Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 December 2004 to 26 April 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
yes

Test animals

Species:
human
Strain:
other: not applicable
Sex:
female
Details on test animals and environmental conditions:
not applicable

Administration / exposure

Type of coverage:
open
Vehicle:
acetone
Duration of exposure:
See in Details on in vitro test system below.
Doses:
See in Details on in vitro test system below.
No. of animals per group:
Not applicable.
Details on study design:
See in Details on in vitro test system below.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Human Skin Samples:
Seven samples of full-thickness breast human skin were obtained from patients (aged 19 to 68 years old), who gave informed consent for their skin to be taken for scientific research purposes, prior to undergoing routine surgery at the Plastic Surgery Unit, St Johns Hospital, West Lothian NHS Trust, Livingston, UK. The skin was transferred to Inveresk stored on ice and cleaned of subcutaneous fat and connective tissue using a scalpel blade. The skins were washed in cold running water and dried using “blue roll” tissue paper. The sample was then cut into smaller pieces (where appropriate), wrapped in aluminium foil, put into self sealing plastic bags and stored at ca 20°C until required. The age and sex of the donor and site from which the skin was taken were recorded.

- Preparation of Split Thickness Skin Membranes:
When required, the human skin samples were removed from storage and allowed to thaw at ambient temperature. The thickness of the uncut 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 at a setting equivalent to 200-400 µm depth using a Zimmer electric dermatome. The membranes were then laid out onto aluminium foil and the thickness of the membranes measured using a micrometer.

PRINCIPLES OF ASSAY
- Flow Through Diffusion Cell Apparatus:
An automated flow through diffusion cell apparatus (Scott/Dick, University of Newcastle upon Tyne, UK) was used (see photograph overleaf). The flow through cells were placed in a steel manifold heated via a circulating water bath to maintain the skin surface temperature at ca 32ºC. The cells were connected to multi channel peristaltic pumps from their different 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 0.25 mL. The peristaltic pumps were adjusted to maintain a flow-rate of ca 1.5 mL/h.

- Receptor Fluid:
A tissue culture medium containing bovine serum albumin (ca 5%, w/v), glucose (ca 1%, w/v), streptomycin (ca 0.1 mg/mL) and penicillin G (ca 100 units/mL) was used as the receptor fluid for the tritiated water barrier integrity and test item permeability assessments.

- Solubility of Test Item in Receptor Fluid:
HBCD has a solubility in water of 65.2 ppb, a toluene solubility of >5% (w/w) and an acetone solubility of >1% (w/w). Therefore, any normally used receptor fluid (OECD Guideline 428) will not have sufficient HBCD solubility. Hence, a physiological receptor fluid was chosen. If absorption was high, then HBCD would be found in the skin layers and would therefore be included in the dermal delivery figure.

- Flow Through Diffusion Cell Preparation:
Sections of split thickness skin membrane, ca 1.5 x 1.5 cm, were cut out, positioned on the receptor chamber of the diffusion cell, containing a magnetic flea, and the donor chamber was tightened into place with screws. The cells were then placed in the heated manifold and connected to the peristaltic pump. The Variomag magnetic stirrer was switched on to mix the contents of the receptor chamber. An equilibration period of ca 15 min was allowed while receptor fluid was pumped through the receptor chambers at ca 1.5 mL/h. The effluent was then collected for ca 30 min and retained as blank samples for use in the tritiated water barrier integrity assessment.

- Barrier Integrity Assessment:
Tritiated water (250 µL, radioactivity ca 100,000 d.p.m.) was applied to the surface of each skin sample and the donor chamber occluded. Penetration of tritiated water was assessed by collecting hourly fractions for 2 hours and analysing the fractions by liquid scintillation counting. Permeability coefficients (kp) were calculated for each skin sample. Any human skin sample exhibiting a kp greater than 2.5 x 10 3 cm/h was excluded from subsequent absorption measurements. A cross reference of skin sample number and donor and its corresponding tritiated water permeability coefficient (kp) was performed. At the end of the 2-h period, residual tritiated water was removed from the skin surface by rinsing with water (ca 2 mL) and the skin was dried with tissue paper. An equilibration period was allowed prior to collection of the pre-dose sample which was collected for ca 30 min.

TEST METHOD
- Formulation of Test Preparation:
HBCD Composite (0.03744 g) was weighed into a 2 mL volumetric flask. [14C] HBCD in acetone stock solution (200 µL) was transferred into the volumetric flask. The volumetric flask was made up to the 2 mL line with acetone and mixed by inversion until the test item had dissolved. Seven 6.4 µL aliquots were taken into scintillation vials, mixed with 10 mL scintillant and analysed by liquid scintillation counting. The aliquots were homogeneous with a coefficient of variation of 3.47%. The mean mass of [14C]-HBCD in each 6.4 µL aliquot was 136.24 µg. Therefore, it was calculated that 30.1 µL would contain 640 µg. To ensure optimal delivery, it was decided to apply the test preparation to the skin in five 6.0 µL aliquots within ca 15 min. This test preparation was accepted for dosing.

- Application of Test Preparation:
The test preparation was applied over the stratum corneum surface of the exposed skin using an M25 Gilson Microman positive displacement pipette set to deliver 6.0 µL, once the acetone had evaporated from the skin surface this was repeated 4 more times until a total of 30 µL (47 µL/cm2) had been applied to the skin. The dosing procedure took a total of ca 15 min to complete. The donor chambers were left open to the atmosphere. To accurately quantify the radioactivity applied to the skin samples, 7 aliquots (30 µL) of the test preparation were collected in Combustocones® in the same manner at the time of dosing. These mock dose samples were used to calculate mass balance.

ANALYSIS
- Analysis of Mock Dose Samples:
The mock dose samples (as above) were analysed by combustion/ liquid scintillation counting. The total mass of [14C] HBCD applied to the skin was determined to be 711.92 µg (1.1 mg/cm2), which was 111.24% of the target application of 640 µg (1 mg/cm2). As this was much higher than anticipated, it was believed that the mock dosing samples may not have been representative of the dosing to the skin. The Combustocones® contain absorptive pads and the skin is non absorptive to solvents. To demonstrate this, 6.0 µL of the dosing solution was applied to six Combustocones® and this was repeated to each Combustocone® 4 times (total volume 30 µL). At the same time, 6.0 µL of the dosing solution was transferred to 20 scintillation vials. Acetone (1 mL) and then scintillant (10 mL) was then added. The Combustocones® were then analysed by combustion/ liquid scintillation counting and the scintillation vial samples were analysed by liquid scintillation counting. The counts for each group of 5 vials were added together. The results showed that the scintillation vials contained only 85.24% of the radioactivity of the combustion samples. A conversion factor of 85.24% was, therefore, applied to the mock dose samples. The corrected total mass of [14C] HBCD applied to the skin was calculated to be 606.84 µg (0.95 mg/cm2), which was 94.82% of the target application of 640 µg (1 mg/cm2).

- Sampling Information:
- Receptor Fluid:
Receptor fluid was collected in hourly fractions from 0 to 8-h post dose and then in 2 hourly fractions from 8-24 h post dose. All receptor fluid samples were mixed with ca. 10 mL scintillation fluid and analysed by liquid scintillation counting.

- Terminal Procedures – 8-h Post Dose:
At 8-h post dose, the exposed skin surface was washed (skin wash 8-h) with ca. 10 mL of a ca. 2% (v/v) soap solution (Radox Supersoap). The skin wash was aspirated with a pipette and collected into a pre weighed vial and mixed with ca 10 mL acetone. The mass of skin wash was weighed and duplicate weighed, ca 1 mL, aliquots taken for analysis by liquid scintillation counting. The pipette tip was cut up and placed into a scintillation vial, mixed with scintillant and analysed directly by liquid scintillation counting. The cell and skin surface was dried with tissue paper swabs (skin swab 8-h). These swabs were pooled and placed in a Combustocone® for subsequent analysis by combustion/ liquid scintillation counting. Initial analysis of the duplicate aliquots for the skin wash 8-h samples indicated that the samples were not homogeneous. The remainder of each sample (ca 18 mL) was divided between 8 vials (7 new vials and the bulk sample vial) and these samples were then analysed directly by liquid scintillation counting. The pipette tip was analysed as described previously. The counts for each portion were added to the counts previously obtained from the duplicate analysis to constitute a total recovery value.

- Terminal Procedures – 24-h Post Dose:
At 24 h post dose (i.e. following a 16-h monitoring period), each diffusion cell was disconnected from the receptor fluid pump lines. The underside of the skin was washed (receptor rinse) with receptor fluid (1-2 mL), which was mixed with scintillation fluid (10 mL) and analysed by liquid scintillation counting. The receptor rinse represented the absorbed test item, which was in the receptor chamber but had not been collected in the 22 to 24-h receptor fluid fraction.

The cell and skin surface were dried with a tissue paper swab (skin swab 24-h) and the cell dismantled. The skin was removed from the cell and dried with a further tissue paper swab. These swabs were pooled and placed in a Combustocone® for subsequent analysis by combustion/ liquid scintillation counting.

The donor and receptor chambers were transferred to pre weighed pots (cell wash) containing a known weight (ca 40 mL) of acetone. The pots containing the cells were left to extract the test item for ca 30 mins and then sonicated for ca 10 mins and then duplicate weighed aliquots (1 mL) were taken for analysis by liquid scintillation counting. The donor and receptor chambers were then removed from the cell wash pots.

The stratum corneum was removed with 20 successive tape strips (Guilbert, Niceday). The first 5 tapes were pooled together in a Combustocone®. This was repeated for tapes 6-10, 11-15 and 16-20. The skin under the cell (unexposed skin) was cut away from the exposed skin with scissors. These samples were placed into Combustocones® for subsequent analysis by combustion/liquid scintillation counting.

- Storage of Samples:
All bulk samples not immediately analysed were stored at ca 20ºC. After analysis, samples were returned to storage at ca 20ºC.

- Combustion Analysis:
Mock dose, skin, tape strip and tissue swab samples were combusted using a Model 307 Tri Carb Automatic Sample Oxidiser (Canberra Packard Limited). The resultant 14CO2 generated was absorbed in Carbo Sorb® (8 mL) and mixed with Permaflour®E+ scintillation fluid (10 mL). Combustion efficiency and carry over were checked at the start of each run of 30 samples by combusting quality control standards containing Spec ChecTM 14C. Combustion efficiency was within the range of 97-103%.

- Quantification of Total Radioactivity:
All samples, except for the tritiated water samples, were counted for 5 min together with representative blanks using a liquid scintillation analyser (Packard 2100 TR) with automatic quench correction by external standard. Representative blank sample values were subtracted from sample count rates to give net d.p.m. per sample. Prior to analysis, samples were allowed to stabilise with regard to light and temperature. The tritiated water samples were treated as above, except that they were subject to liquid scintillation counting for 1 min only.

- Limit of Reliable Measurement:
A limit of reliable measurement of 30 d.p.m. above background has been instituted in these laboratories. Any occasions where results arose from data below the limit of reliable measurement have been noted in the results section of the report.

CALCULATIONS
- Permeability Coefficient (kp) of Water:
Cumulative absorption of tritiated water was calculated for each skin sample by summing the net d.p.m. for each hourly fraction from 0 to 2-h. The slope of the absorption versus time curve from 0 to 2-h (i.e. 3 data points) was calculated by linear regression to give an absorption rate (d.p.m./cm2/h).

Absorption rate (d.p.m./cm2/h) = slope (d.p.m./h) / exposed area (cm2)

This was then converted to the permeability coefficient (kp) from the dose application rate of tritiated water as follows:

Kp = absorption rate (d.p.m./cm2/h) / application rate (d.p.m./cm3)

- Absorption of Radiolabelled Test Item (Flux and Percentage Absorbed):
The absorbed dose was calculated from each individual sample (receptor fluid samples were given as cumulative absorbed dose) radioactivity (d.p.m.), specific activity (SA) and dose area as follows:

Absorbed dose (µg equiv./cm2) = sample radioactivity (d.p.m.) / (SA (d.p.m./µg equiv.) x exposure area (cm2))

In addition, the percentage absorbed dose was also calculated for each sample as follows:

Absorbed dose (%) = sample radioactivity (d.p.m.) / applied dose (d.p.m.) x 100%

Results and discussion

Absorption in different matrices:
A total of 9 samples of human skin, obtained from 6 different donors, were dosed topically with [14C]-HBCD in an acetone vehicle. Cell 2 was rejected from the mean ± SD as it had a poor mass balance (less than 90%). Cell 9 was rejected as it had just failed the tritiated water barrier integrity assessment (2.6E-3 cm/h, rejection criterion >2.5E-3 cm/h) and it had a high value in the exposed skin (7.57%, which was outside the mean ± 2SD of the other samples of exposed skin). Therefore, the following results are based on 7 samples of skin obtained from 5 different donors.

At 8-h post dose, 34.62% of the applied dose was washed off (6.98%, 0.19% and 27.45% were recovered in the skin wash 8-h, tip and tissue swab 8-h, respectively). At 24-h post dose, the mean mass balance was 96.67% of the applied dose. The tissue swab 24-h and cell wash contained 5.70% and 23.06% of the applied dose, respectively. Therefore, at 24-h post dose, the dislodgeable dose was 63.37% of the applied dose. The mean total unabsorbed dose was 95.31% of the applied dose. This consisted of the dislodgeable dose, unexposed skin (0.45%) and the radioactivity associated with the stratum corneum (31.49%). The stratum corneum acted as a good barrier to the test item as the bulk of the radioactivity (25.70%) was recovered in the outermost 5 tape strips (tape strips 1 to 5). Considerably less radioactivity was recovered with each of the subsequent 3 groups of tape strips (3.12%, 1.54% and 1.13% in tape strips 6-10, 11-15 and 16-20, respectively). This indicated that the HBCD was on the skin surface and would be anticipated to be in the stratum corneum which would be sloughed off of the skin. The absorbed dose (0.01%) was the sum of the receptor fluid (0.01%) and the receptor rinse (<0.01%). Dermal delivery (1.35%) was the sum of the absorbed dose and the exposed skin (1.34%).

The mass balance, dislodgeable, unabsorbed, dermal delivery and absorbed doses were 916.58, 600.90, 903.76, 12.82 and 0.06 µg.equiv./cm2, respectively. There were two steady state fluxes observed in this study. The first was attained from 0 to 6-h post dose. The mean steady state flux rate over this period was calculated to be 1.36 ng equiv./cm2/h. The second was attained from 6-h to 24-h post dose. The mean steady state flux rate over this period was calculated to be 2.64 ng equiv./cm2/h.
Percutaneous absorptionopen allclose all
Parameter:
percentage
Absorption:
0 %
Remarks on result:
other: 24 hours
Remarks:
Mean Total absorption 0.01% applied radioactivity
Parameter:
percentage
Absorption:
0 %
Remarks on result:
other: 24 hours
Remarks:
Mean Total absorption 0.06 [14C]-HBCD µg equiv./cm2
Conversion factor human vs. animal skin:
not applicable

Any other information on results incl. tables

Table 5. Distribution of Radioactivity (% Applied Dose) at 24-h Post Dose Following Topical Application of HBCD to Human Split-Thickness Skin

  Cell Number and Donor Number Mean SD
Cell 2 0105 Cell 4 0082 Cell 5 0067 Cell 8 0086 Cell 9 0105 Cell 11 0082 Cell 12 0067 Cell 13 0070 Cell 14 0109
Skin Wash 8 h 13.96 4.5 8.17 9.33 6.08 10.67 3.41 8.15 4.66 6.98 2.77
Tissue Swab 8 h 16.36 48.17 13.24 27.86 22.28 25.49 16.07 40.84 20.48 27.45 12.86
Pipette Tips 8 h 0.21 0.01 0.24 0.14 0.14 0.26 0.08 0.2 0.37 0.19 0.12
Tissue Swab 24 h 1.17 2.05 3.68 2.99 1.95 7.85 9.51 7.88 5.93 5.7 2.85
Cell Wash 26.39 10.82 38.18 31.96 26.17 31.31 12.06 14.12 22.95 23.06 11
Dislodgeable Dose 58.09 65.55 63.51 72.28 56.62 75.57 41.13 71.19 54.39 63.37 12.04
Stratum corneum 1-5 18.09 33.4 23.48 20.83 25.67 15.07 42.06 18.58 26.49 25.7 9.31
Stratum corneum 6-10 2.65 1.6 1.71 3.04 2.04 1.77 5.43 3.54 4.74 3.12 1.54
Stratum corneum 11-15 1.53 1.05 0.91 1.38 4.56 1.21 1.16 1.16 3.9 1.54 1.05
Stratum corneum 16-20 0.97 0.59 0.83 1.59 1.47 0.72 1.13 1.09 1.94 1.13 0.49
Stratum Corneum 23.25 36.65 26.92 26.85 33.74 18.78 49.78 24.38 37.08 31.49 10.39
Unexposed Skin 0.14 0.1 0.51 0.64 0.57 0.73 0.28 0.59 0.31 0.45 0.23
Total Unabsorbed 81.47 102.29 90.94 99.77 90.92 95.08 91.19 96.16 91.77 95.31 4.43
Exposed Skin 4.11 0.38 1.49 1.54 7.57 1.53 1.37 1.96 1.14 1.34 0.49
Receptor Fluid 0.01 *0.00 *0.00 0.01 0.01 0 0.01 0.01 0.01 °0.01 0
Receptor Rinse *0.00 *0.00 *0.00 0 *0.00 0 *0.00 0 0 °0.00 0
Total Absorbed 0.01 0 0 0.01 0.01 0 0.01 0.01 0.01 0.01 0
Dermal Delivery 4.12 0.38 1.5 1.55 7.58 1.54 1.37 1.96 1.16 1.35 0.49
Mass Balance 85.59 102.67 92.43 101.32 98.5 96.62 92.56 98.12 92.93 96.67 4.25

Table 6. Cumulative absorption (% Applied Dose) of [14C]-HBCD into Receptor Fluid Dollowing Topical Application of HBCD to Human Split-Thickness Skin

Time (h) Cell Number and Donor Number Mean SD
Cell 2 0105 Cell 4 0082 Cell 5 0067 Cell 8 0086 Cell 9 0105 Cell 11 0082 Cell 12 0067 Cell 13 0070 Cell 14 0109
0 *0.000 *0.000 *0.000 *0.000 *0.000 *0.000 *0.000 *0.000 *0.000 °0.000 0
1 *0.000 *0.000 *0.000 *0.000 *0.000 *0.000 *0.000 *0.000 *0.000 °0.000 0
2 *0.000 *0.000 *0.000 *0.000 *0.001 *0.000 *0.000 *0.000 *0.001 °0.000 0
3 *0.000 *0.000 *0.000 *0.000 *0.001 *0.000 *0.000 *0.000 *0.001 °0.000 0
4 *0.001 *0.000 *0.000 *0.000 *0.001 *0.000 *0.000 *0.001 *0.001 °0.001 0
5 *0.001 *0.001 *0.000 *0.001 *0.001 *0.000 *0.001 *0.001 *0.001 °0.001 0
6 *0.001 *0.001 *0.001 *0.001 *0.001 *0.001 *0.001 *0.001 *0.002 °0.001 0
7 *0.001 *0.001 *0.001 *0.001 *0.002 *0.001 *0.001 *0.001 *0.002 °0.001 0
8 *0.002 *0.001 *0.001 *0.001 *0.002 *0.001 *0.001 0.002 *0.002 °0.001 0.001
10 *0.002 *0.001 0.002 0.002 *0.002 *0.001 0.002 *0.002 0.003 °0.002 0.001
12 *0.002 *0.002 *0.002 0.003 *0.003 *0.001 *0.002 0.003 0.004 °0.002 0.001
14 0.003 *0.002 *0.002 0.004 0.004 *0.001 *0.002 0.003 0.004 °0.003 0.001
16 *0.003 *0.002 *0.002 0.006 0.005 *0.002 0.003 0.004 0.005 °0.003 0.002
18 0.004 *0.002 *0.002 0.007 *0.005 *0.002 0.003 *0.005 0.006 °0.004 0.002
20 0.004 *0.002 0.003 0.009 0.006 *0.002 *0.004 *0.005 0.007 °0.005 0.003
22 0.005 *0.003 0.004 0.011 0.006 *0.002 0.005 *0.006 0.008 °0.005 0.003
24 0.006 *0.003 *0.004 0.012 0.007 0.003 0.005 0.006 0.008 °0.006 0.004

Table 7. Distribution of [14C]-HBCD (µg equiv./cm2) at 24-h Post Dose Following Topical Application of HBCD to Human Split-Thickness Skin

  Cell Number and Donor Number Mean SD
Cell 2 0105 Cell 4 0082 Cell 5 0067 Cell 8 0086 Cell 9 0105 Cell 11 0082 Cell 12 0067 Cell 13 0070 Cell 14 0109
Dislodgeable Dose 550.81 621.56 602.19 685.42 536.82 716.53 389.94 675.01 515.66 600.9 114.17
Stratum Corneum 220.42 347.47 255.22 254.56 319.87 178.04 472.05 231.14 351.56 298.58 98.47
Total Unabsorbed 772.52 969.93 862.27 946.06 862.09 901.52 864.67 911.74 870.13 903.76 42.07
Total Absorbed 0.06 0.03 0.04 0.12 0.07 0.03 0.05 0.06 0.12 0.06 0.04
Dermal Delivery 39.02 3.64 14.2 14.7 71.87 14.57 13.01 18.62 10.98 12.82 4.65
Total Recovery 811.54 973.57 876.46 960.77 933.96 916.09 877.68 930.37 881.11 916.58 40.36

Table 8. Cumulative Absorption (ng equiv./cm2) of[14C]-HBCD into Receptor Fluid Following Topical Application of HBCD to Human Split-Thickness Skin

Time (h) Cell Number and Donor Number Mean SD
Cell 2 0105 Cell 4 0082 Cell 5 0067 Cell 8 0086 Cell 9 0105 Cell 11 0082 Cell 12 0067 Cell 13 0070 Cell 14 0109
0 0 0 0 0 0 0 0 0 0 0 0
1 1.12 1.15 1.83 1.41 2.31 1.14 0.69 1.57 1.89 1.38 0.43
2 2.71 2.26 3.07 2.24 5.11 2.28 3.02 3.69 5 3.08 1.01
3 3.22 3.28 3.07 3.58 7.47 3.27 4.02 4.68 8.02 4.27 1.74
4 5.74 4.5 3.38 4.61 10 3.72 4.52 6.39 10.59 5.39 2.48
5 7.17 5.01 4.42 6.47 11.35 4.21 5.42 8.14 12.48 6.59 2.93
6 8.62 5.97 6.16 8.08 12.62 5.44 6.45 10.3 14.63 8.15 3.31
7 11.02 7.37 8.07 10.8 14.88 6.44 8.06 12.28 18.42 10.2 4.15
8 14.37 8.42 9.21 13.14 16.98 8.09 9.31 17.68 21.02 12.41 5.11
10 18.57 11.13 14.93 22.88 20.78 8.99 15.15 22.08 30.85 18 7.65
12 21.05 15.04 19.16 31.48 24.51 9.55 18.73 27.02 37.28 22.61 9.73
14 26.29 17.85 20.93 41.24 37.37 11.86 22.62 31.76 42.27 26.94 11.74
16 30.65 19.34 21.88 55.52 44.03 14.75 28.43 42.58 47.76 32.89 15.71
18 36.2 20.84 23.05 70.93 47.62 15.78 33.17 45.72 61.04 38.65 21.24
20 41.56 22.42 29.5 88.14 52.59 17.87 36.26 47.98 66.33 44.07 25.43
22 48.5 24.4 35.77 103.17 58.99 19.3 43.11 52.4 73.15 50.18 29.49
24 53.96 26.94 36.78 117.68 64.58 24.11 48.18 57.16 79.02 55.7 33.24

*Results calculated from data less than 30 d.p.m. above background

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

 

Cell 2 was rejected from Mean and SD due to mass balance being less than 90%

Cell 9 was rejected as it had high tritiated water barrier integrity value and a high exposed skin value

Applicant's summary and conclusion

Conclusions:
In conclusion, following topical application of [14C]-HBCD to human split thickness skin in vitro, the absorbed dose and dermal delivery were 0.01% (0.06 µg.equiv./cm2) and 1.35% (12.82 µg.equiv./cm2) of the applied dose, respectively. At 8-h post dose, 34.62% of the applied dose was removed from the skin by washing and drying. At 24-h a further 28.76% was recovered in the 24-h skin drying and cell wash. Therefore, the dislodgeable dose was 63.37% of the applied dose. The stratum corneum contained a further 31.49% of the applied dose. The bulk of this (25.70%) was recovered in the first 5 tape strips. Since the bulk of the stratum corneum associated material was found in the first 5 tapes strips, this indicated that the [14C] HBCD was on the surface of the skin and that the stratum corneum was an efficient barrier to [14C]-HBCD penetration.
Executive summary:

Hexabromocyclododecane (CAS No. 25637‑99‑4), also known as HBCD, is a brominated flame retardant that is managed under the American Chemistry Council – Brominated Flame Retardant Industry Panel (ACC‑BFRIP) consortium.

 

HBCD is a homogeneous mixture of three diastereomers (alpha, beta and gamma). The gamma is present at the highest percentage level of the three when manufactured. HBCD has very low solubility in most common solvents.

 

As part of the safety evaluation of HBCD, a study was conducted to assess the rate and extent of absorption of HBCD following topical application of HBCD to human skin.

 

The split-thickness skin membranes were mounted into flow-through diffusion cells. Receptor fluid (tissue culture medium containing bovine serum albumin, glucose, streptomycin and penicillin G) was pumped underneath the skin at a flow rate ofca 1.5 mL/h and a tritiated water barrier integrity test performed. The skin surface temperature was maintained at ca 32°C throughout. All skin samples with a tritiated water permeability coefficient (kp) less than 2.5 x 10-3 cm/h were accepted for use.

 

[14C]‑HBCD was applied in an acetone vehicle at ca 47 µL/cm2in 5 applications of 6.0 µL over a ca 15 min period to human split‑thickness skin membranes mounted in flow‑through diffusion cells in vitro. The [14C]‑HBCD could not be applied as the powder as the mass to be applied (ca 640 µg, 1 mg/cm2) could not be accurately dispensed. Therefore, [14C]-HBCD was applied as a solution using acetone as the vehicle. The test preparation ([14C]-HBCD in acetone) could not be applied in a single volume application because the solubility of HBCD in acetone was too low. The acetone evaporated rapidly from the skin surface leaving behind the [14C]‑HBCD.

 

Absorption was assessed by collecting receptor fluid in hourly fractions from 0 to 8-h post dose and then in 2‑hourly fractions from 8 to 24-h post dose. At 24-h post dose, the exposure was terminated by washing and drying the skin. The stratum corneum was then removed by successive tape stripping. All samples were analysed by liquid scintillation counting.

 

A summary of the results is provided in the table below:

  Mean SD
Target HBCD Application Rate (µg/cm2) 1000 -
Actual HBCD Application Rate (µg/cm2) 950 -
Dislodgeable Dose (µg equiv./cm2) 600.9 114.17
Unabsorbed Dose (µg equiv./cm2) 903.76 42.07
Absorbed Dose (µg equiv./cm2) 0.06 0.04
Dermal Delivery (µg equiv./cm2) 12.82 4.65
Mass Balance (µg equiv./cm2) 916.58 40.36
Dislodgeable Dose (% Applied Dose) 63.37 12.04
Unabsorbed Dose (% Applied Dose) 95.31 4.43
Absorbed Dose (% Applied Dose) 0.01 0
Dermal Delivery (% Applied Dose) 1.35 0.49
Mass Balance (% Applied Dose) 96.67 4.25

In conclusion, following topical application of [14C]-HBCD to human split‑thickness skin in vitro, the absorbed dose and dermal delivery were 0.01% (0.06 µg.equiv./cm2) and 1.35% (12.82 µg.equiv./cm2) of the applied dose, respectively. At 8-h post dose, 34.62% of the applied dose was removed from the skin by washing and drying. At 24-h a further 28.76% was recovered in the 24-h skin drying and cell wash. Therefore, the dislodgeable dose was 63.37% of the applied dose. The stratum corneum contained a further 31.49% of the applied dose. The bulk of this (25.70%) was recovered in the first 5 tape strips. Since the bulk of the stratum corneum associated material was found in the first 5 tapes strips, this indicated that the [14C]‑HBCD was on the surface of the skin and that the stratum corneum was an efficient barrier to [14C]-HBCD penetration.