Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Sep. 08, 2004 to Sep. 20, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidance document for the conduct of skin absorption studies No. 28
Deviations:
no
GLP compliance:
yes
Remarks:
(according to UK principles of GLP relating to OECD Principles of GLP)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
UNLABELED TEST MATERIAL
- Name of test material: N,N-Bis-2-(hydroxyethyl)-p-phenylenediamine as the sulphate monohydrate salt; N,N-Bis (Code # 993-997-16)
- TSIN # GTS03849
- Substance type: Pure active substance
- Physical state: Off-white powder
- Stability under test conditions: Not reported
- Storage condition of test material: Ambient temperature in the dark
- Solubility: 35 w/v% in water

LABELLED TEST SUBSTANCE
- Name of test material: 14C-N,N-Bis-2-(hydroxyethyl)-pphenylenediamine as the sulfate monohydrate salt
- Substance type: Pure active substance
- Physical state: Solid
- Specific activity: 80 mCi/mmol; 2.96 GBq/mmol
- Locations of the label: Ring-14C
- Stability under test conditions: Not reported
- Storage condition of test material: - 20°C
Radiolabelling:
yes

Test animals

Species:
other: Human dermatomed skin membranes

Administration / exposure

Duration of exposure:
30 min
Doses:
1:1 w/w formulation containing 2.5% N,N-Bis sulfate monohydrate and developer at the nominal rate of 20 mg/cm2 (500 μg N,N-Bis.SO4.H2O /cm2 or 346 μg N,N-Bis/cm2)
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: The hair dye formulation (Nice’n Easy tint) containing nominal 5% N,N-Bis sulphate monohydrate was applied as 1:1 w/w dilutions with two different developers (a peroxide developer and a placebo developer), both resulting in a nominal final dose concentration of 2.5% N,N-Bis sulphate monohydrate. 14C-radiolabelled N,N-Bis sulfate monohydrate was incorporated into the doses to give in the region of 1 x 10(8) to 1 x 10(9) dpm/mL. The radiolabelled test substance was incorporated in the dose to measure the penetration of test substance.
For details on preparation of formulations, refer to ‘Any other information on materials and methods incl. tables’ section.
- Method of storage: not reported

APPLICATION OF DOSE: The mixed formulations were applied to 12 human dermatomed skin membranes, mounted in glass diffusion cells, at a nominal rate of 20 mg/cm2 for 30 min. The doses were applied to the skin and spread over the surface using small glass rods and the weight of the applied dose recorded after spreading.

OBSERVATION PERIOD: 48 h

TEST SITE
- Area of exposure: 2.54 cm2

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: After a contact period of 30 min, the dose was washed from the surface of the skin using natural sponges soaked in 3% Teepol.
- Time after start of exposure: 30 min

SAMPLE PREPARATION AND SAMPLING PROCEDURE
- Receptor fluid samples: At recorded intervals (0.5, 1, 2, 4, 6, 24, 29 and 48 h), 0.5 mL samples of the receptor fluid were taken for analysis by LSC. The volume of fluid in the receptor chamber was maintained by the addition of 0.5 mL fresh receptor fluid to the chamber immediately after the removal of each sample.
- Sponges: After the 0.5 h sample had been taken, excess formulation was washed from the skin surface by gently swabbing with a series of sponges (approx. 1 cm3) pre-wetted with 3% Teepol. Decontamination was shown to be complete following assessment of residual radioactivity levels on the skin surface with a Geiger counter. Another 2 sponges, pre-wetted with water, were used to further swab the surface. All the sponges were combined and digested in Soluene 350. The digests were made up to a recorded volume and a sample taken for analysis.
- Tape strips: To assess penetration through the stratum corneum, successive layers of the stratum corneum were removed by the repeated application of adhesive tape (eg Scotch 3M Magic Tape, 1.25 cm wide). A strip of adhesive tape was pressed onto the skin surface and then carefully peeled off to remove the stratum corneum. This process was repeated to a maximum of 21 strips. The adhesive strips were soaked Soluene 350 to extract any test material. The first 5 strips were extracted individually, with the remaining strips combined in groups of 4 and each group extracted. The extracts were sequentially numbered and analysed by LSC.
- Residual skin: The dosed area of the remaining skin (2.54 cm2) was carefully cut away from the receptor chamber and digested in Soluene 350 and the digest analysed. The remaining skin from the outer edge of the receptor chamber (flange region), which had been held under the donor chamber, was also removed and digested in Soluene 350 and the digest analysed.
- Donor chambers: After the final receptor fluid sample had been taken, the remaining fluid in the receptor chamber was discarded and the chamber rinsed with fresh receptor fluid which was also discarded. The donor chamber was carefully removed, washed with PBS/A and the sample analysed for N,N-Bis by LSC.

ANALYSIS
- Method type(s) for identification: Samples collected during this study were analysed by liquid scintillation counting on Packard 3100TR, using QuantaSmart – 1.31 software. Counting period was 6 min or to a 0.5% standard deviation of the count. Scintillation fluid used was Optiphase ‘Hi-Safe’ 3.
- Limits of detection and quantification: The limits of quantitation (LOQ) were 0.006 and 0.005 μg/mL (≡ 0.010 and 0.008 μg/cm2) for the peroxide and placebo developer mixes, respectively.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Human whole skin samples obtained from surgery or post mortem. Cells were selected such that each application was represented by 12 intact membranes from 8 different subjects.
- Ethical approval if human skin: Not reported
- Type of skin: Extraneous tissue was removed from the whole skin samples
- Preparative technique: Dermatome
- Thickness of skin: 400 μm
- Membrane integrity check: Yes, the membrane integrity was determined by the measurement of the electrical resistance across the skin membrane. Membranes with a measured resistance of <10kΩ were regarded as having a lower integrity than normal and not used for exposure to the test substances.
- Storage conditions: Approx. -20°C on aluminium foil
- Justification of species, anatomical site and preparative technique: In vitro techniques employing glass diffusion cells have been shown to predict percutaneous penetration of chemicals in vivo (OECD Guidance document No. 428).

PRINCIPLES OF ASSAY
- Diffusion cell: 24 glass diffusion cells (12 each for peroxide and placebo developer). However, only 11 diffusion cells were utilized for Placebo developer mix results as, the data indicated that the skin membranes had been damaged during the 0.5 h decontamination process.
- Receptor fluid: Phosphate buffered saline
- Solubility of test substance in receptor fluid: 370 mg/mL
- Test temperature: 32 ± 1°C (maintained in a water bath)
- Humidity: Not reported
- Occlusion: No
- Reference substance(s): Not reported

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
PEROXIDE DEVELOPER MIX
- Skin wash: The greatest proportion of the applied N,N-Bis was removed from the surface of the skin by the washing procedure at 0.5 h (337 μg/cm2; 97.2%), with only a minimum of 0.328 μg/cm2 (0.095%) being removed by the later procedure at 48 h.
- Flange: 0.023 μg/cm2 (0.007%)
- Donor chamber: 0.228 μg/cm2 (0.066%)
- Receptor fluid: 0.073 μg/cm2 (0.021%)
- Stratum corneum: 0.495 μg/cm2 (0.143%)
- Remaining epidermis/dermis: 0.035 μg/cm2 (0.010%)
The mean systemically available proportion of the dose (mean amount penetrated (receptor fluid)+ amount remaining in epidermis/dermis) = 0.108 µg/cm2 (0.031% of dose)

PLACEBO DEVELOPER MIX
- Skin wash: The greatest proportion of the applied N,N-Bis was removed from the surface of the skin by the washing procedure (348 μg/cm2; 101%), with only a minimum of 0.857 μg/cm2 (0.248%) being removed by the later procedure at 48 h.
- Flange: 0.042 μg/cm2 (0.012%)
- Donor chamber: 1.341 μg/cm2 (0.388%)
- Receptor fluid: 0.092 μg/cm2 (0.027%)
- Stratum corneum: 0.615μg/cm2 (0.178%)
- Remaining epidermis/dermis: 0.096 μg/cm2 (0.028%)
The mean systemically available proportion of the dose (mean amount penetrated (receptor fluid) + amount remaining in epidermis/dermis) = 0.188 µg/cm2 (0.054% of dose)
Total recovery:
PEROXIDE DEVELOPER MIX
- Total recovery: The overall mean recovery of N,N-Bis was 338 µg/cm2 (97.5%).
- Recovery of applied dose acceptable: Yes
- Results adjusted for incomplete recovery of the applied dose: No
- Limit of detection (LOD): Not reported
- Quantification of values below LOD or LOQ: Any calculated values below these LOQs were reported as the
PLACEBO DEVELOPER MIX
- Total recovery: The overall mean recovery of N,N-Bis was 351 µg/cm2 (102%).
- Recovery of applied dose acceptable: Yes
- Results adjusted for incomplete recovery of the applied dose: No
- Limit of detection (LOD): Not reported
- Quantification of values below LOD or LOQ: Any calculated values below these LOQs were reported as the
Percutaneous absorptionopen allclose all
Key result
Dose:
20 mg/cm2 of formulation containing 2.5% of test substance
Parameter:
percentage
Absorption:
0.021 %
Remarks on result:
other: 48 h
Remarks:
Absorption in receptor fluid after 30 min exposure to peroxide developer mix
Key result
Dose:
20 mg/cm2 of formulation containing 2.5% of test substance
Parameter:
percentage
Absorption:
0.01 %
Remarks on result:
other: 48 h
Remarks:
Absorption in epidermis/dermis after 30 min exposure to peroxide developer mix
Key result
Dose:
20 mg/cm2 of formulation containing 2.5% of test substance
Parameter:
percentage
Absorption:
0.027 %
Remarks on result:
other: 48 h
Remarks:
Absorption in receptor fluid after 30 min exposure to placebo developer mix
Key result
Dose:
20 mg/cm2 of formulation containing 2.5% of test substance
Parameter:
percentage
Absorption:
0.028 %
Remarks on result:
other: 48 h
Remarks:
Absorption in epidermis/dermis after 30 min exposure to placebo developer mix

Any other information on results incl. tables

Results of cutaneous absorption from peroxide developer mix:

The fastest rate of penetration rate for N,N-Bis from the peroxide developer mix (0.039 μg/cm2/h) was during the first hour after application. After 6 h the penetration process was effectively complete.The mean penetrated amount of N,N-Bis at 0.5 h was 0.015 μg/cm2 (0.004% of dose) which increased to 0.035 μg/cm2 (0.010%) at 1 h, 0.057 μg/cm2 (0.016%) at 6h and was 0.073 μg/cm2 (0.021%) at the end of the experiment (48 h).

The results of the cutaneous absorption after 48 h with 12 human skin samples treated with 20 mg/cm2 of formulation containing 2.5% of test substance in peroxide developer mix is provided in the below table:

Table 1: Summary of the cutaneous absorption of 2.5% N,N-Bis in peroxide developer mix (Study # 53731)

Amount of N,N-Bis in:

µg/cm2 (mean ± S.D. n=12)

% dose (mean ± S.D. n=12)

 

Flange (48 h)

0.023 ± 0.006

0.003 ± 0.007

Donor Chamber (48 h)

0.228 ± 0.358

0.066 ± 0.103

Skin Wash (0.5 h)

337 ± 5.88

97.2 ± 1.70

Skin Wash (48 h)

0.328 ± 0.076

0.095 ± 0.022

Stratum Corneum (48 h)

0.495 ± 0.234

0.143 ± 0.068

Remaining Epidermis/Dermis (48 h)

0.035 ± 0.016

0.010 ± 0.005

Receptor Fluid (48 h)

0.073 ± 0.047

0.021 ± 0.014

Total balance (recovery)

338 ± 5.81

97.5 ± 1.68

 

Results of cutaneous absorption from placebo developer mix:

The fastest mean penetration rate for N,N-Bis from the placebo developer mix occurred during the half hour of exposure (0.007 μg/cm2/h). After 1 h the rate reduced to 0.002 μg/cm2/h which was maintained until 48 h. The mean penetrated amount of N,N-Bis at 0.5 h was 0.004 μg/cm2 (0.001% of dose), which increased to 0.007 μg/cm2 (0.002%) at 1 h, 0.016 μg/cm2 (0.005%) at 6 h and 0.092 μg/cm2 (0.027%) by 48 h.

The results of the cutaneous absorption after 48 h with 11 human skin samples treated with 20 mg/cm2 (2.5% of test material) in placebo developer mix is provided in the below table:

Table 2: Summary of the cutaneous absorption of 2.5% N,N-Bis in placebo developer mix (Study # 53731)

Amount of N,N-Bis in:

µg/cm2 (mean ± S.D. n=11*)

% dose (mean ± S.D. n=11*)

 

Flange (48 h)

0.042 ± 0.032

0.012 ± 0.009

Donor Chamber (48 h)

1.341 ± 1.806

0.388 ± 0.523

Skin Wash (0.5 h)

348 ± 5.21

100.7 ± 1.51

Skin Wash (48 h)

0.857 ± 0.662

0.248 ± 0.192

Stratum Corneum (48 h)

0.615 ± 0.301

0.178 ± 0.087

Remaining Epidermis/Dermis (48 h)

0.096 ± 0.127

0.028 ± 0.037

Receptor Fluid (48 h)

0.092 ± 0.115

0.027 ± 0.033

Total balance (recovery)

351 ± 4.55

102 ± 1.32

 

*The results for one diffusion cell from the placebo developer mix experiment has not been reported, as the data indicated that the skin membranes had been damaged during the 0.5 h decontamination process.

Peroxide developer mix

The overall mean recovery of the test substance during this experiment was 97.5%. The penetrated amount of the test substance (receptor fluid + dermis/epidermis) at the end of the experiment (48h) was 0.108 ± 0.064 μg/cm2 (range 0.037-0.252 μg/cm2) corresponding to 0.031±0.014% dermal absorption of the applied dose.

Placebo developer mix

The overall mean recovery of the test substance during this experiment was 102%. The penetrated amount of the test substance (receptor fluid + dermis/epidermis) at the end of the experiment (48h) was 0.188 ± 0.242 μg/cm2 (range 0.049-0.497 μg/cm2) corresponding to 0.054±0.07% dermal absorption of the applied dose.

Applicant's summary and conclusion

Conclusions:
Peroxide developer mix
The overall mean recovery of the test substance during this experiment was 97.5%. The penetrated amount of the test substance (receptor fluid + dermis/epidermis) at the end of the experiment (48h) was 0.108 ± 0.064 μg/cm2 (range 0.037-0.252 μg/cm2) corresponding to 0.031±0.014% dermal absorption of the applied dose.

Placebo developer mix
The overall mean recovery of the test substance during this experiment was 102%. The penetrated amount of the test substance (receptor fluid + dermis/epidermis) at the end of the experiment (48h) was 0.188 ± 0.242 μg/cm2 (range 0.049-0.497 μg/cm2) corresponding to 0.054±0.07% dermal absorption of the applied dose.

Executive summary:

Cutaneous absorption (in vitro) study of 2.5% N,N-Bis-2-(hydroxyethyl)-p-phenylenediamine sulphate monohydrate(N,N-Bis) using human skin preparations was performed following OECD Guideline 428.

Two experiments with two different developers (peroxide dilution or placebo dilution) were performed on 12 diffusion cells per application. Extraneous tissue was removed from human whole skin samples of eight different subjects obtained from surgery or post mortem. A minimum of 2 replicate samples from each subject were taken where possible.

1:1 w/w formulation containing 2.5%N,N-Bis sulfate monohydrate and developer at the nominal rate of 20 mg/cm2 (500 μg N,N-Bis.SO4.H2O /cm2 or 346 μg N,N-Bis/cm2) was applied to 24 skin (12 skin per developer) samples for 30 min and subsequently washed off by gently swabbing with sponges soaked in 3% Teepol.

The content of test substance was determined in the receptor fluid in the pretreatment sample as well as at recorded intervals of 0.5, 1, 2, 4, 6, 24, 29 and 48 h after test substance application. To assess the penetration through the stratum corneum, successive layers of the stratum corneum were removed by the repeated application of adhesive tape. The tape strips and residual skin were extracted separately and analyzed for the amount of test material. The radioactivity in different matrices (flange, donor chamber, skin wash at 0.5 and 48 h, stratum corneum, remaining epidermis/dermis, receptor fluid) was quantified by means of a liquid scintillation counter.

The majority of the test substance from both placebo and peroxide developer mix was removed from the surface of the skin by the first washing process at 0.5 h, with only a minimum being removed by the later skin wash procedure at 48 h.

The absorption from peroxide developer mix in different matrices were:

- Skin wash: 337 μg/cm2 (97.2%)

- Flange: 0.023 μg/cm2 (0.007%)

- Donor chamber: 0.228 μg/cm2 (0.066%)

- Receptor fluid: 0.073 μg/cm2 (0.021%)

- Stratum corneum: 0.495 μg/cm2 (0.143%)

- Remaining epidermis/dermis: 0.035 μg/cm2 (0.010%)

The overall mean recovery of N,N-Bis was 97.5%

The absorption from placebo developer mix in different matrices were:

- Skin wash: 348 μg/cm2 (101%)

- Flange: 0.042 μg/cm2 (0.012%)

- Donor chamber: 1.341 μg/cm2 (0.388%)

- Receptor fluid: 0.092 μg/cm2 (0.027%)

- Stratum corneum: 0.615 μg/cm2 (0.178%)

- Remaining epidermis/dermis: 0.096 μg/cm2 (0.028%)

The overall mean recovery of N,N-Bis was 102%.

The amounts of N,N-Bis-2-(hydroxyethyl)-p-phenylenediamine found in the upper skin were not considered as biologically available.

During the first hour of exposure, N,N-Bis penetrates the skin faster from the peroxide developer mix than from the placebo developer mix.

The amounts of test substance removed from the surface of the skin by the washing procedure or extracted from the flange area and the amount absorbed to the stratum corneum was regarded as systemically unavailable.

Based on above, it was concluded that 0.108±0.064 µg/cm2 (0.031%) of N,N-Bis-2-(hydroxyethyl)-p-phenylenediamine sulphate monohydrate in a hair dye formulation containing 2.5% N,N-Bis in peroxide developer mix was considered as systemically available (receptor fluid: 0.073 µg/cm2, remaining epidermis/dermis: 0.035 µg/cm2) in the in-vitro cutaneous absorption through human skin.

0.188±0.242 µg/cm2 (0.054%) of N,N-Bis-2-(hydroxyethyl)-p-phenylenediamine sulphate monohydrate in a hair dye formulation containing 2.5% N,N-Bis in placebo developer mix was considered as systemically available (receptor fluid: 0.092 µg/cm2, remaining epidermis/dermis: 0.096 µg/cm2) in the in-vitro cutaneous absorption through human skin.

This cutaneous absorption (in vitro) is classified as acceptable, and satisfies the guideline requirements of the OECD 428 method.