Abstract
Background
Accurate patch testing is reliant on proper preparation of patch test allergens. The stability of patch test allergens is dependent on several factors including vapor pressure (VP).
Objective
This investigation reviews the VP of American Contact Dermatitis Society Core Allergens and compares stability predictions based on VP with those established through clinical testing.
Methods
Standard references were accessed for determining VP in millimeters of mercury and associated temperature in degrees celsius. If multiple values were listed, VP at temperatures that most approximate indoor storage conditions (20°C and 25°C) were chosen. For mixes, the individual component with the highest VP was chosen as the overall VP, assuming that the most volatile substance would evaporate first. Antigens were grouped into low (≤0.001 mm Hg), moderate (<1 to >0.001 mm Hg), and high (≥1 mm Hg) volatility using arbitrary cutoff values.
Conclusions
This review is consistent with previously reported data on formaldehyde, acrylates, and fragrance material instability. Given lack of testing data, VP can be useful in predicting patch test compound stability. Measures such as air-tight multidose reagent containers, sealed single-application dispensers, preparation of patches immediately before application, and storage at lower temperatures may remedy some of these issues.
Patch testing is critical in the diagnosis of allergic contact dermatitis. In the United States, T.R.U.E. TEST, which covers only a very limited of number of allergens, is the only patch test currently approved for use by the Food and Drug Administration. Comprehensive patch testing typically involves the use of commercially available allergens applied in Finn or IQ chambers. Commercially available allergens are typically prepared in either petrolatum or aqueous vehicles and packaged in syringes or bottles. There are 2 major conditions of concern regarding stability of allergens: (1) the stability of the reagent as supplied and stored according to manufacturer's instructions and (2) the stability of the reagent once placed in the patch test chambers. Unfortunately, these data are lacking for the majority of commercially available patch test allergens. Many patch testing allergens come not as pure substances but as mixtures consisting of solutes dissolved in a solvent or a colloid/suspension of 1 or more allergens dispersed in a vehicle such as water.
Several studies have evaluated stability of specific contact allergen reagents and were recently reviewed by Joy and colleagues.1 These studies are summarized in Table 1 and include thiurams,2 p-toluene diamine,3 diisocyanates,12,16 limonene hydroperoxide,13 fragrances,14,15 triglycidyl isocyanurate,11 methyldibromo glutaronitrile,5 acrylates,9,10 corticosteroids,8 as well as other allergens.4,6,7,10 Data from these studies and others suggest that allergen vapor pressure (VP) is one of several important factors in predicting allergen stability. Volatility is the tendency for a nongaseous substance to vaporize spontaneously. This tendency can be measured in VP where the higher the VP, the more volatile the substance.17 Greater volatility is thought to correlate with shorter shelf-life of a patch testing compound as more of it vaporizes from purchased stock or prepared solutions to the ambient air. Thus VP can be used as an approximation to stratify the stability of patch testing compounds in the absence of stability data. The purpose of this study was to document VPs of the American Contact Dermatitis Society (ACDS) Core Allergen Series.18 The stability of patch test allergens in mixtures is a nonspecific term used to clinically mean how much of the allergen within the mixture is lost with time and not available for testing purposes. The source of this allergen loss is multifactorial and may include vaporization to air, chemical degradation, absorption into a porous storage container, and/or adsorption of an adherent film on container walls.
Table 1. Publications Documenting Allergen Stability by Chemical Concentration Analysis or Clinical Testing.
| Reference, Allergen(s) | Conditions | Results |
|---|---|---|
| Bergendorff and Hansson2 | Fresh, 3 mo |
Concentration: NP Clinical: Tested 10 known thiuram-sensitive patients, all reacted to both |
| Thiuram | ||
| Geier et al3 | Fresh, 2 mo, 6 mo, 21 mo |
Concentration: 1% fresh; 0.2% 2 mo; 0.1% 6 mo; 0.01% 21 mo Clinical: Tested 177 patients; 24 reacted to ≥1 κ = 0.81–0.86 (very good); 1 patient +++ 9 mo and negative to 14 mo and 18 mo; 4 patients + 2–11 mo |
| p-Toluene diamine | ||
| Lembo et al4 | Fresh, 6 y |
Concentration: Thin layer chromatography testing acceptable Clinical: Tested 26 sensitized patients (1–7/allergen) All reacted to both |
| Balsam of Peru | ||
| Cobalt | ||
| Colophony | ||
| Ethylenediamine | ||
| Mercaptobenzothiazole | ||
| Nickel | ||
| Potassium dichromate | ||
| Vioform | ||
| Disperse yellow 3 | ||
| Formaldehyde | ||
| Gruvberger et al5 | Fresh, 1 y (4 concentrations) |
Concentration: All 4 identical concentration; no degradation Clinical: NP |
| Methyldibromo glutaronitrile | ||
| Bar et al6 | 40 y |
Concentration: NP Clinical: 15 known positive patients tested Paraphenylenediamine, 5/5 patients positive Benzocaine, 2/2 patients positive Balsam of Peru, 1/3 patients positive Epoxy, 3/4 patients positive |
| Paraphenylenediamine | ||
| Benzocaine | ||
| Balsam of Peru | ||
| Epoxy | ||
| Mose et al7 | 2 h, 8 h, 24 h, 48 h, 72 h, 7 d |
Concentration: Cinnamal (50% Fridge; 90% RT) Eugenol (30% Fridge; 75% RT) Van der Bend: 980% Fridge and RT ok MMA (960% Fridge; 0% RT) 2-HEMA (960% Fridge; 0% RT) 2-HPA (ok Fridge; 960% RT) Van der Bend: G20%; all 3 Fridge ok Clinical: NP |
| Cinnamal | RT, Fridge | |
| Eugenol | IQ, IQ Ult, Van der Bend | |
| MMA | ||
| 2-HEMA | ||
| 2-HPA | ||
| Isaksson et al8 | 1y |
Concentration: Tixocortol pet and eth, stable all conditions Budesonide pet and eth, stable all conditions Hydrocortisone-17 butyrate eth, freezer ok; RT e 3 mo Clinical: NP |
| Tixocortol | Pet, Eth | |
| Budesonide | RT, Fridge, Freezer | |
| Hydrocortisone-17 butyrate | ||
| Goon et al9 | 4 mo | |
| 2-HEMA | RT, Fridge, Freezer |
Concentration: 2-HEMA, EGDMA, TREGDAVall temperatures ok MMA 8 d RT, 10 d Fridge 2-HPA 21 d RT, 3 mo Fridge All rapid loss in IQ Clinical: NP |
| EGDMA | IQ, Finn | |
| TREGDA | ||
| MMA | ||
| 2-HPA | ||
| Siegel et al10 | In-use, unexpired and expired, allergens from patch test clinics |
Concentration: Nickel, acceptable concentration Formaldehyde, acceptable concentration but loss occurred with storage Methyl methacrylate e56% Glutaraldehyde 27% to 45% Clinical: NP |
| MMA 2% | ||
| Nickel | ||
| Formaldehyde | ||
| Glutaraldehyde 1% | ||
| Erikstam et al11 | 3y |
Concentration: 30% Clinical: Patient tested negative to allergen but positive to product |
| Triglycidyl isocyanurate | ||
| Frick-Engfeldt et al12 | 1y |
Concentration: MDI failed all 3 PMDI better, but only freezer acceptable Clinical: NP |
| MDI (diphenylmethane-4,4-diisocyanate) | RT, Fridge, Freezer | |
| PMDI (polymeric diphenylmethane diisocyanate) | ||
| Nilsson et al13 | 6 wk |
Concentration: Oxidation products unstable α-Tocopherol stabilizer in pet causes degradation of hydroperoxides Use nonstabilized oxidized d-limonene Good for 6 wk Clinical: NP |
| d-limonene | ||
| Mowitz et al14 | 0, 4, 8, 24, 72, 144 h |
Concentration: All Fridge 9 RT 4/7 decreased by 920% within 8 h at RT All except amyl cinnamal decreased by 920% by 144 h F Slightly better in FM than individually Clinical: NP |
| Fragrance mix I | RT, Fridge | |
| Components | 20 mg Finn open | |
| 30 mg IQ with plastic cover | ||
| Hamann et al15 | 0, 8 h, 9 d 5 -C, 25 -C, 35 -C |
Concentration: 8 h, ok 9 d 7 decreased by 30% at 35 -C; decreased by10% at 25 -C; decreased G5% at 5 -C Clinical: NP |
| Lyral | ||
RT, room temperature; Pet, petroleum; Eth, ethanol; NP, not performed.
Methods
Chemical Abstract Series Registry (CAS) numbers were obtained from the Chemotechnique Diagnostics, Patch Test Products & Reference Manual 201419 and used to search for published VPs from the following online chemical databases: United States National Library of Medicine, National Center for Biotechnology Information, PubChem Open Chemistry Database,20,21 Chemical Book 22 (an online data set of materials safety data sheets), and Chemical Laboratory Information Profiles23 (a database of the American Chemical Society detailing physical and safety information on select chemical compounds originally published in the Journal of Chemical Education). Vapor pressure can be obtained from experimental data and estimated/extrapolated using the Antoine equation:24
where P indicates vapor pressure; T indicates temperature; and A, B, and C indicate substance-specific constants.
Small discrepancies can be found between the databases, and when these were encountered, values from the PubChem followed by CLIP databases were utilized. Vapor pressure and associated temperature were recorded as millimeters of mercury and degrees celsius, respectively. If multiple VP values were listed, VP at temperatures that most approximate indoor storage conditions (20°C and 25°C) were chosen. As VP varies nonlinearly with temperature per the Clasius-Clapeyron equation, VP at temperatures much higher than patch testing temperatures are largely irrelevant:25
where P indicates vapor pressure at T; T indicates temperature, ΔAHvap indicates enthalpy of vaporization specific for substances; and R, gas constant (8.3145 J/(mol I K).
For allergen mixes, the individual component with the highest VP was chosen as the overall VP, based on the assumption that the most volatile substance would evaporate first. Documented VPs less than 0.001 mm Hg were listed as <0.001 mm Hg and assumed to be clinically equivalent.
Given the ambient and mild conditions during patch testing, it is unlikely that solid compounds will sublimate from solid to gas phases. Thus, VP was not useful with solids in suspension or compounds that do not dissolve in the vehicle.26 Some metals salts are not soluble in petrolatum (eg, nickel sulfate), and therefore, VP is not helpful in predicting stability. To be comprehensive, we included VP for all allergens in the ACDS Core Allergen Series, including metal salts.
There are few established guidelines for volatility, and as such, categorical cutoff values for volatility are subjective 27; relevance for patch test preparations is the amount of intact allergen present in the patch test chamber, and the temperature in which patch testing is usually performed. After discussion, we used the consensus cutoff values of 1 mm Hg or higher for high volatility, less than 1 to greater than 0.001 mm Hg for moderate volatility, and 0.001 mm Hg or less for low volatility at 25°C given data availability.
Results
Table 2 lists data for the ACDS Core Allergens with published VPs organized within categories of high, medium, and low volatility in alphabetical order. Data for acetone, ethanol, and water vehicles were included as reference points. Allergens with VP data at high temperatures were included if data were not available at ambient or near-ambient temperatures. Allergens without reported VPs were excluded from Tables 2 and summarized in Table 3. For mixtures, available data for each of the components were listed but for the purpose of stratification, the component with the highest VP was used as the overall VP. However, concentration of individual components of a mixture (eg, fragrance mix I) is not reported by the supplier.19
Table 2. ACDS Core Allergens in Grouped by Volatility and in Alphabetical Order19–23.
| ACDS Core Allergen | CAS Number | VP, mm Hg | Temperature,°C | Database |
|---|---|---|---|---|
| High volatility (VP >1 mm Hg) | ||||
| 3-(Dimethylamino)-1 propylamine | 109-55-7 | 5 | 20 | Chem book |
| Ethyl acrylate | 140-88-5 | 38.6 | 25 | Chem book |
| 9.98 | 30 | PubChem | ||
| Formaldehyde | 50-00-0 | 3890 | 25 | PubChem |
| Glutaraldehyde | 111-30-8 | 17 | 20 | PubChem |
| Methyl methacrylate | 80-62-6 | 29 | 20 | PubChem |
| 38.5 | 25 | |||
| N,N-Diphenylguanidine | 102-06-7 | 1.26 | 20 | PubChem |
| Sorbitan sesquioleate | 8007-43-0 | 42 | 20 | Chem book |
| Vehicle, acetone | 67-64-1 | 184 | 20 | Chem book |
| 231 | 25 | PubChem | ||
| Vehicle, ethanol | 64-17-5 | 43.5 | 20 | PubChem |
| Vehicle, water | 7732-18-5 | 17.5 | 20 | CLIP* |
| Medium volatility (VP <1 to >0.001 mm Hg) | ||||
| 2,6-Ditert-butyl-4-cresol (BHT) | 128-37-0 | 0.01 | 20 | PubChem |
| 4-Chloro-3-cresol (PCMC) | 59-50-7 | 0.005 | 20 | PubChem |
| Amyl cinnamal (fragrance mix I) | 122-40-7 | 0.004 | 25 | PubChem |
| Benzyl alcohol | 100-51-6 | 0.099 | 20 | PubChem |
| 0.094 | 25 | |||
| Chloroxylenol (PCMX) | 88-04-0 | 0.002 | 25 | PubChem |
| Cinnamal (cinnamic aldehyde in fragrance mix I) | 104-55-2 | 0.029 | 25 | PubChem |
| Cinnamyl alcohol (fragrance mix I) | 104-54-1 | <0.01 | 25 | Chem book |
| Citral (fragrance mix II) | 5392-40-5 | 0.091 | 25 | PubChem |
| Citronellol (fragrance mix II) | 106-22-9 | 0.02 | 25 | PubChem |
| Ethyl cyanoacrylate | 7085-85-0 | 0.31 | 20 | PubChem |
| <2 | 25 | |||
| Eugenol (fragrance mix I) | 97-53-0 | 0.022 | 25 | PubChem |
| Hydroxyethyl Methacrylate | 868-77-9 | 0.126 | 25 | PubChem* |
| Isoeugenol (fragrance mix I) | 97-54-1 | 0.014 | 25 | PubChem |
| Geraniol (fragrance mix I) | 106-24-1 | 0.03 | 25 | PubChem |
| Methylisothiazolinone | 2682-20-4 | 0.062 | 25 | PubChem |
| Phenoxyethanol | 122-99-6 | 0.01 | 20 | PubChem |
| 0.007 | 25 | |||
| Propylene glycol | 57-55-6 | 0.08 | 20 | PubChem |
| 0.13 | 25 | |||
| Sorbic acid | 110-44-1 | 0.01 | 20 | Chem book |
| Low volatility (VP <0.001 mm Hg) | ||||
| 2-Bromo-2-nitropropane-1,3-diol | 52-51-7 | <0.001 | 20 | PubChem |
| 1,3-Diphenylguanidine (carba mix) | 74-31-7 | <0.001 | 25 | PubChem |
| 2-Ethylhexyl-4-methoxycinnamate | 5466-77-3 | <0.001 | 25 | PubChem |
| Benzocaine | 94-09-7 | <0.001 | 25 | PubChem |
| Benzophenone-3 (2-hydroxy-4-methoxybenzophenone) | 131-57-7 | <0.001 | 25 | PubChem |
| Benzophenone-4 (2-hydroxy-4-methoxybenzophenone-5-sulfonic acid) | 4065-45-6 | <0.001 | 25 | PubChem |
| Butylparaben (paraben mix) | 94-26-8 | <0.001 | 25 | PubChem |
| Cetyl alcohol (cetyl stearyl alcohol mix) | 36653-82-4 | <0.001 | 25 | PubChem |
| Cobalt(II) chloride hexahydrate | 7791-13-1 | <0.001 | 25 | CLIP* |
| Coumarin (fragrance mix II) | 91-64-5 | <0.001 | 25 | PubChem |
| DL α-tocopherol | 10191-41-0 | <0.001 | 25 | PubChem |
| DMDM hydantoin | 6440-58-0 | <0.001 | 25 | PubChem |
| Epoxy resin-bisphosphenol A | 1675-54-3 | <0.001 | 25 | PubChem |
| Ethylparaben (paraben mix) | 120-47-8 | <0.001 | 25 | PubChem |
| Farnesol (fragrance mix II) | 4602-84-0 | <0.001 | 25 | PubChem |
| Iodopropynyl butylcarbamate† | 55406-53-6 | <0.001 | 30 | PubChem |
| Methyldibromoglutaronitrile | 35691-65-7 | <0.001 | 25 | PubChem |
| Methylparaben (paraben mix) | 99-76-3 | <0.001 | 25 | PubChem |
| p-Phenylenediamine† | 106-50-3 | <1 | 21 | PubChem |
| 1.08 | 100 | |||
| Propylparaben (paraben mix) | 94-13-3 | <0.001 | 25 | PubChem |
| p-tert-butylphenol (p-tert-butylphenol formaldehyde resin)† | 98-54-4 | 0.23 | 50 | PubChem |
| Quaternium-15 | 51229-78-8 | <0.001 | 25 | PubChem |
| Stearyl alcohol (cetyl stearyl alcohol mix) | 112-92-5 | <0.001 | 25 | PubChem |
| Tetraethyl thiuram disulfide (carba mix) | 97-77-8 | <0.001 | 25 | PubChem |
| Tetramethyl thiuram disulfide (carba mix) | 137-26-8 | <0.001 | 25 | PubChem |
| Tosylamide (tosylamide formaldehyde resin) | 70-55-3 | <0.001 | 25 | PubChem |
| Triclosan | 3380-34-5 | <0.001 | 20 | PubChem |
Vapor pressure listed with a maximum of 3 significant figures. VP < 0.001 mm Hg considered as negligible.
Discrepancy between PubChem, CLIP, and/or Chem book.
Data not available at 20°C or 25°C but available at much higher temperatures. Assumed low volatility since VP low at high temperatures.
Table 3. ACDS Core Allergens for Which VP Data Were Not Available19–23.
| ACDS Core Allergen Nickel(II) sulfate hexahydrate | CAS Number 10101-97-0 | Comments |
|---|---|---|
| Myroxylon pereirae | Not available | |
| Hydroxycitronellal (fragrance mix 1) | 107-75-5 | Data from other components of fragrance mix 1 available |
| Oakmoss absolute (fragrance mix 1) | 90028-68 | Data from other components of fragrance mix 1 available |
| Neomycin sulfate | 1405-10-3 | |
| Budesonide | 51333-22-3 | |
| p-tert-butylphenol formaldehyde resin | Not available | Data for p-tert-butylphenol used |
| Potassium dichromate | 7778-50-9 | |
| Zinc dibutyldithiocarbamate (carba mix) | 136-23-2 | Data from other components of carba mix available |
| Zinc diethyldithiocarbamate (carba mix) | 14324-55-1 | Data from other components of carba mix available |
| Dipentamethylene thiuram disulfide (thiuram mix) | 94-37-1 | Data from other components of (thiuram mix available |
| Tetramethyl thiuram monosulfide (thiuram mix) | 97-74-5 | Data from other components of thiuram mix available |
| Diazolidinyl urea | 78491-02-8 | |
| N-cyclohexyl-N-phenyl-4-phenylenediamine (black rubber mix) | 101-87-1 | Data from other components of black rubber mix available |
| N-isopropyl-N-phenyl-4-phenylenediamine (black rubber mix) | 101-72-4 | Data from other components of black rubber mix available |
| Imidazolidinyl urea | 39236-46-9 | |
| N-cyclohexyl-2-benzothiazylsulfenamide (mercapto mix) | 95-33-0 | |
| Dibenzothiazyl disulfide (mercapto mix) | 120-78-5 | |
| 2-mercaptobenzothiazole (mercapto mix) | 149-30-4 | |
| 2-(4-morpholinyl mercapto)-benzothiazol (mercapto mix) | 102-77-2 | |
| Methylchloroisothiazolinone/methylisothiazolinone mix | 55965-84-9 | Data from methylisothiazolinone available |
| Tixocortol-21-pivalate | 55560-96-8 | |
| Mercaptobenzothiazole | 149-30-4 | |
| Colophony | 8050-09-7 | |
| Ethylenediamine dihydrochloride | 333-18-6 | |
| Lanolin alcohol (Amerchol 101) | 8027-33-6 | |
| Bacitracin | 1405-87-4 | |
| Dibucaine | 85-97-0 | |
| Parthenolide | 20554-84-1 | |
| Lidocaine | 137-58-6 | |
| Gold sodium thiosulfate | 10233-88-2 | |
| Disperse blue 106 (disperse blue m124/106 mix) | 68516-81-4 | |
| Disperse blue 124 (Disperse blue m124/106 mix) | 61951-51-7 | |
| Hydrocortisone-17-butyrate | 13609-67-1 | |
| Hexyl cinnamic aldehyde (fragrance mix 2) | 101-86-0 | Data from other components of fragrance mix 2 available |
| Lyral (fragrance mix 2) | 31906-04-4 | Data from other components of fragrance mix 2 available |
| Cocamidopropyl betaine | 61789-40-0 | |
| Diethyl thiourea (mixed dialkyl thiosureas) | 105-55-5 | |
| Dibutyl thiourea (mixed dialkyl thiosureas) | 109-46-6 | |
| Oleamidopropyl dimethylamine | 109-28-4 | |
| Decyl glucoside | 141464-42-8 | |
| Amidoamine | Not available | |
| Melaleuca/tea tree oil | 68647-73-4 | |
| Chlorhexidine digluconate | 18472-51-0 | |
| Propolis | 85665-41-4 | |
| Tosylamide formaldehyde resin | 1338-51-8 | Data for tosylamide used |
| Alantolactone (sesquiterpine lactone mix) | 546-43-0 | |
| Costunolide (sesquiterpine lactone mix) | 553-21-9 | |
| Dehydrocostus lactone (sesquiterpine lactone mix) | 477-43-0 | |
| Cocamide DEA | 68603-42-9 | |
| Benzalkonium chloride | 63449-41-2 | |
| Ylang-Ylang | 8006-81-3 | |
| Anthemis nobilis extract (Compositae mix 2) | 84649-86-5 | |
| Chamomilla recutita extract (Compositae mix 2) | 84082-60-0 | |
| Achillea millefolium extract (Compositae mix 2) | 84082-83-7 | |
| Tanacetum vulgare extract (Compositae mix 2) | 84961-64-8 | |
| Arnica montana extract (Compositae mix 2) | 68990-11-4 | |
| Parthenolide (Compositae mix 2) | 20554-84-1 | |
| Dimethylol dihydroxy ethylene urea (ethyleneurea melamine formaldehyde mix) | 1854-26-8 | |
| Melamine formaldehyde (ethyleneurea melamine formaldehyde mix) | Not available | |
| Triamcinolone | 76-25-5 | |
| Clobetasol-17-proprionate | 25122-46-7 | |
| Disperse orange 3 | 730-40-5 | |
| Jasminum officinale oil | 8031-01-4 | |
| White petrolatum | 8009-03-8 |
Volatile Allergens
Based on VP alone, the following allergens are predicted to be the least stable (high volatility): formaldehyde, acrylates (hydroxyethyl methacrylate, ethyl acrylate, and methyl methacrylate), sorbitan sesquioleate, glutaraldehyde, N,N-diphenylguanidine, and 3-(dimethylamino)-1 propylamine (DMAPA). Those with moderate volatility include propylene glycol, methylisothiazolinone, fragrance-related allergens (benzyl alcohol, citral, cinnamic aldehyde, cinnamal, eugenol, citronellol, isoeugenol, cinnamyl alcohol, amyl cinnamal, and geraniol), phenoxyethanol, and chloroxylenol.
Nonvolatile Allergens
The following allergens are predicted to be relatively stable (>0.001 mm Hg VP): formaldehyde-related allergens (quaternium-15, DMDM hydantoin, tosylamide formaldehyde resin, bronopol), paraben mix constituents, rubber allergens (carba mix ingredients and N,N-diphenyl-p-phenylenediamine), 2 fragrances (coumarin and farnesol), 3 sunscreens (benzophenone-3 and -4 as well as 2-ethylhexyl-4-methoxycinnamate), and also benzocaine, epoxy resin, p-phenylenediamine (PDA), methyldibromoglutaronitrile, cetyl alcohol, stearyl alcohol, triclosan, and tocopherol.
Discussion
This study documents the VPs of ACDS Core Allergens. While the most conclusive studies involve analysis of “in use” allergens and patch test preparations, these data are not available for most allergens. The information published herein provides additional information to clinicians regarding 1 parameter that may affect the stability of common allergens. The volatility of an allergen should also be considered when compounding allergens within the dermatology clinic for diagnostic use.
Vapor pressure is defined by the Occupational Safety and Health Administration of the US Department of Labor in the Code of Federal Regulation as “a measure of a liquid's propensity to evaporate. The higher the VP, the more volatile the liquid and, thus, the more readily the liquid gives off vapors.” 17 More theoretically,it is the pressure of a vapor in thermodynamic equilibrium with its condensed phases (liquid and solid)in a closed system.In such a system, although there is constant change among the gaseous, liquid, and solid phases; there is no net change. Another way to think about VP is that it essentially measures the tendency for an atom or molecule to escape into the gaseous phase from condensed phases.26 For example, a vacuum container, at time 0, contains liquid waterat20°C. Because the VP of liquid water at 20°C is approximately 760 mm Hg and the ambient pressure is 0 (vacuum), liquid water will immediately vaporize until VP is achieved or all of the liquid water has vaporized, whichever occurs first. Once at equilibrium, any additional application of energy such as heat will cause the liquid phase to change into gas phase, forming gas bubbles. As VP varies positively with volatility, the higher the VP, the higher the volatility and the higher the rate of loss of the substance from condensed to gaseous states.17
Based on our findings on VP alone, formaldehyde, acrylates (hydroxyethyl methacrylate, ethyl acrylate, and methyl methacrylate), and propylene glycol were predicted to have shorter shelf lives. This is consistent with previous research. Siegel et al10 found that formaldehyde content measured in reagents obtained from a single patch test clinic and directly from the supplier was consistent with the label stated content upon receipt at the laboratory. Upon re-assay after 1 year of undisturbed storage under refrigerated conditions, the formaldehyde reagents supplied in a syringe container had formaldehyde losses of 41% and 67%, while that supplied in an opaque plastic dropper had lost 31% of the formaldehyde from the water vehicle. While significant losses were observed for both container types, this preliminary observation suggests that a more air-tight container may help preserve volatile allergen integrity. The Antoine and Cassius-Clapeyron equations and other theories of thermodynamics were developed under the assumption of a closed system at equilibrium.24–26 In real-world conditions, such as with patch testing compound storage and use, a closed system is not realistic; however, a pseudo-steady state can be achieved. With semiclosed systems such as a leaky container, steady state would be achieved where there is a steady diffusion of volatile gasses from the counter but with minimal change in the partial pressure within the container. This only occurs if there is an equal loss of patch test compounds from the solution to the gas. Thus, with time, there can be significant losses, especially if VP is high, providing a rationale for more air-tight storage methods.
Issues with acrylate allergen stability, specifically methyl methacrylate, were first documented by Kanerva et al.28 They documented false-negative or questionable patch test results with methyl methacrylate obtained from 2 different manufacturers; one had nondetectable methyl methacrylate levels and the other only 25% of the labeled amount. Goon et al9 studied methyl methacrylate stored in syringes and IQ chambers. They documented that methyl methacrylate stored in syringes at room temperature lost more than 20% of the labeled concentration within 2 weeks. When stored at −16°C, the loss was less than 20% at day 128, but increased to more than 20% of the initial concentration by 6 months. Loss was more rapid in IQ chambers under all conditions. Hypothesized reasons included evaporation or spontaneous polymerization. Siegel et al documented that the concentration of methyl methacrylate in stored syringes varied from the tip to the plunger of the syringe. The concentration at the tip of the syringes averaged 42% less than subsequent aliquots, suggesting that the major source of loss was due to volatility.10 In addition, both Goon et al9 and Siegel et al10 observed significant loss of methyl methacrylate during compounding with petrolatum. During this process, petrolatum must be heated to higher than 65°C to melt the petrolatum, which can cause significant loss of allergen from volatilization as VP increases nonlinearly to increasing temperature.
Based on VP, volatility should not be a factor in the stability of paraben mix constituents, rubber allergens (carba mix ingredients and N,N-diphenyl-p-phenylenediamine), 2 fragrances (coumarin and farnesol), and 3 sunscreens (benzophenone-3 and -4 as well as 2-ethylhexyl-4-methoxycinnamate), and also benzocaine, epoxy resin, methyldibromoglutaronitrile, cetyl alcohol, stearyl alcohol, and tocopherol. This is consistent with previous publications. Gruvberger et al5 tested 4 different concentrations of methyldibromoglutraonitrile at 1 year and found that all 4 were stable. Both patients tested to 40-year-old benzocaine, and 3 of 4 patients tested to 40-year-old epoxy resin reacted on patch testing.6 We found no previous reports of stability testing to paraben mix, carba mix, coumarin, farnesol, cetyl alcohol, stearyl alcohol, sunscreens, or tocopherol.
Limitations
There are several limitations to these data. Vapor pressure data were unavailable for many ACDS core allergens. Also, numerous assumptions were made with regards to volatility and stability. First, VP is only one of the many chemical and physical aspects of allergen formulations, which may affect the accuracy of patch testing: self-polymerization, air reaction (including oxidation), carrier incapability, irritation, percutaneous penetration, as well as the presence of stabilizers or components within the mixes that may alter patch test results.29,30 For example, based on VPalone, PDA has very low volatility at 25°C. However, it is very unstable in aqueous preparations as it is readily air oxidized to a brown heterogeneous mixture, whereas it is stable in petrolatum, likely due to protection from air.31,32 Moreover, the vehicles themselves, as well as other constituents of a patch test preparation will likely contribute to the real-world volatility of a substance and potentially alter the patch test article stability.
Second, VP is based on the pure/neat chemical and thermodynamic equilibrium, not real-world conditions where solvent and other solutes may alter the volatility of the allergen. Third, there are few standard data on what cutoff value imparts “stability.” The World Health Organization made a determination between a very volatile organic compound, volatile organic compound, and semivolatile organic compound based on a substances' boiling point and not VP.33 Boiling point is related to both vapor and atmospheric pressure, with the higher the VP, the lower the boiling point at a given atmospheric pressure. Finally, many ACDS Core Allergens are mixtures; overall VP could be affected by the partial pressures of each component, the interactions of each component with other components and/or with the solvent. We made the assumption that the stability of a mixture was that of the most volatile component.
Summary
This review provides additional information to clinicians on potential stability issues based on VP and is consistent with previously reported data on the instability of formaldehyde, acrylates, and fragrance materials. Specific reported stability data for a given allergen supersedes predictions based on VP. However, given the lack of experimental stability data of patch testing compounds, reliance on VP as a proxy for volatility may be a helpful tool for clinicians when compounding a patch test reagent or assessing the probability of a potential false-negative test.
The stability of commercial patch test allergens continues to be of concern as it impacts not only clinical diagnostic accuracy but also reliability of epidemiologic data reported in the literature. Potentially, patch test reagent stability/storage issues due to volatility may be minimized by use of more air-tight multidose reagent containers, sealed single application dispensers, storage at lower temperatures, and reliable beyond-use date labeling of multidose containers by patch test reagent suppliers. In addition, in an effort to minimize risk of false-negatives, allergens known to be highly volatile such as fragrances and those within the predicted high and medium volatility range should not be aliquoted and prepared until just before the application.
Acknowledgments
The authors have no funding.
Disclaimer: This material is the result of work supported with resources and the use of facilities at the Minneapolis Veterans Affairs Medical Center. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs, the National Institute for Occupational Safety and Health, or the US government.
Footnotes
The authors have no conflicts of interest to declare.
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