Document emGNg9aG9voY2gBQJXp4Mkr1G

; v.iV -fc*' vk-o- _y Copy Fro* v- Stroop For Information of Members of Medical Advisory /....-wittee and Subcommittee oo Carcinogenicity Hay 6,1954 ESSO LABORATORIES STARTARC OIL DEVELOPMENT CM*ANY F. 0. BOX 51, LINDEN, H. J. May 4, 1954 70: Members of the Subcommittee on Carcinogenicity . Gentlemen: I am attaching a copy of a manuscript forwarded to me by pr. Horton entitled "Carcinogenesis of the Skin: I. Basic Methods Employed in Testing Complex Hydrocarbon Type Tars and Oils, and the Development of a Quantitative Scale to Express Their Relative potencies to Mice". This paper has been written by Dr. Horton and his associate, Dorothy T. Denman. It is his plan, I believe, to submit this paper to Cancer Research for publication, although he has not yet Indicated to me Just to which Journal he plans to submit it for publication. Dr. Horton has Indicated that any date in May except the period May 11-20 would be all right for a meeting of the Subcommittee. After having reviewed the manuscript, I believe a meeting of the Subcommittee with Dr. Horton would be advisable, and would suggest that this meeting be held after each of you has had asqple time to reviev the manuscript both personally and possibly with some of your technical people, so that at the time of the meeting with Dr. Horton we will be in a position to express all suggestions to him. With this in mind I tentatively propose the dates at Friday and Saturday, May 21 and 22, or Thursday and Friday, May 27 and 28 for a meeting with the Kettering group to discuss this manuscript. I would appreciate it if each of you would let me know as soon as possible . which of these two dates would be satisfactory so that final arrange ments can be made with the Kettering group. As soon as your wishes are in, I shall send you definite notice of the meeting dates. Sincerely yours, /s/ R. E. Eckardt R. E. ECKARDT, M. D. REE:ilk cc: Members of the Medical Advisory Committee Dr. A. Wesley Horton API 05770 EB 005515 SC-API-2529 r/tB thfobmation only HOT FOR PUBLICATION . API RESEARCH PROJECT C-1 . At Kettering Laboratory - University of Cincinnati (1946-1954) n . Manuscript of paper submitted for revlev prior to publication. Title: "Carcinogenesis of the Skin, I" Proposed for publication in "Cancer Research" II Status of Mmuacrlpt Msy 4, 1954 - Received from Kettering Laboratory. my 6, 1954 - Copies mailed to Subcommittee on Carcinogenicity (and to MAC). API 05771 EB 005516 CARCINOGENESIS OF THE SKIN/ I. Basic Methods Employed jesting Complex Hydrocarbon-type Tars and Oils, and the Develop ment of a Quantitative Scale to Express Their Relative Potencies to Mice. by A. Wesley Horton and Dorothy T. Denman The investigation of skin cancer in this Laboratory has been concerned with the estimation of the extent of the potential hazard of this disease in certain industries. As in any other aspect of the general field of toxicology, it must be recognized that a specific hazard is the product of several variables, two of which assume dominant Importance. These may be termed conven iently the physiological activity of the material or mixture in question, and the conditions of exposure. The latter aspect in volves the practices of personal and industrial hygiene. This paper will be devoted primarily to the development of reproducible methods of determining the first variable, the level of activity, or relative carcinogenic potency, of hydrocarbon-type oils and tars. Mice were chosen as the primary test species since neo plasma of the skin, comparable In certain respects to those of man in the same tissue, can be reproduced in these animals under suit able experimental conditions. Certain specific materials, which have induced an abnormally high incidence of cancer of the skin of men as a result of repeated exposure in their occupation, have been found capable of producing epitheliomas in the skin of mice. Conversely, in our experience certain inbred mice, such as the C3H strain, have not developed "spontaneous" skin tymors, nor have ' API 05772 EB 005517 -2- they reacted by akin tumor formation to repeated experimental spplication of a number of materials, including water, benzene, white mineral oil, etc., which are presumed to be non-careinogenic t0 the skin of man on the basis of extensive practical experience. The specification, "suitable experimental conditions", in the previous paragraph was used advisedly. It is evident from perusal of the literature, that too often the problem of excessive mortality due to the toxicity of the material under examination, due to . as well as that the occurrence of incidental disease among the experimental animals, has been met only by increasing the number of animals on teat. Such an approach ignores the probability that the survivors of these conditions are not at all representative of the population involved with respect to their susceptibility to carcinogenesis. Hence, stress will be laid upon the necessity for standards of selection and care of the experimental animals which till maintain their health at as high a level as possible. EXPERIMENTAL METHODS Most of our work has been carried out with male mice of ' the C3H strain, purchased from the Jackson Memorial Laboratory, Bar Harbor, Maine* A number of experiments have also been carried out with mala mice of the CFW strain obtained from Carworth Farms, but the results were not as reproducible as those derived from the C3H strain. Although the difference in genetic homogeneity ls undoubtedly a factor, the fact that the C3H mice are more docile Jprobably contributes to the reproducibility of experiments in which they are employed. The frequent occurrence of bites and scratches < 1 t I i \ \1 { j ' API 05773 EB 005518 -3 - 0f the akin of the CFW mice, inflicted by their companiona, probably results in occasional direct intraepithelial introduction of car cinogens. Added to this complication is the possibility that wound-healing may play an accelerating role in carcinogenesis (1), iyhe same trouble has been experienced in recent experiments with Swiss males, although the females seem to be more congenial. The mice were received at six to eljht weeks of age and placed under observation for approximately four weeks before use in any experiment. To control Infestation of the animals with mites or lice (which when uncontrolled affects adversely their health), the mice were dusted routinely with Aramite1 by the sup plier prior to shipment and then twice again with the same miticlde during the period of observation in this laboratory. Any group in which there was evidence of poor health during this period was withheld from use In teats in which quantitative results were needed* In all experiments described herein, the mice were fed on Purina Laboratory Chow and water, without restriction. At six months of age, C3H mics fed -on this diet and subjected to no adverse t experimental procedure reach an average weight of about 29-31 g., ehlle corresponding CPF' mice weigh about 33-*35 g Since growth of both the hosts and.their tumors might vary with the temperature of their surroundings, an effort has been made to maintain a relatively constant temperature of 75-2 in the animal rooms. For a giver experiment, a group of 20 to 30 mice was divided into three to five cages. The animals were assigned in dividual numbers for identification and marked by clipping their toea* .i i Aramite - lw, U. S. Rubber Company, Naugatuck, Conn. ' . [ .API 05774 EB 005519 -4 - The exposure of the mice to any given material involved repeated applications upon the interscapular region. In preparation for the application of dosages of 100 mg. of a material, the fur was left intact, but when the dosage was to be less than 100 mg., 1the fur was removed by means of electric clippers. A small camel's hair brush of a selected size to deliver the desired dosage was used to apply the material, which was then simply allowed to spread according to its individual physical characteristics. Applications were repeated in accordance with a predetermined schedule (one, two, or three times each week) until the animals died. Care was taken to keep the dosage and the location of each application as constant as possible throughout the experiment. Normal growth of the mice could not be maintained in testing certain of the materials, particularly those of high potency, .when the experiment involved three 100 mg. applications each week. Hence, when there was some physicochemical or biological evidence that a given sample was likely to have a relatively high carcino genic potency to the mice (equivalent to that of a solution of 0.15 percent, or greater, of methylcholanthrene in benzene), the material was applied at a lower frequency, usually once each week. In the case of materials of lower potency, more frequent applications were required to obtain sufficient tumors. Hence, when toxicity of such samples proved a serious problem at the 100 mg. level, the dose was reduced, in some cases to as low as 5 mg. pax* application. API 05775 EB 005520 -5- Experience has shown that, even when the mortality among experimental group was low, the time of appearance of tumors was 3{jmatlmes delayed seriously by conditions of variable or uncertain origin which also interrupted the normal growth of the animals. As s neasure of such conditions, therefore, the mice were weighed at frequent intervals throughout the experiments. Since early observations gave evidence that interruptions of growth were usually the result of intercurrent infections of low severity affecting most of the animals in a given cage, the weighing of individual animals was discontinued in favor of determining the average weight of the survivors in each group. Various precautions, aueh as regular cleaning and sterilization of cages, feeders and water bottles, decontamination (with a bacteriostatic agent) of gloves used to handle animala from one cage before proceeding to ' another, and isolation of sick mice, were taken to minimize infec tions and to prevent their spread from one group to another. Throughout each experiment, the animala were examined at least once each week hy one of us (D.T.D) and the presence and time of appearance of any abnormal gross changes in the skin were recorded. Epilation and crusting of small areas were frequently noted and, in the early stages of tests on solutions of greater than 0.2 percent of methylcholanthrene or benzopyrene in benzene, large swellings, of the type previously described by Cramer (2), and small ulcerated areas, were occasionally observed. In response to the application of solutions of these synthetic carcinogens. Papillomas eventually developed on the skin of practically all of . API 05776 EB 005521 -6 - th0 surviving animals except in the case of the lowest level of concentration tested (0,01 percent). In general, the Induced neo plasms progressed through the familiar pattern from papilloma to squamous cell carcinoma, with gross evidence of invasion. In the first test of a given material, sections of any epidermal tumors produced were prepared for microscopic examination. The date, of which each typical papilloma reached the arbitrary size of 1 mm. in diameter and elevation was recorded as tbs "time of appearance" of the papilloma. In addition, the dte on which it was grossly apparent that the turner was invading the subcutaneous tissues was noted (rolled border, generally accom panied by cratering necrosis of the center). When a mouse developed such an apparently malignant tumor, it was killed. Usually, the others were maintained on test until death from "natural causes" occurred. The data, thus obtained, were recorded on graphs such as those shown in Figures 1 through 4* ' As experience was gained in associating the physical and chemical characteristics of various types of materials with their relative toxicity and with their ability to induce tumors of the akin in C3H mice, certain criteria were developed for selecting optimum conditions for any given biological teat. As a general practice, a schedule of application was chosen which would lead to induction of papillomas in an average time of not less than 12 weeks. Die restriction was based on repeated observations that this average time was unduly variable in experiments In which the severity of exposure resulted in more rapid induction of tumors. API 05777 -7 - cn the other hand, experimental conditions which involved very long periods of exposure before tumors were Induced proved to be even more unsatisfactory than those associated with very short latent periods, because cf the increased possibility of inter current infections or other unidentified interferences with the normal growth of the mice. Even when these Interruptions resulted in little or ho loss of animals, the irregular effects on the growth of the induced tumors made it very difficult to interpret the results of the experiment. Therefore, as a general rule the frequency and severity of the applications were scheduled to produce an average latent period as close to 15 weeks as possible. When the materials were low in potency, this obviously meant the use of as severe conditions as were compatible with the normal growth and survival of the mice. INTERPRETATION OF DATA OBTAINED In general, under these experimental, conditions one of two results has been obtained in response to the applications of any one of a series of materials; i.e., most of the animals which lived long enough developed tumors of the skin at the site of the application, or none of them.did so. The outcome of the experiments, therefore, has been essentially unequivocal, and the relative potencies of the different active materials could be expressed in terms of the relative rates of induction of tumors. API 05778 EB 005523 T -8The calculation of the "mean time of appearance cf tumors" involved the determination, by the method of least squares, of the linear function which best fitted the plot of the cumulative frequency of tumor response (in probits) versus the time (in weeks) after first application (shown on the right side of Figures 1 through , The latter coordinate is equivalent to cumulative dosage. It is to be noted that, for these experiments involving repeated applications of carcinogenic materials to the skin, the response varied linearly with the dosage itself, and not with the logarithm of dosage. It Is assumed that the exponentially decreasing rate of appearance of tumors usually observed following single or limited numbers of exposures to carcinogens (3) was not seen in these ex periments because the more resistant animals in any given group were given a larger effective dosage than were the more susceptible. Calculation of Cumulative Frequencies of Response The convention is-adopted (after Bliss, 4.) that, at the midpoint of the period between two given observatlona, the number of mice bearing tumors is equal to the average of the numbers at the times of the obser vations. At the midpoint of each period during .which one or more mice develop their first papilloma, a calcu lation is made of the cumulative percentage of the 'effective group of mice" which bear tumors on that late. The "effective group" at a given time is defined is the sum of the number pf survivors plus the number of API 05779 EB 005524 -9- mica which died previously after developing tumors of the skin, except that after the appearance of a tumor In the "average" mouse this sum Is held constant. The "average" mouse Is designated by the median In a simple arithmetic array of the times of appearance of the first tumor in each animal. I ' As an example, the calculations used to obtain . the cumulative frequencies of tumor response from the 1 i data of Figure 3 are shown In the following table.: ! t EB 005525 TABLIi 1. . TIME* OP OBSERVATION OF NEW POSITIVES MIDPOINT OF PERIOD BETWEEN OBSERVATIONS, x. TOTAL NUMBER NUMBER OP CUMULATIVE OP NEW . MICE BEARING RESPONSE, POSITIVES, TUM0RS,**TR + (ZR + R'l R R + ZR 2 EFFECTIVE NUMBER OF MICE FREQUENCY OF RESPONSE, * (probits) 12x5 20 1 0.5 18 3.08 1 1*5 24 1 2U.5 25 1 13x5 34 2 15x5 36 3 1 38 31x5 2 wV> 39 1M 2 UoJ 4ix5 00 M 42 46.5 1 47 2 * Number of Mreeke after first application. 2 3 5 8 10 12 13 15 1-5 18 3.62 2.5 4.0 18 1x21 i -> 16 kxii 6.5 16 kxl6 9.0 16 5.16 11.0 16 5xit2 12.5 16 5x2 14.0 16 6.15 &fJs}o8r8eE.?aeisi!.*1 th tumors at the EB 005526 11 - ralculatlon of Mean Time of Appearance of Tumors. 3Ti 1. Assume that the tumor response in problts-, r, la a linear function of the time, x, or r = mx + b 2. Determine constants, m and tv by method of least squares (5) Normal equations: Zrx = mix3 + bZx . . r 2 mix + 9b (Values of x In weeks and r In problts are under lined In proceeding table) H4.85.67 = 10,698.25m + 300.5b 42.28 = 300.5m + 9 b b - O.96O m z 0.112 s slope of dose-response relationship . .*. r = .112x .960 3. x z time of 5-probit. response z 36.1 weeks s, standard deviation of mean, s x minus time of 4-problt response 8.9 weeks. CLASSIFICATION OF EXPERIMENTAT. results on the basis of the health OF THE ANIMALS In the appraisal of data derived from these experiments, consideration has been given to the following apparent relationchips between the average growth and survival of the mice and the Bata "time of appearance" of their epidermal tumors. . _ API 05782 EB 005527 T - 12 - 1. If,In the course of an experiment, an Intercurrent infection overtakes a group of mice at about the time when papgliomas would be expected to appear (on the basis of other comparaile experiments), there is usually a delay In the mean time of appearance of tumors. The infection need not be lethal to any of the mice, a change in sign of the slope of the average growth curve frequently being an indication of such a condition (see Figure 1), It is believed that, in many such instances, the dice would probably be unaffected were their resistance not diminished somewhat by systemic toxic effects Induced by the hydrocarbons being applied upon their skin. Thus, untreated con trols may not show any symptoms of the infection. tfot Infrequently, as the animals recover from such dis turbances, tumors grow to measurable size in a large proportion of the animals in a relatively short time. The slope of the plot of the frequency of response versus time is-then exceedingly steep. In other words, the standard deviation of the mean time of appear ance of tumors in the group is much smaller than normal (see Figure 2). Thus, it could be assumed mistakenly, if it were not for the evidence from the weight curves of the presence of uncon trolled variables, that such an experiment had defined the statistical limits of the mean better than another in which such disturbances had not occurred. API 05783 EB 005528 - 13 - 2. In aome experiments the cumulative toxic effects cf tJie material applied, or chronic infections, or a combination of both, result in prolonged inhibition of the growth of the mice, ^ number of examples have been observed in which the average weights have increased for a few weeks, but became stationary at a point 10. percent or more below that of adult controls, and then, in many instances, gradually declined (see Figure 3) Thus., whether or not these conditions Influence the timing of the initial changes II resulting in the neoplasms they may retard the growth of the in duced tumors to visibly discernible size and, accordingly, lengthen the apparent average period of exposure required for the induction of tumors by the material under test. In such experiments, the standard deviation of this mean time is frequently much greater than normal. This somewhat extended discussion of the apparent relation ships between the Irregularities in growth due to the toxicity of < applied materials and to intercurrent infections, and the rate of Induction of tumors, does not relate to the originality of this observation but rather from our recognition of its Importance in experiments in which various materials are being compared. Occasion al mention of the phenomenon has appeared in the literature on experimental carcinogenesis (.l.e., 6,7,8) and, in the field of chemotherapy of cancer, recognition has been given to the necessity of considering the systemic toxicity of a material in evaluating the specificity of its apparent "anticarcinogenic" effects (i.e., 9). API 05784 EB 005529 thr, the relationships between general health and susceptibility ^"spontaneous" tumors in man are currently receiving attention. uenc *s beHvd that, in the experimental assay of the relative potencies-of various materials or of the susceptibility of various jpeciee or strains of one species to a given material, information on the health and survival of the groups of animals being compared essential to any quantitative interpretation of the data. The results of all experiments described herein have been examined, therefore, in the light of these relationships. Each one has been classified according to the estimated significance of such effects in the following manner. Class A. Those characterized by normal growth and survival of the mice at least until the time when most of the animals have developed papillomas (see Fig.!*) Class B. Those in which all or most of the mice lived long enough to develop tumors, but In which there was . evidence (.from average weights} of Intercurrent infection at times prior to or during the period when the new growths were appearing. So long as recovery from the infection was prompt, and no obvious skewing of the distribution of the times of appearance of tumors occurred, it was felt that reliable estimates of the mean time of appearance could reasonably be made (see Figure 1). However, when no tumors had appeared prior to the onset of an Infection, and when with recovery tumors developed API 05785 EB 005530 - 15 - somewhat precipitously, the reliability of the apparent mean time of appearance was not determin able, and therefore such tests were classified X (see Figure 2), Class X. Those characterized by poor growth (as discussed in Section 2 above) or high rate of mortality. The confidence limits of the apparent mean time for the induction of tumors could not be calculated by stand ard statistical methods, because of .inability to assign proper weight to the effects of the uncon trolled variables. Several types of moderately toxic materials failed to yield experiments of the Class A category. On the other hand, experimental results of the frankly unsatisfactory type (Class X) were usually found to be avoidable through the exercise of appro priate care in the initial choice and the subsequent handling of the mice. .. REFERENCE STANDARDS To provide a background of reference which might permit a more nearly quantitative interpretation of the mean times of appearance of tumors induced in C3H mice by certain complex oils and tars under various conditions of exposure, a number of experi ments have been carried out over the past five years employing solutions of the synthetic carcinogens, 3-methylcholanthrene, benzo[a]pyrene, and 7,12-dimethylbenz [a]anthracene, in various solvents; It has been found that one of the essential requirements API 05786 E3 005531 - 16 - j.or obtaining reproducible and logically consistent results is the tintenance of the animals in a good state of health as measured growtn curves. Thus, in Table II, the results of Experiments show the precision of the determination of the mean time, x, when Class B experiments are involved. Similarly, it has been found that even in the case of a complex material, so long as the conditions of the experiments permitted reasonably normal growth 0f the animals, the repeatability of the determination of the mean time, x, has been quite satisfactory(within 10%). To determine the effect of variations in the level of concentration and in the frequency of exposure, comparable experiments were conducted with other solutions of methylcholanthrene in benzene, and the results are shown in Table II. . The purity of the polycyclic hydrocarbon is obviously an important consideration in obtaining consistent results. The usual chemical criteria are necessary, but not always " sufficient, requirements. In one case in particular, the methylcholanthrene, as received, had a sharp melting point, 176-7#C., and no indication was apparent by Its ultraviolet absorption spectrum of any contamination. Yet, on repeated bioaasay, it proved to be excessively irritating and much more rapidly carcinogenic than the normal material. Un fortunately the quantity available did not permit deter mination of a satisfactory procedure for purification. Since no further sample with such unusual potency has been obtained, bloassay remains the only way of acertainlng that the methylcholanthrene is free of the material responsible for this activity. API 05787 EB 005532 i EXPERIMENTS INVOLVING THE APPLICATION OP SOLUTIONS OP METHYLCHOLANTHRENE IN uENYENE UPON THE SKIN OP C3H MICE EXPERIMENT NUMBER CONCENTRATION OP METHYLCHOL- ANTHRENE IN SOLUTION 1% by weight) DOSAOE OF SOLUTION PER APPLICATION (mg.) NUMBER OP APPLI CATIONS PER WEEK DOSAGE OP METHYLCHOLANTHRENE PER UNIT AREA1 PER WEEK (mg.) MEAN TIME OP APPEARANCE OP TUMORS, x (weeks) CLASSIFI CATION 201 0.012 100 3 0.036 72.6 X 202 0.04J4 100 3 0.132 30.4*3.8* B 203 0.086 ioo 3 0.258 29.9*2.2 B 203 0.086 100 3 0.258 26.3*3.4 B 204 0.172 100 3 0.516 15-7*1.6 B 204 0.172 20 3 0.516 16.4*1.1 B 204 0.172 20 3 0.516 16.61.2 B 204 , 0.172 20 ` . 3 0.516 15.51.1 a 205 0.345 100 3 1.035 ... 11.0*1.9 B 205 0.345 100 2 0.69 15.1*1.5 B 205 0.345 100 1 0.345 22.4+2.7 B 205 0.345 100 3 1.035 14.7 X 206 0.69 100 1 0.69 17.8 X 206 0.69 100 3 1.98 6.7*4.! B 1 Area of akin covered by .100 mg. of solution of methylcholanthrene In benzene. 8 fiducial limits. \ EB 005533 TABLE II. (page 2) EXPERIMENTS INVOLVING THE APPLICATION OP SOLUTIONS OP METHYLCHOLANTHRENE IN BENZENE . UPON THE SKIN OP C3H MICE EXPERIMENT NUMBER CONCENTRATION OF METHYLCHOL ANTHRENE IN SOLUTION by weight) DOSAGE OP SOLUTION PER APPLICATION (mg.) NUMBER OP APPLI CATIONS PER WEEK DOSAGE OP METHYLCHOLANTHRENE PER UNIT AREA1 PER WEEK . (mg.) mean time OP APPEARANCE OP TUMORS, 3T (weeks) CLASSIFI CATION 207 1.15 100 3 209 0;517 20 1 3.45 0.517 6.9 18.31.9* X B 209 0.517 100 1 0.^17 16.22.7 B 209 0.517 100 3 219 0.115 100 3 1.551 0.345 10.4 24.0 X1 X aH> 220 1.034 100 1 1.034 '9.7*1.6 B 238 0.23 20 2 0.46 18.7*2.3 B 238 0.23 100 2 0.46 16.4*2.0 B 243 0.15 100 3 0.45 17.9*2.0 A 243 0.15 20 3- 0.45 18.2*2.1 B 268 0.287 20 2 0.574 17.6 X 1 Area of akin covered by 100 mg. of solution of methylcholanthrene in benzene. m 03 8 I# fiducial limits. . API 05789 - 19 - In Table II it is apparent that variation in the dosage . solution per application from 20 to 100 mg., other factors ^8Bajnlng constant, had no effect on the rate of induction of tumors methylcholanthrene in benzene. Similar results have been observed #hen solutions of benzopyrene in benzene were used as the carcinogenic stimulus. This constancy of potency with variation in the quantity ' 0f material applied (or area of skin exposed) also holds for some complex oils and tars, but for others there is significant change . 0f potency with changes in the quantity used for each application. W xhe chemical basis for these differences will be discussed in a later paper. . The values in column $, Table II, were calculated from the concentration of methylcholanthrene in the solutions and the ' number of applications per week. It has been determined experl ' mentally that variations in the concentration of the carcinogen did not affect the area covered by a given weight of the solutions. It was further assumed that the dosage per unit area was not slgnifi- icantly different when 20 or 100 mg. of the same solution were . and applied. Comparison of the data in columns 5/6 shows a consistent . Inverse relationship between the dosage of carcinogen per unit area per week and the mean time of appearance of tumors. The data (omitting Class X experiments) are plotted in Figure 5 together with the curve of the corresponding hyperbolic function (calculated by the method of least squares), (x - 3.7) (d + 0.10) s 8.2 - ,or, x a ------8--.!-2--------- +3.7 (d + 0.10) . ) API 05790 EB 005535 T - 20 - | is interesting to note the similarity between this equation a^d that calculated from theoretical considerations of the mechanism carcinogenesis by Iversen and Arley (10), t-L. = -i-- + T, where k and T are constants, P kce . The constant, 0,10, In the experimental equation might be inter preted as a correction required by the definite, though very small, probability of the "spontaneous" excitation of a cell of the dorsal akin of a C3H mouse to the rate of proliferation necessary for the it development of a new growth. Similar experiments have been carried out with solutions of two other commonly used carcinogenic hydrocarbons, benzopyrene and 7,12-dimethylbenzanthracene. The results are shown in Table III, together with some data on solutions of methylcholanthrene in sol vents other than benzene. As is evident from the data. It has been difficult to obtain satisfactory experiments with benzopyrene on C3H mice. The growth of the animals-has tended to be extremely erratic. Even In tests in which the average weight eventually reached 30 g., the curve was, more often than not, interrupted at critical times by sharp downward breaks. In such experiments, recovery from-these periods of poor health was sometimes accompanied by the appearance of tumors in a large percentage of the animals in a relatively short time, (relationship exemplified in Figure 2). It will also- be noted that, even at high levels of concentration, the mean time of appearance of tumors was in no case less than 20 weeks. . API 05791 EB 005536 .. TABLB 111 EXPERIMENTS INVOLVING THE APPLICATION OP SOLUTIONS OF SYNTHETIC CARCINOGENS. IN VARIOUS SOLVENTS UPON THE SKIN OP C3H MICE DOSAGE 01 CARCINOGEN PER UNIT AREA* PER WEEK (m g.) CONCENTRATION OF CARCINOGEN IN SOLUTION {% b y w e ig h t) EXPERI MENT CARCIN NUMBER OGEN1 SOLVENT 215 DMBA Benzene 0.115 DOSAGE OF SOLUTION PER APPLI CATION (mg.) NUMBER OP APPLI CATIONS PER WEEK 100 3 0.345 MEAN TIME OP APPEARANCE OP TUMCRS, XT (weeks) 15.63.3a RELATIVE CARCINO GENIC CLASSIPI- POTENCY, CATION Pur B 0.19 214 DMBA Benzene 0.345 100 3 1.035 7.4i.l B 0.62 244 BP Benzene 0.172 100 3 0.516 25.1*2.5 B 0.10 244 BP Benzene . 0.172 20 2$2 BP Benzene 0.322 20 3 1 0.516 0.322 ^27.3 ^35.0 X (0.09) X (0.18) Hro 252 BP Benzene 0.322 20 3 0.966 V23.0 X (0.11) 252 BP Benzene 0.322 . 100 3 0.966 ^20.1 X (0.13) 282 BP Benzene 0.092 20 3 0.276 X 264 BP White oil 0.175 20 3 0.525 ^25.4 X (0.10) 270 BP White oil 0.35 20 3 1.05 *v/21.5 X (0.12) 265 BP Cotton- 0.50 seed oil 20 3 1.50 X 286 BP Cotton- 1.00 20 seed oil 281 MC Cottonseed 0.042 100 3 3 3.00 0.126 A/20i4 ^41.0 B(X) B (O.13) 0.047 1 carcinogens are V12-dimethylbenz fiO anthracene,DMBA;benzof) pyrene, BP;3jnethylcholanthrene# MC. Area of skin covered by 100 mg. of solution of mothylcholanthrene in benzene. $$ fiducial limits. 01 A . " 2.2 - * of reference standards as a scale of relative pctekcy op complex . TARS AND OILS Various methods of expressing the relative potencies of the pure polycyclic carcinogens and complex materials have been reviewed by Badger (11). Many of these are simply rough classifi cations based upon a particular investigator's personal experience. The Iball index (12) and a recent adaptation (13) attempted a more quantitative approach, involving the use of the reciprocal of the mean time of appearance of tumors. Such methods are useful in com paring results obtained in a given laboratory using a standardized and inflexible schedule of application of various materials. How ever, experimental techniques differ significantly from one research organization to the next, thus rendering the comparison of data on this basis rather difficult. Further, as was pointed out . previously, in this article, it was sometimes necessary in the current investigation to change either or both the frequency and severity of exposure to obtain satisfactory conditions for the measurement of the potency of a sample while minimizing its toxic side effects. Hence, a scale of relative potencies which would permit such flexibility in experimental design was needed. The use of the data on relative rates of Induction of tumors by the pure carcinogens, methylcholanthrene and benzopyrene, as external standards of reference, provided a practical solution to this problem. The latter is one of the important contributors to the potency of cracked residua (14); the former has not been identified in complex tars and oils but might be regarded as a EB 005538 API 05793 - 23 - fepresentatiTe of the 4.-ringed carcinogens supposed to be present (15,16) T^us the mean tlo of appearance of tumors induced by 3ome complex tar or oil may be translated into a value of Relative Carcinogenic Potency, Pjjq ( potency as compared to methylehol- anthrene), by use of Figure 5 The dosage of methylcholanthrene corresponding to the given mean time is either read off the graph or calculated from the equation, ; d = 82 - 0.10 . * - 3.7 Then, the value of the relative potency, PM(j, for the complex material in question la PjjC Z d for an experiment involving one application each week, s for an experiment involving two appllcatibna each week, 2 s d, for an experiment Involving three applications each week 3 Thus, by comparing the results of teats on complex mater ials with those involving methylcholanthrene as an external standard of reference, one may obtain the relative carcinogenic potencies of the different materials, even though the experiments may have in volved differing frequencies of applications. It should be noted, however, that the description of the potency of a complex oil can not be considered complete until it has been tested at two apprec iably different dosages, l,e., 20 mg. and 100 mg. per application, unless negative results were obtained at the higher level of dosage in a test in which the mice survived and grew at a normal rate. EB 005539 API 05794 - 24 - yliu8/ a-given oil may be much more potent than another under sflvere conditions of exposure, but the former may actually have ^0jiier potency than the latter if the comparison is carried out under mild conditions. Chemical research on such oils has led to . ^ understanding of this apparent paradox, and the matter will be dealt with in a later publication. The technique of relating results on complex materials to those on standard synthetic carcinogens should facilitate compar ison of results from different laboratories. The synthetic com pound chosen should, of course, be comparable to the type of carcin ogens supposed to be present in the complex mixtures. Under appro priate conditions of dosage and exposure. It must be capable of Inducing tumors at least as rapidly as the most potent of the samples to be tested. Thus, benzopyrene has not proved to be a suit able standard for work with C3H mice, since it will not induce tumors sufficiently rapidly even at high levels of concentration. - 7,12 -Dimethylbenzfclanthracene on the other hand, is very sat isfactory from this standpoint, but has the disadvantage of requir ing special care In handling to prevent its oxidation to the non- carcinogenic 7,12-photooxide (7). . It should be reemphasized that this consideration of a quantitative scale of potencies applies only to the results of experiments which may be classified as satisfactory (A and B by our criteria). Those in our Class X have only a limited value. If . positive results are obtained, the material may be classified qualitatively as carcinogenic; if survival la good and most of the nice eventually develop tumors (X classification based on certain irregularities in growth) the estimation of the apparent potency may be useful as a minimum value. - ------------------------------- - API 05795 EB 005540 - 25 - PELATIVE potencies of polycyclic aromatic compounds The data available to the writers on experiments involving repeated applications of solutions of various polycyclic carcinogens in solvents such as benzene, acetone, and white mineral oil of high viscosity, upon the skin of mice. Indicate that the average rate 0f induction of papillomas is independent of the quantity of sol ution used in each application so long as it exceeds 10 mg. The controlling variables are the frequency of application and the dosage of carcinogen per unit area of the akin. The latter will depend upon the.concentration of carcinogen in the solution applied and the relative spreading coefficient of the solution. . Solutions of polycyclic hydrocarbons in different solvents have different capacities for spreading over the skin of a mouse because of variations in such physical properties as volatility. Interfacial tension, and viscosity. Since much of the previous experimental testing of polycyclic compounds for their carcinogen icity to the skin has been carried out with solutions in benzene, this solvent was used for the' reference standards described herein. An intensive investigation of the effects of variation of the com position of the solvent has also been carried out and will be described in a later paper. By comparing data on the rate of induction of tumors by solutions of known concentration of various aromatic hydrocarbons in benzene under suitable experimental conditions, with such data *s those shown in Table II and Figure $, it should be possible to Arrive at logical estimates of the relative carcinogenic potencies . . . API 05796 EB 005541 T - 26 - tjie different compounds. By Inference, the relative potency of 0+ ' etby]>cholanthrene on the PMC scale would be 100. Corresponding yjierical values for benzopyrene and-7,12-dimethylbenzanthracene * then be derived from the data of Table III. Thus, since O.345 percent 7,12-dlmethylbenzanthracene produced tumors at the same rate as 0.62 percent methylcholanthrene under comparable conditions, tjie relative potency of this dimethyl- derivative la p*MC --" 100 x 0.62 = 160 0.345 prom Experiment 215, the relative potency, Pjk;0, for 7, 13dimethyl- benzanthracene la 165.. Bie higher value Is preferred at the present time, since, if autoxldation of the hydrocarbon oaused any reduction In activity, it might reasonably be assumed that the effect would have been greater in Experiment 215* Similarly, the relative potency of benzopyrene on this scale is 57 - 65 (Experiments 244, 252, and 202). The data from <1 Experiment 252 Involving three applications each week cannot be used since apparently the maximum effective dosage of thia carcin ogen had been reached at a lower level. Thus, as a general, rule the determination of the relative potency of. any given compound should be baaed upon the results of experiments at at least two different concentrations, which yielded significantly different aean times of appearance of tumors under otherwise comparable conditions.' At least one and preferably both of these should Beet our classification A or B from the standpoint of growth and survival. . API 05797 EB 005542 LITERATURE CITED jj) Pullinger, B. D., J Path. Beet., 301 (1943)* j2) Cramer, W. and Stowell, R. E., J. Nat. Cancer Inat., , 369 (1942). <j) Bryan, W.R. and Shlrakin, M.B., J.Nat.Cancer Inst., 1, 807 (194D. (U) Bliss jj) Whittaker, E. and Robinson, G., The Calculus of Observation, London, Blackie and Sons, Ltd., 1924., p. 209, (6) Watson, A. P. and aellanby, E., Brit. J. Exp. Path., 11,267 0-930). (7) Bradbury, J. T., Bachmann, W.E., and Lewisohn, M.G., Cancer Re search, !L, 685 (1941)* . (8) Cook, J.W. and Kennaway, E.L. (9) . (10) Iversen, S. and Arley, N., Acta Pathol. Microbiol. Sc and,, 21, 1 (1950). (11) Badger, G.M., Brit. J. Cancer, 2, 309 (1948). (12) Iball, J., Am. J, Cancer, 2l 188 (1939J. (13) Blending, P.H., King, W.H., Priestley, W., and Rehner, J., : Arch, Ind. Hyg. Occ. Med., Ifc, 335 (195D 04) Tye, R., Graf, M.J., and Horton, A.W., (Anal. Cham., 1954)* 05) Berenblum, I. end Schoental, R., Brit. J. Cancer, 1, 157 (1947) 06) Fischer, H.G.M., Priestley, W., Eby, L.T., Wanless, G.G., and Rehner, J., Arch. Ind. Hyg. . Occ. Med., 1^, 315 (195D API 05798 EB 005543 EXPERIMENTS involving the application op materials upon the skin op C3K MICE LEQEND Symbols used In connection with average weight curvea; ^ Time of death (from disease) of l th tumor-free mouse, i Time of death (from disease) of i th tumor-bearing mouse ^ or T Time mouse killed. * Painting discontinued for 1 week at each Indicated time. Gross and microscopic pathology: 0 Time of appearance of first papilloma in Lth mouse. When this symbol is first used for a given mouse.after Its death, the presence of non-invading carcinoma (Intra-eplthellal) dr small areas of benign neoplasm was determinable only by hlstopathology. . Time of appearance of gross changes indicative of malig nancy In tumor of L th mouse. Diagnosis confirmed except In oases'where no tissue section available. When this symbol la first used for a given mouse after Its death, the Invasive malignancy of the tumor was determinable only by hlstopathology. [florQ No tissue section available for hlstopathology because of extensive post-mortem decomposition or cannibalism. API 05799 EB 005544 KIOUHK 1. EXPERIMENT 220, SOLUTION OK METHYLCHOLANTHHENE, I.O34 PERCENT IN ilEN'itiNE Cage 1, 10 Mice A STARTING DATE: . 5/3/50 STRAIN OP MICE: .* ij >ti C3H 30 fe26 to 26 i. v- - I NUMBER OP APPLICATIONS? WEEK: 1 DOSAGE OF SOLUTION PER. REPLICATION: 100 mg. I . A*' MEAN TIME OP APPEARANCE"-bP TUMORS: l0.11.6 ii'M-y CO Uh 30 4 8 12 Cage 2, 10 Mica 8128 ocwe< w 26 *< 24- m co *>0 ooU1 uo> CXI oo 22 20 HS nl 8 12 16 TIME 20 21* 28 32 36 AFTER FIRST PAINTING AVERAGE HEIGHT OP SURVIVORS (gram a) FXCiimu 2 EXPERIMENT 26Q, SOLUTION OF METHYLCHOLANTHHENE, 0.21*7 PERCENT IN BtfflZ'ENE Cage 1, 7 Mice STARTING DATE: STRAIN OP MICE: ui-' l/l4/i3 C3H-h ft o $ 8>k> NUMBER OP APPLICATIONS EA<$P;fcEEK: t feM 1 2 APj&lpATION: 20 mg. !-.'t i>V' ; ojfiy tjMORS: 17.61.1 4 8 12 28b Cage 2, 6 Mice rn gpgW . CLASSIFICATION: : X 26. 24 22ir-t m.i*i Jti.ii- ---- 12 16 20 24 Cage 3, 7 Mice I3 7,3/74^ II 2t) 32 8, 8 > /7ki7l ou03i j--j--i--j___ii___0-1*n1n/rmiHw... i--jl,-- 4 8 12 16 20 24 32^ X--------A.-----------X. - 2fl 32 O TIME AFTER FIRST PAINTING (we ) i ! V * oom) ONIUIYd HHU 25 8*i *1*1 0*1 95 gif, 02 *12 02 t-r- i" r-f--i--i--r 'i ' r "i--i--1-------------- 95 25 8*1 l Cft 95 1 -T--"--T--~--T-'--"-'feT i (aJ I IcJ O U 3 I d V awii 25 82 *T2 02 91 21 / / A*12 <N o i0n0 o ft, < -92 }2> OOTW L '5 e30. <J. 95 . 2.5 T-9!*1 r--. i-0r+-Tjjj-9r5- j^2r5 82 *12 02 91 21 8 *1 s / A /< -f[2M HQw >2ToJ co J2S 5mion CO 111 0n2w i *1i*2 ' i 0i2 i OOTW 9 42 8t0 i9frTTM2i1--v8vi ---*1i--r - |o5co NOI J.V0IJISSV70 (80*0) 5.0Wd`AONaiOd DlulibNIOHVO 3AIJ.V13H flm C0T!M0IJ.V0^JJ^, .^.^ixmos ao aovsoa 55>uajii nova PN$y|ipt'i<Mv do Haflwntj T ijBlr-i ** wi'j:*30IW 30 NIVXR S':-, nx'/a oHiiHViF L:____________________ ..... _ __ HJSIP- *mvoixnvj.vo-uou **U iNawin:a<jxk - c aunuia *12 2 ^-92 / 92 - 35 otw L 'I 80 ,yps m !r< m oo o ion 2 00 i P1GUHE ."'4 l EXPERIMENTS INVOLVING THE APPLICATION OP SOLUTIONS OP METHYLCHOLKN'ilifi &E IN bKrtZENE UPON THE SKIN OP C1H MICE ' DOSAGE OP NETKYLCHOLANTHRENE ( m g ./s ta n d a r d Area-if/W eek) Mvo* Uol 00 o . 4 8 12 16 * Aroa covered by 100 mg. of the solution. gnR IHFORMATIOH OHLY ROT FOR PUBLICATION API RESEARCH PROJECT M>1 At Kettering Laboratory - University of Cincinnati (1946-1954) Manuscript of paper submitted for revlev prior to publication. Title: "Carcinogenesis of the Skin, I" Proposed for publication in "Cancer Research" Status of Manuscript May 4, 1954 - Received from Kettering Laboratory. May 6, 1954 Copies mailed to Subcommittee on Carcinogenicity (and to MAC). B 005550 API 05805 4 CARCINOGENESIS OF THE SKIN) I. Basic Methods Employed in Testing Complex Hydrocarbon-type Tara and Oils, and the Develop ment of a Quantitative Scale to Express Their Relative Potencies to Mice, by A. Weeley Horton and Dorothy T. Denman The investigation of akin cancer in thia Laboratory has been concerned with the estimation of the extent of the potential hazard of this disease in certain industries. As in any other aspect.of the general field of toxicology, it must be recognized that a specific hazard is the product of several variables, two of which assume dominant Importance. These may be termed conven iently the physiological activity of the material or mixture in question, and the conditions of exposure. The latter aspect in volves the practices of personal and industrial hygiene. This paper will be devoted primarily to the development of reproducible methods of determining the first variable, the level of activity, or relative carcinogenic potency, of hydrocarbon-type oils and tara. Mice were chosen as the primary test species since neo plasms of the akin, comparable in certain respects to those of man in the seme tissue, can be reproduced In these animals under suit able experimental conditions. Certain specific materials, which have induced an abnormally high incidence of cancer of the skin of men as a result of repeated exposure in their occupation, have been found capable of producing epitheliomas in the skin, of mice. . Conversely, in our experience certain Inbred mice, such as the C3H strain, have not developed "spontaneous" skin tumors, nor have - API 05806 EB 005551 - -2- they reacted by akin tumor formation to repeated experimental application of a number of materials. Including water, benzene, white mineral oil, etc,, which are presumed to be non-carclnogenlc to the skin of man on the basis of extensive practical experience. The specification, "suitable experimental conditions", in the previous paragraph was used advisedly. It is evident from perusal of the literature, that too often the problem of excessive mortality due to the toxicity of the material under examination, due to as well as that the occurrence of incidental disease among the experimental animals, has been met only by increasing the number of animals on test. Such an approach ignores the probability that the survivors of these conditions are not at all representative of the population Involved with respect to their susceptibility to carcinogenesis. Hence, stress will be laid upon the necessity for standards of selection and care of the experimental animals which will maintain their health at as high a level as possible. EXPERIMENTAL METHODS Most of our work has been carried out with male mice of the C3H strain, purchased from the Jackson Memorial Laboratory, Bar Harbor, Maine, A number of experiments have also been carried out with male mice of the CFW strain obtained from Carworth Farms, but the results were not as reproducible as those derived from the C3H strain. Although the difference in genetic homogeneity is undoubtedly a factor, the fact that the C3H mice are more docile probably contributes to the reproducibility of experiments in which they are employed. The frequent occurrence of bites and scratches API 05807 EB 005552 0f the'skin of the CFW mice, inflicted by their companions, probably results in occasional direct intraepithelial introduction of car cinogens. Added to this complication is the possibility that w0und-healing may play an accelerating role in carcinogenesis (1), ifhe same trouble has been experienced in recent experiments with gwlss males, although the females seem to be more congenial. The mice were received at six to ei.;ht weeks of age and placed under observation for approximately four weeks before use in any experiment. To control infestation of the animals with mites or lice (which when uncontrolled affects adversely their health), the mice were dusted routinely with Aramite1 by the sup plier prior to shipment and then twice again with the same miticide during the period of observation in this laboratory. Any group in which there was evidence of poor health during this period was withheld from use in tests in which quantitative results were needed. In all experiments described herein, the mice were fed on Purina Laboratory Ckow and water without restriction. At six months of age, C3H mice fed on this diet and subjected to no adverse experimental procedure reach an average weight of about 29-31 g.,. r. . while corresponding CT^ mice weigh about 33-35 g. Since growth of both the hosts and.their tumors might vary with the temperature of their surroundings, an effort has been made to maintain a relatively constant temperature of 75-2 In the animal rooms. For a glvec experiment, a group of 20 to 30 mice was divided into three to five cages. The animals were assigned in dividual numbers for identification and marked by clipping their toes. 1 Aramite - 15W, U. S. Rubber Company, Naugatuck, Conn. . . . . API 05808 EB 005553 The exposure of the mice to any given material involved rCpeated applications upon the interscapular region. In preparation for the application of dosages of 100 mg. of a material, the fur iras left Intact, but when the dosage was to be less than 100 mg., (he fur was removed by means of electric clippers. A small camel's hair brush of a selected size to deliver the desired dosage was ussd to apply the material, which was then simply allowed to spread according to its individual physical characteristics. Applications were repeated in accordance with a predetermined schedule (one, two, or three times each week) until the animals died. Care was taken to keep the dosage and the location of each application as constant as possible throughout the experiment. . Normal growth cf the mice could not be maintained in . testing certain of the materials, particularly those of high potency, when the experiment Involved three 100 mg. applications each week. Hence, when there was some physicochemical or biological evidence that a given sample was likely to have a relatively high carcino- I genic potency to the mice (equivalent to that of a solution of I 0.15 percent, or greater, of methylcholanthrene in benzene), the material was applied at a lower frequency, usually once eiach week. In the case of materials of lower potency, more frequent applications were required to obtain sufficient tumors. Hence, when toxicity of such samples proved a serious problem at the 100 mg. level, the dose was reduced, in some cases to as low as 5 g. par application. API 05809 EB 005554 -5- Experience has shown that, even when the mortality among ftn experimental group was low, the time of appearance of tumors was sometimes delayed seriously by conditions of variable or uncertain origin which also interrupted the normal growth of the animals. As a measure of such conditions, therefore, the mice were weighed at frequent intervals throughout the experiments. Since early observations gave evidence that interruptions of growth were usually the result of intercurrent infections of low severity affecting most of the animals in a given cage, the weighing of individual animals was discontinued in favor of determining the average weight of the survivors in each group. Various precautions, such as regular cleaning and sterilization of cages, feeders and water bottles, decontamination (with a bacteriostatic agent) of gloves used to handle animala from one cage before proceeding to another, and Isolation of sick mice, were taken to minimize infec tions and to prevent their spread from one group to another. Throughout each experiment, the animala- were examined at least once each week by one of us (D.T.D.) and the presence and time of appearance of any abnormal gross changes in the skin were recorded. Epilation and crusting of small areas were frequently noted and, in the early stages of tests on solutions of greater . than 0.2 percent of methylcholanthrene or benzopyrene in benzene, large swellings, of the type previously described by Cramer (2), and small ulcerated areas, were occasionally observed. In response to the application of solutions of these synthetic carcinogens, papillomas eventually developed on the skin of practically all of .. API 05810 EB 005555 T -6j tIie surviving animals except in the case of the lowest level of concentration tes<:ec* (0.01 percent). In general, the induced neopiasQS progressed through the familiar pattern from papilloma to squamous cell carcinoma, with gross evidence of invasion. In the first test of a given material, sections of any epidermal tumors produced were prepared for microscopic examination. The date of which each typical papilloma reached the arbitrary size of 1 mm. in diameter and elevation was recorded as (he "time of appearance" of the papilloma. In addition, the date 0n which it was grossly apparent that the turner was Invading the ' subcutaneous tissues was noted (rolled border, generally accom panied by cratering necrosis of the center)When a mouso developed such an apparently malignant tumor, it was killed. Usually, the others were maintained on test until death from "natural causes" occurred. The data, thus obtained, were recorded on graphs such as those shown in Figures 1 through ).. As experience was gained in associating the physical and | chemical characteristics of various types of materials with their I relative toxicity and.with their ability to induce tumors of the skin in C3H mice, certain criteria were developed for selecting optimum conditions for any given biological test. As a general practice, a schedule of application was chosen which would lead to induction of papillomas in an average time of not less than 12 weeks. The restriction was based on repeated observations that this average time was unduly variable in experiments in which the severity of exposure resulted in more rapid induction of tumors. API 05811 EB 005556 7- the other hand, experimental conditions which involved very long periods of exposure before tumors were induced proved to be even more unsatisfactory then those associated with very short latent periods, because cf the increased possibility of inter current infections or other unidentlfled interferences with the ' normal growth of the mice. Even when these interruptions resulted in little or no loss of animals> the irregular effects on the erowth of the induced tumors made it very difficult to interpret the results of the experiment. Therefore, as a general rule the frequency and severity of the applications were scheduled to produce an average latent- period as close to 1$ weeks as possible. Vlhen . the materials were low in potency, this obviously meant the use of as severe conditions as were compatible with the normal growth and survival of the mice. INTERPRETATION OF DATA OBTAINED . In general, under these experimental conditions one of two results has been obtained In response to the applications of any one of a series of materials, l.e., most of the animals which lived long enough developed tumors of the skin at the site of the application, or none of them did so. The outcome of the experiments, therefore, has been essentially unequivocal, and the relative potencies of. the different active materials could be expressed in terms of the relative rates of induction of tumors. I I I I iiii i API 05812 EB 005557 -8- The calculation of the "mean tine of appearance- of tumors" i involved the determination, by the method of least squares, of the linear function which best fitted the plot of the cumulative frequen cy of tumor response (in probits) versus the time (in weeks) after tha first application (shown on the right side of Figures 1 through k) # The latter coordinate is equivalent to cumulative dosage. It is to be noted that, for. these experiments Involving repeated applications of carcinogenic materials to the skin, the response varied linearly with the dosage itself, and not with the logarithm of dosage. It is assumed that the exponentially decreasing rate of appearance of tumors usually observed following single or limited numbers of exposures to carcinogens (3) was not seen in these ex periments because the more resistant animals in any given group were given a larger effective dosage than were the more susceptible. Calculation of Cumulative Frequencies of Response The convention is adopted (after Bliss, 1;) that, at the midpoint of the period between two given obser vations, the number of mice bearing tumors is equal to the average of the numbers at the times of the obser vations. At the midpoint of each period during which one or more mice develop their first papilloma, a calcu lation is made of the cumulative percentage of the "effective group of mice" which bear tumors on that date. The "effective group" at a given time is defined as the sum of the number of survivors plus the number of API 05813 EB 005558 9 nice which died previously after developing tumors of the skin, except that after the appearance of a tumor in the "average" mouse this sum is held constant. The "average" mouse is designated by the median in a simple arithmetic array of the times of appearance of the first tumor in each animal. As an example, the calculations used to obtain 1 the cumulative frequencies of tumor response from the data of Figure 3 are shown in the following table: 4 API 05814 EB 005559 TIME* OP OBSERVATION . OF NEW POSITIVES MIDPOINT OF PERIOD BETWEEN OBSERVATIONS, x. NUMBER OF NEW POSITIVES, R' TOTAL NUMBER OF CUMULATIVE MICE BEARING RESPONSE, TUMORS,M 2R + (ZR + R') R + ZR 2 EFFECTIVE NUMBER CF MICE FREQUENCY OF RESPONSE, z (probits) 11*5 0.5 18 3.08 20 1 1 2x5 24 1 1.5 I8 . 2 2tuS 25 i 34 25x5 36 2 3 2.5 18 h31 3 4.0 16 4*33 5 6.5 16 ' 4.76 8 , 3B 32x5 2 9.0 10 16 5x16 28^5 11.0 16 5x42 H>*0. 39 41.5 2 12 12.5 16 5x28 0UO0) 42 UM1 47 46.5 1 2 13 14.0 15 16 6.15 * Number of weeks after first application. **ZRtJethf ^l0Q}T8?e?^i8R.wlth tumors at EB 005560 I T - calculation of Mean Time of Appearance of Tumors. 3f: 1. Assume that the tumor response In probits, r, Is a linear function of the time, x, or r s nuc + b 2. Determine constants, m and b7 method of least squares (5). Normal equations: Zrx = mxa + bZx r z mix + 9b . (Values of x In weeks and r In problts are under lined In proceeding table) . 11*85.67 = 10,688.25m + 300.56 1*2.28 = 300,5m + b s 0.960 9b m : 0.112 s slope of dose-response - relationship .*. r = .112x + .960 . 3. x = time of 5-probit' response = 36.I weeks s, standard deviation of mean, ' = x minus time of 4-problt response 8.9 weeks. . CLASSIFICATION OP EXPERIMENTAL RESOLTS ON THE BASIS OF THE HEALTH OF THE ANIMALS In the appraisal of data derived from these experiments, consideration has been given to the following apparent relation ships between the average growth and survival of the mlOe and the nean "time of appearance" of their epidermal tumors. API 05816 EB 005561 12 - 1. If, in the course of an experiment, an intercurrent infecticn overtakes a group of mice at about the time when pap illomas would be expected to appear (on the basis of other comparaile experiments), there is usually a delay in the mean time of appearance of tumors. The infection need not be lethal to any of the mice-, a change in sign of the slope of the average growth curve frequently being an Indication of such a condition (see Figure 1). It is believed that, in many such instances, the nice would probably be unaffected were their resistance not diminished somewhat by systemic toxic effects Induced by the hydrocarbons being applied upon their skin. Thus, untreated con trols may not show any symptoms of the infection. Not Infrequently, as the animals recover from such dis turbances, tumors grow to measurable size in a large proportion of the animals in a relatively short time. The slope of the plot of the frequency of response versus time is then exceedingly steep. In other words, the standard deviation of the mean time of appear ance of tumors in the group is much smaller than normal (see Figure 2). Thus, it could be assumed mistakenly, if it were not for the evidence from the weight curves of the presence of uncon trolled variables, that such an experiment had defined the statistical limits of the mean better than another in which such disturbances had not occurred. API 05817 EB 005562 - 13 - 2. In some experiments the cumulative toxic effects of the material applied, or chronic infections, or a combination of tjcth, result in prolonged inhibition of the growth of the mice. p number of examples have been observed in which the average weights have increased for a few weeks, but became stationary at a point 10 percent or more below that of adult controls, and then, in many instances, gradually declined (see Figure 3) Thus, whether or not these conditions influence the timing of the initial changes resulting In the neoplasms they may retard the growth of the in duced tumors to visibly discernible size and, accordingly, lengthen the apparent average period of exposure required for the induction of tumors by the material under test. In such experiments, the standard deviation of this mean time is frequently much greater than normal. . This somewhat extended discussion of the apparent relation ships between the irregularities in growth due to the toxicity of applied materials and to Intercurrent Infections, and the rate of Induction of tumors, does not relate to the originality of this observation but rather from our recognition of its importance in experiments in which various materials are being compared. Occasion al mention of the phenomenon has appeared in the literature on experimental carcinogenesis (l.e., 6,7,9) and. In the field of chemotherapy of cancer, recognition has been given to the necessity of considering the systemic toxicity of a material in evaluating the specificity of its apparent "anticarcinogenicn effects (i.e., 9) . API 05818 EB 005563 - Ill - curther, the relationships between general health and susceptibility ^"spontaneous" tumors in man are currently receiving attention. Hence, it is believed that, in the experimental assay of the relative potencies of various materials or of the susceptibility of various species or strains of one species to a given material, information on the health and survival of the groups of animals being compared is essential to any quantitative interpretation of the data. The results of all experiments described herein have been examined, therefore, in the light of these relationships. Each one has been classified according to the estimated significance of such effects in the following manner. Class A. Those characterized by normal growth and survival of the mice at least until the time when most of the animals have developed papillomas (see Fig.U.). Class B. Those in which all or most of the mice lived long enough to develop tumors, but.in which there was evidence (from average weights) of intercurrent infection at times prior to or during the period when the new growths were appearing. So long as recovery from the infection was prompt, and no . obvious skewing of the distribution of the times of appearance of tumors occurred, it was felt that - reliable estimates of the mean time of appearance could reasonably be made (see Figure 1). However, when no tumors had appeared prior to the onset of an infection, and when with recovery tumors developed API 05819 EB 005564 - 15 - somewhat precipitously, the reliability of the apparent mean time of appearance was not d'etermin able, and therefore such tests were classified X (see Figure 2), Class X. Those characterized by poor growth (as discussed in Section 2 above) or high rate of mortality. The confidence limits of the apparent mean time for the induction of tumors could not be calculated by stand . ard statistical methods, because of inability to assign proper weight to the effects of the uncon trolled variables. Several types of moderately toxic mateirials failed to yield experiments of the Class A category. On the other hand, experimental results of the frankly unsatisfactory type (Class X) were usually found to be avoidable through the exercise of appro priate care in the initial choice and the subsequent handling of the mice. ` . REFERENCE STANDARDS . To provide a background of reference which might permit a more nearly quantitative interpretation of the mean times of appearance of tumors induced in C3H mice by certain complex oils . and tars under various conditions of exposure, a number of experi ments have been carried out over the past five years employing solutions of the synthetic carcinogens, 3-methylcholanthrene, . benzo[a)pyrene, and 7,12-dimethylbenz [a]anthracene, in various solvents. It has been found that one of the essential requirements API 05820 EB 005565 16 - f0T obtaining reproducible and logically consistent results is the ^air-tenance of the animals in a good state of health as measured by growth curves. Thus, in Table II, the results of Experiments 205 show the precision of the determination of the mean time, x, when Class B experiments are Involved. Similarly, it has been found that even in the case of a complex material, so long as the conditions of the experiments permitted reasonably normal growth of the animals, the repeatability of the determination of the mean time, x, has been quite satisfactory(within 10%). To determine the effect of variations in the level of concentration and in the frequency of exposure, comparable experiments were conducted with other solutions of methylcholanthrene in benzene, and the results are shown in Table II. The purity of the polycyclic hydrocarbon-is obviously an important consideration in obtaining consistent results. The usual chemical criteria are necessary, but not always sufficient, requirements. In one case in particular, the methylcholanthrene, as received, had a sharp melting point, 176-7c., and no indication was apparent by its ultraviolet absorption spectrum of any contamination. Yet, on repeated bioassay, it proved to be excessively irritating and much more rapidly carcinogenic than the normal material. Un fortunately the quantity available did not permit deter mination of a satisfactory procedure for purification. Since no further sample with such unusual potency has been obtained, bioassay remains the only way of acertainlng that the methylcholanthrene is free of the material responsible for this activity. API 05821 EB 005566 TABLE II EXPERIMENTS INVOLVING THE APPLICATION OP SOLUTIONS OF METHYLCHOLANTllRENE IN oENZENH UPON THE SKIN OP C3H MICE EXPERIMENT NUMBER CONCENTRATION OP METHYLCHOL- ANTHRENE IN SOLUTION .($ by weight) DOSAGE OF SOLUTION PER APPLICATION (mg.) NUMBER OP APPLI CATIONS PER WEEK DOSAGE OP METHYLCHOLANTHRENE PER UNIT AREA1 PER WEEK (mg.) . MEAN TIME OP APPEARANCE OP TUMORS, x (weeks) CLASSIFI. CATION 201 202 203 203 204 204 204 0.012 0.044 0.086 0.086 0.172 0.172 0.172 100 100 100 100 100 20 20 3 3 3 3 3 3 3 0.036 0.132 0.258 0.258 0.516 0.516 0.516 72.6 30.413.8* 29.9 2.2 26.3-3.4 15.7*1.6 16.4H.1 16.61.2 X B B B B B B I -vl i 204 0.172 20 3 0.516 . 15.51.1 U 205 0.345 100 ` 3 1.035 ll.oi.9 B 205 0.345 100 2 0.69 15.1*1.5 B 205 0.345 . 100 l 0.345 22.412.7 B 205 0.345 100 3 1.035 14.7 X 206 0.69 100 1 0.69 17.8 X 206 0.69 100 3 1.98 6.714.1 B EB 005567 1 Area of skin covered by 100 mg. of solution of methylcholanthrene in benzene. n> * fiducial limits. TABLE II (page 2) EXPERIMENTS INVOLVING THE APPLICATION OP SOLUTIONS OP METHYLCHOLANTHRENE IN BENZENE UPON THE SKIN OP C3H MICE EXPERIMENT NUMBER CONCENTRATION Ob' METHYLCHOL- ANTHRENE IN SOLUTION [i by weight) DOSAGE OP SOLUTION PER APPLICATION (")......... NUMBER OF APPLI CATIONS PER WEEK DOSAGE OF METHYLCHQLANTHHENE PER UNIT AREA1 PER WEEK (mg.) MEAN TIME OP APPEARANCE OP TUMORS, 7 (weeks) CLASSIFI CATION 207 1 209 209 1.15 100 3 0.517 20 1 0.517 100 1 3.45 0.517 0.517 8.9 18.3+1.9* 16.22.7 209 219 220 238 1 238 0.517 0.115 1.034 0.23 0.23 100 100 100 20 . 100 3. . 1.551 3 0.345 l 1.034 2 O.46 2 0.46 10.4 24.0 9.7*1.6 18.7*2.3 16.4*2.0 243 ' 243 0.15 0.15 100 3 0.45 . 17.9*2.0 20 3 0.45 18.2*2.1 268 0.287 20 2 0.574 17.6 1 Area of akin covered by 100 mg. of solution of methylcholanthrene in benzene. 8 fiducial limits. X B B X X B B B A B X API 05823 EB 005568 (a - 19 - In Table II It is apparent that variation In the dosage 0f solution per application from 20 to 100 mg., other factors remaining constant, had no effect on the rate of induction of tumors by methylcholanthrene in benzene. Similar results have been observed when solutions of benzopyrene in benzene were used as the carcinogenic stimulus. This constancy of potency with variation in the quantity of material applied (or area of skin exposed) also holds for some complex oils and tars, but for others there is significant change of potency with changes in the quantity used for each application. The chemical basis for.these differences will be discussed in a later paper. . The values in column 5 Table II, were calculated from the concentration of methylcholanthrene in the solutions and the number of applications per week. It has been determined experi mentally that variations in the concentration of the carcinogen did not affect the area covered by a given weight of the solutions. It was further assumed that the dosage per unit area was not signifi cantly different when 20. or 100 mg. of the same solution were and applied. Comparison of the data in columns 5/6ahowa a consistent Inverse relationship between the dosage of carcinogen per unit area per week and the mean time of appearance of tumors. The data (omitting Class X experiments) are plotted in Figure 5 together with the curve of the corresponding hyperbolic function (calculated 1 by the method of least squares). i (x - 3.7) (d + 0.10) =8.2 82 or. X = + 3*7 (d + 0.10) API 05824 EB 005569 - 20 It Is interesting to note the similarity between this equation and that calculated from theoretical considerations of the mechanism of carcinogenesis by Iversen and Arley (10), ts + f, where k and T are constants. P kc0 The constant. 0.10, in the experimental equation might be inter preted as a correction required by the definite, though very small, probability of the "spontaneous" excitation of a cell of the dorsal skin of a C3H mouse to the rate of proliferation necessary for the development of a r*w growth. Similar experiments have been carried out with solutions of two other commonly used carcinogenic hydrocarbons, benzopyrene and 7,12-dimethylbenzanthracene. The results are shown in Table III, together with some data on solutions of methylcholanthrene in sol vents other than benzene. As is evident from the data, it has been difficult to obtain satisfactory experiments with benzopyrene on C3H mice. The growth of the animals has tended to be extremely erratic. Even in tests in which the average weight eventually reached 30 g., the curve was, more often than not, interrupted at critical times by sharp downward breaks. In such experiments, recovery from these periods of poor health was sometimes accompanied by the appearance of tumors in a large percentage of the animals in a relatively short time (relationship exemplified in Figure 2). It will also be noted that, even at high levels of concentration, the mean time of appearance of tumors was in no case less than 20 weeks. EB 005570 ^-- * TABLE III EXPERIMENTS INVOLVING TOE APPLICATION OP SOLUTIONS OP SYNTHETIC CARCINOGENS . IN VARIOUS SOLVENTS UPON THE SKIN OF C3H MICE a M CONCENTRATION OF CARCINOGEN IN SOLUTION {% b y w e ig h t) ii EXPERI MENT CARCIN NUMBER OGEN1 SOLVENT DOSAGE OF SOLUTION PER APPLI CATION (mg.) NUMBER OF APPLI CATIONS PER WEEK n > e) WH30 MEAN TIME gj *-3 MO W> OF 0 m0 SHipp oO s APPEARANCE OF TUMCRS, wSuiz&no 7 (weeks) RELATIVE CARCINO GENIC CLASSIFI POTENCY, CATION ___ PMC 215 DMBA Benzene 0.115 100 3 0.345 15.63.33 B 0.19 214 244 244 252 252 252 DM BA BP BP BP BP BP Benzene Benzene Benzene Benzene Benzene Benzene 0.345 0.172 0.172 0.322 0.322 0.322 100 100 20 20 .20 100 3 3 3 1 3 3 1.035 0.516 0.516 0.322 0.966 0.966 7.4^1.1 25.1*2.5 ^27.3 ^35.0 V23.0 si/20.1 B 0.62 B 0.10 X (0.09) X (0.18) X (0.11) X (0.13) 282 264 ' 270 BP Benzene 0.092 BP White oil 0.175 BP White oil 0.35 20 20 . 20 3 3 3 0.276 0.525 1.05 . ^25.4 ^/21.5 X X (0.10) X (0.12) j 285 BP Cotton- 0.50 20 3 1.50 i seed oil X EB 005571 > 286 BP Cotton- 1.00 20 3 3.00 V20.U B(X) (0.13) *HG UO1 seed oil 281 MC Cottonseed 0.042 100 3 0.126 -^41.0 B 0.047 1 carcinogens are1'/', 12-dimethylbenz [gl anthracene,DMBA;benzof) pyrene,BP;3-methylcholanthrane, UC. CfsOj Q\ a Area of skin covered by 100 mg. of solution of methylcholanthrene in benzene. 3 fiducial limits. . i T 5 # 1 i . rrcf OF REFERENCE STANDARDS AS A SCALE OF RELATIVE POTENCY OP COMPT.nnr TARS AND OILS Various methods of expressing the relative potencies of . the pure polycyclic carcinogens and complex materials have been reviewed by Badger (11). Many of these are simply rough classifi cations based upon a particular investigator's personal experience. The Iball index (12) and a recent adaptation (13) attempted a more quantitative approach, involving the use of the reciprocal of the . mean time of appearance of tumors. Such methods are useful in com paring results obtained in a given laboratory using a standardized and inflexible schedule of application of various materials. How ever, experimental techniques differ significantly from one research organization to the next, thus rendering the comparison of data on this basis rather difficult. Further, as was pointed out previously in this article, it was sometimes necessary in the current investigation to change either or both the frequency and severity of exposure to obtain satisfactory conditions for the measurement of the potenoy of a sample while minimizing its toxic side effects. Hence, a scale of relative potencies which would permit such flexibility in experimental design was needed. The use of the data on relative rates of induction of tumors by the pure carcinogens, methylcholanthrene and benzopyrene, as external standards of reference, provided a practical solution to this problem. The latter la one of the important contributors to the potency of cracked residua (14); the former has not been identified in complex tars and oils but might be regarded as a API 05827 I EB 005572 - 23 - representative of the ^-ringed carcinogens supposed to be present {1^,16). Thus, the mean time of appearance of tumors induced by some complex tar or oil may be translated into a value of Relative Carcinogenic Potency, P^q ( potency as compared to methylcholanthrene), by use of Figure 5. The dosage of methylcholanthrene corresponding to the given mean time is either read off the graph or calculated from the equation. d = _9'2 - 0.10 . * - 3.7 Then, the value of the relative potency, PM(,, for the complex material in question is PMC = d, for an experiment Involving one application each week. = for an experiment involving two applications each week. s d, for an experiment involving three applications each weelc 3 Thus, by comparing the results of tests on complex mater- ials with those involving methylcholanthrene as an external standard of reference, one may obtain the relative carcinogenic potencies of the different materials, even though the experiments may have involved differing frequencies of applications. It should be noted, however, that the description of the potency of a complex oil cannot be considered complete until it has been tested at two appreclably different dosages, l.e., 20 mg. and 100 mg. per application, unless negative results were obtained at the higher level of dosage in a test in which the mice survived and grew at a normal rate. API 05828 EB 005573 T Thus* a given oil may be much more potent than another under aavere conditions of exposure, but the former may actually have a lower potency than the latter if the comparison is carried out i under mild conditions. Chemical research on such oils has led to an understanding of this apparent paradox, and the matter will be dealt with in a later publication. The technique of relating results on complex materials to those on standard synthetic carcinogens should facilitate compar ison of results from different laboratories. The synthetic com pound chosen should, of course, be comparable to the type of carcin ogens supposed to be present in the complex mixtures. Under appro priate conditions of dosage and exposure, it must be capable of Inducing tumors at least as rapidly as the most potent of the samples to be tested. Thus, benzopyrene has not proved to be a suit able standard for work with C3H mice, since it will not Induce tumors sufficiently rapidly even at high levels of concentration. i 7,12 -Dimethylbenz{)anthracene on the other hand, is very sat i isfactory from this standpoint, but has the disadvantage of requir ing special care in handling to prevent its oxidation to the. non- . carcinogenic 7,12-photooxide (7)t .' It should be reemphasized that this consideration' of a quantitative scale of potencies applies only to the results of experiments which may be classified as satisfactory (A and B by our criteria). Those in our Class X have only a limited value. If positive results are obtained, the material may be classified qualitatively as carcinogenic; if survival is good and most of the mice eventually develop tumors (X classification baaed on certain irregularities in growth) the estimation of the apparent potency may be useful as a minimum value. - API 05829 EB 005574 - 25 - P.ELATIVE POTENCIES OF POLYCYCLIC AROMATIC COMPOUNDS The data available to the writers on experiments involving repeated applications of solutions of various polycyclic carcinogens in solvents such as benzene, acetone, and white mineral oil of high viscosity, upon the skin of mice, indicate that the average rate of induction of rapillomas is independent of the quantity of sol ution used in each application so long as it exceeds 10 mg. The controlling variables are the frequency of application and the dosage of carcinogen per unit area of the akin. The latter will depend upon the concentration of carcinogen in the solution applied and the relative spreading coefficient of the solution. Solutions of polycyclic hydrocarbons in different solvents . have different capacltiea for spreading over the skin of a mouse because of variations in such physical properties as volatility, interfacial tension, and viscosity. Since much of the previous experimental testing of polycyclic compounds for their carcinogen icity to the skin has been carried out with solutions in benzene, this solvent was used for the reference standards described herein. An intensive Investigation of the effects of variation of the com position of the solvent has also been carried out and will be described in a later paper. . By comparing data on the rate of Induction of tumors by solutions of known concentration of various aromatic hydrocarbons in benzene under suitable experimental conditions, with such data as those shown in Table II and Figure 5 it should be possible to arrive at logical estimates of the relative carcinogenic potencies . API 05830 EB 005575 t . - 26 - 0f the different compounds. B7 Inference, the relative potency of nethylcholanthrene on the scale would be 100. Corresponding numerical values for benzopyrene and 7,12-dimethylbenzanthracene may then be derived from the data of Table III. Thus, since 0.345 percent 7, 12-dlmethylbenzanthracene produced tumors at the same rate as 0.62 percent methylcholanthrene under comparable conditions, the relative potency of this dimethyl- derivative Is P = JL x 0.62 =180 MC 0.345 From Experiment 215, the relative potency, Pjj(j, for 7,12-dimethyl-, benzanthracene is 165. The higher value is preferred at the present time, since, if autoxldatlon'of the hydrocarbon caused any reduction in activity, it might reasonably be assumed that the effect would have been greater in Experiment 215. Similarly, the relative potency of benzopyrene on this scale is 57 - 65 (Experiments 244, 252, and 282). The data from Experiment 252 involving three applications each week cannot be used since apparently the- maximum effective dosage of this carcin ogen had been reached at a lower level. Thus, as a general rule the determination of the relative potency of any given compound should be baaed upon the results of experiments at at least two different concentrations, which yielded significantly different mean times of appearance of tumors under otherwise comparable conditions. At least one and preferably both of these should meet our classification A or B from the standpoint of growth and survival. API 05831 EB 005576 T - 27 ^ LITERATURE CITED | (i) Pul linger, B. D.,. J. Path. Bact., , 301 (1943). I (2) Cramer, W. and Stowell, R. E., J. Nat. Cancer Inst., Z, 369 (1942). ' (3) Bryan, W.R. and Shimkin, M.B., J.Nat.Cancer Inst., 1, 807 (1941). | (4) Bliss (5) . Whittaker, E. and Robinson, 6., The Calculus of Observation, London, Blackle and Sons, Ltd., 1924# P. 209. (6) Watson, A. P. and mellanby, E., Brit. J. Exp. Path., 11,267 0-930). (7) Bradbury, J. T., Bachmann, W.E., and Leirisohn, M.G., Cancer Research, 1, 685 (194D. (8} Cook, J.W. and Kannaway, E.L. (9) . (10) Iversen, S. and Arley, N., Acta Pathol. Microbiol. Scand., 21, 1 (1950). . (11) Badger, G.M., Brit. J. Cancer, 2, 309 (1948). . 4^ (12) Iball, J., Am. J. Cancer, 188 (1939). | (13) Blanding, F.H., King, W.H., Priestley, W., and Rehner, J., j Arch, Ind. Hyg. Oco. Med., {, 335 (1951). ! (14) Tye, R,, Graf, M.J., and Horton, A.W., (Anal. Chea., 1954)* (15) Berenblua, I. and Schoental, R., Brit. J. Cancer, 1, 157 (1947). I (16) Fischer, H.G.M., Priestley, W., Eby, L.T., Wanless, G.G., and ' Rehner, J., Arch. Ind. Hyg. Occ. Med., ifc, 315 (1951). API .05832 EB 005577  experiments involving the application op materials upon the skin op C3H MICE LEGEND Symbols uaed In connection with average weight curvea; ^ Time of death (from disease) of Lth tumor-free mouse. I Time of death (from disease) of tth tumor-bearing mouse ^or T Time mouse killed. e Painting discontinued for 1 week at each indicated time. Gross and microscopic pathology: [H Time of appearance of first papilloma in i th mouse. ^ When this symbol is first used for a given mouse after its death, the presence of non-invading carcinoma (intra-eplthellal) or small areas of benign neoplasm was determinable only by hlstopathology. Time of appearance of gross changes indicative of malig nancy in tumor of ith mouse. Diagnosis confirmed . except in cases where no tissue section available. When this symbol is first uaed for a given mouse after its death; the Invasive malignancy of the tumor was determinable only by hlstopathology. rfi.or& No tissue section available for hlstopathology because ^ of extensive post-mortem decomposition or cannibalism. API 05833 EB 005578 I FIGURE 2 EXPERIMENT 268,. SOLUTION OF METHYLCHOLANTHHENK, 0.287 PERCENT IN UENZENE l/l4/^3 C3H-h o . 20 mg. 17.61.1 X m CD > oH ioioonno OU0C01J . cn (suae*) ONIL'NIYd X S H I d U3UV 3 W I X 35 ft+i nn on gc sc os. ns o? -T - ]- I f 9$ ?5 on nn on 9c sc 03 ns os 91 si IP kJ"|fl EP liT 1 1 9 0--1--1--r- S / -ns / h 95 z$ on nn on 9c gc. 9_ s -1-- - I- -1--I III - I--gp--jyv-ji--r ns o? w~r .\ \ Ss -9S US?< OOTW L `C 88bD 9i si 0 n "T-------I------- I------- 1------- I------- I------- 1*- s /N msS 1/ % 'C -, i \A i1 ^ iNOIXVOIdTSSVIO 1 (eo'o) 0Wd4AON3iod oiwaooNiouvo aAixvaau % am coi^Noiivonsd'' >iaj Noixmos ao aovsoci ?.: naaM nova knotxvondav do Haawmi Htc :aoiw ao Niwaxs tS/1/o :axva onixhvxo 15 ' 'Av/* - oro "aaMOvko xonvoixAnvivo -iion '7nT*xNawfusiaxil"CTiunoid 8 -3 &3o A in )S<| 0fW 9 *Z 3d0 loci 9'T ' 5T' 3 01 u to ns 2 93. /' 03 eofw Z 'T eiBO DC EB 005581 API 05836 ________________ ~3=r LC8S0 IdV KIGURE 4 EXPERIMENT 243, SOLUTION OF METHYLCHOLANTHRENE, 0.15 PERCENT IN BENZENE i AVERAGE WEIGHT OF SURVIVORS (gram s) EB 005582 TIME DOSAGE OP METHYLCIIOLANTHRENE (m g ./S ta n d a rd A re a */W e e k ) EXPERIMENTS INVOLVING THE APPLICATION OP SOLUTIONS OP METHYLCHOLhNTHKENE IN bEN'XENE oU1 0u0 <p jjr * j!/i Ca^' 0L Copy From D. V. Stroop to Recipients of the Kettering Manuscript With Copies of Suaaary for Attachment to Manuscript Which Bad Been Fully Prepared for shipment Prior to Receipt of _______ May 10, 1954 UNIVERSITY CP CINCINNATI The Kettering Laboratory College of Medicine - Eden Avenue Cincinnati 19, Ohio . . . May 8, 1954 fc t Mr. D. V. Stroop, Director Technical Service Division American Petroleum Institute 50 West 50th Street Nev York 17, Nev York Dear Mr. Stroop: Attached are the stencils of the summary of the paper on "Carcinogenesis of the Skin". These pages should he Inserted in the manuscript just before the bibliography. Thanks again for your cooperation in these matters. Sincerely yours, /s/ Wes A. Wesley Horton AHB:mJg cc: Dr. R. E. Eckardt Enc. API 05839 EB 005584 - 27 - ' SUMMARY . . Repeated applications of solutions of carcinogenic hydro carbons upon the dorsal skin of male mice of the C3H strain under suitable experimental conditions result in the induction of epithel iomas in essentially all of the exposed animals. The average period of exposure (x) to solutions of 3-nethylcholenthrene In benzene required to induce papillomas of the skin of these mice varies inversely with the dosage (d) of the carcinogen per unit area per . week, according to the following relationship: d + 0.10 For experiments involving combinations of synthetic carcinogens with non-accelerating solvents, the mean time of induction of tumors is Independent of the total area of skin exposed, so long as a certain minimum is exceeded (about 10 sq. mm.). The good health of the mice, as measured by their growth, has proved to be an essential factor in obtaining reproducible results. . A quantitative scale for expressing the relative carcino genic potencies of various synthetic carcinogens and complex tars and oils has been derived, using the experiments on solutions of methylcholanthrene in benzene as reference standards. Utilizing the relationship between the dosage of methylcholanthrene and the rate of induction of tumors shown above, a value for the relativepotency, F)jq, is obtained. The numerical value of the P^c of a tar or oil is equal to the level of concentration, in percent by weight, of the solution of methylcholanthrene in benzene which would induce papillomas at the same rate as the material in question under - API 05840 EB 005585 - 28 comparable experimental conditions. The potencies, PMC0, of pure compounds, relative to that of methylcholanthrene, taken as 100, may be derived in a similar manner. From the data in this paper, the relative potencies of 712-dimethylbenz [a].anthracene and benzo []pyrene are 180 and 60, respectively. API 05841 EB 005586 \ - 27 SUMMARY Repeated applications of solutions of carcinogenic hydro carbons upon the dorsal skin of male mice of the C3H strain under suitable experimental conditions result in the induction of epithel iomas in essentially all of the exposed animals. The average period of exposure (x) to solutions of 3-Bethylcholanthrene in benzene required to induce papillomas of the skin of these mice varies Inversely with the dosage (d) of the carcinogen per unit area per week, according to the following relationship: d + 0.10 For experiments involving combinations of synthetic carcinogens with non-accelerating solvents, the mean time of induction of tumors, is Independent of the total area of skin exposed, so long as a certain minimum is exceeded (about 10 sq* mm.). The good health of the mice, as measured by their growth, has proved to.be an essential factor in obtaining reproducible results.. A quantitative scale for expressing the relative carcino genic potencies of various synthetic carcinogens and complex tars and oils has been derived, using the experiments on solutions of methylcholanthrene in benzene as reference standards. Utilizing the relationship between the dosage of methylcholanthrene and the rate of induction of tumors shown above, a value for the relative potency, Pjjq, Is obtained. The numerical value of the of a tar or oil is equal to the level of concentration, in percent by weight, of the solution of methylcholanthrene in benzene which would induce papillomas at the same rate as the material in question under - API 05842 EB 005587 - 28 comparable experimental conditions. The potencies, PMC of pure compounds, relative to that of methylcholanthrene, taken as 100, may be derived in a similar manner. From the data in this paper, the relative potencies of 7,12-dimethylbenz [a]anthracene and benzo[]pyrene are 180 and 60, respectively. API 05843 I* EB 005588