# Presentation: 711020

## Scientific Basis for Removal of the EPA’s 10X Database Uncertainty Factor

**Review of *****in vivo***** and *****in vitro***** metabolism / hydrolysis of SO****2****F****2**

## ADME of Sulfuryl Fluoride

**ADME of Sulfuryl Fluoride**

- Primary route of degradation is via
- 		two-step hydrolysis (base catalyzed):

- Authors showed hydrolysis of SO2F2 requires high pH (HO- ion)
-  - SO2F2 hydrolysis half-life in basic water = 3 min    * (Cady 1974)*
-       - hydrolysis _even faster_ with other nucleophiles (ie:  amines, phenolate, CN-)
-          - primary site of albumin adduction = phenolic moiety of tyrosine *(Zhang 2007)*
-  - FSO3 hydrolysis half-life in basic water = 20 hr       *(Jones, 1968)*

## Uptake, Metabolism, and Elimination of SO2F2 Mendrala et al., 2005

- _Methods_
- Fischer 344 rats
- Nose-only inhalation chambers
- Single 4-hour exposure
- Exposure concentrations of **30 or 300 ppm**
- Radiolabeled (35S labeled) or cold SO2F2
- Measured radioactivity and radiochemical analysis

## Uptake, Metabolism, and Elimination of SO2F2  Mendrala et al., 2005

- _Results_
- 35SO2F2 rapidly absorbed via inhalation exposure
- Estimated absorption based on total absorbed radioactivity compared to calculated internal dose
- Rats exposed to 30 ppm absorbed 14.0% of inhaled SO2F2
- Rats exposed to 300 ppm absorbed 12.5% of inhaled SO2F2
- Radioactivity rapidly cleared from plasma
- Radiolabel excreted as fluorosulfate and sulfate

## Uptake, Metabolism, and Elimination of SO2F2 Mendrala et al., 2005

- _Results (continued)_
- Radiochemical profiles did not provide evidence of parent molecule in the blood
- Fluorosulfate and sulfate identified in blood and urine
- Increased fluoride detected in blood, urine and tissues

## ADME in vivo study (35SO2F2) 

*Data from Mendrala et al. (2005)*

- ADME *in vivo* study (35SO2F2)
- Rapidly absorbed, metabolized and eliminated in rat
- No parent compound found in blood or tissues
- 35S-labeled SO4 and FS primary urine and blood metabolites

- FSO3

- SO4

***30 ppm***

## ADME in vivo study (35SO2F2) 

*Data from Mendrala et al. (2005)*

- ADME *in vivo* study (35SO2F2)
- Rapid elimination of metabolites, primarily in urine

***300 ppm***

***30 ppm***

- FSO3

- F

- SO4

- FSO3

- F

- SO4

## Retention of 35S-radioactivity 5-6x higher in lung than plasma

- Retention of 35S-radioactivity 5-6x higher in lung than plasma

- Plasma time-course of 35S-radioactivity

*Data from Mendrala et al. (2005)*

- Tissue 35S-radioactivity 7 days post-exposure

- Conclusions:
- 	SO2F2 rapidly hydrolyzed in portal-of-entry tissue, affording higher 35S-residues than other tissues

***300 ppm***

***30 ppm***

***300 ppm***

***30 ppm***

## ADME of Sulfuryl Fluoride

**ADME of Sulfuryl Fluoride**

**Summary**
- Test material rapidly absorbed
- Hydrolyzed quickly to FSO3 and F
- consistent with known chemical hydrolysis rates
- Tissue residues indicate portal-of-entry hydrolysis
- FSO3 and F rapidly eliminated
- some secondary hydrolysis of FSO3 to SO4

## ADME of Sulfuryl Fluoride

**ADME of Sulfuryl Fluoride**

**Analytical Methods used in support of in vitro and in vivo metabolism/toxicokinetic studies**
- Parent compound (SO2F2) extracted into hexane extraction from blood, followed by headspace GC/MS
- LOD = 0.084 μg/mL blood
- Fluorosulfate (FSO3) analyzed in blood and tissues via aqueous extraction, followed by ion chromatography-MS (IC/MS) analysis
- LOD = 0.15-0.43 μg/gram tissue
- Fluoride (F) analyzed in blood and tissued via aqueous extraction, followed by either IC/MS or microdiffusion-ion specific electrode (MD/ISE) analysis
- LOD = 0.027 μg/gram tissue
- FSO2-albumin adducts analyzed in blood and lavage fluid via trypsin digestion of albumin protein, followed by signature peptide LC/MS/MS method
- LOD = 0.32 μg/mg albumin

**Notes:**
SF in Blood:  matrix stds, 0.5-1 mL sample size
	methods: no detectable SF in blood 15 min after fortification at 850ppb  (LOD = 84ppb, so at least 3 t1/2's of degradation == t1/2 <= 5 min)
SF in NL & LL:  matrix stds, 1-2 mL sample size

FS in blood/tissues:  matrix spikes, 0.4-1 g sample size (homogenates)

F in blood/tissues:  DKA to each micro-diffused sample + matrix spikes, 1 g sample size

FSO3-albumin in plasma/NL/LL:  matrix spikes, 0.1g plasma, 2mL NL, 2.4mL LL sample sizes

## ADME in vitro, as part of Genetic Toxicity studies

- ADME *in vitro*, as part of Genetic Toxicity studies
- RLCAT:  		100% hydrolysis of  SO2F2  ->  FSO3 + F
- (1115 - 27,806 ppm)    	4% hydrolysis of  FSO3  ->  SO4 + F
-  					added liver S9 did not alter metabolism
- MLA TK+/-:  	90% hydrolysis of  SO2F2  ->  FSO3 + F
- (1160 - 25,000 ppm)	3% hydrolysis of  FSO3  ->  SO4 + F
- 		  			added liver S9 did not alter metabolism
- Conclusions:
- 	SO2F2 rapidly hydrolyzed by rat lympocyte or mouse lymphoma cells
- 	Liver S-9 did not affect metabolite profile
- 	FSO3 is metabolically stable in vitro
- 	Hydrolysis rapid but no accurate rates in mammalian tissues needed for PBPK model development

## Hydrolysis of Sulfuryl Fluoride

- PBPK models require more specific data to accurately predict SO2F2 hydrolysis and FSO3 and F- dosimetry.
- 	- Also provides a better understanding of the *in vivo* kinetic data.
***Which tissues are responsible for hydrolysis of SO******2******F******2****** and FSO******3******?***
***What are the rates of hydrolysis in these tissues?***

## In vitro Methods

**Selected Tissues**
- Liver homogenates (rat and human donor)
- Classic metabolizing tissue
- Portal of entry tissues (perfused to remove blood)
- Lung homogenates (rat and human donor)
- Nasal tissues and nasal lavage fluid (rat)
- Whole blood (rat and human donor)
- Red blood cells (rat)
- Plasma (rat)
**SO****2****F****2**** and FSO****3**** Substrate Concentrations**
- 87 ppm or 870 ppm SO2F2 in headspace for 1 hr
- Results in 4 or 40 ppm concentration;
- Estimated blood levels from concentration that approximated a 30 or 300 ppm exposure to SO2F2 vapor (assuming 14% absorption)
- 4 or 40 ppm FSO3 incubated for 1 hr
**Analysis**
- Determine the levels of SO2F2, FSO3, and F after 1 hr.

## In vitro RESULTS:Loss of SO2F2 with various rat tissues and cell fractions

*In vitro* **RESULTS**:***Loss of SO******2******F******2****** with various rat tissues and cell fractions***

- Blood and its components were primarily responsible for loss of SO2F2.

## In vitro RESULTS:Hydrolysis of SO2F2 with Adult and PND 10 Rat Blood, and Adult Human Blood

## In vitro Summary 

- No loss of parent SO2F2 in saline-perfused nasal or lung tissues, or lung lavage fluid
- Minimal loss of parent SO2F2 in classic metabolizing tissues:  liver and lung
- 80% decrease in SO2F2 levels in presence of whole blood and red blood cell fraction
*In vitro* hydrolysis leads to _stoichiometric_ increase in FSO3 and F
- The secondary hydrolysis of FSO3 to fluoride did not occur *in vitro.*
-  No hydrolysis with blood, liver or lung tissue from adult rats or human donors, when added directly to these tissues.
- This is consistent with relatively slow *in vivo* hydrolysis of iv-dosed FSO3 in rats

## In vitro Conclusions 

- These *in vitro *studies, taken together with the *in vivo* studies, increase the understanding of the sites of hydrolysis of SO2F2
- The results of this *in vitro* study suggest that blood within the local respiratory tissues may be important for hydrolysis because no parent SO2F2 has been detected systemically following *in vivo *exposures up to 300 ppm
*In vitro* time-course experiments will be performed to determine the gas-liquid transfer and hydrolysis rate constants
- Improve hydrolysis rate estimates used in the current PBPK models for SO2F2, FSO3, and F estimation.