[4-(2-hydroxyethoxy)-1,3-phenylene]diammonium sulphate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 october 2004 to 10 May 2005

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

This study was performed in accordance with the following documents:
OECD Guideline for Testing of Chemicals, Draft New Guideline 428: Skin Absorption:
In Vitro Method (2002).
OECD Environmental Health and Safety Publications Series on Testing and
Assessment No. 28. Guidance Document for the Conduct of Skin Absorption Studies
(2004).
Basic Criteria for the In Vitro Assessment of Percutaneous Absorption of Cosmetic
Ingredients. SCCNFP/0750/03 Updated, 20/10/03.
COLIPA (1995). Cosmetic Ingredients: Guidelines for Percutaneous Absorption/
Penetration. The European Cosmetic and Perfumery Association.
Diembeck W, Beck H, Benech-Kieffer F, Courtellemont P, Dupuis J, Lovell W, Paye M,
Spengler J, Steiling W. Test Guidelines for In Vitro Assessment of Dermal Absorption
and Percutaneous Penetration of Cosmetic Ingredients. Food and Chemical
Toxicology (1999) 37: 191-205.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(2,4-diaminophenoxy)ethanol dihydrochloride

EC Number:

266-357-1

EC Name:

2-(2,4-diaminophenoxy)ethanol dihydrochloride

Cas Number:

66422-95-5

Molecular formula:

C8H12N2O2.2ClH

IUPAC Name:

2-(2,4-diaminophenoxy)ethanol dihydrochloride

Details on test material:

Test item : 2-(2,4-diaminophenoxy)ethanol dihydrochloride
EC number : 266-357-1
Batch number : 7
Purity : >98%

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Non-Radiolabelled Test Item:
2,4-Diamino Phenoxyethanol HCl, batch no. 0120022, with a stated titre chemical purity of 98.7%, was supplied by L’Oréal Recherche, Direction Internationale de
l’Evaluation de la sécurité, 1 Avenue Eugène Schueller, 93600 Aulnay Sous Bois, France.
The test item was stored at ca +4 °C in the dark under nitrogen gas.
INCI name: 2,4-Diamino Phenoxyethanol HCl
Colipa Code: A042
Chemical name: 1-β-Hydroxyethyloxy-2,4-diamino-benzene dihydrochloride
CAS Registry Number: 66422-95-5
Description: A light grey to light pink powder
Molecular weight: 241.12
Log Po/w: -0.9 (CLogP v4.2 – C.Hansch)
Solubility: 10% (w/w) in water


RADIOLABELLING INFORMATION
- Radiolabelled Test Item:
2-(2,4-Diamino[ring-U-14C]phenoxy)ethanol hydrochloride, batch no. CFQ13910 Batch 1, 111 MBq (3 mCi) was supplied in 6 containers by Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK. A Certificate of Analysis was supplied with the radiolabelled test item stating the following:
Specific activity: 2.18 GBq/mmol, 59 mCi/mmol
Molecular weight: 243.0
Radiochemical purity: 98.6% (by high performance liquid chromatography, 17 June 2004)
Storage condition of test material: The test item was stored at ca -20°C in the dark.

FORMULATION
The following formulations were prepared by L’Oréal Laboratoires de Développement Coloration Capillaire, 92217, Clichy, France and were stored at ca +4 °C in the dark.
The formulations were dispatched from L’Oréal Recherche, Direction Internationale de l’Evaluation de la sécurité, 1 avenue Eugène Schueller, 93600 Aulnay Sous Bois.
Formulation 1: Formulation containing 2,4-Diamino Phenoxyethanol HCl at 3.59%, (w/w) and PPD of ca 1.8% (w/w) to which [14C]-2,4-Diamino Phenoxyethanol HCl was added. This was labelled “A042 3.59 g% couplage”, batch no. 1011997.
Formulation 2: Formulation containing 2,4-Diamino Phenoxyethanol HCl at 3.59%, (w/w) to which [14C]-2,4-Diamino Phenoxyethanol HCl was added. This
was labelled “A042 3.59 g%”, batch no. 1011996.
Formulation 3: Placebo formulation. This was labelled “Placebo”, batch no. 495952.
Formulation 4: Developer (hydrogen peroxide, ca 6%). This was labelled “Developer”, batch no. B021.

OTHER MATERIALS
Aquasafe 500 liquid scintillation fluid was obtained from Zinsser Analytic, Maidenhead, UK.
Carbo-Sorb® CO2 absorbing fluid, Permafluor®E+ scintillation fluid and Spec-ChecTM-14C were supplied by Canberra Packard Limited, Pangbourne, UK.

Radiolabelling:

yes

Test animals

Species:

other: Human skin

Details on test animals or test system and environmental conditions:

Full-thickness human skin samples (4 breast, 3 abdomen) were obtained from patients (aged 19 to 58 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 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
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.

Administration / exposure

Type of coverage:

other:

Doses:

Dose: 2%

Oxidative Test Preparation
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]-2,4-Diamino Phenoxyethanol HCl were removed from ca -20°C storage and allowed to reach ambient temperature. Formulation 1 was then
added to each vial (499.51 mg and 498.74 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 water 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 2,4-Diamino Phenoxyethanol HCl in the formulation was calculated to be 3.95% (w/w). The concentration was 98.68% of the
target concentration of 4.0% (w/w). The formulation was homogeneous with a coefficient of variance (CV) of 2.87%.

2.Preparation of Oxidative Test Preparation
Three aliquots of formulation (197.13 mg, 197.47 mg and 196.10 mg) were transferred into new vials. Immediately prior to dosing, Developer (196.41 mg, 198.82 mg and 194.59 mg) was added to the aliquots, respectively. The contents of each vial were mixed using a metal micro spatula and vortexing for ca 20 sec.

Non Oxidative Test Preparation
1. Preparation of Non Oxidative Formulation
All preparation procedures stated below, up until the addition of the degassed water, were carried out in a nitrogen atmosphere. Two vials of [14C]-2,4-Diamino Phenoxyethanol HCl were removed from ca -20°C storage and allowed to reach ambient temperature. Formulation 2 (500.42 mg and 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 10 mL volumetric flasks and made up to the 10 mL volume with water and mixed. Duplicate 250 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 2,4-Diamino Phenoxyethanol HCl in the formulation was calculated to be 3.91% (w/w). The concentration was 97.77% of the
target concentration of 4.0% (w/w). The formulation was homogeneous with a CV of 2.82%.

2. Preparation of Non Oxidative Test Preparation
Three aliquots of test formulation (197.58 mg, 196.00 mg and 197.10 mg) were transferred into new vials. Immediately prior to dosing, degassed water (197.60 mg,
195.83 mg and 197.03 mg) was added to the vials. The contents of each vial were mixed using a metal micro spatula and vortexing for ca 20 sec.

Prior to addition of the developer or degassed water, three 10 μL weighed aliquots of both formulations were taken and mixed with injection solution (1.8 mL). The concentration of 2,4-Diamino Phenoxyethanol HCl was then confirmed using the HPLC method.
By HPLC, the concentration of 2,4-Diamino Phenoxyethanol HCl in the oxidative formulation was calculated to be 4.05% (w/w) or 40.53 mg/g (CV, 0.46%). The concentration of 2,4-Diamino Phenoxyethanol HCl in the non oxidative formulation was calculated to be 3.92% (w/w) or 39.18 mg/g (CV, 0.34%). For the absorption study, all calculations are based on the concentration obtained by liquid scintillation counting.

Confirmation of Radiochemical Purity of [14C]-2,4-Diamino Phenoxyethanol HCl The solutions in mobile phase prepared for the concentration confirmation
(Section 4.11) were used to determine the radiochemical purity of [14C]-2,4-Diamino Phenoxyethanol HCl in the two 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]-2,4-Diamino Phenoxyethanol HCl was confirmed
by co-chromatography with authentic non-radiolabelled 2,4-Diamino Phenoxyethanol HCl. The radiochemical purity of [14C]-2,4-Diamino Phenoxyethanol HCl in the
oxidative and non oxidative formulations were determined to be 99.5% and 99.1%, respectively.

Details on in vitro test system (if applicable):

An automated flow-through diffusion cell apparatus (Scott/Dick, University of Newcastle-upon-Tyne, UK) was used. 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. 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

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.
The solubility of 2,4-Diamino Phenoxyethanol HCl is 10% (w/w) in water. Prior to commencement of the study, it was calculated that if absorption was 100% in 1 h (worst case scenario), then this would be less than the solubility in water. Therefore this receptor fluid was deemed to be acceptable for this test item.
The stability of the test item in the receptor fluid was not assessed. This was not deemed necessary as the radiolabelled test item, which will have been absorbed
through the skin into the receptor fluid would be analysed by liquid scintillation counting.

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. 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 Assessment
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. A cross reference of skin
sample number and donor and its corresponding tritiated water kp is presented in Appendix 11. 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
pre-dose sample which was collected for ca 30 min.

Skin Temperature Confirmation
The skin temperature of one of the cells that had not been dosed was also recorded at the time of dosing and then periodically over the next 24 h.

Application of Test Preparation
1. Oxidative Test Preparation
Dosing was divided into three due to the short exposure time (30 min) and the time taken to wash each sample. A separate vial of test preparation, as prepared in
Section 4.10.1, was used for each separate dosing period. The test preparation was applied over the surface of the stratum corneum of the exposed skin using an M25 Gilson Microman positive displacement pipette set to deliver 14.3 μL corresponding to ca 12.8 mg (ca 20 mg/cm2). The donor chambers were left open to the atmosphere. To accurately quantify the radioactivity applied to the skin samples, seven 14.3 μL aliquots of test preparation were taken for each dosing
occasion. These were analysed by liquid scintillation counting.
By radioactivity, the concentration of 2,4-Diamino Phenoxyethanol HCl in the test preparation was calculated to be 2.02% (w/w). The mean application rate of the
oxidative test preparation was calculated to be 21.22 mg/cm2 (target 20 mg/cm2).

2. Non Oxidative Test Preparation
Dosing was divided into three due to the short exposure time (30 min) and the time taken to wash each sample. A separate vial of test preparation, as prepared in
Section 4.10.2, was used for each separate dosing period. The test preparation was applied over the surface of the stratum corneum of the exposed skin using an M25 Gilson Microman positive displacement pipette set to deliver 14.2 μL corresponding to ca 12.8 mg (ca 20 mg/cm2). The donor chambers were left open to the atmosphere. To accurately quantify the radioactivity applied to the skin samples, seven 14.2 μL aliquots of test preparation were taken for each dosing
occasion. These were analysed by liquid scintillation counting. By radioactivity, the concentration of 2,4-Diamino Phenoxyethanol HCl in the test preparation was calculated to be 1.92% (w/w). The mean application rate of the non oxidative test preparation was calculated to be 20.45 mg/cm2 (target 20 mg/cm2).

Sampling Information
1 Receptor Fluid
Receptor fluid was collected in hourly fractions from 0-24 h. All receptor fluid samples were mixed with scintillation fluid (10 mL) and then analysed by liquid scintillation counting.
2. Terminal Exposure Procedures (30 min Post Dose)
At 30 min post exposure, each skin was washed with ten successive 320 μL aliquots of water using a 1 mL automatic pipette set to deliver 320 μL. Each aliquot was aspirated once. The pipette tip was removed and replaced by a fresh pipette tip. A single 320 μL aliquot of sodium dodecyl sulphate (SDS) solution in water (2% w/v) was applied to each skin. This was aspirated 3 times. Each skin was washed with a further ten successive 320 μL aliquots of water with a single aspiration between each aliquot. The pipette tips were retained for subsequent analysis. The water and SDS solution were pooled in one skin wash vial per skin sample and water (ca 10 mL) was added. Duplicate weighed aliquots (1 mL) were removed from each skin wash vial, mixed with scintillation fluid (10 mL) and analysed by liquid scintillation counting. The pipette tips were mixed with scintillation fluid (10 mL) and analysed directly by liquid scintillation counting.
The skins were dried with tissue swabs. The swabs were placed into Combustocones® for subsequent analysis by combustion/ liquid scintillation counting.
3. Terminal Monitoring Procedures (24 h Post Dose)
At 24 h post dose, ie after a 23.5 h monitoring period, each diffusion cell was disconnected from the receptor fluid pump lines. The underside of the skin was rinsed
(receptor rinse) with receptor fluid (1-2 mL), which was mixed 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 into the 23-24 h receptor fluid fraction.
The donor and receptor chambers were dismantled and the skin removed. The donor and receptor chambers were transferred into a pre-weighed pot (cell wash) containing water (ca 40 mL) which had previously been weighed. The solvent was mixed and left to extract for ca 30 min. The pot was placed in a sonic bath for ca 15 min during this time. The donor and receptor chambers were removed from the cell wash pot and then duplicate weighed ca 1 mL aliquots of cell wash were taken for liquid scintillation counting analysis.
The skin was dried as previously described and then the 24 h tissue swab was analysed as described for the 30 min tissue swab samples. The stratum corneum was removed with ca 20 successive tape strips (Guilbert, Niceday) leaving the epidermis visibly intact. Where epidermis was removed no further tape strips were taken. The tape strips were placed in groups of 5 into Combustocones® for subsequent combustion/ liquid scintillation counting analysis. The skin under the cell flange
(unexposed skin) was cut away from the exposed skin with scissors. These samples were placed into Combustocones® for subsequent combustion/ liquid scintillation counting analysis.

Results and discussion

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not examined

Absorption in different matrices:

Absorption Study – Oxidative Test Preparation
A total of 12 samples of human skin obtained from 5 different donors were dosed topically with [14C]-2,4-Diamino Phenoxyethanol HCl in the oxidative test preparation of a hair dye formulation (2%, w/w). Cells 5, 8, 10 and 17 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 = 8). The distribution of radioactivity at 30 min and 24 h post dose is provided in Table 1.
The mean total recovery was 94.73% of the applied dose at 24 h post dose. At the end of the 0.5 h exposure period, 88.39% of the applied dose was removed during the washing process (82.06% in the 0.5 h skin wash, 5.52 % in the 0.5 h tissue swab and 0.81% in the pipette tips).
At 24 h post dose, ie after a 23.5 h monitoring period, 0.28% of the applied dose was removed from the skin in the 24 h tissue swab. The cell wash contained 1.01% of the applied dose. Therefore at 24 h post dose, the dislodgeable dose was 89.68% of the applied dose. The total unabsorbed dose was 94.31% of the applied dose. This consisted of the dislodgeable dose and the radioactivity associated with the stratum corneum (4.56%) and unexposed skin (0.07%). 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 (0.03%) was made up from the receptor fluid (0.03%) and the receptor rinse (<0.01%). Dermal delivery (0.41%) was made up from the absorbed dose and exposed skin (0.39%).
The distribution, by mass, of [14C]-2,4-Diamino Phenoxyethanol HCl at 24 h post dose is shown in Table 2. The total recovery, dislodgeable dose, unabsorbed dose, absorbed dose and dermal delivery were 401.34, 379.97, 399.60, 0.11 and 1.74 μg equiv./cm2, respectively.
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]-2,4-Diamino Phenoxyethanol HCl 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]-2,4-Diamino Phenoxyethanol HCl in the non oxidative test preparation after the addition of water (2% 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 3. The mean total recovery was 101.12% of the applied dose at 24 h post dose.
At the end of the 0.5 h exposure period, 92.15% of the applied dose was removed during the washing procedure (86.27% in the 0.5 h skin wash, 5.58% in the 0.5 h tissue swab and 0.30% in the pipette tips).
At 24 h post dose, ie after a 23.5 h monitoring period, 0.06% of the applied dose was removed from the skin in the 24 h tissue swab. The cell wash contained 1.89% of the applied dose. Therefore at 24 h post dose, the dislodgeable dose was 94.11% of the
applied dose. The total unabsorbed dose was 99.44% of the applied dose. This consisted of the dislodgeable dose and the radioactivity associated with the stratum corneum (4.56%) and unexposed skin (0.77%). The absorbed dose (0.75%) was
made up from the receptor fluid (0.74%) and the receptor rinse (0.01%). Dermal delivery (1.68%) was made up from the absorbed dose and exposed skin (0.92%).
The distribution, by mass, of [14C]-2,4-Diamino Phenoxyethanol HCl at 24 h post dose is shown in Table 4. The total recovery, dislodgeable dose, unabsorbed dose, absorbed dose and dermal delivery were 397.02, 369.63, 390.47, 2.94 and
6.55 μg equiv./cm2, respectively. 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]-2,4-Diamino Phenoxyethanol HCl in non oxidative test preparation was effectively removed from the skin surface by the washing procedure employed.

Comparison of [14C]-2,4-Diamino Phenoxyethanol HCl in Oxidative and Non Oxidative Test Preparations
A statistical comparison of [14C]-2,4-Diamino Phenoxyethanol HCl in both oxidative and non oxidative test preparations of hair dye formulations was carried out using an unpaired T-Test (Table 5).
When comparing the results as % applied dose, dislodgeable dose, dermal delivery and total absorbed dose were significantly higher for the non oxidative test preparation than for the oxidative test preparation (p < 0.05, p < 0.01 and p < 0.05, respectively). When comparing results calculated as amounts (μg equiv./cm2) 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 < 0.01 and p < 0.05,
respectively).
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 2,4-Diamino Phenoxyethanol HCl and PPD
leading to the formation of (coloured) compounds of higher molecular weight, whose absorption would be predicted to be lower than 2,4-Diamino Phenoxyethanol HCl. 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 2,4-Diamino Phenoxyethanol HCl available for penetration.

Any other information on results incl. tables

Table 1- Distribution of Radioactivity (% Applied Dose) at 24 h Post Dose Following Topical Application of [14C]-2,4-Diamino Phenoxyethanol HCl in Oxidative Test Preparation (2%, w/w) to Human Split-Thickness Skin

	Cell 1 0084	Cell 3 0.092	Cell 4 0.097	Cell 5 0.105	Cell 6 0.103	Cell 8 0.084	Cell 10 0.092	Cell 11 0.097	Cell 12 0.105	Cell 15 0.084	Cell 17 0.092	Cell 20 0.103	Mean	SD
Skin Wash Tissue Swab 0.5h Tissue Swab 24h Pipette Tips Cell Wash	87.97  7.92  0.62  1.25 1.07	88.08  2.19  0.05  0.66 0.23	79.59  5.39  0.09  0.64 1.33	77.36  4.67  0.04  0.51 0.93	78.45  7.71  0.19  0.27 1.08	74.49   6.29  0.30  0.79 1.36	79.37  3.15  0.09  0.36 0.40	81.41  4.86  0.60  0.56 1.68	79.90  8.93  0.06  0.56 0.30	81.10  2.93  0.38  0.30 1.03	73.55  2.18  0.05  0.14 1.22	79.95  4.22  0.29  2.27 1.34	82.06  5.52  0.28  0.81 1.01	3.79   2.45  0.23  0.66 0.50
Dislodgeable dose	98.84	91.21	87.04	83.52	87.70	83.23	83.37	89.11	89.76	85.73	77.14	88.07	89.68	4.06
Stratum corneum unexposed skin	5.55 0.04	2.57 0.00	4.25 0.01	3.62 0.11	4.08 0.19	5.47 0.31	2.50 0.00	5.05 0.00	4.41 0.06	6.36 0.25	2.33 0.29	4.21 0.01	4.56 0.07	1.13 0.10
TOTAL UNABSORBED	104.43	93.79	91.30	87.25	91.97	89.01	85.87	94.16	94.23	92.35	79.75	92.28	94.31	4.23
exposed skin	0.14	0.08	0.66	0.92	0.43	0.11	0.08	0.18	0.26	0.82	0.22	0.52	0.39	0.27
receptor fluid	*0.03	*0.01	*0.02	*0.06	*0.01	*0.02	*0.01	*0.03	*0.09	0.01	*0.01	*0.00	°0.03	°0.03
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	0.03	0.01	0.02	0.06	0.01	0.02	0.01	0.03	0.09	0.01	0.01	0.00	0.03	0.03
DERMAL DELIVERY	0.17	0.09	0.68	0.98	0.44	0.12	0.09	0.21	0.35	0.83	0.24	0.53	0.41	0.26
Mass balance	104.60	93.88	91.98	88.23	92.40	89.14	85.96	94.37	94.58	93.18	79.99	92.81	94.73	4.09

Cells 5, 8, 10 and 17 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

Table 2 - Distribution of [14C]-2,4-Diamino Phenoxyethanol HCl (μg equiv./cm2) at 24 h Post Dose Following Topical Application of the Oxidative Test Preparation (2%, w/w) to Human Split-Thickness Skin

	Cell 1 0084	Cell 3 0.092	Cell 4 0.097	Cell 5 0.105	Cell 6 0.103	Cell 8 0.084	Cell 10 0.092	Cell 11 0.097	Cell 12 0.105	Cell 15 0.084	Cell 17 0.092	Cell 20 0.103	Mean	SD
Dislodgeable dose	437.99	380.83	369.62	370.11	366.18	353.44	369.44	378.39	374.78	357.96	341.84	373.98	379.97	24.54
Stratum corneum	24.60	10.75	18.05	16.05	17.04	23.23	11.09	21.45	18.41	26.56	10.32	17.86	19.34	4.90
TOTAL UNABSORBED	462.78	391.59	387.69	386.65	384.00	377.99	380.54	399.86	393.45	385.58	353.43	391.87	399.60	26.01
TOTAL ABSORBED	0.12	0.06	0.10	0.26	0.04	0.07	0.05	0.12	0.38	0.05	0.07	0.02	0.11	0.12
DERMAL DELIVERY	0.75	0.39	2.88	4.33	1.82	0.52	0.38	0.89	1.46	3.47	1.05	2.24	1.74	1.08
TOTAL RECOVERY	463.53	391.98	390.58	390.97	385.82	378.50	380.92	400.75	394.90	389.05	354.48	394.11	401.34	25.52

Cells 5, 8, 10 and 17 rejected from mean and SD due to low mass balance (outwith 100 ±10%)

Table 3- Distribution of Radioactivity (% Applied Dose) at 24 h Post Dose Following Topical Application of [14C]-2,4-Diamino Phenoxyethanol HCl in Non Oxidative Test Preparation (2%, w/w) to Human Split-Thickness Skin

	Cell 29 0088	Cell 31 0092	Cell 33 0103	Cell 34 0097	Cell 36 0088	Cell 38 0092	Cell 39 0084	Cell 40 0103	Cell 41 0097	Cell 43 0088	Cell 45 0092	Cell 46 0084	Mean	SD
Skin Wash	92.97	84.00	85.69	78.30	85.73	90.46	85.01	79.32	75.27	97.39	90.47	90.67	86.27	6.49
Tissue Swab 0.5h	5.79	5.68	7.48	9.11	6.10	3.65	6.63	2.44	6.32	1.31	7.50	4.91	5.58	2.22
Tissue Swab 24h	0.03	0.02	0.05	0.15	0.06	0.10	0.14	0.05	0.03	0.01	0.03	0.05	0.06	0.05
Pipette Tips	0.19	1.04	0.54	0.02	0.23	0.33	0.10	0.15	0.31	0.06	0.55	0.12	0.30	0.29
Cell Wash	0.78	2.02	1.15	2.80	1.61	1.36	2.27	3.86	5.01	0.16	0.36	1.33	1.89	1.43
Dislodgeable dose	99.76	92.76	94.92	90.38	93.73	95.89	94.15	85.82	86.95	98.93	98.90	97.08	94.11	4.54
Stratum corneum	2.37	6.02	3.04	6.42	5.94	2.97	5.80	7.27	4.82	1.05	3.63	5.47	4.56	1.91
unexposed skin	0.01	0.36	0.01	0.16	0.40	0.03	0.24	7.19	0.76	0.01	0.01	0.05	0.77	2.03
TOTAL UNABSORBED	102.14	99.14	97.96	96.96	100.07	98.89	100.19	100.28	92.53	99.99	102.54	102.59	99.44	2.79
exposed skin	0.16	0.51	0.98	3.32	1.25	0.94	0.30	1.52	0.54	0.19	0.61	0.78	0.92	0.86
receptor fluid	0.81	0.93	*0.06	*1.01	1.68	*0.09	0.80	0.27	*2.82	0.21	0.15	0.02	°0.74	°0.83
receptor rinse	0.00	0.02	0.00	0.01	0.04	0.01	0.05	0.00	0.02	0.00	0.00	0.00	0.01	0.02
TOTAL ABSORBED	0.81	0.95	0.07	1.02	1.72	0.10	0.85	0.27	2.84	0.21	0.15	0.02	0.75	0.84
DERMAL DELIVERY	0.98	1.46	1.04	4.34	2.96	1.04	1.15	1.79	3.38	0.40	0.77	0.80	1.68	1.23
Mass balance	103.12	100.60	99.01	101.30	103.04	99.93	101.35	102.07	95.90	100.39	103.31	103.40	101.12	2.18

* = 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

Table 4- Distribution of [14C]-2,4-Diamino Phenoxyethanol HCl (μg equiv./cm2) at 24 h Post Dose Following Topical Application of the Non Oxidative Test Preparation (2%, w/w) to Human Split-Thickness Skin

	Cell 29 0088	Cell 31 0092	Cell 33 0103	Cell 34 0097	Cell 36 0088	Cell 38 0092	Cell 39 0084	Cell 40 0103	Cell 41 0097	Cell 43 0088	Cell 45 0092	Cell 46 0084	Mean	SD
Dislogeable dose	392.91	355.19	380.83	346.09	369.16	384.75	377.77	328.63	342.43	389.63	378.71	389.51	369.63	21.37
Stratum corneum	9.32	23.05	12.18	24.57	23.39	11.90	23.28	27.83	18.98	4.15	13.89	21.93	17.87	7.34
TOTAL UNABSORBED	402.27	379.64	393.04	371.29	394.14	396.77	401.99	383.98	364.41	393.82	392.64	411.63	390.47	13.49
TOTAL ABSORBED	3.20	3.63	0.27	3.89	6.77	0.41	3.43	1.04	11.19	0.81	0.58	0.08	2.94	3.30
Dermal delivery	3.84	5.58	4.19	16.61	11.68	4.18	4.62	6.86	13.31	1.56	2.94	3.21	6.55	4.72
TOTAL RECOVERY	406.11	385.22	397.23	387.90	405.82	400.95	406.62	390.84	377.71	395.38	395.57	414.84	397.02	10.55

Table 5 - A Comparison of [14C]-2,4-Diamino Phenoxyethanol HCl in Oxidative and Non Oxidative Test Preparations: A Statistical Analysis Using an Unpaired T-Test

	P values and significance
	% applied dose		µg.equiv/cm²
dislogeable dose	0.01957	*	0.16547	NS
dermal delivery	0.00527	**	0.00583	**
total absorbed	0.01313	*	0.01362	*

NS = Not significant

* = p < 0.05

**= p< 0.01

*** = p< 0.001

Applicant's summary and conclusion

Conclusions:

The amounts of 2,4-diaminophenoxyethanol dihydrochloride considered as absorbed from a typical oxidative hair colouring mixture containing 2,4-diaminophenoxyethanol dihydrochloride at a final concentration of 2.0% were estimated to be 1.74 ± 1.08 μg/cm2 (0.38 – 4.33 μg/cm2).
The amounts of 2,4-diaminophenoxyethanol dihydrochloride considered as absorbed from a non-oxidative mixture containing 2,4-diaminophenoxyethanol dihydrochloride at a final concentration of 2.0% were estimated to be 6.55 ± 4.72 μg/cm2 (1.56 – 16.61 μg/cm2).
However, the maximum dermal absorption of 4.33 μg equiv/cm², observed under oxidative conditions in a typical hair dye formulation, will be used for the calculation of the Margin of Safety.

Executive summary:

2,4-Diamino Phenoxyethanol HCl is a hair dye compound developed by L’Oréal Recherche. 2,4-Diamino Phenoxyethanol HCl is a coupler used in oxidative hair dye formulations. As part of the safety evaluation of the product, an in vitro study was required to assess the rate and extent of absorption of 2,4-Diamino Phenoxyethanol HCl following the topical application of the hair dye to human skin under in-use conditions. For this purpose, the hair dye was tested under oxidative conditions. 2,4-Diamino Phenoxyethanol HCl was incorporated into a typical oxidative hair dye formulation at ca 4.0% (w/w) associated to the primary intermediate para-phenylenediamine (PPD) at ca 1.8% (w/w) before mixing with developer (1:1, w/w) to give a final concentration of 2,4-Diamino Phenoxyethanol HCl of ca 2.0% (w/w). This will be referred to as the oxidative hair dye test preparation. 2,4-Diamino Phenoxyethanol HCl was also tested under non oxidative (“water”) conditions, in order to evaluate the absorption of the coupler (2,4-Diamino Phenoxyethanol HCl) itself. 2,4-Diamino Phenoxyethanol HCl was incorporated into the same typical oxidative hair dye formulation (without primary intermediate) at ca 4.0% (w/w) and then mixed with water (1:1, w/w) to give a final concentration of 2,4-Diamino Phenoxyethanol HCl of ca 2.0% (w/w). This will be referred to as the non oxidative hair dye test preparation. The study was conducted according to the OECD principles of Good Laboratory Practice and was performed following the SCCNFP, COLIPA and draft OECD guideline for skin penetration studies and the OECD guidance document. 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]-2,4-Diamino Phenoxyethanol HCl 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]-2,4-Diamino Phenoxyethanol HCl in the two test preparations was 400 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.

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

Formulation / Test Preparation	Oxidative	Non Oxidative
Target 2,4-Diamino Phenoxyethanol HCl Concentration in Formulation (% w/w) Actual 2,4-Diamino Phenoxyethanol HCl Concentration in Formulation (% w/w) Target 2,4-Diamino Phenoxyethanol HCl Concentration in Test Preparation (% w/w) Actual 2,4-Diamino Phenoxyethanol HCl Concentration in Test Preparation (% w/w) Target application rate of Test Preparation (mg/cm2) Actual application rate of Test Preparation (mg/cm2)	4.00 3.95 2.00 2.02 20.0 21.2	4.00 3.91 2.00 1.92 20.0 20.5
2,4-Diamino Phenoxyethanol HCl (% Applied Dose)	(Mean ± SD)
Dislodgeable Dose	89.68 ± 4.06	94.11 ± 4.54
Unabsorbed Dose *	94.31 ± 4.23	99.44 ± 2.79
Absorbed Dose **	0.03 ± 0.03	0.75 ± 0.84
Dermal Delivery ***	0.41 ± 0.26	1.68 ± 1.23
Mass Balance	94.73 ± 4.09	101.12 ± 2.18
2,4-Diamino Phenoxyethanol HCl (μg equiv/cm2)	(Mean ± SD)
Dislodgeable Dose	379.97 ± 24.54	369.63 ± 21.37
Unabsorbed Dose *	399.60 ± 26.01	390.47 ± 13.49
Absorbed Dose **	0.11 ± 0.12	2.94 ± 3.30
Dermal Delivery ***	1.74 ± 1.08	6.55 ± 4.72
Mass Balance	401.34 ± 25.52	397.02 ± 10.55

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

** Absorbed dose = receptor fluid + receptor rinse

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