Benzyl benzoate

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published study

Data source

Reference

Reference Type:

publication

Title:

In vivo percutaneous absorption of fragrance ingredients in rhesus monkeys and humans

Author:

Bronaugh RL, Wester RC, Bucks D, Maibach HI a& Sarason R

Year:

1990

Bibliographic source:

Food and Chemical Toxicology, Vol 28, Issue 5, 36-373

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Dermal absorption in monkey skin in vivo.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl benzoate

EC Number:

204-402-9

EC Name:

Benzyl benzoate

Cas Number:

120-51-4

Molecular formula:

C14H12O2

IUPAC Name:

benzyl benzoate

Test material form:

other: liquids

Details on test material:

[14C]benzyl benzoate (7.2 mCi/mmol, purity 97%)

Radiolabelling:

yes

Test animals

Species:

monkey

Strain:

other: Rhesus

Sex:

female

Details on test animals or test system and environmental conditions:

Four female rhesus monkeys were used. The captive bred monkeys were 10, 14, 19 and 19 years old.

Administration / exposure

Type of coverage:

other: non-occluded, and two forms of occlusive dressing

Vehicle:

acetone

Duration of exposure:

24 hour

Doses:

Six adult human volunteers each received a single 20µL dose (0.91µCi) of[14C)diethyl maleate in acetone by topical application to 2.5 cm2 of forearm skin (chemical dose = 4µg/cm2). The site was then occluded by using a polypropylene chamber.

Monkeys were treated with each compound, applied to a lightly clipped 1cm2 area of abdominal skin at a concentration of 4 µg/cm2. The vehicle used was acetone (10-20 µl/cm2) or skin moisturiser in a volume of 10 or 30 mg/cm2.

No. of animals per group:

Four

Control animals:

no

Details on study design:

The human in vivo skin absorption studies were performed according to the procedure of Bucks et al. Six adult human volunteers each received a single 20µL dose (0.91µCi) of[14C)diethyl maleate in acetone by topical application to 2.5 cm2 of forearm skin (chemical dose = 4µg/cm2). The site was then occluded by using a polypropylene chamber. After 24 hr, the occlusive chamber was removed and the site of application was cleanedusing a standardised washing procedure (washing the application site with a liquid soap (dishwashing detergent) and water solution (50: 50, v/v) followed by a water rinse and a second soap and water wash followed by two rinses with water. The wash solution was applied to a rayon-cotton ball attached to a pair of curved forceps. The application site was wiped with the washing-solvent-laden rayon-cotton ball) and was then re-occluded with a new plastic chamber for the remaining 6 days.

Percutaneous absorption in humans was determined by measuring the radioactivity excreted in the urine samples collected each day, using liquid scintillation counting and then correcting the amount of radioactivity for complete excretion with a parenteral correction factor.

[14C]Diethyl maleate was administered parenterally to four monkeys and urinary excretion of the dose was 90.9 ±1.4%. Human topical values were divided by this percentage to give total percutaneous absorption.

Monkey skin absorption studies were conducted according to the procedure of Wester and Maibach (1975). Four female rhesus monkeys were used for each study. The captive bred monkeys were 10, 14, 19 and 19 years old. Each compound was applied to a lightly clipped 1cm2 area of abdominal skin at a concentration of 4 µg/cm2. The vehicle used was acetone (10-20 µl/cm2) except where noted that a skin moisturiser was used in a volume of 10 or 30 mg/cm2. The monkeys were restrained in metabolic chairs for 24 hr, at which time the application site was washed with soap and water as described above to remove residual material. The animals were then placed in metabolism cages to continue the urine collection for an additional 4 days. The amount of absorbed compound in the urine was determined by liquid scintillation counting, and results were corrected for total absorption as above by using a parenteral correction factor obtained in separate experiments.

The application sites were occluded by one of two different methods. In some experiments, a plastic wrap (Saran Wrap) was held in place over the application site with adhesive tape. In other experiments, a glass chamber was glued to the skin around the application site with silicone cement and the top of the chamber covered with Parafilm.

The relative volatility of the fragrance materials was determined. Each compound was applied, in 15 µL acetone, to a teflon disc (surface area = 1cm2). After the acetone had evaporated (in less than 5 min) the discs were added to scintillation cocktail at 5 min, 3 hr and 24 hr to determine the remaining radioactivity.

The octanol-water partition coefficient (Ko/w) for benzoin was determined by adding approximately 1 µCi (14C]benzoin to a flask containing 5 ml each of octanol and water. The flask was shaken overnight to achieve an equilibrium distribution between the two liquids. Aliquots of each liquid were removed and radioactivity was determined by liquid scintillation counting.

Details on in vitro test system (if applicable):

Not applicable

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

Benzyl benzoate dermal absorption measured in vivo in Rhesus monkeys was circa 70%. This value was significantly higher than human skin membrane measurements conducted under non-occluded conditions

Total recovery:

The means ± SEM of these values for urinary excretion of the dose obtained in four monkeys were as follows:
benzamide, 77.4 ±4.3%; benzyl alcohol, 56.5 ±7.7%; benzoin, 87.4 ±4.4%; benzyl acetate, 65.9 ±3.1%; benzophenone, 92.6 ±3.4% and benzyl benzoate, 65.3 ±13.4%.

Conversion factor human vs. animal skin:

Percutaneous absorption in humans was determined by measuring the radioactivity excreted in the urine samples collected each day, using liquid scintillation counting and then correcting the amount of radioactivity for complete excretion with a parenteral correction factor.

[14C]Diethyl maleate was administered parenterally to four monkeys and urinary excretion of the dose was 90.9 ±1.4%. Human topical values were divided by this percentage to give total percutaneous absorption.
The amount of absorbed compound in the urine was determined by liquid scintillation counting, and results were corrected for total absorption as above by using a parenteral correction factor obtained in separate experiments.

The means ± SEM of these values for urinary excretion of the dose obtained in four monkeys were as follows:
benzamide, 77.4 ±4.3%; benzyl alcohol, 56.5 ±7.7%; benzoin, 87.4 ±4.4%; benzyl acetate, 65.9 ±3.1%; benzophenone, 92.6 ±3.4% and benzyl benzoate, 65.3 ±13.4%.

Any other information on results incl. tables

The absorption of the fragrance diethyl maleate was comparedin vivoin human and monkey studies after its application in an acetone vehicle. Occlusion of the site of application with either plastic wrap or a glass chamber covered with Parafilm resulted in a similar two-to three-fold increase in absorption. The absorption of diethyl maleate through human skin under occluded conditions was not significantly different from permeation of the compound through occluded monkey skin. The time course of absorption of diethyl maleate through human and monkey skin showed that absorption was essentially complete within 24 hr with monkey skin. However, when diethyl maleate was applied to human skin, a significant amount (17% of the amount absorbed) was excreted during the second day after application. Comparisons of diethyl maleate absorption in monkey and human dermis under occludedconditions were used as the basis for equating absorption of the remaining test compounds in monkey skin assays with in vivo human absorption under occluded conditions.

 

The chemical properties of the homologous series of benzene fragrances are given in Table 1. These compounds have low molecular weights, ranging from 108 to 212 and the octanol-water partition coefficients range from 4.4 (benzamide) to 9333 (benzyl benzoate).

 

The relative volatility of the fragrances was measured to assist in the interpretation of the skin penetration results. Evaporation was determined at various times after application of each fragrance to a teflon disc with a surface area of approximately 1cm²(Table 2). For three compounds (benzyl alcohol, benzyl acetate and benzyl benzoate), loss of radioactivity from the teflon disc occurred in the first 5 minutes after application, possibly during evaporation of the acetone vehicle. After 3 hr, maximum evaporation had occurred for benzyl alcohol (75%) and benzyl benzoate (45%).

 

For the other compounds, maximum loss due to evaporation was not evident until the 24-hr measurement.

The evaporation ofbenzophenone was the most complete with a loss of 98.9% of the applied radioactivity within 24 hr.

 

Values were obtained for percutaneous absorption through non-occluded and occluded monkey skin (Table 3). Penetration through non-occluded skin was greater than 30% of the applied dose for all test compounds, but it was difficult to make comparisons because of the differences in the rates of evaporation of the fragrances.

Therefore, the application sites were occluded, initially with plastic wrap fixed to skin with adhesive tape. For two compounds (benzyl acetate and benzoin), absorption was actually less in the occluded studies; this prompted the use of a chamber glued to the skin to support an occlusive covering. Absorption values obtained when the glass chamber was used for occlusion were uniformly higher than values for non-occluded skin. When evaporation of the fragrances was prevented, no significant difference in percutaneous absorption was observed (ANOVA) for the test compounds.

 

The absorption of three fragrances was also examined after their application in a moisturizing lotion. Benzyl alcohol and benzyl benzoate were applied to skin in 10 mg lotion/cm²and the absorption of each fragrance appeared to be increased when applied in the lotion compared with absorption after application in the acetone vehicle (the increase was significant for benzyl benzoate, t-test, P < 0.05). Benzyl acetate was applied in an excess amount of lotion (30 mg/cm²), which resulted in no significant increase in absorption when compared with absorption after application in an acetone vehicle (t-test, P > 0.05).

 

Table1: chemical properties of the tested fragrance

Compound	R group	Ko/w	Water solubility (g/L)	Molecular weight
Benzamide	ONH2	4.4	13.5	121
Benzyl alcohol	H2OH	7.4	40	108
Benzoin	HOHOCC6H5	22.5	0.3	212
Benzyl acetate	H2OOCCH3	91.2	Practically insoluble	150
Benzophenone	OC6H5	1514	Insoluble	182
Benzyl benzoate	H2OOCC6H5	9333	Insoluble	212

 

Table 2: percentage losses from a 1 cm²Teflon disc

Compound	Percentage loss of fragrance from Teflon disc after
	5 min	3 hr	24 hr
Benzamide	0	2.5	21.6
Benzyl alcohol	10.3	75.2	78.7
Benzoin	0	8.3	28.9
Benzyl acetate	3.9	27.5	35.0
Benzophenone	0	56.8	98.9
Benzyl benzoate	2.3	44.5	44.7

An acetone solution of each fragrance was applied to a Teflon disc, the acetone evaporated within 5 minutes

 

Table 3: Percutaneous absorption of fragrances in the Rhesus monkey

Compound
	Unoccluded site	Site occluded with
		Plastic wrap	Glass chamber
Benzamide	46.5	85.1	73.2
Benzyl alcohol	31.6	56.3	79.9
Benzoin	48.5	42.6	77.2
Benzyl acetate	34.6	17.3	78.7
Benzophenone	43.8	68.7	68.6
Benzyl benzoate	57.0	71.2	64.7

 

Applicant's summary and conclusion

Conclusions:

Occluded topical application in vivo to monkey skin resulted in dermal absorption of benzyl benzoate of circa 70%.

Executive summary:

The percutaneous absorption of fragrance components including benzyl benzoate (14C-radiolabelled) was determined in vivo in monkeys. Absorption through occluded skin was high (approximately 70% of the applied dose in 24 hr). No correlations were seen between skin penetration of the compounds investigated and their octanol-water partition coefficients. Under unoccluded conditions skin penetration of the fragrances was reduced and there was great variability between compounds, presumably because of variations in the rates of evaporation from the site of application. The data suggest that humans may have significant systemic exposure to these fragrance materials.