Letters to the Editor pubs.acs.org/crt
Response to the Letter to the Editor Regarding Our Article (Natsch et al., 2015)
T
problem. However, in the current absence of evidence for relevant exposure, we also considered it crucial to search for and openly discuss alternative explanations within the scientific community. To point 1 of Karlberg et al.: The aim of the study was in no way to negate the ability of hydroperoxides to form specific adducts and to induce specific allergy. We clearly state in the abstract in the first sentence “Hydroperoxides can act as specific haptens...” This is a given fact, clearly established in the literature and widely reviewed. We agree that more citations could have been provided. This fact should be well known to those working in the area and was therefore not expanded on for reasons of limited space since the article was originally written in the form of a rapid report presenting a hypothesis. However, prominently stating this fact in the abstract should be sufficient to indicate we did not want to negate this well-proven fact. Hence, investigating reactions with other amino acids in our view is not needed. Regarding the comment referring to comparison to oxidation triggered by UV intensity of natural sunlight, we recognize that this is a potentially interesting area for further research. To point 2 of Karlberg et al.: Regarding the exposure data, the statement “Concentrations of linalool hydroperoxides >100 μg/g were detected in the perfumes recalled from consumers” is a gross oversimplification of our study. Within a sample of 39 aged perfumes, one perfume exceeded 100 μg/g, while the hydroperoxide was detectable in 31 samples with a geometric mean in the positive samples of 14 μg/g,11 and even much lower levels of limonene hydroperoxide were found.12 The prevalence of positive reactions following patch testing is high. It is therefore not convincing that the finding on a single aged perfume of a sample of 39 can explain the high number of patch testing positive reactions within the clinical population. Citing a single extreme value 10-fold off the geometric mean to describe a study on a sample of 39 values is unusual scientific practice. Expressed as dose-per-area, which is the well-established dose metric in skin sensitization,13 our data yield a typical exposure from an aged fragrance of 0.031 μg/cm2 for the mean in positive samples and 0.29 μg/cm2 for the single sample out of 39 with a dose >100 μg/g. These values are significantly (182and 19-fold) below the lowest dose which triggered a reaction in a single patient in the ROAT after prolonged exposure. Finally, even if that maximal value was able to elicit a reaction, it could still not explain widespread induction of sensitization. Regarding the ROAT study,14 it was not cited in the current study but closely discussed in our study analyzing the exposure scenarios. 11 The published ROAT has two important limitations when used to derive an elicitation dose: (1) Patients were simultaneously exposed to multiple doses on the same arm with a maximal concentration of 3% fully oxidized linalool in a cream. This is above the used levels of perfumes in typical
o the Editor: Our paper was clearly presented as a hypothesis, and we refer in multiple instances including the title to the possibility that oxidative events may be a possible alternative explanation for the high prevalence of positive reactions recorded at high patch test concentrations. We have not indicated that our data would prove such a possible link, but the published clinical and exposure data leave several questions open so that alternative explanations should be openly discussed. A special focus is given below on oxidized linalool, for which most detailed data are available. First of all, we need to be aware of the history of the research on linalool hydroperoxides. This work has not started from a clinical problem of enhanced sensitivity of patients to linaloolcontaining products: it started from the chemical synthesis of a linalool hydroperoxide (3-hydroperoxy-3,7-dimethylocta-1,6diene),1 which was shown to be allergenic in the LLNA but was never found in oxidized linalool.2 Later, linalool was subjected to forced oxidation over several months, and a different, naturally formed, hydroperoxide ((E)-7-hydroperoxy3,7-dimethylocta-1,5-dien-3-ol) was isolated, which proved to be equally sensitizing in the LLNA.3 Specific adduct formation with this hydroperoxide was repeatedly shown.4,5 On the basis of these animal and in vitro data, a diagnostic test was developed.6,7 In a clinical setting, increasing doses were tested on patients, with an increasing and very high prevalence of positive reactions when the dose was increased.7−9 However, no exposure data were ever generated in this research field so that currently there is no established link between a patch test reaction and the presence of the hydroperoxide in products actually used by a patient. Thus, there is currently no established link between the diagnostic test and disease or exposure. Certainly, the absence of an established link is no indication that this link does not exist, but it does trigger the need to carefully investigate further the understanding and interpretation of the diagnostic data and the potential source of reactions observed following patch testing. (Note: It had been claimed that the use of linalool/limonene containing products is indicative of the relevance of patch tests and relevant exposure to the hydroperoxides.10 However, at least for the case of linalool since up to 90% of cosmetic products contain linalool, using cosmetic products is almost synonymous with using linalool containing products. Vice versa, avoidance of linalool will automatically lead to avoidance of the majority of cosmetic products. The presence in products used by the patients of sensitizing hydroperoxides at levels relevant to induce sensitization thus still needs to be established.) To close this gap, we started to investigate exposure by hydroperoxides due to fragranced products,11,12 and these exposure data were carefully analyzed and compared with the clinical doses and doses used in animal tests. We will refer to this analysis below. For us, a significant source of exposure would be the first and most logical hypothesis to explain the positive reactions and hence was our first approach to the © 2015 American Chemical Society
Published: October 23, 2015 2082
DOI: 10.1021/acs.chemrestox.5b00423 Chem. Res. Toxicol. 2015, 28, 2082−2084
Chemical Research in Toxicology
Letters to the Editor
cibility). Whether these figures are a true reflection of the reproducibility is not known, but with only these available figures16 in mind, the frequency of concomitant reactions to different hydroperoxides/oxidized materials in Table 3 in the study by Christensson et al.17 is actually high, with frequencies of concomitant reactions in the same range as repeated reactions to the same patch test material (31−67%). More specifically, reaction to any of the three limonene preparations informs us in a similar way on the probability for reactions to oxidized linalool as it informs us about reactions to the other two limonene preparations. Reaction to limonene-2-OOH is more (only positives counted) or equally (doubtfuls counted as positives) predictive for sensitivity to oxidized linalool than to oxidized limonene, despite the fact that limonene-2-OOH is contained in oxidized limonene, and not in linalool. Our conclusion from Christensson et al.17 would not be that linalool and limonene sensitivity are unrelated but that rather they indeed are related. However, a firm conclusion is not possible due to the limited number of subjects, different concentrations of test materials, and limited information on patch test reproducibility. One would need blind, independent retesting with the multiple materials at equimolar doses to strongly conclude that observed reactions are specific and indeed a proof of specificity of the observed allergies. To Point 4 of Karlberg et al.: We think the data are clearly explained and that the graph was correctly interpreted by Karlberg et al. We compared frequency of positive reactions to the two oxidized materials tested on the same patients in different clinical centers. Unfortunately, the original studies had reported concomitant reactions to other allergy markers but had presented no data on concomitant reactions to oxidized terpenes at the individual level. Thus, data are at the “population” level, meaning here the test population in the clinics and not the test individuals. We indicated that it will be important to present these data at the individual level, but it will be equally important to include both positive and doubtful reactions, as it was noted that “interpreting and classifying(oxidized) terpene patch test reactions, particularly distinguishing irritant from allergic reactions, is more difficult than with other allergens.”9 Also, to validate the diagnostic test, reproducibility must be studied further to verify how often a positive/doubtful reaction will again lead to a positive/doubtful reaction to the same material on replicate testing. Only with this background information will an interpretation of frequency of concomitant reactions at individual levels be possible. Currently, large efforts are ongoing to validate analytical methodologies to detect hydroperoxides in products, including blind and parallel method comparison studies.18 In parallel, better validation of the patch test with hydroperoxides will be essential, which must also include blind testing and parallel testing as is currently done for the analytical methods. Luckily in science, there is a test to resolve every dispute, and the thorough research on oxidized linalool has provided us with the necessary tool: the unnatural hydroperoxide1 with a chemical structure significantly differing from the natural hydroperoxide but with almost equal sensitization potential. There has been no exposure to this non-natural compound in the population. Hence, we would expect zero positive reactions and zero cross-reactions with the natural hydroperoxide. Comparing these two agents on the same patients in repeated or parallel tests will teach us about reproducibility and specificity and possible cross-sensitization to hydroperoxides with no prior exposure.
commercial creams, and perfumes never consist of 100% linalool. Thus, both levels of linalool in the cream and levels of oxidation products in linalool will always be much lower in commercial products. (More specifically, skin creams and lotions typically contain 0.3−0.6% perfume, in rarer instances 1%. Only fine fragrance creams, a niche product, may contain >1%. Out of a perfume, typically 2.5%−5% is linalool, and in some perfumes, level may reach 10−20%. Thus, an exposure to 0.5% linalool from skin cream is clearly at the upper end, more typical would be 0.05%. In the ROAT study, linalool contained 19% hydroperoxide, while in aged perfumes 0.66% of linalool was typically in its hydroperoxide form, if it was detected at all.11 Hence, a level of 0.0033% of linalool hydroperoxide in a skin cream would rarely be surpassed, while the ROAT study tested 0.56%, a 170-fold excess based on these conservative estimations.) Thus, the statement “The concentrations used in the ROAT study were in accordance with concentrations of fragrance terpenes expected in common consumer products” is certainly not correct when referring to the hydroperoxide investigated here. To further address this question, studies to investigate true linalool hydroperoxide levels in commercial creams were initiated in our laboratory and will be reported elsewhere. (2) Simultaneous exposure in adjacent skin sites to high doses will always put a question mark on reactions seen at lower concentration, as the immune system is highly interconnected with immunological effector cells migrating in and out of the skin; thus, simultaneously treated adjacent skin sites on the same person cannot be considered as independent experimental replicates. Thus, at the top dose in the ROAT the daily exposure was 113.6 μg/cm2/day for up to 21 consecutive days, a daily dose which is only 3.5-fold below the EC3 in the LLNA, and with a cumulative dose exceeding the dose triggering the LLNA EC3. It is interesting to note that patient 1 who started to react strongly to the 0.3% dose, but only after 14 days of consecutive exposure, had started the study with a patch test at 6% oxidized linalool rated as + , but he became highly sensitive at the end of the study (+++ down to 0.7% and + at 0.07% of oxidized linalool). Thus, active enhancement of the sensitization state due to the concomitant application of excessive levels of linalool hydroperoxide appears a possibility, and while application of such a dose may be questionable for various reasons, it is certainly not useful to derive a finite elicitation level for a pre-existing sensitization state. In the case of this clinical picture, we would not question sensitization to specific haptens formed by linalool hydroperoxides in that patient at the end of the study. To Point 3 of Karlberg et al.: We completely agree that in animal studies, whereby animals were specifically induced with hydroperoxides, specific allergic reactions are triggered as nicely demonstrated by the careful structure−activity relationships15 cited by Karlberg et al. Also, the data cited on colophony indicate that there indeed appears to be no general crossreactivity between colophony and the oxidized fragrance terpenes. However, the data on different oxidized fragrance terpenes are far less clear-cut, especially as the cited studies did not discuss nor take into account published reproducibility figures of patch tests to the oxidized materials. The only information we found on this subject is from ref 16 where 50% reproducibility of the reaction to oxidized limonene was found upon retesting and even clearly lower reproducibility when testing with the hydroperoxide fraction (33% reprodu2083
DOI: 10.1021/acs.chemrestox.5b00423 Chem. Res. Toxicol. 2015, 28, 2082−2084
Chemical Research in Toxicology
Letters to the Editor
Only with these validations will we find out whether the strong conclusion made by Karlberg et al. in their graphical abstract, namely, that cross-sensitization does not exist, is the final truth.
(11) Kern, S., Dkhil, H., Hendarsa, P., Ellis, G., and Natsch, A. (2014) Detection of potentially skin sensitizing hydroperoxides of linalool in fragranced products. Anal. Bioanal. Chem. 406, 6165−6178. (12) Kern, S., Granier, T., Dkhil, H., Haupt, T., Ellis, G., and Natsch, A. (2014) Stability of limonene and monitoring of a hydroperoxide in fragranced products. Flavour Fragrance J. 29, 277−286. (13) Kimber, I., Dearman, R. J., Basketter, D. A., Ryan, C. A., Gerberick, G. F., McNamee, P. M., Lalko, J., and Api, A. M. (2008) Dose metrics in the acquisition of skin sensitization: Thresholds and importance of dose per unit area. Regul. Toxicol. Pharmacol. 52, 39−45. (14) Andersch Bjorkman, Y., Hagvall, L., Siwmark, C., Niklasson, B., Karlberg, A. T., and Brared Christensson, J. (2014) Air-oxidized linalool elicits eczema in allergic patients - a repeated open application test study. Contact Dermatitis 70, 129−138. (15) Brared Christensson, J., Matura, M., Backtorp, C., Borje, A., Nilsson, J. L., and Karlberg, A. T. (2006) Hydroperoxides form specific antigens in contact allergy. Contact Dermatitis 55, 230−237. (16) Matura, M., Goossens, A., Bordalo, O., Garcia-Bravo, B., Magnusson, K., Wrangsjo, K., and Karlberg, A. T. (2003) Patch testing with oxidized R-(+)-limonene and its hydroperoxide fraction. Contact Dermatitis 49, 15−21. (17) Brared Christensson, J., Hellsen, S., Borje, A., and Karlberg, A. T. (2014) Limonene hydroperoxide analogues show specific patch test reactions. Contact Dermatitis 70, 291−299. (18) IDEA (2015) http://www.ideaproject.info/uploads/Modules/ Documents/20150615---idea-hydroperoxyde-tf-meeting---finalminutes-02-09-2015.pdf (accessed Oct 1, 2015).
Andreas Natsch*,† Roger Emter† Remo Badertscher† Gerhard Brunner† Thierry Granier† Susanne Kern† Graham Ellis‡ †
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Biosciences, Analytical Chemistry and Process Research Chemistry, Givaudan Schweiz AG, Ueberlandstrasse 138, CH-8600 Duebendorf, Switzerland ‡ RAPS Fragrance Toxicology, Givaudan International SA, 5 Chemin de la Parfumerie, CH-1214 Vernier, Switzerland
AUTHOR INFORMATION
Corresponding Author
*Tel: +41 44 824 21 05. Fax: +41 44 824 29 26. E-mail:
[email protected].
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REFERENCES
(1) Bezard, M., Karlberg, A. T., Montelius, J., and Lepoittevin, J. P. (1997) Skin sensitization to linalyl hydroperoxide: support for radical intermediates. Chem. Res. Toxicol. 10, 987−993. (2) Sköld, M., Börje, A., Matura, M., and Karlberg, A. T. (2002) Studies on the autoxidation and sensitizing capacity of the fragrance chemical linalool, identifying a linalool hydroperoxide. Contact Dermatitis 46, 267−272. (3) Sköld, M., Börje, A., Harambasic, E., and Karlberg, A. T. (2004) Contact allergens formed on air exposure of linalool. Identification and quantification of primary and secondary oxidation products and the effect on skin sensitization. Chem. Res. Toxicol. 17, 1697−1705. (4) Kao, D., Chaintreau, A., Lepoittevin, J. P., and Gimenez-Arnau, E. (2011) Synthesis of Allylic Hydroperoxides and EPR Spin-Trapping Studies on the Formation of Radicals in Iron Systems as Potential Initiators of the Sensitizing Pathway. J. Org. Chem. 76, 6188−6200. (5) Kao, D., Chaintreau, A., Lepoittevin, J. P., and Giménez-Arnau, E. (2014) Mechanistic studies on the reactivity of sensitizing allylic hydroperoxides: Investigation of the covalent modification of amino acids by carbon-radical intermediates. Toxicol. Res. 3, 278−289. (6) Matura, M., Sköld, M., Börje, A., Andersen, K. E., Bruze, M., Frosch, P., Goossens, A., Johansen, J. D., Svedman, C., White, I. R., and Karlberg, A. T. (2005) Selected oxidized fragrance terpenes are common contact allergens. Contact Dermatitis 52, 320−328. (7) Christensson, J. B., Matura, M., Gruvberger, B., Bruze, M., and Karlberg, A. T. (2010) Linalool - a significant contact sensitizer after air exposure. Contact Dermatitis 62, 32−41. (8) Brared Christensson, J., Andersen, K. E., Bruze, M., Johansen, J. D., Garcia-Bravo, B., Giménez Arnau, A., Goh, C. L., Nixon, R., and White, I. R. (2012) Air-oxidized linalool-a frequent cause of fragrance contact allergy. Contact Dermatitis 67, 247−259. (9) Audrain, H., Kenward, C., Lovell, C. R., Green, C., Ormerod, A. D., Sansom, J., Chowdhury, M. M., Cooper, S. M., Johnston, G. A., Wilkinson, M., King, C., Stone, N., Horne, H. L., Holden, C. R., Wakelin, S., and Buckley, D. A. (2014) Allergy to oxidized limonene and linalool is frequent throughout the UK. Br. J. Dermatol. 171, 292− 297. (10) Brared Christensson, J., Andersen, K. E., Bruze, M., Johansen, J. D., Garcia-Bravo, B., Gimenez Arnau, A., Goh, C. L., Nixon, R., and White, I. R. (2014) Positive patch test reactions to oxidized limonene: exposure and relevance. Contact Dermatitis 71, 264−272. 2084
DOI: 10.1021/acs.chemrestox.5b00423 Chem. Res. Toxicol. 2015, 28, 2082−2084
