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Background  Most 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.    Methods  Rat NR8383 alveolar macrophages were exposed to 18 inorganic nanomaterials, covering AlOOH, BaSO 4 , CeO 2 , Fe 2 O 3 , TiO 2 , ZrO 2 , and ZnO NMs, amorphous SiO 2  and graphite nanoplatelets, and two nanosized organic pigments. ZrO 2  and amorphous SiO 2  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, H 2 O 2  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/m 3 ) or passive.    Results   In vitro  data reflected the STIS categorization if a particle surface area-based threshold of <6000 mm 2 /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.    Conclusions  The 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 material  The online version of this article (doi:10.1186/s12951-016-0164-2) contains supplementary material, which is available to authorized users.

An in vitro alveolar macrophage assay for predicting the short-term inhalation toxicity of nanomaterials