APPENDIX A: Test Methodology: BALF Study

Screening study to identify effects characteristic for 'lung overload'

Test methodology: BALF Study

Proposal to use early indicators (e.g. biochemical, cytological) as screen for functional/pathological changes in the lung in combination with histopathological evaluation of the respiratory tract and potential lung burden measurements.

Test System: Nose Only

Avoid uptake via other routes (oral due to cleaning of fur, dermal exposure related), especially with regard to "nano”.

Test Species: Rat

Most sensitive species (argument of "intrinsic” biological safety factor) for AOP relevant key events in "particle lung overload” / unique concerning lung tumour development as endpoint

Exposure Duration: fixed exposure period + various recovery periods

An exposure period of 5 to 7 days seems sufficient to induce early indicators of inflammation. Different durations of recovery (e.g. 1, 3, and 7 days up to a maximum of 4 weeks post exposure) to inform about reversibility or progressive course of alterations should be considered.

Exposure Dose: 3 different concentrations + control

Exposure doses of 0, 1, 5 and 10 mg/m3 will allow establishing different lung burdens and dose-response analysis. Modified exposure concentrations to investigate lower or higher exposures may be applicable.

Parameters:

Histolpathology of respiratory tract (e.g. whether inflammation is granulomatous or not)

Lung burden measurements (if applicable)

Biochemical / Cytological Parameters (selection of endpoints)

- Lactate Dehydrogenase (LDH)

- Alkaline Phosphatase (AP)

- γ-Glutamytransferase (γ-GT)

- Total protein

- N-acetylglucosamidase

- ß-Glucuronidase

- Total cell count

- Differential cell count

Additionally, cell proliferation marker as well as marker for genotoxicity (e.g. 8-OH-dG; OGG1) and oxidative stress may be included.

The advantages of such a study design can be seen in its screening character, flexibility, possibility to differentiate between a progressive or reversible course of effects. Depending on the design, dose (lung burden) – response analysis are possible.

Additionally, by including respective (negative, positive) control substances with known toxicity profile, comparative benchmark analysis may be possible.