Battistoni M , Bacchetta R , Di Renzo F , Metruccio F , Menegola E .

	graphical abstract
	Fig. 1. Morphological appearance of NF stage 46 larvae before (A-B-C-D) and after fixation and acridine orange staining according to Zucker et al. [51] (A’-B’-C’-D’). Magnification 20X (A-B-C-D); magnification 32X (A’-B’-C’-D’). A: lateral view of a control larva with normal phenotype. Note the dorsal neural tube (NT), the well-expanded gill basket (GB), the normal eye (*), the intestine (I) and the oral opening (arrow). A': frontal view of a larva with normal phenotype stained with acridine orange. The naris (o) are well distinguishable, as well as the well-expanded oral cavity (‘’). B: lateral view of a NANO β-carotene 3 ng/mL larva with malformed phenotype with round-shape head (NT). B': frontal view of a malformed larva with round-shape head stained with acridine orange. The nasal pits (o) are well formed but in a different inclination due to the round-shape head. The oral cavity present marked reduction accompanied by oedema (arrow). C: lateral view of a larva exposed to NANO β-carotene 7,5 ng/mL with severely malformed phenotype with round-shape and coerced head. Note the extremely reduced oral cavity (>), the abnormal shape of the eye (<<) and the presence of oedema in the cardiac region (*). C': frontal view of a severely malformed larva with round-shape and coerced head stained with acridine orange. Note the nasal pits barely visible (o), the abnormal eyes (<<) and the altered oral cavity (arrow). D: lateral view of a larva exposed to NANO β-carotene 30 ng/mL with extremely malformed phenotype with funnel shaped oral cavity (>). Note the not linear and swollen neural tube (NT), the abnormal eye with half-moon shape (<<) and the oedema in the cardiac region (*). D': frontal view of an extremely malformed larva with funnel shaped oral cavity stained with acridine orange. Note the irregular nasal pits (o), the half-moon shape eye (>>) and the oral cavity, almost a small split (arrow).
	Fig. 2. Evaluation of the benchmark doses (BMDs) for Benchmark-response (BMR) at 50 % of the different categories (malformations, severe malformations and extreme malformations) observed in larvae exposed to NANO β-carotene. Malformations: continuous dose-response curve; Severe malformations: dashed dose-response curve; Extreme malformations: dotted dose-response curve.
	Fig. 3. Evaluation of the relative potency factor (RPF) of the NANO formulation (dose-response curve with crosses) in respect to the BULK form (dose-response curve with triangles) of β-carotene. RPF was calculated at the effective concentration at 50 % (EC50). RPF of the nano-encapsulated β-carotene was 6*10−4 (BMD modelling by using PROAST software).
	Fig. 4. Frontal histological sections of NF stage 46 control and exposed larvae. A: Dorsal view of a control larva with normal phenotype. Note the well-expanded ethmoid cartilage (E) in the anterior portion of the head and laterally to the olfactory mucosa (o). The eyes (*) and the well-developed intestine (I) are visible. Magnification 25 × .A': Detail of the eye: note the pigmented layer (*) and the photoreceptor, bipolar cell and multipolar layers of the retina (>) Magnification 400 × . B: Dorsal view of a larva exposed to NANO β-carotene 3 ng/mL with malformed phenotype. Note the anterior schisis (black arrow) and the moderate oedema in the mesenchyme of the oral cavity (*). Magnification 25 × .B': Detail of the eye: note the slight folding at the level of the layers of the retina (<). Magnification 100 × . C: Dorsal view of a larva exposed to NANO β-carotene 7,5 ng/mL with severely malformed phenotype. Note the anterior schisis (§) and the opened branchial basket (*). Magnification 25 × .C': Detail of the eye: note the prominent folding of different layers of the retina (>>) and the abnormal shape of the crystalline (arrow). Magnification 100 × . D: Dorsal view of a larva exposed to NANO β-carotene 7,5 ng/mL with extremely malformed phenotype. Note the ethmoid cartilage with elongated and flattened shape (E) and the oedematous mesenchyme (*). Magnification 25 × . D': Detail of the anterior portion: note the disorganized tissues at the level of the nasal pits (>) and at the eye level with no centrally positioned crystalline (§) and very folded retinal layer separated by the pigmented layer (#). Magnification 100 × .
	Fig. 5. Morphological appearance of a NF stage 37 control larva before (A) and after fixation and acridine orange staining according to Zucker et al. [51] (B, C), with the corresponding histological sections (D, D’, E, E’). A: Lateral view of a control larva with normal phenotype. Note the well-developed eye (*). Magnification 10 × . B: Lateral view of a control larva with normal phenotype stained with acridine orange. Note the eye pigment circularly positioned and the centrally positioned crystalline (*).Magnification 40 × . C: Frontal view of a control larva with normal phenotype stained with acridine orange. Note the round-opened stomodeum (s) and the forebrain bulges (t). Magnification 40 × . D: Dorsal section of a control larva. Note the optical nerve (>) and the eye structure with centrally positioned crystalline and retinal layer already differentiated (*). Magnification 100 × . D': Detail of the eye. Note the crystalline with different layers (*) and the regularly organized retinal layers (>). Magnification 400 × .E: Ventral section of a control larva. Note the well-introflexed stomodeum (>>). Magnification 100 × .E': Detail of E. Note the stomodeal ectoderm (black >) and the pharyngeal endoderm (red >) which are about to merge. Magnification 400 × .
	Fig. 6. Morphological appearance of NF stage 37 larvae exposed to NANO β-carotene before (A) and after fixation and acridine orange staining according to Zucker et al. [51] (B, C), with the corresponding histological sections (D, D’, E, E’). A: Lateral view of a larva exposed to NANO β-carotene 15 ng/mL with alteration at the cephalic portion. Note the half-shape of the eye (>). Magnification 10 ×  .B: Lateral view of a larva with alteration at the cephalic portion stained with acridine orange. Note the pigmented layers in the upper side of the eye (>). It is not possible to note the crystalline. Magnification 40 × . C: Frontal view of a larva with alteration at the cephalic portion stained with acridine orange. Note the narrowed stomodeum (<). It is not possible to note the forebrain bulges. Magnification 40 × . D: Dorsal section of a larva exposed to NANO β-carotene 15 ng/mL. Note the compromised eye structure with reduced crystalline (>) and with over folded retinal layers (arrow). Magnification 100 × .D': Detail of the eye. Note the reduced crystalline (>) and the disorganized layers of the retina (*). Magnification 400 × . E: Ventral section of a larva exposed to NANO β-carotene 30 ng/mL. Note the reduced stomodeum (>>). Magnification 100 × .E': Detail of the reduced stomodeum (>). Magnification 400 × .
	Fig. 7. X. laevis embryos at NF stage 26 immunostained with antibody anti-CRABP (C-D'). Magnification: 25X, 40 × .A, A’: X. laevis control embryo. Note the immunostained areas at the level of the oral cavity (o), of the frontal region (§), of the otic vesicle (>), of the optic vesicle (*), and at the level of the branchial arches with separate neural crest migratory flows (>>). B, B': X. laevis embryos exposed to NANO-β carotene 30 ng/mL. Note the altered migration of the neural crest cells: the migration streams appear fused forming a ventrally indistinct immunostained mass with fusion regions (<<).

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