Abstract
BackgroundMost in vitro studies investigating nanomaterial pulmonary toxicity poorly correlate to in vivo inhalation studies. Alveolar macrophages (AMs) play an outstanding role during inhalation exposure since they effectively clear the alveoli from particles. This study addresses the applicability of an in vitro alveolar macrophage assay to distinguish biologically active from passive nanomaterials.MethodsRat NR8383 alveolar macrophages were exposed to 18 inorganic nanomaterials, covering AlOOH, BaSO4, CeO2, Fe2O3, TiO2, ZrO2, and ZnO NMs, amorphous SiO2 and graphite nanoplatelets, and two nanosized organic pigments. ZrO2 and amorphous SiO2 were tested without and with surface functionalization. Non-nanosized quartz DQ12 and corundum were used as positive and negative controls, respectively. The test materials were incubated with the cells in protein-free culture medium. Lactate dehydrogenase, glucuronidase, and tumour necrosis factor alpha were assessed after 16 h. In parallel, H2O2 was assessed after 1.5 h. Using the no-observed-adverse-effect concentrations (NOAECs) from available rat short-term inhalation studies (STIS), the test materials were categorized as active (NOAEC < 10 mg/m3) or passive.ResultsIn vitro data reflected the STIS categorization if a particle surface area-based threshold of <6000 mm2/mL was used to determine the biological relevance of the lowest observed significant in vitro effects. Significant effects that were recorded above this threshold were assessed as resulting from test material-unspecific cellular ‘overload’. Test materials were assessed as active if ≥2 of the 4 in vitro parameters undercut this threshold. They were assessed as passive if 0 or 1 parameter was altered. An overall assay accuracy of 95 % was achieved.ConclusionsThe in vitro NR8383 alveolar macrophage assay allows distinguishing active from passive nanomaterials. Thereby, it allows determining whether in vivo short-term inhalation testing is necessary for hazard assessment. Results may also be used to group nanomaterials by biological activity. Further work should aim at validating the assay.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0164-2) contains supplementary material, which is available to authorized users.
Highlights
Most in vitro studies investigating nanomaterial pulmonary toxicity poorly correlate to in vivo inhala‐ tion studies
Particle sizes measured with tracking analysis in H2O were larger than the corresponding primary particle size (PPS)
In the present study, the in vitro NR8383 alveolar macrophage (AM) assay evaluating extracellular release of lactate dehydrogenase (LDH), GLU, tumour necrosis factor α (TNF-α) and H2O2 under standardized conditions using protein-free culture medium proved highly accurate in distinguishing active from passive inorganic NMs or nanosized organic pigments
Summary
Most in vitro studies investigating nanomaterial pulmonary toxicity poorly correlate to in vivo inhala‐ tion studies. To promote the collection of relevant data for the safety assessment of a representative set of NMs, the Organisation for Economic Co-operation and Development Working Party on Manufactured Nanomaterials (OECD WPMN) has launched a Sponsorship Programme for the testing of manufactured nanomaterials [6, 7]. This programme further aims at establishing the role of 3Rs methods for NM testing [6]. NM lung burden and potential for extra-pulmonary translocation may be investigated in the STIS [9,10,11]
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