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: GLP 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:
other: liquid
Details on test material:
Cold product:
- Name of test material (as cited in study report): Hexylene glycol
- Synonym: 2-Methyl-2,4-pentanediol
- Analytical purity: 99.95% & 99.88%
- Purity test date: 25 April 2013
- Lot/batch No.: A1TX6K010101 & A20CEP010101
- Expiration date of the lot/batch: 25 April 2015
- Storage condition of test material: at room temperature and protected from humidity and heat, in a dry, cool and well ventilated place

Radiolabelled product:
- Name of test material (as cited in study report): [14C]-Hexylene glycol
- Purity test date: 21 January 2013
- Lot/batch No.: CFQ41689
- Radiochemical purity (if radiolabelling): 99.6%
- Specific activity (if radiolabelling): 67 mCi/mmol (2.48 GBq/mmol)
- Locations of the label (if radiolabelling): C5 & C6
- Expiration date of radiochemical substance (if radiolabelling): no data
- Storage condition of test material: at +4°C and protected from humidity in a dry, cool and ventilated place
Radiolabelling:
yes

Test animals

Species:
human

Administration / exposure

Vehicle:
unchanged (no vehicle)
Duration of exposure:
24 hours
Doses:
- Nominal doses: 8.6 mg/cm²
- Actual doses: 8.04 mg/cm² (62843 Bq)
- Dose volume: 30 µL

Results and discussion

Percutaneous absorption
Dose:
8.04 mg/cm² (62843 Bq)
Parameter:
percentage
Absorption:
2.59 %
Remarks on result:
other: 24 h
Remarks:
The total absorbed dose was 2.59 ± 1.66%. The mean absorption rate varied between 0.027% and 0.030% of the applied dose/cm2/hour (corresponding to 6.75 and 7.63 µg-eq/cm2/hour) and was found globally constant between 7 and 24 hours after application

Any other information on results incl. tables

- Receptor fluid, receptor chamber, donor chamber (in vitro test system): The mean cumulative amount of (14C)-Hexylene glycol (n = 5; ± SD) recovered in the receptor fluid over the 24-hour period, including the final receptor chamber washes (mean 0.12 ± 0.08% of the radioactive dose) was low at 2.08 ± 1.56% of the radioactive dose (range from 0.84 to 4.66%).

- Skin preparation (in vitro test system): At the end of the 24-hour exposure period, the vast majority of the radioactive dose was recovered in the skin swabs and donor chamber rinses with a mean of 101.5 ± 6.2% of the applied dose, representative of the unabsorbed dose.

- Stratum corneum (in vitro test system): A low level of radioactivity was recovered in the two surface tape strips (0.04 ± 0.01% of the dose, considered as unabsorbed dose) and in the ten following stripping performed to remove the stratum corneum (0.11 ± 0.05% of the dose). The quantity of radioactivity recovered from the exposed skin with a mean of 0.41 ± 0.11% of the applied dose was low and the radioactivity recovered in the flange skin (fraction of skin not exposed as hidden by the wall of the cell) was very low at 0.16 ± 0.07% of the dose. Radioactivity from flange skin areas, although resulting from a test item diffusion from the exposed area was considered as unabsorbed dose.

- Total recovery: 93.2% to 112.9% of the applied radioactive dose.

- Recovery of applied dose acceptable: yes

- Results adjusted for incomplete recovery of the applied dose: no

- Limit of quantification (LOQ): For the different biological matrices, blank values corresponding to the mean value of radioactivity (dpm per aliquot) measured in pre-dose matrices (sample collected before dosing for receptor fluids),or in substitution matrices (ethanol/water 50/50 for rinsing of donor compartment, ethanol for rinsing of receptor compartment, alkaline solution for skin and THF for strip) were determined.

Any measurement of radioactivity (dpm) lower than two times the blank value was reported as BLQ (Below the Limit of Quantification).

Blank value (dpm/aliquot, see table below) was subtracted from each raw measurement of radioactivity (dpm/aliquot) and the value of radioactivity presented is a corrected value. Consequently, the limit of quantification (corrected value) was defined as equal to the blank value.

Applicant's summary and conclusion

Conclusions:
The absorption of (14C)-Hexylene glycol through human skin was defined on the basis of mean absorption rate, percentage dose recovered in the tape strip and in the skin membrane (at 24 hour post-dosing) and percentage of the dose in the receptor fluid. The mean recovery of radioactivity was considered complete (mean of 104.3 ± 5.0%) in five of eight diffusions cells. The mean total directly absorbed radioactivity (receptor fluid + rinsing) was relatively low at 2.08 ± 1.66% and the absorbable dose (remaining radioactivity in the skin + strips) was very low at 0.55 ± 0.15%. The mean absorption rate of Hexylene glycol through human skin varied between 0.027% and 0.030% of the applied dose/cm2/hour (corresponding to 6.75 and 7.63 µg-eq/cm2/hour) and was found globally constant between 7 and 24 hours after application.
Executive summary:

The in vitro percutaneous absorption and distribution of (14C)-Hexylene glycol (14C-HG), was evaluated in human dermatomed skin (Fabreguettes, 2014). For this purpose,14C-HG was applied to excised human skin mounted in flow-through diffusion cells. This study was performed according to OECD guideline 428 and the principles of Good Laboratory Practices. Dermatomed skin samples (thickness between 0.372 and 0.452 mm) from four human donors were maintained in eight flow-through diffusion cells at 32 ± 2°C. Prior to absorption experimentation, the integrity of the skins was tested by measuring the Trans-Epidermal Water Loss (TEWL). Then,14C-HG formulation (mixture of non labelled and14C-HG) was applied to the skin samples at a dose-volume of 10 µL/cm² (i.e. 8.63 mg/cm²) and the cell was occluded with an aluminium sheet to prevent the losses by evaporation. The skin samples were exposed to the test item formulation for 24 hours, after which the remaining formulation was washed off the skin using cotton swabs dampened with an ethanol/water solution (1:1, v/v). Receptor fluid samples were collected at hourly intervals for the duration of the study (24 hours). As Hexylene glycol is described as freely miscible with water, the solubility of the test item in the receptor fluid (0.9% NaCl) was not tested and considered not to be rate limiting to the absorption process. At the end of the study, the skin was washed to remove excess of formulation, the exposed area was tape stripped with 3M scotch tape to remove stratum corneum and then the skin samples were fractionated into the exposed skin area and non-exposed area (flange skin). The total radioactivity of the skin swabs and donor chamber rinses, biological samples (exposed and flange skin areas), receptor fluid and receptor chamber rinses was measured by direct Liquid Scintillation Counting (LSC) for receptor fluids and rinses, by LSC after extraction with appropriate solvent for swabs and tape strip and by LSC after homogenization and solubilization with an ethanol/potassium hydroxide solution for exposed and flange skin samples.

The radiochemical purities of14C-HG and applied formulation were found, respectively at 92.1% and 92.2%, respectively and considered acceptable for the percutaneous absorption test. The overall radioactivity recovery of the applied dose of14C-HG was considered complete in all cells with values ranging from 93.2% to 112.9% of the applied radioactive dose. Three cells were excluded of the mean calculation: cell No.5 which had a recovery outside the range of 90-110% and an anomalous TEWL value and cells Nos. 3 and 6 presented an anomalous profile (leak/unexpected deterioration of the skin suspected). The mean cumulative amount (calculated from five cells) of (14C)-HG recovered in the receptor fluid over the 24-hour period (i.e.dose directly absorbed), including the final receptor chamber washes (mean 0.12 ± 0.08% of the radioactive dose) was relativelylow at 2.08 ± 1.56% ofthe radioactive dose. The kinetic profile of radioactivity in the receptor fluid was characterized by an increase of absorption between 0 and 7 hours to reach a level of 0.08 ± 0.07% of the applied dose/hour. Then, a mean constant absorption 0.08/0.09 ± 0.07% of the dose/hour was observed between 8h and 24h post-dose corresponding to a mean absorption rate varying from 0.027% to 0.030% of the dose/cm²/hour (equivalent to 6.75/7.63 µg-eq/cm²/hour, respectively).

At the end of the 24-hour exposure period, the vast majority of the radioactive dose was recovered in the material washed off the skin (skin swabs) and donor chamber with a mean of 101.5 ± 6.2% of the applied dose, representative of the unabsorbed dose.

A low level of radioactivity was recovered in the two surface tape strips (0.04 ± 0.01% of the dose) and in the ten following stripping performed to remove the stratum corneum (0.11 ± 0.05% of the dose). The quantity of radioactivity recovered from the exposed skin with a mean of 0.41 ± 0.11% of the applied dose was low and the radioactivity recovered in the flange skin (fraction of skin not exposed as hidden by the wall of the cell) was very low at 0.16 ± 0.07% of the dose. Radioactivity from flange skin areas, although resulting from test item diffusion from the exposed area was considered as unabsorbed dose.

The mean (± SD) distribution of radioactivity (% of dose) is summarized in the table below:

 

Donor compartment

Skin compartment

Receptor compartment

Total recovery

Rinsing + swabs

Surface tape strip

Flange Skin

Exposed skin strip

Exposed skin

Receptor fluid

Receptor rinsing

 

101.5 ± 6.2%

0.04 ± 0.01%

0.16 ± 0.07%

0.11 ± 0.05%

0.41 ± 0.11%

1.96 ± 1.48%

0.12 ± 0.08%

 

Unabsorbed dose:
101.7 ± 6.2%

Absorbable dose:
0.55 ± 0.15%

Dose directly absorbed:
2.08 ± 1.56%

104.3 ± 5.0%

Total absorbed: 2.59 ± 1.66%

 

 

In conclusion, the absorption of (14C)-HG through human skin was defined on the basis of mean absorption rate, percentage dose recovered in the tape strip and in the skin membrane (at 24-hour post-dosing) and percentage of the dose in the receptor fluid. The mean recovery of radioactivity was considered complete (mean of 104.3 ± 5.0%) in five of eight diffusions cells. The mean total directly absorbed radioactivity (receptor fluid + rinsing) was relatively low at 2.08 ± 1.56% and the absorbable dose (remaining radioactivity in the skin + strips) was very low at 0.55 ± 0.15%. The mean absorption rate of14C-HG through human skin varied between 0.027% and 0.030% of the applied dose/cm²/hour (corresponding to 6.75 and 7.63 µg-eq/cm²/hour) and was found globally constant between 7 and 24 hours after application.