Skin reactions to irritants assessed by non-invasive bioengineering methods

Pathophysiological components of irritant contact dermatitis caused by 3 chemically-different irritants were investigated. 20 healthy volunteers were patch tested with sodium lauryl sulphate, nonanoic acid and hydrochloric acid on the flexor side of the upper arm. The skin response was evaluated after 24, 48 and 96 h by visual scoring and measured by the following bioengineering methods: transepidermal water loss measurement, electrical conductance for measurement of skin hydration, laser Doppler flowmetry for measurement of cutaneous blood flow and 20 MHz ultrasound A-scan for measurement of skin thickness. In spite of homogeneous inflammatory responses, significant differences in the severity of the injury to the skin barrier function caused by the different irritants were found. Also significant differences between irritants were found in the time course of development of maximum irritant reactions. Bioengineering methods indicating inflammatory responses (measurement of blood flow and skin thickness) were helpful in quantifying the irritant response in general, while bioengineering methods indicating epidermal damage (measurement of TEWL and electrical conductance) were helpful in classifying the individual irritants.     

Multicentre study for the development of an in vivo model to evaluate the influence of topical formulations on irritation

Although skin protective products to prevent irritant skin reactions are in wide use, neither standardized test models to prove differences in efficacy exist, nor has the quality or the reproducibility of results been evaluated in a multicentre approach. This should be mandatory when developing or testing skin care products. Therefore, we have designed a multicentre study in an approach to find a standardized test procedure for the evaluation of skin protective products. In this irritation study, a repeated short-time occlusive irritation test (ROIT) with a standardized protocol has been evaluated in 2 phases (12 days and 5 days protocol) in 4 (n=20) respectively 6 (n=33) skilled centres. The skin reaction was induced by 2 irritants (0.5% aq. SLS and toluene, 2x a day for 30 min). Its modification by 3 different cream bases with different hydrophilicity was analyzed. The irritation was monitored by bioengineering methods (TEWL measurement, colorimetry) and by clinical scoring. The evaluation showed that significant results could already be achieved with the 5-day protocol. Furthermore, in spite of the expected inter-centre variations due to heterogeneity of the individual threshold of irritation, interpretation of clinical score, and inter-instrumental variability, the ranking of the vehicles regarding reduction of the irritant reaction was consistent in all centres.     

Effect of a moisturizer on skin susceptibility to NiCl2

The aim of this study was to evaluate whether a moisturizer used on normal skin can increase skin response to allergens. Twelve nickel-allergic volunteers applied a lipid-rich moisturizer on the upper arm 3 times daily for 7 days, while the other upper arm served as a control. A control group followed the same treatment protocol. Following treatment with moisturizer, patch tests with 1% NiCl2 aqueous solution were applied on each upper arm. After 24 and 72 h, skin reactions were evaluated blinded by clinical scoring, and by bioengineering methods measuring transepidermal water loss, skin colour and skin thickness. In the nickel-allergic group the strength of patch-test reactions was increased on the moisturizer-treated arm as evaluated by clinical scoring after 24 h and by measurement of transepidermal water loss and skin thickness after 72 h. In the control group, no significant differences were found. Our findings show that threshold values for elicitation of allergic reactions in already sensitized individuals may be influenced by use of lipid-rich moisturizers.     

Effect of moisturizers on skin susceptibility to irritants

Moisturizers are used for the treatment of dry and irritated skin. The benefit of moisturizers when used on normal skin has recently been challenged, since an earlier study indicated that the increased hydration that follows long-term use of moisturizers on normal skin may facilitate penetration of irritants. The aim of the present study was to evaluate short-term use of 2 different moisturizers used on normal skin: cream A (high lipid content) and B (moderate/low lipid content). Nineteen healthy volunteers applied the moisturizers on the upper arm/forearm 3 times daily for 5 days, while the other upper arm/forearm served as symmetrical control. The day after moisturizer treatment was stopped the skin was challenged with a patch test of sodium lauryl sulphate. Skin reactions were evaluated by bioengineering measuring methods and clinical scoring. Skin response to sodium lauryl sulphate was increased on moisturizer-treated arms compared to controls for one of the moisturizer (cream A), while this was not statistically significant for the other moisturizer (cream B). Data confirm previous indications that some moisturizers when used on normal skin may increase skin susceptibility to irritants.     

Barrier function of the skin in a repetitive irritation model and influence of 2 different treatments*

Background/aims: The aim of this study was to develop and characterize an experimental model of cumulative irritant contact dermatitis using the repetitive application over 2 weeks of 2 different irritative stimuli, sodium lauryl sulphate (SLS) in low concentration and toluene. Further, it was intended to look at the effect of 2 different treatments which could influence the regeneration of the skin barrier, as already investigated in preliminary short-term experiments. Methods: Occlusive short-time daily application of the irritants (SLS 30 min; toluene 10 min) was performed. After removal of the occlusive patch, the irritated areas were treated with 2 different preparations, Lotio Alba Aquosa and vaseline (white petroleum jelly). Bioengineering techniques were used to precisely measure the skin condition. Results: A mild irritation was obtained under these experimental conditions. The bioengineering measurements allowed us to differentiate both irritants through their different influences on trans-epidermal water loss and skin hydration. Treatment with vaseline did not significantly influence the course of the irritation, but Lotio Alba Aquosa clearly potentiated it, whereby different effects were noticed for both irritative stimuli. Conclusions: The use of bioengineering techniques in this model of mild cumulative irritation allowed us to differentiate the action of both irritants, showing that they act through different mechanisms. The results of the treatments pointed out the need to treat an irritation with the right product for the right time period.     

Skin reactions to irritants assessed by polysulfide rubber replica

Skin damage after application of experimental irritants was evaluated under blind conditions and by the use of polysulfide rubber replica. Closed patch tests with 7 different irritants, solvents and empty chambers were applied to 16 volunteers, and the skin damage was evaluated visually and by a replica technique after 24, 48 and 96 h. We found that 3 of the irritants (sodium lauryl sulphate, hydrochloric acid and croton oil) were capable of causing specific and significantly different patterns of skin damage. The patterns could be divided into papular (hydrochloric acid, croton oil) and non-papular (sodium lauryl sulphate, sapo kalinus, sodium hydroxide) types. Nonanoic acid caused a non-paular pattern, but propanol, used as a solvent, by itself also produced a non-papular pattern. The time between application of the irritant and appearance of the characteristic alteration in the skin surface differed, depending on the irritant applied.     

Cutaneous response to irritants

We evaluated the role of pre-existing dermatitis in the response to irritants by patch testing the skin of 40 healthy volunteers and the uninvolved skin of 480 subjects for 2 days. These latter were affected by active atopic dermatitis, psoriasis, eczema with positive and negative patch test reactions, urticaria and generalized pruritus. A first panel containing 15 micro L of aq. solutions of disodium laureth sulfosuccinate (NaLSS) 5% and 10%, potassium cocoate (KCC) 5%, potassium oleate (KOL) 5%, zinc coleth sulphate (ZnCS) 5%, sodium mireth sulphate (NaMS) 5%, sodium cocoamphoacetate (NaCCAA) 3% and 5%, was simultaneously applied to 1 site on the upper back. The results, scored by visual assessment, were compared to those observed when testing on the opposite side a second panel containing 15 micro L of aq. solutions of 3 well-known irritants, benzalkonium chloride (BAK) 1%, sodium lauryl sulphate (SLS) 1%, and dimethylsulphoxide (DMSO) 10%. Whilst the substances of the first panel and DMSO gave, on the whole, a scarce number of positive responses in all the tested groups, more evident differences in number, percent and mean intensity of the positive responses to BAK and SLS were found between the different groups. Although some of them seemed statistically significant, when the same values were evaluated by means of chi2 and Student t-test, they did not differ in a statistically significant way from the values found in healthy subjects. The results of this study seem to indicate that the substances of the first panel have a chemical structure that makes them quite safe in real-life conditions. In contrast, BAK and SLS have chemical properties that condition the number and intensity of the responses, making the role exerted by the pre-existing dermatosis quite marginal. In particular, there is no proof that the healthy skin of active atopic subjects is the most susceptible to the irritating effects of the tested substances.     

Testing for irritation with a multifactorial approach: comparison of eight non-invasive measuring techniques on five different irritation types

BACKGROUND: Non-invasive bioengineering methods are widely used in the assessment of irritant skin reactions. OBJECTIVES: To assess the ability of eight non-invasive measurement techniques to distinguish changes in skin conditions over time, these changes being induced by five different irritants. METHODS: The following techniques were compared in a multivariate analysis: laser-Doppler perfusion imaging (LDI), laser-Doppler flowmetry (LDF), transepidermal water loss (TEWL), visual scoring (VS), colorimetric measurements (Chromameter CR 200 a* and L* scales), Mexameter Hb scale (Mexa Hb) and capacitance (Corneometer CM 820). Irritants tested were sodium lauryl sulphate 2% (SLS), tape stripping (TS), tretinoin 0.05% (TRET), ultraviolet (UV) exposure to 30 W m(-2) UVB/95 W m(-2) UVA, and dithranol 0.5% (DIT). Measurements were performed at baseline and after 24, 48 and 72 h. The study was conducted on the upper back of 11 healthy volunteers of both sexes aged 27-51 years. RESULTS: For DIT it was possible to discriminate over time with CR 200 a* and L*, VS, LDI, LDF and Mexa Hb. In SLS discrimination over time was seen with TEWL and LDF. Discrimination in TS was demonstrated for TEWL, VS, CR 200 a*, CM 820, LDF, LDI and Mexa Hb. In TRET discrimination ability was seen for LDI, LDF, Mexa Hb and VS. For UV it was possible to discriminate using VS, TEWL, LDF, LDI and Mexa Hb. CONCLUSIONS: Different irritation patterns need different measurement modalities in order to give optimal discrimination over time.     

Skin irritation typing and grading based on laser Doppler perfusion imaging

Background/aims: Vasodilation with increased cutaneous perfusion is an essential part of an irritant inflammatory response. The aim of the present study was to investigate the usefulness of the high-resolution laser Doppler perfusion imaging (HR-LDPI) technique for investigating irritant skin reactions. Irritants may elicit clinically different reactions due to different skin penetration profiles and different modes of irritant action on the exposed tissue.
Methods: Twelve subjects were tested on the forearms using 24 h occlusive application of three concentrations of the irritants sodium lauryl sulphate (SLS) and nonanoic acid (NON) and with the topical acne drug all-trans retinoic acid (RA). Cutaneous blood flow at baseline, the increase in cutaneous blood flow and the skin area having increased perfusion were measured on day 2, day 3 and day 5.
Results: Based both on measurement of mean perfusion and area with increased perfusion, it was possible to differentiate between different clinical irritation grades on any study day. The area with increased perfusion exceeded the area with clinically visible skin reactions for irritant reactions of grade ¹/₂ and above. Irritant reactions for individual irritants could furthermore be typed using HR-LDPI. It was possible to differentiate between vehicle treatment and the different dose levels of the irritant compounds. A correlation was found between clinical scores for the individual irritants and the mean flow and the area with increased flow. The individual irritants could be differentiated due to different time courses of their skin irritation.
Conclusion: Laser Doppler imaging was found to be an important new method for characterization and grading of the inflammatory response of single exposure irritant reactions. However, standardised study procedures cannot be emphasised enough in order to obtain reliable and useful data.     

Irritants in combination with a synergistic or additive effect on the skin response: an overview of tandem irritation studies

Sodium lauryl sulfate (SLS) has often been chosen as a model for irritant contact dermatitis (ICD) to study the effect of irritants in combination (1-14). Recently 'tandem', or sequential, exposures with SLS have been performed to study the mechanism of skin barrier impairment in ICD (1-6, 15). The assessment of reactions have been documented with visual scoring, transepidermal water loss (TEWL), skin colour reflectance measurements, skin blood flow; among which TEWL has been noted as the most sensitive value (16). The matched control groups were treated with either a single exposure to a single irritant or in tandem with the same irritant repeatedly. Synergistic and additive effects have been reported for various tandem pairs of irritants, however, the mechanism for both remains unclear. The results of tandem irritation studies were evaluated to define and investigate the responses produced and deduce a possible mechanism of action. Clinical ramifications, albeit complex, are discussed.     

Long-term repetitive sodium lauryl sulfate-induced irritation of the skin: an in vivo study

Skin may adapt to topical irritants through accommodation. This study focuses on long-term exposure to irritants and attempts to demonstrate accommodation. Sodium lauryl sulfate (SLS) induced irritant contact dermatitis at 3 concentrations (0.025% to 0.075%). Distilled water, acetone and an empty chamber served as controls. Experimental compounds were applied to forearms of 7 healthy volunteers for 24 hr before replacing by a fresh chamber for 6 non-consecutive weeks over 103 days. Possible accommodation was quantified by visual scoring (erythema and dryness) and by bioengineering parameters: transepidermal water loss (TEWL), capacitance, chromametry and laser Doppler flowmetry (LDF). Significant erythema, dryness, elevated TEWL, skin colour reflectance and LDF values occurred during the exposure periods. Upon repeat exposure, an immediate and augmented response in erythema, TEWL, skin colour reflectance and LDF developed. However, irritant skin changes were not sustained. Irritation parameters return to baseline after cessation of exposure. There was no evidence of sustained irritation or accommodation after the last exposure. Study findings do not document sustained accommodation or adaptive hyposensitivity after long-term repetitive irritant exposure under these test conditions. Alternative models should be developed to prove or disprove the accommodation hypothesis.     

Which bioengineering assay is appropriate for irritant patch testing with sodium lauryl sulfate?

For testing with sodium lauryl sulphate (SLS), measurements of transepidermal water loss (TEWL) and cutaneous blood flow with laser Doppler (LD) are considered to be the most reliable methods. The aim of this study was to determine which method of measurement should be preferred when conducting SLS testing under varying conditions. Patch testing with SLS at different concentrations and exposure times was performed. TEWL values were compared with those of LD. TEWL values showed distinct changes at low SLS concentrations and short application periods. By contrast, higher SLS concentrations were necessary to increase LD values. Short application of patches changed TEWL rather than LD values. When evaluating SLS patch testing by bioengineering methods, TEWL measurement appears to be more suitable for a test procedure that provokes mild skin reactions (SLS concentration <1%), whereas LD measurement is more appropriate to evaluate pronounced skin reactions (SLS concentration >or=1%).     

The irritant effect of different metalworking fluids

The irritant effect of different water-based cutting fluids (CFs) on the skin was investigated in healthy subjects ( n = 10) using 2 non-invasive bioengineering methods. Transepidermal water loss (TEWL) was measured by an evaporimeter EP1 (Servo Med), skin blood flow (SBF) was recorded with a laser Doppler flowmeter (MBF 3D, Moor Instruments England). Additionally all test sites were evaluated by visual scoring before measurement. 3 cutting fluids A, B and C from different manufacturers were tested at use concentrations of 4% or 5%. Single 24-h patch tests and repeated patch tests were performed on the volar side of the right forearm. Measurements were carried out before and after exposure to assess basal values and the skin response to CF. Tests with CFs A and C resulted in significant increase in TEWL after 24 h and after cumulative treatment, compared with controls ( p < 0.01). The TEWL values for CF B did not differ from the water probe. An increase in SBF was found only for CFs A and C after cumulative patch testing ( p < 0.01). In spite of their similar alkalinity, the 3 CFs showed different irritant skin reactions, due to their chemical components. This was verified particularly by measurement of TEWL.     

An immunohistochemical analysis of cytokine expression in allergic and irritant contact dermatitis

The purpose of this study was to investigate whether the specific and non-specific inflammatory responses to allergens and irritants give rise to immuno-histochemical detectable differences in the cytokine profile in the skin. Skin biopsies taken at 0, 6, 24 and 72 h from contact allergic reactions to nickel and from irritant reactions to sodium lauryl sulphate were analysed. The main finding was that the dermal cells expressed similar patterns of cytokines (IL-1alpha, IL-1beta, IL-2, IL-4, IL-6 and IL-10) in both types of contact reaction at 72 h. However, two differences were observed. Staining for the IL-1 receptor antagonist was more prominent in the dermis at the late stages of the allergic reaction compared with the late stage of the irritant reaction. The other difference was an increased interferon-gamma staining of infiltrating mononuclear inflammatory cells in the dermis in the sodium lauryl sulphate group compared with the nickel group. A more rapid general onset of cytokine production was found in the sodium lauryl sulphate group than in the nickel group. The main conclusion of this study was that after 6 h the cytokine patterns did not differ between the specific and the non-specific inflammatory responses in the skin.     

Fruit acids and sodium hydroxide in the food industry and their combined effect with sodium lauryl sulphate: controlled in vivo tandem irritation study

BACKGROUND: Cutaneous exposure to a variety of irritants has been extensively studied in recent years. Nevertheless, knowledge of the induction of irritant dermatitis, especially by mild irritants at low doses and for a short duration of exposure, is still incomplete. OBJECTIVES: To quantify the irritant effects and barrier disruption properties of ascorbic acid (ASC), acetic acid (ACA) and sodium hydroxide (NaOH), particularly in combination with an anionic detergent, sodium lauryl sulphate (SLS). METHODS: In a tandem repeated irritation test, the irritants were applied for 30 min twice daily for 4 days to the skin of the mid-back of 19 healthy volunteers of both sexes. We used bioengineering techniques for measurement of transepidermal water loss (TEWL) and skin colour reflectance, as well as visual scoring. RESULTS: Repeated application of ASC and ACA caused a moderate increase in TEWL and erythema. The sequential application of ASC or ACA and SLS enhanced these effects. NaOH induced a strong reaction when applied both occlusively and nonocclusively as well as in combination with SLS, with an early onset of the inflammatory signs, leading to discontinuation of the application on the third day in most of the test fields. Notably, the irritant effect of NaOH was not as marked when applied sequentially with SLS. CONCLUSIONS: Our results demonstrate that concurrent application of an anionic detergent and a mild acidic irritant can lead to disruption of the barrier function which, although not additive, is still considerable. The combined application of SLS and mild acids does not prevent SLS-induced irritation. Furthermore, we showed that NaOH in low concentrations may also act as a potent irritant but that its effect is not enhanced by SLS. The necessity of adequate skin protection and reduction of contact with substances that are potentially barrier disruptive and irritant, e.g. in the food industry, is emphasized, not only when handling detergents, but also when processing food products.     

Differential irritant skin responses to tandem application of topical retinoic acid and sodium lauryl sulphate: II. Effect of time between first and second exposure

In clinical practice, the cutaneous exposure to chemical irritants such as surfactants and topical drugs is frequent. Topical all-trans retinoic acid (RA) is often associated with irritation and induces epidermal changes similar to those produced by sodium lauryl sulphate (SLS). Using bioengineering techniques, e.g. assessing transepidermal water loss (TEWL), capacitance and chromametry, we investigated the variations of the skin response to SLS and RA and to both chemicals applied sequentially, allowing different time periods (from 1 h to 2 weeks) between applications of SLS and RA. Both chemicals caused irritation as assessed by visual scoring, but the values from the objective variables differed at different time periods. TEWL increased dramatically shortly after applying SLS but the increase was delayed after RA. After applying SLS, the capacitance generally decreased then returned to basal values; treatment with RA produced an overall increase. Only the results from chromametry were similar. After tandem application, the drugs were synergistic for all variables except capacitance, showing an antagonistic interaction for skin hydration. These results suggest that non-specific skin irritation profoundly reflects different mechanisms of action at tissue level. With sequential application, SLS injury modified the response to RA for at least 1 week after applying SLS. These late effects of detergents should be considered when studying irritant chemical interactions and in developing strategies for the management of occupational and other irritant dermatitis.     

Contact thermography for assessment of skin damage due to experimental irritants

Irritant dermatitis after application of experimental irritants was studied by means of contact thermography. Sixteen healthy persons were patch-tested, using the following irritants: Sodium lauryl sulphate, benzalkonium chloride, nonanoic acid, hydrochloric acid, croton oil, sapo kalinus and sodium hydroxide. A main finding was that croton oil after 24 h caused a warm skin lesion, and sodium lauryl sulphate after 96 h caused a cold skin lesion. This study emphasizes the differences in the skin reactions to different irritants.     

Less skin irritation from alcohol-based disinfectant than from detergent used for hand disinfection

BACKGROUND: The benefit of alcohol-based disinfectant used on normal skin has been debated. OBJECTIVES: The objective of the present study was to compare the effects of repeated exposure to an alcohol-based disinfectant, a detergent and an alcohol-based disinfectant/detergent alternately for 10 days, including noninvasive measurements in the evaluation. Skin reactivity in irritated skin after a 4-week interval was also evaluated. MATERIALS AND METHODS: Detergent, disinfectant and alternate disinfectant and detergent were applied twice daily every 10 min for 1 h to the ventral upper arms and forearms of 17 healthy volunteers. A control area was included. After 4 weeks an SLS patch was applied to each area. Irritant reactions were quantified with a visual score recording and measurements of transepidermal water loss (TEWL) and skin colour were performed on days 1, 5, 11, 38 and 40. RESULTS: On day 5 the detergent caused a higher visual score than either disinfectant applied alone or alternate disinfectant and detergent, P < 0.05. On day 11 detergent and alternate disinfectant and detergent caused a higher score than disinfectant, P < 0.05. An increased irritant response for detergent compared with disinfectant alone and alternate disinfectant and detergent was confirmed by TEWL evaluations on days 5 and 11, P < 0.001, while no significant difference was found by colour measurement. After 4 weeks, when evaluated by colour measurement, significantly less response to the SLS patch was found on the disinfectant-treated area compared with the control area and detergent area, and a similar trend was found for TEWL, although it was not statistically significant. CONCLUSION: Alcohol-based disinfectant caused less visible skin irritation and less skin barrier disruption than the use of detergent. The alternate use of detergent and disinfectant caused less irritation than the use of detergent, and a possible interaction between the two irritants was not indicated. After 4 weeks there was a tendency for decreased skin reactivity in the skin areas which had 4 weeks previously been exposed to disinfectant.     

Combined effects of irritants and allergens. Synergistic effects of nickel and sodium lauryl sulfate in nickel- sensitized individuals

Knowledge of the combined effects of irritants and allergens is of interest with respect to accurate risk assessment. The threshold for elicitation of allergic contact dermatitis in previously sensitized individuals may theoretically be markedly influenced by the simultaneous presence of irritants and allergens. Combined exposures have, however, only been studied infrequently. In the present study, the combined effect of an irritant and an allergen was evaluated in a dose-response designed experimental study. 20 nickel-sensitized subjects were exposed to patch testing with varying concentrations of NiCl2 (nickel chloride) and sodium lauryl sulfate (SLS) alone and in combination. Evaluation of skin reactions was performed by colorimetry, measurement of transepidermal water loss and clinical evaluation, and the data were analyzed by logistic dose-response models. A synergistic effect was found of combined exposure to NiCl2 and SLS, as compared to each of the substances applied separately, as evaluated by colorimetry and clinical scoring. This means that the effect produced by the combined exposure was substantially greater than the effect produced by either of the substances alone. A synergistic effect of combined exposure on skin barrier impairment was not found, since the barrier function is significantly influenced by SLS-exposure only and not by NiCl2. Concentration limits are used by industry and government agencies to protect consumers. The present results clearly illustrate that elicitation thresholds and concentration limits may be influenced considerably by combined exposure to allergens and irritants.     

Electrical impedance as a potential tool to distinguish between allergic and irritant contact dermatitis

BACKGROUND AND OBJECTIVE: The allergic contact reaction is a model reaction for studying the cell-mediated immune system of the skin. In this study we use a noninvasive method, electrical impedance (IMP), to compare nickel (Ni) allergic contact reactions with an irritant contact reaction induced by sodium lauryl sulfate, which has already been carefully evaluated with this method. For this purpose, we included only Ni- and sodium lauryl sulfate-induced reactions of very similar appearance. METHODS: Various concentrations of Ni sulfate in distilled water were applied on the volar aspect of the forearms of 33 adult women who were allergic to Ni. Assessments were made using visual scoring, a new IMP technique, and transepidermal water loss 3 and 7 days later. RESULTS: In the 19 patients who completed the study, 3 of the 4 impedance indices were significantly lower at the sodium lauryl sulfate sites than at the Ni sites on day 3; ie, the mean magnitude (MIX) (P < or = .001), imaginary part (IMAX) (P < or = .001), and real part (RIX) indices (P < or = .01). Unlike the irritant reactions, no significant increases in transepidermal water loss occurred in the allergic contact reactions. This may be because, in reactions of the studied magnitude, an allergic contact reaction does not significantly affect the epidermis because the inflammatory process is located deeper in the dermis than an irritant reaction. CONCLUSION: This study suggests that IMP, as used herein, is suitable for distinguishing between contact reactions of allergic and irritant nature. Although pathophysiologic events in the tissue studied significantly modify impedance patterns, little is known about how to interpret the structural and chemical changes underlying these patterns. Studies are needed to determine the relation between anatomic or pathophysiologic parameters, and the findings using IMP and other established methods, such as chemical extraction and histopathology.