1H-1,2,4-triazole, sodium salt

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Experimental Starting Date: December 20, 2010 Experimental Completion Date: January 15, 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

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)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,4-TRIAZOLE sodium salt

IUPAC Name:

1,2,4-TRIAZOLE sodium salt

Details on test material:

Internal Test Item Number: S 11465 11
The test item and the information concerning the test item were provided by the sponsor.
Identity: 1,2,4-TRIAZOLE sodium salt
Batch No.: 10.36
Aggregate state at
room temperature: solid
Color: white
Purity: 99.0 %
Stability in solvent: not indicated by the sponsor
Storage: At room temperature, moisture protected
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 or test system 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). 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 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 1,2,4-TRIAZOLE sodium salt using skins from six different donors 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 1,2,4-TRIAZOLE sodium salt 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 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:

1,2,4-TRIAZOLE sodium salt showed penetration into the viable skin layers and the receptor fluid out of the test item dilution with 5.82 ± 2.93 µg/cm2 (0.134 ¿ 0.073 % of applied dose)

Any other information on results incl. tables

 Resu

Summary of the Method Validation

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

Linear:                            From 10.0 up to 4001 ng/mL for 1,2,4-TRIAZOLE sodium salt (reference material), validated by analysing calibration series in PBS.

Accuracy:                                     The accuracy expressed as recovery was between97.7 % and 109 % in PBS.

Precision:                                    The intra-day precision in PBS expressed as CV was between 2.88 % and 6.04 % 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 1,2,4-TRIAZOLE sodium salt is granted up to 24 hours in PBS and 1,2,4-TRIAZOLE sodium salt 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 Annex 1

Analytical Results1

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 1,2,4-TRIAZOLE sodium salt 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:    Summary of results for the test item of all chambers measured

Experiment 1
	Chamber
	1	2	3	4	5	6
Amount of 1,2,4-TRIAZOLE sodium salt applied (µg/cm^2)^1	3858	4154	4450	4011	4169	4456
Total amount of 1,2,4-TRIAZOLE sodium salt measured (µg)	4493	4332	4301	4254	4341	4453
Recovery (%)	112	101	95.1	106	103	99.4
Total absorption of 1,2,4-TRIAZOLE sodium salt (µg/cm^2)^2	9.86	5.21	9.52	4.02	9.33	8.40
Total absorption (%)^3	0.256	0.125	0.214	0.100	0.224	0.189
Experiment 2
	Chamber
	1	2	3	4	5	6
Amount of 1,2,4-TRIAZOLE sodium salt applied (µg/cm^2)^1	4635	4615	4652	4668	4651	4795
Total amount of 1,2,4-TRIAZOLE sodium salt measured (µg)	4669	4787	4812	4836	4977	4709
Recovery (%)	98.9	104	103	103	105	96.9
Total absorption of 1,2,4-TRIAZOLE sodium salt (µg/cm^2)^2	3.72	1.70	3.62	3.43	7.73	3.31
Total absorption (%)^3	0.080	0.037	0.078	0.073	0.166	0.069

1: amount of 1,2,4-TRIAZOLE sodium salt present in 5 mg test item without pipette washing solution

2: the value is the sum of the amount of 1,2,4-TRIAZOLE sodium salt measured in the receptor solution and in the skin extract (epidermis and dermis) of each diffusion cell.

3: percent total absorption = (total amount of 1,2,4-TRIAZOLE sodium salt penetrated/absorbed (µg/ cm²) *100)/ amount of applied 1,2,4-TRIAZOLE sodium salt (µg)

 

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:

1,2,4-TRIAZOLE sodium salt showed penetration into the viable skin layers and the receptor fluid out of the test item dilution with 5.82 ± 2.93 µg/cm2 (0.134 ¿ 0.073 % 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 thestratum corneum(adsorption).

The test item1,2,4-TRIAZOLE sodium saltwas assessed for its potential for dermal absorption on human skin. The relevant compound measured was 1,2,4-TRIAZOLE sodium salt.

Two experiments with the test item were performed on human skin samples under static conditions with 12 diffusion cells. In total a number of six different donors 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 1,2,4-TRIAZOLE sodium saltin receptor solution and extraction solutionis given up to at least 4001 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 1,2,4-TRIAZOLE sodium salt content.

The samples from the skin dermal absorption assay were analysed by LC-MS/MS for the presence of 1,2,4-TRIAZOLE sodium salt. All chambers met the acceptance criteria and, therefore, 12 chambers were used for the analysis of 1,2,4-TRIAZOLE sodium salt.

1,2,4-TRIAZOLE sodium salt 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, 1,2,4-TRIAZOLE sodium salt is considered to has penetrated the skin.

	1,2,4-TRIAZOLE sodium salt
Amount of 1,2,4-TRIAZOLE sodium salt	Expressed as µg/cm^2of skin surface mean±S.D. (n = 12)			Expressed as % of dose mean±S.D. (n = 12)
Amount applied	4426	±	303	100
Washing + SN solution	4512	±	263	102	±	4.53
EXR	0.520	±	0.301	0.012	±	0.008
Epidermis (isolated after 24 hours)	1.61	±	1.18	0.037	±	0.027
Dermis (isolated after 24 hours)	0.676	±	0.554	0.016	±	0.014
Receptor fluid	3.53	±	1.61	0.082	±	0.040
Recovery	4580	±	247	102	±	4.42
Bioavailable portion (receptor fluid + epidermis + dermis)	5.82	±	2.93	0.134	±	0.073

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

The quantity of1,2,4-TRIAZOLE sodium salt absorbed is 5.82± 2.93µg and the quantity of 1,2,4-TRIAZOLE sodium salt not absorbed is 4512± 263µg.