Potency assay of antibodies against rabies. A report on a collaborative study

The relative potencies of a number of rabies immunoglobulin preparations were estimated in an international collaborative study comprising eight laboratories in five countries. Two assay methods were used: a virus neutralization test in mice (MNT) and a virus neutralization test in cell culture (RFFIT). Differences between the results obtained by the two methods, which have been reported, could not be generally corroborated. The results indicate that in some laboratories the MNT cause difficulties and give results different from those obtained by RFFIT. In other laboratories such difficulties are not encountered. The results seem to indicate that the RFFIT is a more reliable method than the MNT.     

Assessment of the performance of the Ames II assay: a collaborative study with 19 coded compounds

Nineteen coded chemicals were tested in an international collaborative study for their mutagenic activity. The assay system employed was the Ames II Mutagenicity Assay, using the tester strains TA98 and TAMix (TA7001-7006). The test compounds were selected from a published study with a large data set from the standard Ames plate-incorporation test. The following test compounds including matched pairs were investigated: cyclophoshamide, 2-naphthylamine, benzo(a)pyrene, pyrene, 2-acetylaminofluorene, 4,4'-methylene-bis(2-chloroaniline), 9,10-dimethylanthracene, anthracene, 4-nitroquinoline-N-oxide, diphenylnitrosamine, urethane, isopropyl-N(3-chlorophenyl)carbamate, benzidine, 3,3'-5,5'-tetramethylbenzidine, azoxybenzene, 3-aminotriazole, diethylstilbestrol, sucrose and methionine. The results of both assay systems were compared, and the inter-laboratory consistency of the Ames II test was assessed. Of the eight mutagens selected, six were correctly identified with the Ames II assay by all laboratories, one compound was judged positive by five of six investigators and one by four of six laboratories. All seven non-mutagenic samples were consistently negative in the Ames II assay. Of the four chemicals that gave inconsistent results in the traditional Ames test, three were uniformly classified as either positive or negative in the present study, whereas one compound gave equivocal results. A comparison of the test outcome of the different investigators resulted in an inter-laboratory consistency of 89.5%. Owing to the high concordance between the two test systems, and the low inter-laboratory variability in the Ames II assay results, the Ames II is an effective screening alternative to the standard Ames test, requiring less test material and labor.     

Inter-laboratory variability in Ames assay results

The Ames test is widely used in the screening of chemicals and compounds for potential carcinogenic effect. There is, however, considerable inter-laboratory variability in results from this assay. Using data from the RTI Collaborative Study of the EPA Ames Test Protocol, we show that their reported standard errors of estimates of mutagenicity fall far short of capturing day-to-day or laboratory-to-laboratory variation. We estimate the factors by which the standard errors must by inflated to account for these sources of variation. The laboratory protocol and previous studies suggest that much of this variation may be caused by factors that are relatively constant within days (e.g. technician, incubation temperature, S9 liver homogenate preparation) but vary over days and across laboratories. Therefore, such variation might be reduced through use of a reference compound tested on the same day and under the same conditions as the test chemical. This conjecture was, however, not supported by analyses that considered the positive control compound and a pure chemical as possible reference assays.     

Statistical evaluation of inter- and intra-laboratory variations of the Ames test, as related to the genetic stability of Salmonella tester strains

A statistical analysis was performed on the data resulting from an international collaborative study of the Ames test according to a standardized experimental protocol, which involved the comparative testing of 4NQO (4 doses), in 3 separate experiments for each of the 38 participating laboratories, by using a common reference (R) culture and in-house laboratory (L) cultures of 5 strains of S. typhimurium. Despite some toxicity phenomena recorded at the highest dose of 4NQO, the majority of the dose-response curves in individual laboratories were linear on a bi-log scale and their mean values fitted a linear regression framework. Scattering of data around mean values of laboratories was Gaussian-like even at the highest dose of 4NQO, toxic effects being expressed as a dose-related increase of variance. A weighted least-square analysis could therefore take into account toxic effects without resorting to a sophisticated non-linear model incompatible with log transformation. Various analytical approaches--e.g. the weighted estimates of linear regression parameters, a multifactor (laboratory, experiment, dose, culture of each strain) analysis of variance with all the possible interactions, the assessment of correlations in individual laboratories and of coefficients of variation for induced and spontaneous mutability--could detect some statistically significant differences between L and R cultures. However, at a critical evaluation on an individual basis, only few of these differences, without any peculiar involvement of given strains, were convincing in view of the existence of real phenomena of genetic drift. Therefore, on the whole, the genetic drift of Salmonella tester strains appears to lend a negligible contribution to the considerable inter- and intra-laboratory variability detected in this study. With a background variability between replications averaging 26%, a dose-related variability was evident both between experiments (28-54%) and between laboratories (44-127%).     

An interlaboratory study of an EPA/Ames/Salmonella test protocol

7 laboratories participated in a collaborative study to evaluate an EPA standard protocol for the Ames test. The study utilized Salmonella typhimurium (strains TA98 and TA100) and 3 metabolic activation levels (0%, 2%, and 10% S9 in the S9 mix). 6 pure chemicals and 2 complex mixtures were tested as coded unknowns. Ability to obtain qualitative results in agreement with published data was less (% agreement) than that reported in an earlier study (% agreement) by de Serres and Ashby (1981) in which each laboratory used its own protocol. The conclusion from analysis of the quantitative data from this interlaboratory Ames study was that both intralaboratory and interlaboratory variations were substantial. Results for the same substance varied by an order of magnitude or more (CV of 115%) when the mutagenic response was measured as the slope of the dose response in revertants/microgram. Taking interlaboratory variation into account, one chemical must be more than an order of magnitude more mutagenic than another (ratio of slopes greater than 10) to have only an even chance of finding a statistically significant difference between the two chemicals at the 5% level. Such large variations must be taken into account when evaluating Ames/Salmonella data.     

Structure-activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters

Sulfonic acid esters are considered as potentially alkylating agents that may exert genotoxic effects in bacterial and mammalian cell systems. One possible source of human exposure stems from drug synthesis when the salt-forming agents methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid are used together with alcoholic solvents such as methanol, ethanol and propanol. In this study computer-assisted structural considerations and in vitro approaches (Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, and the micronucleus test using L5178Y mouse lymphoma cells) were used to assess the genotoxic properties of 19 sulfonic esters. While all esters may be principally active as genotoxicants based on the presence of the sulfonate moiety, the statistical correlative multiple computer automated structure evaluation (MCASE) system (MC4PC version 1.0) using the Ames mutagenicity A2I module (version 1.54), rank-ordered the activity of the benzenesulfonic acid esters in the Ames test negligible due to an inactivating modulator and a deactivating fragment, whereas the methane- and toluenesulfonic acid esters were predicted to be positive in this test. In the Ames test, with the exception of the p-toluenesulfonic acid ethyl and iso-butyl esters, all compounds came out positive in Salmonella strain TA100. Methanesulfonic iso-propyl, sec-butyl and benzenesulfonic acid iso-propyl ester also showed mutagenic potential in strain TA98. In general, differences between results seen in Ames tests performed with or without metabolic activation were rather small. In L5178Y mouse lymphoma cells, benzenesulfonic acid n- and iso-butyl ester and p-toluenesulfonic acid iso-butyl ester did not increase the number of cells containing micronuclei. The other esters were positive in this micronucleus test; however, methanesulfonic acid iso-butyl ester was found to be only weakly positive at excessively cytotoxic concentrations. These compounds were generally found to be more potent with regard to micronucleus induction when tested without metabolic activation (20 h treatment). In conclusion, the iso-propyl esters of the three sulfonic acids under study were found to be the strongest mutagens, either when tested in the Ames test or the micronucleus assay, whereas p-toluenesulfonic acid iso-butyl ester was the only compound shown to be devoid of a genotoxic potential in both tests.     

Evaluation of the repeatability and reproducibility of European suspension test methods for antimicrobial activity of disinfectants and antiseptics.

A collaborative study to determine the precision of the 1987 Method of test for the antimicrobial activity of disinfectants in food hygiene is described. The repeatability and reproducibility of the test was found to vary according to the nature of the test organism, the type of disinfectant product and the skill of the operator. Results indicate that significant differences in microbicidal effect (ME values) occur within test laboratories between test periods as well as between laboratories, and that much of this variability derives from apparently random variations in the resistance of test strains from day to day and test period to test period. Indications are that although the test is sufficiently reliable to be used as a standard method, adequate test replication must be specified to distinguish borderline pass from borderline fail disinfectant concentrations. The implications of the results in relation to current work on the development of unified European test methods for disinfectants is discussed.     

Standardizing global gene expression analysis between laboratories and across platforms

To facilitate collaborative research efforts between multi-investigator teams using DNA microarrays, we identified sources of error and data variability between laboratories and across microarray platforms, and methods to accommodate this variability. RNA expression data were generated in seven laboratories, which compared two standard RNA samples using 12 microarray platforms. At least two standard microarray types (one spotted, one commercial) were used by all laboratories. Reproducibility for most platforms within any laboratory was typically good, but reproducibility between platforms and across laboratories was generally poor. Reproducibility between laboratories increased markedly when standardized protocols were implemented for RNA labeling, hybridization, microarray processing, data acquisition and data normalization. Reproducibility was highest when analysis was based on biological themes defined by enriched Gene Ontology (GO) categories. These findings indicate that microarray results can be comparable across multiple laboratories, especially when a common platform and set of procedures are used.     

An evaluation of the repeatability and reproducibility of a surface test for the activity of disinfectants

A collaborative study was carried out to determine the precision of a disinfectant surface test method which is currently under consideration for development as a harmonized European standard surface test. Results indicate that significant variation in microbicidal effect occurs both within and between test laboratories despite careful standardization of test conditions, but that the variability may be less than that associated with suspension tests. Indications are that much of this variability derives from random variations in the resistance of the test strains from day to day and, most particularly, from test period to test period both within as well as between laboratories. It is concluded that although the test may be sufficiently reliable to be used as a standard method, adequate replication must be specified to distinguish borderline pass from borderline fail concentrations.     

Evaluation of the repeatability and reproducibility of European suspension test methods for antimicrobial activity of disinfectants and antiseptics

A collaborative study to determine the precision of the 1987 Method of test for the antimicrobial activity of disinfectants in food hygiene is described. The repeatability and reproducibility of the test was found to vary according to the nature of the test organism, the type of disinfectant product and the skill of the operator. Results indicate that significant differences in microbicidal effect (ME values) occur within test laboratories between test periods as well as between laboratories, and that much of this variability derives from apparently random variations in the resistance of test strains from day to day and test period to test period. Indications are that although the test is sufficiently reliable to be used as a standard method, adequate test replication must be specified to distinguish borderline pass from borderline fail disinfectant concentrations. The implications of the results in relation to current work on the development of unified European test methods for disinfectants is discussed.     

The Ames Salmonella/microsome mutagenicity assay

The Ames Salmonella/microsome mutagenicity assay (Salmonella test; Ames test) is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances that can produce genetic damage that leads to gene mutations. The test employs several histidine dependent Salmonella strains each carrying different mutations in various genes in the histidine operon. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When the Salmonella tester strains are grown on a minimal media agar plate containing a trace of histidine, only those bacteria that revert to histidine independence (his(+)) are able to form colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner. The Ames test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs. The test is also used for submission of data to regulatory agencies for registration or acceptance of many chemicals, including drugs and biocides. International guidelines have been developed for use by corporations and testing laboratories to ensure uniformity of testing procedures. This review provides historical aspects of how the Ames was developed and detailed procedures for performing the test, including the design and interpretation of results.     

A collaborative study on the use of single radial immunodiffusion for the assay of rabies virus glycoprotein

The single radial immunodiffusion (SRD) technique has been applied to the assay of the glycoprotein content of rabies vaccines produced in cell cultures. Fourteen laboratories in seven countries participated in a collaborative study to evaluate the reproducibility of the SRD technique; some laboratories also examined vaccines in the mouse protection (NIH) test and by enzyme immunoassay. Good agreement was found between potency estimates using the SRD technique: the geometric coefficients of variation for combined potency estimates of all laboratories were about 10%. SRD assays appear to have a role for the in vitro assay of antigen content of vaccine and could complement results obtained in in vivo assays which are subject to wide variability.     

An international collaborative study of an anti-infectious bursal disease virus reference serum

Fourteen laboratories participated in a collaborative study of a freeze-dried preparation of anti-infectious bursal disease virus serum to assess the suitability of the serum as a standard for use in the infectious bursal disease virus neutralization test. Ten laboratories carried out micro-virus neutralization tests and six carried out plaque reduction tests, two laboratories carrying out both tests. When titres were expressed as a proportion of that obtained for a reference preparation there was a marked reduction in variation between results from different laboratories. The use of a reference preparation was therefore of value when comparing results from different laboratories. It is proposed that the reference preparation used in this study be used as a standard to facilitate the comparison of results from different laboratories. The proposed standard contains by definition 10,000 UK units.     

A procedure for the statistical evaluation of Ames Salmonella assay results. Comparison of results among 4 laboratories

Ames Salmonella test data collected in our laboratory and 3 National Cancer Institute contract laboratories were analyzed to study the distribution of experimental errors associated with the test. It is shown that the Poisson distribution is not appropriate, and that the power transformation model Y = (revertants/plate)lambda, with lambda = 0.2 as estimated by the methods of Box and Cox, produced a measurement scale on which the experimental errors could be adequately described by a normal (Gaussian) distribution with a constant variance. The modeling procedure enables one to properly use analysis of variance, regression analysis, and Student's t test to analyze Ames Salmonella test results, and well-known statistical quality control procedures to monitor laboratory performance. The method detects weak mutagenic activity and measures the amount and uncertainty of the increase in revertants/plate. The development of the power transformation model is discussed and examples of its use in the interpretation of Ames Salmonella assay results are included.     

A collaborative study to examine the sensitivity and reproducibility of assays for the detection of DNA in biologicals derived from continuous cell lines.

This report describes the results of a collaborative study which was set up to examine the sensitivity and reproducibility of DNA assays performed by different laboratories. Fifteen laboratories analysed 12 samples containing different amounts of DNA and protein. The study revealed a high degree of variability between laboratories in determining the DNA content of the test samples.     

麻疹减毒活疫苗国家参考品的制备及标定

为了制备麻疹减毒活疫苗国家参考品,选用国内麻疹疫苗生产株沪191制备麻疹疫苗参考品。生产过程中严格控制精密性、水分含量,对候选参考品进行鉴别试验、水分含量、病毒滴度及无菌检查等检验。检验合格后组织进行候选参考品病毒滴度协作标定,共有5个实验室参加了协作标定。协作标定完成后,对实验室内变异、实验室间变异及国际参考品在不同实验室间的变异进行了分析。此外还对疫苗进行了热稳定性和实时稳定性分析。结果显示经5个实验室协作标定后,麻疹减毒活疫苗国家参考品的滴度为4.96±0.26 lgCC ID50/m l,实验室内部变异在1.09%～4.64%之间,实验室间变异为2.62%。国际参考品在不同实验室间的变异为4.19%。稳定性考核数据表明制备的参考品具有较好的稳定性,符合作为麻疹减毒活疫苗国家参考品的要求,滴度为4.96±0.26 lgCC ID50/m l。     

A collaborative assay of the proposed third British Reference Preparation for Pertussis Vaccine and of the relative potencies of the second International Standard and the second British Reference Preparation for Pertussis Vaccine

A collaborative assay has been carried out to estimate the mouse protective potency of a freeze-dried preparation of Bordetella pertussis (88/522) intended to serve as the third British Reference Preparation for Pertussis Vaccine (third BRP). The opportunity was also taken of reassessing the relationship between the second International Standard for Pertussis Vaccine and the second British Reference Preparation for Pertussis Vaccine (second BRP). Workers in nine laboratories took part in the study and together completed 19 assays which were considered to be statistically valid. Based on the results of the study it is proposed that ampouled preparation code number 88/522 be established as the third BRP with an assigned potency of 50 IU per ampoule. The evidence of this study also suggests that the relationship between the second International Standard for Pertussis Vaccine and the second BRP has not changed significantly since they were originally established.     

A collaborative cytogenetics study to measure and minimize interlaboratory variation

Six cytogenetics laboratories joined in a collaborative study of rat chromosome aberrations to measure interlaboratory variation in results of standardized procedures and to devise methods to minimize interlaboratory differences. A preliminary workshop was held to resolve scoring differences, to develop a joint protocol and common glossary, and to reach agreement on uniform reporting methods. Osborne-Mendel rats from a common source were sent to each laboratory. Triethylenemelamine (TEM) was used at doses of 100, 200, 300 and 400 μg/kg to induce clastogenic effects; results were compared to those of a control group of untreated animals. Femoral bone marrow cells were evaluated with the scorers unaware of the dosage. Final results showed highly significant dose effects with the test compound, and most laboratories showed a similar pattern of dose response. This study illustrates that rat cytogenetic analysis can be an effective test system for evaluation of a compound for mutagenic potential, particularly for the index reflecting the proportion of abnormal cells, but that results should be interpreted cautiously when arbitrary values are assigned for some of the categories being analyzed, as was done in this project for the category of severely damaged cells.     

Collaborative study of mutagenicity with Salmonella typhimurium TA102.

Thirty compounds of various chemical classes were investigated for mutagenicity in a collaborative study (3 laboratories) using Salmonella typhimurium TA102. With 5 compounds, namely hydrazine sulfate, phenylhydrazine, hydralazine, glutardialdehyde and glyoxal, mutagenicity was detected by all laboratories. Formaldehyde was assessed as weakly mutagenic in only 1 of 3 laboratories. The remaining 24 agents were uniformly described as non-genotoxic in TA102. In spite of the overall good qualitative agreement in the mutagenicity results between the 3 laboratories some quantitative discrepancies occurred in the dose response of the mutagenic compounds. Varying inter- and intra-laboratory differences in the spontaneous rate of revertants were obtained. The usefulness of the tester strain TA102 in routine mutagenicity testing is discussed.     

A study in test reproducibility between laboratories: Report of a Pseudomonas Working Party

The results are reported of a collaborative study in laboratories of 17 tests commonly used for pseudomonads, together with statistical analysis of the results in the form of analyses of variance. The studies involved 59 strains. The following tests showed good or reasonably good consistency between different laboratories: motility if checked by a Craigie tube, growth at 4 C, production of fluorescin, oxidation of glucose and sucrose, gluconate oxidation, hydrolysis of aesculin, arginine and casein, and gelatin hydrolysis by the tube method. The following were less satisfactory: shape, size and arrangement of organismis, growth at 42 C, oxidase, acid from lactose, reduction of nitrate, acetic acid from ethanol, gelatin hydrolysis by the plate method, and the egg yolk reaction. The following showed poor consistency or noticeable difficulties in performing or interpreting results of the test: growth at 37 C, the denitrification test of Stanier, Palleroni and Doudoroff, and hydrolysis of urea. However, with several of the unsatisfactory tests the replicates within a laboratory showed much better reproducibility. This (together with other evidence on the importance of exact control of variables such as temperature, and time of reading, and the occurrence of mutants in some strains) suggests that careful attention to standardization may give much better testing methods. The considerable value of statistical analyses in such work is discussed.