Benzalkonium chloride in sensitization assays
As well as producing an irritant response in predictive testing, benzalkonium chloride has also been shown to induce responses that would classify it as an allergen in sensitization assays. There are a variety of predictive test procedures used for determining the sensitization potential of chemicals, and benzalkonium chloride has been reported to elicit a positive response in certain of these, although not by all authors. It is not uncommon for strong irritants to produce false-positive reactions in sensitization assays, e.g. SLS, a widely used and well-known irritant, has been reported to give false-positive results in the local lymph node assay (LLNA) (13, 18, 19). In addition, searching questions have been asked concerning the interpretation of results from guinea-pig predictive tests, notably the maximization test (20, 21). Consequently, it is questionable whether these results truly demonstrate that it is a skin sensitizer.
In many cases, benzalkonium chloride elicits few positive responses indicating that it may have sensitization potential but not offering clear evidence that this is the case. In a guinea-pig optimization test reported by Maurer (5), arguably the most sensitive (but not necessarily specific) method ever developed, benzalkonium chloride elicited 11 of 20 positive responses by intradermal challenge at a concentration of 0.1% and 4 of 19 positive responses via epicutaneous challenge at a concentration of 10%. Thus, although apparent positive responses were elicited suggesting a possible sensitization potential, those elicited by epicutaneous application, the correct route of administration, were still too low to classify this material as a skin sensitizer according to the European criteria for the classification of substances (6). Goh (22) reported benzalkonium chloride as being a moderate sensitizer using a modified Beuhler technique, with 20% of guinea pigs showing a response at the challenge phase. Woolhiser et al. (13) investigated the sensitization and irritancy potential of potent chemical sensitizers and irritating agents including benzalkonium chloride at 0.5, 1, 2, 3 and 5% in both a murine LLNA and a murine ear-swelling irritancy assay. In the murine ear-swelling assay, benzalkonium chloride elicited an irritation dose–response that peaked at 5%, while at 2.0% lymph node proliferation in these animals peaked, which the authors suggested could indicate the possible sensitization potential of benzalkonium chloride. Taken together, these tests thus suggest a possible sensitization potential but lack any absolute proof that this chemical is an allergen. In fact, when examining benzalkonium chloride performance in the LLNA test, the results are conclusive, Basketter et al. (19) reported benzalkonium chloride as giving a negative response. It should be noted, however, that a non-standard version of this assay gave contrasting results, showing a marginally positive result (23).
Many assays are being developed to investigate the nature of a chemical and are designed to discriminate between allergic and irritant responses. Recent work has shown that it is possible to discriminate between allergens and irritants by investigating the expression of interleukin-1α (IL-1α) and IL-8. Reconstructed human epidermis was used as an in vitro model to discriminate between 5 skin sensitizers and 3 skin irritants that included benzalkonium chloride (24). From these results, it was proposed that chemicals with a sensitization potential elicit an increase in IL-8 that is higher than that in extracellular IL-1α elicited, whereas skin irritants are chemicals which elicit an increase in extracellular IL-1α which is equal to or greater than the that in IL-8. Benzalkonium chloride fitted this profile and under this particular test regime/end point would be classified as an irritant. This work reflects that completed by others (25, 26) who showed that IL-1α was upregulated by both allergens and irritants.
Gerberick et al. (23) investigated the use of B220+ marker to discriminate between allergens and irritants in the LLNA. Benzalkonium chloride is often reported to produce proliferation in the LLNA, thus resulting in possible classification as an allergen. However, when using the B220+ marker to discriminate further between irritants and allergens, benzalkonium chloride was classified as an irritant in 12 of 14 separate experiments.
The increased expression of IL-1β by epidermal Langerhans' cells (LCs) following exposure of mice to contact allergens has also recently been investigated as a strategy to identify skin sensitizers. It has been demonstrated that culture of human blood-derived LC-like cells with selected allergens stimulates the expression of IL-1β, while under the same conditions of exposure, application of skin irritants such as SLS and benzalkonium chloride has been shown to have no effect on IL-1β expression (27–29).
Thus, this combined in vivo and in vitro evidence clearly identifies benzalkonium chloride as an irritant. Indeed, under the EU scheme (6), higher concentrations are classified as corrosive to skin.
Benzalkonium chloride and allergy in reported case studies
Sensitization to benzalkonium chloride has been reported as a result of occupational exposure (30) in physicians, nurses, dentists and veterinarians, and allergic reactions to a variety of different products containing this chemical have been reported, including detergents/antiseptics (30–34), antifungal creams (35, 36), ophthalmic preparations (4, 37–39), plaster of Paris (40, 41), as a denaturant of ethanol (42) and in toothpastes (43).
Contact sensitivity to benzalkonium chloride appears to be reported most commonly for ophthalmic preparations. Herbst and Maibach (39) reviewed the literature to identify an ophthalmic tray for testing contact allergy suspected from ophthalmic preparations and reported a total of 16 cases of positive patch tests to benzalkonium chloride taken from previously reported cases studies (6 different papers) at concentrations ranging from 0.005 to 0.13%. As such, they recommend routinely using benzalkonium chloride in patch testing for ophthalmic product-related allergy at 0.1%. Both Klein et al. (38) and Cox (37) report cases of ACD associated with benzalkonium chloride-containing eye drops.
When used for diagnostic patch testing, benzalkonium chloride is generally used at 0.1% aq. However, this concentration is often reported to elicit irritant responses and, when Brasch et al. (44) tested 1775 patients with 0.1% benzalkonium chloride, they found that this concentration yielded more irritant reactions than allergic responses. Therefore, it has been suggested that true allergic reactions can be obtained only by testing with a concentration of 0.01% aq. (1, 31, 44), and it is possible that many of the reported allergic responses to benzalkonium chloride are in fact irritant responses that have been misinterpreted. It is accepted that patch test reactions are often misinterpreted as being allergic, particularly when a weak reaction is elicited. Ale and Maibach (45) recommend repeating the patch test to rule out false-positive reactions and also to perform a use test, as also suggested by others (38). Ale and Maibach detailed many different measures which help to determine the clinical relevance of patch test reactions. These measures should help to confirm the true nature of the reaction to the material in question. When examining the case reports detailed in this review, it is clear that many of the documented allergic responses should have been investigated further.