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HYBENX® Oral Tissue Decontaminant

Technical Dossier

Design History Section

Updated

December 2014

EPIEN Medical, Inc. HYBENX Oral Tissue Decontaminant

Product Design History v.2.0 August 2014

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This document contains the Design History and Product Specifications Section of the

European Technical Dossier for HYBENX Oral Tissue Decontaminant as required by the

Medical Device Directive 93/42/EEC for a Class I medical device product. It begins with

an Introduction of the product followed by a presentation of related Background

information. The Product Design Goals are provided next. This is followed by reviews of

the HYBENX Chemistry. Efficacy and Safety studies are then summarized within the

context of product design. The final section summarizes the anticipated Clinical Uses of

the product. A Bibliography of selected literature references organized by subject area is

also provided.



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Table of Contents

Introduction to HYBENX Oral Tissue Decontaminant (4)

Background Summary (7)

Product Design Goals (10)

Product Specification and Chemical Characterization (13)

Viscosity (19)

Hygroscopicity (21)

Desiccation Activity of HYBENX (25)

Acidification Activity (32)

SEM Assessment (40)

Solvation and Neutralization (48)

Safety Evaluation/Studies (49)

ADME Studies (49)

Canine Vital Pulp Exposure (52)

Canine Periodontal Tissue Exposure (54)

Biocompatibility Testing (56)

Literature Review of Safety Issues (58)

Safety Evaluation Conclusions (60)

Efficacy Evaluation/Studies (61)

Disclosing Solution Study (62)

MBEC Assays (65)

Porcine Wound Biofilm Studies (70)

USC Microbial Biofilm Disruption Assays (73)

Clinical Evaluation/Studies (77)

Review of Potential Clinical Dental Applications (79)

Selected Bibliography of Background Scientific Literature (82)



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INTRODUCTION TO HYBENX ORAL TISSUE DECONTAMINANT

HYBENX Oral Tissue Decontaminant (HOTD) is a liquid topical agent intended for routine use by dentists and hygienists for enhanced oral tissue surface cleansing. HOTD is designed to be delivered as a focal irrigation to tissue surfaces involved in a dental procedure. HOTD irrigation is indicated whenever a dental practitioner determines that application of an adjunctive cleansing rinse of this type would be beneficial for the patient. An application of HOTD can be performed quickly and easily during any type of dental procedure ranging from routine prophylaxis to complex surgical procedures.

HOTD was not designed to be used as a therapy for any specific disease in the oral cavity. Its intended use is not tied to any particular clinical treatment outcome. HOTD was designed to be used routinely as a supplemental rinse and debriding agent for enhanced cleansing of dental plaque, necrotized tissues and other infectious matter from clinically important surfaces in the oral cavity during standard dental procedures.

In order to understand this product it is important to first recognize that in some respects HOTD is unlike other professional cleansing and rinsing products currently used in the dental industry. Typical oral cleansers are most often simple aqueous solutions of antiseptic agents that function primarily as passive mechanical rinses. They may secondarily provide an antiseptic benefit from the exposure of the tissue to the antiseptic constituents during application. HOTD also acts primarily by a mechanical rinsing action. However, it has a higher density and viscosity than standard oral rinses. For this reason it can better remove debris from a dental surface due to more intense mechanical shear force generated by the product. HOTD also has a unique secondary or subordinate mechanism of action. It enhances the detachment of pathological material from oral substrates during the application by exerting a self-limited superficial denaturing action onto oral surfaces on contact. This denaturing activity assists the mechanical action of the product in removing the targeted pathological matter. HOTD enhances the release of infectious materials and biofilm off of tissue surfaces through its secondary mechanism of action whereas the other types of dental rinses usually carry away only the debris that has already been mechanically detached.

Enhancing the detachment of pathologic materials from oral tissue surfaces means that more of it can be rinsed and evacuated away during dental procedures. This results in a level of cleanliness that is much better than what is typically achieved with standard mechanical cleansing methods alone. It is intended that HOTD should be available for use during any type of professional dental procedure because an adjunctive application of the product to enhance cleaning could potentially be beneficial at any time.

HOTD is most often applied to both hard and soft oral tissue surfaces with an irrigation syringe and blunt plastic applicator tip but other application techniques are also acceptable. The product is left in contact with the tissue for 10 – 60 seconds and is then rinsed away with water and an evacuator. The entire cleansing treatment usually requires only minutes to complete. HOTD achieves its enhanced cleansing action within seconds after application by denaturing and coagulating plaque and necrotized infectious tissues on oral cavity surfaces. The denaturation and coagulation activity is generated by a selective and self-limited process of contact desiccation. The contact desiccation process



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is provided by concentrated liquid sulfates and sulfonates in the HOTD. Denatured coagulated materials readily detach from oral cavity surfaces and can be easily rinsed away. In other words, HOTD works by rapidly absorbing so much water from plaque biofilm and other pathologic materials on contact that they immediately collapse and start to contract together. They eventually contract to a point where they start to curl up onto themselves in a manner that also pulls them away from the surface and breaks any attachments. They eventually become fully detached by this process and are easily evacuated.

Application of HOTD is not intended to be used as a replacement for any standard dental procedure, but rather it is intended to be used solely as an adjunctive topical treatment to any and all standard dental procedures. HOTD is also not intended as a replacement or competitor for any other currently used anti-plaque dental product. Rather HOTD is used to supplement and enhance the benefits that are achievable through use of any professional mechanical cleaning method and all traditional anti-plaque products. HOTD is intended to make the outcome of all dental treatments, procedures and protocols better by simply and quickly enhancing the thoroughness of dental plaque biofilm removal and infectious tissue debridement at the site of application. HOTD is intended for professional use only.

A significant technical challenge in the HOTD design process resulted from the decision to develop the product as a general purpose tool to assist in tissue cleansing in all types of dental procedures. It was not practical to do a traditional clinical study using formal clinical endpoints to demonstrate the safety and efficacy of HOTD in all of the various standard dental procedures in which it might be applied. A more reasonable approach was taken that demonstrated the efficacy of HOTD as a plaque biofilm cleanser through a limited series of direct observations of HOTD activity using a variety of advanced imaging techniques. Safety data was similarly obtained by direct observation techniques using related studies. Two pilot clinical studies were performed to evaluate the exposure of the product to human mucosal and periodontal tissues by limited measurement of traditional short term clinical outcomes. Additional post-marketing clinical studies are ongoing.

Depending upon the clinical situation an application of HOTD can also provide secondary benefits that are clinically significant but still subordinate to the primary indication for the product. For example, the desiccating activity of the HOTD enables it to both coagulate and seal minor lesions in oral soft tissues while at the same time it removes excess edema fluid from inflamed and swollen tissues. Both of these actions either combined or individually tend to make patients more comfortable after a procedure is completed. By desiccating and denaturing the organic component of calculus deposits HOTD facilitates their removal either manually or with powered instrumentation. By expediting calculus removal an HOTD rinse makes a practitioner more efficient and leaves the patient less traumatized and more comfortable. Other examples of potential secondary benefits from the application of HOTD can be developed for a number of different dental conditions. The product is designed so that dental practitioners can adapt it for adjunctive use during the treatment of the dental conditions that occur most often in their practice in a way that it is compatible with their established treatment protocols.



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The importance of maximizing the cleanliness of oral cavity tissue surfaces to the achievement of optimum clinical results is an established principle in dentistry. HOTD was intended to be regarded by the dental profession as a universal adjunctive cleaning tool. We formulated the product so that individual dental practitioners could use it to enhance their performance of those professional techniques that are the most important for their specific practice. After practitioners have learned the mechanism of action of HOTD and acquired some basic experience in the application of HOTD to both healthy and diseased tissues in the oral cavity, they can then develop their own guidelines for use of the product to best enhance patient outcomes within their own individual practice setting.

Recent clinical research suggests that some oral cavity microbes may play a role in causing or aggravating diseases beyond the mouth. Linkages between oral tissue microbial disease and systemic conditions such as diabetes and coronary artery disease are becoming established. These findings mandate that dental practitioners continue to improve their clinical procedures so as to do everything possible to control and minimize all possible risks that might arise from the accumulation of plaque pathogens in the oral cavity. It has now been shown that antiseptics and antibiotics have limited ability to suppress many of the microbes that are living within dental plaque. They have virtually no effect on the structural components of dental plaque either. The risk of developing additional antibiotic resistant microbes in the oral cavity is also a challenge that needs to be addressed. A closely related concern is that standard mechanical plaque removal techniques can contribute to the overall infection control problem under the right circumstances. Splatter from ultrasonic scaling equipment has been shown to create airborne droplets consisting of pieces of plaque with viable pathogens that can spread throughout an entire clinic. Antibiotics and antiseptics do very little to reduce this risk. Removing as much of the plaque and microbes as possible by a non-mechanical means before scaling, as could be done with an HOTD application, could limit this problem. HOTD was designed to be a safe and cost-effective response to the challenges that will now confront dental practitioners as the true extent of the risk to both oral and systemic health that derives from dental plaque microbes is becoming better known. HOTD was designed to be complementary to all current standard anti-plaque procedures in its method of application and mechanism of action. When HOTD is used routinely in an adjunctive fashion its plaque-removing benefits will be at least additive and in some cases synergistic with the standard anti-plaque methods in minimizing the risks associated with dental plaque pathogens. In the simplest terms, HOTD was designed to be another tool for use by dental practitioners to help them deal with new responsibilities that are being created by our expanded knowledge of plaque microbe pathogenicity. HOTD should be regarded as providing new treatment options for meeting those new responsibilities.



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BACKGROUND SUMMARY

The association between accumulations of dental plaque microbes with the development of progressive oral diseases has been established for a very long time. Plaque microbes are a major part of the etiology of caries and gingivitis. If caries and gingivitis are not treated appropriately they can lead to more serious consequences involving the invasion of plaque microbes deeper into the oral tissues. On the other hand it is also known that the self-healing capabilities of oral cavity tissues are robust. If the dental plaque and plaque microbes are removed thoroughly and in a timely fashion from diseased areas of the mouth any damaged oral tissue will usually be able to quickly heal itself. In the oral cavity aggressive tissue cleansing limits microbial disease progression and facilitates natural tissue healing.

Procedures for therapeutic and preventive dental care usually include one or more steps in the process that are intended either for removing plaque itself or for eradicating plaque microbes or for limiting new plaque formation. However, dental plaque and its microbes have a number of physical and physiologic properties that make effective removal and control both difficult and time consuming. For example, dental plaque has extreme visco-elastic characteristics such that it binds tenaciously to the surfaces of hard tissues and cannot be easily detached mechanically or washed away completely with antiseptic oral rinses. The supragingival and subgingival surface anatomy of the tooth adds to this problem as it contains many anatomically blocked areas that may be covered by plaque but they are not accessible to standard mechanical plaque removal techniques. As a consequence of just the extreme adhesive properties of plaque and the limitations created by tooth surface anatomy it is impossible to completely remove plaque biofilm from teeth using any of the standard mechanical professional techniques. Some methods are better than others. Some techniques simply smear the plaque around on the tooth surface while not removing very much of it. Essentially all currently used professional methods are less than totally satisfactory.

Another problem with the achievement of satisfactory cleansing of oral tissue surfaces by established professional techniques derives from the fact that the current chemotherapeutic agents used to control plaque have limited efficacy due to the microscopic structure of plaque and the metabolism of the microbes within it. In recent years it has been demonstrated that dental plaque has a structure which is identical to a form of microbial colonization now referred to as a microbial biofilm. Microbial biofilms are commonly found growing on wet surfaces. Within a microbial biofilm the microbes of different species live together in clusters within a layer of a sticky irregularly-shaped polysaccharide gel material that is secreted by the microbes themselves. The polysaccharide material, referred to collectively as the biofilm matrix, forms a structure with many pores and channels that are filled with water and are thought to serve as conduits for microbial waste and nutrients. It is now established that the various microbes in plaque biofilm live together in a community relationship and that they regulate their own colony and the activity of the biofilm as a whole through various microbe-to-microbe communication systems. It is speculated that the polysaccharide gel matrix may be protective of the microbes within it. It is believed to be possible that the matrix limits



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the ability of chemotherapeutic agents to reach the microbes at a high enough concentration to be an effective anti-plaque therapy.

It has been established that many of the microbes within a microbial biofilm are actually functioning in a unique resting metabolic condition referred to as a microbial persister state. The microbial persister state appears to be a type of physiologic hibernation with a slow metabolic rate that makes biofilm microbes relatively invulnerable to the standard antiseptic and antibiotic actions of commonly used antimicrobial agents. The agents most commonly used by practitioners today were designed to attack microbes in a non-persister metabolic condition because they were developed before the existence of the persister microbes had been discovered. As a result their activity level against persister microbes is minimal. For example, it has been demonstrated repeatedly that oral rinses containing chlorhexidene have almost no ability to kill microbes within plaque biofilm. The dental plaque that is easily accessible and the non-persister planktonic microbes in the oral cavity can usually be removed to a large extent by standard clinical procedures and products. By contrast at the other extreme, the plaque biofilm that contains many persister microbes or that is located in relatively inaccessible areas such as furcations and fissures is much more difficult to clean and manage by standard mechanical techniques and with standard anti-microbial products alone. A different clinical approach is necessary to manage the more difficult dental plaque biofilm formations and the persister biofilm microbes.

Microscopic imaging technologies that were originally developed to study environmental biofilms have now been adapted to elucidate the structure of dental plaque biofilm in great detail. Some of the key discoveries of that work, relative to the topic of discussion here, relate to the composition and structure of the extracellular matrix that encloses and supports the plaque biofilm microbial colonies. This work revealed that the substance of the matrix is an aqueous gel that is formed from varying proportions of microbial polysaccharides and water that is absorbed into it. As noted above the matrix gel is formed into irregular columnar structures ranging up to several hundred microns in height. Groups of columnar structures eventually blend together in a complex pattern that resembles the framework of natural sponges only on a microscopic scale. The basic framework incorporates interconnected pores and sponge-like channels within the gel. The channels serve to connect the matrix interior to the surface for the purpose of transporting nutrients and waste for the inner colonies of microbes.

A critical feature to note about the plaque biofilm structure from these studies is that there is no protective layer of cells or any other such material, such as an epithelial layer, that covers the surface of the plaque biofilm matrix and protects the underlying biofilm structures from the environment. In other words, the research imaging data suggests that the plaque biofilm matrix and the microbial colonies within it are dependent on the maintenance of a stable microenvironment, including a stable amount of moisture in that microenvironment in order to maintain the integrity of the matrix gel-like structure. The polysaccharide gel of the matrix just like any polysaccharide gel requires water to maintain its shape. Disturbing the microenvironment by removing water from within the gel itself and from the connecting pores and channels would cause the entire plaque structure to collapse, precipitate and detach from the tooth surfaces. The microbes



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themselves would then be vulnerable to desiccation since oral cavity microorganisms have not developed any type of protection from this type of microenvironment stress.

It appears that since dental plaque biofilm and its microbes are located within the oral cavity they are not usually at risk of injury from a microenvironment disturbance and therefore they have developed no defense for it. This creates an opportunity to develop a technology that would exploit this vulnerability to changes in the microenvironment in order to create a unique anti-plaque biofilm therapy. If a product were developed that used the plaque biofilm vulnerability to drying, for example, to remove it, and if such a product were shown to be safe and effective on human tissues it could be used as an adjunctive treatment to virtually all standard dental cleansing methods and all chemical cleansing products because it would act by a unique mechanism, that is drying of a biofilm surface on contact. Since such a product would utilize a different mechanism from any currently used for plaque biofilm control, it could be positioned as synergistic to any and all standard methods of plaque removal and not competitive with any of them. The difficulty of course is with the problem of how to create a technology to safely and aggressively dry the microenvironment of a localized surface in an area that is constantly wet such as the oral cavity so as to selectively denature pathological organic material.

EPIEN Medical, Inc. manufactures and distributes two different products for treating common mucosal ulcers in the oral cavity. Either one could provide a basis for the development of a new product that could remove plaque biofilm with focal surface drying as the mechanism of action. The first product, Debacterol, is made from a substituted natural wood extract. It is currently only distributed to healthcare professionals in the U.S.A. The second product, HYBENX Oralmedic, is made from purified pharmaceutical grade reagents and it is sold to professionals and directly to consumers outside of the U.S.A. Both of these products use the water-absorbing activity of sulfate and sulfonates as their principle mechanism of action. Intense water absorption on contact, or contact desiccation, of the necrotized tissue on the base of an ulcer causes that tissue to denature, precipitate and coagulate. As the necrotized tissue coagulates it is formed into a thin membranous layer of dead tissue that covers the base of the ulcer and acts much like an eschar on skin. This eschar-like membrane makes the ulcer instantly painless and keeps it pain free by protecting the tissue under the bed of the ulcer from further environmental irritation until it can heal itself.

Since both of these oral ulcer products act by absorbing water from the microenvironment of the ulcer bed either one of their formulations could serve as a starting point for developing a desiccating product for plaque biofilm denaturation and removal. However, the chemistry of the HYBENX Oralmedic product, since it is based on purified reagents, is easier to control and manipulate. For that reason, the formulation of the HYBENX Oralmedic product was a better choice as starting material for the development of the HYBENX Oral Tissue Decontaminant product. The HOTD product development process consisted essentially of using the same components that are in HYBENX Oralmedic ulcer product to create a formulation that was more appropriately optimized for tissue cleansing of plaque biofilm and infectious necrotized tissues rather than for sealing oral ulcers.



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PRODUCT DESIGN GOALS

As noted in the sections above a critical component of virtually all professional dental procedures is the need to remove as much dental plaque biofilm and infectious matter from the tissue surfaces at the procedure site as possible. While the standard professional cleansing procedures in use today are clinically very beneficial they are not regarded as entirely satisfactory in that they do not completely remove all of the potentially infectious material. Failure to completely remove all of the infectious material, plaque or tissue, is associated with shorter disease-free intervals and a higher risk of progressive disease. The limitations of the currently used methods are due somewhat to the fact that dental plaque biofilm and the plaque microbes have some physical and metabolic properties that limit their susceptibility to the standard mechanical and chemical removal techniques in use today. The standard techniques are good, but they are usually incomplete for a variety of reasons. Either they require significant training to perform well or they can be very difficult to perform in some patients or they often require a good deal of practitioner time if they are to be done correctly.

The principal goal for the development of HOTD was to create a product that could be a safe, effective and easily applied adjunctive Oral Tissue Decontaminant and tissue surface debriding agent that would overcome the limitations of existing techniques and products as listed above. The product was to be designed as an adjunctive because, as noted above, current techniques are generally effective and HOTD acts by a mechanism that is different and should be synergistic. While a practitioner may not be willing to abandon a familiar useful technique completely, they are more comfortable with adding a supplemental treatment for known supplemental benefits. Therefore, early goals were to be sure that the product was compatible with application as an adjunctive to any and all standard dental procedures and that it effectively compensated for the deficiencies of the other procedures.

HOTD was to be safe for application to both soft and hard tissues of the oral cavity. While it is expected that the product will remove plaque and damaged tissues, it must not harm healthy soft tissue or teeth. The product should have a wide margin of safety so that someone with limited experience could safely use it. Hygienists as well as dentists should be able to use the product safely and comfortably. HOTD should be as selective as possible for plaque and pathologic materials. It should be as self-limiting as possible to enhance the margin of safety even further. Finally the product needs to be chemically stable and predictable. The formulation should be non-toxic for the patient and the practitioner.

HOTD was designed to be effective at coagulating and detaching plaque and necrotic tissue from a surface without oxidizing or acidifying the tissue. In other words, the product was to be designed to render microbes and molecules inactive by changing their form through changing their structure. It was not designed as something that would actually burn or destroy organic matter. HOTD was to be designed as a liquid with specific physical characteristics so that it could easily be delivered onto surfaces that are not usually reached by the typical mechanical cleaning methods such as furcations and fissures.



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In summary, the design goals for HOTD were all based around an idea for an adjunctive plaque biofilm removing rinse and infectious tissue debriding agent that would overcome some of the limitations of existing techniques and products. It was to be selective, self-limiting and safe for healthy tissues. Most importantly, it was designed so that practitioners would have another option for cleansing tissue during a procedure that was safe, easy, comfortable and synergistic with their existing standard procedures. HOTD was to be designed as a product that any practitioner would use during any and all types of standard dental procedure in order to make the results of that procedure better by making the tissue surfaces cleaner and better able to heal themselves. HOTD was not to be designed as a treatment for any particular dental condition or disease state. It was not intended to produce a specific clinical outcome by itself.

There are a number of very distinct advantages to bringing the HOTD contact desiccation technology to the problem of dental plaque biofilm and plaque biofilm microbe removal. We can briefly touch on some of them here. First, as described repeatedly above, the mechanism of action of this product is unique in dentistry therefore application of HYBENX should be compatible as a synergistic adjunctive cleansing treatment to virtually any standard professional mechanical or chemical procedure in the oral cavity. Second, since the mechanism of action is based on a physical change to the microenvironment the probability that any species of microbe would be able to become resistant to HOTD is very remote. Also since there is a physical mechanism of action there is no issue of finding an organism with less than full sensitivity to the action of the product. Third, the product is selective for pathologic material since it is dependent on the removal of water from tissue and healthy non-pathologic tissues simply will not give up their water to HOTD. Likewise the product is effectively self limited in that once all of the susceptible tissues have been desiccated and denatured following an application of the product there will be no tissue left that is capable of being desiccated by the HOTD. Fourth, because of the mechanism of action HOTD removes not only the pathogenic organisms but it also removes the matrix. In addition the product coagulates and facilitates inactivation and removal of necrotic tissues and other tissue components that prolong inflammation. In other words HOTD does a superior job in reducing the overall microbial load and load of pathologic material that the natural healing systems of the oral cavity would otherwise have to remove and in that way it expedites natural healing. Fifth, as a semi-viscous dense liquid HOTD has properties that help it to be delivered into spaces and onto surfaces that might otherwise never be reached by other mechanical or chemical techniques. There are many other specific advantages related to the details of specific dental conditions, but the main points have been highlighted above. For example, we could consider all of the secondary clinical benefits such as enhanced patient comfort, reduction in edema and enhanced local hemostasis that all derive from the contact desiccation activity but are subordinate to the primary indication of plaque removal.

The dental literature from the past decade shows that a lot of research is being done to develop antibiotic and antiseptic-like products for the persister microbes in the plaque biofilm. Additional work is also continuing on the genetics of the biofilm microbes and the unique metabolism of persister plaque pathogens. Much of this work seeks to find ways to exploit weaknesses in the signaling mechanisms that purportedly control the replication rates of plaque microbes. While this work is quite interesting it is obvious that



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there will not likely be any successful dental plaque product emerging from these efforts any time soon. The enormous variety of species of plaque microbes involved and the complexity of their relationships with each other means that the development of effective drugs for use in this area is at best decades away in the future. The HOTD anti-plaque technology concept represents an engineered physical approach to a massive clinical problem that will exploit a simple direct manipulation of the plaque biofilm microenvironment to produce a safe, effective, inexpensive user friendly anti-plaque product with numerous subordinate clinical benefits that is available for use now.



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SPECIFICATION AND CHEMISTRY OF HYBENX ORAL TISSUE DECONTAMINANT

HYBENX Oral Tissue Decontaminant (HOTD) is made by creating a blend of the two aqueous reaction products that are derived from the aromatic sulfonation of hydroxybenzene and hydroxymethoxybenzene under proprietary aromatic sulfonation reaction conditions. The two reaction products are used in their entirety to create the final product without any further purification or isolation steps.

The chemical reaction known as the sulfonation of aromatics is a well characterized reaction in the scientific literature as it is a reaction that is widely used in chemical synthesis in a variety of industries. Most notably it is the first step in the synthesis of laundry detergent and many textile dyes. In this reaction a free sulfate group in aqueous solution forms a bond with a carbon atom within an aromatic ring through a chemical mechanism referred to as electrophilic substitution. The sulfonation substitution may take place at one or more different positions within an aromatic ring depending on the reaction conditions and starting reagents. This leads to the possibility that multiple sulfonation isomers may be formed during any one sulfonation reaction. The proportion of each sulfonation isomer that is formed in an aromatic sulfonation reaction depends on the specific reaction conditions employed, such as the concentration of the starting reactants, the temperature of the reaction and the length of time that the reaction conditions are maintained. Since these reaction conditions can be carefully manipulated and tightly controlled, the composition of the sulfonation reaction products is highly reproducible.

The various sulfonation isomers and mixtures of isomers produced in sulfonation reactions will have different chemical and physical properties. These differences in properties can be exploited to develop sulfonation reaction conditions which produce reaction products and blends of reaction products that have unique properties that are desirable for the manufacture of useful materials such as HOTD. As noted above, the sulfonation reaction of hydroxybenzene with sulfuric acid is capable of producing mixtures of multiple isomers of hydroxybenzenesulfonic acid (CAS No. 1333-39-7) depending on the sulfonation reaction conditions. Likewise, the sulfonation reaction of hydroxymethoxybenzene with sulfuric acid can produce mixtures of multiple isomers of hydroxymethoxybenzenesulfonic acid (CAS No. 50855-43-1) depending on the reaction conditions. The general reaction scheme for these sulfonation processes is shown in the next two figures immediately below.



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In the figure above the line that indicates the chemical bond that is formed between the sulfur atom of the sulfuric acid molecule and the aromatic ring structure is shown to be terminating in the center of the aromatic ring rather than at a specific carbon atom position on the ring itself as you would expect to see in a typical reaction equation. The termination of the sulfur-aromatic bond in the center of the ring is considered to be a convenient shorthand way of indicating to the reader that this bond could be formed between the sulfur and any one of the ring positions depending on the conditions present at the time the reaction occurs. In other words, it is a type of abbreviation of the reaction notation that makes it unnecessary to draw every individual isomer.

OH

Hydroxybenzene

H

H

H

H

H

+ H2SO4 =

OH

Hydroxybenzenesulfonic Acid (HBSA) Isomers

H

H

S

H

H

O

OH

O

+ H2O

OH

Hydroxymethoxybenzene

OCH3

H

H

H

H

+ H2SO4 =

OH

Hydroxymethoxybenzenesulfonic Acid (HMBSA) Isomers

OCH3

H

S

H

H

O

OH

O

+ H2O



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The sulfonation reaction of hydroxybenzene with sulfuric acid under the reaction conditions used to manufacture HOTD is known to reliably generate the two specific sulfonation isomers of hydroxybenzenesulfonic acid shown in the diagrams below.

OH

HO3S

4-Hydroxybenzenesulfonic Acid

CAS No. 98-67-9

OH

HO3S SO3H

4-Hydroxy-Benzene-1,3-Disulfonic Acid

CAS No. 96-77-5

The sulfonation reaction of hydroxymethoxybenzene with sulfuric acid under the conditions used to manufacture HOTD is known to reliably generate the three specific sulfonation isomers of hydroxymethoxybenzenesulfonic acid shown in the diagrams below.

OH

OCH3

HO3S

3-Hydroxy-4-Methoxy-Benzenesulfonic Acid

CAS No. 879-98-1

OH

OCH3HO3S

4-Hydroxy-3-Methoxy-Benzenesulfonic \Acid

CAS No. 7134-11-4

OH

OCH3

HO3S

SO3H 5-Hydroxy-4-Methoxy-Benzene-1,3-Disulfonic

Acid

CAS No. N.A.



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EPIEN Medical developed HOTD through a series of experiments wherein the specific parameters of the sulfonation reactions described above were optimized to produce sulfonation reaction products with the desired physical properties. The two reaction products are used in their entirety and blended together without any further purification or isolation procedures to make HOTD. The final step in manufacturing HOTD is to add a colorant so the product is clearly visible on teeth and tissues in the oral cavity.

As previously noted, EPIEN Medical has developed HOTD by using a specific blend of sulfonation isomers that can be reliably produced as long as the sulfonation conditions are the same each time. The resulting blend is a concentrated semi-viscous liquid product that has physical properties, such as viscosity and density that make the product useful as an oral cavity surface cleanser.

Quantitative analysis of the sulfonated compounds of HOTD has been performed using a variety of standard chromatographic and other analytical chemistry techniques. Results of these analyses have confirmed that the final HOTD product mixture contains mono-sulfonated and bis-sulfonated hydroxybenzenesulfonic acid (HBSA) and mono- and bis-sulfonated hydroxymethoxybenzenesulfonic acid (HMBSA) together with free sulfuric acid and water. In addition, qualitative mass spectrometric analysis was performed on HOTD by two independent consulting laboratories. The results of these analyses confirmed the results of the internal chromatographic analyses.

The chromatograms presented below display the results of an HPLC chromatographic analysis of the component sulfonation reaction products and the final product mix of HOTD. The chromatograms have been put in an overlaid position to better demonstrate the relationship of the components to the final mixture. The technique used was a combined Ion Exchange/Reverse Phase Gradient on a Dionex 3000 Ion Chromatograph with a Hamilton PRP-X100 column and UV/Vis detection.



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Chromatographic Analysis of HOTD by Combined Ion Exchange/Reverse Phase HPLC The top tracing is the chromatogram of the first reaction product from the sulfonation of hydroxybenzene. By mass spectrographic analysis and comparison with standard retention times Peak 1 was identified as the monosulfonated isomer 4-hydroxybenzenesulfonic acid and Peak 2 was identified as the bisulfonated isomer 4-hydroxybenzene-1, 3-disulfonic acid. The middle tracing is the chromatogram of the reaction product from the sulfonation of hydroxymethoxybenzene. By mass spectrographic analysis and comparison with standard retention times Peak 3 was identified as the monosulfonated isomer 4-hydroxy-3-methoxybenzenesulfonic acid, Peak 4 was identified as another monosulfonated isomer3-hydroxy-4-methoxybenzenesulfonic acid and Peak 5 was identified as the bisulfonated isomer 5-hydroxy-4-methoxybenzene-1, 3-disulfonic acid. The bottom tracing is a chromatogram of the final product mixture which is made by combining the two sulfonation reaction products. The peaks of the final product are numbered to correspond with the component tracings above.

OH

HO3S

Peak 1

OH

HO3S SO3H

Peak 2

OH

OCH3

HO3S

Peak 3

OH

OCH3HO3S

Peak 4

OH

OCH3

HO3S

SO3H

Peak 5

Titration Curve for HPBR

2H

1

1

3

2

3

4

4

5

5

HMBSA

0.

5.

10

15.

20.

25.

30.

35.

40

45

50.

-1

0

10

20

30

40

50

60

70

80

90

HBSA

Final Mix



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The figure above shows a Titration Curve of the final HOTD product mixture. The analysis is performed by first combining 0.1 gram of HOTD with 50 grams of water and stirring vigorously for about 1 minute. Then a 0.25 M NaOH titrant solution is added to the product/water mixture in small increments with vigorous stirring while measuring the pH. The black line tracing shows the pH of the mixture as a function of the amount of titrant added. This tracing is used to determine the equivalence point. The equivalence point is the point on the graph that shows the most rapid change in pH per unit of titrant added. This is the point where all of the acid protons have finally been completely neutralized by the titrant and now free base from the titrant is accumulating which causes a very rapid swing in the pH value. It is determined by plotting the first derivative of the pH tracing which is shown by the blue tracing. The position of the blue peak in the first derivative line marks the equivalence point. The position of the equivalence point is extrapolated to the titrant line by a dotted red line. The point of intersection of the dotted red line with the titrant line indicates volume of titrant required to neutralize the acid in the product. The volume of titrant to the equivalence point multiplied by the titrant concentration provides the quantity of acid in the original 0.1 gram sample. From that number you multiply by 10 to get the milli-equivalents of acid that are in a gram of HOTD. It is important to recognize that HOTD does not have a valid pH value. The term and definition of pH is only valid for dilute acid solutions and not for concentrated acidic material such as HOTD. Therefore, the acid content of HOTD is expressed as milli-equivalents per gram of product in the specification and not as pH. Note however that when performing a titration curve a small sample of the product is diluted significantly and then the pH of that diluted product is used in the acid measurement by titration. In other words, the pH shown on the titration curve graph is not the pH of HOTD because the concept of pH does not apply to HOTD. The pH recorded on the titration curve graph is from a diluted sample of HOTD which is at a point of dilution where the concept of pH applies and is valid for analytical purposes only.

Equivalence Point Line

1st Derivative Line Titration Curve (pH)



Page 19

HOTD Viscosity

As noted above HOTD is a blend of the reaction products from two separate aromatic sulfonation reactions (the HBSA reaction and HMBSA reaction). These reaction products were originally designed to be of different viscosities so that the viscosity of the final product could be adjusted by varying the percentages of each component in the final blend. In the studies shown in the chart below the viscosity of various blends of the two reaction mixtures was found to be dependent upon the proportion of each one. The choice for making HOTD was a compromise blend consisting of 60% of the HBSA reaction product and 40% of the HMBSA reaction product by weight. Viscosity was determined in this experiment with a Brookfield DV-I+ Viscometer @ 25°C using sample processing and analysis techniques that are standardized for that instrument.

Test Mix % HBSA

Reaction Product %HMBSA

Reaction Product Viscosity(cPs) @25°C

#1 100 0 690 #2 90 10 740 #3 80 20 790 #4 70 30 845

#5 HOTD 60 40 960 #6 50 50 995 #7 40 60 1050 #8 30 70 1130 #9 20 80 1200

#10 10 90 1270 #11 0 100 1350



Page 20

In the next set of studies on viscosity the dependency of the viscosity of HOTD on temperature and on the absorption of water was evaluated. The rationale behind these studies is that while HOTD is initially delivered onto tissue surfaces at ambient temperature and 100% concentration, the HOTD quickly rises to body temperature and absorbs moisture from the environment once it is in the oral cavity. Understanding the changes in the physical properties of HOTD that occur within the oral cavity helps to choose the blend of components that will give the optimal performance in rinsing. In the tables below the change in viscosity of HOTD is shown as a function of temperature on the left and as a function of diluting the product with water at 37°C on the right.

The data in these tables support the proposition that as HOTD is initially delivered as a tissue surface cleanser it is at a relatively high viscosity which would aid with a mechanical rinsing action and dislodgment of tissue debris. Once the HOTD is warmed toward body temperature and absorbs small amounts of water from the tissue surface the viscosity will decrease. The decrease in viscosity allows the product to spread easily into all parts of the targeted area such as through furcations and into cracks, pits and fissures.

Undiluted HOTD @X°C Viscosity (cPs) 20°C 1460 25°C 960 30°C 655 35°C 460 37°C 400 40°C 300

HOTD @X% Dilution Viscosity (cPs)@37°C 0% Added Water 400 1% Added Water 350 2% Added Water 310 3% Added Water 275 4% Added Water 230 5% Added Water 185 6% Added Water 160 7% Added Water 150 8% Added Water 130 9% Added Water 115

10% Added Water 100



Page 21

HOTD Hygroscopicity

The HOTD components consist primarily of free sulfate and sulfonated compounds. Many different sulfate compounds are known to have a very strong water absorption activity and some are widely used as commercial desiccants in industrial applications. Sulfates in general can reversibly absorb and desorb water depending on the specific environmental conditions. The mechanism by which the sulfates that comprise HOTD can absorb water and the environmental conditions that are required for that to occur are well described in the chemistry literature. Sulfates reversibly absorb and desorb water because water and sulfate have a unique complementary polarity and geometry of the electrostatic charge on their respective surfaces. These surface charges are such that they strongly favor the formation of a thermodynamically favored linkage called a hydrogen bond between them. Water molecules will form hydrogen bonds that are of varying strength with other molecules besides sulfates including with other water molecules, but the characteristics of the hydrogen bonding that occurs with sulfates is exceptional in strength, quality and quantity.

Water molecules will also readily desorb from the sulfate surface if the environmental conditions are changed because there are no irreversible covalent bonds formed between the water and sulfate surfaces. For example, water can be readily removed from sulfates by simple heating. The attraction between the water and sulfate surfaces can be modeled as being similar to the attraction between opposite poles of magnets. The water-sulfate attraction in HOTD is such that it provides for effective dehydration and precipitation of unprotected organic materials such as plaque biofilm when applied properly, but it lacks sufficient dehydrating potential to do any damage to normal healthy oral cavity tissues when used as directed.

As noted above the term “hydrogen bond” refers to a particular type of electrostatic attraction that occurs between the positive and negative poles of molecules that form electrostatic dipoles. Some molecules, such as water, are constructed in such a way that they form dipoles where atoms on one side of the molecule have a positive charge on the surface (the positive pole) and atoms on the other side of the molecule have a negative charge on the surface (the negative pole). The charge on the surface is not a full unit charge, such as one would find on an individual cation or anion (such as Na+ or Cl-) but is a partial charge. An example is shown here in a molecular model of water.



Page 22

The red sphere represents the oxygen atom of water and the white spheres represent the hydrogen atoms. The structure of the water molecule is such that it forms an electrostatic dipole, as discussed above, where the surface of the hydrogen atoms has a partial positive charge (indicated by the symbol δ+) and the oxygen atom surface has a partial negative charge (indicated by the symbol δ-). These charges form an electrostatic dipole that is similar in concept to a magnet which has a negative pole and a positive pole.

The negative and positive poles of the water molecule dipole are attracted to each other and within a sample of water the individual molecules tend to arrange themselves so that the negative and positive sides face each other in the water sample. The molecules are attracted to each other by dipole-dipole interaction that is similar to the poles of magnets being attracted to each other but they do not come into direct contact in the sense that they trade electrons between molecules nor do they create any new molecules through the dipole-dipole attraction. The interaction between the hydrogen atoms with the positive surface on one water molecule with the oxygen atom and its negative surface on another water molecule is known as a “hydrogen bond”. This is not equivalent to a covalent bond which is defined in chemistry as an exchange of electrons between atoms. Hydrogen bonds are much weaker than covalent bonds, they are readily reversible and their formation does not lead to the creation of new molecular entities.

Adsorption of water by molecules such as sulfuric acid and sulfonic acid groups is another example of an electrostatic attraction due to the dipole structure of the water molecule leading to the formation of hydrogen bonds. The positive surface of the hydrogen atoms on the positive dipole of the water molecule is attracted to the negative surface charge on the surfaces of the four oxygen atoms of the sulfate group (SO4

-2). This interaction is similar to the dipole-dipole interaction that happens between water molecules, but in this case it is between a water molecule and a sulfate molecule. The situation can again be thought of as similar to the positive pole of a magnet being attracted to the negative pole of a magnet. Because of its dipole structure, water molecules are known to form a type of water “shell” around a sulfate group with the water molecules assuming a certain ring structure that is determined by the physical shape of the molecule and the orientation of the dipoles. A picture of this water shell formation around a sulfate group is shown in the molecular model below. The hydrogen

Hydrogen Bonds Formed Between Water Molecules



Page 23

atoms are indicated by white spheres, the oxygen atoms by red spheres and the single sulfur atom of the sulfate group at the center of this water shell is indicated by a yellow sphere. The hydrogen bonds (i.e. dipole interactions) are shown by dotted lines between the white spheres and the red spheres. The dipole interactions in this water shell not only occur between the water molecules and the oxygen atoms of the sulfate group, but also just between the water molecules themselves. It is the attraction between the water’s positive hydrogen pole and the sulfate negative oxygen surface that enables the sulfate group to act as a desiccant. Sulfates that come into contact with water molecules will attract them through this electrostatic process. But once again, these hydrogen bonds are not strong ones. They do not lead to the formation of new molecular structures and they are readily reversible by heating. This is why desiccants that are based on this mode of action can be easily regenerated



Page 24

The use of sulfonated aromatics in HOTD provides essential qualities to the product beyond the water absorbing action of the sulfate groups. As already described, the aromatic components make the material dense, viscous and sticky. These properties make the product a better mechanical rinsing agent and they make it much easier to apply and control on wet tissue than a simple aqueous solution of a non-aromatic sulfate. An acidic sulfate mixture is used in HOTD rather than a blend of neutral sulfate metal salts, because the acidic forms are much more soluble than the neutral salts. This means that it is possible to achieve a more concentrated solution of sulfates if the solutes are in acid form. The HOTD was designed to be a product that makes it practical and safe to briefly apply concentrated solutions of sulfonic and sulfuric acid onto tissues in the oral cavity

The details of the mechanism by which dental plaque biofilm and infectious tissue debris in the oral cavity can be detached from the oral cavity surface by being desiccated is well described in the scientific literature. The biological structures that form all of our tissues are themselves formed from large complex macromolecules. These macromolecules are created from large protein polymers together with polysaccharides and lipids. These polymers consist of long chains of constituent monomers held together in specific sequences by covalent bonds. In order to form functional macromolecules, these long polymer chains must fold over onto themselves in specific ways, repeatedly, until they eventually take on a well defined globular character. Once they have assumed these globular shapes, they can function as “normal” components of tissue structures by interacting with other globular macromolecules.

In order for the normal physical state of these macromolecules to persist, the physical environment of the tissue where they reside must be normal and stable. Disruptive physical forces in the environment lead to disruption of the physical forces that keep the macromolecules in their normal shape. The state of hydration is critical to the stability of tissue components. Desiccation of tissues will disrupt macromolecular interactions and lead to denaturation and precipitation within tissue surfaces as water is removed.

When HOTD comes into contact with a tooth, it selectively desiccates whatever material is on the tooth surface. This surface desiccation disrupts the physical structure of the macromolecules by disrupting the physical interactions that are dependent on hydration, such as hydrogen bonding and hydrophobic interactions. The desiccated tooth surface components precipitate and re-aggregate at the molecular level. This process in turn leads to a loss of attachment of the precipitated material to the tissue surface. And this is essentially the mechanism for the primary mode of action of HOTD at the level of the tooth surface.



Page 25

The Desiccation Activity of HOTD

HOTD contains sulfates and sulfonates which are known to be strong water absorbers. A relative quantitative assessment was made of the water absorbing properties of HOTD using standard techniques for measuring hygroscopicity. Hygroscopicity is the property that allows a material to absorb moisture from air. Determining the hygroscopicity levels of test materials is considered one way to indirectly approximate the relative potential of in vivo water absorption activities of materials. To assess hygroscopicity, weighed samples of test materials are placed into an environmental chamber with controlled temperature and humidity in vessels of uniform defined shape and size. The amount of water absorbed by the test material from the air, or the amount of water released by the test material into the air, in a defined environment is determined by weighing the sample containers at specified time intervals. The weight change of the sample in a given unit of time indicates the direction and level of hygroscopicity of the test material under the conditions of the study. In the first study, the level of hygroscopicity of the various blends of the two HOTD component products was determined after 24 hours at 40°C and 75% Relative Humidity. The data below show substantial variability in the hygroscopicity levels depending on the proportion of the two reaction components. The 60% HBSA/40% HMBSA mixture that is used to make HOTD provides it with the ability to absorb 69.4% of its own weight as water after 24 hours at the conditions specified. This information can then be used to compare the properties and modes of action of HOTD to other materials that are evaluated under the same conditions.

Test Mix % HBSA

Reaction Product %HMBSA

Reaction Product % Wgt Change

@ 24 Hrs #1 100 0 +51.0 #2 90 10 +59.5 #3 80 20 +63.4 #4 70 30 +65.8

#5 HOTD 60 40 +69.8 #6 50 50 +77.7 #7 40 60 +70.6 #8 30 70 +57.0 #9 20 80 +53.0

#10 10 90 +52.3 #11 0 100 +49.7

In the next study, the hygroscopicity of intact HOTD was compared to the hygroscopicity of samples of HOTD that had been pre-diluted with pure water to the HOTD concentration levels listed on the left side of the table below. Then the hygroscopicity of some familiar materials was determined as shown on the right side of the table. Multiple aliquots of each test sample were prepared in separate weighing bottles. The bottles were maintained at 40°C and 75% relative humidity. The bottles were weighed at the start of the experiment and after 24 hours. The data are expressed as mean % weight gain (black) or weight loss (red) relative to the weight at the zero time point. Intact HOTD gained 69.4% of its starting weight as water and the serial water dilution samples gained proportionally less as indicated down to the 50% dilution mark. HOTD samples that were



Page 26

pre-diluted beyond the 50% dilution mark were no longer hygroscopic and lost weight in the environmental chamber as water evaporated from those samples under the conditions of this experiment. The changes seen in hygroscopicity with the pre-dilution of HOTD by water confirm that HOTD has a very significant water absorption capacity (69.4%) at a moderate rate (over 24 hours) and that the water absorption process is reversible depending upon the condition of the test material (at dilutions >50%) and the conditions of the environment (40°C and 75% relative humidity).

The Sulfuric Acid NF sample in the experiment absorbed approximately ½ the amount of water from the environment as HOTD at 24 hours. QuikClot is a granular mineral wound dressing material that has as a primary mode of action the absorption of water from blood and it showed substantial hygroscopicity in this assay. Two materials that are well known as tissue denaturants, Silver Nitrate and Salicylic Acid do not show any hygroscopicity in this assay suggesting that these materials do not use water absorption as a mode of action to denature tissues. The hygroscopicity data in this table supports the conclusion that HOTD has a mode of action that involves water absorption. The hygroscopicity data also suggests that HOTD is likely to have a similar mode of action to QuikClot, but it has a mode of action that is very different from materials such as Silver Nitrate and Salicylic Acid.

Hygroscopicity Assay-All Samples at 40°C/75% RH (% Starting Weight from Time 0’)

24 Hrs 48 Hrs 72 Hrs 100% HOTD +69.4 +91.6 +101.8

90% HOTD in water +22.6 +40.6 +49.5 80% HOTD in water +17.3 +28.8 +37.2 70% HOTD in water +11.8 +19.6 +25.4 60% HOTD in water +7.7 +11.7 +14.9 50% HOTD in water +1.2 +1.6 +2.5 40% HOTD in water -4.9 -8.6 -11.1 30% HOTD in water -10.6 -18.1 -24.3 20% HOTD in water -16.8 -26.4 -38.4 10% HOTD in water -23.8 -41.8 -62.1 5% HOTD in water -25.8 -45.4 -63.9 1% HOTD in water -23.7 -42.3 -61.3

Sulfuric Acid NF +32.1 +56.4 +76.8

Salicylic Acid -0.2 -0.2 -0.2 Ca Phenol Sulfonate +0.2 +0.1 +0.1

Ca Guaiacol Sulfonate 0.0 0.0 0.0 Aluminum Sulfate +5.1 +5.1 +5.0

35% Ultra Etch Gel -10.5 -15.2 -16.3 35% Phosphoric Acid -2.4 -3.4 -4.3 Conc. Phosphoric Acid +27.2 +42.3 +51.4

AgNO3 0.0 0.0 0.0 Saline -13.9 -28.8 -37.6



Page 27

The next experiment was designed to determine if the absorption of water by HOTD is a reversible process, like a sponge, and not an irreversible process or a one-way bond-forming reaction. Multiple samples of intact, undiluted HOTD were placed in an environmental chamber that was maintained at 40°C and 75% Relative Humidity and they were allowed to absorb water for 24 hours. The specimens were then transferred to another chamber that was also maintained at 40°C, but the humidity was left to be at ambient room levels. The chart below depicts the mean weight of the HOTD samples as they were first placed in the high humidity chamber and absorbed water (labeled as Absorption phase in the chart) and then tracks the weight decrease as the bottles were moved into the low ambient humidity chamber and water evaporated from the bottles (labeled as Desorption phase in the chart). The chart depicts 4 cycles of this Absorption-Desorption process which is uniform and repeatable with each cycle. At the end of the last cycle the HOTD that was in the vials was subjected to analysis by the established HOTD QC Procedure. It was shown that the process of water Absorption-Desorption as depicted in the graph did not change the composition of the HOTD. This data shows that the Absorption of water by HOTD is a completely reversible process that does not involve any irreversible changes in the composition of the HOTD material.

Absorption and Desorption of Water Vapor by EPIEN ROOT CANAL CLEANSER

-20.

00.

020

.040

.060

.080

.0

0 20 40 60 80 100 120 140 160 180 200

Time (hours)

Mas

s In

crea

se (%

by

mas

s)

Absorption AbsorptionDesorption Desorption Absorption Absorption DesorptionDesorption



Page 28

In the remainder of the hygroscopicity studies that are summarized here the performance of HOTD as a water absorber was compared to the performance of QuikClot as a water absorber. As noted above QuikClot is a granular form of alumina-silicate that has been cleared for marketing as a wound dressing. The manufacturer of QuikClot established that water absorption by hydrogen bonding is the primary mode of action of the product. A series of head-to-head comparisons of QuikClot with HOTD using in vitro water absorption experiments was used to establish that HOTD has an equivalent mechanism of action.

It is not possible to apply QuikClot and HOTD samples onto an oral cavity surface in the same way since one is a liquid and the other is a powdered solid. Therefore the water absorption mechanism of QuikClot cannot be compared directly to HOTD in the oral cavity. Similarly, it would be impractical to compare the performance of HOTD to QuikClot in the control of brisk arterial hemorrhage. As a practical alternative the process by which HOTD absorbs water was compared to the process by which QuikClot absorbs water in a series of experiments utilizing a highly simplified in vitro model of blood and plaque dehydration. Although this approach appears simplistic, the model was developed to provide uncomplicated evidence of the equivalency of the water absorbing properties of the two products.

In this model system a Large Sample Well and a Small Sample Well were crafted so as to both fit within a closed glass test chamber. A sample of one of the two water absorber products was placed in the Large Sample Well and the Small Sample Well was filled with either an aliquot of an Artificial Plaque Matrix material or an aliquot of Citrated Bovine Whole Blood. The Artificial Plaque Matrix is an in-house proprietary mixture of bovine plasma, agar and egg white that is blended to have physical characteristics of dental plaque matrix. The Citrated Bovine Whole Blood was obtained from commercial sources. The water absorbents in the Large Sample Well were not allowed to directly contact the matrix or blood in the Small Sample Well, but they shared the same closed air space within the glass test chamber. The test absorbent in the Large Sample Well could absorb water from the material in the Small Sample Well only by absorption of moisture from the shared closed air space. The sealed chambers were placed at ambient room temperature for 24 hours and then the weight change of the absorbent in the Large Sample Well from water absorption and the weight change of the target material in the Small Sample Well from water loss were determined and recorded. For one of the control arms of the study, samples of HOTD and QuikClot were “Pre-Exposed” to a high humidity chamber for one day before being used in this test system. The impact of their pre-exposure to high humidity on subsequent water absorption in the sealed test chamber was evaluated as another comparison of the properties of HOTD and QuikClot.



Page 29

Exp Large Sample Well

(n=5) Mean Wgt Chg @24

Hrs Small Sample Well

(n=5) Mean Wgt Chg @24

Hrs

1 100% HOTD +10.02% Art. Plaque Matrix -89.90%

100% HOTD +13.29% Bovine Whole Blood -82.01%

2 Pre-Exposed HOTD +4.08% Art. Plaque Matrix -30.51%

Pre-Exposed HOTD +2.68% Bovine Whole Blood -22.26%

3 QuikClot® +14.38% Art. Plaque Matrix -89.79%

QuikClot® +14.52% Bovine Whole Blood -82.01%

4 Pre-Exposed QuikClot +1.56% Art. Plaque Matrix -18.60%

Pre-Exposed QuikClot +2.62% Bovine Whole Blood -14.45%

5 100% Silver Nitrate +3.86% Art. Plaque Matrix -16.36%

100% Silver Nitrate +2.68% Bovine Whole Blood -24.64%

6 Saline Control -0.91% Art. Plaque Matrix -3.43%

Saline Control -0.73% Bovine Whole Blood -2.28%

The data in the table above show that HOTD and QuikClot can absorb water from an Artificial Plaque Matrix and Bovine Whole Blood in an identical fashion in this modified hygroscopicity assay system (Experiments #1 & #3). Both the HOTD and QuikClot samples that had been Pre-Exposed to a humid environment did not absorb as much water and did not produce as much change in their target wells (Experiments #2 & #4). Note also that the pure Silver Nitrate sample demonstrated minimal hygroscopic effect in this assay. This data provides additional support for stating that the mode of action of HOTD in vivo includes tissue dehydration and that it shares a similar tissue desiccating mode of action with QuikClot.

The pictures below demonstrate the typical results seen in the comparisons of water absorption activities of HOTD and QuikClot in the protocol described above. The first row of pictures below compares the changes produced in bovine whole blood by HOTD and QuikClot from Time=0 to Time=24 Hrs (Experiments #1 & #3). Note that HOTD and QuikClot produced the same changes in the whole blood samples with obvious dehydration and detachment of the dried blood material from the small sample well surface.

Time= 24 Hrs Time= 0 Hrs

QuikClot Blood QuikClot Blood


Blood BlooHPBR HPBR



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The pictures in the next row below demonstrate the comparison of changes in the bovine whole blood produced by 24 hours of exposure to HOTD and QuikClot materials that had both been pre-exposed to a high humidity environment for a full day before the start of the 24-hour test chamber period (Experiments #2 & #4). Both the HOTD and QuikClot show reduced water absorption in this assay. The whole blood samples were not changed substantially because of the pre-exposure water absorption by both test absorbents.

The pictures in the next row below demonstrate a comparison of the changes produced in a sample of the Artificial Plaque Matrix by the HOTD and QuikClot from Time=0 to Time= 24 Hours in the test chamber (Experiment #1 & #3). Note that the changes produced by both water absorbents are consistent with dehydration of the samples and detachment from the Small Sample Well surface. Also the results with both water absorbents are essentially identical.

The last row of pictures below demonstrates the comparison of changes in the Artificial Plaque Matrix produced by 24 hours of exposure to HOTD and QuikClot materials that had been pre-exposed to a high humidity environment for a full day before the start of the 24-hour test chamber period (Experiments #2 & #4). The appearance of the Artificial Plaque Matrix was not significantly changed when the HOTD and QuikClot could no longer absorb as much water in this test chamber. This supports the proposition that the mode of action of both products includes water absorption by similar mechanisms because the results were the same for both in this test.


QuikClot Matrix QuikCloMatrix


Matrix Matrix HPBR HPBR

Time= 24 Time= 0 Hrs

QuikClot Blood QuikCloBloo

Time= 24 Time= 0 Hrs

Blood Blood HPBR HPBR


Matrix Matrix HPBR HPBR


QuikClot Matrix QuikClot Matrix



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The next experiment used a combination of the protocols from two prior hygroscopicity assays. In this experiment each of five replicate Large Sample Wells contained a known quantity of HOTD and another five contained a known quantity of QuikClot. Each of ten total Small Sample Wells contained a known quantity of the Citrated Bovine Whole Blood. As in the previous protocol, one of each of the Large and Small Sample Wells were placed together in one of the air-tight test chambers and maintained at ambient room temperature for 24 hours. After 24 hours the chambers were opened and the change in weight of the Large Sample Wells was determined as an indication of the amount of water that was absorbed from the Bovine Whole Blood by the HOTD or QuikClot. The next step in this experiment was to take all of the Large Sample Wells with the HOTD and the QuikClot from the enclosed air-tight chambers and place them in a 40°C chamber that was open to ambient humidity. The HOTD and QuikClot in these Large Sample Wells were allowed to desorb water by evaporation until each Large Sample Well returned its original weight. Then each of the HOTD and QuikClot Large Sample Wells was placed back into a sealed test chamber with a fresh sample of Bovine Whole Blood as in the first experiment and the process of incubation, weight measurement and water desorption was repeated. The entire process was repeated two more times for a total of four cycles of water absorption-desorption by each of the five HOTD Samples and five QuikClot Samples. The data is expressed as mean weight gained in the Large Sample Wells at the end of the absorption phase of each cycle. \

Cycle # Mean Wgt Chg of HOTD Mean Wgt Chg of QuikClot 1 +13.29% +14.52% 2 +10.57% +7.75% 3 +11.22% +7.24% 4 +10.01% +6.17%

The data in the table above shows that HOTD absorbs and desorbs water in a reversible pattern from the Bovine Whole Blood in a manner and on a scale that is equivalent to QuikClot. This indicates that the water absorption process for HOTD is a physical process, similar to QuikClot, and does not involve an irreversible or bond-forming reaction.



Page 32

Evaluation of the Acidification Activity and Acid-Base Reaction Potential of HOTD The terms Enamel Erosion and Dentin Erosion refer to damage to tooth enamel and dentin caused by loss of hydroxyapatite crystal from their surfaces due to the mineral dissolving action that acidic liquids have on hydroxyapatite. HOTD is comprised of aromatic sulfonic acids, sulfuric acid and water so it is appropriate to consider if its mode of action includes acidification of tooth surfaces leading to acid erosion. Acidifying protons require the availability of free water in order to be delivered from an acid into their surroundings and it is known that concentrated sulfuric acid and its derivatives are very hygroscopic and actively absorb free water. Concentrated sulfates tie up the free water in their environment in a manner which limits the release and transfer of acid protons. Concentrated sulfuric acid must be substantially diluted with water in order for it to readily acidify its surroundings. The acidifying properties of HOTD on tissue cannot be reliably predicted from these first principles, however, since it is comprised of a mixture of acids. Therefore it was necessary to perform a series of assays to evaluate whether HOTD had any acidifying activity when put in contact with tooth surfaces which would lead to acid erosion. The potential of an acidic liquid material to cause surface damage to tooth enamel and crystal is commonly determined using a well established model system based on testing samples of enamel and dentin prepared from bovine incisors. A variety of parameters can be measured as indicators of erosive damage to the test samples. Among the most sensitive indicators are measurements of: 1) free calcium ions released from tooth surface mineral, 2) changes in surface hardness as determined by micro-indentation assays and 3) changes in the microscopic appearance of test sample surfaces as seen by scanning electron microscopy. All three of these assays were used in various experiments with the bovine tooth model that were designed to evaluate the potential of HOTD to cause damage to enamel and dentin when it is applied for its intended use. In the initial studies the erosive potential of HOTD was compared to other commonly used dental products as well as to erosive materials found in foods and beverages. The impact of product concentration and free water concentration on the erosive potential of a sample of HOTD was studied in later experiments. Proper preparation of the bovine tooth test samples is a multi-step process. Whole trimmed bovine mandibles were obtained from a local meat packing company on the day of slaughter. Incisors were cut from the mandible using a diamond saw on a rotary tool. The teeth were then cleaned, processed and stored according to ISO Technical Specification 11405-2003 Dental Materials – Testing of Adhesion to Tooth Structure. Uniform cylindrical disks of the bovine incisor were prepared as the standard test specimen. These disks were prepared in a manner that left either the enamel surface from the outside of the tooth or dentin from inside the tooth exposed to the test substances in subsequent assays (see the chart on the next page to follow how the samples were harvested and prepared for analysis). Tooth sample harvest was performed with diamond blades on table saws and a diamond hole-saw on a drill press. The samples were then mounted on a plastic stick to make it easy to manipulate.



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Preparation of Bovine Enamel and Dentin Samples for Acid Erosion Testing

STEP 1

Cut Tooth Sections from Bovine Incisors

STEP 2-a

Seal Tooth Samples with Enamel Surface Left Exposed Only

STEP 2-b

Seal Tooth Samples with Dentin Surface Left Exposed Only

STEP 3

Mount Tooth Sample on Holder with Epoxy with the Exposed Surface (Either Enamel or Dentin) Facing Up for Testing



Page 34

For the Calcium Ion Release Assays the tooth samples were prepared as 5mm diameter disk-shaped pieces that were cut from the incisor buccal surface as described. They were very lightly ground on a fine grit disk in order to clean the surface while avoiding the removal of tooth mineral. In order to test HOTD on dentin, the bottom dentin surfaces of the cylindrical pieces were left exposed. After being prepped and mounted the disks of test enamel and dentin were immersed in a test erosive or test control solution for 2 minutes at 37°C. The quantity of calcium ion released from the tooth test surface into the test solution was the determined by Ion Chromatography-HPLC using a Dionex 3000 Ion Chromatography System. The results are expressed as nanomoles of calcium ion released per square millimeter of sample surface per minute of exposure time. In order to determine the changes in surface hardness caused by test material exposure, the baseline surface hardness of tooth samples can be measured prior to the acid exposure step with a micro-indentation assay method. All of the samples can then be exposed to control solutions or erosive test material solutions for varying times. After a water rinse the change in the hardness of the test sample following the exposure to the test solutions can be determined with the same micro-indentation method. The change in hardness is expressed as a percentage increase or decrease in hardness. Hardness testing data is also reported sometimes as absolute hardness numbers when one test material exposure is being compared directly to another test material exposure without the use of an untreated control sample. Both of these methods were used in the studies being reported here. All of the hardness testing was done internally with a Buehler MicroMet 5124 Micro-Indentation Hardness Tester using a Vickers indenter. The Scanning Electron Microscopy (SEM) analyses were performed with duplicate samples from both the calcium release assays and the hardness assays. The images derived from the SEM studies were compared to the calcium release data and the hardness data. The SEM samples were analyzed both as wet samples in a low vacuum mode and as sputter-coated samples in high vacuum mode using a JEOL JSM-6460 LV Scanning Electron Microscope. Results for Initial Calcium Release Assays The first experiment compared the calcium ion release produced by a 2 minute exposure of enamel and dentin to HOTD and to 10% Citric Acid solution which is marketed as a commercial root canal cleanser and etchant using the samples and methods described above. The chart below shows the results.



Page 35

The data in the chart indicates that compared to 10% Citric Acid solution, HOTD in minimally erosive of enamel and dentin as measured by the amount of calcium ion release created by the exposure. The erosive effect of HOTD on dentin and enamel appears to be about the same, while the citric acid has a much more erosive effect in general and on dentin in particular. In the next experiment the calcium ion release produced by exposure of enamel to HOTD for 2 minutes was compared to exposure to 1% lactic acid and 1% citric acid. The amount of lactic acid in a typical sample of commercial yogurt is approximately 1% and the amount of citric acid in a typical commercial orange juice product is approximately 1%, so these tests were in effect comparing an exposure to HOTD to the equivalent of an exposure to commonly encountered food acids. The data in the next chart shows that HOTD is less erosive of enamel than these two organic acids at levels commonly found in foods when using a calcium release assay to measure the effects.

0

5

10

15

20

25

Cal

cium

rele

ase

(nm

ol m

m-2

min

-1)

2-Minute Exposure to Bovine Enamel

Calcium Ion Release from Enamel Surface Mineral

HPBR

Citric Acid (orange juice)

Lactic Acid (yogurt)

0

20

40

60

80

100

120

Enamel Dentin

Cal

cium

rele

ase

(nm

ol m

m-2

min

-1)

2-Minute Exposure to Enamel and Dentin

Calcium Ion Release from Enamel and Dentin Surface Mineral

HPBR 10% Citric Acid



Page 36

In the next experiment, the hardness change produced in bovine enamel specimens was studied after exposure to HOTD and 10% Citric Acid for an extended period of time. The data in the chart below shows that exposure to HOTD produces no change in the hardness of enamel as measured in a micro-indentation assay for 30 minutes while the 10% Citric Acid solution produced a dramatic loss of hardness that began at the start of the exposure time and continued throughout the experiment.

In the final experiment in this initial group of erosion studies, the impact of exposure of HOTD and Citric Acid on dentin was evaluated in a similar manner as above using the micro-indentation method. Once again, exposure of dentin to HOTD produced no changes in hardness through the end of the experiment. However, the damage to the dentin from just 2 minutes of exposure to the 10% Citric Acid was so severe that the dentin mineral was too soft to obtain any type of micro-indentation result. The dentin material was made into a liquid and would not hold an indentation that was visible in the microscope so a Vickers number could not be determined.

0

50

100

150

200

250

300

350

400

Initial 2 minutes 5 minutes 10 minutes 15 minutes 30 minutes

Har

dnes

s (H

V)

Exposure Time

Hardness of Bovine Enamel

HPBR 10% Citric Acid

0

10

20

30

40

50

60

70

80

Initial 2 minutes 5 minutes 10 minutes

Har

dnes

s (H

V)

Exposure Time

Hardness of Bovine Dentin HPBR 10% Citric Acid



Page 37

Since the effects of HOTD on enamel and dentin were essentially undetectable by hardness testing additional experiments were done to further test the effects of extended exposure on enamel. The data in the table below demonstrates that when enamel is exposed to 10% Citric Acid it suffers significant reduction in hardness almost immediately, where as the exposure of enamel for up to 90 minutes produced no significant change in hardness as measured by the micro-indentation method.

In the next experiment the effect of pre-diluting HOTD with water on its ability to produce acid erosion effects in enamel were studied. Recall that at the beginning of this section we discussed the fact that sulfates are hygroscopic and they can limit the amount of acidification that occurs in their surroundings by the fact that they absorb the free water from the environment that would otherwise be involved in transfer of acid protons. In this experiment the same methods of sample preparation and exposure were employed. Both calcium ion release and hardness by micro-indentation were evaluated as a function of exposure to HOTD that had been serially diluted with water in an effort to determine the effect of the added water on the acidification of the surrounding area. The table below shows the results of a 1-minute exposure time.

%HOTD in Water

Ca++ Release nmol/mm2/min

Hardness Vickers #

72Hr Hygroscopicity % Wgt Gain

1% 136 292 -61 5% 609 180 -64

10% 243 198 -62 20% 199 209 -38 30% 52 213 -24 40% 41 238 -11 50% 45 329 3 60% 47 325 15 70% 46 331 25 80% 42 331 37 90% 42 n.d. 50

100% 48 339 102

Note that with increasing dilution the calcium release and hardness are unchanged until the dilution level reached approximately 30% when it starts to show an effect of the dilution. The peak of the calcium release and the minimum hardness are both at the 5% diluted sample level. In other words we see the maximum effect of acid erosion from the HOTD when the product has been diluted by water to a ratio of 20:1. The chart above

Test Material 5 Minute Exposure

10 Minute Exposure

15 Minute Exposure

30 Minute Exposure

60 Minute Exposure

90 Minute Exposure

10% Citric Acid -53% -66% -71% -72% Not Done Not Done 100% HOTD Not Done Not Done -0.7% -0.8% +0.6% -0.3%



Page 38

also demonstrates that the peak of the hygroscopic activity of HOTD is at the opposite end of the dilution scale from the erosion end of the dilution scale. This finding strongly supports the proposition that the mechanism of action of undiluted HOTD is based on absorption of water and not on an acid-base reaction. The chart below is a plot of the hygroscopic activity and the acidification activity of HOTD as a function of its concentration in water as a way of understanding its properties. Undiluted HOTD (far right of chart) has high levels of water absorption as measured by Hygroscopicity (green line) and a low level of surface acidification as measured by Calcium Release from Enamel (red line). At 5% HOTD concentration in water, as shown at the left side of the chart, the water absorption activity is almost non-existent, but at the same time tooth surface acidification activity is at its peak as measured by Calcium Release. This demonstrates that the mode of action of undiluted HOTD on the root canal surface definitely involves water absorption from the surface but not any mechanism involving acid reactions or tissue acidification

0

100

200

300

400

500

600

-‐50

-‐25

0

25

50

75

100

0 10 20 30 40 50 60 70 80 90 100

Calcium Release (nmol·∙mm2·∙min)

Hygroscopicity(% mass increase)

HPBR Concentration (% by mass)

Hygroscopicity and Acidification by Serial Dilutions of HPBR



Page 39

By way of comparison and perspective, a similar experiment to the one above was carried out with phosphoric acid due to that fact that phosphoric acid has a similar structure to sulfuric acid, phosphorous and sulfur are next to each other in the periodic table and aqueous solutions of phosphoric acid are already widely used as professional dental etchants. This table also demonstrates that Phosphoric acid with the lower levels of hygroscopicity produce more erosion as measured by calcium ion release and hardness changes. In other words, while the pattern is not identical, the properties of phosphoric acid relative to hygroscopicity and acid erosion, and the effects of water dilution, are very similar to HOTD.

Phosphoric Acid % Ca++ Release nmol/mm2/min

Hardness Vickers #

48Hr Hygroscopicity % Wgt Gain

1% 229 283 -63% 2% 277 n.d. -62% 5% 589 255 -61%

10% 930 224 -60% 20% 1113 213 -38%

30% 1081 231 -24%

35% 1241 232 -18%

40% 979 250 -13% 50% 990 263 4% 60% 512 269 18% 70% 168 288 32% 85% 80 319 46%



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Scanning Electron Microscopy in Erosion Assessment The four SEM images below compare the amount of acid erosion produced on the surface of standardized samples of bovine incisor enamel by undiluted HOTD at 9.5 mEq/gm, 35% phosphoric acid etchant gel, 9.5 mEq/gm nitric acid and 9.5 mEq/gm hydrochloric acid. The erosive materials were applied for varying amounts of time based on the amount of erosion anticipated for each product. Undiluted HOTD produced essentially no visible erosion after 2 minutes of exposure. Exposure of the enamel for 15 seconds to a 35% phosphoric acid etchant generated enough erosion to reveal the outlines of the enamel rods on the surface of the tooth. Exposure of the tooth to nitric acid and hydrochloric acid at concentration levels of 9.5 mEq/gram for 1 minute produced a substantial amount of erosion as indicated by the dramatic changes in the appearance of the enamel surface and exposure of the enamel rods as shown in the pictures below. This data demonstrates that the hygroscopic properties of the components used to make HOTD result in a product that is far less erosive than other mineral acids at a comparable concentration.



Page 41

The SEM images in the panel below were taken from the bovine enamel specimens that were used for the HOTD water dilution series studied by calcium ion release as described in the table above. The pictures are labeled sequentially according to the concentration of the HOTD that was used on that specimen. The sample that was exposed to the lowest concentration/highest dilution of HOTD is labeled as Picture #1 and the sample that was exposed to the highest concentration/lowest dilution of HOTD is labeled as Picture #12. Each picture label also shows the total acidity level of the diluted HOTD sample that was used in the testing of the sample in that picture. For example, the 0.095 mEq/gm label on Picture #1 means that 0.095 mEq/gm was the total acid level of the diluted HOTD that was applied to the sample in the picture before the SEM picture was taken. Finally each label has in parentheses the percentage of HOTD in the dilution that was applied to the sample. The erosion of enamel by serially diluted HOTD as measured by calcium ion release was not substantial in this study until the dilution was at or below the 10% HOTD concentration where the total acidity level of the HOTD is at 0.95 mEq/gram (Picture #3 below). The calcium release peaked with exposure to a dilution of the product at 5% concentration and that level appeared to produce the most substantial changes in the appearance of the enamel surface by SEM (Picture #2 below). Erosion was not seen by SEM of the enamel surface after exposure at the highest concentration levels of the product (Picture #12 below). These SEM findings are consistent with the calcium release data and the hardness testing data. Undiluted HOTD does not appear to acidify its environment because it is not seen to produce acid erosion of tooth surfaces. When HOTD is significantly diluted with water it readily produces acidification of its environment as indicated by the erosive changes it produces on tooth surfaces.



Page 42

The SEM images in the panel below were taken from the bovine enamel specimens that were used for the study of the effect of HOTD dilution on hardness testing that is described in the sections above. The pictures are labeled sequentially according to the concentration of the HOTD that was used on that specimen as was described for the pictures in the panel on the previous page above. The sample that was exposed to the lowest concentration/highest dilution of HOTD is labeled as Picture #1 and the sample that was exposed to the highest concentration/lowest dilution of HOTD is labeled as Picture #10. In the micro-indentation assay data, the erosion of enamel by diluted HOTD was again not substantial until the dilution was below the 10% HOTD concentration level where the total acidity is at the 0.95 mEq/gram level or lower. The microindentation assay peaked with exposure to a dilution of the product at 5% concentration and that level appeared to produce the most substantial changes in the appearance of the enamel surface by SEM (See Picture #2). Erosion was not seen by SEM of the enamel surface after sample exposure at the highest concentration levels of the product (See Picture #10) where the total acidity level is 9.5 mEq/gm.



Page 43

The SEM images in the panel below were taken from the bovine enamel specimens that were exposed to serial dilutions of pure sulfuric acid at dilution levels that were comparable to the dilution levels of HOTD in total acidity that were evaluated above. Once again the pictures are labeled sequentially according to the concentration of the sulfuric acid that was used on that specimen. The sample that was exposed to the lowest concentration/highest dilution of sulfuric acid is labeled as Picture #1 and the sample that was exposed to the highest concentration/lowest dilution of HOTD is labeled as Picture #10. The erosion of enamel by diluted sulfuric acid appeared to peak with exposure to a dilution of the product at the concentration 0.95 mEq/gram total acidity. That level appeared to produce the most substantial changes in the appearance of the enamel surface by SEM including the appearance of calcium sulfate crystals on the enamel surface. Erosion was not seen by SEM of the enamel surface after exposure at the highest concentration levels of the sulfuric acid as was true at the highest concentrations of HOTD.



Page 44

In the next series of three SEM studies, samples of bovine dentin were exposed to serial dilutions of HOTD and sulfuric acid as was done with the bovine enamel samples in the previous series of experiments. Calcium ion release and hardness testing was not done on these dentin samples since the impact of HOTD dilution in those assays was already done with enamel in the previous study set and that data was felt to be sufficient to understanding the significance of HOTD dilution on the acidification properties of the material. The samples of dentin in this study were prepared as the standard disks. As with the prior examples, the pictures are labeled sequentially according to the concentration of the HOTD that was used on that specimen. The sample that was exposed to the lowest concentration/highest dilution of HOTD is labeled as Picture #1 and the sample that was exposed to the highest concentration/lowest dilution of HOTD is labeled as Picture #12. Each picture label also shows the total acidity level of the diluted HOTD sample that was used in the testing of the sample in that picture. For example, the 0.095 mEq/gm label on Picture #1 means that 0.095 mEq/gm was the total acid level of the diluted HOTD that was applied to the sample in the picture before the SEM picture was taken. Finally each label has in parentheses the percentage of HOTD in the dilution that was applied to the sample. Once again the images show that the greatest acidification/erosion effect happens at product dilutions greater than 10% (Pictures #3, #2 and #1). At the higher HOTD concentration there is no apparent erosion of the surface mineral (Picture #12)



Page 45

The study results depicted in the next panel of SEM images below was from an experiment that was conducted in a manner similar to the dentin study described immediately above. One difference between the studies was that in this case the samples that were used were not the uniform sample disks as described previously, but rather much larger square pieces of bovine tooth with a larger exposed area of dentin than was available on the disk-shaped samples. The purpose of changing the sample type was to be able to be able to inspect a greater surface area in the SEM than was available with the standard disk samples and in that way to get more and better information about the effects of the HOTD dilutions on dentin. These SEM images again show the expected changes in erosive properties of the HOTD with dilution. Minimal erosive effects are seen in Picture #10 where the HOTD that was applied to the surface was undiluted. The images taken from samples that were exposed to the HOTD at higher dilutions showed erosion (Pictures #1 and #2) and calcium sulfate crystal formation from surface erosion (Picture #3).

#1–0.095 mEq/g(1%)

#2–0.475 mEq/g(5%)

#3–0.95 mEq/g(10%)

#4–1.9 mEq/g(20%)

#5–2.85 mEq/g(30%)

#6–3.325 mEq/g(35%)

#7–3.8 mEq/g(40%)

#8–4.75 mEq/g(50%)

#9–5.7 mEq/g(60%)

#10–9.5 mEq/g(100%)



Page 46

The experimental results in the panel of pictures below is taken from a study that is a parallel study to the one documented above where the effect of serial dilutions of sulfuric acid where applied to bovine enamel samples in preparation for SEM examination. In this study sulfuric acid samples were prepared at dilutions in water so that they had a total acidity level that was the same as the comparable levels of HOTD. The SEM images are labeled as described above in the other SEM picture sets. Once again the effect of dilution on the ability of sulfuric acid to acidify and erode the dentin surface is demonstrated in this image set. There are minimal erosive effects from sulfuric acid at the highest concentration (Picture #10) which is equivalent in total acid level to undiluted HOTD. The most dramatic erosive effects are seen at the highest water dilutions (Pictures #3, #2 and #1).



Page 47

Summary of the HOTD Enamel and Dentin Erosion Studies

The calcium ion release test data, the hardness test data and the SEM images all confirm that undiluted HOTD is minimally erosive of enamel and dentin which means that this material will not harm hard tissue surfaces in the oral cavity. The data also show that significant dilution of the HOTD material is required in order to make the product behave in an erosive manner. This data not only demonstrates the safety of the HOTD product, but it also supports the idea that the subordinate mechanism of action of HOTD which leads to its enhanced ability to remove dental plaque and tissue debris is based on water absorption and not an acid-base reaction of any kind.



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Exothermia on Solvation and Neutralization of HOTD

In this set of experiments a large excess of HOTD, more than 200x a typical application amount, and equivalent amounts of Sulfuric Acid NF were applied repeatedly to freshly cut surfaces of calves liver. The temperature increase on the surface of the tissue over background was monitored using an IR video camera with temperature measurement capabilities. Two screen shots with examples that are typical of the results obtained from this experiment are shown below. The data from this experiment indicates that the solvation and neutralization of HOTD by contact with tissue is exothermic. HOTD contact raises the temperature of the tissue wherever direct contact occurs.

In the next set of experiments the heat release generated by the solvation and neutralization of HOTD was compared to Sulfuric Acid NF in a Parr Solution Calorimeter using standard techniques at room temperature. In the solvation-only experiments, the temperature rise was measured as a test sample was introduced into deionized water only. In the solvation-neutralization experiments, the temperature rise was measured as the test sample was introduced into a 0.25 M Sodium Hydroxide solution which contained an excess of neutralizing base. The quantitative data indicate that in this test system, solvation only and solvation/neutralization of HOTD is substantially exothermic, but only a fraction as much as with Sulfuric Acid NF in the same test system

Sample Solvation only

Solvation & Neutralization

Sulfuric Acid NF (97% Purity)

208.8 Cal/gm SA

577.4 Cal/gm SA

HOTD (9.5 mEq/gm HSO3

-) 37.8

Cal/gm OM 172.8

Cal/gm OM

In this screen shot from a similar video of the surface of the calves’ liver you see the white area at the center of the field where a large excess of HPBR was applied. This time the data column at the far left of the screen indicates that the baseline temperature of the liver surface was 71.6°F (min) and the maximum temperature achieved was 88.4°F (max) following the application. This indicates that solvation and neutralization of HPBR by tissue causes heat to be delivered into the tissue, but obviously, not anywhere near as much as with the application of pure sulfuric acid.

In this screen shot of the surface of the calves’ liver you see the white area at the center of the field where a large excess of Sulfuric Acid NF was applied. The data column at the far left of the screen indicates that the baseline temperature of the liver surface was 71.6°F (min) and the maximum temperature achieved was 172.5°F (max) following the application. This demonstrates that solvation and neutralization of sulfuric acid by contact with animal tissue is exothermic and can raise the temperature of tissue wherever the sulfuric acid is applied.



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SAFETY EVALUATION

This section summarizes the studies that were done for safety evaluation of HOTD. It includes standardized biocompatibility testing, animal testing and clinical trials of two EPIEN Medical products that are very similar to HOTD and have related formulations. The content of this safety evaluation is consistent with what is required for products that are used infrequently on any individual patient and applied topically for a very limited time with minimal if any product absorption. Canine Oral Mucosa Metabolism and IV ADME OF C14-labeled HOTD Two separate studies were carried out in beagle dogs to evaluate the metabolism of HOTD in standard canine model systems. C14-labeled HOTD was prepared by GE Medical using C14-labelled hydroxybenzene and C14-labeled hydroxymethoxybenzene. The studies were conducted by MPI Research, Mattawan, MI. The first study was designed to test whether or not HOTD is metabolized by oral soft tissues. After the dogs were anesthetized replicate lesions were created in the oral mucosa of two dogs using a skin punch biopsy tool to create 4 mm diameter by 1 mm depth cavities in the buccal mucosa surface. An aliquot of the C14-labelled HOTD was placed into the punch biopsy sites of each dog with a syringe and blunt cannula. The heads of the dogs were positioned such that they would retain HOTD in the wound site for an extended period of time without spillage. The material was maintained on the wounded tissues for 15 minutes in the first dog and for 30 minutes in the second dog. Following the HOTD contact period, the dogs were sacrificed and the oral wound tissue that contained the C14-labelled HOTD and some surrounding healthy mucosal tissue were harvested with a wide and deep scalpel incision. The C14-labelled HOTD in those tissues was recovered by standard tissue homogenization and extraction methods. The recovered material was analyzed by a Reverse Phase-HPLC chromatography method with a radioactivity detector. The chromatogram of the C14-labeled material that was recovered from the mucosal wounds was compared to a chromatogram of fresh C14-labelled HOTD product that had not been put on tissue. The chromatograms were evaluated for peak patterns that would indicate the presence of newly formed C14-labeled metabolites of HOTD. The chromatograms demonstrated that the peak patterns and peak retention times for the two chromatograms were identical. This demonstrates that there was no metabolism of HOTD occurring in the surface mucosa or submucosa of the tissue from the wound sites. If there had been any metabolism at the wound site, new compounds would have been formed and these would have been seen as peaks with different shapes, different patterns and with different retention times from the ones in the control materials.



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This is a chromatogram of fresh C14- HPBR that has not been on tissue. This chromatogram demonstrates the usual peak pattern of the sulfonated components of HPBR analyzed by Reverse Phase HPLC with a Hamilton PRP-X100 column. These peaks were detected by a radioactivity detector. The large peaks at the start and end of the pattern are hydroxybenzenesulfonic acid isomers (HBSA). The middle three peaks are hydroxymethoxybenzenesulfonic acid (HMBSA) isomers.

This chromatogram is representative of the Reverse Phase HPLC analyses of the C14-labeled material that was recovered from the C14-HPBR-treated oral mucosa wound sites after 15 and 30 minutes of labeled product contact with the mucosal and submucosal tissues. The peaks seen in this chromatogram show a pattern that is identical to the original C14-labeled HPBR product as shown in the chromatogram above. This indicates that HPBR is not metabolized by soft tissues of the oral cavity.



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The second study in this set consisted of determining absorption, distribution, metabolism and elimination (ADME) kinetics following an intravenous injection of acid-neutralized C14-labelled HOTD in three beagle dogs by standard techniques. The first table on the left below shows the whole blood levels of C14-labelled HOTD over time in one dog. The other two dogs demonstrated a similar pattern. The second table on the right below summarizes the % recovery of radiolabel from the various components of elimination. The data in the two tables indicates that the majority of the radiolabeled material is very rapidly cleared from the blood of the three dogs via the kidneys and into the urine. Trace amounts continue to be excreted in a multiphase pattern of excretion that is seen with products that are cleared by the kidneys

% Recovery of Label at Elimination Components Sample Time 101M 102M 103M Mean Cage Rinse 8 hr 1.44 4.02 1.8 2.42

Cage Rinse 24 hr 1.27 9.09 0.46 3.61

Cage Rinse 48 hr 0.11 0.84 0.07 0.34

Subtotal 2.82 13.96 2.33 6.37 Cage Wash 72 hr 0.14 0.41 0.11 0.22

Cage Wipe 72 hr 0.23 0.9 0.19 0.44

Feces 0-8 hr 0 0.2 *0.00 0.07 Feces 8-24 hr 1.03 0 1.09 0.71 Feces 24 - 48 hr 0.39 0.77 0.9 0.69 Feces 48 - 72 hr 0.17 0.08 0.11 0.12

Subtotal 1.6 1.06 2.1 1.58 Urine 0-8 hr 87.34 61.58 83.56 77.49 Urine 8-24 hr 5.41 9.7 6.52 7.21 Urine 24 - 48 hr 0.82 1.71 0.4 0.98 Urine 48 - 72 hr 0.24 0.34 0.3 0.29

Subtotal 93.8 73.33 90.79 85.97 Total 98.67 89.73 95.62 94.67

WHOLE BLOOD LEVELS Canine Subject Time Conc. (µg/g)

101M Predose 0 101M 5 min 5.78 101M 10 min 4.21 101M 20 min 2.84 101M 40 min 1.76 101M 1 hr 1.22 101M 2 hr 0.55 101M 6 hr 0.14 101M 24 hr 0.04



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CANINE VITAL PULP HOTD EXPOSURE STUDY This study is an assessment of the effects of exposing vital pulp tissue to contact with HOTD in a canine model of Vital Pulp Exposure. In this study, three dogs were used in total. Following standard protocols for Vital Pulp Exposure studies established in the dental literature, 16 teeth in each dog had a vital pulp exposure created by drilling a hole into the buccal surface using a high-speed handpiece and dental bur until hemorrhage was seen. Eight pulp exposures in teeth in the right mandible and maxilla in each dog were treated using a standardized regimen consisting of an application of a commercial calcium hydroxide product (Dentsply Dycal) followed by restoration with a commercial zinc oxide/eugenol cement (Dentsply IRM). The eight left side exposures in each dog were treated with an application of HOTD for one minute, followed by a one minute saline rinse, followed by restoration with the same commercial zinc oxide/eugenol cement. No calcium hydroxide was used on the HOTD test exposures on the left side lesions. One dog was euthanized and the teeth on which the experimental procedures were performed were collected for analysis at 7 days post treatment. Another dog was processed the same way at 21 days post treatment and the third dog at 60 days post treatment. The teeth were prepared for analysis at the University of Minnesota Dental School using a so-called “hard tissue” technique in which histologic sections of teeth can be prepared for microscopic analysis without using a specimen decalcification process. This technique preserves more histological detail of the tissue than the standard decalcification methods, but it requires extensive additional processing steps and much more time to complete A histological analysis of the study sections, specifically comparing and contrasting the appearance of the tissue from the two treatment groups at the various time points was performed by Dr. Michael Rohrer, Director of the Division of Oral Pathology, University of Minnesota School of Dentistry. Dr. Rohrer evaluated each section for the appearance of tissue damage, such as necrosis and inflammation, as well as looking for evidence of tissue repair and new tooth tissue growth. The key findings of the report were:

1) 25% of the pulps from the standard calcium hydroxide treatment group were devitalized while the none of the pulps treated with HOTD were devitalized

2) The HOTD treatment group showed earlier formation of new replacement dentin at the site of the exposure injury than the calcium hydroxide treatment group

Vital pulp is a very sensitive tissue as indicated by the number of pulps in this study that became devitalized by contact with the calcium hydroxide as part of the standard control treatment regimen. The fact that no pulps were devitalized by contact with HOTD in any of the dogs indicates that HOTD should be safe for use in any dental procedure that may put the product in contact with any sensitive, inflamed or damaged oral cavity tissues.



Page 53

The fact that the HOTD -treated pulps supported more rapid dentin repair indicates that HOTD did not damage the ability of the pulp repair cells to repair the pulp exposure site. The pictures above illustrate the procedures used and the type of histological specimens that were available for analysis in the Vital Pulp Exposure study. The three photographs across the top show the key steps in this experimental procedure. In picture 1 a burr hole has just been drilled into a tooth until bleeding is apparent. The bleeding indicates penetration of the hole into the pulp chamber and exposure of vital pulp. In picture 2 HOTD is placed into a burr hole for one minute with an irrigation syringe and small irrigation applicator tip. After one minute the HOTD was rinsed from the burr hole with saline using an identical irrigation syringe. Picture 3 shows the burr hole after it was filled with a zinc oxide-eugenol temporary restoration material. The two microphotographs in the lower part of the box above show two of the histological sections that were prepared, one from a tooth that was harvested at 7 days post procedure and the other that was harvested at 60 days post procedure. The burr hole filled with the restoration material (black substance) is visible in the 7-day specimen on the left. The labels indicate the key features in this slide. Note that there is minimal evidence of any repair tissue forming to cover over the burr hole in the slide on the left from the tooth collected at 7 days post procedure. Contrast that feature with the slide on the right from tissue that was collected at 60 days post burr-hole formation. In the 60-day slide there is an abundant amount of new dentin that has formed to repair the damage to the tooth. The new dentin at this stage appears as clusters of spheres that stain orange-brown with this particular histological preparation method.



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CANINE PERIODONTAL TISSUE HOTD EXPOSURE STUDY This study was conducted in a canine model system to evaluate the reaction of external tooth surfaces, gingival tissue and tissue in a periodontal sulcus to contact with HOTD. Twelve beagle dogs with periodontal disease ranging from moderate to severe were divided at random into three treatment groups. All dogs received their group-specific treatment on the same day. The four dogs in the first group received a one-time ultrasonic supragingival and subgingival scaling treatment only with a Cavitron ultrasonic scaling instrument. The four dogs in the second group of the study received the same one-time ultrasonic scaling treatment as the dogs in the first group, but immediately after the scaling procedure was completed they also received an adjunctive one-time application of HOTD. The product was delivered with an irrigation syringe to the entire surface of each tooth, to tissue along the gingival margin and into any periodontal pockets that were identified. The product was in contact with the teeth and soft tissues for 60 seconds and was then water rinsed and evacuated away. The four dogs in the third group received treatment consisting of a one-time application of HOTD only. They did not receive any scaling treatment. The HOTD was applied to these dogs as it was to the dogs in the second group. One dog from each treatment group was euthanized and the study tissue was collected at four different time points: 1) immediately post-treatment, 2) at 4 days post-treatment, 3) at 2 weeks post-treatment and 4) at 4 weeks post-treatment. The study tissue that was collected consisted of the complete mandible and maxilla with the teeth and attached gingival tissue in place. The tissue specimens were submitted for processing and analysis to the Hard Tissue Research Lab at the University of Minnesota School of Dentistry. Tissue specimens for histological analysis were harvested in two ways. In the first method teeth with periodontal lesions on the buccal or lingual side of a tooth were harvesting by cutting through the jaw bone, soft tissue and tooth in a buccal-lingual orientation through the center of the lesion. In the second method pairs of teeth with a lesion in the interproximal space were identified and the teeth harvested by cutting through the jaw bone, tooth and soft tissue in such a way as to go through the middle of the interproximal lesion and the tooth on either side in a mesial-distal orientation. All harvested sections were then processed by the “hard tissue” method without decalcification as described for the Vital Pulp Exposure Study above. A histological analysis of the tissue sections, specifically comparing and contrasting the appearance of the periodontal tissues in the three treatment arms at the various time points was performed by Dr. Michael Rohrer, Director of the Division of Oral Pathology, University of Minnesota School of Dentistry. The key findings of the report stated that:

1) The treatment of tooth surfaces with HOTD appeared to aid in the removal of dental plaque by histological examination.

2) There was no histological evidence of any kind that application of HOTD



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caused any harm of any kind to the soft or hard tissues of the oral cavity wherever it was applied.

The three pictures across the top of the box above illustrate the technique used to treat the dogs in the third treatment group of the study – the dogs that were treated with an application of HOTD, but did not received ultrasonic scaling. The top picture on the left shows a dog with severe periodontitis before any treatment. The teeth show a very significant accumulation of calculus and there is widespread inflammation of the gingival tissues. The middle picture shows the application of HOTD. The HOTD was allowed to be in contact with the tooth surface and with the periodontal pocket tissue for about 1 minute before it was rinsed away with saline. The picture on the top right shows the same view of the same dog four days later. There is grossly apparent improvement and some visible healing. There is no grossly apparent damage from the HOTD on the teeth or soft tissue. The two lower pictures show two views of the same tissue section. This is a section cut with a buccal-lingual orientation through gingiva, tooth and jaw bone showing an area that was irrigated with HOTD four days before tissue harvest. The section viewed with normal light shows no evidence of ongoing pathology with no apparent necrosis or inflammation. The image on the lower right is the same section as on the lower left but viewed with polarized light. The polarized light view allows for more sensitive detection of new bone growth and remodeling in these specimens.



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HOTD BIOCOMPATIBILTY ASSESSMENT

In order to assure the biocompatibility of HOTD five biocompatibility tests were performed in accordance ISO 10993-1 standards. Testing was conducted by AppTec Laboratory Services, St. Paul, Minnesota, in accordance with Good Laboratory Practices. HOTD by design contains sulfuric acid and is potentially caustic, especially when diluted with water. Sulfuric acid at the concentrations used in the HOTD formulation are not suitable for testing under the routine conditions stipulated in some of the protocols for the required biocompatibility assays because the acid in the product could become irritating to tissues and/or damaging to tissue culture cells. This would make the results of the biocompatibility tests into a potential demonstration of the corrosive potential of sulfuric acid, but they would definitely not be an accurate assessment of the biocompatibility of the materials. In other words, the biocompatibility of a product is unrelated to the caustic potential of a product, but the caustic properties of the product could interfere with biocompatibility testing because caustic activity and non-biocompatibility can give the same results in some of the biocompatibility assays. In order to assess the biocompatibility of the material under the stipulated conditions of the required assays, a sample of acid-neutralized HOTD was prepared. An aliquot of HOTD was diluted with water then neutralized with a sodium hydroxide solution to pH 7.0. In effect this sample contained the acid anions of HOTD as a solution of the sodium salts instead of the acid form and the subsequent testing measured the biocompatibility of the sodium-acid anion product derived from the neutralization procedure. Although this procedure is not a direct assay of the intact HOTD this approach was necessary in order to separate the corrosive behavior of the product from any potential biocompatibility problems so that any biocompatibility could be identified. SUMMARY OF THE BIOCOMPATIBILITY TEST RESULTS FOR HOTD CYTOTOXICITY HOTD was tested for cytotoxicity using the ISO Agarose Overlay using L-929 Mouse Fibroblast Cells method. Under the conditions of the study, HOTD was scored at “1” which is considered to be non-toxic under the conditions of this test. SENSITIZATION HOTD was tested for allergenic potential or sensitization using the ISO Guinea Pig Maximization Sensitization Test method. Under the conditions of the study, none of the animals treated with HOTD were observed with a sensitization response greater than zero. None of the negative control test animals challenged with control vehicle was observed with a sensitization response. The test article was found to be non-sensitizing.



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MUTAGENICITY HOTD was tested to evaluate the mutagenic potential using the Bacterial Mutagenicity Test – Ames Assay. The plate incorporation assay was conducted using Salmonella typhimurium strains. The test article was assayed as a series of dilutions. Under the conditions of the study, the test article did not induce substantial increases in reversion rates of the type that are associated with mutagenesis. No substantial test article toxicity was noted that may have interfered with the detection of mutagens. HOTD was determined not to have caused an increase in point mutations, exchanges or deletions. HOTD is considered non-mutagenic. IRRITATION HOTD was tested for skin irritation using the ISO Primary Skin Irritation test method. Skin irritation testing was conducted using adult albino rabbits. Under the conditions of the study, HOTD was found to be a non-irritant. The primary irritation index scores for the test article were determined to be negligible INTRACUTANEOUS REACTIVITY HOTD was tested to determine if any chemicals leeched from the product cause dermal irritation using the ISO Intracutaneous Reactivity test method. Testing was conducted using adult albino rabbits. Under the conditions of the study, HOTD was found to be a non-irritant. The difference in the mean test and control scores of the dermal observations were less than 1.0.



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REVIEW OF LITERATURE REFERENCES ON HOTD SAFETY ISSUES

Potential Caustic Properties of HOTD

Jelenko C: Chemicals the “Burn” J Trauma 14(1):65-72, 1974. This paper discusses the meaning of the term “chemical burn” as it applies to various classes of caustic chemicals. The point made is that the meaning of “burn” is different depending on the chemical that one is discussing as various chemical groups can damage tissues by interacting with specificity with different chemical groups. Sulfuric acid is noted to be a potent desiccant which produces a hard eschar of dehydrated, coagulated, necrotic tissue on contact through intense desiccation whose size and depth depend on dose and duration. Penner GE: Acid Ingestion: Toxicology and Treatment Ann Emerg Med 9:374-379, 1980. This paper discusses the effects of the ingestion of various acids on the esophagus and stomach. Sulfuric acid produces a coagulation necrosis which leaves shallow hard eschars on the surface of the esophagus that tends to not cause long term problems. Depending on the study, 6% to 20% of acid ingestions reported have lead to the formation of an esophageal lesion. Ingestion of large quantities of sulfuric acid is known to have lead to the formation of gastric damage including ulceration of the gastric wall. The pathophysiology of sulfuric acid lesions in the stomach is thought to include the damage caused by the exothermia generated by the hydration and neutralization of large quantities of sulfuric acid in the gastric space. Salzman M, O’Malley RN: Updates on the Evaluation and Management of Caustic Exposures Emerg Med Clin N Am 25:459-476, 2007. This article provides an update on the topic of ingestion of caustic substances. The factors that have been identified as the key factors in the determining the risks of injury from a caustic ingestion include the pH, the total amount of titratable acid, the physical state, the tissue contact time, the total quantity of material and the concentration of the caustic materials in total. The damage to the esophagus and stomach depend on the material and the quantity and the contact time. The damage can range from nothing to extensive tissue necrosis depending upon the above noted factors.



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Potential Genotoxicity/Toxicology of HOTD Hamaguchi F, Tsutsui T: Assessment of Genotoxicity of Dental Antiseptics

Jpn J Pharmacol 83:273-276, 2000. Investigators studied a variety of dental liquid agents to find out if any of them would induce unscheduled DNA synthesis (UDS) in a model system using Syrian Hamster embryo cells. This UDS model has been proposed as one model system to help determine which dental compounds may be genotoxic and possibly carcinogenic. One of the compounds tested was p-phenolsulfonic acid solution. P-phenolsulfonic acid is one of the products of one of the sulfonation reactions that are used to make HOTD, the sulfonation of hydroxybenzene. P-phenolsulfonic acid is therefore a component of HOTD. This solution is used by dentists in Japan for endodontic procedures. It is said to help to open up root canal systems by irrigation. The study showed that p-phenolsulfonic acid did not induce UDS.

Yamaguchi F, Tsutsui T: Cell-Transforming Activity of Fourteen Chemical Agents Used in Dental Practice in Syrian Hamster Embryo Cells

J Pharmacol Sci 93:497-500, 2003. This article describes a similar study to the one reported immediately above and it was done by the same research group in Japan. In this study p-phenolsulfonic acid did not cause cell transformation of any kind in the SHE cell model system.

Miyachi T, Tsutsui T: Ability of 13 chemical agents used in dental practice to induce sister-chromatid exchanges in Syrian hamster embryo cells Odontology 93:24-29, 2005.

This article describes a similar study to the two reported immediately above and it was done by the same research group in Japan. In this model system, p-phenolsulfonic acid was said to induce sister-chromatid exchange. The significance of this finding was questioned however because 11/13 of the tested materials gave the same results. Also, a simple increase in the ion concentration in the tissue culture solution of the SHE cells was also noted to induce sister-chromatid exchange. Therefore the significance of these findings relative to the potential genotoxic properties of p-phenolsulfonic acid is questionable.

Hagiwara M, Watanabe E, Barrett JC, Tsutusui T: Assessment of genotoxicity of 14 chemical agents used in dental practice: Ability to induce chromosome aberrations in Syrian hamster embryo cells.

Mutation Research 603:111-120, 2006. This article describes a similar study to the three reported immediately above and it was done by the same research group in Japan. In this study the p-phenolsulfonic acid did not induce any chromosomal aberrations of any kind in the SHE model system.



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CONCLUSIONS ABOUT HOTD DERIVED FROM THE SAFETY EVALUATION

1. Consideration of HOTD as a Caustic Agent HOTD is essentially sulfuric acid in a viscous matrix. The acid level of HOTD at 9.5 mEq/gram is just less that ½ the acid concentration of Concentrated Sulfuric Acid ACS. Since the product acts by desiccation, the acid level by itself is not the key factor, but rather the intensity of the desiccation that is produced by that acid level. Damage to the gastrointestinal tract by ingestion of sulfuric acid requires a variable but significant amount of material. In most cases of sulfuric acid ingestion there is no damage to the GI tract. If damage does occur it is most often in the form of small shallow hard eschars on the esophageal surface that heal on their own with no specific treatment. Given that application of HOTD will be in very small quantities and the application will be controlled by trained dental practitioners that likelihood that harm will occur from caustic damage to healthy tissues by a misapplication of the product is very small.

2. Consideration of HOTD as a cytotoxic, genotoxic or mutagenic agent There is a very limited amount of information available in the scientific literature about the cytotoxic, genotoxic and mutagenic properties of the components of HOTD. Sulfuric acid has long been suspected of being a carcinogen in situations with a long term exposure, such as in an industrial setting, but definitive data is lacking. The only available literature that was found on studies of the genotoxicity of a component of HOTD was the data in the four Japanese articles reviewed above that included information about p-phenolsulfonic acid solutions. One study showed a potential genotoxic effect but for a variety of reasons the significance of that data is unclear. Standardized biocompatibility data results indicate that the product is safe.



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EFFICACY EVALUATION

As noted in the Introduction to this Design History HOTD was not designed as a therapy for a specific disease. Its intended use is not tied to any particular clinical treatment outcome. HOTD was designed as a supplemental rinse and debriding agent for enhanced cleansing of dental plaque and other infectious matter from clinically important surfaces in the oral cavity as needed. HOTD works in a different manner from typical oral rinses. It actively detaches pathological material from its substrate during the rinsing application by exerting a superficial denaturing action onto oral surfaces that aggressively assists the mechanical action of the product in removing the targeted material. HOTD is an active cleanser that lifts infectious materials and biofilm off of the tissues whereas the other types of dental rinses just passively carry away only the material that has already been mechanically detached and is lying loose on the tissue surfaces. A significant technical challenge in the HOTD design process resulted from the decision to develop the product as a general purpose tool to assist in tissue cleansing in all types of dental procedures. It was not practical to do a traditional clinical study using formal clinical endpoints to demonstrate the safety and efficacy of HOTD in all of the various standard dental procedures in which it might be applied. A different approach was taken that demonstrated the efficacy of HOTD as a plaque biofilm cleanser through a limited series of studies based upon direct observation using various imaging techniques that documented the cleanser action of HOTD. Two pilot clinical studies of Epien products with formulations related to HOTD were performed to evaluate the exposure of the product to human mucosal and periodontal tissues by limited measurement of traditional short term clinical outcomes.

Summaries of the anti-plaque efficacy studies are presented below.



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DISCLOSING SOLUTION DEMONSTRATION A screening assay for anti-plaque activity was performed with HOTD using freshly harvested specimens of the anterior portion of bovine mandibles with incisors and a standard dental disclosing solution. The results were determined by observing staining by the disclosing solution. The image below shows a photo of a full set of bovine incisors before any exposure to the disclosing solution. The photo below shows the same specimen after exposure to a disclosing solution followed by a water rinse.

The picture below shows the result of treating the stained teeth that were on the right hand side of the picture above with HOTD for 2 minutes followed by a water rinse. The photo in the last row below shows the same specimen above after it has been re-stained with disclosing solution for 20 minutes. Note that the teeth which had been treated with HOTD do not pick up the disclosing solution stain as they did prior to the HOTD treatment.



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A similar disclosing solution evaluation was then performed on human subjects. The pictures below outline the results of one of the treatment procedure. The picture above on the left shows incisors pre-treatment. On the right side a standard application of dental disclosing solution is applied and then rinsed with water. The picture above on the left shows the results of disclosing solution application followed by a water rinse on the mandibular incisors. The right side picture shows the application of HOTD. The product was applied to the surface of the teeth with an irrigation syringe equipped with a plastic applicator tip. After approximately 1 minute the product is rinsed away with water and evacuated. The picture above on the left shows that the HOTD application removed the disclosing solution stain, presumably by removing the dental plaque itself. The picture on the right shows a reapplication of the disclosing solution stain post-HOTD rinse. The picture above shows the results of attempting to re-disclose the dental plaque. The surface did not re-stain because the plaque was removed by the HOTD treatment.



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It is important to note that HOTD is not intended to be used as a primary plaque removing agent by itself in the manner shown in this demonstration. It is intended to be used as an adjunctive to other dental procedures and other cleansing methods. The results displayed in the evaluations noted above do not represent an example of a clinically indicated application of this product. They were provided here simply to demonstrate the efficacy of the product as a cleanser through a simple direct observation technique.



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INNOVOTECH MBEC ASSAYS FOR ANTI-PLAQUE ACTIVITY Innovotech Laboratories in Canada provides a unique testing platform that was designed to screen materials for anti-biofilm activity. In their system pure strain microbial biofilms are grown on plastic pegs that are mounted on the inner surface of the cover of a 96-well plate so that one peg lines up with each of the 96 wells when the cover is in place on the plate. In order to grow a biofilm on each peg the wells of the plate are filled with the appropriate microbial growth media and inoculated with a microorganism that is known to form a biofilm. The cover of the plate is put onto the plate in such a way that one of the pegs projects into the inoculated media in each one of the wells. After a period of time a microbial biofilm will form over the surface of the peg. Once the biofilms have grown sufficiently thick on the pegs they can be lifted from the wells by lifting the cover. The cover is rinsed after it is lifted from the plate and then it is placed on a second plate that has its wells filled with control solutions and test materials that are to be screened for anti-biofilm activity. After a suitable incubation period, the pegs are removed again by lifting the cover. The individual pegs are collected by snapping them from the underside of the plate cover. They can then be processed and analyzed in two different ways. First, they can be fixed for examination by scanning electron microscopy. Second, they can be sonicated to first disrupt the biofilm from the pin and the remaining viable bacteria from the pin can be quantified by standard microbiological quantitative culture techniques. Since dental plaque is now known to be a form of microbial biofilm, it was decided to test the anti-biofilm activity of HOTD in the Innovotech system as a screen of its anti-plaque properties. The proposition was that any material that was effective against pure strain biofilms in the Innovotech assay may work well as an anti-plaque agent against polymicrobial dental plaque in vivo. Innovotech first evaluated the activity of the HOTD with biofilms from several strains of Pseudomonas aeruginosa. Then they repeated the assays with biofilms of two fungi, Aspergillus niger and Candida tropicalis. The SEM studies showed that HOTD produced a change in the appearance of the biofilms that is consistent with desiccation, precipitation, collapse and detachment of the biofilm, from the surface. The quantitative culture data indicated that HOTD completely eradicated viable microorganisms from the surface of the pins. In a final set of experiments Innovotech tested HOTD for activity against the spore-forming bacteria Bacillus subtillus in a standardized bactericidal culture assay system. Once again the HOTD successfully eradicated the microbes at a level comparable to the HCl positive control treatment. Samples of the SEM photographs of the surface of the treated pins and tables of culture data are presented below.



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The Innovotech MBEC SEM images in the panel below show an untreated control Pseudomonas biofilm on a 96-well plate pin on the left and a HOTD-treated Pseudomonas biofilm in the right image. The image of the biofilm in the treated specimen are consistent with the absorption of water from the matrix resulting in precipitation and collapse of the biofilm matrix leading to reduced attachment of biofilm microbes to the pin surface. The chart below contains the data from the quantitative assay of the anti-plaque activity of HOTD using the Pseudomonas biofilms. The quantitative culture data show that a 10 second exposure of any of the four different Pseudomonas biofilms to HOTD was enough to totally remove all of the viable organisms from the surface of the test pin. This is indicated by a 7 log reduction in the number of bacteria/mL in the wells that received the HOTD treatment.

Log Reduction of P. aeruginosa biofilms using clinical isolates from wounds and PA01 at contact times of 5 seconds through 300 seconds

P. Aeruginosa PA01 P. Aeruginosa HR84 P. Aeruginosa HR156 P. Aeruginosa HR148 Contact Time HYBENX Oral Tissue Decontaminant

5 sec 6.51 6.47 6.76 5.70 10 sec 7.06 6.70 6.81 7.24 15 sec 6.24 6.69 6.30 6.42 20 sec 6.13 7.23 2.51 2.71 25 sec 6.49 6.74 7.08 2.98 30 sec 7.76 6.78 6.78 6.02 60 sec 6.89 6.74 7.34 6.65

300 sec 7.13 6.77 6.74 7.04 Log 10 reduction numbers generated by subtracting the log 10 CFU of the treatment well from the log 10 CFU of the growth



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The Innovotech MBEC SEM images in the panel below show an untreated control Candida biofilm on a 96-well plate pin on the left and a HOTD-treated Candida biofilm in the right image. The image of the biofilm in the treated specimen are consistent with the absorption of water from the matrix resulting in precipitation and collapse of the biofilm matrix leading to reduced attachment of biofilm microbes to the pin surface.

The chart below contains the data from the quantitative assay of the anti-plaque activity of HOTD using the Candida biofilm. The quantitative culture data show that a 10 second exposure of any of the four different Candida biofilms to HOTD was enough to totally remove all of the viable organisms from the surface of the test pin. This is indicated by a 5 log reduction in the number of bacteria/mL in the wells that received the HOTD treatment which is the same reduction seen with the positive bleach control samples.

Candida Tropicalis Biofilm - Log Reduction from 0.9% Saline Control Time (sec) 5 10 15 20 25 30 60 300

HOTD 1.90 4.95 5.22 5.08 5.15 4.88 4.69 5.01 1% Bleach 4.93 4.95 5.22 5.08 5.15 4.88 4.69 5.01



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The Innovotech MBEC SEM images in the panel below show an untreated control Aspergillus biofilm on a 96-well plate pin on the left and a HOTD-treated Aspergillus biofilm in the right image. The image of the biofilm in the treated specimen are consistent with the absorption of water from the matrix resulting in precipitation and collapse of the biofilm matrix leading to reduced attachment of biofilm microbes to the pin surface.

The chart below contains the data from the quantitative assay of the anti-plaque activity of HOTD using the Aspergillus biofilm. The quantitative culture data show that a 10 second exposure of any of the four different Aspergillus biofilms to HOTD was enough to totally remove all of the viable organisms from the surface of the test pin. This is indicated by a 4 -5 log reduction in the number of bacteria/mL in the wells that received the HOTD treatment which is the same reduction seen with the positive bleach control samples at the later time points.

Aspergillus niger Biofilm - Log Reduction from 0.9% Saline Control Time (sec) 5 10 15 20 25 30 60 300

HOTD 1.90 4.16 4.59 4.03 4.22 4.20 4.34 4.36 1% Bleach 0.91 1.35 2.37 2.42 4.22 4.20 4.34 5.36



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The final set of Innovotech experiments tested HOTD for activity against the spore-forming bacteria Bacillus subtillus in a standardized bactericidal culture assay system. SEM work was not done in conjunction with this experiment. Once again the HOTD successfully eradicated the microbes at a level comparable to the HCl positive control treatment.



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PORCINE WOUND BIOFILM STUDIES AT THE UNIVERSITY OF MIAMI The next set of experiments consists of studies done on a porcine model of infected partial thickness skin wounds at the University of Miami Medical School in the Dermatology Department. In their model system small partial thickness skin wounds are created on the backs of swine with a modified keratome. The wounds are then infected with pure cultures of either a bacteria or fungus under conditions that support development of a biofilm on the wound surface. Once the biofilm has had a chance to become established, the wound receives treatment in the form of a topical application of the test materials. Then the treated tissue at the wound site is harvested by standard techniques and the number of residual viable microorganisms is determined by culture techniques. The HOTD formulation was tested against wound biofilms derived from Methicillin Resistant Staph Aureus (MRSA), Pseudomonas aeruginosa and Acinetobacter baumannii. The data show that the treatment of the wounds with HOTD for 1 minute produces a 3 to 5 log reduction in recovery of viable pathogens. The data is summarized in the bar graph presentations below. Although this study was performed outside the oral cavity, the target tissue was infected with a microbial biofilm that has the same basic structure as dental plaque. This study measures HOTD activity against a biofilm structure and biofilm microbes. One advantage to performing these tests outside of the oral cavity is that the environment around the wound post-treatment can be maintained in a dry condition. This facilitates collection of tissue samples for histological analysis that confirm the desiccation activity of HOTD by showing desiccated tissue. Tissue specimens from HOTD studies in the oral cavity do not usually show evidence of residual tissue desiccation because the tissues become immediately rehydrated once the HOTD is rinsed. Photomicrographs of some of the histology specimens are presented below. On the left are low and medium power views of a control wound tissue section that shows a layer of thick inflammatory debris on the surface consistent with an infection by a microbial biofilm. On the right are pictures on an identical section post treatment with HOTD. Note how the inflammatory debris on the surface has been precipitated, coagulated and collapsed into a thin layer and the underlying dermis shows hyperchromicity consistent with tissue desiccation.



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The chart below shows the results of an experiment in which experimental porcine skin wounds were infected with a strain of MRSA that formed a biofilm over the wound. The experiment compares the number of viable organisms left on the tissue after treatment with HOTD or Mupirocin compared to untreated control. The experiment looked at tissue collected on the first day after treatment and on the second day after treatment. The red-colored bar is the HOTD data and the tan -colored bar is control data. The next chart below shows the results of a similar experiment in which experimental porcine skin wounds were infected with a biofilm of Acinetobacter baumanii. In this chart the tan-colored bar is the HOTD data, the blue-colored bar is control data and the purple bar is from data for wound tissue treated with silver sulfadiazene.



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The next chart below shows the results of another similar experiment in which experimental porcine skin wounds were infected with a biofilm of Pseudomonas aeruginosa. In this chart the red-colored bar is the HOTD data, the tan-colored bar is untreated control data and the green-colored bar is from data for wound tissue treated with silver sulfadiazene.



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USC DENTAL ORAL MICROBIAL BIOFILM DISRUPTION ASSAYS This next data set was produced in the laboratory of Dr. J.W. Costerton in the Biofilm Center at the Dental School at USC. Dr. Costerton performed three experiments on human dental plaque samples using protocols that had been established in his laboratory for anti-biofilm activity screening. The data from these experiments consists of direct observation of anti-biofilm activity through use of advanced imaging technologies. This data set contains primarily SEM images and fluorescent confocal scanning laser microscopy images. The summaries of theses assays are presented below. SALIVARY BIOFILM LIVE/DEAD STAIN ASSAY In this experiment the effect of exposing a human mixed microbe biofilm derived from human saliva samples to HOTD was studied. Human saliva was first spread onto sterile glass slide coverslips. The coverslips were placed into dishes of growth supporting microbial media and the dishes were maintained in the appropriate incubator environment for 14 days. The media was exchanged once daily. After 14 days the coverrslips were washed. Half of them were treated for two minutes with an application of HOTD which was then removed with a water rinse. The other half was simply rinsed again for two minutes in saline as a control sample. After the incubation period all the slides were stained with the Invitrogen Live/Dead Cell Viability Assay System stain. This stain makes live microbes fluoresce green and dead microbes fluoresce red. After treatment the slides of both groups were examined by fluorescent confocal scanning laser microscopy. This technique allows you to visualize discrete “slices” through the biofilm as the plane of focus it adjusted from the top to the bottom of the thickness of the biofilm. The results of this study showed that all of the cells throughout the entire thickness of the control specimen were alive and stained green. However, all of the microbes in the HOTD-treated biofilm were dead from top to bottom. According to the staff of the Biofilm Center this kind of complete killing is very unusual. Typically antiseptics and antibiotics in this assay will kill only the microbes that are on the very top surface of the biofilm layer. This demonstrates that HOTD can effectively desiccate through the full thickness of a typical biofilm layer of mixed human microbes.



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HUMAN EX VIVO ROOT CANAL BIOFILMSTUDY In the next set of experiments illustrated below HOTD was evaluated as a root canal cleanser in standard ex vivo root canal preparation procedures. Freshly extracted human teeth were clamped to a lab bench apparatus and standard root canal shaping and cleaning protocols were performed on the teeth as if they were still in the patient. Upon completion of the ex vivo cleaning procedures, the teeth were fractured along the length of the root canal and split to allow examination of the canal surface by scanning electron microscopy. The SEM pictures immediately below show the root canal surface of a saline-treated control specimen in the upper row and pictures of the surface of a root canal that had been irrigated with HOTD during the ex vivo procedure in the lower row. In comparison to the saline-treated control surface, the HOTD-treated surface shows less dentinal debris in general and less of the very fine microbial plaque debris at the openings of the dentinal tubules.



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In the third experiment, Dr. Costerton exposed cut sections of the root of an extracted tooth from a periodontitis patient with heavy root surface plaque to either a saline rinse or to an HOTD rinse and then performed SEM on the rinsed areas. The pictures below show the root surface that was exposed to saline at levels of increasing magnification. These pictures show thick plaque covering the entire surface of the root section. The long strands of tangled string-like material seen in the SEM picture at the highest magnification in the far right lower panel are created by the SEM sample preparation procedure from shrinkage of the gel-like substances in the plaque matrix. The spheres and rods that are seen clustered around those strands are the bacteria that created the plaque.

UNTREATED CONTROL -‐ TOOTH ROOT SURFACE PLAQUE



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The SEM pictures in the panel below show the surface of a cut root section from the same tooth that is shown in the pictures above, however, this cut section was exposed to an HOTD rinse before the SEM sample preparation was done. These pictures are also arranged by increasing magnification. In comparison to the pictures of saline-exposed plaque, these pictures show that the HOTD-exposed plaque looks as if it has been dehydrated. At high power in the far right panel the SEM picture shows that the matrix substance and the microbes appear to have been aggregated together into a coagulum by dehydration. The plaque aggregate has cracked and is detaching from the root surface. Clean root surface is visible under the plaque in areas where it has completely detached from the surface. THE SIGNIFICANCE OF THE COSTERTON USC DATA As is well known, Dr. Costerton is considered one of the pioneers of current biofilm technology. He demonstrates here the type of “direct observation” technology that is necessary to evaluate the efficacy of ant-plaque and anti-biofilm agents on the most sensitive levels rather than depending upon secondary clinical indictors and outcomes.

TOOTH ROOT SURFACE PLAQUE TREATED WITH HPBR



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LIMITED CLINICAL PILOTS OF HOTD RELATED FORMULATIONS As previously noted in the BACKGROUND section of this document the HOTD formulation was initially derived from the formulations of two EPIEN products that had been developed for the treatment of oral ulcers 1) Debacterol and 2) Oralmedic. Both of these products have a mechanism of action that is dependent upon desiccation and denaturation of the necrotized tissue on the bed of an oral mucosal ulcer which leads to the formation of a temporary protective barrier of coagulated tissue. Debacterol is sold only to health care practitioners and only in the USA. It is made from the reaction product that is derived from blending a phenolic beechwood distillate (pharmaceutical grade wood creosote) with fuming sulfuric acid under controlled conditions. Several million applications of Debacterol have been performed by dental practitioners in the USA over the past 20 years without any adverse incidents reported to either he company or to the regulatory authorities. A formal study of Debacterol in the treatment of aphthous ulcers was performed at the University of Minnesota Dental School. The results were published in 1998 (see Rhodus N, Bereuter J: An evaluation of a chemical cautery agent and an anti-inflammatory ointment for the treatment of recurrent aphthous stomatitis: a pilot study. Quintessence INT. 1998 29(12):769-773. ). In that study Debacterol was shown to be a safe and effective agent for relieving pain and facilitating healing of recurrent aphthous ulcers. Oralmedic is the name of the oral ulcer product that EPIEN Medical currently markets directly to consumers as well as to healthcare professionals outside of the US. Oralmedic is manufactured from the same component reagents as HOTD. It is in effect a slight more dilute form of HOTD. Oralmedic is described as “contains HYBENX”. The performance of Oralmedic in the treatment of aphthous stomatitis was evaluated in a formal trial at the Eastman Dental Institute in London, England. The results of the clinical trial were published in 2009 (see Porter SR, Al-Johani K, Fefele S, Moles DR: Randomised controlled trial of the efficacy of HYBENX in the symptomatic treatment of recurrent aphthous stomatitis. Oral Diseases 15:155-161. 2009). The results of the study demonstrated that the HYBENX-based formulation of Oralmedic was safe and effective for the treatment of recurrent aphthous stomatitis. Oralmedic provided for immediate pain relief and it may have facilitated healing. Since Oralmedic was initially cleared for marketing in the EU in 2002 well over 1 million applications of the product have been delivered safely and effectively. To date the HOTD product has been evaluated in only one small pilot clinical safety trial involving a traditional clinical protocol with traditional clinical endpoints. The study was performed at the University of Verona Italy by Dr. Giorgio Lombardo in the Periodontics Department of the School of Dentistry. Dr. Lombardo compared the standard clinical markers of periodontal lesion status following a traditional periodontitis treatment trial protocol. He compared the periodontal lesions over time in patients who received standard ultrasonic subgingival debridement only to similar patients who had received a onetime irrigation of their periodontitis lesions with HOTD immediately post completion of ultrasonic subgingival debridement. The patients received their final follow-up



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examination of their periodontal lesions at 90 days post treatment. The differences in the traditional markers of periodontal lesion status (pocket depth, attachment level, bleeding on probing, plaque index, gingivitis index, etc.) between day 1 and day 90 were compared in the two treatment groups. The results demonstrated that application of HOTD to periodontal lesions immediately post ultrasonic debridement was not associated with any adverse effects. The only statistically significant parameter that was different between the two groups at 90 days post treatment was bleeding on probing. The group that received the single HOTD rinse post ultrasonic debridement had significantly less bleeding on probing at 90 days. A draft manuscript describing the study in detail has been prepared by Dr. Lombardo with the title Ultrasonic debridement in association with HYBENX in the initial treatment of chronic periodontitis. The manuscript is currently in preparation and pending publication. The few clinical studies that have been done with HOTD-like formulations have shown that they can be used safely and effectively in the treatment of oral ulcers. As noted in other sections of this document HOTD has not been designed as a disease-specific therapy and its efficacy has not been evaluated clinically as a specific disease treatment. Demonstration of efficacy has been based on direct observation of the products ability to remove plaque biofilm and biofilm microbes in a variety of in vivo and ex vivo assays.



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CLINICAL USES HOTD was designed as a supplemental rinse and debriding agent for enhanced cleansing of dental plaque and other infectious matter from clinically important surfaces in the oral cavity. HOTD was not designed as a therapy for a specific disease. Its intended use is not tied to any particular clinical treatment outcome. The importance of maximizing the cleanliness of oral cavity tissue surfaces to the achievement of optimum clinical results is a well established principle in dentistry. HOTD was intended to be regarded by the dental profession as a universal adjunctive cleaning tool. We formulated the product so that individual dental practitioners could use it to enhance their performance of those professional techniques that are the most important for their specific practice. After practitioners have learned the mechanism of action of HOTD and acquired some basic experience in the application of HOTD to both healthy and diseased tissues in the oral cavity, they can then develop their own guidelines for use of the product to best enhance patient outcomes within their own individual practice setting. Recent clinical research suggests that oral cavity microbes may have a pathological role beyond the mouth. Linkages between oral tissue infection and systemic conditions such as diabetes and coronary artery disease are becoming established. These issues mandate that dental practitioners do everything possible to control and minimize all possible risks that might arise from pathogens in the oral cavity. As noted previously antiseptics and antibiotics have limited ability to suppress microbes that have become established within a biofilm and they have no effect on the biofilm matrix structure itself. The risk of developing antibiotic resistant microbes is also a challenge that needs to be addressed. Another major concern is that standard mechanical plaque removal techniques can contribute to infection control problems. Splatter from ultrasonic scaling equipment has been shown to create airborne droplets consisting of biofilm with viable pathogens that was carried throughout an entire clinic. When HOTD is used in an adjunctive role it is uniquely able to overcome the limitations of established standard anti-plaque methods. HOTD is a liquid and it can be irrigated onto any surface in the oral cavity that can be reached by a liquid. It not only removes microbes, but it also attacks and denatures the entire plaque matrix which makes it more difficult for pathological microbes to become reestablished. Because the product acts by physically disrupting the microenvironment of plaque and necrotic tissues, resistance to the product cannot develop. On the following two pages a table is presented that presents some basic examples and guidelines for the use of HOTD in the performance of specific professional procedures. The information in these tables is intended to suggest the range of possible applications of the product. It is not meant to be used as specific instructions in the application of HOTD in the manner of a package insert. As noted above, HOTD is an adjunctive therapy that is intended to be adaptable to the needs of individual practitioners as they look for ways to optimize their performance.



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For all dental procedures, HOTD is applied with an irrigation syringe and a cannula for 10-60 seconds followed by rigorous rinsing with water and high speed evacuation. HOTD enhances the efficacy of standard hygiene procedures by removing residual components of plaque biofilm that are left behind in pits, fissures, and crevices of tooth structures and gingival tissues after standard mechanical dental procedures. The following table includes suggestions for how HOTD can be implemented into routine dental procedures.

This Table is continued on the next page.

Procedure Condition HOTD is Used? Benefits of HOTD Concerns Significance

Oral Prophylaxis

Routine cleanings, mild

to moderate gingivitis

Prior to cleaning: Apply HOTD one quadrant at a time to tooth and gingival tissues.

Removes superficial plaque biofilm, softens calculus, and removes active biofilm from splatter created during cleaning.

Up to 1 mL of HOTD per quadrant

Allows for faster and more efficient routine cleanings.

After cleaning: Apply HOTD one quadrant at a time to tooth and gingival tissues.

Removes deep and residual plaque biofilm, seals dentinal tubules, and reduces tooth sensitivity.

Scaling and Root

Planing

Advanced gingivitis and Periodontal

disease

Prior to Treatment: Apply HOTD to affected tooth and gingival tissues.

Removes superficial plaque biofilm— especially from inaccessible areas, softens calculus, enhances and hastens instrumentation techniques, removes active biofilm from splatter created during SRP.

Up to 1 mL of HOTD per quadrant

Allows the general practitioner a greater degree of confidence to adequately maintain patients with periodontal disease. Increased patient comfort

After treatment: Apply HOTD to affected tooth and gingival tissues.

Reduces post-treatment bleeding and sensitivity, lessens inflammatory mediators, and enhances patient comfort.

Dental Restoration

Dental Caries, Crown

Preparations, Tooth Fractures

Apply HOTD after cavity preparation and prior to application of a primer. HOTD can be used instead of a hypochlorite rinse.

Removes dentinal smear layer, eliminates residual biofilm from the inside of the cavity preparation, debrides loose necrotic debris, reduces hemorrhaging at the treatment site, reduces the occurrence of bonding failures, and greatly reduces sensitivity.

Up to 0.5 mL of HOTD applied to the inside of the dental cane site.

Increased patient comfort and reduces the risk of restoration failure.



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Examples of Clinical Procedures Using HOTD - Continued

The statements contained in the above chart reflect the opinions of dental practitioners who have evaluated HOTD. These statements have not been reviewed by regulatory agencies in Europe, Canada, or the United States. HOTD has not been approved for marketing in the US by the FDA. It is sold outside of the United States only. Having been cleared for marketing as a Class I Medical Device in the EU and Canada, HOTD is now available to dental practitioners in those jurisdictions as an adjunctive focal irrigation solution intended for the removal of dental plaque biofilm. HOTD is intended to be used as supplied at its full original concentration. Diluting with water and/or combining HOTD with another product will render it ineffective and potentially harmful.

Procedure Condition HOTD is Used? Benefits of HOTD Concerns Significance

Vital Pulp Treatment

Reversible Pulpitis

Apply HOTD rinse, and cover the pulpal tissue

with a glass ionomer followed by the restoration

material.

Removes plaque biofilm contamination of the exposed pulp, removes dentinal smear

layer,

Up to 1 mL of HOTD applied to the treatment area. HOTD is contra- indicated for use

with calcium hydroxide paste.

Increased patient comfort and

reduces the risk of restoration failure.

Dental Implant

Maintenances

Apply HOTD to gingival tissue

around the implant site.

Removes superficial plaque biofilm, reduces

instrumentation damage to the implant, reduces

inflammation, and shrinks the surrounding tissue to the

implant by reducing edema. HOTD does not affect the

implant.

Up to 1 mL of HOTD applied to

oral mucosa surrounding the

implant.


lessens the risk of biofilm

complications at the implant site.

Endodontic Treatment

Irreversible Pulpitis

After shaping the canal with citric acid or EDTA, apply HOTD to the root canal

instead of hypochlorite. Complete the procedure as

normal.

Removes plaque biofilm contamination from very

inaccessible areas--including dentinal tubules, enhances restorations by drying the

canal and improving the seal for the restoration material,

reduces biofilm-related complications, the viscous

nature of HOTD makes it less likely to leak through the root

apex into bony tissues.

Up to 1 mL of HOTD applied to the inside of the

root canal. HOTD is contra- indicated for use with indicated

calcium hydroxide paste.

Makes a hypochlorite rinse unnecessary and

allows the practitioner to

work without fear of periapical

extravasation of hypochlorite.

Extraction sites

and soft tissue

wounds

Various conditions

Using an application

syringe, apply HOTD to a swab

and treat the affected area with the HOTD-coated

swab.

Reduces inflammation and edema of the wound site

denaturing and coagulating the superficial oral tissue of

the wound site. Lessens hemorrhaging at the wound

site.


lessens the risk of biofilm

contamination of the wound area,

and reduces

complications.



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SELECTED BIBLIOGRAPHY OF SCIENTIFIC LITERATURE REFERENCES General Microbial Biofilm

Costerton WJ: The Biofilm Primer Springer Series on Biofilms Vol.1 Springer, 2007 Wilson M, Devine D: Medical Implications of Biofilms. Cambridge, 2003 Pace JL, Rupp ME, Finch RG: Biofilms, Infection and Antimicrobial Therapy CRC Taylor & Francis, 2005 Ghannoum M, O’Toole GA: Microbial Biofilms ASM Press, 2004

Dental Plaque as Microbial Biofilm

Scheie AA, Petersen FG: The Biofilm Concept: Consequences for Future Prophylaxis of Oral Diseases? CritRevOralBiolMed 15(1):4-12, 2004. Marsh P: Dental Plaque as a biofilm and a microbial community – implications for health and disease. BMC Oral Health 6 (Suppl 1):514, 2006 Thomas JG, Nakaishi LA: Managing the complexity of a dynamic biofilm. JADA 137:10S-15S, 2006 Stoodley P, Wefel J, Gleseke A, deBeer D, von Ohle C: Biofilm Plaque and hydrodynamic effects on mass transfer, fluoride delivery and caries. JADA 139:1182-1190, 2008

Dental Erosion

Duckworth RM: The Teeth and Their Environment. Monographs in Oral Science Vol.19 Karger, 2006 Ciba Foundation Symposium #206: Dental Enamel John Wiley & Sons, 1997 Eliades G, Watts DC, Eliaders T: Dental Hard Tissues and Bonding Springer, 2005



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Lussi A: Dental Erosion From Diagnosis to Therapy Monographs in Oral Science Vol.20 Karger, 2006

Sulfonation of Aromatics

Cerfontain H: Mechanistic aspects in aromatic sulfonation and desulfonation Interscience Publishers - John Wiley, New York 1968 Cerfontain H, Coenjaarts N J, Koeberg-Telder A: Aromatic Sulfonation. 103. Sulfonation and sulfation on reaction of 1, 2-dihydroxybenzene and its methyl ethers in concentrated aqueous sulfuric acid Recl. Trav.Chim. Pays-Bas 107(4):325-220, 1988 Cerfontain H, Lambrechts HJA: Aromatic Sulfonation. Part 91. The sulfonation of anisole, phenol, phenyl methanesulphonate, potassium phenyl sulphate and a series of methyl-, bromo-, and chloro-substituted anisoles and phenols in concentrated aqueous sulphuric acid. J. Chem. Soc. Perkin Trans II 1985, p.659

Hydrogen Bonding

Marcus Y: Ion Solvation. John Wiley & Sons, 1985 Marcus Y: Ion Properties. Marcel Dekker, 1997 Jeffrey GA: An Introduction to Hydrogen Bonding Oxford, 1997 Marechal Y: The Hydrogen Bond and the Water Molecule Elsevier, 2007

Morra M: Water in Biomaterials Surface Science John Wiley & Sons, 2001

Tissue Denaturation, Protein Denaturation

Collins KD, Washabaugh MW: The Hofmeister effect and the behavior of water at interfaces Quart.Rev. Biophys. 18:323-422, 1985 Franks F: Protein stability: the value of “old literature”. Biophys. Chem. 96:117-127, 2002



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Pace C, Shirley BA, McNutt M, Gajiwala K: Forces contributing to the conformational stability of proteins. The FASEB Journal 10:75-83, 1996 Wright NT, Humphrey JD: Denaturation of Collagen via Heating: An irreversible rate process. Ann. Rev Biomed Eng. 4:109-128, 2002 Yang A-S, Honig B: Structural origins of pH and Ionic strength effects on protein stability J Mol Biol 237:602-614, 1994 Fink AL, Calciano LJ, Goto Y, Kurotsu T, Palleros DR: Classification of acid denaturation of proteins: Intermediates and unfolded states. Biochemistry 33:12504-12511, 1994.

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