Irritant action due to physico-chemical parameters of test solutions

Fd Chem. ~xic'i Vol. 23. No. 2. pp. 299 302, 1985

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IRRITANT ACTION D U E TO P H Y S I C O - C H E M I C A L PARAMETERS O F TEST S O L U T I O N S D. WALZ Biozentrum. University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Abstract--The irritant action of various buffer solutions was determined by the mouse skin test. All results for isosmolal solutions differing in initial pH values and buffer capacities could be satisfactorily represented by a general model which could be used to predict the irritancy of solutions with unphysiological pH values in contact with mucous membranes. The results obtained with hyperosmolal solutions are as yet too limited for such a general statemet~t, but it appears that increasing osmolality gradually overrides the effect of the pH value. The relevance of the results to the prediction of irritant potentialities for man has been assessed by comparison with results of rabbit-eye tests carried out by Friedenwald et al. (Archs Ophthal.. N . Y 1944, 31. 279).

Introduction The phenomenon of topical irritancy arises from several interactions between biological substrates ('biomatter') and the irritant substances which, in terms of pharmacodynamics, are mostly unspecific. These interactions can be divided into two groups. The first group comprises all direct interactions, such as chemical reactions between irritant substances and the constituents of biomatter that change the integrity of the biomatter sufficiently for the typical signs of irritancy: such as oedema, induration and necrosis, to appear. The second group is related to just one. very important, constituent of biomatter--water. Obviously, the physico-chemical parameters of the aqueous phase surrounding all biological structures are crucial for the structures' proper functioning. Changes in these parameters, due to the presence ot; substances that do not directly interact with biomolecules, can also lead to alterations in the integrity of biomatter, thus causing the signs of irritancy. Interactions belonging to the first group are determined by the molecular properties of the irritant substances and are therefore substance specific. Hence. predictions about irritant potentialities are difficult even when the molecular characteristics such as chemical reactivity are known. In contrast to this, interactions of the second group are specific only for water, and changes in its physico-chemical parameters arising from potentially irritant substances can easily be determined by in vitro measurements. Thus, provided the irritant action of the altered physico-chemical parameters of water is known, irritancy due to this type of interaction should be reliably predictable. We have therefore started a systematic investigation of these parameters and report here results pertinent to pH and buffer capacity, together with some preliminary results on osmolality,

Experimental The test solutions were prepared with various buffer substances which either occur in biological 299 FCT 23:2-J

systems (glycine, glycylglycine, lysine, histidine, glutamate, phosphate, lactate and acetate) or are known to be well tolerated from their use in numerous isolated-tissue, cell or organelle preparations. Those selected from the latter category ('Good buffers') were N-morpholinoethanesulphonic acid (MES), Nmorpholinopropanesulphonic acid (MOPS) and Ntris(hydroxymethyl)methylglycine (tricine). The osmolality was adjusted either to 300 mOsM with NaCl, or to different values with an ionic agent (NaC1, NaCHaSO a or Na2SO4) or a nonionic agent (sorbitol, glycerol or methanol). Details, including the composition of the buffer solutions and the determination of the physico-chemical parameters, are given elsewhere (Walz & Bucher, 1982). The irritant action of the test solutions was assayed by the mouse skin test (Walz, 1985). Control values were obtained by assaying solutions similar in composition to the test solutions but with a pH value of 7 and an osmotality of 300 mOsM.

Results The values for the intensity of the oedematous reaction, R, determined with isosmolal buffer solutions follow well defined dose-response curves with respect to both the buffer capacity, fl, and the initial pH value, pH o (Fig. 1). However this was the case only for those solutions where the composition of buffer substances yielded an approximately constant buffer capacity in the pH range from pH o to 7. If buffer solutions with variable capacity in this pH range were chosen, the R values still complied with dose-response curves with respect to the initial buffer capacity fl(PHo) but the relation with respect to pH o became ambiguous. Thus, the curve for phosphate buffers with pH o = 11 was almost identical with that for amino acid buffers with pH 0 = 10 (Walz & Buch~r, 1982). Since the pH dependence of fl in the range from pHo to 7 is rather different for the two buffer systems, this finding clearly indicates that in the whole pH range and not just at its initial value, fl(pHo) is relevant tO the irritant action.

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Fig. 1. Dose-response relationships between the intensity of the oedematous reaction, R, and the constant buffer Capacity, fl, for initial pH values, pH0, of (a) 2.5, (b) 3, (c) 11.5 and (d) 11. The buffer capacity is a measure of the change in pH to be expected upon addition of a certain amount of acid or base to the solution, i.e. the larger the buffer capacity the smaller the pH change. In view of the finite rate at which neutralizing equivalents can be supplied to the wheal by the counteracting measures of the surrounding tissue, it follows that the greater the buffer capacity of the solution in the wheal at the original pH, the longer is the biomatter exposed to that pH. Let the function s(pH) indicate the susceptibility of biomatter to different pH values, which shall be normalized to 1 for the highest susceptibility to be expected. The average effective concentration of H ÷ ions, ( C n ) , can then be derived from (cH) =

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Fig. 2. Irritant action of (a) H + ions and (b) OH- ions. The intensity of the oedematous reaction, R, was determined for different buffer solutions, These values are plotted against the average effective concentration (c.) (pHo <7) and (Cox) (pH 0 > 7), respectively, which were calculated with equations 1 and 2. The curve represents equation 3. Values for the parameters in these equations are: 7 = 0.1, pKo = 4, pK~ = 8.7, pK2 = ll.5;nx = I , K n = 25mM;non = 2, Kon= 7.2 m~ The buffer solutions, which contained amino acids,'Good buffers'(see Experimental), phosphate, acetate, HCI or NaOH, had various buffer capacities and intial pH values between 1.8 and 12; the osmolality was adjusted to 300 mOsM with NaCI. we were able to represent satisfactorily all experimental data obtained with different buffer systems (Fig. 2). The values for the parameters in equations 2 and 3 (see legend to Fig. 2) emerged from computer simulations with a parameter-fitting program (Walz & Bucher, 1982). Figure 3a presents some results on the irritant action due to osmolality for nonionic and ionic agents at neutral pH. The nonionic agents, selected as having the same structural element (CH-OH),, were sorbitol, glycerol and the basic compound of this series, methanol. It appears that the irritant action decreases with decreasing molecular weight of the agent and was undetectable with the smallest compound. A reasonable interpretation of this phenomenon takes into account that the tissue permeability of compounds with similar structure increases with decreasing molecular weight, substantially shortening the time the compound remains in the wheal and thus the duration of the osmotic stimulus. The ionic agents share the cation, sodium, but have various anions. Again, differences in permeability of the latter could be responsible for the different dose-response curves but, in the case of sulphate, an effect of the greater ionic strength compared with that for the monovalent salts cannot be excluded. The interplay of the two irritant factors, unphysiological pH value and osmolality, is illustrated in Fig. 3 for sorbitol and NaC1 as the nonionic and ionic agent, respectively. When the irritant action of a pH

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Fig. 4. Irritant action of solutions of unphysiological pH value on mucous membranes. The curve is calculated by equations 2-4. The two sets of points represent data obtained by Friedenwald et al. (1944) following 10-min irrigation of the eyes of anaesthetized rabbits with approximately isosmolal buffer solutions of different pH values: for intact epithelium (0) and after mechanical removal of the corneal epithelium (©). value is relatively low under isosmolal conditions, the effects of increasing osmolality appear to be simply additive. However, for greater values of pHinduced irritancy under isosmolal condition, this additivity no longer holds, irrespective of the type of osmotic agent or buffer system. The irritant action remains the same or even decreases at intermediate osmolalities and rises again towards similar values

The satisfactory representation of experimental data for unphysiological pH values obtained with various buffer systems under isosmolal conditions by means of equations 1-3 (see Fig. 2) demonstrates the feasibility of the investigation outlined in the introductory section. It should be pointed out that the function s(pH), expressing the susceptibility of biomatter to different pH values was included in the initial computer simulations as a set of discrete values, s(pHi) which were adjusted to yield the best fit. It then turned out that these values could be well represented by the function given in equation 2 which, in fact, has a physical meaning since s(pH) is equivalent to the fraction of the protonated form of one acid-base pair (pK0) in the acidic range and the fraction of the non-protonated forms of two acidbase pairs (pK1 and pK2) in the alkaline pH range. The values of these pKs, i.e. 4.0, 8.7 and 11.5, come close to those of carboxyl, sulphydryl and amino groups, respectively. Hence it is legitimate to conclude that the main component of biomatter susceptible to unphysiological pH values consists of proteins and that its susceptibility increases the more the carboxyl groups are protonated for the acidic pH range or the more the sulphydryl and amino groups are dissociated for the alkaline pH range. The maximum value of 1 for s(pH) in both pH ranges was deliberately chosen and does not mean that the number of amino plus sulphydryl groups is equal to the number of carboxyl groups; the ratio of amino to sulphydryl groups, however, should be meaningful and indeed comes out to a reasonable value, 9:1. Equations 1-3 can thus be considered to provide a general model expressing the irritant action of unphysiological pH values on biomatter under isosmolal conditions. As outlined elsewhere in this issue (Walz, 1985), the contact time between an irritant substance and the mucous membrane is relatively short. The integration in equation 1 can therefore be omitted, and only the initial concentrations of H ÷ or O H - ions are relevant. These can be expressed by the pH value of the solution. Thus, instead of equation 1, we obtain for the average effective concentrations: (ca> = 10-pns(pH) for pH < 7

. . (4a)

and (CoH) =

10pn - 14s(pH) for pH > 7

. . . (4b)

Note that (cn> and (Con) have the dimension mol/litre since pH is defined by the molar concentration of H + ions. The irritant action on mucosae of solutions with unphysiological pH values is then fully described by means of equations 2--4 as shown in Fig. 4 and, in principle, no further experiments with any test are needed to assess this action, since it can be predicted by the model. Unfortunately, this model holds only for isosmolal conditions, and most

302

D. WALZ

products used in daily life have larger osmolalities. The irritant action of the latter parameter, however, is generally not additive (see Fig. 3) in contrast to previously postulated ideas (Bucher, Bucher & Walz, 1981). We are therefore concentrating on attempts to understand the irritant action of osmolality as well as the effect of unphysiological pH values; this should then enable us to generalize the model. The relevance of our results to the prediction of potential irritancy from accidental contact of substances with h u m a n mucous membranes could in principle be figured out by comparison with results from the rabbit eye test since the latter, although based on very weak scientific grounds, is at present considered to provide the necessary information for such predictions. However, because of the unreliability of the results of this test (Weil & Scala, 1971), such a comparison is b o u n d to fail (Walz & Bucher, 1982). Instead, the results of Friedenwald, Hughes & H e r r m a n n (1944), which were obtained for isosmolal buffer solutions with a much more careful procedure, can serve as a reference. As is evident from Fig. 4, the

two sets of data almost coincide, thus corroborating the validity of the prediction made from our model. Acknowledgement--This work was encouraged and financially supported by the Swiss Federal Office of Health. Section of Toxicology. REFERENCES

Bucher K., Bucher K. E. & Walz D. (1981). The topically irritant substances: Essentials--bio-tests--predictions. Agents & Actions 11, 515. Friedenwald J. S., Hughes W. F. Jr & Herrmann H. (1944). Acid-base tolerance of the cornea. Archs Ophthal.. N. Y 31,279. Walz D. (1985). Quantitative assessment of irritation in the mouse skin test. Fd Chem. Toxic. 23, 199. Walz D. & Bucher K. E. (1982). The quantitative assessment of topical irritancy and its application to unphysiological pH-values. Agents & Actions 12, 552. Weil C. S. & Scala R. A. (1971). Study of intra- and interlaboratory variability in the results of rabbit eye and skin irritation tests. Toxic. appl. Pharmac. 19, 276.