Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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 according to a valid guideline and the study was conducted under GLP conditions.

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:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identity: 2,2‘-dimethyl-2,2’-azodipropiononitrile
Chemical name: 2,2‘-dimethyl-2,2’-azodipropiononitrile

AZDN Batch No.: 6867
Aggregate state at room temperature: solid (powder)
Color: white
Purity: 99.9 %
Stability in solvent: not determined by the sponsor.
Log Kow: 1.1 (at 25°C)
Storage: 2-8 °C in the fridge, light protected
Molecular weight: 164.2 g/mole
Expiry date: December, 2010
Radiolabelling:
no

Test animals

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

Administration / exposure

Type of coverage:
other: non occluded
Vehicle:
unchanged (no vehicle)
Duration of exposure:

After 24 hours of incubation the test item was removed by washing each skin sample nine
times with 1 mL ACN. Than 1 mL receptor solution was added for the impedance measurement of the skin after 24 hours. All washing solutions (WL) were collected and combined in a 20 mL flask, together with the SN solution (1 mL) and the flask was filled up to the mark with ACN afterwards.

Doses:
A limited amount of the test item corresponding to realistic in use conditions was applied to the surface of the skin. According to the guideline cited the application of the test item to the skin should mimic realistic conditions. The test item was weighed and approx.
5 mg/cm2 were applied to the skin.

5 mg of the prepared test item were applied on the skin in the respective donor chamber and were also used as the reference value for calculating the mass balance and the percentage of penetration.
No. of animals per group:
Not Applicable, In Vitro study
Control animals:
no
Details on study design:
Design of the Study

The test item was analysed in two independent experiments with 6 replicates each under static conditions. The volume of each sample was determined by measuring the weight difference of the vessels used for sampling. After checking the skin integrity, the test item was left on the skin for 24 hours under non-occluded conditions in a practice relevant manner. Then the test item was washed off with ACN. After the penetration experiment, some of the skin samples were stripped 10 (2 x 5 times) with a 1 cm2 piece of Tesa¿ film (see 8.11, page 18). The stripes of Tesa¿ film were extracted. The remaining skin samples were separated by heat separation in epidermis and dermis and each skin compartment was extracted separately.


The amount of AZDN was determined in the receptor solutions of the different time points, in the different pipette washing solutions, in the combined washing solutions used to remove the test item formulation together with the solutions removed from the donor chambers after 24 hours (WL+SN)), and in the skin membrane extracts as weil as in the reference solutions (REF, one per chamber).

Controls with benzoic acid and 2-Ethylhexyl trans-4-methoxycinnamate were used to check the performance of the skin penetration system at least once a year. For the data of the latest control run see annex 5.


Sample preparation with internal standard

100 µL internal standard (IST3) were added to 1000 µL of each sample solution
(calibration, QC samples and study samples).

1- Controls

Benzoic acid and 2-Ethylhexyl trans-4-methoxycinnamate were used as positive and negative control substances known to permeate or not permeate the skin to demonstrate the performance of the system. These checks were regularly performed at least once a year. The latest data are included in this report in Annex 5.

2- Test System

Human skin was provided dermatomized by Biopredic, Rennes (France).
According to the guidelines cited whole skin free of any adipose tissue was used. The surface of the skin in contact to the test item was 1.0 cm². For two chambers the skin of one donor was used in order to assure that the study has included four donors.



3- Integrity of the Skin

The integrity of the skin was determined by measuring the impedance (resistance to alternating current) before the treatment and at the end of the experiment after 24 hours. All of the diffusion chambers are equipped with platinum electrodes connecting to a conductometer (LF 330, WTW 82362 Weilheim). Skin damages are indicated by a relevant increase in conductivity, which also is reflected in a change of the penetration kinetics of the test item. The intact skin generally has a conductivity of < 900 µS/cm. The exact values depend on the receptor solution used.


4- Dose Selection

A limited amount of the test item corresponding to realistic in use conditions was applied to the surface of the skin. According to the guideline cited the application of the test item to the skin should mimic realistic conditions. The test item was weighed and approx.
5 mg/cm2 were applied to the skin.


5- Experimental Performance

For the determination of dermal absorption/ percutaneous penetration of the test item, the skin samples were mounted in glass flow-through diffusion chambers with a diameter of
1.135 cm. These chambers are subdivided in an upper part (donor chamber) and in a lower part (receptor chamber). The receptor solution (PBS) was placed in the receptor chamber of each diffusion cell. At the beginning of the experiment each donor chamber was filled with 1 mL receptor solution to determine the impedance of the skin. Before the start of the treatment the blank samples (chamber blank - K Bl) from the receptor chamber were also collected.

After measuring the impedance, the receptor solution was removed from the donor chamber and depleted. Before the application the skin was dried using cellulose.

Procedure for test item dilution:

5 mg of the prepared test item were applied on the skin in the respective donor chamber and were also used as the reference value for calculating the mass balance and the percentage of penetration.

Each weighting boat was washed in 2 ml extraction solution (PWL). The receptor chamber temperature is kept at 32°C ¿ 1°C.
After 24 hours of incubation the test item was removed by washing each skin sample nine
times with 1 mL ACN. Than 1 mL receptor solution was added for the impedance measurement of the skin after 24 hours. All washing solutions (WL) were collected and combined in a 20 mL flask, together with the SN solution (1 mL) and the flask was filled up to the mark with ACN afterwards.



6-Stripping

After the incubation period the exterior regions of the isolated skin pieces were removed using a scissor and extracted with 2 mL extraction solution (EXR). Then the stratum corneum of the isolated skin pieces (chamber 1 and 2 of experiment 1 and chamber 1 of experiment 2) was stripped using Tesa¿ film transparent, No. 57370-00002, five times. The skin piece of chamber 2 (experiment 2) was only stripped 3 times before the epidermis was


removed from the dermis. The epidermis of the skin pieces of the other chambers were easily removed from the dermis using forceps, so stripping could not be applied without removing the whole epidermis instead of the stratum corneum alone. The Tesa¿ film pieces were collected in a tube filled with 2 mL extraction solution used for the LC-MS/MS analysis and extracted for analysis as described below. After the first stripping procedure the isolated skin pieces (chamber 1 of experiment 1 and 2) were stripped another 5 times. The skin piece of chamber 2 (experiment 1) was only stripped 2 times before the epidermis was removed from the dermis. The Tesa¿ film pieces were collected in another tube filled with 2 mL extraction solution used for the LC-MS/MS analysis and extracted for analysis as
described below.

7-Separation of the skin membranes


The isolated skin piece was wrapped in aluminium foil, covered with an approx. 140 g weight and placed on a heating plate for 20–30 seconds at approx. 50 °C. Then the
”upper skin” (= remaining stratum corneum + upper stratum germinativum) was gently pealed off the ”lower skin” (= lower stratum germinativum + upper dermis) using forceps. The separation is not necessarily complete, but the procedure gives valuable information on the distribution of the test items in the skin. The two skin compartments were separately extracted for analysis as described below. Heat separation was only applied to the skin of chamber 1 (experiment 1) and chamber 1 (experiment 2). For the other chambers the epidermis was easily removed from the dermis using forceps.

8.Extraction of the skin

At the end of the separation procedure reference item that had penetrated into the skin was quantified by extraction with 2 mL extraction solution. The extraction was performed sealed at room temperature overnight or for at least 8 hours. The skin extracts were stored at -20°C, if not analysed immediately. AZDN was analysed as the relevant compound.

As a control for all experiments, one skin sample was extracted without prior treatment with the test items in order to determine a possible influence on the analytics of the skin itself. This skin sample was not used in parallel in a diffusion chamber or treated like the skin samples in the experiment, but stripped and the remaining skin extracted in the same way as the samples.

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Percutaneous absorption
Dose:
5 mg
Remarks on result:
other: 24h
Remarks:
AZDN showed penetration into the viable skin layers and the receptor fluid out of the test item dilution with 0.480+/- 0.220 µg/cm2 (0.010 +/-0.005 % of applied dose).

Any other information on results incl. tables

Summary of the Method Validation

 The following limits and aspects were tested :

 Linear Range:    From 0.505 up to 1004ng/mL for AZDN (reference material),validatedbyanalysing calibration series in receptor solution and in extraction solution.

 Accuracy:  The accuracy expressed as recovery was between

89.6% and 106% in receptor solutionand between

91.3%and101%inextractionsolution.

 Precision:  The intra-day precision in receptor solution expressed as  CV  was between 1.85%  and 8.80%  and in extraction solution between 1.35% and 12.1% over the whole calibration range.

Lower Limit of Quantitation:    0.5ng/mL

Limit of Detection:    0.25ng/mL

Specificity/Selectivity:  The chromatographic system showed no injection carry- over and no change in retention time.

Stability in Matrix:  The stability for AZDN is granted up to 24 hours in both receptor solution and extraction solution and AZDN can bestored by freezing (determination after 24hours at room temperature, in the auto sampler (24hours at 8°C) and after one freeze/thaw cycle (at-20°C)).

Integrity of theSkin

 

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.

 

Summary of results for the test item of all chambers measured

 

 

Experiment 1

 

 

Chamber

1

2

3

4

5

6

Amount of AZDN

applied (µg/cm2)1

 

4912

 

4957

 

4786

 

4897

 

4925

 

5024

Total amount of AZDN

measured (µg)

 

5260

 

5248

 

5250

 

4844

 

5401

 

4992

Recovery(%)

105

103

105

96.7

109

97.5

Total absorption of AZDN (µg/cm2)2

 

0.542

 

0.373

 

3.695

 

0.958

 

0.498

 

0.566

Total absorption (%)3

0.011

0.008

0.077

0.020

0.010

0.011

 

Experiment 2

 

 

Chamber

1

2

3

4

5

6

Amount of AZDN

applied (µg/cm2)1

 

4948

 

4752

 

4680

 

4737

 

4901

 

3483

Total amount of AZDN

measured (µg)

 

4692

 

4393

 

4394

 

2991

 

4498

 

4795

Recovery(%)

91.5

87.0

86.7

60.7

88.7

97.1

Total absorption of AZDN (µg/cm2)2

 

0.423

 

0.249

 

0.353

 

1.689

 

0.192

 

0.649

Total absorption (%)3

0.009

0.005

0.008

0.036

0.004

0.019

1: amount of AZDN present in 5 mg test item

2:the value is the sum of the amount of AZDN measured in the receptor solution and in the skin extract

(epidermis and dermis) of each diffusion cell.

3:percent total absorption=(total amount of AZDN penetrated/absorbed (µg/cm2)*100)/ amount of applied

AZDN (µg)

Grey shading indicates chambers not used for further calculations.

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described permeability test and under the experimental conditions reported, AZDN showed penetration into the viable Human skin layers and the receptor fluid out of the test item dilution with 0.480 +/- 0.220 µg/cm2 (0.010 +/-
0.005 % of applied dose).


Executive summary:

The test item AZDN (CAS No. 78 -67 -1) was assessed for its potential to permeate human skin in a GLP guideline study (OECD 428). AZDN was the measured compound. For the test item 12 replicates were analysed. 5 mg of the test item were applied to each chamber for 24 hours and then removed by washing each Human skin sample nine times with 1 mL extraction solution. PBS was used as the receptor fluid. The conductivity across the skin samples of each chamber was measured before treatment and after the sampling. No abrupt change in conductivity, indicating a loss of barrier properties of the skin, occurred in any chamber up to the maximal duration of the experiment, except for chamber 3 in experiment 1, where removal of the upper part of the skin could be observed after the experiment. The samples were analysed by LC-MS/MS for the presence of AZDN. For the test item two experiments with 6 replicates each were performed under non- occluded conditions. Two chambers did not meet the acceptance criteria (chamber 3 in experiment 1; chamber 4 in experiment 2) and, therefore, 10 chambers were used for the analysis of AZDN.

In total 4 donors were used in this study. In conclusion, it can be stated that during the described permeability test and under the experimental conditions reported, AZDN showed penetration into the viable skin layers and the receptor fluid out of the test item dilution with 0.480 +/- 0.220 µg/cm2 (0.010 +/- 0.005 % of applied dose).

 

 

 

AZDN

Amount of AZDN

Expressed as µg/cm2of skin surface mean±S.D. (n = 10)#

Expressed as % of dose mean±S.D. (n = 10)#

Amount applied

4748

±

456

100

 

 

Washing + SN solution

4552

±

624

95.8

±

8.04

Stratumcorneum

(isolated by stripping) + EXR

 

0.013

 

±

 

0.015

 

0.000

 

±

 

0.000

Epidermis

(isolated after 24 hours)

 

0.004

 

±

 

0.005

 

0.000

 

±

 

0.000

Dermis

(isolated after 24 hours)

 

0.026

 

±

 

0.023

 

0.001

 

±

 

0.001

Receptor fluid

0.451

±

0.206

0.010

±

0.005

Recovery

4852

±

368

96.2

±

7.69

Bioavailable portion (receptor fluid + epidermis + dermis)

 

0.480

 

 

±

 

0.220

 

0.010

 

 

±

 

0.005

SN = Solution of the donor chamber

EXR = exterior region of the skin

  In conclusion, it can be stated that during the described permeability test and under the experimental conditions reported, AZDN showed penetration into the viable skin layers and the receptor fluid out of the test item dilution with 0.480 +/- 0.220 µg/cm2 (0.010 +/- 0.005 % of applied dose).