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: December 27, 2010 Experimental Completion Date: February 03, 2011
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.

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
Details on test material:
Internal Test Item Number: S 11462 11
The test item and the information concerning the test item were provided by the sponsor.
Identity: 1,2,4-Triazole
Name for report: TRIAZOLE 1.2.4
Batch No.: 10.77
Aggregate state at room temperature: solid
Color: white
Purity: 99.98 %
Stability in solvent: not indicated by the sponsor
Storage: At room temperature
Expiry date: June 2011
The test item is equal to ‘in-use’ preparation or test preparation and is also used as reference item (test substance) in the study.
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:
Vehicle:
unchanged (no vehicle)
Duration of exposure:
After 24 hours of incubation the test item was removed by washing each skin sample six times with 1 mL H2O:MeOH (70:30, v/v). Then 1 mLreceptor 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 H2O:MeOH (70:30, v/v) afterwards.
Doses:
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.
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 percutaneous absorption and/or penetration properties of the test item topically applied on human skin. Two independent experiments were performed with the test item TRIAZOLE 1.2.4 using skins from five differentdonors in total.

Relevance of the Test System
Experimental procedures have been designed to assess the percutaneous absorption of chemicals through human skin (1). Drugs and other chemicals are routinely applied on the surface of skin to assess product efficacy and dermal toxicity. The percutaneous absorption of chemicals is a major safety concern, but there is no species whose cutaneous diffusion barrier has been shown to be exactly identical to that of man.

Design of the Study
Flow through chambers as shown in Fig. 1 (please see “illustration picture graph section) were used

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 H2O:MeOH (70:30, v/v). After the penetration experiment, the skin samples were separated using forceps in epidermis and dermis and each skin compartment was extracted separately.

The amount of TRIAZOLE 1.2.4 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 well as in the reference solutions (REF, one per chamber). The weighted amount for the application to each chamber was used to calculate the mass balance.
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 please see attached see annex 5.

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Percutaneous absorption
Dose:
5 mg of the test item were applied to each chamber
Remarks on result:
other: 24
Remarks:
During the described permeability test and under the experimental conditions reported, TRIAZOLE 1.2.4 penetration into the viable skin layers and receptor fluid out of the test item dilution with 132 ± 71.0 µg/cm2 (2.73 ± 1.45 % of applied dose)

Any other information on results incl. tables

Results

Summary of the Method Validation

The following limits and aspects were tested (see attached Annex 3):

LinearRange:From 10.0 up to 4004 ng/mL for TRIAZOLE 1.2.4 (reference material), validated by analysing calibration series in PBS.

Accuracy:The accuracy expressed as recovery was between 100 % and 104 % in PBS

Precision:The intra-day precision in PBS expressed as CV was between 1.78 % and 6.58 % over the whole calibration range.

Lower Limit of Quantitation:10.0 ng/mL

Limit of Detection:8.00 ng/mL

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

Stability in Matrix:The stability for TRIAZOLE 1.2.4 is granted up to 24 hours in PBS and TRIAZOLE 1.2.4 can be stored by freezing (determination after 24 hours at room temperature, in the autosampler (24 hours at 8°C) and after one freeze/thaw cycle (at -20°C)).

 

 

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.

Details can be found in the attached Annex 1.

 

Analytical Results [1]

The concentrations given in the tables were rounded values.

If the concentration of the sample was below the lower limit of quantitation (LLOQ = 10.0 ng/mL), the value was replaced by this value. If the concentration of the sample was not detectable, the value was replaced by the limit of detection (LOD = 8.00 ng/mL). This gives the corrected concentration for the calculation of the dermal absorption.

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 TRIAZOLE 1.2.4 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.

 

Summary of Results

Table 4: Summary of results for the test item of all chambers

Experiment 1

 

Chamber

1

2

3

4

5

6

Amount of TRIAZOLE 1.2.4
applied (µg/cm2)1

4855

4943

4883

4856

5003

4910

Total amount of TRIAZOLE 1.2.4
measured (µg)

4791

5450

4949

5237

4829

5004

Recovery (%)

97.4

109

99.0

105

93.6

100

Total absorption of TRIAZOLE 1.2.4
(µg/cm2)2

199

535

290

82.0

130

140

Total absorption (%)3

4.10

10.8

5.94

1.69

2.59

2.85

Experiment 2

 

Chamber

1

2

3

4

5

6

Amount of TRIAZOLE 1.2.4
applied (µg/cm2)1

4896

4781

4698

4902

4658

4796

Total amount of TRIAZOLE 1.2.4
measured (µg)

4794

4954

5098

5082

4750

4670

Recovery (%)

95.5

99.5

101

101

94.1

91.8

Total absorption of TRIAZOLE 1.2.4
(µg/cm2)2

87.6

808

123

43.3

76.9

152

Total absorption (%)3

1.79

16.9

2.61

0.883

1.65

3.17

1: amount of TRIAZOLE 1.2.4 present in 5 mg test item without the pipetting washing solution (PWL)

2: the value is the sum of the amount of TRIAZOLE 1.2.4 measured in the receptor solution and in the skin extract (epidermis and dermis) of each diffusion cell.

3: percent total absorption = (total amount of TRIAZOLE 1.2.4 penetrated/absorbed (µg/ cm2) *100)/ amount of applied TRIAZOLE 1.2.4 (µg)

grey shading indicates outliers


[1]All 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:
The described permeability test and under the experimental conditions reported, TRIAZOLE 1.2.4 showed penetration into the viable skin layers and the receptor fluid out of the test item dilution with 132 ± 71.0 µg/cm2(2.73 ± 1.45 % of applied dose).
Executive summary:

Dermal absorption 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 item quantity left in the stratum corneum (adsorption).

The test item TRIAZOLE 1.2.4 was assessed for its potential for dermal absorption on human skin. The relevant compound measured was TRIAZOLE 1.2.4.

Two experiments with the test item were performed on human skin samples under static conditions with 12 diffusion cells. In total a number of five different donors (six different donors used but one removed from calculations due to significant different penetration detected) were used in this study.

5 mg of the test item were applied on each skin sample, left on the skin for 24 hours and then removed by washing each skin sample six times with 1 mL H2O:MeOH (70:30, v/v).

PBS was used as receptor solution. The solubility of TRIAZOLE 1.2.4 in receptor solution and extraction solution is given up to at least 4004 ng/mL as the calibration curves show. The dermal absorption 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 skin compartments were separated by using a forceps, and were extracted for their TRIAZOLE 1.2.4 content.

The samples from the skin dermal absorption assay were analysed by LC-MS/MS for the presence of TRIAZOLE 1.2.4. Also all chambers did meet the acceptance criteria, two chambers (chamber 2 in both experiments) were removed from the calculation because the determined penetration was extremely higher than the other. Both chambers contained skin from the same donor and, therefore, 10 chambers were used for the analysis of TRIAZOLE 1.2.4.

TRIAZOLE 1.2.4 was detectable in all samples relevant for dermal absorption, i.e. in the skin extracts and in the receptor fluid sample after 24 hours. Thus, TRIAZOLE 1.2.4 is considered to has penetrated the skin.

 

 

TRIAZOLE 1.2.4

Amount of TRIAZOLE 1.2.4

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

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

Amount applied

4946

±

336

100



Washing + SN solution

4580

±

264

94.5

±

5.06

EXR

24.9

±

47.4

0.511

±

0.973

Epidermis
(isolated after 24 hours)

8.25

±

6.13

0.170

±

0.125

Dermis
(isolated after 24 hours)

6.91

±

7.09

0.143

±

0.146

Receptor fluid

117

±

62.0

2.41

±

1.27

Recovery

4920

±

182

97.9

±

4.19

Bioavailable portion
(receptor fluid + epidermis + dermis)

132

±

71.0

2.73

±

1.45

SN = Solution of the donor chamber
EXR = exterior region of the skin

The quantity of TRIAZOLE 1.2.4 absorbed is 132 ± 71.0 µg and the quantity of TRIAZOLE 1.2.4 not absorbed is 4605 ± 311 µg.

In conclusion, it can be stated that during the described permeability test and under the experimental conditions reported, TRIAZOLE 1.2.4 showed penetration into the viable skin layers and the receptor fluid out of the test item dilution with 132 ± 71.0 µg/cm2(2.73 ± 1.45 % of applied dose).