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Toxicological information

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:
2019-09-10 to 2019-12-20
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
2004-04-13
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
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cobalt
EC Number:
231-158-0
EC Name:
Cobalt
Cas Number:
7440-48-4
Molecular formula:
Co
IUPAC Name:
cobalt
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: powder
- Particle size distribution:
D10: 1.12 µm
D50: 1.93 µm
D90: 3.42 µm
D95: 3.92 µm
D100: 5.44 µm
Specific details on test material used for the study:
Particle characterisation:
The general particle size of the test item was assessed using a scanning electron microscope (SEM, Hitachi TM4000Plus) coupled with energy dispersive X-ray spectroscopy (EDS, Oxford Instruments, Aztec ONE).

Particle size was typically with 150 - 190 nm with the occasional particle measuring approx. 500 - 600 nm.
Radiolabelling:
no

Test animals

Species:
other: not applicable, since this is an in vitro study
Details on test animals or test system and environmental conditions:
Not applicable, since this is an in vitro study

Administration / exposure

Type of coverage:
open
Vehicle:
other: artificial sweat
Duration of exposure:
8 and 24 hours
Doses:
nominal concentration: 100 mg/cm²
actual concentrations (8 hour exposure; 3 donors): 100.184 - 102.384 mg/cm² (average: 179.7 ± 1.1 mg (179.6 mg cobalt))
actual concentrations (24 hour exposure; 3 donors): 100.915 - 101.706 mg/cm² (average: 179.4 ± 0.5 mg (179.3 mg cobalt))
No. of animals per group:
not applicable
Details on study design:
DOSE PREPARATION
- Preparation of artificial sweat: the artificial perspiration solution was prepared according to the reference test method EN1811:2011. The solution involved mixing urea (0.1%), sodium chloride (0.5%) and lactic acid (0.1%) in water (>18.2 MΩ). The pH of the artificial sweat solution was then adjusted to pH 6.5 using diluted
ammonium hydroxide.

- Preparation of test item and dosing: prior to each experiment, the test item was pre-weighed into 2 mL Eppendorf tubes at 177 + 5 mg. The experiment was initiated by the application of 177 μL of artificial sweat using a calibrated positive
displacement pipette. Immediately after the application of sweat, the preloaded Eppendorf tubes containing the compound were dispensed onto the skin surface and the cell shaken lightly to ensure full coverage of the skin surface.

Amount of sweat dosed: 177 μL per 1.77 (skin area cm2) = 100 μL/cm2
Amount of compound dosed: 177 mg per 1.77 (skin area cm2) = 100 mg/cm2
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin /Type of skin: full thickness human skin from 3 adult donors (1 male / 2 females; age range 41-50 years; African american or Caucasian) obtained during an elective surgical procedure (purchased from Zenbio (North Carolina, USA) via Amsbio (Oxfordshire, UK). All the skin samples received were excised from the abdomen. A total of 3 x ~100 cm2 sections of skin were purchased per donor. Upon receipt the skin was immediately stored at -20 °C within a temperature monitored freezer until required.

Please refer to the field "Attached background material" for information on the donors.

- Ethical approval if human skin: yes, approval was sought from the National Research Ethics Service and the Health Research Authority.

- Preparative technique: A section (~100 cm2) of each donor’s skin was taken out of frozen storage and allowed to thaw before the skin was prepared for dermatoming. The skin was placed onto a bespoke mount and the skin dermatomed to 500 μm using a Humeca D80 dermatome (Eurosurgical, Surrey, UK). The dermatomed sections of skin where visually inspected for any overt damage and then placed onto an aluminium foil sheet in preparation for cutting the sections to size. Once cut, each skin section was assigned a cell number and the appropriate treatment group and the thickness of each skin section
measured using digital callipers (Tooled-up, Middlesex, UK) and recorded.

Skin thickness (8 hour exposure): 460 - 550 µm
Skin thickness (24 hour exposure): 500 - 580 µm

- Membrane integrity check: before the individual skin sections were included into the study, a barrier integrity test was performed using an LCR (inductance, capacitance and resistivity) meter (model 821, ISO-TECH, UK) using a frequency of 0.1 kHz and a voltage of 1 to measure the resistance of the skin (kΩ). The resistivity of the skin was measured by firstly filling the receptor chamber with 0.1 % saline solution and applying 1 mL of 0.9% saline to the skin surface. The resistivity was then measured by placing one probe at the donor side and the other probe on the receptor side (via the sampling port). The results were
compared with a deliberately damaged skin section where the resistivity had to be at least double that of the damaged section to be included into the study. The saline on the skin surface was gently removed using a cotton swab and the receptor fluid within the receptor chamber removed using a needle and syringe. The receptor chamber was then topped up with the appropriate receptor fluid
and the volume recorded. The diffusion cell was then connected to the heated manifold.

Integrity of skin membranes (8 hour exposure)
Resistivity: 15.102 - 21.925 kΩ

Resistivity of damaged skin:
Donor 1: 8.5685 kΩ
Donor 2: 6.6371 kΩ
Donor 3: 5.2075 kΩ

Integrity of skin membranes (24 hour exposure)
Resistivity: 15.041 - 24.888 kΩ

Resistivity of damaged skin:
Donor 1: 7.3510 kΩ
Donor 2: 5.6606 kΩ
Donor 3: 4.7825 kΩ

PRINCIPLES OF ASSAY
- Diffusion cell: static skin diffusion cells were purchased from PermeGear (Illinois, USA) and were based on the design of the Franz diffusion cell. Each diffusion cell comprises an upper (donor) and lower (receptor) chamber, with an area available for diffusion of 1.77 cm2. Dermatomed skin sections were placed
between the two chambers, with the epidermal surface facing the donor chamber and the ensemble securely clamped. Each diffusion cell was then placed in a Perspex™ holder above a magnetic stirrer which constantly mixed the receptor fluid using a (12 x 6 mm) Teflon™-coated iron bar placed within the receptor chamber. The receptor chambers were of the jacketed variety to enable the flow of warm (35°C) water using a circulating water heater (TC-120, Grant instruments, Cambridge, UK). Once assembled and the sampling port capped with an Eppendorf tube (to prevent evaporation of the receptor fluid), the diffusion cells were left in situ for an equilibration period of up to 24 hours.

- Receptor fluid: a series of preliminary studies were performed to identify the most suitable receptor fluid for the test compound. Citrate-phosphate buffer pH 4.0 is the most effective receptor fluid. However, ethylenediaminetetraacetic acid (EDTA) was added to the receptor fluid to increase binding. The addition of EDTA drastically increased the amount of test compound collected in the supernatant. To ensure dissociation from the EDTA, all receptor fluid samples were acid digested.

Preparation of receptor fluid:
Citric acid receptor solution was prepared by weighing 1.92 ± 0.002 g of citric acid into a 100 mL volumetric flask. This was then topped up with approximately 90 mL of water (>18.2MΩ). The solution was thoroughly sonicated until the solids had fully dissolved and then made to volume (0.1M citric acid solution). Sodium phosphate receptor fluid was prepared by the addition of 2.84 ± 0.05 g of anhydrous sodium phosphate dibasic into a 100 mL volumetric, sonicated after the addition of 90 mL water and then made to volume. A total of 30.9 mL of the 0.1 M citric acid was transferred into a fresh volumetric and 19.2 mL 0.2 M sodium phosphate solution was added. The pH of the solution was recorded and adjusted to pH 4.0 ± 0.1 using 0.2 M sodium phosphate to increase the pH or 0.1 M citric acid to decrease the pH. This was then made up to the final volume with water (>18.2 MΩ) and mixed by inversion until visually homogenous and the final pH checked, recorded and adjusted if necessary. The volumes stated were scaled up or down depending on requirements. Ethylenediaminetetraacetic acid, Disodium Salt Dihydrate (EDTA) was added at the appropriate amount to achieve a 10% solution. A magnetic stirrer was then added, and the solution left to stir
overnight at 35°C (AREX heating magnetic stirrer VELP scientifica setting 7) until the solution was fully dissolved.

- Experimental procedure (8 hour exposure and 24 hour exposure):
After the equilibration period (up to 24 hours), any air bubbles were removed from the diffusion cells and the skin temperature checked using an infrared camera (FLIR Model P620 camera; 8 hour exposure: 30.4 - 32.4 °C; 24 hour exposure: 30.7 - 32.1 °C ). Baseline receptor fluid samples were withdrawn (750 μL) from each diffusion cell and placed into a centrifuge tube. Upon acquisition of the baseline receptor fluid sample, the same volume of fresh receptor fluid (citrate phosphate buffer pH 4 with 10% EDTA) was used to replenish each cell.

8 hour exposure:
Following acquisition of the baseline sample, the artificial perspiration solution (177 μL) was pipetted onto the skin surface and the test item was immediately applied. The cell was shaken to ensure full coverage of the test item on the skin surface. The application of sweat and cobalt dosing was staggered between the different cells adhering to the treatment randomisation. Receptor fluid samples were acquired at regular intervals by removing (and replenishing 750 µL) from each diffusion cell pre-dose (baseline) and 1, 2, 4, 6, 8, 12, 16, 20 and 24 hours after test item application. The test substance remained in situ on the skin surface for 8 hours. At 8 hours, the test items were removed by tipping the diffusion cell (where applicable) into an empty vial, taking care not to introduce an air bubble in the receptor fluid (if occurred, the cell was tilted to remove the bubble). The skin surface was then washed by applying 1 mL of water and applying a cotton swab to absorb the water and test compound. This step was repeated and at the third 1 mL wash, the residual fluid was aspirated using a 1 mL positive displacement pipette. The donor chamber was transiently removed and swabbed, and the skin surface swabbed with a further wet and dry swab. The swabs, rinses and pipette tip were placed into vials for subsequent analysis. The experiment continued to 24 hours with regular receptor fluid sampling. At 24 hours, the receptor fluid within the receptor chamber was removed into an empty glass vial. The skin was removed and placed into a petri dish and stored at -70°C in preparation for cryosectioning. Furthermore, receptor fluids, wash and swaps, pipette tips were stored under ambient laboratory conditions prior to digestion.

24 hour exposure:
Following acquisition of the baseline sample, the artificial perspiration solution (177 μL) was pipetted onto the skin surface and the test item was immediately applied. The cell was shaken to ensure full coverage of the test item on the skin surface. The application of sweat and cobalt dosing was staggered between the different cells adhering to the treatment randomisation. The compounds remained in situ on the skin surface for 24-hours whilst the receptor fluid samples were acquired at the stated regular intervals by removing (and replenishing) 750 µL from each diffusion cell pre-dose (baseline) and 1, 2, 4, 6, 8, 12, 16, 20 and 24 hours after test item application. After the final receptor fluid sample (24 hours), the test item was removed (where applicable) by tipping the diffusion cell into an empty vial. The skin surface was then washed by applying 1 mL of water and applying a cotton swab to absorb the water and test compound. This step was repeated and at the third 1 mL wash, this was aspirated using a 1 mL positive displacement pipette. The donor chamber was removed, and the skin surface swabbed with a further wet and dry swab. The swabs, rinses and pipette tip were placed into vials for subsequent analysis. Once the skin was washed, the remaining receptor fluid was collected into a glass vial for analysis. The skin was removed and placed into a petri dish and stored at -70°C in preparation for cryo-sectioning. Furthermore, receptor fluids, wash and swaps, pipette tips were stored under ambient laboratory conditions prior to digestion.

- Skin sectioning:
The skin was sectioned using a cryotome (OTF5000, Bright Instruments Ltd. UK) using a tungsten carbide blade (228 x 38 x 6mm, 50234, Bright Instruments) set to an angle of 25° to the sample block. Both the specimen and chamber temperatures were set to -28°C with a quick-freezer plate.

Prior to skin sectioning, approx. 1.5 mL of optimal cutting temperature embedding compound (OCT) was placed on the sample holder and allowed to freeze on the quick-freezer plate. The frozen OCT embedding compound was then sectioned to achieve a flat surface. The skin was then taken out of the -70°C and left at room temperature to defrost. Once defrosted, the skin area that was available for diffusion was excised using a 1.6 cm diameter biopsy punch. The skin was then placed stratum corneum side down onto a flat surface and a small amount of OCT applied to assist with ensuring the skin is flat. The pre-sectioned OCT sample holder was then briefly removed from the cryostat and firmly placed down onto the OCT covered skin to adhere the skin is fixed flat to the sample holder. A further amount of OCT was then placed onto the skin surface and the sample placed onto the quick-freezer plate until all the embedding compound had frozen. Once frozen, the OCT was sectioned until the skin was exposed.

The initial five sections (10 μm each) where acquired and placed into separate vials for analysis (samples 1 - 5). The following 5 x 10 μm sections were amalgamed and analysed as sample 6 and this was repeated to produce sample 7. The remaining skin (~350 μm) section was placed into a separate vial to produce sample 8. The skin surrounding the initial punch biopsy (excess skin) was used to produce sample 9. Due to the difficulty sectioning skin laterally the number of sections were recorded in order to quantify the depth of sectioning in the unlikely event for the skin section were to shear. If the section sheared, the depth of the section was calculated and sectioning continued. The blade was cleaned after sectioning each sample with an ethanol wipe which was stored in a 50 mL Falcon tube for future analysis if required.

- Sample digestion:
Prior to quantification using ICP-OES, samples underwent acid digestion to ensure the test items were dissolved for quantification.

Receptor fluid samples:
Prior to digestion, all of the collected receptor fluid samples were spiked with 20 μL of internal standard yttrium and transferred into microwave digestion tubes and 1.5 mL of 70% nitric acid was added. Following digestion using a Mars 6 microwave digestive system (CEM Corporation; protocol is based on an adaption of US EPA 3015A(1)), the sample was transferred into a volumetric flask and the microwave digestion tube rinsed with 5 mL of water and the rinsing added to the volumetric flask. The volumetric was then made to volume (10 mL) using water and the sample transferred into a 25 mL falcon tube in preparation for ICP-OES analysis.

Receptor fluid standards:
To prepare the stock, 500 ± 5 mg of test compound were weighed into an Eppendorf tube. The compound was then transferred into a microwave tube and the post weight recorded to calculate the exact amount of compound. Then, 750 μL of receptor fluid, 100 μL yttrium and 5 mL of 70% nitric acid were added to the microwave tube and digested using a Mars 6 microwave digestive system, as stated above. After digestion, the solution was transferred into a volumetric flask. The microwave tube was rinsed with 10% HNO3 (>18.2 MΩ) and the rinse also transferred to the same volumetric flask which was then made to volume with 10% HNO3 (>18.2 MΩ). These stocks were then diluted to produce an eighteen-point calibration curve ranging from 0.005 – 2000 μg/mL.

Swab samples:
All of the collected swab samples were spiked with 100 μL of internal standard yttrium and transferred into a microwave digestion tube and 5 mL of 70% nitric acid was added and underwent digestion using a Mars 6 microwave digestive system, as stated above. Following digestion, these samples were decanted into a volumetric flask and the microwave tube rinsed with water (>18.2 MΩ) and the rinse added to the same volumetric which was subsequently made to volume. The sample was thoroughly mixed by inversion and an aliquot of the sample transferred into a falcon tube in preparation for ICP-OES analysis. The samples were further diluted 1:10 with 10% nitric acid to ensure samples fell within the calibration range of the ICP-OES analysis.

Swabs standards:
500 ± 5 mg of the test item was placed into a microwave tube. Four cotton swabs were added to the cobalt along with 100 μL of yttrium (2 μg/mL). For the digestion, 5 mL of 70% nitric acid was then added, the microwave tube sealed and digested using a Mars 6 microwave digestive system, as stated above. Once the stock samples had cooled down, they were then transferred into volumetric flask, the microwave tube rinsed with type 1 dH2O (placed into the volumetric containing the stock). The volumetric was then made to volume with type 1 water. This produced a stock of 10 mg/mL of the test item. This was then diluted to produce an eighteen-point calibration curve ranging from 0.005 – 2000 μg/mL.

Excess dose and Pipette tips:
Where applicable, excess dose samples were recovered from the skin diffusion cell wash at either 8 or 24 hours (dependent upon experiment). The excess dose was captured by tipping the diffusion cell into a vial. Each sample (where it was collected) was transferred from the glass tube into a microwave tube. The glass vial was rinsed with 2 mL of 70% nitric acid and this was also transferred into the microwave tube. A further 3 mL of nitric acid was added with 100 μL of yttrium internal standard. The microwave protocol was as described above. Once microwaved, the samples were transferred into a volumetric flask. The microwave tube was rinsed with water and this was also transferred into the same flask. The solution was then made to volume.

Considering the high concentrations of these excess dose samples, they were further diluted 1:10 with water prior to analysis. These were quantified against non-matrix standards (please refer to "Standard preparations" below).

Pipette tips were placed into 15 mL falcon tubes and each sample spiked with 20 μL of yttrium. Each tip was then rinsed using 1.5 mL of 70% nitric acid. The rinse solution was transferred into a volumetric flask and made to volume with water.

Skin samples:
Skin samples were sectioned using a cryotome, as described above. The skin samples were collected in glass vials. The samples were then spiked with 20 μL of yttrium and digested by the addition of 1 mL of 70% nitric acid and the samples placed onto a roller mixer for 2 hours to ensure complete digestion. The samples were transferred into a microwave tube and the glass vial rinsing with
0.5 mL of 70% nitric acid and the contents transferred to the same microwave tube. The samples were then acid-digested as outlined for receptor fluid samples above.

Following complete digestion, the solution was transferred into a volumetric flask. The microwave tube was rinsed with water and the content added to the volumetric flask which was then made to volume with 18.2mΩ water.

A set of skin blanks were prepared for each donor by the addition of a section of dermatomed skin (500 μm approx., 3 x 3 cm skin) into a microwave tube and prepared as described for the samples.

Skin standards:
The skin standards were prepared by weighing approx. 500 mg of the test compound into Eppendorf tubes. The test compounds were then transferred into a microwave tube. Each Eppendorf was re-weighed to record the actual amount of compound transferred into the microwave tube. Then, a section of dermatomed skin (500 μm approx. 3 x 3 cm skin), 100 μL yttrium internal standard and 5 mL of 70% nitric acid were added to the microwave tube and then acid-digested using a Mars 6 microwave digestive system, as stated above. Following the digestion, the solution was transferred into a volumetric flask. The microwave tube was rinsed with water and content also transferred into the same flask, which was then made to volume with water. All further dilutions were prepared with 10% nitric acid. Separate standard curves were prepared for each donor for each test item.

- Standard preparation:
Each analysis run comprised one 17-point calibration curve (ranging from 0.005 – 1000 μg/mL) which was produced using a reference cobalt solution in 10% nitric acid solution to serve as the run validation reference and to quantify the interspersed quality control samples (500, 10, 0.5 μg/mL). A series of blanks solutions were also interspersed within the run to (i) obtain the instrument baseline and (ii) to detect and reduce any potential carry over from the samples.
Each run also included an 18-point calibration curve, produced using a stock solution of the respective matrix being analysed for the test item (ranging from 0.005 – 2000 μg/mL). This matrix calibration curve was used to quantify the amount of cobalt within the samples and this was also compared with the
reference calibration curve to demonstrate any potential matrix effects.

- ICP-OES analysis and parameters:
All digested samples were analysed on an iCap 7200 Duo ICP-OES (ThermoFisher Scientific) instrument, coupled with an ASX-560 Autosampler (Teledyne Cetac) and a ThermoFlex 900 recirculation chiller (ThermoFisher Scientific). The instrument was controlled, and samples analysed using the proprietary software Qtegra (version 2.7.2425.65).

The analytical method for cobalt in the receptor fluid, skin, swabs and excess dose were developed and validated under the current study. The following limits of detection (LOD) and quantification (LOQ) were determined:

LOD: approx. 0.007 - 0.009 µg/mL
LOQ: 0.017-0.022 μg/mL

Recovery: 97.7 - 104.0 %
Accuracy: 100 ± 15 % (relative standard deviation: <10 %)

Quality control samples (QCs) were produced from the reference cobalt solution at 0.5, 10 and 500 μg/mL. These QC samples were interspersed throughout the run and typically bracketed samples within each analytical run. QC samples were checked after each analytical run and had to be within 20% of the stated concentration to pass.

Matrix interference: samples of blank receptor fluid, skin and swabs were spiked with a known amount of cobalt metal powder and compared to un-spiked samples. No interferences were detected. Considering the protocol for skin sectioning, there was a need to assess if the skin and the optimal cutting compound (OCT) could interfere. Therefore, the following samples were analysed (due to the potential for varying quantities): skin only, OCT only, skin and OCT, cobalt metal powder, OCT spiked with cobalt metal powder, skin and OCT spiked with cobalt metal powder and skin spiked with cobalt powder. There were no interferences detected in any of the skin sample iterations and the amounts
of cobalt recovered.

CALCULATIONS
The intensity for each sample was quantified against its respective calibration curve. This was converted to amount (μg) by taking into consideration cobalt purity.

The absorbed dose was calculated for each individual receptor fluid sample acquired and then was calculated as cumulative absorbed dose. This was plotted over time to generate penetration profiles for each of the test items.

Absorbed cobalt (μg/cm) = Absorbed cobalt (µg) / Exposure area (1.77 cm²)
In addition, the percentage absorbed dose was also calculated for each sample as follows: Percentage of applied dose (%) = (Absorbed cobalt (mg) / Dose applied (µg)) x 100.

Data presented in results, tables and figures are those after subtraction of the appropriate blank samples unless otherwise stated.
Each analytical run analysed using Microsoft Excel 2016.

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
8 hour exposure:
One cell sample of donor 2 was excluded from analysis due to damage from 4 hours. Over the 24-hour penetration profile, no cobalt metal powder absorbed into the receptor fluid. The majority of the applied dose was removed by the washing procedure at 8 hours post exposure (93.26 ± 3.89%; 167.47 ± 6.99 mg Co) with a smaller fraction recovered within the skin (0.07 ± 0.04%, 0.13 ± 0.07 mg Co). Cobalt metal powder was recovered from the first two skin sections (20 μm). Individual penetration profiles along with the full tabulated dose distribution for each diffusion cell can be found in the field "Attached background material" below.

24 hour exposure:
There was no cobalt detected penetrating through human skin over 24 hours. Most of the applied dose was recovered in the wash process (93.45 ± 1.01%, 167.58 ± 1.81 mg Co). Approximately 0.1% (0.18 mg Co) was recovered from the skin of which, 0.056 ± 0.057% (100.4 μg) was recovered from the first 10 μm and 0.005 ± 0.006% (8.96 μg) recovered in the deepest layer (350 μm). Data for each individual diffusion cell can be found in the field "Attached background material" below.


Overall, the amount of test item recovered from the skin available for absorption (excess skin excluded) tends to suggest an increase in the amount of test item in the skin with time, as shown below:

Total amount in skin (excluding excess skin) at 8 hours: 85.78 µg
Total amount in skin (excluding excess skin) at 24 hours: 159.54 µg

Expressed as µg/cm2 (8 hours): 48.54 µg/cm2
Expressed as µg/cm2 (24 hours): 90.28 µg/cm2

Furthermore, based on a study by Fischer et al. (2015)*, the dose of cobalt required to elicit a response (based upon the absorption of cobalt chloride) is between 0.663 – 1.95 μg cobalt/cm2. The test item exceeds the range reported by Fischer et al (2015)*, as can be seen below:

8 hour exposure: 48.52 µg cobalt/cm2
24 hour exposure: 90.24 µg cobalt/cm2

Using the same data as reported above (48.52 and 90.24 µg cobalt/cm2 for 8 and 24 hour exposure, respectively) and subtracting the amount of test item recovered from the first top 2 skin sections (20 μm) the cobalt concentrations still all exceed the reported threshold value reported by Fischer et al., 2015, as can be seen below:

8 hour exposure: 5.74 µg cobalt/cm2
24 hour exposure: 19.20 µg cobalt/cm2

The rationale for subtracting the first 20 μm was to remove any compound that was potentially retained within the stratum corneum and therefore only quantifying the amounts within the skin.

Lastly, the potentially absorbed dose calculated as the sum of the total amount cobalt recovered with the receptor fluid and skin available for absorption (first 20 mm omitted) was as follows:

8 hour exposure: 5.74 µg cobalt/cm2
24 hour exposure: 19.20 µg cobalt/cm2

*Reference:
- Fischer LA, Johansen JD, Voelund A, Liden C, Julander A, Midander K, et al. Elicitation threshold of cobalt chloride: analysis of patch test dose-response studies. Contact Dermatitis. 2016;74(2):105-9.
Total recovery:
8h: Recovery of applied dose (8h) acceptable: yes (ca. 93%).
24h: Recovery of applied dose (24h) acceptable: yes (ca. 94%).

Applicant's summary and conclusion

Conclusions:
The dermal absorption of cobalt metal powder was investigated after applying 100 mg/cm2 to human skin in the presence of artifical sweat. The compound was either applied to the skin for an 8-hour or 24-hour exposure period before being washed from the skin surface. Each piece of skin was sectioned using a cryostat to determine the depth of penetration of cobalt.

Generally, the largest proportion of the applied dose was consistently recovered from the wash from both 8- and 24-hour exposures (93.26 % and 93.45 %, respectively).

The potentially absorbed dose calculated as the sum of the total amount cobalt recovered with the receptor fluid and skin available for absorption (first 20 µm of skin omitted) was as follows:

8 hour exposure: 6 µg cobalt/cm² (equivalent to 0.006% of the applied dose)
24 hour exposure: 19 µg cobalt/cm² (equivalent to 0.02% of the applied dose)

The rationale for subtracting the first 20 μm was to remove any compound that was potentially retained within the stratum corneum and therefore only quantifying the amounts within the skin. However, it is not evident from the data if the compound had diffused through the stratum corneum into the underlying skin or had deposited deep within the hair follicles.