Sodium 2-hydroxyethanesulphonate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

Parallel Artificial Membrane Permeation Assay (PAMPA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2022-2023

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

other: Passive permeation

Qualifier:

no guideline followed

Principles of method if other than guideline:

The passive permeation of SI was assessed in the Parallel Artificial Membrane Permeation Assay (PAMPA; Kansy et al. (1998) J. Med. Chem. 41, 1007).
Permeation experiments were carried out in a Multiscreen 96 well tray (donor) covered by a 96-well Multiscreen Immobilon (acceptor). The donor plate was filled with test compounds and reference compounds. Three refence compounds were included (Ceftriaxone, Guanabenz, Carbamazepine), each known for their low, medium and high permeability, respectively. The transport study was initiated by applying 150 µL PBS-buffer to the acceptor plate. After 15 – 16 h of diffusion at room temperature, the contents of the acceptor and donor plate were collected and quantified using LC-MS detection. The permeability of SI was expressed as flux% and recovery%.

GLP compliance:

no

Type:

other: Flux % (with lipid layer)

Results:

0%

Type:

other: Flux % (without lipid layer)

Results:

107.8%

Type:

other: Mean Recovery % (with lipid layer)

Results:

97.2%

Type:

other: Mean Recovery % (without lipid layer)

Results:

131.1%

Details on absorption:

For SI, no permeability across lipophilic lecithin membranes was observed (0% flux).

Recoveries, i.e. the sum of test item recovered from the donor and acceptor compartment were high in presence (97.2%) or absence (131.1%) of lipid layer. Furthermore, free flux of SI through the plates without lipid layer was observed. Therefore, unspecific binding properties of SI towards the test materials / filter device or chemical instability is unlikely and SI can be classified according to the pre-defined classification scheme as low permeable.

Recovery values for internal controls ceftriaxone, guanabenz and carbamazepin were high (=102%) and permeabilities of reference items were classified according to the pre-defined classification scheme (ceftriaxone showed a low permeation with 0.02% flux, guanabenz a medium permeation with 53% flux and carbamazepine a high flux rate of 106%).

The validity of the experiment was confirmed by a post experimental membrane integrity test (Lucifer yellow permeation), which evidenced the integrity of the filter membrane and lipid layer (i.e. flux rates < 5%).

Table 4 - PAMPA permeation of test and reference items (n=3)

Test / reference items	condition	Flux %	SD	%CV	Mean Recovery %	SD	Comments
SI	With lipid layer	0.0	0.0	n.a.	97.2	8.3	Low permeable
	Without lipid layer	107.8	16.0	14.9	131.1	20.7	Control
Ceftriaxone	With lipid layer	0.02	0.02	89.6	102.1	6.2	Low permeable reference
Guanabenz		53.3	3.7	7.0	176.0	7.1	Medium-high permeable reference
Carbamazepine		105.7	7.4	7.0	148.5	11.2	High permeable, reference accepted

 

Conclusions:

Based on the results of a parallel artificial membrane permeation assay, the flux of SI was concluded to be 0%. This indicates that SI is not able to passively permeate a lipid layer, and it can therefore be considered as a low permeable compound.

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 July 2022 - 29 October 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 28. Guidance Document for the Conduct of Skin Absorption Studies

Version / remarks:

2004

Deviations:

no

Principles of method if other than guideline:

The dermal uptake value used for risk assessment purposes was calculated based on the Guidance on Dermal Absorption (EFSA Journal, 2017, 15(6): 4873).

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

Carbon-14 is the isotope of choice in metabolic studies. The position of labelling is one which is considered to be metabolically stable.

Species:

human

Sex:

not specified

Details on test animals or test system and environmental conditions:

Samples of full-thickness human skin (abdominal) were obtained from four female donors aged 33 to 55 years old. Skin samples were obtained from Tissue Solutions Limited and BioIVT. The samples arrived at Charles River deep frozen on dry ice and were stored in a freezer set to maintain a temperature of -20°C until used in the study.

Type of coverage:

open

Vehicle:

other: Ultrapure water

Duration of exposure:

8 hours

Doses:

Single dose

No. of animals per group:

12 samples obtained from 4 different donors (3 samples per donor)

Details on in vitro test system (if applicable):

PREPARATION OF SPLIT-THICKNESS SKIN
Human skin samples were removed from -20°C storage and allowed to thaw at ambient temperature. The thickness of the full-thickness skin membranes was measured using a micrometer. Split-thickness membranes were prepared by pinning the full-thickness skin, stratum corneum uppermost, onto a raised cork board and cutting with an electric dermatome (Zimmer®) at a setting equivalent to 200-400 µm depth. The thickness of the membranes was measured using a micrometer. Membranes were then wrapped in foil, placed into a self-sealing bag and stored in a freezer, set to maintain a temperature of -20°C, for a maximum period of two months.

DIFFUSION CELL APPARATUS
Equipment and Settings:
An automated flow-through diffusion system (McGregor/Toner) was used. The flow-through diffusion cells were placed in a steel manifold heated via a circulating water bath set to maintain the skin surface temperature at 32°C ± 1°C. The cells were connected to multi-channel peristaltic pumps from their afferent ports with the receptor fluid effluent dropping via fine bore tubing into scintillation vials on a fraction collector. The surface area of exposed skin within the cells was 0.64 cm2. The receptor chamber volume was 0.33 mL.

Receptor Fluid:
The receptor fluid chosen for use in this study was phosphate buffered saline receptor fluid containing ca 5% (v/v) human serum, amphotericin B (10 mL of a 250 µg/mL solution per liter of receptor fluid), streptomycin (ca 0.1 mg/mL) and penicillin (ca 100 units/mL).

The maximum concentration of test item in the receptor fluid was estimated at a level assuming 10 x 10% absorption in 1 h following application of the concentrate formulation to skin. Radiolabelled test item was radiodiluted to an appropriate level. Then ca 60.8 mg (Sodium Isethionate) was transferred into each of two 25 mL volumetric flasks. Receptor fluid was added to the first flask, and solvent of known high test item solubility (positive control) was added to the second flask. The solutions were mixed for ca 1 h at ca 32°C and then centrifuged at ca 2000 g for ca 5 min. A minimum of three replicate aliquots were taken from the resultant supernatant and were analysed by liquid scintillation counting. The acceptable coefficient of variation (CV) was <5% and the acceptability of CV >5%.

Flow-Through Diffusion Cell Preparation:
Split-thickness skin samples were removed from -20°C storage and allowed to thaw at ambient temperature. Sections of human split-thickness skin membrane, ca 1.5 x 1.5 cm, were cut and positioned on the receptor chamber of the diffusion cell. The donor chamber was tightened into place with screws and the prepared cells were then placed in the heated manifold and connected to the peristaltic pump. An equilibration period of ca 15 min was allowed while receptor fluid was pumped through the receptor chambers at 1.5 mL/h ± 0.15 mL/h.

Application of Test Preparation to Human Skin:
Sodium [14C]-Isethionate in ultrapure water was applied evenly over the surface of the exposed skin of 12 split-thickness samples of human skin using a Rainin MR25 positive displacement pipette set to deliver ca 6.4 µL (10 µL/cm2). The donor chambers of the cells were not occluded. Seven representative aliquots of Sodium [14C]-Isethionate in ultrapure water were dispensed into vials at the time of dosing, mixed with Scintanol and analysed by liquid scintillation counting.
The results of representative aliquots for Sodium [14C]-Isethionate in ultrapure water are:
- Target test item concentration: 570 g/L
- Mean test concentration: 571 g/L
- CV test item concentration: 0.38 %

Time point:

8 h

Concentrate / Dilution:

dilution

Dose:

10 µL (570 g/L)/cm2

Parameter:

percentage

Absorption:

0.09 %

Remarks on result:

other: Potentially Absorbable Dose

Time point:

8 h

Concentrate / Dilution:

dilution

Dose:

10 µL (570 g/L)/cm2

Parameter:

percentage

Absorption:

0.17 %

Remarks on result:

other: Calculated value for risk assessment purposes

Conversion factor human vs. animal skin:

n.a.

Overall, the absorption profiles looked similar for the majority of samples, with absorption of Sodium [14C]-Isethionate increasing to 24 h post dose. For Cell 2, the trend was similar however the absorption profile was significantly higher. The mean absorption calculated throughout the course of the experiment was above the LoRM for all timepoints; however, multiple individual cell values were below the limit of reliable measurement (LoRM) at various timepoints throughout the experiment. Cell 2 was rejected as skin damage at dosing was suspected due to an abnormally high absorption profile compared to its donor group.

The mass balance for most individual samples was >95% and <110%, except Cell 1 (93.73%). Cell 1 was included in results as total absorbed dose was comparable to other cells. The following results are provided as mean values (n = 11).

The mean mass balance was 96.73% of the applied dose at 24 h post dose. The majority of the applied dose was washed off at 8 h post application (3.97%, 92.64% and <0.01% recovered in the skin wash, tissue swab and pipette tips, respectively). At 24 h post dose, a further 0.02% was recovered in the donor chamber wash. The material recovered in the donor wash was almost certainly material that was dislodged from the skin during the washing procedure. Therefore, the total dislodgeable dose was 96.63% of the applied dose. The mean total unabsorbed dose was 96.66% of the applied dose. This consisted of the dislodgeable dose, unexposed skin (<0.01%) and the radioactivity associated with the stratum corneum (0.03%). The absorbed dose (0.05%) was the sum of the receptor fluid (0.04%), receptor chamber wash (0.01%) and receptor rinse (<0.01%). The exposed skin (0.02%) was the sum of the epidermis (0.01%) and dermis (0.01%). Dermal delivery (0.07%) was the sum of the absorbed dose and the exposed skin. 

More than 75% of the absorption occurred within the first half of the experiment. As defined in the EFSA Scientific Opinion – Guidance on Dermal Absorption 2017; 15(6): 4873, this result suggests complete absorption. However, the majority of the receptor fluid values obtained were below the limit of reliable measurement, so potentially absorbable dose was calculated. The potentially absorbable dose was 0.09%.
The distribution of Sodium [14C]-Isethionate, by mass, at 24 h post dose.
The mass balance, total dislodgeable dose, unabsorbed dose, absorbed dose and dermal delivery and potentially absorbable dose were 5525, 5519, 5521, 2.62, 3.84 and 5.01 μg equiv./cm2, respectively. 

 

Post dose washing efficiency (8 hours):

Overall, most of the applied dose recovered in tissue swabs 1-5 was removed with tissue swab 1. Thereafter, there was a general decrease in the percentage of the applied dose removed with tissue swabs 2-5. In the final tissue swab, a mean of <0.01% of the applied dose was detected, indicating that the washing process was efficient at removing the applied dose.

 

Results in tabular format

	Recovery (%)		Recovery (ug equiv/cm2)
	Mean	SD	Mean	SD
Stratum Corneum*	0.021	0.051	1.173	2.913
Epidermis	0.010	0.022	0.592	1.245
Dermis	0.011	0.032	0.627	1.834
Receptor Fluid	0.046	0.038	2.622	2.159
PAD^	0.088	0.124	5.013	7.109
DD~	0.067	0.077	3.840	4.401
Mass Balance	96.73	1.81	5525.20	103.17

*Tape strips 3-20; ^Potentially absorbable dose ~Dermal Delivery

 

 

Conclusions:

Using the results of an in vitro skin absorption study with sodium [14C]-isethionate, performed according to OECD guideline 428 and GLP principles, the dermal absorption value for risk assessment for SI was calculated to be 0.17% (based on the Guidance on Dermal Absorption (EFSA Journal, 2017, 15(6): 4873)).

Executive summary:

An in vitro skin absorption study was performed according to OECD guideline 428 and GLP principles with sodium [14C]-isethionate (SI). A total of 12 samples of human split-thickness skin membranes obtained from 4 different donors were dosed topically with Sodium [14C]-Isethionate in ultrapure water.

The mean mass balance was 96.73% of the applied dose at 24 h post dose. The majority of the applied dose was washed off at 8 h post application (3.97%, 92.64% and <0.01% recovered in the skin wash, tissue swab and pipette tips, respectively). At 24 h post dose, a further 0.02% was recovered in the donor chamber wash. The material recovered in the donor wash was almost certainly material that was dislodged from the skin during the washing procedure. Therefore, the total dislodgeable dose was 96.63% of the applied dose. The mean total unabsorbed dose
was 96.66% of the applied dose. This consisted of the dislodgeable dose, unexposed skin (<0.01%) and the radioactivity associated with the stratum corneum (0.03%). The absorbed dose (0.05%) was the sum of the receptor fluid (0.04%), receptor chamber wash (0.01%) and receptor rinse (<0.01%). The exposed skin (0.02%) was the sum of the epidermis (0.01%) and dermis (0.01%). Dermal delivery (0.07%) was the sum of the absorbed dose and the exposed skin.

The total absorbed dose, dermal delivery, potentially absorbable dose and mass balance were 0.05% (2.62 μg equiv./cm2), 0.07% (3.84 μg equiv./cm2), 0.09% (5.01 μg equiv./cm2) and 96.73% (5525 μg equiv./cm2) of the applied dose, respectively.

The dermal absorption to be used for risk assessment purposes was calculated based on the Guidance on Dermal Absorption (EFSA Journal, 2017, 15(6): 4873) as 0.17%.

Description of key information

Absorption values

An in vitro skin absorption study was performed with sodium [14C]-isethionate, according to OECD guideline 428 and GLP principles. Based on the results it is concluded that the value for dermal absorption for SI for risk assessment purposes is 0.17%. 

The oral absorption value was based on theoretical assessment. Although the moderate molecular weight of the chemical (ca. 150) and its high water solubility (534 g/L at 20°C) would favor uptake, its low log P value (-4.6) indicates that SI is strongly hydrophilic. This implies that uptake via passive diffusion will be limited (if any) and no uptake will take place via micellular solubilisation. In addition, it is expected that after oral uptake SI will dissociate to a sodium ion (Na+) and a counter ion, which will have a sulphonate group (SO3-). It is generally thought that ionized substances do not readily diffuse across biological membranes. This conclusion is confirmed by the results of a Parallel Artificial Membrane Permeation Assay (PAMPA) performed with SI, in which no penetration of SI through an artifical membrane was observed.

Taking all these factors into consideration, it is concluded that the oral uptake of SI will not exceed 50% and this value is therefore used in the risk assessment. 

 

In vitro ADME studies

The in vitro ADME characteristics of Sodium isethionate (SI), i.e. its chemical stability in aqueous solution, metabolic stability with human hepatocytes, potential to penetrate an artificial membrane (PAMPA), stability in plasma, blood and buffer, binding to plasma proteins and binding to red blood cells (blood partitioning) was studied. SI was shown to be stable in phosphate buffered saline (PBS, pH 7.4) at 37°C during 16 h of incubation. No degradation occurred. Metabolic stability in hepatocytes was tested by incubation of SI at 0.5, 2 and 10 μM with cryopreserved primary hepatocytes from human. SI was shown to be stable during the investigated time, resulting in ≥ 85.2% remaining parental compound, respectively (after 120 min of incubation. The PAMPA method was applied to determine the passive permeation of the test item. Using the standard PAMPA assay procedure, no passive permeation of the test item SI was observed (flux rate of 0%), while the recovery of the compound was high (97%), indicating that non-specific binding to the test system is not an issue. Moreover, SI was highly stable with human plasma. No degradation occurred during 4 h. In blood, test item was relatively stable, some degradation occurred with 77.4 ± 4.9% remaining compound after 4 h of incubation. SI showed low affinity for plasma proteins, indicated by a PPB value of 21.7 ± 8.5. Furthermore, recovery and stability of SI were determined to be high (93.6 and 99.9% remaining compound, respectively). Blood partitioning of test item, i.e. assessment of the distribution of a compound in red blood cells and plasma, was performed using fresh whole human blood. The KRBC/Plasma ratio in the range of 1 (i.e. 1.12) implicates low affinity of SI for red blood cells.

This information was used in the PBK modelling (attached in section 13.2).

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

0.17

Absorption rate - inhalation (%):

100