A sensitive mouse lymph node assay with two application phases for detection of contact allergens

Ikarashi, Yoshiaki; Tsuchiya, Toshie; Nakamura, Akitada

doi:10.1007/bf01974070

Cited by 37 publications

(12 citation statements)

References 36 publications

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“…Therefore, it was difficult to evaluate the sensitivity of the ex vivo approach based on these findings. Other reports (Ikarashi et al, 1993;Sailstad et al, 1995) evaluating contact hypersensitivity in ex vivo models used dietary vitamin A or Freund's complete adjuvant in an attempt to increase assay performance. Although these approaches appeared to improve assay sensitivity, providing vitamin A as a dietary supplement or using adjuvants may not be practical in a routine assay, and the introduction of an adjuvant may lead to increased falsepositives (Buehler, 1996;Kimber et al, 2001;Kligman and Basketter, 1995).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of anex vivo murine local lymph node assay: multiple endpoint comparison

Piccotti¹,

Knight²,

Gillhouse³

et al. 2006

J. Appl. Toxicol.

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The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity.

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Section: Discussionmentioning

confidence: 99%

Evaluation of anex vivo murine local lymph node assay: multiple endpoint comparison

Piccotti¹,

Knight²,

Gillhouse³

et al. 2006

J. Appl. Toxicol.

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“…Another example of a relatively conservative potential modification is consideration of strains of mice other than CBA (43). However, other suggestions call for more significant changes to the standard protocol (37, 44–53). Such modified assays have not been evaluated fully or validated formally, and, although some of these approaches are not necessarily without merit, they will not be considered further here.…”

Section: Conduct Of the Llnamentioning

confidence: 99%

The local lymph node assay: past, present and future

et al. 2002

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The local lymph node assay (LLNA) was developed originally as a method for the identification of chemicals that have the potential to cause skin sensitization and allergic contact dermatitis. The assay is based on an understanding that the acquisition of contact sensitization is associated with, and dependent upon, the stimulation by chemical allergens of lymphocyte proliferative responses in skin-draining lymph nodes. Those chemicals that provoke a defined level of lymph node cell (LNC) proliferation (a 3-fold or greater increase compared with concurrent vehicle controls) are classified as skin sensitizers. Following its original inception and development, the LLNA was the subject of both national and international interlaboratory collaborative trials, and of very detailed comparisons with other test methods and with human skin sensitization data. The assay has now been validated fully as a stand-alone test for the purposes of hazard identification. In recent years, there has been a growing interest also in the use of the LLNA to assess the potency of contact allergens and in risk assessment. There is reason to believe that the extent of skin sensitization achieved is associated with the vigour of LNC proliferation induced in draining nodes. Given this relationship, the relative potency of skin sensitizing chemicals is measured in the LLNA by derivation of an EC3 value, this being the concentration of chemical required to provoke a 3-fold increase in the proliferation of LNC compared with controls. Experience to date indicates that relative potency as determined using this approach correlates closely with what is known of the activity of skin sensitizing chemicals in humans. In this article, we review the development, evaluation and validation of the LLNA for the purposes of hazard identification, and the more recent application of the method for evaluation of potency in the context of risk assessment. In addition, we consider what new applications and modifications are currently being investigated.Key words: allergic contact dermatitis; hazard identification; local lymph node assay; lymphocyte proliferation, potency assessment; risk assessment; skin sensitization. C Blackwell Munksgaard, 2002. C Blackwell Munksgaard, 2002. Accepted for publication 28 October 2002Skin sensitization resulting in allergic contact dermatitis is an important occupational disease, and certainly the most common manifestation of chemical allergy in humans. There is a need therefore to identify the hazards and risks of allergic contact dermatitis that may be associated with exposure to chemicals at work, at home and in the environment. Conventionally, toxicological assessments of this kind are conducted (conceptually at least) in 2 phases, wherein potential hazards are first identified and then the likelihood of those hazards translating into human health risks is assessed. The latter risk assessments are predicated upon consideration of the nature of the hazard (of particular importance being an appreciation of potency) and the likel...

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“…33 Furthermore, it must be appreciated that the majority of non-sensitizing skin irritants fail to elicit positive responses in the LLNA. 6,12,33,[34][35][36] Another issue that is raised sometimes is the performance of the LLNA with metal allergens, and in particular nickel salts. Nickel is a common cause of allergic contact dermatitis and nickel salts are positive in the guinea pig maximization test.…”

Section: Interpretation Of Llna Datamentioning

confidence: 99%

“…Among the latter is a method employing an aggressive treatment regime incorporating repeated topical exposure of mice to the test chemical, together with intradermal injections of the material in adjuvant. [47][48][49] Increased sensitivity has been claimed for this method and if that is indeed the case then it will have been achieved at the cost of some of the most attractive features of the standard LLNA.…”

Section: Procedural Modifications Alternative Endpoints and Related mentioning

confidence: 99%

Local lymph node assay: use in hazard and risk assessment

1999

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The murine local lymph node assay (LLNA) was developed as an alternative method for the identification of chemicals that have the ability to cause skin sensitization and allergic contact dermatitis. The assay now has been evaluated extensively in the context of both national and international inter‐laboratory collaborative trials and has been the subject of detailed comparisons with guinea pig test methods and human skin sensitization data. On the basis of these evaluations the LLNA has been endorsed recently by the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) as a stand‐alone method for skin sensitization testing. The assay offers a number of important benefits compared with conventional guinea pig test methods, among these being provision of an objective and quantitative endpoint. Moreover, the LLNA provides advantages in the context of animal welfare; compared with guinea pig tests, fewer animals are required and these animals are subject to less trauma. It is important now that the validation status of the LLNA is recognized and the method applied widely so that these advantages may be realized. Hazard identification represents only the first step in the risk assessment process. A full toxicological evaluation of skin sensitization activity requires an understanding of relative potency. Guinea pig methods do not lend themselves readily to assessment of potency, and interest recently has focused on the utility of the LLNA for this purpose. Contained within this review article are brief descriptions of the history of the LLNA and the immunobiological basis for the method, together with detailed accounts of the conduct and interpretation of the assay. Procedural modifications to, and alternative endpoints for, the LLNA are considered also. Finally, the current regulatory status of the LLNA is summarized and the application of the method for the purposes of defining relative potency and developing risk assessments is reviewed. Copyright © 1999 John Wiley & Sons, Ltd.

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A sensitive mouse lymph node assay with two application phases for detection of contact allergens

Cited by 37 publications

References 36 publications

Evaluation of anex vivo murine local lymph node assay: multiple endpoint comparison

Evaluation of anex vivo murine local lymph node assay: multiple endpoint comparison

The local lymph node assay: past, present and future

Local lymph node assay: use in hazard and risk assessment

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