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Which kind of drugs get absorbed through epidermis?

Which kind of drugs get absorbed through epidermis?


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Some drugs such as nicotine can be administered through skin. I thought the layers of skin are designed to prevent in-flow of any chemical/germs. Not all drugs get absorbed in this fashion. So do drugs like nicotine have any specialities(such as a special chemical group) that fool the sentries of skin? A link to an article/site would be very helpful, or even a book recommendation.


According to a review (Prausnitz2004), marketed drugs with a dermal route of administration tend to fulfil the following criteria :

  • Small molecular size (< 500 Da)
  • High lipophilicity
  • Small required dose (up to ~milligram)

This is a consequence of the structure of the skin (with a lipophilic stratum corneum, and low diffusion speed) and practical concerns (everything else equal, a higher dose would need more surface area).

Beyond "pure" diffusion, there has been much work on improving drug delivery through other means : chemical enhancers (ethanol or DMSO being classical member of this class), medical devices (microneedle patch, ultrasonic device), and other more exotic methods mentionned in the paper.

These criteria are also mentionned in (GoodmanGillman2006, chapter 26), with emphasis on concentration gradient being the driving force behind diffusion across the skin. This chapter also contain a list of commonly used transdermal/dermatological drug, and their use.


Cell-based in vitro models for dermal permeability studies

Francisca Rodrigues , Maria Beatriz P.P. Oliveira , in Concepts and Models for Drug Permeability Studies , 2016

3.8.1 Introduction

Skin absorption processes are useful to evaluate and understand safety aspects of chemicals, xenobiotics, and cosmetic formulations. The main aim of skin absorption is the opportunity to deliver drug substances to the skin and to the systemic circulation ( Schaefer, Hansen, Schneider, Contreras, & Lehr, 2008 ). Therefore, knowledge of dermal absorption phenomena is relevant for safety issues as well as therapeutic aspects. Changes in regulatory requirements and social views on animal testing have incremented the development of reliable alternative tests for predicting the skin absorption potential of new compounds and evaluating dermal permeability. Although some procedures on conducting skin permeation studies are reported, no formal standardization is available ( EC, 2004, 2006 FDA, 1997 ). The assessment of dermal permeation is a primary procedure for evaluating a substance and classifying it as hazard, particularly in the cosmetics and pharmaceuticals industries ( Cotovio et al., 2010 ). Skin is a multilayered biomembrane with particular absorption characteristics that respond differently to different substance classes.

Skin is a dynamic, living tissue as such, its absorption characteristics are susceptible to constant change. Upon contact with the skin, molecules penetrate into the dead stratum corneum and can subsequently reach the viable epidermis, the dermis, and the vascular network ( EC, 2004 ). Dermal absorption is influenced by many factors such as physicochemical properties of the substance, the vehicle, possible occlusion, substance concentration, the exposure pattern, or even skin particularities in different body parts ( EC, 2004 ).

A vast number of studies in the past have compared in vitro and in vivo methods for measuring dermal absorption. They concluded that, if properly conducted, in vitro measurements can be used to predict in vivo absorption ( OECD, 2004a ). Organization of Economic Cooperation and Development (OECD) Guidance Document 28 for the conduct of skin absorption studies allows the use of reconstructed human skin if its application leads to absorption results similar to those obtained with excised human skin ( OECD, 2004a ).

Also, in line with the Seventh Amendment deadlines, the European Union bans in vivo skin permeation assessment of ingredients for cosmetic purposes. Alternative methods are needed and require validation by the European Center for the Validation of Alternative Methods (ECVAM). In response to that, the OECD adopted a guideline for assessment of skin corrosion based on reconstructed human epidermis (RHE). In 2004, OECD Technical Guidance also stated that skin equivalents can be used for skin permeation assays ( OECD, 2004a ).

For ethical reasons, fundamental skin absorption data cannot normally be obtained by conducting in vivo studies ( Schaefer et al., 2008 ). According to European Union and Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals guidelines, animal testing for skin studies should be performed only if all other approaches have failed to provide a conclusive result. Therefore, other techniques must be used to obtain desired information, such as in vitro penetration and permeation studies. Concerns about the test’s predictivity and reproducibility, plus animal welfare and political pressure in Europe, prompted a search for alternative test methods ( Spielmann et al., 2007 ). Since the early 1990s, several models of the skin have been developed, characterized, validated, and accepted as valid replacement methods for animal experimentation.

Thus, in vitro models based on skin equivalents available on the market for permeation studies are described below.


Drug therapy in special situations

Derek G. Waller BSc (HONS), DM, MBBS (HONS), FRCP , Anthony P. Sampson MA, PhD, FHEA, FBPhS , in Medical Pharmacology and Therapeutics (Fifth Edition) , 2018

Absorption and distribution

Drug absorption across the gut wall is not greatly affected by ageing, although bioavailability may be increased due to reduced first-pass metabolism. Older people tend to have a lower lean body mass and a relative increase in body fat compared with young adults. The apparent volume of distribution ( Vd) of water-soluble drugs such as digoxin may therefore be lower in the elderly, and a smaller loading dose would be needed. Conversely, lipid-soluble drugs may be eliminated more slowly because of their increased Vd resulting from increased body fat.


2. Transdermal Drug Delivery (TDD)

TDD is a painless method of delivering drugs systemically by applying a drug formulation onto intact and healthy skin [2,5]. The drug initially penetrates through the stratum corneum and then passes through the deeper epidermis and dermis without drug accumulation in the dermal layer. When drug reaches the dermal layer, it becomes available for systemic absorption via the dermal microcirculation [8,9]. TDD has many advantages over other conventional routes of drug delivery [10,11,12]. It can provide a non-invasive alternative to parenteral routes, thus circumventing issues such as needle phobia [2]. A large surface area of skin and ease of access allows many placement options on the skin for transdermal absorption [5]. Furthermore, the pharmacokinetic profiles of drugs are more uniform with fewer peaks, thus minimizing the risk of toxic side effects [2]. It can improve patient compliance due to the reduction of dosing frequencies and is also suitable for patients who are unconscious or vomiting, or those who rely on self-administration [13]. TDD avoids pre-systemic metabolism, thus improving bioavailability [2,4]. With reference to the use of the skin as a novel site for vaccination strategies, this organ is known to be replete with dendritic cells in both the epidermal and dermal layers which play a central role in immune responses making TDD an attractive vaccination route for therapeutic proteins and peptides [3,14]. The requirement for an inexpensive and non-invasive means of vaccination, especially in the developing world [3,14,15], has given rise to substantial research focused on the development of simple, needle-free systems such as TDD for vaccination purposes. This theme will be explored further in Section 4.5.2 of this review.


Contents

When a chemical comes in contact with the mucous membrane beneath the tongue, it is absorbed. Because the connective tissue beneath the epithelium contains a profusion of capillaries, the substance then diffuses into them and enters the venous circulation. [1] In contrast, substances absorbed in the intestines are subject to first-pass metabolism in the liver before entering the general circulation.

Sublingual administration has certain advantages over oral administration. Being more direct, it is often faster, [ quantify ] and it ensures that the substance will risk degradation only by salivary enzymes before entering the bloodstream, whereas orally administered drugs must survive passage through the hostile environment of the gastrointestinal tract, which risks degrading them, by either stomach acid or bile, or by enzymes such as monoamine oxidase (MAO). Furthermore, after absorption from the gastrointestinal tract, such drugs must pass to the liver, where they may be extensively altered this is known as the first pass effect of drug metabolism. Due to the digestive activity of the stomach and intestines, the oral route is unsuitable for certain substances, such as salvinorin A.

Pharmaceutical preparations for sublingual administration are manufactured in the form of:

  • Sublingual tablets—tablets which easily melt in the mouth, dissolve rapidly and with little or no residue. Nitroglycerine tablets are an example, the anti-emetic ondansetron is another.
  • Sublingual strips—similar to tablets in that they easily melt in the mouth and dissolve rapidly. Suboxone is an example of medication that comes in a sublingual strip.
  • Multi-purpose tablets—Soluble tablets for either oral or sublingual (or buccal) administration, often also suitable for preparation of injections, Hydrostat (hydromorphone) and a number of brands of morphine tablets and cubes.
  • Sublingual drops—a concentrated solution to be dropped under the tongue, as with some nicocodeine cough preparations,
  • Sublingual spray—spray for the tongue certain human and veterinary drugs are dispensed as such. —effects a metred and patient-controlled-rate combination of sublingual, buccal, and oral administration, as with the Actiq fentanyl lozenge-on-a-stick (lollipop).
  • Effervescent buccal or sublingual tablets—this method drives the drug through the mucous membranes much faster (this is the case in the stomach with carbonated or effervescent liquids as well) and is used in the Fentora fentanyl buccal tablet.

Almost any form of substance may be amenable to sublingual administration if it dissolves easily in saliva. Powders and aerosols may all take advantage of this method. However, a number of factors, such as pH, molecular weight, and lipid solubility, may determine whether the route is practical. Based on these properties, a suitably soluble drug may diffuse too slowly through the mucosa to be effective. However, many drugs are much more potent taken sublingually, and it is generally a safer alternative than administration via the nasal mucosa. [ citation needed ] This method is also extensively used by people administering certain psychoactive drugs. One drawback, however, is tooth discoloration and decay caused by long-term use of this method with acidic or otherwise caustic drugs and fillers.

In addition to Salvinorin A, other psychoactives may also be applied sublingually. LSD, MDMA, morphine, alprazolam, clonazepam, Valium, and many other substances including the psychedelic tryptamines and phenethylamines, and even recreational cannabis edibles, i.e. (THC) are all viable candidates for administration via this route. [ citation needed ] Most often, the drug in question is powdered and placed in the mouth (often directly under the tongue). If held there long enough, the drug will diffuse into the blood stream, bypassing the GI tract. This may be a preferred method to simple oral administration, because MAO is known to oxidize many drugs (especially the tryptamines such as DMT) and because this route translates the chemical directly to the brain, where most psychoactives act. The method is limited by excessive salivation washing the chemical down the throat. Also, many alkaloids have an unpleasant taste which makes them difficult to hold in the mouth. Tablets of psychoactive pharmaceuticals usually include bitter chemicals such as denatonium in order to discourage abuse and also to discourage children from eating them. [ citation needed ]

Allergens may also be applied under the tongue as a part of allergen immunotherapy.

A relatively new way of administration of therapeutic peptides and proteins (such as cytokines, domain antibodies, Fab fragments or single chain antibodies) is sublingual administration. Peptides and proteins are not stable in the gastro-intestinal tract, mainly due to degradation by enzymes and pH differences. As a consequence, most peptides (such as insulin, exenatide, vasopressin, etc. ) or proteins (such as interferon, EPO and interleukins) have to be administered by injection. Recently, new technologies have allowed sublingual administration of such molecules. Increased efforts are underway to deliver macromolecules (peptides, proteins and immunotherapies) by sublingual route, by companies such as Novo Nordisk, Sanofi and BioLingus. [3] Sublingual delivery may be particularly effective for immuno-active medicines, due to the presence of immune-receptor cells close to the sublingual area.

The sublingual route may also be used for vaccines against various infectious diseases. Thus, preclinical studies have found that sublingual vaccines can be highly immunogenic and may protect against influenza virus [4] [5] and Helicobacter pylori, [6] but sublingual administration may also be used for vaccines against other infectious diseases.


Contents

Injections are classified in multiple ways, including the type of tissue being injected into, the location in the body the injection is designed to produce effects, and the duration of the effects. Regardless of classification, injections require a puncture to be made, thus requiring sterile environments and procedures to minimize the risk of introducing pathogens into the body. All injections are considered forms of parenteral administration, which avoids the first pass metabolism which would potentially affect a medication absorbed through the gastrointestinal tract.

Systemic Edit

Many injections are designed to administer a medication which has an effect throughout the body. Systemic injections may be used when a person cannot take medicine by mouth, or when the medication itself would not be absorbed into circulation from the gastrointestinal tract. Medications administered via a systemic injection will enter into blood circulation, either directly or indirectly, and thus will have an effect on the entire body.

Intravenous Edit

Intravenous injections, abbreviated as IV, involve inserting a needle into a vein, allowing a substance to be delivered directly into the bloodstream. [4] An intravenous injection provides the quickest onset of the desired effects because the substance immediately enters the blood, and is quickly circulated to the rest of the body. [5] Because the substance is administered directly into the bloodstream, there is no delay in the onset of effects due to the absorption of the substance into the bloodstream. This type of injection is the most common and is used frequently for administration of medications in an inpatient setting.

Another use for intravenous injections includes for the administration of nutrition to people who cannot get nutrition through the digestive tract. This is termed parenteral nutrition and may provide all or only part of a person's nutritional requirements. Parenteral nutrition may be pre-mixed or customized for a person's specific needs. [6] Intravenous injections may also be used for recreational drugs when a rapid onset of effects is desired. [7] [8]

Intramuscular Edit

Intramuscular injections, abbreviated as IM, deliver a substance deep into a muscle, where they are quickly absorbed by the blood vessels into systemic circulation. Common injection sites include the deltoid, vastus lateralis, and ventrogluteal muscles. [9] Medical professionals are trained to give IM injections, but people who are not medical professionals can also be trained to administer medications like epinephrine using an autoinjector in an emergency. [10] Some depot injections are also administered intramuscularly, including medroxyprogesterone acetate among others. [11] In addition to medications, most inactivated vaccines, including the influenza vaccine, are given as an IM injection. [12]

Subcutaneous Edit

Subcutaneous injections, abbreviated as SC or sub-Q, consist of injecting a substance into the fat tissue between the skin and the muscle. [13] Absorption of the medicine from this tissue is slower than in an intramuscular injection. Since the needle does not need to penetrate to the level of the muscle, a thinner and shorter needle can be used. Subcutaneous injections may be administered in the fatty tissue behind the upper arm, in the abdomen, or in the thigh. Certain medications, including epinephrine, may be used either intramuscularly or subcutaneously. [14] Others, such as insulin, are almost exclusively injected subcutaneously. Live or attenuated vaccines, including the MMR vaccine (measles, mumps, rubella), varicella vaccine (chickenpox), and zoster vaccine (shingles) are also injected subcutaneously. [15]

Intradermal Edit

Intradermal injections, abbreviated as ID, consist of a substance delivered into the dermis, the layer of skin above the subcutaneous fat layer, but below the epidermis or top layer. An intradermal injection is administered with the needle placed almost flat against the skin, at a 5 to 15 degree angle. [16] Absorption from an intradermal injection takes longer than when the injection is given intravenously, intramuscularly, or subcutaneously. For this reason, few medications are administered intradermally. Intradermal injections are most commonly used for sensitivity tests, including tuberculin skin tests and allergy tests, as well as sensitivity tests to medications a person has never had before. The reactions caused by tests which use intradermal injection are more easily seen due to the location of the injection, and when positive will present as a red or swollen area. Common sites of intradermal injections include the forearm and lower back. [16]

Intraosseous Edit

An intraosseous injection or infusion is the act of administering medication through a needle inserted into the bone marrow of a large bone. This method of administration is only used when it is not possible to maintain access through a less invasive method such as an intravenous line, either due to frequent loss of access due to a collapsed vessel, or due to the difficulty of finding a suitable vein to use in the first place. [17] Intraosseous access is commonly obtained by inserting a needle into the bone marrow of the humerus or tibia, and is generally only considered once multiple attempts at intravenous access have failed, as it is a more invasive method of administration than an IV. [17] With the exception of occasional differences in the accuracy of blood tests when drawn from an intraosseous line, it is considered to be equivalent in efficacy to IV access. It is most commonly used in emergency situations where there is not ample time to repeatedly attempt to obtain IV access, or in younger people for whom obtaining IV access is more difficult. [17] [18]

Localized Edit

Injections may be performed into specific parts of the body when the medication's effects are desired to be limited to a specific location, or where systemic administration would produce undesirable side effects which may be avoided by a more directed injection.

Injections to the corpus cavernosum of the penis, termed intracavernous injections, may be used to treat conditions which are localized to the penis. They can be self-administered for erectile dysfunction prior to intercourse or used in a healthcare setting for emergency treatment for a prolonged erection with an injection to either remove blood from the penis or to administer a sympathomimetic medication to reduce the erection. [19] Intracavernosal injections of alprostadil may be used by people for whom other treatments such as PDE5 inhibitors are ineffective or contraindicated. Other medications may also be administered in this way, including papaverine, phentolamine, and aviptadil. [20] The most common adverse effects of intercavernosal injections include fibrosis and pain, as well as hematomas or bruising around the injection site. [20]

Medications may also be administered by injecting them directly into the vitreous humor of the eye. This is termed an intravitreal injection, and may be used to treat endophthalmitis (an infection of the inner eye), macular degeneration, and macular edema. [21] An intravitreal injection is performed by injecting a medication through the pupil into the vitreous humor core of the eye after applying a local anesthetic drop to numb the eye and a mydriatic drop to dilate the pupil. They are commonly used in lieu of systemic administration to both increase the concentrations present in the eye, as well as avoid systemic side effects of medications. [21]

When an effect is only required in one joint, a joint injection (or intra-articular injection) may be administered into the articular space surrounding the joint. These injections can range from a one-time dose of a steroid to help with pain and inflammation to complete replacement of the synovial fluid with a compound such as hyaluronic acid. [22] The injection of a steroid into a joint is used to reduce inflammation associated with conditions such as osteoarthritis, and the effects may last for up to 6 months following a single injection. [22] Hyaluronic acid injection is used to supplement the body's natural synovial fluid and decrease the friction and stiffness of the joint. [22] Administering a joint injection [23] generally involves the use of an ultrasound or other live imaging technique to ensure the injection is administered in the desired location, as well as to reduce the risk of damaging surrounding tissues. [24]

Long-acting Edit

Long-acting injectable (LAI) formulations of medications are not intended to have a rapid effect, but instead release a medication at a predictable rate continuously over a period of time. Both depot injections and solid injectable implants are used to increase adherence to therapy by reducing the frequency at which a person must take a medication. [25] : 3

Depot Edit

A depot injection is an injection, usually subcutaneous, intradermal, or intramuscular, that deposits a drug in a localized mass, called a depot, from which it is gradually absorbed by surrounding tissue. Such injection allows the active compound to be released in a consistent way over a long period. [26] Depot injections are usually either oil-based or an aqueous suspension. Depot injections may be available as certain salt forms of a drug, such as decanoate salts or esters. Examples of depot injections include haloperidol decanoate, medroxyprogesterone acetate, [26] and naltrexone. [27]

Implant Edit

Injections may also be used to insert a solid or semi-solid into the body which releases a medication slowly over time. These implants are generally designed to be temporary, replaceable, and ultimately removed at the end of their use or when replaced. There are multiple contraceptive implants marketed for different active ingredients, as well as differing duration of action - most of these are injected under the skin. [28] A form of buprenorphine for the treatment of opioid dependence is also available as an injectable implant. [29] Various materials can be used to manufacture implants including biodegradable polymers, osmotic release systems, and small spheres which dissolve in the body. [25] : 4, 185, 335

Pain Edit

The act of piercing the skin with a needle, while necessary for an injection, also may cause localized pain. The most common technique to reduce the pain of an injection is simply to distract the person receiving the injection. Pain may be dampened by prior application of ice or topical anesthetic, or pinching of the skin while giving the injection. Some studies also suggest that forced coughing during an injection stimulates a transient rise in blood pressure which inhibits the perception of pain. [30] For some injections, especially deeper injections, a local anesthetic is given. [30] When giving an injection to young children or infants, they may be distracted by giving them a small amount of sweet liquid, such as sugar solution, [31] or be comforted by breastfeeding [32] during the injection, which reduces crying.

Infection Edit

A needle tract infection, also called a needlestick infection, is an infection that occurs when pathogens are inadvertently introduced into the tissues of the body during an injection. Contamination of the needle used for injection, or reuse of needles for injections in multiple people, can lead to transmission of hepatitis B and C, HIV, and other bloodstream infections. [33] [34] [35] Injection drug users have high rates of unsafe needle use including sharing needles between people. [36] The spread of HIV, Hepatitis B, and Hepatitis C from injection drug use is a common health problem, [37] in particular contributing to over half of new HIV cases in North America in 1994. [7]

Other infections may occur when pathogens enter the body through the injection site, most commonly due to improper cleaning of the site before injection. Infections occurring in this way are mainly localized infections, including skin infections, skin structure infections, abscesses, or gangrene. [38] An intravenous injection may also result in a bloodstream infection (termed sepsis) if the injection site is not cleaned properly prior to insertion. Sepsis is a life-threatening condition which requires immediate treatment. [16] : 358373

Others Edit

Injections into the skin and soft tissue generally do not cause any permanent damage, and the puncture heals within a few days. However, in some cases, injections can cause long-term adverse effects. Intravenous and intramuscular injections may cause damage to a nerve, leading to palsy or paralysis. Intramuscular injections may cause fibrosis or contracture. [39] Injections also cause localized bleeding, which may lead to a hematoma. Intravenous injections may also cause phlebitis, especially when multiple injections are given in a vein over a short period of time. [40] Infiltration and extravasation may also occur when a medication intended to be injected into a vein is inadvertently injected into surrounding tissues. [41] Those who are afraid of needles may also experience fainting at the sight of a needle, or before or after an injection. [42]

Proper needle use is important to perform injections safely, [43] which includes the use of a new, sterile needle for each injection. This is partly because needles get duller with each use and partly because reusing needles increases risk of infection. Needles should not be shared between people, as this increases risk of transmitting blood-borne pathogens. The practice of using the same needle for multiple people increases the risk of disease transmission between people sharing the same medication. [43] In addition, it is not recommended to reuse a used needle to pierce a medication bag, bottle, or ampule designed to provide multiple doses of a medication, instead a new needle should be used each time the container must be pierced. Aseptic technique should always be practiced when administering injections. This includes the use of barriers including gloves, gowns, and masks for health care providers. It also requires the use of a new, sterile needle, syringe and other equipment for each injection, as well as proper training to avoid touching non-sterile surfaces with sterile items. [44]

To help prevent accidental needlestick injury to the person administering the injection, and prevent reuse of the syringe for another injection, a safety syringe and needle may be used. [45] The most basic reuse prevention device is an "auto-disable" plunger, which once pressed past a certain point will no longer retract. Another common safety feature is an auto-retractable needle, where the needle is spring-loaded and either retracts into the syringe after injection, or into a plastic sheath on the side of the syringe. Other safety syringes have an attached sheath which may be moved to cover the end of the needle after the injection is given. [45] The World Health Organization recommends the use of single-use syringes with both reuse prevention devices and a needlestick injury prevention mechanism for all injections to prevent accidental injury and disease transmission. [45]

Novel injection techniques include drug diffusion within the skin using needle-free micro-jet injection (NFI) technology. [46] [47]

Disposal of used needles Edit

Over half of non-industrialized countries report open burning of disposed or used syringes. This practice is considered unsafe by the World Health Organization. [2]

Due to the prevalence of unsafe injection practices, especially among injection drug users, many locations have begun offering supervised injection sites and needle exchange programs, which may be offered separately or colocated. These programs may provide new sterile needles upon request to mitigate infection risk, and some also provide access to on-site clinicians and emergency medical care if it becomes required. In the event of an overdose, a site may also provide medications such as naloxone, used as an antidote in opioid overdose situations, or other antidotes or emergency care. Safe injection site have been associated with lower rates of death from overdose, less ambulance calls, and lower rates of new HIV infections from unsafe needle practices. [51]

As of 2019, at least ten countries currently offer safe injection sites, including Australia, Canada, Denmark, France, Germany, Luxembourg, The Netherlands, Norway, Spain and Switzerland. In total, there are at least 120 sites operating. [52] Although the United States does not currently have any safe injection sites, some cities such San Francisco, Philadelphia, and Denver are considering opening them, and some localities offer needle exchange programs. [50] In 2018, the California State Assembly passed Assembly Bill 186 to launch a three-year pilot program in San Francisco for California's first safe injection sites, but this bill was not signed by the governor. [53] Colorado and Pennsylvania have also expressed interest in offering safe injection sites. Court rulings in Pennsylvania have determined that safe injection sites are not illegal under federal law. [54]

Many species of animals use injections for self-defence or catching prey. This includes venomous snakes which inject venom when they bite into the skin with their fangs. Common substances present in snake venom include neurotoxins, toxic proteins, and cytotoxic enzymes. Different species of snakes inject different formulations of venom, which may cause severe pain and necrosis before progressing into neurotoxicity and potentially death. [55] The weever is a type of fish which has venomous spines covering its fins and gills and injects a venom consisting of proteins which cause a severe local reaction which is not life-threatening. [56] Sting rays use their spinal blade to inject a protein-based venom which causes localized cell death but is not generally life-threatening. [57]

Some types of insects also utilize injection for various purposes. Bees use a stinger located in their hind region to inject a venom consisting of proteins such as melittin, which causes a localized painful and itching reaction. [58] Leeches can inject an anticoagulant peptide called hirudin after attaching to prevent blood from clotting during feeding. This property of leeches has been used historically as a natural form of anticoagulation therapy, as well as for the use of bloodletting as a treatment for various ailments. [59] Some species of ants inject forms of venom which include compounds which produce minor pain such as the formic acid, which is injected by members of the Formicinae subfamily. [60] Other species of ants, including Dinoponera species, inject protein-based venom which causes severe pain but is still not life-threatening. [61] The bullet ant (Paraponera clavata) injects a venom which contains a neurotoxin named poneratoxin which causes extreme pain, fever, and cold sweats, and may cause arrhythmias. [62]

Plants may use a form of injection which is passive, where the injectee pushes themselves against the stationary needle. The stinging nettle plant has many trichomes, or stinging hairs, over its leaves and stems which are used to inject a mix of irritating chemicals which includes histamine, serotonin, and acetylcholine. This sting produces a form of dermatitis which is characterized by a stinging, burning, and itching sensation in the area. [63] Dendrocnide species, also called stinging trees, use their trichomes to inject a mix of neurotoxic peptides which causes a reaction similar to the stinging nettle, but also may result in recurring flares for a much longer period after the injection. [64] While some plants have thorns, spines, and prickles, these generally are not used for injection of any substance, but instead it is the act of piercing the skin which causes them to be a deterrent. [65]


SKIN EXPOSURES & EFFECTS

It is estimated that more than 13 million workers in the United States are potentially exposed to chemicals that can be absorbed through the skin. Dermal exposure to hazardous agents can result in a variety of occupational diseases and disorders, including occupational skin diseases (OSD) and systemic toxicity. Historically, efforts to control workplace exposures to hazardous agents have focused on inhalation rather than skin exposures. As a result, assessment strategies and methods are well developed for evaluating inhalation exposures in the workplace standardized methods are currently lacking for measuring and assessing skin exposures.

NIOSH has developed a strategy for assigning multiple skin notations (SK) capable of delineating between the systemic, direct and immune-mediated effects caused by dermal contact with chemicals.

OSD are the second most common type of occupational disease and can occur in several different forms including:

  • Irritant contact dermatitis,
  • Allergic contact dermatitis,
  • Skin cancers,
  • Skin infections,
  • Skin injuries, and
  • Other miscellaneous skin diseases.

Contact dermatitis is one of the most common types of occupational illness, with estimated annual costs exceeding $1 billion.

Occupations at Risk

Workers at risk of potentially harmful exposures of the skin include, but are not limited to, those working in the following industries and sectors:

  • Food service
  • Cosmetology
  • Health care
  • Agriculture
  • Cleaning
  • Painting
  • Mechanics
  • Printing/lithography
  • Construction

Anatomy and Functions of the Skin

The skin is the body&rsquos largest organ, accounting for more than 10 percent of body mass. The skin provides a number of functions including:

  • protection,
  • water preservation,
  • shock absorption,
  • tactile sensation,
  • calorie reservation,
  • vitamin D synthesis,
  • temperature control, and
  • lubrication and waterproofing.

Skin Hazards

Causes of OSD include chemical agents, mechanical trauma, physical agents, and biological agents.

  • Chemical agents are the main cause of occupational skin diseases and disorders. These agents are divided into two types: primary irritants and sensitizers. Primary or direct irritants act directly on the skin though chemical reactions. Sensitizers may not cause immediate skin reactions, but repeated exposure can result in allergic reactions.
    • A worker&rsquos skin may be exposed to hazardous chemicals through:
      • direct contact with contaminated surfaces,
      • deposition of aerosols,
      • immersion, or
      • splashes.

      Dermal Absorption

      Dermal absorption is the transport of a chemical from the outer surface of the skin both into the skin and into the body. Studies show that absorption of chemicals through the skin can occur without being noticed by the worker, and in some cases, may represent the most significant exposure pathway. Many commonly used chemicals in the workplace could potentially result in systemic toxicity if they penetrate through the skin (i.e. pesticides, organic solvents). These chemicals enter the blood stream and cause health problems away from the site of entry.

      The rate of dermal absorption depends largely on the outer layer of the skin called the stratum corneum (SC). The SC serves an important barrier function by keeping molecules from passing into and out of the skin, thus protecting the lower layers of skin. The extent of absorption is dependent on the following factors:

      • Skin integrity (damaged vs. intact)
      • Location of exposure (thickness and water content of stratum corneum skin temperature)
      • Physical and chemical properties of the hazardous substance
      • Concentration of a chemical on the skin surface
      • Duration of exposure
      • The surface area of skin exposed to a hazardous substance

      Research has revealed that skin absorption occurs via diffusion, the process whereby molecules spread from areas of high concentration to areas of low concentration. Three mechanisms by which chemicals diffuse into the skin have been proposed:

      1. Intercellular lipid pathway (Figure 1)
      2. Transcellular permeation (Figure 2)
      3. Through the appendages (Figure 3)

      Figure 1: Intercellular lipid pathway

      As shown in Figure 1, the stratum corneum consists of cells known as corneocytes. The spaces between the corneocytes are filled with substances such as fats, oils, or waxes known as lipids. Some chemicals can penetrate through these lipid-filled intercellular spaces through diffusion.

      Figure 2: Transcellular permeation

      As shown in Figure 2, another pathway for chemicals to be absorbed into and through the skin is transcellular, or cell-to-cell, permeation whereby molecules diffuse directly through the corneocytes.

      Figure 3: Through the appendages (hair follicles, glands)

      As shown in Figure 3, the third pathway for diffusion of chemicals into and through the skin is skin appendages (i.e., hair follicles and glands). This pathway is usually insignificant because the surface area of the appendages is very small compared to the total skin area. However, very slowly permeating chemicals may employ this pathway during the initial stage of absorption.

      Contact Dermatitis

      Contact dermatitis, also called eczema, is defined as an inflammation of the skin resulting from exposure to a hazardous agent. It is the most common form of reported OSD, and represents an overwhelming burden for workers in developed nations. Epidemiological data indicate that contact dermatitis constitutes approximately 90-95% of all cases of OSD in the United States. Common symptoms of dermatitis include:


      Conclusion

      Their lipophilic nature and small molecular size makes essential oil constituents great candidates for dermal absorption. In fact, these fragrant molecules are able to enter the bloodstream through such topical applications in quantifiable ways. 7 Many factors influence transdermal absorption. By understanding the science of essential oils and the physiology of the body, we can target our therapies and maximize our aromatherapeutic effects.

      Timothy Miller ND, RA, is a naturopathic doctor and registered aromatherapist. His efforts lie in spearheading clinical aromatherapy instruction. Dr Miller believes in dynamic and engaging teaching techniques while focusing on interesting and clinically-relevant material. The webinar-based series “Clinical Aromatherapy for Medical Professionals” can be found online at www.ncnm.edu/ce. Coursework has been pre-approved for continuing education through the Oregon Board of Naturopathic Medicine.


      Results

      The effect of 8 oncology molecules, selected from first generation EGFRi and pan-kinase inhibitors, which mostly target VEGFR, and second and third generation therapies targeting main mutations relating to first generation treatment resistance, were assessed in vitro using a 3D micro-epidermis model. The drug incubation concentrations (3, 10, 30, 100 nM) were selected to reflect the clinically relevant (unbound) drug exposures (Table 1). The drug impact was assessed by analysis of tissue size and keratinocyte proliferation using Ki-67 staining and keratinocyte differentiation using filaggrin, desmoglein-1 and involucrin staining.

      By covering a large spectrum of concentrations (3 nM to 1 μM), we showed significant changes in the studied parameters, often in a dose-dependent manner. Using such a concentration range allows us to overcome nonspecific interactions of the drugs (e.g. with the plastic support, the extracellular matrix, the proteins in media, etc.) that might interfere in the experiment.

      Increased micro-epidermis size and Ki-67 staining with a concomitant decreased of filaggrin, desmoglein-1 and involucrin expression were considered as a pro-proliferation effect of the tested molecule. On the other hand, a pro-differentiation effect was defined as a decrease of both the micro-epidermis size and Ki-67 staining and an increase of filaggrin, desmoglein-1 and involucrin expression.

      Pan-kinase inhibitors barely impact the micro-epidermis structure and differentiation markers

      Sunitinib had no impact on the epidermis size and sorafenib strongly decreased the size of the epidermis (Table 2). Both pan-kinase inhibitors did not impact the desmoglein-1 and involucrin protein expression and significantly decreased filaggrin protein expression. Of note, this effect of pan-kinase inhibitors was achieved at 100 nM, lower concentrations did not impact the markers followed in the study. Sunitinib was the only TKi assessed that did not shown any toxicity at the highest concentration tested (1 μM). These results indicate that VEGFRi have a pro-proliferation effect on the keratinocytes.

      EGFRi affect epidermal structure and differentiation markers

      Most of the EGFRi tested, including afatinib, lapatinib, and dacomitinib, showed an effect on desmoglein-1, involucrin and filaggrin expression in a dose-dependent manner (Table 2). Gefitinib increased in a dose-dependent manner only the expression of desmoglein-1. Erlotinib and osimertinib did not affect the expression of the junction proteins. For all EGFRi tested, the epidermal toxicity evaluated at 1 μM was significant, interfering with the epidermal development, to the point that no tissue was available for further data analysis. At unbound plasma drug concentrations 3, 10, and 30 nM, all first and second generation EGFRi showed a decrease in keratinocyte proliferation, micro-epidermis size and an increase of the desmoglein-1, involucrin and filaggrin protein expression, evidence of a pro-differentiation effect.

      Interestingly, the osimertinib, a third generation of EGFRi developed to target drug resistance cells but also to provide better drug tolerance, was the only EGFRi which did not show any impact on all parameters except cell toxicity at the higher concentration (1 μM).

      Afatinib affects keratinocyte protein expression, viability and proliferation

      Afatinib treatment resulted in significantly decreased epidermal volume in the 3D reconstructed micro-epidermis model compared to vehicle (Fig. 1a). Involucrin and desmoglein-1 expression were significantly increased at 3, 10, 30 nM in a dose-dependent manner and filaggrin expression was significantly increased at 10 nM and 30 nM in a dose-dependent manner. A higher drug concentration above 1 μM was toxic leading to epidermal necrosis.

      Afatinib decreases the size of the epidermis and increases skin differentiation markers. Micro-epidermises were treated with afatinib at 3, 10 and 30 nM. Drugs and concentrations effect on microepidermis were assessed with different parameters (a) Micro-epidermis volume incubated with afatinib 30 nM. b actin expression intensity, c microepidermis volume, d desmoglein-1 expression, e Involucrin expression and f filaggrin expression. * p < 0.05, ** p < 0.01, *** p < 0.001

      The effect of Afatinib on the epidermal barrier function was assessed on RHE models by measuring the rate of TEWL (Fig. 2). Addition of petrolatum (negative control) led to a significant decrease of the TEWL rate by 48, 77 and 75% respectively on day 1, 2 and 5 following application, compared to untreated control. The surfactant Sodium Dodecyl Sulfoxide (SDS, at 0.5% used as positive control) significantly increased the TEWL rate by 98 77 and 58% respectively on days 1, 2, and 5 following application. Afatinib significantly increased the rate of TEWL by 22% on day 2, while on days 5 and 7 no significant change was observed.

      RHE skin barrier function is deteriorating on day 2 of afatinib treatment. Skin barrier function was assessed by measuring the rate of trans epidermal water loss. Topical application of petrolatum on the RHE was used as negative control and topical exposure to a 0.5% SDS solution on the RHE was used as positive control

      Further results show that afatinib had a significant effect on cell viability in a dose-dependent manner (Fig. 3). On the other hand, afatinib did not show any effect at 2.59 nM and 25.89 nM on cell apoptosis. Taken together these results show that afatinib impairs keratinocyte viability and proliferation in the micro-epidermis model, but it does not induce keratinocyte apoptosis.

      a Keratinocyte viability decreases following exposure to afatinib. b Afatinib does not induce apoptosis in keratinocytes. Keratinocytes were exposed for 24 h to each condition shown. Percentages represent the relative effect compared to vehicle. Staurosporine at 1 μM was used as positive control and correspond to 100% of cell apoptosis. Post-hoc Dunett’s test * p < 0.05, **** p < 0.0001

      Acetaminophen used as control showed no effect on any of the measured parameters including cell toxicity at 1 μM.


      Dermal Penetration

      Dermal penetration testing, also known as percutaneous penetration, measures the absorption or penetration of a substance “through the skin barrier and into the skin” (OECD, 2004a). Dermal penetration studies are conducted to determine how much of a chemical penetrates the skin, and thereby whether it has the potential to be absorbed into the systemic circulation. Dermal penetration is considered to occur by passive diffusion however, biotransformation of the test substance within the deeper viable regions of the skin (metabolism) prior to systemic absorption can occur (OECD, 2004a).

      Dermal toxicity testing, on the other hand, is conducted to assess the local and/or systemic effects of a chemical following dermal exposure. It may provide an indication that the substance penetrates the skin if it produces systemic toxicity, but the amount of chemical absorbed is not quantified by dermal toxicity testing.

      One of the primary roles of the skin is to act as a barrier to protect humans from substances contacted in the environment. Permeation of a substance through the skin depends upon a number of factors, including: the formulation or vehicle in which it is presented to the skin physicochemical properties of the test substance such as lipophilicity (fat solubility), molecular weight, charge, and concentration of the test substance and area and duration of exposure. The qualities of the outermost layers of the skin, the stratum corneum, typically determine the rate of dermal penetration. The biological factors affecting absorption include the site of the body, the integrity of the stratum corneum and thickness of the epidermis in addition to other physiologic determinants such as temperature and local blood flow (OECD, 2004a 2004b WHO, 2006). [For a description of the structure of the skin, see the AltTox section Skin Irritation/Corrosion.]

      Both in vivo and in vitro methods are used for determining the percutaneous penetration of a substance. Selection of the method used may result in obtaining different types of information and relevance of the test results to human dermal exposure. Another factor in the selection of an in vivo versus in vitro test system (or a combination of the two) may be the preference of the national regulatory authority. The in vitro dermal penetration test methods that utilize donated human skin are considered more relevant to the in vivo tests in animals, since they actually utilize resected human skin. It should be noted here that the dermal absorption testing of new cosmetic ingredients in living animals in the European Union is no longer permitted under Regulation (EC) 1223/2009. Therefore, there is a particular need to have scientifically valid alternatives in the area of safety testing of cosmetics. In fact, the testing of cosmetic ingredients using in vitro models has been the norm for a number of years within the European Union (Diembeck et al., 2005 SCCS, 2010).

      The OECD recommends consulting their guidance document, the OECD Guidance Document for the Conduct of Skin Absorption Studies (No. 28, March 5, 2004), to determine the most suitable method for a particular study (OECD, 2004a). A more recent document published by OECD provides practical guidance to facilitate harmonized interpretation of experimental data from dermal absorption studies with pesticides, biocides, and other industrial chemicals (OECD, 2011). Whatever dermal absorption test system is used, its performance in the testing laboratory should be confirmed with reference chemicals showing comparable results to published values (OECD, 2004a WHO, 2006 OECD, 2011).

      The Animal Test (OECD 427)

      Historically, the rat has been the most common species used for in vivo dermal penetration testing, and is still a requirement as part of the safety assessment of pesticides in North America (US EPA, 1998). The in vivo method for determining the penetration of a substance through the skin of an animal and into the systemic compartment is described in OECD Test Guideline (TG) 427 (OECD, 2004b, adopted April 13, 2004). One or more doses of the test material, usually prepared by combining a radiolabeled form of the active with cold material and all the other components of the formulated product, are applied to a measured area of shaved skin of groups of rats for a specified time. Precautions are taken to ensure that the exposure is limited to the dermal route. The exposure time and dose levels are based on expected human exposure. The animals are observed at regular intervals for signs of toxicity, and daily excreta (and sometimes expired air) are collected separately and analyzed for the radiolabeled test substance. Blood is collected at specified times and when the groups of animals are euthanized.

      The mass balance distribution of the radiolabeled test substance in various tissue samples in rats from the application site, excreta, and the body is determined at specific times following dermal dosing. The results are reported as the rate, amount, or percentage of skin absorption of the test material. The full details of the animal procedure can be found in the guidelines and guidance documents (OECD, 2004a OECD, 2004b OECD, 2011). The dermal penetration of substances through rat skin is known to over-estimate human absorption (WHO, 2006). This is not surprising due to the very different morphologic characteristics of the skin between rats and humans. However, the rat in vivo test method is considered to be of some value due to the “generation of systemic kinetic and metabolic information” that may relate to man (OECD, 2004a).

      The Non-Animal Test (OECD 428)

      In vitro methods to determine dermal absorption are described in OECD TG 428, Skin Absorption: In Vitro Method (OECD, 2004c, adopted April 13, 2004). The in vitro method is based on the permeability of a test substance from its formulation applied as a finite dose across human or animal skin preparations. Additional guidance for industry-specific sectors is also now available (SCCS, 2010 EFSA, 2012). In modern day protocols the skin sample is dermatomed to a specific thickness and mounted in a static or flow-through glass diffusion chamber (Heylings, 2014 WHO, 2006 OECD, 2011). This is described in more detail below.

      Historical Aspects of the In Vitro Guideline Development

      Dermal penetration studies using in vitro skin preparations from animals and man were introduced into the regulatory arena in the early 1990s (ECETOC, 1993). They were not subject to a formal validation program, as would occur with a new “alternative” method in modern day method assessment. However, a number of industries had their own in-house validation data often involving human volunteer studies and human skin in vitro. Many publications were emerging as to the value of the alternative approach in this area of toxicology (Scott and Ramsey, 1987 Ramsey et al., 1994). In 1994, ECVAM brought together an international expert group from academia and the pesticide, cosmetic, industrial chemical, and pharmaceutical industries to review the area of percutaneous absorption and to formulate an outline protocol that could be used for the assessment of dermal absorption. This ECVAM Workshop agreed an approach that included a non-animal method for the assessment of dermal absorption (Howes et al., 1996). Almost another decade passed with a number of meetings and conferences driven largely by a toxicology sub-group of the European Crop Protection Association (ECPA), which focused on the issue of stand-alone in vitro methodology and test guideline development. Finally, under the mutual acceptance of data, separate test guidelines for the assessment of dermal absorption in vivo (OECD TG 427) and in vitro (OECD TG 428) were formally agreed by the Member States of OECD and published in 2004.

      The OECD Guidance Document for the Conduct of Skin Absorption Studies (2004a) states that “…skin absorption is primarily a passive process and studies undertaken using appropriate in vitro experimental conditions have produced data for a wide range of chemicals that demonstrate the usefulness of this method. Such methods have found use in, for example, comparing delivery of chemicals into and through skin from different formulations and can also provide useful models for the assessment of risk due to percutaneous absorption in humans.”

      Although non-animal methods for dermal penetration have not been formally validated, the in vitro methods described by OECD TG 428 have been accepted by EU authorities for many years. The use of the non-animal method for dermal absorption by North American Agencies was an area of significant debate during the development of the OECD test guidelines during the late 1990s. US EPA and the Health Canada Pest Management Regulatory Agency (PMRA) were the most reluctant to formally acknowledge acceptance of the in vitro percutaneous penetration methods. However, these Agencies do now accept in vitro data providing it is conducted according to OECD TG 428, but only as a refinement of risk assessments for pesticides using the “triple pack approach” (NAFTA, 2008). Therefore, rat in vivo studies are still required by North American Agencies in 2015 for the registration of pesticide-containing products.

      Although not a formal validation study, an inter-laboratory assessment of in vitro skin absorption methods was conducted in which three compounds were tested in 10 laboratories using standardized protocols (Van de Sandt et al., 2004). Some variability in the results was reported, but the in vitro methods were considered to be robust. Nine labs used human skin, and it was concluded that “variation observed may be largely attributed to natural human variability in dermal absorption and the skin source.” The type of diffusion cell used did not appear to affect the results. Skin thickness did alter the results – only slightly for benzoic acid and caffeine, but significantly for testosterone, where absorption was higher with dermatomed human skin.

      A variety of in vitro procedures using resected skin have been developed for dermal penetration testing. Most of these methods use split-thickness human or animal skin mounted in a Franz glass diffusion chamber (Franz, 1975 Clowes et al., 1994 Heylings, 2014). Human skin is mostly used in these models for the safety assessment of industrial chemicals and pesticides (EFSA, 2012 OECD, 2011), but pig skin is also permitted for the safety assessment of cosmetic ingredients (SCCS, 2010).

      Advantages beyond the fact that live animals are now rarely used for dermal penetration assessment include the following: human skin more closely estimates human exposure the early phase of absorption can be determined replicate measurements can be made using the same skin from the same human donor, or between different human donors for the same dose application exposure conditions can be easily varied and a wider range of physical forms of test substances such as solids, granules, and powders can be easily evaluated (OECD, 2004a OECD, 2011). The primary limitation described for in vitro percutaneous penetration testing is that the metabolic profile of the test substance cannot be determined (for more on in vitro determination of skin metabolism see Dermal Penetration: Emerging Science & Policy). However, if the purpose of the investigation is the determination of the dermal penetration and ultimately the systemic exposure of the test substance from a formulated product, then the in vitro method using resected skin has great utility and has shown to be predictive of dermal absorption in both rats (Scott and Ramsey, 1987) and man (Ramsey et al., 1994 WHO, 2006 OECD, 2011).

      Practical use of the In Vitro Test Guideline OECD 428

      It is important to note that the in vitro studies that use resected skin now use a finite (low volume) dose of the finished or formulated product. Many of the historic in vitro skin penetration studies used infinite (high volume) doses of the test substance, and usually in a vehicle solvent rather than the finished product (Bronaugh and Franz, 1986). This enables a steady-state rate (or flux) of the chemical to be determined. In modern day regulatory studies it is the amount of the test chemical that is measured in the various compartments of a study following application of a finite dose of the finished product that is the figure used in the risk assessment calculations. In most regulatory studies the mass of the test material that has penetrated the skin into the receptor fluid per area of skin is related to the applied concentration in the finite dose. This can be conveniently expressed as the percentage of the dose applied that has penetrated the skin into the receptor at 24 hours, providing a “worst case” daily absorbed dose. The exposure period may be, for example, 30 minutes for a shampoo-in hair dye (but less for a traditional shampoo), or 6 hours to cover a “working day” for an operator spraying a pesticide. A leave-on type cosmetic would, of course, be left on the skin for the full 24-hour exposure period.

      When designing an in vitro study with excised skin, it is important to determine that the nature of the receptor fluid is not rate limiting to the diffusion of the substance from the skin into this phase (OECD, 2011 EFSA, 2012). The amount and rate of test substance accumulating in the receptor chamber and a time course profile are measured, normally by taking samples of the receptor fluid at multiple time points over a 24 hour period. In a regulatory in vitro dermal penetration study, a mass balance recovery of the applied dose is undertaken at 24 hours, in addition to measuring the material in the receptor fluid. This includes the measurement of the amounts of test substance that can be easily removed from the skin surface by normal soap washing at the end of the expected exposure period, plus the amount removed by soap washing at 24 hours.

      In addition to the washed off fraction of the dose applied, the amount adsorbed to the skin surface is determined in sequential tape strips of the stratum corneum. Following this tape stripping procedure, the proportion of the applied dose in the remaining underlying epidermis and dermis is also determined at this 24 hour time point. This provides a total mass balance recovery of the applied substance in each of the diffusion cells. When the test substance of interest is volatile, a modified donor chamber containing porous carbon filters can be used to trap any material evaporating from the skin surface into the headspace above the skin. It is important here not to occlude the skin since this will potentially enhance skin penetration. The tape stripping of the stratum corneum is particularly important, since it permits the quantification of unabsorbed, non-dislodgeable material that would normally be lost by desquamation in man. This tape stripping procedure conducted using resected human skin in vitro has been shown to predict tape stripping in human volunteers very well for a range of formulated products (Trebilcock et al., 1994). The number of strips taken and the inclusion/exclusion of these layers in the calculation of the absorbed dose is provided in the specific industry guidance (SCCS, 2010 EFSA, 2012). A schematic of a typical mass balance/tape stripping procedure in Franz static diffusion cells as used by Dermal Technology Laboratory for dermal absorption studies is shown in the Figure below.

      Since the stratum corneum skin barrier is a non-viable layer of the skin, both fresh and frozen human and animal skin can be used to assess in vitro percutaneous penetration (OECD, 2004c OECD, 2011). Since the method is designed to assess the diffusion or permeation of a chemical through a non-viable barrier, the stratum corneum, it is very important to ascertain that the barrier function is intact prior to dosing the skin. Regardless of the type of skin preparation used, its integrity must be demonstrated prior to the application of the test material. Several methods, including transcutaneous electrical resistance, transepidermal water loss, and tritiated water permeability, are permitted for assessing the integrity of skin samples prior to their use (Davies et al., 2004 OECD, 2004a SCCS, 2010 EFSA, 2012). The electrical resistance method is regarded as the most practical and reliable of these integrity checks. Furthermore, it is important that laboratories conducting these GLP studies have demonstrated that their methods for assessing skin integrity and the dermal absorption of reference compounds are in line with previously published methods (Davies et al., 2004 SCCS, 2010 OECD, 2011 EFSA, 2012 Heylings, 2012).

      Experimental variables that may affect the results of in vitro dermal penetration studies include: the selection of the in vitro test system, the storage and preparation of the skin samples, the incorporation of the radio-labeled test substance, volatility of the test substance, test substance degradation, the homogeneity and stability of the dose preparations, exposure time, occlusion/non-occlusion of the test site on the skin, temperature, removal of test substances at the end of the experiment, sampling and data analysis techniques, and the sensitivity and accuracy of the analytical method (OECD, 2004a OECD, 2011). Many protocol variables have been shown to influence the rate and extent of skin absorption of a test substance. These variables include the use of infinite versus finite dosing, test substance volatility, vehicle of application, exposure time, and leave on versus rinse off of test substance (Brain et al., 1995 Walters et al., 1997). This is exactly why a strict adherence to guidance is required in modern day studies by the various regulatory bodies (SCCS, 2010 EFSA, 2012).

      The test substance retained within the skin (except for the stratum corneum) is included in the calculated absorbed dose, although in the live animal it may eventually be lost by shedding and renewal of the skin. The so-called “systemically available dose” used in a risk assessment from human skin studies includes the proportion of the applied dose that has reached the receptor fluid at 24 hours, plus the amount in the remaining skin following tape stripping. In addition, depending on the type of risk assessment, the time course profile of absorption and expected exposure to the chemical during its use, a proportion of the tape strips of the stratum corneum may also be included as “potentially systemically available” (EFSA, 2012). For example, for a pesticide that demonstrates a slow continuous absorption into the receptor fluid over a 24 hour period, despite a skin surface wash at 6 hours, if the proportion of the applied dose reaching the receptor at 12 hours is below 75% of the amount in the receptor at 24 hours, then the material present in the tape strips following discarding of strip 1 and strip 2 is also included in the “dose absorbed.” This is for reasons of conservatism for this type of product, and so as not to under-estimate dermal absorption (EFSA, 2012). This so-called 2-strip rule for pesticides does not apply to cosmetic ingredients that utilize a slightly different approach for calculation of dermal absorption, albeit using essentially the same methodology (SCCS, 2010).

      The OECD guidance and TG for in vitro percutaneous penetration testing were issued in 2004, but the guidelines are very broad and do not endorse specific protocols or protocol components. An example of some of the experimental variables to be considered with in vitro dermal penetration studies are described in the report assessing the percutaneous penetration of diethanolamine in cosmetic formulations using in vitro human skin (Brain et al., 2005), including the testing of rinse off versus leave in formulation, varying the formulation ingredients, using replicate skin samples from different donors, and comparing permeability in fresh versus frozen skin tissues. Many of the variables described above have been studied in more detail over the years and harmonization of the protocols has improved the reliability, reproducibility, and accuracy of in vitro dermal absorption testing.

      Other Non-animal Methods

      Human cell-based or reconstituted human epidermal (RHE) skin cell models, sometimes called 3-dimensional (3D) skin models, are also used to evaluate dermal penetration. These models retain some capacity to assess the skin metabolism of a test substance, but they are more permeable than the ex vivo human and animal preparations, and are not permitted at this time for the prediction of skin penetration in a human risk assessment (SCCS, 2010 OECD, 2011 EFSA, 2012).

      (Q)SAR models, which are computational models based on molecular structure, have been developed to assess the skin permeability of drugs and chemicals. As with skin culture models, these mathematical models have a utility in the pre-development arena, particularly for pharmaceuticals. There are suggestions for incorporating (Q)SARs for dermal absorption into risk assessments for use in programs such as REACH (Van de Sandt, et al., 2007 Berge, 2009).

      Reconstituted 3D skin and (Q)SAR models for dermal penetration testing are covered further in the Emerging Science & Policy section.

      Global Acceptance of the In Vitro Test Guideline OECD 428

      In vitro dermal penetration methods that utilize resected human skin are now widely used throughout the world. To promote the development of standardized protocols and better agreement of in vitro dermal permeation methods, the Institute for In Vitro Sciences (IIVS) hosted a workshop in Gaithersburg, USA for a group of international stakeholders back in 2005 to discuss the OECD guidance and to make recommendations on implementing specific aspects of the guidance. Recommendations from this meeting are summarized in a poster available on the IIVS website. Also in 2005, the World Health Organization (WHO) International Programme on Chemical Safety convened a meeting of dermal absorption experts in Hanover, Germany. This group produced a comprehensive monograph detailing the knowledge and development in this area. The output of this task group was published the following year as an Environmental Health Criteria (EHC) 235 (WHO, 2006). This includes issues such as selection of the skin model and receptor fluid, best practices for storing frozen skin, barrier integrity assessment methods, and more. Additional recommendations by the workshop participants were that human skin be considered the “gold standard,” that more in vitro data is submitted to regulatory agencies to promote its acceptance, and that some mechanism be developed for sharing the responses of regulatory agencies on submitted in vitro data to relevant stakeholders.

      The regulatory authorities in the European Union now extensively utilize the in vitro human skin method as a stand-alone method as part of the approval process for the registration of new pesticide products. In an attempt to determine the barriers to acceptance of stand-alone in vitro dermal absorption studies by North American authorities a workshop was held in 2012 in Gaithersburg, Maryland, USA, bringing together an international panel of dermal absorption experts with USA and Canadian pesticide regulators and non-governmental representatives. The outcome of the workshop was to build consensus around best practices for the conduct and reporting of in vitro dermal absorption studies for pesticide risk assessment and to increase comparability of in vitro studies across different laboratories. There has certainly been a move towards more extensive use of the in vitro approach particularly by US EPA. For example, a recent review of the pesticide sulfoxaflor utilized the so-called “triple-pack” approach that uses in vitro dermal absorption data generated in rat and human skin to correct the rat in vivo absorption value for man (US EPA, 2012).

      Hopefully, by the end of this decade, there will be universal acceptance of in vitro dermal penetration methods using resected human skin by all nations and all regulatory authorities round the world and we can look back at this area of safety testing where good science and extensive dialogue between the different stakeholders has led to not just the reduction and refinement of animal procedures, but the complete replacement of living animals for the purposes of estimating the absorption of chemicals through the skin.

      AltTox Editorial Board reviewer(s):
      William Dressler, PhD

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      Brain, K.R., Walters, K.A., Green, D.M., et al. (2005). Percutaneous penetration of diethanolamine through human skin in vitro: Application from cosmetic vehicles. Food Chem. Toxicol. 43, 681-690.

      Bronaugh, R.L.,& Franz, T.J. (1986). Vehicle effects on percutaneous absorption: in vivo and in vitro comparisons with human skin. Brit. J. Dermatol. 115, 1-11.

      Clowes, H. M., Scott, R. C., & Heylings J.R. (1994). Skin absorption: flow-through or static diffusion cells. Toxicol. In Vitro 4, 827-830.

      Davies, D.J., Ward, R.J., & Heylings, J.R. (2004). Multi-species assessment of electrical resistance as a skin integrity marker for in vitro percutaneous absorption studies. Toxicol. In Vitro 18, 351-358.

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      ECETOC. (1993). European Centre for Ecotoxicology and Toxicology of Chemicals. Monograph No. 20, Percutaneous Absorption, August 1993.

      EFSA Panel on Plant Protection Products and their Residues (2012). Guidance on Dermal Absorption. EFSA Journal 10 (4), 2665.

      Franz, T. J. (1975). Percutaneous absorption on the relevance of in vitro data. J. Invest. Dermatol. 64, 190-195.

      Heylings, J.R. (2012). Risk assessment. Transdermal and topical drug delivery: Principles and practice, 1st Ed. Benson, H.E. and Watkinson A.C. (eds.). Wiley & Sons, Inc. New Jersey.

      Heylings, J.R. (2014). Diffusion cell design. Topical drug bioavailability, bioequivalence and penetration. V.P. Shah, et al. (eds). Springer Science, New York.

      Howes, D., Guy, R.H., Hadgraft, J., & Heylings, J.R., et al. (1996). Methods for assessing percutaneous absorption. ATLA 24, 81-106.

      North American Free Trade Agreement (NAFTA) (2008). Dermal absorption group position paper on use of in vitro dermal absorption data in risk assessment (unpublished available upon request). pp. 1-3.

      OECD. (2004a). Guidance document for the conduct of skin absorption studies. OECD Series on Testing and Assessment. No. 28. OECD. Paris, France.

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      Ramsey, J. D., Woollen, B. H., Auton, T. R., & Scott, R. C. (1994). The predictive accuracy of in vitro measurements for the dermal absorption of a lipophilic penetrant (Fluazifop-Butyl) through rat and human skin. Fund. Appl. Toxicol. 23, 230-236.

      Scott, R. C., & Ramsey, J. D. (1987). Comparison of the in vivo and in vitro percutaneous absorption of a lipophilic molecule (cypermethrin, a pyrethroid Insecticide). J. Invest. Dermatol. 89, 142-146.

      Trebilcock, K. L., Heylings, J. R., & Wilks, M. F. (1994). In vitro tape stripping as a model for in vivo skin stripping. Toxicol. In Vitro 8(4), 665-667.

      United States Environmental Protection Agency (US EPA) (1998). Health effects test guidelines. OPPTS 870.7600. Dermal Penetration. Washington, DC. Document ID: EPA 712–C–98–350 pp. 1-12.

      US EPA (2012). Sulfoxaflor human health risk assessment. Office of Chemical Safety and Pollution Prevention. Washington, DC. DP No. 382604 pp. 1-176.

      Van de Sandt, J.J., van Burgsteden, J.A., Cage, S., et al. (2004). In vitro predictions of skin absorption of caffeine, testosterone, and benzoic acid: A multi-centre comparison study. Regul. Toxicol. Pharmacol. 39, 271-281.

      Van de Sandt, J.J., Dellarco, M., & Van Hemmen, J.J. (2007). From dermal exposure to internal dose. J. Expo. Sci. Environ. Epidemiol. Suppl. 1, S38-S47.

      Walters, K.A., Brain, K.R., Dressler, W.E., et al. (1997). Percutaneous penetration of N-nitroso-N-methyldodecylamine through human skin in vitro: Application from cosmetic vehicles. Food Chem. Toxicol. 35, 705-712.

      World Health Organization (WHO). (2006). Dermal absorption. Environmental Health Criteria (EHC 235). ISBN 978 92 4 1572 35 4.



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