Interlaboratory validation of the ToxTracker assay: An in vitro reporter assay for mechanistic genotoxicity assessment.

ToxTracker is a mammalian cell reporter assay that predicts the genotoxic properties of compounds with high accuracy. By evaluating induction of various reporter genes that play a key role in relevant cellular pathways, it provides insight into chemical mode-of-action (MoA), thereby supporting discrimination of direct-acting genotoxicants and cytotoxic chemicals. A comprehensive interlaboratory validation trial was conducted, in which the principles outlined in OECD Guidance Document 34 were followed, with the primary objectives of establishing transferability and reproducibility of the assay and confirming the ability of ToxTracker to correctly classify genotoxic and non-genotoxic compounds. Reproducibility of the assay to predict genotoxic MoA was confirmed across participating laboratories and data were evaluated in terms of concordance with in vivo genotoxicity outcomes. Seven laboratories tested a total of 64 genotoxic and non-genotoxic chemicals that together cover a broad chemical space. The within-laboratory reproducibility (WLR) was up to 98% (73%-98% across participants) and the overall between-laboratory reproducibility (BLR) was 83%. This trial confirmed the accuracy of ToxTracker to predict in vivo genotoxicants with a sensitivity of 84.4% and a specificity of 91.2%. We concluded that ToxTracker is a robust in vitro assay for the accurate prediction of in vivo genotoxicity. Considering ToxTracker's robust standalone accuracy and that it can provide important information on the MoA of chemicals, it is seen as a valuable addition to the regulatory in vitro genotoxicity battery that may even have the potential to replace certain currently used in vitro battery assays.

Key Challenges for In Vitro Testing of Tobacco Products for Regulatory Applications: Recommendations for the In Vitro Mouse Lymphoma Assay.

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 traditional tobacco and various tobacco and nicotine next-generation products (NGPs), including Heated Tobacco Products (HTPs) and Electronic Nicotine Delivery Systems (ENDS). This report was developed by a working group composed of attendees of the seventh IIVS workshop, 'Approaches and recommendations for conducting the mouse lymphoma gene mutation assay (MLA) and introduction to in vitro disease models', which was held virtually on 21-23 June 2022. This publication provides a background overview of the MLA, and includes the description of assay conduct and data interpretation, key challenges and recommended best practices for evaluating tobacco and nicotine products, with a focus on the evaluation of NGPs, and a summary of how the assay has been used to evaluate and compare tobacco and nicotine products.

Longitudinal characterization of TK6 cells sequentially adapted to animal product-free, chemically defined culture medium: considerations for genotoxicity studies.

Introduction: In vitro approaches are an essential tool in screening for toxicity of new chemicals, products and therapeutics. To increase the reproducibility and human relevance of these in vitro assessments, it is advocated to remove animal-derived products such as foetal bovine serum (FBS) from the cell culture system. Currently, FBS is routinely used as a supplement in cell culture medium, but batch-to-batch variability may introduce inconsistency in inter- and intra-lab assessments. Several chemically defined serum replacements (CDSR) have been developed to provide an alternative to FBS, but not every cell line adapts easily and successfully to CDSR-supplemented medium, and the long-term effect on cell characteristics remains uncertain. Aim: The aim of this study was to adapt the TK6 cell line to animal-product free CDSR-supplemented medium and evaluate the long-term effects on cell health, growth, morphology, phenotype, and function. This included a provisional assessment to determine the suitability of the transitioned cell line for standardised genotoxicity testing using the "in vitro mammalian cell micronucleus test" (OECD TG 487). Materials and methods: Gradual adaptation and direct adaptation methodologies were compared by assessing the cell proliferation, size and viability every passage until the cells were fully adapted to animal-free CDSR. The metabolic activity and membrane integrity was assessed every 4-8 passages by PrestoBlue and CytoTox-ONE™ Homogeneous Membrane Integrity Assay respectively. A detailed morphology study by high content imaging was performed and the expression of cell surface markers (CD19 and CD20) was conducted via flow cytometry to assess the potential for phenotypic drift during longer term culture of TK6 in animal-free conditions. Finally, functionality of cells in the OECD TG 487 assay was evaluated. Results: The baseline characteristics of TK6 cells cultured in FBS-supplemented medium were established and variability among passages was used to set up acceptance criteria for CDSR adapted cells. TK6 were adapted to CDSR supplemented medium either via direct or gradual transition reducing from 10% v/v FBS to 0% v/v FBS. The cell growth rate was compromised in the direct adaptation and therefore the gradual adaptation was preferred to investigate the long-term effects of animal-free CDSR on TK6 cells. The new animal cells showed comparable (p > 0.05) viability and cell size as the parent FBS-supplemented cells, with the exception of growth rate. The new animal free cells showed a lag phase double the length of the original cells. Cell morphology (cellular and nuclear area, sphericity) and phenotype (CD19 and CD20 surface markers) were in line (p > 0.05) with the original cells. The new cells cultured in CDSR-supplemented medium performed satisfactory in a pilot OECD TG 487 assay with compounds not requiring metabolic activation. Conclusion: TK6 cells were successfully transitioned to FBS- and animal product-free medium. The new cell cultures were viable and mimicked the characteristics of FBS-cultured cells. The gradual transition methodology utilised in this study can also be applied to other cell lines of interest. Maintaining cells in CDSR-supplemented medium eliminates variability from FBS, which in turn is likely to increase the reproducibility of in vitro experiments. Furthermore, removal of animal derived products from cell culture techniques is likely to increase the human relevance of in vitro methodologies.

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 3D reconstructed skin micronucleus assay: considerations for optimal protocol design.

Implementation of the seventh amendment to the EU Cosmetics Directive has driven much research into suitable in vitro alternative assays to support satisfactory risk assessments. One such assay is the reconstructed skin micronucleus (RSMN) assay. First reported in 2006, further development occurred and a standard protocol was published in 2011. To evaluate and optimise the assay at Covance Laboratories, we tested nine chemicals [4-nitrophenol (4-NP), cyclohexanone (CH), 2-ethyl-1,3-hexanediol (2-EHD), methyl methansulfonate (MMS), mitomycin C (MMC), ethyl nitrosourea (ENU), benzo[a]pyrene (BaP), cyclophosphamide (CPA) and vinblastine (VIN)] using the EpiDerm™ 3D skin model (MatTek Corporation®, IVLSL, Bratislava, Slovakia) and compared the data using the standard 48-h treatment regimen and also an emerging 72-h treatment protocol. The EpiDerm™ tissue has reportedly some metabolic capacity but data using 48-h treatments has provided mixed results. Our investigations demonstrate that the two chemicals requiring metabolic activation (BaP and CPA) were negative following the 48-h protocol but were clearly positive following 72-h treatment. Furthermore, Replication Index (RI) data showed higher RI values in vehicle control treatments (indicating increased cell division) across the treatment set following 72-h treatments. A general greater magnitude of micronucleus (MN) induction was also observed following test chemical treatment. These data suggest that the 72-h treatment protocol is more suitable as a standard approach for the detection of clastogenic, aneugenic and metabolically activated chemicals in the RSMN assay. For further assay optimisation, we compare the statistical power of scoring cells from duplicate or triplicate cultures per treatment concentration and provide recommendations.

The genotoxicological assessment of a tobacco heating product relative to cigarette smoke using the in vitro micronucleus assay.

In vitro studies have supported the toxicological evaluation of chemicals and complex mixtures including cigarette smoke and novel tobacco and nicotine products which include tobacco heating products (THP). This new environment requires faster testing, higher throughput and appropriate in vitro studies, to support product innovation and development. In this study, total particulate matter (TPM) from a commercially available THP and a reference cigarette (3R4F) were assessed up to 500 µg/mL using two in vitro micronucleus techniques. V79 and TK6 cells were assessed using conventional OECD 487 manual scoring techniques, whereas, CHO cells were assessed using contemporary, automated high content screening approaches (Cellomics ArrayScan® VTI). V79 cells gave the most consistent response with all three treatment conditions producing a clear positive genotoxic response. Human TK6 cells only produced dose-dependent response, indicative of a weak-positive response. CHO cells demonstrated a positive response with TPM using long (24 h) -S9 conditions. All three cell lines equally demonstrated a negative response with THP TPM up to 500 µg/mL. In conclusion, THP TPM did not increase micronuclei formation above control levels even at doses far exceeding that tested with reference cigarette smoke, in most cases up to 10x the dose delivered compared to that of cigarette smoke. This study supports the growing belief that THPs are less risky than conventional cigarettes and that 21st century screening techniques can be employed to support product design and decision making, as a potential 1st screen prior to more traditional assessments.

Genotoxicity evaluation of tobacco and nicotine delivery products: Part One. Mouse lymphoma assay.

Conduct of the mouse lymphoma assay (MLA) is underpinned by Organisation for Economic Co-operation and Development (OECD) Test Guideline 490 and International Conference on Harmonisation S2(R1) guidance and is a recognised in vitro genotoxicity test battery assay. It has been used on a limited number of occasions for the assessment of some tobacco and nicotine products, such as e-cigarettes and tobacco heating products (THP). The aim of this study was to assess the suitability of the MLA for genotoxicity testing with a variety of tobacco and nicotine products. Total particulate matter (TPM) from a 3R4F cigarette was compared against a commercial electronic cigarette liquid (e-liquid), electronic cigarette (e-cigarette) aerosol matter captured from the same e-liquid, and TPM from a commercial THP. Treatment conditions included 3 h exposures with and without metabolic activation and a longer 24 h exposure without metabolic activation (-S9) at concentrations up to 500 µg/mL. Under all treatment conditions, 3R4F produced a clear positive response with regard to induction of mutation. In contrast, no marked induction of mutation was observed for the e-liquid, e-cigarette aerosol or THP. Additionally, data are presented as a function of nicotine equivalents for comparisons between these different tobacco products and test matrices.

Inclusion of an extended treatment with recovery improves the results for the human peripheral blood lymphocyte micronucleus assay.

The in vitro micronucleus (IVMN) test was endorsed for regulatory genotoxicity testing with adoption of the Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 487 in 2010. This included two equally acceptable options for extended treatment in the absence of metabolic activation: a treatment for 1.5-2.0 cell cycles with harvest at the end of treatment (Option A) or treatment for 1.5-2.0 cell cycles followed by recovery for 1.5-2.0 cell cycles prior to harvest (Option B). Although no preferences were discussed, TG 487 cautions that Option B may not be appropriate for stimulated lymphocytes where exponential growth may be declining at 96 h after phytohaemagglutinin (PHA) stimulation. Following revision of TG 487 in 2014 and 2016, emphasis has been placed on using Option A. Given the purpose of the IVMN assay is to determine both clastogenic and aneugenic potential, the authors believe the assay is compromised if an extended treatment with recovery is not included for sensitive detection of certain classes of chemical. In this study, average generation time (via bromodeoxyuridine incorporation) of human peripheral blood lymphocytes (HPBL) was measured up to 144 h after PHA stimulation. In addition, the HPBL micronucleus (MN) assay was performed using Option A and B treatment schedules. Cytotoxicity (replication index) and MN induction were determined following treatment with 14 chemicals. The data demonstrate that lymphocytes actively divide beyond 96 h after PHA stimulation. Furthermore, MN induction was only observed with some aneugenic chemicals and nucleoside analogues in HPBLs following extended treatment with a recovery period. For the majority of chemicals tested the magnitude of MN induction was generally greater and MN induction was observed across a wider concentration range following the Option B treatment schedule. In addition, steep concentration-related toxicity following treatment without recovery is more common, making selection of suitable concentrations (within regulatory toxicity limits) for MN analysis challenging.

Genotoxicity evaluation of tobacco and nicotine delivery products: Part Two. In vitro micronucleus assay.

In this study, a variety of test matrices from tobacco and nicotine delivery products were assessed against a 3R4F Kentucky reference cigarette using the in vitro micronucleus assay. Testing was conducted using two Chinese hamster cell lines (CHO and V79), and a human lymphoblastoid cell line (TK6), in accordance with established guidelines. Total particulate matter (TPM) from a 3R4F Reference cigarette was compared to an electronic cigarette e-liquid, electronic cigarette TPM and TPM from a commercial tobacco heating product using a standard and an extended treatment condition with recovery period. Cells were assessed with 3R4F TPM prior to assessment of the other tobacco and nicotine product test matrices. These cell lines gave varied responses to 3R4F TPM with the most robust response using V79 cells. The use of an extended exposure/recovery period was seen to increase assay sensitivity for CHO and V79 cell lines but was less clear for TK6 cells. Negative responses were observed for all products except 3R4F across all treatment conditions in V79 cells. The most potent response to cigarette smoke was following extended treatment with recovery, suggesting this may be a more appropriate treatment for the future assessment of tobacco and nicotine product test matrices.

Relationships between p53 status, apoptosis and induction of micronuclei in different human and mouse cell lines in vitro: Implications for improving existing assays.

Accumulated evidence has shown that in vitro mammalian cell genotoxicity assays produce high frequencies of "misleading" positive results, i.e. predicted hazard is not confirmed in in vivo and/or carcinogenicity studies [1], raising the question of relevance to human risk assessment. A recent study of micronucleus (MN) induction [2] showed that commonly used p53-deficient rodent cell lines (CHL, CHO and V79) gave a higher frequency of "misleading" positive results with 9 non-DNA reactive, Ames-negative and in vivo negative chemicals [3] than human p53-competent cells (blood lymphocytes, TK6 and HepG2 cell lines). This raised the question of whether these differences were due to p53 status or species origin. This present study compared human versus mouse and p53-competent versus p53-mutated function. The same 9 chemicals were tested for induction of MN in mouse lymphoma L5178Y (mutated p53), human TK6 (functional p53) and WIL2-NS (TK6 related, with mutated p53) cells. Six chemicals provided clear positive increases in MN frequency in at least one cell type. L5178Y cells yielded clear positive responses with more chemicals than either TK6 or WIL2-NS, indicating origin rather than p53 functionality was most relevant. Apoptosis induction (measured via caspase-3/7) was also investigated with clear differences in the timing and extent of apoptosis induction between mouse and human cells noted. With curcumin in TK6 cells, induction of caspase-3/7 activity coincided with MN induction, whereas for L5178Y cells, MN induction occurred in the absence of increased caspase activity. By contrast, with MMS in TK6 cells, MN induction preceded increased caspase-3/7 activity. These data suggest that MN induction by "misleading positive" genotoxins in p53-competent human cell lines may result from apoptosis, whereas in p53-defective rodent cells such as L5178Y, MN induction may be independent of apoptosis.

Creation and validation of a novel body condition scoring method for the magellanic penguin (Spheniscus magellanicus) in the zoo setting.

This research aims to validate a novel, visual body scoring system created for the Magellanic penguin (Spheniscus magellanicus) suitable for the zoo practitioner. Magellanics go through marked seasonal fluctuations in body mass gains and losses. A standardized multi-variable visual body condition guide may provide a more sensitive and objective assessment tool compared to the previously used single variable method. Accurate body condition scores paired with seasonal weight variation measurements give veterinary and keeper staff a clearer understanding of an individual's nutritional status. San Francisco Zoo staff previously used a nine-point body condition scale based on the classic bird standard of a single point of keel palpation with the bird restrained in hand, with no standard measure of reference assigned to each scoring category. We created a novel, visual body condition scoring system that does not require restraint to assesses subcutaneous fat and muscle at seven body landmarks using illustrations and descriptive terms. The scores range from one, the least robust or under-conditioned, to five, the most robust, or over-conditioned. The ratio of body weight to wing length was used as a "gold standard" index of body condition and compared to both the novel multi-variable and previously used single-variable body condition scores. The novel multi-variable scale showed improved agreement with weight:wing ratio compared to the single-variable scale, demonstrating greater accuracy, and reliability when a trained assessor uses the multi-variable body condition scoring system. Zoo staff may use this tool to manage both the colony and the individual to assist in seasonally appropriate Magellanic penguin nutrition assessment.

A method for assessment of the genotoxicity of mainstream cigarette-smoke by use of the bacterial reverse-mutation assay and an aerosol-based exposure system.

To date there are no widely accepted methods for the toxicological testing of complex gaseous mixtures and aerosols, such as cigarette smoke, although some modifications to the standard regulatory methods have been developed and used. Historically, routine testing of cigarettes has primarily focused on the particulate fraction of cigarette smoke. However, this fraction may not accurately reflect the full toxicity and mutagenicity of the smoke aerosol as a whole, which contains semi-volatiles and short-lived products of combustion. In this study we have used a modified version of the bacterial reverse-mutation (Ames) assay for the testing of mainstream smoke generated from 3R4F reference cigarettes with a Vitrocell(®) VC 10 exposure system. This method has been evaluated in four strains of Salmonella typhimurium (TA98, TA100, YG1024 and YG1042) and one strain of Escherichia coli (WP2 uvrA pKM101) in the absence and presence of a metabolic activation system. Following exposure at four concentrations of diluted mainstream cigarette-smoke, concentration-related and reproducible increases in the number of revertants were observed in all four Salmonella strains. E. coli strain WP2 uvrA pKM101 was unresponsive at the four concentrations tested. To quantify the exposure dose and to enable biological response to be plotted as a function of deposited mass, quartz-crystal microbalances were included in situ in the smoke-exposure set-up. This methodology was further assessed by comparing the responses of strain YG1042 to mainstream cigarette-smoke on a second VC 10 Smoking Robot. In summary, the Ames assay can be successfully modified to assess the toxicological impact of mainstream cigarette-smoke.

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.

Mouse lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe and the United States, met on September 9, 2005, in San Francisco, CA, USA. This meeting of the MLA Workgroup was devoted to reaching a consensus on issues involved with 24-h treatment. Recommendations were made concerning the acceptable values for the negative/solvent control (mutant frequency, cloning efficiency and suspension growth) and the criteria to define an acceptable positive control response. Consensus was also reached concerning the use of the global evaluation factor (GEF) and appropriate statistical trend analysis to define positive and negative responses for the 24-h treatment. The Workgroup agreed to continue their support of the International Committee on Harmonization (ICH) recommendation that the MLA assay should include a 24-h treatment (without S-9) in those situations where the short treatment (3-4 h) gives negative results.

Novel blockade of protein kinase A-mediated phosphorylation of AMPA receptors.

The phosphorylation state of the glutamate receptor subtype 1 (GluR1) subunit of the AMPA receptor (AMPAR) plays a critical role in synaptic expression of the receptor, channel properties, and synaptic plasticity. Several Gs-coupled receptors that couple to protein kinase A (PKA) readily recruit phosphorylation of GluR1 at S845. Conversely, activation of the ionotropic glutamate NMDA receptor (NMDAR) readily recruits dephosphorylation of the same GluR1 site through Ca2+-mediated recruitment of phosphatase activity. In a physiological setting, receptor activation often overlaps and crosstalk between coactivation of multiple signaling cascades can result in differential regulation of a given substrate. After investigating the effect of coactivation of the NMDAR and the Gs-coupled beta-adrenergic receptor on GluR1 phosphorylation state, we have observed a novel signal that prevents PKA-mediated phosphorylation of GluR1 at serine site 845. This blockade of GluR1 phosphorylation is dependent on cellular depolarization recruited by either NMDAR or AMPAR activation, independent of Ca2+ and independent of calcineurin, protein phosphatase 1, and/or protein phosphatase 2A activity. Thus, in addition to the typical kinase-phosphatase rivalry mediating protein phosphorylation state, we have identified a novel form of phospho-protein regulation that occurs at GluR1 and may also occur at several other PKA substrates.

Mouse lymphoma thymidine kinase gene mutation assay: follow-up meeting of the International Workshop on Genotoxicity Testing--Aberdeen, Scotland, 2003--Assay acceptance criteria, positive controls, and data evaluation.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe, and the United States, met on August 29, 2003, in Aberdeen, Scotland, United Kingdom. This meeting of the MLA Workgroup was devoted to reaching a consensus on the appropriate approach to data evaluation and on acceptance criteria for both the positive and negative/vehicle controls. The Workgroup reached consensus on the acceptance criteria for both the agar and microwell versions of the MLA. Recommendations include acceptable ranges for mutant frequency, cloning efficiency, and suspension growth of the negative/vehicle controls and on criteria to define an acceptable positive control response. The recommendation for the determination of a positive/negative test chemical response includes both the requirement that the response exceeds a defined value [the global evaluation factor (GEF)] and that there also be a positive dose-response (evaluated by an appropriate statistical method).

Decreased intestinal polyp multiplicity is related to exercise mode and gender in ApcMin/+ mice.

Moderate-intensity treadmill running can alter male Apc(Min/+) mouse polyp formation. This purpose of this study was to examine whether exercise mode differentially affects Apc(Min/+) mouse intestinal polyp development in male and female mice. Male and female Apc(Min/+) mice were randomly assigned to control, treadmill (18 m/min; 60 min/day; 6 days/wk), or voluntary wheel running (24-h access) groups. Nine weeks of training decreased total intestinal polyps by 29% in male treadmill runners (66 +/- 9; P = 0.038) compared with male controls (93 +/- 7). The number of large polyps (>/=1-mm diameter) were also reduced by 38% in male treadmill runners (49 +/- 6; P = 0.005) compared with male controls (79 +/- 6). Treadmill running in female Apc(Min/+) mice and wheel running in both genders did not affect polyp number or size. Spleen weight decreased in male treadmill runners (91 +/- 9 mg; P = 0.011) and wheel runners (75 +/- 6 mg; P = 0.004) compared with controls (141 +/- 13 mg). Plasma IL-6 was reduced by 96% in male treadmill runners (1.2 +/- 0.6 pg/ml) and 78% in male wheel runners (6.6 +/- 3.3 pg/ml) compared with control mice (27.9 +/- 2.8 pg/ml; P < 0.05). Female mice responded similarly with an 86% decrease in plasma IL-6 with treadmill running (3.2 +/- 1.2 pg/ml) and 90% decrease with wheel running (2.9 +/- 2.0 pg/ml) compared with control mice (21.1 +/- 5.3 pg/ml; P < 0.05). The crypt depth-to-villus height ratio in the intestine, an indirect marker of intestinal inflammation, decreased by 21 (P = 0.024) and 24% (P = 0.029), respectively, in male and female treadmill runners but not wheel runners. Physical activity-induced attenuation of intestinal polyp number and size is dependent on exercise mode and differs between genders. The modulation of systemic and intestinal inflammation may also depend on exercise mode.

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.

Mouse lymphoma thymidine kinase gene mutation assay: follow-up International Workshop on Genotoxicity Test Procedures, New Orleans, Louisiana, April 2000.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Test Procedures held a second harmonization meeting just prior to the U.S. Environmental Mutagen Society Meeting in New Orleans, LA, in April 2000. The discussion focused on several important aspects of the MLA, including: 1) cytotoxicity measures and their determination, 2) use of a 24-hr treatment, 3) the ability of the assay to detect aneugens, and 4) concentration selection. Prior to the meeting the group developed Microsoft Excel Workbooks for data entry. Ten laboratories entered their data into the workbooks (primarily as coded chemicals). The Excel Workbooks were used to facilitate data analysis by generating an extensive set of graphs that were evaluated by the meeting participants. Based on the Workgroup's previous agreement that a single cytotoxicity measure should be established for both the microwell and soft agar versions of the assay, the Workgroup analyzed the submitted data and unanimously agreed that the relative total growth (RTG) should be used as the cytotoxicity measure for concentration selection and data evaluation. The Workgroup also agreed that the various cytotoxicity measures should be calculated using the same methods regardless of whether the soft agar or microwell version of the assay was used. In the absence of sufficient data to make a definitive determination, the Workgroup continued to endorse the International Committee on Harmonization recommendation for the use of 24-hr treatment and made some specific 24-hr treatment protocol recommendations. The Workgroup recognized the ability of the MLA to detect at least some aneugens and also developed general guidance and requirements for appropriate concentration selection.

Ice-Water Immersion and Cold-Water Immersion Provide Similar Cooling Rates in Runners With Exercise-Induced Hyperthermia.

OBJECTIVE: To assess whether ice-water immersion or cold-water immersion is the more effective treatment for rapidly cooling hyperthermic runners. DESIGN AND SETTING: 17 heat-acclimated highly trained distance runners (age = 28 +/- 2 years, height = 180 +/- 2 cm, weight = 68.5 +/- 2.1 kg, body fat = 11.2 +/- 1.3%, training volume = 89 +/- 10 km/wk) completed a hilly trail run (approximately 19 km and 86 minutes) in the heat (wet-bulb globe temperature = 27 +/- 1 degrees C) at an individually selected "comfortable" pace on 3 occasions 1 week apart. The random, crossover design included (1) distance run, then 12 minutes of ice-water immersion (5.15 +/- 0.20 degrees C), (2) distance run, then 12 minutes of cold-water immersion (14.03 +/- 0.28 degrees C), or (3) distance run, then 12 minutes of mock immersion (no water, air temperature = 28.88 +/- 0.76 degrees C). MEASUREMENTS: Each subject was immersed from the shoulders to the hip joints for 12 minutes in a tub. Three minutes elapsed between the distance run and the start of immersion. Rectal temperature was recorded at the start of immersion, at each minute of immersion, and 3, 6, 10, and 15 minutes postimmersion. No rehydration occurred during any trial. RESULTS: Length of distance run, time to complete distance run, rectal temperature, and percentage of dehydration after distance run were similar (P >.05) among all trials, as was the wet-bulb globe temperature. No differences (P >.05) for cooling rates were found when comparing ice-water immersion, cold-water immersion, and mock immersion at the start of immersion to 4 minutes, 4 to 8 minutes, and the start of immersion to 8 minutes. Ice-water immersion and cold-water immersion cooling rates were similar (P >.05) to each other and greater (P <.05) than mock immersion at 8 to 12 minutes, the start of immersion to 10 minutes, and the start of immersion to every other time point thereafter. Rectal temperatures were similar (P >.05) between ice-water immersion and cold-water immersion at the completion of immersion and 15 minutes postimmersion, but ice-water immersion rectal temperatures were less (P <.05) than cold-water immersion at 6 and 10 minutes postimmersion. CONCLUSIONS: Cooling rates were nearly identical between ice-water immersion and cold-water immersion, while both were 38% more effective in cooling after 12 minutes of immersion than the mock-immersion trial. Given the similarities in cooling rates and rectal temperatures between ice-water immersion and cold-water immersion, either mode of cooling is recommended for treating the hyperthermic individual.