Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

The food contaminant acetamide is not an in vivo clastogen, aneugen, or mutagen in rodent hematopoietic tissue.

Acetamide (CAS 60-35-5) is classified by IARC as a Group 2B, possible human carcinogen, based on the induction of hepatocellular carcinomas in rats following chronic exposure to high doses. Recently, acetamide was found to be present in a variety of human foods, warranting further investigation. The regulatory body JECFA has previously noted conflicting reports on acetamide's ability to induce micronuclei (MN) in mice in vivo. To better understand the potential in vivo genotoxicity of acetamide, we performed acute MN studies in rats and mice, and a subchronic study in rats, the target species for liver cancer. In the acute exposure, animals were gavaged with water vehicle control, 250, 1000, or 2000 mg/kg acetamide, or the positive control (1 mg/kg mitomycin C). In the subchronic assay, bone marrow of rats gavaged at 1000 mg/kg/day (limit dose) for 28 days was evaluated. Both acute and subchronic exposures showed no change in the ratio of polychromatic to total erythrocytes (P/E) at any dose, nor was there any increase in the incidence of micronucleated polychromatic erythrocytes (MN-PCE). Potential mutagenicity of acetamide was evaluated in male rats gavaged with vehicle control or 1500 mg/kg/day acetamide using the in vivoPig-a gene mutation assay. There was no increase in mutant red blood cells or reticulocytes in acetamide-treated animals. In both acute and sub-chronic studies, elevated blood plasma acetamide in treated animals provided evidence of systemic exposure. We conclude based on this study that acetamide is not clastogenic, aneugenic, or mutagenic in vivo in rodent hematopoietic tissue warranting a formal regulatory re-evaluation.

Development of an adverse outcome pathway for chemically induced hepatocellular carcinoma: case study of AFB1, a human carcinogen with a mutagenic mode of action.

Adverse outcome pathways (AOPs) are frameworks starting with a molecular initiating event (MIE), followed by key events (KEs) linked by KE relationships (KERs), ultimately resulting in a specific adverse outcome. Relevant data for the pathway and each KE/KER are evaluated to assess biological plausibility, weight-of-evidence, and confidence. We aimed to describe an AOP relevant to chemicals directly inducing mutation in cancer critical gene(s), via the formation of chemical-specific pro-mutagenic DNA adduct(s), as an early critical step in tumor etiology. Such chemicals have mutagenic modes-of-action (MOA) for tumor induction. To assist with developing this AOP, Aflatoxin B1 (AFB1) was selected as a case study because it has a rich database and is considered to have a mutagenic MOA. AFB1 information was used to define specific KEs, KERs, and to inform development of a generic AOP for mutagen-induced hepatocellular carcinoma (HCC). In assessing the AFB1 information, it became clear that existing data are, in fact, not optimal and for some KEs/KERs, the definitive data are not available. In particular, while there is substantial information that AFB1 can induce mutations (based on a number of mutation assays), the definitive evidence - the ability to induce mutation in the cancer critical gene(s) in the tumor target tissue - is not available. Thus, it is necessary to consider the patterns of results in the weight-of-evidence for KEs and KERs. It was important to determine whether there was sufficient evidence that AFB1 can induce the necessary critical mutations early in the carcinogenic process, which was the case.

Quantitative analysis of the relative mutagenicity of five chemical constituents of tobacco smoke in the mouse lymphoma assay.

Quantifying health-related biological effects, like genotoxicity, could provide a way of distinguishing between tobacco products. In order to develop tools for using genotoxicty data to quantitatively evaluate the risk of tobacco products, we tested five carcinogens found in cigarette smoke, 4-aminobiphenyl (4-ABP), benzo[a]pyrene (BaP), cadmium (in the form of CdCl2), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in the mouse lymphoma assay (MLA). The resulting mutagenicity dose responses were analyzed by various quantitative approaches and their strengths and weaknesses for distinguishing responses in the MLA were evaluated. L5178Y/Tk (+/-) 3.7.2C mouse lymphoma cells were treated with four to seven concentrations of each chemical for 4h. Only CdCl2 produced a positive response without metabolic activation (S9); all five chemicals produced dose-dependent increases in cytotoxicity and mutagenicity with S9. The lowest dose exceeding the global evaluation factor, the benchmark dose producing a 10%, 50%, 100% or 200% increase in the background frequency (BMD10, BMD50, BMD100 and BMD200), the no observed genotoxic effect level (NOGEL), the lowest observed genotoxic effect level (LOGEL) and the mutagenic potency expressed as a mutant frequency per micromole of chemical, were calculated for all the positive responses. All the quantitative metrics had similar rank orders for the agents' ability to induce mutation, from the most to least potent as CdCl2(-S9) > BaP(+S9) > CdCl2(+S9) > MeIQ(+S9) > 4-ABP(+S9) > NNK(+S9). However, the metric values for the different chemical responses (i.e. the ratio of the greatest value to the least value) for the different chemicals ranged from 16-fold (BMD10) to 572-fold (mutagenic potency). These results suggest that data from the MLA are capable of discriminating the mutagenicity of various constituents of cigarette smoke, and that quantitative analyses are available that can be useful in distinguishing between the exposure responses.

Genotoxicity of 2-bromo-3'-chloropropiophenone.

Impurities are present in any drug substance or drug product. They can be process-related impurities that are not completely removed during purification or are formed due to the degradation of the drug substance over the product shelf-life. Unlike the drug substance, impurities generally do not have beneficial effects and may present a risk without associated benefit. Therefore, their amount should be minimized. 2-Bromo-3'-chloropropiophenone (BCP) is an impurity of bupropion, a second-generation antidepressant and a smoking cessation aid. The United States Pharmacopeia recommends an acceptable level for BCP that is not more than 0.1% of the bupropion. Because exposure to genotoxic impurities even at low levels is of significant concern, it is important to determine whether or not BCP is genotoxic. Therefore, in this study the Ames test and the in vitro micronucleus assay were conducted to evaluate the genotoxicity of BCP. BCP was mutagenic with S9 metabolic activation, increasing the mutant frequencies in a concentration-dependent manner, up to 22- and 145-fold induction over the controls in Salmonella strains TA100 and TA1535, respectively. BCP was also positive in the in vitro micronucleus assay, resulting in up to 3.3- and 5.1-fold increase of micronucleus frequency for treatments in the absence and presence of S9, respectively; and 9.9- and 7.4-fold increase of aneuploidies without and with S9, respectively. The addition of N-acetyl-l-cysteine, an antioxidant, reduced the genotoxicity of BCP in both assays. Further studies showed that BCP treatment resulted in induction of reactive oxygen species (ROS) in the TK6 cells. The results suggest that BCP is mutagenic, clastogenic, and aneugenic, and that these activities are mediated via generation of reactive metabolites.

Development of a framework for risk assessment of dietary carcinogens.

Although there are a number of guidance documents and frameworks for evaluation of carcinogenicity, none of the current methods fully reflects the state of the science. Common limitations include the absence of dose-response assessment and not considering the impact of differing exposure patterns (e.g., intermittent, high peaks vs. lower, continuous exposures). To address these issues, we have developed a framework for risk assessment of dietary carcinogens. This framework includes an enhanced approach for weight of evidence (WOE) evaluation for genetic toxicology data, with a focus on evaluating studies based on the most recent testing guidance to determine whether a chemical is a mutagen. Included alongside our framework is a discussion of resources for evaluating tissue dose and the temporal pattern of internal dose, taking into account the chemical's toxicokinetics. The framework then integrates the mode of action (MOA) and associated dose metric category with the exposure data to identify the appropriate approach(es) to low-dose extrapolation and level of concern associated with the exposure scenario. This framework provides risk managers with additional flexibility in risk management and risk communication options, beyond the binary choice of linear low-dose extrapolation vs. application of uncertainty factors.

PBPK modeling to evaluate maximum tolerated doses: A case study with 3-chloroallyl alcohol.

Introduction: A physiologically based pharmacokinetic (PBPK) model for 3-chloroallyl alcohol (3-CAA) was developed and used to evaluate the design of assays for the in vivo genotoxicity of 3-CAA. Methods: Model development was supported by read across from a published PBPK model for ethanol. Read across was motivated by the expectation that 3-CAA, which like ethanol is a primary alcohol, is metabolized largely by hepatic alcohol dehydrogenases. The PBPK model was used to evaluate how two metrics of tissue dosimetry, maximum blood concentration (Cmax; mg/L) and area under the curve (AUC; mg-hr/L) vary with dose of 3-CAA and with dose route (oral gavage, drinking water). Results: The model predicted that oral gavage results in a 6-fold higher Cmax than the same dose administered in drinking water, but in similar AUCs. Predicted Cmax provided the best correlation with severe toxicity (e.g., lethality) from 3-CAA, consistent with the production of a reactive metabolite. Therefore, drinking water administration can achieve higher sustained concentration without severe toxicity in vivo. Discussion: This evaluation is significant because cytotoxicity is a potential confounder of mutagenicity testing. The PBPK model can be used to ensure that studies meet OECD and USEPA test guidelines and that the highest dose used is not associated with severe toxicity. In addition, PBPK modeling provides assurance of target tissue (e.g., bone marrow) exposure even in the absence of laboratory data, by defining the relationship between applied dose and target tissue dose based on accepted principles of pharmacokinetics, relevant physiology and biochemistry of the dosed animals, and chemical-specific information.

Key challenges for in vitro testing of tobacco products for regulatory applications: Recommendations for dosimetry.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across tobacco and various next-generation products (NGPs) including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDSs). This publication was developed by a working group of the workshop members in conjunction with the sixth workshop in that series entitled "Dosimetry for conducting in vitro evaluations" and focuses on aerosol dosimetry for aerosol exposure to combustible cigarettes, HTP, and ENDS aerosolized tobacco products and summarizes the key challenges as well as documenting areas for future research.

A comparison of cigarette smoke test matrices and their responsiveness in the mouse lymphoma assay: A case study.

No cigarette smoke test matrix is without limitation, due to the complexity of the starting aerosol and phase to phase dynamics. It is impossible to capture all chemicals at the same level of efficiency, therefore, any test matrix will inadvertently or by design fractionate the test aerosol. This case study examines how four different test matrices derived from cigarette smoke can be directly compared. The test matrices assessed were as follows, total particulate matter (TPM), gas vapour phase (GVP), a combination of TPM + GVP and whole aerosol (WA). Here we use an example assay, the mouse lymphoma assay (MLA) to demonstrate that data generated across four cigarette smoke test matrices can be compared. The results show that all test matrices were able to induce positive mutational events, but with clear differences in the biological activity (both potency and toxicity) between them. TPM was deemed the most potent test article and by extension, the particulate phase is interpreted as the main driver of genotoxic induced responses in the MLA. However, the results highlight that the vapour phase is also active. MLA appeared responsive to WA, with potentially lower potency, compared to TPM approaches. However, this observation is caveated in that the WA approaches used for comparison were made on a newly developed experimental method using dose calculations. The TPM + GVP matrix had comparable activity to TPM alone, but interestingly induced a greater number of mutational events at comparable relative total growth (RTG) and TPM-equivalent doses when compared to other test matrices. In conclusion, this case study highlights the importance of understanding test matrices in response to the biological assay being assessed and we note that not all test matrices are equal.

Mutagenicity of 11 cigarette smoke condensates in two versions of the mouse lymphoma assay.

Cigarette smoke condensate (CSC) is genotoxic in nearly all assays in which it has been tested. In this study, we investigated the mutagenicity of 11 CSCs using the microwell and soft-agar versions of the mouse lymphoma assay (MLA). These CSCs were prepared from commercial or experimental cigarettes, 10 of them were produced using International Organisation for Standardisation (ISO) conditions and one CSC was generated using intense Massachusetts Department of Public Health (MDPH) conditions. In the presence of rat liver S9, the L5178Y/Tk(+/-) mouse lymphoma cells were treated with 11 CSCs at different concentrations (25-200 µg/ml) for 4 h. All CSCs resulted in dose-dependent increases of both cytotoxicity and mutagenicity in both versions of the MLA. The mutagenic potencies of the CSCs were calculated as mutant frequency per microgram CSC from the slope of the linear regression of the dose-response curves and showed no correlations with the tar yield of the cigarette or nicotine concentrations of the CSCs. Comparing two CSCs produced from the same commercial cigarettes using two different smoking conditions, the one generated under ISO conditions was more mutagenic than the other generated under intense conditions on a per microgram CSC basis. We also examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for the mutants induced by 11 CSCs. The most common type of mutation observed was LOH with chromosome damage spanning less than ∼34 Mbp. These results indicate that the MLA identifies different genotoxic potencies among a variety of CSCs and that the results from both versions of the assay are comparable.

Suitable top concentration for tests with mammalian cells: mouse lymphoma assay workgroup.

The Mouse Lymphoma Expert Workgroup of the International Workshop for Genotoxicity Tests (IWGT) met in Basel, Switzerland in August of 2009. The Workgroup (WG) was tasked with discussing the appropriate top concentration for non-pharmaceuticals that would be required for the conduct of the mouse lymphoma assay (MLA) when sufficient cytotoxicity [to between 10 and 20% relative total growth (RTG)] has not been attained. The WG approached this task by (1) enumerating the various regulatory decisions/use for MLA data, (2) discussing the appropriate assays to which MLA data and assay performance should be compared and (3) discussing all the proposals put forth concerning the top concentration for non-pharmaceuticals. In addition, one of the members presented a summary of a re-evaluation of the National Toxicology Program MLA data using the IWGT harmonized guidance that was underway as a separate (non IWGT) activity, being conducted by two members of the Expert WG. The WG was asked to vote on each of the various proposals for top concentration for when cytotoxicity is not concentration limiting. While there was general agreement that the top concentration for non-pharmaceuticals should be re-evaluated and likely lowered from the current recommended levels, there was no agreement on a specific new recommendation.

Detection of Loss of Heterozygosity in Tk-Deficient Mutants from L5178Y Tk+/--3.7.2C Mouse Lymphoma Cells.

The mouse lymphoma assay (MLA), a forward mutation assay using the Tk+/--3.7.2C clone of the L5178Y mouse lymphoma cell line and the Thymidine kinase (Tk) gene, has been widely used as an in vitro genetic toxicity assay for more than four decades. The MLA can evaluate the ability of mutagens to induce a wide range of genetic events including both gene mutations and chromosomal mutations and has been recommended as one component of several genotoxicity test batteries. Tk-deficient mutants often exhibit chromosomal abnormalities involving the distal end of chromosome 11 where the Tk gene is located, in mice, and the type of chromosome alteration can be analyzed using a loss of heterozygosity (LOH) approach. LOH has been considered an important event in human tumorigenesis and can result from any of the following several mechanisms: large deletions, mitotic recombination, and chromosome loss. In this chapter, the authors describe the procedures for the detection of LOH in the Tk mutants from the MLA, and apply LOH analysis for understanding the types of genetic damage that is induced by individual chemicals.

Revisiting the bacterial mutagenicity assays: Report by a workgroup of the International Workshops on Genotoxicity Testing (IWGT).

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to obtain consensus on unresolved issues associated with genotoxicity testing. At the 2017 IWGT meeting in Tokyo, four sub-groups addressed issues associated with the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471, which describes the use of bacterial reverse-mutation tests. The strains sub-group analyzed test data from >10,000 chemicals, tested additional chemicals, and concluded that some strains listed in TG471 are unnecessary because they detected fewer mutagens than other strains that the guideline describes as equivalent. Thus, they concluded that a smaller panel of strains would suffice to detect most mutagens. The laboratory proficiency sub-group recommended (a) establishing strain cell banks, (b) developing bacterial growth protocols that optimize assay sensitivity, and (c) testing "proficiency compounds" to gain assay experience and establish historical positive and control databases. The sub-group on criteria for assay evaluation recommended that laboratories (a) track positive and negative control data; (b) develop acceptability criteria for positive and negative controls; (c) optimize dose-spacing and the number of analyzable doses when there is evidence of toxicity; (d) use a combination of three criteria to evaluate results: a dose-related increase in revertants, a clear increase in revertants in at least one dose relative to the concurrent negative control, and at least one dose that produced an increase in revertants above control limits established by the laboratory from historical negative controls; and (e) establish experimental designs to resolve unclear results. The in silico sub-group summarized in silico utility as a tool in genotoxicity assessment but made no specific recommendations for TG471. Thus, the workgroup identified issues that could be addressed if TG471 is revised. The companion papers (a) provide evidence-based approaches, (b) recommend priorities, and (c) give examples of clearly defined terms to support revision of TG471.

Evaluation of mutagenic mode of action in Big Blue mice fed methylphenidate for 24 weeks.

Methylphenidate hydrochloride (MPH), a widely prescribed pediatric drug for attention deficit hyperactivity disorder, induced liver adenocarcinomas in B6C3F1 mice exposed to 500 ppm in feed for 2 years (Dunnick and Hailey (1995) [14]). In order to determine if the induction of liver tumors was by a mutagenic mode of action, groups of male Big Blue (BB) mice (B6C3F1 background) were fed diets containing 50-4000 ppm MPH for 4, 12, or 24 weeks. At sacrifice, the livers were removed and the cII mutant frequency (MF) and spectrum of cII mutations were determined. In addition, the frequencies of micronucleated reticulocytes (MN-RETs) and normochromatic erythrocytes (MN-NCEs) were measured in peripheral blood erythrocytes as was the Hprt MF in splenic lymphocytes. Food consumption and body weight gain/loss were recorded weekly for each animal. The levels of MPH and RA were determined immediately before sacrifice in the serum of mice fed MPH for 24 weeks. A significant loss in body weights (p

In Vitro Mouse Lymphoma Cell (L5178Y Tk+/- -3.7.2.C) Forward Mutation Assay.

The in vitro mouse lymphoma cell assay (MLA) is one of the most widely practiced assays in genetic toxicology. MLA detects forward mutations at the thymidine kinase (Tk) locus of the L5178Y (Tk+/- -3.7.2C) cell line derived from a mouse thymic lymphoma. This assay is capable of detecting a wide range of genetic events including point mutations, deletions and multilocus, chromosomal rearrangements, mitotic recombination and nondisjunction. There are two equally accepted versions of the assay, one using soft agar cloning and the second method using liquid media cloning in 96-microwell plates. There are two morphologically distinct types of mutant colonies recovered in the MLA; small and large colony mutants. The induction of small colony mutants is associated with chemicals inducing gross chromosomal aberrations, whereas the induction of large mutant colonies is generally associated with chemicals inducing point mutations. The source and karyotype of the cell line as well as the culture conditions are important variables that could influence the assay performance. The assay when performed according to the standards recommended by the International Workshops on Genotoxicity Testing (IWGT) and the Organization of Economic Cooperation and Development Test Guideline 490 is capable of providing valuable genotoxicity hazard information as part of the overall safety assessment process of various classes of test substances.

Critical review of styrene genotoxicity focused on the mutagenicity/clastogenicity literature and using current organization of economic cooperation and development guidance.

Styrene is an important high production volume chemical used to manufacture polymeric products. In 2018, International Agency for Research on Cancer classified styrene as probably carcinogenic to humans; National Toxicology Program lists styrene as reasonably anticipated to be a human carcinogen. The genotoxicity literature for styrene and its primary metabolite, styrene 7,8-oxide (SO), begins in the 1970s. Organization of Economic Cooperation and Development (OECD) recently updated most genotoxicity test guidelines, making substantial new recommendations for assay conduct and data evaluation for the standard mutagenicity/clastogenicity assays. Thus, a critical review of the in vitro and in vivo rodent mutagenicity/clastogenicity studies for styrene and SO, based on the latest OECD recommendations, is timely. This critical review considered whether a study was optimally designed, conducted, and interpreted and provides a critical assessment of the evidence for the mutagenicity/clastogenicity of styrene/SO. Information on the ability of styrene/SO to induce other types of genotoxicity endpoints is summarized but not critically reviewed. We conclude that when styrene is metabolized to SO, it can form DNA adducts, and positive in vitro mutagenicity/clastogenicity results can be obtained. SO is mutagenic in bacteria and the in vitro mouse lymphoma gene mutation assay. No rodent in vivo mutation studies were identified. SO is clastogenic in cultured mammalian cells. Although the in vitro assays gave positive responses, styrene/SO is not clastogenic/aneugenic in vivo in rodents. In addition to providing updated information for styrene, this review demonstrates the application of the new OECD guidelines for chemicals with large genetic toxicology databases where published results may or may not be reliable. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Horizon scanning for novel and emerging in vitro mammalian cell mutagenicity test systems.

The induction of gene mutation within a DNA sequence can result in an adverse impact, altering or preventing gene function. Therefore, in vitro evaluation of mutagenicity is an essential component of the toxicological screening process. A variety of mutagen screening tools are routinely used in genetic toxicology, which are based on selected reporter genes. These assays are however typically labour intensive and impractical for high throughput screening. Considering this, the IWGT (International Workshops on Genotoxicity Testing) sub-group on Novel & Emerging in vitro Mammalian Cell Mutagenicity Test Systems undertook a literature search to identify new approaches for mutation detection. This review therefore focused on identifying new approaches for mutation detection that have the potential for use as a future genotoxicity screening tool. A comprehensive literature review identified genome-wide loss-of-function screening tools, next generation sequencing (NGS) mutation characterisation and fluorescence-based mutation detection methods as having significant promise as an emerging in vitro mammalian cell mutagenicity test system. Each of the technologies considered was assessed for its capacity to report on a wide array of heritable mutagenic changes, necessary to cover the full spectrum of genetic events imparted by substances with a broad range of modes of action. Of the technologies evaluated, NGS techniques exhibited the greatest advantages for use in a genotoxicity testing setting. However, it is important to note that the emerging techniques identified could not facilitate routine mutagenicity testing in their current format and require substantial additional optimisation and tailoring before they could be utilised as an in vitro mammalian cell mutagenicity test system. Additionally, new mammalian cell mutation test systems must be able to accurately and reliably detect and quantify rare events; hence any new system would require careful validation. Nevertheless, with further development emerging technologies such as NGS could become important in establishing more predictive and high-throughput regulatory hazard screening tools of the future.

Recommended criteria for the evaluation of bacterial mutagenicity data (Ames test).

A committee was constituted within the International Workshop on Genetic Toxicology Testing (IWGT) to evaluate the current criteria for a valid Ames test and to provide recommendations for interpretation of test results. Currently, determination of a positive vs. a negative result is made by applying various data evaluation procedures for comparing dosed plates with the concurrent solvent control plates. These evaluation procedures include a requirement for a specific fold increase (2- or 3-fold, specific to the bacterial strain), formal statistical procedures, or subjective (expert judgment) evaluation. After extensive discussion, the workgroup was not able to reach consensus recommendations in favor of any of these procedures. There was a consensus that combining additional evaluation criteria to the comparison between dosed plates and the concurrent solvent control plates improves test interpretation. The workgroup recommended using these additional criteria because the induction of mutations is a continuum of responses and there is no biological relevance to a strict dividing line between a positive (mutagenic) and not-positive (nonmutagenic) response. The most useful additional criteria identified were a concentration-response relationship and consideration of a possible increase above the concurrent control in the context of the laboratory's historical solvent control values for the particular tester strain. The workgroup also emphasized the need for additional testing to resolve weak or inconclusive responses, usually with altered experimental conditions chosen based on the initial results. Use of these multiple criteria allowed the workgroup to reach consensus on definitions of "clear positive" and "clear negative" responses which would not require a repeat test for clarification. The workgroup also reached consensus on recommendations to compare the responses of concurrent positive and negative controls to historical control distributions for assay acceptability, and the use of control charts to determine the validity of the individual test.

Demonstrating laboratory proficiency in bacterial mutagenicity assays for regulatory submission.

The bacterial reverse mutation test is a mainstay for evaluation of mutagenicity predicting the carcinogenic potential of a test substance and is recommended by regulatory agencies across the globe. The popularity of the test is due, in part, to the relatively low cost, rapid results and small amount of test material required compared to most other toxicological tests as well as the near universal acceptance of the toxicological significance of a clear positive or negative result. Most laboratories follow the Organization for Economic Cooperation and Development Test Guideline 471 (TG471) or national guidelines based on TG471. Regulatory agencies in most countries are obligated to consider results from tests which meet the recommendations laid out in TG471. Nonetheless, laboratories unfamiliar with the test sometimes have trouble generating reliable, reproducible results. TG471 is a test guideline, not a detailed test protocol. A group of experts from regulatory agencies and laboratories which use the assay has assembled here a set of recommendations which if followed, will allow an inexperienced laboratory to acquire proficiency in assay conduct. These include recommendations for how to create a cell bank for the 5 Salmonella typhimurium/Escherichia coli strains and develop a laboratory protocol to reliably culture each strain to ensure each culture has the characteristics which allow adequate sensitivity for detection of mutagens using the test as described in TG471. By testing compounds on the provided lists of positive and negative test substances, the laboratory will have surmounted many of the problems commonly encountered during routine testing of unknown chemicals and will have gained the experience necessary to prepare the detailed protocol needed for performing the test under Good Laboratory Procedures and the laboratory will have generated the historical positive and negative control databases which are needed for test reports which adhere to TG471.

Genotoxic effects of acrylamide and glycidamide in mouse lymphoma cells.

In addition to occupational exposures to acrylamide (AA), concerns about AA health risks for the general population have been recently raised due to the finding of AA in food. In this study, we evaluated the genotoxicity of AA and its metabolite glycidamide (GA) in L5178Y/Tk(+/-) mouse lymphoma cells. The cells were treated with 2-18 mM of AA or 0.125-4 mM of GA for 4 h without metabolic activation. The DNA adducts, mutant frequencies and the types of mutations for the treated cells were examined. Within the dose range tested, GA induced DNA adducts of adenine and guanine [N3-(2-carbamoyl-2-hydroxyethyl)-adenine and N7-(2-carbamoyl-2-hydroxyethyl)-guanine] in a linear dose-dependent manner. The levels of guanine adducts were consistently about 60-fold higher across the dose range than those of adenine. In contrast, no GA-derived DNA adducts were found in the cells treated with any concentrations of AA, consistent with a lack of metabolic conversion of AA to GA. However, the mutant frequency was significantly increased by AA at concentrations of 12 mM and higher. GA was mutagenic starting with the 2mM dose, suggesting that GA is much more mutagenic than AA. The mutant frequencies were increased with increasing concentrations of AA and GA, mainly due to an increase of proportion of small colony mutants. To elucidate the underlying mutagenic mechanism, we examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for mutants induced by AA or GA. Compared to GA induced mutations, AA induced more mutants whose LOH extended to D11Mit22 and D11Mit74, an alteration of DNA larger than half of the chromosome. Statistical analysis of the mutational spectra revealed a significant difference between the types of mutations induced by AA and GA treatments (P=0.018). These results suggest that although both AA and GA generate mutations through a clastogenic mode of action in mouse lymphoma cells, GA induces mutations via a DNA adduct mechanism whereas AA induces mutations by a mechanism not involving the formation of GA adducts.