ISO 4962:2024

Nanotechnologies — In vitro acute nanoparticle phototoxicity assay

This document specifies a procedure to evaluate acute phototoxicity of nanoparticles (NPs), suspended in cell culture media, by measuring the relative reduction in cellular viability under near ultraviolet (UVA) exposure (315 nm to 400 nm). The measurement is intended to assess the potential for acute phototoxicity of NPs by comparing NP photoactivity in vitro to a positive control chlorpromazine. It is not designed to predict other joint effects of nanomaterials and light, such as genotoxicity, photo-allergy or photo-mutagenicity. The method is also applicable to NP aggregates and agglomerates.

Nanotechnologies — Essai in vitro de phototoxicité aiguë des nanoparticules

General Information

• Status: Published
• Publication Date: 04-Nov-2024
• ICS: 07.120 - Nanotechnologies
• Technical Committee: ISO/TC 229 - Nanotechnologies
• Drafting Committee: ISO/TC 229/WG 3 - Health, Safety and Environmental Aspects of Nanotechnologies
• Current Stage: 6060 - International Standard published
• Start Date: 05-Nov-2024
• Due Date: 05-Nov-2024
• Completion Date: 05-Nov-2024

Standards Content (Sample)

International
Standard
ISO 4962
First edition
Nanotechnologies — In vitro acute
2024-11
nanoparticle phototoxicity assay
Nanotechnologies — Essai in vitro de phototoxicité aiguë des
nanoparticules
Reference number
© ISO 2024
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Published in Switzerland
ii
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms, definitions, symbols and abbreviated terms . 1
3.1 Terms and definitions .1
3.2 Abbreviated terms .2
4 Test method . 2
5 Materials and equipment . 3
5.1 Materials .3
5.1.1 Reagents .3
5.1.2 Cell line .3
5.1.3 Controls .4
5.2 Apparatus .5
5.2.1 Laminar flow cabinet, standard biological hazard .5
5.2.2 Incubator (37 °C, 95 % humidified, 5 % CO /air) .5
5.2.3 Inverted phase contrast microscope .5
5.2.4 Centrifuge .5
5.2.5 Water bath .5
5.2.6 Multiple well plate reader .5
5.2.7 Tissue culture flasks .5
5.2.8 24 multi-well plates with flat bottom .5
5.2.9 Flat bottom 96-well black polystyrene microplate .5
5.2.10 8-channel pipette, 20-200 μl Hemocytometer .5
5.2.11 Conical tube .5
5.2.12 Reservoir .5
5.2.13 Vortex mixer .5
5.2.14 Refrigerator .5
5.2.15 Freezer .5
5.2.16 UV crosslinker (capable of light emission at λ=365 nm), as a UVA source.
Irradiation of the test plate should be between 4 and 6 mW/cm . .5
5.2.17 UV-VIS spectrophotometer, capable of measurements in the wavelength range
from 300 to 800 nm. .5
5.2.18 UV power meter, capable of measurements in the wavelength range from 315
to 400 nm. .5
6 Sample preparation . 5
7 Preparations . 6
7.1 General .6
7.2 Culture medium .6
7.3 Preparation of cell stock culture .6
7.4 Verify viable cell growth . .6
7.5 Irradiation conditions .8
7.5.1 UVA source .8
7.5.2 Light dose (insolation) measurement .8
7.6 Multiple well plate reader .8
8 Measurement procedure . 8
8.1 Cell seeding (Day 1) .10
8.2 Incubation of cells with the positive control and NP suspension (Day 2) .10
8.3 UVA exposure (Day 3) .11
8.4 Cell viability assay (Day 4) . .11
8.5 Evaluation of artefacts due to possible NP interferences with the MTS assay (in the
dark) . 12

iii
8.6 Data analysis . 13
9 Report . 14
9.1 Test report .14
9.2 Report data format . 15
10 Precision .15
10.1 Repeatability . 15
10.2 Reproducibility . 15
Annex A (informative) Schematic diagram of 96-well plate position .16
Annex B (informative) Verification of plate reader uniformity . 17
Annex C (informative) Dispersing procedure for TiO nanoparticles in DMEM .18
Annex D (informative) Results of the inter-laboratory test . 19
Bibliography .22

iv
Foreword
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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 committee. International organizations,
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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
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or appl
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