Undecan-4-olide

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From March 15, 2001 to May 29, 2001

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted similarly to OECD test guideline No. 428 with deviations: only 30 minutes exposure and one concentration (not justified). Recovery 100 +/-15 % instead of 100 +/- 10 % as required by the guideline. See guidance document on dermal absorption (OECD 2004). γ-Caprolactone, as a linear saturated 4-hydroxycarboxylic acid derived-lactones, is considered adequate for read-across purpose.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hexan-4-olide

EC Number:

211-778-8

EC Name:

Hexan-4-olide

Cas Number:

695-06-7

IUPAC Name:

5-ethyldihydrofuran-2(3H)-one

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): GCL
- Physical state: clear liquid
- Storage condition of test material: at room temperature

Radiolabelling:

no

Test animals

Details on test animals or test system and environmental conditions:

Not applicable

Administration / exposure

Vehicle:

water

Duration of exposure:

30 minutes

Doses:

- Dose volume: 200 µL of the test item, diluted at 10 % in water

No. of animals per group:

Not applicable

Control animals:

yes

Remarks:

positive control: caffeine

Details on study design:

See below

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: from a local slaughter-house (D-64395 Brensbach)
- Type of skin: porcine ears
- Preparative technique: the outer ear region was washed and cleaned with cold water. After carefully shaving, the skin was removed by dissection.
- Thickness of skin: approximately 400 µm
- Membrane integrity check: integrity of the skin was monitored over the entire duration of the study measuring conductivity across the skin prior to treatment and at each sampling time. All of the six donor and receptor chambers are equipped with platinium electrodes connecting to a conductometer. Skin damage is indicated by a sudden increase in conductivity or unsteady slopes of the measured values over time. The conductivity usually is in a range from 100-500 µS with aquous solutions depending on the thickness of the skin barrier and the area of the platinum electrodes. The maximal conductivity with gross damage to the skin or without skin is 2-5 mS.
- Storage conditions: in a freezer until use (within three months)
- Justification of species, anatomical site and preparative technique: the porcine skin taken from the outer ear of the animals is considered to be a good model for human skin

PRINCIPLES OF ASSAY
For the determination of the absorption of the test item the skin was mounted in glass flow-through diffusion chambers with a diameter of 1.135 cm and a volume of 1.5 mL (donor chamber). These chambers are subdivided in an upper part (donor chamber) and a lower part (receptor chamber). The receptor chambers are filled with saline (0.9 % NaCl), pH 3.0 at the beginning of the experiment. Saline was pumped through the chambers using a 6-channel peristaltic pump with a flow rate of 1-2 mL per hour and chamber (approx. 100 mL per hour at the maximum flow rate to collect the blank sample). The buffer solution of the receptor chamber was collected in plastic vials. The vials were replaced according to the sampling times and stored at -20 °C until analysis. In both experiments, the donor chamber was filled with 200 µL of the liquid test item and covered with Parafilm. The whole system was set up in an incubator adjusted to 32 °C. After 30 minutes of incubation the test item was removed off the skin by washin three times with 1 mL of saline. The three washing solutions of each individual chamber were pooled prior to analysis. Following the washing procedure the donor chambers were filled with 1 mL of saline to determine the impedance of the skin throughout the residual duration of the experiment. After 24 hours the solution in the donor chambers was also collected and stored at -20 °C until analysis.
The collecting vials were changed after 0, 0.5, 1, 2, 4, 6, 8 and 24 hours and analysed. The rate of absorption was calculated by determination of the test item concentration in the collecting vials. The amount of penetrated test item found in the receptor solution plus that found in the lower skin extracts are considered as penetrated respectively absorbed. Since the epidermis was separated from the dermis, the amount ofound in the upper skin extract is considered not to have passed the skin.
Test item bound to epidermis or deeper skin layers was quantified by extraction of both skin layers with 0.1 % TFA in water. HPLC was used to determine the concentration of the test item in the receptor vessels, the combined saline wash-off from each donor chamber and the skin extracts.

SEPARATION OF THE SKIN MEMBRANES
The isolated piece of skin was wrapped in aluminium foil, covered with an approx. 140 g weight and placed on a heating plate for 20-30 seconds at approx. 40-50°C. The the "upper skin" (= stratum corneum + upper stratum germinativum) was gently pealed off the "lower skin" (= low stratum corneum + upper dermis) using forceps. The separation is not necessarilly complete, but the procedure gives valuable information on the distribution of the dye in or on the skin. The two skin layers were separately extracted prior to analysis as described below.

EXTRACTION OF THE SKIN MEMBRANES
Test item bound to the skin membranes was quantified by extraction with 2 mL of 0.1 % TFA in water. The extraction was performed light protected for at least 8 hours or overnight at room temperature. The skin extracts were stored at -20 °C if not analysed immediately.


ANALYSIS
- Method type(s) for identification HPLC
- Limits of detection and quantification: Limit of quantitative determination = 500 µg/mL
Limit of detection with a detection probability of 90 % = 100 µg/mL
Limit of detection with a detection probability of 50 % = 50 µg/mL

Results and discussion

Absorption in different matrices:

See Table 7.1.2/1
No measurable and reproducible permeation through the skin occurred at any time point within the time frame in both experiments. The lowest detection limit under the conditions reported is 50 µg/mL in both experiments. The maximal possible, calculated flux of the test item across the skin barrier is 1926.0 µg/cm² in the first and 1995.2 µg/cm² in the second experiment. Together with the lower skin extracts the worst case considerations of penetrated test item result in 2012.5 µg/cm² in the first and 2072.5 µg/cm² in the second assay. It has to be emphasized that the values of the flux without the lower skin extracts are purely calculatory and do not reflect true penetration. Whenever the measured signal did not exceed the background noise in HPLC analysis, the flux is calculated on the basis of the lowest concentration of the calibration curve. Small but detectable amounts of the test item were identified in the lower skin extracts. LC/MS measurements performed by the sponsor resulted in a penetrated amount of 577 µg/cm² (2.9 %) of the applied test item over the 24 h time span in the first experiment.

Total recovery:

- Total recovery: Experiment 1: 96.1 % (+/- 5.61); Experiment 2: 84.6 % (+/-9.97)
- Recovery of applied dose acceptable: yes, within 100 +/- 15 %
- Limit of detection (LOD): 50.0 µg/mL
- Quantification of values below LOD or LOQ: 50 µg/mL is assumed as the maximal concentration

Percutaneous absorption

Dose:

20 mg/cm²

Parameter:

percentage

Absorption:

2.9 %

Remarks on result:

other: 24

Remarks:

First experiment

Conversion factor human vs. animal skin:

none

Any other information on results incl. tables

No measurable and reproducible permeation through the skin occurred at any time point within the time frame of both experiments. The lowest detection limit under the conditions reported is 50 µg/mL in both experiments. The maximal possible, calculated flux of the test item across the skin barrier is 1926.0 µg/cm² in the first and 1995.2 µg/cm² in the second experiment. Together with the lower skin extracts the worst case considerations of penetrated test item result in 2012.5 µg/cm² in the first and 2072.5 µg/cm² in the second experiment. It has to be emphasised that the values of the flux without the lower skin extracts are purely calculatory and do not reflect true penetration. Whenever the measured signal did not exceed the background noise in HPLC analysis, the flux is calculated on the basis of the lowest concentration of the calibration curve. Small but detectable amounts of the test item were identified in some samples of the lower skin extracts. LC/MS measurements performed by the Sponsor resulted in a penetrated amount of 577 µg/cm² (2.9 %) of the applied test item over the 24 h time span in the first experiment.

A positive control with caffeine is used to check the performance of the skin penetration system every 3 month.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described permeability test and under the experimental conditions reported, γ-Caprolactone did not penetrate the skin in detectable amounts. Together with the lower skin extracts, a worst case considerations assuming concentrations just at the lowest limit of quantification detection in the total volume of the receptor chamber solutions drawn at all time points, results in an upper limit of 2012.5 µg/cm² of the test item (10.06 %of the applied amount) in the first experiment and 2072.5 µg/cm² of the test item (10.36 % of the total amount) in the second experiment. Data generated and supplied by the sponsor indicate a penetrated amount of 577 µg/cm² (2.9 %) during the 24 hour time span of the first experiment

Executive summary:

The potential of γ-caprolatone to permeate porcine skin was assessed in this study, conducted similarly to OECD test guideline No. 428 and in compliance with GLP.

20 mg of test item were transferred into each donor chamber. Saline was slowly pumped through the receptor chambers with a flow rate of 1 to 2 mL per hour. The liquid leaving the receptor chambers was collected and the fractions were collected 0.5, 1, 2, 4, 6, 8 and 24 hours following the application of the test item. The blank samples (at 0 hour) were collected immediately after filling the donor chambers at the maximal flow rate of the pump. The conductivity across the skin samples of each chamber was measured at the same intervals. 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 experiments.

The test item was removed after 30 minutes by rinsing with saline. After 24 hours the skin discs were removed and separated into epidermal and deeper skin layers by heat-treatment. Both skin fractions were extracted and analyzed. Test item localized in the deeper layers of the skin was judged as penetrated, test item localized in the epidermal layers was considered not to have passed the skin.

The samples were analyzed by HPLC.

The mean recovery of the test item was 96.1 % in the first and 84.6 % in the second experiment.

No measurable and reproducible permeation through the skin occurred at any time point within the time frame in both experiments. The lowest detection limit under the conditions reported is 50 µg/mL in both experiments. The maximal possible, calculated flux of the test item across the skin barrier is 1926.0 µg/cm² in the first and 1995.2 µg/cm² in the second experiment. Together with the lower skin extracts the worst case considerations of penetrated test item result in 2012.5 µg/cm² in the first and 2072.5 µg/cm² in the second assay. It has to be emphasized that the values of the flux without the lower skin extracts are purely calculatory and do not reflect true penetration. Whenever the measured signal did not exceed the background noise in HPLC analysis, the flux is calculated on the basis of the lowest concentration of the calibration curve. Small but detectable amounts of the test item were identified in the lower skin extracts. LC/MS measurements performed by the sponsor resulted in a penetrated amount of 577 µg/cm² (2.9 %) of the applied test item over the 24 h time span in the first experiment.

A positive control with caffeine is used to check the performance of the skin penetration system every 3 month.

 

In conclusion, it can be stated that during the described permeability test and under the experimental conditions reported, γ-Caprolactone did not penetrate the skin in detectable amounts. Together with the lower skin extracts, a worst case considerations assuming concentrations just at the lowest limit of quantification detection in the total volume of the receptor chamber solutions drawn at all time points, results in an upper limit of 2012.5 µg/cm² of the test item (10.06 %of the applied amount) in the first experiment and 2072.5 µg/cm² of the test item (10.36 % of the total amount) in the second experiment. Data generated and supplied by the sponsor indicate a penetrated amount of 577 µg/cm² (2.9 %) during the 24 hour time span of the first experiment.