AN ABSTRACT OF THE THESIS OF

Michael Mezei for the Ph. D. (Name)

in Pharmaceutical Science presented on (Major)

(De ree) qt-7_

(Date)

Title: DERMATITIC EFFECT OF NONIONIC SURFACTANTS

Abstract approved: Robert W. Sager, Ph.

Selected nonionic surface active agents, incorporated in various

ointment bases, were applied to normal rabbit skin daily in an attempt

to determine their chronic toxicity. The dermatitic effects of these

surfactant preparations were evaluated by three methods: gross ob-

servation, histological examination and biochemical techniques. The

results indicated that the tested nonionic surfactants have a distinct

potential to irritate rabbit skin, and cause histological and biochemi-

cal changes in the skin to which they are applied.

It was apparent that the polyoxyethylene ether types of surfac-

tants have the highest capacity to produce dermal reactions. These

substances caused thickening, scaling and fissuring of the skin. They

induced histological changes: hyperplasia, acanthosis, and various

necrosis of the epidermis, edema and inflammation of the dermis.

The biochemical changes measured were also the greatest with this

type of surfactants. The metabolic measurements indicated a two

or three -fold increase in the oxygen uptake of skin samples treated

C yL9/

with the polyoxyethylene ether types of surfactants. The phospholipid

content of skin samples treated with ten percent polyoxyethylene ether

96 was increased by 47 -80 percent on the basis of phosphorus content

after four days of application.

The sorbitan fatty acid esters and polysorbates also had unde-

sirable influences on rabbit skin. Depending on the type and concen-

tration of these surfactants, they also produced various degrees of

erythema, hyperkeratinization and desquamation of the skin, hyper -

plasia of the epidermis and inflammation of the dermis. Undiluted

polysorbate 80 and ten percent polysorbate 60 produced severe necro-

sis of the upper epidermis and a high number of inflammatory cells

in the dermis. The oxygen uptake of skin samples treated with sorbi-

tan fatty acid esters or polysorbates showed a two, three and four-

fold increase, depending on the length of the treatment, the concen-

tration and the type of agent used. Polysorbate 85 and sorbitan trio -

leate (ten percent in petrolatum) induced a 26 -53 percent and 27 -58

percent increase, respectively, in phosphorus content derived from

phospholipids of the rabbit epidermis.

Morphological and biochemical changes induced by the above

three types of surfactants resembled those of various skin diseases.

Results of laboratory studies of irritants applied to animal skin are

not always reliable for predicting the effects of similar materials on

human skin. However, the general similarities between the properties

of rabbit skin treated with surfactants and those of human skin in

chronic dermatitis lead to a postulation that surfactants may play

an important role in production of external dermatitis of the hands,

which is one of the most common dermatoses in our modern North

American society.

DERMATITIC EFFECT OF NONIONIC SURFACTANTS

by

Michael Mezei

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the

degree of

Doctor of Philosophy

June 1967

APPROVED:

Professor of Pharmaceutical %fence in charge of maj

Head of Department of Pharm. ceutical Science

Dean of Graduate School

Date thesis is resented ¡ p ,..) -} 1.(71,Y

Typed by Kay Smith for Michael Mezei

\- p

ACKNOWLEDGMENT

I would like to express my sincere gratitude and thanks to

Dr. Robert W. Sager, under whose direction this thesis was pre-

pared; to Dr. William D. Stewart, for his help in the evaluation of

microscopic sections; to Dr. Catherine Mezei, for her advice con-

cerning the techniques of determinations of lipids and DNA; to Dr.

Robert W. Newburgh, for letting me use research facilities of

Science Research Institute.

The financial assistance provided by the Oregon State University

Research Council is also acknowledged.

TABLE OF CONTENTS

Page

I. INTRODUCTION 1

Statement of the Problem 1

Literature Review 2

Definition and Application of Surfactants 2

Physiological Properties of Selected Nonionic Surfactants 4

General Effects 4 Effects on the Skin 6

Effects on Enzymes and Isolated Tissues 10 Purpose of the Study 11

II. MATERIALS AND METHODS 14

Surface Active Agents 14 Experimental Animals 14 Preparation and Application of Surfactants 14 Biopsies for Microscopic Examinations 16 Skin Respiration Measurements 17 Determination of Lipid Composition 18 Determination of Cholesterol 23 Determination of DNA 24

III. RESULTS AND DISCUSSION 27

Gross Observations 27 Histological Evaluation 35 Biochemical Methods 45

Respiratory Metabolic Activity 46 Total Phospholipid Content 55 Composition of Phospholipids 68 Cholesterol Content 70 Deoxyribonucleic Acid Content 76

IV. SUMMARY

V. CONCLUSIONS

BIBLIOGRAPHY

APPENDIX

79

84

87

98

LIST OF FIGURES

Figure Page

1 Various degrees of irritation after one week of treatment. 42

2 Photomicrograph of control skin. 42

3 Photomicrograph of skin treated with sorbitan monooleate in 100% concentration. 43

4 Photomicrograph of skin treated with polysorbate 80 in 100% concentration. 43

5 Photomicrograph of skin treated with polyoxyethy- lene ether 52 in 5% concentration. 44

6 Photomicrograph of skin treated with polyoxyethy- lene ether 30 in 60% concentration. 44

7 The oxygen consumption of control and treated skin samples three to thirteen days after application of surfactant. 50

8 The oxygen consumption of control and treated skin samples thirty to eighty -one days after application of surfactant. 51

9 Percent increase of lipid phosphorus calculated on the basis of DNA content of skin sample. 64

10 Percent increase of lipid phosphorus calculated on the basis of wet weight of skin sample. 65

11 Changes of cholesterol content calculated on the basis of DNA content of skin sample. 73

12 Changes of cholesterol content calculated on the basis of wet weight of skin sample. 74

LIST OF TABLES

Table Page

1 Data of surfactants used. 15

2 Gross observations after three days of application. 31

3 Gross observations after ten days of application. 32

4 Gross observations after 30 days of application. 33

5 Microscopic observation after ten days of application. 40

6 Microscopic observation after 30 days of application. 41

7 Phospholipid content of rabbit epidermis (µg P/100 µg DNA) after four days of treatment. 60

8 Phospholipid content of rabbit epidermis (µg P/100 mg wet weight) after four days of treatment. 61

9 Phospholipid content of rabbit epidermis (µg P/ 100µg DNA) after ten days of treatment. 62

10 Phospholipid content of rabbit epidermis (µg P/100 mg wet weight) after ten days of treatment. 63

11 Relative percent composition of the four major phospholipids of rabbit epidermis. 69

12 Cholesterol content of rabbit epidermis (µg Choles- terol/100 µg DNA) after four and ten days of treatment. 71

13 Cholesterol content of rabbit epidermis (µg choles- terol/100 mg wet weight of skin) after four and ten days of treatment. 72

14 DNA content related to wet weight (µg DNA/ 100 mg wet weight) after four and ten days of treatment. 78

DERMATITIC EFFECT OF NONIONIC SURFACTANTS

I. INTRODUCTION

Statement of the Problem

In the past twenty years there has been an enormous increase

in the utilization of synthetic surfactants as household and industrial

cleansing products, and as solubilizers and emulsifiers in both

pharmaceutical and cosmetic preparations. Consequently, because

of this frequency of contact among the general population, the effects

of these surfactants on the human skin has become of extreme im-

portance. There have been many speculations that the increasing

number of cases of contact dermatitis are associated with the in-

creased use of surfactants as detergents or as household cleaners.

Evidence has been presented that surface active agents cause

denaturation of keratin (van Scott and Lyon, 1953; Light, 1963; and

Schuppli, 1964) and have several harmful effects on the horny layer

of skin as defatting agents, by removal of lipids and other substances

(Barail, 1954; Blank, 1955; Szakall, 1955; Paschoud and Schmidli,

1955; Steigleder, 1961; Burckhardt, 1963; Carrie, 1964). Detailed

and extensive studies, however, failed to prove that synthetic house-

hold detergents are responsible for the large increase of dermatitis

and hand eczema cases (Jambor and Suskind, 1955; Jambor, 1955;

Plüss, 1953; Ferguson and Rothman, 1959; Suskind et al. , 1963).

One of the hypotheses upon which the present study was based is

that if the increased number of hand dermatitis cases in females can-

not be explained by the increased use of synthetic household deter-

gents, it is possible that the surfactants present in hand lotions and

other cosmetic and dermatological preparations could be the offending

agents. Surfactants are usually present in cosmetic and dermatologi-

cal preparations in concentrations varying from one to 15 percent.

If these surfactants are not indifferent to the skin, their action may

be more pronounced when they are applied to the skin as cosmetic

or dermatological products. The duration of contact between skin

and surfactants is much longer when they are applied in the form of

an ointment, cream or lotion than if they are used as detergents.

The contact between skin and household detergents is usually of

short duration, and the detergents are rinsed off with water. Sur-

factants incorporated into ointments, creams or lotions are applied

to the skin more regularly and are not removed immediately; pene-

tration of these agents into the skin is therefore more likely enhanced.

Literature Review

Definition and Application of Surfactants

The term surface active agent or surfactant is applied to

chemical compounds which have the ability to modify the surface and

2

3

interfacial properties of liquids in which they are dissolved. Mate-

rials which produce this phenomenon are known by many other names;

according to their use they may be called detergents, solubilizing

agents, wetting agents, emulsifiers, anti - foaming agents, etc. In

pharmaceutical practice, surfactants are used mainly because of

their emulsifying or solubilizing function.

An emulsifying agent may help the pharmaceutical formulator

to readily mix incompatible substances such as water, oily or waxy

materials, and water soluble and oil soluble ingredients into a stable

and pleasing dosage form. In addition to appearance characteristics,

surfactants may improve the taste and flavor of the final product,

and make it more acceptable to the patient. A further, and probably

more significant advantage of surfactants is that they may influence

therapeutic efficacy by increasing directly or indirectly the absorp-

tion or penetration of medicaments.

The solubilizing function of surfactants enables the pharmacist

to produce "transparent emulsions ", i. e. , clear products containing

water soluble and oil soluble medicaments in stable, homogenous

mixtures.

According to their chemical characteristics, surfactants can

be classified in three major groups: (1) anionic, (2) cationic, and

(3) nonionic. Surfactants of all three types are widely used through-

out industry. The nonionic type surfactants, claimed to be the least

4

toxic, are widely used in pharmaceutical formulations, especially in

dermatological or cosmetic products.

Physiological Properties of Selected Nonionic Surfactants

General Effects

The body utilizes the principle of surface active agents in its

production and use of bile salts, which aid in the absorption of fat and

fat soluble vitamins. Phospholipids, because of their hydrophilic and

hydrophobic character, are often referred to as surfactant lipids

(Benson, 1966). Dipalmityl lecithin is the predominant surfactant in

the lung that stabilizes the lipoprotein foam at the pulmonary alveolar

epithelium, maintaining airway stability by preventing the collapse of

alveoli at low lung volumes. The role of phospholipids as surfactants

in the lung has been recently reviewed by Avery and Said (1965),

Clements (1965), Buckingham et al. ,. (1966), and Mendenhall, Men-

denhall, Jr. , and Tucker (1966).

One may question, however, whether synthetic surface active

agents may interfere with the normal cellular physiology of tissues

with which they come in contact. Several studies have been carried

out to determine the toxicity of synthetic surfactants. These studies

involved acute, subacute, and chronic experiments designed to deter-

mine effects of long -term and acute exposure, both orally and topi-

cally. A number of experiments were also carried out in vitro,

to study the effects of surfactants on isolated enzyme activity.

Hopper, Hulpieu and Cole (1949) tested most of the commonly

used surface active agents on mice by oral and intravenous adminis-

tration. All three classes of surfactants, the anionic, the cationic,

and the nonionic were used. They found that the lethal dose for the

different agents varied, but that the nonionic agents, namely, the

polysorbates, were the least toxic. Draize and Kelley (1952) and

Hazelton (1952) tested surfactants topically on rabbit eyes and con-

firmed the conclusions drawn by the other researchers, that the

order of toxicity from most to least is, (1) cationics, (2) anionics,

and (3) nonionics. The dermal toxicity of these surfactants (Draize,

Woodard and Calvery, 1944) was found to be in the same order of

toxicity.

The first detailed study with nonionic surfactants, namely,

with polysorbates, was reported by Krantz et al. (1951). They

showed by extensive animal studies that Polysorbate 80 is innocuous

when given orally, even in relatively high dose. Oral administration

of Polysorbate 80. (4. 5 -6 gm. per day) to more than 100 human sub-

jects for periods up to four years has revealed no clinical evidence

of ill effect, alteration of metabolic fate and blood chemistries,

change in excretion of water soluble vitamins, or evidence of damage

to liver, kidneys or hematopoietic system. In a more recent study,

Larson et al. (1963) also found no adverse effect in subacute and

5

chronic oral toxicity tests by some nonionic surfactants.

A more comprehensive review of acute, subacute and chronic

toxicity of orally administered nonionic surfactants was published

this year by Elworthy and Treon (1967).

The general conclusion of all these studies was that the ester

type nonionic surfactants (sorbitan fatty acid esters and polysorbates)

are not toxic if taken orally. As a result of these studies all of the

sorbitan fatty acid esters and some of the polysorbates (e. g. poly-

sorbates 20, 60 and 80) are permitted as a direct food additive in

many countries; the polyoxyethylene ethers, however, are not

generally recognized as such (World Health Organization, 1964).

Effects on the Skin

A number of studies have been carried out with nonionic sur-

factants to detect if any specific changes would occur after local

application to the skin. Most of these studies used the patch test

and /or in vitro methods.

Treon (1962, 1963) conducted standard patch tests with a

large number of nonionic surfactants, including those that were used

in the present study. He reported no irritation with human skin and

only mild irritation on rabbit skin in cases of sorbitan trioleate,

polysorbate 80, and polyoxyethylene ethers 52, 56, and 72. The

other nonionic surfactants tested caused no irritation in rabbit skin.

6

All of the surfactants were used in concentrations of 60 percent and

100 percent.

In an in vitro procedure, Choman (1963) tested the same types

of nonionic surfactants in various concentrations on excised calf and

human skin. He reported that irritation or cellular structural altera-

tion did not occur.

Opdyke, Snyder and Rubenkoenig (1964) also reported that

synthetic surfactants incorporated in household detergent products

do not sensitize human and guinea pig skin, cause undue irritation of

the eyes, or lead to the development of skin tumors. One and ten

percent solutions of polysorbate 80, tested by means of the occluded

patch test technique on albino guinea pig skin produced no demon-

strable histological changes (Gisslen and Magnusson, 1966).

Contrary to these negative findings, there are several reports

claiming a variety of inflammatory reactions and even carcinogenic

effects of some of the same nonionic surfactants.

Surfactants present in detergents tested by patch test techniques

on human skin produced irritant reactions more easily on the appar-

ently normal skin of subjects with eczema than in normal subjects

(Bettley, 1964 a, b). Nonionic surfactants, e. g. polysorbate 80, and

other detergent products have been found to increase the permeability

of skin to water and various water solutes (Bettley, 1965). Aqueous

solutions of polysorbate 60 applied to sheep skin daily in various

7

8

concentrations (1 -20 percent) produced marked hyperplasia and some

hypertrophy of the interfollicular epidermis within five days, and

inflammatory reaction was observed in the dermis (Chapman and

Short, 1965). Nonionic surfactants, incorporated into ointment

preparations, induced macroscopic and microscopic changes, as

well as an increase in the respiratory metabolic activity of the rabbit

skin (Mezei et al., 1966).

The most deleterious effect of surfactants, promotion of skin

tumors, was reported by several investigators. Long -term in vivo

studies with mouse skin indicated that sorbitan monolaurate (Setälä,

Setälá and Holsti, 1954), polysorbate 60 (Shubik, Della Porta and

Spencer, 1959; Della Porta et al., 1960 a, b; and Shubik, 1961) and

other nonionic surfactants (Setälä, 1956; Merenmies, 1959) promote

skin tumors. Most of these surfactants were used undiluted and were

applied with a glass dropper on an area of skin approximately 2 x 2

cm., that had been clipped free of hair. This area of skin was pre-

treated by a single application of 9, 10- dimethyl -1, 2- benzanthracene

(DMBA) usually as a 1.0 or 1. 5 percent solution in mineral oil. The

first application of promoting agent was given one week after the

single application of carcinogen (DMBA) and thereafter the promoting

agent (surfactant) was applied once or twice daily. This treatment

continued for more than a year. The average latent period was

25 -30 weeks.

9

In another extensive study Setala, Niskanev and Nyyssonen

(1962) proved that many of the sorbitans and polysorbates produced

tumors without the pre- treatment with DMBA to tumor- susceptible

mice, but tumors did not occur with a different strain of mice.

The type of skin tumors, produced under both the promoting

and producing treatment with sorbitans and polysorbates, was simi-

lar to that obtained by croton oil treatment; they were mostly benign

papillomas, many of which regressed, and only a few carcinomas

were found. Croton oil is considered the most active promoting

agent in mouse skin carcinogens. While the effective dose of sorbi-

tans or polysorbates to produce tumors might be several times higher

than that of the croton oil, the biological effects of the two different

treatments showed many close similarities.

The effect of polysorbate 80 on the in vitro metabolism of

tumor cells (Ehrlich- Lettré Ascites carcinoma) has been studied by

Ka}r (1965 a, b). He reported that polysorbate 80 produced marked

permeability changes of the cell membranes. The oxygen uptake was

reduced by 50 percent. The incorporation of formate into protein and

deoxyribonucleic acid (DNA) decreased, but it increased into nuclear

ribonucleic acid (RNA). Incorporation of 32P into the phospholipids

increased greatly and was found to be localized in phosphatidyl

serine.

10

Effects on Enzymes and Isolated Tissues

Other biochemical studies with nonionic surfactants indicated

that surfactants influence enzymatic reactions. Wilsmann (1963),

using saccharase and phosphatase as model enzymes, demonstrated

that surfactants inhibited the activity of these enzymes, and that the

degree of inhibition was parallel to the results of patch tests on the

skin. Polysorbate 80 inhibited the activity of the DPNH cytochrome

c reductase and also the activity of the DPNH oxidase of heart muscle

preparations (Polgár, 1962 a, b). Gaylor et al, (1966), reported that

polysorbate 80 inhibited the demethylation of lanosterol to cholesterol

in a cell -free preparation of rat skin epidermis.

Intravenous injections of nonionic surfactants (e. g. polysor-

bates, Triton X -100 and Triton WR -1339) induced hyperlipemia, a

sustained elevation of plasma cholesterol, phospholipids and trigly-

cerides in dogs, mice and rabbits (Hirsch and Kellner, 1956; Fried-

man and Byers, 1957; and Courtice, 1962). Experimental data have

been reported and several theories advanced to explain the mechanism

of surfactant induced hyperlipemia. These surfactants might in-

crease the biosynthesis of lipids, might interfere with the degrada-

tion or excretion of lipids (Minard, 1953; Franz and Hinkelman, 1955;

and Hirsch and Kellner, 1956), or the hyperlipemia could be second-

ary to the action of surfactants on the physical and chemical properties

11

of plasma lipoproteins (Scanu and Oriente, 1961; Courtice, 1962; and

Scanu, 1965).

Imanushi, Momotani and Isemura (1965) have studied the

effects of surfactants on the conformation of proteins. They found

that the change in optical rotatory dispersion of proteins by the

addition of surfactants was due to destruction of the original con-

formation.

Polysorbate 20, 60 and 80 in 0. 1 -1.0 percent solutions inhibited

nerve conduction in several isolated nerve fibers, e. g. rat phrenic

nerve, frog sciatic nerve, and single myelinated nerve fiber of toads

(Watanabe, 1958).

Purpose of the Study

The dermatitic effects of synthetic surfactants have been the

subject of much research (Hopper, Hulpieu and Cole, 1949; Krantz

et al., 1951; Draize and Kelley, 1952; Hazelton, 1952; van Scott and

Lyon, 19 53; Plüs s, 19 53; Blank, 19 55; Szakall, 19 55; Jambor and

Suskind, 1955; Jambor, 1955; Ferguson and Rothman, 1959; Steig-

leder, 1961; Suskind et al., 1963; Treon, 1963; Choman, 1963;

Burckhardt, 1963; Opdyke, Snyder and Rubenkoenig, 1964; Bettley,

1964 a, b; Carrié, 1964; Bettley, 1965; Chapman and Short, 1965).

Most of these studies, however, have been limited to surfactants

present in detergents, in household cleaners, or in aqueous solutions.

12

All of these investigations used in vitro and/or patch test techniques

with the exception of skin cancer studies (Setälä, Setälä and Holsti,

1954; Setälä, 1956; Merenmies, 1959; Shubik, Della Porta and

Spencer, 1959; Della Porta, 1960 a, b; Shubik, 1961), where undiluted

forms or aqueous solutions of surfactants were applied to mouse skin

frequently for a long period of time.

The primary purpose of this project has been to study the

chronic effects of some selected nonionic surfactants incorporated

in various ointment bases on rabbit skin. In contrast to the in vitro

and patch test techniques used in most of the previous investigations,

attempts were made to reproduce the frequent application of derma -

tologic and cosmetic preparations by the general public.

It is generally recognized that the toxicologist relies heavily

upon the results of well -planned experimental studies with laboratory

animals to provide the information upon which he bases his predic-

tions as to the effects a given material may have on humans. The

potential hazard of human skin contact with a chemical is frequently

determined from data obtained from the application of the chemical

to the skin of experimental animals. The literature records exam-

ples of the use of a number of mammalian species for such studies.

Since the publication of Draize, Woodard and Calvery (1944), how-

ever, the white rabbit is the sole animal species recognized for such

tests in the regulations under the Federal Hazardous Substances

13 Labeling Act (21 CFR 191). Industrial toxicologists in the United

States have restricted themselves largely to this single species for

the routine screening of skin effects caused by chemicals or chemical

formulations. Laboratory studies on white rabbit skin have yielded

results which have been quite reliable for predicting the capacity of

a chemical to cause primary irritation and epithelial hyperplasia

upon human skin (Rowe and Olson, 1965). The present study was

limited to laboratory studies on animals only, and the New Zealand

white rabbit was selected as the experimental animal.

Previous studies on the cutaneous toxicity of chemical agents

have been based on experimental procedures that are empirical in

nature and arbitrary in evaluation. They rely mainly on gross ob-

servations, very rarely on microscopical evaluations. They reveal

little or nothing about the biochemical disturbances at the cellular

level that actually initiate the injury.

The aim throughout this investigation has been to design and to

apply more definitive and rational test procedures, to evaluate the

chronic toxicity of nonionic surfactants, not only in terms of macro-

scopic and microscopic observations, but to investigate biochemical

changes by more exact and accepted biochemical assay methods.

Measurements and observations were designed to include morphologi-

cal evaluation on both the macroscopic and microscopic level, oxygen

consumption, lipid composition, and deoxyribonucleic acid (DNA)

content of the treated and control rabbit skin.

14

II. MATERIALS AND METHODS

Surface Active Agents

Three main groups of nonionic surfactants were selected for

the study: a) partial esters of sorbitan fatty acids, b) partial esters

of polyoxyethylene sorbitan fatty acids, polysorbate series, and

c) polyoxyethylene ethers. A detailed list of these surfactants is in

Table I. These surfactants were used both in undiluted and in diluted

forms, using distilled water, hydrophilic ointment U.S. P. , hydro-

philic petrolatum U.S. P., and white petrolatum U. S. P. as diluents.

Experimental Animals

A total of sixty -three New Zealand white rabbits of both sexes,

six to twelve weeks old, served as experimental animals. They

were individually housed in hanging screen- bottom cages and main-

tained on Purina Rabbit Chow and water ad libitum. The rabbits

(no. 51 -63) used for skin lipid studies wore harness -type restrainers

(Newmann, 1963), the others (rabbits no. 1 -50) had no restraining

devices.

Preparation and Application of Surfactants

The surfactants were incorporated into the proper diluents by

standard pharmaceutical procedures to obtain preparations in

Table 1. Data of surfactants used.*

Chemical Composition** a. Partial ester of sorbitan fatty acids

Sorbitan monolaurate Sorbitan monostearate Sorbitan monooleate Sorbitan trioleate b. Partial esters of polyoxyethylene

sorbitan fatty acids

Polyoxyethylene (20) monolaurate

Polyoxyethylene (20) monostearate

Polyoxyethylene (20) monooleate

Polyoxyethylene (20) trioleate

sorbitan

s orbitan

s or bitan

sorbitan

c. Polyoxyethylene ethers Polyoxyethylene Polyoxyethylene Polyoxye thyle ne Polyoxyethylene Polyoxyethylene Polyoxyethylene

(4) lauryl ether (2) cetyl ether (10) cetyl ether (2) stearyl ether (2) oleyl ether (10) oleyl ether

Official Name

Sorbitan monolaurate Sorbitan monostearate Sorbitan monooleate Sorbitan trioleate

Polysorbate 20

Polysorbate 60

Polysorbate 80

Polysorbate 85

Polyoxyethylene Polyoxyethylene Polyoxyethylene Polyoxyethylene Polyoxyethylene Polyoxyethylene

ether 30 ether 52 ether 56 ether 72 ether 92 ether 96

Commercial Name

Batch Lot Number

Span 20 541 Span 60 3474C Span 80 947 Span 85 2681C

Tween 20 904

Tween 60 617

Tween 80 745

Tween 85 7211B

Brij 30 182 Brij 52 6310B Brij 56 7615B Brij 72 6312B Brij 92 101 Brij 96 6628B

These surfactants were purchased from the manufacturer, Atlas Chemical Industries, Inc., Wilmington, Del. Batch and lot numbers were given by the manufacturer. More information in Appendix.

*

-

16

concentrations ranging from one to 100 percent. The trunks of the

animals were clipped free of hair with an electric hair clipper

(Oster model A2, size 40), and divided into eight areas in rabbits

no. one to 50, and six areas in rabbits no. 51 to 63. On each animal

one area was left untreated, and one area was treated with only an

ointment base to act as control sites. Two days after the hair was

removed, about 0. 3 gm. of one of the various preparations employed

was evenly applied and then gently rubbed in for three seconds with

a hard rubber sptatula to the center of the appropriate area, once

a day. The site treated with a particular preparation was randomly

varied with different animals to exclude any influence of body area.

After every ten days of treatment, hair clipping was repeated on all

areas and followed by a day of non -treatment to allow recovery from

any mechanical damage.

Biopsies for Microscopic Examinations

For the evaluation of histological changes, biopsy specimens

were taken from the representative sites after application for ten

and 30 days and at the completion of the experiment. After each

biopsy, that area was discontinued for use as an experimental site.

With forceps, the skin was elevated, and a full thickness biopsy was

taken by scissors. The specimens were kept in a ten percent formol-

saline solution until routine histological slides were prepared,

17

stained with hematoxylin and eosin, and examined microscopically.

Skin Respiration Measurements

Oxygen consumption of the treated and control skin samples

was determined by the direct Warburg method as described by Um-

breit, Burris and Stauffer (1964). The animals were killed by frac-

turing the neck. The test areas were washed quickly with water to

remove the remainder of the substances previously applied, and the

samples were taken with the aid of the Castroviejo keratotome

(Blank, Rosenberg and Sarkany, 1961) set to cut 0.2 mm. thickness

of skin. The skin slices were cut to small pieces with cold scissors

and were immediately weighed on an analytical balance (referred to

as wet weight), then transferred to Warburg flasks containing 3.0 ml.

of Kreb's Ringer Phosphate - Glucose (KRP -G) solution (Umbreit et

al., 1964) and 0. 2 ml. of 20 percent KOH solution in the center well.

The measurement was carried out at 37o after a 30- minute equili-

bration period. At the end of the measurement, the samples were

removed from the flasks, rinsed in distilled water and placed in

preweighed crucibles. Dry weights of the samples were obtained

by drying in an oven at 105o to constant weight. The oxygen consump-

tion was calculated in microliters per milligram of skin (dry) per

hour: this value is the QO2. For measuring the oxygen consumption

of a small, easily obtainable sample, attempts were made to use a

18

micro respirometer, the so- called Differential Capillary Respir-

ometer, which was designed by Cruickshank (1954). It is essentially

a closed system formed of two chambers, a reaction and a compensa-

tion chamber, connected by a precalibrated capillary tube containing

one drop of indicator fluid. One ml. of KPR-G solution was placed

in a glass dish 6 mm. deep and 2 cm. in diameter in each of the

chambers. These dishes rested on circles of filter paper which

covered the bottoms of the chambers and were soaked with 0.2 ml. of

20 percent KOH solution. A 0. 2 mm. thick skin sample weighing

10 -20 mg. was placed in one of the glass dishes. The sample was

floating on the surface of the medium. The apparatus was bolted

together with the gassing tap in the open position. The respirometer

was placed on a rack in a constant temperature bath set at 370, with

the ends of the gassing tubes projecting above the water. Readings

were commenced after 30 minutes (allowance for thermal equilibrium)

when the gassing tap was turned so as to seal the chambers. At the

end of the measurement the dry weight of the samples was determined

as described above with the Warburg method.

Determination of Lipid Composition

The lipid content was determined by thin layer chromatographic

and spectrophotometric methods. For this portion of work

rabbits no. 51 to 63, which were wearing harnesses developed by

-

19

Newmann (1963), were used. Only one representative member of

each of the three types of surfactants was used: sorbitan trioleate,

polysorbate 85 and polyoxyethylene ether 96. This selection was

based upon the results of gross and microscopic evaluation, where

sorbitan trioleate and polysorbate 85 had the highest irritation poten-

tial within their groups. Both surfactants are oleic acid esters.

Polyoxyethylene ether 96 is an oleyl ether. These selected surfac-

tants were incorporated in white petrolatum in ten percent concentra-

tion, and were applied to the appropriate areas daily. The other

three areas on the rabbit's back were reserved for controls:

(1) untreated skin a, (2) untreated skin b, and (3) skin treated with

the ointment base only. Untreated skin b was used as a control to

determine whether the surfactant penetrated into the skin influenced

the extraction of lipids and DNA, which could also be a reason for

finding different amounts of lipids and perhaps DNA in control and

treated skin. To this area one of the surfactant preparations was

applied only once: 15 minutes before the sample was taken. This

single application provided some surfactant in the skin homogenate,

but did not induce any measurable biological changes. After killing

the animal, the test areas were quickly washed with cotton soaked

in ether to remove not only the substances applied previously, but

the surface lipid content also. Skin samples were taken with the aid

of the Castroviejo keratotome set to cut a 0. 1 mm thick skin slice,

-

20

immediately dipped in liquid nitrogen, and kept there until the wet

weight was determined on an analytical balance. The weight of each

sample was around 100 mg. The elapsed time between removing

and weighing the sample was not more than 20 -30 minutes.

Immediately after weighing, the skin sample was homogenized

in a glass homogenizer and extracted with 2. 0 ml. of chloroform -

methanol 2:1 according to Folch, Lees and Sloane Stanley (1957).

The extraction was repeated twice to ensure complete removal of

lipids that are soluble in this solvent. After each extraction the

suspension was centrifuged, and the clear supernatants were com-

bined (Extract A). The residue was set aside for DNA extraction.

Non -lipid contaminants were removed from extract A with

0.2 volume of 0.05 percent CaC12 solution, After centrifugation,

the upper phase and any material occurring at the interphase were

removed and discarded. The washed chloroform - methanol layer

was taken to dryness under reduced pressure using a Buchler Rotary

Evapo -Mix flash evaporator. The test tubes containing the dry resi-

due were placed in a vacuum desiccator containing KOH at the bottom.

The desiccator was evacuated and placed in a refrigerator at 40

overnight. The dry residue of extract A was redissolved in a small

amount of chloroform - methanol 2:1, filtered through glass wool into

a volumetric flask and the volume was made up to 2. 0 or 5.0 ml.

(Solution A). Aliquots from these solutions were used to determine

21

the phosphorus and cholesterol content and phospholipid composition.

Total phosphorus content was determined by the spectrophoto-

metric method of Fiske and SubbaRow as modified by Bartlett (1959).

Aliquot samples (0.2 or 0.5 ml.) were placed in an oven at 500 until

all the solvent had evaporated. Ten normal sulfuric acid, 0. 5 ml. ,

was added to all the test tubes, including tubes for reagent blanks

and inorganic phosphorus standards, 1, 2, 3, and 4 µg.

The tubes were placed in an oven for three hours at 180.

Following this digestion, the tubes were removed from the oven,

allowed to cool, and 2 -3 drops of 30 percent hydrogen peroxide were

added. The tubes were then returned to the oven and digested for an

hour at 180 °. To those tubes that still contained undigested material,

a further 2 -3 drops of 30 percent hydrogen peroxide were added and

digested for an additional hour at 180°. This step was repeated until

all the samples were clear and colorless solutions. To each of these

solutions 4. 6 mi. ammonium molybdate solution (prepared by mixing

1 ml. of ammonium molybdate five percent solution with 22 ml. distilled

water) and 0. 2 ml. Fiske - SubbaRow Reagent were added and thorough-

ly mixed. All tubes were placed in a vigorously boiling water bath

for seven minutes. The samples were removed from the water bath

and mixed again. The absorbancy of samples was read at 830 mµ

wave length on the Beckman D. U. Spectrophotometer.

The phospholipid composition was determined by a thin layer

.

22

chromatographic (TLC) method. There have been several procedures

reported for the qualitative and quantitative determination of phospho-

lipids from various tissues by TLC (Marinetti, 1962; Parker and

Peterson, 1965; Privett et al., 1965; Rouser et al., 1965; Rouser,

Siakotos and Fleischer, 1966). The phospholipid content of skin was

also studied by means of TLC (Wheatly et al., 1964; Wheatley, 1965;

Nicolaides, 1965). Attempts were made to follow some of the pro-

cedures described, but in most cases the presence of surfactants in

the samples interfered. After experimentation the following modified

method was employed.

The adsorbant for TLC was Silica Gel G (E. Merck, A. G.

Darmstadt, Germany). Thirty grams of Silica Gel G were mixed

with 63 ml. of distilled water and spread on 20 x 20 cm. glass plates

in 0.25 mm thickness. The plates were dried at room temperature

and were activated for one hour at 1050 just before use. Aliquots. of

Solution A were applied along with standard solutions of known phos-

pholipids with a microliter syringe as narrow streaks on 2 cm. wide

lanes, 2 cm. from the bottom of the plate. The plates were subjected

to ascending chromatography in a closed glass developing tank which

contained ZOO ml. of chloroform -methanol -distilled water, 75:22 :3

(Solvent A). This solvent was allowed to rise to 10 cm. from the

starting line. The plates were removed from the developing tank

and after ten minutes (to allow for the evaporation of Solvent A) were

23

exposed to iodine vapor for 30 seconds. The spots were immediately

outlined with the point of a needle and were identified by simultaneous

chromatography of reference phospholipids of lecithin (L), lysoleci-

thin (LL), phosphatidylethanolamine (PE), and sphingomyelin (SPH)

spotted on each plate.

After the iodine had evaporated from the plate, each outlined

spot and one blank spot were removed by vacuum aspiration tech-

nique (Matthews, Pereda and Aguilera, 1962). The extracting solvent

used in this step was 15 ml. of chloroform -methanol- water, 65:40 :5

(Solvent B). The phosphorus content of each sample was determined

as described before by the modified Fiske- SubbaRow method.

Determination of Cholesterol

The cholesterol content of skin samples was determined with

minor modifications of the procedures used by Hanel and Dam (1955).

Aliquot samples of 0. 5 or 0.1 ml. were taken from extract A and

dried at room temperature in glass stoppered volumetric flasks of

2.0 ml. The following reagents were added to each of these volu-

metric flasks: 0. 8 ml. chloroform, 0.4 ml. zinc chloride solution,

and 0. 4 ml. of acetyl chloride. The contents of the flasks were then

mixed by shaking, and the flasks were placed in a water bath at 65°

for exactly 15 minutes. The flasks were then cooled in ice water and

the volume was made up to 2.0 ml. with chloroform. The light

24

absorbance of these red colored solutions was measured within 30

minutes at 528 mµ in a Beckman D. U. spectrophotometer, against a

blank solution containing only the above reagents. A stock solution

of cholesterol (0. 1 mg /ml) in chloroform was used to prepare a

series of standard cholesterol solutions (from 10 µg to 60 µg). This

method was found to be much more sensitive than the generally used

Liebermann - Burchard reaction. With this method, however, special

care must be taken to exclude water, which interferes with the analy-

sis. Because of this only fresh reagents were used. The zinc chlor-

ide solution was prepared as follows: 40 g anhydrous zinc chloride2

were fused in a porcelain dish over flame, quickly ground and dis-

solved in 153 ml. glacial acetic acid.

Determination of DNA

The DNA content of skin samples was determined by a proce-

dure based upon the technique used by Santen and Agranoff (1963).

The residue obtained after the extraction of lipids was washed

with 5 ml. 95 percent ethanol to prevent formation of gummy residue

upon direct addition of five percent "trichloroacetic 'acid (TCA). The

residue was extracted twice with 5 ml. five percent ice cold TCA, the

supernatants were discarded and the residue was washed again with

5 ml. ethanol, then with 5 ml. ether. The supernatants were dis-

carded and the residue was dried at room temperature for one hour.

25

The dried residue was hydrolyzed with 5 ml. 1 N potassium hydroxide

at 37o for 16 hours. To the hydrolysate, 0.5 ml. 70 percent HC104

was added on an ice bath. Thorough suspension of the residue by

frequent mixing with the Vortex mixer during 20 minutes insured a

complete precipitation of the DNA and proteins. Then the samples

were centrifuged and the supernatant was discarded. The residue

was then washed twice with cold 1 N perchloric acid. The DNA was .

obtained from the residue by two successive extractions at 800 with

5 ml. 1 N perchloric acid for 30 minutes. These supernatants were

combined and made up to 25.0 ml. with IN perchloric acid. The

DNA was read directly at wavelengths 268.5 mµ and 285 mil in the

Beckman D. U. spectrophotometer using 1 N perchloric acid as blank

solution.

In order to correct for interfering ultraviolet- absorbing sub-

stances, a "tissue breakdown products" extract was prepared accord-

ing to a modification of the method of Santen and Agranoff (1963).

Lipid and trichloroacetic acid soluble materials were extracted from

a skin sample as described above. Two 30 minute extractions with

frequent stirring at 800 in 1 N perchloric acid removed the nucleic

acids. The residue was washed with distilled water, ethanol, ab-

solute ethanol, and ether and then dried. Treatment according to

the extraction of ribonucleic acid (RNA) and DNA was resumed with

alkaline hydrolysis. The perchloric acid - soluble fraction minus

26

the RNA, and the hot perchloric acid fraction minus the DNA were

isolated. The absorption at 285 and 268.5 mµ's was determined for

the hot perchloric acid treated fraction. From these data a value of

A285 O. 849 was obtained. Using this value the corrected ab- A 268.5

sorbancy of DNA present in skin samples and the amount of DNA

was calculated as described by Santen and Agranoff (1963).

-

27

III. RESULTS AND DISCUSSION

The effects of surfactant preparations on rabbit skin were

evaluated by three methods:

1. Gross observation

2. Histological examination

3. Biochemical methods

Gross Observations

Gross observations were made and recorded daily, but repre-

sentative tables are made up only for intervals of 3, 10, and 30 days

after starting application. The criteria for estimating macroscopical

changes are indicated on a scale of + to + + + +; +erythema, edema,

+ +thickening, + ++ hyperkeratinization, desquamation and + + ++ forma-

tion of fissures and open lesions. These above characteristics can

be further subdivided to slight, moderate and strong reactions. The

slight and moderate degrees of these dermal reactions are rated as

+ to + + + +; and the strong, more intense reactions are indicated by

underlining these designations (+ to ++++). No visible changes are

recorded as: O. The results of these observations are illustrated

in Tables 2, 3 and 4.

Table 2 shows the results after three days of application. The

first gross changes, namely, erythema and edema, hyperkeratiniza-

tion and desquamation, began to appear where certain of the

28

polyoxyethylene ethers were being applied, even in a dilution of ten

percent. Irritation was also observed, but to a lesser degree, where

the undiluted 100 percent concentrations of polysorbates were applied.

At the site of application of ten percent concentrations of polysorbates,

only a slight erythema was noticeable. With the exception of sorbitan

monolaurate and sorbitan trioleate, no changes were seen at the

sorbitan areas. The data show that the surfactants belonging to the

polyoxyethylene ether series produced irritation even after three

days of application, and in a dilution as low as one percent. As the

treatment continued, a degree of irritation became apparent in most

treated areas. The skin treated with polyoxyethylene ethers of all

concentrations showed thickening and scaling, with marked induration

and fissuring. A type of crust formation, shown microscopically to

consist of necrotic and sloughing epidermis, often developed after

one week of treatment with various concentrations of polyoxyethylene

ehters.

The various degrees of irritation after one week of treatment

are illustrated by Figure 1 on page 42, Area 2, treated with sorbitan

monolaurate ten percent in petrolatum showed very severe erythema

( +) (beet redness); area 4 treated with only petrolatum U. S. P. showed

very slight erythema ( +) (barely perceptible); area 6 treated with

polyoxyethylene ether 56 ten percent in petrolatum showed formation

of fissures and open lesions ( + + + +); area 8 treated with polyoxyethylene

29

ether 56 one percent in petrolatum showed thickening and desquama-

tion ( + + +).

Table 3 records observations made after ten days of applica-

tion, when the areas treated with surfactants belonging to the

sorbitan series began to show some irritation. An interesting ob-

servation was the increased rapidity of hair growth, chiefly in areas

of application of the polysorbates. The inflamed necrotic and irre-

versibly damaged epidermis sloughed off after two or three weeks

of polyxoyethylene ether application, but re- epithelization of the

surface followed rapidly,

The results of surfactant treatment as observed macroscopic-

ally after 30 days of daily application are summarized in Table 4.

All the undiluted surfactant preparations caused severe irritation,

hardening, thickening of the skin, desquamation, and in most cases

severe fissure formation. In the diluted forms only the polyoxyethy-

lene ethers produced the most severe irritation of fissuring, even

in a dilution of five percent in hydrophilic ointment.

Among the sorbitans only the undiluted sorbitan trioleate pro-

duced severe forms of irritation, namely, hardening thickening and

fissuring of the skin. Undiluted polysorbates 60, 80 and 85 also

produced this highest degree of irritation; only polysorbate 20 proved

to be less irritant. The diluted forms of sorbitans and polysorbates

had much less irritation potential than polyoxyethylene ether type of

..

30

Table 2. Gross observations after three days of application.

Sub- stance 100%

60% in 10% in 5% in 1% in Water H. O. . H. P. Water H. O. H. P. Pet. Water H. O, H. P. Pet. Pet.

S-20 k + 0 + 0

S-60 0 0 0 0

S-80 + 0 0 0

S-85 + + +

T-20 + 0 + + +

T-60 + + + + + +

T-80 + + + 0 + +

T-85 + + + + + +

B-30 +++ ++ +++ + ++ ++ +

B-52 ++ ++ ++ + ++ + + +

B-56 ++ ++ ++ + + ++ + + + +

B-72 + + ++ ++ + ++ +

B-92 +++ ++ +++ ++ +

B-96 ++ + + +

H. O. 0

H.P. 0

Pet. 0

Table 3. Gross observations after ten days of application.

Sub- 60% in 10% 5% in 1% in stance 100% Water H. O. H. P. Water H. O. H. P. Pet. Water H. O. H. P. Pet. Pet. S-20

S-60

S-80

++

++

++

+

+

+

+

+

+

+

+

+

+

+

5-85 ++ + +

T-20 ++ + + + +

T-60 ++ + + + + +

T-80 ++ + ++ 0 +

T-85 +++ + ++ ++ + +

B-30 ++++ +++ ++++ ++ +++ ++++ ++

B-52 ++++ +++ ++++ ++ +++ ++ ++ ++ ++

B-56 ++++ ++++ ++++ +++ ++++ ++++ ++ ++ +++ +++

B-72 +++ ++ ++ ++ ++ ++ +

B-92 ++++ ++++ ++++ ++++ ++

B-96 ++++ +++ +++ +++

H. O. 0

H. P. +

Pet. + '

Table 4. Gross observations after 30 days of application.

Substance 100% 60% in 10% in 5% in

Water H. O. H. P. Water H. O. H. P. Water H. O. H. P.

S-20 +++ ++

S-60 ++ ++ +-

S-80 +++ + +

S-85 ++++

T-20 ++ + +

T-60 ++++ + +

T-80 ++++ ++

T-85 ++++ +++

B-30 ++++ ++++ ++++ ++ +++

B-52 ++++ ++

B-56 ++++ ++++ +++ ++

B-72 +++ +++ +++ ++ ++

B-92 ++++ ++++ ++++

B-96 ++++

H. O. +

H. P. ++

34

surfactants; they produced only erythema, thickening and hardening

of rabbit skin.

The results illustrated in Tables 2, 3, and 4 show that the

polyoxyethylene ethers have the highest irritation potential. The

greatest degree of irritation resulted after 10 -15 days of treatment.

After this period of time the fissured and necrotic epidermis sloughed

off and the new epidermis that formed showed somewhat more resist-

ance to surfactant treatment. Polysorbates generally had a higher

potential to irritate the skin than did sorbitan fatty acid esters. The

degree of irritation was dependent mainly upon the concentration of

surfactant and on the time of treatment.

The above results are in conflict with previous reports (Treon,

1962, 1963; Choman, 1963; and Gisslén and Magnusson, 1966). In

these reports no irritation was reported with sorbitan monolaurate,

sorbitan monostearate, sorbitan monooleate, polysorbate 20, poly -

sorbate 60, polysorbate 80, polyoxyethylene ether 30 and polyoxy-

ethylene ether 92. Only mild irritation was reported with sorbitan

trioleate, polysorbate 85, polyoxyethylene ether 52 and polyoxyethy-

lene ether 72. The results of the present experiments indicated that

each of the above listed surfactants produced some degree of irrita-

tion. The difference in the degree of irritation, which during this

investigation was found to be considerable, and which in some in-

stances resulted in complete destruction of edpidermis, may be due

35

to the different methods by which the surfactants were applied. The

above investigators used in vitro (Choman, 1963) and patch test tech-

niques (Treon, 1962, 1963; and Gisslén and Magnusson, 1966). The

method used in the present study was an attempt to reproduce the

frequent application of dermatologic and cosmetic preparations by

the general public. It could be concluded from the above differences

in results of the two application methods, that the closed patch test

applied for 48 hours (a single application) is less reliable as a meas-

ure of capacity of an irritant than a daily application of the test mate-

rial for a period of at least ten days.

Histological Evaluation

The cellular structure of skin appears simple on microscopic

examination, but in reality it is a surprisingly complex organ. Its

complexity can be emphasized by the fact that the skin surpasses the

liver and other internal organs in the multiplicity of its functions.

Studies in modern electron microscopy and histochemistry are re-

vealing the cellular location of these activities and associating struc-

ture with function.

The skin can be described in terms of the epidermis, dermis,

and epidermal appendages (Figure 2). The epidermis consists of

four layers: 1) stratum germinativum or basal layer of single cells

with hyperchromatic nuclei; 2) stratum spinosum or the rete malpighii,

36

a thick "prickle" cell layer; 3) stratum granulosum, a thin granular

layer consisting of diamond shaped cells filled with keratohyaline

granules; 4) stratum corneum, a surface layer consisting of flat

keratinized cells which have lost their nuclei.

The dermis or corium interdigitates with the epidermis by

means of the papillae. The dermal papillae vary greatly in anatomi-

cal location and under conditions of disease as to length, width,

vascularity, fluid content and density of collagen.

The epidermal appendages are the sweat and sebaceous glands

and the hair follicles.

Pathological processes may develop in any of these elements.

In the present study, the epidermis as a whole was examined micro-

scopically to determine variations in thickness, and then in turn at

the various layers of the epidermis, at the dermal papillae, the

dermis, and finally the epidermal appendages, for visible signs of

abnormal staining, change in structure, or cellular infiltration.

To make the histological evaluation more objective, the final

examinations were carried out by Wm. D. Stewart, M. D. Clinical

Instructor in Dermatology in the Faculty of Medicine, University of

British Columbia, Vancouver, B. C. All the biopsy specimens were

identified only by a code number. Dr. Stewart, experienced in the

histopathology of skin, gave an evaluation of all sections without

knowing the code key.

37

Altogether 186 biopsies were taken from the 478 tested areas.

The microscopic changes in the treated areas generally corresponded

to the degree of irritation observed grossly.

Table 5 illustrates the microscopic observations after ten days

of treatment. Surfactant preparations belonging to the sorbitan and

polysorbate series, with the exception of polysorbates 80 and 85,

produced only a slight degree of inflammation: a small number of

inflammatory cells in the upper dermis (See Figure 3). In the cases

of polysorbates 80 and 85 and in the diluted forms of polyoxyethylene

ethers, the number of inflammatory cells increased; more poly -

morphonuclear and round cells were noticed throughout the dermis

(Figure 4). All preparations of polyoxyethylene ethers produced a

pronounced inflammation with numbers of polymorphonuclear and

round cells in the dermis and acanthosis in the epidermis (Figure 5).

The concentrated forms of these preparations produced varying

degrees of superficial necrosis. In these instances the normal

cellular structure of the epidermis was replaced by an eosinophilic

amorphous mass, which was thoroughly infiltrated by polymorpho-

nuclear leucocytes (Figure 6).

In some cases, necrosis of the epidermis was observed with

massive destruction of epidermal nuclei, disruption and disorienta-

tion of cellular structure down to and through the basal layer. The

sebaceous glands and hair follicles usually showed retention of

38

cellular structure. The dermis contained a very large number of

inflammatory cells. In other cases only necrosis of the outer layer

of cells and of the stratum corneum was observed, with varying col-

lections of inflammatory cells in the dermis. The most severe form

of necrosis was found after polyoxyethylene ether 92 treatment,

where, in addition to the complete necrosis of the epidermal layer,

some necrosis of parts of the dermis, hair follicles and sebaceous

glands was also observed. This necrosis process was followed in

all instances by re- epithelization from the remaining appendages.

Table 6 shows the results of microscopic examination of

biopsies taken after one month of treatment. Here the degree of

inflammation and the extent of structural changes are more evident.

The samples taken from areas to which polyoxyethylene ethers

were applied for a one -month period showed many inflammatory

cells in the dermis and acanthosis in the epidermis with various

degrees of necrosis. In these cases, there was little difference in

the microscopic picture of samples taken after ten days and 30 days

of treatment. The process of re- epithelization with reformation of

basal cell layers was actively progressing to varying degrees in all

instance s.

There was considerable change in the microscopic picture of

skin samples treated with polysorbates and sorbitans for a period

of one month as related to that of the ten days treatment.

Notes to Tables 5 and 6

Description of microscopic evaluation:

Inflammation From minimally increased numbers of inflammatory

+ to + + ++ cells in the dermis, chiefly perivascular, to marked

degrees of polymorphonuclear and round cell infil-

trates throughout the depth of the dermis.

Acanthosis An irregular acanthosis to the normally flat, thin

(H) and regular epidermis.

Necrosis A complete destruction of the normal cellular struc-

(N) ture of the epidermis, replaced by an eosinophilic

amorphous mass thoroughly infiltrated by poly -

morphonuclear leucocytes. This process involved

hair follicular epidermis . and sebaceous glands in

severe cases. It was followed in all instances, when

the rabbit lived, by re- epithelization from the

remaining appendages.

Table 5. Microscopic observation after ten days of application.

60% in 10% in 5% in Substance 100% Water H.O. H. P. Water H. O. H. P. Water H.O. H. P.

S -20 + 0 0

S -80 + 0 0

S -85 +

T-20 + + + 0

T-60 + + + +

T-80 ++ + + +

T-85 ++ + + +

B-30 ++++N ++++N ++++N ++ ++H

B-52 +++N ++ +++H

B-56 +++N +++ ++H ++ ++

B-72 +++ ++ ++ ++ ++H

B-92 ++++N ++++N ++++N

H. O. 0

H. P. 0

None (untreated skin) 0

I

Table 6. Microscopic observation after 30 days of application.

60% in 10% in 5% in

Substance 100% Water H. O. H. P. Water H. O. H. P. Water H. O. H. P.

S -20 + 0 0

S -80 +++

S -85 ++H

T-20 ++ + +

T -60 ++H ++++N ++

T -80 +++NH ++ ++ ++

T -85 +++H ++

B-30 ++++NH ++++N ++++N ++

B-52 +++ ++

B-56 ++N ++N +++H +++H +++ +++H

B-72 +++ +++H ++H ++ ++

B-92 ++++NH +++H ++++NH

H. O. +

H. P. +

None (untreated skin) 0

`P r

+

Figure 1. Various degrees of irritation after one week of treatment (Rabbit No. 43; discussed on page 28).

Figure 2. Photomicrograph of control skin, plain hydrophilic ointment appli- cation only ( -).

. ' ' ,i r .

y.

Figure 3. Photomicrograph of skin treated with sorbitan monooleate in 100% concentration; a slight hyperplasia of the epidermis. There is a small number of inflammatory cells in the upper dermis ( +).

Figure 4. Photomicrograph of skin treated with polysorbate 80 in 100% concentration; there is irregular acanthosis of the epidermis, and edema and inflammation in the dermis (++).

= . J

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Figure 5. Photomicrograph of skin treated with polyoxethylene ether 52 in 5% concentration. There is acanthosis and hyperplasia of the epidermis, together with some edema and inflammation of the dermis (4-H-).

Figure 6. Photomicrograph of skin treated with polyoxethylene ether 30 in 60% concentration; the epidermis is destroyed. It is replaced by eosinophilic staining material filled with many polymorphonuclear leuco- cytes. These cells extend to the dermis and in some instances dip down into the sebaceous glands and hair follicles. There are scattered polymorphonuclear leucocytes and indication of edema in the dermis (+-F- f-f-)

13

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45

Polysorbate 60 ten percent in hydrophilic ointment produced severe

necrosis of the upper epidermis and a high number of inflammatory

cells in the dermis. Polysorbate 80 was the other member of this

group of surfactants which also produced superficial necrosis and

acanthosis in the epidermis with somewhat smaller numbers of

inflammatory cells in the dermis. Sorbitan monolaurate and poly

sorbate 20 seemed to be the least irritative, producing only a very

small number of inflammatory cells in the dermis.

Biochemical Methods

The previous two types of evaluation, gross observation and

microscopic examination, can result in rather relative values.

There are no exact, standard scales by which macroscopical and

microscopical inspections can be recorded. Erythema, hyperkera-

tinization, desquamation or fissuring can be well defined, but the

degree of these pathological changes as observed by each individual

investigator may vary rather significantly, Erythema may manifest

itself by slight pink to beet red color; edema may be barely percepti-

ble or may produce a raised area more than 1 mm in height at the

site of treatment; thickening, hardening, desquamation and fissuring

of the skin are also hard to express in exact values.

In the present study of the effect of surfactants on rabbit skin,

a need was felt for more exact and objective data which could be

46

measured by standard and well accepted biochemical means. The

secondary aim in using biochemical methods was to seek the reasons

and the causes of microscopical and macroscopical changes. There-

fore, the following determinations were used:

(a) Respiratory Metabolic Activity

(b) Total Phospholipid Content

(c) Phospholipid Composition

(d) Cholesterol Content

(e) Deoxyribonucleic Acid Content

Respiratory Metabolic Activity

All the active functions of the skin depend upon the provision

of an adequate supply of energy obtained from the energy - yielding

reactions of respiratory metabolism. There is good evidence that

the pathways and the main steps in energy metabolism in the skin

proceed as in other tissues, namely by glycolysis of carbohydrates,

oxidation of the products of glycolysis, lipids and amino acids via

the Kreb's citric acid cycle, and finally, a transfer of electrons

through the cytochrome system to molecular oxygen (Lorinez, and

Stoughton, 1958; Gilbert, 1962; Rosett and Fogg, 1962; and Yardley

and Godfrey, 1963).

The method, used to determine the respiratory activity of skin,

is to measure the oxygen uptake by portions of excised skin in vitro,

47

immediately after removal. One of the most difficult problems in

this technique is in the preparation of the sample for the measure-

ment of oxygen uptake in an isolated, morphologically well defined

and chemically unaltered form. Skin is a very heterogenous organ

as far as cellular structure and biological function are concerned.

The oxygen consumption of the epidermis is significantly higher than

that of the dermis. The connective tissue and attached adipose tissue,

as well as the horny layer, have no measurable oxygen uptake. The

first problem was to provide samples with uniform structure, where

the proportion of epidermis to dermis and to other metabolically

inert (e. g. hair and keratin) tissue components were the same for

all the samples.

In some previous skin respiratory measurements (Griesemer

and Gould, 1954; Brooks, Godefroi and Simpson, 1963; Fitzgerald

and Klein, 1964) this was provided by separating the epidermis layer

from the dermis and measuring the oxygen uptake of epidermis only.

The separation of epidermis is very time - consuming, and it is highly

probable that this process may impair enzymatic reactions which

directly or indirectly influence the uptake of oxygen when measured.

Epidermis in these studies was separated by exposure to ammonia

gas, or by exposure to heat (Baumberger, Suntzeff and Cowdry,

1942), by enzymatic method: digestion with trypsin (Klein and

Fitzgerald, 1962) or by mechanical means: stretching the

48

skin (Gilbert, Mier and Jones, 1963; and Spruit, 1964).

In the present study in order to minimize any artifact in the

results which could be derived by impairing metabolic reactions or

by using various types of tissue elements (epidermis, dermis, horny

layer) in different proportions, the Castroviejo Keratotome (Blank

et al., 1961) was used to obtain uniformly thin skin samples through-

out the experiments. The Castroviejo electrical keratotome is used

mainly in ophthalmology, but recently has been found to be of value

in dermatological research.

The oxygen uptake of treated and control skin samples was

determined by two methods. The results obtained by using the

Cruickshank differential capillary respirometer were inconsistent

and consequently unreliable. This could possibly be due to the fact

that the 0.2 mm skin slice, weighing 10 -20 mg. and placed in the

medium floating on the surface, basal layer pointing downward, may

be submerged during the measurement and apparently the slow dif-

fusion of oxygen through the medium and throughout the tissue could

limit the normal oxygen uptake. Furthermore, there is no stirring

or shaking of the vessels during the measurement; consequently the

supply and the exchange of oxygen in the medium is more limited

than on the surface. In some cases submerged samples were ob-

served after the instrument was opened at the completion of the

experiment, while in other cases the samples were still floating on

49

the surface of the medium (KRP -G solution).

The use of the differential capillary respirometer was dis-

continued and the oxygen uptake was measured by the direct Warburg

method. In this method, the oxygen uptake by living tissues is

measured in a constant volume respirometer, by absorbing the

carbon dioxide, liberated during metabolism, continuously in alkali

during the determination. The essence of the method is to hold the

gas and fluid volumes constant and to measure the change in pressure

when the amount of oxygen changes. The change noted on the mano-

meter is solely due to the oxygen uptake by the tissues. The absorp-

tion of oxygen by the tissue takes place almost entirely from the oxy-

gen in solution. In order to prevent the rate of oxygen diffusion into

the liquid from becoming a limiting factor, (e. g. the uptake rate is

higher than the diffusion rate) the vessels are shaken continuously.

The results obtained by the direct Warburg method were con-

sistent and reproduceable. They are illustrated in Figures 7 and 8.

Figure 7 represents the QO2 values of the control and treated

skin samples obtained from 13 rabbits at a period of 3 to 15 days

(one rabbit a day): 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days

after starting application. Figure 8 illustrates the QO2 values ob-

tained from the second group of rabbits, 15 in number, within the

period of 30 to 81 days after starting the application. During this

period the samples were taken 30, 31, 40, 42, 43, 44, 45, 50, 51,

QO2 values (4l 02 /mg. /hr. )

1.0 2.0 3.0 4.0

Untreated skin

Ointment base

*S - 20

S - 60

S - 85

J

+++++$+.11

oo At? o

T-20 o T- 60 OA

T-85 óAo o

B - 30 AA o

B - 52

B - 56

B - 72

B -92 o B -96

1. 0 2.0 3.10 4.10

Figure 7. The oxygen consumption of control and treated skin samples three to thirteen days after application of surfactant.

Note: Control, or ointment base alone

100% surfactant See abbreviation on page

o 10% surfactant in ointment base

1% surfactant in ointment base

50

1 l 1

+

.

i

4+y+ -0!

o o A

Ii o

ó o o *

o

01

AAE

0

4

Untreated skin

Ointment base

S - 20

S - 60

S - 80

S - 85

T-20

T-60

T - 80

T - 85

B - 30

B - 52

B - 56

B -72

B -92

B -96

Q02 values (µl 02/mg. /hr. )

1.0 2.0 3.0 4.0 5.0

51

6.0

+++++++

++.:4----.4' t +

o o

e s o

o o 00

o

ópo ó 'AI

p p)

O o op°0op

pp

0o

2.10 3. 0 4 -0 5.10 6. 0

Figure 8. The oxygen consumption of control and treated skin samples thirty to eighty --one days after application of surfactant.

Note: + Control, or ointment base only

100% surfactant o 10% surfactant in

ointment base

A 60% surfactant in ointment base

0 5% surfactant in ointment base

*

co

0 O a

1 '0

I I I i

52

52, 55, 58, 77, 78, and 81 days after starting the application.

In all instances the surfactant treated skin consumed two, three,

or four times as much oxygen as the control skin, depending upon the

length of time of treatment, the concentration and the type of surface

active agent used. Though the results are quite scattered, the gen-

eral pattern of oxygen uptake by the treated skin samples resembles

the results of gross and microscopic observations. The only ex-

ception was that skin samples treated with polyoxyethylene ethers

did not always consume more oxygen than those treated with sorbi-

tans and polysorbates. The probable explanation for this might be

that samples here contained more keratinized and necrotic cells,

as is indicated in Tables 2 - 6, than skin samples treated with

sorbitans and polysorbates. These tissue elements consumed no

oxygen but were represented in the wet weight of samples to which

oxygen uptake was referred.

The wide range in the results might be due to biological varia-

tions, but more likely to variations in tissue components present in

samples used. The various degrees of hyperkeratinization and of

hyperplasia of the epidermis have been indicated by gross and

microscopic examinations. Since the wet weight of skin samples

was used as a reference standard, any change in the metabolically

different skin components would also result in a change in the oxygen

uptake measured. Hyperkeratinization would mean a reduction,

..

53

hyperplasia of the epidermis an increase in oxygen consumption.

The response of rabbit skin to treatment with surfactants proved to

be almost uniform, according to gross and microscopic observations

where the exact degree of hyperplasia or hyperkeratinization could

not be stated. Throughout this investigation a uniform thickness of

skin (0.2 mm) has been used to measure oxygen consumption, but

this layer of skin could have had various amounts of epidermis and

dermis, which could also be responsible for the spread in the results

The overall results of oxygen consumption measurements,

however, proved that there was a definite increase in the amount of

oxygen consumed by the skin samples treated with any of the surfac-

tant preparations.

A specific explanation for the increased respiration rate due

to the topical application of surfactants has not yet been found. There

have been several studies on the effect of surfactants on enzyme acti-

vity in vitro, and in other tissues than skin. Wilmsmann (1963)

reported inhibitory effects of anionic surfactants. He used sacchar-

ase and phosphatase as model enzymes and found that the catalytic

activity of these enzymes with respect to sucrose (at 35o and at

pH 4. 0) and to disodium phenylphosphate (at pH 7. 0 and at room

temperature) was inhibited to a certain extent by different anionic

surfactants. He found that a correlation existed between the inhibi-

tory effect and skin compatability: increasing inhibition corresponded

54

to increasing skin irritation.

Polgár (1962 a, b) reported the inhibitory effect of polysorbate

80 on the activity of DPNH cytochrome c reductase and that of DPNH

oxidase in heart muscle preparations. Räihä and Koskinen (1964)

found that a nonionic surfactant, Triton X -100, had an activating

effect on the alcohol dehydrogenase in the supernatant fraction of a

liver homogenate which is not associated with the release of the

enzyme from the nuclear and mitochondrial fractions.

On the basis of these results, it is not clear whether Triton

X -100 releases alcohol dehydrogenase associated with other particles

in the cell, activates bound enzymes, or influences some enzyme

inhibitor.

According to these studies, surfactants can directly influence

enzymatic reactions. Indirectly, a surfactant can alter enzymatic

activity by several ways, which, at the present, are all very much

in the speculative stage; it can activate a bound enzyme by releasing

the enzyme, by disruption of intracellular particles (Wattiaux and

de Duve, 1956); it can influence some enzyme inhibitors, and it can

solubilize a substrate making it more available for reaction. It is

possible that the effect on enzyme activity is partly an indirect one

and it may be due to permeability changes of cell membranes induced

by surfactants. The effects of surfactants on cell membranes, and on

a smaller scale, on the membrane of the mitochondrion, are very

55

likely due to their hydrophilic and lipophilic character, surfactants

generally possessing a particular affinity for membranous structures

(Rockstroh and Zapf, 1966). They might be able to disturb the bal-

ance of cell systems by altering membrane permeability. As a result

of this affinity, a nonionic surfactant can (1) interact by hydrophobic

bonding, being a macromolecule itself, (2) interact with hydrogen

bonding, because of the strong hydrogen- bonding affinity of the ether

oxygens of the polyoxyethylene chains, (3) disturb the micellar struc-

ture of phospholipids, forming micelles, or replacing some phos-

pholipid molecules in the continuous lipid micelles, and (4) interact

with water molecules participating in these micelles - just to mention

a few possibilities which may result in changes in membrane struc-

ture and, consequently, functional changes and metabolic disorders.

On the basis of this postulation, that the primary site of action

of surfactants is at the biological membranes, it was decided to

search for ways to test this possibility. It was apparent that proving

this hypothesis with direct methods would be difficult, since there is

no way at the present time to study membrane structures at the

molecular level.

Total Phospholipid Content

The structure of biological membranes is based upon a lipid-

protein positional relationship. The classical Danielli -Dayson (1935)

56

model of the membrane and the more recent "unit membrane" hypo-

thesis of Robertson (1966) both suggest a bimolecular phospholipid

leaflet as the backbone of membrane structure. The phospholipids

in aqueous media are aggregated to form micelles, which are

"sandwiched" between two layers of protein. The stability of a mem-

brane structure is insured by internal cohesion forces. The phospho-

lipids are bonded to one another by hydrophobic bonds; the polar por-

tion of phospholipid molecules oppose the protein layer. Lipids are

also bonded to the protein layer by hydrophobic interaction. The

reactive partners in these hydrophobic interactions are the phospho-

lipid molecules in micellar form, and protein in the form of a macro -

molecular complex (Green and Tzagoloff, 1966). Recent reports and

reviews by Kavanau (1966), Green and Tzagoloff (1966), Vandenheuvel

(1966), van Deenen (1966), Benson (1966), and by Korn (1966) describe

the structure and function of membranes in a more detailed manner.

All of these descriptions are highly speculative in nature; there is no

proven theory for the exact nature of membranes at the molecular

level.

On the basis of these hypotheses it can be stated, however, that

a qualitative and /or quantitative change in the lipid composition of a

tissue might indicate structural changes in membranes, especially

if these changes were induced by substances which more likely act

on the membranes.

57

In studies of skin lipid analysis, as in metabolic studies, the

greatest problem has been to provide skin samples in chemically

unaltered form with well defined anatomical structure.

Skin lipid samples have usually been divided into three cate-

gories (Nicolaides and Kellum, 1965): (1) epidermal lipid samples,

(2) lipid samples from various sebaceous type glands or from the

dermis, and (3) lipid samples from the skin surface. The lipid

composition of the above three types of human skin samples recently

has been reviewed by Nicolaides (1965).

The human surface lipid was found to be an exceedingly complex

lipid mixture, composed mainly of nonpolar lipids: fatty acids in

free and in esterified form, sterols, paraffins, wax alcohols and

other unidentified components. Lipids from the dermis were mainly

influenced by the residual subcutaneous fat which was very difficult

to separate. Human sebaceous gland lipids showed a pattern similar

to that of human surface lipids.

Lipids of human epidermis were analyzed by Nicolaides (1965)

and other investigators (reviewed by Nicolaides, 1965) in which the

separation of epidermis from dermis was achieved by chemical and

physical methods (Baumberger et al., 1942; Gilbert et al., 1963 and

Brooks et al., 1963). In order to avoid any artifact induced by these

separation techniques, Nicolaides (1965) attempted to use the Castro -

viejo keratotome (Blank et al., 1961), but the thinnest section of skin

..

58

he could regularly obtain was 0.2 mm thick, which contained 10 -25

percent dermal contamination.

In the present work 0. 1 mm thick skin slices were cut from

rabbits, which contained the epidermis and occasionally a trace of

dermis, as was found by microscopic examination of several sam-

ples. This epidermis - dermis separation was achieved by cutting off

the whole skin of the test areas with the aid of a scalpel and stretch-

ing it over a square powder jar. The keratotome could then easily

cut 0. 1 mm skin slices from the lightly stretched skin. The surface

lipids were extracted with ether before separating the epidermis

from the dermis. This gave a well isolated and chemically unaltered

epidermis sample for lipid analysis.

There have been only indirect methods reported for the deter-

mination of lipid composition of the human epidermis (Dawson, 1960;

Gerstein, 1963; Carruthers, 1964; and Nicolaides, 1965). Phospho-

lipids, free and esterified cholesterol and fatty acids have been found

to be present in human epidermis.

The present investigation was mainly concerned with the

measurement of quantitative changes in the phospholipid content of

the rabbit epidermis.

The results are indicated in Tables 7 -10 and in Figures 9 and

10. All phospholipid components which were studied showed a con-

siderable increase, as determined on the basis of their phosphorus

59

content in skin samples which had been treated with surfactant pre-

parations.

The total phospholipid content of control and treated rabbit skin

after four days of treatment is shown by Tables 7 and 8. Using DNA

content as the reference standard, it was found that the phosphorus

(P) content of the control skin samples (untreated skin a and b) was

within the range of 7. 44 -10. 84µg P/ 100µg of DNA. In skin samples

which were treated with the ointment base only, which were also

considered as controls, the range was somewhat higher: 9. 26- 11. 24µg

P/ 100µg DNA. Among the treated skin samples the one which was

treated with polyoxyethylene ether 96 (10 percent in petrolatum)

showed the largest increase in phosphorus content; the range was

11.52 -15. 20µg P /100µg DNA, which represented 47 -80 percent in-

crease as illustrated by Figure 9.

Treatment with polysorbate 85 and sorbitan trioleate also

induced considerable increase in phosphorus content, 26 -53 percent

and 27 -57 percent, respectively. If - the phosphorus content is re-

ferred to the wet weight of the sample, it is found that the same

pattern exists (see Figure 10 and Table 8). The phosphorus content

of treated skin samples also showed an increase, but the spread in

the results was somewhat larger. The increase of phosphorus,

calculated according to the wet weight for samples treated with

petrolatum, polysorbate 85, sorbitan trioleate and polyoxyethylene

.

.

.:

.

.

Table 7. Phospholipid content of rabbit epidermis (µg P/100 µg DNA) after four days of treatment.

Substance Rabbit Number

51 52 53 54 55 56 57 58 59

None (untreated skin a) 8.56 8. 60 8. 83 10. 24 9.01 8. 45 7. 86 7.84 7. 44

None (untreated skin b) 9.73 8. 23 7. 62 10. 84

Petrolatum 9.59 9.86 9.26 11.24 9.80

Polysorbate 85 10.78 12.07 12.89 14. 30 12. 64 12.5 11, 16 11.52 11. 41

Sorbitan trio- leate 13.17 13.54 13.97 13.67 13.48 11.71 9.98 11.42 11.20

Polyoxyethylene ether 96 14.54 15.20 15.04 17.31 14.51 15.28 11.52 11.80 11.67

Table 8. Phospholipid content of rabbit epidermis (µg P/100 mg wet weight) after four days of treatment.

Substance Rabbit Number

51 52 53 54 55 56 57 58 59

None (untreated skin a) 22.46 20. 84 20. 89 26.20 27. 55 26. 18 25. 74 26. 08 23. 85

None (untreated skin b) 22.26 22.98 18.0 28.20 27.11 27.98

Petrolatum 25.61 21.13 23.79 27.88 28.98 30.07

Polysorbate 85 32.43 25.67 26.99 34.51 33.71 37.16 40.05 39.67 36.35

Sorbitan trio- leate 34.78 28.58 26.86 33.54 46.78 42.41 44.56 35.95 34.74

Polyoxyethylene ether 96 35. 51 32.25 30. 49 44. 86 43.5 49.72 44.20 38.37 36. 76

Table 9. Phospholipid content of rabbit epidermis (µg P/100 µg DNA) after ten days of treatment.

Rabbit Number Substance 60 61 62 63

None (untreated skin 8. 24 7.26 8.25 8.28

Petrolatum 8. 83 7. 85

Polysorbate 85 10.64 8. 6 10. 04 11.42

Sorbitan trioleate 11.12 9.30 9.76 10.50

Polyoxyethylene ether 96 10.74 8.88 10.41 11.0

Table 10. Phospholipid content of rabbit epidermis (µg P/100 mg wet weight) after ten days of treatment.

Substance Rabbit Number

60 61 62 63

None (untreated skin) 20. 37 26.0 34. 63 36. 50

Petrolatum 23. 56 26. 54

Polysorbate 85 33. 76 39.40 48. 57 51.58

Sorbitan trioleate 25.14 43. 34 44. 78 47.92

Polyoxyethylene ether 96 35.79 39.77 51.54 53.20

Perc

ent

incr

ease

of

lipid

pho

spho

rus

64

0.0

o 00

®

00 O O

T

Figure 9. Percent increase of lipid phosphorus calculated on the basis of DNA content of skin sample.

Note: Skin samples treated with:

P = white petrolatum T = polysorbate 85, 10% S = sorbitan trioleate, 10% B = polyoxyethylene ether 96, 10%

= after four days of treatment 0= after ten days of treatment

100-,

80-

60-

40-

20-

O

00 O

P

x m

.°c

b P,

b

ó

C

á

S B

Perc

ent

incr

ease

of

lipid

pho

spho

rus

65

100-

80-

60-

40-

207

o

o

o °o 0

o

o 0 O

o

o o

P T

Figure 10. Percent increase of lipid phosphorus calcu- lated on the basis of wet weight of skin sample.

Note: Skin samples treated with:

P = white petrolatum T = polysorbate 85, 10% S = sorbitan trioleate, 10% B = polyoxyethylene ether 96, 10%

= after four days of treatment O = after ten days of treatment

S

66

ether 96, was within the range of 1 -15 percent, 23 -56 percent,

28 -70 percent, and 46 -89 percent, respectively.

Tables 9 and 10 show the results of a similar treatment but

for a longer period of time. After ten days of treatment the increase

in phosphorus was found to be 19 -38 percent, 18 -35 percent, and

22 -33 percent on the basis of DNA content; and 40 -66 percent, 24 -67

percent, and 46 -76 percent on the basis of wet weight of sample,

from the treatment with polysorbate 85, sorbitan trioleate and poly -

oxyethylene ether 96, respectively (see Figures 9 and 10). The

increase of phospholipid phosphorus was considerably smaller than

that found after four days of treatment, if calculated on the basis of

DNA present in the sample, but did not differ too much if wet weight

was used as the reference. A probable explanation could be that

the DNA content was also increased (Table 14, page 78). Conse-

quently, the reference standard became greater in the case of DNA,

and probably changed nothing in the case of wet weight.

The probable reason for the increase of phospholipids is that

the surfactants damaged the biological membranes by either rupturing

the membranes or replacing certain phospholipid molecules in the

continuous phospholipid micelles present in the membranes. To

repair this damage, a higher rate in the biosynthesis of certain

phospholipids should be expected, in order to regenerate the original

membrane structure. Phospholipid molecules present in ruptured

-

67

membranes and those that are possibly replaced by surfactants are

measured along with the newly formed molecules during the analysis

of skin tissue.

An investigation of the effects of polysorbate 80 on the in vitro

metabolism of cells, reported by Kay (1965), should be mentioned

here to support this hypothesis. Treatment with polysorbate 80

induced a marked increase in cell permeability in preparations of

Ehrlich- Lettré ascites cells. An extraordinary increase (up to

almost 300 percent) in the incorporation of 32P into phospholipid

fractions was also observed in cell preparations containing one

percent polysorbate 80.

The above phenomena might also be explained by the assumption

that the increase in permeability is due to structural changes (dis-

ruption of membranes). The rapid incorporation of 32P into phospho-

lipids after the surfactant treatment suggests a reconstitution of cell

membrane material.

A further study of the rate of biosynthesis of phospholipids in

rabbit and in human skin treated with surfactant preparations should

prove the above hypothesis.

Another well known phenomenon, namely, that surfactants

generally increase percutaneous absorption of various substances

(Bettley, 1965), could be explained partly by permeability changes,

due to structural changes in skin "barrier" membranes.

68

Composition of Phospholipids

Results of TLC studies for the determination of phospholipid

components indicated that the four major phospholipids of the rabbit

epidermis are: lecithin, phosphatidyl ethanolamine, sphingomyelin,

and lysolecithin.

The amount of phosphorus recovered from the above phospho-

lipid TLC spots was generally 80 -88 percent of the total phosphorus

present in the sample. Occasionally, however, the recovered amount

was greater than that of the total phosphorus measured directly by

spectrophotometric techniques. A possible explanation for this dif-

ference is that the Silica Gel G blank spots also contained small

amounts of phosphorus and it was not always possible to use a corre-

sponding size of blank spots for correcting this. Because of the

irregular differences between the TLC recovered and the spectro-

photometrically determined total phosphorus, the percent composition

of the above four phospholipids was calculated from the amount of

total phosphorus recovered from the thin layer plates. In this man-

ner, the percent composition refers only to the relative proportions

of the four phospholipids, and does not represent the actual percent

composition. The unidentified phospholipids, which probably compose

10 -20 percent of the total phospholipid content, were disregarded.

The relative percent composition of the four major phospho-

lipids is shown in Table 11. According to these data, there was no

Table 11. Relative percent composition of the four major phospholipids of rabbit epidermis.

Rabbit No.

LECITHIN

Skin Sample

PHOSPHATIDYL ETHANOLAMINE

Skin Sample U. P. T. S. B. U. P. T. S. B.

51 42 44 44 42 48 31 28 29 34 27

52 46 46 46 42 47 27 31 29 35 24

53 40 40 44 40 35 28 30 34 35 38

54 45 47 42 46 37 27 27 30 30 41

55 48 48 51 44 42 27 26 24 28 32

Range 40-48 40-48 42 -51 40 -46 37 -48 27 -31 26 -3 1 24 -34 28 -35 24 -41

Rabbit No.

SPHINGOMYELIN

Skin Sample

LYSOLECITHIN

Skin Sample U. P. T. T. B. U. P. T. S. B.

51 16 16 15 11 15 11 12 12-: 13 10

52 13 15 15 14 19 14 8 10 9 10

53 16 16 15 13 18 16 14 7 12 9

54 15 13 14 14 14 13 13 14 10 8

55 14 14 10 15 14 11 12 15 13 12

Range 13-16 13-16 10-15 11-15 14-19 11-16 8-14 7-15 9-13 8-12

Note: U = Untreated Skin

P = Skin treated with white petrolatum

T = Skin treated with polysorbate 85

S = Skin treated with sorbitan triole ate

B = Skin treated with polyoxyethyle ne ether 96

0

70

apparent change in the distribution pattern of these phospholipid com-

ponents which would be due to surfactant treatment. This statement,

however, cannot be considered conclusive because the small number

of data are rather scattered, and because the TLC technique used is

not sensitive enough. In order to arrive at a more reliable conclusion,

a larger number of data should be collected using silica gel which is

free of phosphorus. A two- dimensional TLC method would ensure a

more complete separation of lysolecithin and sphingomyelin, than was

possible in the present study.

There has been only one report (Schwartz et al. , 1960) on the

phospholipid composition of whole rabbit skin. In that report lecithin

accounted for 50 percent of the lipid phosphorus. The amounts of

other lipid components, phosphatidyl ethanolamine, phosphatidyl

serine, lysolecithin, sphingomyelin and polyglycerophosphatides were

not indicated.

Cholesterol Content

Among the nonpolar lipids, only cholesterol was studied.

Tables 12 and 13 indicate the cholesterol content of control and sur-

factant treated rabbit skin. The percent changes of cholesterol con-

tent, due to treatment with surfactants, are illustrated by Figures 11

and 12. It appears from these figures that in most cases there is an

increase in cholesterol content of the surfactant treated rabbit skin.

Table 12. Cholesterol content of rabbit epidermis (µg Cholesterol/ 100µg DNA) after four and ten days of treatment.

Four days treatment Rabbit Number

Ten days treatment

Substance 54 55 56 57 58 59 60 61 62 63

None (untreated skin) 111 109 152 100 134 90 139 86 104 122

Petrolatum 146 139 160 144 101

Polysorbate 85 180 138 139 95 120 117 178 109 88 100

Sorbitan trioleate 147 130 134 88 190 132 178 120 124 126

Polyoxyethylene ether 96 142 121 155 111 175 207 167 111 118 104

-

Table 13. Cholesterol content of rabbit epidermis (µg cholesterol /100 mg wet weight of skin) after four and ten days of treatment.

Four days treatment Number

Ten days treatment

Substance 54 55 56 57 58 59 60 61 62 63

None (untreated skin) 284 356 360 277 457 419 321 309 363 359

Petrolatum 360 411 416 - 419 369

Polysorbate 85 436 367 413 307 413 369 565 500 408 473

Sorbitan trioleate 361 450 485 379 598 408 405 561 568 562

Polyoxyethylene ether 96 424 365 505 405 569 653 557 503 550 506

itabbit

Perc

ent

chan

ges

of c

hole

ster

ol

73

90-

80-

70-

60-

40-

30-

20-

10-

0-

-10-

-20

OO

o o

o O

o o

o

o

T S

O O o

o

Figure 11. Changes of cholesterol content calculated on the basis of DNA content of skin sample.

Note: Skin samples treated with:

P = white petrolatum T = polysorbate 85, 10% S = sorbitan trioleate, 10% B = polyoxyethylene ether 96, 10%

= after four days of treatment O = after ten days of treatment

50- f

130%

If

P B

ó x

'8u.

b9

W

Perc

ent

chan

ges

of c

hole

ster

ol

74

80-

70-

60-

50-

40-

30-

20-

10-

0-

-10,

-20

O

00

o

0

O

0

0

0

O

O

0 0

o

P T

Figure 12. Changes of cholesterol content calculated on the basis of wet weight of skin sample.

Note: Skin samples treated with:

P = white petrolatum T = polysorbate 85, 10% S = sorbitan trioleate, 10% B = polyoxyethylene ether 96, 10%

= after four days of treatment O = after ten days of treatment

O ;4 a) +,

á ó ú ,H 0

á an 0 , U

-4' ai U k a)

S B

75

The changes due to the treatment with petrolatum, polysorbate 85,

sorbitan trioleate and polyoxyethylene ether 96 are 15 -30 percent;

-12 -76 percent; -3 -82 percent; and 3 -74 percent, respectively. The

above changes were calculated on the basis of the wet weight of skin

samples. If cholesterol content is related to the DNA content of skin

samples, the changes in cholesterol range between 4 -31 percent,

-18 -67 percent, -12 -47 percent, and -15 -130 percent after treatment

with petrolatum, polysorbate 85, sorbitan trioleate, and polyoxyethy-

lene ether 96, respectively.

These results are rather scattered, which is partly due to

biological variations. The other possible reason for the wide range

in results is that the epidermis samples, which were used for lipid

studies, may have been contaminated with cholesterol derived from

the skin surface lipids. The cholesterol content of rabbit skin surface

lipid is estimated to be ten percent (Nicolaides, 1965). During this

experiment an attempt was made to remove the surface lipid by

washing it off with ether before the epidermis sample was obtained

with the aid of the keratotome. It is possible that the removal of

cholesterol with ether washing was not complete and uniform in each

case. It is very difficult to eliminate this source of error, since a

more prolonged ether washing would remove not only surface lipids,

but lipids from the stratum corneum and from other layers of epider-

mis. It is also possible that the removal of surface lipid from areas

76

of untreated skin was more complete than from other areas which

were covered by a protective film of the ointment base applied pre-

viously.

Because of the foregoing wide range in results, the effect of

surfactants on the cholesterol content of rabbit epidermis cannot be

considered conclusive, but present data suggest that treatment with

surfactant preparations resulted in an increase of the cholesterol

content of rabbit epidermis.

Deoxyribonucleic Acid Content

The wet weight is generally used with caution as a reference

standard in biological studies, especially when the tissue studied is

heterogenous in nature. An attempt was made in this study to use

samples with uniform structure. Skin samples of 0.1 mm thick

slices could, however, contain different proportions of the four layers

of epidermis. Slices of 0.2 mm thickness, besides these variations,

could contain different portions of epidermis and dermis.

In order to find a more reliable reference standard than the

tissue wet weight, the DNA content was determined of each sample

that was analyzed for lipid content. DNA content is used to indicate

the viable cell numbers.

On the basis of DNA content, the results of lipid determinations

show patterns similar to those found when the wet weight was used as

77

a reference. There was, however, a distinct difference between the

results, calculated on the basis of the two reference standards, if

the increase of phosphorus in skin samples treated for a period of

ten days was compared. The percent increase of phosphorus is

smaller using DNA as a reference, but the range of these percentages

is narrower than if wet weight is used as the reference standard. The

smaller spread in results indicates that DNA may be more reliable

as a reference standard; but the wet weight still can be used as a

practical and useful standard of reference, especially if the DNA con-

tent is also influenced by the test procedure.

Table 14 shows the comparison of the two reference standards,

DNA content, as related to wet weight of the samples, shows an in-

crease, especially after ten days of treatment. This might be due to

irritation of the skin, caused by the surfactant treatment, which could

initiate a higher metabolic and mitotic activity. An increased rate in

cell proliferation, and hyperplasia of the epidermis, are often par-

alleled with an increased rate of DNA synthesis and an increase in

DNA content (Barka, 1965; Fukujama, Nakamura and Berstein, 1965;

deBersaques, 1966; and Hell and Hodgson, 1966).

Table 14. DNA content related to wet weight (µg DNA/ 100 mg wet weight) after four and ten days of treatment.

Four days treatment Rabbit Number

Ten days treatment

Substance 52 53 - 54 55 56 57 58 59 60 61 62 63

None (untreated skin a) 232 236- 256 306 309 340 342 325 247 358 420 440

None (untreated skin b) 279 241 193 250 244 346 -

Petrolatum 214 257 248 296 261 - 291 310 --

Polysorbate 85 213 209 241 267 296 359 343 315 317 458 462 473

Sorbitan trioleate 211 192 245 347 362 429 315 310 227 466 459 445

Polyoxyethylene ether 96 212 203 359 427 325 469 325 315 330 452 464 486

79

IV. SUMMARY

Selected nonionic surface active agents, incorporated in various

ointment bases, were applied to normal rabbit skin daily in an attempt

to determine their chronic toxicity. The dermatitic effects of these

surfactant preparations were evaluated by three methods: gross ob-

servation, histological examination and biochemical techniques. The

results indicated that the tested nonionic surfactants have a distinct

potential to irritate rabbit skin, and to cause histological and bio-

chemical changes in the skin to which they are applied.

It was apparent that the polyoxyethylene ether type of surfac-

tants have the highest capacity to produce dermal reactions. The

local toxicity of polysorbates and sorbitan fatty acid esters was found

to be somewhat less, but the degree of toxicity varied with the indi-

vidual surfactant within these groups.

Results of gross observations indicated that preparations con-

sisting of 60 percent or more polyoxyethylene ether produced severe

erythema, hyperkeratinization and desquamation within three days.

Treatment with one and five percent preparations of polyoxyethylene

ethers resulted in moderate and severe erythema and in some cases

thickening and hardening of the rabbit skin. The intensity of these

dermal reactions was increased as the treatment continued. The

skin treated with polyoxyethylene ethers of all concentrations showed

severe erythema, thickening and scaling with marked induration and

80

fissuring. The irritation potential of sorbitan fatty acid esters and

polysorbates was less than that of the polyoxyethylene ethers. After

three days of application polysorbate preparations of all concentra-

tions produced a slight to moderate erythema. With the exception of

sorbitan monolaurate and trioleate, which produced moderate to

strong erythema, no changes were seen at the sorbitan areas until

five to seven days of treatment. Among the sorbitan fatty acid esters

and polysorbates, sorbitan trioleate and polysorbate 85 had the highest

irritation potential. Undiluted forms of these surfactants produced

fissuring and scaling of the skin after 20 -25 days of treatment.

The microscopical changes in the treated areas generally corre-

sponded to the degree of irritation observed grossly. All prepara-

tions of polyoxyethylene ethers produced inflammation with a number

of polymorphonuclear and round cells appearing in the dermis and

acanthosis in the epidermis. The concentrated forms of these pre-

parations (60 and 100 percent) produced varying degrees of superficial

necrosis. In some cases, necrosis of the epidermis was observed

with massive destruction of epidermal nuclei and disruption and dis-

orientation of cellular structure down to and through the basal layer.

The necrosis process was followed in all instances by re- epithelization

from remaining appendages. Surfactant preparations belonging to the

sorbitan fatty acid esters and polysorbates, with the exception of

polysorbates 80 and 85, produced a slight degree of inflammation

81

in the upper dermis after ten days of treatment. There was a greater

number of inflammatory cells observed in samples taken from sites

treated with polysorbates 80 and 85. After one month of treatment,

polysorbate preparations and undiluted sorbitan trioleate caused

hyperplasia and acanthosis in the epidermis with various degrees of

inflammation in the dermis. Undiluted polysorbates 80 and 85 pro-

duced severe necrosis of the upper epidermis and a high number of

inflammatory cells in the dermis. Sorbitan monolaurate and poly -

sorbate 20 appeared to be the least toxic, producing only a very small

number of inflammatory cells in the dermis and no change in the epi-

dermis.

Most of the previous studies on cutaneous toxicity of chemical

agents have used only the above two evaluation methods; i. e. , gross

observation and microscopical examination. These experimental

procedures are empirical in nature and arbitrary in evaluation.

There are no exact, standard scales by which macroscopical and

microscopical inspections can be recorded.

In the present study, besides the above two evaluation methods,

an attempt was made to evaluate the effects of selected surfactants

by more exact and objective data obtained by standard biochemical

assay methods.

Measurements of the respiratory activity of rabbit skin indi-

cated that, in all instances, the surfactant treated skin consumed

82

two, three or four times as much oxygen as the control skin. A

specific explanation for the increased respiration rate has not been

found. A hypothesis was proposed that the effect of surfactants on

the respiratory enzymes is partly an indirect one, which derives

from the effect of surfactants on cell membranes.

Surfactants, generally possessing a particular affinity for

membraneous structures, may disturb the balance of cell systems

by inducing structural and, consequently, permeability changes in

the biological membranes. An indirect method, the measurement of

quantitative changes in phospholipid content, was applied to test this

possibility. Skin samples used for lipid determinations represented

mainly the epidermis and occasionally a trace of dermis, as was

found after microscopic examinations of the 0.1 mm thick skin slices.

The surfactant treatment induced a considerable increase in

the total phospholipid content of rabbit epidermis. After four days

of treatment the largest increase in lipid phosphorus (47 -87 percent)

was obtained in the case of polyoxyethylene ether 96, ten percent in

petrolatum. Ten percent concentrations of polysorbate 85 and sorbi-

tan trioleate produced 26 -53 percent and 27 -57 percent increases,

respectively, in lipid phosphorus. The DNA content of the sample

served as a reference standard in the above experiments. A similar

pattern in the increase of phosphorus was obtained if the wet weight

of skin sample was used as reference standard, except that the ranges

83

of results were somewhat larger. After ten days of treatment the

increase in lipid phosphorus content was considerably smaller than

that found after four days of treatment if calculated on the basis of

DNA, but did not differ significantly if wet weight was used as a

reference. The comparison of the two reference standards indicated

an increase in DNA content as related to wet weight, which should

explain the above differences in the results calculated on the basis

of DNA and wet weight.

Results of experiments designed to study possible changes in

phospholipid composition, as well as changes in cholesterol content,

were not conclusive.

84

V. CONCLUSIONS

In most of the previous investigations the prophetic skin patch

test technique has become a routine procedure for the safety evalua-

tion of materials applied to the skin. The results of gross observa-

tions reported in this thesis are in conflict with previous reports

concerning the dermal toxicity of the surfactants tested. The differ-

ence in the intensity of local irritation, which, during this investiga-

tion, was found to be considerably higher than in previous reports,

may be due to the different methods by which the surfactants were

applied. A single application (as in the patch test technique) may

produce no visible dermal reactions, but repeated application (as

used in this investigation) could result in skin responses of various

intensity. Cosmetic and dermatological preparations containing

these surfactants are applied to skin regularly and are intended to

remain in contact with the body for a prolonged period of time.

It could be concluded from the above differences in results of

the two application methods, that the patch test technique is a less

reliable procedure for predicting cutaneous toxicity of substances

applied to skin regularly than a daily application of the test material

for a period of at least ten days.

Morphological changes induced by these surfactants resembled

those of various skin diseases; e. g., many forms of chronic

S5

dermatitis are characterized by redness, thickening, fissuring and

scaling of the skin. There were nonspecific microscopic changes

found in the surfactant treated rabbit skin which might conceivably

be present in human skin in cases of chronic dermatitis; acanthosis

and hyperplasia of the epidermis, edema and infiltration of the der -

mis by inflammatory cells.

The phospholipid content of psoriatic human epidermis,

measured as total lipid phosphorus, was reported to be 68 percent

higher than that of normal human epidermis (Gerstein, 1963). A

comparable increase in lipid phosphorus of the surfactant treated

rabbit epidermis over that of the normal rabbit epidermis was

found during this investigation.

Diseased human skin (in cases of psoriasis and in various

dermatitis) generally consumes more oxygen than normal skin

(Mezei and Stewart, 1964 -) measured by the same technique as was

used in this investigation. An apparent increase in oxygen uptake by

the rabbit skin treated with surfactant is shown in the present study.

The purpose of this study was to investigate the chronic toxicity

of these surfactants on rabbit skin. Results of laboratory studies of

irritants applied to animal skin are not always reliable for predicting

the effects of similar materials on human skin. The general simi-

larities between the properties of rabbit skin treated with surfactants

and those of human skin in chronic dermatitis, however, lead to

86

postulation and association.

It is distinctly possible that a careful study of surfactant

treated human skin, using methods described in this thesis, could

well indicate a part being played by these nonionic surfactants in

the production of external contact dermatitis of the hands, which is

one of the most common dermatoses in our modern North American

society.

87

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APPENDIX

98 APPENDIX

Supplemental Information for the Chemical Composition of Surfactants listed in Table I.

A better understanding of the chemical composition of these

surfactants is gained by a review of their method of preparation.

a. The production of partial esters of sorbitan fatty acids

(sorbitan series) involves anhydrization of sorbitol to

hexitans and h;úxides and their esterification with the

appropriate fatty acid.

HEXITOL (SORBITOL)

CHZ OH

HC-OH

HO-CH

HC-OH

HC-OH

CH2OH

+HOOCR -7

-H20

HEXITANS and I4EXIDES

CH2 2

CH-CH2OH

HO-CH CH-OH 0 %.' CH

H HO-CH CH C2

, i 1

CH CH-OH s SH

HO-CH CHOH

CH2 OI-CH-çH2 OH

IIOH

O ., CH2 CH-CH2OOR

E

HO-CH CH-OH Cl- HO -CH CH

OH CH2 /CH

HO-CH -CH-OH 0

1

CH CH-CH-CH O OH OOCR

PARTIAL ESTERS OF SORBITAN

FATTY ACIDS

O 2 1 - CH-OOCR

R - the residue of a long chain fatty acid

>

/O I I

i

r \

O -

OH

I

99

b. The polysorbates are essentially polyoxyethylene (ethylene

oxide) derivatives of the sorbitan fatty acid esters; the un-

esterified hydroxyls are available for reaction with ethy-

lene oxide:

SORBITAN FATTY ACIDESTER

O 00' ` CH2 CH-CH2O0CR HO-CH CHOH

HO-CH CHOH 2 iH- -CH2O0CR

CH''''' O áH 1

OH

V

ETHYLENE OXIDE O

+"N" CH2 - CH2

POLYOXYETHYLENE SORBITAN FATTY ACIDS (POLYSORBATE)

CH CH-0-(CH2CH20)-H

6-(CH n3

-(CH2CH-0).-F1 n2

O-(CH2 -CH 2-0)ri -0)-H 1

i -(CH -CH 20)ri 1

CH CH-O(CH2CH2O) -H

1

n3

CH*`4%., /-CH-CH200CR

o 1

O(CH2 -CH 2-0)nH n2

n1 +n2+n3 = "N"

In the above types of surfactants (a and b), the hydrophilic

and hydrophobic groups are connected by an ester linkage. The esters

formed by the reaction of the fatty acids with polyhydric alcohols.

1 I

2

I

I

O 2 CH-CH 24

DOCR

I I

\

100

c. The third class of nonionic surfactants used in this study

are prepared by reacting ethylene oxide with a fatty alcohol

to produce polyoxyethylene ethyl ethers.

ETHYLENE

ALCOHOL OXIDE POLYOXYETHYLENE ETHYL ETHER

O

+"NCH -CH [RCOH] 2

2 [RCOCH2CF1-O-(CH2-CH2 O) - CH2 CH2-O]

"N"-2

R - the residue of alcohol

The alcohols used to prepare this type of surfactant are Lauryl

(C12H25 OH) , Cetyl (C16H330H) and Stearyl (C18H370H) saturated

alcohols; and Oleyl (C 18H350H) an unsaturated alcohol.

The number in brackets (in Table I) indicates chain length

of polyoxyethylene portion ( "N ").