Study of the sensitising potential of various textile dyes using a biphasic murine local lymph node assay

Disperse dyes, which are suitable for dyeing synthetic fibres, are responsible for the great majority of allergic contact dermatitis (ACD) cases to textile dyes. The aim of the present study was to investigate the sensitising potential of various disperse dyes using a biphasic protocol of the local lymph node assay (LLNA). Briefly, mice were shaved over a surface of approximately 2 cm² on their backs and treated using a “sensitisation-challenge protocol”. The shaved surface was treated once daily on days 1–3 with 50 μl of the test solution. Animals remained untreated on days 4–14. On days 15–17, mice were treated with 25 μl of the test solution on the dorsum of both ears. Mice were killed on day 19 with deep CO₂ anaesthesia, the lymph nodes prepared and various end points, such as ear thickness, ear punch weight, lymph node weight, lymph node cell count and the proportion of various lymphocyte subpopulations, were determined by flow cytometry. The results were compared to control group treated with the vehicle alone. Our results showed that almost all of the tested textile dyes caused a significant increase in lymph node cell count and lymph node weight. We also observed an increase in ear thickness and ear punch weight in most of the concentrations tested for various textile dyes. We observed a decrease in CD4+ and CD8+ cells and an increase in CD19+, CD45+ and CD45+/1A+ cells in most of the cases, which is characteristic for allergens. The CD4+/CD69+ cells increased in only few experiments mainly with Disperse Blue 124 and Disperse Blue 106. Based on our results, the disperse dyes could be arranged in four groups on the basis of their sensitising potency in the following decreasing order (in parenthesis: lowest concentration causing a significant increase in lymph node cell number): group 1, strong: Disperse Blue 124 and Disperse Blue 106 (0.003%); group 2, moderate: Disperse Red 1 and Disperse Blue 1 (3%); group 3, weak: Disperse Orange 37 and Disperse Blue 35 (10%); and group 4, very weak: Disperse yellow 3 and Disperse Orange 3 (increase at 30% or no increase at 30%). In conclusion, our study shows that the biphasic LLNA protocol was proficient enough to study the sensitisation potential of tested textile dyes and provides data allowing to discriminate them according to their potency.     

Investigation of the sensitising and cross-sensitising potential of textile dyes and β-lactam antibiotics using a biphasic mice local lymph node assay

We used a modified protocol of the murine local lymph node assay (LLNA) to study the cross-sensitising potential of (a) textile dye disperse yellow 3 and its metabolite 2-amino-p-cresol, (b) two antibiotics, penicillin G and cefotiam. The test substances were applied in a biphasic manner, i.e. first on the shaved skin of the back followed by application on the dorsal side of the ears after 2 weeks. The end-points analysed included thickness and weight of an ear-biopsy, weight and cell number of the draining lymph node, and lymphocyte cell surface markers analysed by flow-cytometry. Disperse yellow 3 and its metabolite significantly altered the various end-points at both the tested concentrations (0.5 and 1%), thus demonstrating the sensitising potential of the two substances. The cross-sensitisation study showed significant modulation in the tested variables in the treated group as compared to the control, signifying cross-sensitisation potential of the two substances. Penicillin G and cefotiam showed significant changes in various end-points, pointing towards their sensitising potential. However, even at 50% concentration of the β-lactams no significant change in any end-point indicating absence of cross-reactivity of the antibiotics was noticed. We conclude that a biphasic, modified protocol of the LLNA is a suitable approach to test for a cross-reactivity potential of two related compounds.     

Development of a non-radioactive endpoint in a modified local lymph node assay.

A murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for contact sensitization testing. Although the LLNA appears to be a little less sensitive than the most stringent of guinea pig assays, it provides a rapid, objective, quantitative and cost-effective method for screening strong contact sensitizers and has advantages with respect to animal welfare. However, a potential disadvantage is the need for the use of radioactive material. We have reported previously that an ex vivo assay based on similar principles to the original in vivo LLNA, but using a non-radioactive endopoint, was valid for the prediction of strong sensitizers. This ex vivo assay was not sensitive enough to allow prediction of moderately potent ones. In this study, we propose a new parameter, Corrected IL-2 Index (CII), for the prediction of moderate sensitizers. To obtain CII the IL-2 release in the supernatant of the cell culture is corrected for lymph node weight ratio and ratio of CD4-positive subset. We found that CII predicted the allergenicity of moderate sensitizers, including the ones recommended by the OECD in guideline 406, such as mercaptobenzothiazole and hexyl cinnamic aldehyde. The allergenicity of metal salts, such as potassium dichromate, ammonium tetrachloroplatinate and cobalt chloride, was also predicted by the CII. We conclude that the use of CII as an index significantly increases the sensitivity of the ex vivo method so that moderate sensitizers may also be detected.     

Assessment of the sensitizing potential of textile disperse dyes and some of their metabolites by the loose-fit coculture-based sensitization assay (LCSA)

Certain textile disperse dyes are known to cause allergic reactions of the human skin. Here, we examined 8 disperse dyes and 7 products of azo-cleavage of these dyes in an in vitro assay. We used the loose-fit coculture-based sensitization assay (LCSA) of primary human keratinocytes and of allogenic dendritic cell-related cells for combined testing of the sensitizing and irritative properties of these substances. The obtained data were compared to data generated in a modified version of the local lymph node assay by our working group. Disperse Blue 1 (DB1), p-nitroaniline (pNA) and p-aminoacetanilide (AAA) showed no sensitizing potential under our experimental conditions. Disperse Blue 124 (DB124), Disperse Yellow 3 (DY3), Disperse Orange 37/76 (DO37), Disperse Blue 106 (DB106), Disperse Red 1 (DR1), 2-amino-p-cresol (ApC), Disperse Orange 3 (DO3) and 2,6-dichloro-4-nitroaniline (DCh) were categorized as extreme sensitizers. Para-phenylenediamine (pPD) was categorized as strong sensitizer, and 2-amino-5-nitrothiazole (ANT) and 2-(N-ethylanilino)-ethanol (EAE) as weak sensitizers. All dyes, except for DB1, and ApC turned out to be strong irritants. DB1, ANT and DCh showed only weak irritative potential. PPD, pNA, EAE and AAA did not show any irritative effect at the concentration range tested. These results correlate with data derived from the modified version of LLNA and human data. Therefore, the LCSA represents a suitable test system to simultaneously analyse two crucial properties of substances relevant for allergy induction.     

Ranking of allergenic potency of rubber chemicals in a modified local lymph node assay.

A modified local lymph node assay (LLNA) with ex vivo tritium thymidine (3H-TdR) labeling of the proliferating lymph node cells was used for determination of the allergenic potency of chemicals used in the production of rubber for latex medical gloves. Fifteen chemicals known to induce contact hypersensitivity reactions in man, including various thiuram, carbamate, and benzothiazole compounds, and one amine were tested. The EC3 (effective concentration inducing a 3-fold increase in proliferation of lymph node cells [Stimulation Index, SI = 3]) was calculated with nonlinear regression analysis, including a bootstrap method for determination of the 5-95% confidence interval of the EC3 value. This procedure identified 14 out of the 15 chemicals tested as sensitizers, while for one chemical, ZDBC, no EC3 could be calculated due to low responses and a lack of a dose-response relationship in the data obtained. The ranking order of the chemicals with increasing EC3 values (and thus decreasing allergenic potency) was found to be in the following order: ZDEC < TMTD < TETD < ZPC < ZDMC < MBTS < PTD < TMTM < MBT < MBI < PTT < ZMBT < TBTD < DEA < ZDBC. Our results indicate that the chemicals of choice for use in the production of natural rubber latex products would be for the thiuram compounds, TBTD; for the carbamates, ZDBC; and for the benzothiazoles, ZMBT. However, one has to be aware that besides potency, the total amount of residual chemical present in the final product is also important for allergy induction.     

The local lymph node assay and potential application to the identification of drug allergens

The local lymph node assay (LLNA) is a method for the identification of skin sensitization hazard. The method is based upon measurement of proliferative responses induced in draining lymph nodes following topical exposure of mice to the test chemical. More recently the LLNA has also been used for the evaluation of relative skin sensitization potency in the context of risk assessment. Idiosyncratic drug reactions resulting from the stimulation of allergic or autoimmunogenic responses are poorly understood but represent an important clinical problem. In this article, the potential utility of the LLNA, either in a conventional modified configuration, to provide information of value in assessment the potential for systemic allergenicity is considered.     

Evaluation of skin sensitization potential of jet fuels by murine local lymph node assay

Jet A and JP-8 are the major jet fuels used in civilian and military (US Air Force) flights, respectively. JP-8+100 is a new jet fuel recently introduced by the US Air Force. Besides lung exposure, skin is the potential route of exposure to jet fuels. The purpose of the present study was to investigate the skin sensitization potential of jet fuels (Jet A, JP-8 and JP-8+100) using murine Local lymph node assay (LLNA). Female CBA/Ca mice (8-12-weeks-old) were used in the study. Dinitrochlorobenzene (DNCB, 0.25% w/v) and paraaminobenzoic acid (PABA, 2.5% w/v) were used as positive and negative control, respectively and acetone: olive oil (4:1, AOO) was used as the vehicle (control). All three jet fuels caused a proliferative activity significantly greater than the control (P<0.01). Our results demonstrate that JP-8 is a weak skin sensitizer [stimulation index (SI)=3.17]. The SI of Jet A and JP-8+100 were 2.44 and 2.38, respectively, hence are not considered as skin sensitizers. Interestingly, the SI of JP-8 with butylated hydroxytoluene (BHT) was consistently lower than JP-8, though the difference was not statistically significant (P>0.05). BHT, which is an antioxidant additive of JP-8+100, reduced the skin sensitization potential of JP-8. Furthermore, the lower SI of JP-8+100 could be partially attributed to the presence of BHT. The findings reported here suggest that care should be taken to minimize dermal exposure to jet fuels especially JP-8 to avoid skin sensitization.     

The local lymph node assay in practice: a current regulatory perspective

Following the formal acceptance of the local lymph node assay (LLNA) as an Organization for Economic Cooperation and Development (OECD) guideline in April 2002, the UK Health and Safety Executive (HSE) informed notifiers that this was now the method of choice for the assessment of skin sensitization potential under the EU notification scheme for new industrial chemicals (NONS). This paper summarizes the experience of the HSE for the 2-year period immediately following the issuing of this statement, during which 48 LLNA study reports were assessed for notification purposes. The issues discussed here include adherence to the OECD guideline, interpretation of results, and classification outcomes. Generally, notifying laboratories followed the OECD guideline successfully, with regard to the sex/ strain/numbers of mice used, the precise process used for measurement of cell proliferation, and the use of recommended vehicles and positive controls. Initially, use of the individual animal approach (measuring the cell proliferation in each animal rather than for a pooled dose group) highlighted problems caused by technical inexperience, but these were overcome by practice. Toxicity or irritation were found to be minor factors in dose selection; more important was the choice of vehicle to correctly maximize the test substance concentration, while maintaining appropriate application properties. Contrary to concerns that the LLNA would prove to be less sensitive or more sensitive than the traditionally used Guinea Pig Maximization Test (GPMT), the proportion of new substances classified as skin sensitizers was within the range observed in previous years. Although the sample size is relatively small, the experience of the HSE indicates that the LLNA is satisfactory for routine regulatory use.     

Evaluation of skin sensitization potential of melatonin and nimesulide by murine local lymph node assay.

Melatonin is a good candidate for transdermal delivery considering its short plasma half life, low molecular weight and a favorable octanol:water partition coefficient. Nimesulide is a nonsteroidal anti-inflammatory agent used orally and rectally for inflammatory disorders. The objective of this study was to investigate the skin sensitization potential of melatonin and nimesulide using the standard murine local lymph node assay (LLNA). Melatonin (0.5, 2.5, 5.0 and 10.0%, w/v) and nimesulide (0.5, 2.5, 5.0 and 10.0%, w/v) dissolved in acetone:olive oil (4:1, AOO) was applied (25 microl) on the dorsal surface of each ear of female CBA/Ca mice for three consecutive days. On the sixth day, [3H]methyl thymidine was administered intravenously and the uptake of [3H]methyl thymidine (dpm) by the draining lymph nodes was determined by established methods. Dinitrochlorobenzene (DNCB, 0.25%, w/v) and para-aminobenzoic acid (PABA, 2.5%, w/v) were used as positive and negative control, respectively. The mean dpm obtained with melatonin and nimesulide treatment at all concentrations were not significantly different (P>0.05) from that of AOO. The stimulation index (SI) values of melatonin and nimesulide at different concentrations were close to 1. The results of the present study using the standard LLNA approved by US Interagency Coordinating Committee in the Validation of Alternative Methods (ICCVAM) indicate that melatonin and nimesulide are not skin sensitizers. However, since LLNA has shown false negatives with many drugs, clinical trials are certainly needed to exclude the possibility of a weak or delayed type skin sensitization reaction. Further studies using modified LLNA procedures (extended exposure, alternative vehicle systems, pre-abrasion, etc.) may be useful in identifying the weak or delayed type skin sensitization reactions.     

Comparison of mouse strains using the local lymph node assay

The local lymph node assay (LLNA), as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), only allows for the use of CBA mice. The objective of these studies was to begin to assess the response of chemical sensitizers in the LLNA across six strains of female mice (C57BL/6, SJL/J, BALB/c, B6C3F1, DBA/2 and CBA). The moderate sensitizer alpha-hexylcinnamaldehyde (HCA) was chosen as the test chemical, while toluene diisocyanate (TDI) and 2,4-dinitrofluorobenzene (DNFB) were evaluated at single concentrations as positive controls. Draining lymph node cell proliferation following acetone exposure varied across strains. SJL mice had a significantly higher degree of proliferation with 2111 d.p.m./2 nodes. The remaining five strains demonstrated responses which ranged from 345 to 887 dpm/2 nodes. DBA/2, B6C3F1, BALB/c and CBA mice had essentially equal levels of lymph node proliferation following exposure to the three chemicals. While C57BL/6 mice gave similar results as CBA mice following DNFB and HCA administration, the LLNA response to TDI was considerably lower. SJL mice provided low stimulation indexes (SI) values for all three chemicals evaluated. Regardless of the level of LLNA response, all six mouse strains identified the sensitization potential of HCA, TDI or DNFB. Based on these studies, DBA/2, B6C3F1 and BALB/c mice are good choices for continued evaluation as additional mouse strains for use in the LLNA.     

Measurement of allergenic potency using the local lymph node assay

Chemicals that can act as contact allergens have been identified successfully using guinea-pig models. However, contact allergy is still common, probably because of, at least in part, failures of risk assessment. A new method, the local lymph node assay, replaces the guinea-pig as a tool for hazard identification and offers the real prospect of accurate prediction of allergen potency, the missing link in skin sensitization risk assessment.     

Investigation of the dermal sensitization potential of various essential oils in the local lymph node assay.

Essential oils are commonly used fragrance ingredients. The oils themselves are complex mixtures, which may contain naturally occurring contact sensitizers. The local lymph node assay was used to evaluate the dermal sensitization potential of basil, citronella, clove leaf, geranium, litsea cubeba, lemongrass, and palmarosa oils. Three of the major components--citral, eugenol, and geraniol--were included to investigate any difference in sensitization potential arising from their exposure in a mixture. Each fragrance material was tested at five concentration ranging from 2.5% to 50% w/v in 1:3 ethanol:diethyl phthalate. The stimulation index (SI) values were calculated for each dose level, an SI > or = 3 was considered a positive response. The estimated concentration (EC3) required to elicit a positive was calculated and taken as a measure of relative potency. The EC3 values and potency classification for basil, clove leaf, litsea cubeba, lemongrass and palmarosa oils were calculated to be <2.5% (> or = moderate), 7.1% (weak), 8.4% (weak), 6.5% (weak) and 9.6% (weak), respectively. Citronella and geranium oils were negative. The individual components citral, eugenol and geraniol resulted in EC3 values of 6.3%, 5.4% and 11.4%, respectively. In general, the potency of each essential oil did not differ significantly from that observed for its main individual component.     

Draining lymph node cell activation in guinea pigs: comparisons with the murine local lymph node assay

The local lymph node assay in the mouse is a novel predictive test for the identification of contact sensitizing chemicals. The purpose of the studies described was to determine whether a similar local lymph node assay could be performed successfully in guinea pigs; currently the species of choice for assessment of sensitizing potential for regulatory purposes. Ten sensitizing chemicals (oxazolone, picryl chloride, 2,4-dinitrofluorobenzene, benzocaine, cinnamic aldehyde, 2,4,-dinitrothiocyanobenzene, p-nitrosodimethylaniline, formaldehyde, p-phenylenediamine and cyanuric chloride) and equal concentrations of sodium lauryl sulphate were examined in a guinea pig local lymph node assay. Animals received three consecutive daily applications of various concentrations of the test chemical on the dorsum of both ears. Control animals were untreated. Five days following the initiation of exposure, draining auricular lymph nodes were excised and weighed. Suspensions of lymph node cells (LNC) were prepared and cultured for 24 or 48 h and proliferation measured by incorporation of [3H]thymidine. Exposure to at least one concentration of all sensitizing chemicals, other than benzocaine, induced proliferation by draining LNC. Responses were higher at 24 h rather than 48 h. Evidence is presented that guinea pig LNC proliferation may be enhanced or maintained by addition to culture of an exogenous source of the T cell growth factor interleukin 2 (IL-2). Draining lymph node weight was increased following exposure to some sensitizing chemicals but, compared with LNC proliferation, provided a less sensitive correlate of lymph node activation. Exposure to sodium lauryl sulphate failed to induce changes in either lymph node weight of LNC proliferation. Data are compared with three-day murine local lymph node assays performed concurrently. The available information indicates that the local lymph node assay may be performed in guinea pigs.     

Prior exposure to organophosphorus and organochlorine pesticides increases the allergic potential of environmental chemical allergens in a local lymph node assay

The dysregulation of immune functions by some pesticides leads to various immune disorders, including immunodeficiency, tumorigenesis, allergies, and autoimmunity. This study's primary objective was to examine the relationship between immune disorders and the immunosuppression induced by immunosuppressive pesticides. We focused on the modulation of allergic potential by the organophosphorus pesticide parathion, organochlorine pesticide methoxychlor, phenoxyacetic acid herbicide 2,4-d-butyl, and benzoic acid fungicide eugenol, as detected by a local lymph node assay (LLNA), which was developed initially for hazard identification of skin sensitization. Parathion and methoxychlor are immunosuppressive chemicals, and 2,4-d-butyl and eugenol are contact allergens. After the immunosuppressive characteristics of parathion and methoxychlor were confirmed in a pilot study, 4-week-old mice were orally administered parathion (0, 0.4, 1.2mg/kg) or methoxychlor (0, 100, 300 mg/kg). Four weeks after the last administration, an LLNA was conducted using 2,4-d-butyl (0%, 2.5%, 5%, and 10%) and eugenol (0%, 5%, 10%, and 25%). In addition, detailed analysis of their auricular lymph nodes for number of surface antigen expression of T cells and local cytokine production were performed using 5% 2,4-d-butyl and 5% eugenol treatment groups. EC3 values (estimated concentration to yield a stimulation index of 3) of 2,4-d-butyl and eugenol decreased markedly in parathion- and methoxychlor-pretreated groups. Parathion- and methoxychlor-pretreated groups induced marked increase in number of surface antigen expression of T cells and levels of Th1 cytokines (IFN-γ, TNF-α, and IL-17) produced by ex vivo restimulated lymph node cells. According to our results, the allergic potentials of 2,4-d-butyl and eugenol are increased by prior exposure to parathion and methoxychlor.     

Use of the local lymph node assay in assessment of immune function

The murine local lymph node assay (LLNA) was originally developed as a predictive test method for the identification of chemicals with sensitizing potential. In this study we demonstrated that an adapted LLNA can also be used as an immune function assay by studying the effects of orally administered immunomodulating compounds on the T-cell-dependent immune response induced by the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). C57Bl/6 mice were treated with the immunotoxic compounds cyclosporin A (CsA), bis(tri-n-butyltin)oxide (TBTO) or benzo[a]pyrene, (B[a]P). Subsequently, cell proliferation and interferon-gamma (IFN-gamma) and interleukin (IL)-4 release were determined in the auricular lymph nodes (LNs) after DNCB application on both ears. Immunosuppression induced by CsA, TBTO and B[a]P was clearly detectable in this application of the LLNA. Cytokine release measurements proved valuable to confirm the results of the cell proliferation assay and to obtain an indication of the effect on Th1/Th2 balance. We believe to have demonstrated the applicability of an adapted LLNA as an immune function assay in the mouse.     

Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers

The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [³H]methyl thymidine. A more recent non‐isotopic variation of the assay utilizes bromodeoxyuridine (BrdU) incorporation in vivo. To further improve the utility of this assay, we developed an ex vivo BrdU labeling procedure eliminating the need for in vivo injections. The results of this assay correctly identified a strong sensitizer (i.e., trimellitic anhydride) as well as weak/moderate sensitizers (i.e., eugenol, cinnamaldehyde and hexylcinnaminic aldehyde). As anticipated, neither non‐sensitizers isopropanol and lactic acid nor the false negative chemical nickel II sulfate hexahydrate induced a positive threshold response in the assay. The results of this assay are in close agreement with those of the in vivo LLNA:BrdU‐enzyme‐linked immunosorbent assay labeling procedure. We also used the ex vivo BrdU LLNA procedure to evaluate ammonium hexachloroplatinate, ammonium tetrachloroplatinate and cis‐diamminedichloroplatinum(II) and the assay correctly identified them as sensitizers based on the calculation of EC₂ values. We conclude that this ex vivo BrdU labeling method offers predictive capacity comparable to previously established LLNA protocols while eliminating animal injections and the use of radioisotope. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Appraisal of the sensitising potential of orally and dermally administered Mercaptobenzothiazol by a biphasic protocol of the local lymph node assay

Mercaptobenzothiazole (MBT) is used while manufacturing natural rubber products. Our study deals with assessing its allergenic potential following dermal and oral routes of exposure, using a biphasic local lymph node assay (LLNA). Female Balb/c mice were treated with MBT (dermally 3, 10, 30% concentrations in DMSO; orally 1, 10, 100 mg/kg doses in corn oil) on the back (dermal study) or through oral administration (oral study) on days 1–3 followed by auricular application of 3, 10 and 30% concentrations, respectively, on days 15–17. End points determined on day 19 included ear thickness, ear punch weight, lymph node weight, lymph node cell count, and lymphocyte subpopulations (CD4+, CD8+, CD45+). After dermal application of 3% or 10% solution, a significant increase in cell count and lymph node weight along with significant decrease in CD8+ cells was observed. After initial oral administration of 1 mg/kg, we noticed a significant amplification in cell count. Following oral administration of 10 mg/kg, we observed a similar increase in cell count and lymph node weight. The results of our study show that the modified biphasic LLNA protocol can be used to study the sensitising potential of a compound also following the oral route of exposure.     

Ethanol and diethyl phthalate: vehicle effects in the local lymph node assay

The vehicle in which an allergen is presented to the skin has been recognized to have an effect on the skin-sensitizing potency of the allergen. Typical vehicles used to evaluate the skin sensitization potential of fragrance materials include ethanol, diethyl phthalate, or a combination of the two. The authors conducted a series of studies to evaluate each of these vehicles for their utility in the murine local lymph node assay and to investigate the potential differences in skin sensitization resulting from their use. Four fragrance materials were tested in four different vehicles. The test materials were p-t-butyl-alpha-methylhydrocinnamic aldehyde, geraniol, eugenol, and hydroxycitronellal. The vehicles were diethyl phthalate, 1:3 ethanol:diethyl phthalate, 3:1 ethanol:diethyl phthalate, and ethanol. Each of the fragrance materials was tested at five dose levels ranging from 0.3% to 50% w/v. In all four vehicles, each material tested elicited positive responses, exhibiting weak to moderate skin sensitization potential. Overall, p-t-butyl-alpha-methylhydrocinnamic aldehyde exhibited the most potency, followed by eugenol, geraniol, and hydroxycitronellal. The sensitization potential of both p-t-butyl-alpha-methylhydrocinnamic aldehyde and geraniol was greatest when the vehicle was ethanol. The sensitization potential of eugenol was greatest in 3:1 ethanol:diethyl phthalate, but the sensitization potential for hydroxycitronellal was greatest in 1:3 ethanol:diethyl phthalate. The strength of the sensitization response was observed to vary with the vehicle; however, the results did not show any clear pattern of one vehicle over another regarding skin sensitization.