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Miral 500 CS (CAS# 42509-80-8), an organophosphorus insecticide, has been widely used in Columbia to fumigate coffee plantations. Therefore, there is extensive human exposure to this pesticide. Miral's mutagenic and genotoxic activities, however, are not known. In this study, such activities of the pesticide were evaluated using the Salmonella TA98/S9 test and the chromosome aberration assay in bone marrow cells of Swiss albino CD1 male mice. All doses tested with Salmonella in the presence of S9 mix (3.2, 16, 80, 400 and 2000 micrograms/plate) induced a mutagenic response that was three times the spontaneous mutation frequency. The mutagenic response without S9 was twice the spontaneous frequency. Based on a 4-day treatment (i.p.) of mice with Miral, the median lethal dose (LD50) and the maximum tolerated dose (MTD) were 912.5 mg/kg and 730 mg/kg, respectively. A significant dose-dependent cell cycle delay (r2 = 0.85, p < 0.01) was observed in bone marrow cells when mice were treated for 24 h with 73, 146, 219, 292, 365, 438, 511, 584, 657 and 730 mg/kg. Significant increase in mitotic indices (p < 0.02) and chromosome aberrations (p < 0.05) were induced in bone marrow cells, when mice were treated for 18 h with the highest dose 511 mg/kg. Our results indicate that Miral is a mutagenic compound in Salmonella and is capable of inducing chromosome aberrations at high doses in mice. Additional genotoxicity studies in farmers exposed to Miral should be conducted to determine the potential human health risk resulting from chronic low-dose exposures to this pesticide.

The use of biological markers in the evaluation of human exposure to hazardous agents has increased rapidly in recent years. Because 1,3-butadiene is a mutagenic carcinogen, existing occupational levels of exposure may be appropriately evaluated using somatic cell mutation as a biomarker. Previously, we have described a biomarker study of workers in a butadiene monomer plant (Ward et al., 1994). We now report results from a second study of the same group of workers, conducted after plant modernization, and present preliminary results from a study of exposures in a styrene butadiene rubber (SBR) plant. Air levels of butadiene were determined using either charcoal tubes with air pumps or passive badge dosimeters. The quantity of a butadiene metabolite in the urine was used as a biomarker of exposure and the mutagenic effects of exposure were measured using the autoradiographic hprt mutant lymphocyte assay. In all three studies, the frequencies of hprt mutants were significantly elevated in workers from the areas of highest exposure when compared to workers from lower exposure areas or non-exposed subjects. The concentration of the urinary metabolite was significantly increased in high-exposed workers in the first study of monomer plant workers but not in the second. In the first monomer plant study, historical air concentrations of butadiene were higher in the production units than in the central control unit. While concurrent determined air concentrations were not elevated in the second monomer plant study, they were elevated in high exposure areas in the SBR plant study. Mutant frequencies in the lower-exposure and the non-exposed groups were consistent with historical values for non-smoking individuals who were not exposed to known mutagens. The use of biomarkers, including the hprt mutant lymphocyte assay, may be of great value in determining an appropriate occupational exposure limit for butadiene.

An integrated population monitoring study was initiated to investigate whether occupational exposure to current low levels of butadiene is mutagenic to workers. Ten exposed workers (mean production area concentration of 3.5 ppm) and 10 matched plant controls (mean exposure to 0.03 ppm) were selected and blood samples were collected for our study. The standard cytogenetic assay was used to determine chromosome aberration frequencies. In addition, a challenge assay was used to determine response to gamma-rays as an indication of DNA repair deficiencies. In the latter assay, cells were exposed to gamma-rays at the G1 phase of the cell cycle in vitro and the frequencies of chromosome aberrations in the first post-irradiation metaphase cells were quantitated. Based on results of the cytogenetic assay, the exposed group had a higher frequency of cells with chromosome aberrations and higher chromatid breaks per 100 cells compared with the control. However, the difference was not significant (p > 0.1). With the challenge assay, the exposed group had a higher frequency of aberrant cells (p < 0.04), chromatid breaks (p < 0.05), deletions (p < 0.07), and dicentrics (p < 0.02) than the controls. In addition, the dicentric frequencies from workers were significantly correlated with the presence of a butadiene metabolite [1,2-dihydroxy-4-(N-acetylcysteinyl-S)butane] in urine with a correlation of coefficient of 0.6 (p < 0.01). Two outliers were identified and our interpretation of their responses will be discussed. This study indicates that the workers had exposure-induced mutagenic effects. Together with the observation of gene mutation in a subset of the present population, this study indicates that the current occupational exposure to butadiene may not be safe to workers.

Benzene is a widely used chemical and common environmental contaminant. It is carcinogenic in man and animals and is genotoxic in mice, rats, and occupationally exposed humans at doses above one part per million. In order to evaluate the genotoxic effects of prolonged exposures to very low concentrations of benzene, we exposed CD-1 mice to benzene by inhalation for 22 h per day, seven days per week for six weeks at 40, 100 and 1000 parts per billion (ppb). Additional groups were exposed to purified air or were housed in standard plastic cages. The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The weighted mean variant (mutant) frequencies (Vf) of female mice (three per group) were 7.2 x 10(-6) at 0 ppb; 29.2 x 10(-6) at 40 ppb; 62.5 x 10(-6) at 100 ppb and 25.0 x 10(-6) at 1000 ppb. The Vf of unexposed mice housed in standard cages was 13.2 x 10(-6). In male mice the same pattern of response was observed, but the increases in Vf in response to benzene were not as great. In both sexes of mice, the increases at 40 and 100 ppb were significantly greater than at 0 ppb (P less than 0.05). The increase in Vf with exposure to 100 ppb and the decline at 1000 ppb parallel the results observed for chromosome damage in spleen lymphocytes from the same animals (Au et al., Mutation Res., 260 (1991) 219-224). These results indicate that sub-chronic exposure to benzene at levels below the current Occupational Safety and Health Administration Permitted Exposure Limit may induce gene mutations in lymphocytes in mice.

Cytogenetic analysis has been used for several years in attempts to detect the mutagenic effects of suspect agents. Yet there remain many questions related to statistical analysis. While several procedures have been and are being used, many unfortunately are not always appropriate for the types of data actually obtained. Two general analysis approaches are discussed together with ways to insure that the test assumptions are reasonably met. Because assay sensitivity is a major concern, the sample size issue is discussed with examples for specific situations.

Using the dominant lethal assay, the ability of n-butyl glycidyl ether to induce mutations in male mice was investigated. No significant dose-related changes either in pregnancy rates or in average number of implants per pregnant female were found. However, while the results were not altogether conclusive, there was evidence of a significant increase in fetal death rates by the end of the first week after the highest dosage was administered.

While underlying biological mechanisms responsible for sister chromatid exchange (SCE) formation are not fully understood, scientists worldwide are increasingly using SCEs in the evaluation of excess risk from exposure to chemical and biological agents. SCEs are being used as endpoint measures of cell damage in many types of experimental and nonexperimental investigations. The former includes both simple and complex randomized experiments using both animals exposed in vivo and cells exposed in vitro as experimental units. The latter includes the important, yet potentially misleading, human case-control studies in which a group of humans who are or have been exposed to some agent are compared with a selected nonexposed group on SCE frequency. As more is learned about those factors which result in SCE variations, the assay techniques and study protocols can be adjusted to enhance study sensitivity and to minimize potential bias. Although research concerning sample sizes and statistical analysis methods has been conducted, more is needed. Search for other sources of intersubject variations in SCEs should continue so that such sources can be controlled in future studies, particularly the human exposure types. A number of experimental designs are presented and contrasted with their nonexperimental counterparts. Statistical methods are summarized and sample size options are given for the human and animal exposure studies and for the studies in which cells are exposed in vitro.