Standard Active Last Updated: Jul 19, 2022 Track Document
ASTM E3351-22

Standard Test Method for Detection of Nitric Oxide Production In Vitro

Standard Test Method for Detection of Nitric Oxide Production In Vitro E3351-22 ASTM|E3351-22|en-US Standard Test Method for Detection of Nitric Oxide Production In Vitro Standard new BOS Vol. 14.02 Committee E56
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Significance and Use

5.1 This test method is designed to evaluate nanomaterial capacity to induce nitric oxide production by macrophages.

5.2 Activated macrophages generate large quantities of NO. NO generated from activated macrophages is a cytostatic/cytotoxic agent (3-6).

5.3 The production of NO in excessive amounts leads to the generation of peroxynitrite by its spontaneous reaction with superoxide. Peroxynitrite causes tissue injury through its capability to damage lipids, proteins, and DNA (2).

5.4 NO is a proinflammatory mediator and it is an important marker for activation of inflammation (5, 6).

5.5 Testing the capacity of a nanomaterial to induce NO production in vitro helps in predicting the nanomaterial’s biocompatibility through anticipating and understanding the potential problems that might be encountered during its in vivo administration.


1.1 This test method delivers a protocol for a quantitative measure of nitrite (NO2), a stable end-product of nitric oxide (NO), in cell culture medium due to exposure to nanomaterial(s).

1.2 NO has a critical role in several pathological conditions in addition to its role in many physiological processes.

1.3 This test method uses murine macrophage cell line RAW 264.7 as an in vitro model.

1.4 The nitrite is measured in the cell culture medium by a colorimetric analysis using Griess reagent as shown in Fig. 1.

FIG. 1 Summary of Nitric Oxide Production Assay

Summary of Nitric Oxide Production AssaySummary of Nitric Oxide Production Assay

1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.

1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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Book of Standards Volume: 14.02
Developed by Subcommittee: E56.08
Pages: 10
DOI: 10.1520/E3351-22
ICS Code: 11.100.10