Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 2012-09-10 Experimental Completion Date: 2012-09-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Guideline conform GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
acc. to §19 German Chemikaliengesetz

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-oxybis[5,5-dimethyl-1,3,2-dioxaphosphorinane] 2,2'-disulphide
EC Number:
223-829-1
EC Name:
2,2'-oxybis[5,5-dimethyl-1,3,2-dioxaphosphorinane] 2,2'-disulphide
Cas Number:
4090-51-1
Molecular formula:
C10H20O5P2S2
IUPAC Name:
2-[(5,5-dimethyl-2-sulfanylidene-1,3,2λ⁵-dioxaphosphinan-2-yl)oxy]-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinane-2-thione
Test material form:
not specified
Radiolabelling:
no

Test animals

Species:
other: Pig skin
Strain:
not specified
Sex:
not specified
Details on test animals or test system and environmental conditions:
Not Applicable, In Vitro study

Administration / exposure

Type of coverage:
other: non-occluded
Vehicle:
unchanged (no vehicle)
Duration of exposure:
After the exposure period of 24 hours the test preparations were removed by washing each skin sample nine times with 1 mL extraction solution. The washing solutions (WL) were collected and combined in a 50 mL flask which was filled up to the mark with extraction solution. The SN solution was combined with the WL solutions before analysis as well.
Doses:
Treatment
A limited amount of the test preparation corresponding to realistic in use conditions was applied to the surface of each skin sample. According to the guideline cited the application of the test preparation to the skin should mimic realistic conditions. Approx. 5 mg/cm2 of the test preparations 1, Test Item 1, and 3, Test Item 2 (corresponds to approx. 865 µg test substance per cm2, theoretical value), were applied to the skin. 10 µL of test preparation 2, Test Item 3, (corresponds to approx. 5220 µg test substance per cm2, theoretical value) were applied to the skin. The applicators were cleansed using 4 mL extraction solution (PWL).
No. of animals per group:
Not Applicable, In Vitro study
Control animals:
no
Details on in vitro test system (if applicable):
Aims of the Study
The experiments were performed to obtain information about the dermal delivery of the test preparations topically applied on porcine skin. One independent experiment with six replicates each was performed with each of the three test preparations, Test Item 1, Test Item 2 and Test Item 3 using skins from at least three different donors in each experiment. The analysed compound was Test Item 1..

Reasons for the Study
Experimental procedures have been designed to assess the dermal delivery of chemicals through ex vivo human skin (1). Drugs and other chemicals are routinely applied on the surface of skin to assess product efficacy and dermal toxicity. The dermal delivery of chemicals is a major safety concern and in previous studies porcine skin was shown to be the closest animal model for in vitro dermal delivery studies . In this project the skin was taken from the outer ear region which gave a very good prediction for human skin.

Design of the Study
For the determination of the dermal delivery of the test substance skin pieces were mounted onto Franz chambers and after checking the skin integrity, a finite dose of the test preparations or ‘in-use’ preparations was applied onto the skin and was left on the skin for an exposure period under non-occluded conditions in a practice relevant manner. Afterwards the test preparations were removed by washing the skin and, depending on the exposure time, the dermal delivery was monitored for an additional time frame.
The study was performed under static conditions.



Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Percutaneous absorptionopen allclose all
Dose:
5 mg of test item 1
Parameter:
percentage
Absorption:
0.029 %
Remarks on result:
other: 24
Remarks:
test item 1 (100% submission substance): penetration into the viable skin layers and the receptor fluid out of the test preparation with 1.33±1.71 µg/cm2(0.0268±0.0382 % of applied dose)
Dose:
11.6 mg of test item 2
Parameter:
percentage
Absorption:
0.017 %
Remarks on result:
other: 24 h
Remarks:
test item 2 (45% submission substance): penetration into the viable skin layers and the receptor fluid out of the test preparation with 0.683±0.579 µg/cm2(0.0168±0.0144 % of applied dose)
Dose:
5 mg of test item 3
Parameter:
percentage
Absorption:
0.049 %
Remarks on result:
other: 24 h
Remarks:
test item 3 (17% submission substance): penetration into the viable skin layers and the receptor fluid out of the test preparation with 0.0195±0.0102 µg/cm2(0.0488±0.0266 % of applied dose).

Any other information on results incl. tables

Results

Summary of the Method Validation

 Summary of the Method Validation

Linear Range:                        

 From 0.0996 up to 125 ng/mL for Test Item 1, validated by analysing calibration series in receptor solution and in extraction solution.

Accuracy for Test Item 1:  

The accuracy expressed as recovery was between98.8 % and 109.3 % in receptor solution and between 96.8 % and 103.4 % in extraction solution.

Precision for Test Item 1:  

The intra-day precision in receptor solution expressed as CV was between 4.51 % and 7.98 % in receptor solution, and in extraction solution between 2.76 % and 6.46 % over the whole calibration range.

Lower Limit of Quantitation:    0.1 ng/mL for Test Item 1 in both matrices

Limit of Detection:                     0.08 ng/mL for Test Item 1 in receptor solution and 0.05 ng/mL in extraction solution.

Specificity / Selectivity:               Chromatograms fromblanks (without and with internal standard) and fortified sampleswere checked to assess the specificity and selectivity of the analytical method. No changes in retention times were observed and the chromatographic system showed no significant injection carry-over (less than 0.5 %).

Stability in Matrix:                     The stability for Test Item 1 is granted up to 24 hours in both receptor solution and extraction solution and the reference item can be stored by freezing (determination after 24 hours room temperature, 24 hours in the autosampler and after one freeze/thaw cycle).

 

 Integrity of the Skin

The integrity of the skin was demonstrated prior to application and after the last sampling. The conductivity prior to the experiment was in the acceptable range of < 900 µS/cm for all skin samples used.

Analytical Results

The concentrations given in the table were rounded values.

The volumes (mL) of the receptor solution samples were obtained by measuring the sample vials after sampling with the assumption that the weight corresponds directly to the volume. For the remaining samples the corresponding solvent volume used was taken as the sample volume.

The total amount of reference item present in each sample is calculated by multiplying the corrected concentration of the sample with the corresponding volume.

Samples in receptor solution and in extraction solution, respectively, were measured against the corresponding calibration curves.

    Results Summary for Test Item 1, test preparation 1

Test Item 1,

Test preparation 1

Chamber

1

2

3

4

5

6

Amount of Test Item 1
applied (µg)1

5233

4533

4712

4888

4402

4590

Total amount of Test Item 1
measured (µg)

5351

4505

5301

4616

4921

4040

Recovery (%)

102.2

99.4

112.5

94.4

111.8

88.0

Dermal Absorption of Test Item 1
(µg/cm2)2

0.879

0.132

0.0597

2.66

4.16

0.0774

Dermal absorption (%)3

0.0168

0.00292

0.00127

0.0543

0.0946

0.00169

1: amount of reference item present in 5 mg test preparation

2: the value is the sum of the amount of reference item measured in the receptor solution and in skin extracts of the epidermis and dermis of each diffusion cell.

3: percent dermal absorption = (total amount of reference item penetrated/absorbed (µg/ cm2) *100)/ amount of applied reference item (µg)1

 

 


Results Summary for Test Item 2, test preparation 2

 

Test Item 2,

Test preparation 2

Chamber

1

2

3

4

5

6

Amount of Test Item 1
applied (µg)1

4159

4021

3994

4143

4074

4082

Total amount of Test Item 1
measured (µg)

3565

3788

3586

3977

3535

3493

Recovery (%)

85.7

94.2

89.8

96.0

86.8

85.6

Dermal Absorption of Test Item 1
(µg/cm2)2

0.109

1.52

0.780

0.450

0.0831

1.15

Dermal absorption (%)3

0.00262

0.0378

0.0195

0.0109

0.00204

0.0282

1: amount of reference item present in 10 µL test preparation

2: the value is the sum of the amount of reference item measured in the receptor solution and in skin extracts of the epidermis and dermis of each diffusion cell.

3: percent dermal absorption = (total amount of reference item penetrated/absorbed (µg/ cm2) *100)/ amount of applied reference item (µg)1

 

 Results Summary for Test Item 3, test preparation 3

 

Test Item 3,

Test preparation 3

Chamber

1

2

3

4

5

6

Amount of Test Item 1
applied (µg)1

41.7

39.1

40.2

39.1

39.7

40.1

Total amount of Test Item 1
measured (µg)

46.5

54.0

43.0

39.9

41.2

39.9

Recovery (%)

111.5

138.2

106.9

101.9

103.8

99.3

Dermal Absorption of Test Item 1
(µg/cm2)2

0.0164

0.0161

0.0104

0.0366

0.0136

0.0205

Dermal absorption (%)3

0.039

0.0411

0.0258

0.0935

0.0343

0.0511

1: amount of reference item present in 5 mg test preparation

2: the value is the sum of the amount of reference item measured in the receptor solution and in skin extracts of the epidermis and dermis of each diffusion cell.

3: percent dermal absorption = (total amount of reference item penetrated/absorbed (ng/ cm2) *100)/ amount of applied reference item (ng)1

grey shading indicates an outlier not used for calculation (the chamber was excluded because of the high recovery value)


1All concentrations given are rounded values, but calculation was done with non-rounded values. In case of re-calculation minor discrepancies may occur.

 

 

 





Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that under the reported conditions, the dermal delivery of Test Item 1 out of test preparations Test Item 1, Test Item 2 and Test Item 3 were as follows:
Test Item 1: 1.33 ± 1.71 µg/cm² (0.0286 ± 0.0382 % of applied dose)
Test Item 2: 0.683 ± 0.579 µg/cm² (0.0168 ± 0.0144 % of applied dose)
Test Item 3: 0.0195 ± 0.0102 µg/cm² (0.0488 ± 0.0266 % of applied dose)
Executive summary:

Dermal delivery of a substance defines the amount of the test item absorbed by the skin (absorption) and the amount, which has penetrated the entire skin. This amount does not include the test substance quantity left in thestratum corneum(adsorption).

Test Item 1, Test Item 2 and Test Item 3 (100%, 45% and 17.9% of submission substance, respectively) were assessed for their potential for dermal delivery on pig skin.

One experiment with each of the three test preparations were performed on frozen dermatomized pig skin samples after thawing under static conditions with 6 diffusion cells each. The thickness of the skin used was between 430 - 450 µm. For the evaluation of each experiment data from 3 different donors were used.

For the first and the third test item, Test Item 1 and Test Item 3, about 5 mg were applied on each chamber. For the second test item Test Item 2, 10 µL were applied on each of the six chambers. After 24 hours the test preparation was removed by washing each skin sample nine times with 1 mL acetonitrile (ACN).

PBS/EtOH (80:20 v/v) was used as receptor solution. The solubility of Test Item 1 in receptor solution is given up to at least 55 ng/mL as determined in Harlan CCR Study 1463001 (non-GLP). The dermal delivery was monitored over 24 hours under non-occluded static conditions.

The conductivity across the skin samples of each diffusion cell was determined before treatment and after the sampling as a measure of skin integrity.

The stratum corneumwas separated by tape stripping from the remaining epidermis and dermis. The tape strips (2 strips per sample) were pooled and extracted for analysis, respectively. The remaining skin compartments were separated by heat separation using forceps and also extracted for their content of the reference item.

Controls with Benzoic acid (positive) and 2-Ethylhexyl trans-4-methoxycinnamate (negative) on human skin are used to check the performance of the skin penetration system at least once a year.

The samples from the skin dermal delivery assay were analysed by LC-MS/MS for the presence of Test Item 1. 6 chambers each met the acceptance criteria for Test Item 1 and Test Item 2, and were used for the analysis of Test Item 1. 5 chambers were used for the evaluation of the penetration properties Test Item 1 of Test Item 3.  

Test Item 1 was detected in any sample relevant for dermal delivery as there were the receptor fluid, the extracts of epidermis and dermis in all experiments with each of the test preparations.

The measured samples which were below the limit of quantification (<LLOQ) or detection (<LOD) were replaced as a worst case consideration for the calculations by the corresponding value. Test Item 1 is considered to have a low penetration potential.


 


 

Test Item 1

Amount of Test Item 1

Expressed as µg/cm2of skin surface mean±S.D. (n = 6)#

Expressed as % of dose
mean
±S.D. (n = 6)#

Applied Dose

4726

±

298

100

 

 

Unabsorbed Dose

4787

±

503

101.3

±

10.6

Adsorbed Dose
Stratum corneum

(isolated by stripping)

0.807

±

1.26

0.0171

±

0.0267

Absorbable Dose
Epidermis
(isolated after 24 hours)

0.113

±

0.181

0.00227

±

0.00344

Absorbable Dose
Dermis
(isolated after 24 hours)

0.931

±

1.56

0.0205

±

0.0355

Absorbed dose

0.283

±

0.689

0.0058

±

0.0141

Recovery

4789

±

503

101.4

±

9.6

Dermal Delivery
(receptor fluid + epidermis + dermis)

1.33

±

1.71

0.0286

±

0.0382

 

Test Item 2

Amount of Test Item 1

Expressed as µg/cm2of skin surface mean±S.D. (n = 6)#

Expressed as % of dose
mean
±S.D. (n = 6)#

Applied Dose

4079

±

65.0

100

 

 

Unabsorbed Dose

3656

±

187

89.6

±

4.57

Adsorbed Dose
Stratum corneum

(isolated by stripping)

0.437

±

0.466

0.0107

±

0.0114

Absorbable Dose
Epidermis
(isolated after 24 hours)

0.0917

±

0.108

0.00228

±

0.00271

Absorbable Dose
Dermis
(isolated after 24 hours)

0.568

±

0.548

0.0140

±

0.0136

Absorbed dose

0.023

±

0.031

0.0006

±

0.0008

Recovery

3657

±

187

89.7

±

4.5

Dermal Delivery
(receptor fluid + epidermis + dermis)

0.683

±

0.579

0.0168

±

0.0144

# only valid values with a recovery of >85% and < 115 % were used.
samples <LOD were replaced by value of LOD (0.08 ng/mL in receptor solution and 0.05 ng/mL in extraction solution)
samples <LLOQ were replaced by value of LLOQ (0.1 ng/mL in receptor and extraction solution)

 

Test Item 3

Amount of Test Item 1

Expressed as µg/cm2of skin surface mean±S.D. (n = 5)#

Expressed as % of dose
mean
±S.D. (n = 5)#

Applied Dose

40.2

±

0.969

100

 

 

Unabsorbed Dose

42.1

±

2.80

104.7

±

7.0

Adsorbed Dose
Stratum corneum

(isolated by stripping)

0.00135

±

0.00174

0.00337

±

0.00433

Absorbable Dose
Epidermis
(isolated after 24 hours)

0.00243

±

0.00373

0.00590

±

0.00890

Absorbable Dose
Dermis
(isolated after 24 hours)

0.00387

±

0.00548

0.00967

±

0.0136

Absorbed dose

0.0132

±

0.012

0.0333

±

0.0300

Recovery

42.1

±

2.79

104.7

±

4.72

Dermal Delivery
(receptor fluid + epidermis + dermis)

0.0195

±

0.0102

0.0488

±

0.0266

# only valid values with a recovery of >85% and <115% were used.
samples <LOD were replaced by value of LOD (0.08 ng/mL in receptor solution and 0.05 ng/mL in extraction solution)
samples <LLOQ were replaced by value of LLOQ (0.1 ng/mL in receptor and extraction solution)

 

The amount extractable from test preparation 3, Test Item 3, was lower than the theoretical content. Only 40.2 µg/cm²± 0.969 was found as applied amount, instead of approx. 865 µg/cm². This was most probably due to the fact that during the production of the wadding Test Item 1 was fixed on the wadding material. Because of this a correction factor of 0.0455 was calculated for the evaluation of the mass balance.

 

In conclusion, it can be stated that during the described permeability test and under the experimental conditions reported, Test Item 1 (100 % submission substance) showed penetration into the viable skin layers and the receptor fluid out of the test preparation with 1.33±1.71 µg/cm2(0.0286±0.0382 % of applied dose).

Test Item 2 (45 % submission substance) showed penetration into the viable skin layers and the receptor fluid out of the test preparation with 0.683±0.579 µg/cm2(0.0168±0.0144 % of applied dose). Test Item 3 (17 % submission substance) showed penetration into the viable skin layers and the receptor fluid out of the test preparation with 0.0195±0.0102 µg/cm2(0.0488±0.0266 % of applied dose).