(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Aug. 14, 2006 to Aug. 31, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Reference 2

Reference Type:

secondary source

Title:

Percutaneous absorption of hair dyes: An interlaboratory comparison of in vivo and in vitro data with rat and pig

Author:

Beck, H.; Bracher, M.; Bartnik, F. G.

Year:

1994

Bibliographic source:

IN VITRO TOXICOL 7, 305-312

Reference 3

Reference Type:

publication

Title:

Updated OPINION ON 2-Amino-4-hydroxyethylaminoanisole sulfate

Author:

Scientific Committee on Consumer Safety

Year:

2009

Bibliographic source:

European Comission Directorate-General for Health & Consumers

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

yes

Remarks:

Only 8 samples used from 3 donors (instead of 6 required from at least 3 differents donors) but sufficient for OECD TG 428 requirements

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Environmental Health and Safety Publications, series on testing and assessment No. 28. Guidance Document for the conduct of skin absorption studies. Paris, March 2004

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Test Guidelines for In Vitro Assessment of Percutaneous Absorption and Penetration of Cosmetic Ingredients. Colipa Task Force Cutaneous Penetration. Food and Chemical Toxicology 37,191 - 205 (1999)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to Swiss Ordinance based on OECD principles of GLP

Test material

Test material information

Constituent 1

Reference substance name:

(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

EC Number:

280-734-8

EC Name:

(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

Cas Number:

83763-48-8

Molecular formula:

C9H14N2O2.H2O4S

IUPAC Name:

(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

Constituent 2

Reference substance name:

Lehmann Blau

IUPAC Name:

Lehmann Blau

Constituent 3

Reference substance name:

2-amino-4-hydroxyethylaminoanisole sulfate

IUPAC Name:

2-amino-4-hydroxyethylaminoanisole sulfate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

UNLABELLED TEST SUBSTANCE
- Name of test material: Lehmann blue; 2-Amino-4-hydroxyethylaminoanisole sulfate (Code# A000157)
- TSIN: WR23081
- Substance type: Pure active substance
- Physical state: Pale grey powder
- Stability under test conditions: The substance is considered to be stable for more than 12 years if stored dry and protected from light at room temperature
- Stability in solution: Stability of test stock solution (5 weight %) was tested by HPLC over a period of 7 days when the solution is stored at room temperature in the absence of light.
In water: (t = 0h: 100%; 6h: 93.8%; 2d: 95.1%; 7d: 79.7%) low degradation
In DMSO: (t = 0h: 100%; 6h: 98.8%; 2d: 92.0%; 7d: 80.5%) low degradation
- Storage: At room temperature, protected from light and moisture
- Solubility: 10 g/L in water pH 2.8 (>5 weight% pH 8)
1 weight% in acetone/water 1:1 (pH 2.1)
9-10 weight% in DMSO
0.2 weight% in ethanol

LABELLED TEST SUBSTANCE
- Name of test material: [ring-U-14C] Lehmann blue
- Specific activity: 851 MBq/mmol; 23 mCi/mmol
- Storage: - 20˚C in the absence of moisture, light and air
Color cream formulation with 3% 2-Amino-4-hydroxyethylamino-anisole sulfate and equimolar amounts of reaction partner (WR18247)
Identity: Color cream with PyrazoI DHE and Lehmannblau
Batch N°: DTF68081
Recipe N°: 85903070
Appearance of cream formulation: Beige cream
Aggregate state at room temperature: Paste-like
Storage: At room temperature
Supplier: F. Biolley, RD-IPC, COSMITAL SA
Preparation Date: 26.07.2006
Expiration Date: 26.01.2008

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch no. 57
- Expiration date of the lot/batch: July 2005
- Purity test date: 19 February 2004

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 98.8%
- Specific activity: 10 mCi /mL
- Locations of the label: [U-14C] Uniform labelling of the aromatic ring system
- Expiration date of radiochemical substance: not specified but analysis was performed the 30 May 2006

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light and moisture
- Stability under test conditions: 82% recovery after 3 days in the receptor fluid
- Solubility and stability of the test substance in the solvent/vehicle: 82.6 mg/ml (pH=7.30) in receptor fluid
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The color cream formulation containing 1.5% of test substance was prepared by mixing 1.5 g of Color cream formulation (containing 3% of test substance), equimolar amounts of reaction partner (WR18247), 40 µL of partially radiolabeled test substance and 1.5 g of Welloxon Perfect 6%.
- Preliminary purification step (if any): not specified
- Final dilution of a dissolved solid, stock liquid or gel: not specified
- Final preparation of a solid: not detailed

FORM AS APPLIED IN THE TEST (if different from that of starting material) in color cream formulation

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Radiolabelling:

yes

Remarks:

ring-U-14C

Administration / exposure

Duration of exposure:

30 minutes

Doses:

400 mg of the formulation (100 mg/cm2), containing 1.5% 2-Amino-4-hydroxyethylamino anisole sulfate (1.5 mg of test substance/cm2)

Details on study design:

DOSE PREPARATION:
- Method for preparation of dose suspensions: The color cream formulation containing 1.5% of test substance was prepared by mixing 1.5 g of Color cream formulation (containing 3% of test substance), equimolar amounts of reaction partner (WR18247), 40 µL of partially radiolabeled test substance and 1.5 g of Welloxon Perfect 6%.
The detailed composition of color cream formulation is provided in the study report.

APPLICATION OF DOSE: After checking integrity, 12 skin samples were covered with 400 mg of an oxidative hair dye formulation containing 1.5 of test substance on 4 cm2 for 30 minutes in two consecutive series (6 skin samples for each series).

TEST SITE
- Area of exposure: 4 cm2

REMOVAL OF TEST SUBSTANCE
- Washing procedures: The formulation was removed by extensive washing in five steps (2 x 4 mL of water, 1 x 4 mL of shampoo,, 2 x 4 mL of water)
- Time after start of exposure: 30 minutes

SAMPLE COLLECTION
- Spatula: After application of the final formulation, the spatula was dipped into 1 mL of ethanol and rinsed with 4 mL of water. This solution was analyzed for radioactivity.
- Receptor fluid: The receptor fluid was sampled after 16, 24, 40, 48, 64 and 72 hours.
- Rinsings: 15 µL of rinsing solution was used for analysis of test substance content.
- Separation of Skin compartments: After disassembly of the skin samples from the Teflon chambers, the skin samples were wrapped in aluminum foil and put on a hot surface (80 to 90°C) upside down (i.e. lower skin upside) charged with a small weight of approximately 40 g. After 15 to 60 seconds, the packed skin sample was removed from the hot surface and unwrapped. The upper skin was separated from the lower skin using forceps and the two compartments were extracted for analysis.

SAMPLE PREPARATION
- Spatula: The water solution (in which the spatula dipped in ethanol was rinsed) was diluted with 15 mL of scintillation cocktail and the radioactivity was quantified using a scintillation counter.
- Receptor fluid: An aliquot of collected receptor fluid fraction (i.e. 5 mL) was mixed with 15 mL of scintillation cocktail for radioactivity analysis.
- Skin samples: The skin samples were processed as follows:
Extractions of the skin compartments: Each of the skin compartments was dissolved overnight in 2 mL aqueous KOH solution (10M) at 80°C. 200 µL of the resulting solution were mixed with 200 µL of concentrated hydrochloric acid and 3 mL of the scintillation cocktail. The radioactivity was quantified with a scintillation counter.
Extraction of the aluminium foil: After the separation of the skin compartments, the aluminium foil was dipped overnight into 1 mL of a solution containing 5% methanol in water. Thereafter, the aluminum foil was removed and the extract was diluted with scintillation cocktail. The radioactivity was quantified with a scintillation counter.
- Rinsings: 15 µL of each of the rinsing fractions were diluted with 3 mL scintillation cocktail and the radioactivity was quantified.
- Storage procedure: All samples (application formulation, rinsings, receptor fluid fractions and skin compartment extracts) were stored at -20 °C, if not processed immediately. Samples used for the determination of the stability in receptor fluid were stored at room temperature until analysis.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting (LSC; Packard Tricarb 2250CA)
- Validation of analytical procedure: A calibration curve was established with aliquots of the solution of the radiolabeled test substance. The calibration curve was linear (R2 = 0.9939) up to 1'200'000 dpm.
- Limits of detection and quantification: The limit of detection of test substance is the value for the dpm found for the blank sample. A priori, this value is in the range of 10 to 50 dpm. However 100 dpm may be considered as significant above the background. With respect to the applied formulation containing 1.5% of unlabelled test substance this would correspond to:
Absolute limit of detection [LOD]: 10.14 ng of test substance

Details on in vitro test system (if applicable):

Two independant experiments were performed with 6 skin samples.

SKIN PREPARATION
- Source of skin: Skin samples from 6 pigs (3 pigs were used in each test series) weighing 110 - 120 kg were obtained from Butchery Kunzli, CH-1724 Praroman-Le-Mouret
- Type of skin: Split thickness skin samples obtained from back and flanks. The preparations were composed of the stratum corneum, the stratum germinativum and part of the dermis containing blood vessels.
- Preparative technique: Dermatome
- Thickness of skin: 0.85 ± 0.16 mm(measured with a micrometer)
- Membrane integrity check: Yes, by tritiated water with scintillation counting of one hour fractions over a time period of four hours.
- Storage conditions: Stored at - 20 °C until use
- Justification of species, anatomical site and preparative technique: Among several animal species, pig skin from flank and back represents a good model for human skin.

PRINCIPLES OF ASSAY
- Diffusion cell: Six permeation chambers (Teflon-chambers) with 9.1 cm2 surface were used. At the beginning of the experiment, thawed skin samples were mounted dermal side down in permation chambers.
- Receptor fluid: Receptor fluid was composed of 0.14 M NaCl, 2 mM K2HPO4, 0.4 mM KH2PO4, 100 IU penicillin/mL, 76 IU streptomycin/mL, 0.3% Ascorbic acid, 0.4% Sodium sulfite and 3% of Ethanol( pH = 7.3)
- Solubility of test substance in receptor fluid: 92.6 mg/mL in receptor fluid
- Stability of test material in receptor fluid: 82% recovery after 3 days (50 mg/mL, in the presence of
0.3% ascorbic acid and 0.4% sodium sulfite}
- Flow-through system: Yes, the experiment was performed using a flow through system with a constant flow of the receptor fluid of 5 mL/h
- Test temperature: 32 ± 2°C
- Relative humidity: 20.5 to 34.9% (first series), 24.9 to 32.2% (second series)
- Occlusion: No
- Reference substance: Yes, mannitol was tested to assess the performance and reliability of the test system.
- Principle diffusion barrier: Stratum corneum was identified as the principal diffusion barrier, the integrity of which was controlled in each experiment by 3H2O penetration characteristics.

Results and discussion

Absorption in different matrices:

- Rinsing solution (after 30 minutes): 92.69 ± 3.46% or 1340.92 ± 50.57 µg/cm2; After the removal of applied test substance with water and shampoo after 30 minutes, the recovered test substance was found in majority in the rinsing solutions.
- Receptor fluid: Within 72 hours 0.028 ± 0.015% or 0.409 ± 0.223 µg/cm2 of test substance had penetrated into the receptor fluid.
With respect to the receptor fluid, only small amounts of test substance were detectable within the first fractions collected during 72 hours indicating that no further test substance is available from a potential reservoir of the skin compartments.
- Skin preparations: After 72 hours , small amounts of test substance was found in the upper skin 0.162 ± 0.057% or 2.352 ± 0.824 µg/cm2 and in the lower skin 0.021 ± 0.015% or 0.303 ± 0.219 µg/cm2
Under the assumption that a depot effect is absent, a maximum amount of 0.712 ± 0.313 µg/cm2 of test substance is considered as biologically available (n=8, three donors; receptor fluid + lower skin; 0.409 + 0.303 µg/cm2).

Total recovery:

- Total recovery: 96.59 ± 3.53% (or 1446.71 ± 7.86 µg/cm2)
- Recovery of applied dose acceptable: Yes
- Results adjusted for incomplete recovery of the applied dose: Yes, total recovery is corrected for losses on tips.
- Limit of detection (LOD): 10.14 ng of test substance as the absolute limit of detection with respect to the applied formulation containing 1.5% test substance.

Percutaneous absorption

Percutaneous absorption 1

Dose:

100 mg/cm2 formulation containing 1.5% of test substance (1.5 mg /cm2)

Parameter:

percentage

Absorption:

0.028 %

Remarks on result:

other: 72 hours

Remarks:

Absorption in receptor fluid within 72 hours exposure

Percutaneous absorption 2

Dose:

100 mg/cm2 formulation containing 1.5% of test substance (1.5 mg /cm2)

Parameter:

percentage

Absorption:

0.021 %

Remarks on result:

other: 72 hours

Remarks:

Absorption in lower skin after 72 hours exposure

Percutaneous absorption 3

Key result

Time point:

72 h

Dose:

100 mg/cm2 formulation containing 1.5% of test substance (1.5 mg/cm2)

Parameter:

percentage

Absorption:

0.211 %

Remarks on result:

other: 72% absorption rate including upper skin, lower skin and receptor fluid

Percutaneous absorption 4

Key result

Time point:

72 h

Dose:

100 mg/cm2 formulation containing 1.5% of test substance (1.5 mg/cm2)

Parameter:

amount

Absorption:

3.065 other: µg/cm2

Remarks on result:

other: absorption amount including upper, lower skin and receptor fluid

Any other information on results incl. tables

Reference control: The mean value for the penetration of mannitol through pig skin preparations calculated for all samples was 0.76 ± 0.49% of the applied dose (n=12, water as vehicle).

Skin integrity: The integrity of each skin preparation was determined by examination of penetration characteristics with tritiated water. The skin integrity values for the eight skin samples taken into consideration for the calculation of the mean were within the limit of acceptance (≤ 2.0%).

SCCS Comments

- The amount applied (100 mg/cm²) is very high compared to the normal value of 20 mg/cm².

- The "upper skin" (stratum corneum + upper stratum germinativum) was separated from the "lower skin" (lower stratum germinativum and upper dermis). This does not allow calculating the values in (i) stratum corneum (SC), (ii) epidermis without SC and (iii) dermis separately, as requested by the SCCP.

- The study authors do not consider the amount measured in the "upper skin" as biologically available. However, the "upper skin" contains a large part of the epidermis (not only the stratum corneum), therefore the mentioned value of 0.712 μg/cm² is an underestimation.

- Overall, it is clear that the study authors did not take into account the SCCP requirements (SCCP/0970/06). With only 8 samples in total from 3 donors (instead of the required minimum of 6 evaluable samples, from each of at least 3 donors) and an incorrect separation of the skin, the exact dermal absorption cannot be deduced from this study.

Considering the above, a worst case scenario for dermal absorption of 2-amino-4 - hydroxyethylamino anisole sulfate may be its amount found in the receptor fluid together with its amounts present in the upper as well as in the lower skin. Thus, the dermal absorption of 2-amino-4-hydroxy-ethylamino-anisole sulphate may be considered as 3.064 + 2 x 0.88 = 4.82 μg/cm².

Dermal absorption of 2-amino-4-hydroxy-ethylamino-anisole sulphate

Amount of WR 23081 in:	µg/cm2(mean ± S.D, n = 8)			%* (mean ± S.D, n =8 )
Receptor fluid (72 hours)	0.409	±	0.223	0.028	±	0.015
Lower skin (72 hours)	0.303	±	0.219	0.021	±	0.015
Upper skin (72 hours)	2.352	±	0.824	0.162	±	0.057
Systemic bioavailability**	3.064	±	0.88	0.211	±	0.061
Rinsing solution (after 60 min.)	1340.92	±	50.57	92.69	±	3.46
Total balance (recovery)***	1446.71	±	7.86	96.59	±	3.53

 

* Corrected for individual applied dose;

**sum of receptor fluid + lower skin + upper skin

*** Total is corrected for losses on handling

Applicant's summary and conclusion

Conclusions:

Under the conditions of this in vitro cutaneous absorption study of 2-Amino-4-hydroxyethylamino anisole sulfate, a maximum amount of 3.065 ± 1.266 µg/cm2 and 0.21 ± 0.087 % of test substance is considered as biologically available (including upper skin, lower skin and receptor fluid).

Executive summary:

The cutaneous absorption (in-vitro) of 1.5% 2 -Amino-4 -hydroxyethylamino anisole sulfate in a typical hair dye formulation in the presence of hydrogen peroxide and a reaction partner (1-Hydroxyethy1 4,5-Diamino Pyrazole Sulfate) was determined following methods according to the OECD Guideline 428 (Skin Absorption: In Vitro Method).

Pig skin samples (from back and flanks) were used as the test system. Skins from 6 pigs (skin of 3 pigs was used in the 1^stseries of experiments and skin of other 3 pigs was used in the 2^ndseries of experiments), weighing 110 – 120 kg were obtained from Butchery Kunzli, CH-1724 Praroman-Le-Mouret, Switzerland. Two skin samples were obtained from each pig. In total, 12 skin samples were used in this study.

The integrity of each skin preparation was determined by examination of penetration characteristics with tritiated water. The skin integrity values for the eight skin samples taken into consideration for the calculation of the mean were within the limit of acceptance (≤ 2.0%). After checking the skin integrity, 400 mg of the typical hair dye formulation (100 mg/cm2) containing 1.5% test substance (i.e. 1.5 mg/cm2) was applied to 12 skin samples for 30 minutes in two independent experiments including 6 skin samples each. After completion of exposure period, the samples were washed off with water and shampoo and the amount of test substance in the rinsing solutions was determined.

The receptor fluid was sampled at 16, 24, 40, 48, 64 and 72 hour intervals to determine the test substance concentration. At the termination of the experiment, the skin was heat-treated to separate the upper and lower skin compartments. The different matrices (rinsing solution, receptor fluid, lower skin and upper skin) were processed and analyzed by means of a scintillation counter to determine radioactive concentrations. 

The majority of the test substance was recovered in the rinsing solutions (92.69 ± 3.46% or 1340.92 ± 50.57 µg/cm2) after 30 minutes. Small amounts of test substance were detected in the upper skin (0.162 ± 0.057% or 2.352 ± 0.824 µg/cm2), in the lower skin (0.021 ± 0.015% or 0.303 ± 0.219 µg/cm2) and in the fractions of the receptor fluid collected within 72 hours (0.028 ± 0.015% or 0.409 ± 0.223 µg/cm2).

The mass balance (total recovery) was 96.59 ± 3.53% (or 1446.71 ± 7.86 µg/cm2) of the dose applied to the pig skin samples.

With respect to the receptor fluid, only small amounts of test substance were detectable within the first fractions collected during 72 hours indicating that no further test substance is available from a potential reservoir of the skin compartments. With such an absorption profile, it was assumed that a depot effect is absent.

Under the conditions of this in vitro cutaneous absorption study of 2-Amino-4-hydroxyethylamino anisole sulfate, a maximum amount of 3.065 ± 1.266 µg/cm2 and 0.21 ± 0.087 % of test substance is considered as biologically available (including upper skin, lower skin and receptor fluid).

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