*4.3. Image Analysis*

The image data were analyzed with ROVER (ABX, Radeberg, Germany; v. 3.0.46h). The organs were identified and manually delineated using 3D volumes of interest (VOI). The CT data were used for anatomical orientation and for image registration with the PET data. Relevant source organs like brain, gall bladder, large intestine, small intestine, stomach, heart, kidneys, liver, lungs, pancreas, red marrow (backbone, pelvis, sternum), spleen, thyroid, testes, skeleton (bone), and the urinary bladder were delineated and the time–activity data transformed into percentage of injected dose (%*IDorgan*) with Equation (2).

$$\%ID\_{\text{organt}} = \frac{A\_{\text{organt}} \times c\_{\text{scant}}}{A\_{0t}} \text{ [\%]} \tag{2}$$

where *Aorgant* is the activity in the organ at the time *t*; *cscant* is a calibration factor representing the theoretical body activity (derived from a whole body mask of a volume that equals the body dimentions of the animal) decay corrected to *t* and divided by the imaged body activity in each image frame at the time *t*, and *A*0*<sup>t</sup>* is the injected activity decay corrected to *t*0.

#### *4.4. Incorporation Dosimetry*

The dose calculation has been described by our group in detail before [14–16]. Briefly, due to differences in weight, size, and metabolic rates between the animal species and the human volunteers, it is necessary to map the preclinical extracted biodistribution data to human circumstances. Thus, the animal biokinetic data (time scale and *%ID* values) were adapted to the human circumstances according to [22] to fit the human weight, size, and metabolic rates (Equations (3) and (4)).

$$t\_{\text{humanu}} = t\_{\text{animal}} \left[ \frac{m\_{\text{lnmax}}}{m\_{\text{annual}}} \right]^{0.25} \tag{3}$$

$$\left[\%ID\right]/\sigma\text{gan}\_{\text{human}} = \left[\%ID\right]/\left[\text{g}\_{\text{animal}}\times\frac{m\_{TB\_{\text{initial}}}\left[\text{g}\right]}{m\_{TB\_{\text{human}}}\left[\text{g}\right]}\times m\_{\text{off}\,\text{S}^{\text{univ}}\_{\text{human}}}\left[\text{g}\right] \tag{4}$$

The TIACs were estimated by exponential fitting and the dosimetry estimation was performed using OLINDA/EXM software (v. 2.1). Finally, organ doses (OD) were estimated and multiplied by tissue weighting factors as published in ICRP103 [20].

#### **5. Conclusions**

By extrapolation of the preclinical dosimetry data obtained in piglets, the effective dose as a measure of the overall radiation risk upon iv application of about 300 MBq [18F]FACH to humans is estimated to be 3.7 mSv. However, as frequently observed for other PET tracers, the preclinical dosimetry underestimates the ED to humans by up to 40%. Accordingly, we conclude that the ED to humans by [18F]FACH can be expected to be about 20.6 μSv/MBq. With a systemic application of 300 MBq, PET imaging with [18F]FACH would yield an effective dose of about 6.2 mSv to human subjects. This value is well within the range of other 18F-labeled radiopharmaceuticals. Despite the study-specific overestimation of the contributions of bone and the urinary bladder to the ED, this risk assessment encourages the transference of [18F]FACH from preclinical to clinical study phases to further assess the suitability of this new radiopharmaceutical for PET imaging of oncological diseases. **Supplementary Materials:** The following data are available online: Tables S1–S3: %ID values of the three piglets after i.v. injection of [18F]FACH. Table S4: Detailed results of the dose calculation for the three animals: organ equivalent doses and effective dose contributions involving the tissue risk factor wT of ICRP103. Figures S1–S3: All time-integrated activity curves with fitting functions and fit goodness parameters (R-squared and squared error) of all organs and systems of organs that could be identified as taking up the tracer for all included animals.

**Author Contributions:** B.S., M.K., M.S., P.B., W.D.-C., and O.S. conceived and designed the experiments; M.K., W.D.-C., M.T., N.T.J., C.D., T.S., and B.S. performed the experiments; B.S., M.K., N.T.J., and O.S. analyzed the data; B.S., M.K., M.S., T.S., W.D.-C., O.S., and P.B. drafted the manuscript; M.S., R.-P.M., and P.B. designed the molecule and performed the organic chemistry; M.S., B.W., and R.T. performed the radiosynthesis; B.W., M.S., R.T., and F.-A.L. performed the metabolite analysis; T.S. handled the animals, conducted and maintained their anesthesia, and took all the blood and urine samples. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by ALEXANDER VON HUMBOLDT FOUNDATION, grant number 7211172142 and the APC was funded by STRAHLENSCHUTZSEMINAR IN THüRINGEN E.V., grant number F2015\_06.

**Acknowledgments:** We are very thankful to K. Franke, A. Mansel, and S. Fischer for providing [18F]fluoride.

**Conflicts of Interest:** The authors declare no conflicts of interest.
