B. J. Abbott,1 J. L. Hartwell,1 J. Leiter3 R. E. Perdue, Jr.,3 and S

SCREENING DATA FROM THE CANCER CHEMOTHERAPY NATIONAL SERVICE CENTER SCREENING LABORATORIES. XXXVU. PLANT EXTRACTS

B. J. Abbott,1 J. L. Hartwell,1 J. Leiter3 R. E. Perdue, Jr.,3 and S. A. Schepartz1

SUMMARY

Data are reported on 1197 plant extracts which were tested in the primary screens of the Cancer Chemotherapy National Service Center. Almost all the materials were tested in Sarcoma 180, Adeno- carcinoma 755, Leukemia 1210, and KB cells in culture; some tests were also carried out in Dunning leukemia ascites, Lewis lung carcinoma, Human sarcoma HS1, and Melanotic melanoma. Data are re ported only on materials which have not demonstrated sufficient activity in these systems to warrant fur ther investigation. The number of test systems in the screening program has recently been reduced; the reasons for the reduction are discussed.

INTRODUCTION This report is a continuation of the series of publications of Cancer Chemotherapy National Service Center (CCNSC) screening data, and is the ninth report dealing with plant extracts. Data are included on systems incorporated into the primary screening spectrum a few years ago as well as those used ex clusively until that time. The presentation of data on these systems has been made possible by the re duction of all information to a unified line summary, which facilitates data storage and retrieval by an IBM 1410 computer. A substantial change in data presentation was made recently to permit the inclusion of the newer tumor systems.

The following description of materials and methods, etc. , is similar to that of earlier publications, which first discussed preparation of plant extracts (18) and which first discussed the inclusion of the new test systems (19).

MATERIALSANDMETHODS

Service Screening Laboratories

All the test data reported were obtained under contracts with the CCNSC. Test procedures are prescribed in a set of protocols followed by all contractors. Reports of the experiments are submitted to the CCNSC for review and statistical evaluation. The summary for each experiment gives the code identi fication of the screening laboratory; these codes are listed later.

Source of Plant Extracts All plant materials were collected under the auspices of the New Crops Research Branch of the U. S. Department of Agriculture (USDA)under a transfer of funds from CCNSC. Botanists from the USDA named the plants and also submitted information on the location and date of the collection; a specimen of each collection is maintained by the USDA. Extraction was carried out by the Wisconsin Alumni Research Foundation under contract with the CCNSC.

In Vivo Procedures Tumors and Host Animals. Almost all the materials reported in this publication have been screened in three transplanted mouse tumors: Sarcoma 180 (S180), Mammary adenocarcinoma 755 (Ca755), and an

1 National Cancer Institute, National Institutes of Health, USPHS, Bethesda, Maryland 20014. 2 National Library of Medicine, USPHS, Bethesda, Maryland 20014. 3 Crops Research Division, Agriculture Research Service, U. S. Department of Agriculture, Beltsville, Maryland.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories ascitic form of leukemia 1210 (L1210). These are well-known transplantable tumors and are adequately described elsewhere (5, 14, 17). The Screening Panel of the CCNSC selected these tumors on the basis of experience available in 1955; great weight was given to the ability of these screens to select almost all the materials then considered useful against cancer in man.

Since it was also recognized that these test systems might not be the optimal ones to predict for clinical effectiveness, considerable effort was made to develop and evaluate a variety of additional sys tems. As a result of this work more systems in mice, rats, hamsters, and embryonated eggs were incor porated into the screen. A new material of unknown activity was tested in L1210 and 2 other test systems which were selected on a random basis. If quantities were limited, fewer test systems had to be used. Materials related to active agents were not tested in a random group of systems but rather in the system sensitive to the parent compounds, normally by multidose testing. In selecting additional test systems, an effort was made to find systems which were different from the original systems in their drug sensitivity and which could be used as reliable screens from the standpoint of reproducibility.

After sufficient experience was gained in the evaluation of new agents against this random screen, a correlation study was undertaken to evaluate the predictability, if any, of these test systems against cancer in humans (16). This retrospective analysis of 88 drugs which have been evaluated clinically showed that, of the 45 compounds with definite clinical activity, the activity of 33 was predicted by lymphoid leukemia L1210, 9 by Walker 256 (intramuscular), 1 by Dunning ascites leukemia and 2 by Ca755. As a result of this evaluation, many of the test systems previously used as primary screens have now been discontinued as unproductive. Emphasis is now placed on screening new agents in the L1210 and Walker 256 tumors. Data on drugs tested in the various tumors will be published in this and future issues even though these tumors are no longer used. In addition, the procedures described here are those in effect when the compounds were tested, not necessarily the procedures in effect at the present time.

The test systems which are or have been used as primary screens are given below, along with the host used for propagation and test:

Tumor Host for Propagation Host for Test

Adenocarcinoma 755 C57BL/6 female mouse BDFXmouse (C57BL/6 female x DBA/2 male) Cloudman melanoma (S91) DBA/2 BDFi mouse Ehrlich ascites Randombred albino mouse Randombred albino mouse Friend virus leukemia DBA/2 mouse BT>71mouse Hepatoma 129 C3H/He mouse C3H/He (or hybrid) mouse Lewis lung carcinoma C57BL/6 mouse mouse Lymphoid leukemia 1210 DBA/2 mouse mouse Osteogenic sarcoma HE10734 C3Hf mouse C3H/He mouse Sarcoma 180 Randombred albino mouse Randombred albino mouse Dunning leukemia ascites Fischer/344 rat Fischer/344 rat Human sarcoma HS1 Suitable randombred rat Suitable randombred rat Murphy-Sturm lymphosarcoma Suitable randombred rat Suitable randombred rat Walker 256 carcinosarcoma Suitable randombred rat Suitable randombred rat (intramuscular) Adenocarcinoma of duodenum Hamster Hamster Adenocarcinoma of endometrium Hamster Hamster Adenocarcinoma of small bowel Hamster Hamster Melanotic melanoma Hamster Hamster Human sarcoma HS1 Embryonated egg Embryonated egg

Preparation of Plant Materials. Currently, a single aqueous/ethanol extract is made for each plant sample by treating approximately 100 gm of finely ground dried plant with 300 ml of anhydrous ethanol and 300 ml of distilled water stirred for one hour at room temperature. The extract is then filtered through cheesecloth and evaporated to remove the alcohol. The alcohol-free filtrate is then freeze-dried to a

NOVEMBER 1966 (Part 1303

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. B. J. Abbott, J. L. Hartwell, J. Leiter, R. E. Perdue, Jr., and S. A. Schepartz powder. However, for some of the samples reported here, two extracts were prepared as follows: Aqueous extracts were made by stirring or shaking the finely ground plant sample with water (5-6 ml/gm) at room temperature for at least one hour. In some cases, such as fresh fruits and bulbs, the sample was ground and extracted simultaneously in a high-speed blender. The extract was decanted through a fast filter, concentrated under reduced pressure at 40Â°C or less, and lyophilized.

The alcoholic extracts were made by combining a portion of the original dried plant material with 2-3 parts of 95% ethanol, and stirring, or by using a Soxhlet extractor. The material was filtered and the residue extracted a second time. The 2 filtrates were then combined, and concentrated to a semisolid consistency under reduced pressure at 40Â°C or less.

The lyophilized aqueous extracts were dissolved in saline or suspended in 0. 5% methylcellulose (MC) or carboxymethylcellulose (CMC). Alcoholic extracts were dissolved in a small quantity of 95% ethanol and then suspended in MC or CMC. Occasionally alcoholic solutions were diluted with saline or with MC solution to a concentration not exceeding 2% alcohol.

Because of the gummy nature of these extracts, satisfactory suspension of the material was not always possible. Also marked changes in toxicity occasionally occurred when there was a long interval between successive tests. These facts must be considered in evaluating the data.

Test Procedures. The detailed protocols for each of the tumor systems given above have been published (23), and they will therefore not be repeated here. Copies of these protocols are available on request.4 These protocols include the methods in use at the time of testing the materials reported here. For a discussion of some of the earlier procedures, one may refer to a previous publication in this series (20).

Tumors are transplanted under aseptic procedures including sterilized instruments and hooded areas. Fragments or suspensions are sampled for sterility in every experiment by using thioglycollate broth tubes. If bacterial growth occurs within 48 hours, the entire experiment is discarded.

The details of the treatment procedure for each test are now included with each line of summary data (see Results). It is therefore unnecessary to discuss these procedures in detail. In general, the materials are injected intraperitoneally, except that endocrine substances are given subcutaneously. Normal dosage schedule is one injection daily. The number of days of treatment varies with the test system. The solid tumors are evaluated by weight. Tumor Weights are reported in grams for rat tumors and milligrams for all others. The ratio of the mean weights of tumors in treated animals to that in controls (T/C x 100 = x percent) and respective body weight changes of the animals in the treated and control .groups are recorded. For L1210 the mean survival time of animals is calculated. Ã€T/C value for L1210 tests is the mean survival time (in days) of the test group divided by the mean survival time of the control group, expressed as a percent. The Dunning leukemia systems utilized median survival times.

Deaths are recorded in all groups. A maximum tolerated dose for a single experiment is defined as the highest dose which produces 2 or fewer deaths in 6 animals or 3 or fewer deaths among 10 animals. 6 In L1210 and the Dunning ascites, deaths before Day 6 are considered nonleukemic and form the basis for toxicity evaluations. The equivalent day for the solid Dunning leukemia is Day 11. When a toxic result (greater than 2/6 or 3/10 deaths) is observed, the test is repeated at an appropriately lower dose until the maximum tolerated dose is reached. In addition, if the T/C value for survival studies is less than 85%, the dose is considered too high and is reduced. 4 From Chief, Drug Evaluation Branch, Cancer Chemotherapy National Service Center, National Cancer Institute, Bethesda, Maryland 20014. 6 In these tumor studies, the maximum tolerated dose is considered to be the LD10. which is determined once a sufficient number of tests are done. On a single test with 6 or 10 animals, however, one cannot differentiate 2/6 or 3/10 deaths from the LDi 0. These limits (2/6 or 3/10) are therefore used as the upper limits of lethality on a single experiment.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Effect of Hydroxylamine and Related Compounds on Growth of Transplanted Animal Tumors Control of the quality of the tumor systems in all laboratories is aided by the establishment of limits for deaths, "no-takes", and mean tumor weight (or survival time) among control animals. Control deaths exceeding 10$ are considered excessive. Experiments in which the control animals fall outside of limits are evaluated and some or all tests are repeated as necessary. Additional quality control is imposed by the inclusion of a "positive" control substance as one test on at least every other control group. Tests of such materials thus amount to about 2$ of all tests performed. The positive controls used for each test system are listed in the detailed protocols (23). A dose of the positive control producing a substantial but not overwhelming response has been selected. This is done to enable one to see changes in susceptibility of tumor lines to the drugs, as well as to detect any technical errors which might occur. In addition to the use of the quality control measures just described, other efforts are made to standardize the test results among all laboratories. Among the procedures begun to help standardize the tumors is a frozen tumor bank. A characterized, reliable line of each tumor is distributed from the bank to all screening laboratories at specific intervals. Thus the genetic drift is minimized and the results at different laboratories should be more reproducible. Cell Culture Procedures Cell Lines. The cell line used for routine screening is KB(Eagle), derived from a carcinoma of the nasopharynx (9). It was selected because of its rapid and reproducible growth as a monolayer culture. The work of Eagle and Foley in testing the cytotoxicity of a number of compounds against several cell lines indicates that all cells grown in monolayer culture behave similarly in their response to drugs (11, 12). When the CCNSC cell culture program first started, all compounds were tested against Chang's liver (4) and some against HeLa (15) in addition to KB. An analysis of the data on more than 1000 compounds dem onstrated that less difference in response occurred between 2 cell lines used simultaneously than with successive tests using a single cell line. Thus it was decided to use one cell line, KB, for all routine tests.

Stock cultures on glass are cultivated on basal medium (Eagle) [8] plus 10$ calf, human, or any other suitable serum. The cells are maintained in a state of rapid growth by frequent subculture, generally every 3 or 4 days. The cultures are refed 24 hours before use on test.

Test Procedures. The original procedure employed was similar to that described by Eagle and Foley (11). Cells were removed from glass either with trypsin or Versene, or by mechanical scraping. Dispersed cells were centrifuged and resuspended in complete medium, or diluted to an inactive concen tration of dispersing agent. About 50, 000 cells in 1 ml of medium were implanted in a series of replicate 15-mm screw-cap culture tubes, and the tubes incubated at a 10Â°angle at 37Â°C. After 24 hours the orig inal medium was removed and fresh medium containing the drug was added. The cultures were refed at 72 hours and the protein content was determined 1 or 2 days thereafter according to the method of Oyama and Eagle (22).

A simplification of this procedure described by Smith et al. (27), or a modification thereof, has been used since 1959. In this procedure, the suspension of cells is diluted to a concentration of 10-20 lag of cell protein per ml. Approximately 3. 9 ml of the cell suspension is added to each culture tube to which 0. 1 ml of drug solution or suitable control material had already been added. The test is ended after 72 hours; thus it is a more rapid test and eliminates the need for intermediate feeding of the cultures. The modification of this procedure, which is used in some experiments, provides a 24-hour period for the cells to attach before the drug is added with fresh medium. All these methods give equivalent results and the only critical factor is the degree of cell multi plication. An experiment is considered satisfactory if the final cell protein is at least 6 times the initial amount. Plant extracts are not sterilized but are handled aseptically. They are dissolved in water or saline if possible. If the necessary concentration cannot be attained in these solvents, ethanol, dimethyl formamide, and other solvents are used. In such cases the final concentration of solvent is less than the amount known to affect the cell growth (28).

NOVEMBER 1966 (Part Ã•) 1305

Routine testing of compounds is done at 100, 10 and 1 ^g/ml. A material with an EDBO of less than l (ig/ml (see calculations described later) is retested at lower concentrations; one with an EDSO, above 100 pg/ml is considered inactive and is not retested. In some cases, 5 dose levels at closer intervals are used when a more precise end point is desired.

The determination of cytotoxicity is based on the inhibition of cell protein synthesis. Measure ments include the initial protein per tube (C^, the final protein in control tubes (C) and the final protein in drug-treated tubes (T). The initial protein is determined on an aliquot of the inoculum or by sacrificing a series of control tubes at the time of drug addition, depending on the test procedure used. Internal controls include a protein standard (bovine serum albumin) and a group of tubes containing medium but no cells. The latter are needed because it was found that 20-30 |ag of protein from the medium adhere to each glass tube. The equivalent of a T/C value, in this case (T-CO)/(C-CO), is determined for each dose level. It is assumed that this response varies linearly with the log of the concentration and is a straight line within defined limits of response (plotting a considerable amount of data has shown this to be true). A computer program for the IBM 1410 has been developed to calculate an estimated ED50i> the dose which inhibits protein synthesis to 50$ of controls. A slope is also calculated, representing the change in re sponse for a 10-fold change in concentration.

Approximately 50 to 75 materials (n) are tested simultaneously at 3-5 dose levels against a single group of controls. Each dose level is run in duplicate tubes, and the number of controls is equal to 27rT.

EXPERIMENTALDESIGN

In Vivo Systems

Establishment of Test and Control Groups. In developing each of the test systems used in the primary CCNSC screen, the relative reproducibility of each system was used to establish reasonable test and control groups. Earlier studies had shown that satisfactory reproducibility at a reasonable cost re quired 10 animals each in a treated group and a control group for S 180 and L1210, and 16 animals each for treated and controls with Ca755.

It has been shown in testing of many materials simultaneously against a single control (13) that an optimum use of animals can be made if: (a) the number of control animals is increased by the square root of the number of treatments to be compared with that control, and (b) the sum of the reciprocals of the number of animals in the treated group and the control group is equal to what a simple treatment- control comparison would give, i. e. , equal to (1/10 + 1/10) for S180 and L1210, and (1/16 + 1/16) for Ca755. Thus, in the usual S180 experiment with 25 materials tested against a single control, there are 6 animals per test and 6/TÂ§ = 30 animals for the control group (1/6 + 1/30 = 1/10 + 1/10). A similar experiment for Ca755 uses 10 animals per treatment and 43 per control (1/10 + 1/43 = 1/16 + 1/16). For purposes of simplicity, new tumor systems were assigned to either the S 180 design or the Ca755 design depending on their relative reproducibility. Thus, the Lewis lung carcinoma uses 6 animals per test while S91 uses 10.

The animals are assigned to the control and experimental groups by acceptable randomization techniques. Various randomization procedures are used, but all are based on standard published tables of random numbers such as the Kendall-Smith tables or the Rand Corporation "Million Random Digits. "

Selection of Materials. The in vivo screening system used by the CCNSC was designed to select, for further examination, materials that have more than a specified minimum antitumor activity in at least one animal tumor system. Without prior knowledge of the true activity of materials presented to the screen, it was not possible to set up a screening mechanism which would select a fixed percentage of active materials from all the materials tested. By establishing minimum biologic standards of activity, and examining the earlier experience of screeners who had used the tumors CCNSC now uses, estimates were made of how many materials would be declared suitable for additional study. The preliminary cal culations showed that each tumor would select for further examination 1-5$ of the materials submitted.

To accomplish this selection for the original solid tumors, S 180 and Ca755, a multistage system was established. Derived from the early work of Wald (29, 30) in sequential analysis and, in part, some

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories recent work of Armitage (1, 2), a scheme was developed resembling the one in use at Lederle Laboratories (designed by Charles Dunnett) [6, 7], Detailed descriptions are given by Armitage and Schneiderman (3) and Schneiderman (24). A material passes the 1st test if it produces a T/C of less than 54$. It is then retested at the same dose. The product of the T/C values (expressed as a decimal) for the 1st 2 tests must be less than 0. 20 or the material is rejected. If the material passes the 2d stage it is tested a 3d time. For a material to pass the 3d stage, the product of the T/C values for the 3 tests must be less than 0. 08. As a result of the sequential system, most work is done on the most active materials. A clearly inactive material is rejected after only one test. The borderline material, which has passed the low hurdle of T/C = 0. 54, will usually fail on the 2d trial which requires a (geometric) mean T/C of 0. 45 (i. e. , 55$ inhibition as opposed to only 46$ inhibition). The 3d trial imposes a still tougher limit by requiring an average T/C of 0. 42 (58$ inhibition) for all 3 trials. Truly active materials will go through 3 trials, fol lowed by a minimum of 3 confirmation tests, as explained later. Based on this experience with synthetic compounds, a 2-stage sequential scheme was introduced for plant materials. Activity of a material pass ing the sequential test is confirmed by multidose experiments done with 2 extracts of the plant material. As the large number of additional systems were added to the screen, it was apparent that different sequential schemes would be necessary, depending on the reproducibility and sensitivity of the system. For simplicity, all systems were assigned to 1 of 2 schemes, either the one used for S180 and Ca755, or a somewhat more "liberal" scheme. These schemes are summarized below according to the criteria in effect when the compounds reported here were tested:

SCHEME

Type 1 Type 2

Stage 1: T/C 5 0.44 Stage 1: T/C ^ 0. 60 Stage 2: T/C S 0. 19 Stage 2: T/C ^ 0. 22

TESTSYSTEMASSIGNEDTO EACHSCHEME

Adenocarcinoma 755 Cloudman melanoma (S91) Ehrlich a scite s Solid Friend virus leukemia Hepatoma 129 Human sarcoma HS1 (rat) Lewis lung carcinoma Murphy-Sturm lymphosarcoma Osteogenic sarcoma HE10734 Walker 256 carcinosarcoma (intramuscular) Sarcoma 180 Adenocarcinoma of the small bowel Adenocarcinoma of the duodenum Melanotic melanoma Adenocarcinoma of the endometrium Human sarcoma HS1 (embryonated egg) It should be emphasized that these acceptance levels far exceed those required for a significance test such as the "t" test, which is often used. A significance test has been considered a necessary but not sufficient criterion for acceptance, since it accounts only for the variation among test objects run simultaneously. Detailed statistical analysis of CCNSC data indicates that the variation within a test is exceeded by the variation between tests run at different times. In such cases a with in-experiment sig nificance test must not be used. The operating characteristics of these screens do not provide a sharp distinction between "active" and "inactive". Therefore the activity of plant extracts passing the 2-stage sequence must be con firmed. Fresh extracts of the plant material are tested at 4 doses, 1-the same as the sequential dpse, 1 higher, and 2 lower. The S180 and Ca755 doses are about 0. 2 and 0. 3 log apart, respectively. Two dose-response experiments are normally done with 2 different extracts. To be confirmed in most solid tumors, a material must show reproducible activity (T/C = 42$for S180) at a nontoxic dose. For a ma terial to be confirmed in Ca755, a reproducible therapeutic index of 2 is necessary; the index is LDio/(T/C42^). A reproducible T/C of <10$ at a nontoxic dose is also sufficient for confirmation. Host weight loss, as described later, is considered in establishing the nontoxic dose at this stage.

NOVEMBER 1966 (ParÃ 1307

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. B. J. Abbott, J. L. Hartwell, J. Leiter, R. E. Perdue, Jr., and S. A. Schepartz The L1210 and Dunning ascites system is a 2-stage sequential scheme. A sequential system was established with 125$ as the minimum T/C value for the 1st test. This value represents an increase of 25$ in the mean (median for Dunning ascites) survival time of test animals over controls. To pass the 2d test, a material must produce an effect such that the product of the T/C values of the 2 tests (expressed as a decimal) is a i. 56. The confirmation procedure consists of a dose-response experiment at doses both higher and lower than the dose at which the sequential tests were done. A material is considered confirmed if at least one dose from 2 extraction samples produces a T/C = 125$. Extracts which pass the 2-stage sequential test and meet the criteria in the confirmation test are considered "active" and fractionation and isolation work are recommended.

Up to this stage of testing, no effects of toxicity (other than deaths) are considered in evaluating the compounds. Since it is well known that weight loss influences the growth of many tumors, an evalua tion of this effect has been made for compounds passing the confirmation procedure. The difference in weight change between the treated and control animals has been considered rather than the weight change of the treated animals alone.

Despite intensive efforts, it has not yet been possible to develop a completely general solution to the influence of weight change (or weight loss) on tumor growth, although all solid tumors are af fected. As an interim measure, specific weight change differences for each solid tumor system have been considered evidence of toxicity. Prior experience of CCNSC and other investigators with these tumors was considered in establishing these criteria (23). The recent publication by Skipper ^ta_l. (26) on the use of a "Specificity Test, " indicates that a method may now be available for determining this effect more definitively. Since this method does not draw a conclusion for each test but rather uses all data to draw a general conclusion for a given test material, it tends to minimize the variation in this phenomenon from one experiment to another. This method is now used to aid in the evaluation of com pounds beyond the confirmation stage. Performance Characteristics. The major concerns one has with a screening system are its reproducibility and its stability. The standard deviation of the T/C from experiment to experiment is the most meaningful measure of reproducibility. For the sample sizes used in these studies, the "between-test" standard deviation of the T/C values is 0. 13 (in log units) for both S180 and Ca755. The sample sizes for the S180 and Ca755 were adjusted to their present levels to equalize the between-test standard devia tions. A log standard deviation of 0. 13 means this: given a T/C on a material, one would expect that the next trial of this material would give a T/C (95$ of the time) between (T/C^ /I. 82 and (T/C^ x 1. 82. For example, if the first T/C is 60$, one should not be surprised to see a second T/C on this material anywhere between 33 and 109$. The log standard deviation for the new systems has ranged from about 0. 11 to 0. 25. It was on the basis of this standard deviation that the experimental groups and sequential schemes were assigned. CCNSC results and those of other investigators using these tumor systems have the same order of reproducibility. The variation in T/C from test to test is a result of the inherent variability of the animal systems. Two successive T/C values which are close to each other are not a result of unusual reproducibility of the material, but are merely a chance occurrence, whose frequency could be readily computed. To ascribe any special qualities to a material because of a pair of closely similar T/C values is to misread the re sults of the animal screen. Cell Culture Selection of Materials. Originally cell culture tests were done on a random sample of compounds that were also being tested against rodent tumors to correlate cytotoxicity and activity in one of thetu- mors. This was an extension of the work of Eagle and Foley (10). Preliminary data on about 2000 com pounds (24), showed that there appeared to be a good correlation which improved as the degree of cyto toxicity increased. On the basis of this study, cell culture tests were extended and all plant extracts were tested in vitro as well as in vivo. Cell culture was also used specifically for testing very small quantities of materials to select those which should be prepared in larger quantities for in vivo testing. Changes planned in the in vivo screening procedures will likely reduce the percentage of materials tested initially in cell culture, but such testing will not be eliminated entirely.

1308 CANCER RESEARCH VOL. 26

For selecting plant extracts for isolation and further evaluation in vivo, a level was set initially to choose 10% of the compounds tested. This 10% end point, which should enhance the expectation of in vivo activity (about 20% probability), is 20 jig/ml. A 2-stage testing system was chosen. A material passes the 1st stage if the EDSOis S 30 ^g/ml. In order to pass the 2d stage, a compound must have an arithmetic mean from the first 2 tests of S 20 ^g/ml. A material passing this 2d stage is confirmed by a 2d extraction of plant material. These criteria have recently been tiahtened to select as actives only materials with an EDSO= 10 p.g/ml, which provides a yield of 1-2%.

Performance Characteristics. An estimate of the reproducibility of the ED50 was made based on tests of a positive control compound at 4 laboratories; the number of replicates at each laboratory ranged from 10 to 47. Since the ED50 is a logarithmic function, as mentioned earlier, the standard deviation of the log EDSo was calculated and was equal to 0. 208. This value means that, given an ED50 (A)on a material, one would expect (95%of the time) to obtain a value between A/2. 61 and A x 2. 61. These tests were carried out at 5 dose levels at 0. 3 log intervals. The reproducibility under these conditions is sub stantially better than that previously calculated on the basis of tests with 3 doses at 1. 0 log intervals (21).

RESULTS Data on plant extracts which failed the CCNSC primary screens or confirmation tests are presented. In general the materials were tested in 3 rodent tumor systems and cell culture. The ni vivo data include 1 toxic result (if any toxicity occurred) as an aid in determining dosage for other investigators using these materials.

The format of the data summary has been revised to provide additional information, including route of administration, vehicle, day of first injection (day of tumor implantation is defined as Day 0), frequency of injection, total number of injections, and day of sacrifice. The addition of this information permits the publication of data on all systems now used as CCNSC primary screens. It will also facilitate the publication of data on active compounds, since variations in route, frequency, etc. can now be included as normal computer output. The experiment number, also now included, will aid in the evaluation of the data from the chronologic standpoint. Codes used in this report are given, as well as some additional codes which will appear in future reports. The cell culture results are presented in a new format as an "on-line" print rather than in fixed columns, since these data represent a minor part of the total information reported. This change provides the space needed for the additional information mentioned.

As previously, plant extracts are indexed according to genus and species. The identifications were provided by botanists of the USDA.

NOVEMBER 1966 (Part 2) 1309

1. Armitage, P. Restricted Sequential Procedures. Biometrika, Â¿4:9-26, 1957.

2. . Sequential Tests in Prophylactic and Therapeutic Trials. Quart. J. Med. , 23(9l):255- 74, 1954.

3. Armitage, P., and Schneiderman, M. A. Statistical Problems in a Mass Screening Program. Ann. N.Y. Acad. Sci., Z6(3):896-908, 1958.

4. Chang, R. S. Continuous Subcultivation of Epithelial-like Cells from Normal Human Tissues. Proc. Soc. Exp. Biol. Med. , .87:440-43, 1954.

5. Clarke, D. A. Mouse Sarcoma 180. In: A. Gellhorn and E. Hirschberg (eds.), Investigation of Diverse Systems for Cancer Chemotherapy Screening. Cancer Res. (Suppl.), 3.:14-17, 1955.

6. Dearborn, E. H. Recent Developments in Methods of Detecting Anti-Cancer Activity. Acta UniÃ³ Intern. Contra Cancrum, _15bis:76-84, 1959.

7. Dunnett, C. W. Statistical Theory of Drug Screening. In: de Jonge (ed. ), Quantitative Methods in Pharmacology, pp. 212-31. Amsterdam: North Holland Publishing Co. , 1961.

8. Eagle, H. Nutrition Needs of Mammalian Cells in Tissue Culture. Science. 122:501-4, 1955.

9. . Propagation in a Fluid Medium of a Human Epidermoid Carcinoma, Strain KB. Proc. Soc. Exp. Biol. Med., Eii:362-64, 1955.

10. Eagle, H. and Foley, G. E. Cytotoxicity in Human Cell Cultures as a Primary Screen for the Detec tion of Anti-Tumor Agents. Cancer Res. , 18:1017-25, 1958.

11. . The Cytotoxic Action of Carcinolytic Agents in Tissue Culture. Am. J. Med:, 21:739- 49, 1956.

12. . The Cytotoxicity of Anti-Tumor Agents for Normal Human and Animal Cells in First Culture Passage. Cancer Res. , 18:1012-16, 1958.

13. Finney, D. J. Statistical Method in Biological Assay. New York: Hafner, 1952. 14. Gellhorn, A., Kells, A., and Calino, M. Mammary Adenocarcinoma 755, Glioma 26, and Brown- Pearce Carcinoma. In: A. Gellhorn and E. Hirschberg (eds. ), Investigation of Diverse Systems for Cancer Chemotherapy Screening. Cancer Res. (Suppl. ), 1:38-43, 1955.

15. Gey, G. E., Coffman, W. D., and Kubicek, M. T. Tissue Culture Studies of the Proliferative Capac ity of Cervical Carcinoma and Normal Epithelium. Cancer Res. , 12.:264-65, 1952. 16. Goldin, A., Serpick, A. A., and Mantel, N. A Commentaryâ€”Experimental Screening Procedures and Clinical Predictability Value. Cancer Chemotherapy Rept., Â¿0:173-218, 1966.

17. Law, L. W., Dunn, T. B., Boyle, P. J., and Miller, J. H. Observations of the Effect of a Folie Acid Antagonist on Transplantable Lymphoid Leukemias in Mice. J. Nat. Cancer Inst., Â¿0:179-92, 1949.

18. Leiter, J., Abbott, B. J., Bourke, A. R., Fitzgerald, D. B., and Schepartz, S. A. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories. IX. Plant Extracts. Cancer Res. (Suppl.), 22_(No.7, part 2):609-738, 1962.

1310 CANCER RESEARCH VOL. 26

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories 19. Leiter, J., Abbott, B. J., and Schepartz, S. A. Screening Data from the Cancer Chemotherapy Na tional Service Center Screening Laboratories. XXI. Cancer Res. (Suppl. ), 2_i(No. 11, part 2): 1093- 1152, 1964. 20. Leiter, J., Bourke, A. R., Fitzgerald, D. B., Schepartz, S. A., and Wodinsky, I. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories. VII. Cancer Res. (Suppl.), .22.(No. 4, part 2):221-353, 1962.

21. Leiter, J. , Macdonald, M., and Schepartz, S. A. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories. X. Cell Culture Cytotoxicity Tests. Cancer Res. (Suppl.), li(No. 8, part 2):837-41, 1962.

22. Oyama, V. I., and Eagle, H. Measurement of Cell Growth in Tissue Culture with a Phenol Reagent (Folin-Ciocalteau). Proc. Soc. Exp. Biol. Med. , Â¿1:305-7, 1956.

23. Protocols for Screening Chemical Agents and Natural Products Against Animal Tumors and Other Bio logical Systems. Cancer Chemotherapy Rept. , _25.:l-66, 1962.

24. Schepartz, S., Macdonald, M., and Leiter, J. The Use of Cell Culture as a Presumptive Screen for Antitumor Agents. Proc. Am. Assoc. Cancer Res. , .3:265, 1961.

25. Schneiderman, M. A. Statistical Problems in the Screening Search for Anticancer Drugs by the Na tional Cancer Institute of the U. S. In: de Jonge (ed.), Quantitative Methods in Pharmacology, pp. 232-47. Amsterdam: North Holland Publishing Co. , 1961.

26. Skipper, H. E., Wilcox, W. S., Schabel, F. M. , Jr., Laster, W. R., Jr., and Mattil, L. Experi mental Evaluation of Potential Anticancer Agents. X. A Specificity Test for Distinguishing False Positives. Cancer Chemotherapy Rept. , .29;1-62, 1963.

27. Smith, C. G., Lummis, W. L., and Grady, J. E. An Improved Tissue Culture Assay. I. Method ology and Cytotoxicity of Anti-Tumor Agents. Cancer Res. , .19:843-46, 1959.

28. . An Improved Tissue Culture Assay. II. Cytotoxicity Studies with Antibiotics, Chemi cals, and Solvents. Cancer Res. , J_9:847-52, 1949.

29. Statistical Research Group, Columbia University. Sequential Analysis in Inspection and Experi mentation. Applied Mathematics Panel, National Defense Research Committee, 1945.

30. Wald, A. Sequential Analysis. New York: John Wiley and Sons, 1947.

NOVEMBER 1966 (Part e) 1311

ABBREVIATIONSFOR PLANT PARTSAND EXTRACTS

Bk Bark Bu Bulb FI Flower Fr Fruit If Inflorescence Lf Leaf PI Entire Plant Px Entire Plant Without Roots Rb Root Bark Rh Rhizome Rt Root Sb Stem Bark Sd Seed St Stems Tu Tuber Tw Twigs Uc Corm Wr Wood of Roots Ws Wood of Stems

AQ Aqueous Extract AQ/EtOH Aqueous/Ethanol Extract EtOH Ethanol Extract

SOURCE CODE

532A Mr. Philip Derse Wisconsin Alumni Research Foundation 506 Walnut Street P. O. Box 37 Madison, Wisconsin 53701

1312 CANCER RESEARCH VOL. 26

EXPLANATIONOFABBREVIATIONSANDCODES IN TABLE1

HOST DOSE REG. (Continued) 1 - Swiss 7 - Once a week 2 - BDF! A - Single dose only 51 - Fischer 344 rat B - Ad libitum in diet 70 - Syrian hamster C - Ad libitum in water 80 - Embryonated egg D - Other 90 - Cell culture DAY 1st INI. (Day of First Injection) TEST SYS. (Test System) Numbers 1 to 9 = day of first injection CA - Adenocarcinoma 755 A = Day 10; B = Day 11; C = Day 12; etc. DA - Dunning leukemia ascites HI - Human Sarcoma HS1 KB - KB (Eagle) NO. OF INJ. (Number of Injections) LE - Lymphoid leukemia 1210 Numbers 01 to 99 = total number of injections LL - Lewis lung carcinoma A = ad libitum MM - Melanotic melanoma Z = until death SA - Sarcoma 180 DAYOF SAC. (Day of Sacrifice) LAB (Screening Laboratory) Numbers 01 to 99 = day experiment is completed by 1 - Microbiological Associates, Incorporated sacrificing animals 2 - Hazleton Laboratories Z = until death 5 - Wisconsin Alumni Research Foundation 6 - Arthur D. Little, Incorporated DOSE 8 - Southern Research Institute 16 - University of Miami In mg/kg unless otherwise indicated M = (Â¿g/kg G = g/kg EXPT. NO. (Experiment Number) Experiment Identification Number SURVIVORS VEHICLE Number of animals surviving out of number started on tests as defined in individual protocols. 1 - Methylcellulose (MC) 2 - Saline WT. DIFF. (Animal Weight Difference) 3 - Acid diluted with saline 4 - Steroid suspending solution Average weight change of treated host minus average 5 - Alkali diluted with saline weight change of control host (expressed in grams 6 - Olive oil, sesame oil, peanut oil and exclusive of tumor weight). 7 - Other 8 - Carboxymethylcellulose (CMC) TUMOR WT. OR SURVIVAL(TEST/CONTROL) 9 - Water (1) Tumor weight - The mean tumor weight of the test A - Normal media animals (T); the mean tumor weight of the control B - Propylene glycol animals (C). The units are specified in the C - Acetone individual protocols. D - Alcohol E - Dimethyl formamide (2) Survival (Days) - The mean or median survival F - Dioxane time of the test animals (T); the mean or median H - Acid diluted with CMC survival time of the control animals (C). Y - Alkali diluted with CMC PERCENT ROUTE (Route of Administration) Percent = treated/control x 100. 1 - IP (Intraperitoneal) Blank when toxic. 2 - SC (Subcutaneous) 3 - Oral 4 - Other Change of response for each one-log change of dose. 5 - IV (Intravenous) 6 - IM (Intramuscular) EDK 7 - Oral following fasting The dose, in (j.g/ml, that inhibits growth to 50% of DOSE REG. (Dose Regimen) control growth; expressed as factors of appropriate powers of 10, i. e. 1. 1 x 10(-1) = 0. 11 1 - Once a day 2 - Twice a day 3 - Three times a day 4 - Every other day L = less than 6 - Twice a week or every third day M = more than

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1318 CANCER RESEARCH VOL. 26

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. B. J. Abbott, J. L. Hartwell, J. Leiter, R.E. Perdue, Jr., and S. A. Schepartz

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~~NOVEMBER 1966 (Part 1329~~

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~~1330 CANCER RESEARCH VOL. 26~~

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~~NOVEMBER 1966 (Part S) 1331~~

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~~1332 CANCER RESEARCH VOL. 26~~

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-D

~~I >~~

~~3 D~~

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~~COH COrHScoin ZÂ¡iUlCM IOcoco IOCQ(M IO(Qn~~

~~CO CO coCM (M CO IMIONo IOtsCO IOCN IONOl toN~~

~~NOVEMBER 1966 (Part i) 1333~~

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~~CÂ« IOP.CM IONin IOrÂ»IO tOP>~~

~~1334 CANCER RESEARCH VOL. 26~~

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~~(MIOKOl (MIOin~~

~~NOVEMBER 1966 (Part 2) 1335~~

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~~1336 CANCER RESEARCH VOL. 26~~

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~~NOVEMBER 1966 (ParÃi) 1337~~

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~~1338 CANCER RESEARCH VOL. 26~~

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~~--i>~~

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~~XSÃŒUJ01 COIOtsCM~~

~~NOVEMBER 1966 (Part 1339~~

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top.â€¢*

~~1340 CANCER RESEARCH VOL. 26~~

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~~NOVEMBER 1966 (ParÃÃ¨) 1341~~

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~~tonO 10BCOCOsISin IOBOÃco~~

~~to top.â€¢9-~~

~~1342 CANCER RESEARCH VOL. 26~~

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~~nvOâ€¢tHfHfH0I<0VÂ«co~~

~~NOVEMBER 1966 (Part 1343~~

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~~NOVEMBER 1966 (ParÃ2) 1345~~

~~Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. B. ./. Abbott, J. L. Hartwell, J. Leiter, R. E. Perdue, Jr., and S. A. Schepartz~~

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~~~~1346 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 1347~~~~

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~~~~1348 CANCER RESEARCH VOL. 26~~~~

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~~~~Â«O OCMnwaxo4Â«uÂ»CM-H o o O O zz 01 CM ni N n n IO IO~~~~

~~~~NOVEMBER 1966 (Part 1349~~~~

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~~~~1300 CANCER RESEARCH VOL. 26~~~~

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~~~~0zz2EgzzluCO~~~~

~~~~NOVEMBER 1966 (Part 2) 1351~~~~

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~~~~1352 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 1353~~~~

~~Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. li. J. Abbott, J. L. Hartwell, J. Leiter, R. E. Perdue, Jr., and S. A. Schepartz~~

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~~~~1354 CANCER RESEARCH VOL. 26~~~~

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories

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~~~~NOVEMBER 1966 (Part Ãª) 1357~~~~

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~~~~1358 CANCER RESEARCH VOL. 26~~~~

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~~~~1360 CANCER RESEARCH VOL. 26~~~~

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~~~~1362 CANCER RESEARCH VOL. 2C~~~~

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~~~~NOVEMBER 1966 (Part 2) 1363~~~~

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~~~~1364 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part g) 1365~~~~

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~~~~1366 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 2) 1367~~~~

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~~~~coIOtxin COIOcn~~~~

~~~~1368 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part g) 1369~~~~

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~~~~1370 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 1371~~~~

~~Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. B. ,J. Abbott, J. L. Hartwell, J. Leiter, R. E. Perdue, Jr., and S. A. Schepartz~~

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~~~~1372 CANCER RESEARCH VOL. 26~~~~

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the Cancer Chemotherapy Xational Service Center Screening Laboratories

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~~~~1374 CANCER RESEARCH VOL. 26~~~~

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the-Cancer Chemotherapy National Service Center Screening Laboratories

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~~~~(MIO00fHfIOconinIONo MinCMÂ«Ã§aOÃ¬IOIOCx jp>!EZIHco~~~~

mIONco inIONcn IO[V-P*

~~~~NOVEMBER 1966 (Part 1375~~~~

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~~~~1376 CANCER RESEARCH VOL. 26~~~~

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~~~~IOinIONp- IOtoNco~~~~

~~~~NOVEMBER 1966 (Part 1377~~~~

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~~~~1378 CANCER RESEARCH VOL. 20~~~~

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~~~~1380 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER I960 (Part 1381~~~~

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~~~~1382 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Pari 2) 1383~~~~

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~~~~IOÂ«1IOtaNÂ«HIONpHIO(M&a00IO~~~~

~~~~1384 CANCER RESEARCH VOL. 26~~~~

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~~~~-i4*f0Â«Mugaâ€¢Mewâ€¢~~~~

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~~~~ODCOCM â€¢^iN gzzcnAB~~~~

~~~~NOVEMBER 1966 (Part 2) 1385~~~~

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~~~~1386 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Pori 1387~~~~

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~~~~1388 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Pari 2) 1389~~~~

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~~~~1390 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part g) 1391~~~~

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~~~~1392 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 2) 1393~~~~

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~~~~1394 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part a) 1395~~~~

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~~~~PÂ»ION01 NIONâ€¢Â«â€¢ toNIO IOâ€¢* IOp.CO~~~~

~~~~1396 CANCER RESEARCH VOL. 26~~~~

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~~~~A N(BngIONo~~~~

~~~~NOVEMBER 1966 (Part 2) 1397~~~~

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~~~~~~1398 CANCER RESEARCH VOL. 26~~~~~~

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~~~~~~O 3 Ã›C ULUâ€¢MÂ»â€¢t54Cibtu5~~~~~~

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~~~~~~NOVEMBER 1966 (ParÃg) 1399~~~~~~

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~~~~~~Hâ€¢si 3~~~~~~

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~~~~~~â€”1zz>â€¢EdzzIIIo~~~~~~

~~~~~~ODItodBIStoo~~~~~~

~~~~~~1400 CANCER RESEARCH VOL. 26~~~~~~

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~~~~~~NOVEMBER 1966 (Part i) 1401~~~~~~

~~~~Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. B. J. Abbott, J. L. Hartwell, J. Leiter, R. E Perdue, Jr., and S. A. Schepartz~~~~

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~~~~~~1402 CANCER RESEARCH VOL. 26~~~~~~

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~~~~~~NOVEMBER 1960 (Part 1403~~~~~~

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~~~~~~1404 CANCER RESEARCH VOL. 26~~~~~~

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~~~~~~NOVEMBER 1966 (ParÃÃ) 1400~~~~~~

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~~~~~~1406 CANCER RESEARCH VOL. 26~~~~~~

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~~~~~~NOVEMBER 1966 (Part 1407~~~~~~

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~~~~~~140S CANCER RESEARCH VOL. 26~~~~~~

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~~~~~~nn00COIONo nfH IOIO00n(MCOIN00 IOIOoH>(M(OION~~~~~~

~~~~~~NOVEMBER 1966 (Part 1409~~~~~~

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~~~~~~cotoNco coIO01 coIOo CO10tNo coIOINâ€¢H~~~~~~

~~~~~~1410 CANCER RESEARCH VOL. 26~~~~~~

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~~~~~~z>Â¡i00 a^CO acoto aOkCOco~~~~~~

~~~~~~Â«0toNco coION.â€¢* totO~~~~~~

~~~~~~NOVEMBER 1966 (Part 1411~~~~~~

~~~~~~â€¢*uEoMIOCO CO Eointorv co to~~~~~~

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~~~~~~NrHcorH N rHrHPOP- rH N rH P- rHNrHCM MIHcoP> rH IS rH rH N rHinâ€¢Hoo rHNrHoo rH M â€¢*â€¢Q"sÂ«Â° rHco rH rHoo~~~~~~

~~~~~~cninco CMco cninPOoo oo oo ro cnIOCMcoco cntuco co oo cnIOco oo co oo oo oocntu co cocntO CMCMenIO~~~~~~

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~~~~~~co(VrH IOrÂ»o IOin~~~~~~

~~~~~~1412 CANCER RESEARCH VOL. 26~~~~~~

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~~~~~~3 e~~~~~~

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~~~~~~00 IONCv~~~~~~

~~~~~~NOVEMBER 1960 (Part g) 1413~~~~~~

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~~~~~~1414 CANCER RESEARCH VOL. 26~~~~~~

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~~~~~~COIOp.O COION~~~~~~

~~~~~~NOVEMBER 1966 (Part 1415~~~~~~

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~~~~~~IOrÂ»in IOINO~~~~~~

~~~~~~1416 CANCER RESEARCH VOL. 26~~~~~~

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~~~~~~NOVEMBER 1966 (Part g) 1417~~~~~~

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~~~~1418 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 1419~~~~

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~~~~1420 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 1421~~~~

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~~~~1422 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 1423~~~~

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~~~~1424 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 1425~~~~

~~Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. B. J. Abbott, J. L. Hartwell, ,1. Leiter, R. E. Perdue, Jr., and S. A. Schcpartz~~

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~~~~1426 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 1427~~~~

~~~~Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. B. J. Abbott, J. L. Hartwell, J. Leiter, R.E. Perdue, Jr., and S. A. Schepartz~~~~

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~~~~1428 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (ParÃÂ¿g) 1429~~~~

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~~~~1430 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 1431~~~~

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~~~~1432 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (ParÃ2) 1433~~~~

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories

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~~~~1438 CANCER RESEARCH VOL. 26~~~~

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~~~~DCXziUJoi op.tv~~~~

~~~~NOVEMBER 1966 (Part . 1439~~~~

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~~~~1440 CANCER RESEARCH VOL. 26~~~~

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~~~~coeNNtH CONl>-CM cooINft.CO ONtN00~~~~

~~~~NOVEMBER 1966 (Part 1441~~~~

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~~~~01oÂ£â€¢IÂ».CO~~~~

~~~~1442 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 2) 1443~~~~

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories

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~~~~NOVEMBER 1966 (ParÃ2) 1445~~~~

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~~~~1446 CANCER RESEARCH VOL. 26~~~~

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~~~~NOVEMBER 1966 (Part 2) 1447~~~~

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~~~~MIO IO1 Â« IO IO eÃ¯ oa> P. 1448 CANCER RESEARCH VOL. 26~~~~

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the Cancer Chemotherapy \ational Service Center Screening Laboratories

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~~~~1400 CANCER RESEARCH VOL. 26~~~~

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~~~~1452 CANCER RESEARCH VOL. 26~~~~

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories, XXXVII. Plant Extracts

~~~~B. J. Abbott, J. L. Hartwell, J. Leiter, et al.~~~~

~~~~Cancer Res 1966;26:1302-1453.~~~~

~~~~Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/26/11_Part_2/1302~~~~

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