Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Remarks:
In Vitro Dermal Penetration Study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 February 2018 to 3 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
OECD Guideline 428: Skin Absorption In Vitro Method (Adopted 13th April 2004)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.45 (Skin Absorption: In Vitro Method)
Version / remarks:
Council Regulation (EC) No. 440/2008, Method B.45; Skin Absorption: In Vitro Method, Brussels, May 2008.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EFSA Panel on Plant Protection Products and their Residues (PPR); Guidance on Dermal Absorption: EFSA Journal 2017; 15(6):4873.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance Document 28: Guidance Document For the Conduct of Skin Absorption Studies (5 March 2004)
Deviations:
no
GLP compliance:
yes

Test material

Test material form:
liquid: viscous
Details on test material:
Name: Disflamoll TKP-P and Kronitex TCP mixture (1:1)
Batch/lot number: CH3/08086 / 2015053500
Physical form/appearance: Colourless liquid
Chemical purity
Test compound contains mixture of the following component:
m,m,m-Tricresyl phosphate 32.6%
m,m,p-Tricresyl phosphate 43.3%
m,p,p-Tricresyl phosphate 19.7%
p,p,p-Tricresyl phosphate 3.2%
Unknown components 1.2%
Expiry: 8 August 2018
Storage conditions: Room temperature
Safety precautions: Known reproductive toxicity. Usual precautions for a chemical substance undergoing regulatory testing (standard PPE and avoid release to the environment)
Specific details on test material used for the study:
General information
Chemical name: Tricresyl phosphate
CAS No: 1330-78-5
Molecular formula (free-base): (CH3C6H4O)3PO
Safety precautions: Known reproductive toxicity. Usual precautions for a chemical substance undergoing regulatory testing (standard PPE and avoid release to the environment).

Radiolabelled compound
Name: Tri-[ring-U-14C]cresyl phosphate
Source/supplier: Pharmaron UK Ltd.
Batch/lot number: CFQ43176
Physical form/appearance: Solution in pentane
Molecular weight: 370.3
Specific activity: 60 mCi/mmol
Radiochemical purity: 98.9%
Storage conditions: ca. -20˚C
Expiry date: Radiochemical purity was measured pre- and post- application
Safety precautions: Usual precautions for a chemical substance undergoing regulatory testing

Radiolabelled compound re-purified batch
Name: Tri[14C]cresyl phosphate
Source/supplier: Pharmaron UK Ltd.
Batch/lot number: CFQ43489
Physical form/appearance: Solution in pentane
Molecular weight: 370.2
Specific activity: 58 mCi/mmol
Radiochemical purity: >99.9%
Storage conditions: ca. -20˚C
Expiry date: Radiochemical purity was measured pre- and post- application
Safety precautions: Usual precautions for a chemical substance undergoing regulatory testing
Radiolabelling:
yes

Test animals

Species:
other: Dermatomed abdominal human skin samples
Strain:
other: Not applicable
Sex:
female
Details on test animals or test system and environmental conditions:
Dermatomed abdominal human skin samples (340 - 406 μm, obtained from Biopredric from 4 female donors aged 36 to 68 years old were used in this study. The skin was received and stored frozen at c.a. -20°C following receipt.
Formulations were applied to the skin from four donors in duplicate (total 8 replicates per test substance concentration).
The experiment was conducted using cells with an in-line flow through receptor chamber, with a 0.64 cm2 exposed skin area, with a receptor compartment volume of 0.254 mL, and with a flow rate of 1 mL/hr. Receptor compartment fluid consisted of 0.9% saline (w/v) + 5% tween 80 (v/v) (integrity check) or 0.9% saline (w/v) + 5% tween 80 (v/v) + 40% Ethanol (v/v) (main dermal absorption experiment).

Administration / exposure

Type of coverage:
open
Vehicle:
other: Mineral Oil
Remarks:
[14C]-labelled test compound was radiodiluted with non-radiolabelled test compound as considered appropriate and diluted with mineral oil (obtained from Sigma-Aldrich) to prepare application solutions of 100, 40 and 5% Tricresyl phosphate (v/v)
Duration of exposure:
Skin was exposed to three concentrations of the test substance formulation for 8 hours.
Doses:
[14C]-Tricresyl phosphate (100%, v/v) formulation preparation
[14C]-Tricresyl phosphate (40%, v/v) formulation preparation
[14C]-Tricresyl phosphate (5%, v/v) formulation preparation
No. of animals per group:
8 replicates per test concentration
Control animals:
no
Details on study design:
Test compound/receptor fluid solubility assessment
Prior to conducting the main in vitro dermal penetration experiment the solubility of the test item in the following receptor fluids was assessed. The following receptor fluids were tested at different concentrations of [14C]-Tricresyl phosphate:
0.9% saline (w/v) + 5% bovine serum albumin (BSA) (w/v)
0.9% saline (w/v) + 10% bovine serum albumin (BSA) (w/v)
0.9% saline (w/v) + 5% tween 80 (v/v)
0.9% saline (w/v) + 5% polyethylene glycol (v/v)
0.9% saline (w/v) + 5% 2-hydroxypropyl-β-cyclodextrin (w/v)
Triplicate aliquots of 950 μL for each receptor fluid were spiked with 50 μL of [14C]-Tricresyl phosphate dose formulation to obtain solutions at target concentration of 5% v/v TCP, triplicate aliquots of 975 μL for each receptor fluid were spiked with 25 μL of [14C]-Tricresyl phosphate dose formulation to obtain solutions at target concentration of 2.5% v/v TCP, and triplicate aliquots of 987.5 μL for each receptor fluid were spiked with 12.5 μL of [14C]-Tricresyl phosphate dose formulation to obtain solutions at target concentration of 1.25% v/v TCP.
The vials were mixed at room temperature for at least 1 hour before triplicate aliquots were taken, one from the top, middle and bottom of the liquid, for liquid scintillation counting to determine the concentration of [14C]-Tricresyl phosphate. The vials were then left to stand for approximately 1 hour and a second set of triplicate aliquots were taken, one from the top, middle and bottom of the liquid, for liquid scintillation counting to determine the concentration of [14C]-Tricresyl phosphate.

Additional test compound/receptor fluid solubility assessment
Initial review of the solubility assessment suggested the test compound may not be soluble in the receptor fluids initially tested, therefore further method development experiments were performed by adding ethanol to suitable receptor fluids to investigate solubility of the test compound before further solubility tests were performed.
Additional solubility tests were performed at lower concentrations (0.6 and 1.2% (v/v) final concentration of TCP) in 0.9% saline (w/v) + 5% tween 80 (v/v) with increasing concentrations of ethanol (10%, 20% and 40%).
Following review of the additional solubility tests in 0.9% saline (w/v) + 5% tween 80 (v/v) with increasing concentrations of ethanol, it was concluded that adequate solubility could not be confirmed at the concentrations tested. Additional solubility tests were therefore performed at lower concentrations (0.15, 0.3 and 0.6% (v/v) final concentration of TCP) in 0.9% saline (w/v) + 5% tween 80 (v/v) + 40% Ethanol (v/v).
Each of three target concentrations were made in triplicate using one of the following mixtures: 598.5 μL of receptor fluid and 400 μL of ethanol were spiked with 1.5 μL of [14C]-Tricresyl phosphate dose formulation to obtain solutions at target concentration of 0.15% v/v TCP, 597 μL of receptor fluid and 400 μL of ethanol were spiked with 3 μL of [14C]-Tricresyl phosphate dose formulation to obtain solutions at target concentration of 0.3% v/v TCP, and 594 μL of receptor fluid and 400 μL of ethanol were spiked with 6 μL of [14C]-Tricresyl phosphate dose formulation to obtain solutions at target concentration of 0.6% v/v TCP.
The vials were mixed at room temperature for at least 1 hour before triplicate aliquots taken, one from the top, middle and bottom of the liquid, for liquid scintillation counting to determine the concentration of [14C]-Tricresyl phosphate. The vials were then left to stand for approximately 1 hour and a second set of triplicate aliquots were taken, one from the top, middle and bottom of the liquid, for liquid scintillation counting to determine the concentration of [14C]-Tricresyl phosphate.

Preparation of dosing formulations for application to skin
During this study radiolabelled trial formulations were initially prepared and analysed and results were reviewed before proceeding with the main preparation.

[14C]-Tricresyl phosphate (100%, v/v) formulation preparation
100 μL of radiolabelled Tricresyl phosphate solution in pentane was aliquoted into a pre- weighed vial and the pentane removed under N2 (~0.544 μL, [14C]-Tricresyl phosphate remaining in vial), 499 μL of non-radiolabelled Disflamoll TKP-P and Kronitex TCP mixture (1:1) was weighed into the same vial to prepare the 100% v/v formulation.

[14C]-Tricresyl phosphate (40%, v/v) formulation preparation
100 μL of radiolabelled Tricresyl phosphate solution in pentane was aliquoted into a pre- weighed vial and the pentane removed under N2 (~0.544 μL, [14C]-Tricresyl phosphate remaining in vial), 199 μL of non-radiolabelled Disflamoll TKP-P and Kronitex TCP mixture (1:1) was weighed into the same vial. The resulting formulation was mixed well and 300 μL of mineral oil was added to prepare the 40% v/v formulation.

[14C]-Tricresyl phosphate (5%, v/v) formulation preparation
100 μL of radiolabelled Tricresyl phosphate solution in pentane was aliquoted into a pre-weighed vial and the pentane removed under N2 (~0.544 μL, [14C]-Tricresyl phosphate remaining in vial), 24.5 μL of non-radiolabelled Disflamoll TKP-P and Kronitex mixture (1:1) was weighed into the same vial. The resulting formulation was mixed well and 475 μL of mineral oil was added to prepare the 5% v/v formulation.

Formulation analysis
The homogeneity of the dose formulations were determined by measuring the radioactive concentrations of aliquots of undiluted formulation, or aliquots of formulation diluted in ethanol, prior to LSC (depending on concentrations of radioactivity).
Aliquots of the formulations were also diluted in ethanol, if appropriate, prior to determination of radiochemical purity by chromatographic analysis (HPLC-RAD).
Details on in vitro test system (if applicable):
In vitro dermal penetration experiment
Receptor fluid
0.9% saline (w/v) + 5% tween 80 (v/v) was used for the integrity check experiment and 0.9% saline (w/v) + 5% tween 80 (v/v) + 40% ethanol (v/v) was used for the main in vitro dermal penetration experiment for all concentrations of the formulation, and was continuously pumped at approximately 1 mL/hour.

Skin preparation integrity check
The skin was checked for physical damage prior to being mounted in the test system. The system was then allowed to warm to approximately 32 ± 1ºC prior to a topical application of 18 μL tritiated water (~10 μCi/mL) on the skin in each cell. Receptor fluid (0.9% Saline (w/v) + 5% tween 80 (v/v)) was collected for analysis pre-dose, 0 - 0.5, 0.5 - 1 and 1 - 2 hours after application. Each sample was mixed with 3 mL Gold Star (Meridian Biotechnologies Ltd.) scintillant and counted on a liquid scintillation counter. After the last sample time-point the tritiated water was desorbed by rinsing with 0.9% saline (w/v). The skin was maintained for use at approximately 32 ± 1°C in the test system overnight to allow wash out of tritiated water.

Application to the skin
On the day of application to skin, the receptor fluid was replaced with 0.9% saline (w/v) + 5% tween 80 (v/v) + 40% ethanol, the system was allowed to equilibrate and the skin was warmed up to 32 ± 1°C.
A set volume of 6.4 μL (10 μL/cm2) of each formulation was applied to the skin from four human donors in duplicate. The three concentrations of the test substance formulation prepared were as far as reasonably possible evenly distributed across the surface of the skin using a positive displacement pipette.

Temperature
The approximate skin temperature was monitored throughout the experiment and the temperature was maintained at 32 ± 1°C.

Exposure and sampling
Skin was exposed to three concentrations of the test substance formulation for 8 hours. At 8 hours post-application, the dose was removed from the test skin by washing the area with a 3% aqueous solution of liquid Dove soap, followed by rinsing with water. If considered necessary, the two step washing processes was repeated and skin sites washed using cotton swabs in an attempt to remove any visible residues of the formulation and also dried with a cotton swab. All washings, swabs and pipette tips were retained for quantitative radiochemical analysis (QRA), the radioactivity recovered from these samples have been summed and reported as skin wash. Skin was maintained in cells for an additional 16 hours (up to 24 hours after application).
Following application of the test formulations samples of receptor fluid were collected at the following time intervals over a 24 hour sampling period:
0-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-22 and 22-24 hours after application. = 16 samples
Receptor fluid aliquots were combined with 4 mL of Gold Star (Meridian Biotechnologies Ltd.) scintillant and counted on a liquid scintillation counter.

Terminal procedures
Following collection of the 24 hour sample the test system was dismantled and all components retained for analysis to determine a mass balance.
The remaining terminal receptor fluid was transferred into an appropriate container and stored at room temperature prior to quantitative radiochemical analysis (QRA).
The skin was removed and skin fractionation was performed at the termination of the study. Tape strips (Standard D-Squame, CuDerm Corporation) were used to remove the stratum corneum layer of the skin samples from each test cell. Up to twenty individual strips were used and the first ten strips analysed separately. The number of tape strips required to remove the stratum corneum varied between different skin samples and tape stripping was ceased when the strips failed to adhere to the skin surface. The tape strips were solubilised using Soluene 350 (Perkin Elmer) and taken for quantitative radiochemical analysis (QRA).
The remaining skin was solubilised using GoldiSol (Meridian Biotechnologies Ltd.) and analysed by liquid scintillation counting.
The remaining flow-through cell, which consisted of the donor chamber and receptor chamber were placed in separate containers, to which an appropriate volume of ethanol was added. The extraction solutions were weighed and duplicate aliquots taken for quantitative radiochemical analysis (QRA).
Following both collection and analysis, any residual samples were stored at room temperature.

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
Not examined
Percutaneous absorptionopen allclose all
Key result
Time point:
24 h
Dose:
[14C]-Tricresyl phosphate 100% v/v formulation
Parameter:
percentage
Absorption:
2.45 %
Remarks on result:
other: +/-1.49
Key result
Time point:
24 h
Dose:
[14C]-Tricresyl phosphae 40% v/v formulation
Parameter:
percentage
Absorption:
1.24 %
Remarks on result:
other: +/-0.67
Key result
Time point:
24 h
Dose:
[14C]-Tricresyl phosphate 5% v/v formulation
Parameter:
percentage
Absorption:
6.81 %
Remarks on result:
other: +/-2.03
Conversion factor human vs. animal skin:
Not applicable; human skin.

Any other information on results incl. tables

Test compound/receptor fluid solubility assessment

A bank of receptor fluids were tested prior to the main dermal absorption experiment in an attempt to identify which one would not limit absorption.

 

Initial review of the solubility assessment suggested that the test compound may not be soluble in the receptor fluids tested and therefore method development experiments were performed by adding solvent to suitable receptor fluids to assess the solubility of test compound in the receptor fluids before further solubility test were performed.

 

Additional solubility tests were performed at lower concentrations. They were 1.2 and 0.6% (v/v) final concentration of TCP with the addition of ethanol to selected receptor fluid as concluded from the method development experiments.

 

Following review of the additional solubility tests in 0.9% saline (w/v) + 5% tween 80 (v/v) with increasing concentrations of ethanol, it was concluded that adequate solubility could not be confirmed at the concentrations evaluated and therefore further solubility tests were performed at lower concentrations (0.15, 0.3 and 0.6% (v/v) final concentration of TCP) in 0.9% saline (w/v) + 5% tween 80 (v/v) + 40% Ethanol.

 

For solubility in the receptor fluid to be considered acceptable, it should be at least 10 times higher than the amount of test compound in the receptor fluid at the end of an in vitro experiment.

 

The following receptor fluid was selected for the main in vitro dermal penetration experiment in consultation with the Sponsor study monitor:

0.9% saline (w/v) + 5% tween 80 (v/v) + 40% Ethanol

 

Following review of the dermal penetration experiment data the solubility of [14C]-Tricresyl phosphate in the above receptor fluid was considered acceptable (i.e., not limiting absorption and at least 10 times higher than expected (maximum) concentration of the test compound in the receptor fluid at the end of the in vitro study) when compared with the results of the solubility test data for the selected receptor fluid. To be able to confirm the receptor fluid used was not rate limiting to absorption, concentrations of up to 3.51 mg/mL (0.3% (v/v) final concentration of TCP) would be acceptable. The highest concentration observed was 3.66 μg/mL therefore; the solubility was shown to be 959 times higher than the amount of test compound in the receptor fluid at the end of the in vitro experiment.

 

Formulation preparation and analysis

Ensuring a suitable representation of the Tricresyl phosphate formulation at the three different concentrations selected could be prepared in the laboratory, trial formulations were first prepared and the results reviewed before proceeding with the main preparation.

 

Isomeric mixture containing [14C]-Tricresyl phosphate was formulated for the dermal absorption experiment and analysed.

 

The variability between the radioactive counts on the day of application was less than 5% indicating homogenous formulations had been prepared. The radiochemical purity of the radiolabelled dose formulations applied to skin were considered acceptable at >99%.

 

Integrity check

To avoid over-prediction of the dermal absorption by the use of impaired skin preparations tritiated water ([3H]-H2O) was applied to determine the skin preparation integrity. As detailed in the study plan skin samples were deemed acceptable if the rate of penetration was not greater than 2.5 x 10-3 cm/h. The observed rate of [3H]-H2O penetration was in the range of 0.70 x 10-3 to 3.68 x 10-3 cm/h. Two samples, [14C]-Tricresyl phosphate 40% v/v donor 1 replicate 2, and [14C]-Tricresyl phosphate 5% v/v donor 1 replicate 1 did not meet the acceptance criteria for skin barrier integrity (<2.5 x 10-3 cm/h) and therefore these replicates have been excluded from any subsequent mean and standard deviation calculations. The skin barrier integrity for the remaining samples was less than 2.5 x 10-3 cm/h and therefore considered acceptable.

 

Main dermal absorption

Isomeric mixture containing [14C]-Tricresyl phosphate formulated as [14C]-Tricresyl phosphate 100% v/v, [14C]-Tricresyl phosphate 40% v/v, and [14C]-Tricresyl phosphate 5% v/v was applied to dermatomed abdominal skin from four human donors in duplicate (total 8 replicates per test concentration).

 

[14C]-Tricresyl phosphate 100%, v/v formulation

The distribution of radioactivity at 24 hours following 8 hour exposure is shown in Table 5. The mean total recovery of radioactivity following application of the [14C]-Tricresyl phosphate 100%, v/v formulation to dermatomed human skin was 99.93 ± 0.89 %. At 24 hours post-application, a mean of 0.07 % of the applied radioactivity was detected in the receptor fluid. The mean estimated percentage absorbed and potentially absorbed were 2.45 and 5.42 % respectively.

 

At 24 hours post-application, a mean of 8.97 μg equivalents/cm2 was detected in the receptor fluid (Table 11). The mean estimated concentration absorbed and potentially absorbed were 275 and 605 μg equivalents/cm2. And in Table 18, less than 75% of absorption into the receptor fluid occurred within the first half of the experiment.

 

[14C]-Tricresyl phosphate 40%, v/v formulation

The mean total recovery of radioactivity following application of the [14C]-Tricresyl phosphate 40%, v/v formulation to dermatomed human skin was 101.83 ± 5.26 %. At 24 hours post-application, a mean of 0.06 % of the applied radioactivity was detected in the receptor fluid. The mean estimated percentage absorbed and potentially absorbed were 1.24 and 2.82 % respectively.

 

At 24 hours post-application, a mean of 4.14 μg equivalents/cm2 was detected in the receptor fluid (Table 12). The mean estimated concentration absorbed and potentially absorbed were 61.8 and 139 μg equivalents/cm2. And in Table 18, less than 75% of absorption into the receptor fluid occurred within the first half of the experiment.

 

[14C]-Tricresyl phosphate 5%, v/v formulation

The mean total recovery of radioactivity following application of the [14C]-Tricresyl phosphate 5%, v/v formulation to dermatomed human skin was 99.92 ± 8.37 %. At 24 hours post-application, a mean of 0.69 % of the applied radioactivity was detected in the receptor fluid. The mean estimated percentage absorbed and potentially absorbed were 6.81 and 10.43 % respectively.

 

At 24 hours post-application, a mean of 3.77 μg equivalents/cm2 was detected in the receptor fluid. The mean estimated concentration absorbed and potentially absorbed were 36.3 and 55.4 μg equivalents/cm2 respectively. Less than 75% of absorption into the receptor fluid occurred within the first half of the experiment.

 

Summary

Summary of the recovery data table (mean ± standard deviation)

Formulation applied

Total recovery (%)

Dislodged dose (%)1

Dose directly absorbed (%)2

µg TCP directly absorbed /cm2

Dose potentially absorbed (%)3

µg TCP potentially absorbed /cm3

[14C]-Tricresyl phosphate 100%, v/v

99.93 ± 0.89

94.51 ± 3.52

2.45 ± 1.49

275 ± 166

5.42 ± 2.90

605 ± 323

[14C]-Tricresyl phosphate 40%, v/v*

101.83 ± 5.26

99.01 ± 5.57

1.24 ± 0.67

61.8 ± 32.3

2.82 ± 1.29

139 ± 63

[14C]-Tricresyl phosphate 5%, v/v**

99.92 ± 8.37

89.49 ± 8.76

6.81 ± 2.03

36.3 ± 10.8

10.43 ± 1.99

55.4 ± 10.6

Results are expressed as % radioactivity applied

1Percentage dislodgeable dose includes skin wash, donor chamber wash, tape strip 1 and tape strip 2

2Percentage dose directly absorbed includes receptor fluid, receptor chamber wash and skin

3Percentage dose potentially absorbed includes receptor fluid, receptor chamber wash, skin and stratum corneum (tape strips 3-20)

*[14C]-Tricresyl phosphate 40% v/v, Donor 1 replicate 2 did not meet the acceptance criteria for skin barrier integrity (<2.5 x 10-3cm/h) and therefore samples have been excluded from the mean and standard deviation

**[14C]-Tricresyl phosphate 5% v/v, Donor 1 replicate 1 did not meet the acceptance criteria for skin barrier integrity (<2.5 x 10-3cm/h) and therefore samples have been excluded from the mean and standard deviation 

Applicant's summary and conclusion

Conclusions:
The dermal penetration of an isomeric mixture containing Tricresyl phosphate has been investigated using dermatomed abdominal human skin samples and [14C]-labelled test compound. Experiments were conducted after application of Tricresyl phosphate formulated at three different concentrations for 24 hours following exposure for 8 hours to dermatomed abdominal skin from four human donors in duplicate (total 8 replicates per test concentration).
The barrier integrity of the skin used for each test vessel was determined using tritiated water. The rate of absorption varied from 0.70 x 10-3 to 3.68 x 10-3 cm/h. Two samples, [14C]-Tricresyl phosphate 40% v/v formulation donor 1 replicate 2, and [14C]-Tricresyl phosphate 5% v/v formulation donor 1 replicate 1 did not meet the acceptance criteria for skin barrier integrity (<2.5 x 10-3 cm/h) and therefore these samples have been excluded from any subsequent mean and standard deviation calculations. The skin barrier integrity for the remaining samples was less than 2.5 x 10-3 cm/h and was therefore considered acceptable.
At 24 hours, following an 8 hour exposure to [14C]-Tricresyl phosphate 100% v/v formulation, a mean of less than 75% of absorption into the receptor fluid occurred within the first half of the experiment and 94.51% (10,529 μg equivalent/ cm2) of the applied dose was considered dislodgeable. The percentage directly absorbed which includes the amount recovered from the receptor fluid, receptor chamber wash, and skin was 2.45% (275 μg equivalent/ cm2), and the potentially absorbed dose which also includes the amount recovered from the receptor fluid, receptor chamber wash, skin and tape strips 3-20 was 5.42% (605 μg equivalent/ cm2).
At 24 hours, following an 8 hour exposure to [14C]-Tricresyl phosphate 40% v/v formulation, a mean of less than 75% of absorption into the receptor fluid occurred within the first half of the experiment and 99.01% (4,834 μg equivalent/ cm2) of the applied dose was considered dislodgeable. The percentage directly absorbed which includes the amount recovered from the receptor fluid, receptor chamber wash, and skin was 1.24% (61.8 μg equivalent/ cm2), and the potentially absorbed dose which also includes the amount recovered from the receptor fluid, receptor chamber wash, skin and tape strips 3-20 was 2.82% (139 μg equivalent/ cm2).
At 24 hours, following an 8 hour exposure to [14C]-Tricresyl phosphate 5% v/v formulation, a mean of less than 75% of absorption into the receptor fluid occurred within the first half of the experiment and 89.49% (475 μg equivalent/ cm2) of the applied dose was considered dislodgeable. The percentage directly absorbed which includes the amount recovered from the receptor fluid, receptor chamber wash, and skin was 6.81% (36.3 μg equivalent/ cm2), and the potentially absorbed dose which also includes the amount recovered from the receptor fluid, receptor chamber wash, skin and tape strips 3-20 was 10.43% (55.4 μg equivalent/ cm2).
Executive summary:

The in vitro dermal absorption of radiolabelled Tricresyl phosphate that is representative of the commercially available mixture has been investigated using human skin. The extent of absorption was assessed following topical application of an isomeric mixture containing [14C]-Tricresyl phosphate that was formulated at dose preparations of [14C]- Tricresyl phosphate 100% v/v, [14C]-Tricresyl phosphate 40% v/v, and [14C]-Tricresyl phosphate 5% v/v and applied to dermatomed abdominal skin from four human donors in duplicate (total 8 replicates per test concentration).

 

The integrity of the human skin used for each test vessel was confirmed by assessing the permeability of tritiated water prior to the experiment.

 

The doses were applied to the skin surface of appropriate cells. Aliquots of receptor fluid made up of 0.9% saline (w/v) + 5% tween 80 (v/v) + 40% Ethanol (v/v) were collected for analysis at 0-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-22 and 22-24 hours after application. Approximately 8 hours after application, the dose formulation was removed from the test skin by washing the area using a 3% soap solution. Following the 24 hour sampling time-point, the test system was dismantled and the skin retained for solubilisation and analysis of radioactivity. Skin fractionation was performed by tape stripping and strips were retained for solubilisation and analysis of radioactivity. The remaining flow-through cell donor and receptor chambers were washed with ethanol and the washings retained for analysis of radioactivity and mass balance determination.

 

A summary of the recovery data (mean ± standard deviation) expressed as percentage and mass absorbed at 24 hours following 8 hour exposure is shown below:

 

Formulation applied

Total recovery (%)

Dislodged dose (%)1

Dose directly absorbed (%)2

µg TCP directly absorbed /cm2

Dose potentially absorbed (%)3

µg TCP potentially absorbed /cm3

[14C]-Tricresyl phosphate 100%, v/v

99.93 ± 0.89

94.51 ± 3.52

2.45 ± 1.49

275 ± 166

5.42 ± 2.90

605 ± 323

[14C]-Tricresyl phosphate 40%, v/v*

101.83 ± 5.26

99.01 ± 5.57

1.24 ± 0.67

61.8 ± 32.3

2.82 ± 1.29

139 ± 63

[14C]-Tricresyl phosphate 5%, v/v**

99.92 ± 8.37

89.49 ± 8.76

6.81 ± 2.03

36.3 ± 10.8

10.43 ± 1.99

55.4 ± 10.6

Results are expressed as % radioactivity applied

1Percentage dislodgeable dose includes skin wash, donor chamber wash, tape strip 1 and tape strip 2

2Percentage dose directly absorbed includes receptor fluid, receptor chamber wash and skin

3Percentage dose potentially absorbed includes receptor fluid, receptor chamber wash, skin and stratum corneum (tape strips 3-20)

*[14C]-Tricresyl phosphate 40% v/v, Donor 1 replicate 2 did not meet the acceptance criteria for skin barrier integrity (<2.5 x 10-3cm/h) and therefore samples have been excluded from the mean and standard deviation

**[14C]-Tricresyl phosphate 5% v/v, Donor 1 replicate 1 did not meet the acceptance criteria for skin barrier integrity (<2.5 x 10-3cm/h) and therefore samples have been excluded from the mean and standard deviation 

 

At 24 hours, following an 8 hour exposure to [14C]-Tricresyl phosphate 100% v/v formulation, a mean of less than 75% of absorption into the receptor fluid occurred within the first half of the experiment and 94.51% (10,529 μg equivalent/cm2) of the applied dose was considered dislodgeable. The percentage directly absorbed which includes the amount recovered from the receptor fluid, receptor chamber wash, and skin was 2.45% (275 μg equivalent/cm2), and the potentially absorbed dose which includes the amount recovered from the receptor fluid, receptor chamber wash, skin and tape strips 3-20 was 5.42% (605 μg equivalent/cm2).

 

At 24 hours, following an 8 hour exposure to [14C]-Tricresyl phosphate 40% v/v formulation, a mean of less than 75% of absorption into the receptor fluid occurred within the first half of the experiment and 99.01% (4,834 μg equivalent/cm2) of the applied dose was considered dislodgeable. The percentage directly absorbed which includes the amount recovered from the receptor fluid, receptor chamber wash, and skin was 1.24% (61.8 μg equivalent/cm2), and the potentially absorbed dose which includes the amount recovered from the receptor fluid, receptor chamber wash, skin and tape strips 3-20 was 2.82% (139 μg equivalent/cm2).

 

At 24 hours, following an 8 hour exposure to [14C]-Tricresyl phosphate 5% v/v formulation, a mean of less than 75% of absorption into the receptor fluid occurred within the first half of the experiment and 89.49% (475 μg equivalent/cm2) of the applied dose was considered dislodgeable. The percentage directly absorbed which includes the amount recovered from the receptor fluid, receptor chamber wash, and skin was 6.81% (36.3 μg equivalent/cm2), and the potentially absorbed dose which includes the amount recovered from the receptor fluid, receptor chamber wash, skin and tape strips 3-20 was 10.43% (55.4 μg equivalent/cm2).

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