Document YGpnQZv6odeGX7aw7O7Db0mBE

Rashtni S. Nair F A C T O R S IN T H E E V A L U A T IO N O F 200 NATIONAL CAN CER IN STITUTE CARCINOGEN BIOASSAYS Kenneth C . Chu, Cipriano Cueto, Jr., Jerrold M. Ward National Toxicology Program, National Cancer Institute, Bethesda, Maryland In determining the carcinogenicity o f a chemical tested in a National Cancer institute (NCi} bioassay, the following criteria are considered: ( I ) the adequacy o f the bioassay data, (2) the presence o f significantly increased incidences o f tumors, (3) the adequacy o f the number o f animals at risk o f developing tumors, (4) the adequacy o f the dose o f chemical administered, (5) the etiology and pathogenesis o f the lesions, and (6) other factors that may influence an evaluation, such as a shortened latency period for tumor formation in dosed animals or the stability o f the chemical. A decision tree for evaluating these factors is presented. A summary o f the results o f 200 NCi carcinogen bioassays is also reported. These procedures are presented in the hope that they may serve as discussion points for future developments in the field. IN T R O D U C T IO N It has been estimated that many human cancers are caused by chemical and environmental factors (Boyland, 1969; Higginson, 1972; Doll, 1977,.1978). A number of methods have been used to determine the potential health risk of chemicals to humans. These methods include epidemiologic studies, animal experiments (carcinogen bioassays), and in vitro studies. The most direct approach is to determine the effects of chemicals on humans. However, the testing of chemicals in humans is ethically unacceptable. After an accidental or unintentional exposure of humans to chemicals there may be a latency period of 20-30 yr (Doll, 1971, 1978) before the appearance of any carcinogenic effect, which precludes an immediate epidemiologic evaluation. Although in vitro tests show promise for the future, the carcinogen bioassay is presently the standard method for determining potential carcinogenic risks to humans. For more than 8 yr the National Cancer Institute (NCI) has been directing such tests. The many discussions with members of the NCI Data Evaluation Group are greatly appreciated. Dr. R. A . Grtesemer provided additional insight into the subject matter. , Supported in part by Public Health Service contract NOl-CP-43350 to Tracor Jitco, Inc., and N 0 1-CP-43257 to E G & G/Mason, Inc. Requests for reprints should be sent to Kenneth C. Chu, National Toxicology Program, Room 3-A-06, 7910 Woodmont Avenue, Bethesda, Maryland 20205. 251 Journal of Toxicology and Environmental Health, 8:251-280,1981 Copyright 1981 by Hemisphere Publishing Corporation 0098-4108/81/060251-30$2.25 ^ U During this period, NCI has tested more than 200 chemicals in carcinogen bioassays with two species of rodents. The vast majority of the . results of these studies are published in the NCI carcinogenesis technical report series. There have been more than 190 reports on 200 chemicals. Because of the importance of these reports in assessing the potential health hazards to humans, we will discuss the general criteria used in evaluating the carcinogenicity of a chemical tested in an NCI carcinogen bioassay. This paper represents the views of the authors and does not' necessarily reflect the views of NCI. M ATERIALS AND METHODS The NCI Carcinogen Bioassay ; In a typical NCI chronic bioassay for carcinogenicity (Sontag et al., 1976; Page, 1977), the test chemical is administered continuously for 18-24 mo to groups of 50 male and female (C57BL/6N X C3H/HeN) F1(B 6C 3F1) hybrid mice and for 20-24 mo to groups of 50 male and female Fischer 344 rats. On past occasions, Osborne-Mendel and SpragueDawley rats have also been used. The dosing period may be followed by a 3-6 mo observation period. Generally, for each species, strain, and sex used there are a control group and two treated groups, one given the maximum tolerated dose (MTD), termed the high-dose group, and the other given half the MTD, termed the low-dose group. The MTD is taken as "the,,highest dose that causes no more than a 10% weight decrement, as compared to the appropriate control groups, and does not produce mortality, clinical signs of toxicity, or pathologic lesions (other than those that may be related to a neoplastic response) that would be predicted to shorten the animal's natural life span" (Sontag et al., 1976), as determined from the subchronic study. This dose is used to increase the sensitivity of the test by giving the animals as large a dose as possible, consistent with longevity. During the study, the animals that die are necropsied. At the end of the observation period, all surviving animals are sacrificed and necropsied. Diagnoses of lesions in any of the 25-30 tissues (Sontag et al., 1976) taken from each animal are made by pathologists by microscopic examination. General Evaluation of the Bioassay For the purpose of evaluation, an NCI carcinogen bioassay can be divided into four separate experiments. Each involves the high-dose, low-dose, and control groups of a species, strain, and sex. Thus there is an experiment on the male mice, the female mice, the male rats, and the female rats. Correspondingly, the carcinogenicity of a chemical is assessed for each experiment with a species, strain, and sex. For each experiment, one of four possible conclusions can be drawn IT AU Is in f the nica! icals. mLiai id in logen ; not it al.# y for HeN) e and rague- I by a d sex n the d the taken :nt, as oduce those ted to mined ly of t with to the :d and et al., >scopic EVALUATION OF NATIONAL CANCER INSTITUTE CARCINOGEN BIOASSAYS 2S3 for a species, strain, and sex: (1) the compound is carcinogenic, (2) the compound is a suspected carcinogen, (3) the compound has not been shown to be carcinogenic, or (4) the data are inadequate for further analysis and do not permit a determination of the carcinogenicity of the test chemical {inadequate test). Guidelines for evaluating an NCI carcinogen bioassay on a species, strain, and sex are presented in Fig. 1. V Evaluating a Bioassay One of the first steps in evaluating a bioassay is to determine whether the data are adequate for analysis and reporting. Irregularities in the r I ' I S: FIG U R E 1. Guidelines for evaluating an NCI carcinogen bioassay. 'mm 254 K. C. CHU E T A t f ! experimental design or in the performance of the bioassay that bias the^ tumor incidences in the controls and/or the dosed groups can make I further evaluation meaningless. Two major causes of invalid bioassay data, are procedural and operational problems. The first problem occurs when the tumor incidence in the control group is unusually high due to flaws in the execution of the bioassay. For example, in the case of methiodal sodium, an unusually high incidence of leukemia was found in the control, group as well as the dosed groups. These high, incidences were, on further investigation, linked to the ip route of administration and/or the injection, vehicle, not the chemical. Consequently, the bioassay was considered, inadequate. The second problem occurs when the dosed animals have-a: high degree of autolysis, preventing histopathologic examination of the tissues. Specifically, in the bioassay of imidazole mustard a large number of animals were autolyzed, so that from each group of 25 animals only 5 to 10 animals could be evaluated. The data from this study were considered inappropriate for further analysis and were not reported in the NCI technical report series. If the bioassay data are adequate for analysis, the next step is to determine whether there are any chemically induced carcinogenic effects-- that is, whether there are significantly increased incidences of tumors in the dosed groups as compared to the controls. This complex process involves evaluations by pathologists, statisticians, and toxicologists and is discussed later. If there is a significant effect, additional complicating factors may alter the finah conclusion about a chemical. For example, if there is a survival problem with the controls, the tumor incidences in the controls may be artificially low, since they were not at risk of developing tumors for as long a period as the dosed animals. In these cases, it may be appropriate to compare the tumor incidences in the dosed groups with historical data on laboratory controls with similar longevity, if other variables are similar. This may change the nature of the evidence so that it supports, not a carcinogenic effect, but only a suspected carcinogenic effect. Another complicating factor is the stability or reactivity of the test chemical. For example, during the bioassay of dicofol (NCI-TR-90,. 1978), the chemical changed from a solid to a liquid during storage. If such a change suggests some type of decomposition, it would affect the conclusion' drawn from the bioassay data. In the case of dicofol, although the test substance pro duced hepatocellular carcinomas in 11% of the male mice, the substance was considered a suspect carcinogen, rather than a carcinogen, because its exact identity was not known. These examples show that even-, though there is a significant positive effect, complicating factors may affect the final conclusion on an experiment Even when there are no significant positive effects, there may be evidence suggestive of a carcinogenic effect. For example, there may be no -1 & EVALUATION OF NATIONAL CANCER INSTITUTE CARCINOGEN BIOASSAYS 255 statistically significant tumors, but a progression of proliferative lesions leading to a neoplasm may be seen in the dosed animals. In another case, a rare tumor may be observed in several dosed animals. In a third case, there may be a positive effect in only the low-dose group while the survival in all groups is similar. All these cases may lead to the conclusion that the test chemical is a suspected carcinogen, depending on the particular circumstances. If there are no positive or suggestive effects, then one must examine the survival of the animals. A primary issue is the number of animals at risk of developing a tumor relative to the number initially on test. The basic concern is that a sufficient number of animals remain alive long enough to be at risk of forming late-developing tumors. To be considered adequate, an experiment that has not shown a chemical to be carcino genic should have groups of animals with greater than 50% survival at 1 yr (52 wk). Fo r example, in the bioassay of methylchloroform (NCI-TR-3, 1977) no positive results were found. However, the survival in most groups was poor. O f the Osborne-Mendel rats used, 32 of 50 (62%) low-dose males, 36 of 50 (72%) high-dose males, 24 of 50 (48%) low-dose females, and 21 of 50 (42%) high-dose females died within 1 yr after the start of the study. O f the mice, 21 of 50 (42%) low-dose males, 25 of/5<J (50%) high-dose males, 9 of 50 (18%) low-dose females, and 20 of \40 (40%) high-dose females died within 1 year after the start of the study.'Because of this poor survival and the absence of positive results, the study was considered inadequate for a determination of the carcinogenicity of the chemical. On the other hand, if a study is positive, the survival criteria are not applicable. For instances, in the dibromochloropropane (DBCP) study (NCI-TR-28, 1978) the survival of the dosed mice was less than 50% at 1 yr, but the animals died from stomach tumors induced by DBCP. The bioassay is adequate and the compound is carcinogenic in spite of the low survival rate of the animals. Another major concern with studies that show no significant positive results is the adequacy of the administered dose. In NCI bioassays where > Ihe objective is to determine whether a chemical is carcinogenic at the MTD, the concern is whether the animals actually receive the MTD. In these studies, a dose is considered adequate if there is a detectable weight loss of up to 10% in a dosed group relative to the controls. For example, the doses of chlorpropamide administered to Fischer 344 rats were considered adequate since there was a 10% weight depression in the dosed rats compared to the controls (NCI-TR-45, 1978). The administered dose is also considered an MTD if the dosed animals exhibit clinical signs or severe histopathologic toxic effects attributed to the chemical, so that larger doses of the chemical may be inappropriate or life-threatening. An example is the bioassay of photodieldrin (NC1-TR-17, 1977) in B6C3F1 mice and Osborne-Mendel rats. There was no depression of mean body weights in the dosed animals compared to the controls, and there was no' effect of the chemical on mortality of the dosed animals. However, there were chemically induced clinical signs--convulsions and hyperexcitability-- . n in the dosed rats and male mice. Thus the dose administered to these m animals was considered adequate. In addition, doses are considered adequate if the dosed animals show a slightly increased mortality com pared to the controls. For example, in the bioassay of emetine (N CI-TR43, 1978), the male mice did not exhibit any significant tumor incidences, clinical signs, or weight depression attributed to the chemical. However, there was a decrease in survival in the animals fed 1.6 mg/kg (higher doses' led to greater mortality) relative to the controls. The median survival of this dosed group was 72 wk, compared to 80 wk for the controls. This difference in survival time was considered to indicate that a sufficient dose had been administered. Finally, doses of chemicals given as 5% of the diet are generally considered adequate (Sontag et al., 1976) whether or not there are any chemically related effects, since larger doses may interfere with the nutrition and survival of the dosed animals. The bioassay of diaryl anilide yellow (NCI-TR-30, 1978) is a prime example. There were no chemically related lesions, weight depression, mortality, or other adverse effects, but the doses were considered adequate since the chemical was administered as 5 and 2.5% of the diet. -Si Just as there is concern about administering an insufficient dose, there is also concern that too large_ a dose may be given. Thus the toxicity of the chemical may cause the dosed animals to die before they can develop tumors. For example, when emetine (NCI-TR-43, 1978) was administered to male mice at 6.4 and 3.2 mg/kg, almost all the animals died before the end of the first year. Therefore a dose of 1.6 mg/kg was administered to a third group of mice. The operational and procedurai parts of a bioassay are generally evaluated by the toxicologists. Their evaluations are added to those of the pathologists and statisticians in making the final evaluation of an experiment. Interpretation by the Pathologists A principal role of the pathologist is to decide whether there are any chemically induced lesions. Before the tumor data are interpreted, the histopathologic diagnoses are checked for proper protocols, data recording, and consistency. All pathology reports are reviewed by a team of pathologists, and discrepancies are resolved before acceptance for any further evaluation (Ward et al., 1979). If the data are considered unevaluatable (i.e., incomplete) or uninterpretable, the experiment is termed inadequate to determine the carcinogenicity of the compound. The pathologists review the microscopic slides, make histopathologic diagnoses, and document the findings. They inspect the tumor and nontumor incidence tables and write up the interpretation of the findings. The pathologist's conclusions are based on (1) a review of the EVALUATION OF NATIONAL CANCER INSTITUTE CARCINOGEN BIOASSAYS 257 incidence of the lesions, (2) knowledge of the normal variation in the incidence of the lesions, and (3) an opinion on whether the lesions are induced by the chemical. The latter is determined by answering several questions: 1. Are the lesions morphologically different from naturally occurring lesions? 2. Are the lesions rare in control animals? 3. What are the pathogenesis and histogenesis of the lesions (e.g., are toxic lesions, hyperplasias, dysplasias, adenomas, and carcinomas of a tissue related)? The histogenesis of a neoplastic lesion is its progression from its nonneoplastic origin, often hyperplasia, to a metastatic cancer that kills the animal. For example, the morphology and histogenesis of rat liver tumors were reviewed by Squire and Levitt (1975). Beginning with foci of cellular alteration (hyperplasia), the lesion progresses to neoplastic nodule, to hepatocellular carcinoma, and eventually to metastatic carcinoma. In certain cases, the morphology of a chemically induced lesion can be differentiated from that of a naturally occurring lesion, e.g., "induced" squamous cell carcinoma of the lung as compared to the usual "spon taneous" alveolar-bronchiolar tumor. From these types of considerations, the pathologist draws a conclusion about the significance of the tumors seen in the experiment. For example, in the bioassay of captao in B6C3F1 mice (NCI-TR-15, 1978), there was no statistically significant increase in duodenal tumors; the incidences were 1 in 43 of the low-dose and 3 in 46 of the high-dose male mice, and 0 in 49 of the low-dose and 3 in 48 of the high-dose female mice. However, the pathologists felt that this rare tumor was related to administration of the chemical. The chemical was called a suspected carcinogen. Another study is in progress to confirm this finding. A major issue in determining the carcinogenicity of a compound is the nature of the significant tumor types (Goodman et al., 1979; Ward et al., 1979). If malignant tumors or a combination of malignant and benign tumors are produced, then the compound is considered carcinogenic to the animals. If the significant result is only the production of benign tumors, then the compound may pose a potential health hazard and is termed a suspected carcinogen or a carcinogen, depending on the nature of the benign tumor. For example, 2,4-dinitrotoluene (NCI-TR-54, 1978) was considered a suspected carcinogen since it induced only benign tumors (fibromas of the skin and subcutaneous tissue in male Fischer 344 rats and fibroadenomas of the mammary gland in females). Ideally, a distinction should be made between truly benign tumors, which never progress to malignancy, and tumors that are in a benign state according to histo pathologic criteria at the time of diagnosis. Scientific judgments in this area are limited by inability to predict the biological behavior of a lesion on the basis of morphological criteria, but Ft appears that there are few, if any, truly benign tumors in rodents. If this were true, all chemicals t h a t induce benign tumors would be termed carcinogens. Statistical Analysis " 'i A statistical analysis of tumor incidences is performed to determine :;whether the proportion of dosed animals with a tumor is different from " the proportion of tumor-bearing animals in the controls. Accordingly, the i incidence of neoplastic lesions is expressed as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined. For example, if 50 animals were 1 necropsied but only 49 liver tissues were examined, the denominator for the proportion with liver tumors, such as hepatocellular carcinomas, is 49. However, when gross examination was required to detect lesions for histological examination (e.g., skin or mammary gland) or when lesions could have appeared at multiple sites (e.g., lymphomas), the denominator is the number of animals necropsied. In almost all NCI bioassays, there are no serial sacrifices (no animals are killed at predetermined intervals before the termination of the bioassay), so that the reported "time to tumor" is the time of death of the animal with the tumor, i.e., the time to the detection of the tumor. If the tumor is life-threatening, then the time of death may be a good approximation for the time of tumor appearance. This is the case for leukemias and lymphomas, gastrointestinal cancers that lead to peritoneal metastases, and upper respiratory tract tumors that are associated with asphyxiation or pneumonia. In addition, if the tumors are observable and the time of their appearance is recorded, as for neoplasms of the skin or mammary gland, then the time to tumor appearance can be obtained. On the other hand, tumors that are not immediately life-threatening are generally detected at terminal sacrifice or at the death of the animal if the chemical is toxic. The tumor incidences and time of death of each animal are used in analyzing the significance of tumor incidences. The basic statistics associ ated with the analysis of these ratios are presented here; a full discussion of the statistical methodology for analyzing bioassay ;data has been presented elsewhere (Gart et al,, 1979). Tumor incidences can be analyzed by the one-tailed Fisher, exact test (Cox, 1970), which is used to compare the tumor incidence of a control group to that of a group of dosed animals at a given dose leveK When the control is compared to k dosed groups pairwise, a correction to ensure an overall significance level of 0.05 is made. The Bonferronr . inequality (Miller, 1966) requires that the p value for any comparison be less than or equal to 0.05/& to be significant at an overall level of 0.05, where k -- 2 in most cases. Tumor ratios can also be analyzed by the Cochran-Armitage test for a EVALUATION OF NATIONAL CANCER INSTITUTE CARCINOGEN BIOASSAYS 259 linear trend in proportions with continuity correction (Thomas et al., 1977). Under the assumption of a linear trend, this test determines whether the slope of the dose-response curve is different from zero at the one-tailed 0.05 level of significance. This method also provides a two-tailed test of departure from linear trend. When time-to-tumor data are available, life-table methods are also appropriate (Gart et al., 1979; Thomas et al., 1977). A scheme for the initial evaluation of these statistical tests is given in Table 1. These evaluations are based on the assumption that there was adequate and approximately equal survival in each group. The scheme is based on observing how statistical results are finally resolved in the 200 NCI reports. It is presented as an empirical formulation and does not necessarily reflect present or future evaluations. It is stressed that the scheme may be useful as a preliminary evaluation of the statistical tests. Discussions by the pathologists, toxicologists, and sta tistica l are necessary for a final evaluation. Although these initial evaluations are, in general, independent of the spontaneous tumor rate, there is a major exception. This is when the low-dose comparison to controls is significant, while the high-dose compar ison is not significant. Jf the tumor type is rare, there may be statistical evidence for a chemically related effect. If the low-dose comparison is highly significant, there may be limited evidence for an effect.' For example, in the bioassay of lindane (NCI-TR-14, 1978), the only significant result was hepatocellular carcinomas and neoplastic nodules in male mice, with 5 in 49 of the pooled controls, 19 in 49 of the low-dose animals, and 9 in 46 of the high-dose animals. The incidence in the low-dose animals was statistically significant [p = 0.001); however, the mean incidence of hepatocellular carcinomas and neoplastic nodules in historical control animals at this laboratory was 23%, with spontaneous incidences as high as 35-40%. Thus it was concluded that the results could not be clearly associated with the chemical, and the substance was called a suspected carcinogen. The heavy reliance on statistical evidence to support a positive effect poses the problem of assessing the error rates associated with these statistics. A recent report on error rates in bioassay results (Fears et al., 1977) reaches a number of informative conclusions. A false positive result can arise because the tumor incidence in the dosed group is greater than that in the controls due to sampling variations in the spontaneous tumor incidence rate and not to the chemical. In effect, a false positive result occurs because the dosed group has a random tumor incidence that is statistically significant. A rare tumor has less chance of occurring than a common tumor and therefore has less chance of causing a false positive. Accordingly, the chance of a false positive result occurring is highly dependent on the spontaneous tumor incidence rate. In general, tissue sites with spontaneous tumor rate above 5% have considerably higher chances 260 K . C. CHU E T A U | _T_A__B_L_E__1__. __In_i_t_ia_l_ Evaluations of the Statistical Tests j_ Stat ist ica f Cochran-Arrait a g e (1 ) F i sh er exact(2) F i sh er exact(2) C o n c I u s i o n (3) <1 1~5 % > 5X linear trend l o w - do se c o m p . hi gh - d o s e comp. Si Si HSi Si HSi P " May be positive statistical evidence for carcinogenicity L = May be limited statistical evidence for carcinogenicity - = May be insufficient statistical evidence for carcinogenicity (1) In the linear trend n- n indicates n o n - s i g n i f i c a n t result or significant result with a significant departure statistic. (2) For the Fisher exact test means test is not sign if ic an t. (3) <1X = tumor types with <1ii sp on t a n e o u s tumors; 1 - 57. ~ tumor types with 1-5ii s p o n t a n e o u s tumors; other = tumor types >5 % sp on ta ne ou s tumors. Si= s i g n i f i c a n t result: .001 < P < .025 ; HSi = hi gh ly significant result P <.001. (<) Ma y be s i g n i f i c a n t if the tumor type is rare and the b i no mi al p r o b a b i l i t y is si gn i f i c a n t see text. of generating false positives. The spontaneous primary tumod rates for untreated animals used in the NCI testing program, which are given in Table 2, show that there are only one to five sites with an incidence at least this high. For determining the overall false positive rate, Gart et al. (1979) proposed the formula 1 -- ( 1 --p) f, where t is the number of target sites with spontaneous rates above 5% and p is the nominal ' --j ^ 3 ,? a s = ^ l8 ,a s ? I *B P- I -D -o ki--n in T A B L E 2. Percent Spontaneous Primary Tumors in Untreated Species Used at NCI for Carcinogen Bioassays SPECIES MOUSE STRAIN B6C3F1 SEX > MALE FEMALE NUMBER NECROPSIED (1) 354 3 3617 ORGAN/TISSUE BRAIN < IX . IX SKIN 3.1 1.7 MAMMARY GLAHD -- 1.3 CIRCULATORY SYSTEM (2) 2.9 2.4 LUNG/BROKCH1/TRACHEA 13.7 5.2 LIVER 24.6 4.7 PANCREAS <. 1 <. 1 STOMACH .4 .4 IHTESTINES (3) .5 .2 KIDNEY .3 <. 1 URINARY BLADDER PREPUTIAL GLAND LA) --<. 1 <. 1 -**- TESTES (5> .4 NA OVARY NA .9 UTERUS NA 1.6 PITUITARY .3 3.6 ADRENAL 1.4 .6 THYROID 1.0 1.7 PANCREATIC ISLETS .4 .2 BODY CAVITIES .4 .3 LEUK EMIA/LYMPHOMA 10.3 20.6 RAT FISCHER 344 MALE FEMALE 2960 2924 .SX .6X 7.a . 3.2 1.5 20.9 .7 .4 3.0 1.9 2.2 1.9 .2 -" .3 .2 .6 .3 .5 .2 . 1 .3 2.4 1.8 82.3 NA HA .4 NA 17.0 14.7 34.9 12.4 5.2 8.2 6.8 3.9 .8 2.6 .4 19.9 13.4 RAT 05B`-MHDL MALE FEMALE 270 270 8.9 3.7 4. 1 1.5 1. 1 -- .4 3.3 .4 1.6 .7 NA NA 7.4 10.4 9.6 3.0 1.9 3.3 5.9 28.5 2.6 .7 1.9 -*" .4 .4 2.6 --1.2 NA 1.5 3.7 20.0 10.0 11.1 1.9 .4 1.8 RAT RAT SPR.-DAU. CHAR. RIV. CD MALE FEMALE MALE FEMi 440 205 184 184 7X .5X 3.2 3.4 -1.-4 39.0 --- --- .2 -- .5 -- * .2 --- --.2 --1.0 ---- 2.0 NA NA -- * NA 4.4 5.9 40.0 .7 2.9 .9 2.0 .5 .5 .5 1.9 4.8 0.5 2.7X 7.1 .5 2.2 1.6 .5 -- -- 1.6 .5 3.9 NA NA 33.2 7.6 3.8 2.7 2.2 3.3 1.6X 3.3 45. 1 1.6 2.2 .5 .5 -- ----- NA .5 3.8 57.6 4.3 --- 3.3 tl) STUDIES TERMINATED AT 21-25 MONTHS FOR MICE AND 23-25 MONTHS FOR RATS. (2) HEMANGIOMA AND HEMANGIOSARCOMA. (3) DUODENUM, JEJUNM, ILEUM, CEDUM AND COLON. (A) CLITORAL GLAND IH FEMALES. (5) SEMINAL VISICLE AND TESTIS. uM v .... I f f 262 K . C C H U ET A L i significance level. If there are 50 animals in the control and dosed groups and the Fisher exact test yields a p of 0.028 for 5 dosed animals with a i. tumor compared to zero in the controls, the false positive rate for female F344 rats is less than 0.134 (Gart et al., 1979). N A possible false positive is characterized by several features. The bioassay of the four experiments has a statistically significant result in only one species, strain, and sex. More specifically, the p value of the result is in the range 0.005-0.05 and the tumor type has a spontaneous rate above 5%. On the other hand, a statistically significant result in a species, strain, and sex may be considered an effect of the chemical if the v tumor type is rare, the incidence in the dosed group is much higher than the spontaneous incidence, or the significance of the tumor is confirmed in the opposite sex or a different species. ; As stated earlier, the scheme in Table 1 may be useful for initial evaluations. Discussions by the pathologists, toxicologists, and statisticians will determine the final conclusion. Under certain circumstances, the pathologist has indicated that an incidence of a rare tumor is significant, but this result cannot be verified by the statistical methods discussed due to the small sample size. To quantify the assertion made by the pathologist, one can calculate the binomial probability that the observed number or a greater number of tumors will occur (Fears et al., 1977). If one assumes that the tumor incidence is binomially distributed, the probability of obtaining R tumor in N animals is given by: N \p ' V - p Y where p is the laboratory historical tumor incidence. When this probability was less than 0.001, it was used to indicate that a substance may b e a suspected carcinogen in the particular species, strain, and sex. In other circumstances, the pathologist may indicate that certain types of tumors for a particular species, strain, and sex may have spontaneous incidences that are so high and variable that the use of these tumors as end points is difficult. In these cases, statistically significant results may be nullified. For an extensive discussion of the variability of common naturally occurring lesions, see Tarone et al. (1981). Two examples in F344 rats are interstitial cell tumors of the testes in males, which have an incidence range of 70-100%, and pituitary chromophobe adenomas in females, which have an incidence range of 30-50%. ' In certain other circumstances, discussions between the scientists may indicate that statistically significant results are not meaningful since the incidences in the dosed groups are not above the normal range of incidences in controls in this laboratory. Results that are statistically significant would then be interpreted as not important. This was the case mmmm. EVALUATION OF NATIONAL CANCER INSTITUTE CARCINOGEN BIOASSAYS 263 in the bioassay of malathion (NCI-TR-24, 1978) in male mice. There was a significant dose-related trend in the incidence of hepatocellular carcinomas, with 5 in 49, 7 in 48, and 11 in 50 of the control, low-dose, and high-dose animals, respectively. However, several control groups from the same laboratory had incidences of 35-40%, which are comparable to the incidences in the dosed male mice. The principles described in the sections above were used to evaluate some 200 chemicals. Table 3 shows chemicals that were positive in both sexes of both species; Table 4, chemicals that were positive in at least one sex of both species; and Table 5, chemicals that were positive in one species. In Table 6 the chemicals with suspected carcinogenic effects are listed. Finally, chemicals not shown to be carcinogenic are listed in Table 7 and chemicals with inadequate bioassays are given in Table 8. DISCUSSION. The purposes of this paper are threefold: (1) to explain some of the factors involved in determining the carcinogenicity of a test chemical; (2) to present summary results for 200 chemicals recently evaluated by the National Toxicology Program at N CI; (3) to explain the principles underlying the bioassay procedures. The first two objectives have been addressed, and the principles of testing will now be discussed. The appropriateness of carcinogen bioassays-is based on several char acteristics of carcinogenicity. (1) Almost all human chemical carcinogens (except possibly As) are animal carcinogens. (2) It is ethically un acceptable to perform carcinogenicity tests on the human population. (3) A number of human carcinogens were first detected as animal carcinogens, i.e., vinyl chloride, Be, Cd, and diethyIstilbestrol (DES). (4) Due to the small groups of animals, usually 50-100, used in carcinogen bioassays (designed to detect 5-10% tumor incidences in the dosed groups as compared to none in the controls), the bioassays are relatively insensitive. From these facts the following premises for carcinogen bioassays have been developed. (1) Long-term effects of toxicity studies in animals are applicable to humans. (2) Due to the relative insensitivity of bioassays, exposure of experimental animals to high doses of toxic agents is a necessary and valid method of assessing potential carcinogenic hazards in humans. In the present analysis, the evaluation of the carcinogenicity of a chemical is based on the following premises. (1) A carcinogen is any agent that induces malignant or malignant and benign tumors in a valid carcinogen bioassay. (2) Any agent that induces only benign tumors in a valid carcinogen bioassay may be a carcinogen or a suspected carcinogen, depending on the etiology and pathogenesis of the lesions. (3) The NCI carcinogen bioassays (three groups of 50 animals per sex and species) are relatively insensitive, since they are designed to detect levels of tumor T A BLE 3. NCI Carcinogen Bioassays with Positive Evidence for Carcinogenicity in Both Sexes of Both Species Tested NCI NO. CHEMICAL NAME CAS NUMBER REPORT STATUS ANIMAL ROUTE ORGAN SITES STRAIN C03093 3-Atnino-9-Ethylcarbazolo, Hydrochlori do 132-32-1 C03797 o-Aniaidino Hydrochlor do 139-29-2 COO 191 C O 3292 Chlordccortc (Kepone) 193-50-0 9-Chloro-o-Phenylonedi am)no 95-83-0 C0Z982 p-Crealdino 120-71-8 C03258 Cupforron 135-20-6 CO 1989 '' 2,9-Diaminoanisole Sulfato 39156-91-7 (615-05-9) CO 0500A Di bromochloropropano 96- 12-8 C00522A 1,2-Dibromoathana 106-93-9 CO 05 11 t,2-Di chioroat tv no 107-06-2 REPORT 93 PUBLISHED ); REPORT 89 PUBLISHED REPORT A PUBLISHED REPORT 63 PUBLISHED REPORT 192 PUBLISHED REPORT 100 PUBLISHED REPORT 89 PUBLISHED REPORT' 28 PUBLISHED REPORT 86 PUBLISHED REPORT 55 PUBLISHED F399 B6C3FI FEED F399 D6C3F1 FEED OSB-MDL B6C3F1 F399 B6C3F1 FEED FEED F399 B6C3F1 FEED F399 B6C3F1 FEED F399 B6C3F1 FEED OSB-MDL CAV B6C3FI OSB-MDL GAV B6C3F 1 OSB-MDL GAV B6C3F1 LIVER. HEPATOCELLULAR TUMORS SKIH/SUBCUTAHEOUS TISSUE 2YMDAL S GLAND UTERUS NON-SARCOMA KIDNEY BLADDER* URINARY THYROID FOLLICULAR LIVER. HEPATOCELLULAR TUMORS BLADDER, URINARY FORESTOMACH LIVER, HEPATOCELLULAR TUMORS BLADDER, URINARY NASAL LIVER, HEPATOCELLULAR TUMORS NEMANGIOSARCOMA LIVER. HEPATOCELLULAR TUMORS FORESTOMACH ZYMBAL S GLAND HARDERIAN GLAND SKIN/SUBCUTANEOUS TISSUE THYROID FOLLICULAR ZYMBAL S GLAND THYROID C-CELL PREPUTIAL GLAND FORESTOMACH MAMMARY GLAND FORESTOMACH HEMAHGIOSARCOMA LIVER, HEPATOCELLULAR TUMORS LUNG FORESTOMACH NEMANGIOSARCOMA SO riSRC.'lA MAMMARY GLAND LUNG UTERUS SARCOMA 'RAT MOUSE M F 'M F PP P PP P * PP P PP P PP PP PP PP PP PP PP PP P PP P PP PP PP P PP P P P P PP PP P P PP PP PP P PP P P PP PP V-" '''Vi*; foT DCnol HA nHn-nm UATER r.i fibroma MAMMARY-GLAND --- LUNG UTERUS SARCOMA P PP P .-y-i/ ' J -p i ' W; ' ; v'J>V'!!". ? CQ3689B CO 1923 p-Di oxana 1Z3-91-! 2-Methyl-t-ni troanthraquinone 1Z9-15-7 REPORT 80 PUBLISHED REPORT 29 PUBLISHED OSB-MDL B6C3F1 F344 B6C3F1 WATER FEED NASAL LIVER, HEPATOCELLULAR TUMORS SO FIBROMA LIVER, HEPATOCELLULAR TUMORS F0REST0MACH BLADDER, URINARY HEMANGIOSARCOMA Pp p Pp P s s pp pp COZ006 Mlchler's Ketone 90-94-8 REPORT 181 PUBLISHED F344 B6C3F1 FEED LIVER, HEPATOCELLULAR TUMORS HEMANGIOSARCOMA Pp p p CO 166 1 Phenoxybcnzamino Hydrochloride 63-92-3 REPORT 72 SPR-DAW IP/IJ PUBLISHED B6C3F1 PERITONEAL SARCOMA P p pp CO 13 10B Procarbazine 366-70-1 REPORT 19 PUBLISHED SPR-DAW B6C3F1 IP/IJ NASAL LYMPHOMA MAMMARY GLAND LUNG UTERUS NON-SARCOMA Pp Pp p ss pp p C00453 Sulfallate 95-06-7 REPORT 115 PUBLISHED OSB-MDL B6C3F1 FEED ' FORESTOMACH MAMMARY GLAND LUNG P pp p CO 17 07 4,4'-Thiodianiline REPORT 47 F344 FEED 139-65-1 PUBLISHED B6C3F1 LIVER, HEPATOCELLULAR TUMORS P pp COLON P THYROID FOLLICULAR :P p p p ZYMBAL S GLAND Pp UTERUS NON-SARCOMA p CO 1649 Thlo-TEPA 52-24-4 REPORT 58 PUBLISHED SPR-DAW B6C3F1 IP/IJ SKIN/SUBCUTANEOUS TISSUE HEMATOPOIETIC SYSTEM NASAL UTERUS NON-SARCOMA Pp P ss s p pp C02335 o-Toluidine Hydrochloride 636-21-5 C03Z70 Tris (2,3-Dibro-nopropyl) Phosphate 126-72-7 REPORT 153 PUBLISHED *it REPORT 76 PUBLISHED F344 B6C3F1 F344 B6C3F1 FEED FEED SPLENIC SARCOMA MESOTHELIOMA SQ FIDROMA BLADDER, URINARY MAMMARY GLAND ~ HEMANGIOSARCOMA LIVER, HEPATOCELLULAR TUMORS KIDNEY LUNG FORESTOMACH LIVER, HEPATOCELLULAR TUMORS pp p p p p p \p pp p pp pp F344 = Fischer 344 rat OSB-MDL = Osborno Mendel rat SPR-DAW = Sprague Dawlcy rat GAV = Gavaqe IP/IJ = Intraperiton eal fn ject ion P = Positive evi dene o for carcinogenicity S = Suqqeative evidence for c a r d noqenici ty N = Not shown to be carcinogenic I = Inadequate tost w* PLW-Lj*'JUUJ mm TABLE 4. NCI Carcinogen Bioassays with Positive Evidence for Carcinogenicity in at Least One Sex o f Both Species Tested NCI NO. CHEMICAL NAME CAS NUMBER CO 1876 2-Ami noanthraqui none 117-79-3 CO 190 1 1-Ami no-2-Mothylanthraqui nono 82-28-0 C02686 Chloroform 67-66-3 CD3838 CO 3305 3-lChloromathyl)Pyri dine Hydrochloride 6959-48-4 4-Chloro-m-Phenylenedi amine 5131-60-2 C02302 2,4-D ami notoluene 95-80-7 CO 185$ Hydrazobcnzeno 122-66-7 CO 1627 ICRF-I59 2 14 16-87-5 CO 1638 Iaophosphami do 3778-73-2 ^ CO 1990 4,4,-Methylenebis(N,N-Dimethyl)Benzen- amine 101-61-1 C03021 1i5-NaphthalenodiamfnQ 2243-62-1 REPORT STATUS ANIMAL ROUTE ORGAH SITES STRAIN REPORT 144 PUBLISHED REPORT 111 PUBLISHED REPORT 0 PUBLISHED REPORT 95 PUBLISHED REPORT 85 PUBLISHED REPORT 162 PUBLISHED REPORT 92 PUBLISHED REPORT 78 PUBLISHED REPORT 32 PUDLISHED REPORT 186 PUBLISHED REPORT 143 PUBLISHED F344 B6C3F1 F344 B6C3F1 OSB-MDL B6C3F1 F344 B6C3F1 F344 B6C3F1 F344 B6C3F1 F344 B6C3F1 SPR-DAW B&C3F1 SPR-DAW D6C3F1 F344 B6C3F1 F344 B6C3FI FEED FEED GAV GAV FEED FEED FEED IP/IJ IP/IJ FEED FEED LIVER, HEPATOCELLULAR TUMORS LYMPHOMA KIDNEY LIVER. HEPATOCELLULAR TUMORS KIDNEY LIVER, HEPATOCELLULAR TUMORS FORESTOMACH ADRENAL MEDULLA LIVER, HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS SQ FIBROMA MAMMARY GLAND LYMPHOMA LIVER, HEPATOCELLULAR TUMORS ZYMBAL S GLAND MAMMARY GLAND UTERUS NON-SARCOMA LYMPHOMA LYMPHOMA UTERUS HON-SARCOMA MAMMARY GLAND THYROID FOLLICULAR LIVER, HEPATOCELLULAR TUMORS CLITORAL GLAND UTERUS SARCOMA THYROID FOLLICULAR LIVER, HEPATOCELLULAR TUMORS LUNG THYROID C-CELL RAT MOUSE MF MF N P PP P P PP N P N P N P PP P P PP P P N P 5 P S S P PP PP N P N P S H P H P N P PP SP H P P PP P P \P )f3 " [ldp(HIIlJ 22*1-62-1 PUBLISHED UTERUS SARCOMA THYROID FOLLICULAR LIVER, HEPATOCELLULAR TUMORS LUNG THYROID C-CELL C03792 Nithiazlde 139-95-6 C02766 Nitr1lotriacotic Acid (NTA) 139-13-9 CD 1967 5-Ni troacenaphthene 602-87-9 CO 1934 5-NItro-o-Ani si dine 99-59-2 REPORT 1*6 PUBLISHED REPORT 6 PUBLISHED REPORT 118 PUBLISHED REPORT 127 PUBLISHED F355 B6C3F1 F355 . B6C3F1 F354 B6C3F1 F344 B6C3F1 FEED FEED FEED FEED SKIH/SUBCUTANEOUS TISSUE LIVER* HEPATOCELLULAR TUMORS BLADDER* URINARY KIDNEY ZYMBAL S GLAND LUNG CLITDRAL GLAND MAMMARY GLAHD LIVER, HEPATOCELLULAR TUMORS OVARY LIVER, HEPATOCELLULAR TUMORS SKIN/SUBCUTANE0U5 TISSUE ZYMBAL S GLAND CLIT0RAL GLAHD N P PS N P P N PP PP P P N P P PP PP P SP CO D420B Ni trofen 1836-75-5 C02244 CO 1672 P-Nitrosodiphenylami no 156-10-5 Phenazopyridtne Hydrochloride 136-50-3 CO 1558 Phenester in 35*6-10-9 REPORT 26 OSB-MDL FEED PUBLISHED B6C3F1 REPORT 190 PUBLISHED REPORT 99 PUBLISHED F344 B6C3F1 F344 B6C3F1 FEED FEED REPORT 60 SPR-DAW GAV PUBLISHED B6C3F1 C50 157 Reserpi ne 50-55-5 REPORT * F344 FEED IH REVIEW B6C3F1 C02904 2,5,6-Trichlorophenol 86-06-2 REPORT 1^5 F344 FEED PUBLISHED D6C3F1 C02299 2 ,* >5-Tr imethylan i1ine 137-17-7 REPORT 160 F344 FEED PUBLISHED B6C3FI C03781 Tri methylphosphate 512-56-1 REPORT 81 PUBLISHED F344 B6C3F1 FEED F3*4 = Fischer 3** rat OSB-MDL = Osborne Mendel rat 5PR-DAU = Sprague Dawley rat GAV = Gavaqe IP/IJ = IntraperItonea1 injection P = Positive evidence for carcinogenicity PANCREAS ISLET-CELL LIVER, HEPATOCELLULAR TUMORS HEMANGIOSARCOMA I P PP P LIVER, HEPATOCELLULAR TUMORS NN PP COLON LIVER, HEPATOCELLULAR TUMORS PP N P MAMMARY GLAND LUNG LYMPHOMA HEART SARCOMA N P PP PP PP ADRENAL MEDULLA SEMINAL VESICLE MAMMARY GLAND N P S P HEMATOPOIETIC SYSTEM LIVER, HEPATOCELLULAR TUMORS N P PP LIVER, HEPATOCELLULAR TUMORS LUNG PP P n` P SQ FIBROMA UTERUS NON-SARCOMA N P N P S = Suqqostive evi dence for care inoqcni c ity N = Not shown to be carcinogenic I = Inadequate test s TABLE 5. NCI Carcinogen Bioassays with Positive Evidence for Carcinogenicity In Only One Species NCI HQ. CHEMICAL NAME CAS HUMBER REPORT STATUS ANIMAL ROUTE ORGAN SITES STRAIN CO 1536 Acronyc ino 7008-42-6 C00D44 Aldrin 309-00-2 CO 1887 C03963 C03065 C03736 3-Am Ino-4-EtHoxyacelan i1 1do 17026-8 1-2 4-Ami no-2-H itrophenol 119-34-6 2-Amino-5-Ni trothiazolo 121-66-4 Ant lino Hydrochlortdo 142-04- 1 CO 1569 C0Z926 5-Azacyt1di no 320-67-2 Azobonzeno 103-33-3 CO 0077 C00055 CO 0099 Captan 133-06-2 Chlorambon 133-90-4 Chlordano 57-74-9 C0040SA CO 0 102 C02051 ChlorobenzllaEo 510-15-6 Chlorothalont1 1897-45-6 5-CHIoro-o-Tololdlna 95-79-4 PORT 49 PllQL ISHED REPORT 21 PUOLISHED REPORT 112 PUOLISHED REPORT 94 PUBLISHED REPORT 53 PUBLISHED REPORT 130 PUBLISHED REPORT 42 PUBLISHED REPORT 154 PUBLISHED REPORT 15 PUBLISHED REPORT 25 PUBLISHED REPORT 8 PUBLISHED REPORT 75 ' PUBLISHED REPORT 41 PUBLISHED REPORT 187 PUBLISHED SPR-DAW B6C3F1 05B-MDL B6C3F1 F344 D6C3F1 F344 B6C3F1 F34 4 B6C3F1 F344 B6C3F1 SPR-DAW B6C3F1 F344 B6C3F1 OSB-MDL D6C3F1 OSB-MDL B6C3F1 OSD-MDL B6C3F1 OSB-MDL B6C3F1 OSB-MDL B6C3F1 F344 B6C3F1 IP/IJ FEED FEED FEED FEED FEED IP/IJ FEED FEED FEED FEED FEED FEED FEED OSTEOSARCOMA PERITONEAL SARCOMA MAMMARY GLAND THYROID FOLLICULAR ADRENAL CORTEX LIVER, HEPATOCELLULAR TUMORS THYROID FOLLICULAR BLADDER, URINARY HEMATOPOIETIC SYSTEM HEMANGIOSARCOMA MULTIPLE SITES, SARCOMA SPLENIC SARCOMA HEMATOPOIETIC SY5TEM ABDOMINAL CAVITY SARCOMA MULTIPLE SITES, SARCOMA OSTEOSARCOMA DUODENUM LIVER, HEPATOCELLULAR TUMORS FIBROUS HISTIOCYTOMA THYROID FOLLICULAR LIVER, HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS KIDNEY HEMANGIOSARCOMA . RAT MOUSE nF MF p pP P sS s NN PS N P P PP P II PP P S KH HN II N P H P HN KH HH I P NH PS SP S S HN PP H ''N, PP PP NH P if, COO 102 C02051 Chlorothalonl1 1897-45-6 5-Chloro-o-Toluldind 95-79-4 REPORT 41 PUBLISHED REPORT 167 PUBLISHED ' If'fr * C02368 C03565 C02993 CO 1716 4-Chl oro-o-Tollii dl ne Hydrochlorids 3165-93-3 C.I. Vat Yellow 4 128-66-5 m-Crsldna 102-50-1 Dapsone 80-08-0 CO 0555 CO 3827 P f p*-DDE 72-55-9 Diami nozlda 1596-64-5 000466 C03516 C02175 Di cofol 115-32-2 H N*-Di ethylthiaurea 105-55-5 3 f3*-Di methoxybenzi di na44 *-Diisocyanata 91-93-0 CO 1865 2j4-Dinitrotoluena 121-14-2 C54557 C54579 C54568 CO 1570 Direct Black 38 1937-37-7 Direct Blue 6 2602-46-2 Direct Drown 95 16071-86-6 Estradiol Mustard 22966-79-6 C02857 Ethyl Tcllurae '30145-38-1 COO 180 Heptachlor 76-44-8 REPORT 165 PUBLISHED REPORT 134 PUBLISHED REPORT 105 PUBLISHED REPORT 20 PUBLISHED REPORT 131 PUBLISHED REPORT 83 PUBLISHED REPORT 90 PUBLISHED REPORT 149 PUBLISHED REPORT 128 PUBLISHED REPORT 54 PUBLISHED REPORT 108 PUBLISHED REPORT 108 PUBLISHED REPORT 106 PUBLISHED; REPORT 59 PUBLISHED REPORT 152 PUBLISHED REPORT 9 PUBLISHED 05B-MDL ' FEED B6C3F1 FEED B6C3F1 KIDNEY HEMANGIOSARCOMA pP NN NH LIVER, HEPATOCELLULAR TUMORS F3AA B6C3F1 FEED HEMANGIOSARCOMA F3AA B6C3F1 FEED LYMPHOMA F3AA GAV B6C3F1 BLADDER, URINARY F3A4 86C3F1 FEED SPLENIC SARCOMA PERITONEAL SARCOMA OSB-MDL FEED B6C3F1 ' LIVER, HEPATOCELLULAR TUMORS F3AA B6C3F1 FEED UTERUS NON-SARCOMA UTERUS SARCOMA LIVER, HEPATOCELLULAR TUMORS OSB-MDL FEED B6C3F1 LIVER, HEPATOCELLULAR TUMORS F344 FEED B6C3F t THYROID FOLLICULAR F3A A B6C3F1 FEED LYMPHOMA ZYMBAL S GLAND 5KIN/SUBCUTANE0U5 TISSUE UTERUS HOH-SARCOMA F3't<i D6C3F1 FEED SKIN/SUBCUTANEOUS TISSUE MAMMARY GLAND F3A A B6C3F1 FEED LIVER, HEPATOCELLULAR TUMORS F3A A B6C3F1 FEED LIVER, HEPATOCELLULAR TUMORS F3A A D6C3FI FEED LIVER, HEPATOCELLULAR TUMORS SFR-DAW GAV D6C3FI LUNG HEMANGIOSARCOMA FORESTOMACH LYMPHOMA F3A A B6C3FI FEED MESOTHELIOMA HARDERIAN GLAND OSB-MDL FEED B6C3F1 LIVER, HEPATOCELLULAR TUMORS NH NN PP N P P HN N P P NN PP PP PP P P P P PP PP N P NN H S NN PP PP N P IN NN PP N S N P NN NN NN NN NN HN Ps PP PP PP PS PP 270 T A BLE 5. NCL Carcinogen Btoassays with Positive Evidence for Carcinogenicity in Only One Species (continued) NCI NO. CHEMICAL NAME CAS NUMBER i1 REPORT 5IATUS ANIMAL ROUTE ORGAN SITES STRAIN C04604A CO 1547 CO 1478 Hexachloroothano 67-72-1 IPD 3458-22-8 Lasi ocarpine 303-34-4 CO 1978 CO 19 12 C00420A C02222 C O 3076 3*-Ni fcro-p-Acetophonefcido 1777-84-0 6-Ni trobensimidazola 94-52-0 Hi trofen 1836-75-5 2-N ifcro-p-Phanylenedi amine 5307-14-2 3-Nitroproptonic Acid 504-88-t C02880 N-Ni trosodiphenylamino 86-30-6 CO 1843 5-Hi tro-o-Toluidine 99-55-8 C01445A NTA Trisodium SaU.H20 18662-53-8 C02824 CQ4126 C03850 Pipcronyl Sulfoxide 120-62-7 PWalolactono 1955-45-9 p-Quinono Dioxime 105-11-3 -- REPORT 68 PUBLISHED REPORT 18 PUBLISHED REPORT 39 PUBLISHED REPORT 133 PUBLISHED REPORT 117 PUBLISHED REPORT 184 PUBLISHED REPORT 169 PUBLISHED REPORT 52 PUBLISHED REPORT 164 PUBLISHED REPORT 107 PUBLISHED REPORT 6 PUBLISHED ' REPORT 124PUBLISHED REPORT 140 PUBLISHED REPORT 179 PUBLISHED OSB-MDL B6C3F1 SPR-DAU B6C3F1 F344 F344 B6C3F1 F344 B6C3F1 F344 B6C3F1 F344 B6C3F1 F344 B6C3F1 F344 B6C3F1 F344 F344 D6C3F1 F344 B6C3F1 F344 B6C3F1 GAV IP/IJ FEED FEED FEED FEED FEED GAV FEED FEED FEED FEED GAV FEED LIVER, HEPATOCELLULAR TUMORS PERITONEAL SARCOMA LIVER, HEPATOCELLULAR TUMORS LIVER, ANGIOSARCOMA LYMPHOMA LIVER, HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS PANCREAS ISLET-CELL BLADDER, URINARY SQ FIBROMA HEMANGIOSARCOMA LIVER, HEPATOCELLULAR TUMORS KIDNEY URETER BLADDER. URINARY -LIVER, HEPATOCELLULAR TUMORS FORESTOMACH BLADDER, URINARY RAT MOUSE MF MF NN PP SS PP PP PP P NH NN NN NN H P S PP S NN N P PP PP N P NN * NN PP PP PP P P HN PP N P N P N N- HN C03559 C09580A C O 0 160 ti1,2,2-TetrachIoroethane 79-39-5 Tetrachloroothylene 127-18-9 Tetrachlorvlnphos 961-11-5 CO 1581 C00259 B-TGDR 789-61-7 Toxaphene 8001-35-2 C09579 li 1,2-Trichloroethane 79-00-5 C09596A Trichloroethylene 79-01-6 C0099Z Trifluralin 1582-09-8 M C02186 Tr imethyl-th 1DUra 2989-77-2 F399 = Fischer 399 rat OSB-MDL = Osborna Mendel rat SPR-DAW = Sprague Dawley rat GAV = Gavaqe IP/IJ = Intraperitoneal injection P = Positive evidence for carcinogenicity S - Suggestive evidence For carcinogen icity H = Hot shown to be carcinogenic I ~ Inadequate test REPORT 27 PUBLISHED REPORT 13 PUBLISHED REPORT 33 PUBLISHED REPORT 57 PUBLISHED REPORT 37 PUBLISHED REPORT 79 PUBLISHED REPORT 2 PUBLISHED REPORT 39 PUBLISHED REPORT 129 PUBLISHED FJ** FEED B6C3F1 BLADDER URINARY N KN P DSB-MDL B6C3F1 OSB-MDL B6C3F1 OSB-MDL BCC3F1 SPR-DAU B6C3F1 DSB-MDL B6C3F1 OSB-MDL B6C3F1 OSB-MDL B6C3F1 OSB-MDL B6C3F1 F3A4 B6C3F1 GAV GAV FEED IP/IJ FEED GAV GAV FEED FEED LIVER HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS THYROID C-CELL ADRENAL CORTEX LIVER. HEPATOCELLULAR TUMORS ZYMBAL S GLAND THYROID FOLLICULAR LIVER, HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS ADRENAL LIVER, HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS LUNG FORESTOMACH THYROID FOLLICULAR NN II N S 5 SP PP PP PS II SS NN NH NN N P PP PP SP PP N P S s NN TABLE 6. NCI Carcinogen Bioassays with Evidence Suggestive o f a Carcinogenic Effect NCI NO. CHEMICAL NAME CAS NUMDER 003247 C046 15 C0375S C02664 CD 0066 Acetohexami tie . 968-8 1-0 Allyl Chloride 107-05-1 p-Anisidine Hydrochlorido 20265-97-8 A r o c h l o r - 1254 27323-18-8 Azi nphosnethyl 8 6 t 50-0 C03521 IK-Benzotriazole 95-14-7 CO3598 C02039A Butylated Hydroxytoluene (8HT) 128-37-0 p-Chloroan 1 ino 106-47-8 C033I6 CO 043 1 2-Chloro-p-Phenylened ami ne Sulfate 61702-44-1 Cloni tralid 1420-04-8 C0202B C03667S Dtbutyltin Di actate 1067-33-0 2,7-Dchlorodi bonzo-p-Dioxin (DCDD) 33857-26-0 C04535 11 1-DIchloroethane 75-34-3 C00113A C045Z4 Dlchlorvoa 62-73-7 N.N'-Di cyclohexylthiourea REPORT STATUS 1REPORT 50 PUBLISHED REPORT 73 PUBLISHED REPORT 116 PUBLISHED REPORT 38 i'ODL ISKED REPORT 69 PUBLISHED REPORT 88 PUBLISHED REPORT 150 PUBLISHED REPORT 189 PUBLISHED REPORT 113 PUBLISHED REPORT 91 PUBLISHED REPORT 183 PUBLISHED REPORT 123 PUBLISHED REPORT 66 PUBLISHED REPORT 10 PUBLISHED REPORT 56 ANIMAL ROUTE ORGAN SITES STRAIN F344 B6C3F1 OSB-MDL B6C3F1 F344 B6C3F1 F344 FEED GAV FEED FEED OSB-MDL FEED B6C3F1 F344 B6C3F1 FEED F344 B6C3F1 F344 B6C3F1 FEED FEED F344 B6C3F1 OSB-MDL B6C3F1 FEED FEED F344 B6C3F1 OSB-MDL B6C3F1 PEED FEED OSB-MDL GAV B6C3F1 OSB-MDL B6C3F1 F344 FEED FEED LEUKEMIA FORESTOMACH PREPUTIAL GLAND LIVER. HEPATOCELLULAR TUMORS GASTROINTESTINAL TRACT THYROID FOLLTCULAR PANCREAS ISLET-CELL BRAIH LUNG LUNG SPLENIC SARCOMA HEMANGIOSARCOMA BLADDER. URINARY MAMMARY GLAND FORESTOMACH UTERU5 NON-SARCOMA LYMPHOMA LIVER, HEPATOCELLULAR TUMORS HEMANGIOSARCOMA MAMMARY GLAND HEMANGIOSARCOMA UTERUS NON-SARCOMA ESOPHAGUS RAT MOUSE MF MF S NN N S sS ss N s s s5 NN N S s5 N S s K s NN N S s NN N NN SS NN NN N S H S sS NN rN NN N 5 S s N S N S NN C00113A C0A52A Diehlorvos^, 62-73"? H* H -- 0 1c y c l o h e x y l I H lourdA REPORT 10 PUBLISHED REPORT 56 F364 s Fischer 3A6 rat OSB-MDL - Osborno Mendel rat SPR-DAU = Sprague Dauley rat 6AV = Gavaqe IP/IJ = Intraperitoneal injection P = Positive evidence for carcinogenicity S = Sugqestive evidence for carcinogenicity H = Hot shown to be carcinogenic I = Inadequate test PUBLISHED REPORT 21 PUBLISHED REPORT 132 PUBLISHED REPORT 156 PUBLISHED REPORT 103 PUBLISHED REPORT 70 PUBLISHED REPDRT 16 PUBLISHED REPORT 23 PUBLISHED REPORT 5 PUBLISHED REPORT 135 PUBLISHED REPORT 131 PUBLISHED OSB-MDL B&C3F1 fbaa FEED FEED -- -- ES0PHAQU3 HH H * H S NH JWlV-'Af: B6C3F1 o s b -m d l B6C3F1 F3AA B6C3F t F3AA B6C3F1 F3AA B6C3FI OSB-MDL B6C3F t OSB-MDL B6C3F1 FEED FEED FEED FEED FEED FEED OSB-MDL B6C3F1 F34<i B6C3F1 FEED FEED F3AA B6C3F1 OSB-MDL B6C3F t GAV FEED THYROID FOLLICULAR LIVER* HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS LIVER, HEPATOCELLULAR TUMORS SKIN/SUBCUTANEOUS TISSUE ADREHAL CORTEX HEMANGIOSARCOMA THYROID C-CELL LIVER, HEPATOCELLULAR TUMORS GASTROINTESTINAL TRACT LIVER, HEPATOCELLULAR TUMORS LUNG THYROID FOLLICULAR S HN HN NH NN SS S s Ss I S NN H S N S N S N S N S NN HN NN I S N S NN C0S67 3 CO 36 56 A DI az3t3n3o-^n 1-3 D i b o n r o - p - D I ox Ift 262-12-4 COO 124 COO 135 Dimethoate 60-51-5 C02255 Z> 4-Dimethoxyaniline Hydrochloride 54 150-69-5 C50055 Dimethyl Terephthalate 120-6 1-6 CO 0395 Di oxathion 78-34-2 CQ3974 EDTA Trisodium Salt (Trihydrate) 150-38-9 CO 056 6 Endosulfan 115-29-7 COO 157 Endrtn 72-20-8 CO 16 94 Ethfonamide 536-33-4 C030 10 Formulated Fenaminoaulf 140-56-7 C02653 Hexachlorophene 70-30-4 C0456S Iodoform 75-47-8 C02891 Lead Dlmcthyldithi ocarbamate 19010-66-3 CO 0204 CO3361 Lindane 56-89-9 Lithocholic Acid 434-13-9 C00215A Malathi on 121-75-5 C50000 dl-Mcnthol 89-78-1 CO 04 97 Methoxychlor 72-43-5 C02971 Methyl Parathion 298-00-0 CO 0544 Mexacarbate 315-18-4 REPORT 137 PUBLISHED REPORT 122 PUBLISHED F3A'* DGC3F1 OSB-MDL BGC3F1 FEED FEED ^ i!l.:-atWVVV?;V REPORT Z2 F3G4 PUBLISHED FEED REPORT $ OSB-MDL FEED PUBLISHED B6C3F1 REPORT 171 F36G FEED PUBLISHED B6C3F1 REPORT 121 F3<*4 PUBLISHED B6C3F1 FEED REPORT 125 OSB-MDL FEED PUBLISHED B6C3F1 REPORT 11 F3G4 FEED PUBLISHED B6C3F1 REPORT 62 OSB-MDL FEED PUBLISHED D6C3F1 REPORT 12 OSB-MDL FEED PUBLISHED B6C3F1 REPORT A6 OSB-MDL FEED PUBLISHED B6C3FS REPORT 101 F365 FEED PUBLISHED B6C3FT REPORT 0 PUBLISHED F3gg FEED REPORT 110 OSB-MDL GAV PUBLISHED D6C3F1 REPORT 151 PUBLISHED F3<i B6C3F1 FEED REPORT H PUBLISHED OSB-MDL FEED B6C3F1 REPORT 175' F34A GAV PUBLISHED B6C3F1 REPORT Z6, OSB-MDL FEED PUBLISHED 1 B6C3F1 REPORT 98' F3AA PUBLISHED , B6C3F1 FEED REPORT 35 OSB-MDL FEED PUBLISHED B6C3F1 REPORT 157 PUBLISHED F3`i`t B6C3F1 FEED REPORT 167 OSB-MDL FEED PUBLISHED B6C3F1 HH NN HN HN v- ' a-- HN NN HH NN NN NN NN HH H H NH NN IH IH NH HN KH NN NH HN NH HH HH NN NH NH HN NN HN HH NN HH HH HN HH NH HN HH HN 276 TA BLE 7. NCL Carcinogen Bioassays with No Significant Evidence for Carcinogenicity (continued). NCI HO. CHEMICAL HAME CAS HUMBER C03281 CO 19^5 CO 1956 CO 396 1 C02Z1 1 CO1665B H -(1-Naphthyl)Ethylenndi ami no.2MCI 1665-25-6 6-Nitroanthrani1 ic Acid 6 19- 17-0 1-Ni tronaphthaleno 86-57-7 6-H tro-o-Phcnylonedi amino 99-56-9 BQta-Hitrostyrene and Styrene 102-96-5 100-62-5 HTA Trisodium Salt.H20 18662-53-8 REPORT STATUS { REPORT 168 PUBLISHED REPORT 109 PUBLISHED REPORT 66 PUBLISHED REPORT 180 PUBLISHED REPORT 170 PUBLISHED REPORT 6 PUBLISHED ANIMAL STRAIN F 366 B6C3F1 F366 B6C3F1 F366 D6C3F1 F366 B6C3F1 F366 B6C3F1 F366 B6C3F1 ROUTE ORGAN SITES FEED FEED FEED FEED GAV FEED C00619A CO 176 1 C03930 CO 3952 C02233 C020 17 C00599 C03612 C03601 C02813 Pentachloron itrobenzeno 82-68-8 Phenform in 1 16-86-3 p-Phenylenedi amine Di hydrochloride 626- 18-0 1-Phenyl-3-t1ethyI-5-PyrazoIone 89-25-8 N-Pheny1-p-Phenylenedi ami no 10 1-56-2 1-Phenyl-2-Thiouroa 103-85-5 Photodi eldri n 13366-73-9 . Phthalamido 88-96-0 Phthalic Anhydrido ...-85-66-9 Piporonyl Butoxido 51-03-6 REPORT 61 PUBLISHED REPORT 7 PUBLISHED REPORT 176 PUBLISHED REPORT 161 PUBLISHED REPORT 82 PUBLISHED REPORT 168 PUBLISHED REPORT 17 PUBLISHED REPORT 161 PUBLISHED REPORT 159 PUBLISHED REPORT 120 PUBLISHED OSB-MDL B6C3F1 F366 B6C3F1 F366 B6C3F1 F366 B6C3F1 F366 B6C3F1 F366 B6C3F1 OSB-MDL B6C3F1 F366 B6C3F1 F366 B6C3F1 F366 B6C3F1 FEED FEED FEED FEED FEED FEED FEED FEED FEED FEED RAT MOUSE MF MF NH NN HN HN HN HN NH HN HH KH H H' N H NH HN NN HN HN NN NH NN HN HH NH HN HH NH NN NN HN NH NN KN H H HI WVifOli r l u i i t i i I ^ n m i y y i i w C02813 Plperonyl Butoxldo 91-03-6 C50Q22 Sodium Diethyldtthtocarbamats 168-18-5 Suif Isoxazole 127-69-5 C06557 C O 3032 C02959 C06260 C03327 C0 1763 CO 1832 C06637 3-Sulfolene 77-79-2 2,3,5,6-Tetrachloro-6-Ni troanisole 2638-88-2 Tetraethylthiuram Disulfide 97-77-8 Titanium Dioxide 13663-67-7 1309-63-3 Tolazamide 1156-19-0 Tolbutamide 66-77-7 2,5-Toluenediamine Sulfate 6369-59-1 Trichlorofluoromethane 75- 69-6 C00260 Triphenyltin Hydroxide 76- 87-9 CO 1729 1-Tryptophan _______________ 7 3 -22-3__________________________ F366 = Fischer 366 rat OSB-MDL = Osborne Mendel rat SPR-DAH = Sprague Dawley rat GAY = Gavage IP/IJ = Intraperitoneal injection H = Hot shown to be carcinogenic I = Inadequate test PUBLISHED' REPORT 120 PUBLISHED B6C3F1 F344 B6C3F1 FEED REPORT 48 PUBLISHED REPORT 77 PUBLISHED REPORT 172 PUBLISHED REPORT 138 PUBLISHED REPORT 102 PUBLISHED REPORT 114 PUBLISHED REPORT 166 PUBLISHED REPORT 97 PUBLISHED REPORT 51 PUBLISHED REPORT 31 PUBLISHED REPORT 126 PUBLISHED REPORT 106 PUBLISHED REPORT 139 PUBLISHED REPORT 71 PUBLISHED F344 B6C3F1 F344 B6C3F1 F344 B6C3F1 F344 B6C3F1 FEED FEED FEED GAV OSB-MDL GAV B6C3F1 F344 FEED B6C3F1 F344 FEED B6C3F1 F344 B6C3F1 FEED F344 B6C3F1 FEED F344 FEED B6C3F1 F344 B6C3F1 FEED OSB-MDL GAV D6C3F1 F344 FEED B6C3F1 F344 FEED B6C3F1. HN NN HH H1 KN IN HH HN NH NN HN HH HN HH HH HH NN NN MN HH HN NH NN HN II HH KH NH NN NN 278 TABLE 8. NCI Carcinogen Bioassays That Arc Inadequate NCI NO. CHEMICAL NAME CAS NUMDER REPORT STATUS ANIMAL ROUTE STRAIN C02108 002095 CO 1514 C02120 COZ 13 1 C04591A CO 1821 CO 1605 C50259 C02 142 CO 3 190^ C 0 2084 C02153 C04626A COZ 119 Acotam idc 60-35-5 NO TCH RPT; INCONCLUSIVE Ad ipamidc 628-94-4 NO TECH RPTi INCONCLUSIVE A dr{aroyci n 23214-92-8 NO TECH RPT; INCONCLUSIVE 1-Arqinine Glutamato 4320-30-3 NO TECH RPT; INCONCLUSIVE N-Butylurca 592-31-4 NO TECH RPTi INCONCLUSIVE Carbon Disulfide . 75-15-0 NO TECH RPT; INCONCLUSIVE NtN-Dimethyl-p-Nitrosoaniline 13B-89-6 NO TECH RPT; INCONCLUSIVE Emotino 483-18-1 REPORT 43 PUBLISHED Hexamcthylmclamine 645-05-6 NO TECH RPT; INCONCLUSIVE H ex an air.ide 628-02-4 NO TECH RPTi INCONCLUSIVE Mothansulfon-m-Ani si do, 4'-(9-Acridinyl- HO TECH RPTi amino)-, monohydrochlor ido UMK057 9 IHCONCLUSIVE Nitrous Acid, Sodium Salt 7632-00-0 NO TECH RPTi INCONCLUSIVE p-Tolyluroa 622-5 1-5 NO TECH RPTi IHCONCLUSIVE 11 1, 1-1r ichloroothane 71-55-6 REPORT 3 PUBLISHED Urea 57-13-6 NO TECH RPT; IHCONCLUSIVE F344 B6C3F1 F344 B6C3F1 F344 D6C3F1 F34 4 B6C3F1 F344 B6C3F1 OSB-MDL B6C3F1 F344 D6C3F1 SPR-DAU B6C3F1 F344 B6C3F1 F344 B6C3F1 F34 4 BC3F1 B6C3F1 F344 B6C3F1 05B-MDL D6C3F1 F344 D6C3F1 FEED FEED IP/IJ FEED FEED GAV FEED IP/IJ IP/IJ FEED IP/IJ FEED FEED GAV FEED F344 = Fischer 344 rat OSB-MDL = Osborno Mondai rat SPR-DAU = Spraquo Daulcy rat GAV = Gavaqo IP/IJ = Intraperitoneal Injection ,1 i.V.^ Ii +*3* -4m O LL C !S3 c .=" 5 I.O:OInCD.LDOQcC o --*O l>. it o l/l CC3T-c*' . JoCt|i Q<3|ilD4" t-f4 04li/UL<- . /H EV AL UA TI ON O F NA TI ON AL C A N C E R INSTITUTE C A R C I N O G E N BIOASSAYS 279 incidences higher than 10% when there are no tumors in the control group; as a consequence, negative results do not necessarily mean that the test chemical is not a carcinogen. (4) Any substance that is deemed to be a carcinogen or a suspected carcinogen is a potential hazard to humans unless proved otherwise. The extent of the health risk to humans may be a matter for further research. These premises are scientific issues. Increasing knowledge about chemical carcinogenesis may validate, alter, or eliminate any of them. As a consequence, the design, conduct, and evaluation of the carcinogen fr bioassay will undergo an evolutionary process. We hope that the procedures described in this paper will serve as discussion points for further developments of rodent bioassays. REFERENCES Boyland, E. 1969. The correlation of experimental carcinogenesis and cancer in man. 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Neoplastic and nonneoplastic lesions in aging (C57BL/6N X C3H/HeN}F1 (B6C3F1) mice. } . Natl. Cancer Inst. 63:849-854. Received January 29, 1980 Accepted December 15, 1980