Fatty acids, C16-18, sodium salts

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication which meets basic scientific principles.

Data source

Reference

Reference Type:

publication

Title:

The percutaneous absorption of some anionic surfactants

Author:

D. Howes

Year:

1974

Bibliographic source:

J. Soc. Cosmet. Chem. 26, 47-63 (1975)

Report date:

1974

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The percutaneous absorption of some [14C] labelled anionic surfactants has been measured in vivo in rats, after both consumer-type applications and applications of longer duration, and the results have been compared with those from in vitro studies using isolated rat skin and human epidermis.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

hexadecanoic acid (C16:0)

IUPAC Name:

hexadecanoic acid (C16:0)

Constituent 2

Reference substance name:

octadecanoic acid (C18:0)

IUPAC Name:

octadecanoic acid (C18:0)

Details on test material:

Surfactants used:
- Decanoic acid (C10:0)
- Dodecanoic acid (12:0)
- Tetradeccanoic acid (C14:0)
- Hexadecanoic acid (C16:0)
- Octadecanoic acid (C18:0)
All test substances were converted to their sodium salts by neutralization to pH 9.5 with sodium hydroxide.
These acids were also obtained [1-14C] labbeld. These labelled acids were incorporated into a model soap system.

- Sodium [1-14C] dodecyl sulphate; 5.11 µCi/mg (14C-SDS), sodium [1-14C] dodecoyl isethionate; 1.7 µCi/mg (14C-SDI) and sodium p-1-[1-14C] dodecylbenzenesulphonate; 8.5 µCi/mg (14C-DOBS) were sythesized.

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: Colworth-Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

weight: 100-120g

Administration / exposure

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

In vitro penetration through rat skin:
Female rats were clipped to exposed dorsal skin 24 h before cervical dislocation. The skins were excised and mounted in 2.5 cm diameter penetration cells. 0.25 ml of the [14C] surfactant solution was pipetted onto the epidermal surface of the skin and 10.0 ml of saline was added to the sampling compartment against the dermis. The cells were kept in a warm room at 37°C throughout the experiment and the saline was magnetically stirred continuously. The saline was monitored hourly for 14 by removing 1.0 ml and replacing with fresh saline maintaining the volume of 10.0 ml in the sampling compartment. After 24 h the epidermal surface was washed with excess of distilled water and was monitored for 14C.

In vitro penetration through human epidermis:
Female abdominal skin samples obtained at autopsy were frozen and stored at -70°C. Samples of the skin were allowed to thaw out and were heated at 58°C for 2 min and the epidermis removed in sheets. The epidermal samples were mounted in 1 cm diameter penetration cells. Saline containing 0.012% Penicillin and 0.01% Streptomycin was placed in contact with both surfaces of the sample and the cells was measured and only cells with a resistance greater than 50000 ohm were used. The saline from the corneum surface was removed and 0.1 ml of the [14C] surfactant solution was placed on the corneum. 1.0 ml aliquots of the saline in the sampling compartment (8.0ml) were monitored for 14C at 0.5, 1, 2, 3, 4, 6, 7, 8, 24 and 48 h, each time 1.0 ml fresh saline was added to maintained the volume at 8.0ml. At the end the corneum was washed with excess of distilled water and was monitored for 14C.

Turnover of surfactants:
The turnover of each [14C] labeled surfactant was measured by injecting 3 animals intraperitoneally and 3 animals subcutaneously with 0.1 or 0.5 ml of surfactant solution. the animals were then placed in sealed metabolism cages where urine, faces and expired air were collected and monitored for 14C. After 6 or 24 h the animals were killed by cervical dislocation. The carcasses of the animals were homogenized in an Atomix blender and aliquots of the homogenate were freeze dried.

Percutaneous absorption:
The hair from animals`backs was removed with fine bladed clippers 24 h before topical application. Only animals with visibly undamaged skin were used in the topical studies and all animals were lightly anaesthetized during the treatment.
Topical application of 0.1 or 0.5 ml of the [14C] test solution was made from a microlitre syringe on to an area of skin (7.5 or 10 cm²) previously marked out on the animal’s back with a felt-tipped pen. The solution was lathered over the treatment area with a rounded glass rod for 1 min during application. After 15 min contact with the skin the animal was inverted over a 6-inch diameter funnel and the excess of the test solution was rinsed off with distilled water at 37°C. After about 50 ml of water had been used the treated area of skin was lightly drawn over the top of the funnel to squeeze excess of rinse water from the skin. This process was then repeated and the skin dried with paper tissues. The animals were then fitted with either restraining collars or non-occlusive protection patches and placed in metabolism cages for collection of excreta as described above.
Before homogenizing the carcasses, the protective patch was removed and the treated area of skin was excised and frozen between glass plates. Punch autopsies from the frozen skin were monitored for 14C.

Duration and frequency of treatment / exposure:

In vitro penetration through rat skin: 24 h
In vitro penetration through human epidermis: 48 h
Turnover of surfactants: 6 or 24 h
Percutaneous absorption: 15 min

Doses / concentrations

Remarks:

Doses / Concentrations:
In vitro penetration through rat skin: 0.25 ml of the [14C] surfactant solution
In vitro penetration through human epidermis: 0.1 ml of the [14C] surfactant solution
Turnover of surfactants: 0.1 or 0.5 ml of the [14C] surfactant solution
Percutaneous absorption: 0.1 or 0.5 ml of the [14C] surfactant solution

No. of animals per sex per dose / concentration:

no data

Control animals:

not specified

Positive control reference chemical:

no data

Details on study design:

see above

Details on dosing and sampling:

see above

Statistics:

no data

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

see below

Details on distribution in tissues:

see below

Details on excretion:

see below

Metabolite characterisation studies

Metabolites identified:

not specified

Any other information on results incl. tables

In vitro penetration of [14C] surfactants through rat skin and human epidermis:

The results show no meassurable penetration of SDS, SDI, DOBS or the C18:0 soap through rat skin up to 24 h after application, but 0.2 µg/cm² of the C16:0 soap had penetrated at 24 h. Some 7.5 µg of the C10:0, C12:0 and C14:0 soaps had penetrated per cm² at 24 h but the results were not significantly different for the three soaps. For the 3 soaps which penetrated the skin there was a lag time of 1 h before any measurable penetration occured, but after this the rate of penetration steadily increased. At the end of the experiment, i.e. 24 h after apllication, between 60 and 70% of the applied [14C] soaps and [14C] SDI were rinsed from the skin and 30 -40% was associated with the skin. the [14C]SDS and DOBS were less easily rinsed from the skin as only 30% was recovered in the rinsings and 70% reamined associated with the skin.

The results from hte human epidermis experiments showed no measurable penetration of the [14C] DOBS and no measurable penetration of the [14C] SDS until 24 h after application when the rate of penetration was rapidly increasing so that at 48 h, 87.2 +/-24.1 µg/cm² had penetrated. the [14C]SDI showed a steadily increasing rate of penetration up to 48 h. The penetration of the [14C] soaps showed different rates of penetration which ranked C12:0 > C10:0 >C14:0 >C16:0 >C18:0. The in vitro penetration of the C16:0 soap through human skin was 0.3 µg/cm2 after 24 hours and of the C18:0 soap 0.1 µg/cm2. All of the surfactants which penetrated the epidermis showed increasing rates of penetration over the duration of the experiment which probaly reflects the ssurfactant/stratum corneum interaction and the breakdown of the barrier properties. This effect was most marked for the SDS where no penetration was detected during the first 8 h of contact. all of the epidermal samples showed some degree of swelling after 48 h contact but this was most marked with the SDS treated samples.

The amount of [14C] surfactant adsorbed to the epidermis was highest for the SDS where some 75% of the applied 14C was not removed by rinsing. for the other surfactants 30 -50% of the applied 14C was retained in the epidermis after rinsing.

In vitro penetration os surfactants through human epidermis and rat skin (µg/cm²):

			Time after application (h)
			Rat skin			Human epidermis
[14C] surfactant		Conc. (mg/ml)	2	6	24	2	6	24	48
Soaps	C10:0	1.2	0.2+/-0.1	1.1+/-0.2	8.6+/-3.4	0.2+/-0.1	1.3+/-0.6	16.8+/-5.1	-
	C12:0	1.3	0.2+/-0.1	1.0+/-0.3	7.2+/-3.7	0.4+/-0.2	4.9+/-0.2	31.7+/-8.4	-
	C14:0	1.5	0.2+/-0.1	1.0+/-0.2	6.9+/-3.0	<0.1	0.6+/-0.2	9.6+/-3.6	-
	C16:0	1.7	<0.1	<0.1	0.2+/-0.1	<0.1	<0.1	0.3+/-0.2	-
	C18:0	1.8	<0.1	<0.1	<0.1	<0.1	<0.1	0.1+/-0.1	-
SDS		7.3	<0.1	<0.1	<0.1	<0.1	<0.1	3.9+/-3.6	87.2+/-24.1
SDI		9.8	<0.1	<0.1	<0.1	0.4+/-1.7	3.0+/-1.7	8.4+/-3.4	30.1+/-13.6
DOBS		1.2	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1

Turnover of surfactants in the rat:

The rate and route of excretion of 14C from intraperitoneally administered [14C] surfactant solutions were the same as that from subcutaneously administered solutions. The recoveries are given below:

		Dose		% Apllied dose
[14C] surfactant		µCi	mg	CO2	Urine	Faeces	Carcass
Soaps	C10:0	7.25	1.2	57+/-5	<0.1	<0.1	37+/-6
	C12:0	10.49	1.3	65+/-7	<0.1	<0.1	30+/-7
	C14:0	8.13	1.5	5+/-3	2.1+/-1.2	<0.1	85+/-9
	C16:0	7.74	1.7	21+/-4	<0.1	<0.1	71+/-8
	C18:0	8.59	1.8	38+/-9	<0.1	<0.1	56+/-16
SDS		16.60	7.3	1.5+/-0.4	77+/-4	2.6+/-0.7	15+/-3
SDI		8.29	9.8	80+/-7	2.7+/-0.2	1.7+/-0.5	12+/-5
DOBS		8.69	1.2	<0.1	78+/-4	1.5+/-0.6	22+/-5

These results showed that at 6 h after administration the C10:0 and C12:0 soaps were readily metabolized and the main route of excretion was 14CO2. The C14:0 soap was readily incorporated into the body and the 14C excretion was slow. the C16:0 and C18:0 soaps showed some metabilism with subsequent 14CO2 excretion but most of the 14C was recovered in the carcass at 6 h. For both the [14C] SDS and DOBS most of the administered 14C was recovered in the urine at 24 h after dosing. the [14C] SDI was metabolized and most of the dose was recovered as 14CO2 at 24 h indicating the breakdown of the isethionate ester link. From the results the route of excretion of 14C surfactant giving the most sensitive indication of percutaneously abssorbed surfactant was indicated.

Absorption of [14C] sopas through rat skin in vivo:

The amount of the [1 -14C] labelled soaps penetrating through 7.5 cm² of treated skin was calculated from the levels of 14C recovered in the expired CO2, urine, faeces and in the carcass.

Traces of 14C were seen in the epidermis from all the soaps but only with the C12:0 and C14:0 soaps could detectable amounts of 14C be seen in the upper regions of the dermis. The amounts of [14C] soap present in the skin at 6 h after application were between 2 and 5 µg/cm² of skin, but there were no statistically significant differences between the 5 sopas even after prewashing regime with unlabelled soap solution.

		Amount penetrating over 7.5 cm² of skin (µg)
		Number of prewashes with 300 mM soap solution
[1-14C] Soap	Application (µg)	0	1	3
C10:0	116	1.78 +/-0.70	2.99+/-1.71	8.92 +/-4.26
C12:0	131	5.06 +/-2.59	5.29 +/-3.44	9.04 +/-2.57
C14:0	150	2.04 +/-0.39	1.52 +/-0.42	1.60 +/-0.18
C16:0	167	0.53 +/-0.18	0.55 +/-0.17	0.63 +/-0.16
C18:0	184	0.53 +/-0.14	0.36 +/-0.13	0.35 +/-0.02

The results showed an increase in the penetration of C10:0 and C12:0 and no significant chnage for the other soaps.

Absorption of [14C] SDS, SDI and DOBS through rat skin in vivo:

Autoradiography of the skins sshowed heavy deposition of all 3 of the surfactants on the skin surface and in the upper regions of the hair follicles. Only [14C]SDS was seen in the lower regions of the hair follicles but some of the autoradiograms showed visible amounts in the dermis.

The recoveries from all the tissue and excreta samples examined for 14C are shown below:

Surfactant	Application (µg)	Area of skin treated (cm²)	Rinsings (µg)	Skin levels (µg/cm²)	Protective patch (µg)	Penetration (µg/cm²)
[14C] SDS	3640	10	1929 +/-90	202+/-37	36+/-16	0.26+/-0.09
[14C] SDI	4900	10	4297+/-353	75+/-18	5	<0.3
[14C] DOBS	250	7.5	135+/-27	11+/-4	<2	<0.1

These results show that only small amounts of the applied surfactants penetrate the skin although conssiderable amounts are deposited on the skin. the level of 14C in the expired CO2 of the [14C] SDI treated animals was very low and from these levels the amount penetrating were shown to be <0.3 µg/cm but >0.1 µg/cm². No 14C was detected in any of the excreta from the DOBS treated animals.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Because of the short test time of the turnover of [14C] surfactants in rats of only six hours, a bioaccumulation potential cannot be judged based on this study results. The turnover of the C16:0 and C18:0 soaps were slow. But for a statement of the bioaccumulation potential, the test would have to run longer than 6 hours.

Executive summary:

The percutaneous absorption of some [14C] labelled anionic surfactants has been measured in vivo in rats, after both consumer-type applications and applications of longer duration, and the results have been compared with those from in vitro studies using isolated rat skin and human epidermis.

In vitro penetration through rat skin:

Rat skin was excised and mounted in 2.5 cm diameter penetration cells. 0.25 ml of the [14C] surfactant solution was pipetted onto the epidermal surface of the skin and 10.0 ml of saline was added to the sampling compartment against the dermis. The cells were kept in a warm room at 37°C throughout the experiment for 24 hours.

In vitro penetration through human epidermis:

The human epidermal samples were mounted in 1 cm diameter penetration cells. 0.1 ml of the [14C] surfactant solution was placed on the corneum. 1.0 ml aliquots of the saline in the sampling compartment (8.0ml) were monitored for 14C at 0.5, 1, 2, 3, 4, 6, 7, 8, 24 and 48 h, each time 1.0 ml fresh saline was added to maintained the volume at 8.0ml.

Turnover of surfactants:

The turnover of each [14C] labeled surfactant was measured by injecting 3 animals intraperitoneally and 3 animals subcutaneously with 0.1 or 0.5 ml of surfactant solution. The animals were then placed in sealed metabolism cages for 6 or 24 h, where urine, faces and expired air were collected and monitored for 14C.

Percutaneous absorption:

Topical application of 0.1 or 0.5 ml of the [14C] test solution was made from a microlitre syringe on to an area of skin (7.5 or 10 cm²) previously marked out on the animal’s back with a felt-tipped pen. After 15 min contact with the skin the animal was inverted over a 6-inch diameter funnel and the excess of the test solution was rinsed off with distilled water at 37°C. The animals were then fitted with either restraining collars or non-occlusive protection patches and placed in metabolism cages for collection of excreta as described above.

Results:

The results from the excised rat skin experiments showed penetration of the shorter chain length soaps, where the permeability constants were 2.5-3.9 gcm min -1 for the C10: 0, C12:0 and C14: 0 soaps at 24 h after application, but the penetration of the other surfactants was not measurable. No autoradiographic studies on these skin samples were performed and little can be deduced from these results as to the distribution of the [14C] surfactants in the skin. The observed rate of penetration will depend upon the time required for equilibration of the skin samples in the cell and the interaction between the skin and the surfactant. It is likely that some

penetration occurred through the stratum corneum in most of the samples but, whereas in the in vivo state it would be removed in the peripheral blood supply, in the in vitro state the dermis has to be traversed.

From the rat skin data some deposition of surfactant on the skin surface could be predicted but the amounts of SDS, SDI, DOBS, C18: 0 and C16: 0 soaps penetrating from a 15 min wash and rinse would be very small. The C10: 0, C12:0 and C14: 0 soaps had permeability constants of 3 µ cm/min in vitro so that from a 15 min wash and rinse with a 6 mM solution a penetration of between 0.05 and 0.1 µg/cm would be predicted.

From the human epidermis studies in vitro only small amounts of the C10: 0, C12: 0, C14:0 soaps and the SDI would be likely to penetrate from a 15 min wash and rinse in vivo. The low penetration rates of the C16: 0 and C18: 0 soaps and DOBS and the very long lag time before SDS penetrates suggests that little or none of these would penetrate from a 15 min wash and rinse in vivo.

The in vitro penetration of the C16:0 soap through human skin was 0.3 µg/cm2 after 24 hours and of the C18:0 soap 0.1 µg/cm2.

The turnover of the [14C] surfactants in the rat showed that there was no significant difference in the rate or route of excretion of 14C given by intraperitoneal or subcutaneous administration. It was thus thought valid to assume that [14C] surfactant penetrating the skin and entering the blood stream would be excreted at a similar rate. The turnover of the C14: 0, C16: 0 and C18: 0 soaps was slow but for the other [14C] surfactants levels of 14C in the excreta could be used as good indications of percutaneously absorbed material.

Penetration of the [14C] soaps in vivo followed the same order as those obtained with excised human epidermis, i.e. C12: 0>C10:0>C14: 0> C16:0 = C18:0. The actual amounts of soap which penetrated from the 15 rain wash and rinse applications to untreated skins with the 6 mM soap solutions ranged from 0.674 +/- 0.34 µg/cm² for the C12:0 to 0.7 +/- 0.02 µg/cm² for the C18:0. These amounts are considerably higher than those predicted from the in vitro study with excised rat skin. Prewashing the skin with 300 mM model soap solution - approximately 7.5% w/v solution which is similar to that found during consumer use, increased the permeability of the skin, especially for the C10:0 and the C12:0 soaps.

The SDI penetration in vivo was below our limits of accurate measure- ment in this study, i.e. < 0.3 µg/cm² penetrated from a 15 min waash and rinse.

The penetration of the DOBS isomer was below our limits of detection (0.1 µg/cm²) for all experiments.

Thus the in vivo studies show that all of these [14C] surfactants penetrate rat skin with the exception of the [14C] DOBS, the solubility of which was very low. From the in vivo penetration data presented, it can be seen that there is an order of magnitude difference between the most penetrating of the soaps (C12:0 -0.6 µg/cm²) and the least penetrating (C18:0 -0.07 µg/cm²) when applied as 6 mM solutions. The penetration of the synthetic surfactants from 25 mM solutions showed that some 0.25 µg of SDS and 0.15 µg of SDI penetrated per cm² of skin. Thus, provided a linear relationship between the amount penetrating and concentration of these surfactants in the applied solution exists, then the C12:0 soap is about ten times as penetrating as SDS or SDI which penetrate at similar rates to the C18:0 soap.

Autoradiography of the treated skins from the 15 min wash and rinse applications showed deposition of surfactant on the skin surface and in the hair follicles especially at their entrances. This deposition suggests that penetration occurs both transepidermally and via the hair follicles which have been regarded as the main source of penetration for applications of short duration. The presence of 14C in the epidermis and upper dermis at 6 h after application of the C10:0 and C12:0 soaps shows the penetration of these soaps but gives no indication when they penetrated. Penetration may have occurred only during the 15 min washing time but penetration may also have taken place from the labelled soap deposited on the skin surface. The fact that the rate at which 14CO2 was recovered from the animals washed with C12:0 soap was slightly slower than from animals injected with C12:0 soap may be a reflection of the route of administration but is probably due to the fact that penetration occurs from the [14C] soap deposited on the skin.