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Standards Development
This technical specification (TS) provides an optimized BALF analysis method in inhalation toxicity studies of nanomaterials. This technical specification will include the collection and preparation method for the BALF analysis in the aspect of inflammatory cell counts and levels of lactate dehydrogenase, total protein, and inflammatory mediators. In addition, this TS includes nanomaterial-specific considerations, such as inflammation patterns, proposed inflammatory mediators, and interferences in measurements. This TS is optimized for half-lung lavage of Sprague-Dawley (SD) rats but also provides information for the lavage volume of different strains and volume-determining parameters.
• OECD guidelines for mammalian toxicity testing have been undergoing revisions to make them
applicable to nanomaterials.
• In 2018, documents for inhalation toxicity tests using mammals, TG 412 (subacute) and 413 (chronic),
were revised to allow for the application of nanomaterials. One of the mandatory tests among the
revisions is the analysis of bronchoalveolar lavage fluid (BALF).
• The OECD guidance document on inhalation toxicity testing, GD39, mentions the BALF assay but
lacks standardized test conditions and procedures. This appears to result in large differences in
experimental data between laboratories.
• Standardized BALF analysis methods are crucial for accurate and reproducible assessment of
nanomaterial inhalation toxicity.
• An optimization of BALF analysis methods is necessary for inhalation toxicity tests of nanomaterials
because, unlike general chemicals, nanomaterials have different toxicities depending on their
physicochemical properties (e.g., particle size, shape, aggregation, surface chemistry). Therefore, it is
highly likely that the safety assessment results for a specific nanomaterial will not be applicable to other
nanomaterials with the same elemental composition.
• Particle-to-particle comparison of toxicity test results for nanomaterials is essential, and it is
necessary to demonstrate the toxicological equivalence of materials based on the correlation between
physicochemical properties and toxicity.
• To demonstrate toxicological equivalence, it is necessary for the toxicity testing methods conducted
by different institutions or laboratories to be similar. However, there is no international standard for
BALF analysis methods yet, so there are differences between laboratories in the methods of collecting
and analyzing lavage fluid, and comparison is impossible.
• Differences in experimental results between laboratories can arise from several factors related to
bronchoalveolar lavage (BAL) fluid collection and analysis. These factors include BALF collection
methods (tools used for alveolar lavage, type and amount of lavage fluid, collection method), BALF
analysis methods (cell collection post-processing methods, cell analysis), etc.
• This document focuses on investigating the effects of several factors on alveolar lavage (BAL)
parameters, including the presence or absence of massage, the number of flushing cycles, the type of
medium used for BAL and cell resuspension, and the condition of the resuspension medium. These
factors were found to significantly influence the number of recovered alveoli, the concentration of total
protein in BAL fluid, and the quality of slide smears.
• Standardizing BALF analysis through ISO TS development is crucial for harmonizing global testing
practices and ensuring data comparability. This standardization can also lead to cost reductions for
inhalation toxicity tests, which are currently expensive worldwide.
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