N-1-naphthylaniline

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Sep 2020 to 19 Mar 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

Adopted: 13 April 2004

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-1-naphthylaniline

EC Number:

201-983-0

EC Name:

N-1-naphthylaniline

Cas Number:

90-30-2

Molecular formula:

C16H13N

IUPAC Name:

N-phenylnaphthalen-1-amine

Details on test material:

Radiolabelled Test Item:
- Chemical name: [phenyl-U-14C]phenyl-1-naphthalen-1-amine
- Name of test material (as cited in the study report): [phenyl-U-14C]PANA
- Batch (Lot) Number: 11172GXR004-4
- Expiry Date: Not indicated
- Physical Description: Pale brown solid
- Radiochemical Purity: 98.8%
- Chemical Purity: 99.1
- Specific Activity: 9.59 MBq/mg (259.1 μCi/mg)
- Supplier: Selcia

Non-radiolabelled Test Item:
- Chemical name (IUPAC, synonym or trade name): N-1-naphthylaniline (PANA)
- Name of test material (as cited in the study report): PANA
- Physical Description: Pink crystalline powder
- Batch (Lot) Number: 47/XII/18
- Best Before Date: 04 April 2021
- Purity/Composition: 99.88%
- Storage Conditions: Ambient

Radiolabelling:

yes

Test animals

Species:

other: Human Skin Donors

Administration / exposure

Type of coverage:

open

Vehicle:

other: neat compound, rubber matrix and base oil for lubricants

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. See any other information on material and methods for further details.

Doses:

- Nominal doses: 1 % and 5 % in base oil for lubricants, 100 % as neat powder and 0.5 % and 3.5 % in rubber matrix
- Actual doses: 1.04 % (w/v), 5.34 % (w/v), 100 % (w/w), 0.409 % (w/w), 3.17 % (w/w)
- Actual doses calculated as follows: Seven representative weighed discs per dose were collected throughout dosing, seven representative aliquots of test preparation 1 and 2 (1 and 5% in base oil for lubricants) were dispensed into vials at the time of dosing and seven representative aliquots of the test preparation 3 (100% w/w) were collected immediately after dosing. All representative aliquots were analysed by liquid scintillation counting.
- Dose volume: 10 µL/cm² for 1% and 5% in base oil for lubricants, 5 mg/cm² for the 100% neat powder and 1 disc per skin sample for the 0.5% and 3.5% in rubber matrix (diameter of 20 mm and thickness 1120-1220 µm and 1150-1470 µm, respectively)
- Rationale for dose selection: in line with the ECHA final decision, several small dosages representative of the in-use conditions were chosen

No. of animals per group:

A total of 12 samples of human split thickness skin membranes obtained from four different donors were used per test preparation.

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions:
After assessment of the radiochemical purity the radiochemical was diluted with non-radiolabelled PANA using ethanol (which was evaporated afterwards). The concentration and homogeneity of the mixture was assessed by liquid scintillation counting.
For test preparation 1 and 2, radiolabelled PANA was incorporated into a base oil for lubricants at a concentration of approx. 1 and 5 %, respectively.
For test preparation 4 and 5, radiolabelled PANA at a concentration of 0.5 and 3.5 % incorporated into a rubber disc by homogeneous mixing into a rubber mix. After mixing, the compounds are pressed into 1 mm thick mixture skins and discs with a diameter on 20 mm are punched out. Rubber disc containing 0.5 % and 3.5 % PANA respectively were applied to the skin samples.

APPLICATION OF DOSE:
Test preparation 1 and 2 were applied evenly over the surface of the exposed skin of 12 split thickness samples of human skin using a positive displacement pipette set to deliver ca 31.4 µL (10 µL/cm²). For [phenyl U 14C]-PANA in Test Preparation 3, water (31.4 µL) was applied to wet the skin surface. Test Preparation 3 was weighed into weighing boats (ca 15.7 mg) then tapped onto the skin surface and distributed evenly using a glass rod (application rate of 5 mg/cm²)
For test preparation 4 and 5 the skin surface was wetted prior to dosing with water (10 µL/cm²) and a single disc was applied using tweezers to a minimum of 12 skin samples. A glass rod was used to press down the disc to the skin.

VEHICLE
- Justification for use and choice of vehicle: rubber matrix and base oil for lubricants, representative of in-use conditions

TEST SITE
- Preparation of test site: The glass static diffusion cells were placed in a manifold on a magnetic stirrer plate heated via a circulating water bath to maintain the skin surface temperature at 32°C ± 1°C. The actual cell temperatures (ranging from 31.1°C to 32.6°C) were calibrated prior to mounting the skin membranes.
Magnetic stirrer bars were placed in the receptor fluid chambers which were filled with receptor fluid. Once thawed, sections of split thickness skin (ca 3 x 3 cm) were cut and mounted in the diffusion cells between the donor and receptor chamber. The donor chamber was tightened into place with a clamp. Cells were visually checked to ensure no cells were leaking and no air bubbles were present in the receptor fluid chamber.

Skin samples were allowed to equilibrate at 32°C ± 1°C for a minimum of 5 min. PBS (3 mL) was then added to the donor chamber and the skin samples were allowed to equilibrate for a further minimum of 30 min. The electrical resistance was then measured using a Tinsley Databridge (Model 6401) set at low voltage alternating current, 1000 Hz with a maximum voltage of 300 mV root mean squared (rms) in the parallel equivalent circuit mode. Any skin sample exhibiting a resistance less than 4.0 kΩ was excluded from subsequent absorption measurements. The PBS was removed from the skin surface; the skin was rinsed with water and dried with a tissue swab.
- Area of exposure: The surface area of exposed skin within the cells was 3.14 cm² with a receptor chamber volume of ca 10 mL (nominal).

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

SAMPLE COLLECTION
Prior to dosing, a 300 µL sample of receptor fluid was removed from the receptor chamber collection arm. The receptor fluid volume was then maintained by the addition of fresh receptor fluid up to the calibration line on the receptor chamber collection arm. Following sample collection, the receptor chamber collection arm was sealed with Parafilm® to prevent evaporation of receptor fluid.
Receptor fluid aliquots were collected at 1, 2, 4, 8 and 12 h post dose as described above. All receptor fluid samples were analysed by liquid scintillation counting.

- Terminal procedure:
At 24 h post dose, the cell were dismantled and the donor chamber were retained for analysis (donor chamber wash). Skin was removed from the cell and placed on a piece of tissue paper to remove any remaining receptor fluid from the underside of the skin. The tissue was placed in the receptor wash pot for that particular cell.
The stratum corneum was removed with 20 successive tape strips. The skin sample was rotated 90° after each tape strip. Rotation was stopped if the epidermis/dermis junction became fragile or if epidermis was removed. Each tape was analysed individually by liquid scintillation counting.
The skin under the cell flange (unexposed skin) was cut away from the exposed skin. Each exposed skin sample was wrapped in a piece of clingfilm. A ca 200 g weight was heated to ca 65°C in a water bath and placed onto the epidermal surface for ca 90 s. The epidermis was then separated from the dermis using a scalpel. The clingfilm was retained for analysis.
Bulk receptor fluid (ca 10 mL) was removed from each receptor chamber and retained for analysis.
The receptor chambers were rinsed with ethanol (40 mL). The solvent was pooled as a single sample into the pre weighed receptor wash pot and sample weights were taken.
Total radioactivity was analysed in all samples of skin, receptor fluid, and washes by liquid scintillation counting.

SAMPLE PREPARATION
- Storage procedure: All samples were stored at ambient temperature for analysis. Following analysis, bulk samples were stored in a freezer set to maintain a temperature of -20°C.
- Preparation details: Skin samples were solubilised using Solvable® prior to analysis by liquid scintillation counting.

ANALYSIS
- Method type(s) for identification: All samples prepared in scintillation fluid were subjected to liquid scintillation counting, together with representative blank samples, using a Liquid Scintillation Analyser with automatic quench correction by an external standard method.
- Limits of detection and quantification: A limit of reliable measurement of 30 d.p.m. above background has been instituted

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Eight donors were obtained from Tissue Solutions Ltd and two donors were obtained from BioIVT. The samples arrived at Charles River frozen and were stored in a freezer set to maintain a temperature of 20°C until used in the study.
- Donor information: 10 donors (male and female) aged 32 to 58 years old
- Ethical approval if human skin: Ethical approval for receipt and use of human skin has been obtained (Lothian Local Research Ethics Committee, REC Reference No. 06/S1101/19 and Glasgow Royal Infirmary REC, REC Reference No. 08/S0704/30). Tissue is regularly sourced from hospitals and tissue banks.
- Type of skin: full-thickness human skin (abdomen)
- Preparative technique: 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.
A static diffusion cell system (PermeGear Inc) was used.
- Thickness of skin (in mm): split-thickness range from 0.38 to 0.4 mm
- Membrane integrity check: Skin samples were allowed to equilibrate at 32°C ± 1°C for a minimum of 5 min. PBS (3 mL) was then added to the donor chamber and the skin samples were allowed to equilibrate for a further minimum of 30 min. The electrical resistance was then measured using a Tinsley Databridge (Model 6401) set at low voltage alternating current, 1000 Hz with a maximum voltage of 300 mV root mean squared (rms) in the parallel equivalent circuit mode. Any skin sample exhibiting a resistance less than 4.0 kΩ was excluded from subsequent absorption measurements. The PBS was removed from the skin surface; the skin was rinsed with water and dried with a tissue swab.
- Storage conditions: -20°C
- Justification of species, anatomical site and preparative technique: The human has been chosen for the safety evaluation as workers and consumers will be exposed during handling of the test item or the use of products containing the test item.


PRINCIPLES OF ASSAY
- Diffusion cell: A static cell diffusion system was used.
- Receptor fluid: Phosphate buffered saline (PBS) containing polyoxyethylene 20 oleyl ether (PEG, ca 6%, w/v), sodium azide (ca 0.01%, w/v), streptomycin (0.1 mg/mL) and penicillin (100 units/mL) with the pH confirmed and adjusted to 7.4 ± 0.1 if required.
- Solubility of test substance in receptor fluid: The target concentration (2.198 mg/mL) represented the maximum concentration of test item in the receptor fluid based on the entire applied dose being absorbed in 24 h from the 3.5% (w/w) rubber discs. As 83.78% of the target concentration was recovered, the receptor fluid was accepted for use on the study. The maximum absorption (equivalent to 5.11 µg/mL, Cell 5) was greatly below this level in the study, showing the receptor fluid was not rate limiting.
- Static system: The static diffusion cells was positioned in a steel manifold and heated via a circulating water bath to maintain a skin surface temperature
- Test temperature: 32 +/- 1 °C
- Occlusion: The donor chambers were not occluded and left open to the atmosphere

Results and discussion

Signs and symptoms of toxicity:

no effects

Dermal irritation:

no effects

Absorption in different matrices:

- Skin wash:
Test preparation 1: 20.03 ± 9.14 % applied dose;
Test preparation 2: 30.28 ± 15.35 % applied dose;
Test preparation 3: 32.58 ± 7.60 % applied dose;
Test preparation 4: 0.07 ± 0.03 % applied dose;
Test preparation 5: 0.13 ± 0.10 % applied dose
- Skin test site:
Test preparation 1: 94.60 ± 3.24 % applied dose;
Test preparation 2: 93.84 ± 23.96 % applied dose;
Test preparation 3: 94.76 ± 1.49 % applied dose;
Test preparation 4: 98.05 ± 1.26 % applied dose;
Test preparation 5: 100.57 ± 1.50 % applied dose
- Skin, untreated site:
Test preparation 1: °0.40 ± °0.69 % applied dose;
Test preparation 2: °0.30 ± °0.88 % applied dose;
Test preparation 3: °0.01 ± °0.01 % applied dose;
Test preparation 4: °0.00 ± °0.00 % applied dose;
Test preparation 5: °0.00 ± °0.00 % applied dose
- Receptor fluid, receptor chamber, donor chamber (in vitro test system):
Receptor fluid:
Test preparation 1: 1.78 ± 0.85 % applied dose;
Test preparation 2: 1.66 ± 0.54 % applied dose;
Test preparation 3: 0.08 ± 0.04 % applied dose;
Test preparation 4: 0.22 ± 0.06 % applied dose;
Test preparation 5: 0.24 ± 0.07 % applied dose
Receptor chamber:
Test preparation 1: °0.28 ± °0.16 % applied dose;
Test preparation 2: 0.22 ± 0.17 % applied dose;
Test preparation 3: °0.02 ± °0.01 % applied dose;
Test preparation 4: °0.03 ± °0.01 % applied dose;
Test preparation 5: °0.03 ± °0.01 % applied dose
- Stratum corneum (in vitro test system):
Stratum Corneum 1-2:
Test preparation 1: 0.24 ± 0.20 % applied dose;
Test preparation 2: 0.37 ± 0.33 % applied dose;
Test preparation 3: 0.24 ± 0.18 % applied dose;
Test preparation 4: 0.01 ± 0.01 % applied dose;
Test preparation 5: 0.01 ± 0.01 % applied dose
Stratum Corneum 3-20:
Test preparation 1: 0.65 ± 0.44 % applied dose;
Test preparation 2: 1.14 ± 1.16 % applied dose;
Test preparation 3: 0.18 ± 0.11 % applied dose;
Test preparation 4: 0.02 ± 0.03 % applied dose;
Test preparation 5: 0.03 ± 0.02 % applied dose


°=Mean includes results calculated from data less than 30 d.p.m above background

Total recovery:

- Total recovery:
Test preparation 1: 100.49 ± 1.39 % applied dose;
Test preparation 2: 100.16 ± 23.47 % applied dose;
Test preparation 3: 95.96 ± 1.30 % applied dose;
Test preparation 4: 98.38 ± 1.24 % applied dose;
Test preparation 5: 100.93 ± 1.51 % applied dose
- Recovery of applied dose acceptable: Mass balance should be within 85-115%. However, if the mass balance was below 85% and the loss can be explained, the samples may also be accepted.
- Results adjusted for incomplete recovery of the applied dose:
The mass balance for most individual samples was within 100 ± 15% except for 4 samples for test preparation 2: Cell 37 (137.01%), Cell 38 (55.79%), Cell 39 (138.06%) and Cell 40 (73.04%). IncIt is thought that an error occurred during the wash for this study, as if the tissue swab values were swapped with the cells’ donor pair mass balance for each cell would be acceptable. Incorrect dosing has also been excluded as the same pipette and pipette setting were used throughout dosing. For these reasons, cells have been included in calculations..
- Limit of detection (LOD): A limit of reliable measurement of 30 d.p.m. above background has been instituted in these laboratories.
- Quantification of values below LOD or LOQ: Counts that are below 30 d.p.m. above background represent a true value. This means that data are recorded with values that are less than the limit of reliable measurement and marked.

Percutaneous absorption

Percutaneous absorption 1

Time point:

24 h

Dose:

1%

Parameter:

percentage

Absorption:

5.6 %

Percutaneous absorption 2

Time point:

24 h

Dose:

5%

Parameter:

percentage

Absorption:

6.4 %

Percutaneous absorption 3

Time point:

24 h

Dose:

100%

Parameter:

percentage

Absorption:

0.66 %

Percutaneous absorption 4

Time point:

24 h

Dose:

0.5%

Parameter:

percentage

Absorption:

0.35 %

Percutaneous absorption 5

Time point:

24 h

Dose:

3.5%

Parameter:

percentage

Absorption:

0.38 %

Conversion factor human vs. animal skin:

not applicable

Applicant's summary and conclusion

Conclusions:

Following topical application of [phenyl U 14C]PANA to human skin in vitro dermal absorption after accounting for variability between replicates is 0.38% (17.6 μg equiv./cm²) of the applied dose in rubber discs (ca 3.5%, w/w), 0.35% (1.91 μg equiv./cm²) of the applied dose in rubber discs (ca 0.5%, w/w), 5.6% (5.84 μg equiv./cm²) of the applied dose in Test Preparation 1 (ca 1%, w/v), 6.4% (34.4 μg equiv./cm²) of the applied dose in Test Preparation 2 (ca 5%, w/v), and 0.66% (33.4 μg equiv./cm²) of the applied dose in Test Preparation 3 (ca 100%, w/w).

Executive summary:

The test item, N‑1‑naphthylaniline (PANA) is used as an antioxidant in various oils such as lubricants and in the production of rubber. As part of the safety evaluation of PANA, a study was conducted to assess the absorption following dermal application in accordance with the OECD 428 and under GLP compliance. Exposure to the test item could occur during handling of the pure compound and during processing, for example, after dilution in a base oil for lubricants or after incorporation into a rubber matrix. Concentrations of PANA tested within the scope of this study represented the neat product (ca 100% (w/w) moistened with water); and relevant in-use concentrations diluted in base oil for lubricants (ca 5% (w/v) and ca 1% (w/v)). Due to the use pattern of the test item, a rubber matrix was selected to generate a relevant and technically feasible test sample representing a worst-case sample of the test item using a simplified manufacturing method based on the actual industrial processing (ca 3.5% (w/w) and ca 0.5% (w/w) in a rubber disc).

 

Split‑thickness human skin membranes were mounted into static diffusion cells in vitro. Receptor fluid was added to the receptor chamber (nominal volume 10 mL, nominal exposure area 3.14 cm²). The skin surface temperature was maintained at 32°C ± 1°C throughout the experiment. An electrical resistance barrier integrity assessment was performed and any skin sample exhibiting resistance lower than 4.0 kΩ was excluded from subsequent absorption measurements.

 

The rubber discs and Test Preparation 1-3 were applied to human split‑thickness skin membranes from four to six different donors and the cells were left open to the atmosphere. Test item stability following dosing was confirmed by high performance liquid chromatography (HPLC).

 

Percutaneous absorption was assessed by collecting receptor fluid at 1, 2, 4, 8 and 12 h post dose. At 8 h post dose, the exposure period was terminated by removing the rubber disc (if applicable) then 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 tissue paper. At 24 h post dose, the skin was then removed from the static cells, the stratum corneum tape stripped and the skin divided into exposed and unexposed skin. The exposed skin was heat separated into dermis and epidermis samples. The bulk receptor fluid was collected from the receptor chamber and retained for analysis. The donor chambers and receptor chambers were rinsed with ethanol and the samples retained for analysis. The skin samples were dissolved with Solvable™ tissue solubilizer. All samples were analysed by liquid scintillation counting.

Following topical application of [phenyl U 14C]PANA to human skin in vitro, dermal absorption after accounting for variability between replicates is 0.38% (17.6 μg equiv./cm²) of the applied dose in rubber discs (ca 3.5%, w/w), 0.35% (1.91 μg equiv./cm²) of the applied dose in rubber discs (ca 0.5%, w/w), 5.6% (5.84 μg equiv./cm²) of the applied dose in Test Preparation 1 (ca 1%, w/v), 6.4% (34.4 μg equiv./cm²) of the applied dose in Test Preparation 2 (ca 5%, w/v), and 0.66% (33.4 μg equiv./cm²) of the applied dose in Test Preparation 3 (ca 100%, w/w).