Assessment of skin sensitization under REACH: A case report on vehicle choice in the LLNA and its crucial role preventing false positive results.
In the EU, chemicals with a production or import volume in quantities of one metric ton per year or more have to be tested for skin sensitizing properties under the REACH regulation. The murine Local Lymph Node Assay (LLNA) and its modifications are widely used to fulfil the data requirement, as it is currently considered the first-choice method for in vivo testing to cover this endpoint. This manuscript describes a case study highlighting the importance of understanding the chemistry of the test material during testing for 'skin sensitization' of MCDA (mixture of 2,4- and 2,6-diamino-methylcyclohexane) with particular focus on the vehicle used. While the BrdU-ELISA modification of the LLNA using acetone/olive oil (AOO) as vehicle revealed expectable positive results. However, the concentration control analysis unexpectedly revealed an instability of MCDA in the vehicle AOO. Further studies on the reactivity showed MCDA to rapidly react with AOO under formation of various imine structures, which might have caused the positive LLNA result. The repetition of the LLNA using propylene glycol (PG) as vehicle did not confirm the positive results of the LLNA using AOO. Finally, a classification of MCDA as skin sensitizer according to the Globally Harmonized System (GHS) was not justified.
Product Safety Department, BASF SE, 67056 Ludwigshafen am Rhein, Germany. Electronic address: nico.watzek@basf.com.
Nico
N
Watzek
Product Safety Department, BASF SE, 67056 Ludwigshafen am Rhein, Germany.
Franz
F
Berger
Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen am Rhein, Germany.
Susanne Noreen
SN
Kolle
Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen am Rhein, Germany.
Tanja
T
Kaufmann
Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen am Rhein, Germany.
Matthias
M
Becker
Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen am Rhein, Germany.
Bennard
B
van Ravenzwaay
D000096:Acetone / Q000737:chemistry; D000485:Allergens / Q000737:chemistry / Q000145:classification / Q000633:toxicity; D000818:Animals; D003514:Cyclohexylamines / Q000737:chemistry / Q000145:classification / Q000633:toxicity; D017449:Dermatitis, Allergic Contact; D005079:Excipients / Q000737:chemistry*; D005260:Female; D006241:Haptens / Q000737:chemistry / Q000145:classification / Q000633:toxicity; D023441:Local Lymph Node Assay; D008808:Mice, Inbred CBA; D000069463:Olive Oil / Q000737:chemistry; D019946:Propylene Glycol / Q000737:chemistry; D012680:Sensitivity and Specificity
D000485:Allergens; D003514:Cyclohexylamines; D005079:Excipients; D006241:Haptens; D000069463:Olive Oil; D000096:Acetone; D019946:Propylene Glycol
AOO; Adverse Outcome Pathway; BrdU-ELISA; False positive results; Local Lymph Node Assay; Skin sensitization; Test substance stability in the vehicle