2. However, preliminary data indicate that the pink mutation rate of mutable clone 6 to chemical mutagens (EMS, DBE) may be approximately ten times as high as that of clone o2, and comparable to that for a Tradescantia interspecific hybrid clone (No. 443o) which has been previously found to exhibit a mutation rate simiIar to that of mutable clone 6 for X-rays and EMS. Ongoing research is directed at studying dose-response curves under various conditions and at determining the basis for the differential responses of these clones to chemical mutagens and ionizing radiation. Research supported in part by the U.S. Atomic Energy Commission and in part b y the National Institute of Environmental Health Sciences. Abbreviations : DBE, dibromoethane; I~MS, ethyl methanesulfonate.

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2 SPARROW, A. H., AND L. A. SCHAIRER, Biology Department, Brookhaven National

Laboratory, Upton, N.Y. zz973 (U.S.A.).

Mutagenic response of Tradescantia to treatment with X-rays, EMS, DBE, ozone, SO2, N~O and several insecticides The increase in frequency over normal background of pink or colorless mutant cells or groups of cells in petals and/or stamen hairs of mature flowers of variolls blueflowered Tradescantia clones m a y be used advantageously as a monitor or test system for induced genetic iniury. The responses of two different clones of Tradescantia (clone o2 and 4430) heterozygous for flower color have been studied and compared after treatments with X-rays, chemical mutagens and suspected mutagens. Significant increases in mutation rate after exposures as low as 250 millirad of X-rays and 3 ppm of gaseous ethyl methanesulfonate (EMS) or z,2-dibromoethane (DBE) attest to tile efficacy of the Tradescantia stamen hair systems as a monitor of mutation rate. Data on pink somatic mutation rates will be presented for plants treated as noted above and for plants treated with several pesticides and air pollutants. Significant increases in mutation rate above spontaneous levels were observed for ozone, SOs, and N~O but the data suggest that they are at best weak mutagens in this system since the maximum response obtained was little more than twice the spontaneous rate. Preliminary results were also obtained on the mutagenicity of four insecticides (dip or spray T E P P and the systemics Systox, lV[eta-systox R and Temik) used at concentrations recommended by the manufacturers. Of these only T E P P was found to produce a statistically significant increase (at the 1% level) in the mutation rate. These conclusions on the insecticides should be considered only tentative since satisfactory dose-response data have not yet been obtained. Research supported in part by the U.S. Atomic Energy Commission and in part by the National Institute of Environmental Health Sciences. Abbreviation: ppm, parts per million.

3 I-IICKEY, R. J. *, R. C. CLELLAND

*, D. E. BOYCE* AND E, J. BowERs**, * The Wharton, Scllool and ** Anthropology Department, University of Pennsylvania, Philadelphia Pa. 19174 (U.S.A.).

Atmospheric sulfur dioxide, nitrogen dioxide and lead as mutagenic hazards to human health The air pollutants, sulfur dioxide and nitrogen dioxide, if present in sufficient concentrations, are ocular and respiratory irritants. Studies have been conducted to investigate the possibility that these chemicals, in concentrations commonly found in many metropolitan regions, are also seriously hazardous to the health of human populations. The role of atmospheric lead was also considered.

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Regression techniques applied to multichemical data for 38 metropolitan regions have indicated that concentrations of NO~ [i.e., e(NQ) ] and SO~ are statistically significant predictors for mortality rates for several categories of diseases of ageing including total cancer, cancer of the respiratory organs, and arteriosclerotic heart disease. Similarly c(NO~) and c(Pb) were significant predictors for mortality rates for infant deaths ( ~ I yr.) and congenital malformations, while c(NQ) was a significant predictor for birth rate. Typically, more than half the variance in the criterion was "explained" by atmospheric concentrations of chemicaI pollutants. Similar results were obtained using age-sex-"race" specific subsets of these same populations. A possible explanatory theory for these results is presented. It assumes that the fundamental mechanisms involved are principally genetic, both somatic and intergenerational. On aqueous solution, SO2 and NO2 yield the weak acids, H.~SO,~and HNO~, both of which can function as mutagens. Bisulfite (HSQ-) adds reversibly to the 5,6-double bonds of cytosine (C) and uracil (U). Such adducts of C moieties may alter DNA function, while adducts of C and U may alter RNA function. The mutagenic character of H N Q is well known. Moreover, excessive lead bound to DNA and to RNA in place of the "normal" complements of bound metals may also alter their function.

4 MALISZEWSKI, W. M. B., AND GYULh FlCSOR, Department of Biology, Western Michigan University, Kalamazoo, Mich. 49001 (U.S.A.).

Streptozotocin-induced univalents in male mice Cytoxan and Trenimon have been shown to increase the frequency of univalents during diakinesis and metaphase I (E. SCHLEIERMACHER,in F. VOGELAND G. R6HllBORN (Eds.), Chemical Mutagenesis in M~mmals a~¢dMan, Springer, New York, 197o, pp. 317-34I ). The purpose of the present investigation was to determine if the broadspectrum antibiotic SZN may also increase the frequency of univalents. TEM and EMS were included for comparison. Random-bred Upjohn strain Swiss albino male mice of 25 g bodyweight were dosed with 0.5, i.o, i. 5 and 5 #M/g SZN 24 or 48 h prior to sacrificing. Other mice were similarly dosed with 0.5 #M/g TEM or with 5 and IO l~M/g EMS. Chromosome preparations were prepared by the air-drying method of EVANS. Diakinetie spreads were scored for bivalents and univalents. TEM was found to induce the most univalents while ElVIS did not increase the frequency of univalents over water-injected controls. SZN induced univalents of intermediate frequency between the controls and TEIVI. The frequency of SZN-induced univalents was independent of dose. This investigation was supported by a Faculty Research Grant to G.F. Abbreviations: EMS, ethyl methanesulfonate; SZN, streptozotocin; "IEM, triethylenemelamlne,

5 ROTH, DANIEL, Institute for Environmental Medicine, NYU Medical School, New York, N.Y. 10016 (U.S.A.).

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Disparate photosensitized damage in D N A at two latitudes Sunlight possesses mutagenic potential via several pathways, one by direct damage to genetic material, others through the mediation of sensitizers or chemically active photoproducts, and still others involving the competitive inhibition o~ DNArepairing enzymes specific for both radiation and chemicaMnduced damage. The DNA-damaging capability of solar radiation was studied under standardized exposure conditions in sites at 28 and 45 degrees north latitude. Acetone {Ac) or dihydroxyacetone (DHA) was admixed with calf thymus DNA to sensitize thymine photodimerization in high yield. The extent of the resultant sensitized actinic-induced dimerization was estimated in terms of the associated denaturing effect on DNA. An acridine probe was employed to measure this. The activity of DHA as a sensitizer was found to be proportional to the total actinic irradiance at the two sites, whereas Ac was both relatively and absolutely more active at 28 degrees north latitude despite a lower actinic irradiance there. The difference spectrmn for Ac absorbanee with reference to DHA showed a peak in the UV-B (29o-32o nm), suggesting that the disparity may have been caused by the presence of atmospheric el~romophores possessing low absorptivities in this range (e.g. : ozone, unidentified pollutants). A contributory role may have been played by Rayleigh and aerosol scattering of light. These findings indicate that the known action spectrum for the toxic effects of actinic irradiation (UV-B) corresponds more closely to the absorption spectrum of Ac than of DHA. They also demonstrate in more general terms that photosensitized genetic damage can vary not only with the particular sensitizing agent, but also with the responses of each to local variations in the irradiating milieu.

6 Vm, B. K., Department of Biology, University of Nevada, Reno, Nev. 895o7 (U.S,A.).

Somatic crossing over in Glycine max (L.) Merrill: Differential response to ~l-l-emitted #-particles and ~°Co-emitted ~-rays The varieties T2I 9 and L65-I237 of soybean have dark green, light green and golden yellow leaves because of the gene combinations YI~Yzl, YzlYzz and y~y~z, respectively. The heterozygous plants are found dotted with dark green, yellow and double (dark green-yellow) spots on the two simple and rarely the first compound leaf. The other two genotypes are usually devoid of spots. It has been suggested that t h e twin or double spots on tl~e leaves of Y~Yzz plants result from somatic crossing over. The single spots may originate from the failure of one or the other component of the double spots. Some single spots may result from chromosomal disturbances like deletions, numerical inequalities and point mutations. When seeds of variety L65-I237 are treated with aH~O (5-zoo #Ci/ml), the frequency of all types of spots is increased with the relative proportions of these spots remaining comparable to those in the control. This indicates that the processes effected by /%particles from 3H are similar to those effected by some sort of presumed internal mutagen. The treatment of seeds with 5o-75o R of y-rays ("°Co-emitted) increased the frequency of all types of spots but yellow spots increase most in frequency followed by dark green spots. This effect could be due to increased incidence of seg-

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mental losses, numerical inequalities and mutations of the gene Yn. Point mutations and other aberrations increase in parallel. The frequency of spots on the lower surface of t h e control or treated leaves is lower than that on the upper surface suggesting resistance of the spongy mesophyll.

7 CUMMING, R. ]3., AND GARY A. SEGA, Biology Division, Oak Ridge National Labora-

tory, Oak Ridge, Tenn. (U,S.A.).

Initial ethylation of D N A in sperm and testes of the mouse as a function of administered dose of ethyl methanesulfonate Metabolic and incorporation data indicate t h a t most ethylation which will occur in male reproductive cells following a single injection of EMS occurs during the first 4 h postinjection. Methods are now well enough refined to ensure that ethylation measured is on the DNA molecule, and that no significant errors result from contamination of the DNA sample, non-random recovery of I)NA, or from metabolic incorporation of label into the DNA sample. The m a x i m u m percentage of sperm cell ethylation which is in sperm cell DNA is about z8% at doses from ioo to 400 mg/kg and is lower at doses lower than IOO mg/kg. The number of ethylations per nucleotide (E/N) in DNA from sperm in the vasa deferentia and epididymis 4 h after an i.p. injection ranges from approx. 8. Io -8 at 3.3 mg/kg up to L 3 ' Io -4 at 4o0 mg/kg. A least squares fit of the data indicates that the E/N increases with approx, the 1.5 power of the dose. For testicular DNA the E / N increases from 3 . 3 ' I o-~ at 3.5 mg/kg to I.z. Io- ~ at 400 mg/kg and thus E/N is of the same order o5 magnitude for the two sources of DNA. E/N in testicular DNA increases with about the z. 3 power of the dose. These data provide a foundation on which studies correlating molecular dose with germ cell developmental stage or with specific genetic damage patterns can be based. Research jointly sponsored by the National Center for Toxicological Research and by the U.S. Atomic Energy Commission under c o n t r a c t with the Union Carbide Corp. Abbreviations: EMS, ethyl methanesulfonate.

8 SEGA, GARY A., AND R. B. GUMMING, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. (U.S.A.).

Ethylation pattern in mouse sperm as a function of developmental stage and time after exposure to ethyl methanesulfonate A single, intraperitoneal injection of 200 mg/kg of [2-all]ethyl methanesulfonate ([aH]EMS) was given to (C3H × zoz)Fz hybrid males z o - x 2 weeks old at the time of treatment. At I I time points from 4 h to z 6 days after [~H]EMS injection, 4 mice were killed and the sperm from the caput epididymis, caudal epididymis and vasa deferentia were recovered. Sperm ethylations were determined from the specific activity

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of the [3H]EMS and from [2-3H]ethyl activity measured in samples of purified sperm heads by liquid scintillation counting. The ethylations per sperm reach a peak first in the caput epididymis about 2 days postinjection, followed by a peak in the caudal epididymis at about 6 days and finally a peak in the vas sperm at 9-1o days. The ethylation pattern for vas sperm, over the I6-day period studied, closely parallels the dominant lethal pattern measured for the offspring of treated males. The pattern of ethylation indicates that the sperm most heavily ethylated were testicular sperm at the time of treatment and that these ethylated cells could be traced moving through the caput epididymis, the caudal epididymis and the visa deferentia ir~ sequence. Research jointly sponsored by the National Center for Toxicological Research and by the U.S. Atomic Energy Commission under contract with Union Carbide Corp. 9 I(OHN, HENRY I., AND ROGER W. MELVOLD, Harvard Medical School and Shields Warren Radiation Laboratory of the New Englmad Deaconess Hospital, Boston, Mass. 02115 (U.S.A.).

Temporal variations in spontaneous mutation rate of the histocompatibility loci of the mouse The H-test, employing dermal grafts, screens at least 30 and perhaps several hundred loci (the H-system) for histocompatibility mutations in the hybrids BALB/ c × C57BL/6 and C57BL/6 × BALB/c. In San Francisco, the annual mutation rates (gains) of the H-system for the years 1963, I964 and 1965 were, respectively, 13.5, 5.7 and 7.5 mutants per IOOOtested animals, with a weighted mean of 9 per lOOO, based on 2200 animals (D. BAILEY, TransplantatioJL 4 (1966) 482). In Bostonj using lines derived directly from BAILEY'S, and with similar methods, we found annual rates for 1966 through i972 to be consistently below 2.5 per IOOO, with a weighted mean Of 1.o9 per IOOO, based on 82oo tested animals. Some impheations of these findings will be discussed.

10 ONG, TONG-MAN, National Institute of Environmental Health Sciences, Research

Triangle Park, N.C. 27709 (U.S.A.).

Mutagenicity of nitrofuran derivatives in

Neurosporacrassa

Nitrofuran derivatives are commonly used as antibacterial drugs and, in some countries, as food additives. Little is known, however, in regard to the potential mutagenic hazard of these compounds. Several nitrofuran derivatives including nitrofurazone (an antibacterial drug), AF-2 (a food additive), FANFT, SQI8,5o6 and four other closely related compounds were studied in the ad-3 test system in Neurospora

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crassa. Their mutagenicity and the relationship between their chemical structures and nlutagenic activities were evaluated. Conidia from a genetically m a r k e d two-component heterokaryon were treated with different concentrations of n i t r o f u r a n derivatives in sodium-phosphate buffer, pFI 7, at 3 o ° for 2 h in tile dark. At the e n d o f t h e treatment, chemicals were decanted and conidia were washed b y centrifugation. Treated and untreated conidia were analyzed for tile presence of the ad- 3 mutants by the direct method (DE SERRES AND KOL~IARI<, Nature, I82 (I958) I249). The results show t h a t AF-2, FANFT and SQI8,5o6 are p o t e n t mutagens in Neurospora. The ad-3 niutation frequency increased more than I o o - f o l d over the spontaneous m u t a t i o n frequency after the conidia were treated with a o.I m M solution of a n y of these compounds. No significant increase in the ad-3 m u t a t i o n frequency was observed after the conidia were treated with any of the four other nitrofurans. These results seem to suggest t h a t the nitro group at the 5 position and the side chain at tt~e 2 position of furan play important roles in the m u t a g e n i c i t y of nitrofuran derivatives.

11 HOFFMANN, G. R., AND H. V. I~IALLING,N a t i o n a l Institute of E n v i r o n m e n t a l Health Sciences, Research Triangle Park, N.C. 277o9 (U.S.A.).

Detection of 8-azaguanine-resistant m u t a n t s in a N e u r o s p o r a heterokaryon A new system has been developed for t h e detection of forward m u t a t i o n s at the aza- 3 locus in a heterokaryon of Neurosibora crassa. The aza- 3 gene controls resistance to 8-azaguanine in a purine auxotrophic s t r a i n under conditions of limited adenine supplementation. When incorporated into v e g e t a t i v e heterokaryons, the aza-3 gene is recessive to its sensitive allele, Forward mntants in an adenine-requiring heterokaryon carrying the aza- 3 gene are selected in Westergaard's basal m e d i u m containing 0. 5 g D-glucose, 0.5 g Dfructose, 20 g L-sorbose, 2oo mg 8-azaguanine, I.O mg adenine sulfate, and 20 g washed Difeo Bacto-Agar per liter. An i n c r e a s e d frequency of mutants was detected following treatment of conidia with X-rays, ultraviolet light, ethyl methanesulfonate, and the acridine mustard ICP~-ITo. The new system is a specific locus s y s t e m desig-aed to detect a wide variety of genetic alterations. Tile characteristics of t h e mutants detected, continuous expression of mutants with time, and the high m u t a t i o n frequencies obtained will be discussed.

12 WHONG, %VEN-ZONG,AND HERMAN E. BROCKlV[AN,Department of Biological Sciences, Illinois State University, Normal, Ill. 61761 (U.S.A.).

Mutagenicity of ICR-IT0 at low concentrations and high survivals in Neurospora crassa The first report of the mutagenicity o f ICR-I7o noted its p o t e n t mutagenic activity in Drosophila melanogaster (CARLSON AND OSTER, Genetics, 47 (I962) 561-

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576). A high mutagenicity of this agent was next reported in Neurospora crassa (BRoClCMANAND GOBEn, Science, 147 (1965) 75o-751). We have now investigated the mutagenicity of ICR-I7o at low doses that cause little or no killing. Two assays of the mutagenicity of ICR-I7o in conidia of N. crassa were used: forward mutation at the ad-3 region in a heterokaryon and reverse mutation of an ad-3 mutant that is a presumptive frameshift (12-9-17). Typical results of a 4-h exposure of the heterokaryon to ICR-I7 o are: o.I/~g/ml (o.2I #M)--99 % survival, and 21 ad-3 mutants/Io ~ survivois; 0.3 #tg/ml (o.63/zM)--99% survival, and 154 ad-3 mntants/Io ~ survivors. As the spontaneous ad-3 forward mutation frequency is about 4.io-7, these are large induced frequencies. Typical results of a 3-h exposure of 12-9-17 to ICR-I7O are: o.o3/zg/ml (o.o6 #M)--IOO% survival, and 8 revertants/Io~ survivors; o.3 #g/ml (o.63 #M)--94 % survival, and Io 9 revertants/io 7survivors. This strain is stable spontaneously. These results show that low doses of ICR-I7O that cause little or no kiiling can induce significant increases in forward and reverse-mutation frequencies at the ad-3 region of N. crassa. Survivals and mutation frequencies at various doses will be presented. This research was supported by the U.S. Atomic Energy Commission under Contract No. AT(II-I)-I3I 4 with Illinois State University (Report number Coo-I3Z4 24). Abbreviations: ad-3, adenine-3; ICR-I7o, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylarnino) acridine.2 HC1.

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STRAUS,DANIEL S., National Institute of Enviromnental Health Sciences, Research Triangle Park, N.C. 277o9 (U,S.A,),

Detection of tandem gene duplications in Escherichia coli Tandem gene duplications, like other chromosomal rearrangements in bacteria, are believed to arise from aberrant recombinational events. Where suitable methods exist for their detection, tandem duplications have been found to occur in bacteria at high spontaneous frequencies ( > IO-S). The effect of mutagens on the frequency of duplications in the gZyS region of the E. colt chromosome has been studied, using a mutant strain isolated by FoLx A~rD BERG (J. Mol. Biol., 58 (197I) 595). In this strain it is possible to simultaneously monitor the frequency of tandem duplications in the glyS region, and forward mutations in a gene involved in glycine dissimilation. Four mutagens (UV, nitrous acid, the acridine half mustard ICR 37~, and niridazole) have all been found to increase the frequency of tandem gene duplications in the glyS region. The four mutagens were selected because they induce different types of pre-mutational lesions in DNA. The observation that all four mutagens were active in inducing duplications suggests that the stimulation is due to some type of DNA damage (or DNA repair intermediate) common to a variety of mutagenic treatments, such as single-strand breaks or gaps. The genetic activity of niridazole is of interest because this compound is currently being used on an investigational basis for the treatment of schistosomiasis ].n humans. In E. colt, at 1% survival, it induces a I4-fold increase in tandem'duplica-

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tions in gIyS and a I5o-fold increase in forward mutations in the glycine dissimilation gene. The strong mutagenic activity of niridazole in bacteria suggests that it should be tested further for mutagenicity and carcinogenicity.

t4 IGALI, SANDOR, AND I~. C. VON BORSTEL, Department of Genetics, University of

Alberta, Edmonton, Alberta (Canada T6G 2E9).

Induction of mutations and lethality in Saccharomyces cerevisiae after exposure to several indazole analogs of hycanthone Two haploid and one diploid strains of the budding yeast Saccharomyces cerevisiae were tested for survival and mutation induction after treatment with several indazole analogs of the antischistosomal compound hycanthone (IA-2). The indazole analogs were IA-3, IA-5, and IA-6. All experiments were conducted at pH 5.9 for 8 h at 26° where concentration of the mutagen was the variable, and at a concentration of 25o #g/ml where time was the variable. Under these conditions only hycanthone was mutagenic for reversion of hisI- 7 (a base-substitution mutant), lysl-z (an ochre-suppressible mutant), and hom3-IO (a presumptive frameshift mutant). On the other hand, marked differences were seen in the patterns of survival. Except for IA-6, the diploid strain was always more resistant than the haploid strains for the indazole analogs including hycanthone. The survival of yeast cells was not affected at any concentration of IA-6 that was used. IA- 3 is more toxic to yeast ceils than hyeanthone, even though it is not mutagenic. One interesting relation was observed: for the nonmutagenic indazole analog IA-5, doubling of the concentration with time held constant was nearly twice as efficient for killing as doubling of the time of exposure with concentration held constant. This is also true for the non-mutagenic analog IA-4, but the reverse is true for hycanthone (M. G. MEADOWS,Master's Thesis, University of Alberta, x973).

15 ZUBERI, I~lAZ I., AND CrYULA FlCSOR, Department of Biology, Western Michigan University, Kalamazoo, Mich. 49001 (U.S.A.).

Glucose-mediated mutagenicity in Salmonella typhimurium The mutagenicity of SZN, eight SZN analogs and MNU were compared in

Salmonella ~yphimurium. SZN and its analogs carry MNU attached to the carbohydrate moiety at the C-2 position. The C-I analogs tested were co- and/~-methylSZN, c~-ethyl-SZN, /%propyl-SZN, g- and /5-butyl-SZN; in two analogs glucose was replaced by c~- or tS-inositol. When the ability of these compounds to revert the hisG46 auxotroph was compared, they fell into four groups, each differing from the other b y about Io-fold in mutagenicity. The most mutagenic was (i) SZN, followed by (ii) fl-methyl-SZN; (iii) o~-methyl-SZN, c~-ethyl-SZN, fl-propyl-SZN, ~- and/5-butylSZN; (iv) c~- and/3-inositol-MNU and MNU. From these results, it appears that the presence of the glucose moiety is conducive to a high level of mutagenicity of MNU. This is clearest in the case of SZN which is about lO4times more nlutagenic than I~INU. Alterations of the glucose moiety by addition of larger alkyl groups, especially in the

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g-position lead to decreased mutagenicity. When the glucose moiety is replaced by inositols, a level of mutagenicity is noted similar to MNU, indicating that inositols neither increase nor decrease the mutagenicity of MNU. From these results the hypothesis is proposed that the uptake of SZN is mediated by gtucose transport. To further test this hypothesis, the mutagenicity of SZN, ~-methyl-SZN, fl-hutyl-SZN, ~:inositolMNU and MNU was determined in his G46 mutants having normal and defective ceil walls (deep rough strains of Ames). Also, disappearance of SZN, fl-methyl-SZN, aethyl-SZN, ~-butyl-SZN and of ~-inositol-MNU from the media in the presence of bacteria was measured. The experimental results supported the hypothesis. We thank Dr. TETSUO SUAlVlIfor the streptozotocin analogs. R.I.Z. was in part supported by a Departmental Grant from the Upjohn Company and G.F. by a Faculty Research Grant from Western Michigan University. Abbreviations: MNU, N-methyl-N'-nitrosourea; SZN, streptozotoein.

t6 Ii'RANTZ, CHRISTOPHERN., AND ~[-tEINRICHV. MALLING, National Institute of Environ-

mental Health Sciences, Research Triangle Park, N.C. 27709 (U.S.A.).

Metabolism of dimethylnitrosamine to a mutagen by rodent liver microsomes Dimethylnitrosamine (DMN) and diethylnitrosamine are not mutagenic by themselves, but they can be converted by mammalian enzymes to tfighly mutagenie products. Mutagenic activity of these products was detected in tile histidine-requiring strain 046 of Salmo~ella typhimurium by measuring an increase in the frequency of histidine revertants. The activation of DMN is mierosomal, inhibited by SKF525-A, and requires Co-factors, The activation enzyme is induced in mice by pretreatment with phenobarbital and 3-meibylcholanthrene. The mutagenic activity of the reaction products is directly correlated with the metabolic formation of formaldehyde with and without induction by 3-methylcholanthrene and across strains of mice (C3H, DBA]2J, C57BL/6, B6D2FI, B6C3FI). Formaldehyde does not contribute measurably to the mutagenic activity of the reaction products. Thus, the rate-limiting step in the metabolism of DMN to a mutagen is the initial microsomal enzymatic N-demethylation.

17 WEEKES, URSlJLA, FREI~ GLETTEN AND DAVID BRUSICK,Howard University College of Medicine, Washington, D.C. 20001 (U.S.A.). Conversion of dimethyl- and diethylnitrosamine to mutagenic metabolites by microsome fractions from liver, lung and kidney tissues of four mouse strains The production of mutagenic metabolites from the chemical carcinogens di-

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methylnitrosamine (DMNA) and diethylnitrosamine (DENA) was determined by an in vitro mutagenic assay employing a IOO ooo × g cell fraction containing the microsomal enzymes, a buffered reaction mixture and a microbiM indicator population of Salmonella typhimurh~m strain G-46. The degree and rate of mutagen formation over the t r e a t m e n t period was monitored by measuring the induction of reverse mutation in the i n d i c a t o r cells. T h e two test agents DIVINA and DENA have low or negligible mutagenic activity without metabolic activation by mammalian microsomal enzymes especially those a m o n g the mixed function oxidases. The activation capacities of microsome preparations from liver, lung and kidney tissues of BALB/cJ, R F / J , C57B1/6J and H a / I C R mice were determined and compared to the neoplastic susceptibilities to DMNA a n d DENA exhibited by the organs of each mouse strain. Results from this study s h o w e d a good correlation between the metabolic activation capacity of an organ, b a s e d on mutagen production, and its susceptibility to DMNA- and DENAinduced tumors. M u c h of this work was conducted with the support of grants from the U.S. Food a n d Drug Administration and the National Cancer Institute.

18 BRUSICK, DAVID, AND HOPE ANDREWS, Howard University College of Medicine, Washington, D.C. 20001 (U.S.A.).

A comparison of the genetic activity of chemical mutagens in Saccharomyces cerevisiae strains D3, D4 and D5 utilizing in vitro assays with and w i t h o u t hepatic enzyme activation T h r e e diploid strains of Saccharomyces cerevisiae, originally constructed by F. K. Zimmermann, have been employed by a number of investigators as indicator organisms in assays designed to screen environmental agents for their genetic properties. T h i s study compared these strains (D 3, D4 and D5) for their sensitivity as genetic indicators in a series of in vitro tests using compounds t h a t are active directly or produce genetically active intermediates following metabolism. The tests that employed a metabolic system utilized a 9ooo ×g fraction from homogenized mouse liver tissue. T h e cell fraction was incorporated into a buffered reaction mixture containing the test chemical and the indicator organisms. T h e results of the study indicate t h a t these three strains of yeast are capable of detecting genetic activity produced by frameshiit and base-substitution mutagens which a c t directly as well as some agents that produce active metabolites. T h e overall sensitivity of the three strains was similar in these tests. M u c h of this work was conducted with the support of grants from the U.S. Food a n d Drug Administration and the National Cancer Institute.

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19 ELLIS JR., JOHN H., VERNE A. RAY AND HENRY E. HOLDEN, Medical Research Laboratories, PKzer Central Research, Groton, Conn. o634o (U.S.A.).

Comparative studies of 6-chloropurine in host-mediated and in vitro bacterial assays Considerable attention is being given to the reassessment of a genetic test battery originally recomlnnended for inclusion in drug safety evaluation protocols. Direct testing of chemicals on bacteria with and without liver enzyme activation and tests in the host-mediated assay are among those being actively examined. This report deals with the activity of the mutagen, 6-chloropnrine (6-C1P), in both these assay systems. At levels of o.oo4-5.o rag/plate, 6-C1P fails to revert histidine-requiring strains of Salmonella typhimuri,~m, G-46 and TA 1535, to prototrophy. The addition of mouse liver microsomal fraction S- 9, supplemented with TPN and glucose-6-P04, did not produce an increase in reversion frequency. Host-mediated assays were performed orally, subcutaneously and intramuscularly at levels oi 5, IO, 25, 50 and 1oo mg/kg in HAM/ICR Charles River CD-I mice. Significant effects were obtained by all three routes at the 25, 50 and IOO Ing/kg levels using G-46 as the indicator cell. These results demonstrate that an established mutagen can be inactive in direct bacterial tests including models which utilize liver microsomal enzymes for activation, yet be quite active in the host-mediated assay using the same mammal from which these liver enzymes were obtained. Such results indicate the need for a group of point mutation tests, run concurrently, which includes both in vitro and in rive components. The relationship of these results to those obtained with other mammalian test procedures will be discussed.

2O HOLDEN, H. E., V. A. RAY, J. FLORIO A N D M. G. WAHRENBUI~G, Pfizer Central Research, Groton, Conn. o634o (U.S.A.).

In vivo and in vitro cytogenetic studies with 6-chloropurine 6-Chloropurine is a nucleic base analogue which has been used in treatlnent of acute leukemia in man. Despite its relationship to pathways involving DNA synthesis, little is known concerning its mutagenic activity in malnmalian cells. Cytogenetic studies were initiated to evaluate the effects of 6-CP on mouse bone marrow cells in rive and on human lylnphocytes in vitro. For the in rive experiments, groups of five CD-I male mice were given single oral doses of 15o mg/kg and bone marrow samples were obtained at 6, xz, or 24 h post-administration. An additional group was exposed to daily oral doses of 7.5 ing/kg/day for 5 days and sacrificed 6 h after the final dose. All mice were injected i.p. with I mg/kg colchicine 3 11 prior to sacrifice. A total of 50 Inetaphase figures from each mouse was analyzed for chromosome breakage. A maximum response was observed in mice treated for 2¢ h ~ith 15o mg/kg. To establish an in rive dose-response curve, additional mice were treated with ~5, 50, Ioo, 15o, 200 or 250 ing/kg p.o. Bone marrow samples were obtained 24 h after dosing. Significant, dose-related, chromosome damage occurred at doses of 50 mg/kg and

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above. In vitro studies were conducted on short-term human l y m p h o c y t e cultures exposed to various concentrations of the drug in the culture medium f o r 24 h prior to harvest. Chromosome analysis of these cells revealed no drug-related eytogenetic effect at doses of IO to 2oo #g/ml. Tile induction of chromosome d a m a g e i~¢ vivo but not in vitro suggests that 6-CP is converted to a mutagenic d e r i v a t i v e in the intact animal.

21 LEGATOR, MARVIN S., MARIE STOECKEL AND THO~IAS CONNOR, B r o w n University,

Roger Williams General Hospital, Providence, R. I. (U.S.A.).

Techniques for isolating mutagenic substances from urine and blood of treated mammals using histidine auxotrophs of $. Typhimuriurn as the indicator organisms The analysis of body fluids for either direct acting mutagens or active metabolites is a versatile procedure that can be used with experimental animals as well as in studies in man. To extend the utility of this method, two procedures w e r e investigated for concentrating blood or urine from treated animals prior to analysis for active mutagens. In the first procedure blood or urine is lyophilized and reconstituted in a minimum volume of solvents prior to analysis. In the second p r o c e u d r e an Amberlite XAD-2 column is employed to concentrate active substances. A special apparatus was designed to insure the sterility of collected urine samples. To demonstrate the utility of the method, rats were given Ioo mg/kg of niridazole by gavage prior to urine and blood analysis. Mutagenic a c t i v i t y was detected in blood within IO rain after the administration of the c o m p o u n d as well as in a I6-h urine sample. Lyophilization was the method of choice for c o n c e n t r a t i n g blood, while the Amberlite XAD-2 column using methanol to elute the a c t i v e material was the method of choice for urine analysis. TAI538 tester strain of S. typhim¢~rium histidine auxotroph proved to be the most sensitive indicator of m u t a g e n i c activity with niridazole.

22 CONNOR, THOMAS, MARIE $TOECKEL AND MARVIN $. LEGATOR, B r o w n University,

Roger Williams General Hospital, Providence, R. I. (U.S.A.).

Niridazole, a direct acting framesbift mutagen, not affected by microsomal enzyme preparation which can be detected in the host-mediated assay In in vitro tests, using S. typhitn,~rium histidine auxotrophs, t h e antischistosomal agent, niridazole, was found to induce back mutations w i t h tester strains TAI538, TAI534 and TAI964 which specifically detect frameshift m u t a g e n s . The drug was found to retain its mutagenic activity when i n c u b a t e d with active liver homogenates from phenobarbital-treated animals, using TA1538 a repair-deficient strain with a defective lipopolysaccharide coat. In the host-mediated assay with a single oral dose of zo mg/kg, this compound

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was active using TAI538, TAI534 and TAI964 as the indicator organisms. Niridazole is the initial example of a frameshift mutagen whose activity can be detected with the S. typhimurium histidine auxotrophs in the host-mediated assay.

23 HooK, E. B., P. ~BRINSON,J. FECIC, P, GRERNWALD,N. HATCHERAND O. STANECKY, Birth Defects Institute, Burns Care Institute, Cancer Control Bureau, N.Y.S. Department of Health and Department of Pediatrics, Albany Medical College, Albany, N.Y. 12208 (U.S.A.).

A search for chromosomal breakage in individuals exposed to spray adhesives In view of reports from the Consumer Product Safety Commission in August and September 1973 of a 5- to 6-fold increase in the rate of cells with chromosomal damage in individuals exposed to spray adhesives in Oklahoma City, a double-blind study of "exposed" individuals and matched controls was undertaken in Albany, N.Y. 36 metaphases from PHA-stimulated peripheral blood of 22 subjects were studied --IZ "exposed" and I I controls, providing about 400 cells from each group for analysis. Should the expected control rate of about 1.2% have been found in both groups then this would have excluded with 95% confidence a doubling or greater rmte in the exposed compared to the controls. When the code was broken, a rate of 3/398 cells (o.8 ~o) with evidence for breakage was found in the controls,, and 7/397 (I.76%) in the exposed, a "non-significant" difference (chi ~ = o.92) which in fact excludes with 95% confidence a rate in the exposed of five times or greater that in the controls. The study was done over a 2-week period and specimens from controls and exposed could not always be set up and harvested at the same time although the same batch of media was used throughout. In view of the possible significance of the question and the results obtained, individuals with heaviest known exposure were restudied, using simultaneous controls and again using a "blind" approach. As of Dec. IO, z973 the latter study is still in progress and the code not yet broken. In both studies attempts to use metaphases harvested after 48 h of culture were unsuccessful because of sparse growth, so all data are from 72-h cultures. With regard to distinction between gaps and breaks, achromatic areas in a chromatid, no matter how large, were scored as gaps unless there was also an apparent displacement in the chromatid axis associated with this non-staining region. Abbreviation: PHA, phytohemagglutinin.

24 VORSANGER, GARY, AND KURT HIRSCI-II-IORN,Division of Medical Genetics, Department of Pediatrics, Mount Sinai School of Medicine, New York, N.Y. lOO29 (U.S.A.).

Absence of increased chromosome aberrations due to aerosol spray adhesives It was recently reported that there may be a causal relationship between multi-

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ple birth defects and the use of foil art spray adhesives. In addition to several unrelated individuals, two abnormal infants whose parents had used this spray showed increased chromosome breakage. In one family, the parents used the adhesive spray prior to and during the pregnancy. The parents of the second infant used the spray only prior to conception. In a second independent study, a correlation of spray adhesive sales data and multiple birth defects was attempted. The results of the study revealed t h a t in the two areas polled, an increase in the sale of this product was not accompanied by an increase in birth defects. To aid our counselling of prospective parents as well as individuals who had come into contact with this product, we undertook a study on the chromosomal effect of spray adhesives. Peripheral bIood taken from patients and controls was cultured and harvested according to standard techniques and metaphases were scored for chromosome changes. Controls were matched for age, sex and the time at which the preparations were made. To date, we have found no indication t h a t the s p r a y adhesives used are chromosome-breaking agents; i.e. the rate of chromosome aberrations shows no difference between subjects and controls. In addition to peripheral blood, we are undertaking a study of the effect of spray adhesives in another experimental system, Drosophila mela1¢ogaster.

25 WALLENSTEIN,SYLVAN,JOYCEBROBSTAND R. E. BAGDON,Departments of Research Data Services and Biological Research, Sandoz-Wander Inc., Hanover, N.J. 07936 (U.S.A.). Establishment of a positive control and statistical techniques for the d o m i n a n t lethal test in mice Three experiments in Charles River CD-I mice were conducted employing untreated and tricaprylin vehicle controls, N E T E P A , 3.125 to 50 mg/kg, and ethyl methanesulfonate (EMS), 50 to 30o mg/kg, to establish a positive control, assess intralaboratory reproducibility and compare results with data previously reported (EPSTEIN e~ al., Toxicol. Appl. Pharmacol., 17 (197 o) 23-40). The most meaningful index for preimplantation losses caused by M E T E P A is mean number of implants per pregnancy averaged over 8 weeks; significant differences from concurrent controls occurred at I2.5, 15 and 5 ° mg/kg but not at 3.125 mg/kg. Preimplantation losses evoked by EMS did not follow a dose-response relationship. Using mean early deaths per female per week as a mutagenic index (MI), M E T E P A produced a significant and dose-related increase at 12.5, 25 and 5o mg/kg at weeks 1-3 ; the response obtained at 3.125 m g / k g was inconsistent in repeated experiments. EMS did not show a consistent dose-response relationship. MI for untreated and tricaprylin-treated controls ranged from 0.3 to 0. 7 averaged over weeks I to 3 ; the number of control females with 3 or more early deaths ranged from 5 to 6 per experiment. These rates tended to be higher than the control data reported by EPSTEIN (op. cir., 1970 ). These data indicate that a meaningful index of mutagenicity is mean number of early deaths averaged over the

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first 3 weeks. METEPA appears to be an adequate positive control because of its reproducible and dose-related response effect.

26 JORGENSON, TED A., GORDON W. NEWELL, RONALD J. SPANGGORD, DAVID L. KAY AND PETER L. GRIBLING, Stanford Research Institute, Menlo Park, Calif. 94025 (U.S.A.).

Mutagenic activity of triethylenemelamine administered orally in the diet and drinking water Triethylenemelamine (TEM) is the usual reference of choice for mammalian mutagenic tests, including the dominant lethal test. In this test, TEM is generally given as a single i.p. injection. New approaches for evaluating the mu±agenic potential of food chemicals emphasize repeated treatment of the test animals over extended periods. These new procedures have included modifications of the dosage regimen of the reference mutagen. We evaluated the mutagenic activity of TEM administered to adult Swiss Webster male mice for 3 weeks in the diet or for 4 weeks in the drinking water at intake levels =~ o.o8, o.o4, o.o2, o.oi mg/kg/day. Treatment was followed b y a 7-week breeding period, with evaluation of the mutagenic effect at midterm of pregnancy. TEM added to the diet failed to produce a mutagenic response. TEM in the drinking water caused matagenic effects between the first and third weeks of breeding after treatment was discontinued. The mutagenic effect was less intense when TEM was given repeatedly in the drinking water than when it was administered i.p. as a single dose.

27 ARNOLD, D. W., G. L. KENNEDYANDM. L. KEPLINGER,Industrial BIOTEST Laboratories, Inc., Northbrook, Ill. 60062 (U.S.A.).

Comparison of the mutagenic effects of triethylenemelamine on rats and mice in the dominant lethal assay Dominant lethal mutagenic studies were conducted on rats and mice treated with the alkylating agent triethylenemelamine (TEM). Dose levels employed were o.I25, 0.250, 0.375 and 0.500 mg/kg administered as single intraperitoneal injections to the males. The animals were then mated for Io consecutive weeks (rats), or for 8 consecutive weeks (mice). Comparisons of effects between species and stages of spermatogenesis affected were conducted. Effects noted in both species included increased incidence of early death i~ utero, and decreased numbers of implantations and embryos. A dose-correlated response was noted for both species. In rats, at week 3, the numbers of implantations were II.o, 9.7, 5.7 and 5.3 while the numbers of viable embryos at that week were 5.3, 3.9, 0.8 and o.3 from the lowest to the highest dose. The percent of early deaths in mice for the same week were 22.0, 24.9, 33,3 and 44.9 respectively. Effects on mating were noted at all levels for rats, b u t only at the highest

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level for mice. A correlation between treatment and spermatogonial stages affected were noted. The greatest effects were noted in rats during weeks 3 and 4, while in mice, weeks 2 and 3 post-treatment displayed the greatest effects. This corresponds to sperm that was from late to mid-spermatids (rats) or late to early spermatids (mice) at the time of treatment. The effects lasted longer in rats with a more abrupt return to normal seen in mice.

28 MICHEL, THOMAS M., AND MARVIN S. LEOATOR,Brown University, Roger Williams General Hospital, Providence, Rhode Island, (U.S.A.).

A comparative study of mutagen.induced D N A repair cytogenetic alterations in Wv0

synthesis and

The alkylating agent, triethylenemelamine (TEM), was studied for its ability to induce unscheduled DNA (repair) synthesis (UDS) in vivo in rat lymphocytes. Somatic eytogenetie alterations were analyzed (in bone marrow) and compared with UDS as a function of TEM dosage. UDS was evaluated through the use of autoradi0graphy; cytogenetic alterations were studied in Inetaphase bone marrow chromosome preparations. D a t a indicated that the degree of UDS is a direct function of TEM dosage up to a rate-Hmiting concentration, at which point it ceases to be dose-dependent. Except for a deviation at the highest dose level tested, the extent of cytogenetic damage was directly and linearly related to TEM dose. Between the control and intermediate (0.2 mg/kg) dose levels, UDS response increased II-fold while cytogenetic damage showed only a 4-fold increase; this disparity diminished with increasing TEM dose. In the lower dose levels, therefore, the greater relative sensitivity of UDS elevation in the detection of genetic activity m a y be indicated. Patterns of UDS response observed through the in vivo assay developed in this study were ~ound to be analogous to those previously reported for in vitro studies.

29 FARROW, MICHAEL G., W y e t h Laboratories, Inc., Paoli, Pa. 193o1 (U.S.A.).

Improved microculture techniques utilizing the whole blood of the dog, guinea pig, monkey, mouse or rat Improved microculture techniques for chromosome analysis have been developed for five species: dog, guinea pig, monkey, mouse and rat. The advantages of the methods to be reported are: they do not necessitate killing the animals as do bone marrow techniques; they utilize a small amount of whole blood instead of the larger volumes needed in the usual white cell separation techniques; they are simple, rapid and reproducible and can be used either for individual chromosome studies or in the same animal when sequential chromosome analyses are required. All of the methods are based on stimulation of mitosis b y phytohemagglutinin and utilize commercially available chromosome media.

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3O WEBER, ERIKA, KAREN BIDWELL AND MARVIN S. LEGATOR, Brown University, Roger Willianls General Hospital, Providence, R. I. (U.S.A.).

An evaluation of the micronuclei test for routine cytogenetic analysis To determine the feasibility of the micronuclei procedure, for cytogenetic studies, a comparatively weak chromosome breaking agent, trimethylphosphate (TMP) and the potent alkylating agent, triethylenemetamine (TEM) were evaluated. The procedure followed was that of SCHMID et al. ~ with the following modifications: (a) direct flushing of bone marrow with 0.2 ml calf fetal serum, (b) air drying slides for a period of only i h, and (c) the use of p K 6.0 phosphate buffer to dilute both the Wright and Giemsa stains. With this technique a dose-response curve was generated for both TMP and TEM, using mice as the experimental animal. With TMP, a doubling over background was found when a concentration of 0.5 g/kg per day for 5 days was administered. To establish a statistically significant doubling dose over the control, a minimum of five animals must be used with 2ooo polychromatic cells being analyzed per animal. With TEM, activity was found over a dose range of o.I to Io mg/kg. The results obtained with these compounds compare favorably with what has been reported for the standard if, vivo metaphase analysis. ~"

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(I97I) IO3-II8.

31 TAN, JAMES C., Valparaiso University, Valparaiso, Ind. 46383 (U.S.A.).

Application of survey sampling techniques in the evaluation of muta. genicity data in cytogenetic studies This is a study on the theory and practice of survey sampling techniques as applied to the evaluation of mutagenicity data obtained from tight microscopic study of cytological anomalies. Two aspects of the sample design are emphasized : a selection process by which some members (cells) of the population are included in the sample; and an estimation process for computing sample statistics which are sample estimates of population values. The estimation for proportion of a cytological anomaly (nonorientation) in PMC of Solarium simplicifolium is used as an example to compare the accuracy and efficiency of four basic sampling designs: simple random sampling, cluster sampling, systematic sampling, and inverse binomial sampling. An ideal population of 26 873 cells whose positions in a slide are mapped is constructed, and various population values are calculated from this surveyed population. Sample statistics characteristic to each sampling design are computed and compared with the population parameters. The four basic sampling designs are evaluated on the basis of biasness and efficiency as measured by cost function. Their :feasibility of being applied in different mutagenicity studies involving cytological and chromosmnal anomalies will be discussed.