Sodium 4-(methoxycarbonyl)phenolate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics, other

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

See attached read-across justification according to RAAF (chapter 13)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

received: 27. Feb. 1960

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: None-guideline study following a scientifically sound study design (metabolic study) with restricted study reporting.

Objective of study:

metabolism

Qualifier:

no guideline followed

Guideline:

other: performed before guidelines

Deviations:

not applicable

Principles of method if other than guideline:

Methyl p-hydroxybenzoate sodium salt was administered to rabbits and urine collected for 24 hours was examined for metabolites.

GLP compliance:

no

Remarks:

performed before GLP guidelines

Radiolabelling:

no

Species:

rabbit

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

4 male rabbits
body weight: 2.4 - 2.8 kg
Animals were housed in metabolism cages and fed "Okara" (soybean curd residue) only

Route of administration:

oral: gavage

Vehicle:

not specified

Details on exposure:

Methyl p-hydroxybenzoate sodium salt (0.8 g/kg body weight as Methyl p-hydroxybenzoate) was administered by somach tube as a 12% (w/v) solution and a total dose of 8.2 g of Methyl p-hydroxybenzoate was used for 4 rabbits.

Duration and frequency of treatment / exposure:

Single dose

Remarks:

Doses / Concentrations:
800 mg/kg bw

No. of animals per sex per dose / concentration:

4 males

Control animals:

no

Positive control reference chemical:

None

Details on study design:

After application the urine was collected for 24 hours and analysed for metabolites.

Details on dosing and sampling:

Please refer to "Any other information on materials and methods incl. tables"

Statistics:

None

Preliminary studies:

Previous to this study, quantitative determination on the metabolism of Methyl p-hydroxybenzoate in rabbit showed that after a dose of 0.4 g/kg bw of Methyl p-hydroxybenzoate, the excretion was concluded in almost 6 hours, about 1/3 portion being excreted as free p-Hydroxybenzoic acid and the rest was discharged as conjugated form, without the unchanged Methyl p-hydroxybenzoate.

Details on absorption:

Not determined

Details on distribution in tissues:

Not determined

Details on excretion:

No data

Metabolites identified:

yes

Details on metabolites:

The excretion of unchanged Methyl p-hydroxybenzoate was not identified. Although the result could not be quantified, the ether-type glucuronide of p-Hydroxybenzoic acid appeared to be present in greater amount than the ester-type on paper chromatogramm.

No further data

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
The ether-type glucuronide of p-Hydroxybenzoic acid was isolated as Methylacetyl derivative from the urine of rabbits receiving Methyl p-hydroxybenzoate. Ester-type glucuronide was detected in the urine by paper chromatography.
Although the result could not be quantified, the ether-type glucuronide of p-Hydroxybenzoic acid appeared to be present in greater amount than the ester-type on paper chromatogramm.

Executive summary:

Please refer to "Conclusions" and "Preliminary studies".

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

received: 25. Jan. 1961

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Guideline:

other: performed before guidelines

Deviations:

not applicable

Principles of method if other than guideline:

Methyl p-hydroxybenzoate sodium salt was administered to rabbits and collected 24 hours was examined for metabolites.

GLP compliance:

no

Remarks:

performed before GLP guidelines

Radiolabelling:

no

Species:

rabbit

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

3 male rabbits
body weight: 2.6 - 3.2 kg
Animals were housed in metabolism cages and fed "Okara" (soybean curd residue)

Route of administration:

oral: gavage

Vehicle:

not specified

Details on exposure:

A total dose of 7.2 g of Methyl p-hydroxybenzoate (0.8 g/kg bw) was administered as a 12% solution in the form of sodium salt to 3 rabbits.

Duration and frequency of treatment / exposure:

Single dose

Remarks:

Doses / Concentrations:
800 mg/kg

No. of animals per sex per dose / concentration:

3 males

Control animals:

no

Positive control reference chemical:

None

Details on study design:

After application the urine was collected for 24 hours and analysed for metabolites.

Details on dosing and sampling:

Please refer to "Any other information on materials and methods incl. tables"

Statistics:

None

Preliminary studies:

Please refer to Tsukamoto, 1960 (IUCLID file 7.1.1 Basic toxicokinetics, WoE_Metabolism and excretion_rabbit_1960_Tsukamoto)

Details on absorption:

Not determined

Details on distribution in tissues:

Not determined

Details on excretion:

No data

Metabolites identified:

yes

Details on metabolites:

Please refer to "Overall remarks, attachments"

None

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
After application of a single dose Methylparaben to rabbits the main metabolites found in urine were p-Hydroxybenzoic acid, p-Hydroxyhippuric acid and the ether-type glucuronide.

Executive summary:

Please refer to "Overall remarks, attachments" and "Conclusions".

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

accepted: June 1952

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Abstract

Objective of study:

absorption

excretion

Qualifier:

no guideline followed

Guideline:

other:

Principles of method if other than guideline:

Chronical application of Methylparaben to dog for 1 year, determination of urinary excretion.

GLP compliance:

no

Remarks:

performed before GLP guidelines

Radiolabelling:

not specified

Species:

dog

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

No data

Route of administration:

oral: unspecified

Vehicle:

not specified

Details on exposure:

No details stated

Duration and frequency of treatment / exposure:

daily for 1 year

Remarks:

Doses / Concentrations:
1 g/kg bw/d

No. of animals per sex per dose / concentration:

1 dog/dose

Control animals:

no

Positive control reference chemical:

None

Details on study design:

No further details

Details on dosing and sampling:

No further details

Statistics:

None

Preliminary studies:

No data

Details on absorption:

Please refer to "overall remarks"

Details on distribution in tissues:

Not determined

Details on excretion:

The rate of urinary excretion in the dog had increased to such an extent that after 24 hours 96% of the dose was recovered in the urine, whereas in a dog given a single oral dose it was not until 48 hours that the entire amount was accounted for.

Metabolites identified:

not specified

Details on metabolites:

No data

None

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
From this study it appears that Methylparaben is efficiently absorbed, metabolised and excreted by the body. Hydrolysis of the ester linkage and metabolic conjugation provide adequate degradation mechanisms. There was no sign of accumulation.

Executive summary:

Please refer to "excretion" and "overall remarks"

Reference 5

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.

Objective of study:

metabolism

Qualifier:

no guideline available

Guideline:

other: no "in vitro" guideline available

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

Radiolabelling:

no

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.

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

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.

Preliminary studies:

No data

Details on absorption:

n.a., metabolism study

Details on distribution in tissues:

n.a., metabolism study

Details on excretion:

n.a., metabolism study

Metabolites identified:

yes

Details on metabolites:

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

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.

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.

Reference 6

Endpoint:

basic toxicokinetics

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

2009

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Abstract

Objective of study:

absorption

metabolism

toxicokinetics

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

[14C]-label at Phenyl moiety

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

12 male and 12 female rats
no further data

Route of administration:

dermal

Vehicle:

not specified

Details on exposure:

Groups of 12 male and 12 female rats received a single dermal administration of 100 mg/kg bw [14C]-labelled Methylparaben.

Duration and frequency of treatment / exposure:

1 single application

Remarks:

Doses / Concentrations:
100 mg/kg bw

No. of animals per sex per dose / concentration:

12

Control animals:

no

Positive control reference chemical:

None

Details on study design:

Please refer to "Details on exposure" and "Details on dosing and sampling"
Rats were sacrificed 168 h after application and radioactivity analysed in major organs and tissues
Plasma metabolites were investigated by HPLC/UV/radioactivity monitoring

Details on dosing and sampling:

Blood samples were taken at 0, 0.5, 1, 2, 4, 8, 12, 22 and 24 h after treatment
Urine and faeces was examined up to 168 h after application

Statistics:

no data

Preliminary studies:

None

Details on absorption:

Dermal absorption:
14% in males
26% in females

Metabolites identified:

yes

Details on metabolites:

Methylparaben was completely metabolised to 4-Hydroxybenzoic acid. No systemic exposure was detected to Methylparaben 8 h after dermal application.

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
Methylparaben was partially absorbed after dermal administration to rats (14% in males, 26% in females). The plasma concentrations consisted entirely of 4 -Hydroxybenzoic acid

Executive summary:

Groups of 12 male and 12 female rats received a single dermal administration of 100 mg/kg bw [14C]-labelled Methylparaben.Blood samples were taken at 0, 0.5, 1, 2, 4, 8, 12, 22 and 24 h after treatment. Urine and faeces was examined up to 168 h after application. Methylparaben was partially absorbed after dermal administration to rats (14% in males, 26% in females). The plasma concentrations consisted entirely of 4 -Hydroxybenzoic acid (the metabolite).

Reference 7

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

received: 26. Jan. 1956

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Important aspects (application, sampling and analysis) are in line wtih the current OECD guideline. Restricted reporting.

Objective of study:

distribution

excretion

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

4 animals per group

GLP compliance:

not specified

Radiolabelling:

no

Species:

dog

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

no further details

Route of administration:

other: oral and intravenous

Vehicle:

not specified

Details on exposure:

Absorption and Excretion:
A comparison was made between oral and intravenous administration of Methylparaben. Intravenous injections of 50 mg/kg bw were given to a series of 3 or more fasted dogs and blood and urine samples were taken at fixed intervals following the injection until levels fell to 0. Determinations of the compound and its various metabolic products were then made on the samples. A similar study was carried out in which 1 g/kg bw of Methylparaben was given orally in capsules to the dogs.

Distribution:
Dogs were infused intravenously at the rate of 2 mg/kg bw/min. until a total of 100 mg/kg bw of the test item was injected. When 95 mg/kg bw had been given, blood samples were taken. The dog was killed and tissues immediately isolated. The organs were cut up, mixed with an equal quantity by weight of saline in a Waring Blendor, and finely ground in a tissue grinder until mash could be drawn through a syringe. Analsyses were made on the various tissues and the plasma to determine the levels of p-Hydroxybenzoic acid, Methylparaben and "Total".

Duration and frequency of treatment / exposure:

Absorption and Excretion:
Single application
Distribution:
continuous infusion over 50 min.

Remarks:

Doses / Concentrations:
Absorption and Excretion: 50 mg/kg bw i.v.; 1 g/kg bw oral
Distribution: 100 mg/kg bw

No. of animals per sex per dose / concentration:

at least 3 animals/study type

Control animals:

no

Positive control reference chemical:

none

Details on study design:

Please refer to "Details on exposure"

Details on dosing and sampling:

The procedures as finally adopted for use are as follows: A buffer of pH 10-11 was prepared for the medium in which the coupling reaction takes place in order to insure maximum, stable color development (43.9 mL of 0.1 N NaOH + 50.0 mL of 0.1 M H3BO3 diluted to 100 mL).

- Determination of p-Hydroxybenzoic acid in water, urine and plasma
1 mL of plasma or 1 mL urine diluted 1:10, 1 mL of 6 N H2SO4 and 7 mL of absolute Ether were shaken mechanically for 6 min in a glass-stoppered centrifuge tube. The mixture was then centrifuged for 1 min at 1500 rpm and a 4 mL aliquot of the supernatant Ether solution was transferred to a test tube and the Ether removed by evaporation from warm water bath. 4 mL of the buffer were added to the tube and the solution heated for 5 min at 90-100°C to insure complete solution of residue. Upon cooling to room temperature 1 mL of Amino-anti-pyridine reagent (1.36 g/L) and 0.5 mL of Potassium ferricyanide solution (8.67 g of K3Fe(CN)6 and 1.8 mL of concentrated NH4OH/L) were added to the tube. The resulting colored solution was shaken by hand for a few seconds and the transmittance was determined at 490 nm on the Beckman spectrophotometer. Color develops immediately upon shaking and remains stable for at least 1 hour.

- Determination of p-Hydroxybenzoate in water, urine and plasma
1 mL of plasma or urine, 1 mL of satured NaHCO3 and 7 mL of absolute Ether were mechanically shaken for 6 min. in a glass-stopped centrifuge tube. The mixture was then centrifuged for 1 min. at 1500 rpm and a 5 mL aliquot of the Ether phase was evaporated in a glass-stopped centrifuge tube from warm water bath. 1 mL of water and 1 mL of 6 N H2SO4 were added to the residue and the tube heated for 2 hours in a boiling water bath. It had been previously determined that further hydrolysis did not take place after 2 hours. Upon cooling, 7 mL of absolute Ether was added to the tube and the extraction was carried out by mechanical shaking for 6 min. The remainder of the procedure was identical with the p-Hydroxybenzoic acid determination. Calibration curves were run for the Ester using this procedure.

- Determination of "total" material (p-Hydroxybenzoic acid + p-Hydroxybenzoate + metabolic conjugate) in water, plasma and urine
This procedure is identical with the p-Hydroxybenzoic acid determination except that before the addtion of the Ether the acidic phase was heated for 2 hours in a boiling water bath. No standard curves were availble for "total" material, but values were read from the p-Hydroxybenzoic acid curve. By correcting for the aliquots, 75-85% of the added material may be accounted for the procedures.

- Determination of p-Hydroxybenzoate and "total" material in tissues
Satisfactory analyses have been made in liver, spleen, muscle, kidney, brain and pancreas. The organ under study was cut up and added to an equal quantity by weight of saline. After preliminary mixing in Waring Blendors, the resulting suspension was ground in a tissue grinder untl it might be drawn through a syringe. 1 mL of this mixture was used for analysis. The procedures for the determination of p-Hydroxybenzoic acid, p-Hydroxybenzoate and "total" material in tissues are identical with those in plasma except that the Ether aliquot was shaken mechanically in a glass-stoppered centrifuge tube for 6 min with 4 mL of the buffer. The mixture as then centrifuged for 1 min. at 1500 rpm and the supernatant Ether phase removed by suction. Color was then developed in the buffer solution. This double extraction was necessary to remove blank interference.
Standard curves were run in all tissues. Recoveries were in the same range as those from water, urine and plasma.

Statistics:

None

Preliminary studies:

No data

Details on absorption:

Not determined

Details on distribution in tissues:

For details please refer to "remarks on results incl. tables and figures"; table 1

Appreciable Methylparaben is only found in the brain and spleen. High "total" concentrations are found in the liver and kidney and high concentrations of free p-Hydroxybenzoic acid are detected in the kidney. Other than these instances, levels in the various organs are below plasma levels.

Test no.:

#1

Transfer type:

blood/brain barrier

Observation:

slight transfer

Details on excretion:

Excellent recoveries of "total" material were noted from the urine of dogs having received intravenous and oral doses of Methylparaben. These ranged from 85 to 89% indicating that the major portion of the test item which is metabolised and excreted by the body retains the phenolic structure. Most of the material is excreted by 6 hours after injected doses whereas appreciable amounts are still detected in the urine 30 hours after oral ingestion with the greater part of Methylparaben being excreted between 6 and 24 hours. These results, along with data obtained from the plasma concentrations after oral feeding, seem to indicate that absorption is essentially complete.
Please refer to table 2.

Metabolites identified:

yes

Details on metabolites:

Metabolism mechanisms seem to proceed efficiently immediately after ingestion of the test item. The concentration of the p-Hydroxybenzoate in the plasma only rarely surpasses the lower limit of sensitivity of the determinations even immediately following intravenous injections. Significant levels were found only in the plasma of some dogs a few hours after oral administration. The amount of free p-Hydroxybenzoate excreted never exceeds 0.5% regardless the method of dosage. The bulk of the "total" material seems to be excreted in the conjugated form. This conjugation may take place before or after hydrolysis and the biochemical methods employed cannot disitnguish between the 2 metabolic routes. Rapid hydrolysis is indicated since after intravenous doses of the compounds practically as much free p-Hydroxybenzoic acid is excreted as when the acid itself is injected.
After intravenous administration the values for free p-Hydroxybenzoic acid and "total" material in the plasma are similar, but the "total" value remains high for a longer period of time. From this it would seem that the first detoxication mechanism called upon is one of hydrolysis.
Please refer to table 3.

Table 1:  Distribution of Methylparaben and its metabolic product in tissues of dogs, infused intravenously at a rate of 2 mg/kg bw/min for 50 min.

Tissue	Concentration in Tissue (mg / g or mL)
	Concentration of p-Hydroxybenzoic acid	Concentration of Methylparaben	Concentration of “Total”
Liver	74	0	520
Kidney	190	0	520
Brain	14	10	32
Spleen	36	6	56
Pancreas	-	-	-
Muscle	12	0	34
Plasma	155	0	265

Table 2:  Absorption and excretion of Methylparaben

a.      Average of 3 dogs given 1 g/kg bw orally (plasma levels)

Time	mg/mL , Plasma Levels
	Paraben	Free p-Hydroxybenzoic acid	Total
5 min	-	-	-
15 min	0	15	17
30 min	-	-	-
1 hr	0	110	273
2 hr	0	293	437
4 hr	0	553	827
6 hr	0	630	867
24 hr	0	19	77
48 hr	0	0	3

 

b.     Average of 3 dogs given 1 g/kg bw orally (urinary levels)

Time	Dose excreted (accumulative), %
	Paraben	Free p-Hydroxybenzoic acid	Glucuronic acid Conjugate	Total
5 min	-	-	-	-
15 min	-	-	-	-
30 min	-	-	-	-
1 hr	0.003	0.7	0.5	8.1
2 hr	0.005	2.3	2.1	14.4
4 hr	0.005	6.2	4.9	15.5
6 hr	0.014	11.0	28.8	32.0
24 hr	0.014	21.2	33.1	66.1
48 hr	0.014	21.3	35.1	89.0

 

c.      Average of 3 dogs given 50 mg/kg bw i.v. (plasma levels)

Time	mg/mL , Plasma Levels
	Paraben	Free p-Hydroxybenzoic acid	Total
5 min	0	82	115
15 min	0	68	120
30 min	0	48	115
1 hr	0	19	113
2 hr	0	12	61
4 hr	0	4	27
6 hr	0	0	12
24 hr	0	0	0
48 hr	0	0	0

 

d.     Average of 3 dogs given 50 mg/kg bw orally (urinary levels)

Time	Dose excreted (accumulative), %
	Paraben	Free p-Hydroxybenzoic acid	Glucuronic acid Conjugate	Total
5 min	-	-	-	-
15 min	-	-	-	-
30 min	-	-	-	-
1 hr	0.03	8.4	4.3	14.1
2 hr	0.03	12.4	9.5	28.6
4 hr	0.08	18.1	28.5	67.3
6 hr	0.10	20.6	28.9	80.0
24 hr	0.58	20.6	32.0	85.0
48 hr	0.58	20.6	32.0	85.0

Table 3:  Rate of hydrolysis of Methylparaben by dog tissue mashes. Test item concentration 100 mg/mL, tissue diluted 1:200 and reaction carried out at room temperature

Tissue	Time [min]	Compound hydrolysed [%]
Liver	3	100
	30	100
	60	100
Brain	3	0
	30	0
	60	0
Kidney	3	72
	30	100
	60	100
Muscle	3	0
	30	16
	60	18
Spleen	3	0
	30	0
	60	0
Pancreas	3	0
	30	0
	60	0
Plasma	3	0
	30	0
	60	0
Whole blood	3	0
	30	0
	60	0

 

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
High plasma levels and urinary output of free and conjugated p-Hydroxybenzoic acid (metabolite of Methylparaben) in dogs indicate that hydrolysis of the ester linkage and metabolic conjugate constitute the main paths of alteration for Methylparaben. Excretion via urine is almost complete 48 hours after application.

Executive summary:

Please refer to "Distribution in tissues", "Excretion" and "Details on metabolites"

Reference 8

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Study period:

Sep 2019 - Sep 2020

Reliability:

1 (reliable without restriction)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; female animals were non-pregnant and nulliparous
Age at the start of the treatment period: approximately 11-12 weeks old
Body weight at the allocation of the animals to the experimental groups: males: 304 – 431 g (mean: 383.05 g, ± 20% = 306.44 – 459.66 g) females: 182 – 252 g
(mean: 223.70 g, ± 20% = 178.96 – 268.44 g)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals are bred for experimental purposes. This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.
Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking
water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals / sex / group / cage in IVC cages
(type IV, polysulphone cages) on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and
afterwards archived at Eurofins Munich
- Adequate acclimatization period (at least five days)

Route of administration:

oral: gavage

Vehicle:

other: 1% hydroxyethyl-cellulose

Details on exposure:

1% hydroxyethyl-cellulose (viscosity 80-125 cP, 2% in water at 20 °C)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

80 male and 80 female animals were used for the study (10 animals per group for each test item and each concentration).

Details on dosing and sampling:

The study was conducted with 2 groups per test item. Animals were treated orally by gavage. Methyl 4-hydroxybenzoate, Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate or Butyl 4-hydroxybenzoate was administered once. Due to the high number of animals and the sampling time points the dosing and the blood sampling of all test items was not taken place in the same week. Propyl 4-hydroxybenzoate were tested in parallel with Butyl 4-hydroxybenzoate in study week 1 and Methyl 4-hydroxybenzoate were tested in parallel with Ethyl 4-hydroxybenzoate in study week 5. At the end of the test all surviving animals were euthanised.
In all groups the test item formulations was administered once by gavage. The application volume was 5 mL/kg body weight.
For pharmacokinetic evaluation after single oral administration, blood was sampled in the week before administration and at different time points following the administration Four time points was investigated per rat. The investigated time points are 5 min, 10 min, 15 min, 30 min, 60 min, 4 h and 8 h after administration.

Type:

distribution

Type:

metabolism

Details on distribution in tissues:

individual plasma concentrations of Methyl 4-hydroxybenzoate were all above the quantification limit (10 ng/mL) until 8 hours after dosing. Mean maximum plasma concentrations and overall exposure increased with increasing doses. This increase was less than dose-proportional for Cmax in females and males, roughly dose-proportional for AUC0-t in females, and more than dose-proportional for AUC0-t in males. In general, mean maximum plasma concentrations and overall exposure appeared to be higher in females than in males. Within the first hour after dosing mean plasma concentrations had decreased to 1.6 - 5.1% of the mean maximum concentrations. The exposure over the first 1 h after dosing (AUC0-1h) was between 55.4 and 75.4% of mean AUC0-t(in both genders).

Metabolites identified:

yes

Details on metabolites:

Mean Cmax of 4-Hydroxybenzoic acid were generally observed between 10 and 30 min after dosing of Propyl 4-hydroxybenzoate, Butyl 4-hydroxybenzoate, Methyl 4-hydroxybenzoate or Ethyl 4-hydroxybenzoate with the exception of Butyl 4-hydroxybenzoate after 1000 mg/kg where highest concentrations were observed at the last sampling time point (8 hours). In all treatment groups, individual animals showed 4-Hydroxybenzoic acid concentrations above the
BLQ (130 ng/mL) from 10 min until 8 hours after dosing. The highest exposure of 4-Hydroxybenzoic acid was observed in males and females after dosing of Methyl 4-
hydroxybenzoate followed by Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate, and
Butyl 4-hydroxybenzoate. The highest mean maximum plasma concentration was found
in females after dosing of Ethyl 4-hydroxybenzoate followed by Methyl 4-
hydroxybenzoate, Propyl 4-hydroxybenzoate, and Butyl 4-hydroxybenzoate, and in
males after dosing of Methyl 4-hydroxybenzoate followed by Ethyl 4-hydroxybenzoate,
Propyl 4-hydroxybenzoate, and Butyl 4-hydroxybenzoate. Generally, mean maximum
plasma concentrations and overall exposure of 4-Hydroxybenzoic acid increased with
increasing dose of the test items. There appeared to be no obvious trend for sex
differences in Cmax and AUC0-t after dosing of either test item.

Conclusions:

On the basis of this single oral dose toxicity study in rats with Methyl 4- hydroxybenzoate, Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate and Butyl 4-
hydroxybenzoate with male and female Wistar rats with a dose level 500 mg/kg body weight and 1000 mg/kg body weight Including Pharmacokinetic the following
conclusions can be made:
The test item was well tolerated by all animals. No mortality or clinical signs of toxicity were observed during the entire study period. After a single oral administration of 500 or 1000 mg/kg, Methyl 4-hydroxybenzoate, Ethyl 4-hydroxybenzoate, Propyl 4-hydroxy-benzoate and Butyl 4-hydroxybenzoatewere rapidly absorbed with mean maximum plasma concentrations observed between 5 and 15 min post dosing. Afterwards, mean concentration-time profiles revealed a multiphasic
behaviour with a rapid decline up to 1 hour and a plateau close to the detection limit between 1 and 8 hours. The course of the mean plasma concentrations did not allow extrapolation of the apparent terminal phase and thus, reliable estimation of t1/2 and AUC0-inf was not possible. Fast decline of all four tested parabens was accompanied by rapid onset of 4- Hydroxybenzoic acid indicating an efficient and comparable metabolism. In fact, 1 h after dosing mean plasma concentrations of all four test compounds had decreased to less than 10% of the maximum concentration. Furthermore, a substantial portion of the overall exposure was seen within the first hour after dosing.

Executive summary:

The purpose of this study was to obtain information on the pharmacokinetic of Methyl 4- hydroxybenzoate, Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate and Butyl 4- hydroxybenzoate in rats after oral administration. The study was conducted with 2 groups per test item. Animals were treated orally by gavage once. The 8 groups comprised 10 male and 10 female Wistar rats. The test item formulation was prepared freshly on the day of administration. The test item was dissolved in 1 % aqueous hydroxyethyl-cellulose, the vehicle used in this study Dose volumes were adjusted individually based on body weight measurement. The following doses were evaluated:

Low Dose: 500 mg/kg body weight

High Dose: 1000 mg/kg body weight

The animals were observed precisely during the test period for signs of toxicity. Blood was collected at different time points to investigate the pharmacokinetic. At the end of the test all surviving animals were euthanised.

No mortality or clinical signs of toxicity occurred in any of the experimental groups and the mean body weight values were in a normal range. Propyl 4-hydroxybenzoate (Groups 1 and 2): Individual concentrations above the quantification limit (10 ng/mL) were seen for most of the females until 4 hours after dosing of 500 or 1000 mg/kg with detectable concentrations found in 2 animals at the latest sampling time point of 8 hours. In males, for some of the animals plasma levels were BLQ 15 min after dosing already, but others showed detectable plasma concentrations up to 4 hours after dosing at 500 mg/kg and up to 8 hours (1 animal only) after dosing of 1000 mg/kg. In females, mean maximum plasma concentrations (Cmax) and overall exposure (AUC0-t) were lower at the higher dose whereas in males a roughly dose-proportional increase in Cmax and AUC0-t was observed between 500 and 1000 mg/kg. One hour post dosing mean plasma concentrations had already decreased to 2.1 - 8.5% of the mean maximum concentrations and the exposure over the first 1 h after dosing (AUC0-1h) represents between 25.9 and 63.3% of the overall exposure during the sampling interval (AUC0-t) (in both genders). Butyl 4-hydroxybenzoate (Groups 3 and 4): Most of the females showed concentrations above the quantification limit (10 ng/mL) until 8 hours after dosing of 500 or 1000 mg/kg. Some of the males had levels BLQ at 1 hour after dosing already but others showed detectable levels until 8 hours after dosing of 500 or 1000 mg/kg. Mean Cmax increased less than dose-proportionally with dose in males and females, and AUC0-t revealed a roughly dose-proportional increase with doses between 500 and 1000 mg/kg with higher mean Cmax and AUC0-t in males than in females. One hour post-dosing mean plasma concentrations were only between 4.3 and 9.1% of the mean maximum concentrations. Further, AUC0-1h was between 42.5 and 54.5% of mean AUC0-t (in both genders). Methyl 4-hydroxybenzoate (Groups 5 and 6): Individual plasma concentrations of Methyl 4-hydroxybenzoate were all above the quantification limit (10 ng/mL) until 8 hours after dosing. Mean maximum plasma concentrations and overall exposure increased with increasing doses. This increase was less than dose-proportional for Cmax in females and males, roughly dose-proportional for AUC0-t in females, and more than dose-proportional for AUC0-t in males. In general, mean maximum plasma concentrations and overall exposure appeared to be higher in females than in males. Within the first hour after dosing mean plasma concentrations had decreased to 1.6 - 5.1% of the mean maximum concentrations. The exposure over the first 1 h after dosing (AUC0-1h) was between 55.4 and 75.4% of mean AUC0-t (in both genders). Ethyl 4-hydroxybenzoate (Groups 7 and 8): Individual plasma concentrations of Ethyl 4- hydroxybenzoate were all above the quantification limit (10 ng/mL) until 8 hours after dosing. There was no trend seen for dose-dependency of mean maximum plasma concentrations in females and males, and of AUC0-t in males, whereas in females mean AUC0-t tended to increase dose-proportionally. In general, mean maximum plasma concentrations and overall exposure were higher in females than in males. One hour after dosing mean plasma concentrations had decreased to between 2.0 and 5.9% of the mean maximum concentrations and exposure within the first hour after dosing represents between 50.0 and 70.2% of mean AUC0-t. In females, the mean maximum plasma concentration and overall exposure were highest for Methyl 4-hydroxybenzoate followed by Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate, and Butyl 4- hydroxybenzoate. In males, the highest mean Cmax and AUC0-t were also found for Methyl 4-hydroxybenzoate but followed by Butyl 4-hydroxybenzoate, Ethyl 4- hydroxybenzoate, and Propyl 4-hydroxybenzoate (in both genders). 4-Hydroxybenzoic acid (Groups 1 to 8): Mean Cmax of 4-Hydroxybenzoic acid were generally observed between 10 and 30 min after dosing of Propyl 4-hydroxybenzoate, Butyl 4-hydroxybenzoate, Methyl 4-hydroxybenzoate or Ethyl 4-hydroxybenzoate with the exception of Butyl 4-hydroxybenzoate after 1000 mg/kg where highest concentrations were observed at the last sampling time point (8 hours). In all treatment groups, individual animals showed 4-Hydroxybenzoic acid concentrations above the BLQ (130 ng/mL) from 10 min until 8 hours after dosing. The highest exposure of 4- Hydroxybenzoic acid was observed in males and females after dosing of Methyl 4- hydroxybenzoate followed by Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate, and Butyl 4-hydroxybenzoate. The highest mean maximum plasma concentration was found in females after dosing of Ethyl 4-hydroxybenzoate followed by Methyl 4- hydroxybenzoate, Propyl 4-hydroxybenzoate, and Butyl 4-hydroxybenzoate, and in males after dosing of Methyl 4-hydroxybenzoate followed by Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate, and Butyl 4-hydroxybenzoate. Generally, mean maximum plasma concentrations and overall exposure of 4-Hydroxybenzoic acid increased with increasing dose of the test items. There appeared to be no obvious trend for sex differences in Cmax and AUC0-t after dosing of either test item.

On the basis of this single oral dose toxicity study in rats with Methyl 4- hydroxybenzoate, Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate and Butyl 4- hydroxybenzoate with male and female Wistar rats with a dose level 500 mg/kg body weight and 1000 mg/kg body weight Including Pharmacokinetic the following conclusions can be made: The test item was well tolerated by all animals. No mortality or clinical signs of toxicity were observed during the entire study period. After a single oral administration of 500 or 1000 mg/kg, Methyl 4-hydroxybenzoate, Ethyl 4-hydroxybenzoate, Propyl 4-hydroxy-benzoate and Butyl 4-hydroxybenzoatewere rapidly absorbed with mean maximum plasma concentrations observed between 5 and 15 min post dosing. Afterwards, mean concentration-time profiles revealed a multiphasic behaviour with a rapid decline up to 1 hour and a plateau close to the detection limit between 1 and 8 hours. The course of the mean plasma concentrations did not allow extrapolation of the apparent terminal phase and thus, reliable estimation of t1/2 and AUC0-inf was not possible. Fast decline of all four tested parabens was accompanied by rapid onset of 4- Hydroxybenzoic acid indicating an efficient and comparable metabolism. In fact, 1 h after dosing mean plasma concentrations of all four test compounds had decreased to less than 10% of the maximum concentration. Furthermore, a substantial portion of the overall exposure was seen within the first hour after dosing.

Description of key information

Toxicokinetic data on methylparaben and its sodium salt have shown that it is completely absorbed after oral ingestion, hydrolysed to p-Hydroxybenzoic acid, conjugated and excreted in urine. There is no evidence of accumulation.

Additional information

There is only one study available on toxicokinetics for sodium methylparaben (Tsukamoto, 1960). However, there are reliable data on methylparaben which is considered suitable for read-across using the analogue approach.

Sodium methylparaben (Sodium methyl 4-hydroxybenzoate) is the sodium salt of methylparaben (methyl 4-hydroxybenzoate). The substance is highly water soluble (418 g/L), dissociates in aqueous solutions and is hydrolysed to sodium hydroxide and the source substance methylparaben. Based on the assumption that upon oral and dermal administration, sodium methylparaben dissociates and is hydrolysed to methylparaben and sodium hydroxide, it was predicted that after exposure the primary effect is local irritation/corrosion at the site of contact due to sodium hydroxide.

Target and source substance are of low acute toxicity. No systemic or local effects were found up to single doses of 5000 mg/kg bw for sodium methylparaben and 2100 mg/kg bw for methylparaben. A dermal absorption of 80% was calculated for sodium methylparaben using QSAR and the available physico-chemical properties. For methylparaben, the dermal absorption was estimated in an in vitro test to be 84.7% in human skin (Fasano, 2004). 85% as wosrt case consideration.

Therefore, it can reasonably be deduced that no higher amounts than tested in the acute oral toxicity study will be systemically available via the intact skin barrier. Inhalation is of no concern for the target as well as the source substance because of the low vapour pressure.

Available data on sodium methylparaben did not show reactive properties under in-vitro test conditions, i. e. Ames test and gene mutation in mammalian cells. Also, by modelling the likelihood of interactions of sodium methylparaben with skin proteins, no structural alerts were found (DR. KNOELL CONSULT, 2012). Data on methylparaben with respect to genetic toxicity in-vivo and skin sensitisation were all negative and are therefore comparable to that of sodium methylparaben. Thus, it is considered that there is no evidence for generation of the chemically reactive metabolites and for interactions with skin proteins for both the target and the source substance.

The results of several repeated dose toxicity studies indicate a low toxicological concern for methylparaben. Methylparaben caused no systemic toxicity and no effects on fertility or developmental toxicity (NOAEL >1000 mg/kg bw per day). Moreover, toxicokinetic data have shown that methylparaben is completely absorbed after oral and dermal administration, hydrolysed, conjugated and rapidly excreted in urine. Thus, there is no evidence of accumulation.

As bioavailability and metabolism and therefore mammalian toxicity of sodium methylparaben were considered to be comparable to that of methylparaben, the assessment of systemic toxicity based on analogue approach can be considered as justified.

Several studies have been identified that examine the basic toxicokinetics of Methylparaben:

- 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.

- High plasma levels and urinary output of free and conjugated p-Hydroxybenzoic acid (metabolite of Methylparaben) in dogs indicate that hydrolysis of the ester linkage and metabolic conjugate constitute the main paths of alteration for Methylparaben. Excretion via urine is almost complete 48 hours after application of 100 mg/kg bw to dogs.

- 1 mg Methylparaben/kg bw/d was applied orally to a dog for one year. The rate of urinary excretion in the dog had increased to such an extent that after 24 hours 96% of the dose was recovered in the urine.

- Methyl p-hydroxybenzoate sodium salt was administered to rabbits (800 mg/kg bw/d) and urine collected for 24 hours was examined for metabolites. After application of a single dose Methylparaben to rabbits the main metabolites found in urine were p-Hydroxybenzoic acid, p-Hydroxyhippuric acid and the ether-type glucuronide.

- Groups of 12 male and 12 female rats received a single dermal administration of 100 mg/kg bw [¹⁴C]-labelled Methylparaben. The substance was completely metabolised to 4-Hydroxybenzoic acid. No systemic exposure was detected to Methylparaben 8 h after dermal application.

Taking into account the results of all above mentioned ADME-studies, Methylparaben is considered to be metabolized to p-Hydroxybenzoic acid, which is conjugated to form the glucuronide rapidly and excreted completely within 24 hours after application. There is no tendency for bioaccumulation.

Three dermal absorption studies were performed applying Methylparaben:

- The penetration kinetics and first-pass metabolism of Methylparaben in viable rat (n=10 replicates) and human skin (n=13 replicates) has been determined. The active ingredient was formulated as oil-in-water emulsion at a target concentration of 0.8% and 0.4%. rat and human skin. Penetration was followed using [¹⁴C]-labeled active ingredient. The amount of active applied per area skin was approx. 65 µg/cm²and 36 µg/cm². 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.

- 24 hours after the application on human skin (25 µg/cm²) 33.4% of the applied Methylparaben was absorbed; the surface wash removed unabsorbed Paraben, which was 11.5% of the applied dose. The skin was analysed for Methylparaben and a level of 28.6% was recovered. 24 hours after application to minipig skin, 38.6% of the applied Methylparaben had been absorbed; the skin surface wash contained about 13.4%. 23.9% Methylparaben were extracted from the skin. The metabolite 4-Hydroxybenzoic acid was found in the receptor fluid: 18.1% of the applied amount was found.

- Methylparaben penetrated the dorsal guinea pig skin via non-polarstratum corneumlipid lamella as a rate limiting step for skin penetration. The enhancers tested increase skin penetration of Methylparaben by increasing the fluidity ofstratum corneumlipid lamella, which seems to lead to the increase of diffusion coefficient of the test item.

Methylparaben data:

- log K(p) full thickness (dorsal Guinea pig) skin: -0.9 cm/h

- log (p) lipid depleted (dorsal Guinea pig) skin: -2.25 cm/h

The addition of 1% l-Menthol in 15 % Ethanol increased the permeability coefficient of Methylparaben about 16 times. A similar, though weaker, tendency was observed in the presence of 15 % Ethanol itself without l-Menthol. N-dodecyl-2-pyrrolidone stimulated permeation of relatively hydrophilic Methylparaben. From these data can be concluded that Methylparaben penetrates human as well as rat and minipig skin. Up to 79 % of the applied amount is absorbed. Up to 35 % of the absorbed substance is metabolised to p-Hydroxybenzoic acid.