Genetic toxicology in silico protocol.

In silico toxicology (IST) approaches to rapidly assess chemical hazard, and usage of such methods is increasing in all applications but especially for regulatory submissions, such as for assessing chemicals under REACH as well as the ICH M7 guideline for drug impurities. There are a number of obstacles to performing an IST assessment, including uncertainty in how such an assessment and associated expert review should be performed or what is fit for purpose, as well as a lack of confidence that the results will be accepted by colleagues, collaborators and regulatory authorities. To address this, a project to develop a series of IST protocols for different hazard endpoints has been initiated and this paper describes the genetic toxicity in silico (GIST) protocol. The protocol outlines a hazard assessment framework including key effects/mechanisms and their relationships to endpoints such as gene mutation and clastogenicity. IST models and data are reviewed that support the assessment of these effects/mechanisms along with defined approaches for combining the information and evaluating the confidence in the assessment. This protocol has been developed through a consortium of toxicologists, computational scientists, and regulatory scientists across several industries to support the implementation and acceptance of in silico approaches.

In silico toxicology protocols.

The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information.

Report on stage III Pig-a mutation assays using N-ethyl-N-nitrosourea-comparison with other in vivo genotoxicity endpoints.

N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon. Blood was sampled predose (Day-1) and at various time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD592-) and RET(CD592-) frequencies. Consistent with the results from a reference laboratory, RBC(CD592-) and RET(CD592-) frequencies increased in a dose and time-dependent manner, producing significant increases at all doses by Day 15, with similar frequencies seen in both rat strains. ENU also induced small but significant increases in % micronucleated RETs on Days 4 and 29. No significant increases in micronuclei were seen in the liver or colon of the ENU-treated SD rats. Hprt and Pig-a lymphocyte mutation assays conducted on splenocytes from Day 56 F344 rats detected two- to fourfold stronger responses for Hprt than Pig-a mutations. Results from the in vivo Comet assay in SD rats at Day 29 showed generally weak increases in DNA damage in all tissues evaluated. The results with ENU indicate that the Pig-a RET and RBC assays are reproducible, transferable, and complement other genotoxicity endpoints that could potentially be integrated into 28-day repeat dose rat studies.

Defining EMS and ENU dose-response relationships using the Pig-a mutation assay in rats.

In recent years, experimental evidence has accumulated that supports the existence of sublinear dose-response relationships at low doses of DNA reactive mutagens. However, creating the in vivo data necessary to allow for a more detailed dose-response modeling with the currently available tools might not always be practical. The purpose of the current work was to evaluate the utility of the Pig-a gene mutation assay to rapidly identify dose-response relationships for direct acting genotoxicants. The induction of mutations in the peripheral blood of rats was evaluated following 28 days of exposure down to low doses of the direct acting alkylating agents ethyl methane sulfonate (EMS) and ethylnitrosourea (ENU). Using statistical modeling based on the 28-day studies, a threshold for mutation induction for EMS was estimated to be 21.9mg/kg, whereas for the more potent ENU, the threshold was estimated to be 0.88mg/kg. Comparing mutation frequencies from acute and sub-chronic dosing indicated less than additive dose-response relationships, further confirming the possibility of a threshold dose-response relationship for both compounds. In conclusion, the work presented provides evidence that the Pig-a assay might be a practical alternative to other in vivo mutation assays when assessing dose-response relationships for direct acting mutagens and that an experimental approach using fractionated dosing could be used to substantiate a biological mechanism responsible for the observation of a sublinear dose-response relationship.

Potential thresholds for genotoxic effects by micronucleus scoring.

The concept of thresholds in genotoxicity has been open for debate in the last decades. The micronucleus (MN) test contributed to a large extent in understanding the dose-response relationship for aneugens and clastogens. The threshold concept for aneuploidy is well accepted by the scientific community based on the data and for mechanistic reasons. The concept of threshold for clastogens is still challenging. Acceptance is based on a case-by-case basis together with thorough mechanistic understanding of the different steps from the mutagen-target interactions to MN formation for this class of genotoxicants. This review summarises the significant achievements in the assessment of threshold for genotoxins using the MN test and concludes with an overview of knowledge gaps and recommendations.

Flow cytometry peripheral blood micronucleus test in vivo: determination of potential thresholds for aneuploidy induced by spindle poisons.

Non-DNA binding genotoxins (e.g., aneugens), unlike DNA-binding genotoxins, are theoretically expected to show thresholded concentration-effect response curves. This is a major issue in genetic toxicology testing because the identification of thresholds in vivo can provide a safety margin for exposure to a particular compound. In the current study we measured micronucleus induction by flow cytometry to determine the dose-response curves for tubulin interacting agents, a specific class of aneugens. All experiments with aneugens, which include colchicine, vinblastine, vincristine, as well as the clastogen cyclophosphamide (CP) were performed in mice to avoid the splenic elimination of micronucleated reticulocytes, which has been described in rats. Flow cytometry analysis revealed a non-linear dose-dependent increase in micronuclei frequencies for all tested aneugens, and a linear dose response curve for the clastogen, CP. To determine whether micronucleus induction at higher doses was due to chromosome loss (aneuploidy) or chromosome breakage (clastogenicity), flow sorting of the micronucleated reticulocytes and fluorescent in situ hybridization (FISH) with a mouse pan centromeric probe were performed for vinblastine, vincristine, and colchicine. Statistical evaluation of the flow cytometry and FISH data was performed to determine the threshold levels for chromosome loss in vivo. The threshold concentrations for vinblastine, vincristine, and colchicine were found at 0.35, 0.017, and 0.49 mg kg(-1), respectively.

Comparison of the peripheral blood micronucleus test using flow cytometry in rat and mouse exposed to aneugens after single-dose applications.

Detection of clastogenic compounds in the peripheral blood micronucleus test (MNT) in rats is a well-established methodology. However, the results obtained on the induction of micronuclei by aneugens in rat peripheral blood are controversial. Our aim was a comparative evaluation of the peripheral blood flow cytometry MNT in Wistar Han rat and CD1 mouse exposed to three aneugens (vinblastine, vincristine and colchicine) after single-dose applications. In addition, the same compounds were tested in the rat bone marrow MNT. The treatment with vinblastine (0.25, 0.5, 1, mg/kg), vincristine (0.025, 0.05, 0.1 mg/kg) or colchicine (0.7, 1, 1.3 mg/kg) induced no statistically significant increase in MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) in rat peripheral blood. In rat bone marrow, a clear statistically significant increase in MN-PCE was found with vincristine and vinblastine. However, colchicine showed a clear increase in MN-PCE frequency without reaching statistically significant level only at 1 mg/kg. The positive effect in the bone marrow MNT shows that the target organ was exposed to the appropriate concentration levels of the respective aneugens. In mouse, the peripheral blood flow cytometry analysis after the treatment with vinblastine, vincristine and colchicine showed clear statistically significant increase in MN-PCE with all three compounds. The experiments with splenectomized rats treated with vincristine and colchicine were performed and statistically significant increases in MN-PCE were found with 0.05, 0.1, 0.15 mg/kg of vincristine and 0.7 and 1 mg/kg of colchicine. Our results demonstrate that micronucleated cells induced by aneugens are removed from rat peripheral blood by the spleen due to the large size of micronuclei. Based on our data, it is concluded that the flow cytometry peripheral blood MNT after single-dose applications is an appropriate test system for evaluating the genotoxic effects of aneugens in mice. However, in rats peripheral blood MNT aneugen detection might require multiple-dose applications to overwhelm the spleen effect.

In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals.

The use of flow cytometry with rat peripheral blood erythrocytes is expected to increase the sensitivity of the in vivo micronucleus test and allows assessment of the genotoxic effects at doses that may be equal or close to those relevant to human exposure. However, there was a limitation to the use of rat peripheral blood erythrocytes since the spleen selectively removes micronucleated erythrocytes from circulation. In the present study, the selective analysis by flow cytometry of young MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) by use of anti-CD71 antibodies was intended to compensate for the splenic clearance of micronucleated erythrocytes. The young polychromatic erythrocytes have on their surface a specific marker (CD71 antigen) that decreases in density during the maturation process. To investigate the usefulness of the flow cytometric micronucleus analysis combined with anti-CD71 staining of reticulocytes several compounds were tested in acute or sub-chronic treatment regimens. Furthermore, an evaluation was conducted in comparison with the standard rat bone-marrow micronucleus test with additional compounds. The results of acute studies with intraperitoneal application of ethyl methanesulfonate (EMS) (50, 100 and 200 mg/kg) and mitomycin C (MMC) (0.5, 1 and 2 mg/kg), were comparable to data published in the literature. Sub-chronic experiments were performed with cyclophosphamide (CP) (1, 2, 4 and 8 mg/(kg day)), colchicine (6, 8 mg/(kg day)) and mitomycin C (0.1 mg/(kg day)) and showed dose- and time-dependent accumulation of MN-PCEs. Parallel analysis of micronucleus induction in peripheral blood and bone marrow performed with Novartis compounds up to the highest tested dose (5 mg/kg of compound A, 200 mg/kg of compound B and 1250 mg/kg of compound C) showed concordant results. Furthermore, we performed kinetic studies of micronucleus induction in peripheral blood samples obtained at various times after a single treatment with 10 mg/kg CP and with 6 or 8 mg/kg of colchicine. Such experiments gave important supplementary information about the time course of micronucleus induction. Our data suggest that the peripheral blood flow-cytometry micronucleus test can be used for the assessment of micronucleus induction after acute and chronic exposures of rats to chemicals.