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Adaptation of OECD TG 433 (Acute Inhalation Toxicity) to nanomaterials for the requirements under REACH (OECD TG 433)

07/2022-06/2025

Funding programme / funding institution: Bundesministerium für Umwelt, Naturschutz, Bau und Reaktorsicherheit (BMU) - Deutschland

Grant number: UM 22 66 0010

Project homepage: -

Project description:

The REACH Regulation for Chemicals (EC No 1907/2006) requires registrants to submit testing data on acute toxicity for all substances marketed in volumes > 1 tonne/year in the EU (REACH Annex VII, No. 8.5.1.). Recently, the Annexes to REACH, which detail the information requirements, have been revised. In particular, new information requirements related to nanomaterials entered into force on 01.01.2020. Inter alia, REACH now requests inhalation as default route for acute toxicity testing of nanomaterials, thus taking into account that inhalation of nanoparticle aerosols is the most likely and most relevant route of exposure for humans.

The three existing OECD test guidelines for acute inhalation toxicity (OECD TG 403, 433, 436) are equivalent in terms of their regulatory validity. However, none of these test guidelines has been validated for nanomaterials yet. OECD TG 403 is not often applied anymore, because its protocol requires considerably high numbers of animals. Currently, OECD TG 436 appears to be the most frequently used protocol. OECD TG 433 is the latest test guideline of the three. It was developed explicitly to minimise animal numbers and suffering and hence better complying with the 3R principle (“Replace, Reduce, Refine”). OECD TG 433 rather puts the focus on the endpoint “evident toxicity” rather than on mortality (as OECD TG 403 and 436 do). Therefore, this project emphasizes the adaptation of OECD TG 433. However, it is likely that the project results can be readily transferred to the two other test guidelines, especially for OECD TG 436, in particular with limited additional experimental input.

When conducting acute inhalation toxicity studies aerosolization of and exposure to a considerable amount of nanoparticles, without modifying the properties of the test substance or blocking administration devices, is challenging. This applies in particular to nanoparticles of low density, which tend to agglomerate rapidly depending on the surface properties. Therefore, one focus of the project is on experimental investigations aiming to optimize and standardize the preparation and administration of particulate test materials.

Another focus is on conducting animal experiments with the aim of developing valid criteria for the assessment of substance-related toxicity as well as for the evaluation of the severity of sub-lethal adverse effects with special consideration of histopathological findings. The findings of inhalation experiments shall be compared with an alternative exposure method, such as intratracheal instillation. Comparing exposure methods addresses two complementary aspects: Firstly, it allows distinguishing between substance-related effects and non-specific effects such as obstruction of the upper airways resulting from inhalation at high exposure concentrations, because this is bypassed by instillation. Secondly, the suitability of an alternative exposure method in case of technical issues preventing inhalation exposure is investigated.

In parallel, (experimental) work on non-animal (alternative) test methods will be carried out to support findings from animal studies (e.g. when applying grouping) as well as to replace animal studies in the future. Initially, a systematic literature search will be performed on recent developments of alternative approaches (including both, in vitro and in silico approaches) regarding (acute) inhalation toxicity testing. Based on this, the alternative methods to be further developed within the project will be selected.

The applicability of the project results in the context of chemicals regulation and mutual recognition according to the OECD MAD principle require the validation of the results. For the planned submission to the OECD Test Guidelines Programme, the experimental work to adapt OECD TG 433 to nanomaterials will therefore take into account the validation criteria required by the OECD.

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