Acqueous Parachlorophenol: Its Toxicity and Antimicrobial Effectiveness

Loyola University Chicago Loyola eCommons

Master's Theses Theses and Dissertations

1969

John W. Harrison Loyola University Chicago

Follow this and additional works at: https://ecommons.luc.edu/luc_theses

Part of the Medicine and Health Sciences Commons

Recommended Citation Harrison, John W., "Acqueous Parachlorophenol: Its Toxicity and Antimicrobial Effectiveness" (1969). Master's Theses. 2367. https://ecommons.luc.edu/luc_theses/2367

This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Master's Theses by an authorized administrator of Loyola eCommons. For more information, please contact [email protected].

I'I'S TOXICITY AND AN'I'IMICROBIAL EFFECTIVENESS

John Wylie Harrison, B.&., D.M.D.

A Thesis Submitted to the Faculty of the Graduate School

of Loyola University in Partial Fulfillment of

the Requirements for the Degree of

Master of Science

June 1969

lftrary · · Loyora University Medical <:enbl Acknowledgements

There are many people who deserve thanks for

their assistance in the myriad of problems which

inevitably arise in a research project of this na

ture. I can only hope that my sincere gratitude for

their efforts has been made personally evident.

I am particularly indebted to B. Franklin Gurney, B.S., M.s •• D.D.S., F.A.C.D., for suggesting certain

ideas which eventually led to the choice of this par

ticular problem as a research project; and to Norman

K. Wood, D.D.S., M.s., Ph.D., who spent so many hours

examining tissue specimens and constructively criti cizing this treatise.

Finally, there are a few whose contributions have

been so great that all expressions of appreciation seem entirely inadequate. They are:

John V. Madonia, DoD.S., PhoD., my re search advisor, who has shown a sincere interest in

every facet of this project and has been available for advice and encouragement whenever it was needed.

It was because of him that this project never seem ed a burdensome task but was, instead, an enriching and enjoyable experience. I can pay him no higher compliment than to say that he is, in the truest

ii sense of the word, a teachero

My wife, Lloyd, and my children, Michael,

Leigh and Kelly, who have displayed an incredible pat ience over the past two years. Their many sacrifices have evidenced their love and devotion. So much was asked, and even more was given.

iii Curriculum Vitae John w. Harrison was born in Cedartown, Georgia on November 29, 19J4. He graduated from Tuskegee ( Alabama ) High School in June 1953 and entered the University of Alabama the following September.

Upon receiving a Batchelor of Science degree in 1957, he entered the University of Alabama School of Dentistry and graduated in May 1961 with the degree of Doctor of Dental Medicine. In August of 1961, he entered the United States

Army and served tours in Europe and the United Stat es prior to entering, in 1967, a two year graduate program in the Department of Oral Biology of Loy ola University School of Dentistry leading to the degree of Master of Science and a postg~aduate cer tificate in endodontics.

iv Table of Contents

Chapter: Page: 1. Introduction • • 0 • • • • • • • • 1

2. Review of the Literature • • • • • • • 4

A. 1830-1887 • • • • • • • • • 0 • • 4

B. 1888-1909 • • • • • • • • • • • 0 . 6

C. 1910-1939 o o • • • • • 0 0 • 0 • 012

D. 1940-1963 o o •• 0 • • • 0 0 • • .19

E. 1963-1968 o • • • • • • • 0 0 0 • .27

F. 1969 - • • • • • • 0 . • • • • .Jl 3. Methods and Materials • 0 • • • • • .33 A. Toxicity of Aqueous Parachlorophe-

nol •••••••••••••••• 33 B. Antimicrobial Effectiveness • ••• 38

c. Neutralization 0 • • • • • • 0 .43

4. Results o • o • 0 • • 0 • 0 • 0 • 0 A. Toxicity • • • • 0 • • • 0 0 • •

B. Antimicrobial Effectiveness o • • .56

c. Neutralization • • • 0 • • 0 0 • .58

5. Discussion 0 • 0 • • • 0 • • • .61

60 Summary • o • 0 0 0 0 0 • • 0 0 • • .75 7. Bibliography • • • • • • • • • • .76

8. Appendix •• 0 0 • • • • • • • • .92

v 1 CHAPTER I

INTRODUCTION Parachlorophenol, N. F., is commonly used in endodontic therapy in a thirty-five percent concentration in a vehicle of camphor. This mixture is kno'n as camphornted parachlo ropheno1 Cl6l), and is a highly toxic(l20), though effec tive(lo9,i21>, intracanal antimicrobial agent. r Crunphorated parachlorophenol enjoys probably the high est popularity(ll2 ) of all the root canal medications vdth its prime advantages of good clinical effectiveness and ease o:r handling. The question arises: Is it possible to reduce the toxicity of this drug without greatly diminish ing its disinfecting power and effectiveness in root canal therapy? Two factors are of prime importance in answering this question, the vehicle and the concentration of parachlorophenol. The antimicrobial activity of crunphorated parachloro phenol is due to the parachlorophenol (162 ) and the camphor serves merely as a vehicle. The bacterial action of parach lorophenol varies gre2tly with the solvent. In solvents of low dielectric constant, such as alcohol, acetone, ether, xylene, chloroform, etc., parachlorophenol is alleged to exhibit little antiseptic action; while in solvents of high 2 dielectric constant, such as glycerin, nitrobenzene and water, it reportedly exhibits marked antimicrobial activity.(163) .In addition to the above consideration, it has never been established that thirty-five percent is the optimum antimicrobial concentration of parachlorophenol. Tnere is for every disinfectant a maximum concentration beyond which, according to the law of diminishing returns, a proportionate increase in antimicrobial effectiveness is not evidenced. The irritating potential of these agents does, however, normally increase with the concentration. Therefore, as the vehicle camphor possesses toxic qualities<164 ), it would also seem logical to change the vehicle in an attempt to reduce toxicity. Also, as there is no valid scientific evidence that a thirty-five percent concentration of parachlorophenol is necessary for disin fecting purposes, it would seem logical to test microbio logically a reduced concentration. An aqueous vehicle presents the opportunity to test a non-toxic vehicle and a greatly reduced concentration of parachlorophenol. The purposes of this investigation are: (I) To study the toxicity of aqueous pa.rachlorophenol and compare with that of other endodontic antimicrobial agents and (II) To study the antimicrobial effectiveness of aqueous para chlorophenol. CHAPTER II REVIEW OF THE LIT3RATURE

The number of root canal medicaments suggested, tested and used in the past century are legion. Soon after the inau8uration of the antiseptic era in 1867, by Lister, Cl) dentistry adopted his methods for the antimicrobial treatment of root canals in an empiric manner and with little regard to the protection of the periapical tissues. The history of root canal antiseptics and disinfectants is closely related to that of the evolution of the specialty of endodontics and even of dentistry itself. 1830 - 1887 During the early years, arsenic trioxide, phenol and creosote were conunonly used in canals as caustics, obtun dents and, unwittingly, as antiseptics.( 2) Arsenic was the most popular agent used and had been suggested for the purpose of destroying the pulp by Spooner in 1836.(3) The first recorded root canal filling took place "about 1830"(4) when Dr. Edward Hudson of Philadelphia filled the canals of two maxillary incisors. Other records indicate that he had been filling canals prior to 1830 1 perhaps as early as 1809.(5) But, as a rule, no effort was made during this period to remove the pulps or to fill the canals,, and restorations were placed over necrotic pulps with little regard to the consequences. In addition to devitalization of the pulp with caustic drugs, some pulps were 11 knoclced out" by driving wooden points into the root canals. A single blow crushed the pulp tissue and one can only speculate at the number of fractured roots which must have resulted from this practice. Also in vogue during this period was the practice of stuffing the root canals with cotton saturated with creo sote following pulp devitalization. The purpose of the creosote was to serve as a preservative. ~1hite{ 6 ) in 1864 stated that this method has been used for "at least twenty five years" and the creosoted cotton was packed in the canal and left as a root filling. In 1857,, rlatt(7 ) recommended filling root canals with gutta percha,, and more and more practitioners began to condemn the practice of leaving the pulp in the canal to "disintegrate and rot, proving a source of much offense.n(8) The first warning of possible ill-effects which may be caused by caustic drugs in the canal was issued by McMurtrie{S) in 1861. Six years later, McLain(lO) criti- cized the use of creosoted cotton for filling canals and was the first to suggest the possibility of using anti septics in the canal, stating that nshould they do no good, their presence could not possibly exert any bad influence." He recommended a grain of iodine dissolved in half an ounce of chloroform, to which is added a dracbm of creosote. This is the first mention in the literature of the use of an antiseptic for antiseptic purposes in the canal.

After 1870, attempts to save teeth irli th necrotic pulps became common practice. Many operators used gold wires or gold foil for filling the canal but, as the years passed, gutta percha crone more and more into general use,(ll) as did the rubber dam which was introduced in 1864 by Barnum. (l2)

The period from 1870 to 1887 was a period of trial and error and, except for the work of McQuillen(13), Rich(l4 ) and Raymond(lS) in anesthesia, produced little of interest in endodontics. 1888 - 1909 The most exciting and stimulating period in the history of root canal therapy, and likewise dentistry per se, began with the publication of an article by W. D. Miller in 6 l888.Cl ) This epoch-making paper was the first to call attention to the presence and importance of microorganisms in root canals. Long before William Hunter aroused the laity and the medical and dental professions, Miller stated: " ••• each gangrenous tooth pulp may be in itself a center of infection, or it may serve as a channel through which pathogenic bacteria from the oral cavity may invade the tissue surrounding the point of the root, or even obtain entrance into the circulationo 0 In 1890, Miller presented a second paper(l?) which stressed the importance of the decomposition of the pulp as an irritating factor in the treatment of pulpless teeth. These two papers were followed by a third important treatise in 1891 by G. V. Black(lB) in which he pointed out the difference between 1tinfectious matter" and "septic matter." He emphasized that the products of pulpal putre faction or decomposition, even though containing no micro organisms, may be poisonous to the periapical tissues. These three papers initiated the slow change in den tistry from a technical to a scientific profession, and provided a biological foundation for the treatment of root canals. The importance of removing microorganisms and pulpal rennants from the canal was clearly stated, but, as is the case with most changes, almost a quarter of a 8 century passed before the profession as a whole recognized and accepted these facts.<19 ) The papers of Miller and Black initiated the use of antiseptics in root canals. It was the beginning of a parade of intracanal medican1ents which has lasted some eighty years and continues today.

In 1889 1 Black(20) tested the effectiveness of certain antiseptics and found many of the essential oils to be wor~hless against salivary microorganisms. He also demonstrated the greatly diminished antimicrobial activity of these antiseptics in the presence of albumen. Oil of - . cassia and eucalyptol were recommended for root canal therapy. _Dr. A. w. Harlan(21) in 1891 argued ag~inst the current practice of disinfecting canals with phenol, zinc chloride, sulfuric acid or cresote. He reasoned that these· agents coagulate protein and are therefore self- limiting. Harlan thus was the first to advocate the use of a diffusable root canal antiseptic and suggested the possible use of potassium permanganate, chlorinated soda, sodium fluosilicate and certain essential oils. During this period, little or no thought vms given to the toxic effect on the periapical tissues of agents sealed 9 ·1n the canals. In 1892, a mixture of iodoform, oil of cinnamon and finely powdered, ground coffee was suggested and, a year later, Dr. Emil Schreier(23) of Vienna proposed the use of metallic potassium and sodium nto convert the septic contents of the canal into an aseptic condition ••• tt The pryotechnic display following the insertion of metallic sodium and potassium into the moist canal undoubt- edly created some anxious moments for those subscribing to Schreier's therapy. 24 Cassidy< > suggested the intracanal use of formalin in 1894 and Wooley< 25 ) in 1898 advocated "the most powerful antiseptic agents we can find." Pfeifer,, (2o) also in 1898~ re-emphasized the importance ·or thorough debridet::i.ent, of protection of the tooth with a rubber dam and of the use of a diffusable antiseptic such as Blacks 1-2-3 (oil of cassia,, phenol and oil of gaultheria),, eucalyptol or oil of cassia. If these drugs failed, phenol or zinc chloride were rec om.mended.

It was in 1898 that Dr. A. H. Peck(2s, 29 > reported on the first scientif'ic series of experiments on the irritating potential of antiseptics. Cotton pellets, saturated with the test solution, were applied to the skin of the forearm with a rubber cup, held by adhesive tape, to l.O

prevent evaporation. On the basis of his experiments, Peck recommended for canal disinfection the use of beechwood creosote, oil of cloves, oil of bay, Blacks 1-2-3, and eucalyptol. He rejected the intracanal use of oil of cassia (too toxic), oil of peppermint (odor objectionable), 95% phenol (not a "permanent" antiseptic), oil of myrtle (too toxic), oil of gaultheria (not antiseptic), eugenol (not antiseptic), formalin (too toxic) and oil of cajeput (not antiseptic.) Following Peck's report, G. V. J?lack(30) pleaded with the profession to choose their antiseptics " ••• not with relation to their power as a poison to microbes entirely, but ••• (with) especial reference to their action upon the animal tissue to which they are applied". The importance of Peck's work lies in its initiation of the profession's concern with the toxic properties of root canal antiseptics. Two decades later, these tests were confirmed by Willard at Northwestern University.(31) At the turn of the century, other events occurred which were to have profound effects on the future of root canal therapy. Speaking before the Nev; York Institute of Stomatology 32 in 19011 Dr. T. W. Onderdonk< ) proposed "having a culture --

made :from the cotton dressing ••• and with the aid of the bacteriologist, know when we have secured an aseptic root." This was the rirst suggestion that root canals be cultured. The secrets hidden ·below the gums were revealed ror the rirst time when Dr. Otto Walkofr or Munich rirst used the X-ray ror dental purposes two weeks after its discovery in 1895. In the United States, the roentgen ray was employed in root canal therapy in 1896 by c. Edmund Kells and others soon followed his example.(33 ) This new and important diagnostic aid ca~e into general use shortly before 1910. The first paper in English on the use or Novocaine as a local.. anesthetic was published by Part(34) in 1906, a year after its discovery. Like the X-ray, it also crune into general use about 1910.<5 >

And so it was that in the year 1910, dentistry wa~ slowly progressing, blissf'ully unaware of the explosion which was.shortly to take place. Caustic drugs were being sealed in root canals despite the VTarnings of Peck, Black and others.<19) The heavy reliance placed on the use or powerful and highly toxic intracanal medications, combined vri th the rear with which many dentists approached instru mentation of the canals, lead to countless failures and prolonged, painful treatments. Because the canals ·were not properly debrided and because the increasing use o~ X-rays began to reveal the existence of periapical radio- lucencies, the use of more powerful and penetrating antiseptics seemed logical. This overreliance on powerful drugs complicated an already complicated therapy. It is surprising, when one considers the handicaps under which the early practitioners worked, that endodontics was sue- cessful in maintaining any degree of popularity. Anes thesia and the roentgen ray were still in their ir..fancy; root canal instruments were not readily available; pulpal and periapical pathoses were poorly understood and the in tricate anatomy of the root canals had not been investigated. Unfortunately, with the advent of the roentgen ray and the local anesthetic came the ti1eory o.f focal infection.

1910-1939 The event which rocked the dental profession to its very foundations, and placed the field of endodontics in near suspended animation for almost thirty years, took place in Montreal, Canada, on October 3, 1910. On that date, Dr. William Eunter,<35)a physician, blamed oral foci of infections as the etiological factor in many systemic disorders. This was soon .followed by exten sive laboratory and clinical studies; Frank Billings<35 ) or Chicago, 't7ho investigated the relationship between the diseases of rheumatism and arthritis and the infections of the teeth and tonsils. An associate of Billings, Dr. E. c. Rosenow, gave further impetus to the theory by his observations.<37) Almost immediately, the theory was grasped by the medical profession in the hope that many obscure conditions confronting them might be relieved by removal of localized areas of oral infection. The dental profession, being anxious to cooperate in combatting disease, joined the crusade,(38 ) and the clinical application of the theory of focal infection began with the wholesale extraction of "infected" teeth. With few exceptions,<39- 45> the dental and medical practitioners insisted upon the immediate extraction of all pulpless teeth. Oral infections were routinely con- demned in the literature as the cause of almost every systemic disorder of doubtful origin.<46-51} Nodine< 52) in 1919, listed those systemic disorders - which could be caused by oral infections. They included anemia, hysteria, epilepsy, leukemia, nephritis, lichen planus, arthritis, herpes zoster, melancholia and scores of others. J.4

It remained for research in histology and path ology<53-5s) and in microbiology<57, 5s) to place the focal infection theory in its proper perspective; an event which Ingle(59) terms one of the three greatest improvements in the history of endodontics. Along with the improvement of the X-ray and local anesthesia, ca~e the first comprehensive studies of the anatomy of root canals by Guido Fischer,(60) Callahan,(61) and Hess.<62)

Abraham,<63 ) in 1915, introduced Rhizophor, the fibers of the Piassa Palm impregnated with zinc chloride, creosote, thymol and fonnaldehyde, to be used as both a filling material.. and "in pellet form to sterilize the roots of difficult access." Bauchwitz,C54) in the same year, re commended that a mixture of benzoic acid, boric acid and iodoform be heated over an alcohol flarae and the ascend- ing vapor guided into the root canal. Electrosterilization, using a one percent sodium chloride solution, was introduced in 1917 as a method for obtaining canal sterility.(65) Cullen and Taylor<66 ) demonstrated the irritating / ·,..-- - potential of Dakins.. Solution, but found two other commonly used root canal antiseptics, dichlora.r,1ine-T and chlora- mine-T, to be less toxic. Prinz,C27 > in 1919, listed those drugs and drug com- pounds which had been recon~ended and used in root canal therapy. They included: creosote, parachlorophenol, camphorated phenol, lysol cresol, creolin, beta-naphthol, salicylic acid, hydrogen peroxide, zinc chloride, thymol, eugenol, eucalyptol, Black's 1-2-3, sodium dioxide, formaldehyde and sodium chloride in conjunction with electro sterilization. He also discussed in detail the merits and deficiencies of the more commonly used intracanal antiseptics such as phenol, silver nitrate, mercuric chloride, iodoform, iodine solutions, the essential oils and dichloramine-T. The 11 sight and smell" method of determining when to fill a root canal continued to be used until 1919, when LaRoche(67 ) and Coolidge(68 ) elevated the treatment to a scientific level by employing microbiological tests, as 32 suggested by Onde;donk, < > to determine the sterility of pulpless teeth. Price,<69 ) in 1923, reported on the results of antimicrobial and toxicity studies of certain root canal medications and stated that only phenol, an iodine-creosote solution, formalin, formocresol and dichloramine-T consist- ently gave negative cultures, with the latter being the J.6 most effective, but too toxic for use in the canal. li.mong the drugs tested, but failing to give satisfactory results,, were parachlorophenol,, iodine and sulfuric acid. In 1924,, the L.D. Caulk Company int~oduced OsogenfR)(70) The liquid component of this powder-liquid mixture was used separately as an antiseptic and contained potassium iodohy- ·drargyrate which was hailed as the "most powerful inorganic. germicide knovm." It is interesting to note, however,, that the instructions for its use recommended flooding the canal for three or four minutes with phenol prior to placing the Osogen liquid in the canal. Dr. J. R. Blayney in l924,(7l) and again in 1926,, and 1928,,<72> admonished the dental profession not to ask nature to resist chemical necrosis in addition to infection. He emphasized the dangers of using caustic drugs in canals, of forcing these drugs beyond the apex and of failing to completely remove all organic debris and softened dentin. In 1929,, Coolidge(73) stated that drugs which coae- ulate protein have only a shallow penetration and are not qualified for the treatment of infected canals,, and that low surface tension is of value only if the germicide doesn't precipitate protein. He also warned of the consequences of using caustic drugs in the canals and used 17 laboratory animals to test for tissue toxicity.(74) Eugenol, eucalyptol, phenol, formalin, formocresol and chlora..~ine-T were found to be particularly toxic. Kitchin and HortonC75) in 1931, reported the results of an extensive microbiological study comparing the various root canal medicru.~ents. They ranked them in descend ing order of germicidal effectiveness: 1. Osogen{R), 2. Silver .Nitrate with Formalin, 3. Iodine, 4. Ethyl Alcohol, 5. Beechwood Creosote, 6. Hexylresorcinal S.T.-37, 7• Formocresol, 8. Dichloramine-T, 9. Sodium Ricinoleate, 10. Cresatin with Benzene. The proponents of electrosterilization received sup- port from a detailed and meticulous study by Grossman and Appleton,<76 ) which presented evidence that more than twice the antibacterial effect was obtained from an elec- trolyzed chemical solution than from the same solution without electrolysis. A zinc-iodide-iodine solution, the best of some fifty solutions tested, was recomn:.ended with a thirty milliampere-minute dosage for canal sterilizationo Werther(77) and Grossman and Prinz,C7s) in 1932, compared the clinical effectiveness of camphorated parachlorophenol and electrosterilization. The former ranked a poor second, requiring twice as many appointrr1ents to obtain a negative culture. Prinz' and Rickert's text,C79 ) published in 1938, discussed in great detail the antimicrobial and toxic properties of the intracanal medicaments. Despite all the warnings, a large segment of the dental profession in 1939, still did not consider it nee- essary to treat the periapex with the care and delicacy recommended. Evidence of this is seen in the first article suggesting the use of a sulfa drug for endodontic therapy$SO) Instructions called for dissolving sulfanilamide in hot ,water, and when the solution begins boiling it is drawn into a syringe and forced into the canal with sufficient force so that it emerges through the fistula. The dentist was to place a finger over the fistula for the first few seconds to allow pressure to build up. If the affected tooth had n6 fistulous tract, it was. suggested that one be surgically created. The author did not, however, have a total lack of concern for tissue irritation as evidenced by his ·warning to those .dentists with sensitive hands: ttrr the hand is particularly sensitive to heat, a rubber glove can be worn ••• and when (the solution) emerges from. the f'istula, it will be hot enough to cause the patient discomfort and even mild burns could be produced except f'or the fact that the assistant will keep a stream of' cold water flowing over the opening of the fistula." There is little argument that the period between 1910 and 1939 produced tremendous progress in most facets of dentistry, including root canal therapy. Putting this progress into practice was still many years in the future, as the dominant factor in the treatment of pulpless teeth remained the theory of focal infection. 1940 - 1963 As opposed to its quick inception following Hunter's address, the theory of focal infection died a gradual death over many years. Endodontics likewise developed gradually a~ it threw off the shackles of this theory. The knowledge gained by those who continued through the years to practice and do research in the treatment of ~ulp less teeth began to.be used by more and more of the pro- f'ession. During the period from 1940 to 1963, when so many ad- vances were made in endondontic instruments, equipment, diagnostic procedures and therapeutic techniques, it is surprising that no ne~ non-specific root canal medication was introduced, although new combinations of old gerraicides were suggested.(81-83) Indeed, Ingle(s4 ) states that very little true progress has been made in sterilization of the infected root canal since V/alkoff recommended parachlorophenol in 1891. Following Adam's(BO) suggestion of the use of sulfa drugs in root canals, the next two decades found much of the endodontic literature devoted to the use of sulfa drugs

Following ~iorld 1f/ar II, Penicillin became available for dental research and in early 1947, Bender(lOl) reported that radiographic healing appears to occur faster VIith pen icillin therapy because of its non-irritating effect, but warned against lookin8 upon the antibiotic as a panacea for all endodontic problems. Buchbinder(lOO) noted a lack of 22 irritation even when freely _pumping penicillin past the apex, and reported no irritation with eugenol or :renicillin using a modified forearm skin test. In 1948, Stewart(lOS) determined the approximate vol- urnes of medication th&t can be sealed into prepared canals. This was of particular interest in determining the number of units of an antibiotic which could be sealed in a canal. After extensive testing of penicillin, Ostrander, Crowley and Dawson{l09) concluded it was not an adequate root canal an ti septic, and studies from the University of Michigan(llO} and the University of PennsylvaniaC94> demonstrate the isolation of resistant strains of microorganisms, particularly streptococci, from the root canals. Realizing that the intracanal use of penicillin alone was inadequte, Seltzer and Bender(l02) tested antifungal agents; Grossman and Stewart<95) tested a penicillin streptomycin suspension and in 1951 a combination of anti biotics (Penicillin,.Streptomycin and bacitracin with the antifungicide sodiu..~ caprylate) was proposed by Grossman. A clinical study(99) endorsed the antimicrobial effectiveness of this polyn.ntibiotic paste which was given the name PBSC. Although many otner antibiotic combinations were suggested(ll3,ll9,l22,143) PBSC was destined to become one of the two most popular intracanal medications of modern times.<112)

Hedman's< 53 > classic investigation into residual peria pical infection following root canal therapy proved that elimination of demonstrable microorganisms from the canal, as evidenced by two successive negative.cultures, leads to resolution of the infection of the periapical tissues. (114) Auerbach reported in 1953 that seventy-eight percent of infected root canals would yield negative cultures .following mechanical debridement and irrigation with sodium hypochlorite and warned the profession against ovcrreliance on antibiotic therapy. Two years later, this study was con firmed by Ste1nart (llS) who usod hydrogen peroxide and sodium hypochlorite as irrigants. Zeldow and Ingl.e, (ll5 ) however, obtained only 4.6 negative cultures after mechanical in strumentation and sterile water irrigation and attributed the results of Auerbsch and of Stewart to the antibacterial action of the irrigating solutionso The reports of resistant strains of microorgan isms <94, io9, llO) were only the first of the problems en-

countered by the advocates of antibiotics and in 19541 it vras demonstrated that the frequency of false negative cultures .with intracanal antibiotics is as high as thirty-one percent unless incubated for a full week.(ll7 ) This anti- 24 biotic interference with culturing had been described in two earlier studies.(108, 118) In 1957, Victor Dietz(Sl) suggested a combination of

parachlorophenol (25 grams), camphor (50 grams) and meta

cresylaceta te ( 25 grams) which he hailed as the nuni versul." endodontic medicament and originally labeled XP-7. He failed to show any antimicrobial advantage to this compound but did report the remarkable irritational qualities of XP-7_and camphorated parachlorophenol, both causing forearm ulcerations lasting one month and residual scars visible after two years. This agent.is simply a combination of ca.mphorated parachlorophenol and cresatin(R), and is marketed under the trade name Cresanol.(R) 9 In 19581 Rubbo, Reich and Dixson(ll } noted that ca.'llphorated parachlorophenol, beechwood creosote and formocresol produced severe necrosis and ulceration follovdng subcutaneous injection into laboratory animals. This was confirmed by Schilder and .Amsterdam,Cl20) who also sho~ed a high inflammatory potential 1'ollov1ing the intradermal

~njection of polyantibiotic paste (PBSC). Studies comparing the clinical effectiveness of PBSC( 121) and other polyantibiotic combinations<122) with camphorated parachlorophenol indicate no statistically sig~ificant difference~. Sargenti and Richter in 1959 suggested to the American dental profession the use of N2 as a filling material and as a root canal disinfectant. N2 :Medical, the disinfectant, was claimed to be so powerful that neither cultures nor rubber da.i1t protection was necessary during treatment. Though enjoying great popularity in Europe, it has not found favor in the United States. Ehrmann,(123) Guttuso(l24) and others!125) including the .American Dental Association's Council on Dental Therapeutics,(126) warned of the dangers of using this highly toxic paraformaldehyde preparation. Healy<127) sanctioned the use of a drug rotation program during endodontic therapy employing cresol, followed by beechwood creosote and, finally, camphorated parachloro phenol. In 1959, Winkler and van Ameronc;en(l28) reported t/:le results of the most extensive study to date on the i'requency of specific microorganisms infecting vital and necrotic pulp tissue. Streptococci were found to compose some sixty-three percent of the microore;anisms in infected canals, with Streptococcus faecalis being the most frequently isolated _ microorganism in pure culture. This predominance of streptococci had been earlier reported by Ostrander and Crowley.(lll) Sciacky and sultzenu(l29) pointed out the presence of anaerobic microorganisms in ten percent of the infected canals and agreed with those who reported the high fre

quency of streptococci present, Crawford and Shankle(l30) were able to demonstrate the presence of intracanal micro- organisrns'histologically which could not be detected by

culturing. A comparison of oral flora with root canal flora revealed a marked restriction of some oral forms. Beta hemolytic streptococci and sta:Jhylococcus aureus ·were conspicuous by their absence from infected canals. The absence of these common pathogens in canals had been re ported earlier by Hobson.<131) Grossman<132) clinically tested· an .antibiotic, Kanamycin sulfate, in combination with an antifungal agent, Nifuroxine, and suggested that further studies were war"." ranted. Calvin Torneck(l33) surgically inserted polyethylene tubing containing various root canal medications into the connective tissue of hamsters. iie observed histologically the presence of necrosis associated with formocresol, phenol, beechwood creosote, eugenol and PBSC. A less

severe reaction rosul ted v:i th cauphora ted parachlorophenol and cresatin. 27 1963 - 1968

It was in 196~ that endodontics reached the long- sought plateau of specialty status, as conferred by the American Dental Association. This destroyed the last vestiges of resistance by the few who still clung to the theory of focal infection and was the crowning achievement of twenty years of organizational efforts by the American Association of Endodontists. It is perhaps paradoxical that in this same year, there appeared the first significant report questioning the time-honored, scientific principle of culturing root canals. Seltzer, Bender and Turkenkopr<134) at the Uni- versity of Pennsylvania reported no significant difference in successful repair between teeth presenting a negative culture and those presenting a positive culture just prior to filling the canals. The following year, the same authors{l35) bluntly challenged the accepted principles of never filling a canal which demonstrates the presence of microorganisms. Repudiating the conclusions of' Buch binder, C135) Zeldow and Ingle,<137) Oliet(l3S) and other protagonists of .the culture concept, they raised the question of a change in bacteriologic. status ci: the canal between the culture appoinunent and the filling appointment. They observed that sixteen percent of the teeth which yielded a negative culture on the previous visit, yielded pooitive cultures immediately prior to filling the canal. Seltzer, et a1,<139) conflrmed this clinical study with a histologic evaluation of the repair of the periapical tissues of laboratory animals. If one accepts these findings, and considers the high incidence of false negative cultures, serious doubts are indeed raised as to the validity of the conclusions

dra~m from other studies showing the L"Ilportance or· the negative root canal culture.

Also in 19636 a combination or penicillin and strep tomycin with iontophoresis was suggested{l40) for canal sterilization and, in Sweden, the effectiveness of these same antibiotics with the addition of a quarternary am monium compound was tested.(141) Uchin and Parris (142) tested the antimicrobial activity of four commonly used canal medications after varying

time intervals sealed in the car~l and concluded that PBSC maintained a greater effectiveness over a longer period than cresatin, camphorated parachlorophenol or microcide. In 1964, Theilade and Schiott{l43) of Den.~ark found a five percent incidence of yeast in two thousand cultureso In sixty-seven teeth treated with a combination of neomycin 29 and bacitracin, yeast occurred in thirty-seven percent of the cultures. They concluded, therefore, that intracanal treatment with certain antibiotic combinations greatly increase the incidence of yeast. Shovelton,Cl44 ) !nvestigating the occurrence and dis- tribution of bacteria in infected, nonvital teeth, reported their presence in the dentinal tubules of seventy- nine of ninety-seven teeth studied by serial sectioning. The deepest penetration was halfway through the thickness of the dentin and in no section were bacteria seen to reach the cementum. A clinical study at Tufts University(l45) found no significant advantage in sealing camphorated parachloro phenol in the canals of asymptomatic nonvital teeth. Chlorhexidine and Cetrimide were recommended for root canal therapy by Hampson and Atkinson(146) who hailed their antiseptic and detergent properties as well as lack of toxicity. In 1965, Gurney, et a1,Cl47 ) reported on the screen ing of endodontic aitifungal agents and the selection of 5-nitro-2-methyl-furfuryl ether as the best antifungal agent for intracanal use with para-arninotoluene sulfonamide (Microcide(R)). For necrotic pulps, Vasilescu and 30 Ionescu{l48) proposed a combination of neomycin, erythromycin and griseofulvin. Grossman{l49,l50)demonstrated the residual antimicrobial effect of camphorated parachlorophenol after ten days in the canal. He concluded that an inactivator must be used to prevent a false negative culture. Engstrom and Lundeberg,(l5l) in 1966, disagreed with Seltzer, Bender and coworkers,Cl34, 135} and reported a significantly higher success rate after four years with teeth which yielded negative cultures at the time or obtur- ation. In a second study, these authors reported that sixteen percent of two hundred thirty-six teeth tested gave a positive test far> microorganisms followinz two consecutive negative tests, thus agreeing in fact, if not in pri~ciple with Bender; et al. They supported the view or Amerongen who in 1957 stated that the risk of a positive test in endodontic therapy is the same whether proceeded by one or two negative tests. There is, ~~ey conclude, no necessity for two negative tests prior to filling the canals.(152) Reviewing the status of microbiological knowledge as applied in current endodontic therapy, Hobson(l53} and Butler(154) were appalled by the lack of utilization of available information and termed the presently used pro cedures primitive, inadequate Dl1d, worse, misleading. In 1966, benzylkonium chl~ride(lSS) and bot cblora- mine-T (156) were suggested for canal sterilization. The following year Spanberg and Engstrom(lS'l) tested the cyto toxic effect of four intracanal medications on HeLa cells and reported all (tricresol-formalfn, zinc iodide-iodine, Biosept(R) and eugenol) were more toxic than phenol. Grossnan(lSS) also r·eported a severe inflamrnatory response with necrosis associated with the subcutaneous injection of camphorated parachlorophenol in hamsters. In 1967, Fax and Isenberg(159) found there was no single antibiotic of the twelve tested to which one or more microorganisms isolated from root canals were not resistant to one or more of the antibiotics. Frank and co-workers(l6~) reported a significant interappointment reduction in pain by using a sulfathiazole paste in the canals f'ollowing opening and biomechan- ical cleansing. Although the purpose of this paste is only to reduce discomfort, its inherent antimicrobial properties require its mention in this revien. 1969 The present status of root canal chemotherapy is not unlike that vhich existed at the turn of the century. The only significant difference which energes from an objective review of root c~nal chemotherapy since Joseph Lister's time is that most toxic drugs which were not good antiseptics have been reluctantly discarded while those toxic drugs which are good antiseptics remain in general use. The oldest and perhaps most popular(112) of the root canal antiseptic agents is camphorated parachlorophenol. Its chief advantages are in its clinical effectiveness and ease of use, while its major disadvantage is its high degree of toxicity. It is surprising to note that not one study could be found in the dental literature which reports the antimicrobial spectrum of camphorated parachlorophenol. Likewise, the effective concentration of this drug has not been determined against the various microorganisms most frequently iILhabiting the infected root carnl. It is the combination of these factors, the known and the unknown,, which prompted the present study. 33 CHAPTER III

METHODS AND K~TERILLS

A. Toxicity

A comparison of the toxic properties of aqueous para-

chlorophonol and currently used endodontic intracanal medications was studi cd by ti:1c conjunc ti va.l inflammatory

test and bJ intradermul injection into the abdomen of

rabbits. l. Conjunctival Inflamrnatory Response

Eighteen male, white rabbi ts vrnighing two to th..ree

kilograms each were obtained fyom. a cornr.'.lercial laboratory

and housed in individual wire cages.

The test solutions selected vrnre ~Ucrocide A-:~; Cresatin-lP..i-; l;b aqueous paru.chlorophenol .(PCP); 2;; PCP;

canpho1"'a ted parachlorophonol, 1£. F.; and eugenol -lHH:· ..

Physiologic saline was used as a control. At the time of experimentation, each animal r:as placed in a hooded box w:iich allor1sd only the head to

protrude. 0.15 ml. of physiologic salino v:c.s dravm in-

to a tubercul!n syringe, and with the head of the aninal

turned to the appropriate side, the saline vms carefully

~<-?:ovoJ Chcmicul l,Ifg. Co., Inc.; Brooklyn, N. Y. -~:-~·dcrcK; Sh:::.rp and Dolr.10; ·.. o;: t ?oin t, Pa. -~-:~-:H. Bird l~oycr Co., Inc.; Philadelphia, ?a. 34

dropped fro~n about one inch into the cornea of the left

eye. The head of the animal was then turned to the oppo

site side and 0.15 ml. of one of the six test solutions

was similarly dropped into the right eye. Both the le.ft

and right eye were gently held shut for approxi~ately one minute after exposure to the solution to insure its re

tention.,

'l'he left eye of each animal v:as used as a. controi

and the right eye received the test solution. Each of

the six solutions were tested on t'nree animals.

Gross observations and, in most instances color

photographs, were made prior to the application of the

solutions, and after five 1ninutes, five hours, twenty

.four hours and ninety-six hours. 'rl1.e presence and de gree of inflarma tory response was recorded using the· fol

lowing criteria: hyperemia of the conjunctiva, exudation,

edematous s;-.rellins of the conjunctiva:, clouding of the cornea with loss of c_la.rity of the anatomic strL.1tions

of the iris and external swelling of the eye.

Results were recorded usin.::; the following descriptive

terminology:

Very mild - very slight hyperemia (only).

Mild - mild to moderate h;:;-pcrcmia, slight exudation. 35 Moderate - moderate to severe hyperemia,, mild to moderate edematous swelling, cloudiness of cornea with some loss of clarity of iris striations, exudation. Moderate to Severe - severe hyperemia,, moderate swelling of conjunctiva, marked corneal clouding,, exudation. Severe - all criteria severe, external swelling.

2. De~mal Connective Tissue Inflammatory Response Fourteen male rabbits of the type described in the previous study were used in this experiment. In addition to the six test solutions listed above, a five percent sodium hypochlori te solution-::- was tested. Physiologic saline was used as a control.

Each animal was anesthetized by injecting fifty·mg of sodium pentobarbi ta1-::-::- into an ear vein. The animals were then shaved with an electric razor along two three-inch wide strips, one on each side of the midline of the abdo men and extending from foreleg to hindleg. A depilatory-::-~-:~- was used for removal of hair remnants. *Zeni te; Chemway Corp.; nayne, N. J. *"::·Nembutal; Abbot Laboratories; North Chicago, Ill. ~.~-~·Nair; Carter Pro due ts; New York, N. Y. 36 Six injection sites, three on~each side, v1ere selected on each animal and marked with ink. One of the six sites on each animal was used as a control site. In the five test sites on each animal, O.l ml of a dif ferent test solution was injected intraderm.ally using sterile tuberculin syringes Hith 27 gauge needles. In the control site,, 0 .1 ml of physiolozic saline v1as inj ec ted intra:brmally. CJ:·he presence of a wheal at the in jection site indico.ted intradermal deposition of the solutions. The animals were sacrificed by intravenous injection of an overdose of sodium pentobc.rbi tal after tvrnnty-four or sevcnty-tv10 hour intervals. Biopsies of the eighty two injection sites (two sites were not UBed) were im mediately placed into separate coded containers, each with a minimu:n of' twenty volw-;1es of' 10% formalin. Care wa.s taken to include normal tissue as well as tl1e ink marked injection site in each biopsy. A minimu.i.il of five biopsic s, each from a different animal, were obtained for each test solution in the twenty-four hour interval. A minimUL1 of three biopsies., each from a difi'erent animal, ·Herc obtained for each test solution in the seventy-tr.ro hour interval. 37 Following a fixation period of approximately seven days, the biopsies vTere dehydrated and cleared with alcohol and xylol, embedded in paraffin, sectioned and stained with hematoxylin and eosin. A minimum of ten sections were

taken fro~ varying levels of each biopsy to insure obtain ing the area of maximum tissue response. In a blind study conducted by the Department of Oral Pathology, Loyola University School of Dentistry, each coded section was graded on a scale from zero to five ac-

cording to the severity and extent of tissue response to the injected test solution. Criteria used for rating the degree of inflainmatory response were: vasodilatation, mar gination, vascular engorgement, fluid exudation, inflamma tory cell infiltration and extravasation of red blood cells. The presence and degree of tissue necrosis were also re cordedo The ratings for each of the biopsies of a given test solution were totaled, and the mean response deter mined. This mean response was used as the final rating for the solution. A rating between o. 5t and 1 •4i was termed very mil~; between 1.5+ and 2.4-t was termed m:i..ld; between 2. 5i and 3.4f was moderate; and above 3.5i was considered a severe reaction. )8

B. Antimicrobial Effec ti vene s s of Aqueous Parachl orophenol

To determine the effective antimicrobial concentrc.. tion of parachlorophcnol for rnicroorganisms commonly found in infected root c~nals, serial tube dilution studies were conducted.

1. Specific Microorg:::m~

In orO.cr to find the conccntra ti on at v1hich p.:..trachl orophenol exerts antimicrobial activity against u specific microorganism, a minimum of three series of experiments, each with a narrov1ed range, were conducted. Bach series: consisted of tv10 identical experir.1onts to insure accuracy.

Wncn results fror:i the identical experiments did not D.gree, the entire series Vias repea.ted.

A twenty-four hour, turbid growth of the selected test microorganisr:1 v10.s used as an inoculun1 in each exper iment and was prepared by needle inoculc.tion of 10 ml of' sterile broth media from a stocl:: cul-Sure naintained by the Dcpartr:.. ent of Microbiology, Loyola University

School of Dentistryo The twenty-four hour growth was always cultured in the sc:cie media to be used in the experiment. Gram sto.lns were used before and after each experiment to insure the presence of tl.10 test ndcroorgan ism alone. The selected test microorganisms included: 1. Streptococcus faecalis (Microbiology Department; Northwestern University School of Dentistry) 2. Streptococcus mi tis (Microbiology Departnent; Loyola University School of Dentistry) 3. Streptococcus sali varius (Microbiology Department; Loyola University School of Dentistry) 4. Staphylococcus aureus (American Type Culture ·Collection (ATCC) #12600) 5. Staphylococcus epidermidis (ATCC #155) 6. Neisseria catarrhalis (Midwest Culture (MC) #56)

7. Candida albicans (MC/if95) a.· Sarcina lutea (ATCC #272) 9. Corynebacterium diphtheriae (MCr'fo27) 10. Escherichia coli (ATCC #11755) Freshly prepared 1% PCP (l.O gm parachlorophenol in 100 ml of water) was used for each experimental series and strict adherence to sterile microbiological techniques was observed during all procedures. The tube dilution tests consisted of a series of media dilutions of l~~ .PCP, initially involving a broad range of concentrations and subsequently being narrowed 40 t·o a very small range until the endpoint determination could be mo.de to the nearest lXlo-4 gm paracholorphenol per ml. Each succeeding test series therefore confirmed the preceeding series. For example, the initial dilution series with each test microorganism involved a logo.ritbmic progression from a dilution of 1:5(2.Xlo-3 gm parachloro phenol per ml) to 1:54(1.6Xl0-5 gm parachlorophenol per ml). The subsequent series continued to narrow the end point and finally a series with 1 ml dilution increments determined the final endpoint. Endpoint determination for each test microorganism was based on the snallest concentration of parachlorophenol inl~ibiting that microorganism (i.e., the smallest bactericidal or fungicidal concentration). 0.4 ml of a twenty-four hour growth of the test microorganism was inoculated into each of the dilution tubes. Four control tuben, each containing 8 ml of media, were used for each experincnt. Two of the tubes re ceived only the lj~ PCP (1 ml) to test for any precipita- tion or conto:r:iin::.tion. The remaining tHo tubes recei vcd only the test microorganism to insure its capability of grov7th under normal test conditions. 41

The tubes were incubated at 37°c for seventy-two hours, after which the presence or absence of turbidity was noted. FinQl results, however, were based only on the results of subculturing each tube onto a pour plate of sim.ilar media. The pour plates were then incubated for seventy-two hours. The presence of colonies of the test microorganism, is confirmed by gram stains, was considered positive evidence of growth in the correspond ing tubeo

Tryplicase Soyi~ media vms used for nll e.A'}Jeriments except those ·with Co..ndida albicJ..ns, for which Ss..bouraud 1 s~:- media wns used. 2. Positive Ro6t Canal Cultures

In order to determine the ai. tir.:.icrobial cffecti veness of parachlorophenol against clinically virulent r.licro organisms, five positive cultures were selected fron the endodontic clinic of Loyola University School .of Dentistry. Each was either the second or third consecutive positive culture from tee th v1!1ich vrnre relatively into.ct, making salivary contar,1ination less likely.

~=·Bal tiViore Biological Lo.bora tori es; Cockeysville, L'.d. 42 Gram stains of each revealed tha. t cultures //1, f,12 and #5 were pure cultures and v:ere gram positive cocci in short chains. Cultures ://3 and :j/4 were mixed, with the former sJ:1ov;ing large grD..r.J. positive diplococci and smaller grrun positive cocci in short chains. Culture /f4 revealed gram positive cocci in clusters and grum negative cocci appearing singly. No attempt was made to identify the microorgcnisms.

A twenty-four hour growth was prepared by plncing one loopful of -1-'uDC positive culture in 10 ml of sterile

Trypticase Soy broth.

An experimental range of 1:6(1.6X:l0-3 gm PCP/r:ll) 4 to 1:20(5Xl0- gm PCP/ml) was selected on the basis of results from the previous study vii th specific microorganisms. The initial series was conducted as previously described using increments of 4 ml (i.e., 1:6, 1:10,

1:14, etc.).

A second series wn.s then conducted using 1 ml increments to further nurrm·1 the endpoint. Each series consisted of two identical experiments. All procedures, including incubation and subculturins, were conducted as described with the specific ~icroorganiscs. 43 b. Ueutralization of Aqueous Parachlorophcnol

The effect on tr1e antimicrobial a.c ti vi ty of l~~ PCP exerted by certain test solutions, suspensions, necrotic tissue and environmental conditions were tested.

1. Test Solutions a.nd Suspensions

A sterile dentin suspension was prepared by placing one gram of finely pov1dered dentin in 100 ml of dis tilled water. The suspension was autoclaved at 120°c, fifteen pounds pressure, for twenty minutes and v1as agitated daily for three days.

A sterile physiolocic saline solution vms prepared by dissolving 0.85 gu"1 of sodium chloride in 100 ml of dis- tilled water and autoclaving as above.

100 ml of huma.n saliva rms collected and sterilized with a membrane fil tor apparatus-~· using a filter of 0. 45 micron pore size.

Two corn.merci[ll preparations were al so tested, EDTAC·:H:- and Bacto-Asci tic Fluid~HHZ..

Fresh, v1hole, ci truted hwnan blood was obtained from a hospital blood b::i.nk and used experimentally six days after being draxm.

*~illipore Filter; Bedford, Mass. -~~i-?rocoSol Che1::ic2..l Co., Inc.; Fhil2.delphia, Pa. ~-;~:·Difeo Lo.boro.tories, Inc.; Detroit, ~.Iich. 44

Each of the ~bove test preparations were selected because they represent fluids which may be present in a root canal and could possibly affect the activity of

With the exception of the blood, each of the above test solutions and suspensions vrnre checked for sterility by transferring a loopful into separate test tubes containing Trypticase Soy broth and incubQting for seventy- two hours at 37oc. Each tube was then subcultured onto pour platen which were incubated for an additional seventy- two hours: No gro·.vth occurred in any tube:. Streotococcus faecalis was select3d as the test microorganism because previous studies had proved it to be the most resistant of the microorganism~ tested to parachlorophenol, a concentration of 0.00125 gia/r,11 (1:8 dilution) beinc required for bactericidal activity. crowth was used as 0.4 ml of a turbid tw~nty-four hour ~) an inoculu.~ for each experiment. A dilution range from 1:6(1.SXl0-3 g,n1 PCP/ml) to 1:11(9Xl0-4 ) was selected for all except blood .for which dilutions of 1:4.5(2.~\l0-3 em PCP/~l) and 1:3.5(2.8Xl0-3 ) were added. 45

Sterile techniques were used during all experimental procedures and gram stains .v1ere made prior to and following each experiment. The experimental procedures were the same as used in the previously described experiments.

Subculturing and incubating procedures were also id en ti- cal.

With each of t~-ie six prepCJ.ra tions, four experimental tube dilution series were conducted, with each series being repeated o.t lea.st once for confirmation of results.

Series 1 was a control series using l~S PCP prepared tv;enty-four hours prior and not subjected to contact with one of the test preparations.

Series 2 was conducted with ljb PCP which had been placed with one-half its volume of the test preparation (1 PCP:0.5 test preparation) and allowed to stand together twenty-four hours. For example, 20 ml of 17~ PCP nas pipetted into a bottle containing 10 ~;.l of test proparo.- tion, thoroughly a;;i tatcd, und allowed to stand. '11his mixture, already a 1:1.5 dilution of l~b PCP, was used in the experimental series and the desired dilutions e.stablished. .._, Series 3 was une sa:;;1e as above except that equal volumes of 15S PCP and ec.ch test pr0paration were allowed 46 together for twenty-four hours.

Series 4 was again tl-1e same with the exception that

one volume of 1% PCP was placed with tvvo volumes of test. preparation (a 1:2 ratio).

Of the test preparations, only blood required an extra procedure. After twenty-four hours, the FCP-blood mixture was centrifuged for fifteen minutes to remove

the dena turod blood. T'he supernatent ws.s then used as described to obtain the desired dilutions of parachloro phenol.

An additional study vms added for the EDTAC prepo.ra tion because it contains a powerful bactericidal n.gent, cetyl trimethyl o.n.~onium bromide. An identical series of tests as described in series 1, 2, 3 and 4 were con ducted at three additional dilutions (1:15, 1:20 and

1:25)., 2. Necrotic Tissue

The neutralizing effect of necrotic tissue on i;:~ PCP was tested, using Streptococcus faecalis as a test microore;a.nism.

A frog v1as pithed, the legs renoved o.. t the hip joint, and the feet, skin dnd boneo removed. The remaining tissue was suspended by thread in o. capped glass container above 5 ml of vm ter to prevent drying. After tv1enty-four hours, the necrotic tissue was cut into small sections of about 3X3 mm to increase the exposed surface area. Three grams of this tissue was placed in a sterile bottle to which was added 30 ml of l~b PCP. Following thorough agitation, this was allowed to stand for twenty-four hours.

At the end of this period, 1 ml of' the liquid was pipetted under sterlle conditions into each of a series of tubes to obtain dilutions from 1:2(5Xl0-3 gm parach lorophenol per ml) to l:lO(lXl0-3 s111/ml) in 1 ml increments. SF media.;:. was used in this experiment to insure that only the test microorganism, Streptococcu3 faccalis, would survive. Cont.:nlination from the necrotic tissue was thus eliminated so that the results could be properly interpreted. This media contains sodium azide and. is a selective media for the test raicroorganism. All test procedures, controls, incubation and sub culturing were conductea as previously described. A control series, using SF ~edia and 1% PCP which had been prepared twenty-four hours earlier were conducted in the manner described under Series 1 above.

-~·Bal tir1~ore Biolor;ic~:.l Laborn. tori es; Cockc~-sville, :.Id. 48 3. Thermal Extremes

The neutralizing effect of thermn.l extreaes on 1% PCP was tested, using Streptococcus faecalis as the test microorganism in Trypticase Soy media. Sterile techniques were employed in all procedures. Test procedures, controls n.nd subculturing were conducted as previously described.

Twenty ml of freshly prepared l~~ PCP was placed in each of five numbered sterile·screw-c~p bottles. Container

#1 was placed in an incub~tor 37oc, #2 was placed in a rcefrigera tor at 6°C and if3 w:::...s refrigerated at O.J')prox- in:ately. -s0 c. All Vo/ere allovrnd to rei:i~in under these e:::J.vironlliental conditions for tvrenty-four hours. Contain- er #4 was autoclaved at 120°c, fifteen pounds pressure, for twenty minutes and allow0d to cool und remain at room temperature for ~v1enty-four hours. 'J:'hc contents of

Container 1:~5 wa.s used for D. control n.nd reGained at room tenperature for twenty-four hours. Each of the 1% ~CP solutions were used in a separate experimental tube dilution series ~s described previously. A dilution range of 1:5(2Xlo-3 gm PCP/ml) to l:lO(lXl0-3 ) in one ml in- creuents wo..s selected for the sc tests. E2.ch seric s vms conducted twice and gro.rn. stn.:Lns v;ere employed to coni'irm the test micr?organism before and after each experiment. 4. Shelflife The neutralizing ef.fect of prolonged shelfli.fe in dark and clear containers were tested, using Streptococcus fa.ecalis as a test rn.icroorganisrn. Trypticase Soy was used as media and sterile techniques Tiere used in all pro cedures.. Gram stains ·were used before and after each experimental series to confirm the presence only of the test microorganism. Control Series: Freshly prcpar.Jd 1% PCP was tested in dilutions of 1:5 and 1:10 exactly n.s described above.

Light Exposure Serie3: 200 ml of the sm11e l;; PCP preparation used in the Control Series was pipetted into a sterile,clear-glass, screw-cap bottle and.placed on a shelf receiving full light exposure, both artificial and indirect sun light. Portions of this solution nara tested after six r1onths .c.nd t...-rel ve months using t!:1e same pro cedures as the Control Series.

Dark Series : 200 ml of the same l:i; ?CP preparation as used in tho Control Series was pipetted into a sterile, dark-stained, r;lass, screri-cap container and placed on a shelf recei vin;::; minimal ar:ouLts of artificial light. 50

This solution was tested .'.lfter six montJ:1s and twelve months using the sai.:ie procedures as the Control Series. 51

CHAPTER IV

RESULTS

A. Toxicity

1. Conjunctiv:d Infl0;u1x1atory Response

The reaction~ ranged from no response with Microcide to severe responses Tiith c~~phorated parachlorophenol

(CPC) Ql1d eugenol. '.I.1he maximurrr resi::.onses to the irri tating test solutions were noted at the five hour observa tion period. (Fig.I) At the end of ninety-six hours, only those eyes which were tested with CPC and eugenol continued to be inflan::med. In no case did a response occur due to the snline controls~(Fig. 2)

Cresa.tin and l;~ PCP caused only very slight hypcr. emia, while 25; PCP caused a moder a. te inflarru11a tory response after five hours. CPC caused severe inflrumnation after five hours and.continued to produce a moderate response after twenty-four c.nd ninety-six hours. :2ugenol exhibited a moderate response after only five ~inutos and resulted in a severe response at the five hour observation ~ith no improve2ent after ninaty-six hours. Typical responses are seen in Fig. 3 - 9. 52 The inflarrilllatory responses at each observation period are presented in tabul.'.'lr form in Table 1.

Table 1 - Conjunctivo..l Inflammatory Response

Animo.l 5 5 24 96 Solution No. EinutcE:: Hours Hours Hours

l.Cresatin 1 Normal Very mild Very mild Very mild 2 Kormsl "'Jery n1ild Formed 1;orraa.l 3 Nonnal Vory mild Mild l·lormal 2.3s~;cpc 4 Mild Severo Moderate Mild to Moderate 5 Mild Severe Severe Moderate 6 Mild Modera.te to Severe l.1odera te Moderate 3.Microcide 7 Norm.al Normo..l Eormr'.l Hormal 8 Normal Norr:ial lform.n.l Jfor:r.1al 9 Normal Nonna.l Nonno.l Normal 4.Eugenol 10 Modero. te Severe Severe Severe 11 Moderate Severe Severe Severe 12 Mild to Moderate Severo Severe Severe 5. 2;~ PCP 13 Mild Moderate Mild Normal 14 Hormal Hild to Moderate Mild 14 Mild to Moderate Moderate Mild Norms.l 6.l;s PCP 16 Very mild Very r.1ild Very mild Eornrn.l 17 Very oild I.Iild Very mild Normal 18 Very mild V0ry nild Very mild Normal

2. Derm~l Con.nee ti vc r.ri s sue Ir:d'lat"::na tory Response

The saline control sitez presented :::i. broad ranee of

responses from O.Of in t-:.10 biop0i0s to 3 .of (moderate} in three biopsies, with ~ ~ean response of 1 • 5 ~ (mild) 53 in the twenty-four te.st period and l.Of (very mild) in the seventy-two hour period.

The mean respons~ of the tissues to the test solution revealed a mild reaction in the twenty-four hour group to

1% PCP, 21b PCP, microcide and cresa tin. Sodium hypochlor- 1 te provoked a moderate response, v1hereas eugenol and camphorated'parachlorophenol caused severe inflammatory responses. (Fig. 10) In the seventy-two hour group, microcide caused the mildest response with a mean rating of l.O+ (very mild}. ( 2 Q.1) ~ l·:i% PCP and cresa tin were rated as ~ • ' and 2% PCP and sodium hypochlorite caused moderate reactions(2 •5 + and 2.Sf respectively}. E.'ugenol and CPC both received a severe rating of 4.0.f.. (Fig. 11) Fig. 12 - 18 represent typical tissue reactions as observed microscopically. Mean responses as well as the rating given individual biopsy responses are presented in tabular form for the twenty-four hour group in Table 2 and the seventy-two hour group in Table 3. 54

Table 2 - Twenty-four Hour Connective Tissue Infl 8.Lillm tory Re s1)onse

Mean Response Solution Response Biopsy 1;umber 1 2 3 4 5 6 7 8

Oro satin 2.3-f 2.0f 2.0+ 3.0f 3.0-t 1.0-t 3.0f

35% CPC 3.8~ 2. 5.f. 3.5+ 4.5+ 4.5f 4.5-t 3.5+

ld/0 PCP 1.9+ 2.0+ 3.0+ 2.0+ l.O+ 1.0+ 2.0.f. 2.0+ 2.0f

2:;/,o PCP 2.2.f. 4.0+ 3.0f l.Of 2.0+ l.Of 3.0f 2.0+ 2.0f

Microcide 2.3+ 2.0+ 3.0.f. 3.0f 2.0.f. 3.0+ 1.0.f

Eugenol 3.6t 4.0-f 4.5f 2.0+ 4.5.f 3.0+

Na.OCl 3.lf 3.5f l.5f 3.0f 3.5+ 4.0t 3.0f Saline l.5f 3.01' 1.0.f. o.o+ 1.0.J. o.o+ 3.0.f. 3.0+

..._---~-----··-· ,..-.-----·--·--·- 55

Table 3 - Seventy-two Hour Connective Tiseue Inf'lrunm.ato17 Response

Mean Response Solution Response Biopsy ;,rumbcr 1 .2 3 4 5 6 7

Cresatin 2.0f 1.0+ 3.0f 2.0-f 35/b CFC 4.0f 4.5f 4.0f 3.5f

l"'/0 PCP 2.04 2.0t 2.0f 2.0f 2.0f 2/b PCP 2.5;. 2.0f 3.04 3.0f 2.04 Microcide l.Of l.Ot l.Ot l.Of Ruined Eugenol 4.0+ 4.5.J. 3. 5.f. Section NaOCl 2.8f 2.5f 3.5;. Saline l.Of l.Of l.Of l.Ot l.Of l.Ot l.Of 1-.0i I 56

B. Antimicrobial Ei'fectiveness of Aqueous Parachloro phenol

1. Specific LTicroorga.niscis Parachlorophcnol was effective against all ten test microorganisms in a range varying fro!!': a 1. 2Xl0-3 gm per ml concentration to 4Xlo-4 concentration. (Fig. 19-20)

Streptococcus faccolis ~as the most resistant of the

test microorg::i.nisms rfr1ereo..s Candida alblc:."!.ns, Coryncbactc- rium diphthariae, Sarcina lutea and Escherichia coli exhibited much greater sensitivity. The latter three were destroyed in all experiments at a concentration of 4 4.lXl0- gm PCP per rr.l but in tviO experiments exhibited an even gren.ter sensitivity. The effective antinicrobial concentrations of para- cholorphonol fromthe test m:·croorgo..nim:-is ar-:: presented in Table 4. 57

Table 4 - EndDoint Determination - Aqueous Parncrilorophenol

Effective Antimicrobinl Antimicrobial PCP Dilution of Concentration Microorganism l~b PCP (gm/ml)

Streptoeoccus faecalis 1:8 .00125 Streptoeoccus mi tis 1:20 .00050

Streptococcus salivarius 1:22 .00045

Staphylococcus aureus 1:23 .00043

Neisseria catarrhalis 1:16 .00062

Staphylococcus epidermis 1:12 .00083

Candida albicans 1:25 .00040 Corynybact0rium diphtheriac 1:24 .00041

Sarcina lutes. 1:24 .00041

Escherichia coli 1:24 .00041 58 2. Positive Root Canal Cultures For the five selected positive cultures, the effective range of parachlorophenol concentrations varied from 1.1 3 Xl0- gm/ml to 7.lXl0-4 •· (Fig. 21) The antimicrobial concentrations for the test cultures are presented in Table 5. Table 5 Endpoint Determination - Aqueous Paracrtlorophenol

Effective Antimicrobial Antimicrobial PCP Dilution of Concentration Microore;anisms 1% PCP {gm/rri..l)

Positive Canal Culture (I) 1:9 .00111 Positive Cannl Culture (II) 1:10 .00100 Positive Canal Culture (III) 1:14 .00071 Positive Canal Culture (rV} 1:14 .00071 Positive Canal Cultur.e (V) 1:12 .00083

C. Ne.ut1•a.lization of Aqueous Parachlorophenol 1. Test Solutions and Suspensions The control Series 1 experiments gave relatively 59 uniform 'results vJith either a l.lXl0-3 or l.2Xl0-3 gm/ml ·parachlorophenol concentration being effective against the test microorganism. Blood exerted the most profound inhibitory effect with bacterial growth occurring even at a concentration

of 2.8Xl0-3 • In a ratio of tw8 parts suspension to one

part l~; PCP, as tested in Series 4, dentin also inhibited

parachlorophenol ac ti vi ty vii th grov;th occurrinG at a con centration of l.6Xl0-3 grr;/ml. No bacterial growth occurred at the low parachloro

phenol concentration of 4Xlo-4 gm/ml with the EDi'AC test series 2, 3 and 4. The results of test series 2, 3 and 4 with saline, saliva and ascites fluid showed a slight variation in

effective antimicrobial :pnrac;lloropb.enol ccncentra. ti on from l.4Xl0-3 gm/ml (saline: to l.6Xlo-3 (ascites fluid).

The results of ti.1e control and test series of ench

test preparation is s~ovn in Fig. 22.

2. ~ecrotic Tissue

Results of the control series showed parachlorop~enol

to be effective asainst tiJ.o test microorganism c,t a con-

centration of l.L

tissue a concentration of 3.3Xl0-3 gm/ml was required. (Pig. 23) 3. Thernia.l Extremes Thermal extrerlles appeared to have little effect on the antir.licrooial activity of parachlorophonol against the test nicroorginism. The control, incubator and autoclave series showed an effective parac~lorophcnol concentration of l.lXl0-3 gm/ml. The 5oc series and -s0 c series resulted in an of f'ec.tive conce;.1tration of l.2Xl0-3 27:i/ml. (Pig. 23) 4. Sholflife Ho change in the antimicrobic.l activity 9f parachlorophenol against the test r.1icroorganism resulted after prolonged sholflife in either a dark or light-transmitting container. The control series and all test series at six-nonth and tv1el vc-month intorya.ls resulted in an ef.fecti ve para chloropi:1enol concentration of l.2x10-3 gm/ml. (Fig. 23) 61 CHAPTER V DISCUSSION

The elimination of infection from human tissue is a necessary goal based on fundamental biolo;icc.l principles. It is an even more essential procedure in an environrnent in which the natural defense mechanisms of the body are

unable to function. S:..teh an environment is the i•oot canal.

Elimino.tion of infection from the root canal is ..._. accosplished by thorough biomecha..riical cleansing and vne use of intraco.no.l antimicrobial medications. Tho relative merits of eac!1. have beon much discussed, but it is the latter which presently conce~ns us. There is little disagreement as to the requirements of an ideal root canal medica.tion( 2'l, lSS, 159) and there is likewise little disagreement that such a drug docs not at present exist. As the choice of a root canal disinfcc- tant cannot be based on the ideal, there are minimun re quirements which should be met.(l'lO) The disinfectant should: l) be effective, 2) noninjurious to periapicQl tissues, and 3) not infcrfcre with accurate culture techniques after forty-eight hours. 62

It is of interest to note that the most corru~only used root ct'.l..nal disinfectant(ll2}, cmnphorated parachlorophenol,

N.F., meets only the first require~ent, and even that is highly debatable.

The follo 1.'i·ing objective review of the knm11n c.nd un-

knovm factors of this drug, relative to these require~:1ents, raises some interesting points of consideration.

1) Effectiveness: As defined by the liational

Formulary(l6l), canphorated parn.chlorophenol (CPC) con- to.ins not lest: than thirty-three percent or more than thirty-seven percent :parnchlorophcnol and not le:::. s than

sixty-three percent· of' i~wre than sixty-seven percent

coJnphor. It is considered clinically as a highly eff'ec tivc (lo9, lll, 121) 8.ntimicrobin.l a.c;ont.

The antimicrobial activity of CPC is due to the para chlorophenol ( lS2) o.nd the camphor serves merely as a vehicle. Camphor is an irri tn.nt which possesses ~orn.e n.no dyne qualitiesl64 ) and.is of little or no antis0ptic value.

Parachlorophenol per se reportedly has a phenol coeffici ent of 4.0(155), but the bactericidal action varies greatly with the solvent. In solvents of loil dielectric constant such as alcohol, o.cetone, ether, xylene, chloroforrn, 63 etc., parachlorophenol is believed to exert no marked antiseptic action. However, in solvents of high dielectric constant, such as glycerin, nitrobenzene and water, it reportedly exhibits marked antimicrobial activity.(163) CPO was introduced to the dental profession in 1391 by Walkoff <166) and since that time there has been no valid microbiological inyestigation into the antimicrobial activity of this drug against the ·array of microorganisms commonly found in root canals. Most investigators seem concerned primarily with the economics of the nuruber of applications, related to patient visits, required to obtain negative cultures.(121, 122) A critical look at our current endodontic culturing proced~es <153' 154) points out the fallacy in drawing the all too obvious conclusion that a negative culture indicates antimicrobial effectiveness of the intracanal medication.<154, 155) As presently employed, a negative culture indicates only. that we have either: 1) reduced the number of microorganisms, 2) eliminated the microor ganisms, or 3) used an inadequate culturing technique. As we cannot distinguish between these three, we cannot unequivocally state that our intracanal medication has eliminated any or al 1 mic roorgani sE1s from the canal. 64 In addition, Auerbach(ll4 ) and Stewa.rt<115) have shown that over seventy-five percent of the infected root canals will present negative cultures after mech2.nical 'debridernent and irrigation. This is a further indictment of drawing the conclusi8n that negative cultures are directly related to the effectiveness of the intro.canal medication. The use of CPC has been predicated on this ussumption that clinical results, as evidenced by negative cultures, indicate antimicrobial effectiveness. The effective parachlorophenol concentration age.inst the microorge.nisms of the root canal is an unknown quan- .. tity. The question of the concentration of parachlorophenol in CPC becomes suspect, as there is no vru.id ovi-

dence that thirty-fi Ve percent is the optinnh~ concentration. For every disinfectant there is a mnxir.ium concen-

tration beyond which, according to the lav• of diminishing returns, a proportionate increase in nntimicrobinl

activity is not e~idenced. The toxicity, houever, nor mally increases with the concentration.(133)

2} 'l'oxici ty: Pain follo'aing enciodontic therapy may

result from infection, overinstru~entntion, or the penet-

ration of a toxic intracanal medication into t~o periapex.(154, 171) This reason alone is sufficient to wurr- ant the use of a non-toxic medication. If, in addition, the intraca.nal medication is toxic

to the degree of causing coagulation ci' protein, the dis

advantage is cor.ipounded by a concomitant inhibition of antimicrobial n.c ti vi ty. (20) Agents r;hich coagulate pro tein have only a shallon penetration, despite low surface tension, and according to CoolidgoC73 ) are not qun.lified for the treatment of infected canals containine organic material • There is, surprisingly, some di sagreem.ent as to the toxic qualities of CPC. Parachlorophcnol produces toxic manifestations in animals sh1ilar to those produced by pheno1C165), and animal studies (119 , 120) with CPC have dernonstre.ted its high toxicity. Despite this, a leading endodontic text contains the follov.;ing statement ••• "when combined in the proportion of three to seven with gum cariphor, it becomes virtually non-irritating.(170) The results of t:ne present investigation der.r.o:nstrc;i_te the severe toxic qualities of CPC as well as its ~bility to coagulate protein.

In the dernal to:::ici ty test, severe coagulation necrosis wns pointedly evident in the tue~ty-four and 66 seventy-two hour biopsies. Cellular detail as well as the general architectural tissue pattern was completely destroyed in a wide area adjacent to the injection site.

(Fig. 17, 18) The area evidenced coagulation nec~osis, displaying a large pale-staining area lacking organization al arrangement and with an absence of connective tissue or inf'lammator:r cells. Cellular debris was observed in the necrotic area and inflammatory cells were evident in the viable tissues bordering the area.

Menkin(l67) observed that vtlth po~erful irritants :fixation is .the initial phase in the development of severe inf'lammatory reactions, and that this precedes the migra tion of' leucocytes. 3. · Interference with Culturing: In 1965, it was convincingly demonstrated by Grossman(l49) that CPC does, in fact, inhib~t culturing and therefore requires an inhibitor for accurate culturing, as do the antibiotics. Such an inhibitor is not presently available for clinical use. Considering all of the above factors, the present study sought to investigate the possibility of a more advantageous use of parachlorophenol. An aqueous vehicle was chosen because it presented the best opportunity to test parachlorophenol without superimposing toxic or anti

microbial qualities. The results of all tests with Ct.quc

ous parachlorophenol could therefore be interpreted as a manifestation of the solute.

Toxicity of Aaueous ?urachloro:Jhcnol

In sharp contrast to the toxicity displuyed by CPC, l}b aqueous p2.rn.chlorophenol ( l~; PCP) produced only mild

inflo.m:rnn.to!'.y responses and no evidence of necrosis was

observed in uny microscopic section. (F'ig. 14,, 15) Mild

to moderate acute inflammatory cell infiltration with r.1ild

edema vms noted in the twenty-four hour biopsies, nhereas

chronic inflammatory cell infiltration was evident after seventy-two hours.

The conju.."'l.c ti vDl inflc.m;~a tory res pons es ccnf'irr:i.ed. the microscopic evidence of the vast difference in toxicity

po ten tio..l betv1e en i;; ~queous 8.nd 35,~; CQrrphor solutions

of parac~lorophcnol. (Fig. 4, 6)

Both the dcr:::al 2.nd conju~1cti val tests also indicated a slishtly his.-11.er inflamnc.tor:r potcnti!~l for 25; aqueous parachloropl::.enol Vihen cor.:.;:arcd to l~; PCP. (E'ig. 11,, 5)

However, it r:1u.st bo noted that a 'aide range of responses

\7ere observable lEicroscopicJ.lly with o.11 tho test solutions,, 68 as well as with the saline control. (Fig. 10} As a re sult of this, no conclusions should be drawn as to the relative toxicity of the test solutions within a given degree of response. One would not, therefore, be justi fied on the basis of the present der:rn.tl infiar.::.r:1a tory tests to state that 2~b PCP is slightly r~ore toxic than

17; PCP. It may be concluded, however, that contrary to the severe toxic rco.ctions produced by CPC. (F'ig. 17, 18) :::md eue:;enol (F'ig •. 9) a nild. inflo.rnmatory response results from 1% PCP (Fig. 4, 14, 15}, Eicrocide (Fig. 8, 16) and Crcsatin (Fig. 7). Microcide was particularly conspicu ous in its la.ck of inflammatory potential in the conjun ctival tests. (Fig. 1, 2) · Sodium hypochlorite (5%} produced small localized nreas of necrosis in four of six bbpsies in tho twcnty four hour test period. This was :::.ccompanied by moderate to severe edema, extravasation of red blood cells, and moderately severe acute inflatimatory cell infiltration.

In seventy-two hours, a simil~r though less intense re nction wo.s r.:anifested ':;i th chronic infl.::i.r:catory cell infiltration. 69

Only eusenol proved to be as irritating and caustic as CPC, presenting a microscopic picture of severe coagulation necrosis and a conjunctival response of even greater comparative intensity.

Antir.dcro bi al Effec ti vene s s of Aoueous ? arachlorophcnol

Far more surprising t:1an tho relative lack of taxi- city displayed b:r l;& PCP was the antimicrobinl effectiveness displayed by this prcparntion.

The most comprei1onsi ve and well-controlled r.1icrobio- logical study of' the root co.na.l flora vms reported in

1959 by i'finldcr and van .A.mcrongen (128) und later confirmed by other investigators. (1291 l 30) Their stud~r involved the results from 1, 141 positive root canel cultures.

Streptococcus faecalis was found to be ti.1.c most frequently isolated microorganism in pure culture, indicating its resistant nature. This nicroorga.nism t:~lso proved to be the oost persistant, as evidenced by its high frequency in lntur cultures.

Streptococcus f'aocalis proved to be the most resistant test microorganism to p~rachlorophenol. An effective 3 bact~ricidal concentration was 1 .~\.°··10- srn/-,·1 ''" , "mien., . , represented a. 1:8 dilution· of the 1;; PCP. Sta:;-ihylococcus 70

epidorrn.idis and ncisscria co..tarrhalis were effectively destroyed at parachlorophenol concentrations of 8.3Xlo-4 4 gm/ml and 6.2Xl0- gm/ml respectively. All other microor ganisms tested were 2.o:nsitivc to concentrations of 5Xlo-4 grn/ml or less, vlith Cn.ndidn. n.lbicans being the most sensitive of those tested. (Fig. 19, 20) The microorganisms tested were selected as rcpresen- ta..tives of the various groups of organisms isolated by Winkler and van Amerongcn.(123 ) They included: Streptococcus (faocalis, mitis, salivarius) Staphylococcus (aureus, epiderraidis) Gram pOS:l.1,,:LVe. -'- . rods (Cory_neb:icterium diphtheriae)

Gram negative rods (Es c~rnri chi a ~) Gram negative cocci (1foisseria catarrhalis) Gram positive cocci (Sarcina lu tea) Yeasts (Candida albicans) Those m.icroorganisus represent groups of microorsan- isms which comprise about eighty-five percent of those groups isolated. The r,iost important group not tested are anaerobes.(104, 105, 106)

The effectiveness of par3.cnlorophenol against the test microorganisms isolated frorn. positive root c:::.nal cultures agreed vrith the results obtained from the 71 specific microorganisms, relative to the effective range of concentrations.(Fig. 21) The least sensitive microorganism {pure culture #1) required a bactericidal parachlorophenol concentration of l.lXl0-3 gm/ml whereas the most sensitive required a 7.lxlO. -4 gm I r::tl concentration. Neutralization of Aqueous Parachlorophenol Marked inhibition of 1% PCP antimicrobial action was exhibited by blood and necrotic tissue, ostensibly due to protein binding.{20,?3) The ratio of necrotic tissue tested (three grams tissue to 30 ml 1% PCP) did not completely des- troy the bactericidal action, but greatly weakened it.(Fig. 22, 23} This is a common finding vdth most, if not all, root canal antiseptic or disinfectant agents.(1681 l?l) A 2:1 ratio of dentin suspension also evidenced an inhibitory effect. In light of the complete lack of inhibition with a 1:1 ratio and the narrow test range, a true inhibition cannot be assumed. It is also doubtful that such a high ratio of dentin suspension to medication is of any clinical significance; It must also be pointed out that the dentin was obviously greatly altered from its original state because of autoclaving and being ground to a powdery consistency. Neutralization with saline, saliva and ascites fluid 72 was minor. Bactericidal activity required only a slightly increased concentration of parachlorophonol and the results could be a manifestation of the test procedure variables. It is, however, readily apparent that 1% PCP does exhibit bactericidal activity agalnst the test microorganism in the presence of a high concentration of these test solutions. The stability of aqueous parachlorophenol is obvious from the results of the shelflife and thermal extremes experiments. (Fig. 23)

CONCLUSIONS The in vitro antimicrobial activity thus displayed by this low concentration of parachlorophenol poses serious doubts as to the necessity of using the highly toxic

~h.iPty-five percent concentration as used in CPC. This in no v1ay implies that a low concentration. of parachlorophenol ~nuld be as effective in vivo. The liter ature is replete with such "test tube darlings" that !'ailed miserably in the. root canal.(27,29,63,70,79,80,86,95)

One cannot help but wonder, however, if the only real ad vantage in the use of a thirty-rive percent concentration in CPO is "ease of trituration". If the ratio of per- centages of ca~phor and parachlorophenol are varied only slightly the solution will not remain stable and will settle out. It is possible, though as yet un?roved, that 7J the camphor aids the effectiveness by acting as a reser- voir which releases parachlorophenol slowly over an ox- tended period of timeo The camphor is al·so believed to reduce the surface tension and thereby increase the pcne- trability of parachlorophenol.

It is a. microbiolo6ical "rule of thumb" that a fi vo fold increase above the endpoint concentration be used to provide a safety mar[;in for the effects of dilution, neutralization, etc.(173) A l;.b parachloroph.enol concentration provides a nine fold increuse beyond the effective in vitro ccncentration ago.inst tho most resist:int microorganism tested.

The results of' this study indicate thv.t: 1) CF'C is highly toxic, 2) a reduced par•achloropL1enol concentration in a vehicle of water is far less toxic than C:?C and

3) parachlorophenol is D..n effective ~ntimicrobial agent in eztreraely small conce.u.trations in aqueous solution against a ve..ricty of microorg~'!J.izms comnonly found in ti:ie root canal.

If, in fact n.s in theory, it could be este:olished by clinical study tha.t a l;:v or z;; pnrachlorophonol preparation possesses tl1e sar:1c clinical 11 officicmc:rn in producing I ne;;a ti vc cultures as .does ere, t:ie use of ti1e le, tter could no longer be justified. 75 CHAPTER VI

SUI.'If!LARY

A study of the toxicity of aqueous parachlorophcnol as compared to campb.orated purachlor·oph.enol and other currently used endodontic medications Tias conducted. It was determined by conjunc ti vc.l inflamr:i.atory te:::sts Qnd in tradcrmal injection into tho abdomens of rabbits, tho.t a l/~ aqueous pn.rachlorophen.ol .solution is far less toxic than camphorated para.chlorophonol.

The antimicrobio.l effec ti vene s s of o..queous parachlo roph.enol wo..s tes tcd in vitro and it v:as detcr~::irJ.ed. that a l~; solution is effective against a variety of microorgan isms commonly found in infected root c\'.l.D.als. In addition., the inJ1ibitory activity of various environraental condi":' tions on aqueous parachlorophenol v1as tested.

In the light of the results of these tests, it is concluded that a reduced concentration of parnchlorophcnol in aqueous and othor vehicles s:1.ould be further investiga ted in vitro and in vivo for possible future clinic.J.l use. BIBLIOGRAPHY l. Lister, Joseph: On the Antiseptic Principle of the Practice of Surgery, Brit. Med. J., 2:139, Aug. 1867. 2. Prinz, H. and Rickert, U.G.: Dental Materia Medica and Therapeutics, 7th ed., St. Louis, c.v. Mosby Co., l938, P• 29. 3. Spooner, s.: Guide to Sound •reeth, or a Po:eular Treatise on the teeth, New York, Columbia Press Co., l836. 4. Editorial: Who First Filled Nerve Cavities?, D. Cosmos 3:556-557, 1861-62. 5. Black, Arthur c.: Operative Dentistry: A Review of the Past Seventy-five Years, D. Cosmos, 76:61, 1934. 6. White, J.D.: Practical Hints, D. Cosmos, 6:1-2, 1864-65. 7. Watt: Minutes of the Mississippi Valley Association of Dental Surgeons, Dental News Le·tter, 10:2351 1856. 8. Fitch, C·P.: Alveolar Abscess, D. Cosmos, 5:16, · 1863-64. 9. rlicMurtrie, G.: Fang Filling, D. Cosmos, 11:225-226, 1861.

10. McLain, A.F .. : Ro~t Plugging, D. Cosmos, 9:24-27 1 1867. 11. Black, Arthur D.: op. cit., p. 54-55. 12. Dental Chronology, D. Cosmos, 76:150-182, 1934. 13. McQuillen, J. H. : Treatment of Sensitive Den tine, D. Cosmos, 12:393-394, 1870. 77 14. Rich, John B.: Minutes of the Meeting of the New York Odontological Society, D. Cosmos, 24:35-38 1882.

150 Raymond, T. E. H.: Cocaine Used Hypodermically, ~ Items Interest, 8:59-62, 1886. 16. Miller, w. D.: Gangrenous Tooth-Pulps as Centers of Infection, D. Cosmos, 30:213-214, 1888. 17. Miller, w. D.: Decomposition of the Contents of the Dentinal Tubules as a Disturbing Factor in the Treatment of Pulpless Teet~, Ohio J. D. Sc., 10:288, 1890. 18. Black, G. V.: Infections and Septic Matter in Teeth, Oil of Cassia, D. Items Interest, 13:663-665, Nov. 1891.

19. Coolidge, Edgar D.: Past and Present Concepts in Endodontics, J. &~er. Dent. A., 61:677-681, Dec. 1960~ 20. Black, G. V.: Antiseptics, D. Revo, 3:128-147, Feb. 1889. 21. Harlan, A. W.: The Use of Diffusable .Medicaments in and Around the Roots of Teeth, ~ev. 4:466- 475, July, 1890. 22. Steele, Wm. H.: 567 Useful Hints for the Busy Dentist 1892. (As quoted in J. Endodontia 3:37, July 1948.) 23. Schreier, Emil: Treatment of Infected Root Canals with Kalium and Natrium, D. Cosmos, 35:863-866, Septo 1893. 24. Cassidy, J. s.: Iilinutes of the A..'ilerican Dental Asso ciation, D. Cosmos, 36:1018-1019, 1894. 25. Wooley, J. H.: The Application of Heat in Dentistry for the Destruction of Pathogenic Germs, D. Rev. 11:172-175~ 1898. 78 26. Pfeifer, Josephine D.: Alveolar Abscess, D. Rev., 11:363-367, 1898. 27. Prinz, Hermann: What Constitutes Reinfection of Root Canals and How Can it be Prevented? D. Cosmos 61:954-965, 1919. 28. Peck, A. H.: The Essential Oils and Some Other Agents, Their Antiseptic Value, also Their Irritating and Non-irritating ?roperties, D. Rev. 12:593- 6081 1898. 29. Peck, A. H.: Formaldehyde: Its Antiseptic and Irri tating Properties, D. Rev. 11:356-362, 1898. 30. Black, G. Vo: Discussion on Essential Oils, D. Rev. 11:631-633, 1898.

3lo Black, G. V.: Special Dental Patholog_l, 2nd ed. 1 Chicago, hiedico-Dental Publishing Co., 19209 P• 295-298. 32. Onderdonk, T. VJ.: The Treatxent of Unfilled Root Canals, Int. D. J. 22:20-22, 1901.

33. Lufkin, Arthur W.: A History of Dentistr11 Phila.,, Lea & Febiger, 1948, p. 276-277.

34. Lufkin1 Arthur W.: op.· cit. 1 p. 346.

35. Hunter, William: The.Role o~ Sepsis and Antisepsis in Medicine and the Importance of Oral Sepsis as its Chief Cause, D. Re~ister, 65:579, 1911. 36. Billings, Frank: Mouth Infection as a Source of Systemic Disease, J • .A.M.~.:l._., 63:2024-2025, 1914. 37. Rosenow, E. c.: The Relation of Dental Infection to Systemic Disease, D. Cosmos, 59: 485-491, i'.Iay 1917.

38. Hayes, R. L·.: The Pulpless Tooth and Focal Infection,, J. Endodontia, 3:54-57, 1948. 79 39. Crane, Arthur B.: Root Canal Techniaue, Phila., Lea & Febiger, 1920, p.2. 40. Stengel, Alfred: Relation of Dental Affections to Systemic Diseases, D. Cosmos, 61:619-626, 1919.

41. Ingle, John I.: Endodontics, ?hila. 1 Lea and Febiger, 1965, P• 25.

42. Howe, Percy R.: To \'vba t Degree are. Oral Pathological Conditions Responsible for Systemic Diseases? D. Cosmos, 61:33-39, 1919. 43. Johnston, Harry B.: The Improved Status of the Pulp less Tooth, J • .Amer. Dent. A., 25:429-441, 1938. 44. Miller, J. L.: Dental Infection as Cause of Systemic Di.sease, J. Amer. Dent. A., 17:1598, Sept. 1930.

45. Blayney, J. R.: Vvhat Teeth Should be Extracted, J. Amer. Dent. A., 15:1217, July 1928. 46. Hughes, w. R.: Extraction vs. Treatment. D. Cosmos, 61:724, 1919. 47. Epstein, s.: Focal Infection as a Cause of Painf'ul Heel, D. Cosmos, 61:1259, 1919. 48. Wolf, H. F.: The Clinical Picture of Streptococcus Viridans Infection, D. Cosmos, 61:684, 1919. 49. Mead, Sterling V.: The Practical Use of the Dental Radiograph, D. Cosmos, 61:965-971, 1919.

50. Ellett, E. c.: Relation of Foe al Infections of the Teeth to Ophthalmic Conditions, J. Amer. Dent. A., 19:784-787, 1932. 51. Miller, W. C.: V

62. Hess,, ~alter: Formation o:r Root Canals in Human Teeth, J. Amer. Dent. A., 8:704-734, Sept. Part II, P. 790 1 Oct. 1921. 81 63. Abraham: Rhizophor, An Antiseptic Impregnated Veg etable Fiber Point for the Convenient Filling of Root-Canals, D. Cosmos, 57:1302, 1915. 64. Bauchwitz, Stettin: Root Canal Treatment with Sub limating Substances (abs.), D. Cosmos, 57:349-350, 1915. 65. Prinz, Hermann: Electrosterilization of Root Canals, D. Cosmos, 59:373-394, April, 1917. 66. Cullen, Glenn E. and Taylor, Herbert D.: Relative Irritant Properties of the Chlorin Group of Antiseptics, J. Ex_per. Med., 25:681-6991 Dec. 1918. 67. La.Roche, M.: The Importance of Bacterial Findings in Relation to the Treatment of Infected Teeth, J. Allied D. Soc., 13:154-1561 1918. 68. Coolidge, Edgar D.: Diagnosis and Treatment of Conditions Resulting from Diseased Dental Pulps, J.N.D.A., 6:337-341, 1919. 69. Price, w. A.: Dental Infections, Oral and Systemi_q,, Vol. 1, Cleveland, ~enton Publishing ~o., 1923, P• 186-187.• 70. The 1.'i.anagement and Treatment of Putrescent Pulp Canals and .reriapically Infected 'J.'eeth with Osogen, (L. D. Gaulle Co.), rhiiadelpfila, Frankin. Printing Co., 1924. 71. Blayney, J. R.: Some Factors in Root Canal Treat ment, J. Jln1er. Dent. ·A., 11:840-846, Sept. 1924. 72. Blayney, J. R.: The Medicinal Treatment and the Filling of Root Canals, J. luner. Dent. A., 15:239-243, ?eb. 1928. 73. Coolidge, Edgar D.: Studies of Germicides for the Treatment of Root Canals, J. i\rner. Dent. A., 16:698-712, 1929. 74. Coolidge, Edgar D.: Reaction of Dog Tissues to Drugs Used in Root Canal Treatment, J. kn.er. Dent. A., 19:'747-759, lfiay 1932. 82 .75. Kitchin,, P.C. and Horton, B·I.: The Relative Effi ciency from a Bacteriologic Standpoint or Some Materials Used in the Treat~ent of Putres cent Root Canals, J. Amer. Dent. A., 18:1668- 1685, 1931. 76. Grossman, L.I. and Appleton, J.L.T.: Experimental and Applied Studies in Electrosterilization, D. Cosmos, 73:147-160, 1931. 77. Werther, Raymond: Some Observations on the Use of Electrolytic Therapy in the Treatment of Peri apical Inrection, D. Cosmos, 74:328-331, 1932. 78. Grossman, L.I. and Prinz, Hermann: A Comparative Study of Root Canal Therapy with (I) Camphorated Chlorophenol and with (II) Electro-steriliza tion, D. Cosmos, 74:324-327, 1932. 79. Prinz, Hermann and Rickert, U.G.: Dental Materia Medica and Therapeutics, 7th ed., St. Louis, c. V. Mosby Co., !938. 80. Adams, Fred R.: A Preliminary Report on the Use or Sulfanilomide in Root Canal Therapy, D. Items Interest, 61:652-657,, 1939. 81. Dietz, Victor H.: XP-7: A Universal Endodontic Medicament,, Oral Surg., Oral Med. and Oral Path.$ 10:1317-1322, Dec. 1957. 82. Stewart, George G. and Gautieri, Ronald F.: Reduced Inflai.ill11a tory Root Canal Medicament, Oral Surg. 1 Oral Med. and Oral Path.,, 15:715-720, June 1962. 83. Sargenti, A.G. and Richter, S.L.: Rationalized Root Canal Treatr..nent, New York, AGSA Scientific Publications, 1959. 84. Ingle, John I.: op. cit., P• 26. 85. Adams, Fred R•: Hot Solutions of the Sulfonamides, J. Amer. Dent. A., 30:58-68, 1943. BJ 86. Grossman, L.I.: Treatment of Pulpless Teeth with a Concentrated Sulfonamide Solution, J. Amer. Dent. !..!_, 32:1432-1436, 1945. 87. Adams, Fred R.: The Technique of Treating Periapical Infections with Sulfanilamide, D. Items Interest, 63:328-340, April, 1941. 88. Casey, G.J.; Gurney, B.F., and Rapp, G.W.: A new Drug and its Clinical Application, Bur, 47:108, 1947. - 89. Gurney, B.F.: Casey, G.J.; Carroll, R.J•; Restarski, J.S.; Best, E.J. and Conlin, A.: Endodontic Therapy with Para-aminotoluene Sulfonamide Hydrochloride (Benzylog), J. Amer. Dent. A., 56:329-335, 1958. 90. Gurney, B.F. and Best, E.J •. : A Mew and Superior Endodontic Bactericidal A5ent, Part I, Oral Surg., Oral Med. and Oral Path., 12:104-112; Part II, 12:222-228, 1959. 91. Robinson, H.B.G. and Koch, W.E.: Experimental Study of the Efficacy of Penicillin, Sulfathiazole, Sulfanilamide, Beechi:lOod Creosote, Tricresol and Organic Mercurials in Root Canal Therapy, J. Endodontia, 3:41-45, July 1948. 92. Adams, Fred R.: Penicillin in Fulp Canal Therapy, A Preliminary Report, D. Items Interest, 66:1147- 1160, 1944. 93. Auerbach, M.B.: Antibiotics vs. Instrumentation in Endodontics, l;ew York D.J., 19:225-228, May 1953. 94. Grossman, L.I.: Treatment of Infected Pulpless Teeth with Penicillin, J. Amer. Dent. A., 37:141-148, 1948. 95. Grossman, L.I.; and Stewart, G.G.: An Effective Peni cillin-Streptomycin Suspension for Endodontic Treatment, Oral Surg., Oral med. and Oral Path., 2:374-378, 1949. 84 96. Seltzer, S.; Bender, I.B., and Christian, C.: Treatment of Penicillin-Streptomycin Resistant Organisms in the Root Canals of Infected Pulp less Teeth, Oral Surg., Oral Med. and Oral Path, 3:802-806, 1950. 97. Grossman, L.I.: Antibiotics vs. Instrumentation; A Reply, New York State D.J., 19:409~411, 1953. 98. Grossman, L.I.: Preliminary Report on the Use of a Penicillin-Streptomycin Suspension in Endodontia, J. Endodontia, 3:39-40, 1948. 99. Grossman, L.I.; Polyantibiotic Treatment of Pulpless Teeth, J. Amer. Dent. A., 43:265-278, 1951. 100. Buchbinder, B. M.i Laboratory and Clinical Study of Penicillin in Root Canal Therapy, J. Amer. Dent. A., 34:108-114, 1947. 101. Bender, I.B.: Penicillin in Root Canal Therapy: Report of Fifty-Three Cases, J. A.~er. Dent. A., 34-99-108, 1947. 102. Seltzer, S. and Bender, I.B.: The Efficacy of Various Drugs A~ainst Iiionilia Albicans Iso lated from the noot Canal of an Infected Tooth, Oral.Surg., Oral Med. and Oral Patp.., 2:799- 802, 1949. 103. Pear, John R.: Bactericidal Effects of Some Drugs Used in Pulp Canal Therapy, J. Amer. Dent. A., 29:244-248, 1942.

104. Morse, F.W. and Yaifes 1 M.F.; Root Canal Studies: Anaerobic Cultures, J.D. Res., 21:5-8, 1942. 105. Cran, J.A.: Study of the Pathology and Bacteriology or the Pulpless Tooth and its Bearing on Treat ment, IV, Australian J. Dent., 60:161-164 1 1956.

106. Blechman, Harry: Bacteriolo0~ in Endodontic Treat ment, Dental Clinics of .i:;orth America, .t'hila., w:---3. Saunders Co., Nov. 1957, p. 845. 85

107. Grossman, L.I.: Irritating Potentialities of Root Canal Medicaments, Am. J. Ortho. and Oral SurG., 30:564-566, 1944. 108. Stewart, George G.: Determination of the Approxi mate Volumes of Medication Used in Endodontic Treannent, J.D. Res., 27:24-26, 1948. 109. Ostrander, F. D.: Crowley, M.C. and Dawson, John: Clinical Study of the Treatment of Root Canal and Periapical Infections with Penicillin, J. D. Res., 26=!03-407, Dec. 1947. 110. Crowley, M:.c. and Harner, Verna: Penicillin Sensi tivity of Streptococci Isolated from. Root Canal Infections and the Normal Mouth,, J.D. Res., 26:399-401, 1947. ~

111. Ostrander, F.D. and Crowley, M.C.: The Effective ness of Clinical Treatment of Pulp Involved Teeth as Determined by Bacteriological Methods, J. Endodontia, 3:6-12, Jan. 1948. 112. Uchin, Robert A.: A Survey of Endodontics as Taught in the Dental Schools in the United States and Canada (1959-60). Unuublished Data as quoted in Uchin, Robert A. a~d Parris, Leonard: Antibacterial Ac ti vi ty of bndodontic .Medications after Varying Time Intervals i'ii thin the Root Canal, Oral Surg., Oral liled. and Oral P~th., 4:886-890, July 1951. 113. Bender, I.E. and Seltzer, s.: Combination of Anti biotics and Fungicides Used in Treatment of the Infected Pulpless Tooth, J. Amer. Dent. A., 45:293-300, Sept. 1952. 114. Auerbach, M.B.: Antibiotics vs. Instrllillentation in Endodontics, Hew York State D•J., 19:225-228, 1953. 86

115. Stewart, George G.: The Importance of Chemomechan ical Preparation of the Root Canal, Oral Surg., Oral Med. and Oral Path., 8:993-997, Sept. !"955.

116. Inele, J.I. and Zeldow, B.J•: An Evaluation of Mechanical Instrumentation and the Negative Culture, J. Amer. Dent. A. 1 57:471-476, 1958. 117. Bender, I.B. and Seltzer, s.: The Probability of Error of the Negative Culture with the Use o:f Combinations of Antibiotics in Endodontic Treatment 1 Oral, Surg., Oral Med. and ·oral Path., 7:1311-1319~1954.

118. Buchbinder, .r.I. and Bartels, H.A.: Criticism of the Use of Hoot Canal Cultures in Evaluating Antibiotic Therapy, Oral Surg., Oral Med. · and Oral Path., 4:886-890, July 1951.

119. Rubbo, S.D•: Rei.ch,, J. and Dixson, S.: The Use of a Combination of Eeomycin, Bacitracin and Polymyxin in Endodontia, Oral Surf., Oral Med. and Oral Path., 11:878-896, Aug. 958. 120. Schilder, Herbert and Amsterdam, Morton: Inflam matory Potential of Root Canal Medicaments,, Oral Surg.f Oral Med. and Oral Path.,, 12:211~ 221, J?eb. 95"9.

~ '~i21. Ostrander, Fo D.: The Development o:f Antiseptics and Antibiotics for Use in Endodontics, In Transactions of the Second International-Con ference on BnaO

132. Grossman, L.I.: Endodontic Tr~atrnent of Pulpless Teeth, J. Amer. Dent. A., 61:671-676, Dec. 1960. 88

133. Torneck, Calvin D.: Reaction of Hamster Tissue to Drugs Used in Sterilization of the Root Canal, Oral Surg., Oral Med. and Oral Path.,, 14:730- 747, June 1961. 134. Seltzer, S.; Bender, I.E. and Turlrnnkop.f, S., Factors Affecting Successful Repair after Root Canal Therapy, J. Amer. Dent. A., 67: 651-662, Nov. 1963. 135. Bender, I.B.; Seltzer, S. and Turkenkopf, S.: To Culture or Hot to Culture?, Oral Surg., Oral :Med. and Oral Path., 18:5217-540, Oct. 1964. 136. Buchbinder, Mo: A Statistical Comparison o.f Cul tured and Non-cultured Root Canal Cases, J. D. Res., 20:93-96, April 1941. 137. Zeldow, B.J. and Ingle, J.I.: Correlation of the Positive Culture to the Prognosis of Endodon tically Treated Teeth: A-Clinical Study, J. Amer. Dent. A., 66:9-13, 1963. . ~ 138. Oliet, Seymour: Evaluation of Culturing in Endo dontic Therapy: A Preliminary Clinical Report, Oral Surg., Oral Ivied. and Oral Path., 15:727- 730, June l962. 139. Seltzer, S.; Turkenlcopf, S._; Vita, A.; Green, D.; and Bender, I.B.: A liistologic Evaluation of Periapical Repair_Following ?ositive and Negative Root Canal Cultures, Oral Surg., Oral Med. and Oral Path., 17:507-532, 1964.

140. Fiorentini, Sergio and Sambuelli, Mario~ Primi Risultadi Clinici Di una Terapia Jonoforetica e Antibiotica Nella cura dei canali Radicolari, (abs.) D. Abs., 9:26-27, Jan~ 1964. - 141. Strindberg, L.Z.: Lokal Anvandning av Antibiotica vid Konserverande Hotbehandling, (abs.), D. Abs, 9:284~286, May 1964. 89 142. Uchin, Robert A. "Uld Parris, Leonard: Antibacterial Activity of Endodontic Medications af'ter Varying Time Intervals Within the Root Canal , Oral Surg., Oral Med. and Oral Path., 16:608-612, May, l963. 143. Theilade, Else and Schitt, C.R•: Isolering af' Gaersvampe fra Rodkanaler, Tandleagebl., 68:509- 515, Oct. 1964.

144. Shovelton, David s.: The Presence and D~stribution of' Microorganisms w'i thin Non-vital Teeth, Brit. D.J., 117:101-107, Aug. 1964. 145. Pearson, A.H. and Goldman, .M.: The Effect of' Pre medication in Endodontic Treatment, Oral Surg. 2 Oral Med. and Oral Path., 18:372-376, Sept. l964. 146. Hrunpson, E. L. and Atkinson, A. M.: The Relation Between Drugs Used in Root Canal Therapy and the Permeability of the Dentine, Brit2 D. J., 116:546-550, June 1964. 147. Gurney, B. F.; Best, E. J. and Lucotorto, F. M.: Screening and Selection of' an Endodontic Anti :f'ungal Agent, J. D. Res., 44:314-320, Mar. Apr. 1965. 148. Vasilescu, R. and Ionescu, M.: Pasta Antibiotica Pentru Trata~entul Gangrenei Pulpare Simple Si Complicate, Stomat., Bucharest, 12:23-261 Jan.-Feb. 1965. 149. Grossman, L.I.: Residual Effect in Culture Medium of' Camphorated Chlorphenol and PBSC, Oral Surg!..t. Oral Med. and Oral Path., 20:104-107, 1965. 150. Grossman, L.I.: Residual Antimicrobial Activity of Camphorated Chlorphenol, J. D. Res., 42:5831 1963. 151. Engstrom, Bure and Lundeberg, 11.: The Correlation Between Positive Cultures and the Prognosis of Root Canal Therapy after Pulpectomy, Odont. Rev., 16:193-203, Sept. 1965. 90 152. Engstrom, Bure and Lundeberg, M.: The Frequency and Causes of Reversal from Negative to Positive Bacteriological Tests in Root Canal ~aerapy, Odont. T. 1 74:189-195, May-June 1966. 153. Hobson, Pamela: The Bacteriological Problems of Root Canal Therapy, Dent. Pract., 16:43-47, Oct. 1965. 154. Butler, N.P.: Aspects of Endodontic Chemotherapy, J. Irish Dent. A., 12:43-491 April-May 19660 155. Schlomo, S.; Heling, B. and Erb, Adolf: The Use of Benzylkonium Chloride in Root Canal Therapy, J. Oral Med., 21:123-126, July 1966. 156. Kurer, J.: Irrunediate Root Canal Sterilization, Dent. Pract., 16:404-407, July 1966. 157. Engstrom, B. and Spanberg, L.: Studies on Root Canal Medicaments, I. Cytotoxic Effect of Root Canal Antiseptics, Acta Odont. Scand., 25;77- 841 June 1967. 158. Grossman, L.I.: Evaluation of Antifungal Agents for Endodontic Use, J. Dent. Res., 46:215-2171 Jan.-Feb. 1967. 1590 Fox, J. and Isenberg, H.D.: Antibiotic Resistance of Microorganisms Isolated from Root Canals, Oral Surg.f Oral Med. and Oral Path., 23:230- 235, Feb. 967.

160. Frank, A. L.; Glick, D. H.; Weich~an, J.A. and Harvey, H.: The Intracanal Use of Sulfathiazole in Endodontics to Reduce Pain, J. Amer. Dent. A., 77:102-106, July, 1968. 161. National Formulary, 1i/ashington, D. ·c. American Pharma ceutical Association, 12th edo 1 .~965, p. 76. 162. Sommer, Ralph F.; Ostrander, F. Darl and Crowley, Mary C.: Clinical Endodontics, Fhila., W. B. Saunders Co., 1956, P• 149. • 91

163. Engelhardt, W. A.: Biochem. Zentralblatt, 1927: as quoted in Osol, Artner, Pratt, Robertson and Altschule, Mark: The United States Dis ensator and Ph sicians' Pharmacolo, 26th ed., Phila., • B. Lippincott o., 19671 P• 804-805. 164. Osol, Arthur; Pratt Robertson and Altschule, Mark; op. cit., p. 234-235.

165. Osol, Arthur; Prutt, Robertson c-.nd Al tschule, Hark; ?P• cit., P• 804-805. 166. Prinz, Hermann: A su.mn:ary of' Dr. Wo.lkoff' s Dis cussion of Chlorophcnol .::nd Trcat1ncnt of Diseases of the Dental Pulp, Ohio Dent. J'nl., 19: 152-153, ~,~ar. 1899. 167. I.1enkin, V.: IFrr.ianics of Inflan:i:1ation, Hew York, The I;Tn.cmillan Uo., 1950.

168. Somr:ier, Ralph F.; Ostr2-nder, F. Darl and Crowley, Mary C.: Clinical Endodontics, 3rd ed., Philadelphia, ~. B. Saunders Co., 1966 1 p. 178. , 169. Grossman, L.I.: Endodontic Practice, 6th ed., Philadelphia., Lea a'nd l<'ooiger, 1965, p. 249.

170. Sonmcr, Ralph F.; Ostrmdcr, F. Darland CroY1lcy1 Mary c.: ODo cit., Po 182, 183. 171. Incle, John I.: op. cit., p. 488, 498.

172. Flor.line, Alex2.....rider:. Proc. Staff I.Icet., Mayo Clinic, 21:67, 1946.

1730 Davis, B.D.; Duloecco, R.; 'Eisen, H.N.; Gintberg, H. S. and ';Jood, ~'f. B.: Microoiolo'.':-y, i~en York, Harper a.nd Row; Inc.,, 1967, p. 3•14. 92

CON.JUNCT!VAL -INF! AMMATORy RESpONSE 5 HOURS

2 v0 pep 35 /o cpc eugenol

. Test So!utjons

I Mean Response I Variation in Response

Figure 1. Conjunctival Inflammatory Response

After Five Hours. CON !UNCTIYAL INF! AMMATOBY RESPONSE

5 5 24 96 min hrs hrs hrs IIME eugenol - 35°/o c pc 2°/o pc p 1°/o pep cresatin microcide

Figure 2. Conjunctival Inflammatory Response

at Various Time Intervals. 94

Figure 3. Normal Rabbit Conjunctiva

Figure 4o Conjunctival Response Five Hours

After 1% Parachlorophenol . 95

Figure 5o Conjunctival Response Five Hours

After 2% Para chloroph e n olo

Figure 6 . Conj unct i val Response Five Hours

After Ca mph ora ~e d Par achlorophenol Figure ?o Conjunctival Response Five Hours

After Cresatin.

Figure 8. Con j unctival Respon se Five Hours , After Microcid e. ' 97

Figure 9. Conjunctival Respo~se Five Hours

After Eugenolo 98

Connective Tissue Inflammatory Response 45 24 Hours 4.0 severe 3.5

3.0 moderate 2.5 0::: 2.0 0 1.5

sa ine 1°/o 2°/o m1cro PCP PCP cide Test I Mean Response I variation in Response

Figure 10. Connectiv~ Tissue Response After

Twenty Four Hours o 99

CONNECTIYE I ISSUE IN ELAMMATOBy BES pONSE 72 HOURS

saline micro- 1°10PCP cresatin 2°/oPCP NaOCI eugenol 35 °10 cide CPC TEST SOLUTIONS

I Mean response I Variation in response

Figure 11. Connective Tissue Response After

Seventy Two Hours. 100

Figure 12. Saline Control lOOx

Figure lJ. Salirie Control 250x 101

Figure 140 1% Parachlorophenol lOOx

, Figure 15. 1% Parachlorophenol 250x 102

Figure 16. Microcide 250x

Figure 17. Camphorated Parachlorophencl 250x lOJ

Figure · l8. Camphorated Parachlorophenol 250x 104

EFFECTIVE CONCENTRATION OF PCP

~ cone of 1°/o PCP

per CONC

St rept. faeca!js mjtjs salivarius aureus cataccbaljs

Mjcroorganjsms

Figure 190 Effective Concentration of Para-

chlorophenol Against Specific

Microorganisms. 105

E FFECTIYE CONCENTRATION OF PCP

~ cone of 1°/o pep

per CONC

Gm/Ml

.0000

epjderm. albjcans lutea coli

M jc rnorganjsms

Figure 200 Effective Concentration of Para

chlorophenol Against Specific

Microorganisms. 106

EFFECTIVE CONCENTRATION OE pee

.Ql QQL concentration of ]O/o pep

PCP CONC

Gm/ml

Po Sit i ye Boot Can a I Cu It u res

Figure 21. Effective Concentration of Parachloro

phenol Against Positive Root Canal

Cultureso 107

NEUTR_A!.J.Z.AIION __ OF 1 % PCP

0004

PCP CONC.

Gm/Ml

dentin saline saliva E DTAC asc1tes blood TEST SOL UT IONS .CONTROL I 1 PCP-05 Test Sol. I 1PCP-1 Test Sol

I 1 PCP- 2 Test Sol '• Pos. Culture - Neg. Culture

Figure 22. Neutralization of Antimicrobial Effect-

iveness Against Streptococcus faecalis. 108

NEUTRAL! ZATION OF 10/o PCP

pep CONC

Gm/Ml

NECROTIC TISSUE

I Bactericidal Cone for Strept. faeca lis

Figure 23. Neutralization of Antimicrobial Effect

iveness Against Streptococcus faecalis. Approval Sheet

The thesis submitted by Dr. John W. Harrison has been read and approved by three members of the Gradu- ate School Faculty.

The final copies have been examined by the direct- or of the thesis and the signature which appears below verifies the fact that any necessary changes have been incorporated, and that the thesis is now given final approval with references to content, form and mechanical accuracy.

The thesis is therefore accepted in partial fulfillment of the requirements for the degree of Master of Science.

Q.J y Pt~ ,&,tfl.J. fl~ L! 17/!tt7 71*D e ~ ~gnature of Advisor ·