1、BSI Standards Publication PD ISO/TS 19337:2016 Nanotechnologies Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicityPD ISO/TS 19337:2016 PUBLISHED DOCUMENT National foreword This Published Document is the UK implementation of ISO/TS 193
2、37:2016. 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. U
3、sers are responsible for its correct application. The British Standards Institution 2016. Published by BSI Standards Limited 2016 ISBN 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 author
4、ity of the Standards Policy and Strategy Committee on 30 April 2016. Amendments/corrigenda issued since publication Date T e x t a f f e c t e dPD ISO/TS 19337:2016 ISO 2016 Nanotechnologies Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object t
5、oxicity Nanotechnologies Caracteristiques des suspensions de nano-objets utilises pour les tests in vitro valuant la toxicit inherente aux nano-objets TECHNICAL SPECIFICATION ISO/TS 19337 Reference number ISO/TS 19337:2016(E) First edition 2016-03-15PD ISO/TS 19337:2016ISO/TS 19337:2016(E)ii ISO 201
6、6 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in Switzerland All 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
7、internet or an intranet, 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 office Ch. de Blandonnet 8 CP 401 CH-1214 Vernier, Geneva, Switzerland Tel. +41 22 749 01 11 Fax +41 22 749 0
8、9 47 copyrightiso.org www.iso.orgPD ISO/TS 19337:2016ISO/TS 19337:2016(E)Foreword iv Introduction v 1 Scope . 1 2 Normative references 1 3 T erms and definitions . 1 4 Abbreviated terms 2 5 Characteristics and measurement methods 2 5.1 General . 2 5.2 Endotoxin . 2 5.3 Stability of working suspensio
9、ns 2 5.3.1 General 2 5.3.2 Representative size change of secondary particles of nano-objects . 3 5.3.3 Concentration change of nano-objects . 3 5.4 Concentration of metal ions. 3 5.5 Concentration of culture medium components 3 5.5.1 General 3 5.5.2 Proteins 4 5.5.3 Calcium . 4 6 Reporting 4 6.1 Gen
10、eral . 4 6.2 Name of nano-objects and manufacturer 4 6.3 Metallic elements included in the nano-object sample 4 6.4 Culture medium and serum . 4 6.5 Measurement results . 4 6.6 Deviation 5 Annex A (informative) Flow of measurements. 6 Annex B (informative) Measurement and evaluation of stability 7 A
11、nnex C (informative) Measurement of metal ions. 8 Annex D (informative) Measurement of culture medium components 9 Bibliography .10 ISO 2016 All rights reserved iii Contents PagePD ISO/TS 19337:2016ISO/TS 19337:2016(E) Foreword ISO (the International Organization for Standardization) is a worldwide
12、federation of national standards bodies (ISO member bodies). The 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 c
13、ommittee. International organizations, governmental and non-governmental, 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 documen
14、t and those intended for its further maintenance are described in 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,
15、 Part 2 (see www.iso.org/directives). Attention is drawn to the 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
16、of the document will be in the Introduction and/or on the ISO list 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 specif
17、ic terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information The committee responsible for this document is ISO/TC 229, Nanotechnologie
18、s.iv ISO 2016 All rights reservedPD ISO/TS 19337:2016ISO/TS 19337:2016(E) Introduction Before 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
19、screening hazardous materials. This testing provides essential information 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 wat
20、er soluble chemicals. For example, immediately after the introduction 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 th
21、e components of culture medium onto the nano-object surface, or (c) changes 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 teste
22、d nano-object itself or from other uncontrolled sources and to avoid false 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
23、, metal ion release from the nano-objects and impurities (residual from 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 impor
24、tant to carefully assess and describe the characteristics of the suspension 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. Intenti
25、on is that reliable test results on nano-object toxicity could be shared 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
26、 rights reserved vPD ISO/TS 19337:2016PD ISO/TS 19337:2016Nanotechnologies Characteristics of working suspensions of nano-objects for in vitro assays to evaluate inherent nano-object toxicity 1 Scope This Technical Specification describes characteristics of working suspensions of nano-objects to be
27、considered when conducting in vitro assays to evaluate inherent nano-object 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 great
28、er than 100 nm. NOTE This Technical Specification intends to help clarify whether observed toxic effects come from tested nano-objects themselves or from other uncontrolled sources. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and a
29、re indispensable for its application. For dated references, only the edition cited 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 ameboc
30、yte lysate (LAL) test 3 T erms a nd definiti ons For the purposes of this document, the following terms and definitions apply. 3.1 culture medium aqueous solution of nutrients required for cell growth 3.2 secondary particle complex agglomerate/aggregate of primary particle(s), proteins and other med
31、ium components 3.3 stability properties to remain unchanged over a given time under stated or reasonably expected conditions of storage and use for an in vitro toxicity assay 3.4 working suspension suspension prepared for an in vitro assay that includes culture medium and nano-object sample TECHNICA
32、L SPECIFICATION ISO/TS 19337:2016(E) ISO 2016 All rights reserved 1PD ISO/TS 19337:2016ISO/TS 19337:2016(E) 4 Abbreviated terms For the purposes of this Technical Specification, the following abbreviated terms apply. AAS atomic absorption spectrometry BCA bicinchoninic acid C-U/F ultrafiltration ass
33、isted by centrifugation DLS dynamic light scattering FFFF flow field-flow fractionation ICP-AES inductively coupled plasma-atomic emission spectrometry ICP-MS inductively coupled plasma mass spectrometry LD laser diffraction SLS static light scattering TFF tangential flow filtration TOC total organi
34、c carbon U/F ultrafiltration UV-Vis ultraviolet-visible 5 Characteristics and measurement methods 5.1 General To characterize the working suspension for in vitro toxicity assays, it is necessary to determine certain characteristics that might impact the biological system tested. This Clause specifie
35、s essential characteristics of the working suspension, listed below, and measurement methods that are applicable to them. Presence of endotoxins, stability of working suspensions, concentration of metal ions, and concentration of culture medium components. Measurements of those characteristics shall
36、 be made for each dose of working suspensions. The measurement of endotoxin can be made alternatively for the stock nano-object suspension to be dosed. See Annex A for an example of flow of measurements. 5.2 Endotoxin Contamination with endotoxins, part of the outer membrane of Gram-negative bacteri
37、a may significantly alter the results of the in vitro toxicity test. Therefore, it is critical to quantify the concentrations 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
38、 ISO 29701 and the monocyte activation test (MAT). 23 5.3 Stability of working suspensions 5.3.1 General Stability of working 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
39、 gravitational settling of the nano-objects are major issues that may affect the stability of the suspended nano-objects. The 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) si
40、ze of secondary particles of nano-objects and the relative change of the concentration of nano-objects in the working suspension, resulting from gravitational settling during an in vitro toxicity assay, by considering experimental duration required for the in vitro toxicity assay. Evaluation results
41、 of the stability shall be expressed in the unit of per cent (%) over the timescale for in vitro toxicity assay. NOTE ISO/TR 13097 6is recommended as a comprehensive guidance for stability of working suspension. 5.3.2 Representative size change of secondary particles of nano-objects An appropriate m
42、ethod shall be selected to directly measure the representative size change of secondary particles of nano-objects from among dynamic light scattering (DLS), 47laser diffraction (LD) 8and static light scattering (SLS). 9Other methods deviating from this Technical Specification can be used and reporte
43、d in accordance with 6.6. See Annex B for measurements. 5.3.3 Concentration change of nano-objects An appropriate method shall be selected to measure the concentration change of nano-objects suspended in the biological media from among the light scattering, 4710inductively coupled plasma mass spectr
44、ometry (ICP-MS), 111213ultraviolet-visible (UV-Vis) absorption, X-ray transmission 14and the total organic carbon analysis. 15Other methods 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 ions Metal
45、 ions, produced as a result of nano-object test sample dissolution, can contribute to test cell toxicity. The concentration of metal ions in the working suspension shall be measured after separation of particulate matter. Particulate matter can be separated from the ionic fraction by ultra-filtratio
46、n (U/F), ultra filtration assisted by centrifugation (C-U/F) or tangential flow filtration (TFF). The measurement shall be made for all metallic elements that are included in the nano-object sample. An appropriate method shall be selected to measure the metal ion concentrations from among inductivel
47、y coupled plasma-atomic emission spectrometry (ICP-AES), ICP-MS, atomic absorption spectrometry (AAS) and the colourimetric method. Other methods deviating from this Technical Specification can be used and reported in accordance with 6.6. Measurement results of concentrations shall be expressed in t
48、he 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 suspensions. See Annex A for an example of flow of measurements. See Annex C for measurements. 5.5 Concentration of culture medium components 5.5.1 General A
49、 nano-object sample added to a culture medium to generate a working suspension may adsorb components of the culture medium. 1This can result in starvation 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 stabil
