1-Propanaminium, 3-[[9,10-dihydro-4-(methylamino)-9,10-dioxo-1-anthracenyl]amino]-N,N-dimethyl-N-propyl-, bromide (1:1)

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-11-26 to 2008-07-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 428 and GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

481-170-7

EC Name:

-

Cas Number:

502453-61-4

Molecular formula:

Hill formula: C23H30BrN3O2 CAS formula: C23H30N3O2.Br

IUPAC Name:

dimethyl(3-{[4-(methylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl]amino}propyl)propylazanium bromide

Constituent 2

Reference substance name:

Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide

IUPAC Name:

Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide

Details on test material:

- Name of test material (as cited in study report): B119 HC Blue 16
- Molecular formula (if other than submission substance): C23H30N3O2.Br
- Molecular weight (if other than submission substance): 460.42
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Substance type: active
- Physical state: dark blue powder

Radiolabelling:

no

Test animals

Species:

other: donor pigs were used for in vitro experiments with pig skin

Strain:

other: Schweizer Landedelschwein

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: N/A
- Age at study initiation: N/A
- Weight at study initiation: N/A
- Fasting period before study: N/A
- Housing: N/A
- Individual metabolism cages: N/A
- Diet (e.g. ad libitum): N/A
- Water (e.g. ad libitum): N/A
- Acclimation period: N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°C): N/A
- Humidity (%): N/A
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A


IN-LIFE DATES: N/A

Administration / exposure

Type of coverage:

open

Vehicle:

other: Accord Setting Lotion (formulation)

Duration of exposure:

24 hours

Doses:

- Nominal doses: 0.5% B119 HC Blue 16 in Accord Setting Lotion
- Actual doses: 0.1 mg/sq. cm of HC Blue 16
- Actual doses calculated as follows: 80 mg of the formulation (= 20 mg/sq. cm) containing 0.5% B119 HC Blue 16 was applied to the skin samples yielding 0.1 mg/sq. cm of B119 HC Blue 16
- Dose volume: N/A
- Rationale for dose selection: The quantity of formulation and test substance applied to the skin samples imitated use conditions for humans when applying a typical leave-on formulation.

No. of animals per group:

Two independent experiments were performed with 10 diffusion cells in total.

Control animals:

yes

Remarks:

blank formulation (Accord Setting Lotion) was applied to the blank skin sample in each of the two series

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: The formulation (Accord Setting Lotion) containing 0.5% HC Blue 16 was applied as received from the test sponsor.
- Method of storage: at room temperature


APPLICATION OF DOSE: 80 mg of the formulation (Accord Setting Lotion, 20 mg/sq. cm) containing 0.5% B119 HC Blue 16 (0.1 mg/cm2) was applied to each skin sample for 24 hours.


VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle used imitated use conditions for humans.
- Amount(s) applied (volume or weight with unit): 80 mg (20 mg/sq. cm)
- Concentration (if solution): N/A
- Lot/batch no. (if required): Accord Setting Lotion Gr. 3, batch no. DTF488005, recipe no. 85707322
- Purity: N/A


TEST SITE
- Preparation of test site: Thawed skin samples were mounted, dermal side down, in permeation chambers.
- Area of exposure: 4 sq. cm
- % coverage: N/A
- Type of cover / wrap if used: N/A
- Time intervals for shavings or clipplings: N/A


SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: N/A


REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: N/A
- Washing procedures and type of cleansing agent: After 24 hours exposure time, the hair dye formulation was removed by extensive washing in five steps (2x 4 mL of water, 1x 4mL of shampoo, 2x 4 mL of water). Shampoo = "Salon shampoo, Energy, Duftshampoo," Formulation no. 7355034030, batch no. D0150074; diluted 1 + 6 with deionized H20.
- Time after start of exposure: 24 hours


SAMPLE COLLECTION
- Collection of blood: N/A
- Collection of urine and faeces: N/A
- Collection of expired air: N/A
- Terminal procedure: N/A
- Analysis of organs: After disassembly of the skin samples from the teflon chambers, the upper skin was separated from the lower skin using a heat method so that the amount of B119 HC Blue 16 in each skin compartment could be determined.


SAMPLE PREPARATION
- Storage procedure: All samples (application formulation, rinsings, receptor fluid fractions, and skin compartment extracts) were stored at -20 deg. Celsius, if not processed immediately.
- Preparation details: N/A


ANALYSIS
- Method type(s) for identification: HPLC (Alliance system from Waters Associates) equipped with a PDA detector (Waters Associates) controlled by Empower software.
- Liquid scintillation counting results (cpm) converted to dpm as follows: N/A
- Validation of analytical procedure: A calibration curve was established with aliquots of the formulation containing 0.5% HC Blue 16 and was linear up to 500 ng.
- Limits of detection and quantification: The absolute limit of dectection of B119 HC Blue 16 was 3 ng/injection and the absolute limit of quantification was found to be 10 ng/injection.


OTHER: N/A

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Source of pig skin was Butchery Kunzli, CH-1724 Praroman-Le-Mouret.
- Ethical approval if human skin: N/A
- Type of skin: pig skin
- Preparative technique: Split thickness skin samples from backs and flanks were prepared using a dermatome on different dates and from different donors, and stored at -20 degrees Celsius until use. The preparations were composed of the stratum corneum, the stratum germinativum, and part of the dermis containing blood vessels. Prior to the penetration experiment, the thickness of each skin sample was determined using a micrometer.
- Thickness of skin (in mm): 1000 uM
- Membrane integrity check: The integrity of each skin preparation was demonstrated by examining penetration characteristics with tritiated water.
- Storage conditions: -20 degrees Celsius until use
- Justification of species, anatomical site and preparative technique: Pig skin from flank and back represents a good model for human skin.


PRINCIPLES OF ASSAY
- Diffusion cell: Teflon chambers with 9.1 sq. cm surface were used. The upper part of the Teflon chamber was used for fixing the skin sample. The lower part of the chamber contained a cavity for the receptor fluid, and had two connections for the receptor fluid tubings (flow-through assembly).
- Receptor fluid: 0.14 M NaCl, 2 mM K2HPO4, 0.4 mM KH2PO4, 100 IU penicillin/mL, 97 ug streptomycin/mL, and 3% of ethanol
- Solubility of test substance in receptor fluid: 93.612 mg/mL (at pH = 6.8)
- Static system: N/A
- Flow-through system: The experiments were performed using a flow-through system, with a constant flow of the receptor fluid of 5 mL/hour.
- Test temperature: 32 +/- 2 deg. Celsius
- Humidity: First experiment (10.9 to 13.6% relative humidity); second experiment (15.8 to 17.7% relative humidity)
- Occlusion: N/A
- Reference substance(s): mannitol
- Other: The experimental conditions, the quantity applied, the vehicle used, and the time of exposure imitated use conditions for humans when applying a typical hair dye formulation. All assumptions were rather conservative.

Results and discussion

Signs and symptoms of toxicity:

not examined

Remarks:

N/A

Dermal irritation:

not examined

Remarks:

N/A

Absorption in different matrices:

- Non-occlusive cover + enclosure rinse: N/A
- Skin wash: The majority of the test substance could be found in the rinsing solutions (97.563 +/- 3.311 ug/sq. cm)
- Skin test site: N/A
- Skin, untreated site: N/A
- Blood: N/A
- Carcass: N/A
- Urine: N/A
- Cage wash + cage wipe: N/A
- Faeces: N/A
- Expired air (if applicable): N/A
- Serial non-detects in excreta at termination: N/A
- Receptor fluid, receptor chamber, donor chamber (in vitro test system): The test substance was not detectable in the samples within 24 hours.
- Skin preparation (in vitro test system): Small amounts of test substance could be found in the remaining skin (other than the stratum corneum) (0.326 +/- 0.207 ug/sq. cm).
- Stratum corneum (in vitro test system): (i.e tape strips): Small amounts of test substance could be found in the stratum corneum (0.678 +/- 0.471 ug/sq. cm)

Total recovery:

- Total recovery: 98.570% +/- 3.219% (mean +/- S.D., n=10)
- Recovery of applied dose acceptable: yes
- Results adjusted for incomplete recovery of the applied dose: N/A
- Limit of detection (LOD): 3 ng/injection
- Quantification of values below LOD or LOQ: All receptor fluid samples were below the limit of detection, corresponding to 5 ng test substance/sq. cm skin for each receptor fluid sample.

Percutaneous absorption

Dose:

N/A

Remarks on result:

other: N/A

Remarks:

N/A

Conversion factor human vs. animal skin:

N/A

Any other information on results incl. tables

Taking into consideration that the amounts of test substance found in the stratum corneum and the rinsings were not bioavailable, a maximum of 0.331 +/- 0.207 ug/sq. cm of 0.5% HC Blue 16 in Accord Setting Lotion was considered as biologically available (n=10, three donors; receptor fluid + remaining skin; 0.005 ug/sq. cm + 0.326 ug/sq. cm). 

Table: Summary of the cutaneous absorption of 0.5% HC Blue 16 in Accord Setting Lotion

Amount of test substance in:	µg/cm^2 (mean ± S.D., n = 10)			% (mean ± S.D., n = 10)
Receptor fluid (24 hours)	BLD* (0.005)	±	-	0.005	±	-
Remaining skin (24 hours)	0.326	±	0.207	0.326	±	0.207
Stratum corneum (24 hours)	0.678	±	0.471	0.678	±	0.471
Rinsing solution (24 hours)	97.563	±	3.311	97.563	±	3.311
Total balance (recovery)	98.570	±	3.219	98.570	±	3.219
*BLD: below the limit of detection, corresponding to 0.005 ug/sq. cm for the receptor fluid samples

Applicant's summary and conclusion

Conclusions:

After applying 0.5% B119 HC Blue 16 in Accord Setting Lotion to in vitro pig skin preparations under conditions that imitated human use, the majority of the test substance was found in the rinsing solutions at the end of the experiment (24 hours). Small amounts were found in the stratum corneum and in the remaining skin. With respect to the receptor fluid collected, HC Blue 16 was not detectable within 24 hours. The mass balance of the test substance resulted in values of 92.7 to 103.0% recovery for 10 skin samples with acceptable integrity (as measured by the penetration characteristics of tritiated water). Taking into consideration that the amounts found in the stratum corneum and the rinsings were not bioavailable, a maximum of 0.331 +/- 0.207 ug/sq. cm of test substance was considered as biologically available (n = 10, three donors; receptor fluid + remaining skin; 0.005 ug/sq. cm + 0.326 ug/sq. cm). Taking into account the recommendations in applicable guidelines and the acceptance criteria defined in the study plan (at least 8 valid chambers), the study was valid.

Executive summary:

The cutaneous absorption of 0.5 %B119 HC Blue 16 in an Accord Setting Lotion was investigated in vitro, using pig skin preparations, which were continuously rinsed from underneath with physiological receptor fluid at a temperature of 32 ± 2 °C. Two independent experiments were performed with 10 diffusion cells in total. Additionally, a blank formulation was applied to the blank skin sample in each of the two series. For calculations, the mean value of all valid skin samples (n=10) in contact with 0.5 % B119 HC Blue 16 in formulation for the individual dye was used. The integrity of each skin preparation was determined by examination of penetration characteristics with tritiated water resulting in 0.7 to 1.4 % of the applied dose found after 4 hours in the receptor fluids, which was within the limit of acceptance (<=2.0 %) for all skin samples.

After checking the skin integrity, 80 mg of the formulation (= 20 mg/cm2), containing 0.5 % HC Blue 16 was applied to the skin samples (= 0.1 mg/cm2 of test substance) for 24 hours and subsequently washed off with water and shampoo.  The determination of the amount of the dye in the washings (= amount dislodgeable from the skin surface) was performed by HPLC.  At the end of the experiment (after 24 hours), the content of the B119 HC Blue 16 was determined in the receptor fluid by the same method.  At termination of the experiment, the stratum corneum was separated from the remaining skin (= stratum germinativum and upper dermis) using tape strips and the tapes were extracted and the dye content was quantified by HPLC. The amount of the test substance in the remaining skin was extracted and the dye content was quantified by the same HPLC method.

With respect to B119 HC Blue 16, the majority of the test substance could be found in the rinsing solutions (97.563 ± 3.311 ug/cm2). Small amounts of B119 HC Blue 16 could be found in the stratum corneum (0.678 ± 0.471 ug/cm2) and in the remaining skin (0.326 ± 0.207 ug/cm2). With respect to the receptor fluid fractions, the test substance was not detectable in the skin samples collected within 24 hours. The mass balance of B119 HC Blue 16 resulted in values of 92.7 to 103.0 % recovery for all (10) skin samples taking into consideration for the calculation of the mean values.

Taking into consideration, that the amounts found in the stratum corneum and the rinsings were not bioavailable, a maximum of 0.331 ± 0.207 ug/cm² of B119 HC Blue 16 in Accord Setting Lotion was considered as biologically available (n=10, three donors; receptor fluid + remaining skin; 0.005 ug/cm² + 0.326 ug/cm²). Taking into account the recommendations in the applicable guidelines and the acceptance criteria defined in the study plan (at least eight valid chambers), the study was considered to be valid.