Sixth plot of the carcinogenic potency database: results of animal bioassays published in the General Literature 1989 to 1990 and by the National Toxicology Program 1990 to 1993.

This paper presents two types of information from the Carcinogenic Potency Database (CPDB): (a) the sixth chronological plot of analyses of long-term carcinogenesis bioassays, and (b) an index to chemicals in all six plots, including a summary compendium of positivity and potency for each chemical (Appendix 14). The five earlier plots of the CPDB have appeared in this journal, beginning in 1984 (1-5). Including the plot in this paper, the CPDB reports results of 5002 experiments on 1230 chemicals. This paper includes bioassay results published in the general literature between January 1989 and December 1990, and in Technical Reports of the National Toxicology Program between January 1990 and June 1993. Analyses are included on 17 chemicals tested in nonhuman primates by the Laboratory of Chemical Pharmacology, National Cancer Institute. This plot presents results of 531 long-term, chronic experiments of 182 test compounds and includes the same information about each experiment in the same plot format as the earlier papers: the species and strain of test animal, the route and duration of compound administration, dose level and other aspects of experimental protocol, histopathology and tumor incidence, TD50 (carcinogenic potency) and its statistical significance, dose response, author's opinion about carcinogenicity, and literature citation. We refer the reader to the 1984 publications (1,6,7) for a detailed guide to the plot of the database, a complete description of the numerical index of carcinogenic potency, and a discussion of the sources of data, the rationale for the inclusion of particular experiments and particular target sites, and the conventions adopted in summarizing the literature. The six plots of the CPDB are to be used together since results of individual experiments that were published earlier are not repeated. Appendix 14 is designed to facilitate access to results on all chemicals. References to the published papers that are the source of experimental data are reported in each of the published plots. For readers using the CPDB extensively, a combined plot is available of all results from the six separate plot papers, ordered alphabetically by chemical; the combined plot in printed form or on computer tape or diskette is available from the first author. A SAS database is also available.


Background
The Carcinogenic Potency Database (CPDB) is a standardized resource of results of chronic, long-term carcinogenesis bioassays (1)(2)(3)(4)(5). It provides an easily acces-We thank the many researchers who provided additional experimental results for the CPDB, sometimes by having to go back to earlier pathology reports. Susan Sieber and Unnur Thorgeirsson were enor-sible resource with sufficient information on each experiment to permit investigations in many research areas of carcinogenesis. Both qualitative and quantitative information on positive and negative tests are reported. All experiments in the CPDB meet a specific set of inclusion criteria designed to permit the estimation of carcinogenic potency; therefore, reasonable consistency of experimental protocols is assured. Bioassays are included in the database only if the test agent was administered alone, rather than in combination with other substances; if the bioassay included a control group; if the route of administration was diet, water, gavage, inhalation, iv injection or ip injection; and if the length of experiment was at least half the standard lifespan for the species and the duration of dosing one-fourth the standard lifespan.
Many cancer tests do not meet these rules and are not included, e.g., if a rodent test was shorter than 1 year or the duration of dosing was shorter than 6 months, if dosing was not chronic, or if the compound was administered by skin painting or sc injection.
The CPDB is exhaustive because it includes all published tests that meet a set of experimental criteria. There is great diversity in the testing of chemicals reported in the database; while most chemicals have been tested in rats or mice, some have been tested in hamsters, dogs, prosimians, or monkeys. Experiments with 107 different mouse strains and 76 rat strains are included. For a given chemical, the database may have only a single experiment or several experiments. For example, among the 952 chemicals tested in rats, 26% have only one rat test and 53% have two tests; however, 18 chemicals have more than 10 tests. Among the 1230 chemicals in the CPDB, 52% have been tested in only a single species, 45% in two species, and 3% in more than two.
In the CPDB we do not ourselves evaluate whether the results in each experiment provide evidence for carcinogenicity; rather, we report the published opinions of the investigators and the statistical significance of the dose response. We have corresponded with many researchers to clarify their evaluations of particular target sites as well as to obtain additional experimental results; this is indicated in the plot by "pers. comm." in the reference field. The CPDB includes results of all National Cancer Institute/National Toxicology Program (NCI/NTP) Technical Reports published through June 1993, except for a few bioassays of particulates or where the compound was administered by skin painting. Thirty percent (370/1230) of the chemicals included in the CPDB have been tested in NCI/NTP bioassays.
A detailed guide to the plot of the database was included in the first published plot in 1984 (1). It described the contents, field by field, and discussed the sources of data, the criteria for the inclusion of particular experiments and particular target sites, and the conventions adopted in summarizing the literature. Therefore, readers who are not familiar with the CPDB should read the 1984 paper before using the plot in this paper.
The TD50, our numerical index of carcinogenic potency, has been fully described (1,6,7) and may be briefly defined as follows: for a given target site(s), if there are no tumors in control animals, then TD50 is the chronic dose rate in mg/kg body weight/day that would induce tumors in half the test animals at the end of a standard lifespan for the species. Since the tumor(s) of interest often does occur in control animals, TD50 is more precisely defined as the chronic dose rate that will halve the probability of remaining tumor-free throughout the standard lifespan. One reason for choosing TD50 is that it is easy to understand the concept, particularly because of the analogy to LD50. Importantly, TD50 is often within the range of doses tested; thus the experimental results do not have to be extrapolated far to estimate TD50. The TD50 does not indicate anything about carcinogenic effects at low doses, since carcinogenesis bioassays are generally conducted at doses at or near the maximum tolerated dose (MTD). In the CPDB, TD50 values for NCI/NTP bioassays have been estimated using lifetable data, whereas summary analyses have been used for the general literature. [See (8) for a comparison of methods.] The range of statistically significant TD50 values for chemicals in the CPDB that are carcinogenic in rodents is more than 10 millionfold.
In each of the six plot papers, Appendices 1 through 13 are in the same format and provide information for the data in that publication. Appendices 1 through 13 below apply only to the plot presented here. Appendix 1 lists alphabetically the compounds included in the current plot, their common synonyms, and Chemical Abstracts Service (CAS) registry numbers; Appendix 2 provides a list of those same compounds ordered by CAS number. The next several appendices provide codes and definitions required for using the plot: strains of test animal (Appendix 3); routes of administration (Appendix 4); sites of tumor induction (Appendix 5); histopathology of tumors (Appendix 6); notes about the experiment, e.g., survival problems (Appendix 7); dose-response curve symbols (Appendix 8); journal reference codes (Appendix 9); species of test animal (Appendix 10); and author's opinion codes (Appendix 11). Appendices 12 and 13 give full bibliographic information for experiments reported in this plot: a bibliography for the general literature (Appendix 12); and a list of NTP Technical Reports (Appendix 13). Appendix 14 provides an index to chemicals in all six plots of the CPDB, and evaluations of positivity and carcinogenic potency in rats and mice for each chemical.
In the fifth plot paper of the CPDB (5) we updated several analyses reported earlier, in order to reflect data in the CPDB at that time: i.e., the proportion of chemicals that are positive for several datasets, the association between mutagenicity and carcinogenicity, prediction of positivity between species, reproducibility of results in "near-replicate" experiments, and carcinogen identification on the basis of two vs four sex-species groups. When data from this sixth plot are added, results in each of these analyses are similar to those reported in plot 5 (within two percentage points); therefore, we have not presented the updated tables in this paper.

Plot in this Supplement
This sixth plot of the CPDB includes results of 531 long-term, chronic experiments on 182 chemicals. It reports on 50 compounds published in Technical Reports of the NTP between January 1990 and June 1993, and 139 compounds published in the general literature between January 1989 and December 1990. Experiments are reported in rats, mice, hamsters, prosimians, and monkeys. Eighty-seven of the 182 chemicals in this plot were also included in an earlier plot, and we have flagged the chemical names on the plot with a triple asterisk (***). Whereas only a few experiments may be reported here for a given chemical, several experiments may have been reported in earlier plots (e.g. 2-acetylaminofluorene, BHT, formaldehyde, and lead acetate). By using Appendix 14, the reader can identify the earlier plot publications that include data on each chemical as well as information on whether the chemical was tested and evaluated as carcinogenic in each sex of rat and mouse; the most potent TD50 value in rats and mice from all six plots is also reported in Appendix 14.
Chemicals from a variety of sources and with a variety of uses are included in this sixth plot: for example, industrial chemicals (e.g. cadmium chloride, glycidol, rhodamine 6G, and tetranitromethane); food this plot have extensive dose-response data, with more than four dose groups (e.g., 1,3butadiene, diethylstilbestrol, N-nitrosopyrrolidine, and sulfamethazine). The TD50 values for the compounds in this plot fall within the 10 millionfold range reported earlier (1).
For several recent bioassays in which NTP suspected a potential for chemically induced renal tubule neoplasms, multiple additional sections were taken of the kidneys. This is the first CPDB plot to include results on these step sections. For bioassays with kidney step sections in which NTP evaluated the evidence for carcinogenicity as "clear" "some" or "equivocal," we have estimated one TD50 value using data from the standard protocol and one that includes the step section data. On the plot the TD50 including step sections is indicated by the words "with step." Whereas TD50 values for NTP bioassays throughout the CPDB are estimated with lifetable methods, only summary data were available for the kidney step sections. The confidence limits of the summary TD50 value for the step sections are therefore indicated on the plot by the symbol "." (as for all summary values in the CPDB). For example, see TD50 values for kidney tubule adenoma in male mice for tris (2-chloroethyl)phosphate.

Analyses of Bioassays in Nonhuman Primates
A series of lifetime studies of 30 chemicals in cynomolgus and rhesus monkeys have been conducted at the Laboratory of Chemical Pathology, National Cancer Institute (NCI) [SM Sieber and UP Thorgeirsson, personal communication; (34)(35)(36)]. Included in this plot of the CPDB, are lifetable analyses of results on 17 completed studies, 16 of which are rodent carcinogens. The duration of experiments is up to 27 years. We have relaxed some of our standard CPDB rules in order to report these results, for example, because there were often fewer than five animals per sex-species, because controls are from a colony rather than concurrent, and because some positive experiments were shorter than half the standard lifespan. We describe our methods of analysis below, and details of each experiment appear on the plot including exposure and experiment length, special notecodes, and tumor incidence for each site. The first plot of the CPDB included interim results for 7 of these studies, using summary incidence data to estimate TD50 (1). These 7 are indicated with the notecode "j" in this plot.
The published authors evaluated 10 of the test agents as carcinogenic in monkeys: aflatoxin Bl, N-nitrosopiperidine, procarbazine.HC4 urethane, IQ, sterigmatocystin, cycasin and methylazoxymethanol acetate, N-methyl-N-nitrosourea, N-nitrosodipropylamine, and N-nitrosodiethylamine. Seven were not considered carcinogenic: 2acetylaminofluorene, sodium arsenate, N,N-dimethyl-4-aminoazobenzene, 3'methyl-4-dimethylaminoazobenzene, 3-methylcholanthrene, N-methyl-N'-nitro-N-nitrosoguanidine, and N-nitrosodimethylamine. We note that the protocol for most of the negative studies differed from that of all but one of the positive studies in that exposure to the test agent was stopped early in the experiment rather than being administered to death or nearly to death. Three other negative compounds are not included here because they did not meet the inclusion rules of the CPDB: cigarette smoke condensate (a mixture administered by implant); 2,4,9,10-dibenzopyrene and copper chelate of N-OHacetylaminofluorene (administered sc).
Studies are ongoing at NCI for 10 additional compounds; many animals are still alive, and few or no tumors have been observed in those that have died. These studies will be added to the CPDB when completed: adriamycin, melphalan, azathioprine, cyclophosphamide, cyclamate, saccharin, MeIQx, PhIP, DDT, and 2,7acetylaminofluorene.

Special Considerations
(i) Generally, few animals of each sex of either cynomolgus or rhesus monkeys were on test for each chemical, and there was usually only one dose group per experiment. Even combining results for males and females of each species, about half the experiments had 10 or fewer dosed animals, and never more than 22 in a dose group. Therefore, we have combined males and females of each species in our analyses; this results in having at least 5 dosed animals in all experiments but one (in which all of the 4 dosed animals developed tumors). (ii) Whereas experiments with surgical intervention are excluded from the CPDB, laparoscopic examination of the liver was performed every 3 to 6 months, followed by wedge or needle biopsies of observed liver lesions. (iii) We have included experiments that were shorter than our rule of one-half the standard lifespan, when tumors were induced in nearly all animals (cycasin, N-nitrosodipropylamine, IQO; these are indicated on the plot by the symbol "(+)". (iv) A few experiments have been included in which animals were put on test as adults (4

years of age). (v)
Control monkeys for all analyses are from the colony at NCI, which includes breeders, offspring, and some feral monkeys. At any given time, the age of colony control animals ranged from neonate to greater than 25 years. In our lifetable analyses, control animals of each species are included if they lived longer than 8 months of age, the age of the first tumor in any group in these experiments. [For one ongoing study, IQ in cynomolgus, only summary data were available and concurrent vehicle controls were used in our analyses (37).] We note that, compared to rodent bioassays, the tumor incidence rate in control monkeys in this colony is low: for all tumorbearing animals, whether benign or malignant, the tumor rate is 3% (3/99) in dead cynomolgus controls, and 10% (11/108) in rhesus.

Estimation of Average Daily Dose Level
In the CPDB for standard rodent bioassays the dose rate used to estimate TD50 is the daily dose rate for each group (in mg/kg body weight); if dosing is less than the experiment length, then the dose rate is obtained by averaging over the duration of the experiment. For this series of monkey studies we use the same averaging over experiment length; however, the dose rate for the group is calculated based on the average rate for individual animals. We obtained from NCI the total cumulative dose for each animal in mg/kg body weight. Using the age at death, we calculate an average daily dose rate for each animal, and the dose rate for the group (mg/kg/day) is the mean of these individual daily dose rates. This value can be considerably lower than the administered dose level reported by Thorgeirsson et al. (35) because the duration of dosing is often less than the experiment length (sometimes less than one-fourth the experiment length) and because dosing schedules ranged from once every 2 weeks to 5 times per week.
ifetable Analyses Estimates of TD50 are based on lifetable analysis, which adjusts for the differential effects of toxicity among dose groups and for differences between groups in the time pattern of tumor incidence.
On the plot, a TD50 is reported for each site at which a benign or malignant tumor was diagnosed in dosed monkeys. The denominators in each group, whether control or dosed, represent the number of animals alive at the age of the first death with Volume 103, Supplement 8, November 1995 5 the tumor of interest in any group. The numerator on the plot for control animals excludes tumors found at death in animals that lived to be older than the last dosed animal, and such cases are indicated by the notecode "W' on the plot.
Since individual animal pathology reports were made available to us, we were able to calculate a TD50 for combined tumors, just as we do in the CPDB for lifetable analyses of the NCI/NTP rodent bioassays; these are signified in the plot by the code MXB for "Berkeley Mix". For each experiment in monkeys, we estimated a TD50 for "all tumor-bearing animals" ("tba" on the plot) for all benign tumors, for all malignant tumors, and for any animal with either a benign or malignant tumor. The denominators in the tumor incidence fields of the plot for "all tumorbearing animals" represent the maximum number of animals used in any TD50 in the experiment, and indicate the number alive at the first tumor in any group. As for NCI/NTP bioassays, we also estimate a composite TD50 value for all sites that were evaluated as target sites by the published authors (35) This composite value (denoted on the plot by MXB MXB), is reported for four chemicals that were evaluated as having more than one target site in a monkey experiment.
Further analyses are in progress which compare results in rodents and monkeys. For example, the liver is the most frequent target site in monkeys, just as it is in mice and rats.
Addenda and Errata to Earlier Plots A few changes have been made to earlier plots. In plot 1 the chemical name "ethylene glycol" should have been "ethylene glycol, cyclic sulfate." Two code changes are necessary in previously reported results due to typographical errors. The code "cca" (c-cell adenoma) in the fifth plot was used with adrenal gland when the code should have been "coa" (cortical-cell adenoma) for the following chemicals: acetaldoxime, acrolein, acrolein diethylacetal, acrolein oxime, allyl alcohol, cyclohexanone, N-methyldopamine, 0, 0'-diisobutyroyl ester.HCI and 3-nitro-4-hydroxyphenylarsonic acid. In the first plot for N>N-dimethyl-4-aminoazobenzene, the tumor code for liver cell carcinoma should have been "1cc" but was reported as "cca". The author's opinion for urinary bladder papilloma in a test of sodium saccharin in plot 4 (Hasegawa et al., Cancer Research 45:1469-1473 has been changed from + (positive) to blank (none) following recent personal communication with the author. Kidney step section data in the CPDB for NTP bioassays are reported for the first time in this publication. Below we plot step section results for furosemide and nitrofurantoin, which were induded in an earlier plot without this data. The previously published results are included for comparison.       3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  3m24  9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 9.71mg 8.                     TETRAETHYLTHIURAM DISULFIDE  TETRANITROMETHANE  THIOACETAMIDE  THIOCYANATE, SODIUM  THIOURACIL  TITANOCENE DICHLORIDE  TOLUENE  2 434  1993  406  1992  381  1990  392  1992  392  1992  379  1990  377 1990 compilation of results on positivity and potency in rats and mice. Chemical names and common synonyms are listed alphabetically for the 1229 chemicals in the database, Chemical Abstracts Service registry (CAS) number is reported, and the plots that include experimental results on the chemical are listed by plot number.

Positivity
For each chemical a result is reported in male rats (MR), female rats (FR), male mice (MM), and female mice (FM). If there is no experiment in the CPDB for that sex-species group, this is indicated by NT. When all four sex-species groups are NT, the chemical was tested only in a species other than rats or mice (see footnotes g and h below). The classification of positivity is based on a positive result in at least one experiment, and we classify an experiment as either positive or negative on the basis of the author's opinion in the published paper. We use the author's opinion to determine positivity because it often takes into account more information than statistical significance alone, such as historical control rates for particular sites, survival and latency, and/or dose response. Generally, this designation by author's opinion corresponds well with the results of statistical tests for the significance of the dose-response effect. The strongest level of evidence of carcinogenicity in any experiment in the sex-species group is reported in Appendix 14 for each chemical. We indicate whether the compound was tested in each group and list the strongest level of evidence for carcinogenicity based upon any author's evaluation in either the general literature or the NCI/NTP. In the general literature, a (+) indicates a positive author's opinion, and a (-) indicates either that "no opinion" was reported for this experiment or that the opinion was negative. In the NCI/NTP the strongest evaluation is "clear evidence of carcinogenicity" (+). When there was no such evaluation in one of the sexspecies groups, but the compound was tested by NCI/NTP and their evaluation was stronger than "no evidence of carcinogenicity" (-), we indicate whether that NCI/NTP evaluation was "some evidence of carcinogenicity" (P), "equivocal" (E) or "inadequate bioassay" (1). For older NCI/NTP tests the evaluation (A) indicates "associated with carcinogenicity," and we do not interpret this as positive. These evaluations correspond to the opinions reported in our published plots. The abbreviations for positivity in Appendix 14 are as follows: NT No test in the CPDB in this group + The CPDB contains at least one experiment in which the compound was evaluated as a carcinogen by the published author. For NCI/NTP tests, the evaluation was "clear evidence of carcinogenicity." P The strongest level of evidence in the CPDB was an NTP evaluation of "some evidence" of carcinogenicity.
No tests in the CPDB in this sex-species group were evaluated as positive; however, the NCI/NTP test was evaluated as inadequate. A The strongest level of evidence in the CPDB was an NCI/NTP evaluation of "associated with carcinogenicity." E The strongest level of evidence in the CPDB was an NTP evaluation of "equivocal." -All tests in this group were negative. B+ In the only positive test in the sex-species, results were reported only for males and females combined. B-In the only test in the sex-species, results were reported only for males and females combined, and the test was negative.

Carcinogenic Potency
For the purposes of Appendix 14, TD50 values for a chemical are reported only for a species with a positive evaluation of carcinogenicity in at least one test. In any given positive experiment we select the lowest TD50 value from among positively evaluated target sites with a statistically significant dose response (two-tailed p<0.1). If no positive sites have a significant dose response, then we select the most potent (lowest TD50) from among positively evaluated sites with p20.1. This method provides a single TD50 to represent an experiment. For chemicals with more than one positive experiment, we summarize potency in a species by selecting the lowest significant TD50 value from among those representing each experiment. If none is significant, the lowest TD50 is chosen from among the nonsignificant values with a positive author's opinion (see footnote b below). In some experiments, no TD50 could be estimated because all dosed animals had the tumor of interest, and the only data available were for crude percentages of animals with a tumor. For these cases we use the 99% upper confidence limit of TD50 as a replacement for the TD50.
In a series of footnotes, we provide additional information about TD50 values and test results in the CPDB. These are as follows: a The CPDB contains more than one positive test in the species.
Environmental Health Perspectives b The reported TD50 is not statistically significant (i.e., p20.1), and all results evaluated as positive in the species are not significant. c Only an upper bound and no TD50 could be estimated because all dosed animals had the tumor of interest and only summary data were available. The reported value is the 99% upper confidence limit. d All positive results for this species in the CPDB are from tests in which the compound was administered by either intraperitoneal or intravenous injection. e The reported TD50 is from a test in which the compound was administered by intraperitoneal or intravenous injection; however, the CPDB also contains a positive test in this species with a less potent TD50 value from a test where the route was oral or inhalation. f TD50 values from different significant, positive experiments in this species vary by more than tenfold from one another. The most potent TD50 value is reported here. g The CPDB includes tests in a species other than rats or mice, and at least one test is positive. h The CPDB includes tests in a species other than rats or mice, and all tests are negative. i Data on four NCI bioassays are included in Appendix 14 but are excluded from the analyses and tables in this and previous papers: C.l. Direct Black 38, C.l. Direct Blue 6, and C.l. Direct Brown 95, which were only tested subchronically; 3-amino-9-ethylcarbazole. HCland 3-amino-9ethylcarbazole mixture were tested by NCI in one bioassay but we separated them in the CPDB because slightly different chemicals were used for different dose groups; we combined them for our analyses, as NCI had done.      Number  Number  Chemical Name  NT  -NT  NT  NT  NT  +  NT  NT  NT  NT  +  NT  NT  NT  594a   +  +  +  +   -NT  NT  --NT  +  + NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT   NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT  NT -THIODIANILINE  beta-THIOGUANINE DEOXYRIBOSIDE  THIOSEMICARBAZIDE  THIOURACIL  THIOUREA  THIRAM (see TETRAMETHYLTHIURAM DISULFIDE