EFFECT OF OIL OF WINTERGREEN ON SPONTANEOUS TUMORSOF THE MAMMARY GLAND IN MICE

VI. THE DIFFERENT EFFECT OF Two FRACTIONS OBTAINED BY THE

DISTILLATION OF THE TRUE OIL

LEONELL C. STRONG, PH.D.l

Yale U/liversity, School of Medicine

In a series of papers (1-5) data have been presented dealing with theeffect of oil of wintergreen on spontaneous carcinoma of the mammary glandin mice. The fourth and fifth papers are of particular interest for a full un­derstanding of the present data. In the fourth paper it was pointed out thatthe redistilled synthetic methyl salicylate had very little, if any, effect on thegrowth of such tumors. In the fifth paper, on the other hand, it was shownthat the true or natural oil of wintergreen administered in the diet had a pro­nounced effect, slowing up the growth of spontaneous tumors in mice and en­hancing certain retrogressive changes in the tumors. The survival time of thetreated animals after the onset of malignancy was not influenced, however.Histologically tumors in treated animals showed, to a great extent, exaggera­tion of the retrogressive changes recorded by different observers in spon­taneously receding tumors. These changes include large areas of necrotictissue, pyknotic nuclei, and an enhancement of the connective tissue of thetumor. Several of the tumors in treated animals had completely regressed.These effects were brought about when treatment was initiated after themalignant tumors had been discovered.

Except for the presence of impurities in small amounts, there is chemicallyvery little to account for this difference in effect of the two oils. The trueoil of wintergreen consists of more than 95 per cent methyl salicylate. Whileit is well known that the true oil of wintergreen is more stable than the syn­thetic methyl salicylate, due perhaps to the impurities contained in the former,it is questionable whether this could determine the different effect on malig­nant tumor growth.

In order to test the possibility that some impurity present in the naturaloil might be the agent influencing tumor growth, as well as having a possibleeffect on the stability of the oil, a quantity of the natural oil was fractionallydistilled. No attempt was made at a quantitative analysis of the differentfractions. True oil of wintergreen was distilled to complete dryness underreduced atmospheric pressure, in a double column flask (water pump suctionat 15-20 mm. of mercury). Three first crude fractions thus obtained werefurther distilled and similar fractions were added together until finally three

1 This experiment has been made possible by grants from the International Cancer ResearchFoundation and the Anna Fuller Fund. Acknowledgment is also made of aid from the AtypicalGrowth Fund of Yale University. Dr. G. M. Smith has very kindly examined the pathologicpreparations. Dr. R. J. Anderson has granted the use of equipment and given advice for thefractionation of the true oil of wintergreen.

227

228 LEONELL C. STRONG

tENT IMETERS ,,,,,

S 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19WEEKS

OL- _

CHART 1. AVERAGE TUMOR GROWTH RATES }'OR (A) SIXTEEN MICE TREATED WITH THE H FRACTION

(DASH LINE) AND (n) ONE HUNDRED CO"'TROL MICE UF THE SAME STRAIN (SOLID UNE)

The number of weeks following the beginning (S) of the experiment is given along the baseline j the longest linear diameter of the tumor in centimeters along the vertical line. There isobviously no difference between the two sets of data.

PERCENTIIC;[

/,

s I ~ 2~ 3~ 4~ ~~ 6~ 7~ 8~ g~ 10~ 11& 12~ 1M 14~ res 16& 17~ 185 195

DAYS

CHART 2. SURVIVAL Trxtt FOR (A) MICE RECEIVING TIlE H FRACTlON (MSH LINt:) AND (Il)COl"TROLS (SOI.ID UNt:)

The number of days following the onset of malignant growth are I(iven along the base line j

the cumulative mortality data (on a percentage basis) on t.he vertical line. There is no differencebetween the two curves.

fractions were obtained. These were designated as follows: L, the low frac­tion or that part of the true oil which distilled over first; (2) M, the middlefraction; H, the high fraction. The L fraction consisted of approximatelyone-sixteenth of the original oil, and the H fraction of approximately one­twelfth. The M fraction consequently consisted of the greater part of theoil and is no doubt methyl salicylate. Both the Land H fractions also con-

SPONTANEOUS TUMORS OF MAMMARY GLAND IN MICE. VI 229

TABLE I: Data for 16 Mice Receiving the H Fraction of Oil of Wintergreen

SurvivalAge at Time after

Discovery Discovery Age atNumber of Tumor of Tumor Death Metastases

of Mouse (days) (days) (days) in Lungs

115,926 311 6 317117,605 222 100 322 +113,715 430 120 550 +113,650 429 81 510115,882 335 78 413117,832 215 89 304 +113,747 439 35 474113,144 474 66 540115,431 391 54 445115,690 375 45 420116,226 316 26 342112,694 519 82 601 +118,732 281 32 313116,389 421 34 455119,391 278 15 293120,459 256 86 342

Average 355.7 59.3 415.0

tained some methyl salicylate, but, because of the cost of the true oil, no at­tempt was made to purify these two fractions further.

The results obtained with the L and the H fractions are included in thiscommunication. The data for the M fraction will be reported subsequently.

Sixteen mice of the Strong A strain with spontaneous tumors of the mam­mary gland were treated with the H fraction. The oil was administered, asin all preceding experiments in this series, in the diet. Because of the smallquantity of the H fraction available, it became necessary to administer lessoil than would otherwise have been desirable. The initial amount used wasone drop of oil to 2 grams of the oatmeal mixture. The amount administeredvaried from day to day depending upon the weight and physical appearanceof the animal, the dosage being altered in order to give the maximum dosewithout causing loss of weight for want of food intake. The age of the miceat the onset of malignant growth, survival time after the discovery of thetumor, age at death, and occurrence of metastases in the lungs are given inTable I. The average survival time of the sixteen mice receiving the H frac­tion was 59.3 days; that for the controls (100 mice of the same stock) 55.2days. The growth rate of the tumors in mice receiving the H fraction is com­pared to that for the controls in Chart 1. The average growth rates for thetwo sets of animals are the same. The cumulative survival time per unit oftime for the experimental animals receiving the H fraction and for the con­trols is given on Chart 2. The two curves are practically identical.

No distinctive histological feature could be detected in tumors from micereceiving the H fractiori. The tumors appeared to be normal in every respectwhen compared with a similar series of tumors from control animals of thesame inbred strain of mice (Strong A). Four, or 25 per cent, of the micereceiving the H fraction developed metastases in the lungs. These were ani-

230

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2.

LEONELL C. STRONG

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5 234 ~ 6 7 8 9 10 II 12 13 14 I~ 16 17 18 19WEEKS

CUART 3. AVERAGE GROWTH RATES OF THE TUMORS IN (A) THE TJIIRTY-FOUR ANIM,\(.S RECt:l\'l~G

TIlE L OR Low FRACTION (DASH LINE) AND (n) OSE HUNDRED CONTROl.S (SOl.ID I.J~E)

Beginning with the second week. the tumors in the Lvtreated animals lag behind the tumors inthe control mice. By the ninth week the L-treated tumors become stationary, The growth curvefor the treated animals reaches the base line at twenty weeks. The number of weeks following thebeginning (S) of the experiment is given along the base line; the greatest linear dimension of thetumor in centimeters along the vertical line.

PERCENT""E

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:I 15 25 3:1 4:1 ~:I 65 75 85 9~ 105 115 12:1 135 14:1 15:1 16~ 17~ 185 195

CUART 4. SURVIVAl. PERIOD, IN DAYS, AFTER TilE OSSET OF MALIGNANT GROWTH IN (A) THE THIRTY­

FOUR MICE REl:'EIVING TilE L FRACTION (DASH UNE) AND (a) THE CONTROLS (SOLID LINE)

The dash line extends to the 245th day. TIme in days is plotted on the base line; cumulativemortality data on the vertical line. There is a tendency for the experimental animals to outlive thecontrols.

mals that lived 100, 120,89 and 82 days after the onset of malignant growth.This percentage is not significantly different from the percentage of untreatedcontrol animals of the same stock having metastases (32 per cent).

With the L fraction an entirely different result was obtained from thatwith the H fraction. Thirty-four animals of the same A strain were em­ployed and dosage was controlled at the same level as with the H fraction;that is, the initial dose was one drop of oil to two grams of the dry oatmealmixture, variations from this ratio depending upon the weight and the physicalappearance of the mouse. The age of the mice, survival time, age at death,

SPONTANEOUS TUMORS OF MAMMARY GLAND IN MICE. VI 231

TABl.E II: Data for 34 Mice Receiving the L Fraction of Oil of Wintergreen

SurvivalAge at Time after

Discovery Discovery Age atNumber of Tumor of Tumor Death Metastases

of Mouse (days) (days) (clays) in Lungs

117,676 313 22 335117,817 326 70 396117,805 327 40 367117,228 365 41 406116,105 353 50 403118,076 297 22 319118,290 297 139 436 +118,456 290 19 309118,737 285 247 532118,975 253 64 317119,121 256 129 385119,180 285 119 404118,336 318 60 378 +119,867 235 10 245119,390 262 37 299118,707 317 113 430118,378 341 24 365118,337 335 68 403120,432 192 82 274119,811 255 31 286115,927 513 65 578 +120,469 218 70 288120,470 218 110 328117,884 403 134 537118,754 348 55 403117,746 399 98 497119,147 308 126 434119,518 304 161 465118,797 345 12,~ 468119,195 323 78 401 +118,379 389 35 424118,894 353 27 380118,506 328 38 366117,346 458 69 527Average 314.9 75.7 390.5

and incidence of lung metastases are given in Table II. The average sur­vival time for mice receiving the L fraction was 75.7 days. The growth rateof the tumors in these mice and in the controls is given in Chart 3. Thecumulative survival time is shown in Chart 4.

As is shown in Chart 3, the tumors in animals receiving the L fraction weresmaller than their controls beginning the second week after treatment. Onemay say that the treated tumors apparently went through a period of de­pression beginning at this time. Some of the tumors completely disappeared;others, after a period of slower growth, recovered somewhat and then con­tinued to grow for several weeks, at which time they again became stationaryand many actually regressed.

This depressive period was more marked in the younger animals. InChart 5 are presented data on the growth rates of tumors after the mice hadbeen separated into two groups according to age at the beginning of the ex-

232 LEONELL C. STRONG

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ClIART 5. TUMOR GROWTH RATE IN (A) THIRTEEN YOUNG MICE, 192-300 DAYS OF AGE (DASH

LINE) COMPARED TO TUMOR GROWTH RATE IN (8) SEVENTEEN OLD MICE, 301-513DAYS OF AGE (SOLID LINE) 1:11 THE L-TREATED SERIES

The only possible difference is a slight indication of a greater "lag period" in the youngeranimals between the second and ninth weeks. All tumors, irrespective of initial size, are includedin this chart.

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CIIART 6. GROWTH RATES OF TUMORS IN (A) H-TREATED ANIMALS (DASH LINE) COMPARED TO

GROWTH RATES OF TUMORS IN (B) L-TREATED ANIMALS (SOLID LINE)

There is apparently a distinct difference between the average growth curves, appearing withthe third week after the beginning of the experiment.

periment. The dash line represents the mice that were less than 300 daysof age when their tumors were discovered, the average being 257.1 days.These mice survived 83.0 days on an average with their tumors. The solidline represents mice that were more than 300 days of age when their tumorsbecame palpable, the average age at that time being 350.7 days. These micesurvived with their tumors 71.2 days. There is thus a slight tendency forthe younger mice to live longer with spontaneous tumors than do the olderanimals.

SPONTANEOUS TUMORS OF MAMMARY GLAND IN MICE. VI 233

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2 3 4 5 6 7 6 9 10 II 12 13 14 15 16 17 16 19W£IKS

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CHART 7. AVERAGE GROWTH RATES OF TUMORS IN YOUNG ANIMALS

All observations are on mice receiving the L fraction. Comparison is made of (A) the smallertumors, 0.2--{).9 em. (solid line), and (B) the larger tumors, 1.D-l.i em. (dash line), at the beginningof the experiment. The two curves stay practically the same distance apart. The main differenceis that complete regression (reaching the base line) is obtained only with the smaller tumors.

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CHART 8. COMPARATIVE DATA ON TUMOR GROWTH IN (A) OLD MICE, 301-513 DAYS (SOLID LIN!:)

AND (B) YOUNG MICE, 192-300 DAYS (DASH LINE)

Only the growth rates for the smaller tumors (O.2--{).9 em. at beginning of experiment) areused in this chart. There is a slight tendency for the tumors in the younger animals to be smallerthan similarly treated tumors in the older series. Both curves, however, reach the base line.

Growth rate curves of tumors in the two series of experimental animalsare given in Chart 6. It will be noted that, beginning the third week afterthe experiment was started, the two curves are distinct. Tumor growth inthe mice receiving the low fraction lags behind that in mice receiving the Hor high fraction.

A more detailed analysis of the growth rates of tumors in the low-fractiontreated animals is given in Charts 7-12. For this study, the mice were di­vided into two groups depending upon the age at which their spontaneoustumors were discovered. Mice less than 300 days of age constitute theyounger group; mice beyond this age form the older group. The animals are

234 LEON ELL C. STRONG

30

2.5

CtNTIWETERS

S 2 3 4 S II 7 6 9 10 II I 2 13 14 I 5 I II 17 I 6 19WEEKS

CII,\RT 9. GROWTH RATES OF LARGER TUMORS (1.0-1.7 CM. AT BEGINNING OF EXPERIME~T),

IRRESPECTIVE or AGE OF ANIMAL IN (A) THE L-TRt:ATED SERIES (nASIl r.ix») A~D

(8) TilE CONTROL SERIES

There is a difference between the curves. This is a feature that was not obtained with trueoil of wintergreen, indicating that the L fraction is probably more potent than the true oil in in­l1uencing malignancy.

CENT IIotCTt RS

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CHART 10. GROWTH RATES OF TilE SMALLER TUMORS (0.2-0.9 CM. AT THE BEGINNING OF TilE

EXPERIMENT) IN THE OWER MICE

The dash line represents the average growth curve of the tumors in the 15 Lvtreated animals inthis series and the solid line the curve for the 37 controls. The two curves are again different.

further subdivided according to the size of the tumor at the beginning of theexperiment. One class consists of those mice which had tumors from 0.1 to0.9 em. in longest diameter at the time of discovery; the second class con­sists of those animals in which the tumors measured 1.0 em. or more. Thevarious combinations of these classes are explained in the captions of thecharts.

SPONTANEOUS TUMORS OF MAMMARY GLAND IN MICE. VI 235

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5 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19WEEKS

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CENTIMETERS

CUART 11. GROWTU RATES OF TIlE SMALLER TUMORS (0.2-0.9 C"M. AT THE BEGINNING OF THE

EXPERIMENT) IN THE YOUNGER MICE

The dash line shows the average growth rate of the tumors in 7 L-treated mice and the solidline for the 12 controls. The two curves are different.

CENTIMETERS

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CIlART 12. GROWTU RATES OF TilE LARGER TUMORS (1.G-1.7 CM. AT BEGINNING OF EXPERIMENT)

IN TilE YOUNGER MICE

The growth curve for the 6 L-treated animals is represented by the dash line, that for the20 controls by the solid line. The two groups are again different.

Softening and liquefaction of tumors, rarely encountered in control ani­mals, were of common occurrence among the L fraction-treated mice. Inmany instances the tumors become extremely cystic. In four of the mice(118,337; 115,927; 117,884; 118,797) these cystic spaces were drained bysterile puncture. On the sixty-eighth day of continued treatment 7 C.c. of

236 LEONELL C. STRONG

clear reddish fluid was taken from the tumor in mouse 118,337. The mousewas accidentally dropped and died as a result. Her tumor was completelyhollow. The lungs, liver, and kidney were in perfect condition. On thethirty-first day of treatment, the tumor in mouse 115,927 was cystic andwatery. Fluid was drained from the tumor at twelve different times, 13.2 C.c.of liquid being thus obtained (see Fig. 4). The tumor in mouse 117,884 be­came cystic also on the thirty-first day of treatment and was drained on sixoccasions. A watery exudate continually oozed from the remaining mass. Asthis process apparently kept the mouse emaciated, the tissue was removedsurgically. The animal lived 134 days after the discovery of the tumor,

FIGs. 1 AND 2. CROSS-SECTIONS OF TilE SPOXTA~EOllS TIIMORS OCCURRING IN MaeSE 5118,456(I) AND MOUSE 5118,076 (2), BOTH OF WIllCH RECEIVED TilE L FRACTION

These tumors had never been punctured nor manipulated in any way. In Fig. 1 note thecavity near the periphery of the tumor. The central part of the tumor in Fig. 2 consisted of agelatinous non-cellular mass of debris. This tumor was obtained twenty-two days after the be­ginning of treatment.

nearly three times the average span of life for control mice of the same series.The tumor in mouse 118,797 became soft and watery on the fifty-ninth dayof treatment. It was drained at eight different times. The animal livedwithout surgical or other aid 123 days after the onset of the tumor. Thisliquefaction of tumors in mice treated with the L fraction is illustrated inFigs. 1, 2, and 3 (mice 118,456, 118,076, and 118,378). These tumors hadnever been drained or manipulated in any manner. Such extremely hollowed­out tumors have never been seen in control animals, though cystic formationdoes occur to a certain extent in untreated mice.

At the time the tumors became softened and started to regress, severalanimals died. It was thought that this mortality might be due to a toxemiaproduced by the elimination of necrotic or liquefying tumor tissue. An at­tempt was made, therefore, to control the rate of regression by alternating

SPONTANEOUS TUMORS OF MAMMARY GLAND IN MICE. VI 237

the diet containing the L fraction and the regular oatmeal diet. This proce­dure was of an empirical nature and success was not always obtained. If theanimals were returned to the oatmeal diet for too long a time, the tumorsstarted to grow again and did not respond to subsequent treatment as theyhad previously done. Hemorrhage from the surface of the tumor did notoccur as frequently as in mice treated with the true oil of wintergreen. Withthe progress of the experiment improvement in the administration of alternatetreatment with the L fraction and the control oatmeal diet apparently oc­curred, since the survival time for the first 17 mice in the series was 70.9 days;while for the succeeding 17 mice it was 80.6 days.

FIGs. 3 AND 4. CRO~S-SECTIOKS OF Tl'MORS IN MICE RECElVl!\'G THE L FRACTION

Fig. 3. Tumor in mouse 5118,378. This tumor had never been punctured nor manipulatedin any manner. The extensive cavity was filled with a clear, non-cellular liquid. The animal hadbeen under treatment for twenty-four days.

Fig. 4. Tumor from mouse 115,927. The section shows a thin layer of tumor tissue sur­rounding an extensive cavity, which, in the living animal, had been filled with a colorless fluid.The tumor had been drained several times by sterile puncture. This section was obtained sixty­five days after the appearance of the tumor.

Four, or 11.7 per cent, of the mice receiving the L fraction developed me­tastases in the lungs. These were animals which had lived under treatment139, 60, 65, and 78 days. The incidence of metastases is thus lower thanin control mice (32 per cent).

The histologic changes in the tumors in mice treated with the L fraction,resembled, to a great extent, those which had been described in mice receivingthe true oil of wintergreen. The main difference between the two series isthat the changes seem to be more pronounced in the animals receiving theL fraction. In all these tumors great areas of widespread devastating necrosiswere seen. Spicules of calcification were abundant in these centers of nec­rosis. Extensive bands of dense fibrous connective tissue traversed the nee-

238 LEONELL C. STRONG

rotic areas as well as areas where necrobiosis was less pronounced. In manyplaces the blood vessels were intensely engorged with red cells and hemor­rhages into the tissue were frequent. The blood vessels near the necroticareas especially were distended with red cells which had become fused to­gether into hyaline masses, resembling thrombi. These vascular changessometimes took place without an accompanying fibrous stroma change. Inother places the fibrous connective tissue was increased without vascular

FIGS. 5 AND 6. TUMORS IN MICE RECEIVING TIlE L FRACTION

Fig. 5. A lateral view of mouse 118.378 (see also Fig. 3). The tumor. over the left hind leg.was split by a sharp razor and the two halves were pressed apart. disclosing the underlying musclesof the leg. Note the cavity near the periphery in both halves of the tumor.

Fig. 6. A ventral view of mouse 118,456 (see also Fig. 1). The tumor on the ventral aspectof the left hind leg has heen split by a razor and the two halves are pushed apart to show the ex­tensive cavity in each part of the tumor.

change. In some of the tumors, a secondary invasion of lymphocytes hadtaken place. As in the series receiving oil of wintergreen, the increase offibrous connective tissue stroma seemed to have been produced by some sortof stimulation in situ. The large cavities in the tumor of Mouse A 118,337contained serum, blood and lymphocytes. No evidence of abscess formationwas detected in histologic examination of the tumors.

GENERAl. DISCUSSION

The data presented in this paper demonstrate quite clearly that the twofractions of the true oil of wintergreen administered in the diet have a dif­ferent action on the spontaneous tumors of the mammary gland in mice. TheH or high fraction, like the synthetic redistilled methyl salicylate, has no ef-

SPONTANEOUS TUl\IORS OF MAl\L'\IARY GLAND IN MICE. VI 239

fect on the growth rate of the tumors, the number of days that the animalwill live after the onset of malignant growth (survival time), or the histologicstructure of the tumor. The L or low fraction, on the other hand, like thetrue or natural oil of wintergreen, has a pronounced effect: (a) a slowing upof the growth rate of spontaneous tumors, with complete regression of themasses in approximately 11.7 per cent of treated animals; (h) an increase insurvival time of approximately twenty days in a total of fifty-five days: (c)gross and microscopic changes in the structure of the tumors. The effectson the tumors include liquefaction of the tumor tissue, an enhancement orstimulation of dense fibrous connective-tissue stroma, distention and engorge­ment of blood vessels, and extreme necrosis and scattered foci of calcification.For the criteria of effect used in this investigation, it would seem that the lowfraction has a more pronounced biologic action than the true oil of winter­green. It is reasonable, therefore, to assume, that this action is due to a traceof some substance in the oil itself.

CONCLUSIONS

1. Two fractions obtained by the distillation of the true oil of wintergreenhave different effects on spontaneous tumors of the mammary gland in mice.

2. The high fraction has no effect on (1) the growth rate of the tumor,(2) the survival time of the animal, or (3) the histologic structure of thetumor.

3. The low fraction has a pronounced effect, causing (1) slowing of thegrowth rate of tumors, with complete regression in 4 out of 34 animals, (2)increase in the survival time after the onset of cancer, (3) gross and histologicalterations in the tumors.

4. The action of the low fraction appears to be more pronounced thanthe action of the true oil of wintergreen.

5. The difference in effect of the two fractions of the true oil of winter­green depends upon the chemical difference between them, and not upon themethyl salicylate content which they have in common.

REFERENCES

1. STRONG, L. c.: Proc. Soc. Exper. BioI. & Med. 30: 386,1932.2. STRONG, L. C.: Am. ]. Cancer 20: 38i, 1934.3. STRONG, L. c.. Am. ]. Cancer 25: tst, 1935.4. STRONG, L. c.. Am. J. M. Sc. 192: 546, 1936.5. STRO!\G, L. c.. Am.]. Cancer 28: 550,1936.