A GENERAL STUDY OF THE EFFECTS OF LIPIDS UPON THE QUANTITY OF PEROXIDES PRODUCED BY PHOTOSENSITIZED ZINC OXIDE

DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By FREDERICK CLAIRE BLUBAUGH, B.S., B.S. in Phar., M.S. The Ohio State University 1955

Approved by:

Adviser College of Pharmacy ACKNOWLEDGMENT

The appreciation and gratitude of this author is expressed to Dr. Earl P. Guth, Professor, College of Pharmacy, under whose guidance this research has been per- foiroed, for his invaluable aid given to me. To Dr. B. V. Christensen, Dean, College of Pharmacy, for his advice and guidance and for the opportunity to continue by rendering to me the necessary financial assistance. To Drs. Loyd E. Harris, Frank W. Bope, and John W. Nelson of the College of Pharmacy for their invaluable assistance. To Dr. C. A. Randles, Department of Bacteriology, for his services. My deepest appreciation and gratitude to my wife, Mary Catherine, and to the children, Carol, Barbara, Susan, and M a r g a r e t .

ii TABLE OF CONTENTS PAGE INTRODUCTION ...... 1 CALAMINE ...... 4 ZINC O X I D E ...... 5 ZINC OXIDE AS A PHOTOSENSITIZER...... 7 ULTRAVIOLET LIGHT...... 10 PROPERTIES OF THE O I L S ...... 11 OLIVE O I L ...... 14 EXPRESSED ALMOND OIL...... 16 PEACH KERNEL OIL...... 17 CASTOR OIL...... 18 LARD OIL...... 18 COTTONSEED OIL...... 19 SESAME OIL...... 20 LINSEED O I L ...... 21 COD LIVER O I L ...... 22 LIQUID PETROLATUM ...... 24 SQUALENE...... 25 PEROXIDES...... 25 MATERIALS...... 27 ASSAY OF THE OILS...... 29 EXPERIMENTAL: PREPARATION OF SAMPLES...... 30 METHOD OF IRRADIATION ...... 31 iii TABLE OP CONTENTS (CONT'D) PAGE METHOD OP ASSAY ...... 32 IRRADIATIONS ...... 33 DISCUSSION...... 51 SUMMARY...... 82 REFERENCES...... 84

AUTOBIOGRAPHY ...... 88

iv A GENERAL STUDY OP THE EFFECTS OF LIPIDS UPON THE QUANTITY OF PEROXIDES PRODUCED BY PHOTOSENSITIZED ZINC OXIDE

INTRODUCTION Zinc Oxide is a common ingredient of many medicinal products used in the treatment of a wide variety of derma- tological conditions. It is official in the Pharmacopeia of the United States, XIV (41), as Zinc Oxide and in prepar­ ations such as Calamine, Calamine Lotion,! Zinc Oxide Ointment, and Zinc Oxide Paste. In the National Formulary,

IX (3 6), it is found in the form of Prepared Neocalamine, Neocalamine Lotion, Neocalamine Liniment, Neocalamine Ointment, Phenolated Neocalamine Lotion, Calamine Liniment, Calamine Ointment, Phenolated Calamine Lotion, Zinc Oxide Hard Paste, Zinc Oxide Soft Paste, and Zinc Oxide Paste with Salicylic Acid. In commerce Zinc Oxide is used in many products similar in composition to the official products as well as many modifications. In addition physicians prescribe this compound freely in many combinations containing fats, hydrocarbons, water, glycols, phenols, and many other types of materials. Reese (51), Mathias (32), and Minardi (34) have studied the "Formation of Hydrogen Peroxide in Calamine Lotions," "The Effect of Various Phenols on the Formation of Hydrogen Peroxide by Zinc Oxide," and "The Effect of Time on the Hydrogen Peroxide Production by Calamine Lotions," respec­ tively. Their studies were devoted exclusively to suspen­ sion of zinc oxide in water preparations. This study is with emulsion type of preparations containing zinc oxide. All of the emulsions were water in oil type except Mineral Oil wherein the zinc oxide apparently functions as an emul­ sifier due to its particle size. The irradiation procedures employed by Reese, Mathias, and Minardi were modified to make them adapted to the study of the water in oil type of emulsion. Reese (51) showed that peroxides were produced in Cala­ mine Lotions upon irradiation with ultraviolet light. Vari­ ous ingredients such as phenol, glycerin, polyethylene glycols, Spans and propylene glycol alginates were shown to have a pronounced effect upon the quantity of peroxide pro­ duced. The addition of ferric oxide to the lotions was detrimental to peroxide production. Reese also found that the light source for peroxide production could be either an ultraviolet lamp, incandescent lamp, fluorescent lamp, or sunlight. Mathias (32) found wide variations in the quantity of peroxides produced in commercial brands of Calamine Lotions when subjected to ultraviolet irradiation. He studied the effect of adding phenol, resorcinol, resorcinol monoacetate and pyrogallol to the suspension. Although his results indicated that the amount of peroxides could be significantly altered, the evidence indicated that there is an optimum concentration of peroxide beyond which any increase in the amount of "additive" results in a decrease in the quantity of peroxide that could be produced. The effect of the addition of various pharmaceutical adjuvants has been shown by Minardi (3*0 to effect markedly peroxide production. Minardi concluded that the addition of either dioctyl sodium sulfosuccinate and/or sodium car- boxymethylcellulose depressed peroxide production. All previous research has been devoted to "lotion" type preparations containing zinc oxide and calamine. Since zinc oxide and calamine are also incorporated into many oifatments and liniments with either a fixed oil or petrola­ tum, a comparative study of the effect of these materials upon the quantity of peroxides produced by photosensitized zinc oxide was in order. Most of the fixed oils used in dermatological practice were selected for this study.

CALAMINE During the nineteenth century, the teiro "calamine" was applied by mineralogists Indiscriminately to two minerals, scarely distinguishable by their external characteristics, the carbonate and the silicate of zinc (11). The mineralo­ gist applies the term "calamine" to a hydrous zinc silicate while the carbonate of zinc is known as "Smithsonite." (10) When native zinc carbonate is calcined, an impure zinc oxide remains. This product was teiroed "prepared calamine" in the supplement to the British Pharmacopaeia of 1867 (10).

The National Formulary IV, 1916 (38), defined Prepared Cala­ mine as “Native zinc carbonate containing a varying amount of zinc silicate calcined at a moderate temperature; or calcined zinc carbonate, containing a small amount of fer­ ric oxide." The United States Pharmacopeia, XIV (41), defines Calamine as "Zinc oxide with a small amount of ferric oxide."

The Merck Index, 6th Edition (33)* lists Calamine (Prepared Calamine) as zinc oxide with about 0.5$ ferric oxide. Calamine is used in medicine only as an external appli­ cant, being employed as a mild astringent and exsiccant in excoriations and superficial ulcerations (11). Apart from its color, which may have some aesthetic value, Calamine U.S.P.has no special advantage over Zinc Oxide U.S.P. (49). Goodman (19) suggested that zinc oxide be substituted for calamine in lotions and other preparations. While Calamine U.S.P. "is zinc oxide with a small amount of ferric oxide," the British Calamine "is a basic zinc carbonate suitable colored with ferric oxide" (4). Calamine Liniment, N.F. IX, served as the initial phar­ maceutical preparation for this study. It first appeared in the N.F. V, 1926 (37)* and has remained unchanged in all revisions of the N.F. ZINC OXIDE The Pharmacopeia of the United States, XIV (41), states that "Zinc oxide, freshly ignited, contains not less than 99 per cent ZnO." The method of its manufacture or its crystalline structure are not specified. Freedom from im­ purities especially of lead and arsenic are of prime impor­ tance. The commercial production of zinc oxide was started by LeClair and Sorel in France during the latter part of the eighteenth century, employing the prodess now known as the French Process or the two step process. In the United States, commercial production was started by Weatherill and Jones about 1 8 5 5. The American process was based on an observa­ tion made by a workman who noticed white fumes given off from zinc ores while repairing a break in a boiler flue. This process is now known as the American Process or the direct process (39). The French Process involves burning of slab zinc metal (spelter) with air. The zinc metal is heated in a retort to the boiling point. When the zinc vapors come in contact with air, they b u m with a bright yellowish colored flame to produce zinc oxide. These fumes are carried through suit­ able conduits to settling chambers. The physical properties of the oxide may be controlled within certain limits by the rate of oxidation, but the particle shape is characteristic­ ally spherical. The basic principle of the American Process is to vaporize the zinc directly from zinc ores rather than from the metal since the oxide possesses the property of passing from the solid state directly to the vapor state and back again to the solid under suitable temperature conditions (39). The American Process includes the Eastern and the West­ ern Weatherill processes. The Eastern Weatherill process, in which the zinc ore is mixed with anthracite coal before subjecting it to high temperatures, gives predominantly nodular particles. The Western Weatherill process, in which the zinc ore is mixed with bituminous coal, yields acicular particles. The Electro-thermo Process is one in which con­ trol is maintained over the chemical composition of the charge in the ehctric furnade and over the physical proper­ ties of the oxide by regulating the conditions at the oxidizing zone. This process combines the desirable charac­ teristics of both the French and American Processes,produc­ ing a very pure zinc oxide as usually associated with the French Process and the particle sizes and shapes characteris­ tic of both processes (39). Zinc oxide has been official in the U.S.P. since the first edition (48). The British Pharmacopeia, 1885 (3), and the U.S.P V, 1876 (47), recognized zinc oxide prepared from the carbonate. This method of preparation, the Wet Process, is one in which zinc in solution is precipitated from the solution and the zinc carbonate subsequently calcined to zinc oxide. This produces a finer particle size than that produced by the fume processes (Weatherill or Electro- thermo Processes) (39). Zinc Oxide is a white crystalline or amorphous powder. Some of its properties differ depending on the method of its preparation. The solubility of zinc oxide prepared by precipitation is 1.6 x 1 0 and by decomposition of zinc carbonate at 1000° is 2.75 x 10-18 (14). Zinc oxide has been used internally as well as exter­ nally in medical use. Internally, it was given in chorea, epilepsy, whooping cough, spasm of the stomach dependent on dyspepsia, and other similar affections (11). Externally, its use has remained somewhat the same principally as an exsiccant to excoriated surfaces (9). Zinc oxide is widely used in dermatology and is tolerated well in almost every skin disorder. It is a component of most dermatological powders, pastes, shake lotions, drying pastes and cooling pastes (49)i It owes its popularity to a combination of four qualities: lack of toxic reaction, protective action, mild astringent action and it is probably mildly antisep­ tic (1 0 ).

ZINC OXIDE AS A PHOTOSENSITIZER Hydrogen peroxide is formed at light activated zinc oxide surfaces in contact with oxygen, water and miscellan- eous compounds. Baur and Neuweiler (1) first reported that zinc oxide catalyzed formation of hydrogen peroxide. Good- eve (18) confirmed the phenomenon. The often proposed over-all reactions is 2 H2 O + 0 2 — > 2 H20 2, a reaction which involves a large increase in free energy. The light absorp­ tion region of zinc oxide limits the effective wave length for hydrogen peroxide formation to those less than 4000 A. About 4 per cent of the total sun’s energy incident at the earth's surface can be effective in promoting this reaction (52). Aqueous suspensions of zinc oxide, which have been pre­ pared by burning zinc vapor in air (French Process), shows the presence of hydrogen peroxide after irradiation with ultra violet light. Aqueous suspensions of zinc oxide, prepared by burning zinc vapor in air containing reducing gases (American Process), do not show the presence of hydro­ gen peroxide after irradiation. Also, the zinc oxide pre­ pared by the French Process shows no visible fluorescence when treated with ultraviolet light at room temperature while the zinc oxide prepared by the American Process shows a strong yellow fluorescence (5 6). Smith and Hawk (53) concluded in their study of methanol decomposition by zinc oxide that zinc oxide produced from zinc carbonate is more active than zinc oxide prepared by combustion of zinc. Huttig and Goerk (27) concluded that the catalytic activity of zinc oxide depends on the nature of the material from which it was obtained, presumably be­ cause of the nature of the original crystals. Garn (17) concluded that the principal cause of these differences lies in the general method of preparation resulting in difference in the surface characteristics of the ainc oxide. Chari and Qureshi (5) found that sunlight, artificial ultraviolet and visible light up to 4700 A are effective in causing the reaction to proceed. They also reported increased yields of hydrogen peroxide in the presence of organic compounds. Yamahuzi, Nisioeda, and .Ryusi (55), having studied the photochemical formation of hydrogen per­ oxide in the presence of biological sensitizers, suggest that the formation proceeds by three reactions:

2h20 ~ > H2 02 + 2H (I) 2H + 02 H2 02 (II) A + HgO + 02-» AO + H2 02 (III) "A" is the organic stabilizer. Although light energy is sufficient to promote these reactions, the heat produced by (II) and (III) would accelerate reaction (I) (17). The presence of a small concentration of one of the many water soluble easily oxidized compounds (termed "sta­ bilizers," "promoters," or "additives") such as sodium formate, potassium oxalate, phenol, etc. in the zinc oxide- water, oxygen mixture increases the rate of peroxide forma­ tion greatly. In such a system, zinc oxide is a photosensi- 10 tizer for hydrogen peroxide formation. Hydrogen peroxide is formed primarily in the region of light absorption by zinc oxide, 2500 A. There is no apparent net change by zinc oxide. In general, the light activated zinc oxide catalyzes the occurrence of a normal exothermic reaction (5 2). The presence of.' oxygen in the system is necessary for hydrogen peroxide formation. Hydrogen peroxide decomposi­ tion is very rapid in zinc oxide-water-additive mixtures in which the oxygen supply is low. Uiese so called "stabili­ zers" are actually reactants and undergo oxidation simultan­ eous with the hydrogen peroxide formation. The formate ion and the oxalate ion are oxidized to carbonate, while phenol is oxidized to catechol (5 2).

HgO + Og + HCOCT — > HgOg + HC03

ULTRAVIOLET LIGHT Ultraviolet light first received its name because it was adjacent to or beyond the violet limit of the visible spec­ trum. For purposes of convenience, it is divided into three parts in reference to the visible spectrum (3 0). a. Extreme ultraviolet energy (less than 2000 A) is absorbed by most substances, including air. This is the least penetrating of all radiant energy, and must be pro­ duced and studied in a vacuum by means of special photo­ graphic plates, gelatine emulsions being opaque to it. b. Middle ultraviolet energy (2000 to 3000 A) is the important spectral region biologically and therapeutically. While it is not transmitted by ordinary glass, quartz is transparent throughout this range. Water is also an impor­ tant exception permitting transmission of radiant energy. The solar spectrum barely extends into this region, its short wave limit being near 2900 A. Solar energy between 2900 and 3100 A is very valuable in antirachitic action, germicidal action, Vitamin D synthesis, in the production of erythema and of conjunctivitis. The maximum geimicidal action is in the neighborhood of 2600 A, for its conjunc­ tivitis effect, 2500 A, and its erythemal effect, 3000 A. Vitamin D production by irradiating ergosterol is due to energy somewhere between 2500 and 3100 A. c. Near ultraviolet energy (3000 to 3900 A) produces fluorescence and photographic action. Ordinary glass is transparent between 3500 and 3900 A, but fairly opaque at 3100 A. The source of ultraviolet light for this study was a

DAZOR Floating Fixture Ultraviolet Lamp, Model N. U-5 8, equipped with a General Electric UA-3 360 Watt Quarts Photo­ chemical "Ulviarc" Lamp. The radiant energy output of this lamp is greatest at 2537 A, 3131 A, and 3564 A (40).

PROPERTIES OF THE OILS While a great deal of factual information has been ob­ tained during the past century about the fats and the oils, 12 there has been little change in their pharmacological appli­ cations. Their use in external medication has been as emollients to soften the skin and crusts in eczema and psoriasis, as dressings for burns, as vehicles for lini­ ments, etc.(24). The protective action of oils against irri­ tation and absorption was known and practiced by the early Greek physicians (49). Most of the vegetable oils are dermatologically innocu­ ous (24). While some are quite odorless, others have been limited in their dermatological use because of odor. How­ ever, Linseed Oil is of interest because of its high content of linoleic and linolenic acids and their possible benefi­ cial effects on the skin. Cottonseed Oil has been allowed as a substitute for Almond Oil and Olive Oil in toilet preparations primarily because of cost. Sesame Oils unusual stability, makes it highly desirable as a vehicle for medicaments for subcutaneous or intramuscular injections. The fats consist almost entirely of non-volatile, odor­ less esters. They are composed of glycerol combined with organic acids belonging to the aliphatic straight chain type, with few exceptions. These acids almost always have an even number of carbon atoms per molecule, usually between

8 and 24. Constituents occurring in smaller amounts include the phospholipides, sterols, vitamins, antioxidants, pig­ ments, and hydrocarbons as well as other materials less well known and amount to less than 5 per cent of the weight 13 of a fat in most vegetable oils. When a sample of natural fat is saponified and acidu­ lated, the fatty acids are set free as a mixture. A few fats yield mixtures in which one kind of fatty acid consti­ tutes 80 to 90 per cent of the total but these are the exceptions. A large proportion of the total weight of the oil is accounted for by a relatively small number of differ­ ent acids. The principal fatty acids are the unsaturated series with an 18 carbon chain. Oleic, linoleic, and linolenic with one, two, and three double bonds respec­ tively occur most frequently. Laurie, myristic, palmitic and stearic acids in the saturated series are the most wide­ spread. Ricinoleic acid is the principal acid of Castor Oil from which the oil derives many of its characteristic properties. It constitutes 87 per cent of the total fatty acids present. The properties of fatty acids depend upon the chain length of the molecule, the degree of unsaturation, geo­ metrical isomerism in the unsaturated acids, the position of the double bond with respect to the carboxyl group and respect to each other (1 2 ). The minor components are more important than their proportionate weight indicates (12). Plant phospholipides marketed as "lecithin** or "vegetable lebithin" have become an important commercial product. Phytosterols have become an important source of raw materials for the preparation of 14 valuable sex hormones. Tocopherols serve a dual role as a source of Vitamin E and as antioxidants. A highly unsatu­ rated hydrocarbon, squalene, has been found in many of the vegetable oils (1 5) and may have an important role in dermatology. Some of the more common characteristics of the oils, along with their fatty acid composition, are given in Table I.

OLIVE OIL Olive Oil is the fixed oil obtained from the ripe fruit of Plea europaea (41). Formerly it was the most used oil in dermatological preparations but the cheaper oils of Sesame and Cottonseed have been substituted. Olive Oil is used extensively as an emollient and as a food. The methods used for harvesting, storing, and milling of olives to obtain the oil are of great variety. The oils thus produced cover a correspondingly great range in char­ acteristics. Flavor is important in grading the oils but can not be evaluated chemically (12). One of the main deter­ minations relied upon is that of the free fatty acid con­ tent. The highest grade oils show a low acid value which tends to increase as the quality of the oil deoreases. United states Standards for Olive Oil include Grade a (U.S. Fancy), with free fatty acid not more than 1.4 per cent; Grade B (U.S. Choice), with free fatty acid not more than 2.5 per cent; Grade C (U.S. Standard), with free fatty acid 15

TABLE I CHARACTERISTICS OP THE C

COTTON- EXPRESSED PEACH CASTOR SEED CHARACTERISTIC OLIVE ALMOND KERNEL A.O.C.S. A.O.C.S Acid Value 0.2-6 0.1-3 0.4-3

Saponification Value 187-196 188-197 189-194 176-185 189-198 Iodine Value 79-90 93-105 95-110 8 1 -9 1 99-113 Unsaponifiable below (#) 0.5-1.3 0 .4-1.0 0-7 below 1 1 .5 COMPOSITION OP FATTY ACIDS, WEIGHT % OP TOTAL: Saturated* 3.1-5.7 2 .4 Myristic 1.2 0 .5 Palmitic 15.6 21.9 Stearic 2.0 0.3 1.9 Arachidic 0.1 Unsaturated: Oleic 64.6 77 7 .4 3 0 .7 Linoleic 15.0 17-20 3 .1 44.9 Linolenic Ricinoleic 8 7.0 * Compiled from: Eckey, E. W., Vegetable Pats and Oils. Amer Series (12) The Merck Index, 6th Edition (1954) (33) Properties of the Principal Fats, Fatty Oil Their Salts, The Texas Company (50). 15

TABLE I CHARACTERISTICS OP THE OILS*

COTTON­ PRESSED PEACH CASTOR SEED COD LMOND KERNEL A.O.C.S. A.O.C.S. SESAME LINSEED LARD LIVER .1-3 0.4-3 4.0 max. 1.5-215 1 .2

.88-197 189-194 176-185 189-198 188-195 188-196 195-197 180-190 93-105 95-110 81-91 99-113 103-116 170-204 56-74 145-180 below below below 0.54- .4-1.0 0-7 below 1 1.5 1 .8 0 .5-1 .2 2 .6 8 ;, WEIGHT % OF TOTAL: i . 1-5.7 2.4 13 0.5 0 .1 0 .2 1 .0 5.8 21.9 8 .2 5.4 2 6 .0 8.4 0.3 1.9 3.6 11.5 0 .6 0 .1 1 .1 03*i .6

C14 «* 0 .2 77 7.4 30.7 45.3 19 58 C16 =2 0 .0 7-2 0 3.1 44.9 41.2 24 3.5 C18 =29.1 47 C20 =2 5 .4 8 7.O 022 = 9 . 6 W., Vegetable Pats and Oils. American Chemical Society Monograph 2) : Index, 6th Edition (1954) (33) s of the Principal Fats, Fatty Oils, Waxes, Patty Acids, and ts, The Texas Company (50). 16 not more than 3 per centj Grade D, or substandard (12). The U.S.P. XIV maximum free fatty acid content is 2.8 per cent. The saturated fatty acids of Olive Oil consist principal- ly of palmitic acid (1 5 .6 per cent), with a smaller quantity of stearic acid (2 per cent) and traces of myristic (1 .2 per cent) and arachidic acid. Unsaturated fatty acids consist mainly of oleic acid (64.6 per cent) with a smaller quantity of linoleic acid (15 per cent). The glycerides of Olive Oil consist mainly of mixed glycerides, with triolein likely to amount to a little less than half of the total glycerides in any specimen of oil. Minor constituents of Olive Oil include about 0.2 per cent of phytosterol and a relatively small quantity of tocopherol. The unsaturated hydrocarbon squalene occurs in Olive Oil in quantities between 0.1 and 0.7 per cent of the weight of the oil (1 5). Most of the Olive Oil produced in the world is used as food. Olive Oil is also used for a large variety of other purposes, including the manufacture of soaps, textile lubri­ cants, sulfonated oils, cosmetics and pharmaceuticals.

EXPRESSED ALMOND OIL Expressed Almond Oil is the fixed oil obtained from the kernels of varieties of Prunus Amygdalus (41). The oil is reputed to be an excellent emollient for Inflammed condi­ tions of the skin (4 9). 17 This oil has been a product of commerce for centuries. It is recognized and specified for certain uses by the pharmacopeias of several countries (12). Numerous varieties of almonds are grown which may be grouped into two main categories known as sweet almonds and bitter almonds. Bit­ ter almonds contain the glycoside amygdalin which on hydrol­ ysis yields benzaldehyde, hydrocyanic acid, and glucose. This product is called "Oil of Bitter Almond.Vj Sweet almonds yield a fixed oil only and are the ones used as nuts. The characteristics of Apricot Kernel Oil are so similar to those of Expressed Almond Oil that the two can not be differentiated by the usual determinations. The fatty acids of Expressed Almond Oil consist mainly of oleic acid (77 per

cent) and linoleic acid (17 to 20 per cent) with smaller percentages of myristic acid and palmitic acid. The oil consists principally of trioleins and dioleins. The pharmaceutical and cosmetic fields use most of the Expressed Almond Oil for the preparation of lotions and creams.

PEACH KERNEL OIL Peach Kernel Oil is the fixed oil expressed from the kernels of Prunus Persica (41). This oil is relative new to the pharmaceutical field, becoming official in the U.S.P. XII, 1942 (42). The usual characteristics for Peach Kernel Oil are very similar to those of Expressed Almond Oil or Apricot Kernel 18 Oil. The U.S.P. XIV recognizes either Apricot Kernel Oil or Peach Kernel Oil as Persic Oil; further, the U.S.P. XIV recognizes either Expressed Almond Oil or Persic Oil as a component of Rose Water Ointment.

CASTOR OIL Castor Oil is a fixed oil obtained from seed of Ricinus communis (41). It is distinctive in character. Its peculiar composition gives it great versatility. Not only is the oil useful as such $n a variety of compositions but it is cap­ able of chemical transformations into numerous useful deriv­ atives. Caator Oil has arhigher viscosity, specific gravity, hydroxyl value and alcoholic solubility than most vegetable oils (1 2 ). Castor Oil consists almost entirely of triglycerides. The fatty acids include a large proportion of rlcinoleic acid. The proportion of saturated fatty acids is small in comparison to other vegetable oils. The proportion of minor constituents are small. Tocopherol amounts to about 0.5 per cent of the oil. A large amount of Cafetor Oil is consumed in cosmetics because of its superior emollient behavior (24). Its high viscosity and alcohol solubility makes it quite suitable for this purpose.

LARD GIL Lard Oil was official in the U.S.P. VIII (44). When 19 lard, the internal fat of the abdomen of the hog, is kept at low temperatures and subjected to pressure, the stearin is separated from the olein (1 0 ). Lard Oil absorbs oxygen when exposed to the air, devel­ oping ketones and peroxides. Ihis occurs more rapidly if the oil is alkaline. This reaction is accelerated by light and elevated temperatures. Lard Oil possesses emollient and skin penetrating prop­ erties (21). It does not appear to be harmful. However, in the rancid state, it may be irritating to the skin and is of objectionable odor. Further, if rancid, it may react chemi­ cally with the medicaments incorporated in it (2 0 ).

COTTONSEED OIL Cottonseed Oil is the refined fixed oil obtained from seed of cultivated plants of Gossypium hirsutum (41). It is one of the most important of the vegetable oils in American and world commerce. The portion produced in the United States normally amounts to between 35 to 45 per cent of the total world production. The refined oil has relatively good keeping qualities. It is resistant to the development of rancidity due to oxidation. Crude Cottonseed Oil usually is a dark colored oil with a characteristic odorsand flavor. Most of this color is extracted when the phospholipid fraction, amounting to between 1 and 2 per cent, is removed. 20 Cottonseed Oil is classed as semi-drying, the principal fatty acids being linoleic, oleic, and palmitic. The esters consist principally of mixed glycerides. More than

50 per cent of the weight of the oil consists of glycerides containing one saturated acid radical and two unsaturated acid radicals. In the refining process, the phospholipids and gossypol (a yellow phenolic compound) are removed. A considerable portion of the unsaponifiable matter remains in the refined oil. This includes the sterols (0.01 per cent), tocopherols (0.1 per cent) and hydrocarbons (i.e., squalene). The tocopherols have a major influence upon the keeping qu a n ­ tities of the oil. Nearly all the crude Cottonseed Oil produced in the United States is refined for use in edible products. Shortening, cooking and salad oils, and margarine account for more than 95 per cent of the refined oil (12). Pharma­ ceutically, Cottonseed Oil is replacing Expressed Almond Oil and Olive Oil in cosmetic preparations.

SESAME OIL Sesame Oil is the refined fixed oil obtained from the seeds of one or more cultivated varieties of Sesamum indicum (4l). It is probably one of the oldest of the cul­ tivated oil seed crops, having been cultivated in India for centuries (1 2 ). 21 Sesame Oil possesses unusual stability in part because of the antioxidant effect of a phenolic material, sesamol (12). Its presence affords a property to Sesame Oil which makes it highly desirable as a vehicle for medicaments to be subcutaneously or intramuscularly injected. Oleic and linoleic acid are the principal fatty acids of the oil. Linolenic appears to be entirely lacking (12). The saturated fatty acids, palmitic and stearic acids, amount to about 14 per cent of the total fatty acids. The glycerides consist almost entirely of mixed glycerides. Sesame Oil contains a higher percentage of unsaponifi- able matter than is characteristic for most vegetable oils. Substances found in the unsaponifiable matter of the oil are possibly responsible for some of the special properties.

LINSEED OIL Linseed Oil is the fixed oil obtained from the dried ripe seed of Linum usitatissimum (4l). Its high iodine value reflects a high degree of unsaturation of its fatty acid radicals. The properties of Linseed Oil make it highly useful in the manufacture of paints but limits its useful­ ness as an article for human food. Linseed Oil is the standard drying oil. The glycerides of Linseed Oil are the mixed glycerides of linolenic, oleic, and linoleic and saturated acids with linolenic acid as the principal and characteristic acid of 22 the oil. There is marked variability of the composition of the mixed fatty acids (1 2 ). Unsaponifiable material in Linseed Oil amounts to about 1 per cent. Sterols constitute a considerable portion of this matter. The tocopherol content is about 0.1 per cent

(12). Raw Linseed Oil contains in addition a variable pro­ portion of other materials. This material has been referred to as "foots" or "mucilage," a part of which separates from the oil on standing. The composition of' this fraction is principally phospholipids together with carbohydrates and other miscellaneous materials. The "foots" fraction has an affinity for moisture. When it becomes hydrated, it is in­ soluble in the oil. The refining of Linseed Oil is done in part to free the oil of these materials. The principal uses of Linseed Oil are in the manufacture of paints and varnishes, linoleum and oilcloth and printing inks. Because of its odor, its pharmaceutical uses are limited. However, its high content of linoleic and linol­ enic acid with their potential benefit of the skin is of interest (24). Deodorized Linseed Oils however are not stable (1 2 ).

COD LIVER OIL Cod Liver Oil is the partially destearinated fixed oil obtained from fresh livers of Gadus morrhua Linne' and other species of the Family Gadidae. The high grade quality medicinal oils are obtained by treating the livers with steam to rupture the cell membranes. Since exposure of the livers after their removal from the fish for only a few hours results in detrimental action to the quality of the oil, the extraction is done immediately. The separated oil is filtered and bleached. “Diis oil as prepared congeals at low temperatures due to the presence of stearin, which can be removed by chilling to a -10 °, followed by expression (1 0 ). The composition of Cod Liver Oil is very complex. From the studies of Harper and Hilditfeh(23), and Hilditch and Terleski (25), a summary of its composition was given as follows: TABLE II Fatty Acid Composition of a Sample of Cod Liver Oil with a Original Iodine Value of 177.7

______ACID______. ______MOLE PER CENT______

Saturated; 14 carbon chain 1 .8 16 carbon chain 14.0 18 carbon chain 1 .5 Unsaturated;

14 carbon chain 2 .1 16 carbon chain 9 .3 18 carbon chain 26.4 20 carbon chain 2 5 .8 22 carbon chain 1 9 .1 24

Cod Liver Oil contains in the C20 to c22 series approx­ imately 45 per cent of unsaturated acids (2 3,2 5). The Council on Pharmacy and Chemistry of the American

Medical Association (6) reported favorably on the use of Cod Liver Oil in the local treatment of burns, wounds, and ulcers. The mechanism of action is not known but in local infectious diseases, necrotic material is liquefied and growth of granulation tissue and epithelization are stimu­ lated.

LIQUID PETROLATUM Liquid Petrolatum is a mixture of liquid hydrocarbons obtained from petroleum (41). Properly prepared oils are completely innocuous. Since they are not absorbed, they are of value in preserving a bland surface protection coat­ ing on the skin. They are used as substitutes for the true fats in ointments and are advantageous in that they do not become rancid (24). Petrolatum first became official in the U.S.P. VI (46) and was defined as "A semisolid substance, consisting of hydrocarbons, chiefly of the marsh gas series (C16H34; etc.-C32H34J etc.), obtained by distilling off the lighter and more volatile portions from American Petroleum, and purifying the residue." Liquid Petrolatum became official in the U.S.P., VII (45). However, the use of Petrolatum as a base for ointments did not become official until the tenth revision (43). SQUALENE Squalene is the highly unsaturated hydrocarbon found in the unsaponifiable fraction of certain vegetable and animal oils. It was first found in 1910 by Tsujimoto in shark liver oil, and it was reported in 1935 to be present in olive oil and in wheat germ oil. Chemically, its struc­ ture resembles the central part of the carotene molecule but with less unsaturation (1 2 ). CH39=CHCH2CH2C=CHCH2CH2C=CHCH2CH2CH=CCH2CH2CH=CCH2CH2CH=CCH3

CH3 CH3 CH3 CH3 CH3 CH3 Squalene is fairly easily oxidized. It is water insol­ uble but freely soluble in the organic fat solvents. Human sebum, according to Mackenna, Wheatley, and Wormull (31), contains 32 per cent unsaponifiable matter con­ sisting of: 30-40 per cent hydrocarbons, 14-20 per cent normal chain alipatic alcohols and 14-19 per cent choles­ terol. The remainder consists of oxidation products of squalene and noncholesterol sterol-like substances. The hydrocarbon fraction consists of 30-40 per cent- squalene and normal paraffins. Table III, page 26, shows the squalene content that has been reported for the fixed oils used in this study.

PEROXIDES Hydrogen Peroxide was discovered by Thenard in 181 8. It is marketed as a solution in water in concentrations from 26

TABLE III (14,15)

Oil Milligrams per 100 grams of Oil Olive Oil 136 - 708 Expressed Almond Oil 2 Cottonseed Oil 3-15 Sesame Oil 3 - 9 Linseed Oil 4 Cod Liver Oil 31

3 to 30 per cent by weight (32). Solutions of hydrogen per­ oxide deteriorate gradually and are usually stabilized by the addition of acetanilid or a similar substance. Among these organic compounds, Chari and Quershi (5) lists the following in the order of decreasing stability to the photo- decomposition of hydrogen peroxide: phenol, glycerol, acetanilid, ethyl alcohol, ethyl oxide, acetone, benzidine, salicylic acid and oxalic acid. Certain metallic ions, such as Cu++ and Fe^t have been shown by Kiss and Lederer (28) to catalyze its decomposition. Horkheiraer (26) was able to show practically no decomposition of hydrogen peroxide solutions stored at temperatures of 16 to 18° C. for six weeks. Hydrogen peroxide solutions are rapidly decomposed in alkaline media while the presence of mineral acids renders hydrogen peroxide solutions more stable. The organic peroxides may be regarded as derivatives of hydrogen peroxide, H00H, obtained by replacing one or both of the hydrogen atoms by organic radicals. They all have the 0 -0 link in common and practically all their 27 chemistry is associated with this linkage (54). Hydroperoxides are formed by replacing a hydrogen atom on the methylenic group adjacent to the double bond. The structure for one of the isomers of methyl oleate hydroper­ oxides is as follows:

c h 3(c h 2 )6c h c h =c h c h 2 (c h 2)6c o o c h 3 00H MATERIALS The materials utilized in this study were obtained from the following sources: 1. Zinc Oxide. U.S.P. (Merck and Co., Inc., Rahway, New Jersey) 2. Zinc Oxide, U.S.P. (Zoco Brand, Zinc Oxide Company of Canada Limited, Montreal) 3. Calamine (Zinc Oxide, Merck, with 0.5# Ferric Oxide Powder added. The two were blended in a Twin Shell Dry Blender and processed through a Bantam Mikro Pulverizer..) 4. Olive Oil I, U.S.P (Gerber's Pure Virgin Olive Oil, R. Gerber and Co., Chicago) 5. Olive Oil II (A Blend of the Finest Olive Oils, R.U. (Re Umberto), United Pure Food Co., New York)

6. Oil of Sweek Almond, U.S.P. (Expressed) (Orr, Brown and Price, Columbus, Ohio) 7. Peach Kernel Oil (Laboratory Supply, Ohio State University)

8. Castor Oil. U.S.P. Laboratory Supply, Ohio State University) 9. Lard Oil (Orr, Brown and Price, Columbus, Ohio) 10. Cottonseed Oil (Laboratory Supply, Ohio State University) 11.Sesame Oil, U.S.P. XIII )Magners, Mabee, and Reynard, Inc.) 28 12. Raw Linseed Oil (Laboratory Supply, Ohio State University) 13. Cod Liver Oil, U.S.P. (Orr, Brown and Price, Columbus, Ohio) 14. Mineral Oil (Laboratory Supply, Ohio State Uni­ versity) 15. Squalene 90# (Practidal)(Eastman Organic Chemicals, Distillation Products Industiles, Rochester 3, N.Y.) TABLE IV CHARACTERISTICS OP THE OILS AS DETERMINED BY ASSAY*

Oil Class Iodine Number Saponification Number Acid Number

**01ive Oil I I 84.9 193.4 0.97 Olive Oil II I 94.7 1 9 1 .0 0 .6 0 **Expressed Almond Oil I 1 0 1 .6 190.4 0.09 Peach Kernel Oil I 1 0 3 .6 189.9 1.09 ♦♦Castor Oil III 85.9 1 8 0 .2 1.95 Lard Oil IV 36.5 39.8 6.29 ♦♦Cottonseed Oil V 110.3 194.0 0.04 **Sesame Oil V 111.1 188.4 0 .2 2 Linseed Oil VI 154.5 1 9 1 .8 4.80 ♦♦Cod Liver Oil VII 150.7 1 8 6 .1 1.19 ♦U.S.P., XIV, Methods of Assay ♦♦Meet U.S.P., XIV, Specifications 30 EXPERIMENTAL

PREPARATION OP SAMPLES Calamine Liniment, N. P. IX (36), was selected as the basic formula for the study of the effect of selected lipds upon the quantity of peroxides produced by photosensitized Zinc Oxide. Calamine Liniment Calamine...... 80 Gm. Zinc O x i d e ...... 80 Gm. Olive Oil...... 500 ml. Calcium Hydroxide Solution, a sufficient quantity, ______To make ...... 1000 ml. Zinc Oxide was substituted for the Calamine so that the above formula was changed to Zinc Oxide 160 grams, Olive Oil 500 ml., and Calcium Hydroxide Solution q.s. to make 1000 ml. In subsequent studies, Calamine was reinstated in order to determine the effect of ferric oxide upon per­ oxide production. In this first phase of the study the only variable was the oil used. The fixed oils used in dermatological preparations are from many species of plants and animals. They include the non-drying, semi-drying, and drying oils of vegetable origin as well as those obtained from animals. The fixed oils se­ lected for this study included representatives of each of the classes^(2 2 ) of oil utilized in dermatological prepara­ tions. Each of the oils used was tested for purity and identity (See Table IV, page 29). 31 Each oil in an amount equal to Olive Oil in the formula for Calamine Liniment was mixed with zinc oxide. The zinc oxide-oil mixture was placed in a eight ounce prescription square and shaken for 20 minutes on a mechanical shaker. After the initial mixing, Calcium Hydroxide Solution was added to the containers. The containers were closed immew diately. This procedure maintained the strength of the Calcium Hydroxide Solution. The containers were returned to the mechanical shaker for an additional 20 minutes of shaking. When the mixture consisted of two ingredients, i.e., zinc oxide and oil, zinc oxide and Calcium Hydroxide Solu­ tion, or oil and Calcium Hydroxide Solution, the same pro­ cedure of mixing and shaking was employed. Five (5) gram samples of the Zinc Oxide-Liniments were used for irradiation. When only one or two of the components were studied, only that amount of the ingredient represented in a 5 gram sample of Calamine Liniment was irradiated. These amounts were determined on a weight basis. A 5 gram sample was made up of 0 .7 6 grams of zinc oxide, 2 .1 2 grams of oil, and 2.12 ml. of Calcium Hydroxide Solution.

METHOD OF IRRADIATION Five (5) gram samples of the Zinc Oxide-Liniments were weighed into 800 ml. Pyrex beakers. One hundred (100) ml. of distilled water or 100 ml. of 0.2 M sodium formate solu­ 32 tion was added to the beakers. The latter acted as an "additive1* for the photochemical production of peroxides by zinc oxide (52). When only one or two of the ingredients were to be irradiated, the samples consisted only of the amount of those ingredients present in a 5 gram sample. The four 800 ml. beakers were placed in the water bath under the ultraviolet lamp (See page 10). The water bath was cooled by running water. Glass stirrers driven by a Cenco Stirrer Variable Speed motor at 530 RPM were utilized as a means of agitation of the samples during irradiation. The equipment was arranged so that each of the beakers was the same distance from the ultraviolet lamp. The distance

from the lamp to the surface of the liquid was 35 cm. The irradiation unit was completely enclosed within aluminum sheeting. The beakers rested on a false aluminum bottom in the water bath and held in place by a sheet of aluminum containing holes that were the same diameter as the beakers. The level of the water in the bath was kept at a constant level slightly above the contents of the beakers. Irradiation time for all samples was 30 minutes.

METHOD OF ASSAY The iodometric method of Kolthoff (29) as modified by Chari and Quershi (5) was utilized for the determination of peroxides. Peroxides react with iodide in acid medium 33 according to the following reaction:

H2 02 + 21“ + 2H --- > I2 + 2H20 Ammonium molybdate catalyzes this reaction. The irradiated samples were filtered immediately through Whatman No. 1 filter paper, giving a clear filtrate. The first 15 ml. of the filtrate was discarded. To a 50 ml. aliquot of the clear filtrate in an Iodine flask, the fol­ lowing reagents were added in the order given: 10 ml. 2 M sulfuric acid 10 ml. 0.2 N potassium iodide 5 ml. 0.04 N ammonium molybdate The flasks were well shaken and placed in the dark for 10 minutes. The liberated iodine was titrated with 0.01 N sodium thiosulfate, using freshly prepared starch solution as the indicator. IRRADIATI0NS The fixed oils utilized in this study are classified as follows (2 1 )j Class I Non-drying vegetable oils of the Olive Oil type Olive Oil Expressed Almond Oil Peach Kernel Oil Class III Non-drying oils of Castor Oil type Castor Oil Class IV Non-drying animal oil Lard Oil Class V Semi-drying vegetable oils Cottonseed Oil Sesame Oil 34 IRRADIATION UNIT

. MAf • 55 Class VI Drying vegetable oils Linseed Oil Class VII Pish and marine animal oils Cod Liver Oil In addition, Mineral Oil was included. Samples for irradiation consisted of the following ingredients: 1. Each ingredient separately. 2. Emulsions of the Oils by addition of Calcium Hydroxide Solution. 3. Zinc Oxide in combination with the Oils. 4. Zinc Oxide in combination with Calcium Hydroxide Solution. 5. Zinc Oxide Liniments. Zinc Oxide (Merck) was irradiated in combination with each of the listed Oils. Zinc Oxide (Zoco) was irradiated in combination with Olive Oil, Cottonseed Oil and Mineral Oil. Calamine was irradiated in combination with Olive Oil, Expressed Almond Oil, Cottonseed Oil and Mineral Oil. Olive Oil of two different grades are included in this study and are designated Olive Oil I and Olive Oil II (See "Materials," Page 27). The addition of sodium formate not only increased the production of peroxides (See Table VII, page 39) but acted as a "stabilizer," preventing marked destruction of pero­ xides. Hydrogen peroxide was destroyed at an appreciable rate in the absence of sodium formate (See Table V, page 3 6). TABLE V Stability of Hydrogen Peroxide to Ultraviolet Irradiation

Irradiation In Distilled Water In Sodium Formate Solution Time In Micrograms of Percentage Micrograms of Percentage Minutes h 2°2 Present Loss Remaining H2 O2 Present Loss Remainir

0 5,530 0 100 5,876 0 100 10 4,968 10.2 89.8 5,533 5,8 94.2 20 4 ,6 7 8 15.4 84.6 5,516 6.1 93.9 30 4,289 22.5 77.5 5,434 7.5 92.5

u> o\ 37 Thfe irradiation of 100 ml. of distilled water and 100 ml. of a 0.2 M solution of sodium formate produced measur­ able peroxides (See Table VI).

TABLE VI Micrograms of Peroxides Produced after 30 Minutes of Ultra­ violet Irradiation

MEDIUM SAMPLE AVERAGE N0.1 No.2 NO. 3 NO.4

Distilled Mater 71 85 78 60 74 Sodium Formate Solution 156 143 136 133 142

Pour (4) samples of the separate ingredients were ir­ radiated in 100 ml. of distilled water or in 100 ml. of 0.2 M sodium formate solution. The amount of the ingredi­ ent irradiated in each irradiation was the quantity of the ingredient present in a 5 gram sample of the basic formula (See "Preparation of Samples," page 30). The results obtained from the irradiation of the oils are given in Table VIII, page 40. For the results obtained from the irradiation of the Zinc Oxides, Calamine, and Calcium Hydroxide Solution, see Table VII, page 39. Four (4) samples of each of the following were irradi­ ated in 100 ml. of distilled water and in 100 ml. of 0.2 M sodium formate solution. The results are given in the Tables as indicated: 38 Combination Results Oils + Calcium Hydroxide Solution Table IX, page 41 Zinc Oxide (Merck) + Oils Table X, page 42 Zinc Oxide (Zoco) + Oils** Table XII, page 44 Calamine* + Oils** Table XIV, page 45 Zinc Oxide (Merck) + Calcium Table VII, page 39 Hydroxide Solution Zinc Oxide (ZBco) + Calcium Table VII, page 39 Hydroxide Solution Calamine* + Calcium Hydroxide Table VII, page 39 Solution Pour (4) five gram samples of each Zinc Oxide and Cala­ mine Liniment were irradiated in 100 ml. of distilled water and 100 ml. of 0.2 M solution of sodium formate. The re­ sults for Liniments containing Zinc Oxide (Merck) are given in Table XI, page 43; for Liniments containing Zinc Oxide (Zoco), in Table XIII, page 44; for Calamine Liniments, in Table XV, page 45.

IRRADIATION WITH SQUALENE Squalene (See ’’Materials," page 27) is a natural con­ stituent of many fixed oils (See ’’Squalene," page 25). It was added directly to Mineral Oil in concentrations of 0.5, 1, and 2 per cent V/V. Pour (4) samples of each concentra­ tion were irradiated 30 minutes in 100 ml. of a 0.2 M

*Calamine: 0.3& Grams of Zinc Oxide Merck) and 0 .3 8 Grams of Calamine. **See Experimental," page 33 and 35> for the Oils combined -with Zinc Oxide (Zoco) and Calamine. TABLE VII Micrograms of Peroxides Produced after 30 Minutes of Ultraviolet Irradiation Ingredients In Distilled Water In Sodium Formate Solution Irradiated No. 1No. 2 No. 3 No.4 Average No. 1 No. 2 No. 3 No. 4 Average Zinc Oxide (Merck) 707 742 686 742 719 7,942 7,728 8 ,0 2 6 7 ,6 0 6 7 ,8 2 6 0 .7 6 Grams

Zinc Oxide (Zoco) 310 362 297 355 331 2,342 2 ,2 6 7 2,229 2,196 2,259 0.76 Grams

Calamine* 187 205 196 187 194 5,124 5 ,1 6 1 5,273 5 ,0 8 6 5,161 O .7 6 Grams

Calcium Hydroxide 70 70 60 67 67 118 150 126 136 133 Solution, 2.12 ml.

Zinc Oxide (Merck), 318 297 283 293 298 6,259 6,591 6,131 6,874 6,464 0 .7 6 Grams and Calcium Hydroxide Solution, 2.12 ml. Zinc Oxide (Zoco), 220 200 184 226 208 833 898 759 904 849 O .76 Grams and Calcium Hydroxide Solution, 2.12 ml. Calamine*, O .7 6 161 161 161 161 161 4,428 4,009 4,136 4,376 4,247 Grams and Calcium Hydroxide Solution, 2.12 ml. ♦Calamine: O .3 8 Grams of Zinc Oxide (Merck) and O .3 8 Grams of Calamine (See "Mater­ ials," page 27). TABLE VIII Micrograms of Peroxides Produced by 2.12 Grams of the Oil after 30 Minutes of Ultraviolet Irradiation

Oil Class In Distilled Wa1ter In Sodium Formate Solution No. 1 No. 2 No. 3 No. 4 Average No. 1 No. 2 No. 3 No. 4 Average Olive Oil I I 92 60 60 80 73 157 163 177 140 159 Olive Oil II I 93 93 97 89 93 187 187 194 187 189 Expressed Almond Oil I 108 127 105 127 117 150 176 153 168 162 Peach Kernel Oil I 176 180 157 180 173 150 187 150 176 166 Castor Oil III 67 82 67 70 71 142 142 121 150 l4l Lard Oil IV 136 129 129 162 139 226 257 226 236 236 Cottonseed Oil V 99 80 93 86 90 97 111 119 92 105

Sesame Oil V 108 112 105 123 112 168 198 168 205 185

Linseed Oil VI 166 180 166 211 181 198 198 204 204 201 Cod Liver Oil VII 239 243 228 239 237 262 299 299 299 289 Mineral Oil 71 71 73 53 67 113 127 112 93 112

•fcr O TABLE IX Micrograms of Peroxides Produced by a Mixture of 2.12 ml. of Calcium Hydroxide Solution and 2.12 Grams of Oil after 30 Minutes of Ultraviolet Irradiation

Oil Class In Distilled Water In Sodium Formate So]Lution No. 1 No. 2 No. 3 No. 4 Average No. 1 No. 2 No. 3 NO. 4 Average Olive Oil I I 99 93 66 66 81 92 99 106 102 100 Olive Oil II I 93 123 101 97 104 131 131 131 138 133 Expressed Almond Oil I 120 131 135 127 128 175 168 157 168 167 Peach Kernel Oil I 86 93 101 79 89 150 142 150 157 150 Castor Oil III 00 00 00 00 00 135 120 131 135 130 Lard Oil IV 161 203 136 158 164 220 220 242 200 220 Cottonseed Oil V 99 99 99 92 96 158 142 175 158 158 Sesame Oil V 131 112 131 112 121 187 187 179 179 183 Linseed Oil VI 160 160 212 219 188 146 166 226 198 182 Cod Liver Oil VII 105 112 120 112 112 318 284 322 287 304 Mineral Oil 73 53 53 60 60 71 64 66 86 72 TABLE X Micrograxns of Peroxides Produced by a Mixture of O.76 Grains of Zinc Oxide (Merck) and 2.12 Grams of Oil after 30 Minutes of Ultraviolet Irradiation

Oil Class In Distilled Water In Sodium Formate Solution No. 1 No. 2 No. 3 No. 4 Average No. 1 No. 2 No. 3 No. 4 Average

Olive Oil I I 127 141 141 156 141 266 287 266 286 276 Olive Oil II I 153 150 138 131 143 561 587 538 542 556 Expressed Almond Oil I 183 216 187 216 201 3,983 3,777 3,740 3,983 3,871 Peach Kernel I 194 198 206 187 196 344 344 337 355 345 Castor Oil III 150 168 165 180 166 224 232 224 232 228 Lard Oil IV 136 177 145 136 149 194 226 200 204 206 Cottonseed Oil V 104 106 117 104 108 548 612 518 510 547 Sesame Oil V 168 176 168 180 173 643 651 617 617 632 Linseed Oil VI 146 140 140 126 138 371 385 399 315 368 Cod Liver Oil VII 243 280 269 224 254 374 339 339 374 357 Mineral Oil 884 884 898 909 894 6 ,7 0 0 6 ,9 0 2 6 ,9 8 07,015 6,899

-fr ro TABLE XI Micrograms of Peroxides Produced by a Mixture of 0.76 Grams of Zinc Oxide (Merck), 2.12 Grams of Oil, and 2.12 ml. of Calcium Hydroxide Solution after 30 Minutes of Ultraviolet Irradi- tion

Oil Class In Distilled Water In Sodium Formate So ILution No. 1 No. 2 No. 3 No. 4 Average No. 1 No. 2 No. 3 No. 4 Average Olive Oil I I 116 150 120 136 131 1,464 1,669 1,704 1,697 1,634 Olive Oil II I 269 299 251 281 275 2,147 2,244 2,233 2,150 2,194 Expressed Almond Oil I 367 426 367 374 384 3,085 3,321 3,321 3,112 3,210 Peach Kernel Oil I 153 198 203 187 188 1,515 1,590 1,507 1,518 1,533 Castor Oil III 135 131 120 135 130 490 504 531 486 503 Lard Oil IV 123 132 113 120 122 207 232 152 194 196 Cottonseed Oil V 76 86 99 99 90 980 871 814 798 866 Sesame Oil V 542 561 561 561 555 3,048 3,952 3,052 2,992 3,036 Linseed Oil VI 187 183 166 166 175 216 226 200 229 218

Cod Liver Oil VII 172 206 161 187 181 1,141 1,197 1 ,1 8 6 1,204 1 ,1 8 2 Mineral Oil 686 693 679 707 691 3,715 3,727 3,876 3,682 3 ,2 8 6

-pr

Oil Class In Distilled Water In Sodium Formate Solution No. 1 No. 2 No. 3 No. 4 Average No. 1 No. 2 No. 3 No. 4 Average Olive Oil I I 97 97 90 106 98 210 194 178 216 200 Cottonseed Oil V 119 113 145 171 137 507 630 410 662 552 Mineral Oil 297 307 281 323 320 2,041 2,412 1,97022,179 2,179

TABLE XIII Micrograms of Peroxides Produced by a Mixture of O .7 6 Grams of Zinc Oxide (Zoco), 2.12 Grams of Oil, and 2.12 ml. of Calcium Hydroxide Solution after 30 Minutes of Ultraviolet Irradiation

Oil Class In Distilled Water In Sodium Formate Solution No. 1 No. 2 No. 3 No. 4 Average No. 1 No. 2 No. 3 No. 4 Average Olive Oil I I 127 120 129 150 132 507 542 501 581 533 Cottonseed Oil V 165 200 165 203 183 339 354 305 357 339 Mineral Oil 258 246 258 258 255 807 856 717 824 801

4=- -Pr TABLE XIV Micrograms of Peroxides Produced by a Mixture of 0.38 Grams of Zinc Oxide (Merck), 0.38 Grams

Oil Class In Distilled Water In Sodium FormateSo]Lution No. 1 No. 2 No. 3 No. 4 Average No. 1 No. 2 No. 3 No. 4 Average Olive Oil I I 97 112 112 105 107 288 299 344 325 314 Expressed Almond Oil I 168 176 187 187 180 2,169 2,169 2,102 2,102 2,152 Cottonseed Oil V 131 168 150 150 130 1,877 1,945 1,859 1,773 1,864 Mineral Oil 494 471 449 486 475 5,348 5,385 5,385 5,236 5,338

TABLE XV Micrograms of Peroxides Produced by a Mixture of 0.38 Grams of Zinc Oxide (Merck), 0.38 Grams Calamine*, 2.12 Grams of Oil, and 2.12 ml. of Calcium Hydroxide Solution after 30 Minutes of ______- ______Ultraviolet Irradiation______

Oil Class In Distilled Water In Sodium Formate Solution No. 1 No. 2 No. 3 No. 4 Average No. 1 No. 2 No. 3 No. 4 Average Olive Oil I I 150 150 157 161 155 2,356 2,356 2,281 2,356 2,337 Expressed Almond Oil I 812 830 830 729 800 2,319 2,304 2,319 2,225 2,292 Cottonseed Oil V 299 299 262 269 282 1,533 1,496 1,496 1,496 1,505 Mineral Oil 374 355 363 374 367 3,568 3,590 3,441 3,261 3,465 * Calamine: See "]Materials," Page

VJ1-fr 46 solution of sodium formate. The amount of each sample irradiated was equivalent to the amount of Mineral Oil in a 5 gram sample of the basic formula (See page 31). The re­ sults are given in Table XVI, page 47. To the 3 Mineral Oil squalene samples, Calcium Hydrox­ ide Solution was added. Likewise, Zinc Oxide (Merck) was added. Pour (4) samples of each concentration with the Calcium Hydroxide Solution and with the Zinc Oxide (Merck) were irradiated as previously. The results are given for the Calcium Hydroxide-Mineral Oil irradiation in Table ;2CfIIJ,page 48. For the Zinc Oxide-MLneral Oil irradiations, see Table XVII, page 47. Squalene was added directly to Olive Oil I, Cottonseed Oil, and linseed Oil in a 1 per cent concentration, V/V. The oils were irradiated alone and in combination with Zinc Oxide (Merck) and with Calcium Hydroxide Solution. The re­ sults of the irradiation of the oils separately are given in Table XVI, page 47. The results for the Zinc Oxide-Oil irradiations are given in Table XVII, page 47. The results for the irradiation of the emulsions are given in Table XVIII, page 48. Pour (4) samples of each Zinc Oxide-Liniment were irr- diated in 100 ml. of distilled water and in 100 ml. of 0.2 M sodium formate solution. The results are given in Table XIX, page 49. 47 TABLE XVI Micrograms of Peroxides Produced by 2.12 Grams of Oil Con- tainink Squalene

Squalene Oil Class Per cent In Sodium Formate Solution Added No. 1 No. 2 No. 3 No.4 Average

Olive Oil I I 1 200 242 204 220 217 Cottonseed Oil V 1 200 291 194 252 234 Linseed Oil VI 1 307 307 291 300 301 Mineral Oil 0.5 133 166 150 156 151 Mineral Oil 1 146 180 143 189 165 Mineral Oil 2 133 156 123 133 136

TABLE XVII Micrograms of Peroxides Produced by O .7 6 Grams of Zinc Oxide (Merck) and 2.12 Grams of Oil Containing Squalene after 30 ______Minutes of Ultraviolet Irradiation______Squalene Oil Class Per cent In Sodium Formate Solution CM s 0 Added No. 1 • No. 3 No. 4 Average

Olive Oil I I 1 452 420 339 426 409 Cottonseed Oil V 1 2,358 1,922 2,277 2,413 2,243 Linseed Oil VI 1 268 317 246 246 269 Mineral Oil 0.5 6,349 6,515 5,890 6,402 6,289

Mineral Oil 1 7,113 6,881 6,236 6,389 6 ,6 5 5 Mineral Oil 2 5,937 5,604 5,631 5,385 5,639 48

TABLE XVIII

Micrograms of Peroxides Produced by 2 .1 2 Grams of Oil Con­ taining Squalene and 2 .1 2 ml. of Calcium Hydroxide Solution after 30 Minutes of Ultraviolet Irradiation

Squalene Oil Class Per cent In Sodium Formate Solution Added No. 1 No. 2 No. 3 No. 4 Averaj

Olive Oil I I 1 216 187 200 204 202 Cottonseed V 1 258 242 242 226 242 Oil Inseed Oil IV 1 339 355 307 291 323 Mineral Oil 0.5 123 156 106 150 134 Mineral Oil 1 133 176 133 146 147 Mineral Oil 2 90 116 93 100 100 TABLE XIX Micrograms of Peroxides Produced by O .76 Grams of Zinc Oxide (Merck), 2.12 Grams of Oil Con­ taining Squalene, and 2.12 ml. of Calcium Hydroxide Solution after 30 Minutes of Ultraviolet Inadiation Squa Oil Class lene Per Cent In Distilled Water In Sodium Formate Solution No. 1 No. 2 No. 3 No. 4 Average No. 1 No. 2 No. 3 No. # Average Olive Oil I I 1 325 367 337 337 342 1,659 1,883 1,673 1,938 1 ,7 8 8 Cottonseed Oil V 1 224 224 232 250 232 2 ,6 1 6 2,487 2,923 2,713 2,685

Linseed Oil VI 1 232 243 243 243 240 371 355 265 271 316 Mineral Oil 0.5 4,195 4,860 3,809 4,202 4,267 Mineral Oil 1 692 :: 684 699 699 694 4,707 4,773 4,368 4,208 4,514 Mineral Oil 2 4,833 5,372 4,368 4,906 4 ,8 7 0

VO- t r 50 IRRADIATION WITH HUMAN PLASMA Quantities of dried human plasam equivalent to 1 / 1 6, 1/4 and 1 ml. of human plasma were added directly to 100 ml. of ©.2 M sodium formate solution containing 5 gram samples of a Mineral Oil-Zinc Oxide-Calicum Hyeroxide emulsion. The results of irradiation are given in Table XX.

TABLE XX Micrograms of Peroxides Produced by a Zinc Oxide-Mineral Oil-Calcium Hydroxide Bnulsion in 100 ml. of 0.2 M Sodium Formate Solution after the Addition ______of Human Plasma______Amount of Plasma Added Sample to Each Sample No. 1 No. 2 No. 3 No. 4 Average

None 3,715 3,727 3,876 3 ,6 8 2 3,750 1 /1 6 ml. 2,067 2,112 1,906 1,873 1,990 1/4 ml. 2,022 1,699 1,938 1,550 1,802

1 ml. 1,663 1,638 1,356 1 ,6 1 5 1,568 DISCUSSION To quantitatively measure the production of peroxides by photosensitized zinc oxide in pharmaceuticals containing lipid materials, it seemed desirable to develop a means by which maximum production with minimum destruction could be attained. Preliminary investigation showed that 100 ml. of irradiating medium in a 800 ml. Pyrex beaker would permit the best utilization of available irradiating space. Suffi­ cient volume of irradiating medium was also desirable to permit assay of a 50 ml. aliquot (5). Increasing the quan­ tity of irradiating medium to 200 ml. or reducing the volume by one half did not significantly effect the amount of peroxide produced.

Reese (5 1), Mathias (3 2 ), and Minardi (34<) employed magnetic stirrers for agitating zinc oxide-water suspensions. With the introduction of water in oil emulsions, the magne­ tized stirring bars were not satisfactory since the stirring bars became coated with emulsion and ceased to move. To overcome this difficulty, a Cenco Stirrer Variable Speed motor mounted in the top of the irradiating chamber was uti­ lized as the power source. Through a series of pulleys, it became possible to agitate four samples at the same rate of speed during irradiation. The quantities of peroxides produced by irradiation of

Zinc Oxide (Merck) in distilled water for 30 minutes were in order of 700 micrograms (See Table VII, page 3 9). Such 52 quantities are measurable by the method of Kolthoff (29) as modified by Chari and Quershi (5). Chari and Quershi (5) employed glycerol and phenol as organic "stabilizer^* in concentration of 0.1 M. G a m (17) employed sodium formate in a 0 .2 M concentration as an or­ ganic "additive." A 0.2 M solution is a neutral solution. Acetanilid was also reported by Chari and Quershi (5) as an organic stabilizers. In addition, acetanilid is used com- merically to stabilize hydrogen peroxide solutions (7). The latter two were comparatively studied by irradia­ tion of 0.76 grams of Zinc Oxide (Merck) in a 0.2 M solu­ tion of sodium formate and in a 0.01 M solution of acetanilid. Measurable amounts of peroxides produced with­ out Zinc Oxide in 100 ml. of distilled water after 30 min­ utes of ultraviolet irradiation averaged 74 micrograms; in 100 ml. of 0.2 M sodium formate solution, the average was 142 micrograms; in 100 ml. of 0.01 M acetanilid solution,

the average was 85 micrograms of peroxides (See Table VI,

page 3 7). For a series of irradiations containing 0 .7 6 grams of Zinc Oxide (Merck) in 0.01 M acetanilid medium, the average was 2,175 micrograms of measurable peroxides. In 100 ml. of 0.2 M sodium formate solution with the same amount of Zinc Oxide (Merck), the peroxide production averaged 7*826 micrograms (See Table VII, page 39). These amounts repre­ sent significant increases in the quantity of measurable 53 peroxides as a results of the addition of the organic AdditivesSince sodium formate gave the greatest in­ crease, it was selected in preference to acetanilid. The Irradiation time was 30 minutes. This was suffici­ ent time to permit the production of measurable quantities of peroxides by the photosensitized zinc oxide (See Table VII, page 39.

IRRADIATION OP THE OILS Irradiation of the fixed oils for 30 minutes in either of the irradiating media produced measurable quantities of peroxides (See Table VIII, page 40)l An examination of these tables show the oils in different order of increasing perox­ ide production for the two Irradiating media (See Table XXII, page 6£). The reasons for this have not been determined. Oxidation of fats produce a variety of oxidation prod­ ucts (12). These include peroxides, aldehydes, ketones, acids, water, carbon dioxide, hydroxy acids, hydrogen and pdymerized fats. In all probability, the results (See Table VIII, page 40) represent only the peroxides that are water soluble. It is possible to produce by the oxidation of fats peroxides that are water insoluble (54). Most vegetable fats contain antioxidants small concentrations of which re­ tard oxidation rather efficiently. Animal fats contain little natural protection of this kind (12). Therefor^, these values are in excess of those necessary to oxidize the natural antioxidants present. 54 Among the many natural antioxidants occurring in vege­ table lipids are the tocopherols. Refining, bleaching, hydrogenation and deodorization processes ordinarily do not remove much of the tocopherols (12). The tocopherol content of Olive Oil has been given at 0.02 per cent, Castor Oil at 0.05 per cent, Cottonseed Oil at 0.09 per cent, Sesame Oil at 0.06 per cent, and Linseed Oil at 0.1 per cent (12). Oxidation of fats is accelerated by light, its rate being dependent upon the degree of unsaturation and structure of the fatty acids present in the fat (12). Parmer and Sutton (13) showed that methyl oleate hydroperoxide was formed when methyl oleate was partially oxidized at 35° C. under the catalytic influence of ultraviolet light. The purified peroxidic product contained the same amount of unsaturation as was present in the original methyl oleate. Considerable amounts of hydroperoxides can also be found in methyl linoleate even when oxidized at low temperatures with or without light as a catalyst. The lodometric method of assay does not differentiate between hydrogen peroxides and hydroperoxides. Moreover, peroxides can not be detected until all of the antioxidants are oxidized (2). No information was av&ilable as to whether antioxidants had been added to the oils over and above the natural occurring antioxidants. However, the U.S.P., XIV, permits the addition of tocopherols to Liquid Petrolatum in quantities not exceeding 10 parts per million for purpose 55 of stabilization (4l). Since only the filtrate of the irradiated samples were assayed for peroxides, the assays are only a measure of the water soluble peroxides and do not take into account perox­ ides possibly formed but insoluble in water. Subsequent studies will be necessary to determine if any peroxides re­ mained in the oil phase.

IRRADIATION OF OILS WITH CALCIUM HYDROXIDE SOLUTION This phase consisted essentially of the irradiation of emulsions of the oils with Calcium Hydroxide Solution. Since emulsification produces no change in the over-all structure of the unsaturated fatty acid radicles and since the anti- oxidants are not affected, results of peroxide formation should approach those obtained by irradiation of the oils alone. However, the increased alkalinity of the mixture as a result of the addition of the Calcium Hydroxide Solution could result in an increased rate of photodestruction of the peroxides, since it is generally agreed that stability de­ creases with a rise in pH values (33). Comparison of the peroxides produced by irradiation of the emulsions with the peroxides produced by irradiation of the oils alone (See Table XXI, page66 ) reveals no signifi­ cant increase or decrease. While the amounts produced in the sodium formate solution are generally less, the order of the oils with respect to the amount of peroxides pro- duced remain identical (See Table XXII, page *6 7). Some rear­ rangement of the order occurred with distilled water as the irradiating medium. This change in position was evident with the non-drying vegetable oils, Expressed Almond Oil and Peach Kernel Oil. Expressed Almond Oil changed from the 7th position to the 10th position when Calcium Hydroxide Solution was added. Peach Kernel Oil changed from the 9th to the 4th position with the addition of Calcium Hydroxide Solution. The reasons for this has not been determined. A rpacroscopic evaluation of the emulsions formed by the addition of Calcium Hydroxide Solution to the oils showed a wide degree of dispersion, ranging from coarse emulsions to fine emulsions. While the free fatty acids present in the oil react with Ca++ and more slowly saponify some of the esters to form the emulsifier (7)# the amount of free fatty acid initially present (See Table IV, page 29) was not an index to the degree of dispersion obtained. Other ingredients in the oil in addition to the free fatty acids must contribute to the formation of the emulsion. Expressed Almond Oil with an acid value of 0.09 (See Table IV, page 29) gave the finest degree of dispersion. Castor Oil with an acid value of 1.95 showed a very coarse dispersion. Cottonseed Oil was comparable to Olive Oil II, acid values of 0.04 and 0.60 respectively. Olive Oil I gave a finer dispersion than Olive Oil II. Linseed Oil with an acid value of 4.80 was comparable with Olive Oil I with an 57 acid value of 0.97. Peach Kernel Oil gave a fairly coarse dispersion while Sesame Oil was intermediate between Peach Kernel Oil and Castor Oil. Lard Oil and Cod Liver Oil both gave very find dispersions, comparable to Linseed Oil. The fatty glycerides undergo saponification with aque­ ous alkali to form soap and glycerol. This results in a cleavage at the ester group with the formation of the alkyl and acyl derivatives. The range of reaction rates of esters are quite varied (12) and could account for the differences noted in the degree of dispersion of the emulsions. Reac­ tion rates available do not include data pertinent to this phase of the study. Mineral Oil when mixed with Zinc Oxide and Calcium Hydroxide Solution yielded an oil in water emulsion. All other emulsions formed were of the water in oil type.

IRRADIATION OP OILS WITH ZINC OXIDE (MERCK) The irradiation of 0.76 grams of Zinc Oxide (Merck), an equivalent to the amount present in a 5 gram sample of the basic formula, produced 719 micrograms of peroxide when ir­ radiated in distilled water (See Table VII, page 39) and

7 ,8 2 6 micrograms of peroxide when irradiated in sodium formate solution. Mineral Oil mixed with the same amount of Zinc Oxide in the two irradiating media yielded 894 micro­ grams and 6 ,8 9 9micrograms of peroxide (See Table X, page 42). With the substitution of the other oils, considerable reduc­ tion in the amount of measurable peroxides resulted (See Table X, page 42). Measurable peroxides were less than 30 per cent of that obtained from Zinc Oxide-Mineral Oil ir­ radiated in distilled water. With the exception of Expressed Almond Oil, measurable peroxides were less than 10 per cent of that obtained from Zinc Oxide-Mineral Oil irradiated in sodium formate solution. A given quantity of zinc oxide when activated by ul­ traviolet light can catalyze the production of a given quantity of hydrogen peroxide (52). If these two factors are constant, the quantity of hydrogen peroxide produced will likewise by constant unless the role of the catalyst is in some way altered by the addition of a thii>d substance. To determine the effect of varying quantities of zinc oxide on the amount of peroxides, the following experiment was performed: Thirty eight hundredth of a gram (0.38), 0 .7 6 gram, and 1.42 gram of Zinc Oxide (Merck) were irradi­ ated in a 0.2 M sodium formate solution. These gave values of 6,6 8 0, 7,8 2 6, and 8 ,6 7 0micrograms of peroxide respect­ ively. These values show a gradual increase in the amount of peroxides formed but the amount is not in direct ratio of the zinc oxide used. While the amount of peroxide in­

creased approximately 30 per cent, the amount of zinc oxide

used had increased 300 per cent. The addition of Mineral Oil to Zinc Oxide had no marked effect upon the quantity of peroxide produced by photosensi- tized Zinc Oxide as compared to the fixed oils. While Zinc

Oxide with Mineral Oil produced 894 micrograms of peroxide when irradiated in distilled water compared to 719 micro­ grams without Mineral Oil, Zinc Oxide with the fixed oils gave results significantly lower (See Table X, page 42). As previously stated, a certain quantity of zinc oxide when photosensitized can catalyze the production of a specific quantity of peroxide in a specified time. If this same quantity of zinc oxide was photosensitized in the presence of fixed oils, the same quantity of peroxides should be measurable providing the ingredients of the fixed oil did not catalyze the decomposition of peroxides or physically or chemically react with the zinc oxide. Comparing the peroxide produced when the fixed oils were added to zinc oxide with Mineral Oil added to zinc oxide as maximum production possible under the method of irradiation in distilled water, the quantities of peroxide produced in the zinc oxide oil mixtures were less than 254 micrograms

(See Table X, page 42) as compared to 894 micrograms for the Zinc Oxide-Mineral Oil mixture. For similar irradiations but employing sodium formate solution, Expressed Almond Oil produced 3,871 micrograms. The other Zinc Oxide-Oil mix­ tures, however, produced less than 632 micrograms. Zinc Oxide-Mineral Oil under identical conditions of irradiation produced 6,899 micrograms. If such catalytic material is present, the amounts ap- 60 parently vary with the different oils and with different grades of the same oil. The results also indicate that such catalytic substances are not present in Mineral Oil (Com­ pare Tables VII and X, pages 39 and 42). Oils recovered after irradiation of Zinc Oxide-Olive Oil I and Zinc Oxide-Cottonseed Oil mixtures by chloroform extraction showed the same iodine number, saponification number and refractive index as the initial oil. In addition, a quantity of Zinc Oxide equivalent to the amount initially incorporated was recovered from a Zinc Oxide-Olive Oil I mixture. A quantity of Mineral Oil, Olive Oil I, and Cottonseed Oil equivalent to the amount present in the basic formula was added to 100 ml. of a 0.003 per cent solution of hydro­ gen peroxide in water. These mixtures were shaken on a mechanical shaker for 30 minutes after which time an aliquot sample of the filtrate was checked for hydrogen peroxide content. There was no loss of hydrogen peroxide. The same oils were likewise exposed to a 0.015 per cent concentration of hydrogen peroxide and shaken on the mechanical shaker. Again, there was no loss of hydrogen peroxide. If a catalytic substance were present, a decrease in peroxide content should have resulted. Since this was car­ ried out in light resistant prescription squares, two points may be significant. Sufficent time may not have 61 elapsed, or the catalytic substance Is light activated. This phase requires additional study. The arrangement of the oils in the order of the quan­ tity of peroxides produced were significantly altered (See Table XXII, pagetf5’7). This was most evident with the Zinc Oxide-Mineral Oil mixtures. The quantities of peroxides produced with and without zinc oxide indicates no inhibition by the Mineral Oil to the catalytic production of peroxides by zinc oxide. The pH of the filtrate from a slurry of O .7 6 grams of Zinc Oxide (Merck) and distilled water before irradiation was 7.3. After irradiation the pH of the filtrates aver­ aged 7.35. Seven hundred and nineteen (719) micrograms of measurable peroxides were produced. The pH of a slurry of a Zinc Oxide-Mineral Oil mixture before irradiation was 7 .6 and the pH of the filtrates after irradiation averaged,7.4. Eight hundred and ninety four (894) mocrograms of peroxide were produced. The filtrate from a slurry of 0 .7 6 grams of Zinc Oxide (Merck) in sodium formate solution was 8.1 before irradia­ tion and averaged 8 .7 after irradiation with 7 ,8 2 6 micro­ grams of measurable peroxides produced. The pH of a slurry of Zinc Oxide-Mineral Oil before irradiation was 8.2 and after irradiation averaged 8 .7 with 6 ,8 9 9micrograms of peroxide produced. Employing a Zinc Oxide-Olive Oil mixture in distilled 62 water, the pH before irradiation was 7.3 After irradiation, the average pH was 7.0 with 141 micrograms of peroxide pro­ duced. With sodium formate as the irradiating medium, the pH of a mixture was 7*9 before irradiation. The average pH of the filtrate after irradiation was 7.4 with 276 micrograms of peroxide produced.

Peroxide production of the order of 7 to 8 thousand micrograms of peroxide gave a rise in pH values while irra­ diations producing less than 1000 micrograms of peroxide gave pH values lower than the pH of the initial suspensions. The formate ion is oxidized to carbonate (52) simultaneously with the production of hydrogen peroxide. The significance of pH as it effects the production of peroxides needs additional study.

IRRADIATION OP OILS WITH ZINC OXIDE (MERCK) AND LIME WATER The addition of Calcium Hydroxide Solution to the Zinc Oxide-Mineral Oil mixture reduced measurable amounts of per­ oxides from 894 micrograms to 691 micrograms when irradiated in distilled water, and from 6,899 micrograms to 3>286 micrograms when irradiated in sodium formate solution (See Table XXI, page <66). T&ese are significant changes and can be attributed to increased photodecomposition of peroxides in a more alkaline medium.

Kiss and Lederer (2 7) studied the decomposition of hydrogen peroxide in the presence of the Ca++ and reported 63 that this ion had no effect. However, since these decom­ positions took place under ultraviolet light, the effect of the Ca** tiipon stability could be affected. If a substance is present in the fixed oils that cata­ lyzes photodecomposition of hydrogen peroxide or in some way marks the zinc oxide so that it can no longer be photo­ sensitized by ultraviolet light, it may explain the de­ creased production of measurable peroxides (See Table XXI, page 66 for the assay results). On the other hand, Calcium Hydroxide neutralizes the free fatty acids and slowly sapon­ ifies some of the glycerides. In addition, the Calcium hydroxide physically or chemically could react with the un­ known substance which is responsible for the catalytic ef­ fect or the inactivity of the zinc oxide. Further it might be assumed that the attractive forces between the Ca++ and the unknown substance was greater than between the Zn++ or ZnO and the unknown. Consequently, the catalytic or other effects are removed. The photosensitized zinc oxide pro­ duces peroxides without catalytic decomposition. The re­ sults (See Table XXI, page66 ) suggest such a possibility. The results in Table XXI, page 66 were obtained from samples compounded by adding the zinc oxide to the oil be­ fore emulsification with Calcium Hydroxide Solution. Employ­ ing Olive Oil I, the oil was emulsified before the addition of zinc oxide. There was no difference in the amounts of peroxides produced. However, by doubling the quantity of 64 zinc oxide, measurable peroxides average 5,230 micrograms when irradiated in sodium formate solution as compared to 1,634 micrograms when the amount of zinc oxide in the sam­ ple corresponded to the amount present in a 5 grams sample of the basic formula. A corresponding increase in the amount of zinc oxide in the absence of a fixed oil and ir­ radiated under the same conditions increased the measurable peroxides from 7,684 to 8 ,6 7 0micrograms. By doubling the zinc oxide, there was an increase of 3,596 micrograms of peroxides. This increase might be e x ­ plained on.the basis that sufficient zinc oxide was present to react with the unknown substance and to produce addition­ al amounts of peroxide. Arrangements of the last column in Table XXI, page66 , i.e., the Zinc Oxide-Liniments irradiated in sodium formate solution, against a similar arrangement of the acid values of the oils as determined by assay (See Table IV, page 2 9 ), the highest fatty acid value as 1, the two arrangements are identical except for Cottonseed Oil. While this correlation exist, there seems to be no evidence that the free fatty acid content influences the quantity of peroxides produced. If such was the case, there would be greater similarity in the order of arrangements of the oils with respect to the quantity of peroxides produced (See Table XXII, page 467). Table XXI, page (66, reveals the following points of Interest: 65 1. Peroxides are increased in the Zinc Oxide-Liniments when the samples were irradiated in sodium formate solution. It should be noted that two fixed oils, Expressed Almond Oil and Sesame Oil, in addition to Mineral Oil gave values of over 3,000 micrograms of peroxides. 2. The quantity of peroxides produced in sodium forn­ ate solution by a Zinc Oxide-Expressed Almond Oil mixture was decreased upon the addition of Calcium Hydroxide Solu­ tion. If the quantity produced in a Zinc Oxide-Mineral Oil mixture were assumed to be maximum production, i.e., 6 ,8 9 9 micrograms of peroxide, then the amount produced in a Zinc Oxide-Expressed Almond Oil mixture is approximately 50 per cent of the maximum production. However, with the addition of Calcium Hydroxide Solution, the maximum production with a Zinc Oxide-Mineral Oil mixture as the base was reduced by 50 per cent of its former value. If this represents the maximal amount obtainable in a Zinc Oxide-Liniment, the.addi- tion of Calcium Hydroxide Solution, even though it would neutralize the catalytic substance, would not result in in­ creased peroxide production but an actual decreas due to the increased rate of destruction in a more alkaline medium. 3. Fixed oils with similar iodine numbers, saponifica­ tion numbers, and acid values effect the production of per­ oxides by photosensitized zinc oxide differently. 4. Oil from different sources varies in its composition as it effects the peroxide production by photosensitized Zinc oxide. TABLE XXI Comparative Tabulation of the Micrograms of Peroxides Produced by Ultraviolet Irradiation

Irradiation in Distilled Water Irradiation in Sodium Formate Oil Class Oils Oils + Oils + Oils+ZnO+ Oils Oils + Oils + Oils*ZnO+ Alond Ca(0H)2 ZnO Ca(0H)2 Alone Ca(0H)2 ZnO Ca(0H)2

Olive Oil I I 73 meg. 8l meg. 141 meg. 131 meg. 159 meg. 100 meg. 276 meg. 1,634 me Olive Oil II I 93 104 143 273 189 133 556 2,194 Express Almond Oil I 117 128 201 384 162 167 3,871 3,210 Peach Kernel Oil I 173 89 196 188 166 150 345 1,533 Castor Oil III 71 0 166 130 141 130 228 503 Lard Oil IV 139 164 149 122 236 220 206 196 Cottonseed Oil V 90 96 108 90 105 158 547 866 Sesame Oil V 112 121 173 555 185 183 632 3,036 Linseed Oil VI 181 188 138 175 201 182 368 218 Cod Liver Oil VII 237 112 254 181 289 304 357 1 ,1 8 2 Mineral Oil 67 60 894 691 112 72 6,899 3 ,2 8 6 ZnO = Zinc Oxide (Merck) cr\ o\ TABLE XXII A Systematic Arrangement of the Oils with .Respect to the Quantity of Peroxides Produced Lowest Peroxide Value as Number 1 ______Compare with Table XXI______

Oil Class Irradiation in Distilled Water Irradiation in Sodium Formate Oils Oils + Oils + Oils+ZnO+ Oils Oils + Oils + Oils+ZnO+ Alone Ca(0H)2 ZnO Ca(0H)2 Alone Ca(OH)2 ZnO Ca(0H)2

Olive Oil I I 3 3 3 4 4 4 3 7 Olive Oil II I 5 6 •'4 8 8 8 8 8 Expressed Almond Oil I 7 10 9 9 5 5 10 10 Peach Kernel Oil I 9 4 •. 8 ■ 7 6 6 4 6 Cator Oil III 2 1 6 3 3 3 2 3 Lard Oil IV 8 10 ‘ 5 2 10 10 1 1 Cottonseed Oil V 4 3 1 1 1 1 7 4 Sesame Oil V 6 7 7 10 7 7 9 9 Linseed Oil VI 10 11 •2 5 9 9 6 2 Cod Liver Oil VII 11 9 10 . 6 11 11 5 5 Mineral Oil 1 2 11 11 2 2 11 11 seed Oil with Zoco Zinc Oxide when irradiated in distilled water. This has resulted in a rearrangement of the order for irradiation in distilled water from 1 , 2 , and 3 for Zoco Zinc Oxide to the order of 2, 1, and 3 for Merck Zinc Oxide with respect to the quantity of peroxides produced. This seems to indicate that the catalytic or "masking** sub­ stance present in the oils was not the same in all cases. If the substance present in oils was only catalytic in nature, the quantity of measurable peroxides would be less in the same ration with Cottonseed Oil as with Olive Oil I and with Mineral Oil when mixed with Zoco Zinc Oxide as com­ pared to Merck Zinc Oxide. Since this was not the case the surface characteristics of the zinc oxide appear not only of importance to its photosensitization by ultraviolet light but with its potential reaction with the unknown substance pres­ ent in the oil that are responsible for reduced measurable peroxides,

IRRADIATION OP OILS WITH ZINC OXIDE (ZOCO) AND CALCIUM HYDROXIDE SOLUTION The irradiation of a five gram sample of Zoco Zinc Oxide-Mineral Oil-Calcium Hydroxide Solution emulsion aver­ aged 255 micrograms and 801 micrograms of peroxide in the two irradiating media (See Table XIII, page 44). This was in contrast to 691 micrograms and 3 ,2 8 6 micrograms of 70 peroxide for Merck Zinc Oxide under similar conditions (See Table XI, page 43). When Calcium Hydroxide Solution was added to Zinc Oxide (Merck) Mineral Oil mixtures and irradiation, peroxide production was reduced. A similar reduction in measurable peroxides was noted when Calcium Hydroxide Solution was add­ ed to Zoco Zinc Oxide-Mineral Oil mixtures (See Table XXIII, page 71)• As previously stated, these reduced amounts of measurable peroxides'may be attributed to the greater alkalinity of the Liniments resulting from the addition of Calcium Hydroxide Solution. A comparative evaluation of the other two oils points up the possible difference in brands of Zinc Oxide. Employ­ ing djsbilled water as the irradiating medium, the effect of the addition of Calcium Hydroxide Solution to the Zoco Zinc Oxide-Oil mixtures was greater peroxide production. With Merck Zinc Oxide, the effect was less peroxide production (See Table XXIII, page 71). Comparison of the results of samples of Zoco Zinc Oxide-Olive Oil I with and without Calcium Hydroxide Solution irradiated in sodium formate solu­ tion shows 533 and 200 micrograms of peroxides respectively. Merck Zinc Oxide under similar conditions yielded 1634 micro­ grams and 276 micrograms of peroxides (See Table XXIII, page 71). The difference between the two brands of zinc oxide in these respective Liniments was 1,100 micrograms of peroxide, certainly a significant increase. Since Olive TABLE XXIII Comparative Tabulation of Zinc Oxide (Merck) and Zinc Oxide (Zoco) Micrograins of Peroxides Produced by Ultraviolet Irradiation

Irradiated in Distilled Water Irradiated in Sodium Formate Olive Cottonseed Mineral Olive Cottonseed Mineral Merck:

Zinc Oxide + Oil 141 108 894 276 547 6,899 Liniment 231 90 691 1,634 866 3 ,2 8 6 Zoco:

Zinc Oxide + Oil 98 137 302 200 552 2,179 Liniment 132 183 255 533 339 801 72 Oil I was recognized as U.S.P. grade and since both brands of zinc oxide were recognized as U.S.P. grades, the impor­ tance of the recognition of the physical characteristics of zinc oxide by the U.S.P. was further emphasized.

IRRADIATION OP OILS WITH CALAMINE Calamine as used in this study consisted of 0.5 per cent ferric oxide added to Zinc Oxide (Merd$. The basic formula for Calamine Liniment consists of equal parts of zinc oxide and Calamine, or a final concentration of 0.25 per cent ferric oxide in zinc oxide. When the term "Cala­ mine" is used in this thesis, reference is made to the latter. The quantity of Calamine as used was equivalent to the combined weight of both Calamine and Zinc Oxide as stated in the N. F. formula. The irradiation of a quantity of Calamine equivalent to the amount present in a five gram sample of the basic formula in the irradiating media, distilled water and sodium formate solution, produced 194 micrograms and 5»l6l micro­ grams of peroxide respectively. The same quantity of Merck Zinc Oxide without Ferric Oxide produced 719 micrograms and 7,826 micrograms of peroxide respectively (See Table VII, page 39). Red Ferric Oxide, N.F. IX, contains not less than 90

per cent Fe2 C>3 on the basis of the ignited product. Loss on ignition is not less than 3 per cent. In addition, this 73 product may contain up to 1 per cent water soluble substances (36). Within this mixture, there was some substance that reduces the quantity of measurable peroxides when Red Ferric Oxide was added to Merck Zinc Oxide. Comparative results of Merck Zinc Oxide mixed with oils and Merck Zinc Oxide containing Ferric Oxide (Red) mixed with oils generally Indicated a loss of measurable peroxides in the latter. However, Cottonseed Oil seems to be an ex­ ception. Irradiated Calamine Cottonseed Oil samples in sodium formate solution produced 1,300 micrograms more of peroxide than Merck Zinc Oxide Cottonseed Oil samples (See Table XXIV, page 7^). The effect of the addition of Red Ferric Oxide was apparently determined by the nature of the oil present. The complexity of Ferric Oxide and the oils, however, make a possible explanation difficult.

IRRADIATION OF CALAMINE LINIMENTS The addition of Calcium Hydroxide Solution to Calamine- Mineral Oil-mixtures reduced the quantity of peroxides pro­ duced in irradiated samples. This was also noted when Calcium Hydroxide Solution was added to either Merck or Zoco Zinc Oxide-Mineral Oil mixtures (See Tables XXI and XXIII pages 66 and 71). Irradiation of five gram samples of Calamine Liniment, containing Mineral Oil instead of Olive Oil, produced 367 TABLE XXIV Comparative Tabulation of Zinc Oxide (Merck) and Calamine* Micrograms of Peroxides produced by Ultraviolet Irradiation-. Irradiated in Distilled Water Irradiated in Sodium Formate Expressed Cotton- Expressed Cotton- Olive Almond seed Mineral Olive Almond seed Mineral Merck: Zinc Oxide + Oil 141 201 108 894 276 3,871 547 6,899 Liniment 131 384 90 691 1,634 3 ,2 1 0 866 3,286 Calamine: Calamine + Oil 107 108 150 475 314 2 ,1 5 2 1,864 5 ,3 3 8

Liniment 155 800 282 367 2,337 2,292 1,505 3,465

* Calamine: 0.38 grams of Zinc Oxide (Merck) and 0 .3 8 grams of calamine.

-J ■fcr 75 micrograms and 3,465 micrograms of peroxide as compared to

691 and 3,286 micrograms of peroxide for Merck Zinc Oxide Liniment containing Mineral Oil (See Table XXIV, page 74). When a comparison of the oils included in this phase of the study was made against the same oils when mixed with Merck Zinc Oxide and Zoco Zinc Oxide, a reverse of the pat­ tern of the latter two was in evidence with respect to the effect of Calcium Hydroxide Solution on the quantity of peroxides produced. Ferric Oxide appeared to "depress" the detrimental effects caused by Calcium Hydroxide. How­ ever, this observation needs additional study. It is of interest that the amount of peroxide pro­ duced by irradiation samples of Calamine Mineral Oil Lini­ ment were approximately the same as irradiated samples of Merck Zinc Oxide Liniment when the irradiating medium was sodium formate solution (See Table XXIV, page 74). Samples of Official Calamine Liniment, N.F. IX, produced more per­ oxides by ultraviolet irradiation than samples of either Merck Zinc Oxide Liniment or Zoco Zinc Oxide Liniment (See Tables XXIII and XXIV pages 71 and 74). Official Calamine Liniment, after irradiation in distilled water and sodium

formate solution, assayed 155 micrograms and 2,337 micro- grams of peroxide,; With Merck Zinc Oxide, the values were 131 micrograms and 1,634 micrograms. Zoco Zinc Oxide as­ sayed 132 micrograms and 533 micrograms respectively. 76 IRRADIATION WITH SQUALENE Squalene is a natural occurring highly unsaturated hydrocarbon present in the unsaponifiable fraction of many fixed oils of animal and plant origin (15) . Since Mineral Oil does not contain this ingredient, the addition of squalene to Mineral Oil-Zinc Oxide-Calcium Hydroxide Solu­ tion emulsions would indicate its effect if any on peroxide production. Olive Oil is reported to contain 0.1 to 0.9 per cent squalene (15). Using the higher percentage, squalene was added to Mineral Oil (l per cent V/V) before the addition of either zinc oxide or Calcium Hydroxide Solution. The irra­ diation of the Mineral Oil containing 1 per cent squalene produced 165 micrograms of peroxide (See Table XVI, page 47) as compared to 112 micrograms of peroxide without squalene (See Table VIII, page 40). This was in contrast to

151 micrograms and 136 micrograms of peroxide for Mineral Oil containing squalene in 0.5 per cent and 2.0 per cent V/V (See Table XVI, page 47). The addition of Zinc Oxide (Merck) to the Mineral Oil

containing squalene (1 per cent V/V) produced 6 ,6 5 5micrograms of peroxide as compared to 6,899 micrograms of peroxide with­ out squalene added (See Table XXV, page 77). For the other two trials, 0.5 per cent and 2.0 per cent, the amounts were

6 ,2 8 9 and 5>639 micrograms of peroxide (See Table XVIII, page 48). In the Zinc Oxide-Mineral Oil-Calcium Hydroxide TABLE XXV Comparative Tabulation of the Addition of Squalene Micrograms of Peroxides Produced by Ultraviolet Irradiation

Without Saualene With Saualene Substance Cotton- Lin­ Min­ Cotton­ Lin­ Min­ Irradiated Olive seed seed eral Olive seed seed eral Irradiated in Sodium Formate: (1 per cent squalene added) Oil 159 105 201 112 217 234 361 165 Oil+Calcium hydroxide Solu - tion 100 158 182 72 202 242 323 147 Oil+Zinc Oxide (Merck) 276 547 3686,899 409 2,243 269 6,655 Liniment 1,634 866 218 3,286 1 ,7 8 8 2 ,6 8 5 316 4,514

• Irradiated in Distilled Water • (1 per cent squalene added) Liniment 131 90 175 691 342 232 240 694 78 Solution emulsion with 1 per cent squalene, irradiation produced 4,514 micrograms of peroxide as compared to 3>286 micrograms of peroxide without added squalene (See Table XXV, page 77). The two other trials were 4,267 micrograms and 4,870 micrograms of peroxide respectively (See Table XIX, page 49). Squalene undergoes oxidation with the production of hydroperoxides. The original unsaturation remains essen­ tially unchanged (13). The addition of squalene increased the peroxide produc­ tion in the ultraviolet irradiated Zinc Oxide-Mineral Oil- Cabium Hydroxide Solution samples. Highest production in a Zinc Oxide-Mineral Oil mixture resulted in a concentration of 1 per cent V/V. However, upon the addition of Calcium Hydroxide Solution, the Liniment mixture containing 2 per cent squalene was slightly higher. Calcium Hydroxide probably removed the catalytic inhibitors and permitted higher amounts of measurable peroxides, Squalene was added to Olive Oil I, Cottonseed Oil and Linseed Oil in a concentration of 1 per cent V/V. This addition increased the quantity of measurable peroxides for the three oils (See Table XXV, page 77). The results of the addition of squalene to a Zinc Oxide- Cottonseed Oil mixture was an increase of 1,700 micrograms of peroxides. This significant increase was also evident after the addition of Calcium Hydroxide Solution to the Zinc 79 Oxide-Cottonseed Oil mixture. This increase along with the increase exhibited in the case of Olive Oil I and the de­ crease in the case of Linseed Oil cannot be satisfactorily explained on the basis of present knowledge about these fixed oils. Since squalene is a part of the unsaponifiable fraction and has markedly effected the results, it is possible that other fractions of the unsaponifiable matter oould play a similar role, either increasing or decreasing the photo­ sensitizer role of zinc oxide in pharmaceuticals containing a lipid base. All of the above studies were conducted in sodium for­ mate solution with Zinc Oxide (Merck). Irradiation of the Liniment emulsions in distilled water also showed increases in measurable peroxides. Squalene also has the additive effect of possessing surface active activity. Its presence markedly improved the quality of dispersion and stability of Cottonseed Oil emulsions.

IRRADIATION WITH HUMAN PLASMA The addition of human plasma to the irradiating media containing Merck Zinc Oxide-Mineral Oil-Calcium Hydroxide Solution resulted in a marked decrease in measurable per­ oxides. With the addition of the equivalent of l/l6th of a ml. of human plasma, the quantity of peroxides was re- 80 duced from 3 ,7 5 0micrograms to 1,990 micrograms, a reduction of 47 per cent. The addition of the equivalent of l/4th and 1 ml. of human plasma resulted in reductions of peroxide by 52 per cent and 58 per cent respectively. This phase of the study was added to note the effect of a natural body fluid upon the quantity of measurable peroxides. This reduction may be due to catalytic action from the substance added or oxidation of the added substance.

CONTEMPLATED ADDITIONAL STUDY The importance of pH as it effects the quantity of peroxide production in lipid-zinc oxide pharmaceuticals needs evaluation. Since pH increased with increased per­ oxide production, especially noticeable in sodium formate solution,it may be possible to obtain production curves of peroxides during the irradiation period. If certain constituents in the fixed oils are respon­ sible for the reduction in peroxide production when zinc oxide was photosensitized, removal of these substances should enhance peroxide production. The fixed oils could be fractionated and each fraction studied separately with zinc aside or the fixed oils could be recovered from a zinc oxide-oil mixture by the use of selective organic solvents. Oils recovered with the different solvents could again be mixed with zinc oxide for irradiation studies. The effect of the order of mixing and the quantity of 81 zinc oxide added needs additional study. Present informa­ tion indicates that increased peroxide production can be attained by increasing the quantity of zinc oxide over the present amount in the basic formula. Since squalene, a fraction of the unsaponifiable matter present, possesses surface activity, other fractions may be present that also possess similar activity. SUMMARY 1. The addition of fixed oils markedly reduced the quantity of peroxides produced by photosensitized zinc oxide. There was considerable variation in the amount of reduction among the various fixed oils. No correlation was apparent with respect to the amount of reduction and known constants. Oils within the same class showed a wide divergence of depressant action on zinc oxide. 2. Olive Oil from two different sources varied in the amount of depressant action. 3. The addition of sodium formate to the irradiating media increased the peroxide production by zinc oxide when exposed to ultraviolet light. This increased production was markedly reduced by the addition of fixed oils. 4. The addition of Mineral Oil to zinc oxide did not markedly reduce the production of peroxides by zinc oxide. 5. The addition of Calcium Hydroxide Solution to the oil zinc oxide mixtures increased the quantity of peroxides with some oils and decreased the amounts produced with other oils. 6. Zinc oxide from different sources varied in per­ oxide producing ability. This is in all probability asso­ ciated with the method of manufacture. 7. Calamine Liniment, N.F., which contains ferric oxide, produced a greater quantity of peroxide than the

82 83 same preparation without Calamine. The addition of ferric oxide to the Mineral Oil Liniment, however, reduced the quantity of peroxide. 8. The addition of squalene increased peroxide pro­ duction. 9. The addition of human blood plasma reduced per­ oxide production. 10. The quantity of peroxide produced by Calamine Liniment can be greatly increased through the control of the fixed oil used, the source of the zinc oxide, and the addi­ tion of an additive. REFERENCES 1. Baur, E.. and Neuweiler, C., Helv. Chim Acta, 10:901 (1927). 2. Borden's Review of Nutrition Research, "Fats and Fatty Acids in Nutrition," XV: No. 1 (1954). 3. British Pharmacopoeia, Spottis woode and Co., Grace- church Street, London (1 8 8 5). 4. Ibid., Pharmaceutical Press, London (1953). 5. Chari, C. N., and Qureshi, M., "Photochemical Formation of H0O9 from Water. I. In presence of ZnO," J. Indian Chem. Soc., 21: 97 (1944). 6. Council on Pharmacy and Chemistry, A.M.A., "A Status Report on the External Use of Cod Liver Oil," J.A.M.A., 121:759-61 (1943). 7. Crossen, G. F., and Goldner, F. J., Pharmaceutical Preparations,3rd Edition, Lea and Febiger, Philadel­ phia (1952). 8. Crowe, R. W., and Smyth, C. P., "The Low-frequency Dielectric Properties of Some Symmetrical Mixed Triglycerides in the Solid State,V J. Am. Chem. Soc., 73:2040-45 (1951). 9. The Dispensatory of the United States of American, 24th Edition, J. B. Lippincott Company (1947). 10. Ibid., 22nd Edition, J. B. Lippincott Company (1940). 11. Ibid., 7th Edition, Grigy, Elliot and Co., No. 14 N. Fourth Street, Philadelphia (1847). 12. Eckey, E. W., Vegatable Fats and Oils, American Chemi­ cal Society Monograph Series, No. 123, Reinhold Publishing Corporation, New York (1954). 13. Farmer, E. H., and Sutton, D. A., J. Chem. Soc., 119: 543 (1949). 14. Feitknecht, W., and Haberli, E., Helv. Chim. Acta, 33: 922 (1950). 15. Fitelson. J., "The Occurrence of Squalene in Natural Fats, J. Assoc. Official Agr. Chem., 26:506-511 (1943). 84 85 16. Ibid., ‘'Report on the Analysis of Oils, Pats, and Waxes," J. Assoc. Aofficial Agr. Chem., 28:282- 89 (1945). 17. Garn, P. D., "The Catlytic Activity of Zinc Oxide in the Photochemical Formation of Hydrogen Peroxide," Ph.D. Thesis, The Ohio State University (1952). 18. Goodeve, C. P., Trans Faraday Soc., 33:340 (1937). 19. Goodman, H., “Calamine and Zinc Oxide," J.A.M.A., 129:707 (1945). 2 0 . Goodman, L. and Gilman, A., The Pharmacological Basis of Therapeutics, MacMillan, New York (1942).

2 1 . Greenberg, L. A., and Lester, D., Handbook of Cosmetic Materials, Interscience Publishers, Inc., New York (1954). 22. Handbook of Chemistry and Physics, 34th Edition, Chem­ ical Rubber Publishing Co., Cleveland (1952-53). 23. Harper, D. A., and Hilditch, T,. P., "Component Acids and Glycerides of Partly Hydrogenated Marine Animal Oils (ill)," J. Soc. Chem.Ind., 56:322 (1937). 24. Harry, R. G., Cosmetic Material, Vol. II, London, Leonard Hill (1948). 25. Hilditch, T. P. and Terleski, J. T., "Component Acids and Glycerides of Partly Hydrogenated Maine Animal Oils (II)," J. Soc. Chem. Ind., 56:315 (1937). 2 6 . Horkheimer, P., "Stability of Hydrogen Peroxide Solu­ tions." Phar. Ztg., 80:507 (1935). II 27. Huttig, G., and Goerk, H., Z. Anorg. Allgem. Chem., 231:249 (1937). 2 8 . Kiss, A. and Lederer, R., "The Mechanism of the Cataly­ tic Decomposition of Hydrogen Peroxide Solutions by Metal Ions," Rec. Trav. Chim., 46:453-62 (1927). 29. Kolthoff, I. M., "Iodometric Method for Hydrogen Per­ oxide," Z. Anal. Chem., 60:400 (1921). 30. Luckiesh, M., Application of Germicidal, Erythemal and Infared Energy, D. Van Nostrand Company, Inc., New York (1946). 86 31. Mackenna, R. M. B., Wheatley, V. R., and Wormull, A., "Studies of Sebum. II. Some Constituents of the Unsaponifiable Matter of Human Sebum," Biochem. J., 52:l6l-8 (1952). 32. Mathias, M. C., "The Effect of Various Phenols on the Formation of Hydrogen Peroxide by Zinc Oxide," M. S. Thesis, The Ottib State University (1953). 33. The Merck Index of Chemicals and Drugs, Sixth Edition, Merck & Co., Inc., Rahway, N. J. (1952). 34. Minardi, F. C., "The Effect of Time on the Hydrogen Peroxide Production by Calamine Lotions.V M.S. Thesis, The Ohio State University (1954). 35. Modem Drug Encyclopedia, 5th Edition, Drug Publica­ tions, Inc., New York (1952). 3 6. The National Formulary, Ninth Edition, American Pharma­ ceutical Association, Washirgjon, D.C. (1950). 37. Ibid., Fifth Edition, American Pharmaceutical Associa­ tion, Washington, D. C. (1926).

3 8. Ibid., Fourth Edition, American Pharmaceutical Associa­ tion, Washington, D. C. (1916). 39. Outcault, H. E., "Zinc Oxide," Can. Chem. Process Inds., 27:118 (1943). 40. Pamphlet. General Electric Engineering Division, Lamp Department, Cleveland 12, Ohio. 41. Pharmacopeia of the United Stsbes, Fourteenth Revision, Mack Printing Company, Easton, Pa. (1950). 42. Ibid., Twelfth Revision, Mack Printing Company, Easton, Pa., (1942). 43. Ibid., Tenth Revision, J. B. Lippincott Company, Phil­ adelphia (1926). 44. Ibid., Eighth Revision, P. Blakiston's Sons & Comp. Philadelphia (1905). 4 5. Ibid., Seventh Revision, J. B. Lippincott Company, Philadelphia (1894). 46. Ibid., Sixth Revision, William Wood & Company, New York (I8 8 3). 87 47. Ibid., Fifth Revision. J. B. Lippincott Company, Philadelphia (1 8 7 6). 48. Ibid., Wells and Lilly, Boston (1820). 49. Polano, M. K., Skin Therapeutics, Elsevier Publishing Company, New York (1952). 50. Properties of the Principal Fats, Fatty Oils, Waxes, Fatty Acids, and Their Salts, The Texas Company, New York (1952). 51. Reese, D. R., "Formation of Hydrogen Peroxide i n Cala­ mine Lotions," M.S. Thesis, The Ohio State Univer­ sity (1 9 53). 52. Rubin, T. R., Calvert, J. G., Rankin, G. T. and. MacNevin, W., "Photochemical Synthesis of Hydrogen Peroxide at Zinc Oxide Surfaces," Jr. American Chemical Society, 75:2850-53 (1953). 53. Smith, D. F. and Hawk, C. 0., J. Phys. Chem., 32:415 (1928). 54. Tobolsky, A. V., and Mesrobian, R. B., Organic P e r ­ oxides, Interscience Publishers, Inc., New York (1954). 55. Yamahuzi, K., Nisioeda, N., and Ryusi, R.. Blochem Z., 3 0 3:260 (1939).

5 6. Winter, G., "Photo-chemical Activity of Paint Pigments," Nature, 163:326 (1949). AUTOBIOGRAPHY I, Frederick Claire Blubaugh, was b o m in Fort Scott, Kansas, June 25, 1916. I received my secondary school education in the public schools of Fort Scott, Kansas. My undergraduate training was obtained at the Kansas State Teachers College, Pittsburg, Kansas, from which I received the degree Bachelor of Science in 1938. From The Ohio State University, I received the degree Master of Science in 1941. While in residence at The Ohio State University, I acted inthe capacity of a graduate assistant in the Department of Bacteriology during the years 1939-41. I entered military service in 1941 and was discharged in 1946. From 1946 to 1950 I was employed in the pharmaceuti­ cal industry. In 1950, I returned to The Ohio State University, College of Pharmacy, from which I was graduated in 1953, with the degree Bachelor of Science in Pharmacy. In October, 1953, I received an appointment as an Assistant in the College of Pharmacy, The Ohio State University. For the following year, 1954-55, I received an appointment as an Instructor in the College of Pharmacy. I held this position while completing the requirements for the degree Doctor of Philosophy.