Michael L. Garriott

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. Using a protocol previously developed by testing 16 chemicals, this manuscript contributes to the validation of the protocol using 10 additional chemicals. Furthermore, conclusions drawn from the developmental effort regarding the need for an extended exposure in the absence of metabolic activation, the number of cells to be counted, and the preferred statistical procedure for the assay are re-examined. The recommended, validated protocol utilizes cytochalasin B and 4h exposures in the presence and in the absence of metabolic activation, specifies the need to test to a relative survival rate of approximately 50%, requires the counting of 2,000 binucleated cells per treatment concentration, and employs a trend test for statistical analysis of the data.

A protocol for the in vitro micronucleus test

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. The parameters investigated are: the effect of cytochalasin B on the outcome of the assay when conducted with continually growing cell lines; the need for an extended exposure in the absence of metabolic activation; and the number of cells to be counted for a valid assay. In addition, two statistical procedures for the analysis of data from the test are described. The results of the investigation indicate that cytochalasin B does not effect the outcome of the test, that the extended exposure treatment is not necessary, that counting 2000 cells is preferable to counting 1000, and that the data can be appropriately analyzed using a trend test.

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. Compounds selected for evaluation were acetylsalicylic acid, mitomycin C, cyclophosphamide, colchicine, 6-mercaptopurine, and etoposide. With the exception of colchicine, the results obtained were as expected with acetylsalicylic acid yielding negative results and the other compounds yielding positive results. These findings are consistent with those published for mice (MacGregor et al., Fund. Appl. Toxicol., 14, 513-522, 1990) and show that a bone marrow micronucleus test can be successfully integrated into a routine subchronic rat toxicology study.

Genotoxicity studies on the preemergence herbicide trifluralin

This paper reports the results of studies conducted within the Lily Research Laboratories and discusses additional studies with trifluralin that have been reported in the literature

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. In addition, a larger population of cells can be treated and cloned for mutant selection. In order to compare the effectiveness of the test systems, 10 chemicals were evaluated for the induction of forward mutations in the CHO and MLA. Several of these chemicals have been reported as clastogenic; therefore, abbreviated colony sizing was performed to gauge the extent of genetic damage to the MLA cells. Both test systems detected benzo[a]pyrene, mitomycin C, acridine orange, and proflavin, and, with the exception of proflavin, more large colonies were present than small colonies. The suspect clastogen, phenytoin, was not mutagenic in the MLA and produced inconclusive results in the CHO. Ethidium bromide, a clastogen and a bacterial mutagen, was not mutagenic in either the MLA or CHO. Four compounds (p-aminophenol, benzoin, methoxychlor, and pyrene) were positive in the MLA, generally inducing a large number of small colonies, while demonstrating no mutagenic activity in the CHO assay. They have also been shown to be generally nongenotoxic in other test systems. Overall, the modified CHO assay did not appear to be better than the MLA for the detection of mutagenic agents. However, the MLA does appear to have lower specificity.

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. Chemical mitogens and partial hepatectomy have been used to increase HEP proliferation to improve the sensitivity for detection of clastogenic compounds, but these practices raise concerns for the evaluation of drug candidates. The use of 4‐wk‐old rats provides an alternative to mitogenic stimulation because livers from these animals have ∼5.4% of their HEP in S‐phase. HEP were isolated by collagenase perfusion, or from formalin‐fixed tissue, from 4‐wk‐old treated rats. Six compounds were evaluated for the incidence of MN in HEP that were isolated by both methods. The results for MN induction by these compounds were similar for the two methods and confirmed that formalin‐fixed tissue is an acceptable source of cells for evaluating MN induction in HEP. BM polychromatic erythrocytes (PCE) also were harvested at the end of the live phase for each study and then evaluated for the incidence of MN. Diethylnitrosamine and 2‐nitrofluorene induced MN in HEP but had no effect in PCE. 2‐Acetylaminofluorene, cyclophosphamide and 7, 12‐dimethylbenz[a]anthracene did not induce MN in HEP but were positive in PCE. The direct‐acting clastogen, mitomycin C, was positive in both HEP and PCE. These results indicate that this modified liver micronucleus test, using 4‐wk‐old rats, offers an alternative to existing methods that use mitogens or partial hepatectomy to stimulate cell replication. Analysis of MN from formalin‐fixed tissue provides additional flexibility by allowing the investigator to assess MN induction at a later time. Environ. Mol. Mutagen. 29:379–385, 1997

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. In this report the two methods were compared with regard to: (1) spontaneous and induced mutant frequencies; (2) cloning efficiencies; and (3) colony size distributions for mutants. In addition, small and large mutant colonies from microtitre wells were rechallenged for trifluorothymidine (TFT) resistance. In a majority of the cases, cloning efficiency values were higher for the microtitre as were the spontaneous and induced mutation frequency (MF) values. Nevertheless, when responses were compared according to mutation index (fold increase over background MF) the results from the two systems were often similar. More spontaneous small colonies were observed in the microtitre assay. While colony size distribution for induced mutant colonies was compound specific, generally, more small colonies were counted in microtitre. All mutant clones that were rechallenged with TFT demonstrated resistance. Aside from the differences mentioned above, both the microtitre and the soft agar procedures appear equally capable of identifying mutagenic agents.

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. Mitomycin C was used as a micronucleus-inducing agent to elicit a positive response and Lilly compound 303497 was used as an example of a compound negative for the induction of micronuclei. No statistically significant differences were observed between micronucleus counts obtained manually and those obtained by the automated system. The PCE/NCE ratios calculated by the automated system were also similar to those determined from the manually collected PCE and NCE counts for the vehicle and positive controls, however, differences in the ratios were observed in compound treatment groups. These differences were attributed to a larger population of transitional cells in the treated groups. These results confirm that the Loats Automated Micronucleus Scoring System is an acceptable alternative to manual evaluation of mouse bone marrow slides for the incidence of micronucleated PCE.

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). BAP, 2AAF and MMS were mutagenic. The mutagenic responses for BAP, 2AAF and MMS included small colony mutants which have been shown to correlate with chromosomal mutation in the MLA. Colonies ranged from approximately 0.2 to 1.5 mm in size. The mutant frequency (MF) for the AS52 cells treated with BAP and 2AAF exceeded that previously reported for the MLA by 2-fold. In contrast, the MF for AS52 cells treated with MMS was one-third that reported in the MLA. The MF obtained in AS52 cells exceeded that reported for the CHO/HGPRT mutation assay for all 3 compounds. MCR, which produces almost entirely small colonies in the MLA, was negative in AS52 cells as were the MLA chromosomal mutagens AM and BZ. However, AM and BZ have only been reported mutagenic in the MLA. Both are considered nongenotoxic and noncarcinogenic. The results with the latter 3 compounds suggest that the AS52 assay is not as sensitive as the MLA for the detection of compounds identified as chromosomal mutagens.

The in vivo effect of 2,6-xylidine on induction of micronuclei in mouse bone marrow cells.

The ability of 2,6-xylidine to produce chromosome breakage and/or spindle malformation in vivo was evaluated by an assessment of the capacity of the compound to induce micronuclei in bone marrow polychromatic erythrocytes. Male ICR mice were administered a single oral dose of 350, 175 or 87.5 mg/kg of 2,6-xylidine by oral gavage and bone marrow was extracted from the femurs 24, 48 and 72 h thereafter. The frequency of micronuclei in animals treated with 2,6-xylidine was not different from that observed for the corresponding solvent treated controls.