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Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Feb 2020 to 13 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
adopted in April 2004
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2,6-diisopropylphenyl)carbodiimide
EC Number:
218-487-5
EC Name:
Bis(2,6-diisopropylphenyl)carbodiimide
Cas Number:
2162-74-5
Molecular formula:
C25H34N2
IUPAC Name:
N,N'-bis(2,6-diisopropylphenyl)carbodiimide
Details on test material:
Radiolabelled Test Item:
- Chemical name: [phenyl-U-14C]Bis(2,6-diisopropylphenyl)carbodiimide
- Name of test material (as cited in the study report): [14C]Bis(2,6-diisopropylphenyl)carbodiimide
- Batch (Lot) Number: 10312JYC033-3
- Expiry Date: Not indicated
- Physical Description: White solid
- Radiochemical Purity: 99.8%
- Chemical Purity: 99.6%
- Specific Activity: 5.62 MBq/mg
- Storage Conditions: In freezer (≤ -15°C)
- Supplier: Selcia
- Total activity received: 42.2 MBq
- Test item handling: No specific handling conditions required

Non-radiolabelled Test Item:
- Chemical name (IUPAC, synonym or trade name): N,N'-bis[2,6-bis(propan-2-yl)phenyl]methanediimine / Bis(2,6-diisopropylphenyl)carbodiimide / Stabilizer 7000
- Name of test material (as cited in the study report): Bis(2,6-diisopropylphenyl)carbodiimide
- Physical Description: White crystalline powder (determined by Charles River Den Bosch)
- Batch (Lot) Number: 11180058/002
- Purity/Composition: 100.0%
- Storage Conditions: In refrigerator (2-8°C)
- Test item handling: No specific handling conditions required
- Stability at higher temperatures: Not indicated
Radiolabelling:
yes

Test animals

Species:
other: Human Skin Donors
Sex:
female

Administration / exposure

Type of coverage:
open
Vehicle:
other: neat compound and PE polymer powder
Duration of exposure:
The exposure period was terminated at 8 h after dosing. At 24 hours post dose, i.e. after 16 hours monitoring period, each diffusion cell was dismantled and the skin removed.
Doses:
- Nominal doses: 100% and 1% (mixture with PE)
- Actual doses: 96.8% and 0.97%
- Actual doses calculated as follows: Individual doses were accurately determined by collecting (weighed) mock doses at the time of dosing.
- Dose volume: 5 mg/cm²
- Rationale for dose selection: Exposure to the test item can occur during handling of the pure compound and during processing, e.g. the incorporation into a polymer matrix. Concentrations of bis(2,6-diisopropylphenyl)carbodiimide to be tested within the scope of this study represent the neat product and a relevant in-use concentration. Due to the use pattern of the test item, a PE polymer matrix was selected to generate a relevant and technically feasible test mixture representing a worst-case sample of the test item. The dose volume was selected based on the maximum recommended application dose for solids (5 mg/cm², OECD Guideline 428, Section 16). Moistening the test preparations with water is a realistic exposure, as this will allow better contact between the skin surface and the solid, and is intended to mimic the operator sweating during exposure to the test item. This is recommended for solids in the OECD Guidance Document No. 28 (Section 52).
No. of animals per group:
8 samples of skin obtained from at least 4 different donors per dose
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions:
Test Preparation 1:
First, a mixture of non-radiolabelled bis(2,6-diisopropylphenyl)carbodiimide and [14C]bis(2,6-diisopropylphenyl)carbodiimide was prepared. An amount of 1.00039 g of nonradiolabelled bis(2,6-diisopropylphenyl)carbodiimide was weighed into a pre-weighed glass vial. Approximately 5 MBq of radiolabelled test item was added by adding 0.0956 g (approximately 122 μL) from the stock solution Acetone was added until all test item was dissolved. The solvent was then evaporated overnight under a gentle nitrogen gas stream until a near dry powder remained. From the final weight of the vial, a concentration of 96.7% bis(2,6-diisopropylphenyl)carbodiimide was calculated. The remaining powder was grinded and mixed using a spatula. By radioactivity, a concentration of 316097 ± 3751 DPM/mg was determined. The test item was homogeneously distributed within the test preparation with a CV of 1.2%.
Test Preparation 2: First, 9.03 mg bis(2,6-diisopropylphenyl)carbodiimide and 1.00030 g PE powder was weighed into a pre-weighed glass vail. Approximately 5.62 MBq of radiolabelled test item was added by adding 0.1089 g (approximately 139 μL) from the stock solution. Acetone was added for mixing and the solvent then evaporated overnight under a gentle nitrogen gas stream until a near dry powder remained. From the final weight of the vial, a concentration of 0.87% (non-radiolabelled) bis(2,6-diisopropylphenyl)carbodiimide was calculated. The remaining powder was mixed using a spatula and stored at ambient conditions, shielded from direct light until use on the following day. By radioactivity, on the day of application, a radioactive concentration of 302421 ± 111501 DPM/mg was determined. The test item was homogeneously distributed within the test preparation with a CV of 3.7%.

APPLICATION OF DOSE:
A single approximate 5 mg dose of the test preparations (5 mg/cm²) was applied to a minimum of 8 samples of skin obtained from at least 4 different donors. Prior to dose application, the skin surface was dried. The dose formulation was applied to the stratum corneum surface of the skin by weighing the target amount on a small piece of weighing paper, which was then gently ‘poured’ onto the skin surface. Distribution over the skin surface was done by gently tapping against the diffusion cell. Following application, the powder was wetted using water (approximately 1 μL/mg formulation applied, i.e., a 1:1 mixing with water) to mimic sweat on the skin surface or occlusive conditions under clothing. The ‘paste’ was distributed evenly over the skin surface using a glass rod. The donor chamber of the cells was left open to the atmosphere.
Individual doses were accurately determined by collecting (weighed) mock doses at the time of dosing. The dosing procedure was repeated for all other test preparations.

VEHICLE
- Justification for use and choice of vehicle: Due to the use pattern of the test item (exposure to the test item can occur during handling of the pure compound and during processing, e.g. the incorporation into a polymer matrix), a PE polymer matrix was selected to generate a relevant and technically feasible test mixture representing a worst-case sample of the test item.
- Amount(s) applied (volume or weight with unit): please refer to details on test preparation 2
- Lot/batch no.: P28D047
- Purity: 100%

TEST SITE
- Preparation of test site: Prior to dose application, the skin surface was dried.
- Area of exposure: 1 cm²

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: The exposure period was terminated at 8 h after dosing. Commercial hand wash soap (50 μL) was applied to the skin and the soap gently rubbed onto the skin with a cotton swab. The skin was then rinsed with approximately 5 mL of a 2% (v/v) commercial soap solution. The soap solution was applied in aliquots (1 mL) and each aliquot was aspirated with a pipette. The skin was dried with a cotton swab. This process was repeated once.
- Time after start of exposure: The exposure period was terminated at 8 h after dosing.

SAMPLE COLLECTION
Collecting fractions of the receptor fluid at the following time intervals: 0-1, 1-2, followed by 2 h intervals until 24 h after dosing. At 24 h post dose, i.e. after 16 h monitoring period, each diffusion cell was dismantled and the skin removed.

ANALYSIS
- Method type(s) for identification: HPLC-UV-RAD for Determination of the Radiochemical Purity, all radioactivity measurements will be performed by LSC (Liquid scintillation counting)

Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: obtained from elective plastic surgery was purchased from Alphenyx
- Donor information:
Batch number: 19-06-80029, 19-10-80057, 19-03-80016, 19-09-21018
Sex: all Female
Age (yrs): 44, 29, 18, 63
Race: all Caucasian
Site: Breast, Abdominal, Breast, Breast
- Type of skin: Full-thickness skin
- Preparative technique: The skin was removed from the freezer and allowed to thaw to room temperature. The skin was cut to a thickness of ca. 200-400 μm using an electric dermatome. The thickness of the skin was confirmed using a micrometer. The split-thickness membranes was wrapped in aluminium foil and stored in a freezer (≤ -15 °C) for a maximum period of 1 year.
- Thickness of skin (in mm): ca. 200-400 μm
- Membrane integrity check: A tritiated water barrier integrity test was performed. The integrity of all human skin samples was within the acceptability criteria (absorption <1.6% of applied dose of tritiated water).
- Storage conditions: The skin was shipped on dry ice and stored in the freezer (≤ -15 °C) for a maximum period of 1 year
- Justification of species, anatomical site and preparative technique: Human skin has been chosen for the safety evaluation as this is the species that will be exposed to the test item during handling and use. Split-thickness skin membranes are an acceptable test system for predicting absorption of a test item.

PRINCIPLES OF ASSAY
- Receptor fluid: The receptor fluid chosen for use in this study was phosphate buffered saline (PBS) containing polyoxyethylene 20 oleyl ether (6%, w/v), sodium azide (0.01%, w/v), streptomycin (0.1 mg/mL) and penicillin G (100 units/mL). The pH was checked and adjusted to pH 7.4.
- Solubility of test substance in receptor fluid: The solubility of bis(2,6-diisopropylphenyl)carbodiimide in the receptor fluid was 18.2 ± 9.3 μg/mL.
- Flow-through system: The flow-through diffusion cells were positioned in a manifold heated via a circulating water bath to maintain a skin surface temperature of 32 ± 1 °C. The cells were connected to a multichannel peristaltic pump from their afferent ports. Effluent from the cells dropped into vials on a fraction collector via tubing. The surface area of exposed skin within the cells was 1 cm², with a receptor chamber of 0.25 mL. The peristaltic pump speed was adjusted to obtain a standard flow rate of 1.5 mL/h.



Results and discussion

Signs and symptoms of toxicity:
no effects
Dermal irritation:
no effects
Absorption in different matrices:
- Skin wash: 100%: 0.11 ± 0.23 % applied dose, 1%: 0.13 ± 0.05 % applied dose
- Skin test site: 100%: 99.0 ± 9.5 % applied dose, 1%: 96.4 ± 9.3 % applied dose
- Skin, untreated site: 100%: 0.006 ± 0.008 % applied dose, 1%: 0.009 ± 0.014 % applied dose
- Receptor fluid, receptor chamber, donor chamber (in vitro test system):
Receptor fluid: 100%: <0.013 ± 0.004 % applied dose, 1%: 0.02± 0.01% applied dose
Receptor chamber: 100%: 0.004± 0.005 % applied dose, 1%: 0.002± 0.001% applied dose
- Stratum corneum (in vitro test system):
Stratum Corneum 1-2: 100%: 2.26 ± 5.89 % applied dose, 1% 0.48 ± 0.22 % applied dose
Stratum Corneum 1-20: 100%: 2.33 ± 5.90 % applied dose, 1%: 1.95 ± 0.76% applied dose
Total recovery:
- Total recovery: 100%: 101.5± 5.7 % applied dose, 1%: 98.6± 9.8 % applied dose
- Recovery of applied dose acceptable:
100%: Overall recovery slightly outside the OECD criterion of 100 ± 10%. However, the absorption data for this replicate were highly comparable to the other replicates. The higher recovery value is very likely caused by technical difficulties involved with accurately weighing low amounts of solid formulation.
1%: The absorption data obtained for those replicates showing a lower overall recovery were highly comparable to the replicates showing an adequate recovery (>95%). Thus, it can be concluded that any missing recovery is not associated with the absorbed fractions. Variation in the individual recovery values were the result of technical issues during application (i.e., some electrostatic interaction of the PE material) and difficulties involved with accurately weighing low amounts of solid formulation.
- Limit of detection (LOD): LoD rec fluid, receptor fluid wash and skin: 0.001% of the mean applied dose per sample. LoD ts: 0.0003% of mean applied dose per sample (100% dose); LoD ts: 0.0002% of mean applied dose per sample (1% dose)
- Quantification of values below LOD or LOQ: Values below LoD were considered as equal to LoD in the calculations and presented as ‘
Percutaneous absorptionopen allclose all
Time point:
24 h
Dose:
100 %
Parameter:
percentage
Absorption:
0.19 %
Time point:
24 h
Dose:
1 %
Parameter:
percentage
Absorption:
2.2 %

Applicant's summary and conclusion

Conclusions:
In conclusion, following topical application of [14C]bis(2,6-diisopropyl-phenyl)carbodiimide in test preparation 1 (actual concentration 96.8%) and test preparation 2 (actual concentration 0.97%) to human skin in vitro, the absorbed dose was 0.017% (0.76 μg equiv./cm²) and 0.021% (0.009 μg equiv./cm²) of the applied dose, respectively. The 8 h dislodgeable dose was 99.0% and 96.4% of the applied dose for test preparations 1 and 2, respectively, demonstrating consistently high decontamination of the skin. The total dislodgeable dose was 99.1% and 96.5% of the applied dose for test preparations 1 and 2, respectively.
The dermal delivery of [14C]bis(2,6-diisopropyl-phenyl)carbodiimide was 0.07% (2.87 μg equiv./cm²) of the applied dose for test preparation 1 and 0.16% (0.07 μg equiv./cm²) of the applied dose for test preparation 2.
Finally, the mass balance was 101.5% (4551 μg equiv./cm²) of the applied dose for test preparation 1 and 98.6% (44.9 μg equiv./cm²) of the applied dose for test preparation 2.
Based on the same EFSA Guidance, the absorption values should be corrected to account for variability. Based on the number of replicates, a multiple of the standard deviation is added to the mean value (e.g. 0.92 for 7 replicates and 0.84 for 8 replicates). The calculated dermal absorption value for bis(2,6- diisopropylphenyl)carbodiimide from test preparation 1 would then be 0.14 + (0.92×0.05) = 0.19% (rounded to two significant numbers as per EFSA Guidance). After correction to account for variability, the calculated dermal absorption value for bis(2,6-diisopropylphenyl)carbodiimide from test preparation 2 would then be 1.63 + (0.84×0.63) = 2.2%.
Executive summary:

The objective of this study was to determine the dermal absorption of the test Item bis(2,6-diisopropylphenyl)carbodiimide, following OECD Guideline 428 in compliance with GLP. Exposure to the test item can occur during handling of the pure compound and during processing, e.g., the incorporation into a polymer matrix. Concentrations of bis(2,6-diisopropylphenyl)carbodiimide tested within the scope of this study represented the neat product and a relevant in-use concentration. Due to the use pattern of the test item, a PE polymer matrix was selected to generate a relevant and technically feasible test mixture representing a worst-case sample of the test item.

Split-thickness human skin samples were mounted into flow-through diffusion cells (1 cm2). Phosphate buffered saline (PBS) containing polyoxyethylene 20 oleyl ether (PEG; 6%, w/v), sodium azide (0.01%, w/v), streptomycin (0.1 mg/mL) and penicillin G (100 units/mL) with the pH adjusted to 7.4 was used as receptor fluid. The skin surface temperature was maintained at 32 ± 1°C throughout the experiment.

A tritiated water barrier integrity test was performed. The integrity of all human skin samples was within the acceptability criteria (absorption <1.6% of applied dose of tritiated water). The test preparations were applied to human split-thickness skin membranes from four different donors and the cells were left open to the atmosphere. Test item stability during dosing was confirmed by high performance liquid chromatography (HPLC).

Absorption was assessed by collecting receptor fluid in hourly fractions from 0 to 2 h after dosing, then in 2 hourly fractions until 24 h after dosing. At 8 h after dosing, the exposure period was terminated by washing the skin surface with a concentrated commercial hand wash soap followed by rinsing with a dilute soap solution (2%, v/v) and drying the surface with a cotton swab. At 24 h after dosing, the diffusion cell was dismantled. The skin was then removed from the flow-through diffusion cells, the stratum corneum tape stripped and the skin divided into exposed and unexposed skin. All samples were analyzed by liquid scintillation counting.

For test preparation 1 (neat compound), the absorption of [14C]bis(2,6-diisopropylphenyl)carbodiimide into the receptor fluid within the first half of the study was 66 ± 9% of the total absorption into the receptor fluid at 24 h. As this is below 75% as defined in the EFSA Guidance on Dermal Absorption 2017, 15(6): 4873, the potentially absorbed dose is calculated as the sum of the radioactivity recovered in the receptor fluid, the receptor wash (sum of rinse and chamber wash), the exposed skin and the stratum corneum excluding tape strips 1 and 2, i.e., 0.14 ± 0.05%. Based on the same EFSA Guidance, the absorption values should be corrected to account for variability. Based on the number of replicates, a multiple of the standard deviation is added to the mean value (e.g. 0.92 for 7 replicates and 0.84 for 8 replicates). The calculated dermal absorption value for bis(2,6 -diisopropylphenyl)carbodiimide from test preparation 1 would then be 0.14 + (0.92×0.05) = 0.19% (rounded to two significant numbers as per EFSA Guidance).

For test preparation 2 (1% mixture with PE) the absorption of [14C]bis(2,6-diisopropylphenyl)carbodiimide into the receptor fluid within the first half of the study was 55 ± 13% of the total absorption into the receptor fluid at 24 h. As this is below 75%, the potentially absorbed dose is calculated as described before, i.e., 1.63 ± 0.63%. After correction to account for variability, the calculated dermal absorption value for bis(2,6-diisopropylphenyl)carbodiimide from test preparation 2 would then be 1.63 + (0.84×0.63) = 2.2%.

In conclusion, following topical application of [14C]bis(2,6-diisopropyl-phenyl)carbodiimide in test preparation 1 (actual concentration 96.8%) and test preparation 2 (actual concentration 0.97%) to human skin in vitro, the absorbed dose was 0.017% (0.76 μg equiv./cm2) and 0.021% (0.009 μg equiv./cm2) of the applied dose, respectively. The 8 h dislodgeable dose was 99.0% and 96.4% of the applied dose for test preparations 1 and 2, respectively, demonstrating consistently high decontamination of the skin. The total dislodgeable dose was 99.1% and 96.5% of the applied dose for test preparations 1 and 2, respectively. The dermal delivery of [14C]bis(2,6-diisopropyl-phenyl)carbodiimide was 0.07% (2.87 μg equiv./cm2) of the applied dose for test preparation 1 and 0.16% (0.07 μg equiv./cm2) of the applied dose for test preparation 2. Finally, the mass balance was 101.5% (4551 μg equiv./cm2) of the applied dose for test preparation 1 and 98.6% (44.9 μg equiv./cm2) of the applied dose for test preparation 2.