Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-07 - 2014-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Reference substance: MPA
Chemical description (IUPAC): N-[(3-Methyl-1H-pyrazol-1-yl) methyl] acetamide, N-[(5-Methyl-1H-pyrazol-1-yl) methyl] acetamide
EC number: 700-208-8
Molecular formula: C7H11N3O
Molecular weight: 153.2 g•mol-1
Radiolabelling:
no

Test animals

Species:
other: not applicable. in vitro

Administration / exposure

Duration of exposure:
24 hours
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human skin from surgical operations
- Ethical approval if human skin: yes
- Type of skin: abdomen
- Preparative technique: Dermatomization, tape stripping and cryo-sectioning
- Thickness of skin (in mm): mean thickness of around 500 ± 100 µm
- Membrane integrity check: yes, with caffeine
- Storage conditions: cooled to 4 °C
- Justification of species, anatomical site and preparative technique:

PRINCIPLES OF ASSAY
- Diffusion cell: Franz cell
- Receptor fluid: PBS pH 7.4
- Solubility od test substance in receptor fluid: yes as determined
- Static system: yes
- Flow-through system: no
- Test temperature: 32 °C ± 1 °C

Results and discussion

Total recovery:
The recovery of MPA after extraction development were 105.66 %. if the applied amount is set to 100 %. These value is within the set specifications (recovery of 90 % to 110 %). The development of an extraction method for skin was successful.
Percutaneous absorption
Dose:
0.2%
Parameter:
percentage
Absorption:
0.21 %
Remarks on result:
other: 24 hours

Any other information on results incl. tables

Solubility

To analyze the solubility of MPA and to ensure sink conditions during the resorption experiments, one concentration (ca. 1072 µg·mL-1for MPA) in the preselected acceptor medium was prepared and diluted to the linear range for measurement.The solutions were stored for 4 hours at room temperature on a stirrer. Samples were taken in triplicate at time points 0 h, 1 h and 4 h.The following table shows the results of the solubility measurements of MPA in the selected acceptor medium PBS pH 7.4.

Solubility of MPA in PBS pH 7.4

Storage time [h]

0

1

4

Storage temperature

RT

RT

RT

n1 [µg∙mL-1]

1076.60

1062.18

1142.36

n2 [µg∙mL-1]

1080.44

1081.32

1154.58

n3 [µg∙mL-1]

1079.90

1068.38

1177.16

Mean [µg∙mL-1]

1079.0

1070.6

1158.0

SD [µg∙mL-1]

1.7

8.0

14.4

RSD [%]

0.2

0.7

1.2

Theoretical c for t=0 [µg∙mL-1]

1072.44

Appearance

Clear colorless solution with few particles

Clear colorless solution with few particles

Clear colorless solution with few particles

Dissolved [µg∙mL-1]

1079.0 ± 1.7

1070.6 ± 8.0

1158.0 ± 14.4

The measured solubilities ensure sink conditions in the resorption experiments with PBS pH 7.4 as acceptor medium.

Skin quality control experiment (MEA)

In an earlier study the permeability of caffeine from an aqueous solution across full-thickness skin, dermatomized skin, heat-separated epidermis and isolated SC had been studied at Across Barriers to establish quality assurance benchmarks for skin integrity [Bock et al., 2002]. Subsequent quality control studies have been performed on a range of human skin samples.

The following table provides an overview of caffeine permeability through dermatomized skin specimens measured at Across Barriers and includes the Pappvalues determined in the present study.

Comparison of apparent permeability coefficients for caffeine through different dermatomized skin specimens with intact SC. Skin No.617-01-0714 was used in the present study.

Skin number
Mean Papp[cm∙s-1] (n=3)
RSD [%]

061-01-0701

9.38E-08

29

153-01-0104

8.41E-08

28

059-01-0601

8.24E-08

39

060-01-0601

5.63E-08

6

062-01-0701

5.34E-08

18

155-01-0204

5.03E-08

27

209-01-0605

3.82E-08

7

157-01-0304

3.23E-08

27

057-01-0601

3.21E-08

16

150-01-1203

2.54E-08

56

058-01-0601

2.00E-08

7

308-01-1107

1.28E-08

25

239-01-0707

1.04E-08

3

617-01-0714*

6.14E-08

15

*Evaluated as n=2 because one Franz cell was declared as outlier due to a significant higher transport most likely derived from a damaged skin biopsy.

 

The skin tightness is comparable with skins used in the past. The permeation coefficient for caffeine applied as infinite dose is in the same magnitude compared with previous data.

Mass balance of MPA after transport through human skin over a time period of 24 h.

 

MPA
Applied amount [µg·cm-2]

559.61

Amount remaining on skin surface [µg·cm-2]

585.51

Amount in stratum corneum [µg·cm-2]

0.34

Amount in epidermis/dermis [µg·cm-2]

0.00

Amount resorbed [µg·cm-2]

5.53

Sum of absorbed & not absorbed [µg·cm-2]

591.27

Recovery from applied amount [%]

105.66

All calculations and evaluations were performed with the measured amounts of test substance in the test items. 559.61 µg·cm-2MPA was applied (corresponds to 0.1 % (w/v)). The recovery of MPA after extraction development were 105.66 % for MPA if the applied amount is set to 100 %. These value is within the set specifications (recovery of 90 % to 110 %). The development of an extraction method for skin was successful.

Transport of MPA through human skin

The permeation coefficient was calculated from the linear range of the transport curve (time points 4 to 8; 8 h to 24 h). The permeation coefficient was 1.96E-8 cm∙s-1± 5.86E-9 cm∙s-1. After 18 hours MPA could be detected for the first time. For the calculation of the permeation coefficient at least five points are necessary; the fourth time point after 8 h was additionally included. The transported amount after 24 hours was 2.61 µg∙cm-2± 1.03 µg∙cm.

Mass balance reported as µg·cm-2and recovery reported as % for MPA after transport through human skin over a time period of 24 h.

Parameter
MPA
Amount remaining on skin surface [µg·cm-2]
(Not absorbed: residual test items in donor chamber + first two tape strips)

1219.65 ± 30.06

Amount in stratum corneum [µg·cm-2]
(All tape strips except for the first two strips)

2.44 ± 0.65

Amount in epidermis/dermis [µg·cm-2]
(All slices of the skin layers)

0.00

Absorbed into skin [µg·cm-2]
(Sum of stratum corneum + epidermis/dermis)

2.44 ± 0.65

Amount resorbed [µg·cm-2]
(Found in acceptor medium)

2.61 ± 1.03

Sum [µg·cm-2]
(Absorbed + resorbed + not absorbed)

1224.70 ± 31.74

Applied amount [µg·cm-2]

1156.95

Recovery of sum from applied amount [%]
(Absorbed + resorbed + not absorbed)

106

 Penetration of MPA

see attached figure

The maximum absorbed quantity into the skin was 2.44 µg.cm-2for MPA after 24 hours contact with the skin. This corresponds to 0.21 % of the applied dose MPA. The amount of MPA resorbed or transported through the skin was 2.61 µg.cm-2which corresponds to 0.23 % of the applied dose. Therefore, the permeation coefficient for MPA was determined as 1.96E-8 cm∙s-1± 5.86E-9 cm∙s-1. The lag time (first time point where MPA could be detected in the acceptor compartment of the Franz diffusion cell) was eight to ten hours. After the resorption experiment MPA could only be detected in the stratum corneum. It did not penetrate the deeper skin layers in measurable amounts. The sum of absorbed MPA has been calculated by adding the amount MPA found in the stratum corneum to the amount in the epidermis/dermis.

 

Applicant's summary and conclusion

Conclusions:
The study was performed as a risk assessment for the test substance MPA. It was the objective to find out, which amounts of the substance are capable of permeating through and penetrating into the human skin. In practice, the maximum possible exposure concentrations are 0.1 % (w/v) for MPA , that can contaminate human skin. These concentrations are based on the finished mixtures of the substances that will be marketed.
During the study the expected concentration in a product were doubled and the duration of incubation set to 24 h to get a measurable transport of the substances through the skin. Even with the doubled concentration (0.2 % for MPA) and a duration of 24 h a very low transport was determined for MPA (1.96E-8 cm∙s-1 ± 5.86E-9 cm∙s-1). The maximum absorbed quantity into the skin was 2.44 µg.cm-2 after 24 hours contact with the skin. This corresponds to 0.21 % of the applied dose MPA.
Based on these results, it can be stated that there will be a negligible transport of MPA into or through human skin, especially as the real life exposure levels are half of the used ones. Furthermore it can be assumed that the contact time will be much shorter, because the skin will be cleaned faster.

Executive summary:

In the present study, a comparative in vitro examination of the dermal absorption of N-[(3/5-Methyl-1H-pyrazol-1-yl) methyl] acetamide (MPA) was performed according to OECD-Guideline 428. The test item was applied to human skin as follows, as no interferences with analytics were determined:

 

·     Test item A:           1 mL of 0.2 % MPA (w/v) in PBS pH 7.4

The amount of MPA which can be absorbed into the human organism through the skin was determined using human excised skin as an in vitro model for dermal absorption. The resorbed amounts of the test substance were quantified over a time period of 24 hours by analyzing samples from the acceptor compartment of Franz diffusion cells.

After the resorption experiment the amounts of MPA taken up into the skin were determined. For this penetration study the stratum corneum was stripped by the so-called “tape stripping technology”. The strips were collected in two samples. The deeper skin layers were sliced in parallel sections using a cryo microtome. The skin slices were collected in one sample.

 

The used skin was qualified by performing a resorption study with caffeine over a period of 24 hours. Resorbed caffeine was quantified by a method developed and validated at Across Barriers. The resorption data were compared with historical data gained by the testing facility.

 

The analytical method for MPA was obtained from sponsor and adapted. For the quantitative analysis of the test substances the following validation parameters were measured: the system suitability, the linearity and the LLOQ. This was carried out according to EMA Guideline for Bioanalytical method validation (EMEA/CHMP/192217/2009).

The amount of MPA resorbed or transported through the skin was 2.61 µg.cm-2which corresponds to 0.23 % of the applied dose. After the resorption experiment MPA could only be detected in the stratum corneum. It did not penetrate in measurable amounts into the deeper skin layers.

During the study the expected concentrations of 0.1% in a product were doubled and the duration of incubation set to 24 h to get a measurable transport of the substances through the skin. Even with the doubled concentrations (0.2 % for MPA) and a duration of 24 h a very low transport was determined for MPA (1.96E-8 cm∙s-1± 5.86E-9 cm∙s-1). The maximum absorbed quantity into the skin was 2.44 µg.cm-2for MPA after 24 hours contact with the skin. This corresponds to 0.21 % of the applied dose MPA.

Based on these results, it can be stated that there will be a negligible transport of MPA into or through human skin, especially as the real life exposure levels are half of the used ones. Furthermore it can be assumed that the contact time will be much shorter, because the skin will be cleaned faster.