A protocol for the in vitro micronucleus test. II. Contributions to the validation of a protocol suitable for regulatory submissions from an examination of 10 chemicals with different mechanisms of action and different levels of activity.

The in vitro micronucleus test is currently used as a screening assay during the early stages of drug development by pharmaceutical companies to identify chemicals likely to produce positive outcomes in the in vitro chromosome aberration assay. For several reasons the assay is being considered as an alternative to the aberration assay-it requires less laboratory time, less material and less training. However, the current screening protocols are not rigorous enough to fully satisfy concerns about genotoxic safety.

A protocol for the in vitro micronucleus test. I. Contributions to the development of a protocol suitable for regulatory submissions from an examination of 16 chemicals with different mechanisms of action and different levels of activity.

The in vitro micronucleus (IVM) test is currently used as a screen during the early stages of pharmaceutical development to identify chemicals likely to produce positive outcomes in the in vitro chromosome aberration assay. For several reasons, the assay is being considered as an alternative to the aberration assay, but the current screening protocols are not rigorous enough to fully satisfy concerns about genotoxic safety. This manuscript describes the investigation of several protocol parameters to assist with the development of a regulatory guideline for the IVM test.

A comparison of the soft agar and microtitre methodologies for the L5178Y tk +/- mouse lymphoma assay.

The L5178Y tk +/- mouse lymphoma assay (MLA) has been validated as a sensitive and specific test system for the detection of mutagens/clastogens. There are currently two methodologies for performing the MLA: the original soft agar procedure and the newer microtitre procedure. While both procedures are considered acceptable, a limited amount of comparative information exists for the two methods.

An evaluation of micronucleus induction in bone marrow and in hepatocytes isolated from collagenase perfused liver or from formalin-fixed liver using four-week-old rats treated with known clastogens.

The bone marrow (BM) micronucleus (MN) test is a sensitive assay for identifying clastogens. However, some clastogenic compounds and metabolites may never reach the BM. The liver has been suggested as an alternative tissue to BM but adult rat liver has a low mitotic index that increases the difficulty of evaluating hepatocytes (HEP) for MN induction.

An evaluation of the twofold rule for assessing a positive response in the L5178Y TK+/- mouse lymphoma assay.

The L5178Y tk+/- mouse lymphoma assay (MLA) has been in use for more than 15 years as a tool for evaluating the mutagenic potential of various agents. As with other genetic toxicology test systems, one criterion for a positive response has been the requirement of at least a 2-fold increase in mutant frequency (MF) as compared to the respective MF of the solvent controls. More recently, an actual specific increase in MF has been proposed as a criterion for determining a positive response in the MLA; however, this may not be appropriate for laboratories with a low, yet stable, background MF.

Validation of an automated image analysis micronucleus scoring system.

The Loats Automated Micronucleus Scoring System was developed to assist with the evaluation of compounds for the ability to induce micronuclei in mouse bone marrow polychromatic erythrocytes (PCE). This image analysis system can identify PCE as well as normochromatic erythrocytes (NCE) and calculate the PCE/NCE ratio as an index for bone marrow toxicity. Two studies were conducted to provide slides for a comparison of micronucleated PCE values collected manually to those collected by the automated system.

The in vivo rat micronucleus test: integration with a 14-day study.

A 14-day subchronic toxicity study is routinely conducted in Fischer 344 rats at the Lilly Research Laboratories. This study is done to gather preliminary toxicological information about chemical entities showing efficacy in various pharmacological screens. This manuscript describes the validation of a method for evaluating micronuclei in the bone marrow polychromatic erythrocytes of animals from this test in order to obtain additional information about the genotoxic potential of these compounds without incurring the cost of additional animals or the use of additional test article, which is often in limited supply.

The gradient plate assay: a modified Ames assay used as a prescreen for the identification of bacterial mutagens.

Bacterial test systems have been used extensively to identify the mutagenic potential of new compounds. In particular, the Ames test has gained worldwide acceptance and is required by many regulatory agencies to support product registration. The gradient plate assay (GPA) is a modification of the Ames test.

Localization of the xanthine guanine phosphoribosyl transferase gene (gpt) of E. coli in AS52 metaphase cells by fluorescence in situ hybridization.

The purpose of this study was to localize the xanthine guanine phosphoribosyl transferase gene (gpt) to a specific chromosome to investigate its proposed autosomal location in the AS52 cell line. AS52 cells are hgprt-deficient Chinese hamster ovary (CHO) cells which carry a single functional copy of the E. coli gpt gene.

An evaluation of 6 chromosomal mutagens in the AS52/XPRT mutation assay utilizing suspension culture and soft agar cloning.

The AS52/XPRT mutation assay was examined for sensitivity in the detection of chromosomal mutagens. 6 compounds identified as chromosomal mutagens in the mouse lymphoma assay (MLA) were tested for mutagenicity in AS52 cells using suspension treatment and soft agar cloning. The 6 compounds were benzo[a]pyrene (BAP), 2-acetylaminofluorene (2AAF), methylmethanesulfonate (MMS), methyl acrylate (MCR), benzoin (BZ), and p-aminophenol (AM).

A comparison of the CHO/HGPRT+ and the L5178Y/TK+/- mutation assays using suspension treatment and soft agar cloning: results for 10 chemicals.

The mouse lymphoma assay (MLA) and Chinese hamster ovary (CHO) cell assay are sensitive indicators of mutagenicity. The CHO assay has been modified technically to permit treatment in suspension and soft agar cloning comparable to the MLA. This methodology eliminates the risk of metabolic cooperation and the trauma of trypsinization.

The evaluation of methapyrilene for bacterial mutation with metabolic activation by Aroclor-induced, methapyrilene-induced and noninduced rat-liver S9.

The antihistamine methapyrilene (MP) has been shown to be a potent hepatocarcinogen in rats. However, it has demonstrated little genotoxic activity in a wide variety of short-term tests. In this study, Fischer 344 rats were fed a carcinogenic dose of 0.

Genetic toxicology studies with a tumor promoter.

A diuretic antihypertensive agent, SC-33643 (8-[2-ethoxyethyl]-7-phenyl-[1,2,4]triazolo[4,3-c]pyrimidine-5- amine, also known as bemitradine), was tested in the Ames test, in the mouse lymphoma TK +/- mutation assay, in the Chinese hamster ovary cell hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) mutation test and in the CHO chromosome aberration assay with and without metabolic activation. Additionally, the compound was tested in the rat primary hepatocyte unscheduled DNA synthesis (UDS) assay and in the mouse bone marrow micronucleus assay. The results were uniformly negative.

Genotoxic properties of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU).

(E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU)is a 5-substituted 2'-deoxyuridine antiviral compound that inhibits thymidylate synthetase. The selectivity of BVDU for virus-infected cells has been attributed to phosphorylation of BVDU by a virus-induced thymidine kinase. Since the closely related compounds 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine are in vitro and in vivo mutagens, BVDU was tested for genotoxic activity in bacterial and mammalian cell mutation assays as well as in assays measuring DNA damage/repair and clastogenic activity.

Diphenylhydantoin is not genotoxic in a battery of short-term cytogenetic assays.

5,5-Diphenylhydantoin (DPH) is an antiepileptic drug associated with an increase in malformations in infants born to women taking DPH during pregnancy. Positive and negative results have been reported by various investigators for in vivo and in vitro chromosome aberration (CAB) assays, in vivo and in vitro sister chromatid exchange (SCE) assays, and in vivo micronucleus tests (MNT). In this laboratory, DPH was tested in an in vitro CAB assay using Chinese hamster ovary cells with and without an S-9 activation system, an in vivo SCE assay in female CD-1 mice, an in vivo MNT, using both male and female CD-1 mice, and a transplacental micronucleus test.

Expulsion of demecolcine-induced micronuclei from mouse bone marrow polychromatic erythrocytes.

The mouse micronucleus test is a valuable tool for evaluating in vivo chromosome damage produced by test articles in polychromatic erythrocytes of bone marrow. Compounds that are clastogens, such as cyclophosphamide, induce micronuclei that are smaller than those induced by compounds that are spindle poisons, such as demecolcine. In vitro studies have previously shown that the frequency of mitomycin C- and vincristine-induced micronuclei in mouse L-929 cells was reduced due to micronuclear extrusion following treatment with cytochalasin B.

The evaluation of a multiple dosing protocol for the mouse bone-marrow micronucleus assay using benzidine and 2,6-xylidine.

Male ICR mice were treated with 1, 2 or 3 daily doses of either benzidine or 2,6-xylidine. Groups of 5 animals were sacrificed 24 h after the last dose and the bone marrow examined for micronuclei. Benzidine was given at dose levels of 40 and 200 mg/kg and 2,6-xylidine was given at dose levels of 75 and 375 mg/kg.

Mutagenicity evaluation of HC Blue No. 1 and HC Blue No. 2, II. Effect on the in vitro induction of unscheduled DNA synthesis in rat, mouse, rabbit, hamster, and monkey primary hepatocytes.

2 hair dyes, HC Blue No. 1 and HC Blue No. 2, were evaluated for the in vitro induction of unscheduled DNA synthesis (UDS) in primary hepatocytes of rat, mouse, hamster, rabbit and monkey.

Mutagenicity evaluation of HC Blue No. 1 and HC Blue No. 2, I. Effect on the induction of micronuclei in mouse bone marrow cells.

Two hair-dye chemicals, HC Blue No. 1 and HC Blue No. 2, were assessed for the ability to produce chromosome breakage and/or spindle malformation in vivo by evaluating the capacity of these compounds to induce micronuclei in polychromatic erythrocytes of mouse bone marrow.

Pooled inference across sexes for the in vivo micronucleus assay.

The Japanese Environmental Mutagen Society has investigated the extent of sex differences in the in vivo micronucleus assay (Sutou et al., 1986). In light of their findings, this manuscript reexamines the statistical analysis of the assay.

Quantification of unscheduled DNA synthesis by a whole cell counting method.

A procedure was developed for the quantification of the autoradiographic assay for unscheduled DNA synthesis. Relative to commonly used practices for grain counting, this procedure provides a more accurate net nuclear grain count by eliminating the subjectivity currently associated with selection of the areas to be counted for the cytoplasmic background count. Briefly, the object area and aperture area modes of an ARTEK 880 colony counter are used to collect values for the total number of silver grains over a particular cell (nuclear and cytoplasmic counts), as well as for the nuclear and cytoplasmic areas.