Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 15 June to 13 July 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across from a similar substance which has the same main componenT and with a different counter ion that doesn't influence the characteristics related to the specific end-point

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
yes
Remarks:
The EU in vitro validated percutaneous absorption test method (OECD TG 428) requires at least 6 replicates for each product : considering the screening purpose of this study a reduced number of replicates have been used.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
no

Test animals

Species:
human
Details on test animals and environmental conditions:
EX VIVO HUMAN SKIN EXPLANTS
The TRANSKIN model from Biopredic International (8-18 Rue du Professeur Jean Pecker, 35000 Rennes, France) is a fresh ex-vivo HUMAN SKIN explant from abdominal
surgical intervention of 3,8 cm2: it comprises epidermal and dermal layers. The intended use of the biological model is for research purpose only (French Law Art.
L. 1245 CSP) and for standardized in vitro testing of chemicals or formulations. Human skin samples are accepted as test system in the OECD-TG 428: in order to
guarantee adequate reproducibility the tissues are from the same donor, without stretchmarks and with homogeneous thickness. Each tissue has been tested for the presence of viruses, bacteria and mycoplasma (attached technical and safety data sheet).

Administration / exposure

Type of coverage:
open
Vehicle:
other: saline solution
Duration of exposure:
24 h
Doses:
MG OXALATE:
- Formulation: 100 mg/ 3.8 cm^2
- Active dye: 81 mg /3.8 cm^2

MG CHLORIDE:
- Formulation: 100 mg/ 3.8 cm^2
- Active dye: 100 mg /3.8 cm^2
Control animals:
no
Details on in vitro test system (if applicable):
TEST SYSTEM PREPARATION
After arrival in the laboratory the human skin disks were placed in petri dishes (35mm) with maintenance medium (batch MIL305027), following supplier instructions. The day of the test they were removed from the refrigerator and placed at room temperature for 10 minutes before placing them upon the static cells in the thermostatic bath for the percutaneous absorption test.

INSTRUMENTS
- Instruments used during the application of test substance: Diffusion cells by Laboratory Spiral; Thermostatic bath polystat 36 by Fisher Scientific; Analytical Balance XS 204 by Mettler - Toledo; Micropipette-Microman M100 by Gilson; +5°C FRL 360 V-GL -20°C Frigolab 700 BT/1 by Angeloni
- Instruments for analysis: HPLC-MS

DOSE PREPARATION AND APPLICATION DOSE
The applied dose defined for this study has been of 100 mg in order to reach a relative applied dose between 1 and 5 mg/cm^2 (OECD TG 428: section 16). The products have been applied as powder and 50 μL of saline solution have been added to allow a better adhesion of the powder to the skin surface. Each product has been applied for 24h on 4 replicates.

PRINCIPLE OF THE METHOD
The rate of absorption and passage of test chemicals into and through human skin can be modelled in the in vitro percutaneous absorption study.
The test substance is applied to the surface of the skin model separating the two chambers of a diffusion cell: the upper donor compartment where the product is applied and the lower acceptor compartment containing the receptor fluid (saline solution and ethanol, 1:1) placed in the thermostatic bath at 37°C ± 2 °C.
Skin exposure to the test preparation is for the entire duration of the experiment (24h). A period of sampling of 24 hours is required to allow an adequate characterization of the absorption profile. Sampling frequency of the receptor fluid allows to determine the kinetics absorption of the compound.
The amount of test substance in the receptor fluid, skin preparation, skin surface washings are analyzed to quantify the absolute quantity and % of active principle absorbed through the test system. The total test substance localization allows to calculate the percentage of recovery and to quantify the total mass balance in order to validate the assay.

PROCEDURE
The diffusion chamber and the skin are maintained at a constant temperature close to normal skin temperature of 32 °C ± 2 °C (36 °C in the water bath) in constant shaking. 4 mL of receptor fluid are placed in the lower part of the diffusion cell and maintained under constant stirring in the thermostatic bath at 37 °C ± 2 °C. Then the tissues (defrozen) are placed on the top of the lower part, the product applied with a micropipette and then the diffusion cell is closed by incorporating the upper part with a special screw.

At the times of 2h and 4h, 2 mL of the receptor fluid have been sampled with a microsyringe: the samples have been coded and stored at +4 °C for analysis. At each time 2 mL of fresh saline solution have been added in the lower part of the diffusion cell at each time. At 24h all the receptor fluid has been collected, the diffusion cell washed, coded and stored at +4 °C for analysis (total volume 4 mL).

At the end of the penetration study (24h) the skin models have been placed in a Petri dish and the residual cream on the skin surface has been removed with a spatula and put in a Falcon tube coded as sample L. Then the epidermal surface has been rinsed by using fresh receptor fluid and the liquid
has been put together with the residual cream in sample L (total volume 15 mL). With a sharp scalpel the skin has been divided in two pieces (epidermis and dermis) that are separately introduced in a defined volume of saline solution plus ethanol (5 mL). Each sample has been immediately sonicated and placed for 24h at +5°C.

ANALYSIS
Biological samples have been analyzed with a HPLC method by a GLP certified laboratory, Labotech SrL, Via Kennedy, 19 -20059 Vimercate (MI). The raw data of analytical measurements have been reported in a Excel file and processed.
The results are expressed as absolute content of active for cm^2 and also presented as % of active penetrated compared to the applied quantity.
Values of TOTAL MASS BALANCE 90 ± 10-15 % are accepted for study validation following OECD 428 (±10%) and SCCNFP (SCCNFP 0970/06) (±15%) criteria.

STATISTICAL ANALYSIS
According to the accuracy and precision of the analytical method the raw data has been used for calculation in an excel file and the standard deviation (SD) calculated.

Results and discussion

Total recovery:
MALACHITE GREEN OXALATE
- Mean of absorbed dose: 0.015%
- Unabsorbed dose (L-residual): 92.377 %
- Absorbable dose (Epidermal Homogenate + Stratum corneum and Dermal Homogenate): 7.608 % (OE: 6.505% + OD: 1.102%)

MALACHITE GREEN CHLORIDE
- Mean of absorbed dose: 0.017%
- Unabsorbed dose (L-residual): 91.507 %
- Absorbable dose (Epidermal Homogenate + Stratum corneum and Dermal Homogenate): 8.476 % (OE: 6.862% + OD: 1.613 %)
Percutaneous absorptionopen allclose all
Dose:
81000.00
Parameter:
percentage
Absorption:
ca. 0.001 %
Remarks on result:
other: 2h
Remarks:
Absorbed dose MGO
Dose:
81000.00
Parameter:
percentage
Absorption:
ca. 0.001 %
Remarks on result:
other: 4h
Remarks:
Absorbed dose MGO
Dose:
81000.00
Parameter:
percentage
Absorption:
ca. 0.013 %
Remarks on result:
other: 24 h
Remarks:
Absorbed dose MGO
Dose:
100000.00
Parameter:
percentage
Absorption:
ca. 0 %
Remarks on result:
other: 2h
Remarks:
Absorbed dose MGC
Dose:
100000.00
Parameter:
percentage
Absorption:
ca. 0.001 %
Remarks on result:
other: 4h
Remarks:
Absorbed dose MGC
Dose:
100000.00
Parameter:
percentage
Absorption:
ca. 0.017 %
Remarks on result:
other: 24h
Remarks:
Absorbed dose MGC

Any other information on results incl. tables

The results and the TOTAL MASS BALANCE of OXALATE and CHLORIDE salts are reported in Tables: the values reported in the tables include the dosage of Leucomalachite Green metabolite formed during the study.

  MG Oxalate Malachite (81%) Malachite Green 4 Oxalate mcg Total  Mass balance(%)
Test system mg/3.8 cm^2 mcg on 3.8 cm^2 2h 4h  24h  L OE  OD  mcg on 3.8 cm^2   
1379 -1 100.00 81000.00 1.07 2.00 10.25  32598.35  3959.69 811.32  37382.68  46.15 
1379 -2 100.00 81000.00 0.58 0.22  6.32  62179.63  4016.57  1197.32  67400.65  83.21 
1379 -3 100.00 81000.00 0.85  0.12  9.00  88847.20  7246.74  494.93  96598.84 119.26 
1379 -4 100.00 81000.00 0.64 0.67 11.16  81413.73  3441.48  659.22  85526.91  105.59 
                 
                  MEAN 88.55
                  DS 31.93 

MG Chloride Malachite (100%) Malachite Green Chloride mcg Total  Mass balance(%)
Test system mg/3.8 cm^2 mcg on 3.8 cm^2 2h 4h  24h  L OE  OD  mcg on 3.8 cm^2   
1380 -1 100.00 100000.00 0.03 0.74 6.27 7499.38  3821.8 733.31 12061.53 12.06
1380 -2 100.00 100000.00 0.18 0.19  7.14 16803.29 3147.5 885.84  20844.13  20.84
1380 -3 100.00 100000.00 0.00  0.19 8.15 66468.66 1233.8 411.09 68121.88 68.12 
1380 -4 100.00 100000.00 0.00 0.02 8.80 76813.13 4364.2 924.62  82110.79  82.11 
                 
                  MEAN 45.78
                  DS 34.53 

L: residual product after 24h solution

OE: Epidermal Homogenate + Stratum corneum

OD: Dermal Homogenate

Amounts of Leucomalachite dosed in the different compartments: the derivative has not been founded in the receptor fluid.

Leucomalachite (mcg)
Samples OE  OD 
MGO 1379 -1 2770.57 750.51 22.85 
MGO 1379 -2 7324.45 436.35  29.77 
MGO 1379 -3 6338.93 738.84  8.14 
MGO 1379 -4 9340.67 431.46 15.23 
       
MGC1380 -1 460.60 187.18 25.74 
MGC 1380 -2 1586.91 464.37  37.16 
MGC 1380 -3 7418.31 134.05  6.58 
MGC 1380 -4 5315.18 186.48  37.33 

Unabsorbed dose: represents that washed from the skin surface after exposure and any present on that including any dose shown to volatilise from the skin during exposure: 92,377 % for Oxalate satl and 91,507% for Chloride salt.

Absorbed dose: is the mass of test substance reaching the receptor fluid /systemic circulation within a specified period of time: 0,015% for Oxalate salt and 0,017% for Chloride salt

The absorbable dose: represents the mass of test substance present on or in the epidermis after washing: 7,608 % for Oxalate salt and 8,476 % for Chloride salt.

The TMBs calculated for Oxalate salt (88,55±31,93) and for Chloride salt (45,78 ±34,53 ) cannot meet the OECD 428 acceptance criteria (90 ± 10%).

However the study can be considered valid demonstrating that:

- absorption of both MG Oxalate and Chloride is negligible

- formation of Leucomalachite is also negligible and in any case it is not absorbed

- absorption rate of both salts is similar, confirming again their similar behaviour

- in the final percentage of both salts in the OD layer is above 1% and therefore the skin sensitization process may occur

Applicant's summary and conclusion

Conclusions:
The study can be considered valid demonstrating that:
- absorption of both MG Oxalate and Chloride is negligible
- formation of Leucomalachite is also negligible and in any case it is not absorbed
- absorption rate of both salts is similar, confirming again their similar behaviour
- in the final percentage of both salts in the OD layer is above 1% and therefore the skin sensitization process may occur
Executive summary:

The rate of absorption and passage of test chemicals into and through human skin can be modelled in the in vitro percutaneous absorption study. The test substance is applied to the surface of the skin model separating the two chambers of a diffusion cell: the upper donor compartment where the product is applied and the lower acceptor compartment containing the receptor fluid. Skin exposure to the test preparation is for the entire duration of the experiment (24h). Sampling frequency of the receptor fluid allows to determine the kinetics absorption of the compound. The amount of test substance in the receptor fluid, skin preparation, skin surface washings are analyzed to quantify the absolute quantity and % of active principle absorbed through the test system. The total test substance localisation allows to calculate the percentage of recovery and to quantify the total mass balance in order to validate the assay.

Unabsorbed dose: represents that washed from the skin surface after exposure and any present on that including any dose shown to volatilise from the skin during exposure: 92,377 % for Oxalate salt and 91,507% for Chloride salt.

Absorbed dose: is the mass of test substance reaching the receptor fluid /systemic circulation within a specified period of time: 0,015% for Oxalate salt and 0,017% for Chloride salt.

The absorbable dose: represents the mass of test substance present on or in the epidermis after washing: 7,608 % for Oxalate salt and 08,476 % for CLhloride salt.

The TMBs calculated for Oxalate salt (88,55±31,93) and for Chloride salt (45,78 ±34,53 ) cannot meet the OECD 428 acceptance criteria (90 ± 10%).

However the study can be considered valid demonstrating that:

- absorption of both MG Oxalate and Chloride is negligible

- formation of Leucomalachite is also negligible and in any case it is not absorbed

- absorption rate of both salts is similar, confirming again their similar behaviour

- in the final percentage of both salts in the OD layer is above 1% and therefore the skin sensitization process may occur