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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride
EC Number:
916-466-1
IUPAC Name:
Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride
Test material form:
solid: particulate/powder
Details on test material:
Identification : Basic Blue 99
Batch number : RS27988101
3-[(Bromo-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (main) : 50.2a/a%
3-[(Dibromo-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (E) : 14.6a/a% 3-[(Bromo-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (F) : 16.9a/a%
3-[(Bromo-5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (J) : 4.8a/a% 3-[(8-Amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (A) : 1.0a/a% 3-[(Sulpho-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (B) : 2.0a/a%
3-[(Dibromo-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (G): 2.8a/a%
3-[(Sulpho-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (O) :<0.3a/a%
3-[(Bromo-sulpho-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (N) : 2.1a/a%
Tribromo-8-amino-5-hydroxy-1,4-naphthoquinone (L) : 1.3a/a%
Chloride ion : 14.5%
Sulphate ion : 0.1%
Acetate ion : 0.2%
Zinc ion : 5.5%
Water : 4.1%
Radiolabelling:
no

Administration / exposure

Details on in vitro test system (if applicable):
The penetration of the test item was measured in-vitro through dermatomed pig skin from a solution in water and from a standard formulation. The aqueous solution and formulated material both containing 1.09 w/w% basic blue 99 were applied to the dermatomed membranes at a rate of 10ul/cm2 and left unoccluded. The applications were rinsed off after a 0.5 hour contact period with the penetration of basic blue 99 through the membrane being assessed throughout the entire 48 hour exposure period. At the end of the exposure period, the distribution of basic blue 99 in the test system was assessed which included a tape stripping technique to determine the distribution of the dye in the skin preparation. The applications and exposure conditions were designed to simulate potential human dermal exposure to the formulation during normal use and compare the penetration of basic blue 99 from a standard formulation with the penetration from an aqueous solution. Samples collected during the study were analysed by high performance liquid chromatography.

Results and discussion

Percutaneous absorptionopen allclose all
Time point:
48 h
Dose:
10 uL/cm2 aqueous solution containing 1.09% w/w Basic Blue 99
Parameter:
amount
Absorption:
< 0.2 other: ug/cm2
Time point:
48 h
Dose:
10 uL/cm2 aqueous solution containing 1.09% w/w Basic Blue 99
Parameter:
percentage
Absorption:
< 0.18 %
Remarks on result:
other: % of the applied dose
Time point:
48 h
Dose:
10 uL/cm2 formulation containing 1.09% w/w Basic Blue 99
Parameter:
amount
Absorption:
< 0.2 other: ug/cm2
Time point:
48 h
Dose:
10 uL/cm2 formulation containing 1.09% w/w Basic Blue 99
Parameter:
percentage
Absorption:
< 0.18 %
Remarks on result:
other: % of the applied dose

Any other information on results incl. tables

The results of the mass balance and test item distribution are summarised in the table below.

 Test Compartment  Mean Recovery Aqueous Solution/%  Mean Recovery Formulated Material/%
 Donor Chamber & Spreader  2.28  0.910
 Skin Wash after 0.5 hours  64.6  70.5
 Skin Wash after 48 hours  4.37  3.28
 Stratum Corneum  <3.25  <3.25
 Remaining Epidermis/Dermis  3.12  0.549
 Penetrated  <0.18  <0.18
 Total  74.4 -77.8  75.2 -78.7

Applicant's summary and conclusion

Conclusions:
No test item was detected as having penetrated through the skin into the receptor fluid from either of the applications (<0.18% of the applied dose). The vast majority of the test item was removed after a contact period of 0.5h by simple rinsing of the skin surface (65% from the aqueous solution and 71% from the formulated material). The maximum systemically available dose (epidermis/dermis plus receptor fluid, including limit of quantification value as positive values) would be approximately 3.3% for the aqueous solution and 0.7% from the formulated material. The systemic availability of the test item after dermal exposure to the standard formulation or an aqueous solution of the test item would be minimal.
Executive summary:

The penetration of Basic Blue 99 has been measured in vitro through dermatomed pig skin from a solution in water and from a standard formulation. The aqueous solution and formulated material, both containing 1.09%w/w Basic Blue 99, were applied to the dermatomed membranes at a rate of 10 uL/cm2 and left unoccluded. The applications were rinsed off after a 0.5 hour contact period, with the penetration of Basic Blue 99 through the membrane being assessed throughout the entire 48 hour period. At the end of the exposure period, the distribution of Basic Blue 99 in the test system was assessed, which included a tape stripping technique to determine the distribution of the dye in the skin preparation. The applications and exposure conditions were designed to simulate potential human dermal exposure to the formulation during normal use and compare the penetration of Basic Blue 99 from a standard formulation with the penetration from an aqueous solution. Samples collected during this study were analysed by high pressure liquid chromatography. Penetration of Basic Blue 99 through dermatomed pig skin was below the limit of quantification (0.2 ug/cm2) for both the aqueous solution and formulation. The mean recovery of Basic Blue 99 was between 74 -78% from the aqueous solution and between 75 -79% from the formulated material, which was below the target value of 85%. The majority of the applied Basic Blue 99 was washed from the surface of the skin at 0.5 hours (aqueous solution = mean 64.6%, formulated material = mean 70.5%). A further 4.37% and 3.28% of the dose, respectively, was removed from the surface during terminal wash at 48 hours. No Basic Blue 99 was recovered from the tape strips, representing the stratum corneum (total <3.25% of dose) for both the aqueous solution and formulation, however small amounts (mean 3.12% (aqueous solution) and 0.549% (formulation) were recovered from the remaining epidermis/dermis. No test item was detected as having penetrated through the skin into the receptor fluid from either of the applications (<0.18% of the applied dose). The vast majority of the test item was removed after a contact period of 0.5h by simple rinsing of the skin surface (65% from the aqueous solution and 71% from the formulated material). The maximum systemically available dose (epidermis/dermis plus receptor fluid, including limit of quantification value as positive values) would be approximately 3.3% for the aqueous solution and 0.7% from the formulated material. The mean recovery of Basic Blue 99 was slightly less than the target value of 85%, at 78% from the aqueous solution and 79% from the formulated material. The systemic availability of the test item after dermal exposure to the standard formulation or an aqueous solution of the test item would be minimal.