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APPE NISI X H EVALUATION OF DATA FROM SHORT-TERN GENETIC TESTING OF ANTICHOL INERG IC CHEMI CALS by Virginia C. Dunkel, Ph.~. * A number of short-term tests can be used to determine the geno~oxic potential of chemicals. These tests use both pro~"otic and eukaryotic cells and measure such end points as gene muta~elons chromosomal aberrations, and interactions with critical macromolecules. It is widely recognized that no test can detect all genotoxic compounds, and multiple end points are required to provide a reliable assessment of genotoxicity. Information from several tests can be combined to reveal two important toxic effects: c arc inogenesi ~ a nd mutagenest s . There are reports of studies in which short-Berm tests have been used to determine the genotoxic potential of the anti- cholinergic compounds 3-quinuclidinyl benzilate (BZ), atropine, and scopolamine. The objective of this discussion is to evaluate the data available on these compounds, to determine whether they are genotoxic. Such information would aid in the overall assessment of the potential of these drugs to produce long-term adverse health ef feet s . 3-QUINUCLIDI`~L BENZILATE In the reported studies of (BZ), the test systems used have had three different end points: point mutations, chromosomal aberrations, and dominant-la that effects. In the point-mutation studies (1), Saccharomyces cerevisiae was used as the target cell, and the effects of the chemical were tested in a direct in vitro assay without metabolic activation and in a has unmediated modification. In the direct reverse mutation assay, there were increases by a factor of 2-6 in the number of dish revertants and by a factor of 2-7 in the number of trp+ revertants over the spontaneous frequencies. In contrast, there were no reported increases in the number of His+ or tips revertants in the has Mediated assay. Cytogenetic analyses f or chromosomal aberrations were carried out on cells from mice (1) and Chinese hamsters (12) treated with BZ and on human peripheral lymphocytes (1) treated in vitro. It was reported that BZ did not induce tranalocations and did not increase the number of abnormal meeaphases in spermatocytes from male mice. *Chief, Genetic Toxicology Branch, Food and Drug Administration, Washington, DeCe 20204 e

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With bone marrow cells from treated mice, an increase in the frequency of abnormal metaphasea vea report et, but there is no lafonnation on the type of abnormality observed. Similarly, in the studies with human peripheral ly~phocytea, "aberrant cells" were observed at a single concentration of BZ (10-4 M), but again the type of aberration wee not identlflet. Lower concentrations hat showed no effects, and higher concentrations were reported to be toxic. In the studies with bone marrow cells from BZ-treated Chinese hamsters, Sram (2) reported that there was a dose-related increase in the number of gape and that the frequency of breaks was increased, but not dose-related. That the occurrence of gape is not a useful mesaure of the clastogenic potential of a chemical has been substantiated in ocher atutlea (3). In addition, although it was concluded that the frequency of breaks observed vas not dose-related, it appears from the data that there were increasing numbers with increasing dose, which plateaued and then began to decrease at the highest doses tested. The final system used in the evaluatin of BZ was the dominant-lethal aaasy (1). The only effect observer in these studies was a tecresee in the ferellity of the malea at the highest dose tested. Such an effect is indicative only of the toxic effects of the test compound, Overall, at moat a marginal response might be indicated by the results of the point-mutation aaasys with Saccharoeyces cereviaiae and the possible presence of cytogenic effecta--breaks in Chinese hamster bone marrow cells. The Significance of these effects, however, is open to question, ant further studies are required to confirm or negate the original obaervationa. Since these studies were reported, a substantial amount of information has been obtained on the predictive capacity of ahort-term teats, and other assays, Such as of gene mutation in bacteria and maamallan cells and of unscheduled DNA aynthesia, might provide a better base of information for reaching a conclualon about the potential genotoxicity of BZ. SCOPOLAMINE The studies reported on scopolamine are also limited and inconclusive, even though the capacity to induce DNA damage and point mutatlona in bacteria and chromosomal aberrations In "lien cells has been tested. No effects were observer In either the DNA-damage sassy (4) or the point-.utation aaasy in Salmonella (5), but the data available OD neither of these ayateea can be considered sufficient for evaluation. In the DNA-damage ready, the compound ~ a treated at only two doses and only In the absence of an exogenous metabolic activating system. The testing in Salmonella :2~is~rtu~ (the Salmonellamicrosome (teat), although conducted bobby -and without Metabolic actlvatlon, wee at a alugle concentration, and only two teat Strains were used. In testing of a compound for its ability to induce mutations in this aaany, it is necessary to use a range of concentrationa and at least four of the five test strains (6).

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In the studies carried out to determine whether acopolamine had the capacity to induce chromosomal aberrations, three different types of mammalian cells were used: He La ant BSC-K cell lines and human peripheral leukocytes (7). No effects were observed in either the human leukocytes or the BSC-K cells, but chro~atid aberrations were observed in HeLa cells at a final 1: concentration of the compound. The aberrations were gaps ant breaks, but there was no evidence of exchange figures, such as translocations. As Indicated previously, the presence of gaps is not a significant Because of clastogenic potential, ant the presence of breaks in a single cell line toes not establish the mutagenic potential of a chemical. The available data on scopolamine are insufficient, and a conclusion cannot be drawn as to whether this chemical is potentially genotoxic. ATROPINE As is the case with BZ and scopolamine, reports are available on the testing of atropine for les capacity to induce DNA damage and point mutations in bacteria and chromosomal aberrations in mammalian cells. With one exception, these studies are deficient in one respect or another, and no teflnitive conclusion can be drawn from them. The problems include lack of incorporation of a metabolic activation system in assays in which the target cell does not have full metabolic capabilities (4,8); lack of a range of doses up to ant including one at which toxic effects are observed, that reaches the level of solubility, or that is recognized as sufficient for testing (4,9); lack of specification of the dose used (10); and inclusion of a test method, induction of chromosomal aberrations in grasshopper spen~atocytes (113, with which experience in genetic toxicology is limited. In the single study in which there is adequate information, McCann _ al. (12) reported that atropine toes not induce point :eutations in the Salmonella/microsome assay. The compound was tested at up to 5,000,ug/plate, both with ant without metabolic activation, in rest strains that can reveal both base-patr and frameshift mutations. This negative result, however, does cot in itself provide sufficient infoneation to justify a conclusion about the genotoxic potential of the compound. CONCLUSIONS The lack of adequate data, including test results, and limitations in the test systems used preclude ~ definitive assessment of the genotoxic potential of BE, Scopolamine, and atropine. Only through further studies can a conclusion be reaches about the genotoxic potential of these chemicals.

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8. 9- REFERENCES 1. Srao', R.J., K~cerova, M., and Bartode, Z . 19 75: ~tagenic activity of 3-chinuclldylbenzilate. Act. Nerv. Super., 17: 252-253. 2. Sra2e, R.J. 1975: Cytogenic analysis of 3-chinuclidylbenzilate, 3-chinuclidinol ant benaceyzine in bone marrow of Chinese hamater. Act. Nerv. Super., 17:253-254. L:egator, M.S. Palmer, K.A.. and Adler, I-D. 1973: ~ collaborative study of in vivo cytogenetic analysis. I. Interpretation of slite preparations. Toxicol. and Appliet. Pharm., 24:337-350. 4. Fluck, E.R., Poirier, L.A. and Ruelius, Hana, W. 1976: Evaluation of a DNA polyo~ereae-deficient o~utant of 8. Coli for the rapid detection of carcinogena. Chem. Biol. Interactiona, 15: 219-231. 5. Waskell, L. 1978: A study of the mutagenicity of aneathetica and thetr metabolites. ^~. Res., 57:141-IS3. 6. deSerres, F.J. and Shelby, M.D. 1979: Recoomendationa on data production and analysis uaing the Sal~onella/o~icrosome asasy. En~rtron. Mutagen. ~ 1:87-92. Vrba, M. 1967: Chromosoo~enaberrationen, hervorgeruten durch scopolaminum hytrobromat~n. Humangenetick, 6~: 371-373 ~ Ishidate, Jr., M., lIayashi, M., Sawada, M., Matauolca, A., Yoshikawa, K., Ono, M., and Naicadate, M. 1978: Cytotoxicity test on medical druge-chromosome aberration tests wIth Chinese hameter cells in vitro. Eise! Shikenyo Hokoku, 96:55-61. Com~oner, 8. 1976: Reliability of bacterial mutagenesis techniquesto distis~guish carcinogenic and non~carcinoge~c chemicals. UISNTIS PB Report; EPA-600/1-76-022. 10. ttinnich, V., - 4th, M.~., Thompeon, D. and Rornfeld, S. 1976: Detection of ~utagenic activity in huean urine ualng mutant ~ traine of Salmonella typhimurium. Cancer, 38 :1253-1258. 11. Saha, A.K., and Chattopadhyay, S.C. 1977: Studlea on the effect of atropine in the production of chromosome anomaliea in apermatocytic chromosomea of Indian acritit grasahopper. Chromosome Infor~ation Ser~rice, No. 22 pp 3-5. 12. McCann, 3., Choi, E., Yamsaski, B., and Amea, B.N. 1975: Detection of chemical carcinogens as mutagens in the Salmonella/microsome teat; Asasy of 300 cheo~icala. Proc. Natl. Acad e Sci e USA, 72: 5135~5139e 11 4