# Local lymph node assay

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{{Short description|Toxicological test}}
The [murine](/source/murine) '''local lymph node assay''' (LLNA) is an ''[in vivo](/source/in_vivo)'' test for [skin sensitisation](/source/skin_sensitisation).

LLNA has largely superseded the [guinea pig maximisation test](/source/guinea_pig_maximisation_test) and the [Buehler test](/source/Buehler_test). It is considered more scientific and less cruel (lower number of animals; less suffering) and has found broad scientific and regulatory acceptance.

The principle underlying the LLNA is that skin sensitizers induce growth of [lymphocytes](/source/lymphocytes) in the [lymph nodes](/source/lymph_nodes) draining the site of application. Lymphocyte [proliferation](/source/cell_proliferation) can be measured by [radiolabeling](/source/radiolabeling) (quantifying tritiated [thymidine](/source/thymidine)), [bioluminescence](/source/bioluminescence) (quantifying [ATP](/source/Adenosine_triphosphate) content in lymphocytes) or [immunoassay](/source/immunoassay) ([ELISA](/source/ELISA) utilizing an [antibody](/source/antibody) specific for [BrdU](/source/BrdU)).<ref>{{citation | editor1=Michael J. Derelanko | editor2=Carol S. Auletta | title=Handbook of Toxicology | edition=3rd | year=2014 | publisher=CRC Press | isbn=978-1-4398-9014-1}}</ref>

The test material is applied to the ears of mice. Optionally, a tracer substance such as 3H-Methyl-[thymidine](/source/thymidine) or [BrdU](/source/BrdU) is injected intraperitoneally for lymphocyte incorporation. The animals are euthanized and their lymph node cells are removed and analyzed. The ratio of tracer incorporation in lymph nodes from dosed animals is compared to control animals, giving a stimulation index (SI). When the stimulation index exceeds 3 (SI > 3), a relevant sensitizing potential is assumed. In contrast to the classical guinea pig tests, the LLNA provides a quantitative measurement of sensitizing potency of a tested chemical.<ref>{{citation | contribution=Hair Preparations | title=[Ullmann's Encyclopedia of Industrial Chemistry](/source/Ullmann's_Encyclopedia_of_Industrial_Chemistry) | edition=7th | publisher=Wiley | year=2007 | doi=10.1002/14356007.a12_571.pub2 | last1=Clausen | first1=Thomas | last2=Schwan-Jonczyk | first2=Annette | last3=Lang | first3=Günther | last4=Schuh | first4=Werner | last5=Liebscher | first5=Klaus Dieter | last6=Springob | first6=Christian | last7=Franzke | first7=Michael | last8=Balzer | first8=Wolfgang | last9=Imhoff | first9=Sonja | last10=Maresch | first10=Gerhard | last11=Bimczok | first11=Rudolf | isbn=978-3-527-30385-4 }}</ref>

The LLNA may not be appropriate for certain metallic compounds, surfactants, high-molecular-weight proteins, strong dermal irritants, and materials that do not sufficiently adhere to the ear for an acceptable period of time during treatment. There is no absolute conformity in the sensitizing potential of a substance in mouse, guinea pig and human.

== Regulatory ==
The [OECD Guidelines for the Testing of Chemicals](/source/OECD_Guidelines_for_the_Testing_of_Chemicals) guideline No. 429 of 23 July 2010.<ref>{{Cite web|url=http://www.oecd-ilibrary.org/environment/test-no-429-skin-sensitisation_9789264071100-en|title = Test No. 429: Skin Sensitisation: Local Lymph Node Assay| date=22 July 2010 }}</ref> The BrdU-ELISA method and the BrdU-FCM method are described in OECD Test Guideline No. 442B.<ref>{{Cite web |date=2025-06-24 |title=Test No. 442B: Skin Sensitization |url=https://www.oecd.org/en/publications/test-no-442b-skin-sensitization_9789264090996-en.html |access-date=2025-07-16 |website=OECD |language=en}}</ref>

The [REACH Regulation](/source/REACH_Regulation), Annex VII, paragraph 8.3 states "The Murine Local Lymph Node Assay (LLNA) is the first-choice method for ''in vivo'' testing. Only in exceptional circumstances should another test be used. Justification for the use of another test shall be provided."

According to European Guideline OECD 406 Skin Sensitization, the LLNA or the MEST ([Mouse ear swelling test](/source/Mouse_ear_swelling_test)) can be used as a first stage in the assessment of skin sensitization potential. If a positive result is seen in either assay, a test substance may be designated as a potential sensitizer, and it may not be necessary to conduct a further guinea pig test. However, if a negative result is seen in the LLNA or MEST, a guinea pig test (preferably a GPMT or BT) must be conducted.

== References==
<references/>

Category:Allergology
Category:Toxicology
Category:Toxicology tests

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