ICCVAM evaluation of the murine local lymph node assay. Data analyses completed by the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods.

To evaluate the reliability of the murine local lymph node assay (LLNA), a test for allergic contact dermatitis activity, the inter- and intralaboratory consistency statistics (h and k, respectively) were calculated for validation studies testing multiple chemicals. The analysis indicated the absence of excessive variability in the dose calculated to induce a threefold or greater increase in the stimulation index (SI). To assess the appropriateness of using an SI of 3 as the decision criteria for identifying a sensitizing compound, LLNA results based on SI values of 2.0, 2.5, 3.0, 3.5, and 4.0 were compared with guinea pig or human results. The results supported the use of an SI of 3 as the decision criteria. Assay performance was determined by comparing LLNA results to results obtained for guinea pigs or humans. The accuracy of the LLNA was 89% when compared with results from the guinea pig maximization test (GPMT)/Buehler assay (BA). The performance of the LLNA and the GPMT/BA was similar when each was compared to human maximization test results plus substances included as human patch test allergens. The LLNA offered advantages over the GPMT in respect to both the time required to conduct the test and the assay cost.

The proportions of mutagens among chemicals in commerce.

It has been estimated that there are approximately 80,000 chemicals in commerce. Thus, it is not possible to test all these substances for mutagenicity and carcinogenicity; it is possible, however, to test or make estimates from selected subsets of these chemicals. For example, in the U.S. National Toxicology Program (NTP), 35% of the chemicals tested for mutagenicity in Salmonella were positive, as were 52% of the chemicals tested for carcinogenicity in rodents. In contrast, in the U.S. EPA Gene-Tox database, the proportions of chemicals that are Salmonella mutagens is 56%. These and other databases may be biased toward positive responses because they generally have been developed to look at specific structural or use classes of chemicals or chemicals suspected of genetic or carcinogenic activity. To address the question of the proportions of mutagens among all chemicals in commerce, a database of 100 chemicals was created from a random selection of chemicals in commerce. These chemicals were tested for mutagenicity in Salmonella and 22% were mutagenic. The mutagenicity of the 46 highest U.S. production organic chemicals was also compiled; 20% were mutagenic. These values provide a more accurate estimate of the proportions of mutagens among chemicals in commerce than can be derived from published mutagenicity databases.

Statistical modeling and analyses of a base-specific Salmonella mutagenicity assay.

Statistical features of a base-specific Salmonella mutagenicity assay are considered in detail, following up on a previous report comparing responses of base-specific Salmonella (Ames II) strains with those of traditional tester strains. In addition to using different Salmonella strains, the new procedure also differs in that it is performed as a microwell fluctuation test, as opposed to the standard plate or preincubation test. This report describes the statistical modeling of data obtained from the use of these new strains in the microwell test procedure. We emphasize how to assess any significant interactions between replicate cultures and exposure doses, and how to identify a significant increase in the mutagenic response to a series of concentrations of a test substance.

Report and summary of the major conclusions from statistics in genotoxicity testing working group from the International Workshop on Genotoxicity Test Procedures (IWGTP), March 1999.

A working group of five statisticians experienced in the use of statistical methods in mutagenicity reviewed aspects of the statistical analysis of genotoxicity test procedures. Issues discussed included methods for integrating biological importance and statistical significance, the relationship of the experimental unit to the experimental design, and the impact of new developments in statistics and computing. Three major recommendations were made relating to the need for: (1) the effective use of statistical advice in designing interlaboratory and intralaboratory investigations; (2) the development of appropriate experimental designs for new assays; and (3) education and training in the use of statistical methodology in mutagenicity testing. Environ. Mol. Mutagen. 35:260-263, 2000 Published 2000 Wiley-Liss, Inc.

Predicting rodent carcinogenicity using potency measures of the in vitro sister chromatid exchange and chromosome aberration assays.

In vitro sister chromatid exchange (SCE) and chromosome aberration (ABS) tests have been extensively used to identify potential rodent carcinogens. A number of measures of potency were developed to describe in vitro SCE and ABS test results: the dose needed to induce a unit increase over the control; the lowest effective dose; the slope of the ordinary linear regression; the maximum observed slope; and the maximum fold increase over background. The ability of these potency measures to predict the qualitative and quantitative carcinogenicity of chemicals was compared to the predictivity of the qualitative in vitro responses. The results of the analyses showed that the quantitative measures of the SCE or ABS responses only minimally increased the predictivity of carcinogenesis when compared to the predictivity using the qualitative responses.

Statistical methods for the Ames Salmonella assay: a review.

The Ames Salmonella assay remains the most widely used in vitro genotoxicity assay. Several statistical methods have been proposed for its analysis [B.H. Margolin, N. Kaplan, E. Zeiger, Statistical analysis of the Ames Salmonella/microsome test, Proc. Natl. Acad. Sci., 78 (1981) 3779-3783; L.E. Myers, N.H. Saxton, L.I. Southerland, T.J. Wolff, Regression analysis of Ames test data, Environ. Mol. Mutagen., 3 (1981) 575-586; A.G. Stead, V. Hasselblad, J.P. Creason, L. Claxton, Modelling the Ames test, Mutation Res., 85 (1981) 13-27; L. Bernstein, J. Kaldor, J. McCaan, M.C. Pike, An empirical approach to the statistical analysis of mutagenesis data from the Salmonella test, Mutation Res., 97 (1982) 267-281; N.E. Breslow, Extra-Poisson variation in log-linear models, Appl. Stat., 33 (1984) 38-44; J. Wahrendorf, G.A.T. Mahon, M. Schumacher, A nonparametric approach to the statistical analysis of mutagenicity data, Mutation Res., 147 (1985) 5-13; D.G. Simpson, B.H. Margolin, Recursive nonparametric testing for dose-response relationships subject to downturns at high doses, Biometrika, 73 (1986) 589-596; D.G. Simpson, B.H. Margolin, Nonparametric testing for dose-response curves subject to downturns: Asymptotic power considerations, Annals Stat., 18 (1990) 373-390.]. In this paper we review recent literature to see what statistical methods are in fact employed for the analysis of the Ames assay. We then note that these methods can be classified into a common category in the framework of Haynes and Eckardt's mutation induction kinetics model [R.H. Haynes, F. Eckardt, Mathematical analysis of mutation induction kinetics, in: F.J. de Serres, A. Hollaender (Eds. ), Chemical Mutagens, Principles and Methods for Their Detection, Vol. 6, Plenum, New York, 1980, pp. 271-307]. The value in knowing this is that most methods of analysis considered here will likely exhibit common statistical behavior. These analyses are computationally intensive, e.g., [B.H. Margolin, N. Kaplan, E. Zeiger, Statistical analysis of the Ames Salmonella/microsome test, Proc. Nat. Acad. Sci., 78 (1981) 3779-3783], hence the ready availability of computer programs is essential if biologists are to use these methods. We briefly review two statistical software programs that are available in the public domain, and describe in detail a third program, Salm, [B.H. Margolin, N. Kaplan, E. Zeiger, Statistical analysis of the Ames Salmonella/microsome test, Proc. Nat. Acad. Sci., 78 (1981) 3779-3783; B.H. Margolin, B.S. Kim, K. Risko, The Ames Salmonella/microsome assay: Issues of inference and validation, J. Amer. Stat. Assoc., 84 (1989) 651-661]. The Salm program is obtainable through the file transfer protocol (ftp) or using a WWW browser. Finally, we discuss two statistical consequences of naively applying the two-fold rule, a method of analysis employed by a number of researchers.

Prediction of rodent carcinogenicity utilizing a battery of in vitro and in vivo genotoxicity tests.

The primary purpose of this study is to investigate the degree to which we can improve the prediction of rodent carcinogenicity (CA) by combining results from an in vitro and two in vivo genotoxicity tests. We used the Ames Salmonella assay (SAL) for the in vitro test and the micronucleus assay (MNC) and chromosome aberration assay (ABS) in mouse bone marrow cells for the two in vivo tests. We collected complete assay data for 82 chemicals (55 carcinogens and 27 noncarcinogens) from the NTP data base and the IARC monograph series. Our results indicate that: (1) only SAL affects the predictivity of CA, (2) MNC has a strong association with ABS, and (3) SAL predicts ABS. It has been known for some time that once the SAL assay result is available for prediction, other in vitro mutation tests provide little additional information for predicting CA. Our study indicates that the same conclusion holds for CA, SAL, MNC, and ABS.

Sources of variability in data from a positive selection lacZ transgenic mouse mutation assay: an interlaboratory study.

Experimental features of a positive selection transgenic mouse mutation assay based on a lambda lacZ transgene are considered in detail, with emphasis on results using germ cells as the target tissue. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined, with the goal of identifying sources of excess variation in the observed mutant frequencies. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal variability. Data from five laboratories are evaluated in detail. Results suggest only scattered patterns of excess variability below the animal-to-animal level, but, generally, significant excess variability at the animal-to-animal level. Using source of variability analyses to guide the choice of statistical methods, control-vs-treatment comparisons are performed for assessing the male germ cell mutagenicity of ethylnitrosourea (ENU), isopropyl methanesulfonate (iPMS), and methyl methanesulfonate (MMS). Results on male germ cell mutagenesis of ethyl methanesulfonate (EMS) and methylnitrosourea (MNU) are also reported.

Predicting rodent carcinogenicity from mutagenic potency measured in the Ames Salmonella assay.

Many in vitro tests have been developed to identify chemicals that can damage cellular DNA or cause mutations, and secondarily to identify potential carcinogens. The test receiving by far the most use and attention has been the Salmonella (SAL) mutagenesis test developed by Ames and colleagues [(1973): Proc Natl Acad Sci USA 70:2281-2285; (1975): Mutat Res 31:347-364], because of its initial promise of high qualitative (YES/NO) predictivity for cancer in rodents and, by extension, in humans. In addition to the initial reports of high qualitative predictivity, there was also an early report by Meselson and Russell [in Hiatt HH et al (1977): "Origins of Human Cancer, Book C: Human Risk Assessment," pp 1473-1481] of a quantitative relationship between mutagenic potency measured in SAL and carcinogenic potency measured in rodents, for a small number of chemicals. However, other reports using larger numbers of chemicals have found only very weak correlations. The primary purpose of this study was to determine whether mutagenic potency, as measured in a number of different ways, could be used to improve predictivity of carcinogenicity, either qualitatively or quantitatively. To this end, eight measures of SAL mutagenic potency were used. This study firmly establishes that the predictive relationship between mutagenic potency in SAL and rodent carcinogenicity is, at best, weak. When predicting qualitative carcinogenicity, only qualitative mutagenicity is useful; none of the quantitative measures of potency considered improves the carcinogenicity prediction. In fact, when qualitative mutagenicity is forced out of the model, the quantitative measures are still not predictive of carcinogenicity. When predicting quantitative carcinogenicity, several possible methods were considered for summarizing potency over all experiments; however, in all cases, the relationship between mutagenic potency predictors and quantitative carcinogenicity is very weak.

Patterns of amino acid variability in NSI-like and SI-like V3 sequences and a linked change in the CD4-binding domain of the HIV-1 Env protein.

The V3 domain plays a central role in the biology of the HIV-1 Env glycoprotein gp 120 as a dominant target for neutralizing antibodies for some HIV-1 isolates, and as a major determinant in the switch from the nonsyncytium-inducing (NSI) to the syncytium-inducing (SI) form of HIV-1 that is associated with accelerated disease progression. Basic amino acid substitutions are known to play an important role in the SI phenotype. We have used the presence of basic amino acid substitutions in V3 sequences to divide sequences in a large data base into SI-like and NSI-like. We found significant differences in features of sequence variability between these two groups of sequences. Of the thirty-six most frequent substitutions in V3, twenty appear disproportionately among either the SI-like sequences or the NSI-like sequences. The fourteen favored substitutions among the SI-like sequences account for 50% of the twofold increased variability seen in this group. In addition, we found a linked change within the CD4-binding domain of gp 120 downstream of V3. These differences are interpreted in the context of structure, function, and selective pressure. An understanding of these patterns of sequence variability offers the possibility of designing a degenerate SI-specific V3 immunogen to use as a therapeutic vaccine with the hope of slowing or preventing the appearance of SI variants.

Study design and sample sizes for a lacI transgenic mouse mutation assay.

Design features that adjust and account for excess variation in a transgenic mouse mutation assay based on a lacI target transgene from E. coli are considered. These features include proper identification of plate, packaging reaction, and animal identifier codes throughout the experimental and analysis phases of the study, "blocking" of exposed and unexposed animals when preparing and plating multiple packaging reactions from the same genomic DNA sample, separating sectored mutant plaques and complete mutant plaques before performing any quantitative analyses, and testing for sources of excess variation attributable to features of the experimental protocol--such as plate-to-plate (within packaging reactions), packaging reaction-to-packaging reaction (within animals), and animal-to-animal (within study). Control and ethylnitrosourea-treated animal data are presented from a fully designed study in the lacI assay. The study design incorporates many of these experimental principles. Statistical methods to identify excess variability are noted, and the designed study data are used to illustrate the types of variability encountered in practice. A standard statistical test for two-sample testing is highlighted, from which recommendations are made for sample size selection in future studies.

Sources of variability in data from a lacI transgenic mouse mutation assay.

Experimental features of a transgenic mouse mutation assay based on a lacI target transgene from Escherichia coli are considered in detail. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined with the goal of identifying sources of excess variation in the observed mutant fractions. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal (within study) variability. Data from two laboratories are evaluated, using various statistical methods to identify excess variability. Results suggest only scattered patterns of excess variability, except possibly in those cases where genomic DNA from test animals is stored for extended periods (e.g., > 90 days) after isolation from tissues. Further study is encouraged to examine the validity and implications of this time/storage-related effect.

Predicting carcinogenicity by using batteries of dependent short-term tests.

Among the various methods for predicting carcinogenicity from a battery of short-term tests (STTs), the carcinogenicity prediction and battery selection (CPBS) procedure is the most prominent. A major assumption of CPBS is that the STTs used in the prediction are conditionally independent. Results of recent National Toxicology Program studies of four commonly used in vitro STTs contradict this assumption, thereby necessitating modification of CPBS to accommodate dependencies. This is accomplished via log-linear modeling, which then also yields an important dividend: standard errors for the predicted probabilities of carcinogenicity.

Some comments on potency measures in mutagenicity research.

In this article, the measurement of the potency of a chemical or mixture from its dose response in a particular assay is addressed. Attention is focused on data from the Ames Salmonella assay. Three measures of potency are explored and shown to be highly correlated. The presentation then discusses specific areas of research that might benefit from a study of potency.

Cigarette smoking and sperm density: a meta-analysis.

OBJECTIVE: To quantify, through meta-analysis techniques, the association between cigarette smoking and sperm density. METHODS: The logarithm of the ratio of mean sperm density for smokers to that for nonsmokers for the studies included in this meta-analysis was regressed against a constant, an indicator of study population source (infertility clinic patients or normal men), minimum number of cigarettes smoked per day among smokers (< 10, > or = 10), exclusion of azoospermic men (yes/no), number of semen specimens analyzed (one versus two), and blinding of laboratory personnel to the smoking status of the study participants (yes/no). Regression analyses were performed both unweighted and weighted inversely by study size. A qualitative and quantitative assessment of the relationship between the numbers of cigarettes smoked per day and sperm density was performed. RESULTS: Results of the meta-analysis indicate that smokers' sperm density is on average 13% to 17% (95% confidence interval = 8.0, 21.5) lower than that of nonsmokers. No other factors besides cigarette smoking were found to be independent predictors of sperm density. No clear dose-response relationships between the numbers of cigarettes smoked per day and sperm density emerged. Research conducted by the authors supports the findings of the meta-analysis. CONCLUSION: Cigarette smoking is associated with lowered sperm density. The inconsistency in the literature with regard to this conclusion appears to be the result of small sample sizes in most studies.

Cotinine concentrations in semen, urine, and blood of smokers and nonsmokers.

Cotinine levels in the semen, urine, and blood of 88 male smokers and nonsmokers, aged 18 to 35, were analyzed via radioimmunoassay. Detectable cotinine levels were found in all three body fluids, and cotinine levels in all three fluids were highly correlated. Cotinine levels in semen and blood were of similar magnitude; cotinine levels in urine were an order of magnitude or more higher. In all three fluids, cotinine levels increased with an increase in cigarette smoke exposure.

Diet and human leukemia: an analysis of international data.

BACKGROUND: There has been little direct exploration of the relationships between dietary factors and human leukemia; however, a number of literature reports from animal studies and correlation analyses between countries suggest that diet can influence leukemia risk. METHODS: Statistical analyses of international food supply data and leukemia incidence data were performed to define better the diet-human leukemia relationship. Incidence data for lymphoid, myeloid, and total leukemia (all leukemia subtypes combined) from 24 countries with reliable cancer registry data were regressed with estimates of per capita disappearance of macronutrients and alcohol, as well as gross national product and average height. RESULTS: Simple correlation analyses showed that the dietary variables were associated with leukemia in a gender- and site-specific fashion. Males consistently had higher correlations than females. The strongest correlations were found between total calorie intake and both lymphoid and total leukemia incidence, especially among males. Myeloid leukemia in either gender was most strongly associated with gross national product. To control for potential confounding, multiple regression analyses were performed, with all regression models adjusted for gross national product, height, and dietary covariates. Total calorie supply was the only significant explanatory variable for the international variation in lymphoid and total leukemia in these analyses. The calorie-leukemia association was stronger among males than among females. No significant association was observed between myeloid leukemia and any of the dietary variables studied, after adjusting for height and gross national product. CONCLUSION: The findings from this rigorous analysis of international data strengthen and expand the hypothesis based on previous simple correlation analyses and animal experiments that an underlying biological relationship exists between diet, particularly energy intake, and international variations in the incidence of certain types of human leukemia. Possible mechanisms for the calorie-leukemia associations are discussed.

Contributions to the design and statistical analysis of in vivo SCE experiments.

Two issues that arise in the design and statistical analysis of in vivo SCE and similar experiments are considered. First, with regard to analysis, the merits of various methods of data transformation are explored in depth. The conclusion drawn is that common transformations of the type studied here seemingly offer little advantage in the assessment of whether a test agent induces SCE in a dose-related manner. Second, a proposal is made for a method to determine, subject to budgetary constraints, the desired numbers of animals/dose group and cells scored/animal. The approach advocated also lends itself to discussions weighing the gains and losses from possible reductions in the number of animals below the 'desired' levels.

Methodological issues in epidemiologic studies using biologic markers.

Biologic markers are becoming prominent features of many classical epidemiologic studies. Their existence has also modified the character of some epidemiologic research such that the term "transitional epidemiologic studies" may be warranted. In the latter type of study, collaboration between laboratory and epidemiologic investigators is integral to the research. In this paper, goals, characteristics, and examples of transitional epidemiologic research are presented. Pertinent features are highlighted, including sources of misclassification and confounding, and elements of study design useful in reducing these potential biases. Sample size issues, transformation of variables, and sources of variability acquire enhanced importance. The study examples presented are intended to illustrate the altered substrate for these methodological features in studies using biologic markers.