Carcinogenesis Bioassay Results from the National Toxicology Program

This article begins a series of condensations on the National Toxicology Program's (NTP) toxicology and carcinogenesis bioassay results as summarized from the NTP Technical Report collection. This first compilation lists those 40 carcinogenesis bioassay studies (Table 1) that have undergone both NTP staff interpretation and evaluation as well as external peer review and critique. The majority of these studies were initiated by the National Cancer Institute's Carcinogenesis Testing Program, now part of the National Institute of Environmental Health Sciences, National Toxicology Program. Precis on individual NTP Technical Reports will be submitted for publication in Environmental Health Perspectives (EHP) at the same time that the approved reports are delivered for printing. For instance, information and data condensations on four recently printed carcinogenesis bioassay reports [caprolactam, cytembena, di(2-ethylhexyl) adipate, and FD&C Yellow No. 6] appear as separate inserts following this introductory article. Considerably more abbreviated results are published routinely in the NTP Technical Bulletins. More extensively treated manuscripts are being prepared for publication consideration in EHP and in other recognized peer-review journals. This allows not only more rapid and wider dissemination of results generated by the NTP, but permits open interchangebetween scientists/organizations engaged or interested in similar research and testing activities. For orientation purposes, the following overview highlights the National Toxicology Program, the chemical nomination and selection process, and the toxicology and carcinogenesis bioassay program.

This article begins a series of condensations on the National Toxicology Program's (NTP) toxicology and carcinogenesis bioassay results as summarized from the NTP Technical Report collection. This first compilation lists those 40 carcinogenesis bioassay studies (Table 1) that have undergone both NTP staff interpretation and evaluation as well as external peer review and critique. The majority of these studies were initiated by the National Cancer Institute's Carcinogenesis Testing Program, now part of the National Institute of Environmental Health Sciences, National Toxicology Program.
Precis on individual NTP Technical Reports will be submitted for publication in Environmental Health Perspectives (EHP) at the same time that the approved reports are delivered for printing. For instance, information and data condensations on four recently printed carcinogenesis bioassay reports [caprolactam, cytembena, di(2-ethylhexyl) adipate, and FD&C Yellow No. 6] appear as separate inserts following this introductory article. Considerably more abbreviated results are published routinely in the NTP Technical Bulletins. More extensively treated manuscripts are being prepared for publication consideration in EHP and in other recognized peer-review journals. This allows not only more rapid and wider dissemination of results generated by the NTP, but permits open, interchange between scientists/organizations engaged or interested in similar research and testing activities.
For orientation purposes, the following overview highlights the National Toxicology Program, the chemical nomination and selection process, and the toxicology and carcinogenesis bioassay program. The NTP completed its fourth year of operation and first year in permanent status in Fiscal Year (FY) 1982. The NTP was established in November 1978 as a Department of Health and Human Services (DHHS) cooperative effort to coordinate and provide information about potentially toxic chemicals to regulatory and research agencies and to strengthen the science base in toxicology. The need for creation of such a program evolved from increasing scientific, regulatory, and congressional concerns about the human health effects of chemical agents in the environment.
A major reorganization occurred within NIH in FY 1981: the transfer of the carcinogenesis bioassay program from the National Cancer Institute (NCI) to the National Institute of Environmental Health Sciences (NIEHS) was approved by the Secretary, DHHS, in the fourth quarter of FY 1981. Further, at the beginning of FY 1982, the Secretary granted permanent status to the National Toxicology Program.
Under the broad objective of providing the necessary scientific information for prevention of human disease related to chemical exposure, the NTP emphasizes four principal goals: (1) broaden the toxicological characterization of chemicals that are tested; (2) increase the rate of chemical testing (as funding will permit); (3) develop protocols appropriate for regulatory needs; and (4) communicate program plans and results to governmental agencies, the medical and scientific communities, and the public.
Although testing chemicals for toxicity, particularly for carcinogenicity and mutagenicity, remains an important program focus, increasing emphasis over the first four years has been placed on short-term test methods development and validation. A second primary emphasis continues to be   (1 0 Cd the broadening of the testing protocols through addition of other specific studies to the prechronic phases (single, 14-day, and 90or 120-day studies) of the long-term bioassay'and, to a lesser extent, to the chronic phase (104 weeks) of the bioassay.
Accomplishments of NTP programs during FY 1981 and program plans for FY 1982 are described in the NTP Annual Plan for Fiscal Year 1982.

NTP Chemical Nomination and Selection
Because more chemicals are nominated for NTP consideration than can be selected for study, the NTP Executive Committee formulated a set of program guidelines. These resultant eight chemical selection criteria motivate an NTP matrix which operates throughout the NTP. All research, testing and test development/validation efforts start here.
The NTP Executive Committee acts under the principle that industry will test chemicals for health and environmental effects as intended and mandated by Congress under legislative authorities. Therefore, the NTP, using these chemical selection principles, will test: (1) chemicals found in the environment that are not closely associated with commercial activities; (2) desirable substitutes for existing chemicals, particularly therapeutic agents, that might not be developed or tested without federal involvement; (3) chemicals that should be tested to improve scientific understanding of structure-activity relationships and thereby assist in defining groups of commercial chemicals that should be tested by industry; (4) certain chemicals tested by industry, or by others, the additional testing of which by the federal government is justified to verify the results; (5) previously tested chemicals for which other testing is desirable to crosscompare testing methods; (6) "old chemicals" with the potential for significant human exposure which are of social importance but which generate too little revenue to support an adequate testing program (some of these may be "grandfathered" under FDA laws); (7) two or more chemicals together, when combined human exposure occurs (such testing probably cannot be required of industry if the products of different companies are involved); and (8) in special situations, as determined by the Executive Committee, marketed chemicals which have potential for large-scale -and/or intense human exposure, even if it may be possible to require industry to perform the testing.
Most chemicals are nominated and selected for testing because toxicologic information is lacking and because the potential exists for human exposure. Other important criteria include production 193 levels, physical and chemical properties, agency interests, and significance to society. The NTP toxicology testing strategy: identify with assurance the major toxic effects for each chemical studied. This includes (in addition to identifying chemical mutagens and carcinogens) damage to critical target organs such as the reproductive system, lungs, liver, and nervous system. Nominations of chemicals for toxicological testing are submitted by the NTP participating agencies as well as other government agencies, industry, labor, and the public. The nominating source is asked to submit the name of the chemical, the particular toxicological tests desired, the rationale for testing, and to provide the available background data on production, use, exposure, environmental occurrence, and toxic properties in a supporting summary document.
An initial examination determines which proposed chemicals have already been tested, are being tested, are scheduled for test, or have been previously considered and rejected for testing by the NTP or its predecessors.
Literature containing relevant data is assessed and literature summaries are prepared for each chemical. Included in each literature summary are sections on chemical identification and physical properties, surveillance index (production, use, environmental occurrence, and available regulatory status and exposure limits), human exposure and health effects, research hypothesis to be tested, categories of study, and source of and reason(s) for nomination. Chemicals nominated for mutagenicity testing are reviewed only with regard to the available genetic toxicology information.
These summaries are reviewed and evaluated by the Chemical Evaluation Committee (composed of representatives from CPSC, EPA, FDA, OSHA, NCI, NCTR, NIEHS, NIOSH, and NTP)* who recommend the type(s) of testing to be considered. All suggestions and recommendations for future research and testing activities must satisfy at least one of the eight NTP principles of chemical selection.
Announcements listing the chemicals and the recommended types of testing appear in the Federal Register and the NTP Technical Bulletin. tion on completed, ongoing, and planned testing in the private sector. These steps are taken to encourage others outside the immediate program to participate in the NTP evaluation and selection process, as well as to specifically prevent unnecessary duplication. Revised summaries with additional public input are forwarded to the Board of Scientific Counselors for review. The Board evaluates these data and makes recommendations to the Executive Committee.
Final summaries are submitted to the NTP Executive Committee who decide whether to select, defer, or reject the chemicals for testing. Following Executive Committee action, the chemicals are referred by the NTP Steering Committee to one or more participating agencies within the NTP: NIEHS, NIOSH, NCTR. At this stage certain approved chemicals may be identified as being inappropriate candidates for testing as a result of technical or budgetary reasons or in some cases public information describing ongoing testing may only have been submitted following Executive Committee decision. Such chemicals are then returned to the Executive Committee for reconsideration.
All chemicals selected are then tested as time and resources permit.

Toxicology and Carcinogenesis Bioassay
The "standard" two-year carcinogenesis bioassay remains as the most definitive method for detecting chemical carcinogens in animals. The standard protocol as developed by the NCI (and frequently still used by the NTP) typically uses two rodent species (usually Fischer 344 rats and B6C3F1 mice), both sexes, and administration of multiple dose levels (concurrent controls, low dose, and high dose) of a chemical to groups of 50 animals, beginning at weaning and ending after two years. These experiments are designed primarily to determine whether selected chemicals produce cancer in animals. Chemicals tested in the NTP Carcinogenesis Bioassay Program are chosen primarily on the bases of human exposure, available (or lack of) toxicology data, level of production, and chemical structure. Selection per se is not an indicator of a chemical's carcinogenic potential.
Negative results, in which the test animals do not have a greater incidence of cancer than control animals, do not necessarily mean that a test chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set of conditions. Positive results demonstrate that a test chemical is carcinogenic for animals under the conditions of the test and indicate that exposure to the chemical is a potential hazard to humans. The determination of the risk to humans from chemicals found to be carcinogenic in animals often requires a wider analysis which at present extends beyond the purview of these studies.
The results of the bioassay also serve as the reference base for the validation of short-term carcinogenesis assays. Two additional objectives have been identified as priority items: (1) to expand the bioassay experimental protocols to extend and better characterize the toxicologic proffle of chemicals; and (2) to investigate, develop, and validate accurate, less costly, and more rapid methods for detecting carcinogenic potential.
Under the NTP, the carcinogenesis bioassay procedure(s) has been and continues to be changed to meet the objective of a broadened toxicologic characterization of chemicals and, further, to lead or stay abreast of advancing scientific developments. Prior to NTP involvement, the prechronic phases of the bioassay-which include single dose (acute), 14-day repeated dose, and 90to 120-day repeated dose studies-were conducted to determine gross toxicity and general target organ effects at different dose levels as a primary basis for setting appropriate doses for the two-year bioassay studies. Now, the NTP has begun to gather routinely other information related to target organ effect: chemical disposition, fertility and reproduction, urinalysis, and hematology also are obtained from the prechronic studies-especially the 90-day study; certain other specific studies as applicable are included in the chronic two-year studies as well. Once those parameters that may be altered through exposure to the tested chemicals are identified, then suspect chemicals are referred to specific organ system groups for more detailed study of the functional, biochemical, and morphologic effects of the test compounds. Also, wider analysis of the quantitative and comparative absorption, distribution, metabolism, and excretion patterns may be desired. For instance, 28 (70%) of the 40 chemical starts in FY 1980 included specific toxicology studies in the prechronic testing phase. All chemicals started on test in FY 1981 had an expanded design including other select studies. Significantly all chemicals selected for chronic bioassay will be profiled for chemical disposition patterns. The goal is to ensure that all major toxic effects will be identified for each chemical being considered for long-term bioassays.
With this composite information base, the doses for the chronic study are selected. The high dose, termed the estimated maximum tolerated dose (EMTD), represents the highest dose of a chemical or substance given during a chronic study that can be predicted not to alter the treated animals' normal longevity from toxic effects other than carcinogenicity. The low dose ordinarily equals 1/2 EMTD. Other empirical factors include weight gain/food consumption data; for instance, a decrease in weight gain near 10-15% (not associated with a tumorigenic response) is often used as a general indication that the EMTD was achieved.
Prior to commencing the actual long-term carcinogenesis bioassay, all chemicals undergo genetic toxicology testing in at least five in vitro short-term assays: (1) gene mutations in backteria-Salmonella typhimurium microsome; (2) gene mutations in mammalian cells-mouse lymphoma (L5178Y, thymidine kinase); (3) chromosome damage in mammalian cells -cytogenetic damage and sister chromatid exchange (in vitro, CHO); (4) a mammalian cell transformation assay-(BALB/c -3T3); and (5) a direct measure of DNA damage/repair (which does not necessarily result in mutation or transformation)-unscheduled DNA synthesis (rat hepatocytes). These data, together with other prechronic bioassay information, are used by the experimental design groups for preparing appropriate study protocols and are used by staff for assisting in establishing priorities for chemicals queued into the long-term carcinogenesis bioassay. A key decision that must be made at this juncture between the completion of the prechronic phase and the beginning of the chronic study centers directly on whether indeed the lifetime bioassy should be done at all.
Thus, while the lifetime animal bioassay remains the best procedure for determining the carcinogenic potential of chemicals, NTP does not ordinarily use a standardized design. Rather the design is adapted to the special testing needs identified for the particular chemical. The NTP tailors its testing protocols to the particular chemicals based on the results from the prechronic testing phases, on available literature, and on structure-activity rela-195 tions. These new protocols permit better, more specific information to be generated for the tested compounds, which increases the effectiveness of the tests for potential human risk estimations. Such protocols also will be useful as guidelines for testing undertaken by other agencies and by industry. As examples, the NTP continues to pursue actively other design methodologies-increase the number of dose levels, "unbalanced" distribution of animals among dose groups, interim kills, and reduced essential histopathology.

Bioassay Results
During FY 1981, 23 long-term carcinogenesis bioassays were completed and the conclusions and data were evaluated and approved by the NTP staff review committee and subsequently by the external NTP peer review panel. Under the conditions of these carcinogenesis bioassays, 12 (52%) were considered negative, 10 (44%) positive, and 1 (4%) equivocal. One, polybrominated biphenyl (PBB) mixture (Firemaster FF-1), was conducted using a broadened experimental protocol aimed at providing a better dose-response determination (through use of six doses) and at giving more data on toxicologic end points other than induction of cancer. Nontumor toxicities of this PBB included porphyrogenic effects, decreases in serum thyroid hormones, hematotoxicity, enzyme alterations, and hepatotoxicity.
As of February 1982: (1) Twenty-five chemicals have been newly assigned to the testing laboratories (Table 2). (2) Seventy-nine chemicals (89 separate studies) were in the prechronic testing  (Table 3); most include one or more additional toxicology studies in the prechronic testing phase.
(3) Ninety-six chemicals frequent the chronic two-year testing stage (Table 4). There are a number of new or ongoing long-term carcinogenesis studies concerned with chemicals or mixtures of chemicals which humans encounter in occupational settings (benzene, diesel fuel marine, gentian violet, napththalene, Navy fuels JP-5, as examples). (4) Twentynine chemicals (35 separate studies) are in the histopathology phase (Table 5). (5) For 10 chemicals, reports are being written or draft reports are J. E. HUFF in program review (Table 6). Including the new chemical starts, there were 185 bioassays in either the prechronic or the chronic bioassay testing phases at the end of FY 1981 (Tables 3 and 4). (6) In FY 1982, the NTP expects to complete (through peer review) 29 long-term bioassays (Tables 6 and  7). Abstracted from the peer-reviewed approved draft technical reports, Table 1 contains assorted information about the particular chemical carcinogenesis study (CAS Registry Number, peer review date, use, route and dose, contract laboratory, and  asbestos-DMH oral studies). The letter "P" following the Technical Report (TR) number in column two means that the NTP Technical Report has been published; detailed condensations of four carcinogenesis bioassays so designated follow this article. The others settle into various stages of publica-