Technical Report 122

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.


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.