Screening of Organic Compounds for Spermicidal Activity

Screening of Organic Compounds for Spermicidal Activity John H. Holzaepfel, M.D., Roy W. Greenlee, B.S., R. E. Wyant, M.S., and W. C. Ellis, Jr., B.S.

Tms PAPER presents the first extensive study of the spermicidal activity of chemical compounds to be published since the classic works of Baker and Voge. 22 The investigation, in which 581 organic substances were surveyed, has revealed several new classes of spermicidally ac~ive compounds. Evaluation of the spermicidal effectiveness of chemical substances dates back to 1855, when Kolliker14 first studied the effect of var'ious chemical substances on animal sperm. The methods of evaluating spermicidal activity used by investigators since that time have been numerous, e.g., the several procedures of Baker. A useful step toward standardizing spermicidal evaluation technics was made in 1940 by Brown and Gamble, 6 who designed a method for comparing the relative effectiveness of topical contraceptive materials. In 1951 Millman16 presented an excellent review of the many procedures used by various investigators for spermicidal evaluation. Among these, the method of Brown and Gamble is of major importance, since it has received wide us·e and has been accorded medical recognition. 1 The procedures of this "B and G" method are basically simple and manipulatively easy, so that the technic was worthy of consideration as a method for screening spermicides. A modification in the materials and minor changes in the procedure of the method provided us with the screening method used From a cooperative research program of the Department of Obstetrics and Gynecology, College of Medicine, Ohio State University (senior author), and Battelle Memorial Institute. The work was sponsored by the Holland-Rantos Company and was sanctioned by the Planned Parenthood Clinic of Columbus, Ohio. 272

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in this investigation. Its advantages were those of greater speed and simplicity, along with the provision of a standardized pH environment.

METHOD AND MATERIALS The spermicidal evaluation procedure developed by Brawn and Gamble, intended primarily for the evaluation of semisolid contraceptive materials, involves a tenfold dilution of the material being studied. This is done in 2 steps: ( 1) dilution of the fluid or semisolid sample with 4 parts of normal saline; ( 2) mixing of the resulting solution or dispersion with an equal amount ( 5 parts) of human semen. The time required for essentially complete sperm immobilization is recorded, 30 minutes being considered an acceptable time for contraceptive jellies and creams. Since the publication of the method in 1940 and subsequent recognition as a criterion for commercial contraceptive products, the literature has given no indication that the standard B and G method has been used for extensive screening of new spermicides. One fault in its application for this purpose is that as ordinarily used the procedure does not provide for pH control. Acids, by providing low pH conditions, are regularly spermicidal; therefore, acidic substances with no really inherent spermicidal activity may register as spermicides in the Band G method. Actual use may render such substances fully neutralized and thus inactivated by semen, which is decidedly basic (pH 8), with a large buffering capacity. In their original publication, Brown and Gamble reported use of a nutrient isotonic phosphate buffer called BGS * as an alternate diluent liquid. This solution, used interchangeably with normal saline, had the effect of maintaining the basic and nutrient environment of the semen in spite of the necessary dilution. This undoubtedly favored the sperm generally; it also avoided extraneous indications of spermicidal activity that could result merely from pH-lowering effects of acidic substances. The complication of this special BGS diluent, however, was shown to be unnecessary for evaluating commercial materials, which probably were no more than mildly acidic. 7 • 19 In the present screening program, however, the superiority of BGS over normal saline was evident, since compounds of widely varying acidities were to be studied. Therefore, a modified form of the B and G method involving use of the BGS diluent was chosen for general screening purposes. This choice was advantageous;

* Buffered glucose saline, originated by Baker.

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it invoked the widely accepted B and G procedure, yet standardized the environment at a reproducible pH level-essentially that of human semen. PROCEDURE As already indicated, the major modification of the B and G method as us·ed for the screening of organic compounds in this study was the use of the BGS diluent instead of normal saline. However, other simplifications and minor modifications of a practical nature were evolved; we believe that the apparatus used is an improvement. A new type of glassware used made for easier manipulations. The criterion of semen quality chosen was the number of active sperm/cc., which seemed to correlate better with resistance to spermicides than the phthalate criterion used by Brown and Gamble. 6 Since this was a screening procedure, the number of determinations made on each compound was reduced to from 1 to 4 (with different semen specimens ) . In other respects the screening method was identical with the B and G method. A description of the procedure and apparatus used in this work follows. 1. Solutions of candidate spermicides. Only organic compounds that have appreciable solubility (ca 0.1 per cent) in water at pH 8 were studied. The standard concentration of solutions for use was 0.5 per cent, although occasionally 0.25 per cent was used. Other concentrations specified represent known solubility limits. Where solubilities were unknown but were less than 0.5 per cent, a saturated solution was used, as indicated by the figure 0.5 in tabulations of results. Solutions of acidic compounds were adjusted to pH 8 (by addition of NaOH) before use. 2. Semen samples. The semen specimens were obtained from young donors and collected in clean Pyrex test tubes. These samples were used within 4 hours after collection. For this work specimens showing counts of active sperm lower than 40 million/cc. were considered unsuitable. 3. Dilution of sperimicide solutions. Just before use, 5 cc. of each spermicide solution, as described above, was diluted by shaking with 20 cc. of BGS solution. 4. Apparatus. A 100 microliter micropipet,* provided with 20-microliter calibration markings, was substituted for the less convenient appall"atus used by Brown and Gamble. This was manipulated by a pipet control, which consisted simply of a 1 cc. glass tuberculin syringe provided with a ring for thumb manipulation and a rubber-tubing pipet adapter. * Micropipets of this description are supplied from stock by Fisher Scientific Company, Pittsburgh, Pa.

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5. Procedure. The required volumetric measurement of equal portions of diluted spermicide and semen was made by drawing 20 microliters of the former into pipet, admitting a small air bubble, and then drawing in 20 microliters of semen. Mixing was accomplished by expelling the pipet contents onto a microscope slide and withdrawing into the pipet 10 times in rapid succession. The final mixture was covered with a cover slip. The process was repeated to obtain a duplicate. 6. Immobilization time. Each slide was examined immediately by microscope (magnification X 200, dark-field illumination), the slide being scanned for even slight indications of sperm mobility. The period required to assure that all motion had ceased was recorded as the immobilization time. Uncertainties in this procedure led to the adoption of 4 general classifications of the rate of kill: Class I-L5 minutes, rapid or instantaneous kill, high spermicidal activity. Class II-> 5,~ 30 minutes, intermediate rate of kill, moderate spermicidal activity (meets AMA criterion). Class III-> 30, L 100 minutes, slow kill, minor spermicidal activity. Class IV-> 100 minutes, essentially inactive spermicidally. Compounds of Class I were usually rescreened at greater dilutions to find the lowest concentration that gave rapid kill. FINDINGS A total of 581 compounds was included in this study. Although there is considerable overlapping, the compounds screened may be classified under 5 main types: A, acids and alcohols; B, phenols and their simple derivatives; C, nitrogen compounds; D, surfactants of 3 major kinds; and E, iodonium salts. Each type is divided into subtypes for a more exact classification of the substances under study. The numerical distribution of compounds of various subtypes according to spermicidal ratings of the screening test is given in Table 1. It may be seen that roughly 10 per cent of the compounds fell into each of the first 3 spermicidal classes (I, II, and III), whereas the remaining 70 per cent (Class IV) showed no :appreciable activity. Only the 55 compounds of Class I seem to qualify for further consideration as spermicides in practical contraceptive formulations. For this reason, and because of the large number of compounds studied, only those of Class I will be identified in this paper. It may be observed also from Table 1 that

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TABLE 1.

Numerical Distribution of Organic Compounds According to Compound Type and Spermicidal Classification Number of compounds

Subtype number

Spermicidal class Compound type and subtype

II

III

IV

All classes

0

0

2

20

22

0 0 0

0 0 0

1 1 3

18 26 22

19 27 25

0 0 0

0 0 0

1 2 0

30 19 9

21 21 9

3 5 1 4 1

14 30 19 7 17

24 41 24 18 18

I

A. Acids and alcohols (total, 144) 1 2 3 4 5 6 7

Aliphatic monocarboxylic acids ( unsubstituted) Unsaturated and cyclic monocarboxylic acids ( unsubstituted) Polycarboxylic acids ( unsubstituted) Oxygen-substituted carboxylic acids Halogen- and nitrogen-substituted carboxylic acids Miscellaneous acidic compounds Aliphatic polyols

B. Phenols and simple derivatives (total, 125)

8 9 10 11 12

Alkyl- and aryl-substituted monohydric phenols Functionally substituted monohydric phenols Polyhydric phenols and quinones Bisphenolsa Phenol derivatives

2 1 4

4 0

5 5 0 3 0

C. Nitrogen and sulfur compounds (total, 79) 13 14 15 16

Amines and amidines Oxygen-substituted nitrogen compounds Dithiocarbamates, xanthates, and derivatives Other sulfur-substituted nitrogen compounds

1 0

1 1

1 1

18 21

21 23

6

4

3

7

20

1

0

4

10

15

0

7

6

47

60

0

1

8

16

25

4

1

3

11

19

9

5

2

6

22

1

4

9

8

23

10

3

1

8

22

D. Surfactantsb (total, 208) 17 18 19 20 21 22

Anionics" Nonionics-1. Polyoxyethylened derivatives of organic acids Nonionics-2. Ether and ester derivatives of alcohols and thiols• Nonionics-3. Polyoxyethylened derivatives of phenols Nonionics-4. Polyoxyethylenel derivatives of nitrogen compounds Cationics-1. Polyoxyethylened derivatives of amines and amidines

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TABLE 1-(Continued) Number of compounds Subtype number

Spermicidal class Compound type and subtype

I

All

II

III

IV

classes

7

3

4

11

25

0

3

4

5

12

5

1

8

11

25

55

48

78 400

581

D. Surfactantsb (total, 208) 23

24

Cationics-2. Miscellaneous quaternary ammonium salts Cationics-3. Phenolic amines and derived quaternary ammonium saltsu

E. Iodonium salts (total, 25) 25

Diphenyliodonium saltsh Grand totals

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Polyhydric bishydroxyphenyl or bishydroxyphenylmethane derivatives. Mostly proprietary wetting agents, detergents, and emulsifiers. 0 Typically, sodium salts of long-chain organic acids. a Polyoxyethylene derivatives are obtained by reactions with several molecules of ethylene oxide to form a polyether chain. e Mostly including a polyol or polyoxyethylene group. f Typically, polyoxyethylene derivatives of long-chain amides. u Compounds contain a phenolic hydroxyl group. h Mostly ring-substituted derivatives of diphenyl iodonium chloride, ( CaH5) 2l • Cl. a b

....

the 208 surfactants studied provided by far the largest number ( 31) of these active substances. This impressive number includes unavoidable duplications under proprietary names, but several of the materials, notably the alkylthiopolyoxyethylene ethanols, have not been reported previously as spermicides. The remaining compounds of high activity summarized in Table 1 are almost entirely those of 4 subtypes: 10, 11, 15, and 25. It is believed that the spermicidal properties of the latter 3 of these, representing bisphenols, dithiocarbamates (and similar compounds), and iodonium salts, respectively, are also reported here for the first time. It is notable that none of the compounds of type A, acids and alcohols, could qualify as either Class I or II. The sign1ificance of this result will become apparent later. The highly spermicidal compounds of Class I are listed individually in Table 2 in the numerical subtype order used in Table 1. Wherever possible, the concentration reported was the lowest found to give the indicated result; such values were marked with an asterisk. Reasonably exact chemical names have been used to identify all substances. However, the chemical identifica-

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TABLE 2.

Test concentration, per cent

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Summary of Compounds in Class I (High Spermicidal Activity)

Compounda Subtype

Code number

Chemical identification

B. Phenols and simple derivatives

0.5 0.5

8 8

1 2

p-sec-Butylphenol ,B-Naphthol

0.5

9

3

4-Amino-1-naphthol

0.5 0.5 0.5 0.5

10 10 10 10

4 5 6 7

Toluhydroquinone Naphthohydroquinone Hexylresorcinol Octylresorcinol

0.5 0.25* 0.5 0.5

11 11 11 11

8b 9b 10 11

c.

Nitrogen and sulfur compounds

0.25*

13

12

Lauramidine hydrochloride

0.001* 0.002* 0.2 0.5 0.5 0.5

15 15 15 15 15 15

13 14 15 16 17 18

Sodium dimethyldithiocarbamate Sodium diethyldithiocarbamate Dimethylammonium dimethyldithiocarbamate Potassium ethylxanthate Potassium n-butylxanthate Potassium n-amylxanthate

0.5

16

19

Methylene dithiocyanate

0.5* 0.25* 0.5* 0.25*

19 19 19 19

20b 2Jb 22b 23b

D. Surfactants tert-Dodecylthiopolyoxyethylene ethanol Alkoxypolyoxyethylene ethanol Alkylthiopolyoxyethylene ethanol Alkylthiopolyoxyethylene ethanol

0.5* 0.5* 0.5* 0.5* 0.5* 0.5* 0.5*

20 20 20 20 20 20 20

24b 25b 26b 27b 28b 29b SOb

Alkylphenoxypolyoxyethylene ethanol Alkylphenoxypolyoxyethylene ethanol Isooctylphenoxydecaoxyethylene ethanol Isononylphenoxynonaoxyethylene ethanol Isononylphenoxydecaoxyethylene ethanol Alkylphenoxypolyoxyethylene ethanol Alkylphenoxypolyoxyethylene ethanol

Bis(2-hydroxy-5-chlorophenyl)methane 2,2-Bis(p-hydroxyphenyl)butane 2,2-Bis(p-hydroxyphenyl)4-methylpentane 1,1-Bis(2,4-dihydroxyphenyl)octane

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TABLE 2-(Continued) Test concentration, per cent

Compoundsa Subtype

Code number

0.5* 0.5* 0.5*

20 20 21

3lb 32 33b

Isooctylphenoxynonaoxyethylene ethanol Octylresorcinol + IO(EO) condensate• Fatty acid alkylolamide

0.25* 0.2* 0.2* 0.2* 0.25* 0.25* 0.5* 0.25* 0.5* 0.5*

22 22 22 22 22 22 22 22 22 22

34b 35b 36b 37b 38b 39b 40b 4Jb 42b 43

R-NH(CH 2)sNH 2a + 2(EO) condensate• R-NH(CH 2)3 NH 2tl + 5(EO) condensate" R-NH(CH 2)3NH2a + IO(EO) condensate• R-NH(CH 2)3 NH2tl + 15(EO) condensate" Stearylamine + IO(EO) condensate" Stearylamine + 15(EO) condensate• R-NH2• + IO(EO) condensate• R-NH 2• + 5(EO) condensate• R-NH 21 + 15(EO) condensate• Cetylamine + IO(EO) condensate•

0.5

23

44b

0.5 0.4* 0.4*

23 23 23

45b 46b 41b

0.5

23

48b

0.5* 0.25*

23 23

49b 50b

N-Dodecylcarbamylmethyldimethylbenzylammonium chloride Cetyldimethylbenzylammonium chloride n-Octadecyldimethylbenzylammonium chloride Diisobutylphenoxyethoxyethyldimethylbenzylammonium chloride Diisobutylcresoxyethoxyethyldimethylbenzylammonium chloride Alkyldimethylbenzylammonium chlorideu Alkyldimethylbenzylammonium chlorideu

0.10* 0.10* 0.25* 0.10* 0.10*

25 25 25 25 25

51 52 53 54 55

E. Iodonium salts Bis(n-butylphenyl)iodonium chloride Bis(sec-butylphenyl)iodonium chloride Bis(tert-butylphenyl)iodonium chloride Bis(2,3-endotetramethylenephenyl)iodonium chloride Bis( tert-amylphenyl)iodonium chloride

Chemical identification

D. Surfactants

* Minimum concentration for instantaneous kill. Order of presentation and subtype identification same as in Table 1. Commercial product. c "Compound + n(EO)" indicates a condensate between 1 molecule of compound and n molecules of ethylene oxide. Such condensates are characterized by hydrophilic polyoxyethylene chains, the length of which is dependent upon values of n. a "R" represents alkyl groups from tallow-derived fatty alcohols. e "R" represents alkyl groups from soya oil-derived fatty alcohols. t "R" represents alkyl groups from C1s-C24 fatty alcohols. u Alkyl group probably from C12-C1s fatty alcohols. a b

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tion of proprietary materials was sometimes incomplete because relatively little such information was supplied by manufacturers.* An analysis of the results as they apply to each of the 5 majO'r types of compounds studied is given briefly in the following discussion. A. Acids and Alcohols It has been proposed that salts of certain organic acids, specifically those of relatively long-chain fatty acids (soaps), are efficient spermicides. 9 The data obtained for compounds of this kind (represented by type A in Table 1) showed that high spermicidal activity was not demonstrated in the screening method used for this study. Included under subtypes 1 and 2 of type A were saturated and unsaturated fatty acids ranging in chain length from c3 to c24, including stearic, oleic, linolenic, and ricinoleic acid. 17 Since under the seminal pH conditions of the screening method these acids were evaluated as their sodium salts, the results indicate that soaps very generally show negligible spermicidal activity by this procedure. B. Phenols and Simple Derivatives Phenols and quinones have long been recognized as having moderate to high spermicidal activity. Certain monohydric alkylphenols have been patented, 13 but their activity was found by this method to be relatively low. Toluquinone was the most spermicidal organic compound tested by Baker, and both toluhydroquinone and hexylresorcinol exhibited high activity in this study. The latter has been recognized as a useful ingredient of contraceptive formulations, 2 but quinones and hydroquinones appear undesirable on the basis of color development. By the methods of this study, the spermicidal activity of a new class of polyhydric phenols, the bisphenols, was recognized (Table 2, subtype 11). The compounds in reference are bismonohydroxy- or bisdihydroxyphenyl derivatives of methane or substituted methanes, of which the most active example is 2,2-bis-p-hydroxyphenylbutane. In other subtypes, the high activity of octylresorcinol is not surprising, but that of the two naphthalene derivatives, ,8-naphthol and 2-amino-lnaphthol, was unanticipated. The absence of subtype 12, phenol derivatives, from Table 2 is significant. In these compounds the phenolic hydroxyl group had been altered by conversion to an ether, ester, or thiol derivative, with accompanying loss of spermicidal indications. *Chemical identification of surfactants has depended largely on the data collected by McCutcheon (reference 15).

J. W.

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C.

Nitrogen Compounds There is very little information in the literature regarding the spermicidal activity of the subtypes of compounds considered under this heading. Also, to the best of our knowledge, none of the compounds of this group that emerged as having high spermicidal activity have been reported previously in this connection. The compounds of major interest are those of subtype 15. Here, low molecular weight sodium dialkyldithiocarbamates, specifically the dimethyl and diethyl compounds, showed the highest activity of any material studied by this procedure (regardless of type), being effective at test concentrations of 0.001 and 0.002 per cent, respectively. Simple alkylxanthate salts, including the amyl compound, were effective at 0.5 per cent. Higher homologs of both types were relatively inactive. Since both dithiocarbamate and xanthate salts are unstable under acid conditions, other related compounds were also tried. The thiuram disulfides chemiCally derived from the active dithiocarbamates retained only a minor degree of activity; they were in Class II. Two other specific compounds gave indications of high spermicidal activity, although in both their homologs were found by this method to be relatively inactive. These were lauramidine hydrochloride (subtype 13) and methylenedithiocyanate (subtype 16). A few other amidines showed minor activity, but amines themselves appeared to be generally nonspermicidal. D.

Surfactants

The observation that surfactants provided the largest number of materials that evidenced high spermicidal activity in this study was not surprising; a number of known spermicides are representatives of this type of substance. The nature of the spermicidal action of surfactants is not well understood. Many surfactants are nonspermicidal, whereas others are highly spermicidal. Because of this wide variation of spermicidal activity between different compounds of equal surface activity, it has been argued that the effects must be chemical rather than physicaJ.2° However, a small difference in the balance of hydrophilic and hydrophobic character between members of a single class of surfactants may be critical from the standpoint of spermicidal activity. It was surprising that of the 60 anionic surfactants studied, none registered as Class I spermicides. This group, subtype 17, consisted largely of the sodium salts of long-chain sulfates and sulfonates. These are physically simi-

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lar to soaps, which, as already mentioned, are apparently nonspermicidal in this procedure. The 7 compounds of Class II included 2 that are known spermicides, sodium laurylsulfate8 and sodium dioctylsulfosuccinate. 5 Of major interest were the nonionic surfactants, which provided several substances that registered as highly spermicidal by the screening method. These were uniformly polyoxyethylene derivatives, obtained by reaction of several molecules of ethylene oxide with one of long-chain alcohols, phenols, etc. Subtype 18, derivatives of organic acids, gave no Class I spermicides, but several spermicides of Class I activity were found in subtype 19. Use of some of the compounds in both of these subtypes is governed by the patents of Elias, 10 Taub,21 and Ward; 23 however, the 3 alkylthiopolyoxyethylene ethanols indicated in Table 2 represent a new type of spermicidally active surfactant. Subtype 20, substantially alkylphenoxypolyoxyethylene ethanols, has yielded 9 Class I spermicides in this study. These, except for the experimental octylresorcinol condensate (with 10 ethylene oxide units), seem all to be branched-chain Cs or CD alkylphenol condensates, with 9 to 11 units of ethylene oxide. These compounds, as single spermicides, are :finding increasingly wide use in contraceptive jellies and creams. The patents in this field (those of Sander/8 Elias, 11 and Berlinm-4) seem to deal only with their use under special conditions, e.g., to potentiate other spermicides. The essentially neutral nitrogen compounds of subtype 21, largely polyoxyethylene derivatives of fatty amides, gave only 1 active spermicide in this study, as seen in Table 2. However, the mildly basic (cationic) compounds of subtype 22, the polyoxyethylene derivatives of long-chain amines and diamines, included 10 substances of Class I. The activity of these materials appears not to have been recognized previously. Quaternary ammonium salts, subtype 23, are familiar in spermicidal applications. 2 • 24 It was not surprising, therefore, that of 25 such salts of familiar types, 7 were Class I spermicides. An attempt was made to develop a new type of phenolic quaternary ammonium salt spermicide (from phenolic tertiary amines). The intermediates and products comprised subtype 24 (Table 1); no compounds of high spermicidal activity were found. E. lodonium Salts

Iodonium salts, members of a relatively unfamiliar class of ionic compounds, have been shown12 to be bactericidal. However, their spermicidal

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activity was first demonstrated during this study. The 25 compounds studied (comprising subtype 25) were derivatives .of the diphenyliodonium structure, ( C6H5 )2I·Cl. Halogen, carboxyl, and nitrogen derivatives were relatively inactive, but certain alkyl derivatives appeared to have good spermicidal activity. The 5 best of these were Class I spermicides, effective at about 0.1 per cent, as shown in Table 2. SUMMARY

A group of 581 organic compounds of various kinds was screened for spermicidal activity by a simplified screening procedure that was a modification of the familiar Bmwn and Gamble method. -Of these, 55 gave essentially instantaneous kill at aqueous concentrations of 0.5 per cent or less. Several new types of spermicidally active compounds were found, including bisphenols, dialkyldithiocarbamates, alkylxanthates, tetraalkylthiuram disulfides, alkylthiopolyoxyethylene ethanols, and diphenyliodonium salts. REFERENCES I. American Medical Association, New and Nonofficial Remedies 1952. phia, Lippincott, 1952, p. xxxi.

Philadel-

2. American Medical Association, New and Nonofficial Remedies 1952. Philadelphia, Lippincott, 1957, p. 330. 3. BAKER, J. R. The Chemical Control of Conception. London, Chapman and Hall, 1935. 4. BERLINER, V. R. "Spermicidal Composition" U.S. Patent 2,330,846, June 26, 1956. 5. CROSSLEY, M. L. "Vaginal Preparation" U.S. Patent 2,149,240, February 28, 1939. 6. BROWN, R. L., and GAMBLE, C. J. A method of testing the relative spermicidal effectiveness of contraceptives and its application to ten commercial products. Human Fertility 5:97, 1940. 7. BROWN, R. L., and GAMBLE, C. J. The comparative spermicidal powers of fifteen commercial contraceptives. Human Fertility 6:1, 1941. 8. DE VILBISs, L. A. Contraceptive effectiveness of foam-powder and sponge method. ]. Contraception 8:1, 1938. · 9. DICKINSON, R. L., and BRYANT, L. S. Control of Conception. Baltimore, Williams and Wilkins, 1932, p. 49. 10. ELIAS, N. M. "Spermicide" U.S. Patent 2,467,884, Aprill9, 1949. 11. ELIAS, N. M. "Spermicidal Compositions" U.S. Patent 2,777,796, January 15, 1957. 12. GERSHENFELD, L., and WITLIN, B. Iodonium compounds and their antibacterial activity. Am./. Pharm. 120:158, 1948. 13. KLARMAN, E. "Germicidal Preparation" U.S. Patent 1,953,443, April 3, 1934. 14. KoLLIKER, A. Physiologische Studien iiber die Sammenfl.ussigkeit. Z. Wiss. Zool. 7:221, 1855. 15. McCUTCHEON, J. W. Synthetic detergents and emulsifiers, up to date, 1955. Soap and Chemical Specialties, July-Oct., 1955.

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16. MILLMAN, N.

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A critical study of methods of measuring spermicidal action.

Ann.

New York Acad. Sc. 54:806, 1952. 17. SANDER, F. V. "Spermicide" U.S. Patent 2,330,846, October 5, 1943. 18. SANDER, F. V. "Spermicidal Composition" U.S. Patent 2,541,103, February 13, 1951. 19. SHEDLOVSKY, L. Some acidic properties of spermicidal jellies. ]. Contraception 2:147, 1937. 20. SwAYNE, V. R., BEILER, J. M., and MARTIN, G. J. Spermicidal effect of certain physiologically active substances. Proc. Soc. Exper. Biol. & Med. 80:384, 1952. 21. TAUB, A. "Spermicidal Compositions" U.S. Patents 2,623,839, 2,623,840, and 2,623,841, December 30, 1952. 22. VoGE, C. I. B. The Chemistry and Physics of Contraceptives. London, Jonathan Cape, 1933. 23. WARD, W. C. "Vaginal Suppository" U.S. Patent 2,469,618, May 10, 1949. 24. WARD, W. C. "Spermicidal Vaginal Suppositories" U.S. Patent 2,702,779, February 22, 1955.