Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The results of the risk evaluation (see section 9 and 10 of CSR) indicate the need for restrictive risk management measures to control the risk for the dermal route of exposures, therefore we suggest to perform an in vitro skin absorption study (OECD 428) in order to have a more precise indication on undecylenic acid skin absorption as we used a maximum skin absorption factor of 100% for DNEL derivation via the dermal route. The risk evaluation will be therefore fine tuned and more relevant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Undec-10-enoic acid
EC Number:
203-965-8
EC Name:
Undec-10-enoic acid
Cas Number:
112-38-9
Molecular formula:
C11H20O2
IUPAC Name:
undec-10-enoic acid
Test material form:
solid
Radiolabelling:
yes

Test animals

Species:
other: Human skin see below study design

Administration / exposure

Type of coverage:
other: topic
Vehicle:
other: see below study design
Duration of exposure:
8 hours
Doses:
see below study design
No. of animals per group:
see below study design
Control animals:
no
Remarks:
see below
Details on study design:
Integrity of skin membranes

After placing the skin membranes in the diffusion cells (see section 4.3.2), membrane integrity was assessed. To this purpose, the permeability coefficient (Kp) for tritiated water was determined as follows: 200 µL saline (supplemented with 0.01% sodium azide) containing [3H]H2O (16.9 kBq.mL-1) was applied in the donor compartment of the flow-through diffusion cells. The compartments were covered with a glass slide. The receptor fluid consisted of saline (0.9% sodium chloride (w/v)) containing 0.01% sodium azide (w/v). Samples of the receptor fluid (ca. 1.8 mL per hour) were collected every hour up to three hours after application. Tritiated water remaining at the application site was then removed and the skin was dried with cotton swabs. Membranes were kept overnight to allow wash-out of the tritiated water from the skin.

Experimental design
10-Undecenoic acid was topically applied to the skin membranes according to the design below. The exposure time was 8 h and receptor fluid samples were collected from 0-24 h.

Test group A
Group size 8 b
Species Human
Total concentration 10-Undecenoic acid = 989 g.L-1
Mean dose 10-Undecenoic acid applied a =9588 ± 127 µg.cm-2

a A net volume of ca. 6.4 µL of the dose preparations was applied on each skin membrane (0.64 cm2)
b Two membranes from four donors

Dose formulation preparation

The dose formulations was prepared the day prior to application as follows:

-Test group A
-Amount of [14C]10-Undecenoic acid (a) ~0.015 MBq
-10-Undecenoic acid added =0.1757 g (~176 µL)
-Radioactive concentration measured /0.09 MBq.mL-1


a Dissolved in HPLC mobile phase (after purification; see 4.5.2). During evaporation, unfortunately part of the original amount of tracer left after purification was lost. Consequently, the anticipated radioactive dose was lower than anticipated.

For dose formulation A, [14C]10-Undecenoic acid, dissolved in HPLC mobile phase (after purification), was added to a brown glass vial. The solvent was evaporated under N2 gas until dry. The non-radiolabelled test material 10-Undecenoic acid was liquefied in a water bath, set at a temperature of maximally 40oC. When liquefied, the test compound was kept in a water bath at ca. 37oC. A volume of ca. 176 µL (0.1757 g) of 10-Undecenoic acid was added to the dried radioactive residue. The dose formulation was kept at ca. 38oC while mixing, until application. After application, the dose formulation was stored at 2-10ºC.


Test concentration and homogeneity check, and application :

The concentration and homogeneity of [14C]10-Undecenoic acid in the dose formulations was checked by taking random aliquots in triplicate before dosing. For homogeneity, a coefficient of variation lower than 10% was considered sufficient. Random aliquots, in triplicate, were taken again after dose application. Prior to dose application, the skin surface was dried. The dose preparations were applied with a pipette and subsequently spread evenly on the skin surface within the donor compartment using a glass rod (dose volume ca. 10 µL.cm-2). A slightly higher volume than 6.4 µL (i.e. 6.7 µL)1 was applied to account for the expected loss of material during the distribution over the skin surface. Thus, a net volume of approximately 6.4 µL was applied.

Collection of mass balance samples :

Twenty-four hours after application, the mass balance of the test substance was determined from the following samples: receptor fluid samples, skin wash, receptor compartment wash, donor compartment wash, tape strips, and digested skin.

¿ Receptor fluid samples were collected during the following intervals: 0-1 h, 1-2 h, followed by 2-h intervals until 24 hours after application.
¿ Skin wash: After an exposure period of 8 h, the unabsorbed test substance was removed from the application site using a mild soap solution (i.e. 3% Dove in water, warmed up to ca. 45oC; six rinses) followed by two rinses with water. The skin was dried with cotton swabs between each rinse and finally dried with two cotton swabs (i.e. in total ten cotton swabs were collected).
¿ 24 h after application, the diffusion cell was dismantled. Receptor compartments were washed with ethanol. Donor compartments were extracted with ethanol.
¿ Each skin membrane was tape stripped 15 times using D-Squame® Skin Sampling Discs (CuDerm Corporation) and a D-Squame pressure device. Tape strips were collected individually for further analysis. Tape stripping was discontinued in case the epidermis was ruptured.
¿ Skin membranes were digested in a 1.5 M KOH solution with 20% aqueous ethanol for at least 24 h.

Analysis:

Determination of radioactivity

The radioactivity in the study samples was determined by liquid scintillation counting (LSC) using a Canberra Packard Tricarb 3100TR or Tricarb 3110TR scintillation counter as follows:

Dose formulations Ultima Gold™ (Packard) scintillation liquid was added to aliquots of the dose formulation taken just before and directly after dosing, to the glass rods that were used as spreading aid and to pipette tips used for application. Samples were measured by LSC.

Receptor fluid Ultima Gold™ scintillation liquid was added directly to the collected receptor fluid and samples were measured by LSC.

Skin wash The cotton swabs were pooled per skin membrane and extracted with ethanol for at least 24 h at ambient temperature. An ultra-sonication step was used to facilitate the extraction. Ultima Gold™ scintillation liquid was added to weighed aliquots of the extracted cotton swabs and samples were measured by LSC.

Donor compartment The samples were extracted for at least overnight at ambient temperature and sonicated. Subsequently, the samples were mixed with Ultima Gold™ scintillation liquid and measured by LSC.

Receptor compartment Ultima Gold™ scintillation liquid was added directly to the receptor compartment wash and samples were measured by LSC.

Tape stripping Ultima Gold™ scintillation liquid was added directly to individual tape strips for at least overnight extraction and samples were measured by LSC.

Skin membranes Hionic Fluor™ (Packard) scintillation liquid was directly added to the digested skin membranes and samples were measured by LSC.

Blank samples The appropriate scintillation liquid was added to blank receptor fluid samples, blank solution used for digestion of the skin membranes, blank skin wash samples and blank tape strips.

Radio-HPLC analysis of [14C]10-Undecenoic acid

The radiochemical purity of [14C]10-Undecenoic acid in the dose formulation was determined by radio-HPLC analysis according to the following method:

Column Inertsil ODS-2, 250 ¿ 4.6 mm, 5 µm
Mobile phase A: demineralised water + 0.01% formic acid (FA) B: acetonitrile + 0.01% FA
Gradient Time (min) 0 15 30 35 40

Column temperature B (%)
ambient 40 55 55 95 95
Flow rate 1.0 mL.min-1
Detection online radioactivity (¿-ram)
UV detection ¿ = 210 nm

Calculation and interpretation of the results

¿ The cumulative absorption of test substance equivalents was calculated from the receptor fluid samples by the following equation:
¿ Cumulative DPMT = DPMT + ¿(DPMT-1 ... DPM1)
¿ DPMT : radioactivity at sampling time T
¿ DPMT-1 : radioactivity at the sampling time preceding T
¿ DPM1 : radioactivity at the first sampling time
For each receptor fluid sample, background values were subtracted.

¿ The permeability coefficient or Kp value for tritiated water [cm.h-1] was calculated as follows:
¿ Kp = flux constant [µg.cm-2.h-1] / applied concentration [µg.cm-3]
¿ Using the program Microsoft Excel, the ‘slope’ and ‘intercept’ of the virtual line through the linear portion of the absorption curve was calculated. A straight line was mathematically represented by the formula “y = slope × x + intercept”, in which y represents the cumulative absorption and x represents time. This formula was used to calculate the maximal flux and the lag time from the mean values of the skin preparations:
¿ Maximal flux = slope
¿ Lag time = - intercept / slope
¿ For each test group, the following data was calculated:
¿ Percentage of the applied dose reaching the receptor fluid
¿ Amount of the test substance reaching the receptor fluid
¿ RATA: the amount in the receptor fluid at each time point, relative to the total cumulative amount reaching the receptor fluid over 24 h
¿ Recovery of the test substance (% of applied dose) in each compartment
¿ Mass balance of the test substance
¿ The total absorption (absorbed dose): the amount of compound-related radioactivity present in the receptor fluid, the receptor compartment wash, and the skin (excluding tape strips)
¿ The potentially absorbed dose: the amount of compound-related radioactivity present in the receptor fluid, the receptor compartment wash, the skin, and the stratum corneum (except for the first 2 tape strips)

¿ Limits of detection (LoD) for radioactivity in receptor fluid and tape strip samples were used if the radioactivity in the measured sample was below LoD. The LoD was calculated according to the following formula: (v(counting efficiency × average background × counting time)/counting time)/counting efficiency × 3 + average background, where the counting efficiency was 90% (0.9) and the counting time was at least 2 min.
¿ If more than 50% of the amount of test substance in the receptor fluid and/or tape strips samples of a replicate was below the LoD, the cumulative absorption and/or total (cumulative) value for this replicate was presented as ‘< calculated value’.
¿ If more than 50% of the amount of test substance in the samples contributing to the (potentially) absorbed dose was below the LoD, the (potentially) absorbed dose was presented as ‘< calculated value’.
¿ If more than 50% of the replicates showed values below the LoD, the mean value was presented as ‘< calculated value’.




Details on in vitro test system (if applicable):
4.3.1 Preparation of skin membranes

Human skin membranes were prepared from frozen skin samples, present at Triskelion. Human skin, derived from the breast and abdomen, was obtained from four female donors after surgery.

In agreement with the hospital, only skin tissue for which informed consent was given by the donor, was provided to Triskelion. The transportation of the skin to the laboratory was carried out on ice. Subcutaneous fat was removed and the skin was stored at <-18 °C until use.

Upon thawing, the skin was cut to a target thickness of ca. 0.2-0.4 mm (i.e. split-thickness skin membranes) using a Dermatome (25 mm, Nouvag GmbH, Germany). The thickness of all skin preparations was measured with a digimatic micrometer (Mitutoyo Corporation, Japan) and is presented in Appendix 1.

Flow-through diffusion cells and receptor fluid

Approximately 20 h before the start of exposure to the test preparation, the split-thickness skin membranes were placed in 9 mm flow-through automated diffusion cells (PermeGear Inc.,
Riegelsville, PA, USA) to hydrate the skin. The actual mean skin surface temperature was 32 ± 1ºC and exposure was at ambient humidity. Following application of the test preparations, the actual temperature was recorded at 15-minute intervals during the study in one diffusion cell containing a non-exposed skin membrane.



The receptor fluid was pumped at a flow rate of ca. 1.8 mL.h-1 and consisted of phosphate buffered saline (PBS) containing 0.01% sodium azide, (w/v)) supplemented with 6% PEG (polyoxyethylene-20-oleyl ether, v/v), pH ca. 7.2.

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
Integrity of skin membranes

Prior to the determination of the percutaneous absorption of 10-Undecenoic acid, the permeation coefficient (Kp) for tritiated water was determined in human skin membranes.

Skin membranes with a Kp value below the cut-off value of 2.5×10-3 cm.h-1 were selected for the study. The individual data of the absorption of tritiated water through the skin preparations are given in Appendix 1.

Receptor fluid solubility

The solubility of 10-Undecenoic acid in water is reported to be 73.7 µg.mL-1 (at 30oC, see section
4.2.1 for reference). PBS containing 0.01% sodium azide (w/v), was supplemented with 6% PEG (w/v) to further improve the solubility of the test substance in the receptor fluid. In the present study, the maximum absorption of 10-Undecenoic acid into the receptor fluid was 375 µg (i.e. 587 µg.cm-2, replicate A-2) in ca. 40 mL over 24 h, i.e. 9.4 µg.mL-1. Therefore, the solubility in the receptor fluid was considered sufficient.

Furthermore, in the flow-through cells used, the volume of the receptor fluid in the receptor chamber beneath the skin was ca. 0.2 mL, which at a flow rate of ca. 1.8 mL.h-1, was replenished continuously (9 times per hour). Thus, it was assured that the rate of diffusion into the receptor fluid did not become a rate-limiting step (i.e. sink conditions were maintained).



Total recovery:
The mean absorption of 10-Undecenoic acid, applied as neat compound, into the receptor fluid over the 24 h study duration was 412 µg.cm-2, representing 4.31% of the applied dose. The mean maximal flux for the absorption of 10-Undecenoic acid through human skin was
34.7 µg.cm-2.h-1 and the lag time was 4.0 h.

The mean total absorption, defined as the compound-related radioactivity present in the receptor fluid, the receptor compartment wash and the skin membranes (excluding tape strips) was 5.52 ± 1.12% of the applied dose. The mean potentially absorbed dose, which is defined as the compound-related radioactivity present in the receptor fluid, the receptor compartment wash, the skin membranes and the stratum corneum (except for the first 2 tape strips) was 6.12 ± 1.19% of the applied dose.

The mean recovery of 10-Undecenoic acid in human skin was 99.3 ¿ 1.5% of the applied dose.

Less than 75% of the absorption of 10-Undecenoic acid in the receptor fluid over 24 hours occurred within half of the study duration (i.e. 12 hours). For risk assessment, in agreement with the EFSA Scientific Opinion behind the revision of the Guidance Document on Dermal Absorption (2012), it is considered appropriate to include the tape strips (except the first 2 tape strips) in the calculations of the total absorption values (i.e. use the potentially absorbed dose).

Based on the new EFSA guidance on dermal absorption (2017), the mean potentially absorbed should be corrected to account for variability. Based on the number of replicates (i.e. n=8), a multiple (i.e. 0.84) of the standard deviation is added to the mean value. The mean potentially absorbed dose is thus calculated as 7.1%.
Percutaneous absorption
Time point:
24 h
Dose:
9588 ± 127
Parameter:
other: [µg.cm-2]
Absorption:
7.1 %
Remarks on result:
other: In vitro Human skin absorption

Applicant's summary and conclusion

Conclusions:
The mean potentially absorbed dose of Undecylenic acid though in vitro Human skin is thus calculated as 6.1%.
Executive summary:

The study was designed to examine the invitro percutaneous absorption of 10-Undecenoicacid (or Undecylenic acid) through human skin membranes. The test substance was tested as neat compound, which represents the maximal concentration possible when handling the neat compound. Under realistic in-use conditions the test item is handled at high temperatures; therefore, the test substance was applied in its liquid form,i.e.at a temperature ofca.37oC. The objective of the study was to elucidate the extent of percutaneous absorption of the compound-related radioactivity. The contact time was 8 hours,i.e.a normal working day and the post exposure time was 16 hours. In addition to the amount of radioactivity in the receptor fluid, the residues remaining in/on the skin membranes and in the stratum corneum(16 h post exposure) were determined. The study was performed in flow-through diffusion cells.

The mean absorption of 10-Undecenoic acid, applied as neat compound, into the receptor fluid over the 24 h study duration was 412 µg.cm-2, representing 4.31% of the applied dose. The mean maximal flux for the absorption of 10-Undecenoic acid through human skin was 34.7 µg.cm-2.h-1and the lag time was 4.0 h.

 The mean total absorption, defined as the compound-related radioactivity present in the receptor fluid,the receptor compartment was hand the skin membranes(excludingtapestrips) was 5.52 ± 1.12% of the applied dose. The mean potentially absorbed dose, which is defined as the compound-related radioactivity present in the receptor fluid, the receptor compartment wash,the skin membranes and the stratumcorneum(except for the first 2 tape strips) was 6.12 ± 1.19% of the applied dose.

 The mean recovery of 10-Undecenoic acid in human skin was 99.3±1.5% of the applied dose.

 Less than 75% of the absorption of 10-Undecenoic acid in the receptor fluid over 24 hours occurred within half of the study duration (i.e. 12 hours). For risk assessment, in agreement with the EFSA Scientific Opinion behind the revision of the Guidance Document on Dermal Absorption(2012),it is consideredappropriate to include the tape strips(except the first 2 tape strips) in the calculations of the total absorption values (i.e.use the potentially absorbed dose).

 Based on the new EFSA guidance on dermal absorption (2017), the mean potentially absorbed should be corrected to account for variability. Based on the number of replicates (i.e.n=8), a multiple (i.e.0.84) of the standard deviation is added to the mean value. The mean potentially absorbed dose is thus calculated as 7.1%.