Multilaboratory evaluation of 15 bioassays for (eco)toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID

Full Te ms & Condi ions o access and use can be ound a h p://www. and online.com/ac ion/jou nalIn o ma ion?jou nalCode=inan20 Nano oxicology ISSN: 1743-5390 (P in ) 1743-5404 (Online) Jou nal homepage: h p://www. and online.com/loi/inan20 Mul ilabo a o y e alua ion o 15 bioassays o (eco) oxici y sc eening and haza d anking o enginee ed nanoma e ials: FP7 p ojec NANOVALID Olesja M. Bonda enko, Ma gi Heinlaan, Ma iliis Sih mäe, Angela I ask, Imbi Ku e , Elise Joonas, Ani a Jemec, Ma ika Manne s öm, Tuula Heinonen, Rohi Rekulapelly, Shashi Singh, Jing Zou, Ilma i Pyykkö, Damjana D obne & Anne Kah u To ci e his a icle: Olesja M. Bonda enko, Ma gi Heinlaan, Ma iliis Sih mäe, Angela I ask, Imbi Ku e , Elise Joonas, Ani a Jemec, Ma ika Manne s öm, Tuula Heinonen, Rohi Rekulapelly, Shashi Singh, Jing Zou, Ilma i Pyykkö, Damjana D obne & Anne Kah u (2016) Mul ilabo a o y e alua ion o 15 bioassays o (eco) oxici y sc eening and haza d anking o enginee ed nanoma e ials: FP7 p ojec NANOVALID, Nano oxicology, 10:9, 1229-1242, DOI: 10.1080/17435390.2016.1196251 To link o his a icle: h ps://doi.o g/10.1080/17435390.2016.1196251 © 2016 The Au ho (s). Published by In o ma UK Limi ed, ading as Taylo & F ancis G oup. View supplemen a y ma e ial Accep ed au ho e sion pos ed online: 03 Jun 2016. Published online: 28 Jun 2016. Submi you a icle o his jou nal A icle iews: 1012 View ela ed a icles View C ossma k da a Ci ing a icles: 11 View ci ing a icles h p://in o maheal hca e.com/nan ISSN: 1743-5390 (p in ), 1743-5404 (elec onic) Nano oxicology, 2016; 10(9): 1229–1242 !2016 The Au ho (s). Published by In o ma UK Limi ed, ading as Taylo & F ancis G oup.. DOI: 10.1080/17435390.2016.1196251 ORIGINAL ARTICLE Mul ilabo a o y e alua ion o 15 bioassays o (eco) oxici y sc eening and haza d anking o enginee ed nanoma e ials: FP7 p ojec NANOVALID Olesja M. Bonda enko 1 , Ma gi Heinlaan 1 , Ma iliis Sih ma ¨e 1 , Angela I ask 1 , Imbi Ku e 1 , Elise Joonas 1,2 , Ani a Jemec 3 , Ma ika Manne s o ¨m 4 , Tuula Heinonen 4 , Rohi Rekulapelly 5 , Shashi Singh 5 , Jing Zou 6 , Ilma i Pyykko ¨ 6 , Damjana D obne 3 , and Anne Kah u 1 1 Labo a o y o En i onmen al Toxicology, Na ional Ins i u e o Chemical Physics and Biophysics, Tallinn, Es onia, 2 Facul y o Science and Technology, Ins i u e o Ecology and Ea h Sciences, Ta u Uni e si y, Ta u, Es onia, 3 Bio echnical Facul y, Uni e si y o Ljubljana, Ljubljana, Slo enia, 4 The Finnish Cen e o Al e na i e Me hods (FICAM), School o Medicine, Uni e si y o Tampe e, Tampe e, Finland, 5 The Cen e o Cellula & Molecula Biology, Habsiguda, Hyde abad, Telangana, India, and 6 Hea ing and Balance Resea ch Uni , Field o O o-La yngology, School o Medicine, Uni e si y o Tampe e, Tampe e, Finland Abs ac Wi hin EU FP7 p ojec NANOVALID, he (eco) oxici y o 7 well-cha ac e ized enginee ed nanoma e ials (NMs) was e alua ed by 15 bioassays in 4 labo a o ies. The highes es ed nominal concen a ion o NMs was 100 mg/l. The panel o he bioassays yielded he ollowing oxici y o de : Ag4ZnO4CuO4TiO 2 4MWCNTs4SiO 2 4Au. Ag, ZnO and CuO p o ed e y oxic in he majo i y o assays, assumingly due o dissolu ion. The la e was suppo ed by he pa allel analysis o he oxici y o espec i e soluble me al sal s. The mos sensi i e es s/species we e Daphnia magna ( owa ds Ag NMs, 24-h EC 50 ¼0.003 mg Ag/l), algae Raphidocelis subcapi a a (ZnO and CuO, 72-h EC 50 ¼0.14 mg Zn/l and 0.7 mg Cu/l, espec i ely) and mu ine ib oblas s BALB/3T3 (CuO, 48-h EC 50 ¼0.7 mg Cu/l). MWCNTs showed oxici y only owa ds a al eola mac ophages (EC 50 ¼15.3 mg/l) assumingly due o high aspec a io and TiO 2 owa ds R. subcapi a a (EC 50 ¼6.8 mg Ti/l) due o agglome a ion o TiO 2 and en apmen o algal cells. Finally, we cons uc ed a decision ee o selec he bioassays o haza d anking o NMs. Fo NM es ing, we ecommend a mul i ophic sui e o 4 in i o (eco) oxici y assays: 48-h D. magna immobiliza ion (OECD202), 72-h R. subcapi a a g ow h inhibi ion (OECD201), 30-min Vib io ische i bioluminescence inhibi ion (ISO2010) and 48-h mu ine ib oblas BALB/3T3 neu al ed up ake in i o (OECD129) ep esen ing c us aceans, algae, bac e ia and mammalian cells, espec i ely. No ably, ou esul s showed ha hese assays, s anda dized o oxici y e alua ion o ‘‘ egula ’’ chemicals, p o ed e icien also o sho lis ing o haza dous NMs. Addi ional assays a e ecommended o immuno oxici y e alua ion o high aspec a io NMs (such as MWCNTs). Abb e ia ions: 3,5-DCP: 3,5-dichlo ophenol; AAS: a omic abso p ion spec oscopy; AFW: a i icial eshwa e ; AO/EB: ac idine o ange/e hidium b omide; BET: B unaue , Emme and Telle ; CNTs: ca bon nano ubes; DI wa e : deionized wa e ; DLS: dynamic ligh sca e ing; DMEM: Dulbecco’s modi ied Eagle’s medium; FBS: e al bo ine se um; CLP: egula ion on Classi ica ion, Labeling and Packaging; hMSC: human bone ma ow-de i ed mesenchymal s em cells; ICP-MS: induc i ely coupled plasma mass spec ome y; QSAR: quan i a i e s uc u e– Keywo ds Me als, mul i ophic es ba e y, physico-chemical cha ac e iza ion, isk assessmen , solubiliza ion His o y Recei ed 25 No embe 2015 Re ised 8 May 2016 Accep ed 17 May 2016 Published online 22 June 2016 This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i a i es License (h p:// c ea i ecommons.o g/Licenses/by-nc-nd/4.0/), which pe mi s non-com- me cial e-use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed, and is no al e ed, ans o med, o buil upon in any way. Co espondence: Olesja M. Bonda enko, Labo a o y o En i onmen al Toxicology, Na ional Ins i u e o Chemical Physics and Biophysics, Akadeemia Tee 23, Tallinn 12618, Es onia. Tel: +372 639 8362. E-mail: [email p o ec ed] Anne Kah u, Labo a o y o En i onmen al Toxicology, Na ional Ins i u e o Chemical Physics and Biophysics, Akadeemia Tee 23, Tallinn 12618, Es onia. Tel: +372 6 398 373. Fax: +372 639 8362. E-mail: anne.kah u@kb i.ee ac i i y ela ionship; LB: Lu ia–Be ani medium; LOEC: lowes obse ed e ec concen a ion; MBC: minimal biocidal concen a ion; MTT: 3-(4,5-Dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide; MWCNTs: mul i-walled ca bon nano ubes; n.a.: no applicable; n.d.: no de e mined; NPs: nanopa icles; NMs: nanoma e ials; NRU: neu al ed up ake; OECD: O ganiza ion o Economic Co-ope a ion and De elopmen ; pdi: polydispe si y index; PI: p opidium iodide; PVP: poly inylpy olidone; REACH: Eu opean Union egula ion on Regis a ion, E alua ion, Au ho iza ion and Res ic ion o Chemicals; ROS: eac i e oxygen species; SDS: sodium dodecyl sul a e; SEM: scanning elec on mic oscopy; TEM: ansmission elec on mic oscopy; SSA: speci ic su ace a ea; WST-1: 2-(4-Iodophenyl)-3-(4-ni ophenyl)-5-(2,4-disul ophenyl)-2H- e a- zolium; XRD: X- ay di ac ion; XPS: X- ay pho oelec on spec oscopy In oduc ion Taking in o accoun he inc easing numbe o nanoma e ials (NMs) en e ing he ma ke , haza d and isk assessmen o each a ian o NMs and all hei possible de i a i es is no ealis ic. The e o e, he need o he haza d anking and g ouping o NMs is widely acknowledged (A s e al., 2014, 2015; Gebel e al., 2014; Godwin e al., 2015). A comp ehensi e o e iew on NM classi ica ion me hods p oposed by di e en au ho i ies and he ini ial decision-making amewo k we e ecen ly published (A s e al., 2015; Godwin e al., 2015). In o de o classi y NMs, eliable oxici y da a and cos -e icien oxici y assays allowing o pinpoin he haza dous NMs a e u gen ly needed. This would allow o ocus on he mechanisms o oxici y o haza dous NMs o di e en o ganisms, depending on he po en ial use and p oduc ion olume o each NM ype. I is equally impo an o deno e non-haza dous NMs ha a e sa e o use o p o- mo e u he de elopmen o nano echnologies. In line wi h he abo e-men ioned aspec s, he e a e cu en ly se e al ongoing discussions on he need o publishing no only hese nanosa e y s udies, whe e oxic e ec s ha e been egis e ed bu also no- e ec s udies as he po en ial isk om NM exposu es is p obably exagge a ed (Gebel e al., 2014; K ug, 2014; Wa hei & Donne , 2015). Recen ly, OECD has come o he conclusion ha he app oaches o he es ing o adi ional chemicals a e in gene al app op ia e o assessing he NM sa e y, bu should be adap ed o he speci ici ies o NMs (OECD, 2012). Cu en ly, di e en conso ia a e in ol ed in he p ocess o adap ing exis ing OECD oxici y es s o NM es ing (Pe e sen e al., 2015). Howe e , in he absence o s anda dized NM-speci ic es s, indus y and he whole socie y would bene i om simple cos -e ec i e in i o sc eening assays o se e as a s a ing poin o ob aining p elimina y haza d in o ma ion. The ea lie he oxic side-e ec s o NMs will be disco e ed, he mo e ime and de elopmen cos s will be sa ed. Indeed, Choi e al. (2009) es ima ed ha he cos s o he es ing o he exis ing nanopa icles (NPs) could ange om $249 million (p esuming he NPs a e in gene al sa e and equi e simple in i o sc eening assays) o $1.18 billion (p esuming he NPs equi e long- e m in i o es ing) and he comple e oxici y es ing would ake 34–53 yea s. This would no only be a hea y inancial bu den bu also an e hical p oblem, because a la ge numbe o animal expe imen s we e in ol ed. Al e na i e me hods a e inc easingly p omo ed o educe o eplace e eb a e animals in (nano) oxicology expe imen a ion (Ha ung, 2010; Kanda o a & Le as ˇio a, 2011). Va ious da a se s ha e al eady been gene a ed and se e al es ing s a egies ha e been p oposed o he in i o sc eening o NMs wi h he emphasis on mechanism-based high- h oughpu app oaches (Fa cal e al., 2015; Geo ge e al., 2011; Godwin e al., 2015; Nel e al., 2013). Mos o hese high- h oughpu mechanis ic s udies ocused mainly on human cells and didn’ conside po en ial en i onmen al haza d o NMs. Al hough he e is also a ela i ely la ge numbe o nano-eco oxici y s udies a ailable ( e iewed by Adam e al., 2015; Bonda enko e al., 2013a; Chen e al., 2015; Coll e al., 2015; Jackson e al., 2013; Juganson e al., 2015; Vale e al., 2016), only a ew single s udies p o ided da a o a wide ange o en i onmen ally ele an o ganisms and enable o e ie e he mos sui able o ganisms and endpoin s o he en i onmen al haza d es ing o NMs. Nume ous EU esea ch conso ia a e cu en ly dedica ed o nanosa e y. The espec i e EU p ojec s a e consolida ed unde he ‘Nanosa e y Clus e ’ ha in ol es a ound 100 p ojec s including wo lagship FP7 p ojec s NANOVALID and MARINA. The main aim o he NANOVALID p ojec (www.nano alid.eu; 2011–2015) was o de elop a se o eliable e e ence me hods and ma e ials o physico-chemical cha ac e iza ion and haza d iden i ica ion o NMs, whe eas he au ho s o he cu en pape ocused on (eco) oxicological sc eening o NMs. Al oge he 15 di e en es o ganisms and cell lines (6 medically impo an bac e ial species, yeas , alga, p o ozoan, 2 c us acean species, zeb a ish and 3 mammalian cell lines in i o) in ol ing a wide a ie y o cells and ecologically ele an o ganisms we e used. The selec ed eco oxicological o ganisms ep esen bo h, pa icle- inges ing (p o ozoa, c us aceans) and p esumably non-inges ing (bac e ia, algae, ish emb yos) species. All hese es o ganisms a e abundan in he e es ial compa men (bac e ia, isopods), was ewa e ea men plan s (bac e ia, p o ozoa) and na u al wa e bodies (algae, p o ozoa, aqua ic c us aceans, ish). In addi ion, his selec ion ep esen s o ganisms om di e en ophic le els including consume s (p o ozoa, c us aceans, ish), p ima y p oduce s (algae) and decompose s (bac e ia). Fo compa ison, we es ed he oxici y o NMs o mammalian cell lines in i o: human bone ma ow-de i ed mesenchymal s em cells, mu ine ib oblas s and a al eola mac ophages. Bac e ial g ow h and bioluminescence inhibi ion, yeas iabil- i y, algal g ow h, c us acean immobiliza ion, zeb a ish emb yos’ mal o ma ion and se e al assays de ec ing he loss o iabili y o cells (ATP p oduc ion, p opidium iodide s aining, neu al ed up ake, MTT and WST-1 educ ion and ac idine o ange/e hidium b omide s aining) we e used as endpoin s o de e mine he (eco) oxici y o NMs. Fi e o he assays we e conduc ed in acco dance o ISO o OECD guidelines (Table 1). The main goals o his esea ch we e (i) o gene a e oxici y da a o 7 physico-chemically well-cha ac e ized NMs using a sui e o 15 bioassays, (ii) o pinpoin he mos sensi i e bioassays and (iii) o sugges (on he basis o a decision- ee) a e ined in i o es ba e y, comp ising o selec ed in i o bioassays, o (eco) oxici y sc eening and ini ial haza d anking o NMs. Ma e ials and me hods S udy design and pa icipa ing ins i u ions Fou pa ne ins i u ions o he EU FP7 p ojec NANOVALID pa icipa ed in he s udy: Na ional Ins i u e o Chemical Physics and Biophysics (NICPB, Es onia), Uni e si y o Ljubljana (UL, Slo enia), The Cen e o Cellula & Molecula Biology (CCMB, 1230 O. M. Bonda enko e al. Nano oxicology, 2016; 10(9): 1229–1242 India) and Uni e si y o Tampe e (UTA, Finland). Expe imen al se -up is schema ically p esen ed in Figu e S1. The pa ne s we e p o ided wi h NMs o he same ba ch. All he pa ne s ollowed he same p ocedu e o he s o age and p epa a ion o he es suspensions, i no s a ed o he wise in he ‘‘Nanoma e ials’’ sec ion. Chemicals All he chemicals we e a leas o analy ical g ade. AgNO 3 was om J.T. Bake ; ZnSO 4 , 3,5-dichlo ophenol, ATP assay eagen and dilu ion bu e , neu al ed solu ion (3.3 g/l in phospha e bu e ed saline), p opidium iodide and ace ic acid we e om Sigma-Ald ich; CuSO 4 *5H 2 O om Al a Aesa ; Vib io ische i Reagen om Aboa ox (Tu ku, Finland); Daphnia magna’s do man eggs we e om Mic oBioTes s Inc (Belgium), ATP s anda d om BioO bi ; Dulbecco’s Modi ied Eagle’s Medium (DMEM) and Kaighn’s Modi ica ion o Ham’s F-12 om ATCC (UTA) o In i ogen (CCMB), e al bo ine se um (FBS) om Gibco In i ogen, Newbo n Cal Se um om Bioch om Ag, WST-1 Cell P oli e a ion Reagen om Roche o Li e Technologies and ac idine o ange and e hidium b omide om Me ck. NMs and hei cha ac e iza ion Se en NMs we e cha ac e ized and es ed: SiO 2 (NNV-002), Ag (NNV-003), Au (NNV-004), MWCNTs (NNV-006), CuO (NNV- 011), ZnO (NNV-012) and TiO 2 (NNV-013). The Ag NMs and MWCNTs es ed in he cu en s udy we e also used in he EU FP7 p ojec MARINA. NMs we e p o ided cen ally as aqueous suspensions (Au and Ag NMs) o as d y powde s (SiO 2 , TiO 2 , MWCNTs, CuO and ZnO). All he NMs we e a leas 99% pu e acco ding o X- ay di ac ion (XRD), X- ay pho oelec on spec oscopy (XPS) and induc i ely coupled plasma mass spec ome y (ICP-MS). Suspensions o Au and Ag NMs we e p o ided in nominal concen a ions o 60 and 40 000 mg/l, espec i ely. The shape, speci ic su ace a ea (acco ding o BET me hodology) and p ima y size (acco ding o ansmission elec on mic oscopy, TEM) we e measu ed by NM p o ide s (Table 2). P epa a ion o NM suspensions All he s ock suspensions o NMs and he espec i e soluble me al sal s (ionic con ols) we e p epa ed on me al basis o acili a e compa ison o hei oxici ies. The s ock suspensions o NMs (excep MWCNTs, Ag and Au NMs) we e p epa ed a nominal concen a ions o 5000 mg me al/l in s e ile deionized (DI) wa e (pH ¼5.8), homogenized using ul asonic p obe immedia ely a e he p epa a ion o he suspensions (40 W, 4 minu es; 450 Ul asoni ie , B anson Ul asonics Co po a ion, Danbu y, CT) a con inuous mode wi hou empe a u e adjus men and le o wo days o equlib a e. S ock suspensions we e s o ed in he da k a oom empe a u e o up o 2 weeks. To ob ain a s able suspension o MWCNTs, 0.01% T i on X-100 was used as a dispe gan . Gene ally, all pa ne s ollowed he same p o ocols o he dispe sion o NMs (40 W, 4 minu es) wi h he ollowing excep ions: (i) CCMB p epa ed he NM s ock suspensions a 1000 mg me al/l in DI wa e and dispe sed using ul asonic p obe (30 W, 10 minu es; SONICS Vib acell); (ii) UTA p epa ed s ock suspension o MWCNTs a 200 mg/l eshly be o e use and ul asonica ed in a wa e ba h sonica o (40 kHz, 2 hou s; B3510, B anson Ul asonics); (iii) UTA p epa ed he s ock suspension o SiO 2 NMs a 2000 mg/l eshly be o e use and ul asonica ed in a wa e ba h sonica o (40 kHz, 5 minu es; B3510, B anson Ul asonics). As Au and Ag NMs we e p o ided as s able suspensions, hey we e used o es ing wi hou p io sonica ion. Cha ac e iza ion o NMs in suspension Hyd odynamic size (measu ed by dynamic ligh sca e ing, DLS), polydispe si y index (pdi) and z-po en ial we e measu ed a a concen a ion o 10 mg me al/l o Au and 100 mg me al/l o he o he NMs using Mal e n Ze asize (Nano-ZS, Mal e n Ins umen s, UK). Dissolu ion o Ag, CuO and ZnO NMs was quan i ied using a omic abso p ion spec oscopy (AAS). Fo ha , NMs a 10 mg/l we e incuba ed in DI wa e o selec ed oxici y es media o 30 min, 4 h, 24 h o 72 h, ul acen i uged (390 000g, 30– 60 min) and he supe na an was analyzed by GF-AAS in acc edi ed labo a o y o he Ins i u e o Chemis y o Tallinn Uni e si y o Technology (Es onia) using EVS-EN ISO/IEC 17025:2005. The a io o de e mined dissol ed me al o o al nom- inal me al in NMs was designa ed as dissolu ion (%) (Table 3). Toxici y assays The e e ences o and he desc ip ion o he used oxici y assays a e consolida ed in Table 1. Due o he la ge numbe o implemen ed di e en oxici y assays and o a oid edundancy, he de ails o he oxici y es ing p ocedu es can be ound in he e e ed a icles and/o he espec i e guideline documen s. All he modi ica ions o hese p e iously published me hods and/o guidelines a e lis ed in Table 1. Tes ing s a egy Due o he la ge es ing ma ix (7 NMs and 15 es species) he maximum nominal NM concen a ion o be es ed was se o 100 mg/l. In he i s ound o es ing he ollowing concen a ions o NMs we e analyzed: 0.1; 1; 10 and 100 mg me al/l o MWCNTs/l. In case o Au NM (concen a ion 60 mg Au/l in he s ock p o ided), lowe maximum concen a ion (30 mg/l) was used. Abio ic con ols we e un in pa allel o e alua e he po en ial in e e ence o he es ed NMs wi h he oxici y endpoin s. In addi ion, ionic con ols ( espec i e soluble me al sal s) we e used in pa allel o es ima e he ole o dissolu ion o NMs in hei oxici y. The es s we e pe o med a leas in duplica es and in wo independen expe imen s. I he oxici y o NM was no obse ed in he i s ound o es ing, he EC 50 was assigned4100 mg me al/l (in case o MWCNTs mg compound/l). I oxici y was obse ed, mo e ho ough es ing (i.e., lowe concen a ions and 2- old dilu ions) was pe o med o de e mine p ecise EC 50 . Dose- esponse cu es and calcula ion o EC 50 EC 50 and con idence limi s we e calcula ed om he concen a- ion-e ec cu es based on nominal exposu e concen a ions and using he log-no mal model o MS Excel mac o Reg ox (Vindimian, 2005; NICPB, CCMB), MS Excel Reg ession analysis (NICPB, UL) o SigmaPlo Reg ession analysis (UTA). The dose- esponse cu es we e based on a leas six concen- a ions and a leas six concen a ions we e used o he eg ession. The con idence limi s and he numbe o eplica es o each assay a e shown in Table S1. Cons uc ing he decision ee o NMs es ing The decision ee was cons uc ed o selec he bioassays o haza d anking o NMs on he basis o ou benchma ks: du a ion and complexi y o he assay, sensi i i y, s anda diza ion s a us and ime needed o aining o he echnician/ esea che . Fo each o hese ou benchma ks 1 o 3 poin s we e sco ed: 1 poin o desc ibe low pe o mance and 3 poin s o high pe o mance. The mos sui able es s we e selec ed on he basis o he highes o al sco es. Rapidi y and ease o pe o mance we e he c i e ia o DOI: 10.1080/17435390.2016.1196251 In i o oxici y sc eening and haza d anking o nanoma e ials 1231 du a ion and complexi y o he es and he sco ing was based on he pa ne s’ expe ience. Toxici y alues (EC 50 ) we e he c i e ia o he sensi i i y o he es . The highes sco e (3) was assigned i he es was he mos sensi i e o wo o mo e NMs and lowes sco e (1) i he es was ne e he mos sensi i e o NMs (Table 4). S anda diza ion o he es was conside ed a quali y c i e ion and he s anda dized es s (Table 1) we e he e o e assigned he highes sco e (3). Time du a ion was he c i e ion o he aining equi ed o mas e execu ion o he es and was based on he pa ne s’ expe ience. The es s wi h he sho es es ima ed equi ed aining ime we e assigned he highes sco e (3). Resul s Cha ac e iza ion o NMs The physico-chemical cha ac e iza ion da a o analyzed NMs a e shown in Table 2. The p ima y size o NMs (TEM) was in he nano-size ange (1–100 nm, acco ding o Eu opean Commission Table 1. Summa y o (eco) oxicological me hods used in his s udy. Tes name Re e ence Modi ica ion(s) compa ed o e e ence (i any) Bac e ial g ow h inhibi ion assay (all bac e ia excep Vib io ische i) Bonda enko e al., 2013b The ini ial op ical densi y (OD 600 ) o bac e ial suspen- sion was OD 600nm ¼0.07. The 96-well pla es we e incuba ed s a ically a 30 C du ing assay and we e shaken once be o e each measu emen . V. ische i bioluminescence inhibi ion assay ISO, 2010; Ku e e al., 2011 The empe a u e was 20 C ins ead o 15 C s a ed in he ISO guideline as mos o he luminome e s can no be adjus ed below oom empe a u e. Yeas iabili y assay (Saccha omyces ce e isiae) Suppi e al., 2015 No modi ica ions. Algal g ow h inhibi ion assay (Raphidocelis subcapi a a) OECD, 2011 No modi ica ions o he guideline. P o ozoan iabili y assay (Te ahymena e mophila) Jemec e al., 2016 No modi ica ions. C us acean acu e immobiliza ion assay (Daphnia magna) Jemec e al., 2016; OECD, 2004 Di e en ly om OECD guideline, he daphnid neona es used in he assays, we e no ob ained om he in- house cul u e bu ha ched om he daphnia do man eggs. Isopod (Po cellio scabe ) memb ane in eg i y assay (AO/EB s aining) Valan & D obne, 2012 The exposu e o 3 h a oom empe a u e ins ead o 18 h a 4 C was used. The es was pe o med on 24-well pla es ins ead o glass ials. Zeb a ish (Danio e io) emb yo oxici y assay Jemec e al., 2016; OECD, 2013 No modi ica ions o he guideline. Human mesenchymal s em cell memb ane in eg i y assay in i o (PI s aining) Zhang e al.,1999 Tes was ini ia ed 24 h a e exposu e o NMs. 488 nm exci a ion/578 nm emission il e s we e used o ead pla es. Human mesenchymal s em cell mi ochond ial ac i i y assay in i o (MTT educ ion) Mosmann, 1983 Tes was ini ia ed 24 h a e exposu e o NMs and incuba ed wi h es eagen o 4 h. A e cell lysis wi h sodium dodecyl sul a e he abso bance was ead a 570 nm. Mu ine ib oblas BALB/c 3T3 memb ane in eg i y assay in i o (NRU) OECD, 2010 No modi ica ions o he guideline. Mu ine ib oblas BALB/c 3T3 mi ochond ial ac i i y assay in i o (WST-1) Zou e al., 2012 No modi ica ions. Ra al eola mac ophage NR8383 mi ochond ial ac i i y assay in i o (WST-1) Zou e al., 2012 Ra al eola mac ophages NR8383 (ATCC, CRL-2192) ins ead o BALB/c 3T3 desc ibed in he e e ence we e used. Cells we e seeded in o 96-well pla es a densi y 12 000 cells/well. AO/EB, ac idine o ange/e hidium b omide; MTT, 3-(4,5-Dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide; NRU, neu al ed up ake; PI, p opidium iodide; WST-1, 2-(4-Iodophenyl)-3-(4-ni ophenyl)-5-(2,4-disul ophenyl)-2H- e azolium. Table 2. Physico-chemical p ope ies o he s udied nanoma e ials (NMs we e 99% pu e). NMs P o ide Coa ing Shape Speci ic su ace a ea [m 2 /g] P ima y size (TEM) [nm] Hyd odynamic size in DI (DLS) [nm] pdi in DI z-po en ial in DI [mV] SiO 2 * Nanologica No Sphe ical 910 ± 42 212 ± 52 854 ± 38 0.6 37.9 Ag Colo obia PVP-s abilized Sphe ical n.d. 20.4 ± 6.8 132 ± 0.5 0.2 10.6 Au** Inme o Ci a e Sphe ical n.d. 13.3 ± 0.8 23 ± 4 0.2 48 MWCNT Nanocyl No Tubula 265 ± 18 diame e : 10.2 ± 0.5 leng h: 41000*** n.a. n.a. n.a. CuO In insiq Ma e ials No Sphe ical 23 ± 3.7 24.5 ± 2.3 152± 2 0.2 45.4 ZnO Nanoga e No Sphe ical 58 ± 5 13.6 ± 1.7 102 ± 1 0.2 32.1 TiO 2 **** CCMB No Sphe ical 257 ± 47 55 367 ± 60 0.4 14.2 *Mesopo ous; **Acidic suspension (pH ¼5.8); ***Measu ed by SEM; ****Ana ase c ys al s uc u e wi h some u ile p esen acco ding o XRD analysis. DI, deionized wa e ; DLS, hyd odynamic ligh sca e ing; n.a., no applicable; PVP, poly inyl-py olidone; pdi, polydispe si y index; SEM, scanning elec on mic oscopy; TEM, ansmission elec on mic oscopy. 1232 O. M. Bonda enko e al. Nano oxicology, 2016; 10(9): 1229–1242 ecommenda ion; EC, 2012) wi h he excep ion o SiO 2 (p ima y size 212 nm). Howe e , SiO 2 had he po es in he nanome e scale ha ansla ed in o la ge speci ic su ace a ea o 910 m 2 /g (Table 2). As one o he de ini ions o ‘‘nano’’ e e s o he la ge speci ic su ace a ea a he han he small p ima y size (NM is de ined as he ma e ial wi h 60 m 2 /cm 3 olume-speci ic su ace a ea acco ding K eyling e al. 2010), we conside ed SiO 2 as NM. The suspensions o Ag, Au, CuO and ZnO NMs we e s able (pdi ¼0.2; Table 2) in DI wa e . Au NMs had he smalles hyd odynamic size in DI wa e (23 nm), ollowed by ZnO (102 nm), Ag (132 nm) and CuO (152 nm), whe eas SiO 2 and TiO 2 o med isible agglome a es wi h hyd odynamic size o 367 nm (TiO 2 ) and 854 (SiO 2 ) and pdi 0.4. Howe e , all he NMs agglome a ed in he ac ual es media o a ious deg ees, depending on he es media used (Table S2). The su ace cha ges (-po en ial) o NMs in DI wa e anged om 37.9 mV (SiO 2 ) o 45.4 mV (CuO) (Table 2). Howe e , in he ac ual es media all he NMs gained mo e compa able, mos ly sligh ly nega i e, z-po en ials (Figu e 1). Simila phenomenon was obse ed in ou p e ious s udy, whe e z-po en ial o 11 di e en me al oxides a ied om 30 mV o 30 mV in DI wa e bu was compa able in se um-supplemen ed cell cul u e medium (close o he z-po en ial o his es medium, 8.5 mV) (I ask e al., 2015). This may explain mo e p ominen agglome a ion o NMs in he ac ual es media compa ed o DI wa e (Table S2) since he z-po en ial highe han +30 mV o lowe han 30 mV is usually equi ed o main ain he NM dispe sed by cha ge s abiliza ion (Hi chman e al., 2013). Ou dissolu ion analysis (i.e., po en ial o me al-based NMs o elease soluble me al ions) showed ha h ee NMs (Ag, CuO and ZnO) we e pa ly dissol ing in DI wa e . The dissolu ion o CuO, ZnO and Ag a concen a ion 10 mg me al/l a e 24-h incuba ion in DI wa e was 9.2%, 34% and 49.8%, espec i ely (Table 3). In addi ion o DI wa e , dissolu ion was analyzed in ou es media ha we e used in oxici y assays which p o ed he mos sensi i e (lowes EC 50 , Table 4). Rema kably, he dissolu ion o Ag NMs was e y low in all he es media compa ed o DI wa e (Table 3). A he same ime, he dissolu ion o CuO and ZnO NMs was enhanced in BALB/c 3T3 cell cul u e medium (se um-supplemen ed DMEM) compa ed o he o he es media (mine al media wi hou se um), p obably due o se um con en . Toxici y alues and c i e ia o haza d anking o NMs The oxici y o 7 NMs (SiO 2 , TiO 2 , Au, Ag, CuO, ZnO and MWCNTs) was analyzed using 15 bioassays. The highes es ed nominal concen a ion was 100 mg/l. Rep esen a i e dose- esponse cu es on he example o CuO NMs a e shown in Figu e S2. EC 50 alues (exp essed in nominal concen a ions on me al basis) we e calcula ed om he dose- esponse cu es and a e colo -coded in Table 4 o o m a oxici y hea map. Fo he assays wi h yeas S. ce e isiae and c us acean P. scabe he minimal biocidal concen a ion (MBC) and he lowes obse ed e ec concen a ions (LOEC), espec i ely, we e used ins ead o EC 50 . In addi ion, Table 4 con ains oxici y da a o he soluble sal s o Ag, Cu and Zn o es ima e he ole o dissolu ion in oxici y o NMs ha a e p one o solubiliza ion (Ag, CuO and ZnO, espec i ely). Pa allel analysis o solubiliza ion p oduc s o NMs is also ecommended by he Eu opean esea ch ne wo k (Handy e al., 2012). Haza d anking o NMs and soluble me al sal s was pe o med as in ou p e ious s udies (Bonda enko e al., 2013a; Kah u & Dubou guie , 2010): EC 50 alue51 mg/l anked NMs as e y oxic; 1–10 mg/l ¼ oxic;410–100 mg/ Figu e 1. Su ace cha ge (z-po en ial) o i e nanoma e ials in i e di e en es media analyzed a concen a ion 100 mg me al/l using Mal e n Ze asize . Table 3. Dissolu ion (sha e o dissol ed me al o o al me al) o Ag, CuO and ZnO nanoma e ials was de e mined by a omic abso p ion spec oscopy in supe na an s o ul acen i uged 10 mg me al/l NM suspensions a e 30-min, 24-h, 48-h and 72-h incuba ion in deionized wa e (DI wa e ) o es media. DI wa e (yeas , p o ozoan), 24-h dissolu ion, % 2% NaCl (Vib io ische i), 30-min dissolu ion, % OECD202 (Daphnia magna), 24-h dissolu ion, % OECD129 (BALB/c 3T3 cells), 48-h dissolu ion, % OECD201 (alga), 72-h dissolu ion, % Ag 49.8 0.12 0.5 4.7 0.35 CuO 9.2 8.3 1 61.1 0.7 ZnO 34 17.7 28.7 100 13.2 DOI: 10.1080/17435390.2016.1196251 In i o oxici y sc eening and haza d anking o nanoma e ials 1233 Table 4. Toxici y (EC 50 , MBC o LOEC) alues o nanoma e ials exp essed in a hea map. Tes s models Time, medium, endpoin SiO2TiO2Au MWCNT Ag CuO ZnO AgNO3CuSO4ZnSO4 BACTERIA: Esche ichia coli 4 h EC50, LB, g ow h inhibi ion >100 >100 >10 n.d 3,1 >100 67 1,3 >100 52,3 S aphylococcus au eus 4 h EC50, LB, g ow h inhibi ion >100 >100 >10 n.d 5,2 >100 16 2,2 >100 15,5 Bacillus sub ilis 4 h EC50, LB, g ow h inhibi ion >100 >100 >10 n.d 4,5 >100 14 3,0 >100 16,0 Pseudomonas pu ida 4 h EC50, LB, g ow h inhibi ion >100 >100 >10 n.d 3,8 >100 69 2,0 >100 49,5 Pseudomonas ae uginosa 4 h EC50, LB, g ow h inhibi ion >100 >100 >10 n.d 3,2 >100 >100 2,1 >100 >100 Vib io ische i 0.5 h EC50, 2% NaCl, bioluminescence inhibi ion >100 >100 4,8*n.d 2,9 3,4 8,3 1,4 0,3 7,7 YEAST: Saccha omyces ce e isiae 24 h MBC, deionized wa e , iabili y n.d >100 >10 >100 1 >100 20 14>100 ALGA: Raphidocelis subcapi a a 72 h EC50, OECD201 medium, g ow h inhibi ion 83,6 6,8 n.d. n.d. 0.0086** 0,7 0,14 0.0071** 0,011 0.042*** PROTOZOAN: Te ahymena he mophila 24 h EC50, deionized wa e , iabili y >100 >100 >30 >100 3,9 >100 3,9 2,9 n.d. n.d. human mesenchymal s em cells 24 h EC50, comple e medium, memb ane in eg i y (PI) >100 >100 >30 100 6,4 2,2 1,9 n.d. 2,73 1,7 human mesenchymal s em cells 24 h EC50, comple e medium, mi ochond ial ac i i y (MTT) >100 >100 >30 n.d 4,6 >100 16,3 n.d. >100 48 mu ine ib oblas s BALB/c 3T3 24 h, 50% comple e medium, mi ochond ial ac i i y (WST-1) >100 >100 >6 >100 3**** 0,7 9,1 2 2,9 8,7 mu ine ib oblas s BALB/c 3T3 48 h EC50, 50% comple e medium, memb ane in eg i y (NRU) >100 >100 >6 >100 2.8**** 1 6,9 2 6,1 5,3 a mac ophages NR8383 24 h EC50, 50% comple e medium, mi ochond ial ac i i y (WST-1) >100 >100 >6 15,3 9,8 n.d n.d n.d. n.d. n.d. CRUSTACEAN: Daphnia magna 48 h EC50, a i icial eshwa e , immobiliza ion >100 >100 >30 >100 0,003 0,9 1,87 0,001 0,05 1,55 CRUSTACEAN: Po cellio scabe 3 h LOEC, 0.9% NaCl, memb ane in eg i y (AO/EB) n.d >100 n.d n.d 10 100 >100 n.d. 50 >100 FISH: zeb a ish (Danio e io) emb yo 96 h EC50, a i icial eshwa e , le hal mal o ma ions n.d >100 >30 n.d 3 1,6 n.d n.d. 0,35 n.d. 83,6 6,8 4,8 15,3 0,003 0,7 0,14 0,001 0,05 0,042 Vib io ische i a mac ophages D. magna mu ine ib oblas s and R. subcapi a a R. subcapi a a D. magna Colou codes: Lowes EC50: Mos sensi i e es : R. subcapi a a R. subcapi a a NANOMATERIALS SOLUBLE METAL SALTS EC50, MBC o LOEC (mg me al/l o mg MWCNTs/l) l lec e l gnis ,se oy ako P llec elgnis ,se oy a kuE o i ni s e u l u c ll e c na il amm aM smsinag o al u lle c i l u M <1 mg/l = e y oxic >10-100 mg/l = ha m ul >100 mg/l = no classi ied/no ha m ul 1-10 mg/l = oxic *The oxici y o Au NMs o V. ische i was due o acidic pH; **Jemec e al., 2016; ***A uoja e al., 2009; ****Zou e al., 2012. AO/EB, ac idine o ange/e hidium b omide; LB, Lu ia–Be ani; LOEC, lowes obse ed e ec concen a ion; MBC, minimal bac e icidal concen a ion; n.d, no de e mined; PI, p opidium iodide. 1234 O. M. Bonda enko e al. Nano oxicology, 2016; 10(9): 1229–1242 l¼ha m ul;4100 mg/L ¼no classi ied/no ha m ul. MBC (yeas iabili y assay) and LOEC (isopod memb ane in eg i y assay) alues we e anked on simila basis as EC 50 alues. Ve y oxic NMs: Ag, CuO and ZnO Ag, CuO and ZnO we e anked as e y oxic (EC 50 alue51 mg/ l). Thus, hese h ee NMs belong o he Ca ego y Acu e 1 acco ding o EU’s egula ion on classi ica ion, labeling and packaging (CLP) o subs ances and mix u es (EC, 2008) based on hei oxici y o ish (96 h), daphnids (48 h) and algae (72 o 96 h). Indeed, Ag was he mos oxic NM es ed. C us acean D. magna and alga R. subcapi a a ( o me ly known as Selenas um cap ico nu um and Pseudoki chne iella subcapi a) we e mos sensi i e o Ag NMs (24-h EC 50 ¼0.003 and 72-h EC 50 ¼0.0086 mg Ag/l, espec i ely). Thus, he oxici y es s wi h D. magna and R. subcapi a a lagged he oxici y o Ag NMs al eady a mg/l le els, being up o h ee o de s o magni ude mo e sensi i e han all he o he assays used. This demons a es he haza d o Ag NMs o aqua ic li e, i leaching om he consume p oduc s o he wa e bodies e en a e y low concen a ions. In addi ion o high oxici y o Ag NMs o aqua ic c us aceans and alga, he haza dous na u e o Ag NMs was also demons a ed by zeb a ish emb yo oxici y assay (96-h EC 50 ¼3 mg Ag/l). A 10 mg Ag/l, Ag NMs comple ely inhibi ed he ha ching o ish emb yos (Figu e 2). In e es ingly, he oxici y o Ag NMs o bo h mammalian cells in i o and a ious bac e ial cells was obse ed a he same concen a ion ange (3–10 mg/l, Table 4), indica ing he highe oxici y o Ag NMs o non- a ge o ganisms such as c us aceans han o a ge o ganisms (mic obes) (Bonda enko e al., 2013a). In line wi h ou esul s he e a e se e al ecen mul ispecies s udies and nume ous e iews showing po en ial eco oxici y o Ag NMs (Coll e al., 2015; Ga ne e al., 2015; Kwak e al., 2016; Vale e al., 2016). All hese s udies suppo he commen a y ha ‘‘ he Eu opean Commission should be egula - ing nanosil e , no asking o ye ano he epo on i s impac on heal h and he en i onmen ’’ (Hansen & Baun, 2012). ZnO was he second mos oxic NM being especially oxic in acu e assays wi h he aqua ic o ganisms (R. subcapi a a,D. magna,T. he mophila and V. ische i). As in case o Ag NMs, D. magna and especially R. subcapi a a, we e he mos suscep- ible o ZnO NMs (48-h EC 50 ¼1.87 and 72-h EC 50 ¼0.14 mg Zn/l, espec i ely, Table 4). This is in ag eemen wi h he ecen comp ehensi e s udy o Adam e al. (2015) showing ha algae and c us aceans we e mo e sensi i e o ZnO NMs han o he aqua ic o ganisms. The oxici y o ZnO NMs o mammalian cell lines in i o was sligh ly lowe (1.9–16.3 mg Zn/l) depending on he cells used. These esul s a e in ag eemen wi h p e ious epo s by Fa cal e al. (2015) and I ask e al. (2015) who showed using a ious endpoin s ha o mos o he mammalian cell lines EC 50 o ZnO was in he ange o 10–30 mg/l. The oxici y o CuO NMs anged om 0.7 mg Cu/l (mu ine ib oblas s and R. subcapi a a) o4100 mg Cu/l ( a ious bac e ia) depending on es o ganism, oxici y endpoin and es medium used (Table 4). I has been p e iously shown ha he es medium and especially he p esence o o ganic compounds signi ican ly modi ies he oxici y o CuO and specia ion o coppe (Blino a e al., 2010; Ka ¨kinen e al., 2011). Howe e , in some es s wi h unicellula o ganisms (e.g., p o ozoa and yeas ) CuO was no oxic despi e o he use o DI wa e ( ha does no complex Cu ions no induces agglome a ion o NMs) as an exposu e en i onmen . This sugges s ha some o ganisms migh be inhe en ly mo e esis an o CuO. Ala mingly, assays wi h mu ine ib oblas s BALB/3T3 showed he lowes EC 50 alues o CuO, demons a ing ha human cell lines in i o a e highly suscep ible o his NM. We ha e p e iously sugges ed ha di e en ly om mos o he o he cell ypes and o ganisms, mammalian cells migh be suscep ible also o pa icle-speci ic ad e se e ec s o CuO NMs (Bonda enko e al., 2013b) ha is p obably ela ed o in acellula dissolu ion o in e nalized CuO pa icles in he acidic en i onmen o lysosomes (T ojan ho se mechanism; C onholm e al., 2013). High sensi i i y o mamma- lian cells o CuO NMs obse ed in his s udy suppo s his hypo hesis. No ably, he oxici y pa e ns ( oxici y o di e en o ganisms) o dissolu ion-p one NMs (Ag, CuO and ZnO) and he espec i e me al sal s we e almos iden ical (Table 4), sugges ing ha he oxici y o hese NMs was due o dissolu ion. Toxic NMs: TiO 2 TiO 2 exhibi ed oxici y only in he assay wi h algae R. subcapi a a (72-h EC 50 ¼6.8 mg Ti/l). The isual obse a ion o TiO 2 - exposed algae showed ha mos likely he inhibi ion o algal g ow h was due ‘‘en apmen ’’ o he algal cells in o TiO 2 NM agglome a es (Figu e 3c,d). No such e ec was obse ed in case o SiO 2 NMs-exposed algae (Figu e 3a,b), al hough he su ace cha ges o hese wo NMs (17 mV o SiO 2 and 20 mV o TiO 2 , Figu e 1) as well as hyd odynamic sizes (575 nm and 478 nm, espec i ely, Table S2) in he algal es medium we e Figu e 2. Shema ic ep esen a ion o emb y- onic de elopmen (0.2 h and 72 h a e shown) o zeb a ish (Kimmel e al., 1995, ep in ed wi h he pe mission o John Wiley and Sons) (a). Rep esen a i e images o de elopmen o unexposed zeb a ish emb yo (con ol) o exposed o 100 mg/l TiO 2 NMs o o 10 mg/l Ag NMs a e 72 h (b). Red a ow indica e unde eloped emb yo exposed o Ag NMs. Scale ba ¼1 mm. DOI: 10.1080/17435390.2016.1196251 In i o oxici y sc eening and haza d anking o nanoma e ials 1235 Table 5. Ad an ages and limi a ions o he selec ed es assays based on he expe ience gained by he pa ne s o he NANOVALID p ojec . Tes , o ganism Model o Ad an ages Limi a ions Bac e ial g ow h inhibi ion assay (Esche ichia coli, S aphylococcus au eus, Pseudomonas sp. Bacillus sub ilis) Medically impo an pa hogenic bac e ia; En i onmen ally ele an bac e ia Simple and apid es s showing whe he he es ed NMs exhibi gene al oxici y o he gi en bac e ial s ains. G ow h inhibi ion assays a e conduc ed in o ganics- ich media ha may so b o in e e e wi h NMs (agglome a ion/dissolu ion e c). Vib io ische i bioluminescence inhibi ion assay Ma ine bac e ia The assay wi h V. ische i is apid and eliable s anda dized assay (ISO, 2010). Tes is sensi i e o pH ( he es medium is no bu e ed). High salini y (2% NaCl). Yeas (Saccha omyces ce e isiae) iabili y assay The simplies model o euka yo ic cell The use o DI wa e as he es en i onemn elimina es he specia ion- ela ed e ec s o me allic NMs. DI wa e is no a na u al en i onmen and may igge osmo ic s ess in cells. This may change he suscep ibili y o cells o NMs. Algal (Raphidocelis subcapi a a) g ow h inhib- i ion assay En i onmen ally ele an eshwa e p i- ma y p oduce Algal g ow h inhibi ion assay (OECD, 2011) is among he mos sensi i e eco oxicological es s. NMs may in e e e wi h op ical densi y meas- u emen s and shade he ligh necessa y o he g ow h o algae. P o ozoan (Te ahymena he mophila) iabili y assay En i onmen ally ele an euka yo ic o ganism T. he mophila can in e nalize NMs by phagocy osis. DI wa e is no a na u al en i onmen and may igge osmo ic s ess in cells. This may change he suscep ibili y o cells o NMs. C us acean (Daphnia magna) acu e immobiliza- ion assay En i onmen ally ele an eshwa e o ganism One o he mos sensi i e o ganisms used in eco oxici y assessmen o chemicals and D. magna es (OECD, 2004) is among he assays ha a e equi ed by he REACH guidelines o he eco oxici y assessmen . Pa icle- eeding o ganism and hus, e y ele an model o NMs. Tes medium may induce agglome a ion and p ecipi a ion o NMs. High sample olume (50 ml pe concen a ion in one expe imen ) is equi ed. Isopod (Po cellio scabe ) cell memb ane in eg- i y assay (AO/EB) En i onmen ally ele an e es ial o ganism Isola ed diges i e gland is he model o issue cell memb ane pe meabili y. The es p o ides e y speci ic da a on he cell memb ane s abili y. Accu a e aining o s a is equi ed. The me hod is no obus . The physiological s a e o he gland need o be p ecisely desc ibed. Zeb a ish (Danio e io) emb yo oxici y assay En i onmen ally ele an eshwa e o ganism The es is obus and highly aluable o NM es ing in aqua ic en i onmen . Fish emb yos a e anspa en allowing di ec obse a ion o mal o ma ions. The se -up o ea ing he zeb a ish emb yos is echnically challenging. Human mesenchymal s em cell memb ane in eg i y (PI) and mi ochond ial ac i i y (MTT) assays Non-cance ous p ima y de i ed human cells Tes endpoin s a e based on undamen al cellula p ocesses, i.e., mi ochond ial ac i i y (MTT, WST-1) and memb ane in eg i y (NRU and PI). NRU assay wi h mu ine ib oblas s is s anda - dized oxicological assay o chemical es ing (OECD, 2010). Some NMs may in e e e wi h he luo escence and abso bance-based iabili y assays. Mu ine ib oblas BALB/c 3T3 memb ane in eg i y (NRU) and mi ochond ial ac i i y (WST-1) assays Non-cance ous mammalian cell line Ra al eola mac ophage NR8383 mi ochond ial ac i i y assay (WST-1) Non-cance ous mammalian mac ophages Does no equi e washing a e exposu e p io o he assay, and is he e o e sui able o sus- pension cells. Ve y as and easy o pe o m. Some NMs may in e e e wi h he assays. AO/EB: ac idine o ange/e hidium b omide; NRU: neu al ed up ake; PI: p opidium iodide; WST-1: 2-(4-iodophenyl)-3-(4-ni ophenyl)-5-(2,4-disul ophenyl)-2H- e azolium. 1236 O. M. Bonda enko e al. Nano oxicology, 2016; 10(9): 1229–1242