Further evaluation of the local lymph node assay in the final phase of an international collaborative trial

Loveless, Scott E.; Ladics, Gregory S.; Gerberick, G. Frank; Ryan, Cindy A.; Basketter, David A.; Scholes, E.W.; House, Robert V.; Hilton, Jennifer; Dearman, Rebecca J.; Kimber, Ian

doi:10.1016/0300-483x(95)03279-o

Cited by 181 publications

(85 citation statements)

References 30 publications

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“…A good example is DNCB, a chemical that has been extensively tested by multiple methods. Even here, the comparisons are complicated by the fact that different endpoints are being evaluated; local lymph node cell proliferation (LLNA) in the mouse (39), erythema in the guinea pig (40), and skin fold thickness changes in man (33). In spite of these differences, the comparison of the dose per unit area resulting in approximately 1 / 4 to 1 / 2 maximal responses for each method were remarkably similar; 25 (mg/cm 2 ) in the mouse (39) and guinea pig (40) and 16.4 (mg/cm 2 ) in man (33).…”

Section: Comparison Of Exposures In Animal Andmentioning

confidence: 99%

The importance of exposure estimation in the assessment of skin sensitization risk

et al. 2000

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The development of new ingredients and products for the consumer market requires a thorough assessment of their potential for skin sensitization and the possible clinical manifestation of allergic contact dermatitis. The process by which low molecular weight chemicals induce and elicit skin sensitization reactions is complex and dependent on many factors relevant to the ability of the chemical to penetrate the skin, react with protein, and trigger the cell-mediated immune response. These major factors include inherent potency, chemical dose, duration and frequency of exposure, vehicle or product matrix, and occlusion. The fact that a chemical is a contact allergen does not mean that it cannot be formulated into a consumer product at levels well tolerated by most individuals. Many common ingredients (e.g., fragrances, preservatives) are known skin allergens. However, all allergens show dose-response and threshold characteristics. Therefore, one should be able to incorporate these chemicals into products at levels that produce acceptably low incidences of skin sensitization under foreseeable conditions of exposure. The critical exposure determinant for evaluating skin sensitization risk is dose per unit area of skin exposed. Use of this parameter allows for comparative assessments from different types of skin sensitization tests (including cross-species comparisons), and, at least for known potent allergens, there is remarkable similarity in threshold dose/unit area determinations across species. The dose/unit area calculation enables a judgment of the sensitization risk for different product types. This is illustrated using the chemical preservative methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) as a case study.

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Section: Comparison Of Exposures In Animal Andmentioning

confidence: 99%

The importance of exposure estimation in the assessment of skin sensitization risk

et al. 2000

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“…Thus, a potential limitation of the ex vivo LLNA, as with the standard assay, is the possibility of false-positive findings due to irritation or, in the case of citral, over-estimating the sensitization potential. Hexylcinnamic aldehyde is a weak sensitizer in humans (Basketter and Kimber, 2001); however, this compound is a weak to moderate sensitizer in the LLNA (Tables 5 and 6; Loveless et al, 1996;Basketter et al, 1999). This difference in potency classification also may be attributed to an over-estimation due to the irritancy properties of this compound.…”

Section: Discussionmentioning

confidence: 99%

“…Mice (4/group) were treated by topical application with 25 µl of test substance in acetone to the dorsum of both ears on Days 1-3 using a single channel pipetter. Basketter et al, 1999;Ryan et al, 2000; The concentrations tested for each compound listed in Table 1 were based on published reports (Basketter et al, 1994Loveless et al, 1996;Ryan et al, 2000). Control animals were treated with vehicle (acetone) alone.…”

Section: Ex Vivo Llnamentioning

confidence: 99%

Use of an Ex Vivo Local Lymph Node Assay to Assess Contact Hypersensitivity Potential

Piccotti

Kawabata

2008

Journal of Immunotoxicology

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The local lymph node assay (LLNA) is used to assess the contact hypersensitivity potential of compounds. In the standard assay, mice are treated topically with test compound to the dorsum of both ears on Days 1-3. The induction of a hypersensitivity response is assessed on Day 6 by injecting [ 3 H]-thymidine into a tail vein and measuring thymidine incorporation into DNA of lymph node cells draining the ears. The ex vivo LLNA is conducted similarly except lymphocyte proliferation is assessed after in vitro incubation of lymph node cells with [ 3 H]-thymidine, which significantly reduces the amount of radioactive waste. The current study tested the use of this approach for hazard assessment of contact hypersensitivity and to estimate allergenic potency. Female BALB/c mice were treated on Days 1-3 with two nonsensitizers (4 -methoxyacetophenone, diethyl phthalate), three weak sensitizers (hydroxycitronellal, eugenol, citral), one weak-tomoderate sensitizer (hexylcinnamic aldehyde), two moderate sensitizers (isoeugenol, phenyl benzoate), and one strong sensitizer (dinitrochlorobenzene). On Day 6, isolated lymph node cells were incubated overnight with [ 3 H]-thymidine and thymidine incorporation was measured by liquid scintillation spectrophotometry. The ex vivo LLNA accurately distinguished the contact sensitizers from the nonsensitizing chemicals, and correctly ranked the relative potency of the compounds tested. The EC3 values, i.e., the effective concentration of test substance needed to induce a stimulation index of 3, were as follows: 4 -methoxyacetophenone (>50%), diethyl phthalate (>50%), hydroxycitronellal (20.4%), eugenol (13.6%), citral (8.9%), isoeugenol (3.8%), hexylcinnamic aldehyde (2.7%), phenyl benzoate (2%), and dinitrochlorobenzene (0.02%). In addition, low inter-animal and inter-experiment variability was seen with 25% hexyl-cinnamic aldehyde (assay positive control). The results of the ex vivo LLNA in the current study were consistent with published reports using the standard LLNA and provided further

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“…Although we aimed at having responses in the short-term experiments of SI = 2, this was not always achieved. Variations in lymph node responses to sensitizers have previously been reported (Loveless et al, 1996;Dearman et al, 1998;De Jong et al, 2002a). Several factors may be responsible for variability in the performance of the assay including the animals used and minimal variations in the actual dose received by the animals.…”

Section: Exposure To Chemicalmentioning

confidence: 95%

“…The LLNA has the advantage over the guinea pig assays in that objective quantitative results are obtained by measurement of tritiated-thymidine incorporation in proliferating cells. The LLNA has been evaluated extensively and compared to the GPMT and human patch testing (Basketter and Scholes, 1992;Kimber, et al, 1995;Loveless et al, 1996;Gerberick et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Effect of Prolonged Repeated Exposure to Formaldehyde Donors with Doses Below the EC3 Value on Draining Lymph Node Responses

Jong

Klerk

Beek

et al. 2007

Journal of Immunotoxicology

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In the Local Lymph Node Assay (LLNA), a stimulation index of 3 (SI = 3) is established as a threshold value for hazard identification of sensitization. The corresponding EC3 value, the effective concentration inducing a threefold increase compared to controls, can possibly predict threshold levels for sensitization in humans. Exposure to a dose below the threshold dose would not result in an induction of an immune response. Each repeated contact would be considered and viewed as a new contact and as long as the dose is below the threshold there will be no response, even after repeated exposures. However, repeated exposure may result in local accumulation eventually resulting in a dose that induces a response above the threshold for immunization. We investigated lymph node responses after short and prolonged exposure to formaldehyde donors, chemicals that are highly reactive with proteins and may thus persist in the skin. The studies were performed with formaldehyde and formaldehyde releasers (formaldehyde, paraformaldehyde, Quaternium-15, 2-Chloro-N-(hydroxymethyl)acetamide, and hexamethylenetetramine), at concentrations that induce a SI = 2, i.e., below the threshold for hazard identification. For all test chemicals investigated enhanced lymph node responses were obtained when comparing long-term prolonged exposure to short-term exposure, while three of five chemicals induced responses above SI = 3. Our results show that repeated and prolonged exposure to doses below the EC3 value can induce reactions above the SI = 3, the hazard identification threshold for sensitization in mice. So, when discussing the possible use of the EC3 as benchmark for risk assessment, one should consider duration of exposure and the possibility of local accumulation of the chemical under investigation.

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Further evaluation of the local lymph node assay in the final phase of an international collaborative trial

Cited by 181 publications

References 30 publications

The importance of exposure estimation in the assessment of skin sensitization risk

The importance of exposure estimation in the assessment of skin sensitization risk

Use of an Ex Vivo Local Lymph Node Assay to Assess Contact Hypersensitivity Potential

Effect of Prolonged Repeated Exposure to Formaldehyde Donors with Doses Below the EC3 Value on Draining Lymph Node Responses

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