Dose addition in chemical mixtures inducing craniofacial malformations in

A challenge in cumulative risk assessment is to model hazard of mixtures. EFSA proposed to only combine

chemicals linked to a defined endpoint, in so-called cumulative assessment groups, and use the dose-addition

model as a default to predict combined effects. We investigated the effect of binary mixtures of compounds

known to cause craniofacial malformations, by assessing the effect in the head skeleton (M-PQ angle) in 120hpf

zebrafish embryos. We combined chemicals with similar mode of action (MOA), i.e. the triazoles cyproconazole,

triadimefon and flusilazole; next, reference compounds cyproconazole or triadimefon were combined with

dissimilar acting compounds, TCDD, thiram, VPA, prochloraz, fenpropimorph, PFOS, or endosulfan. These

mixtures were designed as (near) equipotent combinations of the contributing compounds, in a range of cumulative

concentrations. Dose-addition was assessed by evaluation of the overlap of responses of each of the 14

tested binary mixtures with those of the single compounds. All 10 test compounds induced an increase of the MPQ

angle, with varying potency and specificity. Mixture responses as predicted by dose-addition did not deviate

from the observed responses, supporting dose-addition as a valid assumption for mixture risk assessment.

Importantly, dose-addition was found irrespective of MOA of contributing chemicals.