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: 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.     

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.     

The mouse lymphoma assay

In this paper, the current status of the protocol for the Mouse Lymphoma Assay is discussed. A brief history describes the events leading to current protocol recommendations. Areas for further development such as cytotoxicity, 24-h treatments, acceptability criteria and statistical analysis are also considered. Recent guidelines are reviewed, and consensus issues from the Mouse Lymphoma workgroup assembled as part of the International Workshop on Genotoxicity Test Procedures (IWGTP) are included. There are two versions of the assay - soft agar and microwell - and both will be discussed. For assay procedures, the emphasis will be on a typical microwell protocol but an attempt will be made to highlight protocol variations between laboratories and between the microwell and agar versions of the assay.     

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.Abbreviations AO acridine orange - BAP benzo[a]pyrene - BZN benzoin - CHO Chinese hamster ovary cell assay - DPH diphenylhydantoin - EB ethidium bromide - EMS ethylmethanesulfonate - 3MC 3-methylcholanthrene - MLA mouse lymphoma asay - MMC mitomycin C - MXC methoxychlor - PAP p-aminophenol - PRO proflavin - PYR pyrene     

Compilation and use of genetic toxicity historical control data

The optimal use of historical control data for the interpretation of genotoxicity results was discussed at the 2009 International Workshop on Genotoxicity Testing (IWGT) in Basel, Switzerland. The historical control working group focused mainly on negative control data although positive control data were also considered to be important. Historical control data are typically used for comparison with the concurrent control data as part of the assay acceptance criteria. Historical control data are also important for providing evidence of the technical competence and familiarization of the assay at any given laboratory. Moreover, historical control data are increasingly being used to aid in the interpretation of genetic toxicity assay results. The objective of the working group was to provide generic advice for historical control data that could be applied to all assays rather than to give assay-specific recommendations. In brief, the recommendations include:     

Comprehensive linkage map of bovine chromosome 27

The results of genotypic data contributed to the International Society for Animal Genetics (ISAG) Bovine Chromosome 27 Workshop are presented. Eight laboratories contributed 23 261 informative meioses from 44 loci. Eighteen loci were typed by at least two laboratories and were used to construct a consensus linkage map. Twenty-one loci were subsequently incorporated into a comprehensive map. The sex-averaged consensus map covered 66.9 cM. The sex-averaged comprehensive map was 75.5 cM, while the female and male maps were 73.1 and 63.7 cM, respectively. Five loci were excluded from the analysis because of ambiguous position in the linkage group and a low LOD score (less than 2.0). Average distance between loci in the comprehensive map was 1.98 cM.     

In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test

The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed, but a consensus regarding acceptability for regulatory purposes could not be reached at that time. Subsequent validation efforts, combined with accumulated published data, demonstrate that blood-derived reticulocytes from rats as well as mice are acceptable when young reticulocytes are analyzed under proper assay protocol and sample size. The working group reviewed the results of micronucleus assays using target cells/tissues other than hematopoietic cells. We also discussed the relevance of the liver micronucleus assay using young rats, and the importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have been integrated into the data bases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not dose-limiting.     

Using statistical analysis for setting process validation acceptance criteria for biotech products

This paper discusses the challenges of setting process validation acceptance criteria for biotech products for cases where using statistical tools is appropriate. Data are analyzed under three different scenarios that are frequently encountered in biotech applications. Scenario A represents the case when a small data set around center point conditions is available for setting acceptance criteria. Scenario B represents the case when a larger data set within normal operation conditions is available for setting acceptance criteria. Scenario C represents the case when a large characterization data set is available for setting acceptance criteria and it is possible to accurately model the impact of operation conditions on performance of the step. Statistical approaches including mean +/- 3SD, tolerance interval analysis, prediction profiler, and Monte Carlo simulation are applied to the different scenarios. Strengths and shortcomings of the different statistical tools are discussed, and the best approach for each scenario is recommended. It is shown that selection of the right statistical approach is a critical first step toward setting appropriate acceptance criteria.     

Comprehensive linkage map of bovine chromosome 11

The results of genotypic data contributed to the International Society of Animal Genetics (ISAG) Bovine Chromosome 11 (BTA11) Workshop are presented. Six laboratories contributed a total of 26 199 informative meioses from 80 loci. Thirty-six loci were typed by at least two independent laboratories and were used to construct a consensus linkage map of the chromosome. The remaining loci were subsequently incorporated into a comprehensive map. The sex-averaged consensus map covered 128.9 cM. The female consensus map was 101.2 cM, while the male consensus map was 129.8 cM. The comprehensive sex-averaged map was 134.2 cM and the average genetic distance between loci was 1.72 cM.     

New achievements in human cell toxicology: the 20th annual workshop on in vitro toxicology

The 20th Annual Workshop on In Vitro Toxicology (Oxford, UK, September 22-24, 2002) was convened as part of a European meeting entitled Human Cell Culture 2002. The meeting was arranged by the Scandinavian Society for Cell Toxicology (SSCT), the European Tissue Culture Society and the British Prostate Group. Two sessions, which are summarised in this report, were devoted to in vitro toxicology: Human Cell Toxicology and The SSCT Free Paper Session. Outstanding experts in the field of toxicology outlined contemporary approaches in toxicity testing in their lectures. Short oral presentations demonstrated a variety of in vitro model systems and methodologies, which can be useful for investigating human toxicity, as well as for studies on mechanisms of toxicity.     

Cytokine release: A workshop proceedings on the state-of-the-science,current challenges and future directions

In October 2013, the International Life Sciences Institute - Health and Environmental Sciences Institute Immunotoxicology Technical Committee (ILSI-HESI ITC) held a one-day workshop entitled, “Workshop on Cytokine Release: State-of-the-Science, Current Challenges and Future Directions”. The workshop brought together scientists from pharmaceutical, academic, health authority, and contract research organizations to discuss novel approaches and current challenges for the use of in vitro cytokine release assays (CRAs) for the identification of cytokine release syndrome (CRS) potential of novel monoclonal antibody (mAb) therapeutics. Topics presented encompassed a regulatory perspective on cytokine release and assessment, case studies regarding the translatability of preclinical cytokine data to the clinic, and the latest state of the science of CRAs, including comparisons between mAb therapeutics within one platform and across several assay platforms, a novel physiological assay platform, and assay optimization approaches such as determination of FcR expression profiles and use of statistical tests. The data and approaches presented confirmed that multiple CRA platforms are in use for identification of CRS potential and that the choice of a particular CRA platform is highly dependent on the availability of resources for individual laboratories (e.g. positive and negative controls, number of human blood donors), the assay through-put required, and the mechanism-of-action of the therapeutic candidate to be tested. Workshop participants agreed that more data on the predictive performance of CRA platforms is needed, and current efforts to compare in vitro assay results with clinical cytokine assessments were discussed. In summary, many laboratories continue to focus research efforts on the improvement of the translatability of current CRA platforms as well explore novel approaches which may lead to more accurate, and potentially patient-specific, CRS prediction in the future.     

Modeling the mouse lymphoma forward mutational assay: the Gene-Tox program database

An SAR model of the induction of mutations at the tk(+/-) locus of L5178Y mouse lymphoma cells (MLA, for mouse lymphoma assay) was derived based upon a re-evaluation of experimental results reported by a Gene-Tox (GT) working group [A.D. Mitchell, A.E. Auletta, D. Clive, P.E. Kirby, M.M. Moore, B.C. Myhr, The L5178Y/tk(+/-) mouse lymphoma specific gene and chromosomal mutation assay. A phase III report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res. 394 (1997) 177-303.]. The predictive performance of the GT MLA SAR model was similar to that of a Salmonella mutagenicity model containing the same number of chemicals. However, the structural determinants (biophores) derived from the GT MLA SAR model include both electrophilic as well as non-electrophilic moieties, suggesting that the induction of mutations in the MLA may occur by both direct interaction with DNA and by non-DNA-related mechanisms. This was confirmed by the observation that the set of biophores associated with MLA overlapped significantly with those associated with phenomena related to loss of heterozygosity, chromosomal rearrangements and aneuploidy. The MLA SAR model derived from the GT data evaluation was significantly more predictive than an SAR model previously derived from MLA data reported by the US National Toxicology Program [B. Henry, S.G. Grant, G. Klopman, H.S. Rosenkranz, Induction of forward mutations at the thymidine kinase locus of mouse lymphoma cells: evidence for electrophilic and non-electrophilic mechanisms, Mutation Res. 397 (1998) 331-335.]. Moreover, the latter model appeared to be more complex than the former, suggesting that the GT induction data was both simpler mechanistically and more homogeneous than that of the NTP.     

Joint report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, Kennett Square, Pennsylvania, 25-27 April 1984

The Third International Workshop on Lymphocyte Alloantigens of the Horse was held on 25-27 April 1984 in Kennett Square, Pennsylvania. Twelve laboratories from five countries participated. The principal purpose of this Workshop was to determine the phenotypic and gene frequencies of the 10 equine lymphocyte antigens (ELA) and a non-ELA lymphocyte antigen, ELY-2.1, in several breeds of horse. A total of 86 alloantisera characterized in previous workshops were tested against lymphocytes from 1179 horses. In addition, several experimental antisera were also tested against the same panel of lymphocytes. As a result of analysis of these data, the Workshop recognized two new equine lymphocyte alloantigens: W11 of the ELA system, and ELY-1.1, an antigen not linked to the ELA system.     

Multiple phenotypes associated with Myc-induced transformation of chick embryo fibroblasts can be dissociated by a basic region mutation.

Chimaeric alleles were constructed to assay the biological functions of an N-terminal deletion and C-terminal mutations which were found in a naturally occurring mutant of feline vMyc, T17. The mutant alleles were assayed for their ability to transform chick embryo fibroblasts in vitro by a number of criteria, namely the ability to induce morphological transformation, an accelerated growth rate and growth in soft agar. Feline cMyc could transform the avian cells, whilst T17 vMyc could not, and the N-terminal deletion was responsible for conferring the primary transformation defect on the mutant protein. The C-terminal mutations which consist of a point mutation adjacent to the nuclear localisation signal and a point mutation/amino acid insertion within the basic region (BR) could, however, dissociate the Myc-induced parameters of transformation. This effect was a specific function of the BR mutation alone, and the mutation could be transferred into avian cMyc with comparable biological consequences. The BR mutation did not disrupt the sequence specific DNA binding activity of the protein in vivo, despite exerting a biological effect. These data suggest a novel phenotype where the mutation may affect a subset of Myc-regulated genes through altered DNA binding specificity or protein-protein interactions.     

Joint Report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, Kennett Square, Pennsylvania, 25–27 April 1984

Summary. The Third International Workshop on Lymphocyte Alloantigens of the Horse was held on 25–27 April 1984 in Kennett Square, Pennsylvania. Twelve laboratories from five countries participated. The principal purpose of this Workshop was to determine the phenotypic and gene frequencies of the 10 equine lymphocyte antigens (ELA) and a non-ELA lymphocyte antigen, ELY-2.1, in several breeds of horse. A total of 86 alloantisera characterized in previous workshops were tested against lymphocytes from 1179 horses. In addition, several experimental antisera were also tested against the same panel of lymphocytes. As a result of analysis of these data, the Workshop recognized two new equine lymphocyte alloantigens: W11 of the ELA system, and ELY-1.1, an antigen not linked to the ELA system.     

Temperature dependency for maintenance of transformation in mouse cells transformed by simian virus 40 tsA mutants.

Mouse embryo fibroblasts and 3T3 cells were transformed by wild-type, tsB4, tsA7, tsA58, and tsA209 simian virus 40. Clones of transformants were generated both in soft agar and in liquid medium by focus formation and at both high and relatively low multiplicities of infection. All transformants were assayed for three phenotypes of transformation: (i) the ability to form highly multinucleated cells in cytochalasin B-supplemented medium, i.e., uncontrolled nuclear division; (ii) the capacity to continue DNA synthesis at increasing cell density; and (iii) the ability to form colonies in soft agar. The great majority of mouse embryo fibroblast transformants generated with tsA mutant virus were temperature sensitive for transformation in all three assays, regardless of the input multiplicity or whether they were generated in liquid medium or soft agar. These transformants exhibited a normal or near-normal phenotype at the nonpermissive temperature of 40 degrees C. All but one of the transformants which appeared transformed at both temperatures were in the A209 group. In contrast to mouse embryo fibroblasts, transformants generated with 3T3 cells and tsA virus were often not temperature sensitive, exhibiting the transformation phenotypes at both temperatures. This phenomenon was more often observed when 3T3 transformants were generated in soft agar. These results, along with other published data, suggest that uncontrolled nuclear division and uncontrolled DNA synthesis are a function of the simian virus 40 A gene. Finally, with the 3T3 transformants, there was often discordance in the expression of transformation among the three phenotypes. Some tsA transformants were temperature sensitive in one of two assays but were transformed at both 33 and 40 degrees C in the remaining assay(s). Other transformants exhibited a normal cytochalasin B response at either temperature but were temperature sensitive in the other assays.     

Influence of capsular neuraminic acid on properties of streptococci of serological group B.

Neuraminic acid is thought to be a critical virulence factor of group B streptococci. The present study was designed to further characterize a previously described type III group B streptococcus and its transposon-mutagenized asialo capsular mutant. The wild-type group B streptococcus grew as short chains with a uniform turbidity and had diffuse colonies in soft agar media. In contrast, the asialo mutant grew in fluid media as a granular sediment, formed significantly longer chains and had compact colonies in soft agar. These differences, possibly related to the surface charge of the bacteria, could also be demonstrated in salt aggregation tests and hexadecane adherence studies. The wild-type group B streptococcus showed hydrophilic, and the asialo mutant hydrophobic surface properties. Removal of neuraminic acid from the wild-type strain changed the surface properties from hydrophilic to hydrophobic. A similar masking effect of capsular neuraminic acid could be observed in adherence and phagocytosis experiments. In contrast to the wild-type strain, the asialo mutant adhered significantly more to buccal epithelial cells and was phagocytosed more by polymorphonuclear leucocytes. These altered properties might possibly be of importance for group B streptococcal pathogenicity.     

Rapid method for permanent slide preparation of colonies in soft agar cultures

A method is presented for preparing permanent microscopic slides from colony-bearing agar layers in soft agar cultures. The main advantages of this technique are its simplicity, rapidity and accurate colony preservation. This method could have broad applications in the human tumor clonogenic assay (HTCA), particularly in the quantitative morphological, cytochemical and immunocytochemical assessment of colonies that form in both control and drug-treated cultures. Thus, this method opens up possibilities for using cytopathological criteria as a quantitative endpoint of the HTCA.     

An ensemble framework for clustering protein-protein interaction networks

MOTIVATION: Protein-Protein Interaction (PPI) networks are believed to be important sources of information related to biological processes and complex metabolic functions of the cell. The presence of biologically relevant functional modules in these networks has been theorized by many researchers. However, the application of traditional clustering algorithms for extracting these modules has not been successful, largely due to the presence of noisy false positive interactions as well as specific topological challenges in the network. RESULTS: In this article, we propose an ensemble clustering framework to address this problem. For base clustering, we introduce two topology-based distance metrics to counteract the effects of noise. We develop a PCA-based consensus clustering technique, designed to reduce the dimensionality of the consensus problem and yield informative clusters. We also develop a soft consensus clustering variant to assign multifaceted proteins to multiple functional groups. We conduct an empirical evaluation of different consensus techniques using topology-based, information theoretic and domain-specific validation metrics and show that our approaches can provide significant benefits over other state-of-the-art approaches. Our analysis of the consensus clusters obtained demonstrates that ensemble clustering can (a) produce improved biologically significant functional groupings; and (b) facilitate soft clustering by discovering multiple functional associations for proteins. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.