The

murine The Old World rats and mice, part of the subfamily Murinae in the family Muridae, comprise at least 519 species. Members of this subfamily are called murines. In terms of species richness, this subfamily is larger than all mammal families ex ...

local lymph node assay (LLNA) is an ''

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...

'' test for

skin sensitisation Skin is the layer of usually soft, flexible outer tissue covering the body of a vertebrate animal, with three main functions: protection, regulation, and sensation. Other animal coverings, such as the arthropod exoskeleton, have different de ...

. LLNA has largely superseded the guinea pig maximisation test and the Buehler test.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 in the

lymph nodes A lymph node, or lymph gland, is a kidney-shaped organ of the lymphatic system and the adaptive immune system. A large number of lymph nodes are linked throughout the body by the lymphatic vessels. They are major sites of lymphocytes that incl ...

draining the site of application. Lymphocyte

proliferation Proliferation may refer to: Weapons *Nuclear proliferation, the spread of nuclear weapons, material, and technology *Chemical weapon proliferation, the spread of chemical weapons, material, and technology * Small arms proliferation, the spread of ...

can be measured by

radiolabeling A radioactive tracer, radiotracer, or radioactive label is a chemical compound in which one or more atoms have been replaced by a radionuclide so by virtue of its radioactive decay it can be used to explore the mechanism of chemical reactions by tr ...

(quantifying tritiaded

thymidine Thymidine (symbol dT or dThd), also known as deoxythymidine, deoxyribosylthymine, or thymine deoxyriboside, is a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine (A) in double-stranded DN ...

bioluminescence Bioluminescence is the production and emission of light by living organisms. It is a form of chemiluminescence. Bioluminescence occurs widely in marine vertebrates and invertebrates, as well as in some fungi, microorganisms including some ...

(quantifying

ATP ATP may refer to: Companies and organizations * Association of Tennis Professionals, men's professional tennis governing body * American Technical Publishers, employee-owned publishing company * ', a Danish pension * Armenia Tree Project, non ...

content in lymphocytes) or

immunoassay An immunoassay (IA) is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes). The molecule detected by the immunoass ...

(

ELISA The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence ...

utilizing an

antibody An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and Viral disease, viruses. The antibody recognizes a unique m ...

specific for

BrdU Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU, BUdR, BrdUrd, broxuridine) is a synthetic nucleoside analogue with a chemical structure similar to thymidine. BrdU is commonly used to study cell proliferation in living tissues and has been st ...

). The test material is applied to the ears of mice. Optionally, a tracer substance such as 3H-Methyl-

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. 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 OECD Guidelines for the Testing of Chemicals (OECD TG) are a set of internationally accepted specifications for the testing of chemicals decided on by the Organisation for Economic Co-operation and Development (OECD). They were first published in 1 ...

guideline No. 429 of 23 July 2010.{{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 The

REACH Regulation Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) is a European Union regulation dating from 18 December 2006. REACH addresses the production and use of chemical substances, and their potential impacts on both human h ...

, 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) 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

Allergology Toxicology