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Encyclopedia of Biopharmaceutical Statistics

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Ames Test
Wherly P. Hoffman a; Michael L. Garriott b a Department of Global Statistical Sciences, Lilly Research Laboratories, Eli Lilly and Company, Greenfield, Indiana, U.S.A. b Department of Investigative Toxicology, Lilly Research Laboratories, Eli Lilly and Company, Greenfield, Indiana, U.S.A. Online Publication Date: 23 April 2003

To cite this Section Hoffman, Wherly P. and Garriott, Michael L.(2003)'Ames Test',Encyclopedia of Biopharmaceutical Statistics,1:1,28

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Ames Test
Wherly P. Hoffman Michael L. Garriott
Lilly Research Laboratories, Eli Lilly and Company, Greenfield, Indiana, U.S.A.

INTRODUCTION Genetic toxicology tests are among the early studies conducted to assess the safety profile of a compound. A battery of tests, each assessing a different genetic endpoint, is typically performed to thoroughly evaluate a given compound since no single test is capable of detecting all mutagens. The tests are designed to determine whether the compound can interact with DNA and lead to the production of gene mutations or chromosomal breakage. Some tests can also detect compounds that interact with components of the mitotic spindle apparatus. The detection of mutagens is important because they may also be either teratogens or carcinogens. Of a battery of short-term genetic assays, the Samonella typhimurium/ microsome test developed by Ames et al.[1,2] is the most commonly used genotoxicity test. Two- and threefold rules and various parametric and nonparametric methods have been applied to the Ames test data. A general review of these analysis methods is the primary purpose of this article. The design, statistical analyses, and interpretation of results of the Ames test will be discussed. Background includes information pertaining to the relevance of the Ames test to the toxicology safety profile. The Design section describes the basic design of the test. The section Analysis Methods for Ames Test Data provides a review of some analysis methods employed for the evaluation of the mutagenicity of a compound. In the Discussion section, we discuss the methods reviewed and some important issues in the evaluation of mutagenicity based on Ames test results, and in the last section we draw concluding remarks to summarize this article.

without too much detail. This section is not intended to provide enough information to conduct the assay; it is intended to provide readers with a general understanding of how the responses are obtained and thus leads naturally into the following sections on the design and statistical methods employed for analysis of the test data. Why the Ames Test When developing a compound for commercial purposes, one has to establish its safety profile. Animal in vivo and in vitro tests are designed to serve this purpose. In general, the testing includes acute toxicity tests in rodents, genetic toxicity tests, developmental toxicity tests, general animal toxicity tests from 30 days up to 1 year in duration, and lifetime carcinogenicity tests in rodents. Of these general tests that are needed to demonstrate the safety of a compound and to ensure its registration worldwide, the most costly and time-consuming test is the carcinogenesis bioassay in rodents. Assays for genotoxicity were developed initially in an attempt to predict in a shorter time frame the eventual outcome of the bioassay. The basic premise revolved around the somatic theory of carcinogenesis, which suggested that mutational events were causative factors in the development of cancers. Additionally, there was evidence that mutagenic events were associated with embryonic mortality, birth defects, and genetic disease. Therefore, the use of genotoxicity tests seemed to have broad application in the overall safety assessment. Although bacterial mutation tests had been used since the 1950s, they were not efficient in the detection of compounds acting by various mutagenic mechanisms. Efforts by Dr. Bruce Ames and his colleagues at the University of California, Berkeley, to develop a more efficient bacterial screening system culminated in 1971 with the test that now bears his name. Despite its utility, it was realized that bacteria are not mammalian cells and that the organization of the genetic material was different such that other short-term mutagenicity tests would be required to ensure the detection of the majority of rodent carcinogens.[3] Although over 200 mutagenicity tests have been developed, few have been well validated, and current regulatory requirements for product registration include only a battery consisting
Encyclopedia of Biopharmaceutical Statistics DOI: 10.1081/E-EBS 120007383 Copyright D 2003 by Marcel Dekker, Inc. All rights reserved.

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BACKGROUND In this section, a brief summary is presented of why the Ames test was developed and how this assay is conducted. The procedure for conducting the Ames test is described

Adapted from WP Hoffman. In: SC Chow and JP Liu, eds. Design and Analysis of Animal Studies in Pharmaceutical Development. New York: Marcel Dekker, 1998, pp. 357 372. 28

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Fig. 1 Description of the conduct of the Ames test.

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of two to four tests of the seven most thoroughly validated standard tests.[46] Of these required tests, the Ames test is the most commonly used and for this reason alone merits discussion. The Ames Test The Ames bacterial mutation test is an in vitro assay in that it does not use live animals; instead, it uses bacterial cells. Typically, the S. typhimurium bacterial tester strains TA1535, TA1537, TA98, and TA100 and an Escherichia coli tester strain WP2uvrA are used in the Ames test. Although WP2uvrA was added to the bacterial test system after the Ames test was developed, it is handled the same as the four S. typhimurium strains in all aspects of the conduct and analysis of the experiment. Therefore, in this article, Ames test will refer to the assay of the four S. typhimurium strains and the E. coli strain. For each strain of the bacteria, a mixture of the proper amounts of three ingredients is first prepared in a tube. See Fig. 1. The three ingredients are: 1) from 10 to 100 million bacterial cells from a particular strain, 2) a compound, which can be a test article, negative control, or positive control, and 3) minimal agar, which includes trace amounts of histidine, tryptophan, and biotin. The mixture is then poured into a plate containing a layer of solidified minimal agar for the growth of revertant colonies. Some compounds are not directly mutagenic in vivo but are metabolized to a compound that is. Therefore, all in vitro genotoxicity tests, including the Ames test, are conducted so that cells are exposed to the test article both in the presence and in the absence of an exogenous metabolic activation system. This metabolic activation system consists of a postmitochondrial supernatant (S9) of homogenized liver tissue from rats. This cell-free extract contains a variety of drug-metabolizing enzymes. The S9 is supplemented with cofactors, salts, and a buffer system collectively known as S9 mix.

Histidine and tryptophan are amino acids essential for the growth of the tester strains of S. typhimurium and E. coli, respectively. The Ames tester strains contain mutations in the genes that allow normal bacteria (prototrophs) to manufacture their own histidine or tryptophan. The tester strains are dependent upon the inclusion of the amino acids into the minimal agar to support their growth. These mutant bacteria are called auxotrophs. The Ames test is often referred to as a reverse mutation assay because what is measured is the reverse mutation rate from histidine/tryptophan dependency (auxotrophy) to histidine/tryptophan independence (prototrophy). Bacterial cells that have undergone the reverse mutational event are called revertants. Trace amounts of histidine and tryptophan are provided at the start of the experiment to sustain the bacterial cells at the beginning and thus to allow the possible expression of reverse mutation events later. After a 48-hr incubation period, the revertant cells will form visible colonies, which are counted. Because reverse mutation may occur without treatment with a mutagen, a negative control is always included in the assay to account for the spontaneous reverse mutation rate. If a compound is not a mutagen, then one would not expect a large increase in the number of revertant colonies compared to the number of spontaneous revertant colonies in the negative control. Therefore, the number of revertant colonies is the response evaluated to determine the mutagenic effect of a test article. A significant increase in revertant colonies on the compoundtreated plates is an indication of a positive response for bacterial mutation.

DESIGN The Ames test procedure is rather strictly defined for regulatory purposes.[79] The assay is typically conducted both with and without metabolic activation for each of the five bacterial strains. For each bacterial strain, five or more concentration levels of a compound and proper concentration levels for the negative and positive controls are determined by scientists. A positive control is required for the validity of the assay but is not included in the evaluation of the mutagenicity of a compound. A negative control is needed to account for spontaneous reverse mutation in the evaluation of the mutagenicity of the test article. Historical control data are useful for further evaluation of positive effects associated with a compound. Each dose level is tested in triplicate, and each of the triplicate plates is prepared independently. Therefore, if five concentrations are selected for each bacterial strain, then a total of 50 sets of triplicate data from the compound (five concentrations, two types of activation, and five strains of bacteria) and 10 sets of triplicate data

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from each of the negative control and positive control are the results of the assay. The purpose of the Ames test is to evaluate the mutagenic effects of a compound. A suitable selection of the concentration levels of a compound for testing is essential. Logarithmically spaced concentration levels are determined by scientists in an attempt to capture an increasing trend in the response. However, if the concentration of the compound is too high, then one would expect a downturn in the number of revertant colonies as a result of toxicity. For determination of the concentration levels for the Ames test, a compound is assayed over a wide range of concentration levels up to 5000 mg/plate for each bacterial strain. Based on the preliminary results, five to seven concentration levels are then selected using a top concentration of 5000 mg/plate, adjusted for toxicity. The highest concentration may be very close to the toxic level.

ANALYSIS METHODS FOR AMES TEST DATA Proper analysis methods should account for various sources of variability in the response. Therefore, one needs to identify different sources of variability in the assay data before considering the analysis. Based on the variability and some assumptions, we discuss various statistical and nonstatistical methods for the analysis of revertant colonies obtained from the Ames test. Edler[10] and Mahon et al.[11] gave a good overview of statistical methods for the Ames Salmonella test. For a more recent review of statistical methods, see Lin.[12] Here we will begin the discussion with some basic information on data in the next section. Then selected methods will be discussed to bring about different approaches for the analysis of Ames test data. The methods discussed in the subsequent sections are modified two- and threefold rules, nonparametric methods, and parametric methods. Understanding Data What is the distribution of the number of revertants from the Ames test? Before suggesting an answer, let us understand the data first and try to identify various sources of variability in it. For each replicate plate, a separate minimal agar mixture of the selected strain of bacterial cells, the test article, and a solution of histidine, tryptophan, and biotin is made in a tube. Therefore, the numbers of revertants from the triplicate plates are independent. Because auxotrophs need to grow in a histidine/tryptophan-sufficient environment, trace amounts of

these amino acids are supplied at the beginning of the test. For each strain of bacteria, variability in the number of revertants can be a result of the number of bacterial cells that are plated at the beginning of the test, the amount of histidine/tryptophan provided initially, the concentration of the test article, the contents of the minimal agar, the amount of S9 mix, the duration of the incubation time, or other factors. In a laboratory, attempts are made to control all factors so that changes in the revertant counts would reflect only the effect of the test article through the concentration levels. To demonstrate that a compound is not mutagenic, one has to test it at sufficiently high concentrations to rule out doubts that the bacterial cells may not have been challenged enough. Pilot studies are usually conducted first to select proper concentration levels for each strain of bacteria. The final selection often includes concentration levels that are very close to the toxicity threshold of the bacterial cells. Consequently, one may observe a downturn in the number of revertants when some of the concentration levels of a compound are higher than the toxicity threshold of the bacterial cells. Because the number of bacterial cells plated initially is large (about 107) and the probability of a cell undergoing a reverse mutation from auxotrophy to prototrophy is small, the resulting revertant counts of such rare events may have a Poisson distribution. Does the number of revertants X follow a Poisson distribution p(X = x, l) where pX x; l lx e l x! for x 0; 1; 2; . . .
1

and l is the mean rate of reverse mutation? One can examine this either graphically or by performing a statistical test. To get a good assessment of the distribution assumption, both approaches require more data than are available from one assay in general. When a sufficient amount of data is available, one can check this Poisson assumption by plotting the sample variance against the sample mean on a logarithmic scale. A linear relationship with slope near one is an indication of Poisson distribution. Or one can perform Fishers dispersion test[13] for a sample of n counts, x1, x2, . . ., xn, which compares the test statistic Pn
i1

xi  x2  x

to a chi-square distribution with n 1 degrees of freedom, where  x is the average of the n revertant counts. Stead et al.[14] reported no overdispersion in their experience, whereas Vollmar[15] and Margolin et al.[16] observed overdispersion. Because each laboratory may have

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its unique features in conducting the assay and controlling the assay environment, an assessment of overdispersion should be performed based on its own historical data, and reevaluation should be performed periodically. If the rate of reverse mutation l from replicate to replicate is not a constant, then the excess of variability in replicates over the mean would suggest that the revertant counts are a random sample from a mixture of Poisson distributions. For ease of further references in later sections, we will follow the parameterization of Margolin et al.[16] If l is a random variable with a gamma distribution, then the revertant counts have a negative binomial distribution   x 1 m pX xjm; c x c 1 x m c 1  1 c1 c m c 1 for x 0; 1; 2; . . .
3

where c  0 and m > 0. The mean of this distribution is m, and the variance is m(1 + cm). The extra variability in the Poisson counts is reflected by the dispersion parameter c. Large values of c indicate inadequacy of modeling without accounting for the extra variability in the sampling. Modified Two- and Threefold Rules The two- and threefold rules declare a given test article to have induced a positive response if a concentrationrelated increase in the number of revertant colonies is at least twice the control count for strains TA98, TA100, and WP2uvrA or three times the control count for strains TA1535 and TA1537.[17] The comparison is based on the average number of revertants from the replicates for the control and each concentration level. The treated averages are compared to the control average. In the event that there is a downturn in the number of revertants owing to toxicity, the evaluation criteria are applied only to the results at the nontoxic level. Nonparametric Methods Vollmar examined the results from the European Collaborative Ames-Test Study 1977 1978, and the plots of log-range vs. log-mean indicated that the number of revertants did not follow a Poisson distribution. Overdispersion was evident between laboratories as well as within each laboratory. It was recommended that nonparametric methods be applied to Ames test results. Two rank-based tests were considered: the Jonckheere test[18] for monotonic concentration effects and the
[15]

Kruskal Wallis test[19] for other effects, including a downturn at the toxic levels. Given 713 Ames test results, the Jonckheere test was superior to the Kruskal Wallis test and was recommended as the routine statistical method for qualitative evaluations. Vollmar pointed out that, in general, three to five replicate samples were necessary for achieving a statistical significance level of 0.05. Wahrendorf et al.[20] proposed a nonparametric approach to the analysis of Ames test data. Assume that there are K concentration levels with nk replicates at the kth level and the total number of replicates is N. In their approach, the concentration levels were arranged in K increasing levels, with K = 1 for the control. For a given j, 1  j  K, the revertant counts from all K concentration levels are assigned to one of the two categories: 1 is the below category for levels 1 j; 2 is the above category for levels j + 1 to K. The revertant counts are ranked from low to high. If there are no concentration-related effects, then the sum of the ranks, Lj, from the below category should be the product of the midrank, (N + 1)/2, and the number of observations in the below category. Define L as the sum of the below categories corresponding to j for 1  j  K 1. If a compound does induce a positive trend in the number of revertants, then the ranks in the above category would be relatively higher than those in the below category. Therefore, positive trends are associated with smaller values of L, and a test statistic for positive trends is L. Wahrendorf et al. provided critical values for L based on Monte Carlo simulations for standard designs in their paper. Incorporating the expectation, E0(L), and variance, Var0(L), of L under the null hypothesis of no concentration-related effects, the resulting standardized test T for the alternative hypothesis of an increasing trend is a one-sided test: E0 L L T p Var0 L
4

where E0(L) and Var0(L) are defined[20] by the total sample N and sj, the number of observations in the below category for a given j as follows: E0 L
K 1 N 1 X sj 2 j1

Var0 L

K 1 N1 X sj N sj 2 j1 K 2 X K 1 X j 1 k j 1

sj N sk

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The T statistic is approximated by a normal distribution. In addition to the test proposed in the foregoing, Wahrendorf et al. also provided a calculation for the probability ^ qj that a revertant count from the below category is smaller than that from the above category for a given j. Under the null hypothesis of no concentration-related effects, ^ qj is expected to have a value of 0.5. This probability gives insight to the change of trends in the revertant counts and is helpful for further understanding of the type and strength of the trends.

Mutagenicity is established by a statistically significant positive slope of the regression line. Chu et al. required a test to have at least five concentration levels, including a negative control, and each with a minimum of two replicates. When concentration levels are approximately equally spaced, instead of adding 1 to all concentration levels to allow for the log transformation on the 0 concentration level, an alternative was recommended by Margolin et al.[24] The concentration level of the negative control is selected so that all concentration levels are equally spaced on the logarithmic scale. Nonlinear regression on revertant counts Although rare events are often modeled by Poisson models, and it seems reasonable to consider the small number of revertants in about 10 million bacterial cells a Poisson random variable, there are statistical concerns in the assumption of simple Poisson distribution. Evidence of extra-Poisson variation was observed by some, but not all. Because replicates are required at each concentration level, accumulating historical control data for examination of the adequacy of the Poisson assumption is essential. Under the Poisson assumption, the revertant counts can be modeled by a generalized linear model[25] with Poisson distribution and logarithmic link. Positive trends in the treated groups can be tested in a sequential fashion by a trend test[26] if identifying the concentration level associated with no observable effect is of interest. For laboratories that exhibit extra-Poisson variation in the historical control data, the generalized linear model can be adjusted using a quasi-likelihood method for the extraPoisson variation. The extra-Poisson variation is accounted for by including an extra factor in the variance of the model. The extra factor can be estimated by the square root of Pearsons chi-square divided by the degrees of freedom. This analysis can be carried out using PROC GENMOD in SAS.[27] If the mean rate for reverse mutation, l, in Eq. 1 is a random variable with gamma distribution, then the posterior distribution of the revertant counts is a negative binomial distribution as in Eq. 3. Thus, the extra-Poisson variation can be incorporated in the modeling and evaluation of the mutagenicity of a compound using a full likelihood approach. Biologically based models Nonmonotonicity observed as a downturn in the revertant counts is not uncommon in practice. Bacterial cells subject to toxicity are inhibited from full expression of mutagenicity. Once a definition of a toxic concentration level is established, then one can eliminate the toxic concentration levels and model the mutagenic effects of a

Parametric Modeling Parametric methods include the following: 1) regression on transformed data, assumed to be normally distributed;[21] 2) nonlinear regression on data assumed to be Poisson distributed, either with or without extra variation, using a full likelihood or quasi-likelihood approach; and 3) biologically based models incorporating extra-Poisson variation.[15,22,23] Linear regression on logarithmically transformed revertant counts Chu et al.[21] at the In Vitro Program of the National Cancer Institute (NCI)/National Toxicology Program (NTP) evaluated the statistical methods for Ames test data based on results from 2362 tests performed in four laboratories on 17 test articles. In defining the screening criteria for adequate data, they defined toxic dose level as any dose level which was greater than the dose eliciting the highest average response and which had every response less than the lowest single response in the highest average response dose level. Evidence of toxicity is a decrease in the number of revertants after reaching the toxic level. Therefore, when a downturn is present in the revertant counts and the peak mean revertant count occurs at concentration level ci, then the toxic levels are those concentration levels, cj, for j > i, that have all revertant counts smaller than the lowest revertant count at concentration level ci. Once the toxic concentration levels are identified, evaluation of the mutagenic effects will be based on data obtained in the nontoxic concentration range. One approach to the statistical evaluation of the mutagenic effects is regressing the logarithmically transformed revertant count on the logarithmically transformed concentration level.[21] Because the control concentration level is 0 and the log transformation of 0 does not exist, the recommendation was to increase all concentration levels by 1. The validity of this regression approach lies in the appropriateness of the normality assumption resulting from the log transformation on the revertant counts.

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compound using the remaining data. An alternative to this two-step modeling approach is to model both mutagenicity and toxicity of a compound simultaneously. Margolin et al.[16] developed biologically based models for modeling the mutagenicity and toxicity while accounting for the extra variation in the Poisson distribution in the revertant counts from the Ames test. The choice of their models depends on the number of generations the bacterial cells have for their histidine/tryptophan supply, the number of generations the compound is mutagenic, and the number of generations the compound is toxic. Two basic assumptions of the biologically based models are that mutagenic and toxic effects are independent, and durations of bacterial cells, being mutagenic and toxic, are not affected by the concentration of the compound. The mean, m, of the negative binomial distribution, is related to the number of bacterial cells exposed to the test article, N0, and the probability of resulting a revertant colony when exposed to the compound at concentration level d, P(d), as m N 0 P d
7

models. Their proposal to contain the Type I error rates was performing a pretest on g = 0 to identify the presence of toxicity. The pretest of H0:g = 0 against Ha:g > 0 is a likelihood ratio test: lg 2La; b; g; c La; b; 0; c
10

where L(a, b, g; c) is the maximum of the log-likelihood of the data. If the toxicity is not supported by the data, then the mutagenicity parameter b will be tested by the likelihood ratio test in Eq. 10 constrained on g = 0: lg 2La; b; 0; c La; 0; 0; c
11

Otherwise, the mutagenicity parameter b will be tested by lg 2La; b; g; c La; 0; g; c

12

We will discuss the two simplest cases, when the bacterial cells have enough histidine/tryptophan supply for just one generation and the compound is mutagenic for one generation. The first case is when the compound is toxic for one generation. The second case is when it is toxic for infinitely many generations. If a compound is toxic only for one generation, then P(d) is P d 1 e
abd

Histidine/tryptophan is necessary for the growth of auxotrophic cells. In the biologically based models described in the foregoing, amino acid diffusion was not considered and each cell will have a local supply of the essential amino acid. However, if the amino acids do diffuse through the plate agar, then the revertant counts should be a function of the amount of histidine/tryptophan as well. Krewski et al.[23] proposed to generalize the biologically based models by Margolin et al.[16] to allow for diffusion of the amino acids in an agar plate.

DISCUSSION The original and modified two- or threefold rules are simple to use and have been compared to other methods in Refs. [21,28]. Chu et al.[21] reported favorable performance of the modified twofold rule based on results from 2362 tests performed in four laboratories on 17 test articles. Define the false positive and false negative as follows: False positive = negative tests determined by consensus of microbiologists that is concluded positive using the decision rule. False negative = positive tests determined by consensus of microbiologists that is concluded negative using the decision rule. Among the methods that gave higher false-positive conclusions than false-negative conclusions is the modified twofold rule, which gave 4.1% false-positive and 1.8% false-negative results and the positive linear trend test described in the section Linear Regression on Logarithmically Transformed Revertant Counts, which gave 20.0% false-positives and 0.4% false-negative results. If the modified twofold rule is supplemented

gd

where a and b are positive and g is nonnegative. However, if it has long-lasting toxicity, then P(d) is Pd 1 eabd 2 egd
9

where [y] + = max(y, 0). The spontaneous reverse mutation rate, mutagenicity, and toxicity are modeled through parameters a, b, and g. The evaluation of the Ames data is based on Models 8 and 9 in combination with Eq. 7 and the negative binomial distribution in Eq. 3 through parameters a, b, g, and c. If there is no toxicity, then g = 0 and Models 8 and 9 are identical. Mutagenicity of a compound is evaluated by testing the null hypothesis of H0:b = 0 against the alternative of Ha:b > 0. Margolin et al.[16] approximated the distribution of the ratio of the maximum likelihood estimate of ^, and its standard error, SEb ^, by a standard normal b, b ^ was obtained based on the Fisher distribution. The SEb information matrix evaluated at the maximum likelihood estimates of a, b, g, and c. However, further research by Margolin et al.[22] identified problems with the two models. Inflated Type I error rates were reported for both

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with a modified threefold rule, one would expect the proportion of false positives to decrease further. For situations where there is no convincing evidence for the assumptions of Poisson distribution, overdispersion in the Poisson distribution, and the normality distribution of the transformed data, the nonparametric methods discussed in the section Nonparametric Methodsnamely, the Jonckheere test and the method proposed by Wahrendorf et al.seem plausible. The latter also provide a descriptive quantity for assessment of the types of increasing trends and the possibility of downturn due to toxicity. An example of revertant counts of strain TA100 in triplicate of (66, 82, 64), (66, 73, 87), (89, 84, 86),(76, 83, 87), (87, 103, 91), and (89, 98, 82) corresponding to the concentration levels of 0, 10, 30, 50, 100, and 300 mg per plate, respectively, was taken from Stead et al.[14] The nonparametric method by Wahrendorf concluded a highly significant mutagenic effect with p value = 0.0028 based on normal approximation and p = 0.0016 based on critical values established by Monte Carlo simulations. If the modified twofold rule were applied to the data, no mutagenic effects would be concluded. Parametric methods with the assumptions of Poisson distribution with or without extra variation and negative binomial distribution of the revertant counts utilize statistical tools in modeling the counts for mutagenicity, toxicity, and dispersion, whereas the modified twofold rule calls only for simple averages of the revertant counts. Although the modified twofold rule demonstrated good agreement with the consensus of the microbiologists in the NCI/NTP study,[21] it was considered too conservative by statisticians in general. It is clear that if the statistical methods are adopted, then there will be a great deal of statistically significant findings than using the modified twofold and threefold rules. To address the differences in the mutagenicity of compounds using statistical evaluation and biological evaluation, several issues merit further discussion here: 1. The use of historical control data. 2. The toxicity threshold of a bacterial cell to a test article. 3. Extra-Poisson variation in the revertant counts. 4. The amount of histidine/tryptophan provided. 5. The relationship between statistical significance and biological significance. Clearly, these issues are all interrelated and, therefore, the discussion may not be in the order listed here. Because, in practice, most laboratories conduct Ames tests in triplicate, it is difficult to identify if extra-Poisson variation is present in the revertant counts within each experiment. A historical database for negative control

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data should be a must for examining the assumption of excessive variation in the revertant counts. In addition, a historical database can provide a base range for the evaluation of compounds that may yield statistical significance that is not biologically important. Because there may be variations among various laboratories, it is best for each laboratory to establish its own database for the historical negative control. The toxicity threshold of a bacterial cell to a test article may be determined using the rule described by Chu et al. or by 50 90% lethality. In other words, the number of revertants in the treated plates has to be reasonably low to indicate a toxic effect. Statistical methods can then be applied without modeling the toxic effects. However, if the rules are not accepted by the regulatory agencies, statistical modeling may be the best way to determine the presence of the toxic effects. Biologically based models for different combinations of mutagenicity and toxicity lead to different models. How one determines the number of generations that the trace amino acid supply will last, the number of generations the compound will be mutagenic, and the number of generations the compound will be toxic is unclear.

CONCLUSION At this point, no consensus has been reached on statistical methods for analyzing and interpreting the Ames data. This is reflected in the ICH guideline for genotoxicity,[5,29,30] which does not mention any statistical methods for the evaluation of the test results. Considering the small set of numbers that the Ames test provides for each bacterial strain, statistical methods designed to evaluate the mutagenic effects should reflect the biological significance to be of any help to microbiologists. Statistical methods that find biologically small increases statistically significant with small p values are not desirable and may slow the development of a compound unnecessarily. Any statistical methods recommended for the routine data analysis for the Ames test should be validated by a collection of compounds with known mutagenicity. A historical database is a must in the evaluation of the mutagenicity of compounds and should be established in each laboratory. When a historical database is not available, nonparametric approaches may be a better choice than parametric methods. Statistical methods should support microbiologists findings when the effects, mutagenic or nonmutagenic, are clear, and should guide scientists when the effects are not clear. Because the modified twofold and threefold rules seem to agree well with the consensus of microbiologists,[21] further work in this area is needed to compare the

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proposed statistical methods with the modified twofold and threefold rules based on known compounds.

REFERENCES
1. Ames, B.N.; Lee, F.D.; Durston, W.E. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 782 786. Ames, B.N.; McCann, J.; Yamasaki, E. Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsome mutagenicity test. Mutat. Res. 1975, 31, 347 364. Casciano, D.A. Introduction: Historical Perspectives of Genetic Toxicology. In Genetic Toxicology; Li, A.P., Heflich, R.H., Eds.; CRC Press: Boca Raton, 1991; 1 12. Auletta, A.E.; Dearfield, K.L.; Cimino, M.C. Mutagenicity test schemes and guidelines: U.S. EPA Office of Pollution Prevention and Toxics and Office of Pesticide Programs. Environ. Mol. Mutagen. 1993, 21, 38 45. Muller, L.; Kikuchi, Y.; Probst, G.; Schechtman, L.; Shimada, H.; Sofuni, T.; Tweats, D. ICH-harmonised guidances on genotoxicity testing of pharmaceuticals: Evolution, reasoning and impact. Mutat. Res. 1999, 436, 195 225. Cimino, M.C. New OECD Genetic Toxicology Guidelines and Interpretation of Results. In Genetic Toxicology and Cancer Risk Assessment; Choy, W.N., Ed.; Marcel Dekker: New York, NY, 2001; 223 248. Claxton, L.D.; Allen, J.; Auletta, A.; Mortelmans, K.; Nestmann, E.; Zeiger, E. Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity. Mutat. Res. 1987, 189, 83 91. Gatehouse, D.; Haworth, S.; Cebula, T.; Gocke, E.; Kier, L.; Matsushima, T.; Melcion, C.; Nohmi, T.; Ohta, T.; Venitt, S.; Zeiger, E. Recommendations for the performance of bacterial mutation assays. Mutat. Res. 1994, 312, 217 233. Organisation for Economic Cooperation and Development (OECD). Bacterial Reverse Mutation Test, No. 471. In OECD Guidelines for Testing of Chemicals; OECD: Paris, 1997. Edler, L. Statistical methods for short-term tests in genetic toxicology: The first fifteen years. Mutat. Res. 1992, 277, 11 33. Mahon, G.A.T.; Middleton, B.; Robinson, W.D.; Green, M.H.L.; Mitchell, I.; Tweats, D.J. Analysis of Data From Microbial Count Assays. In Statistical Evaluation of Mutagenicity Test Data; Kirkland, D.J., Ed.; Cambridge University Press: Cambridge, 1989; 26 65. Lin, K.K. Statistical review and evaluation of in vitro mutagenicity study data. Drug Inf. J. 1997, 31, 335 344. Fisher, R.A. The significance of deviations from expectation in a poisson series. Biometrics 1950, 6, 17 24.

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14. Stead, A.G.; Hasselbald, V.; Greason, J.P.; Claxton, L. Modelling the Ames test. Mutat. Res. 1981, 85, 13 27. 15. Vollmar, J. Statistical Problems in the Ames Test. In Progress in Mutation Research; Kappas, A., Ed.; Elsevier/ North-Holland Biomedical Press: Amsterdam, 1981; Vol. 2, 179 186. 16. Margolin, B.H.; Kaplan, N.; Zeiger, E. Statistical analysis of the Ames Salmonella/microsome test. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 3779 3783. 17. Kier, L.; Brusick, D.J.; Auletta, A.E.; Von Halle, E.S.; Brown, M.M.; Simmon, V.F.; Dunkel, V.; McCann, J.; Mortelmans, K.; Prival, M.; Rao, T.K.; Ray, V. The Salmonella typhimurium/mammalian microsomal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat. Res. 1986, 168, 69 240. 18. Jonckheere, A.R. A distribution-free k-sample test against ordered alternative. Biometrika 1954, 41, 133 145. 19. Kruskal, W.H.; Wallis, W.A. Use of ranks in one-criterion variance analysis. J. Am. Stat. Assoc. 1952, 47, 583 621. 20. Wahrendorf, J.; Mahon, G.A.T.; Schumacher, M. A nonparametric approach to the statistical analysis of mutagenicity data. Mutat. Res. 1985, 147, 5 13. 21. Chu, K.C.; Patel, K.M.; Lin, A.H.; Tarone, R.E.; Linhart, M.S.; Dunkel, V.C. Evaluating statistical analyses and reproducibility of microbial mutagenicity assays. Mutat. Res. 1981, 85, 119 132. 22. Margolin, B.H.; Kim, B.S.; Risko, K.J. The Ames Salmonella/microsome mutagenicity assay: Issues of inference and validation. J. Am. Stat. Assoc. 1989, 84, 651 661. 23. Krewski, D.; Leroux, B.G.; Bleuer, S.R.; Broekhoven, L.H. Modeling the Ames Salmonella/microsome assay. Biometrics 1993, 49, 499 510. 24. Margolin, B.H.; Resnick, M.A.; Rimpo, J.Y.; Archer, P.; Galloway, S.M.; Bloom, A.D.; Zeiger, E. Statistical analysis for in vitro cytogenetic assays using Chinese hamster ovary cells. Environ. Mutagen. 1986, 8, 183 204. 25. McCullagh, P.; Nelder, J.A. Generalized Linear Models, 2nd Ed.; Chapman and Hall: London, 1989; 193 214. 26. Tukey, J.W.; Ciminera, J.L.; Heyse, J.F. Testing the statistical certainty of a response to increasing doses of a drug. Biometrics 1985, 41, 295 301. 27. SAS Institute Inc. The GENMOD procedure; SAS/STAT Users Guide, version 8, SAS Institute: Cary, NC, 1999; Vol. 2, 1363 1464. 28. Margolin, B.H. Statistical studies in genetic toxicology: A perspective from the U.S. national toxicology program. Environ. Health Perspect. 1985, 63, 187 194. 29. International Conference on Harmonisation; Guidance on specific aspects of regulatory genotoxicity tests for pharmaceuticals. Fed. Regist. 1996, 61, 18198 18202. 30. International Conference on Harmonisation; Guidance on genotoxicity: A standard battery for genotoxicity testing of pharmaceuticals. Fed. Regist. 1997, 62, 62472 62475.