1、BSI Standards PublicationPD ISO/TS 19337:2016Nanotechnologies Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicityPD ISO/TS 19337:2016 PUBLISHED DOCUMENTNational forewordThis Published Document is the UK implementation of ISO/TS 19337:2
2、016.The UK participation in its preparation was entrusted to Technical Committee NTI/1, Nanotechnologies.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users ar
3、e responsible for its correct application. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 90824 8 ICS 07.030 Compliance with a British Standard cannot confer immunity from legal obligations.This Published Document was published under the authority of the
4、 Standards Policy and Strategy Committee on 30 April 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dPD ISO/TS 19337:2016 ISO 2016Nanotechnologies Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicityNanote
5、chnologies Caracteristiques des suspensions de nano-objets utilises pour les tests in vitro valuant la toxicit inherente aux nano-objetsTECHNICAL SPECIFICATIONISO/TS19337Reference numberISO/TS 19337:2016(E)First edition2016-03-15PD ISO/TS 19337:2016ISO/TS 19337:2016(E)ii ISO 2016 All rights reserved
6、COPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet
7、, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.
8、orgPD ISO/TS 19337:2016ISO/TS 19337:2016(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Abbreviated terms 25 Characteristics and measurement methods 25.1 General . 25.2 Endotoxin . 25.3 Stability of working suspensions 25.3.1 General 25.3.2 Representative
9、size change of secondary particles of nano-objects . 35.3.3 Concentration change of nano-objects . 35.4 Concentration of metal ions. 35.5 Concentration of culture medium components 35.5.1 General 35.5.2 Proteins 45.5.3 Calcium . 46 Reporting 46.1 General . 46.2 Name of nano-objects and manufacturer
10、46.3 Metallic elements included in the nano-object sample 46.4 Culture medium and serum . 46.5 Measurement results . 46.6 Deviation 5Annex A (informative) Flow of measurements. 6Annex B (informative) Measurement and evaluation of stability 7Annex C (informative) Measurement of metal ions. 8Annex D (
11、informative) Measurement of culture medium components 9Bibliography .10 ISO 2016 All rights reserved iiiContents PagePD ISO/TS 19337:2016ISO/TS 19337:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). Th
12、e work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-gov
13、ernmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described i
14、n the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives). Attention is drawn to the
15、possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO li
16、st of patent declarations received (see www.iso.org/patents). Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as wel
17、l as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 229, Nanotechnologies.iv ISO 2016 All rights reservedPD ISO/TS 19337:2016ISO/TS 19337:
18、2016(E)IntroductionBefore nano-objects enter into the market, their possible impact on human health and the environment needs to be carefully evaluated.In vitro toxicity assays using cultured cells are frequently used as a tool in screening hazardous materials. This testing provides essential inform
19、ation for understanding the mechanisms of biological effects induced by the materials. However, nano-objects require specific considerations with respect to the in vitro toxicity assays, because their behaviour is distinct from water soluble chemicals. For example, immediately after the introduction
20、 of nano-object samples into the culture medium, the nano-objects undergo changes, such as (a) dissolution, which is the dissolving of nano-objects into their ionic counterparts, (b) corona formation, which is the adsorption of the components of culture medium onto the nano-object surface, or (c) ch
21、anges in aggregation/agglomeration state, leading to alteration in particles size and sedimentation. Therefore, it is critical to consider the aforementioned phenomena in clarifying if the observed effects are related to the tested nano-object itself or from other uncontrolled sources and to avoid f
22、alse interpretation of assay results.The rigorous characterization of the working suspension prior and during in vitro toxicity assays is essential to exclude the in vitro experimental artefacts. For example, the corona formation, metal ion release from the nano-objects and impurities (residual from
23、 the nano-object synthesis process) can interfere with some in vitro assays,1producing inaccurate results. Additionally, the formation of agglomerates and aggregates can alter the toxicity of a suspension. Therefore, it is important to carefully assess and describe the characteristics of the suspens
24、ion of nano-objects being tested.This Technical Specification describes the essential characteristics and applicable measurement methods of working suspension containing nano-object samples for in vitro toxicity assays. Intention is that reliable test results on nano-object toxicity could be shared
25、and communicated among stakeholders of nano-objects, such as regulators, general public, manufacturers and end users. This Technical Specification does not describe a procedure for validation of working suspension. ISO 2016 All rights reserved vPD ISO/TS 19337:2016PD ISO/TS 19337:2016Nanotechnologie
26、s Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicity1 ScopeThis Technical Specification describes characteristics of working suspensions of nano-objects to be considered when conducting in vitro assays to evaluate inherent nano-object
27、 toxicity. In addition, this Technical Specification identifies applicable measurement methods for these characteristics.This Technical Specification is applicable to nano-objects, and their aggregates and agglomerates greater than 100 nm.NOTE This Technical Specification intends to help clarify whe
28、ther observed toxic effects come from tested nano-objects themselves or from other uncontrolled sources.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition ci
29、ted applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 29701, Nanotechnologies Endotoxin test on nanomaterial samples for in vitro systems Limulus amebocyte lysate (LAL) test3 Terms and definitionsFor the purposes of this document, t
30、he following terms and definitions apply.3.1culture mediumaqueous solution of nutrients required for cell growth3.2secondary particlecomplex agglomerate/aggregate of primary particle(s), proteins and other medium components3.3stabilityproperties to remain unchanged over a given time under stated or
31、reasonably expected conditions of storage and use for an in vitro toxicity assay3.4working suspensionsuspension prepared for an in vitro assay that includes culture medium and nano-object sampleTECHNICAL SPECIFICATION ISO/TS 19337:2016(E) ISO 2016 All rights reserved 1PD ISO/TS 19337:2016ISO/TS 1933
32、7:2016(E)4 Abbreviated termsFor the purposes of this Technical Specification, the following abbreviated terms apply.AAS atomic absorption spectrometryBCA bicinchoninic acidC-U/F ultrafiltration assisted by centrifugationDLS dynamic light scatteringFFFF flow field-flow fractionationICP-AES inductivel
33、y coupled plasma-atomic emission spectrometryICP-MS inductively coupled plasma mass spectrometryLD laser diffractionSLS static light scatteringTFF tangential flow filtrationTOC total organic carbonU/F ultrafiltrationUV-Vis ultraviolet-visible5 Characteristics and measurement methods5.1 GeneralTo cha
34、racterize the working suspension for in vitro toxicity assays, it is necessary to determine certain characteristics that might impact the biological system tested. This Clause specifies essential characteristics of the working suspension, listed below, and measurement methods that are applicable to
35、them. Presence of endotoxins, stability of working suspensions, concentration of metal ions, and concentration of culture medium components.Measurements of those characteristics shall be made for each dose of working suspensions. The measurement of endotoxin can be made alternatively for the stock n
36、ano-object suspension to be dosed. See Annex A for an example of flow of measurements.5.2 EndotoxinContamination with endotoxins, part of the outer membrane of Gram-negative bacteria may significantly alter the results of the in vitro toxicity test. Therefore, it is critical to quantify the concentr
37、ations of endotoxins in the working suspension. The concentration of endotoxins in the working suspension shall be measured by Limulus amebocyte lysate (LAL) test in accordance with ISO 29701 and the monocyte activation test (MAT).235.3 Stability of working suspensions5.3.1 GeneralStability of worki
38、ng suspension is a key characteristic as it directly impacts the in vitro assay conditions in terms of the dose of the nano-objects to the cells.45Aggregation/agglomeration and gravitational settling of the nano-objects are major issues that may affect the stability of the suspended nano-objects. Th
39、e stability shall be evaluated for the two characteristics, i.e. the relative change of representative 2 ISO 2016 All rights reservedPD ISO/TS 19337:2016ISO/TS 19337:2016(E)size of secondary particles of nano-objects and the relative change of the concentration of nano-objects in the working suspens
40、ion, resulting from gravitational settling during an in vitro toxicity assay, by considering experimental duration required for the in vitro toxicity assay. Evaluation results of the stability shall be expressed in the unit of per cent (%) over the timescale for in vitro toxicity assay.NOTE ISO/TR 1
41、30976is recommended as a comprehensive guidance for stability of working suspension.5.3.2 Representative size change of secondary particles of nano-objectsAn appropriate method shall be selected to directly measure the representative size change of secondary particles of nano-objects from among dyna
42、mic light scattering (DLS),47laser diffraction (LD)8and static light scattering (SLS).9Other methods deviating from this Technical Specification can be used and reported in accordance with 6.6.See Annex B for measurements.5.3.3 Concentration change of nano-objectsAn appropriate method shall be selec
43、ted to measure the concentration change of nano-objects suspended in the biological media from among the light scattering,4710inductively coupled plasma mass spectrometry (ICP-MS),111213ultraviolet-visible (UV-Vis) absorption, X-ray transmission14and the total organic carbon analysis.15Other methods
44、 deviating from this Technical Specification can be used and reported in accordance with 6.6.See Annex B for measurements.5.4 Concentration of metal ionsMetal ions, produced as a result of nano-object test sample dissolution, can contribute to test cell toxicity. The concentration of metal ions in t
45、he working suspension shall be measured after separation of particulate matter. Particulate matter can be separated from the ionic fraction by ultra-filtration (U/F), ultra filtration assisted by centrifugation (C-U/F) or tangential flow filtration (TFF). The measurement shall be made for all metall
46、ic elements that are included in the nano-object sample. An appropriate method shall be selected to measure the metal ion concentrations from among inductively coupled plasma-atomic emission spectrometry (ICP-AES), ICP-MS, atomic absorption spectrometry (AAS) and the colourimetric method. Other meth
47、ods deviating from this Technical Specification can be used and reported in accordance with 6.6. Measurement results of concentrations shall be expressed in the unit of molarity, mass/mass or mass/volume. The measurements can be omitted when a toxic effect is not observed to the cells in the working
48、 suspensions. See Annex A for an example of flow of measurements.See Annex C for measurements.5.5 Concentration of culture medium components5.5.1 GeneralA nano-object sample added to a culture medium to generate a working suspension may adsorb components of the culture medium.1This can result in sta
49、rvation stress to the test cells. The concentration of protein components and calcium, as surrogates for the nutritional components in the solvent shall be measured by setting aside enough time after the addition of nano-object sample to the culture medium. If culture medium components other than protein and calcium that may significantly affect the stability of working suspension for in vitro toxicity assays are known, the concentration of those components shall be measured as well. The measurements can be om
