The murine local lymph node assay (LLNA) is an in vivo test for skin sensitisation.
LLNA has largely superseded the guinea pig maximisation test and the Buehler test.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 draining the site of application. Lymphocyte proliferation can be measured by radiolabeling (quantifying tritiaded thymidine), bioluminescence (quantifying ATP content in lymphocytes) or immunoassay (ELISA utilizing an antibody specific for BrdU). [1]
The test material is applied to the ears of mice. Optionally, a tracer substance such as 3H-Methyl-thymidine or 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. [2]
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.
The OECD Guidelines for the Testing of Chemicals guideline No. 429 of 23 July 2010. [3]
The 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) 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.
The Draize test is an acute toxicity test devised in 1944 by Food and Drug Administration (FDA) toxicologists John H. Draize and Jacob M. Spines. Initially used for testing cosmetics, the procedure involves applying 0.5 mL or 0.5 g of a test substance to the eye or skin of a restrained, conscious animal, and then leaving it for a set amount of time before rinsing it out and recording its effects. The animals are observed for up to 14 days for signs of erythema and edema in the skin test, and redness, swelling, discharge, ulceration, hemorrhaging, cloudiness, or blindness in the tested eye. The test subject is commonly an albino rabbit, though other species are used too, including dogs. The animals are euthanized after testing if the test renders irreversible damage to the eye or skin. Animals may be re-used for testing purposes if the product tested causes no permanent damage. Animals are typically reused after a "wash out" period during which all traces of the tested product are allowed to disperse from the test site.
N,N′-Dicyclohexylcarbodiimide (DCC or DCCD) is an organic compound with the chemical formula (C6H11N)2C. It is a waxy white solid with a sweet odor. Its primary use is to couple amino acids during artificial peptide synthesis. The low melting point of this material allows it to be melted for easy handling. It is highly soluble in dichloromethane, tetrahydrofuran, acetonitrile and dimethylformamide, but insoluble in water.
1,1'-Carbonyldiimidazole (CDI) is an organic compound with the molecular formula (C3H3N2)2CO. It is a white crystalline solid. It is often used for the coupling of amino acids for peptide synthesis and as a reagent in organic synthesis.
Allergic contact dermatitis (ACD) is a form of contact dermatitis that is the manifestation of an allergic response caused by contact with a substance; the other type being irritant contact dermatitis (ICD).
Hydroxybenzotriazole is an organic compound that is a derivative of benzotriazole. It is a white crystalline powder, which as a commercial product contains some water. Anhydrous HOBt is explosive.
A micronucleus test is a test used in toxicological screening for potential genotoxic compounds. The assay is now recognized as one of the most successful and reliable assays for genotoxic carcinogens, i.e., carcinogens that act by causing genetic damage and is recommended by the OECD guideline for the testing of chemicals. There are two major versions of this test, one in vivo and the other in vitro.
N,N′-Diisopropylcarbodiimide is a carbodiimide used in peptide synthesis. As a liquid, it is easier to handle than the commonly used N,N′-dicyclohexylcarbodiimide, a waxy solid. In addition, N,N′-diisopropylurea, its byproduct in many chemical reactions, is soluble in most organic solvents, a property that facilitates work-up.
HATU is a reagent used in peptide coupling chemistry to generate an active ester from a carboxylic acid. HATU is used along with Hünig's base (N,N-diisopropylethylamine), or triethylamine to form amide bonds. Typically DMF is used as solvent, although other polar aprotic solvents can also be used.
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide is a water-soluble carbodiimide usually handled as the hydrochloride.
The Guinea pig maximisation test (GPMT) is an in vivo test to screen for substances that cause human skin sensitisation. It was first proposed by B. Magnusson and Albert Kligman in 1969 and described in their 1970 book Allergic Contact Dermatitis in the Guinea Pig.
The Buehler test is an in vivo test to screen for substances that cause human skin sensitisation. It was first proposed by Edwin Vernon Buehler in 1965 and further explained in 1980.
The Human maximisation test (HMT) is a test method for testing for contact allergens. It was first developed by Albert Kligman in 1966 and updated by Kligman and William Epstein in 1975. The first paper appeared 1966 and was a citation classic in 1985.
Tetramethyl acetyloctahydronaphthalenes is a synthetic ketone fragrance also known as OTNE and by other commercial trade names such as: Iso E Super, Iso Gamma Super, Anthamber, Amber Fleur, Boisvelone, Iso Ambois, Amberlan, Iso Velvetone, Orbitone, Amberonne. It is a synthetic woody odorant and is used as a fragrance ingredient in perfumes, laundry products and cosmetics.
HBTU is a coupling reagent used in solid phase peptide synthesis. It was introduced in 1978 and shows resistance against racemization. It is used because of its mild activating properties.
Mixed lymphocyte reaction (MLR) is a test used by pharmaceutical and biotech organizations to show the safety of a drug or implantable material. It is commonly used as part of the FDA clearance process. Put simply, it is mixing populations of T-lymphocytes together, and measuring the reaction that occurs. Technically, it is an ex-vivo cellular immune assay that occurs between two allogeneic lymphocyte populations. In a one-way MLR, only one lymphocyte population can respond or proliferate. In a two-way MLR, both populations can proliferate. MLR’s are performed to assess how T-cells react to external stimuli. T cells are a type of white blood cell that scans for cellular abnormalities and infections. They are essential to human immunity.
Tetramethylurea is the organic compound with the formula (Me2N)2CO. It is a substituted urea. This colorless liquid is used as an aprotic-polar solvent, especially for aromatic compounds and is used e. g. for Grignard reagents. Tetramethylurea is a colorless liquid with mild aromatic odor. Unusual for an urea is the liquid state of tetramethylurea in a range of > 170 °C.
E-SCREEN is a cell proliferation assay based on the enhanced proliferation of human breast cancer cells (MCF-7) in the presence of estrogen active substances. The E-SCREEN test is a tool to easily and rapidly assess estrogenic activity of suspected xenoestrogens. This bioassay measures estrogen-induced increase of the number of human breast cancer cell, which is biologically equivalent to the increase of mitotic activity in tissues of the genital tract. It was originally developed by Soto et al. and was included in the first version of the OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters published in 2012. However, due to failed validation, it was not included in the updated version of the framework published in 2018.
The mouse ear swelling test is a toxicological test that aims to mimic human skin reactions to chemicals. It avoids post-mortem examination of tested animals.
HCTU is an amidinium coupling reagent used in peptide synthesis. It is analogous to HBTU. The HOBt moiety has a chlorine in the 6 position which improves reaction rates and the synthesis of difficult couplings HCTU and related reagents containing the 6-chloro-1-hydroxybenzotriazole moiety can be prepared by reaction with TCFH under basic conditions. It can exist in an N-form (guanidinium) or an O-form (uronium), but the N-form is generally considered to be more stable for this class of reagent. In vivo dermal sensitization studies according to OECD 429 confirmed HCTU is a strong skin sensitizer, showing a response at 0.50 wt% in the Local Lymph Node Assay (LLNA) placing it in Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Dermal Sensitization Category 1A.
TCFH is an electrophilic amidine reagent used to activate a number of functional groups for reaction with nucleophilies. TCFH is most commonly used to activate carboxylic acids for reaction with amines in the context of amide bond formation and peptide synthesis.