− mouse lymphoma specific gene and chromosomal mutation assay

Mutation Research 394 Ž1997. 177–303

The L5178Yrtkqry mouse lymphoma specific gene and chromosomal mutation assay A phase III report of the U.S. Environmental Protection Agency Gene-Tox Program 1 A.D. Mitchell

a,)

, A.E. Auletta b, D. Clive c , P.E. Kirby d , M.M. Moore e, B.C. Myhr f

a

e

Genesys Research, Incorporated, Research Triangle Park, NC 27709, USA b U.S. EnÕironmental Protection Agency, Washington, DC 20460, USA c Raleigh, NC 27606, USA d Sitek Research, Inc., RockÕille, MD 20850, USA U.S. EnÕironmental Protection Agency, Research Triangle Park, NC 27711, USA f CoÕance Laboratories, Inc., Vienna, VA 22182, USA Received 3 April 1997; accepted 5 June 1997

Abstract The L5178Yrtkqry-3.7.2C mouse lymphoma assay ŽMLA. which detects mutations affecting the heterozygous thymidine kinase Ž tk . locus is capable of responding to chemicals acting as clastogens as well as point mutagens. Improvements in the assay to enhance detection of this spectrum of genetic events are summarized, and criteria for evaluating the data are defined. Using these criteria, the Phase III Work Group reviewed and evaluated literature containing MLA results published from 1976 through 1993. The data base included 602 chemicals of which 343 were evaluated as positive, 44 negative, 18 equivocal, 54 apparently inappropriate for evaluation in this test system with the published protocols, and 142 that were inadequately tested, and thus a definitive call could not be made. The overall performance of the assay is summarized by chemical class, and the outcome of testing 260 chemicals in the MLA is compared with Gene-Tox and National Toxicology Program evaluations of rodent carcinogenesis bioassay results for the same chemicals. Based on the Work Group’s evaluation of published MLA data for chemicals that were considered adequately tested, it is concluded that for most chemicals the L5178Yrtkqry mouse lymphoma assay is eminently well suited for genotoxicity testing and for predicting the potential for carcinogenicity. q 1997 Elsevier Science B.V.

) Corresponding author. Mailing address: Genesys Research, Incorporated, PO Box 14165, Research Triangle Park, NC 27709, USA. Tel.: q1 Ž919. 544-9500; fax: q1 Ž919. 544-9501. 1 This manuscript has been reviewed by the U.S. Environmental Protection Agency Office of Toxic Substances, Pollution Prevention and Toxics, and the U.S. Environmental Protection Agency National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 1 5 - 0

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1. Introduction Historical reviews have been published ŽClive et al., 1983a; Clive, 1987. describing the origin of the target cells and the definition of the L5178Yrtkqry ™ tkyry and tk ory mouse lymphoma assay ŽMLA. which detects mutations Ž tkyry . and allele loss Ž tk ory . affecting the heterozygous thymidine kinase Ž tk . locus in L5178Yrtkqry-3.7.2C cells. The Phase I Gene-Tox report ŽClive et al., 1983a. evaluated MLA data published through 1979 for 48 chemicals that were tested for mutagenesis at the tk locus. Since publication of the Phase I Gene-Tox report, the mouse lymphoma assay has been widely used, and the testing procedures have been refined in a number of laboratories following the demonstration that, in the presence of trifluorothymidine ŽTFT., which is toxic to tkqry cells, the assay is capable of detecting two classes of mutants, distinguished on the basis of colony size ŽClive et al., 1980.. Whereas large colony mutants grow at rates similar to the parental cells and include intragenic events as well as total deletions of the tkq allele, small colony mutants Žapproximately - 0.6 mm in diameter. are associated with slower than normal cellular growth and a variety of genetic damage ranging from intragenic events and entire gene loss to karyotypically visible deletions and rearrangements of the tkqbearing chromosome 11b ŽHozier et al., 1982, 1985; Blazak et al., 1986; Applegate and Hozier, 1987; Applegate et al., 1990.. Hence, in contrast to microbial mutagenesis and eukaryotic cytogenetics assays, the MLA is capable of detecting a broad spectrum of genetic damage including both gene and chromosomal mutations. The capability of the MLA to detect viable chromosomal mutations enhances the utility of the assay because chromosomal mutations occupy a central role in carcinogenesis, and chromosomal alterations are associated with infertility, spontaneous abortion, and congenital malformation in humans. In addition, extensive research to map genetic loci in humans and mice has revealed that the greatest homology exists between human chromosome 17 and mouse chromosome 11, within a highly conserved ; 35 centiMorgan region on the distal 15% of the mouse chromosome. This region contains all of the loci mapped to human chromosome 17.

Because of its ability to detect chromosomal as well as gene mutations, the mutant frequencies seen with the MLA can be quite high. However, the magnitude of mutagenic responses that have been measured in this assay has varied considerably in the published literature, indicating that, in some laboratories, small colony mutants were poorly detected. This presents obvious problems when attempts are made to utilize the current literature data base and has led some observers to the erroneous conclusion that the MLA lacks reliability. Thus, performance of the assay to maximize mutant recovery and detection is emphasized in the following section. The performance section is followed by the Work Group’s definition of response criteria that were used in evaluating published data. Detailed and summary evaluations of the performance of each chemical, and tabulations of the evaluations by chemical class and by the outcome of the rodent carcinogenesis bioassay ŽRCB. are then presented. These evaluations are followed by the Work Group’s recommendations and conclusions concerning the utility of the L5178Yrtkqry mouse lymphoma assay in testing for genotoxicity and potential carcinogenicity.

2. Assay performance Detailed procedures for performance of the MLA and an approach for interpreting the results were published by Clive and associates ŽTurner et al., 1984., shortly after publication of the outcome of the first Gene-Tox MLA review ŽClive et al., 1983a.. Since that time, a number of laboratories have further modified the testing procedures to enhance recovery of the complete spectrum of genetic information that is available from this assay. The following description of assay performance emphasizes aspects of the protocol that are conducive to optimal performance in the testing of most chemicals. Based on unusual chemical or biological properties of a test material, and the quantity of the chemical available for testing, appropriately modified protocols may be necessary and warranted. But with any such modifications, as well as with the basic protocol, it is necessary to conduct the testing with an awareness of the properties of the test material and the capabilities of the assay system and to be alert to possible

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observations during testing that reflect situations that might influence the outcome and thus require careful interpretation of the results. 2.1. Cells and culture medium Sufficient numbers of L5178Yrtkqry-3.7.2C cells in suspension culture are maintained in log phase, with a doubling time of 9–10 h, and they are periodically replenished from frozen stocks of cells whose origin is as close as possible to the original source ŽD. Clive.. Fischer’s Medium for Leukemic Cells of Mice supplemented with 10% horse serum ŽClive and Spector, 1975; Turner et al., 1984. is conventionally used for cell growth. Agar Že.g., BBL agar, ; 0.22% final concentration wMeyer et al., 1986x. is added to the growth medium for cloning. RPMI 1640 medium may also be used, but proper attention must be given to horse serum heat inactivation in order to provide sufficient selection stringency with this alternate medium ŽMoore and Howard, 1982.. Thus, laboratories that use RPMI 1640 use three-fold higher concentrations of TFT Ž3 m grml. to compensate for this potential problem whereas a TFT concentration of 1 m grml is sufficient for mutant selection when Fischer’s is the basic medium. Regardless of the growth medium used, each laboratory should evaluate the stringency of their selection procedures. The osmolality and pH of the medium should be confirmed by the manufacturer or the testing laboratory to be in the physiological range Ž300 " 20 mOsm wFreshney, 1983x and pH 7.0 " 0.4 wBrusick, 1986x., and each lot of horse serum should be tested for its ability to support optimal cell growth in suspension culture Žlow and high cell densities., high plating efficiency, and small colony mutant recovery. With adjustment of the salt concentration, HEPES buffer may be used in addition to bicarbonaterCO2 to counteract some high and low pH effects of chemical treatment and to permit optimal growth with low or high cell densities ŽFreshney, 1983.. 2.2. Cleansing Stock cultures of tkqry-3.7.2C cells are cleansed of spontaneously accumulating mutants within the week preceding each assay ŽTurner et al., 1984., but the cells should not be exposed to test chemicals

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until they have completely recovered from cleansing. The cells grow at longer doubling times during cleansing than they do in non-selective growth medium. Alternatively, cleansed cells may be grown and cryopreserved and new cultures started from the cryopreserved stocks for use in assays. 2.3. Chemical exposure Comparable treatment times are routinely used for testing without and with S9 metabolic activation. Four-hour exposure times are normally used; this timing is recommended as a best compromise between optimal mutant induction, S9 stability, and the avoidance of toxicity caused by longer exposure to the S9 activation systems. Because many chemicals induce chromosomal damage, which is usually associated with cytotoxicity as well as with slower growth rates, and because the induction of gene mutations as well as clastogenesis is associated with cytotoxicity, it is necessary that testing be performed to concentrations that produce significant cytotoxicity before a chemical can be considered nongenotoxic. However, sound biology dictates that treatment conditions should not be so severe as to disturb cellular integrity and function in a manner that might make the results irrelevant to realistic exposures and measurements of cellular responses leading to mutagenesis. Hence, it is generally agreed that ‘positive’ responses seen only in cultures with less than approximately 10% RTG Žrelative total growths cloning efficiency= relative suspension growth wRSGx. are not considered relevant. Exposure concentrations for each assay are usually selected based on the results of preliminary range-finding experiments, and these concentrations should span a range of anticipated survival ŽRTG. from non- or weakly toxic to 10–20% RTG, with the concentrations selected to emphasize the lower RTG values. A chemical showing no mutagenic response must be tested to concentrations giving between 10 and 20% RTG before being declared negative. The use of only a ‘halving’ series of concentrations tends to emphasize non-toxic, rather than cytotoxic, concentrations and, thus, will seldom adequately describe the toxicity curve. The specific cultures cloned in each mutagenesis assay are usually selected from cultures exposed to wider concentration ranges than

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may be necessary, and the cultures are selected for cloning based on a range of toxicities to the growth of the cells in suspension ŽRSG. during the expression period. Preliminary range-finding experiments, if performed, are usually conducted with procedures identical to those of the mutagenesis assay to follow, except that the cultures may not be cloned. However, since cloning efficiencies of treated samples may be increasingly depressed at higher concentrations —particularly with potent mutagens— RSG values are not absolutely predictive of RTG. Thus, some laboratories routinely clone selected cultures from their range-finding experiments. Testing under nonphysiological conditions must be avoided in the MLA and other in vitro mammalian cell assays, as acidic pH shifts, to F 6.5, in the presence of S9 and high salt concentrations have been shown to induce physiologically irrelevant positive results ŽBrusick, 1986.. Conversely, if the pH of the medium used to culture the cells is G 7.5, cell growth in suspension culture may be depressed, and small mutant colonies will grow poorly and not be detected by automated counters. In the absence of information that testing can be accomplished with physiologically relevant exposure conditions, osmolality and pH should be carefully determined under treatment conditions, and documented. If necessary and possible, the exposure medium should be adjusted to return these conditions to the physiological range. Chemicals that have been tested to the limits of physiological exposure conditions, or to the limits of solubility, but to less than a concentration of 5000 m grml or 5 m lrml, and which have not exerted sufficient cytotoxicity, are not interpreted as negative in the absence of a mutagenic response. These chemicals are considered to be ‘not testable’ in the MLA, at least by the ‘standard’ protocol. Under some circumstances it may be appropriate to consider using the lower concentrations and longer treatment times. 2.4. Metabolic actiÕation Metabolic activation systems are used for in vitro testing to mimic the metabolism of whole animals, and a chemical must be adequately tested in the presence and absence of metabolic activation before

declaring it to be nonmutagenic. Methods using Aroclor-induced rat liver S9 in the mouse lymphoma assay, as well as noninduced S9, S9 from other species, microsomes, and rat liver hepatocytes Žboth induced and noninduced. have been described in the literature Že.g., Turner et al., 1984; Mitchell et al., 1988a; Oglesby et al., 1989; Majeska and Matheson, 1990.. Aroclor-induced rat liver S9 is now routinely used. However, other activation systems may be used if information about the in vivo metabolism of a test chemical indicates that an alternate system would be more appropriate. Quality control characterization of the metabolic activation system includes information on enzyme activity, checks for sterility and for activity in the mutagenesis system Žusing negative and positive controls., and evidence that the S9 mixture is minimally toxic to the cells. 2.5. NegatiÕe controls (solÕents) It is often necessary to evaluate solubility of the test chemical not only in various solvents but also after representative dilutions have been added to culture medium, and it may be necessary to evaluate some solvents for effects on the test chemical and the assay system. The objective is to select a solvent that will permit testing of a chemical over the desired concentration range with solvent concentrations Že.g., F 1%. which do not noticeably alter cell growth or mutant frequencies. The solvents most often used include culture medium, phosphate buffered saline, distilled water, DMSO, ethanol, and acetone. If acetone is the solvent, laboratory supplies Žsuch as pipettes composed of plastic. that react with acetone must be avoided. The limit of solubility should also be carefully reassessed during the treatment phase of each assay, with observations documented at the initiation of chemical exposure, after the exposure period, and after the cells have been rinsed to remove the test chemical. If persistent precipitates of the test chemical preclude testing to cytotoxic levels, or to a maximum target concentration in the absence of toxicity, higher solvent concentrations or alternate solvents should be considered. Numerous other solvents are available in addition to the ones most commonly used. For example, Mavournin et al. Ž1990. list 30 additional solvents and mixtures of

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solvents that have been used as vehicles for in vivo micronucleus tests. A number of these solvents and solvent mixtures have been found appropriate for the MLA in testing for regulatory submissions, e.g., for testing lipid-soluble materials. However, none of the publications reviewed by the MLA Work Group cited the use of any of these alternate solvents. 2.6. PositiÕe controls The purpose of a positive control in any assay is to convincingly demonstrate that the system was working properly at the time a test chemical was evaluated. In addition, the concentration used for the positive control should yield a level of survival that is consistent with the criterion used for the test chemical, i.e. it should not be below approximately 10–20% RTG. For this reason, some laboratories routinely use two concentrations of those positive controls that have steep toxicity curves. The choice of positive controls should ensure that the recovery and detection of small colony mutants, the most variable aspect of the mouse lymphoma assay, is sensitively assessed, as indicated by mutant colony sizing. Positive control chemicals that induce high numbers of small colony mutants include methyl methanesulfonate for exposures in the absence of S9 and cyclophosphamide for testing with metabolic activation; both are expected to yield induced mutant frequencies in the range of 750–1500 = 10y6 at RTG G 10%. 2.7. Expression period Newly induced mutants still retain a functional thymidine kinase enzyme, which must be diluted from the cells before TFT-selection can be applied. The expression of newly induced tk mutants is rapid Žcompared to other markers such as hprt ., and it is usually two days or less. On the other hand, because cells giving rise to small colony tkyry and tk ory mutants have slower growth rates, their frequency declines with time ŽMoore and Clive, 1982.. Thus the time allowed for the expression period must be long enough to allow for mutant expression but no longer than necessary because of the decline in the small colony mutant frequency Ždue to the slow growth of cells that yield small colony mutants.. A

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two-day expression period has been found to be an optimal compromise for the detection of maximum mutant frequencies for most concentrations of most mutagens. In order to circumvent the problem of small colony mutant loss during the expression period and to obtain more accurate measures of mutant frequencies, Moore and Clive Ž1982. and, later, Rudd et al. Ž1987. recommended that the cells be plated into microwells or cloned in soft agar immediately after exposure, with TFT in liquid medium added after a sufficient time for expression. This approach is useful for generating more quantitative measurements of mutation frequencies, but it requires quite different criteria for performance and evaluation which have not yet been sufficiently validated to recommend its use for routine testing. 2.8. Cloning for mutant selection High efficiency of small colony mutant recovery in soft agar and the quantitation of small colony mutants are among the most critical aspects of the MLA. Factors that can affect cloning efficiency and small colony mutant recovery and detection include: the accuracy of cell counts and cell dilutions; proper cell dispersal to achieve single cell suspensions prior to cloning; the quality of the medium, serum and agar; incubation conditions; the time provided for colony growth before the colonies are counted and sized; and the precision with which the colonies are enumerated and sized. Often, small colony mutant recovery and detection will be significantly improved if the colonies are allowed to grow for up to 14 days rather than the originally recommended time of 10–11 days ŽTurner et al., 1984.. 2.9. Colony enumeration and sizing Mutant colony sizing provides mechanistic insight into a chemical’s genotoxicity Ži.e., potential clastogenicity.; therefore, colony sizing is performed for those chemicals which demonstrate a positive effect as well as for the negative and positive controls in each experiment. It is the responsibility of each laboratory to determine and report the size ranges of the large and small mutant colonies that are enumerated. Electronic counters used for mutant colony size

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analysis and enumeration should be capable of detecting at least half of the colonies ; 0.1 mm in diameter that can be observed by visual inspection; virtually all colonies of larger size should be detected. The efficiency of small colony detection and enumeration is assessed by sizing representative colonies using a microscope equipped with a micrometer, and it must be determined, for each tested chemical, whether inaccurate counts of small colony mutants could influence the interpretation of the results. Cole et al. Ž1983, 1986. developed a microtiter method to circumvent the possible influence of agar on recovery of small colony mutants and, in the process, obtained a higher efficiency for enumerating these colonies than was then available with electronic colony counters. For this approach, the number of microtiter wells containing no colonies can be counted efficiently with a microtiter plate reader. Microwells containing mutant colonies were observed using an inverted microscope, and the colonies were sized based on a subjective scale. However, image analysis systems are now available for counting and sizing the mutant colonies in microtiter plates. Differences in accuracy between the soft agar and microtiter approaches are negligible if the colonies in agar plates are counted and sized with the same precision Ž; 10 = magnification. as used for the microtiter plates, and this is now possible with recently developed systems. Although some find the microtiter approach to be more time-consuming than the agar cloning method ŽOberly et al., submitted., either approach is acceptable if conducted by experienced investigators and if the small mutant colonies are detected with reasonably high efficiency Ž) 50% at 0.1 mm diameter.. It should be emphasized, however, that the molecular and cytogenetic studies that form the basis for the current interpretation of the bimodal size distribution of mouse lymphoma mutant colonies were based on agar-derived colonies. 2.10. Replicate cultures and experiments Extensive MLA testing has been conducted with replicate cultures and replicate experiments in order to assess experimental variability or to satisfy the requirements of statistical analysis systems. However, because only a relatively small number of cultures Že.g., F 30. can be used without compro-

mising the technical performance of the assay, the use of replicate cultures can preclude testing a sufficient number of concentrations to give confidence that a response is either positive or negative. Instead, it is usually preferable to treat the cultures with as many closely spaced single concentrations as technically feasible for a laboratory, and to assess experimental variability by the shape of the dose response curve. It is not necessary to repeat appropriately conducted experiments that are clearly positive or negative. Furthermore, there is no justification for multiple, exact replications of experiments when a less than definitive concentration range was used in the first experiment. Repeat tests are required to resolve inconclusive results and to correct any deficiencies of the first assay Že.g., inadequate cytotoxicity or solubility problems.. For established laboratories, a very good measure of technical consistency and reproducibility in an assay may be found by comparing negative and positive control values with historical control data from appropriately conducted assays. 2.11. Assay acceptability Absolute cloning efficiencies for solvent controls are expected to be between 80 and 120%; lower and higher cloning efficiencies may be acceptable in individual experiments, especially if a test chemical is unambiguously positive. Published solvent control mutant frequencies from a variety of laboratories span the range of - 20 = 10y6 to ) 100 = 10y6 , but the lower spontaneous mutant frequencies are of concern because of the possibility of poor small colony mutant recovery. For this review of published data, experiments with spontaneous mutant frequencies G 20 = 10y6 were accepted for evaluation, but spontaneous mutant frequencies are currently expected to be consistently higher, such as between 70 and 120 = 10y6 . Lower values may be acceptable if small colony mutant recovery and enumeration are conclusively demonstrated. Spontaneous mutant frequencies which are significantly above 120 = 10y6 are considered valid and even a sign of improved mutant recovery provided that other explanations —such as low absolute cloning efficiencies and inadequate cleansing or selection— have been excluded.

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3. References and criteria for data selection and rejection The Phase III W ork Group evaluated L5178Yrtkqry MLA data published from 1976 through 1993, obtained from 114 peer-reviewed publications, two sets of contract laboratory reports prepared for the U.S. EPA, and a National Toxicology Program ŽNTP. Technical Report. To provide a consistent set of evaluations, test results for the 48 chemicals tested at the tk locus and evaluated in the first mouse lymphoma Gene-Tox review ŽClive et al., 1983a. were included in the data base and reevaluated. The following were excluded from the evaluation: abstracts, data that had been previously published, publications with insufficient MLA data to evaluate system performance, data from loci other than tk and from cells other than 3.7.2C, radiation effects, and the use of 3.7.2C cells for in vivo exposures Že.g., the host mediated assay. or for in vitro chromosomal aberrations or micronucleus testing. Data obtained with the microtiter approach Žwhich contained one unique chemical. was included in the evaluation. Data obtained with a third method in which cells were cloned immediately after exposure, sometimes identified as the in situ approach, was not available in peer-reviewed publications. In total, a data base was obtained that consisted of 602 chemicals tested under one or more activation conditions, and frequently in more than one laboratory. Approximately half of the data base consisted of published results from the blind testing of Žcoded. chemicals for the NTP using a protocol established in the late-1970s for assessing the reproducibility of the assay ŽMitchell et al., 1988a..

4. Data interpretation Scientific judgment and common sense are necessary for the interpretation as well as the performance of the mouse lymphoma assay. Earlier guidance on data interpretation Že.g. Clive et al., 1979, 1983a. indicated that a doubling of the mutant frequency over the solvent control Žspontaneous. frequency was sufficient to evaluate a result as positive, provided that it occurred at reasonable Ž10–90%. RTG and the spontaneous mutant frequency was in the Žthen. nor-

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mal range of 30–80 = 10y6 . However, this guidance was provided before the significance of small colony mutants was fully appreciated, before improvements in assay performance were implemented, and, in retrospect, was based largely on the experience of one laboratory with strongly mutagenic chemicals. Even earlier, in the mid- to late-1970s, statistical analysis approaches were developed for evaluating MLA data Že.g., Lee and Caspary, 1983. which required replicate dosing for each concentration of the test chemical, as well as the controls. To satisfy the replicate dosing requirements of such statistical analysis systems, too often an insufficient number of concentrations were tested to provide convincing evaluations, and too frequently results obtained at very high toxicities resulted in positive evaluations because of apparent statistical significance. When a test chemical is evaluated as mutagenic based on extreme treatments, the evaluation may have only marginal utility for assessing mechanisms of mutagenesis to assist in risk assessment or for predicting the outcome of testing the same chemicals in other biological systems. Thus, the members of the Work Group found no previously used method for evaluating MLA data that they considered to be appropriate in light of current knowledge about the performance and capabilities of the assay, and it therefore became necessary for them to establish new evaluation criteria before the data were reviewed. When the Work Group met to define the evaluation criteria, the experience of one laboratory ŽD. Clive. was that, in general, an absolute increase of 70 = 10y6 over the spontaneous mutant frequency was required for the effect to be significant at p 0.05 within a single experiment and that an even greater response Žapproximately 100 = 10y6 . was required to avoid having a positive or negative call contradicted by a repeat experiment. Group discussions based on these examples and the overall experience of the Work Group in generating and evaluating data led to agreement that data with an IMF G 100 = 10y6 should be evaluated as positive when observed at G 20% RTG. However, the Work Group also recognized that the data to be reviewed were obtained over a time when improvements in assay performance were being identified. Furthermore, even with optimum assay conditions most biological data represent a continuum of responses, with a

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‘gray’ area that some will determine to be positive and others will call negative. Therefore, it was the consensus of this Work Group that such responses should be categorized and considered separately and that two levels of positive responses should be defined based on the observed magnitude of the response. A limited positive response was assigned to results consisting of an IMF of at least 70 = 10y6 at RTG G 10%. The Work Group also defined two levels of negative responses, based on cytotoxicity, and three additional response categories for results that were neither positive or negative. Once defined, the qualitative Gene-Tox evaluation criteria were applied to evaluate the data for each activation condition —without S9 Ž0S9., with induced S9 ŽIS9. and with noninduced S9 ŽNS9. — from each publication. The criteria were then used to obtain summary evaluations for each activation condition with which a chemical was tested Žin one or more publications.. Finally, the criteria were used to obtain an overall evaluation for each chemical. The overall qualitative evaluations were used to assess performance by chemical class and were compared with summary evaluations of chemical carcinogenesis, when available, using the evaluations and response categories defined by Nesnow et al. Ž1986.. 4.1. Response categories for each publication of MLA results (for each actiÕation condition) The positive response categories that were used in evaluating the published data for each activation condition were defined as follows. q q A definitive positive response, a concentration-related increase in MF with an IMF G 100 = 10y6 at RTG G 20% for at least one concentration, and a spontaneous mutant frequency G 20 = 10y6 . However, if the spontaneous mutant frequency was ) 100 = 10y6 , a doubling of the spontaneous frequency was required. q A limited positive response, a concentrationrelated increase in MF with IMF G 70 = 10y6 at RTG G 10% for at least one concentration, with a spontaneous mutant frequency G 20 = 10y6 . Any apparently positive effects observed only below 10% RTG were discounted as lacking biologi-

cal relevance and excluded from consideration. In addition, as most problems in interpretation occur at RTGs - 20%, chemicals found positive only at 10–20% RTG were interpreted with caution and in light of other evidence from the assay such as the presence or absence of a dose response, the absence of technical problems, and reproducibility of the effect. Although the IMF conditions in the limited positive Žq. category were used to evaluate published data, the IMF of the definitive positive Žqq . category is advocated for future evaluations of mouse lymphoma assays conducted with protocols designed to maximize small colony mutant recovery. The Work Group also used two levels of negative responses, based on cytotoxicity, in recognition of the fact that, without significant toxicity or other evidence that a test chemical is capable of reaching the cell nucleus, some chemicals may, with additional information, be reclassified as inadequately tested or untestable in this assay. Thus, the following negative response categories were used. y A cytotoxic negative response, used for chemicals inducing a mutant frequency ŽIMF. F 70 = 10y6 and tested to an RTG of 10–20% in an adequate experiment Že.g., with acceptable spontaneous and positive control values.. s A noncytotoxic negative outcome, used for chemicals adequately tested to 5000 m grml or 5 m lrml, with values of IMF - 70 = 10y6 and RTG ) 20%. Three additional response categories were required to evaluate published data that were neither positive or negative. E Equivocal. A category used to indicate that a chemical fluctuated between being very weakly positive and negative either within an experiment or in repeated experiments. In these situations, further testing, under the same protocol, would not be likely to provide a definitive evaluation. These chemicals may be candidates for additional MLA testing, using different protocols. I Inconclusive or inadequately tested. A category necessary for published data in which insufficient concentrations were tested over the critical range of 10–20% RTG to be able to evaluate the results as clearly positive or negative. This category was also used for experiments with spontaneous mu-

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tant frequencies less than 20 = 10y6 , for apparently negative results with inadequate positive controls, and for apparently negative results for chemicals tested with BUdR as the selective agent. Because of the consensus that the quality and utility of published MLA data is of significance, and to provide some information for retesting these chemicals to obtain conclusive results, test results evaluated as inconclusive or inadequately tested were retained in the data base. But this classification should obviously only be applied to published data, as future testing should be performed until all assay deficiencies have been corrected. a Not testable. A separate category was applied to identify those chemicals that could not be tested in a valid manner in the MLA, at least under the protocols that were used; it may be possible to retest some of the chemicals evaluated herein as a with other solvents or protocols. Chemicals were placed in the a category if they were unstable or insoluble in culture medium or if they caused acidic pH shifts or elevated the osmolality of the medium. Additionally, chemicals were placed in this category if they were known to react with plastic, or were tested with acetone as the solvent in the absence of information that the chemical was tested in nonreactive plastic or glass. Because such experimental outcomes are without biological relevance for assessing mutagenesis, results in this category should not be classified as either negative or positive. Additional information was used to reach consensus evaluations for some of the data and included the experience of the reviewers, an assessment of the overall quality and reliability of data from the testing laboratory, and information about the physical properties of the test chemicals and the capabilities of the MLA, including information that may not have been available at the time the original results were published. 4.2. Response categories used for summary eÕaluations for each actiÕation condition The response categories were applied in a hierarchical manner to summarize the published data for

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each chemical for each activation condition Ž0S9, IS9 or NS9. when evaluations differed for published results from one or more sources. Specifically, when the evaluations of results from different publications did not agree, the more conservative evaluation was usually applied in the hierarchical order of qq ) q) y)s)E ) I ) a, with two exceptions. If at least one publication reported that a chemical was insoluble in a range that was evaluated as nontoxic and negative in another laboratory, when using the same solvent and similar test conditions, a summary evaluation of a was used instead of s . Similarly, a was used instead of an evaluation of qq or q if one publication Žor the chemical literature. suggested that a chemical may have been unstable in culture medium or induced acidic pH shifts, high osmolality, or reacted with plastic over the concentration range tested, and if there was no indication that the testing laboratory had documented these problems or adjusted the protocol to preclude them. In rare instances, such as a chemical having been evaluated as negative without and with activation in one laboratory and positive in the absence of activation when tested in another laboratory, the negative result with activation was not included in the summary. 4.3. Response categories used for the oÕerall summary eÕaluations of each chemical The summary evaluations for each activation condition were then re-summarized to yield an overall evaluation of the performance of each chemical in the MLA. Again, the more conservative evaluations were applied in the order of qq ) q) y)s)E ) I ) a. However, if a summary evaluation of published results was available for only one test condition Žwithout or with activation., and that evaluation was less than positive Žqq or q., an inconclusive ŽI. overall summary evaluation was necessary. 4.4. Response categories carcinogenesis bioassays

used

for

rodent

The Gene-Tox Carcinogenesis Panel for overall evaluation of the carcinogenicity of chemicals ŽNesnow et al., 1986. defined and used eight response categories. Five of these categories were used

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by the MLA Work Group, without modification: sufficient positive ŽSP., limited positive ŽLP., sufficient negative ŽSN., limited negative ŽLN., and equivocal ŽE.. Three additional categories —IŽP., inadequate Žpositive.; IŽN., inadequate Žnegative. and ŽI. inadequate— were combined and used as one category ŽI.. All rodent carcinogenesis evaluations in the review are those of the Carcinogenesis Panel or from application of the Carcinogenesis Panel’s definitions to the q, y, or E summary evaluations of Haseman and Clark Ž1990. for 112 long-term studies carried out in male and female mice and rats by the NTP. Hence, chemicals found negative by the NTP in both mice and rats Žmales and females. were classified as SN; chemicals evaluated as positive in only one species Žwhether one or both sexes. were classified as LP; chemicals that were positive in both species were classified as SP, and if one equivocal response was obtained and all other responses were negative or equivocal, the carcinogenesis result was classified as E.

5. Test performance 5.1. Detailed eÕaluations of published mouse lymphoma assay results Table 1, which was prepared from 890 records of published results, summarizes detailed evaluations of MLA performance for 602 chemicals. Only one set of published data was available for 449 chemicals, but two or more sets of published results were available for 153 chemicals and, usually, the results were reproducible between laboratories. Information provided in Table 1 includes the chemical names and CAS numbers, abbreviated literature citations, and, for each activation condition under which a chemical was tested: the concentration range that was tested, the lowest effective concentration tested for a mutagenic response ŽLECT. or the highest ineffective concentration tested ŽHICT., and the Work Group evaluation. Footnotes have been used to indicate testing with alternate activation systems, e.g., liver microsomes or hepatocytes. When the data were reviewed, mutant frequencies and RTG values were tabulated for each negative control and each concentration identified as the LECT

or HICT. This information was used to verify the consistent application of response criteria but has been omitted from Table 1. The reviewers usually documented rationales for application of the evaluation criteria, particularly for evaluations of E, I, or a, and the reasons for applying the a evaluations are indicated, with footnotes, in the table. When the published record provided chemical concentrations, RTGs, and mutant frequencies only in graphs, the reviewers used their best efforts to estimate actual values. In addition, for consistency in data presentation and to facilitate its analysis, molar concentrations were converted to units per ml, using molecular weights and the densities of liquids for the calculations. 5.2. Summary eÕaluations by actiÕation condition and oÕerall summary eÕaluations Table 2 provides information on the summary evaluations of MLA results for each chemical by activation condition, an overall summary evaluation of the performance of each chemical in the mouse lymphoma assay, the chemical class Žor classes. of each of the chemicals, and the overall evaluations of rodent carcinogenicity for the test chemicals, which were available for 260 chemicals. The 602 chemicals in the data base included 259 Ž43%. that were evaluated as definitively positive, qq, and 83 chemicals Ž14%. evaluated as limited positive responses, q, for a total of 342 Ž57%. positive responses. Only 21 chemicals Ž3%. were evaluated as negative with cytotoxicity, y, and only 23 Ž4%. evaluated as negative without cytotoxicity, s . Thus, only 7% Ž44. of the chemicals were evaluated as negative with or without cytotoxicity. The high proportion of positive responses and the small number of negative responses Žwith and without cytotoxicity. reflect not only the composition of the data base, as publications more frequently contain results for chemicals expected to be positive, but also more stringent requirements for negative responses: testing to 10–20% RTG for a negative with cytotoxicity, or to a concentration of 5000 m grml or 5 m lrml for a negative without cytotoxicity, and the chemical must have been tested in both the absence and the presence of metabolic activation. Thus, when a number of chemicals tested with only one activa-

Table 1 Detailed mouse lymphoma assay results and qualitative evaluations by activation condition for 602 reviewed chemicals

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230 Concentration range, LECT and HICT units are per ml except when % concentrations. b LECT, lowest effective concentration tested; HICT, highest ineffective concentration tested. c qq, Definitive positive; q, limited positive; y, cytotoxic negative; s, noncytotoxic negative; E, equivocal; I, inconclusive; a, not-testable. ŽSee text for complete definitions.. d Testing limited by solubility. e Testing limited by osmolality. f Testing limited by acidic pH shift. g Chemical reacts with plastic. h Chemical rapidly hydrolyzed at neutral pH; only stable at acidic pH. i Rat hepatocytes. j Mouse S9. k Rat S20. l Mouse microsomes. m Tested with microtiter approach. n Inconclusive ŽI. in the presence of formaldehyde dehydrogenase ŽFDH.. o Cytotoxic negative Žy. in the presence of FDH. p Tested to concentrations greater than 5000 mgrml.

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a

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Table 2 Chemical classes, summary MLA a evaluations by activation condition b , overall evaluations, and rodent carcinogenesis bioassay c evaluations for 602 reviewed chemicals

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Table 3 Summary of overall mouse lymphoma evaluations listed by chemical class a Class No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 a b

Class description

Summary evaluation

Acridines, quinacridines, benzimidazoles Acyl and aryl halides, halogenated ether, halohydrins, saturated and unsaturated alkyl halides Aldehydes, anhydrides Alkyl and aryl epoxides Alkyl sulfates, sulfozides, sulfones, sulfonates, organic sulfur chemicals not otherwise classified Anthraquinones, quinones Antibiotics, mycotoxins Aromatic and aliphatic amines, amides and sulfomides Aziridines, nitrogen and sulfur mustards Aromatic azo, azoxy and hydrazo chemicals, diazoalkanes, nitriles, azides Carbamates, ureas, thioureas, dicarboximides Dioxins, xanthenes, thioxanthenes, phenothiazines Halogens and inorganic derivatives, sulfur and nitrogen oxides Hydrazides, hydrazines, triazenes Hydroxylamines, amine-N-oxides Lactones, organic peroxides Mineral fibres Nitroimidazoles, nitrofurans, nitroquinolines, nitroaromatics, nitroalkanes Nitrosamides, nitrosoureas, nitrosogaunidines Nitrosamines Organolead, organomercury, and organophosphorus chemicals, metals and derivatives, phosphoric acid esters, phosphoramides Polycyclic aromatic hydrocarbons, fluorenes, fluorenones Pyrimidine and purine derivatives Steroids Benzene ring Amino acids and derivatives Alkaloids Carbohydrates and derivatives Alcohols and phenols Heterocyclic rings not otherwise classified, unclassified

qq

q

y

q

E

I

a

Class total b

10 47

0 17

0 1

0 0

0 3

0 22

0 12

10 102

10 13 19

1 1 15

0 0 4

0 0 2

0 1 5

3 5 21

0 0 4

14 20 70

8 10 96 8 6 11 11 2 1 7 4 0 13 3 7 24

3 1 26 0 11 6 0 3 2 4 1 0 2 0 0 7

0 1 9 0 4 0 0 0 0 0 0 0 1 0 1 4

0 1 4 0 4 2 0 0 0 0 0 0 1 0 0 4

1 1 3 0 3 1 1 0 0 0 1 0 3 0 0 5

0 5 52 0 9 6 2 1 3 3 3 0 6 0 1 20

6 1 15 1 1 5 1 0 1 0 1 0 2 0 0 11

18 20 205 9 38 31 15 6 7 14 10 0 28 3 9 75

23 9 0 77 4 2 10 50 58

5 0 1 38 0 0 1 13 20

3 0 0 11 0 0 1 6 7

2 0 0 4 2 0 2 3 7

3 0 3 9 0 0 0 7 3

9 6 4 55 5 0 5 32 30

1 1 1 30 1 1 2 5 12

46 16 9 224 12 3 21 16 137

See Tables 1 and 2 and text for definitions. Number of chemicals in class.

tion condition yielded results that were evaluated as positive by the authors Žpresumably requiring no further testing. but were found negative for that

activation condition by Gene-Tox criteria, it was necessary for the overall Gene-Tox evaluation to be I Žinconclusive..

Notes to Table 2: a MLA, mouse lymphoma assay. b Summary evaluations by activation condition: OS9, tested without S9; IS9, tested with induced S9; NS9, tested with noninduced S9. Evaluations are: qq, definitive positive; q, limited positive; y, cytotoxic negative; s , noncytotoxic negative; E, equivocal; I, inconclusive; a, not testable. ŽSee text for complete definitions.. c RCB, rodent carcinogenesis bioassay evaluations using response categories of Nesnow et al. Ž1986.. SP, sufficient positive; LP, limited positive; SN, sufficient negative; LN, limited negative; E, equivocal; I, inadequately tested. d Testing limited by solubility. e Testing limited by osmolality. f Testing limited by acidic pH shift. g Chemical reacts with plastic. h Chemical rapidly hydrolyzed at neutral pH; only stable at acidic pH. i Inconclusive ŽI. in the presence of formaldehyde dehydrogenase ŽFDH.. j Cytotoxic negative Žy. in the presence of FDH. k RCB evaluations from Haseman and Clark Ž1990. using the response categories of Nesnow et al. Ž1986.. ŽThese 9 chemicals were also tested by the NTP for carcinogenicity and a manuscript is in preparation containing the Gene-Tox Carcinogenesis Panel’s evaluations of those testing results..

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The published results for 142 chemicals Ž24%. were evaluated as inconclusive or inadequately tested in the MLA. In addition to obtaining less than a positive result for testing with only one activation condition, historical reasons for approximately onefourth of the chemicals having been inadequately tested included the use of protocols that resulted in a severe underestimation of mutant frequencies, as indicated by the negative and positive controls, and testing too few concentrations to yield a clearly positive or negative result Žoften missing the 10–20% RTG range.. The published results for only 18 chemicals Ž3%. were evaluated as consistently equivocal under the conditions of testing without and with activation, and 54 chemicals Ž9%. were evaluated as inappropriate for testing in the MLA, at least with the solvents that were used and the published protocols. The majority of the not-testable chemicals, 38 of 54, were apparently insoluble, and 8 induced acidic pH shifts, including one chemical that was insufficiently soluble and acidic during exposure of the cells. 5.3. Summary eÕaluations by chemical class The distribution of chemicals by classes reflects the selection of chemicals for testing and is similar to the distribution of chemicals by class in the Gene-Tox carcinogenesis data base. Because 375 of the chemicals in the MLA data base were assigned to more than one chemical class, as was shown in Table 2, 1283 chemical class records are summarized in Table 3 and listed in Table 4. As illustrated in these tables, three or more chemicals were tested in 29 of the 30 chemical classes that are used for Gene-Tox reviews. The single class without representative chemicals was Class 17, mineral fibers, which would not be considered appropriate for evaluation in this assay. Four chemical classes contained over 50% of the chemicals in the data base: Class 8, aromatic amines, aliphatic amines, amides and sulfomides; Class 25, benzene ring chemicals Žthe largest class.; Class 29, alcohols and phenols; and Class 30, heterocyclic rings that were not otherwise classified and other unclassified chemicals. As would be expected from the selection of chemicals, positive responses predominated. For experiments with either positive or negative results, at least

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75% of the responses were positive for 28 of the 29 represented classes, and no negative responses were obtained for chemicals in 14 of the classes. Only in Class 10, which contained 38 aromatic azo chemicals, did the ratio of positive to negative chemicals fall below 3:1. All of the chemicals tested in two of the classes —Class 1, acridines, quinacridines and benzimidazoles Ž10 chemicals., and Class 19, nitrosamides, nitrosoureas and nitrosoquinidines Ž3 chemicals. — yielded definitive positive Žqq . responses, and over 50% of the chemicals included in seven additional classes ŽClasses 3, 4, 9, 12 and 27. yielded responses that were evaluated as qq. Conversely, less than 20% of the responses were qq in Class 10, aromatic azo and azoxy chemicals, hydrazo chemicals, diazoalkanes, nitriles and azides, and Class 14, hydrazides, hydrazines, and triazenes. No steroids, Class 24, were qq. Three classes contained higher percentages of chemicals with inconclusive results than was the norm: Class 23, pyrimidine and purine derivatives Ž6 of 16 chemicals., Class 24, steroids Ž4 of 9 chemicals., and Class 26, amino acids and derivatives Ž5 of 12 chemicals.. One-third of the chemicals in two classes were evaluated as not testable —Class 6, anthraquinones and quinones Ž6 of 18 chemicals., and Class 27, alkaloids Ž1 of 3 chemicals.. Nine chemicals which were classified as steroids, Class 24, included three chemicals that yielded equivocal results, four that yielded inconclusive results, and one that was evaluated as not testable; the ninth chemical was positive Žq.. As has been found for other in vitro test systems, apparently the MLA may not be well suited for evaluating most steroids. The observation that one or more chemicals was evaluated as positive for mutagenesis in each of the 29 represented classes suggests that the MLA is well suited for testing chemicals from most of the chemical classes. However, conclusions can not be reached for those classes that had few Že.g. - 10. representative chemicals Ž8 of 30 classes., and the discriminatory powers of the assay may have been insufficiently assessed for an additional 8 of the remaining 22 classes in which all of the valid MLA test results were positive. As may be found by examination of Table 4, most chemical classes contained relatively few representative chemicals that were tested for carcinogenesis as

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Table 4 Qualitative mouse lymphoma assay and rodent carcinogenesis bioassay evaluations a of reviewed chemicals grouped by chemical class

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a

See Tables 1 and 2 and text for definitions. Testing limited by solubility. c Testing limited by acidic pH shift. d Testing limited by osmolality. e Chemical reacts with plastic. f Chemical rapidly hydrolyzed at neutral pH; only stable at acidic pH. g These chemicals were also tested by the NTP for carcinogenicity and a manuscript is in preparation containing the Gene-Tox Carcinogenesis Panel’s evaluations of those testing results. b

well as in the MLA. Only 12 of 30 classes contained at least 10 chemicals that were tested for carcinogenesis. Although all 12 of these classes contained both positive and negative carcinogenesis results, 10 other classes contained no negative carcinogenesis results. In addition, in only six classes were at least 50% of the chemicals tested for carcinogenesis: Class 2 Ž56 of 102 chemicals., Class 9 Ž7 of 9 chemicals., Class 14 Ž6 of 7 chemicals., Class 18 Ž15 of 28 chemicals, Class 19 Ž2 of 3 chemicals. and Class 22 Ž23 of 46 chemicals.. However, with these caveats, when evaluated by chemical class, the MLA and RCB evaluations of chemicals tested for carcinogenicity were 100% concordant, i.e. positive and negative evaluations agreed, for 18 of the 29 represented chemical classes: Classes 3, 4, 6, 7, 9, 10, 11, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23 and 28, and concordance of positive and negative results was over 75% for six additional classes: Class 2 Ž94%., Class 5 Ž90%., Class 8 Ž95%., Class 25 Ž94%., Class 29 Ž79%. and Class 30 Ž79%.. Four chemical classes, numbers 1, 17, 24 and 27, contained no chemicals that could be evaluated for concordance. Class 12 contained only one chemical that could be evaluated for concordance, hycanthone methanesulfonate, and it was strongly positive in the MLA but evaluated as a limited negative ŽLN . for carcinogenesis. Methotrexate, one of three chemicals that could be evaluated for concordance in Class 26, was strongly

positive in the MLA but was evaluated as negative in the rodent carcinogenesis bioassay. 5.4. Correspondence of MLA results with the outcome of rodent carcinogenicity testing Any summary comparison of results from in vitro testing with the outcome of in vivo testing must be accomplished with the following caveats. First, any such comparison must, of necessity, exclude a large body of information about each chemical, including its physical properties, its metabolism in different species, and the methods used for testing; the latter include the concentrations Ždoses. tested and the exposure conditions, e.g., the activation systems used for in vitro testing and the route of exposure for in vivo tests. Second, any such summary of testing outcomes is constrained by the chemicals included in the evaluation, the adequacy of testing, and the evaluation criteria. Thus, although some members of the scientific community have characterized the mouse lymphoma assay as unreliable Že.g., Purves et al., 1995., this characterization has been based largely on the NTP’s summary evaluation of the performance of four in vitro genetic toxicity tests for predicting rodent carcinogenicity, as first published for 73 chemicals ŽTennant et al., 1987. then later expanded to total 114 chemicals ŽZeiger et al., 1990.. Because the Gene-Tox Work Group’s basic premises

A.D. Mitchell et al.r Mutation Research 394 (1997) 177–303 Table 5 Comparison a of overall mouse lymphoma assay and rodent carcinogenesis bioassay evaluations of 260 chemicals tested for carcinogenicity Mouse lymphoma assay

qq q y s E I a

Rodent carcinogenesis bioassay SP

LP

SN

LN

E

I

57 21 0 0 2 20 8

28 7 0 0 3 13 11

4 1 4 4 6 19 10

1 1 1 1 0 4 1

6 3 1 1 1 2 3

12 1 0 0 1 2 0

a

Overall mouse lymphoma assay and rodent carcinogenesis bioassay evaluations for each chemical listed in Table 2. See Table 2 and text for definitions.

related to acceptability of data and criteria for its evaluation differed significantly from those of the NTP, the Gene-Tox evaluations of MLA results differed significantly from the NTP evaluations for 58 of the 114 chemicals ŽMitchell et al., submitted a.. Table 5 provides a summary comparison of the current Gene-Tox MLA evaluations with Gene-Tox evaluations of rodent carcinogenicity for 260 chemicals, a comparison that is valid only for the current Gene-Tox MLA and carcinogenesis data bases and evaluation criteria. Within these constraints, it may be noted that for both the MLA and the RCB, Ža. the majority of the 260 chemicals were positive, Žb. the majority of the positive responses were sufficient or definitive positives ŽSP or qq ., Žc. there was a greater association of chemicals that were clearly positive in the MLA Žqq . with chemicals providing sufficient evidence of carcinogenesis in the bioassay ŽSP. than for chemicals with limited evidence of a positive response in vitro and in vivo Žq and LP., and Žd. the percentages of noncarcinogens evaluated as E, I, or a in the MLA were twice as high as the percentages of carcinogens with these evaluations. Valid comparisons between MLA and RCB results could not be made for 130 of the 260 chemicals Ž50%., as the chemicals could not be tested in a valid manner in the MLA with the published protocols, or the test results were evaluated as equivocal or inconclusive in either the MLA or the carcinogenesis bioassay, as illustrated in Table 5. However, the

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accuracy of the MLA as a predictive test for carcinogenesis could be assessed for the 130 chemicals that were evaluated as positive or negative for mutagenesis and carcinogenesis, which is a sufficiently large group of chemicals to provide a reasonable level of confidence in the analysis. The 120 chemicals evaluated as positive in the MLA Žqq and q. and that could be compared with RCB results included 113 that were positive in the carcinogenesis bioassay ŽSP and LP.; thus, the MLA was apparently 94% accurate in predicting positive responses in the rodent bioassay. In addition, all of the 113 carcinogens were evaluated as positive in the MLA; therefore, the sensitivity of the assay in detecting carcinogens was 100%. As there were fewer chemicals evaluated as negative in both the MLA and the carcinogenesis bioassay, estimations of negative predictivity and specificity are less accurate. However, as all of the 10 chemicals that were negative in the MLA Žwhen the y and s categories were combined. were negative for rodent carcinogenesis ŽSN and LN., accuracy of the MLA in predicting noncarcinogens was 100%. Specificity of the MLA was 59%, as only 10 of 17 noncarcinogens were negative for mutagenesis. The overall concordance of chemicals that were evaluated as positive or negative in both the MLA and the carcinogenesis bioassay Ž123 of 130 chemicals. was 95%. Little significance can be attributed to the observation that all of the mutagenic noncarcinogens belong to Classes 25, 29 andror 30, as these classes are among the four with the greatest number of representative chemicals. The seven noncarcinogens that were positive in the MLA included piperonyl butoxide, a chemical that was considered to be negative for in vitro aberrations in Chinese hamster ovary cells by Galloway et al. Ž1987.; however, the authors noted that this chemical was immiscible in culture medium, i.e., it could not be tested in a valid manner with the standard NTP in vitro aberration protocol in which cells are grown in monolayer cultures. All six of the other noncarcinogens that were positive in the MLA —2-chloroethanol, 1,2-dichlorobenzene, hycanthone methanesulfonate, 8-hydroxyquinoline, methotrexate, and methyl methacrylate —have been shown to induce chromosomal aberrations in vitro ŽBlazak et al., 1986; Moore et al., 1988a; Waters et

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al., 1988; Ivett et al., 1989; Loveday et al., 1989; Anderson et al., 1990; Mitchell et al., submitted b.. Because of the strong association of clastogenesis with carcinogenesis, it is appropriate to ask whether the seven mutagenic Žand six clastogenic. noncarcinogens were ‘false positives’ for in vitro genotoxicity or ‘false negatives’ for carcinogenesis, as has been discussed by Waters et al. Ž1988.. Three of the seven chemicals were classified as limited negatives ŽLN. for carcinogenesis by Nesnow et al. Ž1986.: hycanthone methanesulfonate, although it had not been tested in rats; methotrexate, although it had not been tested in mice; and piperonyl butoxide although it was equivocal for carcinogenesis in female rats. A fourth chemical, 8-hydroxyquinoline, was classified as a significant negative ŽSN. for carcinogenesis by Nesnow et al. Ž1986.; however, this chemical yielded equivocal results when tested in male rats. Thus, it would appear that, upon reexamination, some would consider the carcinogenicity test results for these four chemicals to be either inadequately tested ŽI. or equivocal ŽE. rather than negative. The other three mutagenic noncarcinogens were classified as SN for carcinogenesis for this MLA review by applying the definitions of Nesnow and associates to the outcome of NTP testing as reported by Haseman and Clark Ž1990.. These chemicals included 2-chloroethanol that was evaluated for carcinogenesis in skin painting studies, 1,2-dichlorobenzene that was administered by gavage, and methyl methacrylate that was tested with inhalation exposures. 2-Chloroethanol was mutagenic in vitro only in the presence of metabolic activation, and it is possible that, due to the pharmacokinetics of this chemical, the skin painting route of administration may have presented insufficient doses for activation and transport to the target tissueŽs., or, alternatively, that toxic effects of 2-chloroethanol may have prevented the observation of tumors due to localized killing of the target cells ŽWaters et al., 1988.. 1,2-Dichlorobenzene was one of the more toxic chemicals tested in vitro, and it is possible that severe toxicity in vivo may have precluded the observation of tumors in test animals, as was postulated for methotrexate and hycanthone methanesulfonate by Waters et al. Ž1988.. Methyl methacrylate polymerizes easily, as was noted by Moore et al. Ž1988a., particularly in the

absence of hydroxyquinone, which serves as an antioxidant. ŽWhen oxidized by peroxide catalysts, methyl methacrylate forms a clear plastic known as Lucite or Plexiglas.. It would seem possible that the mixing of methyl methacrylate with air for inhalation exposures may have permitted polymerization of the monomeric ester, which could have precluded sufficient doses from reaching target tissues. As this last chemical may have been ‘not testable’ by inhalation exposures, the last three of the seven mutagenic noncarcinogens may have been ‘false negatives’ in vivo because of the doses tested, the route of exposure, or test material instability. It may be suggested, therefore, that it is less probable that each of the seven non-concordant chemicals was a ‘false positive’ in the MLA than that it was incorrectly identified as a noncarcinogen, and, if so, positive predictivity, specificity and concordance of the MLA for the 130 chemicals would approach 100%.

6. Conclusions The L5178Yrtkqry mouse lymphoma cell mutagenesis assay, developed a quarter-century ago ŽClive et al., 1972., far exceeded initial expectations for utility and significance of observed effects, particularly when the MLA was shown to detect viable chromosomal mutations as well as gene mutations. Because of improvements in assay performance to enhance the recovery of chromosomal mutants, especially during the last decade, it is now known that the originally defined protocol was relatively insensitive, and the published data has been reviewed with this knowledge. Thus, it was necessary to evaluate approximately one-fourth of the previously-tested chemicals as having been inadequately tested to provide a clear demonstration of a positive or negative result, and these chemicals require retesting with attention to the procedural details and quality control measures that have been described. While the strengths of the MLA are unique, the weaknesses of the assay are those common to all in vitro genotoxicity assays, particularly assays that use mammalian cells. Specifically, in the absence of measurements of pH, osmolality and solubility under testing conditions, ‘false positive’ results can be obtained with nonphysiological testing conditions and

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‘false negative’ results can be obtained for insoluble chemicals. But such results are without biological relevance and, hence, invalid for safety assessment. Therefore, basic protocols can be used to test such chemicals only if the testing conditions can be adjusted to titrate against nonphysiological environments during chemical exposure and only if solvents are used that will permit the test chemicals to remain in solution during the exposure period. For some chemicals, estimated to be less than 2 to 4% of the 602 reviewed chemicals, the latter may not be possible. Thus, predictions of carcinogenicity for the chemicals that remain not testable in the MLA may be dependent upon the outcome of other genotoxicity tests. The MLA has been sufficiently validated to demonstrate that it is sensitive to most classes of chemicals and that it can predict rodent carcinogenicity with a high degree of accuracy. As the more recent approaches to enhance small colony mutant recovery and detection are more widely implemented, and as chemicals that have been inadequately tested are reevaluated with protocols that more completely assess the spectrum of genetic events that can be detected in the MLA, the number of chemicals found clearly negative or positive is expected to increase. The utility of in vitro genotoxicity assays in testing programs, and for regulatory submissions, is based on the accuracy with which the in vitro assays can predict the outcome of exposing animals to a variety of chemicals and, hence, permit the conservation of resources and a reduction in animal usage for predicting effects in humans. This review has shown that the MLA not only provides useful mechanistic information on test chemicals, but also that, when the assays are conducted with acceptable protocols and the results are evaluated with consideration of biological relevance, the MLA is highly predictive of in vitro clastogenesis, in vivo carcinogenesis, and, usually, an absence of carcinogenesis in rodents. Because of the capabilities of the MLA, an in vitro chromosomal aberrations assay is seldom needed for initial genotoxicity screening if the MLA is used to assess gene and chromosomal mutations in vitro. There is ample justification for the current use of the MLA to predict the outcome of testing most chemicals in vivo. It is the consensus view of the

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reviewers that, when appropriate performance and evaluation criteria are applied, the MLA is of high utility and eminently well suited for assessing the spectrum of mutagenesis that can be induced by chemicals in mammalian cells and for accurately predicting the outcome of in vivo tests, including rodent carcinogenesis bioassays. Therefore, both public and private resources will be conserved, and animal usage reduced, by the continued use of the L5178Yrtkqry-3.7.2C mouse lymphoma assay for initial assessments of the potential risks of human exposure to chemicals.

Acknowledgements The section of this report entitled ‘Assay Performance’ was initially drafted for submission as a separate consensus manuscript during a meeting held at Burroughs Wellcome Co., Research Triangle Park, NC, in February, 1989. The meeting was attended by the authors of this MLA Gene-Tox review and by Drs. J. Cole ŽMRC Cell Mutation Unit, University of Sussex, UK., K.L. Dearfield ŽU.S. EPA, Washington, DC., J. Harbell ŽMicrobiological Associates, Inc., Rockville, MD., and V.A. Ray, ŽPfizer, Inc., Groton, CT.. The MLA Work Group appreciates the contributions of the other meeting participants and is responsible for any changes resulting from revision and expansion of the initial, unpublished manuscript. The authors appreciate the assistance of Dr. K.H. Mavournin and the staff of the Environmental Mutagen, Carcinogen and Teratogen Information Center, Oak Ridge, TN, who provided references through 1989 and extracted data from these references, J.T Deahl., Genesys Research, Inc., Research Triangle Park, NC Žnow at Eli Lilly & Company, Greenfield, IN., who conducted literature searches and assisted in extracting data from the more recent publications, and Dr. E.E. Mitchell of Genesys who designed and implemented the MLA data base system that was used for this publication.

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