R. Valencia

Chemical mutagenesis testing in Drosophila. IX. Results of 50 coded compounds tested for the National Toxicology Program.

Results are presented from mutagenesis testing in Drosophila of 20 coded compounds. Two compounds were positive in the sex-linked recessive lethal test, and two were inconclusive. The positive compounds were calcium chromate in adult feeding experiments and ferrocene after adult injection. The two inconclusive compounds were 2,4-diaminophenol dihydrochloride and hexachlorocyclopentadiene. Compounds that produced a positive response were assayed for chromosome breakage using the conventional translocation test. Calcium chromate was negative in the translocation test, and ferrocene was positive. Many of the test compounds were poorly soluble in water, raising questions regarding the effective concentration to which the flies were exposed.

The sex-linked recessive lethal test for mutagenesis in Drosophila melanogaster. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The test for sex-linked recessive lethals (SLRL) in Drosophila melanogaster has been used to detect induced mutations since 1927. The advantage of the test for both screening and hazard evaluation is its objectivity in testing for transmissible mutations in the germ cells of a eukaryote. Statistical criteria for both positive and negative mutagenicity at the highest concentration tested under a particular exposure condition were developed by the Work Group, and a recommended protocol for future testing was agreed upon. For 421 compounds there were sufficient data available in the literature for analysis; 198 compounds were found to be positive and 46 negative at the highest concentration tested. Most experiments had been done for objectives of pure research rather than for deliberately screening for mutagenicity, although many of the 421 chemicals were selected for testing because of suspected mutagenicity. Therefore, the statement of 198 positive and 46 negative should not be taken as an example of the proportion of mutagens in the environment. In three sets of experiments with D. melanogaster that were done specifically for screening, one involving 40 compounds for the Environmental Protection Agency (EPA), the others involving 13 for the Food and Drug Administration (FDA), only 6 mutagens were discovered. After completion of the classification of compounds according to their response in the SLRL test, the compounds were classified as to their carcinogenic response according to the list of Griesemer and Cueto (1980). There were 62 compounds that could be classified as positive or negative for both carcinogenesis and mutagenesis. Of the 62 compounds, there was agreement between the carcinogenesis and mutagenesis classification in 56 (50 positive and 6 negative), or 90% would have been correctly classified as to carcinogenesis from only the SLRL test. Because of inadequate sample size, 177 compounds could not be classified as positive or negative according to the statistical criteria established by the Work Group. This large number of inadequately tested compounds reflects the fact that many of the experiments were not done for screening. Further work is needed on the compounds with inadequate sample size.(ABSTRACT TRUNCATED AT 400 WORDS)

Study of Pesticide Genotoxicity

With a limited supply of arable land supporting an ever-increasing human population, the threat of crop loss to agricultural pests becomes continually more acute. Thus pesticides have become an essential component of modern agriculture. As competing organisms evolve resistance to commonly used agents, new and more effective poisons and repellants must constantly be developed. The fundamental problem in pesticide development is to produce chemicals that act specifically against certain organisms without adversely affecting others. Because of the similarities in the structural, metabolic and genetic components of all life forms, absolute species specificity is frequently difficult to attain. Furthermore, such toxic chemicals improperly used may engender biological effects beyond those for which they were originally manufactured.

An overview of short‐term tests for the mutagenic and carcinogenic potential of pesticides

In the last few years, marked progress has been made in the development of methods for evaluating the mutagenic and carcinogenic potential of pesticide chemicals. The correlation of genetic and related biological activity in short-term tests with carcinogenic activity in whole animals allows the utilization of short-term mutagenicity bioassays to prescreen chemicals for effects related to mutation induction and presumptive carcinogenicity. In addition, bioassays now available can measure directly the chemical transformation of normal cells in culture into cells capable of producing tumors when injected into animals. This paper will review briefly the major types of relevant short-term tests and will develop a rationale for a phased approach to the evaluation of the mutagenic and carcinogenic potential of environmental chemicals. This approach involves the sequential application of bioassays which are organized into a three-level matrix emphasizing first detection, then confirmation, and finally hazard assessment. Chemicals demonstrating positive results in the short-term detection systems and confirmatory bioassays are pursued in higher level whole animal define a negative result. The phased approach should facilitate a cost effective utilization of limited testing resources and provide protection for human health in proportion to the anticipated hazard. Results obtained in evaluating a series of thirty-eight pesticide chemicals according to the phased approach discussed in detail.

The genetic toxicology of Kathon biocide, a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one.

Kathon biocide, an aqueous solution containing a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one in an approximate ratio of 3:1, was tested for mutagenic activity in Salmonella typhimurium, L5178Y mouse lymphoma cells in culture and Drosophila melanogaster. Tests also were conducted for chromosome aberrations in vivo on mouse bone marrow cells, for DNA damage/repair in primary rat hepatocytes in culture, and for morphological transformation in C3H 10T1/2 cells in culture. Kathon biocide produced point mutations in the absence of a rat-liver metabolizing system in bacteria (strain TA 100) and mammalian cells in culture. In the presence of rat-liver metabolizing system a 10-fold higher concentration was required to induce point mutations in mammalian cells in culture. No mutagenic activity was observed with the metabolizing system and S. typhimurium. Negative results were obtained in the sex-linked recessive lethal assay in Drosophila, the in vivo cytogenetic assay in mice, the unscheduled DNA synthesis assay in cultured rat hepatocytes, and the in vitro cell transformation assay.

Comparative mutagenicity testing of a drug candidate, U-48753E: mechanism of induction of gene mutations in mammalian cells and quantitation of potential hazard

U-48753E is a potential human drug which was subjected to a battery of short-term assays for genetic activity. The compound was negative in the Salmonella (Ames) test, the in vitro UDS assay, the mouse bone-marrow micronucleus test and the Drosophila sex-linked recessive lethal assay. However, it was weakly positive in the CHO/HPRT assay in the presence of metabolic activation (S9). The weak positive response might easily have been labeled artifactual since there was no dose response and the dose level producing positive findings varied from experiment to experiment. In addition, the weak positive response was not confirmed in V79 cells. However, a reproducible dose-related increase in mutants was observed in the AS52/XPRT assay in the presence of S9. Metabolism of this drug proceeds through conversion of aliphatic N-methyl groups to formaldehyde. Addition of formaldehyde dehydrogenase to the S9 resulted in elimination of the mutagenicity of the compound in AS52 cells. Thus, the mutants were probably induced by formaldehyde. From the endogenous levels of formaldehyde in human blood, and the limiting potential therapeutic dose levels, the genotoxic hazard associated with U-48753E is marginal. This assessment of risk and its quantitation depend upon an understanding metabolism and exposure limits imposed by known side effects of the drug. This study can serve as a model for quantitative genetic risk assessment when mutagenicity is due to N-demethylation and formation of formaldehyde in situ.

A cytogenetic study of radiation damage in entire genomes of drosophila

Abstract Adult Drosophila melanogaster males, aged 1–2 days, were X-irradiated with 4000 R and mated for approximately 48 h to females containing genetic markers and/or crossover suppressors in all chromosomes. F1 females of the desired genotype were collected and appropriate genetic schemes followed to arrive at balanced stocks of entire treated genomes. Each chromosome of each genome was observed for visibles, lethals, sterility factors and extreme detrimentals. All chromosomes of each genome were examined cytologically for deficiencies, inversions, transpositions, etc. , and to determine the breakpoints of translocations, previously detected genetically. Of the 310 genomes analyzed, 63.2% were found to contain genetic damage. It can be calculated that approximately 2265 genomes were actually irradiated, of which 10.7% transmtted genetic damage to succeeding generations. Inherited damage was tabulated in terms of damaged “sites”, a site being either a point mutation or a chromosome break. The number of sites in any particular genome varied from 1. to 6, there were on the average 1.12 sites per genome, and the average number of sites per chromosome was found to be related to mitotic length. Analyses were made of the interrelationships of the different types of damage by tabulating rearrangements according to cytological nature, viability, fertility and phenotypic expression, of recessive lethals according to cytological nature, and of visibles according to cytological nature, viability and fertility.

THE VERSATILITY OF DROSOPHILA MELANOGASTER FOR MUTAGENICITY TESTING

I trust that I have accomplished my aim in this presentation, to demonstrate that Drosophila mutagenesis testing protocols are varied and are adaptable. The choices are many, at different levels. First, the route of administration can be chosen that is most appropriate for the chemical compound or to duplicate the human exposure route or to obtain data needed for some particular purpose, such as comparative mutagenesis. Second, the genetic test scheme or combination of schemes can be chosen to suit the purpose of the testing (screening, risk evaluation, or comparative mutagenesis). Third, by treating the appropriate fly development stage and managing the mating and brooding procedures, the germ cell stage can be sampled that is the critical one for the mutagenic effect of that compound and the correct one for the end point to be observed. Fourth, in screening, using the SLRL assay, the experiment size can be adjusted in accordance with the test sensitivity desired, the projected use of the data, and time and cost restrictions. In summary, Drosophila melanogaster has long been known as a most versatile organism for genetic studies of all kinds--and we now know that this versatility extends to the evaluation of chemical compounds for mutagenic effects. If it were only a mammal!