Methyl 4-hydroxybenzoate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April to June 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported GLP-compliant guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 4-hydroxybenzoate

EC Number:

202-785-7

EC Name:

Methyl 4-hydroxybenzoate

Cas Number:

99-76-3

Molecular formula:

C8H8O3

IUPAC Name:

methyl 4-hydroxybenzoate

Constituent 2

Reference substance name:

Methylparaben

IUPAC Name:

Methylparaben

Details on test material:

Lot No.: M3146
Physical Characteristics: White powder
Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.
Molecular weight: 152
Formula: C8H8O3
Purity: 99.9%
Octanol-water partition coefficient: 1.87

The radiolabeled test substance was supplied by Amersham Biosciences U.K, Ltd. and assigned Haskell Laboratory No. 22705-84 upon receipt.
Phenyl-14C(U)methylparaben
Code: CFQ13765
Batch: 1
Specific activity: 97µCi/mg
Purity: 99.5%

Test animals

Species:

other: human and rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

A. Rat Skin
Male Wistar rats, approx. 6-8 weeks of age, were supplied by Charles river Laboratories, Inc., Raleigh, North Carolina. Following a required quarantine period, rats were removed from stock and uniquely identified by tail markings. Rats were sacrificed by carbon dioxide asphyxiation, the fur from the dorsal region carefully shaved using clippers, and the skin excised. Skin specimens were identified using Haskell animal number.

B. Human Skin
Samples of human abdominal skin from local surgeons were obtained fresh. The source and identity of the akin sample (sex, anatomical locale and approx. age of donor) was documented in the study recoreds. Skin specimens were identified using unique code.

At the time of harvest both rat and human skin specimens were placed in Hepes-buffered Hanks´balanced salt solution and were maintained on wet ice until prepared for use. All skin specimens were used less than 4 hours following removal from donor.

Administration / exposure

Type of coverage:

other: in vitro study

Vehicle:

other: Oil-in-water

Duration of exposure:

24 hours

Doses:

0.8% Methylparaben solution
approx. 65 µg/cm2

No. of animals per group:

Replicates:
A. Rat: n = 10
B. Human: n= 13

Control animals:

no

Details on study design:

In vitro study, please refer to "Details on in vitro test system" below

Details on in vitro test system (if applicable):

The penetration kinetics and first-pass metabolism of Methylparaben in viable rat and human skin has been determined. The active ingredient was formulated as oil-in-water emulsion at a target concentration of 0.8% and 0.4%. Samples of fresh rat and human skin were dermatomed to approx. 450 µm and mounted stratum corneum uppermost, onto a flow-through diffusion cell system with an exposure area of 0.64 cm2. The underside of each skin specimen was perfused with sterile-filtered Hepes-buffered Hanks´balanced salt solution containing gentimicin (0.05 mg/mL) and bovine serum albumin (3.75%). Each formulated emulsion was applied as a finite dose at a rate of 10 mL/cm2 to rat ( n=10 replicates) and human skin (n=13 replicates). Penetration was followed using [14C]-labeled active ingredient, which was uniformly blended into emulsions. The amount of active applied per area skin was approx. 65 µg/cm2 and 36 µg/cm2 . The applied formulation remained in contact with the skins for 24 hours without occlusion. During 24 hour exposure period, serial receptor fluid samples were collected hourly for the first 6 hours and then every other hour until termination. At the end of the exposure period, the skin surface was washed with a dilute soap solution to remove excess formulation and then tape-stripped to remove the stratum-corneum. Distribution of the applied radiolabeled material was determined by liquid scintillation counting. First-pass metabolism was determined by quantitative analysis of serial receptor fluid samples and the principle metabolite 4-Hydroxybenzoic acid using HPLC-mass spectrometry. Receptor fluid samples representing each formulation, from both rat and human experiments, were also analysed by radiochromatography and LC-MS.

Results and discussion

Signs and symptoms of toxicity:

not specified

Remarks:

in vitro study

Dermal irritation:

not specified

Remarks:

in vitro study

Absorption in different matrices:

Following application of a 0.8% Methylparaben emulsion to viable rat and human skin, a greater amount of total radioactivity penetrated human skin (79.36%) compared to rat skin (54.94%). A major portion of the total radioactivity that had penetrated rat skin was metabolised to 4-Hydroxybenzoic acid (53.9%), with a smaller portion (23.8%) accounted for as unmetabolised Methylparaben. By comparison, a lesser portion of the total radioactivity that had penetrated viable human skin had been metabolised to 4-Hydroxybenzoic acid (35.1%), with the majority (60.3%) accounted for as unmetabolised Methylparaben.

Total recovery:

Material balance
A. Rat skin
Absorbable dose
- Receptor fluid: 54.94 +/- 5.92 %
- Receptor wash: 0.43 +/- 0.20 %
- Skin: 12.23 +/- 5.57 %
=> Total absorbable : 67.61 +/- 6.06 %
Unabsorbed dose
- Skin wash: 17.81 +/- 2.82 %
- Donor chamber: 0.03 +/- 0.01 %
- Tape strips: 5.65 +/- 1.12 %
=> Total unabsorbed: 23.49 +/- 2.40 %
=> Total recovered: 91.09 +/- 5.66 %

B. Human skin
Absorbable dose
- Receptor fluid: 79.36 +/- 15.62 %
- Receptor wash: 0.46 +/- 0.11 %
- Skin: 4.88 +/- 2.01 %
=> Total absorbable : 84.69 +/- 15.46 %
Unabsorbed dose
- Skin wash: 14.65 +/- 8.76 %
- Donor chamber: 0.42 +/- 0.94 %
- Tape strips: 6.13 +/- 12.01 %
=> Total unabsorbed: 21.21 +/- 20.48 %
=> Total recovered: 105.91 +/- 15.10 %

Percutaneous absorption

Percutaneous absorption 1

Dose:

65 µg/cm2

Parameter:

percentage

Absorption:

ca. 67.6 %

Remarks on result:

other: 24 hours

Remarks:

Rat skin

Percutaneous absorption 2

Dose:

65 µg/cm2

Parameter:

percentage

Absorption:

ca. 84.7 %

Remarks on result:

other: 24 hours

Remarks:

Human skin

Conversion factor human vs. animal skin:

no data

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:

These data show, based on dermatomed viable rat and human skin model and analysis of receptor fluid samples, that the dermal bioavailability of Methylparaben from oil-in-water emulsion was greater for human skin than rat skin.
A. Rat skin
- amount penetrated skin: 54.94% 24 hours after application
- 53.9% of the penetrated amount metabolised to 4-Hydroxybenzoic acid

B. Human skin
- amount penetrated skin: 79.36% 24 hours after application
- 35.1% of the penetrated amount metabolised to 4-Hydroxybenzoic acid

Executive summary:

The penetration kinetics and first-pass metabolism of Methylparaben in viable rat and human skin has been determined. The active ingredient was formulated as oil-in-water emulsion at a target concentration of 0.8% and 0.4%. Samples of fresh rat and human skin were dermatomed to approx. 450 µm and mounted stratum corneum uppermost, onto a flow-through diffusion cell system with an exposure area of 0.64 cm2. The underside of each skin specimen was perfused with sterile-filtered Hepes-buffered Hanks´balanced salt solution containing gentimicin (0.05 mg/mL) and bovine serum albumin (3.75%). Each formulated emulsion was applied as a finite dose at a rate of 10 mL/cm2 to rat ( n=10 replicates) and human skin (n=13 replicates). Penetration was followed using [14C]-labeled active ingredient, which was uniformly blended into emulsions. The amount of active applied per area skin was approx. 65 µg/cm2 and 36 µg/cm2 . The applied formulation remained in contact with the skins for 24 hours without occlusion. During 24 hour exposure period, serial receptor fluid samples were collected hourly for the first 6 hours and then every other hour until termination. At the end of the exposure period, the skin surface was washed with a dilute soap solution to remove excess formulation and then tape-stripped to remove the stratum-corneum. Distribution of the applied radiolabeled material was determined by liquid scintillation counting. First-pass metabolism was determined by quantitative analysis of serial receptor fluid samples and the principle metabolite 4-Hydroxybenzoic acid using HPLC-mass spectrometry. Receptor fluid samples representing each formulation, from both rat and human experiments, were also analysed by radiochromatography and LC-MS.

Following application of a 0.8% Methylparaben emulsion to viable rat and human skin, a greater amount of total radioactivity penetrated human skin (79.36%) compared to rat skin (54.94%). A major portion of the total radioactivity that had penetrated rat skin was metabolised to 4-Hydroxybenzoic acid (53.9%), with a smaller portion (23.8%) accounted for as unmetabolised Methylparaben. By comparison, a lesser portion of the total radioactivity that had penetrated viable human skin had been metabolised to 4-Hydroxybenzoic acid (35.1%), with the majority (60.3%) accounted for as unmetabolised Methylparaben.