A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses

Basketter, David A.; Lea, Linda J.; Dickens, Andrea; Briggs, David; Pate, Ian; Dearman, Rebecca J.; Kimber, Ian

doi:10.1002/(sici)1099-1263(199907/08)19:4<261::aid-jat572>3.0.co;2-5

Cited by 215 publications

(130 citation statements)

References 13 publications

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“…The effective concentration of a stimulation index of 3 (EC3) for each experimental group was calculated by linear interpolation (IS, [20][21][22][23][24]. In each case, a stimulation index (SI) relative to the concurrent vehicletreated control was derived.…”

Section: Llnasupporting

confidence: 50%

Evaluation of Allergenicity of Constituents of Myoga Using the Murine Local Lymph Node Assay

Wei

Harada

et al. 2010

Int J Immunopathol Pharmacol

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Myoga (Zingiber Myoga Roscoe) is a perennial plant with a pungent smell from its flower buds. It is native to East Asia and has been reported to cause allergic contact dermatitis. The purpose of this study is to assess the allergenicity of myoga related to its major chemical components, «-pinene, p-pinene, limonene, llmonene oxide and p-phellandrene, which are supposed to be the causative agents of contact dermatitis among myoga cultivators. We performed a toxicity study of the volatile constituents of myoga using the local lymph node assay (LLNA), in which llmonene, limonene oxide and p-phellandrene had positive responses and the EC3 was 35.8%, 8.22%, and 0.54%, respectively. EC3 for both «-pinene and p-pinene was over 100%. Both chemicals failed to induce positive responses in the LLNA. While the maximization rating of limonene, llmonene oxide and phellandrene were evaluated as moderate, extreme, and extreme respectively, u-plnene and p-pinene were evaluated as weak in the previously reported GPMT. The usage ofLLNA was also confirmed by comparing with previously reported GPMT results to detect the allergenicity of myoga constituents. The actual risk of humans developing an allergy to myoga constituents depends on many factors. The concentration ofthe compounds, the frequency and duration of exposure and the condition of the skin are supposed to be important factors. Myoga (Zingiber Myoga Roscoe) is a perennial plant with a pungent smell of its flower buds and is of East Asian origin (1), belonging to the ginger family (Zingiberacea). The Japanese have long eaten the flower buds as a spice or pickle. The technology for cultivating myoga in greenhouses was established in the 1990s, enabling the flower buds to be supplied to markets all year round. Myoga greenhouses have increased in some areas in Japan, while hand dermatitis suspected to be due to the allergenicity to myoga has been recognized among myoga greenhouse cultivators.Minamoto et al. first reported a case study of contact dermatitis from myoga (2-3), before which contact dermatitis from myoga was unknown (4). According to our earlier investigation of myoga cultivators in Japan, 8 of 35 cultivators experienced contact dermatitis during the harvest season (2-3).

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Section: Llnasupporting

confidence: 50%

Evaluation of Allergenicity of Constituents of Myoga Using the Murine Local Lymph Node Assay

Wei

Harada

et al. 2010

Int J Immunopathol Pharmacol

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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%

“…Although the cause of these variable results remains unclear, one possible explanation was the use of acetone as vehicle. Although the authors are unaware of any published data on a vehicle comparison with phenyl benzoate, Basketter et al (1999) showed more robust responses (i.e., higher SI values at similar concentrations) with this compound when tested in acetone-olive oil (4:1, v/v) than were seen in the current study using acetone alone, although similar EC3 values ultimately were obtained.…”

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%

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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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“…Using the EC3 value (the effective concentration inducing a threefold increase in lymph node cell proliferation compared to 240 DE JONG ET AL. controls, SI = 3), various chemicals can be compared with each other for their sensitizing potency, and a ranking order can be determined (Basketter et al, 1999a(Basketter et al, , 1999b(Basketter et al, , 2005aVan Och et al, 2000;De Jong et al, 2002b). Based on such ranking, chemicals might be classified according to their potency in various categories instead of the two categories currently used-sensitizer and non-sensitizer (Dearman et al, 1999;Kimber et al, 2003;Schneider and Akkam, 2004;Basketter et al, 2005a).…”

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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A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses

Cited by 215 publications

References 13 publications

Evaluation of Allergenicity of Constituents of Myoga Using the Murine Local Lymph Node Assay

Evaluation of Allergenicity of Constituents of Myoga Using the Murine Local Lymph Node Assay

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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