Novel statistical approach for evaluating flow cytometric in vitro micronucleus data.

Statistical methods currently recommended for analysis of in vitro micronucleus data are based on small sample sizes. The tests are designed to evaluate linear trends and differences between treated and control samples. When using flow cytometric analysis, >5 times the number of cells are easily evaluated, and the variance estimates from these large samples are small. Application of these recommended tests to large samples resulted in statistically significant outcomes which were not considered to be biologically meaningful. Alternative statistical methods for testing trends and differences among treatments that were either widely used, or sample-size independent, were investigated. Using data from 95 experiments (from 2011-2013) where 19% of the experiments were considered positive, results for the various statistical methods were compared. When using either the recommended or alternate methods, 42-68% of the experiments resulted in statistically significant results (p < 0.05). A new concept was then tested using the same data sets: the "z' factor", designed to identify 'hits' during high throughput screening. Using this simple-to-compute statistic the number of significant calls was reduced to 27%. Then, when combined with a biological criterion based on historical vehicle control data, there was restoration of the original positive frequency (19%). Given the larger sample sizes evaluated using flow cytometry, we have demonstrated that traditional statistical tests may be overly sensitive to small changes in micronucleus induction, and that a simple-to-compute index of separation (z') may be a better tool for analysis, provided that the response is first determined to be biologically meaningful. Environ. Mol. Mutagen. 57:589-604, 2016. © 2016 Wiley Periodicals, Inc.

Biologic markers in hospital workers exposed to low levels of ethylene oxide.

Operators of hospital sterilizers that use ethylene oxide were studied to determine if there was a relationship between exposure and a battery of biological markers. A total of 73 workers from nine hospitals in the United States (U.S.) and one hospital in Mexico City was evaluated for ethylene oxide exposure during four months prior to collection of peripheral blood. The frequency of hemoglobin adducts (p = 0.0006) and sister-chromatid exchanges (SCEs) (p = 0.002) increased with cumulative exposure to ethylene oxide in U.S. subjects when controlling by regression analysis for various confounding factors, including cigarette smoking. Hemoglobin adducts, but not SCEs, were also increased in Mexican subjects (p = 0.0012). Chromosomal micronuclei showed no consistent relationship with exposure. The U.S. study participants were classified by four-month cumulative exposure levels of 10 ppm-h (n = 8), greater than 0 to 32 ppm-h (n = 32) and greater than 32 ppm-h (n = 11) of ethylene oxide exposure. The group with an exposure of greater than 32 ppm-h had an increased frequency of hemoglobin adducts (p = 0.002) and SCEs (p = 0.0001) compared to the nonexposed group. The estimated mean of the 8-h time-weighted average (8-h TWA) exposure levels for the highest U.S. exposure group (greater than 32 ppm-h) was 0.16 +/- 0.007 ppm (mean +/- SD). A similar exposure-related differential was observed in the Mexican subjects for hemoglobin adducts (p = 0.04) but not for SCEs. The latter finding may have been due to longer shipping times for the specimens in the cytogenetic assays. The estimated mean of the 8-h TWA exposure levels for the highest Mexican exposure group (greater than 32 ppm-h) was 0.48 +/- 0.08 ppm. This study is the third to suggest that exposures less than the U.S. OSHA standard of 1 ppm 8-h TWA result in biochemical and biologic changes. It is not known whether these changes may be indicative of increased risk of disease; however, they do appear to reflect exposure to relatively low levels of ethylene oxide. The exact meaning of these changes is unknown.

Comparison of single-, double- or triple-exposure protocols for the rodent bone marrow/peripheral blood micronucleus assay using 4-aminobiphenyl and treosulphan.

Two human carcinogens, 4-aminobiphenyl (4AB) and treosulphan (Treo), were tested in male B6C3F1 mice for the induction of micronuclei in bone marrow and peripheral blood cells by 1-, 2- and 3-exposure protocols. Both compounds tested positive. The magnitude of response with respect to the incidence of micronucleated polychromatic erythrocytes by 2- and 3-exposure protocols was considerably higher than by the single-exposure protocol. The peripheral blood results for Treo were as typically seen with a 24-h delay when compared to the bone marrow. The peripheral blood results for 4AB, however, differed from those expected. The incidence of MN-PCE in peripheral blood of animals exposed to 4AB was significantly greater than seen in the bone marrow in 2- and 3-exposure protocols. There was also an increase in the % PCE at the 60 mg/kg dose level as a function of time. Based on these studies, it is concluded that a step-wise scoring scheme may be the best protocol for rodent micronucleus assay, involving a 3-exposure protocol with single sampling of bone marrow (24 h after the last treatment) and two samplings of peripheral blood (24 h and 48 h after the first treatment). This approach is cost-effective, it limits the number of animals required and provides maximum sensitivity.

Results of tests for micronuclei and chromosomal aberrations in mouse bone marrow cells with the human carcinogens 4-aminobiphenyl, treosulphan, and melphalan.

Three human carcinogens, 4-aminobiphenyl, treosulphan, and melphalan, were tested for the induction of micronuclei or chromosomal aberrations in the bone marrow cells of male B6C3F1 mice. These studies were conducted to provide further information on the in vivo genetic toxicity of compounds known to cause cancer in humans. All three compounds gave positive results in the mouse bone marrow micronucleus test, and melphalan, the only compound tested for aberration induction, was positive in this assay. These results extend the evidence that nearly all known human carcinogens are detected in relatively simple and widely employed short-term in vivo tests.

Comparison of metabolic systems required to activate pro-mutagens/carcinogens in vitro for sister-chromatid exchange studies.

Irradiated Syrian hamster fetal cells (feeder layer) and rat-liver homogenate (S9 mix) were used to compare their capacity to metabolize 3 known promutagens/carcinogens; BaP, 3-MC and DMBA. DNA-damaging potential was determined by the induction of SCE in V79 target cells. The S9 mix (1/20th strength) was toxic to the target cells and reduced the mitotic index by half with an exposure time of 2.5 h. The feeder layer was not toxic to the target cells and, therefore, was included for the duration of the Expt. The test chemicals elicited a dose-response with both activating systems. At similar concentrations of the test chemicals, the cells grown on the feeder layer showed a greater number of SCEs as compared to those activated by the S9 mix.