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Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP, but low recovery rates; therefore, reliability of 2 was assigned

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
not specified
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl salicylate
EC Number:
204-263-4
EC Name:
2-ethylhexyl salicylate
Cas Number:
118-60-5
Molecular formula:
C15H22O3
IUPAC Name:
2-ethylhexyl 2-hydroxybenzoate
Test material form:
other: neat liquid
Details on test material:
chemical name: Benzoic acid, 2-hydroxy-, 2-ethylhexyl ester
Radiolabelling:
yes

Test animals

Species:
human
Strain:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
Full-thickness human female abdominal skin, obtained at autopsy, was frozen, stored at -20 °C, and thawed prior to processing. Following removal of
the subcutaneous fat by blunt dissection, individual portions of skin were immersed in water at 60°C for 45 seconds. The skin was then pinned, dermis side down, on a cork board and the epidermis (comprising stratum corneum and viable epidermis) gently removed from the underlying dermis. The latter was discarded and the -epidermal membrane floated onto the surface of water and taken up onto aluminium foil. The membranes were thoroughly dried and stored flat at -20 °C until used.

Administration / exposure

Type of coverage:
other: diffusion cells
Vehicle:
other: oil-water emulsion and hydroalcoholic formulation
Duration of exposure:
48 hours
Details on in vitro test system (if applicable):
Vehicle preparation
The vehicles used in this study were an oil-in-water emulsion and a hydroalcoholic formulation both containing 5% w/w octyl salicylate plus a trace amount of 3H-sucrose (active), an oil-in-water emulsion and a hydroalcoholic formulation both containing a trace amount of 3 H-sucrose (control), and an oil-in-water emulsion containing 2.7% w/w salicylic acid plus a trace amount of 3H-sucrose (comparator).
All vehicles were manufactured at Unilever Environmental Research Laboratories, Colworth House, Sharnbrook, UK.
Skin preparation:
Full-thickness human female abdominal skin, obtained at autopsy, was frozen, stored at -20 °C, and thawed prior to processing. Following removal of the subcutaneous fat by blunt dissection, individual portions of skin were immersed in water at 60 °C for 45 seconds. The skin was then pinned, dermis side down, on a cork board and the epidermis (comprising stratum corneum and viable epidermis) gently removed from the underlying dermis. The latter was discarded and the epidermal membrane floated onto the surface of water and taken up onto aluminium foil. The membranes were thoroughly dried and stored flat at -20 °C until used.
On the day of use the epidermal membranes were floated onto water from the aluminium foil and taken up onto filter paper supports. The membranes were then mounted onto diffusion cells and trimmed to size. For each vehicle, containing either octyl salicylate or salicylic acid, four to six different donors were used, representing at least two replicates per donor per vehicle.
Diffusion cells:
The prepared epidermal membranes were placed between the two halves of horizontal Franz-type glass diffusion cells, the stratum corneum facing the donor chamber. The cells were designed such that the area available for diffusion was about 1.0 cm2 (range 0.82-1.45 cm2), the exact area being measured for each diffusion cell. Receptor chamber volume varied from 2.38- 3.54 ml, the exact volume being measured for each diffusion cell.
The diffusion cells were immersed in a constant temperature water bath such that the receptor chambers were maintained at 37.0 ± 0.5°C throughout the experiment. This ensured that the skin surface temperature was maintained at 32.0 ± 1 °C. The receptor chamber contents were continuously agitated by small PTFE-coated magnetic followers driven by submersible magnetic stirrers. The receptor chambers of the diffusion cells were filled with a known volume of receptor solution, capped, and allowed to equilibrate to the correct temperature.
Receptor solution:
The solubilities of octyl salicylate in pH 7.4 phosphate buffered saline and in pH 7.4 phosphate buffered saline containing 6% (w/v) Volpo N20 (Bronaugh, 1985) were determined. Briefly, octyl salicylate was shaken with the test media and incubated at 37 °C for 0.5 hour. The resulting mixtures were filtered through 1.0 Whatman membrane filters (PTFE backed) and centrifuged at 14,000 rpm for 5 minutes. The clear solutions were then analysed for octyl salicylate content using the HPLC method described by DiNunzio and Rao Gadde(1990).
The solubility of octyl salicylate in pH 7.4 phosphate buffered saline was found to be approximately 1.0 pg/ml whereas that in pH 7.4 phosphate buffered saline containing 6% (w/v) Volpo N20 was 2.24 mg/ml. The latter solution was therefore selected as the receptor medium.
Application of formulations:
Finite dose in oil-in-water emulsion: A glass-rod was weighed and tared on a 5-place electronic balance. A small amount of formulation was placed on the end on the glass rod and the weight of formulation noted. The formulation was then spread, from the glass rod, evenly over the stratum corneum surface and the rod reweighed to indicate the exact amount of formulation applied to the skin surface. The target application amount was 5 mg/cm2. For the oil-in- water emulsion containing octyl salicylate and sucrose the achieved application weights ranged from 3.52-6.54 mg/cm2 (mean 4.85±0.98). For the oil-in-water emulsion containing salicylic acid and sucrose the achieved application weights ranged from 3.86-7.68 mg/cm2 (mean 5.37 ±1.13). The exact time of application was noted and that time represented 0 time for the run. Three 5-10 mg, accurately weighed, samples of each emulsion system were taken from different regions of the bulk emulsion, and were added to 5.0 ml HiSafe 3 liquid scintillation fluid and counted on a Wallac 1409 liquid scintillation counter.
Infinite dose in oil-in-water emulsion: A 1.0 ml plastic syringe was filled with the formulation and tared together with a glass rod on a five-place balance. Approximately 100 mg of the formulation was then directly displaced onto the skin surface. The formulation was then spread (using the glass rod), evenly over the stratum corneum surface and the rod and syringe reweighed to indicate the exact amount of formulation applied to the skin surface. The target application amount was 100 mg/cm2. The achieved application weights ranged from 78.59-127.00 mg/cm2 (mean 103.68 ± 16.39) and for the oil-in- water emulsion containing sucrose alone the achieved application weights ranged from 103.37-149.80 mg/cm2 (mean 116.79 ± 6.93). The exact time of application was noted and that time represented 0 time for the run.
Finite and infinite dose in hydroalcoholic formulation: At room temperature the hydroalcoholic formulation was solid. Prior to application, the lotion was gently heated in warm water, to melt the consituents, and were maintained in the fluid state throughout the application procedure. The formulations were then applied, using a motorised digital pipette, evenly over the stratum corneum surface. The target application amounts were 5 µI/cm2 for the finite dose and 100 /µl/cm2 for the infinite dose. The achieved application volumes were exactly 100 µl/cm2for the infinite dose and ranged from 4.89-5.20 µI/cm2 (mean 5.06±0.10) for the finite dose application. The exact time of application was noted and that time represented 0 time for the run.
Determination of permeation:
Following application of the vehicles, 200 µl samples were taken from the receptor media at 2, 4, 8, 10, 24, 30, 44 and 48 hours using a digital pipette. The samples were placed in plastic scintillation vials containing 5.0 ml HiSafe 3 liquid scintillation fluid and counted on a Wallac 1409 liquid scintillation counter. The receptor chambers were refilled with fresh temperature- equilibrated receptor medium. Subsequent to the 48 hour sample the following washing procedure was applied to all skin samples and diffusion cells, apart from those exposed to an infinite dose of octyl salicylate in hydroalcoholic formulation: The skin surface was rinsed three times with 2 ml of fresh receptor medium per rinse, the rinsates were pooled for each individual cell and the entire rinsate sampled and counted for the presence of radiolabel. The epidermal membranes, complete with filter paper supports, were placed in individual glass vials together with 1.0 ml OptiSolve fluid. After 48 hours solubilisation, 10 ml HiSafe 3 liquid scintillation fluid was added and the vials were counted on a Wallac 1409 liquid scintillation counter. For those skin samples and diffusion cells exposed to an infinite dose of octyl salicylate in hydroalcoholic formulation a slightly modified washing procedure was employed. This was found to be necessary because of the solid nature of the lotion at 32 °C, as described previously. Thus, 0.5 ml fresh receptor fluid was placed on the skin surface and the skin gently swabbed with a cotton bud. The wash fluid and cotton bud were collected into individual glass scintillation vials and 2.5 ml ethanol added. The vials were vortexed and left to stand for several hours prior to sampling and counting. The cells were dismantled and the donor chambers immersed in 20 ml ethanol for 30 minutes. The ethanol solution was then sampled and counted. The skin was placed into 1.0 ml OptiSolve fluid and left for 48 hours. When the tissue was solubilised, 9 ml ethanol was added, the solution sampled and the sample counted.

Results and discussion

Signs and symptoms of toxicity:
no effects
Dermal irritation:
no effects
Absorption in different matrices:
Flux and recovery of octyl salicylate after application as a finite dose in an oil-in-water emulsion vehicle (mean ± SE):
total absorption over 48 hours was 0.65 ± 0.16% (n = 9);
recovery in wash(%): 36.66 ±5.31;
recovery in skin(%): 17.18 ±1.28;
total recovery(%):54.50 ± 5.47.
Flux and recovery of octyl salicylate after application as an infinite dose in an oil-in-water emulsion vehicle (mean ± SE):
the average total absorption over 48 hours was 0.47 ± 0.22% (n = 11).
recovery in wash(%): 34.13 ±3.09;
recovery in skin(%):11.49 ±1.43;
total recovery(%):46.38 ± 2.18.
Flux and recovery of octyl salicylate after application as a finite dose in a hydroalcoholic formulation (mean ± SE):
total absorption over 48 hours was 0.59 ± 0.09% (n = 12);
recovery in wash(%): 36.21 ±5.98;
recovery in skin(%): 32.77 ±4.74;
total recovery(%):69.57 ±6.84.
Flux and recovery of octyl salicylate after application as an infinite dose in a hydroalcoholic formulation (mean ± SE).:
total absorption over 48 hours was 0.23 ± 0.05%(n = 11);
recovery in wash(%): 44.31 ±7.36;
recovery from donor cap(%): 24.08 ±7.80;
recovery in skin(%): 14.31 ±2.40;
total recovery(%):82.93 ± 6.35.
Flux and recovery of salicylic acid after application as a finite dose in an oil-in-water emulsion vehicle:
total absorption over 48 hours was 1.14 ± 0.23% (n = 11);
recovery in wash(%): 56.73 ±6.02;
recovery in skin(%): 4.79 ±0.41;
total recovery(%):62.67 ± 6.06.




Total recovery:
The distribution of octyl salicylate between the skin surface, skin and receptor medium, is remarkably similar following application as both finite and infinite doses in the oil-in-water emulsion vehicle. Thus, following 48 hour exposure 10-20% of the applied dose was recovered from the skin phase.
This data, taken with the amount that had crossed the skin and the amount remaining on the skin indicates some accumulation of the compound within the stratum corneum. A similar profile was obtained following application of an infinite dose of octyl salicylate in a hydroalcoholic formulation.
There was some dissimilarity following application of octyl salicylate as a finite dose in the hydroalcoholic formulation, since in this case the recovery from the skin appeared relatively high (33%).
In the initial experiment under similar conditions, a flux rate of 7.07 µg/cm2 and recoveries totaling 22.3% of 14C were obtained. Following an improved washing procedure and an evaluation of activity remaining in the donor cap, subsequent to the wash, recoveries were increased to 83% while the flux rate did not alter significantly (11.28 µg/cm2).
Flux and recovery of octyl salicylate after application as a finite dose in an oil-in-water emulsion vehicle: total recovery(%): 54.50 ±5.47;
Flux and recovery of octyl salicylate after application as an infinite dose in an oil-in-water emulsion vehicle:total recovery(%): 46.38 ±2.18;
Flux and recovery of octyl salicylate after application as a finite dose in a hydroalcoholic formulation:total recovery(%): 69.57 ±6.84;
Flux and recovery of octyl salicylate after application as an infinite dose in a hydroalcoholic formulation :total recovery(%): 82.93 ±6.35.;
Flux and recovery of salicylic acid after application as a finite dose in an oil-in-water emulsion vehicle:total recovery(%): 62.67 ±6.06.
Percutaneous absorptionopen allclose all
Dose:
51.58 ± 0.36 µg/cm2
Parameter:
percentage
Absorption:
> 0.49 - < 0.81 %
Remarks on result:
other: 48h
Remarks:
after application as a finite dose in an oil-in-water emulsion vehicle
Dose:
527.54 ± 13.91 µg/cm2
Parameter:
percentage
Absorption:
> 0.25 - < 0.69 %
Remarks on result:
other: 48h
Remarks:
after application as an infinite dose in an oil-in-water emulsion vehicle (
Dose:
51.58 ±0.25µg/cm2
Parameter:
percentage
Absorption:
> 0.5 - < 0.68 %
Remarks on result:
other: 48h
Remarks:
after application as a finite dose in a hydroalcoholic formulation
Dose:
11.28 ± 2.55µg/cm 2
Parameter:
percentage
Absorption:
> 0.18 - < 0.28 %
Remarks on result:
other: 48h
Remarks:
after application as an infinite dose in a hydroalcoholic formulation
Dose:
1.65±0.39µg/cm2
Parameter:
percentage
Absorption:
> 0.91 - < 1.37 %
Remarks on result:
other: 48h
Remarks:
after application as a finite dose in an oil-in-water emulsion vehicle

Applicant's summary and conclusion

Conclusions:
It is concluded that the in vitro human skin permeation of test article is relatively low. Absorption of test article after application as a finite/infinite dose in an oil-in-water emulsion/hydroalcoholic formulation vehicle: 0.65~1.14% of the applied dose.
Executive summary:

In this study, full-thickness human female abdominal skin, obtained at autopsy, was employed to investigate the skin penetration of test article in vitro following GLP regulations.

When applied as a finite dose in an oil-in-water emulsion vehicle, containing 5% w/w octyl salicylate, the average total absorption of 14C over 48 hours was 0.65 ± 0.16% of the applied dose, representing a total flux of 1.58 ± 0.36 µg/cm2. When applied as an infinite dose in the oil-in-water emulsion vehicle the average total absorption of 14C over 48 hours was 0.47 ± 0.22% of the applied dose, representing a total flux of 27.54 ± 13.91 µg/cm2. When applied as a finite dose in a representative hydroalcoholic formulation, containing 5% w/w octyl salicylate, the average total absorption of 14C over 48 hours was 0.59 ± 0.09% of the applied dose, representing a total flux of 1.58 ± 0.25 µg/cm2. When applied as an infinite dose in the hydroalcoholic formulation the average total absorption of 14C over 48 hours was 0.23 ± 0.05% of the applied dose, representing a total flux of 11.28 ± 2.55 µg/cm2.

The penetration of 14C-salicylic acid applied in the oil-in-water emulsion was also determined. When applied as a finite dose the average total absorption of 14C over 48 hours was 1.14 ± 0.23% of the applied dose, representing a total flux of 1.65 ±0.39 µg/cm2. 3 H-sucrose was incorporated into all formulations and provided a marker for membrane integrity. For the results reported, penetration of sucrose was very low indicating that the membranes used were intact throughout the experiment.

The data obtained in this study suggest that the in vitro human skin permeation of octyl salicylate is relatively low. The flux of octyl salicylate and salicylic acid when applied in similar vehicles were remarkably similar over 48 hours (1.58 µg/cm2 and 1.65 µg/cm2 respectively). It is supposed that the 14C label appearing in the receptor fluid may, in both cases, represent salicylic acid. If this is the case then it is possible that the amount of octyl salicylate permeating through the skin is much less than that suggested by the data obtained here.