3,4-dihydro-2H-1,4-benzoxazin-6-ol

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 2004 to 28 April 2005

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

version of 2002

Deviations:

no

Principles of method if other than guideline:

- Principle of test: The test substance, radiolabelled, is applied to the surface of a skin sample separating the two chambers of a diffusion cell. The chemical remains on the skin for a specified time under specified conditions, before removal by an appropriate cleansing procedure. The receptor fluid is sampled at time points throughout the experiment and analysed for the test chemical and/or metabolites. Absorpti on of a test substance during a given time period is measured by analysis of the receptor fluid and the treated skin. The test substance remaining in the skin should be considered as absorbed unless it can be demonstrated that absorption can be determined from receptor fluid values alone. Analysis of the other components (material washed off the skin and remaining within the skin layers) allows for further data evaluation, including total test substance disposition and percentage recovery.

- Short description of test conditions: the hair dye, Hydroxybenzomorpholine, was tested under oxidative conditions. Hydroxybenzomorpholine was incorporated into a typical oxidative hair dye formulation at ca 2.0% (w/w) associated to the primary intermediate para-phenylenediamine (PPD) at ca 1.43% (w/w) before mixing with developer (1:1, w/w) to give a final concentration of Hydroxybenzomorpholine of ca 1.0% (w/w). This will be referred to as the oxidative hair dye test preparation. Hydroxybenzomorpholine was also tested under non oxidative (“water”) conditions, in order to evaluate the absorption of the coupler (Hydroxybenzomorpholine) itself. Hydroxybenzomorpholine was incorporated into the same typical oxidative hair dye formulation (without primary intermediate) at ca 2.0% (w/w) and then mixed with water (1:1, w/w) to give a final concentration of Hydroxybenzomorpholine of ca 1.0% (w/w). This will be referred to as the non oxidative hair dye test preparation.

- Parameters analysed / observed: The integrity of the skin was checked by determination of the permeability coefficient for tritiated water which was < 2.5 x 10-3 cm/h for all selected membranes. [14C]-Hydroxybenzomorpholine was applied in two test preparations (oxidative and non oxidative) to human split-thickness skin membranes mounted in flow-through diffusion cells in vitro. Both oxidative and non oxidative hair dye test preparations were applied at a target application rate for the formulation of ca 20 mg/cm2. The target application rate of [14C]-Hydroxybenzomorpholine in the two test preparations was 200 μg/cm2.
Absorption was assessed by collecting receptor fluid (PBS) samples hourly from 0-24 h post dose (flow rate 1.5 mL/h). At 30 min post dose, the skin was washed with water, sodium dodecyl sulphate (SDS) solution (2% w/v) and water again. The skin was then dried with tissue paper swabs. At 24 h post dose the underside of the skin was rinsed with receptor fluid. The skin was then removed from the flow-through cells, dried and the stratum corneum removed by tape stripping. The remaining skin was divided into exposed and unexposed skin. All liquid samples were analysed by liquid scintillation counting and the remaining samples were analysed by combustion/ liquid scintillation counting.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Radiolabelled test item:
- Source and lot/batch number of test material: 6-Hydroxy[benzene ring-U-14C]benzomorpholine, batch no. CFQ13914 Batch 1, 74 MBq (2 mCi) was supplied in 4 containers by Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK. 98.5%
- Expiration date of the lot/batch: not mentioned
- Purity test date: 5th july 2004
- molecular weight : 153.1

Non-radiolabelled test item:
- Source and lot/batch number of test material: Hydroxybenzomorpholine, batch no. 0508918, with a stated titre chemical purity of 98.3%, was supplied by L’Oréal Recherche, Direction Internationale de l’Evaluation de la sécurité, 1 Avenue Eugène Schueller, 93600 Aulnay Sous Bois, France
- Expiration date of the lot/batch: September 2005
- Purity test date: 31 August 2004
- molecular weight : 113.16

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 98.5% (by high performance liquid chromatography, on 05 July 2004)
- Specific activity: 2.18 GBq/mmol, 59 mCi/mmol
- Locations of the label: The structure and site of labelling one cycle of 6-Hydroxy[benzene ring-U-14C]benzomorpholine.
- Expiration date of radiochemical substance:not metioned

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: All bulk samples not immediately analysed were stored at ca -20°C. After analysis, samples were returned to storage at ca -20oC.[14C]-Hydroxybenzomorpholine were removed from ca -20°C storage and allowed to reach ambient temperature.
- Stability under storage conditions: not controlled
- Stability under test conditions: The concentration and purity of test item in either formulation at 24 h was less than 5% of that at 0 h. This is within the criteria for the limit of acceptance of the HPLC method and therefore the stability of the test preparations over the live phase of the study is considered as acceptable.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
three 10 μL weighed aliquots of both formulations (Oxidative Test Preparation and Non Oxidative Test Preparation) were mixed with injection solution (1 mL).
The concentration and radiochemical purity of Hydroxybenzomorpholine was then determined using the HPLC methods
The concentration of Hydroxybenzomorpholine in the oxidative formulation was calculated to be 2.02% (w/w) or 20.23 mg/g (CV, 1.38%). The concentration of
Hydroxybenzomorpholine in the non oxidative formulation was calculated to be 1.99% (w/w) or 19.88 mg/g (CV, 0.73%). The radiochemical purity of
[14C]-Hydroxybenzomorpholine in the oxidative and non oxidative formulation was determined to be 95.9% and 94.6%, respectively.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:

Radiolabelling:

yes

Species:

other: Human skin

Sex:

female

Doses:

Formulation of Test Preparations

1 Oxidative Test Preparation
1.1 Preparation of Oxidative Formulation
All preparation procedures stated below, up until the addition of the Developer, were carried out in a nitrogen atmosphere. Three dose vials were required to be prepared due to dosing constraints and the nature of the formulation.
Two vials containing [14C]-Hydroxybenzomorpholine were removed from ca -20°C storage and allowed to reach ambient temperature. Formulation 1 was then added to each vial (498.96 mg and 499.16 mg) and the formulation was mixed by sonication and vortexing.
The contents of the vials were transferred to a single dose vial and mixed by sonication and vortexing. To determine the radioactive homogeneity and concentration, six weighed 10 μL aliquots were taken, transferred into 5 mL volumetric flasks and made up to the 5 mL volume with ethanol and mixed. Duplicate 125 μL aliquots were removed from each volumetric flask and scintillation fluid (ca 10 mL) added to each for analysis by liquid scintillation counting.
By radioactivity, the concentration of Hydroxybenzomorpholine in the formulation was calculated to be 2.03% (w/w). The concentration was 101.32% of the target concentration of 2.0% (w/w). The formulation was homogeneous with a coefficient of variance (CV) of 2.31%.

1.2 Preparation of Oxidative Test Preparation
Three aliquots of formulation (191.69 mg, 196.89 mg and 196.78 mg) were transferred into new vials. Immediately prior to dosing, Developer (194.84 mg, 195.68 mg and 196.09 mg) was added to the aliquots, respectively. The contents of each vial were mixed using a metal micro spatula and vortexing for ca 30 sec.

2 Non Oxidative Test Preparation
2.1 Preparation of Non Oxidative Formulation
All preparation procedures stated below, up until the addition of the degassed water (section 2.2), were carried out in a nitrogen atmosphere.
Two vials of [14C]-Hydroxybenzomorpholine were removed from ca -20°C storage and allowed to reach ambient temperature. Formulation 4 (498.76 mg and 499.79 mg, respectively) was added to the vials. The dose was mixed by sonication and vortexing.
The contents of the vials were transferred to a single dose vial and mixed by sonication and vortexing. To determine the radioactive homogeneity and concentration, six weighed 10 μL aliquots were taken, transferred into 5 mL volumetric flasks and made up to the 5 mL volume with ethanol and mixed. Duplicate 125 μL aliquots were removed from each volumetric flask and scintillation fluid (ca 10 mL) added to each for analysis by liquid scintillation counting.
By radioactivity, the concentration of Hydroxybenzomorpholine in the formulation was calculated to be 2.04% (w/w). The concentration was 101.88% of the target concentration of 2.0% (w/w). The formulation was homogeneous with a CV of 0.79%.

2.2 Preparation of Non Oxidative Test Preparation
Three aliquots of test preparation (192.15 mg, 194.85 mg and 189.33 mg) were transferred into new vials. Immediately prior to dosing, degassed water (190.93 mg, 195.48 mg and 188.85 mg) was added to the vials. The contents of each vial were mixed using a metal micro spatula and vortexing for ca 20 sec.

Confirmation of Radiochemical Purity of [14C]-Hydroxybenzomorpholine
The solutions in mobile phase prepared for the concentration confirmation were used to determine the radiochemical purity of [14C]-Hydroxybenzomorpholine in the formulations. This was performed using the same equipment and conditions as used for the method transfer except that the following equipment was also used:
Canberra Packard Radiomatic™Flo-one®\Beta Scintillation Analyser (Model 150TR).
Data was captured by Atlas quantified by integration of peak areas.
The chemical authenticity of the [14C]-Hydroxybenzomorpholine was confirmed by co-chromatography with authentic non-radiolabelled Hydroxybenzomorpholine. The radiochemical purity of [14C]-Hydroxybenzomorpholine in the oxidative and non oxidative formulations was determined to be 98.0% and 98.5%, respectively.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Full-thickness human skin samples (5 breast, 3 abdomen) were obtained from patients (aged 24 to 56 years old),
- Ethical approval if human skin: yes, 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 on arrival at Inveresk cleaned of subcutaneous fat and connective tissue using a scalpel blade. The skin was washed in cold running water and dried using “blue roll” tissue paper. The skin 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.
- When required, 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 (pocket thickness gauge, Mitutoyo Corp, Kanagawa, Japan). Split-thickness membranes were prepared by pinning the full-thickness skin, stratum When required, 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 (pocket thickness gauge, Mitutoyo Corp, Kanagawa, Japan).
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 200-500 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
- Diffusion cell: An automated flow-through diffusion cell apparatus (Scott/Dick, University of Newcastle-upon-Tyne, UK) was used
- Receptor fluid: Calcium and magnesium free phosphate buffer saline (PBS) was used as the receptor fluid. The receptor fluid was degassed in a sonic bath prior to use.
- Solubility od test substance in receptor fluid:
- Flow-through system: The cells were connected to multi-channel peristaltic pumps from their afferent 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
- Test temperature: The flow-through cells were placed in a steel manifold heated via a circulating water bath to maintain the skin surface temperature at ca 32°C
- Other: 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. A 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 barrier integrity assessment
- Barrier integrity : Tritiated water (250 μL, equivalent to 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. 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 of ca 90 min was allowed prior to collection of the predose sample which was collected for ca 30 min.

Absorption Study – Oxidative Test Preparation

A total of 12 samples of human skin obtained from 5 different donors were dosed topically with [14C]-Hydroxybenzomorpholine in the oxidative test preparation of a hair dye formulation (1%, w/w). Cells 7 and 14 were rejected from the mean and standard deviation (SD) values due to low mass balance (outwith 100±10%). Therefore, the following results are provided as mean values (n = 10).

The distribution of radioactivity at 30 min and 24 h post dose is provided in Table 1.

 

Table 1 Distribution of Radioactivity (% Applied Dose) at 24 h Post Dose Following Topical Application of [14C]-Hydroxybenzomorpholine in Oxidative Test Preparation (1%, w/w) to Human Split-Thickness Skin

 

	Cell Number and Donor Number
	Cell 1	Cell 3	Cell 4	Cell 5	Cell 7	Cell 8	Cell 10	Cell 14	Cell 15	Cell 17	Cell 18	Cell 19	Mean	SD
	0102	0084	0101	0091	0094	0102	0084	0094	0102	0084	0101	0091
Skin Wash	92.61	90.70	91.09	94.96	70.65	91.24	84.02	63.69	92.01	78.43	85.50	84.37	88.49	5.14
Tissue Swab 0.5h	2.53	1.07	3.40	0.84	8.22	5.95	4.64	11.94	2.28	12.84	3.73	9.09	4.64	3.78
Tissue Swab 24h	0.04	0.03	0.02	0.01	0.04	0.03	0.01	0.04	0.01	0.02	0.07	0.25	0.05	0.07
Pipette Tips	0.07	0.17	0.08	0.28	0.47	0.48	2.10	0.08	0.23	0.34	0.94	0.55	0.52	0.61
Cell Wash	0.27	0.61	0.57	0.12	2.16	0.90	0.56	2.13	0.13	1.36	0.83	0.64	0.60	0.38
Dislodgeable Dose	95.52	92.58	95.17	96.21	81.53	98.59	91.33	77.89	94.65	92.98	91.06	94.89	94.30	2.33
Stratum Corneum	1.95	2.84	1.21	3.66	3.70	1.76	1.40	5.80	1.05	2.25	1.36	1.06	1.85	0.85
Unexposed Skin	0.06	0.00	0.06	0.00	0.13	0.00	0.01	0.17	0.00	0.01	0.13	0.01	0.03	0.04
Total Unabsorbed	97.53	95.42	96.45	99.88	85.36	100.35	92.74	83.85	95.71	95.24	92.55	95.96	96.18	2.58
Exposed Skin	0.01	0.01	0.27	0.03	0.78	0.02	0.04	0.07	0.02	0.29	0.21	0.03	0.09	0.11
Receptor Fluid	0.05	0.19	0.14	0.21	0.08	0.12	0.09	0.07	0.04	0.07	0.19	0.45	0.16	0.12
Receptor Rinse	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Total Absorbed	0.05	0.19	0.14	0.21	0.08	0.12	0.09	0.07	0.05	0.07	0.19	0.46	0.16	0.12
Dermal Delivery	0.06	0.21	0.40	0.24	0.86	0.14	0.13	0.15	0.07	0.36	0.40	0.49	0.25	0.15
Mass Balance	97.59	95.62	96.85	100.12	86.22	100.49	92.87	84.00	95.78	95.61	92.95	96.45	96.43	2.54

 

 

Cells 7 and 14 were rejected from mean and SD due to low mass balance (outwith 100±10%)

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

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

 

 

The mean total recovery was 96.43% of the applied dose at 24 h post dose.

At the end of the 0.5 h exposure period, 93.65% of the applied dose was removed during the washing process (88.49% in the 0.5 h skin wash, 4.64 % in the 0.5 h tissue swab and 0.52% in the pipette tips).

At 24 h post dose,ieafter a 23.5 h monitoring period, 0.05% of the applied dose was removed from the skin in the 24 h tissue swab. The cell wash contained 0.60% of the applied dose. Therefore at 24 h post dose, the dislodgeable dose was 94.30% of the applied dose. The total unabsorbed dose was 96.18% of the applied dose. This consisted of the dislodgeable dose and the radioactivity associated with the stratum corneum (1.85%) and unexposed skin (0.03%). Those amounts retained by the stratum corneum and unexposed skin at 24 h are not considered to be dermally absorbed and thus do not contribute to the systemic dose. The absorbed dose (0.16%) was made up from the receptor fluid (0.16%) and the receptor rinse (<0.01%). Dermal delivery (0.25%) was made up from the absorbed dose and exposed skin (0.09%).

The absorption profile is provided in Table 2 .

 

 

Table 2 - Cumulative Absorption (% Applied Dose) of [14C]-Hydroxybenzomorpholine into Receptor Fluid Following Topical Application of the Oxidative Test Preparation (1%, w/w) to Human Split-Thickness Skin

 

 

	Cell Number and Donor Number
	Cell 1	Cell 3	Cell 4	Cell 5	Cell 7	Cell 8	Cell 10	Cell 14	Cell 15	Cell 17	Cell 18	Cell 19	Mean	SD
Time (h)	0102	0084	0101	0091	0094	0102	0084	0094	0102	0084	0101	0091
0	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1	0.03	0.01	0.00	0.05	0.04	0.10	0.02	0.01	0.01	0.01	0.01	0.06	0.03	0.03
2	0.04	0.05	0.02	0.07	0.04	0.11	0.04	0.01	0.03	0.02	0.04	0.20	0.06	0.05
3	0.04	0.08	0.03	0.11	0.04	0.11	0.06	0.01	0.03	0.03	0.07	0.26	0.08	0.07
4	0.04	0.10	0.05	0.13	0.05	0.11	0.07	0.01	0.04	0.04	0.08	0.30	0.09	0.08
5	0.04	0.11	0.06	0.14	0.05	0.11	0.07	0.01	0.04	0.04	0.10	0.32	0.10	0.08
6	0.04	0.13	0.07	0.15	0.05	0.11	0.08	0.02	0.04	0.05	0.11	0.34	0.11	0.09
7	0.04	0.14	0.08	0.16	0.05	0.11	0.08	0.02	0.04	0.05	0.12	0.35	0.12	0.09
8	0.04	0.14	0.09	0.17	0.06	0.11	0.08	0.02	0.04	0.05	0.13	0.36	0.12	0.09
9	0.04	0.15	0.10	0.18	0.06	0.11	0.08	0.02	0.04	0.06	0.14	0.38	0.13	0.10
10	0.04	0.16	0.10	0.18	0.06	0.11	0.09	0.02	0.04	0.06	0.15	0.40	0.13	0.11
11	0.04	0.16	0.11	0.19	0.06	0.11	0.09	0.02	0.04	0.06	0.16	0.42	0.14	0.11
12	0.04	0.17	0.11	0.19	0.06	0.11	0.09	0.02	0.04	0.06	0.16	0.43	0.14	0.11
13	0.04	0.17	0.11	0.19	0.06	0.11	0.09	0.02	0.04	0.06	0.17	0.44	0.14	0.12
14	0.04	0.17	0.11	0.20	0.07	0.11	0.09	0.02	0.04	0.07	0.17	0.44	0.14	0.12
15	0.04	0.18	0.12	0.20	0.07	0.11	0.09	0.02	0.04	0.07	0.17	0.44	0.15	0.12
16	0.05	0.18	0.12	0.20	0.07	0.11	0.09	0.07	0.04	0.07	0.18	0.44	0.15	0.12
17	0.05	0.18	0.12	0.20	0.07	0.11	0.09	0.07	0.04	0.07	0.18	0.44	0.15	0.12
18	0.05	0.18	0.12	0.20	0.07	0.11	0.09	0.07	0.04	0.07	0.18	0.45	0.15	0.12
19	0.05	0.19	0.13	0.20	0.07	0.11	0.09	0.07	0.04	0.07	0.18	0.45	0.15	0.12
20	0.05	0.19	0.13	0.21	0.07	0.11	0.09	0.07	0.04	0.07	0.18	0.45	0.15	0.12
21	0.05	0.19	0.13	0.21	0.07	0.12	0.09	0.07	0.04	0.07	0.19	0.45	0.15	0.12
22	0.05	0.19	0.13	0.21	0.08	0.12	0.09	0.07	0.04	0.07	0.19	0.45	0.15	0.12
23	0.05	0.19	0.13	0.21	0.08	0.12	0.09	0.07	0.04	0.07	0.19	0.45	0.15	0.12
24	0.05	0.19	0.14	0.21	0.08	0.12	0.09	0.07	0.04	0.07	0.19	0.45	0.16	0.12

The distribution, by mass, of [14C]-Hydroxybenzomorpholine at 24 h post dose is shown in Table 3. The total recovery, dislodgeable dose, unabsorbed dose, absorbed dose and dermal delivery were 199.30, 194.91, 198.78, 0.32 and 0.52μg equiv./cm², respectively.

 

Table 3 - Distribution of [14C]-Hydroxybenzomorpholine (µg equiv./cm2) at 24 h Post Dose Following Topical Application of the Oxidative Test Preparation (1%, w/w) to Human Split-Thickness Skin

 

	Cell Number and Donor Number												Mean	SD
	Cell1 0102	Cell3 0084	Cell4 0101	Cell5 0091	Cell7 0094	Cell8 0102	Cell 10 0084	Cell 14 0094	Cell 15 0102	Cell 17 0084	Cell 18 0101	Cell 19 0091
Dislodgeable Dose	190.93	194.05	202.71	192.31	170.89	197.06	191.44	165.90	201.61	194.90	182.00	202.12	194.91	6.36
Stratum Corneum	3.89	5.94	2.58	7.32	7.76	3.52	2.93	12.35	2.25	4.71	2.71	2.26	3.81	1.70
Total Unabsorbed	194.94	200.00	205.43	199.64	178.92	200.58	194.38	178.60	203.86	199.64	184.98	204.40	198.78	6.08
Total Absorbed	0.09	0.41	0.29	0.42	0.16	0.23	0.20	0.15	0.10	0.15	0.38	0.97	0.32	0.26
Dermal Delivery	0.11	0.44	0.86	0.48	1.81	0.28	0.27	0.31	0.15	0.76	0.80	1.04	0.52	0.33
Total Recovery	195.05	200.43	206.29	200.11	180.73	200.85	194.66	178.91	204.00	200.39	185.78	205.44	199.30	6.12

   

Cells 7 and 14 were rejected from mean and SD due to low mass balance (outwith 100±10%)

 

 

 The absorption profile, by mass, is provided in Table 4.

 

Table 4 – cumulative absorption (ng equiv./cm²) of [14 C] – Hydroxybenzomorpholine into Receptor Fluid Following Topical Application of the Oxidative Test Preparation (1%, w/w) to Human Split-Thickness Skin

 

Time (h)																										Mean			SD
	Cell Number and Donor Number
	Cell1 0102	Cell3 0084			Cell4 0101		Cell5 0091		Cell7 0094		Cell8 0102		Cell 10 0084		Cell 14 0094		Cell 15 0102		Cell 17 0084		Cell 18 0101		Cell 19 0091
0	0.00	0.00			0.00		0.00		0.00		0.00		0.00		0.00		0.00		0.00		0.00		0.00		0.00			0.00
1	61.93	28.93			5.32		91.11		76.62		197.58		44.31		13.01		25.06		21.33		19.82		122.01		61.74			59.86
2	76.43	114.88			36.75		146.38		87.20		211.87		93.90		23.09		60.12		47.65		86.74		426.10		130.08			116.08
3	80.28	171.18			71.06		215.08		92.95		215.35		121.00		26.71		71.00		65.16		132.25		560.46		170.28			148.70
4	82.36	208.89			102.62		255.23		98.03		217.51		138.01		29.29		76.53		78.72		166.59		632.15		195.86			166.08
5	83.83	238.14			129.38		285.07		102.92		219.40		149.97		31.67		80.04		90.06		197.10		679.09		215.21			177.56
6	85.05	263.01			153.05		308.57		107.61		220.72		158.35		33.84		82.66		100.01		223.28		713.54		230.82			186.15
7	85.97	286.79			173.02		327.44		111.58		221.78		164.96		35.94		84.68		108.46		245.92		740.34		243.94			193.12
8	86.72	302.81			190.47		342.67		115.46		222.70		170.33		37.58		86.07		114.96		265.32		760.67		254.27			198.50
9	87.32	316.71			204.82		354.73		119.00		223.52		174.67		39.09		87.20		120.46		282.87		810.47		266.28			212.43
10	87.80	329.08			217.97		364.74		122.26		224.30		178.71		40.68		88.15		125.16		298.06		858.29		277.22			225.88
11	88.24	339.21			228.03		373.17		125.77		224.89		181.34		42.31		88.98		128.93		310.89		892.74		285.64			235.66
12	88.59	348.35			236.93		379.93		129.75		225.57		183.59		43.78		89.69		132.11		321.82		913.59		292.02			241.58
13	89.00	355.86			236.93		385.59		136.08		226.44		185.47		45.00		90.37		134.71		331.02		928.41		296.38			245.96
14	89.36	363.71			243.82		390.67		139.46		226.92		187.65		46.32		90.94		136.75		338.98		939.46		300.83			249.12
15	89.68	370.20			250.53		394.88		142.24		227.28		188.76		47.61		91.44		139.21		345.83		940.94		303.88			249.53
16	89.96	375.29			255.85		398.73		144.80		227.73		189.97		140.64		91.84		141.02		351.87		946.90		306.92			251.24
17	90.16		379.82	260.65		402.24		147.09		228.22		191.39		141.80		92.28		142.66		356.91		951.00		309.53			252.41
18	90.41		384.41	265.49		405.34		149.42		228.78		192.53		143.02		92.76		144.13		361.43		954.72		312.00			253.46
19	90.68		388.20	271.52		408.17		151.48		229.22		193.43		144.22		93.06		145.50		365.70		958.18		314.37			254.43
20	90.93		391.68	277.60		410.71		153.80		229.60		195.11		145.32		93.57		146.59		369.61		958.18		316.36			254.37
21	91.16		394.87	280.85		413.32		155.70		230.04		196.23		146.50		93.92		147.63		373.01		960.72		318.17			255.12
22	91.33		398.84	283.96		415.80		157.56		230.45		197.16		147.83		94.21		148.49		376.12		963.18		319.96			255.89
23	91.61		401.52	286.86		418.11		159.43		230.74		198.00		149.11		94.56		149.31		378.88		965.69		321.53			256.62
24	91.87		404.36	289.24		420.19		161.40		231.05		198.77		150.47		94.76		150.08		381.34		967.96		322.96			257.31

    Cells 7 and 14 were rejected from mean and SD due to low mass balance (outwith 100±10%)

  

The lag time and steady state flux could not be accurately determined since the test preparation was removed from the skin surface at 30 min post dose.

The data shows that [14C]-Hydroxybenzomorpholine in oxidative test preparation was effectively removed from the skin surface by the washing procedure employed.

 

Absorption Study – Non Oxidative Test Preparation

A total of 12 samples of human skin obtained from 5 different donors were dosed topically with of [14C]-Hydroxybenzomorpholine in the non oxidative test preparation after the addition of water (1%, w/w). None of the cells were rejected. The following results are provided as mean values (n = 12).

The distribution of radioactivity at 30 min and 24 h post dose is provided in Table 5.

 

Table 5  _ Distribution of radioactivity (% applied dose) at 24h Post dose Following Topical Application of [14C]-Hydroxybenzomorpholine in Non Oxidative Test Preparation (1%, w/w) to Human Split-Thickness Skin

 

  

	Cell Number and Donor Number												Mean	SD
	Cell 31 0101	Cell 32 0094	Cell 33 0085	Cell 34 0091	Cell 35 0101	Cell 36 0102	Cell 39 0091	Cell 41 0085	Cell 42 0094	Cell 43 0102	Cell 45 0101	Cell 47 0094
Skin Wash	80.64	85.95	96.70	87.26	93.04	87.65	86.16	102.03	101.97	90.72	95.38	88.25	91.31	6.65
Tissue Swab 0.5h	2.51	2.59	1.02	4.87	1.63	2.01	7.83	0.18	0.56	0.63	0.67	12.60	3.09	3.70
Tissue Swab 24h	0.10	0.05	0.07	0.04	0.02	0.12	0.02	0.01	0.01	0.01	0.17	0.03	0.05	0.05
Pipette Tips	0.43	0.11	0.13	0.14	0.75	0.19	0.09	0.23	0.07	0.67	0.04	0.33	0.27	0.23
Cell Wash	0.76	0.30	0.45	0.12	0.14	0.42	0.21	0.09	0.06	0.06	0.26	0.36	0.27	0.21
Dislodgeable Dose	84.44	89.00	98.37	92.42	95.58	90.39	94.31	102.55	102.67	92.09	96.52	101.57	94.99	5.70
Stratum Corneum	3.47	1.69	1.43	1.04	0.94	1.38	0.76	0.14	0.17	0.18	0.18	1.03	1.03	0.94
Unexposed Skin	0.10	0.03	0.07	0.01	0.01	0.03	0.11	0.02	0.01	0.00	0.15	0.08	0.05	0.05
Total Unabsorbed	88.02	90.72	99.87	93.47	96.53	91.80	95.17	102.71	102.85	92.27	96.84	102.68	96.08	5.07
Exposed Skin	0.74	1.10	0.31	0.21	0.35	0.50	0.54	0.03	0.28	0.04	0.30	0.76	0.43	0.31
Receptor Fluid	3.26	2.16	2.95	1.92	1.62	1.93	3.21	0.56	0.89	0.35	0.56	1.71	1.76	1.03
Receptor Rinse	0.02	0.06	0.01	0.01	0.01	0.02	*0.00	*0.00	*0.00	0.00	0.00	*0.00	°0.01	°0.02
Total Absorbed	3.28	2.22	2.95	1.92	1.62	1.95	3.21	0.56	0.89	0.35	0.56	1.71	1.77	1.03
Dermal Delivery	4.02	3.33	3.27	2.13	1.97	2.45	3.75	0.59	1.17	0.40	0.86	2.47	2.20	1.24
Mass Balance	92.04	94.05	103.14	95.61	98.51	94.25	98.92	103.30	104.02	92.66	97.70	105.15	98.28	4.69

 

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

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

 

The mean total recovery was 98.28% of the applied dose at 24 h post dose.

At the end of the 0.5 h exposure period, 94.67% of the applied dose was removed during the washing procedure (91.31% in the 0.5 h skin wash, 3.09% in the 0.5 h tissue swab and 0.27% in the pipette tips).

At 24 h post dose,ieafter a 23.5 h monitoring period, 0.05% of the applied dose was removed from the skin in the 24 h tissue swab. The cell wash contained 0.27% of the applied dose. Therefore at 24 h post dose, the dislodgeable dose was 94.99% of the applied dose. The total unabsorbed dose was 96.08% of the applied dose. This consisted of the dislodgeable dose and the radioactivity associated with the stratum corneum (1.03%) and unexposed skin (0.05%). The absorbed dose (1.77%) was made up from the receptor fluid (1.76%) and the receptor rinse (0.01%). Dermal delivery (2.20%) was made up from the absorbed dose and exposed skin (0.43%).

The absorption profile is provided in Table 6.

 

Table  6 -  Cumulative Absorption (% Aplied) of [14 C] – hydroxybenzomorpholine into receptor Fluid FollowingTopical Application of the Non Oxidative Test Preparation (1%, w/w) to Human Split-Thickness Skin

 

Time (h)	Cell Number and Donor Number												Mean	SD
	Cell 31 0101	Cell 32 0094	Cell 33 0085	Cell 34 0091	Cell 35 0101	Cell 36 0102	Cell 39 0091	Cell 41 0085	Cell 42 0094	Cell 43 0102	Cell 45 0101	Cell 47 0094
0	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1	0.01	0.01	0.27	0.08	0.01	0.01	0.07	0.17	0.01	0.01	0.00	0.00	0.05	0.08
2	0.10	0.08	0.51	0.40	0.11	0.07	0.36	0.36	0.17	0.07	0.06	0.06	0.19	0.16
3	0.21	0.17	0.66	0.58	0.22	0.14	0.70	0.43	0.35	0.12	0.12	0.16	0.32	0.22
4	0.34	0.28	0.82	0.74	0.33	0.22	1.04	0.47	0.48	0.16	0.17	0.26	0.44	0.28
5	0.48	0.37	0.99	0.87	0.44	0.30	1.35	0.49	0.58	0.20	0.21	0.37	0.55	0.35
6	0.67	0.46	1.16	0.99	0.56	0.39	1.63	0.50	0.65	0.22	0.24	0.47	0.66	0.41
7	0.82	0.63	1.34	1.10	0.66	0.49	1.88	0.52	0.70	0.24	0.28	0.58	0.77	0.47
8	1.00	0.74	1.51	1.20	0.76	0.59	2.08	0.52	0.74	0.26	0.31	0.58	0.86	0.52
9	1.17	0.84	1.66	1.29	0.86	0.69	2.25	0.53	0.77	0.28	0.34	0.68	0.95	0.57
10	1.33	0.94	1.80	1.38	0.95	0.79	2.40	0.53	0.80	0.29	0.36	0.77	1.03	0.61
11	1.58	1.08	1.93	1.45	1.02	0.88	2.52	0.54	0.81	0.30	0.39	0.87	1.11	0.65
12	1.73	1.19	2.05	1.51	1.10	0.98	2.63	0.54	0.83	0.31	0.41	0.99	1.19	0.69
13	1.91	1.28	2.16	1.57	1.16	1.07	2.72	0.54	0.84	0.32	0.43	1.08	1.26	0.73
14	2.06	1.37	2.27	1.62	1.20	1.16	2.80	0.55	0.85	0.32	0.45	1.17	1.32	0.76
15	2.20	1.45	2.37	1.67	1.25	1.23	2.87	0.55	0.86	0.33	0.46	1.28	1.38	0.79
16	2.35	1.53	2.45	1.71	1.29	1.30	2.92	0.55	0.87	0.33	0.48	1.36	1.43	0.82

17	2.48	1.61	2.54	1.74	1.35	1.37	2.97	0.55	0.87	0.34	0.49	1.43	1.48	0.85
18	2.61	1.75	2.61	1.78	1.43	1.47	3.01	0.56	0.88	0.34	0.50	1.48	1.53	0.88
19	2.74	1.83	2.67	1.80	1.47	1.55	3.05	0.56	0.88	0.34	0.51	1.53	1.58	0.91
20	2.85	1.90	2.76	1.83	1.50	1.66	3.08	0.56	0.88	0.34	0.52	1.58	1.62	0.93
21	2.96	1.97	2.82	1.85	1.54	1.73	3.12	0.56	0.89	0.35	0.53	1.62	1.66	0.96
22	3.07	2.03	2.88	1.88	1.57	1.80	3.15	0.56	0.89	0.35	0.54	1.65	1.70	0.98
23	3.17	2.09	2.94	1.90	1.59	1.87	3.18	0.56	0.89	0.35	0.55	1.68	1.73	1.01
24	3.26	2.16	2.95	1.92	1.62	1.93	3.21	0.56	0.89	0.35	0.56	1.71	1.76	1.03

 

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

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

 

The distribution, by mass, of [14C]-Hydroxybenzomorpholine at 24 h post dose is shown in Table 7.

 

table 7 – distribution of [14 C]- hydroxybenzomorpholine (µg equiv./cm²) at 24 h Post Dose Following Application of the Non Oxidative Test Preparation (1%, w/w)to Human Split-Thickness Skin

 

	Cell Number and Donor Number												Mean	SD
	Cell 31 0101	Cell 32 0094	Cell 33 0085	Cell 34 0091	Cell 35 0101	Cell 36 0102	Cell 39 0091	Cell 41 0085	Cell 42 0094	Cell 43 0102	Cell 45 0101	Cell 47 0094
Dislodgeable Dose	179.09	182.68	191.52	196.01	196.20	191.71	193.58	199.65	199.90	195.30	198.12	197.74	193.46	6.51
Stratum Corneum	7.37	3.47	2.79	2.21	1.93	2.93	1.57	0.27	0.32	0.38	0.37	2.02	2.13	1.99
Total Unabsorbed	186.68	186.22	194.45	198.24	198.15	194.69	195.36	199.97	200.24	195.68	198.79	199.91	195.70	4.79
Total Absorbed	6.96	4.57	5.75	4.08	3.33	4.14	6.58	1.10	1.74	0.75	1.15	3.33	3.62	2.14
Dermal Delivery	8.53	6.83	6.36	4.52	4.05	5.20	7.69	1.16	2.28	0.84	1.76	4.80	4.50	2.58
Total Recovery	195.21	193.05	200.81	202.77	202.20	199.89	203.05	201.12	202.51	196.52	200.55	204.71	200.20	3.51

 

 

The total recovery, dislodgeable dose, unabsorbed dose, absorbed dose and dermal delivery were 200.20, 193.46, 195.70, 3.62 and 4.50 μg equiv./cm², respectively.

The absorption profile, by mass, is provided in Table 8. The lag time and steady state flux could not be accurately determined since the test preparation was removed from the skin surface at 30 min post dose.

The data shows that [14C]-Hydroxybenzomorpholine in non oxidative test preparation was effectively removed from the skin surface by the washing procedure employed.

 

Table 8 - Cumulative Absorption (µg equiv./cm2) of [14C]-Hydroxybenzomorpholine into Receptor Fluid Following Topical Application of the Non Oxidative Test Preparation (1%, w/w) to Human Split-Thickness Skin

 

 

Time (h)	Cell Number and Donor Number												Mean	SD
	Cell 31 0101	Cell 32 0094	Cell 33 0085	Cell 34 0091	Cell 35 0101	Cell 36 0102	Cell 39 0091	Cell 41 0085	Cell 42 0094	Cell 43 0102	Cell 45 0101	Cell 47 0094
0	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1	0.01	0.01	0.52	0.16	0.01	0.02	0.15	0.33	0.03	0.01	0.01	0.00	0.10	0.16
2	0.20	0.15	1.00	0.85	0.23	0.14	0.74	0.71	0.33	0.14	0.13	0.12	0.39	0.33
3	0.44	0.35	1.29	1.23	0.45	0.29	1.43	0.83	0.69	0.26	0.24	0.31	0.65	0.44
4	0.71	0.58	1.60	1.56	0.68	0.46	2.13	0.91	0.94	0.35	0.34	0.51	0.90	0.57
5	1.02	0.76	1.93	1.84	0.90	0.64	2.77	0.95	1.12	0.42	0.42	0.71	1.12	0.71
6	1.41	0.94	2.26	2.10	1.14	0.84	3.36	0.98	1.26	0.47	0.50	0.92	1.35	0.84
7	1.74	1.28	2.61	2.33	1.36	1.04	3.85	1.00	1.36	0.52	0.58	1.13	1.57	0.95
8	2.13	1.52	2.95	2.55	1.56	1.25	4.27	1.02	1.44	0.56	0.64	1.13	1.75	1.07
9	2.48	1.72	3.23	2.74	1.76	1.46	4.61	1.03	1.50	0.60	0.69	1.32	1.93	1.16
10	2.82	1.92	3.50	2.92	1.94	1.67	4.92	1.04	1.55	0.62	0.75	1.50	2.10	1.25
11	3.34	2.21	3.76	3.07	2.10	1.87	5.18	1.04	1.58	0.64	0.80	1.69	2.27	1.34
12	3.67	2.45	3.99	3.20	2.25	2.07	5.40	1.05	1.61	0.66	0.84	1.92	2.43	1.42
13	4.05	2.64	4.21	3.33	2.38	2.26	5.59	1.06	1.64	0.67	0.88	2.11	2.57	1.49
14	4.37	2.81	4.42	3.44	2.47	2.45	5.75	1.07	1.66	0.69	0.92	2.27	2.69	1.56
15	4.68	2.98	4.61	3.54	2.56	2.60	5.88	1.07	1.67	0.70	0.95	2.50	2.81	1.63
16	4.98	3.15	4.78	3.62	2.65	2.76	6.00	1.08	1.69	0.70	0.98	2.65	2.92	1.69

17	5.27	3.31	4.94	3.70	2.77	2.91	6.09	1.08	1.70	0.71	1.01	2.77	3.02	1.75
18	5.54	3.59	5.08	3.77	2.94	3.12	6.18	1.08	1.71	0.72	1.03	2.89	3.14	1.81
19	5.80	3.76	5.21	3.83	3.02	3.28	6.26	1.08	1.71	0.73	1.06	2.99	3.23	1.87
20	6.04	3.91	5.37	3.88	3.09	3.52	6.33	1.09	1.72	0.73	1.08	3.07	3.32	1.93
21	6.28	4.04	5.49	3.93	3.15	3.67	6.40	1.09	1.72	0.73	1.10	3.15	3.40	1.98
22	6.50	4.17	5.61	3.98	3.21	3.82	6.47	1.09	1.73	0.74	1.11	3.21	3.47	2.03
23	6.71	4.29	5.72	4.02	3.27	3.96	6.53	1.09	1.74	0.74	1.13	3.27	3.54	2.08
24	6.91	4.43	5.73	4.07	3.32	4.10	6.58	1.10	1.74	0.75	1.15	3.33	3.60	2.12

 

Comparison of [14C]-Hydroxybenzomorpholine in Oxidative and Non Oxidative Test Preparations

A statistical comparison of [14C]-Hydroxybenzomorpholine in both oxidative and non oxidative test preparations of hair dye formulations was carried out using an unpaired T-Test (Table 9).

 

Table 9           A Comparison of [14C]- Hydroxybenzomorpholine in Oxidative and Non Oxidative Test preparations: A Statistical Analysis Using an Unpaired T-Test

Unpaired T-test	P Values and significance
	% Applied Dose		µg.equiv./cm^2
Dislodgeable Dose	0.36134	NS	0.30199	NS
Dermal Delivery	0.00004	***	0.00005	***
Total Absorbed	0.00005	***	0.00005	***

 

NS	=	Not significant
*	=	p < 0.05
**	=	p < 0.01
***	=	p < 0.001

 

When comparing the results as % applied dose, for the dislodgeable dose, there was no significant difference between the two test preparations. However, dermal delivery and total absorbed dose were significantly higher for the non oxidative test preparation than for the oxidative test preparation (p < 0.001 and p < 0.001, respectively). This trend was also seen when comparing results calculated as amounts (μg equiv./cm²).

These differences may have been as a result of the following hypothesis. In the oxidative conditions, in the presence of developer, oxidation and then coupling reactions will have occurred between Hydroxybenzomorpholine and PPD leading to the formation of (coloured) compounds of higher molecular weight, whose absorption would be predicted to be lower than Hydroxybenzomorpholine. In the non oxidative conditions, in the presence of water, with no PPD in the formulation, there would not have been any coupling reactions. Although some auto-oxidation could conceivably take place, this would result in much more hydroxybenzomorpholine available for penetration.

Conclusions:

In conclusion, [14C]-Hydroxybenzomorpholine in oxidative and non oxidative test preparations was applied topically to human skin in vitro. Under the present experimental conditions, for [14C]-Hydroxybenzomorpholine in the oxidative test preparation, most of the applied dose was removed at 30 min post dose (93.65% of the applied dose). At 24 h post dose, a further 0.65% was removed, therefore, the dislodgeable dose was 94.30% of the applied dose. At 24 h post dose, the absorbed dose and dermal delivery were 0.16% (0.32 μg equiv/cm²) and 0.25% (0.52 μg equiv/cm²) of the applied dose, respectively. Under the present experimental conditions, for [14C]-Hydroxybenzomorpholine in the non oxidative test preparation, most of the applied dose was removed at 30 min post dose (94.67% of the applied dose). At 24 h post dose, a further 0.32% was removed, therefore, the dislodgeable dose was 94.99% of the applied dose. At 24 h post dose, the absorbed dose and dermal delivery were 1.77% (3.62 μg equiv/cm²) and 2.20% (4.50 μg equiv/cm²) of the applied dose, respectively. The dermal absorption figure to be taken into consideration for the calculation of the margin of safety is 0.52 μg equiv./cm² for the oxidative test preparation and 4.50 μg equiv./cm² for the non oxidative test preparation.

Executive summary:

Human abdominal or breast skin samples were obtained from eight different female donors subjected to plastic surgery. The skin samples were transferred on ice and kept frozen at -20°C until use.

Skin samples were dermatomed (380-400 μm in thickness) and mounted in flow-through diffusion cells, using calcium and magnesium-free phosphate-buffered saline as the receptor fluid. The integrity of the skin was checked by determining the permeability coefficient for tritiated water (<2.5 x 10-3 cm/h for all selected membranes), and skin was maintained at approximately 32°C. Twenty-four diffusion cells were used in two separate experiments.

In a first experiment (oxidative conditions), a typical oxidative hair dye formulation containing 2% w/w hydroxybenzomorpholine associated with the primary intermediate p-phenylenediamine (PPD) was mixed with the developer (1:1, w/w) to yield a final concentration of 1% w/w hydroxybenzomorpholine. Twenty (20) mg/cm² of this mixture was applied to the skin surface (corresponding to exactly 212.1 μg/cm² of hydroxybenzomorpholine). After 3 0 minutes, the remaining formulation on the skin surface was removed using standardized washing procedures, simulating use conditions. Twenty-four (24) hours after application, the percutaneous absorption of [14C]-Hydroxybenzomorpholine was estimated by measuring its concentration by liquid scintillation counting in the following compartments/samples: skin washes (dislodgeable dose), stratum corneum (isolated by tape strippings), living epidermis/dermis and receptor fluid.

In a separate experiment, a similar experimental procedure was applied to evaluate the percutaneous absorption of hydroxybenzomorpholine in non-oxidative conditions, using a formulation devoid of PPD and mixed with water (1:1, w/w) to yield a final concentration of 1% hydroxybenzomorpholine (about 20 mg/cm² were applied, corresponding exactly to 208.1 μg/cm²).

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

Formulation / Test preparation	Oxidative	Non Oxidative
Target Hydroxybenzomorpholine concentration in formulation (% w/w)	2.00	2.00
Actual Hydroxybenzomorpholine concentration in formulation (% w/w)	2.03	2.04
Target Hydroxybenzomorpholine concentration in test preparation (% w/w)	1.00	1.00
Actual Hydroxybenzomorpholine concentration in test preparation (% w/w)	1.01	1.02
Target application rate of test preparation (mg/cm²)	20.0	20.0
Actual application rate of test preparation (mg/cm²)	21.0	20.4
Hydroxybenzomorpholine (% applied dose)	(Mean +/- SD)	(Mean +/- SD)
Dislodgeable dose	94.30 ± 2.33	94.99 ± 5.70
Unabsorbed dose *	96.18 ± 2.58	96.08 ± 5.07
Absorbed dose **	0.16 ± 0.12	1.77 ± 1.03
Dermal delivery ***	0.25 ± 0.15	2.20 ± 1.24
Mass balance	96.43 ± 2.54	98.28 ± 4.69
Hydroxybenzomorpholine (μg equiv/cm²)	(Mean +/- SD)	(Mean +/- SD)
Dislodgeable dose	194.91 ± 6.36	193.46 ± 6.51
Unabsorbed dose *	198.78 ± 6.08	195.70 ± 4.79
Absorbed dose **	0.32 ± 0.26	3.62 ± 2.14
Dermal delivery ***	0.52 ± 0.33	4.50 ± 2.58
Mass balance	199.30 ± 6.12	200.20 ± 3.51

* Unabsorbed dose = dislodgeable dose + stratum corneum + unexposed skin

** Absorbed dose = receptor fluid + receptor rinse

*** Dermal delivery = exposed skin (except stratum corneum) + absorbed dose

A total of 22 samples of human skin yielded data that could be analysed (10 in oxidative conditions and 12 in non-oxidative conditions). Most of the hydroxybenzomorpholine applied on the skin surface was removed with the skin washes (dislodgeable dose, about 94% and 95% of

the applied dose in oxidative and non-oxidative conditions, respectively), and the total recovery rate was about 96% and 98% in oxidative and non-oxidative conditions, respectively.

The mean amounts of hydroxybenzomorpholine considered as absorbed (dermal delivery) were estimated as follows (sum of the amounts measured in living epidermis/dermis and receptor fluid): 0.52 ± 0.33 μg equiv/cm2 (0.25 ± 0.15% of the applied dose) and 4.50 ±

2.58 μg equiv/cm2 (2.20 ± 1.24% of the applied dose) in oxidative and non-oxidative conditions, respectively.

	Oxidative conditions	Oxidative conditions	Non-oxidative conditions	Non-oxidative conditions
Cutaneous distribution	μg equiv/cm²	% applied dose	μg equiv/cm²	% applied dose
Dislodgeable dose	194.91 ± 6.36	94.30 ± 2.33	193.46 ± 6.51	94.99 ± 5.70
Receptor fluid	0.32 ± 0.26	0.16 ± 0.12	3.62 ± 2.14	1.77 ± 1.03
Dermal delivery *	0.52 ± 0.33	0.25 ± 0.15	4.50 ± 2.58	2.20 ± 1.24

* Dermal delivery = exposed skin (except stratum corneum) + absorbed dose (= receptor fluid + receptor rinse)

Conclusion

Under the present experimental conditions, for [14C]-Hydroxybenzomorpholine in the oxidative test preparation, most of the applied dose was removed at 30 min post dose (93.65% of the applied dose). At 24 h post dose, a further 0.65% was removed, therefore, the dislodgeable dose was 94.30% of the applied dose. At 24 h post dose, the absorbed dose and dermal delivery were 0.16% (0.32 μg equiv/cm2) and 0.25%

(0.52 μg equiv/cm²) of the applied dose, respectively. Under the present experimental conditions, for [14C]-Hydroxybenzomorpholine in the non oxidative test preparation, most of the applied dose was removed at 30 min post dose (94.67% of the applied dose). At 24 h post dose, a further 0.32% was removed, therefore, the dislodgeable dose was 94.99% of the applied dose. At 24 h post dose, the absorbed dose and

dermal delivery were 1.77% (3.62 μg equiv/cm²) and 2.20% (4.50 μg equiv/cm²) of the applied dose, respectively. The dermal absorption figure to be taken into consideration for the calculation of the margin of safety for a typical oxidative hair colouring formulation containing hydroxybenzomorpholine at a final concentration of 1% were estimated to be 0.52 ± 0.33 μg/cm² (0.11 – 1.04 μg/cm²) and 4.50 ± 2.58 μg/cm² (0.84 – 8.53 μg/cm²) in oxidative and non-oxidative conditions, respectively.

Description of key information

In, in vitro Percutaneous Absorption Study using dermatomed human Skin, hydroxybenzomorpholine was tested in oxidative and non oxidative conditions at 1% in formulation.

The amounts of hydroxybenzomorpholine considered as absorbed from a typical oxidative hair colouring formulation containing hydroxybenzomorpholine at a final concentration of 1% were estimated to be 0.52 ± 0.33 μg/cm² (0.11 – 1.04 μg/cm²) and 4.50 ± 2.58 μg/cm² (0.84 – 8.53 μg/cm²) in oxidative and non-oxidative conditions, respectively.

Key value for chemical safety assessment

Absorption rate - dermal (%):

2.2

Additional information

In a formulation containing 1% of hydroxybenzomorpholine, dermal delivery in oxidative conditions was 0.25 ± 0.15 % applied dose, and in non oxidative conditions was 2.20 ± 1.24% applied dose.