Methyl 4-hydroxybenzoate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

received: 2. July 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-guideline study following a scientifically sound study design (mechanistic study) with sufficient study reporting.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline available

Guideline:

other: no "in vitro" guideline available

Deviations:

not applicable

Principles of method if other than guideline:

Comparison of the hydrolysis of Methylparaben by human and minipig skin and liver microsomes and cytosol.

GLP compliance:

not specified

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:

Supplier: Sigma Chemicals Co., Poole, Dorset, UK

Radiolabelling:

no

Test animals

Species:

miniature swine

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

Human breast skin (n = 3) was obtained following breast reduction surgery of healthy female individuals (age 28, 37 and 37). Ethical approval was obtained from the Durham University Hospital, U.K. Human liver (n = 22) was obtained from the liver bank of Toxicology Uint, Newcastle University, U.K.
Liver and dorsal skin from 3 male minipigs (weight 9 +/- 1.1 kg) were obtained from Pfizer, US.

All tissues were stored at -70°C until analysis.

Administration / exposure

Route of administration:

other: in vitro study: Methylparaben was added to microsomal fraction

Vehicle:

physiological saline

Details on exposure:

Subcellular protein (skin, 75 µg or liver, 5 µg) was incubated with Methylparaben (100 µM) for 1 hour at 37°C in phosphate buffer (0.1 M, pH 8.0) in a total volume of 250 µL. The reaction was stopped by addition of 750 µL Methanol containing internal standard 3,4-Dihydroxybenzoic acid (100 µM).

Duration and frequency of treatment / exposure:

1 hour at 37°C

Doses / concentrations

Remarks:

Doses / Concentrations:
100 µM

No. of animals per sex per dose / concentration:

humans: 3 females

minipigs: 3 males

Control animals:

other: blank sample without protein

Positive control reference chemical:

n.a.

Details on study design:

- Subcellular fraction preparation:
Human and minipig skin were cut with a dermatome (350 µm) to obtain the epidermis and minimal thickness of upper dermis. This was done to obtain the highest concentration of esterases, as they are predominantly located at the basal layer of epidermis. Tissue was weighed, minced, and pulverised in liquid nitrogen with a mortar and pestle. Pulverised tissue was added to ice-cold KCl/phosphate buffer (150 mM KCl, 0.1 M K2HPO4, ph 7.4) 1 mL per 100 mg tissue. Tissue was homogenised with an Ultra-turrax homogeniser with 3 x 10 sec bursts. The homogenate was centrifuged for 10 min at 750xg followed by ultra-centrifugation of the supernatant for 10 min at 10,000xg to remove mitochondria, nuclei and cell debris. Further centrifugation of the supernatant for 70 min at 100,000xg separated the microsomes from the cytosol. Cytosol was retained and stored at -70°C. The microsomal pellet was resuspended with buffer and centrifuged at 100,000xg for 70 min and the pellet resuspended in glycrol buffer (10% glycerol, 0.05 M Tris, 0.1 mM KCl, 250 mM sucrose, pH 7.4) and stored at -70°C for analysis. Microsomes and cytosol were prepared from liver by the above method without the liquid nitrogen pulverisation. The samples were frozen at -70°C.

- Determination of Methylparaben by HPLC:
Methylparaben and the internal standard Dihydroxybenzoic acid were analysed by HPLC using a Varian ProStar system with UV detection. A Gemini microbore 5 µmx250 mmx2 mm column was used (Phenomenex). Solvant A was water with 1% formic acid and solvent B was Methanol with 1% formic acid. The gradient was run from 35% B to 95% B over 6 min and held at 95% B for 4 min at a flow rate of 0.4 mL/min. The column was allowed to re-equilibrate for 5 min between runs. Column temperature was maintained at 40°C. Sample injection volume was 10 µL and peaks were detected at 264 nm. Retention times were: 4-Hydroxybenzoic acid 3.5 min, Dihydroxybenzoic acid 4.8 min, Methylparaben 6.2 min.

- Determination of Methylparaben hydrolysis by skin and liver microsomes and cytosol:
After the reaction was stopped (please refer to "Details on Exposure") the samples were vortexed for 10 sec and then centrifuged at 3000xg for 10 min. The supernatant was analysed by HPLC. Protein concentration in the reaction was at a level to give sufficient metabolite for analysis but resulting in less than 10% conversion of parent, to ensure linearitiy of reaction over the 1-hour incubation period. Blank incubation were conducted in the absence of protein to determine spontaneous hydrolysis. In a parallel set of incubations, Paraoxon (1mM), Bis-nitrophenyl phosphate (1 µM) and Loperamide (20 µM) were added to the incubation for 20 min before addition of Methylparaben.

Details on dosing and sampling:

Please refer to "Details on study design"

Statistics:

Enzyme activities were expressed as µmol 4-Hydroxybenzoic acid formed/min/mg protein (n =3, mean +/- S.E.M). Activities were compared by ANOVA followed by the post hoc Dunnett´s test. Activities in the presence of inhibitors were expressed as a percentage of the control.

Results and discussion

Preliminary studies:

No data

Toxicokinetic / pharmacokinetic studies

Details on absorption:

n.a., metabolism study

Details on distribution in tissues:

n.a., metabolism study

Details on excretion:

n.a., metabolism study

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Please refer to "Remarks on results including tables and figures"

Any other information on results incl. tables

Microsomal and cytosolic fractions displayed similar hydrolysis rates for both human and minipig skin. The rates were similar for both microsomal and cytosolic fractions.

a.) hydrolysis of Methylparaben by human/minipig skin

microsomal (human): 0.4 µmol/min/mg protein

microsomal (minipig): appr. 0.4 µmol/min/mg protein

cytosolic (human): 0.55 µmol/min/mg protein

cytosolic (minipig): 0.56 µmol/min/mg protein

Methylparaben hydrolysis rates using liver fractions were significantly higher than for skin microsomes or cytosol. The hydrolysis of the test item by human liver microsomes was also significantly higher (nearly 5 -fold) than minipig liver microsomes (p<0.001).

b.) hydrolysis of Methylparaben by human/minipig liver:

microsomal (human): 260 µmol/min/mg protein

microsomal (minipig): 40 µmol/min/mg protein

cytosolic (human): 24 µmol/min/mg protein

cytosolic (minipig): 14 µmol/min/mg protein

Pre-incubation of human or minipig skin subcellular fractions with Paraoxon (1µM) or Bis-nitrophenyl phosphate (1µM) completely inhibited the hydrolysis of Methylparaben. Loperamide (20 µM) had no effect on the hydrolysis by human or minipig microsomal or cytosolic skin fractions.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: Methylparaben hydrolysis rates using liver fractions were significantly higher than for skin microsomes or cytosol.
Microsomal and cytosolic fractions displayed similar hydrolysis rates for both human and minipig skin. The rates were similar for both microsomal and cytosolic fractions.
Methylparaben hydrolysis rates using liver fractions were significantly higher than for skin microsomes or cytosol. The hydrolysis of the test item by human liver microsomes was also significantly higher (nearly 5 -fold) than minipig liver microsomes (p<0.001).

Executive summary:

Methylparaben was hydrolysed by both liver and skin microsomal/cytosolic fraction of humans and minipigs. The hydrolysis by human esterases was higher than that observed for minipigs.

In this study, significantly different rates of hydrolysis and effect of the inhibitor Loperamide suggest that Methylparaben may be preferentially hydrolysed in the skin by one carboxylase isoform, hCE1 since Loperamide has been shown to specifically inhibit hCE2.