UNIVERSITY OF MEDICINE AND PHARMACY
GR.T.POPA IAŞI
FACULTY OF PHARMACY
SIMONA ALEXANDRINA PINTEA (ARDELEAN)
PhD THESIS
Scientific coordinator
Prof. Univ. Dr. NĂSTASE VICTOR
2011
UNIVERSITY OF MEDICINE AND PHARMACY
GR.T.POPA IAŞI
FACULTY OF PHARMACY
SIMONA ALEXANDRINA PINTEA (ARDELEAN)
EXPERIMENTAL STUDIES OF SOME
NEW SURFACTANTS USED IN SOME
PHARMACEUTICAL PREPARATIONS
PhD THESIS
Scientific coordonator
Prof. Univ. Dr. NĂSTASE VICTOR
IAŞI
2011
2
CONTENTS
I. DATE OF LITERATURE
1. RELATING TO SURFACTANTS GENERALITIES……. 6
2. MECHANISMS OF ACTION AND SURFACTANTS
CLASSIFICATION .............................................................. 12
3. QUALITATIVE AND QUANTITATIVE
DETERMINATION OF SURFACTANTS ........................... 18
4. METHODS FOR ASSESSING THE TOXICITY
LEGISLATION ..................................................................... 22
II. PERSONAL RESEARCH
Motivation and research objectives ....................................... 33
5. MODERN METHODS USED FOR STUDY OF
SURFACTANTS .................................................................. 38
5.1. INVITTOX main protocols used as alternative methods for
testing the harmfulness .......................................................... 38
5.2. Non-invasive methods used for testing the skin surfactant
action ..................................................................................... 50
5.3. Databases ....................................................................... 59
6. SURFACTANTS USEGE FOR ACQUISITION OF
PHARMACEUTICAL FORMULATIONS AND THEIR
TESTING ............................................................................... 67
6.1. Membrane diffusion and use of Franz cells ................... 67
6.2. Practical assessment of the failure of one active ingredient
by Franz cells equipped with cellulose membranes and skin of
rats ......................................................................................... 75
6.3. Rheological analysis of harm and semisolid formulations
with different types of surfactants ......................................... 77
3
7. SURFACTANTS HARMFULNESS ASSESSMENT BY
USING NON-INVASIVE METHODS ................................ 94
7.1. Generalities ................................................................... 94
7.2. Materials and methods ................................................. 96
7.3. Results and discussion ................................................ 100
7.4. Conclusions ................................................................ 109
8. INVITTOX APPLICATION TESTS FOR ASSESSMENT
OF SURFACTANS ............................................................. 110
8.1. Introductory aspects ..................................................... 110
8.2. Materials and methods ................................................. 113
8.3. Results ......................................................................... 117
8.4. Conclusions .................................................................. 142
9. USE OF SURFACTANTS IN SYNTHESIS OF
NANOCAPSULES POLYETHER-URETHANE USED FOR
TRANSDERMAL TRANSPORT ....................................... 143
9.1. Introductory aspects ..................................................... 143
9.2. Scanning electron microscopy (SEM) ……………….. 145
9.3. Differential scanning calorimetry (DSC) ..................... 149
9.4. Materials and methods .................................................. 153
9.5. Results and discussion .................................................. 156
10. NANOCAPSULES TESTING OF SURFACTANTS
WITH NON-INVASIVE METHODS BY ANIMAL MODEL
............................................................................................. 168
10.1. Terms of determining .................................................. 168
10.2. Tewametric determinations ........................................ 169
10.3. pH measurements ....................................................... 172
10.4. Sebum determinations ................................................. 175
10.5. Mexametric determinations ........................................ 178
10.6. Corneometric determinations ...................................... 184
10.7. Conclusions ................................................................. 187
MAIN CONCLUSIONS ...................................................... 188
REFERENCE ....................................................................... 192
4
II. PERSONAL RESEARCH
Motivation and research’s objectives
Surfactants are known for their influence on many
biological membranes, including skin. This lead to changes in
skin qualities, integrity and inherent to changes in its local
and/or system therapeutic activity. From surfactant’s group,
sodium lauryl sulfate (SLS) is of particular importance
because
it changes qualities of biological membranes,
especially skin’s qualities. Using surfactants to
facilitate
dermal absorption is especially useful for solutions, lotions
and gels for the skin resistance is reduced and surfactant hurry
skin penetration. In these cases it has to take account that
surfactants modify or interact with some components of the
skin layer. After a while the steady state is established.
The skin is an intensively studied organ because of the
exposure to environmental factors, cosmetics, etc. Increased
pathological aspects that may occur in skin has accelerated the
need for studying in experimental models [Franz, TJ, 1978,
Wester, R., 1992, Michaels, A., 1975]. SLS diffuses between
the lipid layers of the skin and it liquefies this double layer. In
addition to this disturbance of skin balance and integrity, it is
known that it produces harmful phenomena such irritation,
transdermal water loss as a result of binding to intracellular
5
keratin. SLS is considered a "golden model" of skin irritation
and a reference compound for the study of skin harm. Not in all
cases using ionic surfactants (anionic and cationic) determine
the most intensive skin penetration. Recent studies on
Metotrexat have shown that they are advanced by another
surfactant, Transcutol. An important aspect is that besides
surfactants, as penetration agents there are also used acids,
fatty alcohols and organic solvents.
Surfactants are a class of auxiliary substances which
can be used both in pharmaceuticals and cosmetics , imposing
the need for quality and safety analysis, including non-ionic
surfactant compounds [Franz, TJ, 1975].Although surfactants
are auxiliary components, they have an important role by
contributing to several formulations (emulsion, ointment bases,
emulsion, etc.) [Franz, TJ, 1975, Franz, TJ, 1978].
The category of surfactant compounds includes the
group of non-ionic surfactants being important for people with
sensitive skin with 1 or 4 hypersensitivity type. From this
group there are used polysorbate or tweens, especially Tween
20 and 80. It is well known their very low irritating capacity,
but for a better understanding it is necessary a detailed
observation. Various forms of mixtures of surfactants were also
very developed for their anti-irritant effects. Polysorbate 20
and 80 are used in formulation of biotherapy products for two
6
features: the prevention of surface adsorption and as stabilizers
against protein aggregation.
Modern analysis of forms with surfactants is justified
by their extensive use in cosmetics and secondly by including
sets of measurements readily achievable [Michaels, A., 1975,
Franz, TJ, 1975]. Regarding their applications a comparison
between the compounds may also be useful. Because they are
not included into the category of dangerous substances, animal
testing is not ethically justified while human testing is
desirable.
The use of noninvasive methods for surfactants
analyses and products containing surfactants enable early
detection of their harmfulness. Some of the most popular, are
devices that measure the transdermal water loss, skin irritation
or the amount of local hemoglobin, melanin pigment, pH, skin
hydration, etc. The most popular devices in this aspect are:
Tewa-meter, Dermaspectrometer, cromameter, Mexameter,
Skin-pH meter, Corneometer, Sebumeter etc. Information
provided by such measurements define the quality of body
skin by providing information about stratum corneum
(corneometrie), transdermal water loss (Tewa-meters), local
redness and pigmentation changes (Mexameter). Mexameter is
a narrow-band reflectance spectrophotometer designed to
measure and quantifies erythema (hemoglobin) and melanin as
7
pigmented areas. Its application in the cosmetic field extends to
hyperpigmentation disorders and it is considered sufficiently
sensitive to detect small differences in skin color. Another
important aspect is that it offers the possibility of
reproducibility [Shah, V, 1993, Smith, EW, 1992].
The skin test methods are various, such as different cell
lines in vitro testing, in vivo testing of experimental animals
and human testing. These methods pass through a continuous
analysis and modernization. Animal testing for cosmetics is
limited. These methods have to be accurate and reproducible.
Excessive exposure to physical and environmental agents
favors the appearance of skin photo-ageing. Intervention on the
body skin through aggressive environmental factors such as
formulations with surfactants, excessive exposure to UV
radiation etc., are matters of concern in current research. Many
evaluation can be made on human model [Corbo, M., 1993].
Toxicity
of
surfactants
compounds
may
generate
a
physiological response, could stimulate irritation and involves
impartial changes (local redness and swelling) or subjective
sensations (itching and pain). Even recent data, especially on
the final preclinical studies, show that various safety
determinations of products call on animal testing because the
similarity of evaluation. However, alternative methods are
intensively supported of which the latest INVITTOX
8
test
develops methods on blood cells tests,
embryo egg, etc..
[Vinardell, M.P., 2008].
A large number of people use a range of creams and
ointments especially on cosmetic purposes. However, it
haven’t been tested the impact of their use after or during
exposure to UV, being known that their composition have a
number of potentially harmful factors (surfactants etc.). This is
correlated with the percentage of skin cancers that occur even
when intensive exposure to UV was reduced a certain time
before. The research aims to highlight the skin quality changes
in the presence of an antioxidant through non-invasive methods
such as: corneometer, Tewa-meter and Mexameter.
In this context, in vivo animal models on mice or rats
are easily obtained with deep observations to molecular level
and can generate results well correlated with studies on man.
This study aims to examine the toxicity of common
surfactants and their use in experimental models especially
non-invasive types. This refers to toxicological evaluation
studies. The data presented would like to emphasize the
possibility of assessing surfactants in formula applied on
human skin. The methods of study refers to non-invasive
techniques applied on the skin and new methods like
INVITTOX test: studies on blood cells (RBC red blood cell
test) - INVITTOX test, protocol no. 37 and the embryo egg
9
test (corioalantoidian membrane) - INVITTOX test. protocol
no. 96. For more detailed data about surfactants applications
were used formulation of nano-capsules observing their
differentiation due to the different type of surfactant.
THE MAIN OBJECTIVES SET IN THE THESIS
1. Analysis of pharmaceutical semi-solid formulations with
surfactants. Comparison of some formulations with different
surfactants.
2. Surfactant’s harmfulness study on skin using non-invasive
methods. Tests applied on human skin and on animal skin.
3. Interpretation the harmfulness of some surfactants using the
latest
alternative
INVITTOX
tests.
Applications
on
erythrocytes and embryo egg.
4. Study of the influence of an active substance disposal
depending on surfactant’s type. Applications using Franz cells,
synthetic membrane and derived from rat skin membrane.
5. Surfactants applications in formulation of nanocapsules.
Differentiation depending on the type of surfactant.
10
6.3. Rheological and harmfulness analysis of some semisolid
formulations with different surfactants
6.3.2. Materials and methods
Rheological readings were made with RV-MLV
Rheotest at 25 °C. Saponification index was calculated
according to FRX, followed by a statistical validation of values
(6).
Formulas of semi solid product that has been used for
surfactant 2% testing were taken and adapted from a cosmetic
form (7). The preparation process of semisolid formulations
was heating the two phases (aqueous and oily phase) at a
temperature of about 70 °C and mixing them. The surfactant is
soluble in the aqueous phase. The two phases are mixed until
cooling/refrigerating and solidification.
To determine the stretch capacity there have been used
the formulas from Table no.
3.
Stretch capacity was
determined using Pozo Ojeda and Sune Arbusa extensometer.
We took account the diameter of the circle occupied by 1 g
ointment after pressing with a glass plate (diameter 11 cm)
weighing 54.5 grams (G0). At 1 minute intervals, weights are
placed on the top plate of the extensometer: 50 g (G1), 100 g
(G2), 150 g (G3) and 200 g (G4). The diameter of the circle
11
formed by spreading ointment is read. The area (π r2) is
calculated and denoted S0, S1, S2, S3 and S4. Based on these
of results, extensometrics curves are drawn.
Table 13. Cosmetic formulas analyzed for 100 g base
Formula
Formula no.
Formula no.
no. 1
2
3
Sodium lauryl sulphate
-
-
0,5; 1; 1,5; 2
Tween 20
-
0,5; 1; 1,5; 2
-
0,5; 1; 1,5; 2
-
-
Cetyl alcohol
8
8
8
Cocoa Butter
7
7
7
Vaseline
20
20
20
Preservative solution
65
65
65
Ingredient
D1216 sucrose laurate
To test the flow curves the next formula was used:
Where τ is the tangential pressure (Pa), z is the cylinder
constant and α is the read value (the force that semisolid
opposes to rotation).
In assessing the viscosity the formula is used
12
where η is dynamic viscosity (Pa. s), τ is the tangential pressure
(Pa), and D is the velocity gradient (1 / s or s-1). Rheograms
and flow curves take account of these parameters.
For harmfulness testing we have resorted to three groups of
volunteers, each of five people aged 20-30 years who have
been trained in this test and showed no other pathologies that
interfere with the final result. Also there was no skin
hypersensitivity.
6.3.3. Results and discussion
Rheological analysis of the studied formulation
revealed the following issues presented in Figures 25-31.
13
Fig 25. Rheogram for the three formulations at a concentration
of 0.5 % surfactant
Fig. 26. Yield curve of the analyzed formulations at a
concentration of 0.5%
14
Fig 27. Rheogram for the formulations at a concentration of 1 %
surfactant
Fig. 28. Yield curve of the analyzed formulations at a concentration of
1%
15
Fig. 29. Rheogram of the analyzed formulations at a concentration of
0.5 %
Fig. 30. Yield curve of the analyzed formulations at a concentration of
0.5%
16
Fig 31. Rheogram for the analyzed formulations at a concentration of
2%
Fig. 32. Extensiometric curve for 0.5% cream
The appearance of the skin to three groups of volunteers
after application of patches with the formulas for analysis and
surfactant concentration of 2% maximum for 24 hours.
17
Fig 36. Formula no.1 (A) before; (B) after 12 hours
Fig. 37. Formula no. 2 Tween 20 (A) before; (B) after 12 hours
Fig. 38. Formula no. 3 SLS (A) before; (B) after 12 hours
Surfactants are amphiphilic compounds that form
mono-layers oriented on interface and presents equilibrium
concentration at interfaces higher than those existing in bulk
solution. They show several features, including detergents,
18
foaming, wetting, emulsifying, dispersion and solubility. These
agents are an integral part of dispersed formulating systems for
medicines and cosmetics and can integrate physical stability for
these systems. In general, they are responsible for the
rheological
properties
of
a
new
formula
for
dermatocosmetologic use [Rieger, MM, 1988, Garg, A., 2002].
Rheological characteristic of semisolid pharmaceuticals and
cosmetics products provide important information to facilitate
production and processing. Today, most producers can count
on rheological results to develop products for the customers.
Therefore, an equipment having a reliable rheological
rheometer with relevant and well understood data, is absolutely
necessary for producers of pharmaceuticals and cosmetics
products. The results indicate that semisolid formulations offer
potential and actual benefits, representing a powerful vehicle
for the development and preparation of dermopharmaceutic
and cosmetic preparations which deserves further investigation.
In another study that should be mentioned, visual
assessments were the first comments on the irritation potential
of semisolid formulations.These are shown in Figure 40. The
first images show the main results of patch tests, and the
second images show Proderm analysis. As shown in Figures 39
and 40, the most harmful agent is Tween 80, but the skin
texture and visual appearance are not as important as
19
observable
negative
side
effects.
Maximum
surfactant
concentrations (10%) were applied on the samples.
Fig. 39. Patch test resuls and general aspect of the skin. No important
side effects
(A) Tween 80, (B) Tween 20, (C) normal
Fig. 40. Skin texture anfer the patch removal (Proderm)
(A) Tween 80, (B) Tween 20, (C) normal
6.3.4. Conclusion
Sodium lauryl sulfate is the surfactant that determines
the best rheological properties (flow, viscosity) but also
sucrose laureat leads to qualitative rheological formulations.
Compared to their two surfactants, sodium lauryl sulfate causes
very serious harm.
20
7. HARMFULNESS ASSESSMENT OF SURFACTANTS
BY USING NON-INVASIVE METHODS
Surfactants are among the most common and versatile
constitutive elements that are found in many topical
medications, cosmetics, cleaning agents, soaps and shampoos.
Surfactants are applied as additives in various products because
of their surface and interface activities. Thus, concentrations
used in commercial preparations should avoid side effects such
as skin or eye irritation as well as the other types of secondary
pathologies of their use. For this reason, the search for new
non-irritating surfactants is of great interest and requires the
development of pre-formulating studies. To ensure that
preparations / formulations are harmless for skin and eye, it is
necessary to study the irritation potential of surfactants by
determining their toxicity.
7.2. Materials şi methods
Occlusive bandages, fixing materials and needles were
used. According to the Declaration of Helsinki, all study’s data
were kept confidential. The study’s objectives also targeted
scientific issues. Before starting the study all subjects were
informed in detail about what will happen. Clinically healthy
Caucasian volunteers who agree to participate freely in
21
research were selected. Exclusion criteria were related to skin
diseases that may interfere with the final assessment,
pregnancy status, simultaneously participation in other studies,
tattoos, burns and bedsores in the areas of study. Total number
of volunteers was 25 women divided in five groups.
Participants will be excluded from the group if:
- don’t closely follow the instructions
- get ill or suffer injuries during the study
- they no longer wish to continue the study
Test Description
Testing methodology consists of the following steps: a
certain amount of product (2ml) is applied with a 2 ml syringe
on an occlusive bandage, The bandage impregnated with
cosmetic product is applied on human skin (shoulder or
upperback); simultaneously ,placebo patches (no active
substances) and those who achieved positive control (sodium
lauryl sulfate), are applied on skin; the three types of samples
are coded and randomly applied in a trial- "single blind" type;
occlusive bandage is maintained for 24 hours without any
moisture, observing any possible side effects; after 24 hours,
the occlusive bandage is removed and the product is washed
without any rubbing maneuver; all the skin adverse effects
22
(redness, dryness, edema), are evaluated using COLIPA scores,
any reaction is observed immediately, after 30 minutes, one
hour, 24 and 48 hours after removal of the occlusive bandage;
evaluation was performed by qualified personnel and under the
same lighting conditions; occlusive bandages, adhesive
bandages for fixation, syringes and paper were used for
patches. Studies have been conducted in according to the
ethical principles of the Helsinki Declaration on human
subjects testing, including confidentiality of all records and
documents.
Skin appearance was analyzed using patch tests and
confirmed by analysis with a Proderm device.
Fig. 41 Proderm device used in skin analysing
Toxic / irritating potential assessment of the tested cosmetic
products is visually performed - skin redness and / or dryness is
23
evaluated on a scale in accordance with COLIPA scores (Table
14).
Table 14. COLIPA scores
Dryness
(appearance of
scales)
Erythema
0
Edem
= without erythem 0 = without adverse
effects
0,5 = minimum
0.5 =dryness,
erythem
without scales
1 = diffuse
1 = minor scales
2 = reduced
erythema ,
Uniform redness
3 =strong
erythema,
uniform redness
2 = reduced scales
redness, spot light
redness
4
— = absence
+ = present
3 = severe
desquamation, with
many scales
= extreme redness
Transepidermal transport consist in crossing the intact
stratum corneum (SC), being
intracellular
and
intercellular
two possibilities to pass:
(through
and
among
corneocytes). Crossing model depends on k partition
coefficient of the active substance: hydrophilic substances
would prefer the intracellular crossing while the hydrophilic
substances prefer intercellular way. In the same time, there are
molecules that can use both ways. This method is an indirect
assessment of the integrity of the hydrophilic layer responsible
24
for skin barrier function. There is a continuous diffusion of
water in the body to the stratum corneum and from there to the
environment. Low levels of transepidermal water evaporated
mean better skin function as a barrier and a lower loss of
natural dampening level. Barrier function is easily disturbed by
mechanical or chemical problems. TEWL measurements will
be made only after using products with occlusive bandage for
24 hours. This type of testing is a non-invasive method of skin
analysis, in a
"single blind" trial. The semi-solid formula
prepared for the TEWL tests was compared with a similar one
using a dry extract from the birch bark; 1g / 100 g cream (see
Table 15). This formula has proven to have a skin protection
activity [Urşica, L., 2005, Morell, JLP,1996].
Table 15. Semisolid formula used for sodium lauryl sulfate test
Types of ingredients
Concentration (mg/100g base)
Sodium Lauryl
sulfaTecetylic
alcohol
cocoa butter
Vaseline
Tween/Tween 80
cetylic alcohol
cocoa butter
Vaseline
Preservative solution
xg
8g
7g
25 g
6og
2/lOg
8g
7g
25 g
6og
25
Table 16. Constituents of analyzed formulations
Purified water
U/A cream
with
Anionic
emulsifier
59,59
Polysorbate 60
0
Constituens (%)
U/A
cream
with nonionic
Sodium
Laurel
sulfate
Cetylstearilic
Alcohol
( B type).
emulsified
Liquid paraffin
White Parafin
1.58
Polysorbate Sodium Laurel
60 solution sulfate solution
(3)
(4)
96
98,42
4
1,58
7,88
14.19
15,76
Ethyl phydroxybenzoate
1
2
7.3. Results and discussion
Visual assessment was the first observations about the
irritating potential of these semisolid formulations. The main
tests were: erythem, peeling and swelling. The results are
shown in Table 17.
26
Table 17. Assessing the skin irritation potential (patch- test 24 hours
after COLIPA regulations).Visual Assessment
Tested Eryth desquamatio Edem
% of
sample em
n
subjects
with
0
0
75%
Cream
base
with
vegetal
extract
Observation
The rest (25%) subjects
presented a slight redness,
swelling,
which resolved within one
hour after patch removal.
SLS
1%
0,5
0
80%
20% of tested subjects
presented edema or diffuse
redness. Symptoms
disappeared after 1 hour
after removal of occlusive
patches
SLS
10%
1
0
75 %
25% of tested subjects
presented edema or diffuse
redness. Symptoms
disappeared after 1 hour
after removal of occlusive
patches.
SLS 20
%
3
0
80%
Obviously irritated skin,
erythema persisted for 24,
edem disappeared after
minimum 2 hours after the
patch removal
Controlnot
treated
0
0
100%
-
-
27
Tested Eryth desquama Edem % of
sample em tion
subjects
with
Tween 0
0
80%
20 2 %
Observation
The rest (20%) of subjects had
a mild redness, a very diffuse
edema, which disappeared 20
minutes after patch removal
Tween 0,5
20 10
°/o
0
80%
The rest (20%) of subjects had
a mild redness, adiffuse
edema, which disappeared 1
hour after patch removal
Tween 0
80 2 °/o
0
75 °/o
The rest (20%) of subjects had
a mild redness or a very
diffuse
edema,
which
disappeared 1/2 hour after
patch removal.
80 10
%
1
0
30 %
Control 0
- non
0
-
100%
28
The product irritates the skin,
but not compared with sodium
lauryl sulfate, the erythema
persisted for 2 hours and
disappeared after at least ½
hour after patch removal
-
Fig 42. Evolution of TEWL values after application of a SLS cream
comparative with a base cream
Fig 43. Skin irritation after application of a lauryl sulfate 5% topic
formula (2)
A. after 12 hour, B, after 24 hours
29
Fig 44. Transdermic water lose for the tested formulation
Fig. 45. Hydratation status for the applied formulations
30
Fig. 46. Erythema values (haemoglobin) for the tested formulations
Fig. 47. Measurements realised with mexameter for melanim values
The main observation in this preliminary study is that
the anionic surfactant solution is most damaging formulation.
As presented in the literature, sodium lauryl sulfate is
considered the "golden model" to test for irritation. It could
be important to analyses what type of formulation lead to the
most obvious as well as the is the importance of different
concentrations to skin induced disorders. Data on these
31
compounds are elementary because they are applied on skin
with considerable frequency. Issues relating to transdermal
water lose highlights the presented discussion and show that
a short and constant period of sodium lauryl sulfate low
doses application an causes important harm on skin.
Skin hydration status is influenced by external factors,
such as creams, lotions, and a real evaluation show the
differences in the type of surfactant and the used formulation.
The most important observations in this direction are
related to sodium laurel sulfate’s harm for both formulations
but easier for non-ionic cream. Semisolid formulations
influence the hydration status after a short period of continuous
application. Parameter with a low influence refers to the
melanin values for all formulations. Even if it is detectable by
the same device, Mexameter, erythem is highly influenced by
the anionic surfactant. This comment can be correlated with the
visual appearance of the skin (Figure 43) for a patch test on
sensitive skin indicating that at a lower concentration of the
anionic surfactant (5%) occurs outside major changes(2
erythem score). Analysis of surfactants applied to the skin are
justified mainly by their frequently use. This could be reason
why the science has developed new trends in surfactnt’s
toxicity analysis, such as
embryo-corioalantoidian egg
membrane (HET-CAM), in vitro tests (fibroblasts, etc.). These
32
evaluations
will
complete
the
non-invasive
tests
of
haemorrhage, lyses and coagulation.
7.4. Conclusion
Polysorbate can be considered the most safety
ingredients in semisolid formulations. Polysorbate 20 is a very
protective compound that can be applied in products for
sensitive skin. Long-term applications of polysorbates are a
safe procedure.
Higher concentrations of surfactants such as ionic
compounds are safe for skin and noxious aspect does not have
important meaning. Even non-ionic compounds introduced in
semisolid cosmetic formulations, which are not toxic, require
testing for safety because of their long-term application and
their use in products for sensitive skin or in various biological
separations.
33
8. INVITTOX TEST APPLICATION FOR THE
ASSESSMENT OF SOME MODERN AND CLASSICAL
SURFACTANTS
Evaluation of irritation potential of surfactants by in vitro
and in vivo methods
Cleanliness / hygiene is an old necessity and surfactants
were used for this purpose since ancient times.
Surfactants are amphoteric molecules containing a
hydrophilic half and a lipophilic one that enable them to
interact with molecules such as polar and non-polar.
Surfactants are generally classified according to these
properties, or based on their chemical constitution. Depending
on their load, they are classified as anionic, non-ionic, cationic
or amphoteric surfactants. Due to their amphoteric properties
they can be used as a cleaning agent, foaming, wetting agents,
emulsifiers etc.
When applied to the skin, they can interact with skin
structures, in particular lipid and protein components. Although
surfactants used in products that are removed, are generally
well tolerated, they pose a risk to develop an irritant contact
dermatitis.
Irritant potential is highly dependent on the concentration of
the substance and the pH achieved in the formulation’s
composition [Rhein, LD, 1986; Barandi, L., 2002]. Because of
34
their ability to emulsify and reduce water surface tension,
surfactants can eliminate fat from the skin during washing.
Therefore, the dermal irritant effects manifest especially in skin
dryness and harshness. Rarely, there can be induced even more
severe injuries.
There are several methods used to distinguish regular
detergents in terms of their potential for irritation and to
classify them in terms of risks. Draize eye irritation test has
been the gold standard for many years for eye irritation testing.
Given the concern for animal welfare and limiting their use in
laboratory experiments, alternative methods were developed
such as red blood cell test,
test on egg corioalantoidian
membrane (HET-CAM test) and tests using ocular tissue
model, all to assess the irritant potential of eye /mucous
membranes
induced by surfactants and other substances.
Dermal irritation is often assessed
in humans using
epicutaneos patch testing (ECT, occlusive one-time patch test),
while the soap-room type tests (SCT) can be used to assess
irritation after repeated occlusive exposure to surfactants.
The purpose of this study was to assess the
comparability of test results using different methods. To avoid
the group-dependent inconsistencies, the irritation potential
was assessed for 18 surfactants, using the same stock solution
of surfactants.
35
When labeling is done in according to the occupational
risk classification, the substances are generally tested undiluted
and at pH of the stored raw material. This doesn’t reflect the
conditions of personal hygiene products such as shampoo,
whose pH level usually ranges from 5 to 8. Therefore, in the
study of surfactants solution’s compatibility the assessment
was made at a pH relevant for this type of consumer product.
Exceptions were cases with limitation of the method with a
specific pH, when testing was performed on products adapted
to a pH of 6.5.
Eye irritation potential of surfactants was studied using three in
vitro test systems that are currently used as alternatives to
animal studies, namely RBC test and HET-CAM test.
8.2. Materials and methods
Sodium lauryl sulfate (SLS), Tween 20 and Tween 60
solutions were 5% concentration. They have been offered by
the department of pharmaceutical technology UMFT.
A
vehicle control solution was prepared for application to CAM;
the pure reagent was diluted with distilled water (1:10, v / v).
SDS was administered to CAM and as a model of a positive
control we used a solution of 1 mg / ml in distilled water.
36
HETCAM test
This biological investigation is used to assess the eye
irritation potential for any substance to test, measured by its
ability to induce vascular toxicity on corioalantoidian chicken
membrane [Ribatti, D., 2008]. HETCAM bioassay was
conducted following the ICCVAM recommendations published
in November 2006 in Appendix G and adapted to our
laboratory conditions [Kishore, AS, 2008].
Animals and treatment
Type CD1 Nu/Nu mice have been bought from Charles
River (Sulzfeld, Germany), 4 mice / group. From week 8, the
test solution was applied on the dorsal area: group A. controlonly solvent (water), group B. 5% SLS solution, group C. - 5%
Tween 20 solution group D. 5%, Tween 60 solution, two times
a day. The final results were recorded and centralized after 30
days. Work protocol followed all the rules proposed by healthNIAH National Institute of AnimalHealth: during the
experiment the animals were maintained in standard
conditions:12 hours light-dark cycle, food and water libidum,
temperature 24 °C, relative humidity exceeds 55%.
Mexameter measurements were performed with the aid of the
Courage-Khazakar research device, a MX18 mexa-meter
(Courage-Khazaka Electronic, Cologne, Germany). Mice were
anesthetized with ketamine and xylazine.Continuous
measurement time was 20 seconds.
37
The Protocol sought observations of hemoglobin status
(erythem). The device was applied to the evident affected areas
and kept on skin for 20 seconds. Data were recorded by the
specific software Mexametru MX18 and then expressed in
units. All data were processed as initial and final values
measured in the same area.
Tested compounds and reagents
All surfactants used, except sodium lauryl sulfate (SLS)
were obtained from Cognis Deutschland GmbH & Co. KG
(Germany). A description of these surfactants can be found in
table 19. The same stock solutions were used for all tests (12%
AS, pH 6.5).
Table 19. Description of the surfactants used in this study
Name
The type of surfactant
Chemical structure
SDS (control -)
Anionic Surfactant
C12H25S04Na
NaCI
0,9
% (control -)
-
-
Olive oil(control-) -
-
Sucrose ester
-
-
Cremophor A 6
emulsified
Polyethylene glycol 260
mono(hexadecy
l/octadecyl) ether and 1Octadecanol
38
Cremophor A25
polyethoxylated non-ionic
detergents
Cremophor EL
Used to emulsify and
solubilise oils other and
insolubles substances,
Cremophor is the brand name
for a series of
polyethoxylated non-ionic
detergents. It was developed
for using as an emulsifier for
soluble and oral preparations,
Macro gol (25)cetostearyl
ether. Polyethylene glycol
1100
Mono
(hexadecyl/octadecyl)
ether
Cremophor RH 40 Cremophor is the brand
Macrogol glycerol
name for a series of
hydroxystearat. PEG^40
polyethoxylated non-ionic
castor oil, Polyoxyl 40
detergents. It was developed hydrogenat castor oil
for using as an emulsifier for
soluble and oral preparations,
topical and parenterale
Abnl EM 90
Macro golglycerol
ricinoleate. PEG-3fi
castor oil, Polyoxyl 35
hvdrogenated , castor oil
C1o H6(CHO)2
Isolan GI 34
Polyglyceryl-4 Isostearate
a lipophilic ester of
isostearic acid polyglycerol
optimized. The molecular
structure oft he emulsifier is
similar to natural skin lipids.
ISOLAX GI ® 34 is noted
for its goodstability against
oxidation.
Labrasol
Excipients for preparation
of pharmaceutical
formulations and cosmetics
39
Glyceryl şi
polyethylene glycol
ester.
8.3. Results
Identical stock solutions (12% AS, pH 6.5) were used
for all tests in order to avoid variations due to differences in
test results caused by intrinsic properties of the test.
The concentration of active substance (AS) and pH play
a major role in causing irritation, and AS% pH being adjusted
in accordance with Table 20, depending on the method used
before testing. With the exception of ECT, all tests were
performed in the same month.
Some samples were tested in ECT in the same month
while other samples were tested two months later. Tests were
effectual "double-blind and randomized appropriate.
RBC Test
Test red blood cells is a cell-based photometric test and
was developed to assess the initial reactions that occur in
cellular processes irritation caused by surfactants and
surfactant-based products. The principle is to evaluate the test
RBC membrane disorder and protein distortion caused by
surfactants. To this end, cell membrane damage is assessed by
the amount of hemoglobin, which runs into the supernatant and
protein conformation changes are evaluated by denaturing
hemoglobin. Because the physiological blood pH is 7.4, all
solutions were adjusted to this pH to avoid irritation potential
40
changes only because of pH. Ratio H50 / DI is used as a
measure for the classification of eye irritation in analogy with
the Draize eye irritation test.
Surfactants Dehyton MC, Gluadin WQ, Dehyton ML
Plantacare 2000 Plantacare 818, Gluadin WK, and Plantapon
LC7 were classified as non-irritating surfactants Plantapon
while LC7, Gluadin Plantapon WK and 818 were assigned the
least irritating. PGFAC-S surfactants, Plantapon ACG 50, 138
Plantapon CML and Plantacare 1200 were classified as slightly
irritating surfactants Plantapon SB3, DC Dehyton Dehyton
PK45 were classified as moderate and irritating surfactants
Texapon S K14, Texapon FTA, Texapon K12 N70 Texapon G
were classified as irritants.
HET-CAM Test
HET-CAM test was applied according to the protocol
described by Spielemann ICCVAM and Liebsch (INVITTOX
1992). It is based on observation of the membrane after
application of samples to be analyzed corioalantoide and takes
into account the advantage of using biological tissue - blood
vascular network, to respond promptly in contact with the
compounds analyzed.
Corioalantoidian membrane of chicken embryo is
observed continuously for 5 minutes after application of
41
solutions analyzed. The method could be used for all samples,
they are transparent. Time of occurrence of first sign of
hemorrhage, vascular lysis (ghost vessels) or coagulabilitate is
noted in seconds and used to calculate the index irritability
caused severe reactions was also evaluated.
The results are shown in figure 48 and table 20.
a
42
c
e
43
g
i
44
k
m
45
o
q
46
s
Fig. 48. HET CAM Test, the image of corioalantoidian membrane
before application and five minutes later: SDS (a) before (b) after (c)
NaCl 0.9% (d) olive oil, sucrose ester (e) before (f) after, Cremophor
A6 (g) before (h) after, Cremophor A25 (i) before (j) after, Cremophor
RH40 (k) before (l) after Cremophor EL (m) before (n) after Abril
EM90 (o) before (p) after, Isolan GI34 (q) before (r) after, Labrasol (s)
before (t) after
47
Table 20. Tags irritability, severity and classification of its
irritating effect from HET CAM test surfactants control
samples analyzed
Analized
compound
SDS (control +)
NaCl 0.9 %
(control -)
Olive oil
(control-)
Sucrose ester
Cremophor A6
Cremophor A25
Cremophor RH
40
Cremophor EL
Abril EM 90
Isolan GI 34
Labrasol
Iritability
index
(average)
17.99
0
Effect
severity
(average)
Effect clasification
3
0
Severe reaction
None
3.03
0
None
10.7
17.47
0.625
2.75
5.05
11.45
0.325
0.5
Easy reaction
Between moderatesevere
Very easy reaction
Easy reaction
6.85
6.33
6.33
8.65
1
0.25
0.25
0.625
Easy reaction
Very easy reaction
Very easy reaction
Easy reaction
48
Fig. 49. Irritability index for the series obtained from the
application of surfactants analyzed HET CAM test
Fig. 50. The level of severity of the effect of surfactants
obtained for the series examined the application HET CAM
test
Analyzing the results observed difference between
control samples and analyzed. There is severe reaction caused
by sodium lauryl sulfate (SLS), its toxicity is confirmed by the
effect produced on application of 0.3 ml of 1% in the
corioalantoidian membrane, embryonic death occurred within
20 minutes. In addition to all other specimen treated with SLS
have survived until the next day.
The series of compounds analyzed and Cremophor A6
stands, during the 5 minute observation of the evolution of
49
vascular plexus in contact with the solution applied were
observed microhaemorrhage many areas, also the evaluation of
macroscopic appearance a day after the application of
membrane samples, Cremophor A6 is the only test that
determines the full destruction of the corioalantoidian
membrane (figure 51).
Fig. 51. Specimens treated with Cremophor A6, saline,
Labrasol, Abril EM90, olive oil, sucrose ester and Isolan GI34
24 hours after application of samples
None of the molecules examined was devoid of the
irritating potential. The following compounds were evaluated
as agents for surfactants with very mild irritative reaction:
Cremophor A25, Abril EM 90, Isolan GI34. Sucrose ester,
Cremophor RH 40, Cremophor EL and Labrasol showed a
slightly irritating potential. The compound with the highest
potential irritation classified as moderate to severe effect was
Cremophor A6. This effect was confirmed by the destruction
evoked membrane structure in figure 51.
50
The results show a large difference between positive
control, two surfactants SLS and Tween 20 and Tween 60
tested.
SLS
induces
major
vascular
disorders
the
corioalantoidian membrane, after application of 0.3 ml of
solution (1 mg / ml), a large area was affected by the large
number of microhaemorrhage, death model is registered after
20 minutes of exposure with SLS solution. Other copies have
better viability, resisting until the next day.
The two surfactant solutions evaluated did not produce
severe irritation of the CAM capillary plexus treated. Both
have shown signs of microcoagulation. Tween 20 solution
induced a generalized and severe response, which appeared
later, thus explaining the existence of the lowest irritation score
values. Also, there was no sign of lyses, vasodilatation and
mild bleeding occurred only at the end of observation time. In
terms of Tween 60 solution, there were some areas of
microcoagulation and vascular lyses, but have been recorded
earlier, induces a higher irritative score, although the reaction
severity was classified as mild.
In vivo experiments support the theory irritating aspects
on macroscopic images of the compounds tested (figure 52).
The compound is a landmark irritating SLS, but also those
nonionics (Tweens) have been developed irritation and dry skin
(figures 54 and 55).
51
A. Blank
C.Tween60
B.Tween20
D. SLS
Fig. 54. Evaluation of in vivo surfactant solution observed in
mice with macroscopic images
CD1 Nu / Nu after 30 days of continuous application
300
250
200
150
100
50
0
W
SLS
T60
T20
Fig. 55. Erythema values measured in units compared to the
end of the experiment
(30 days)
52
9. USE OF SURFACTANTS IN SYNTHESIS OF
NANOCAPSULES POLYETHER-URETHANE
TRANSDERMAL USED IN TRANSPORTATION
9.4. Materials and methods
The synthesis of polyether-urethane nanocapsulelor
using a hydroxylic component (diol, polyol or most often their
mixture), a component of isocyanates (a semiprepolimer based
isocyanic Lisin-diisocyanate), solvent and surfactants.
Hydroxyl component comprising a mixture of low molecular
weight diols as 1,2-ethylene, 1,4-butanediol, polyethylene
glycol molecular weight 200 respectively. Isocyanic is a
component-based
semiprepolimer
Lisin-diisocyanate;
semiprepolimer variant is used to avoid mixing problems due
to differences in viscosity between the two components. Use
acetone as solvent and different surfactants.
HO
HO
OH
OH
HO
OH
CH2 CH2 O
1,2-Ethylene-glycol 1,4-Butanediol
Polyethyleneglycol
n
1,6-Hexanediol
O
OH
OCN
NCO
Lysin-diisocianate
Synthesis of isocyanate semiprepolimerului based on
lysine-diisocyanate:
53
Synthesis of polyether-urethane nanocapsules:
54
Table 22. Raw materials mixture ratio
Isocyanate
component
Hydroxil component
Surfactants
Sample
code
LDI
µL
MEG
µL
1,4BD
µL
PEG
200
µL
T20.s
300
80
120
200
T20.a
300
80
120
200
T20.l
300
80
120
200
T20.i
300
80
120
200
T60.A6
300
80
120
200
Tween®60
500 µL
T60.A25
300
80
120
200
Tween®60
500 µL
T60.EL
300
80
120
200
Tween®60
500 µL
T60.RH
300
80
120
200
Tween®60
500 µL
T80.se
300
80
120
200
Tween®80
500 µL
T80.sl
300
80
120
200
Tween®80
500 µL
in 20 mL
acetone
1
2
Tween®20
500 µL
Tween®20
500 µL
Tween®20
500 µL
Tween®20
500 µL
Span85
500 µL
Abril EM90
500 µL
Labrasol
500 µL
Isolan G134
500 µL
Cremophor
A6
20 mg
Cremophor
A25
20 mg
Cremophor
EL
100 µL
Cremophor
RH40
20 mg
Sucrose
ester
20 mg
Sucrose
laurate
20 mg
in 40 mL water
9.5. Results and discussion
Inclusion complexes are analyzed by differential
scanning calorimetry for detecting changes in thermal
properties compared to pure substances. If nanocapsules
55
incorporation, there is a shift of their melting points, boiling
and sublimation or even their disappearance.
Analysis by differential scanning calorimetry was
performed with an apparatus Mettler Toledo DSC 821 status,
version 6.0 (Mettler Inc., Schwerzenbach, Switzerland) with a
heating rate of 5 °C / min and a flow rate of argon of 167 ml /
min (10 l / h). Sample mass was between 2-5 mg and the
temperature range was between 25-300 ºC.
Fig. 61. DSC curve for T20.s nanocapsule sample
56
Fig. 62. DSC curve for T60.EL nanocapsule sample
DSC curves do not show peak's, amorphous substance
having the character and substance decomposition occurs
around the temperature of 280 °C. If products tested are found
HPBCD's disappearance little endothermic (220 ºC) and the
emergence of highly endothermic little sites at around 260 °C,
which probably attests evidence in the whole melting
temperature value. It can not be excluded and a breakdown of
the sample, with a possible reaction between the two
compounds.
Particle morphology was examined using Hitachi 2400S
scanning electron microscope (Hitachi Scientific Ltd, Japan).
We used a thin layer coating apparatus (Bio-Rad SC 502, VG
Microtech, England) to induce electrical conductivity of the
sample surface. The air pressure was 1.3 to 13.0 mPa. The
57
shape and surface morphology of the nanocapsules formed
compounds and their complexes obtained by spray drying
technique are presented in the next figure. Microscopic images
were recorded with a magnification of 10,000 x sample so as to
be visible both picture and a more detailed particulate
substances.
If the review finds that products prismatic crystals,
various sized, smooth surface, is presented as nano crystals of
different sizes, irregular. Regarding the two forms involved in
obtaining inclusion complexes mentioned, there is a significant
difference in particle morphology: some are in the form of
three-dimensional crystals, and irregular size RAMEB consists
of spherical particles, cracked, smooth. If the complexes
obtained by spray drying technique reveals a remarkable
change of the morphology of the substances. SD by products
have an amorphous nature, with spherical particles, some
partially cracked, with a smooth surface. It also notes the
aggregation of spherical particles.
Consequently, the morphology and particle size SD
products are fundamentally different from those of the original
substances, it is impossible highlighting individual crystals
(crystallographic cleavage planes) of diuretics or β-CD. These
observations lead to the estimation of a single phase in SD and
58
therefore products resulting from the formation of inclusion
complex.
Fig. 65. SEM image of T20.s - scale 200 µm
Fig. 66. SEM image of T20.s - scale 50 µm
59
Fig. 67. SEM image of T20.s - scale 100 µm
Fig. 68. SEM image of T60.EL - scale 50 µm
60
Fig. 69. SEM image of T60.EL - scale 20 µm
Fig. 70. SEM image of T60.EL - scale 10 µm
61
Fig. 71. SEM image of T60.EL - scale 200 µm
Fig. 72. SEM image of T60.EL - scale 100 µm
62
Fig. 73. SEM image of T80.sl - scale 50 µm
Fig. 74. SEM image of T80.sl - scale 30 µm
63
Fig. 75. SEM image of T80.sl - scale 100 µm
Fig. 76. SEM image of T80.sl - scale 20 µm
64
Fig. 77. SEM image of T20.s - scale 2 µm
Fig. 78. SEM image of T60.EL - scale 2 µm
65
Fig. 79. SEM image of T80.sl - scale 1 µm
Fig. 80. SEM image of T20.s - scale 1 µm
66
10. NANOCAPSULES TESTING OF
SURFACTANTS WITH NON-INVASIVE
METHODS THROUGH THE ANIMAL MODEL
The experiment to determine the changes occurring in
skin treated with drugs incorporated in polyether-urethane type
nanocapsules were taken into account:
- were used in the experiment 30 mice divided into 10
lots C57BL6j (three mice per group);
- epilated mice were the day before application and
determination that one day before application and
determinations of weeks 4 and 8;
- was applied every time a quantity of 200 ml emulsion
nanocapsules in acetone (conc = 0.01% w / w);
- the witness has applied 200 ml of acetone;
- values were determined after 30 minutes after
application on the skin;
- tables and graphs are processed in the average values
of each batch of three mice;
- groups were noted in table 26 depending on the
surfactants used in synthesis of polyurethane nanocapsules.
Table 26. Batches of mice according to the surfactants noted
Code
T20.s T20.a T20.l T20.i T60.A6 T60.A25 T60.EL T60.RH T80.se
Tween
Tween
Tween Tween
Tween Tween Tween Tween Tween
®20
®20
®20
®20
®60
®60
®60
®60
®80
Surfactant
Abril
Isolan Cremophor Cremophor Cremophor Cremophor Sucrose
Labrasol
Span85
G134
A6
A25
EL
RH40
ester
EM90
Code
Surfactant
T20.s T20.a T20.l T20.i T60.A6
Tween Tween Tween Tween Tween
®20
®20
®20
®20
®60
Span85
T60.A25
Tween
®60
T60.EL
Tween
®60
T60.RH
Tween
®60
T80.se
Tween
®80
Abril
Isolan Cremophor Cremophor Cremophor Cremophor Sucrose
Labrasol
G134
A6
A25
EL
RH40
ester
EM90
67
Fig. 81. Polyaddition reaction for the nanocapsules synthesis
10.2. Tewametric determinations
Table 27. TEWL values for 10 weeks of application-determination
blank
T20.s
T20.a
T20.l
T20.i
T60.A6
T60.A25
T60.EL
T60.RH
T80.se
1
3.1
2.6
2.8
3.2
3.1
2.7
3.1
3.3
2.9
2.7
2
3.5
3
2.8
3
3.3
2.9
3
3.2
2.9
2.7
3
3.8
3.1
3
3.1
3.4
2.9
3.2
3.3
3
2.9
4
3.7
3.2
3.5
3.1
3.6
2.9
3.4
3.4
3.4
2.8
week
5
6
3.8 3.6
3.4 3.3
3.7 4
3.4 3.7
3.2 3.2
3 3.2
3.5 3.6
3.6 3.7
3.6 3.8
3 2.9
7
4.1
3.5
4
3.5
3.4
3.3
3.7
3.8
4.1
3.4
8
3.5
3.5
3.9
3.6
3.7
3.6
4
3.8
4
3.3
9
3.6
3.6
4.1
4
3.8
3.7
4.1
3.8
4.3
3.5
10
3.8
3.9
4
4.3
4.1
3.6
4.3
4
4.3
3.7
Table 28. Statistical characteristics for determined TEWL values
blank
T20.s
T20.a
T20.l
T20.i T60.A6 T60.A25 T60.EL T60.RH
T80.se
Average
3.65
3.31
3.58
3.49
3.48
3.18
3.59
3.59
3.63
3.09
Standard
error
0.0833330.113969 0.1658650.1337080.0997780.1123490.1386040.086217 0.176415 0.11299
Median
3.65
3.35
3.8
3.45
3.4
3.1
3.55
3.65
3.7
2.95
Standard
deviation 0.2635230.360401 0.524510.4228210.3155240.3552780.4383050.272641 0.557873 0.357305
Sample
variance 0.0694440.129889 0.2751110.1787780.0995560.1262220.1921110.074333 0.311222 0.127667
Range
1
1.3
1.3
1.3
1
1
1.3
0.8
1.4
1
Minimum
3.1
2.6
2.8
3
3.1
2.7
3
3.2
2.9
2.7
Maximum
4.1
3.9
4.1
4.3
4.1
3.7
4.3
4
4.3
3.7
68
Fig. 82. TEWL values evolution for the 10 groups
10.3. pH determinations
Table 29. pH values for 10 weeks of application-determination
week
1
2
3
4
5
6
7
8
9
blank
3.1 3.5 3.8 3.7 3.8 3.6 4.1 3.5 3.6
T20.s
2.6
3 3.1 3.2 3.4 3.3 3.5 3.5 3.6
T20.a
2.8 2.8
3 3.5 3.7
4
4 3.9 4.1
T20.l
3.2
3 3.1 3.1 3.4 3.7 3.5 3.6
4
T20.i
3.1 3.3 3.4 3.6 3.2 3.2 3.4 3.7 3.8
T60.A6 2.7 2.9 2.9 2.9
3 3.2 3.3 3.6 3.7
T60.A25 3.1
3 3.2 3.4 3.5 3.6 3.7
4 4.1
T60.EL 3.3 3.2 3.3 3.4 3.6 3.7 3.8 3.8 3.8
T60.RH 2.9 2.9
3 3.4 3.6 3.8 4.1
4 4.3
T80.se
2.7 2.7 2.9 2.8
3 2.9 3.4 3.3 3.5
69
10
3.8
3.9
4
4.3
4.1
3.6
4.3
4
4.3
3.7
Table 30. Statistical characteristics for determined pH values
blank
T20.s
T20.a
T20.l
T20.i
T60.A6 T60.A25 T60.EL T60.RH
Average
3.65
3.31
3.58
3.49
3.48
3.18
3.59
3.59
3.63
Standard
error
0.0833330.113969 0.1658650.1337080.0997780.1123490.1386040.086217 0.176415
T80.se
3.09
0.11299
Median
3.65
3.35
3.8
3.45
3.4
3.1
3.55
3.65
3.7
2.95
Standard
deviation 0.2635230.360401 0.524510.4228210.3155240.3552780.4383050.272641 0.557873 0.357305
Sample
variance 0.0694440.129889 0.2751110.1787780.0995560.1262220.1921110.074333 0.311222 0.127667
Range
1
1.3
1.3
1.3
1
1
1.3
0.8
1.4
1
Minimum
3.1
2.6
2.8
3
3.1
2.7
3
3.2
2.9
2.7
Maximum
4.1
3.9
4.1
4.3
4.1
3.7
4.3
4
4.3
3.7
Fig. 83. pH values evolution for the 10 groups
70
10.4. Sebum determinations
Table 31. Sebum values for 10 weeks of application-determination
blank
T20.s
T20.a
T20.l
T20.i
T60.A6
T60.A25
T60.EL
T60.RH
T80.se
1
0
6
5
2
4
8
9
11
1
0
2
10
9
15
11
12
13
10
18
7
4
3
16
12
13
14
13
14
16
24
8
11
4
25
16
16
17
19
20
21
20
16
17
week
5
6
20 18
19 24
23 22
21 25
21 24
19 22
24 27
22 28
13 22
15 16
7
24
22
26
32
28
21
25
27
26
18
8
31
26
28
26
28
17
29
30
24
17
9
28
31
24
34
26
24
31
31
25
21
10
29
30
25
36
29
28
32
36
27
26
Table 32. Statistical characteristics for determined sebum values
Average
Standard
error
Median
Standard
deviation
Sample
variance
Range
Minimum
Maximum
blank T20.s T20.a T20.l T20.i
20.1 19.5 19.7 21.8 20.4
3.031 2.733 2.280 3.457 2.646
135 537 594 038 591
22 20.5 22.5
23 22.5
9.585 8.644 7.211 10.93 8.369
29 202 873 211 256
91.87 74.72 52.01 119.5 70.04
778 222 111 111 444
31
25
23
34
25
0
6
5
2
4
31
31
28
36
29
71
T60.
A6
18.6
1.839
082
19.5
5.815
688
33.82
222
20
8
28
T60.
A25
22.4
2.617
038
24.5
8.275
801
68.48
889
23
9
32
T60.
EL
24.7
2.295
164
25.5
7.257
946
52.67
778
25
11
36
T60.
RH
16.9
2.930
491
19
9.267
026
85.87
778
26
1
27
T80.
se
14.5
2.436
984
16.5
7.706
419
59.38
889
26
0
26
Fig. 84. Sebum values evolution for the 10 groups
10.5. Mexametric determination
Table 33. Melanin values for 10 weeks of application-determination
blank
T20.s
T20.a
T20.l
T20.i
T60.A6
T60.A25
T60.EL
T60.RH
T80.se
1
465
431
436
439
438
429
431
426
462
450
2
489
453
428
421
440
435
466
422
500
455
3
432
469
450
425
472
468
470
429
486
431
4
395
471
468
438
456
492
495
461
482
469
72
week
5
6
403 462
470 433
471 496
461 482
465 497
494 504
476 508
455 476
477 508
471 486
7
504
459
482
484
496
514
513
492
526
499
8
478
476
509
473
524
518
504
506
530
501
9
495
485
501
498
512
526
530
469
534
514
10
466
499
511
500
503
524
531
484
512
536
Table 34. Statistical characteristics for determined melanin values
blank
Average
Standard error
Median
T20.s
458.9
475.2
T20.l
T20.i
462.1
480.3
T60.A6 T60.A25 T60.EL T60.RH T80.se
490.4
492.4
462
501.7
481.2
11.849476.759684 9.450229.369513 9.5812911.183529.9724069.1893667.74890710.13443
465.5
Standard deviation 37.47132
Sample variance
464.6
T20.a
469.5
476.5
21.37629.88422
467
484
499
499.5
465
504
478.5
29.629 30.298735.3653931.5355229.0593324.5041932.04788
1404.1456.9333893.0667877.8778918.01111250.711994.4889844.4444600.45561027.067
Range
109
68
83
79
86
97
100
84
72
105
Minimum
395
431
428
421
438
429
431
422
462
431
Maximum
504
499
511
500
524
526
531
506
534
536
Fig. 85. Melanin values evolution for the 10 groups
73
Table 35. Erythem values for 10 weeks of application-determination
blank
T20.s
T20.a
T20.l
T20.i
T60.A6
T60.A25
T60.EL
T60.RH
T80.se
1
102
114
121
101
114
124
113
138
126
140
2
123
108
103
94
141
145
117
162
128
149
3
98
111
126
99
145
156
105
170
119
178
week
5
6
135 126
156 168
154 153
114 131
185 172
184 199
136 168
144 147
160 164
197 194
4
114
140
165
102
163
178
109
181
132
168
7
147
189
187
134
179
198
169
159
184
209
8
152
201
190
152
184
210
175
181
182
202
9
169
212
168
169
189
203
172
186
179
201
10
184
194
174
164
200
216
170
178
178
203
Table 36. Statistical characteristics for determined erythem values
blank
Average
Standard error
Median
T20.s
135
159.3
T20.a
154.1
8.89069412.50693 9.17539
130.5
162
159.5
T20.l
T20.i
126
167.2
T60.A6 T60.A25 T60.EL T60.RH T80.se
181.3
143.4
164.6
155.2
184.1
8.91698.392854 9.632189.497602 5.461388.2835857.689025
122.5
175.5
191
152
166
Standard deviation 28.1148439.5503929.0151328.1977126.5405430.4596330.03405 17.2704
Sample variance 790.44441564.233841.8778795.1111
162
195.5
26.19524.31483
704.4927.7889902.0444298.2667686.1778591.2111
Range
86
104
87
75
86
92
70
48
65
69
Minimum
98
108
103
94
114
124
105
138
119
140
Maximum
184
212
190
169
200
216
175
186
184
209
74
Fig. 86. Erythem values evolution for the 10 groups
10.6. Corneometric determinations
Table 37. Corneometric values for 10 weeks of applicationdetermination
blank
T20.s
T20.a
T20.l
T20.i
T60.A6
T60.A25
T60.EL
T60.RH
T80.se
1
20.1
17.7
15.4
16.9
18.4
15.6
15.3
16
17.4
16.6
2
18.3
16.5
17.8
18.6
18.5
17.6
15.9
13.2
18.2
16.5
3
19.4
16.7
18.5
18.8
17.7
17.1
17.4
15.6
16.3
15.6
4
16.2
18.3
20.3
19.6
17.3
19.7
17.8
15.1
14.5
18.3
75
week
5
6
17.8 16.9
17.8 17.4
21.6 21.1
19.1 21.9
16.2 19.5
19.6 22
19.5 18.4
14.2 16.6
15.9 17
18 19.9
7
16.9
19.9
19.8
24.1
20
21.4
20.6
20.3
17.8
19.8
8
18.3
20.1
19.6
23.6
20.1
20.6
20.8
18.8
20.4
20.8
9
21.4
23.8
19.9
20.3
19.9
18.9
21.5
18.7
20.7
22.5
10
21.2
22.5
20
20.1
18.2
18.4
22.7
19.8
21.3
22.1
Table 38. Statistical characteristics for determined corneometric
values
blank
Average
18.65
Standard error
Median
T20.s
19.07
T20.a
19.4
T20.l
T20.i
20.3
18.58
T60.A6 T60.A25 T60.EL T60.RH T80.se
19.09
18.99
16.83
17.95
19.01
0.5756640.7816010.5648990.7192590.4106360.6288350.7720750.771586 0.706360.757254
18.3
18.05
19.85
19.85
18.45
19.25
18.95
16.3
17.6
19.05
Standard deviation 1.8204092.4716391.7863682.2744961.2985461.9885512.4415162.4399682.2337062.394647
Sample variance 3.313889
Range
6.1093.1911115.1733331.6862223.954333
5.9615.9534444.9894445.734333
5.2
7.3
6.2
7.2
3.9
6.4
7.4
7.1
6.8
6.9
Minimum
16.2
16.5
15.4
16.9
16.2
15.6
15.3
13.2
14.5
15.6
Maximum
21.4
23.8
21.6
24.1
20.1
22
22.7
20.3
21.3
22.5
Fig. 87. Corneometric values evolution for the 10 groups
76
10.7. Conclusions
If the values determined from groups of mice subjected
to this experiment, it was observed that the application of
emulsions containing nanocapsules not change the parameters
studied.
77
MAIN CONCLUSIONS
1. Surfactants or compounds with surfactant activity can
generate major changes in the body skin. Their study requires
review especially since it can be part of products to use and it
requires frequent monitoring of their harmfulness. In addition
to new concepts about surfactants exclusion does not refer to
themselves but to their reassessment. Quality products are
undeniable surfactants especially as regards the wording in
which they act as emulsifiers.
2. Modern methods of study of surfactants allow direct
investigation on human skin to provide their harmfulness.
However for a breakdown of their overall action in
dermatological products or finished products requires the study
of complex organisms in vivo (experimental animals, chicken
embryos).
3. Surfactants current research is carried out by non-invasive
methods.
Tewa-meter
corneometer
and
mexameter
measurements are just some of the testing methods applied to
body skin.
4. Anionic compounds such lauryl sodium sulphate appear to
be extremely harmful to the skin even at low doses. This is
78
easily noticeable especially if applied as a solution.
Harmfulness anionic compounds occur primarily by changing
the appearance of skin by irritation and swelling (patch test).
Another visible from the application of surfactants, especially
anionic compounds is fluid status modification skin.
5. Although the rumor of anionic compounds on various ways
the idea of harm they cannot be replaced and ignored because
of the quality products and finished formulations afforded these
formulations. Detailed demonstration of the possibilities
detailed in the current material, different and reproducible
assessment of products with surfactant helps to solve the
problem of testing and clarifying the applicability versus harm.
6. Ionic compounds are not completely devoid of interventions
in terms of changing the skin. This demonstrated through
various
forms
of
surfactant
(semi-solid
formulations,
nanocapsules) leads again to the idea of testing for possible
non-invasive techniques such compound formulations.
7. Surfactants compounds does not change noticeably melanin,
but are reachable by Mexameter detect local changes in
hemoglobin (erythem). In addition influences transdermal
water loss and fluid status of the skin.
79
8. Parameters such as water and hemoglobin transdermal local
expression are important in local harmfulness of topically
applied compounds, including emulsifiers.
9. Surfactants toxicity study using modern methods is
especially timely as the frequency of their use, particularly
cosmetics. INVITTOX techniques can be an important solution
for accurate assessment of harmfulness.
10. A large number of studies have been conducted to find tests
that need to replace experimental animals in tests for eye and
skin safety. Mouse skin test, which is a true test for the body in
its entirety, can be correlated with the CAM assay. A battery of
tests should be established, because no single test cannot meet
all requirements of risk assessment may use a method in vitro
and some of these tests may be simple tests on eggs or embryos
animal skin used as non-invasive measurement techniques.
11.
Surfactants
pharmaceutical
can
be
used
formulations
and
to
produce
modern.
important
They
are
indispensable for some semi-solid formulations, especially
those dermocosmetics market.
12. Application of modern surfactants showing noxious cold
can be a solution in reducing the potential harm that can be
exercised by such compounds.
80
13. Classical surfactants used in individual formulations are
skin irritants and sensitizers, even at low doses of 1-2%.
Combining them with guidelines to help protect the body skin
to reduce harmful local effects.
14. Surfactants can influence the in vitro release of active
principles, whether it be that the cellulose membrane is the skin
membrane failure rats.
15. Nanocapsules are modern formulations in some form
appeals to surfactants variants. Type can influence the structure
and characteristics of the finished shape but not in sharply.
16. Nanocapsules surfactants on skin testing with experimental
animals by rapid non-invasive methods show reduced their
harmfulness.
17. New trends in studies of potentially harmful compounds
refer to the possibility of further use with clear rules.
18.
Characterization
synthesized
by
nanocapsules
different
solubility
based
and
surfactants
pH,
electron
microscopy, differential scanning calorimetry and zeta
potential not least, demonstrates that surfactants influences
only a very small extent the appearance, size, solubilization
and nanocapsules stability.
81
PROPOSALS AND RECOMMENDATIONS
1. Surfactants use in pharmaceutical formulations must be
balanced. This will take into account the idea of using them in
low doses or use of substances to reduce the harm associated
with local government.
2. Surfactants applications can span various fields, including
the formulation of nanocapsules. These formulations can be
manufactured with low doses of surfactant and free of adverse
effects highlights.
3. The application is an easy solution INVITTOX tests and
detailed presentation of the harmfulness of a surfactant.
4. INVITTOX tests may correlate with each other and then to
assess other surfactants detailed toxicity even at low doses.
5. We recommend testing and careful monitoring of surfactant
formulations especially if anionic type.
6. To assess surfactants and their formulations are shown using
non-invasive methods (Mexameter, TEWAmeter etc.) quickly
and accurately rendering the skin degradation.
82
7. We recommend using some type of penetrating power
surfactants giving similar failure when testing the active
principle cellulose membrane or skin of an animal.
8. Surfactants must be taken into account as modern variants of
compounds with emulsifier or pervasive role short of a
remarkable harm.
Elements of originality
Through this document outlines the elements of originality that
may be as follows:
1. The present toxicity studies and studies related to human
skin implemented on experimental animals which use modern
techniques of analysis and investigation.
2. The thesis proposes to address modern testing techniques
and surfactants harmful compounds, INVITTOX tests to
choose from egg embryo test and the test was applied on red
blood cells that a large number of surfactants of different kinds.
Many of these are the last generation.
83
3. The thesis emphasizes the implementation of non-invasive
techniques to study modern methods of quality as the body
skin.
4. Formulation of nanocapsules with different surfactants, the
default state of the art and emphasizes the most important
element of new material in it.
5. Nanocapsules obtained by current synthesis methods have
been investigated by modern methods of analysis on
experimental animals.
6. The study is conclusive in terms of scope and analysis
surfactants from harm.
84
SELECTIVE REFERENCES
1. Addicks, W.J.; Flynn, G.L.; Weiner, N. Validation of a
flow-through diffusion cell for use in transdermal
research. Pharm. Res. 1987, 4, 337–341.
2. Ai R, Dahl JE, Morisbak E, Polyzois GL. Irritation and
cytotoxic potential of denture adhesives. Gerondol
2005; 22:177–183.
3. Al-Bawab, A.; Friberg, S.E., Some pertinent factors in
skin care emulsions. Adv. Colloid Interf. Sci. 2006,
123–126, 313–322.
4. Baranda L. et al.: Correlation between pH and irritant
effect of cleansers marketed for dry skin, Int. Journ of
Dermatol., vol. 41 (8), Aug., p. 494, 2002;
5. Barany E, Lindberg M, Loden M. Biophysical
characterization of skin damage and recovery after
exposure to different surfactants. Contact Dermat 1999;
40:98–103.
6. Barany, E., 2000. Human in vivo skin irritancy testing.
In: Loden, M., Maibach, H.I. (Eds.), Dry Skin and
Moisturizers. CRC Press LLC, Boca Raton, pp. 243–
250.
85
7. Barry,
B.W.;
Grace,
A.J.,
Sensory
Testing
of
Spreadability: Investigation of Rheological Conditions
Operative During Application of Topical Preparations,
J. Pharm. Sci. 1972,61 (3), 335–341.
8. Bellomo, R.; Ronco, C. An Introduction to continuous
renal replacement therapy. In Atlas of Hemodialysis;
Bellomo, R., Baldwin, I., Ronco, C., Thomas, G., Eds.;
Bailliere Tindall: London, UK, 2001; Volume 1, pp. 1–
10.
9. Benassi L, Bertazzoni G, Magnoni C et al. Decrease in
toxic potential of mixed tensides maintained below the
critical micelle concentration: an in vitro study. Skin
Pharmacol. Appl Skin Physiol 2003; 16: 156–164.
10. Bonferoni, M.C.; Rossi, S.; Ferrari, F.; Caramella, C. A
modified
Franz
diffusion
cell
for
simultaneous
assessment of drug release and washability of
mucoadhesive gels. Pharm. Dev. Tech. 1999, 4, 45–53.
11. Breton, P.J., 1999. From microns to nanometers: early
landmarks in the science of scanning electron
microscope imaging. Scanning Microsc. 13-1, 1–6.
12. Budai P, Varnagy L, Fejes S et al. Irritative effects of
some pesticides and a technical component on tissue
structure of the chorioallantoic membrane. Commun
Agric Appl Biol Sci 2004; 69: 807–809.
86
13. Capitanul, A.; Luzy, A. P. ;Esdaile,D. ; et al.,
Comparison of the human skin grafted onto nude mouse
model with in vivo and in vitro models in the prediction
of
percutaneous
penetration
of
three
lipophilic
pesticides, Regul. Toxicol. Pharm. , 2007, 47: 274-287.
14. Caracciolo P.C., de Queiroz A.A.A., Higa O.Z., Buffa
F., Abraham G.A., 2008, Acta Biomaterialia, 4, 976988;
15. Chattaraj, S.C.; Kanfer, I. Release of acyclovir from
semisolid dosage forms: a semi-automated procedure
using a simple plexiglass flow-through cell. Int. J.
Pharm. 1995, 125, 215–222.
16. Chenoweth, D.E.; Cheung, A.K.; Henderson, L.W.
Anaphylatoxin formation during hemodialysis: Effects
of different dialyzer membranes. Kidney Int. 1983, 24,
764–769.
17. Chilcott R, Barai N, Beezer A, Brain S, Brown M,
Bunge A, et al. Inter- and intra-laboratory variation of
in vitro diffusion cell measurements: an international
multi-centre study using quasistandardised methods and
materials. J Pharm Sci. 2005;94:632–8.
18. Clement, P.; Laugel, C.; Marty, J.-P. Influence of three
synthetic membranes on the release of caffeine from
87
concentrated w/o emulsions. J. Controlled Release
2000, 66, 243–254.
19. 1997, (123-124), 169-182.
20. Corbo, M.; Schultz, T.W.; Wong, G.K.; van Buskirk,
G.A. Development and validation of in vitro release
testing methods for semisolid formulations. Pharm.
Tech. 1993, 9, 112–128.
21. Crisante F., Francolini I., Bellusci M., Martinelli A.,
D‟Ilario L., Piozzi A., 2009, European Journal of
Pharmaceutical Sciences, 36, 555-564;
22. Cronin, M.T.D.; Dearden, J.C.; Moss, G.P.; MurrayDickson, G. Investigation of the mechanism of flux
across human skin in vitro by quantitative structurepermeability relationship. Eur. J. Pharm. Sci. 1998, 7,
325–330
23. De Fine Olivarius, F.; Agner, T.; Menne, T., Skin
barrier
function
and
dermal
inflammation.
An
experimental study of transepidermal water loss after
dermal tuberculin injection compared with SLS patch
testing. Br. J. Dermatol. 1993, 129, 554–557.
24. Debbasch C, Ebenhahn C, Dami N et al. Eye irritation
of low-irritant cosmetic formulations: correlation of in
vitro results with clinical data and product composition.
Food Chem Toxicol 2005; 43: 155–165.
88
25. Diembeck
W,
Beck
H,
Benech-Kieffer
F,
Courtellemont P, Dupuis J, Lovell W, et al. Test
guidelines for in vitro assessment of dermal absorption
and percutaneous penetration of cosmetic ingredients.
Food Chem Toxicol. 1999;37:191–205.
26. Djabari Z, Bauza E, Dal Farra C et al. The HET-CAM
test combined with histological studies for better
evaluation of active ingredient innocuity. Int J Tissue
React 2002; 24: 117–121
27. Duggin, G., Softening Skin with Emollient Ingredients,
Manufacturing Chemist 1996, 67 (6), 27–31.
28. Eccleston, G.M., Functions of mixed emulsifiers and
emulsifying waxes in dermatological lotions and
creams, Colloids and Surfaces A: Physicochemical and
Engineering Aspects
29. Edresi, S.; Baie, S., In-vitro and in-vivo evaluation of a
photo-protective kojic dipalmitate loaded into nanocreams. Asian Journal of Pharmaceutical Sciences
2010, 5 (6), 251-265.
30. Eun HC, Suh DH. Comprehensive outlook of in vitro
tests for assessing skin irritancy as alternative to Draize
tests. J Dermatol Sci 2000; 24: 77–91.
31. FDA-SUPAC-SS. Guidance for Industry. SUPAC-SS
Non-sterile Semisolid Dosage Forms. Scale-up and
89
Postapproval Changes: Chemistry, Manufacturing and
Controls. In vitro ReleaseTesting and In Vivo
Bioequivalence Documentation 1997; pp. 19–24.
32. Fernandez A.M., Abraham G.A., Valentin J.L., San
Roman J., 2006, Polymer, 47, 785-798;
33. Franz, T.J. Percutaneous absorption. On the relevance
of In vitro data. J. Invest. Dermatol. 1975, 64, 190–195.
34. Franz T. The finite dose technique as a valid in vitro
model for the study of percutaneous absorption. Curr
Probl Dermatol 1978;7:58–68.
35. Friend, D.; Catz, P.; Heller, J.; Okagaki, M.
Transdermal delivery of Levonorgestrel IV:Evaluation
of membranes. J. Pharm. Sci. 1989, 78, 477–481.
36. Fluhr, J.W.; Kuss, O.;Diepgen, T.; Lazzerini, S.; Pelosi,
A.; Gloor,M.; Berardesca, E.,Testing for irritation with
a
multifactorial
approach:
comparison
of
eight
noninvasive measuring techniques on five different
irritation types, Br. J. Dermatol. 2001,145, 696–703.
37. Frum Y, Eccleston GM, Meidan VM. Evidence that
drug flux through synthetic membranes is assocated
with normally distributed permeability coefficients. Eur
J Pharm Biopharm. 2007;67:434–9.
38. Gallagher, S.J.; Trottet, L.; Carter, T.P.; Heard, C.M.
Effects of membrane type and liquid/liquid phase
90
boundary on in vitro release of Ketoprofen from gel
formulations. J. Drug target. 2003, 11, 373–379.
39. Garg, A.; Aggarwal, D.; Garg, S.; Singla, A.
K.Spreading
of
Semisolid
Formulations
An
Update,Pharmaceutical Technology 2002, 9,84-105.
40. Gettings SD, Lordo RA, Hintze KL et al. The CTFA
Evaluation of Alternatives Program: An evaluation of
in vitro alternatives to the Draize primary eye irritation
test. (Phase III) surfactant based formulations. Food
Chem Toxicol 1996; 34: 79–117.
41. Gogolewski S., 1996, ‚Biomedical polyurethanes”. In:
Arshady R., editor. „Desk reference of functional
polymers. Syntheses and applications”, American
Chemical Society, Washington – DC, 657-698;
42. Goldstein,
J.,
et
al.,
2003.
Scanning
Electron
Microscopy and X-ray Microanalysis, 3rd ed. Plenum
Press, New York.
43. Guan J., Sacks M.S., Beckman E.J., Wagner W.R.,
2004, Biomaterials, 25, 85-96;
44. Hagino S, Itagaki H, Kato S et al. Quantitative
evaluation to predict the eye irritancy of chemicals:
Modification of chorioallantoic membrane test by using
trypan blue. Toxicol In Vitro 1991; 5: 301–304.
91
45. Hagino S, Itagaki H, Kato S, Kobayashi T. Further
evaluation of the quantitative chorioallantoic membrane
test using trypan blue stain to predict the eye irritancy
of chemicals. Toxicol In Vitro 1993; 7: 35–39.
46. Haguenau, F., Hawkes, P.W., Hutchison, J.L., SatiatJeunemaıˆtre, B., Simon, G.T., Williams, D.B., 2003.
Key events in the history of electron microscopy.
Microsc. Microanal. 9, 96–138.
47. Hawkes, P., 2004. Recent advances in electron optics
and electron microscopy. Ann. Fondation Louis de
Broglie 29-1 .
48. Henning A, Schaefer UF, Neumann D. Potential pitfalls
in
skin
permeation
experiments:
influence
of
experimental factors and subsequent data evaluation.
Eur J Pharm Biopharm. 2009;72:324–31.
49. Herh, P.; Tkachuk, J.; Wu, S.; Bernzen, M.; Rudolph
B.,The rheology of pharmaceutical and cosmetic
semisolids,American Laboratory 1998, 1, 12-14.
50. Herzinger T, Korting HC, Maibach HI. Assessment of
cutaneous and ocular irritancy: a decade of research on
alternatives to animal experimentation. Fundam Appl
Toxicol 1995; 24: 29–41.
92
51. Hong J.H., Jeon H.J., Yoo J.H., Yu W.-R., Youk J.H.,
2007, Polymer Degradation and Stability, 92, 11861192;
52. Imokawa,
G.,
Surfactant-induced
depletion
of
ceramides and other intercellular lipids: implication for
the mechanism leading to dehydration of the stratum
corneum, Exog. Dermatol. 2004,3, 81–98.
53. Jiang X., Li J., Ding M., Tan H., Ling Q., Zhong Y., Fu
Q., 2007, European Polymer Journal, 43, 1838-1846;
54. Jones, D.S.; Woolfson, A.D.; Brown, A.F., Texture,
Viscoelastic,
and
Mucoadhesive
Properties
of
Pharmaceutical Gels Composed of Cellulose Polymers,
Int. J. Pharm. 1997,151, 223–233.
55. Joy, D.C., 1991. The theory and practice of highresolution
scanning
electron
microscopy.
Ultramicroscopy 37, 216–233.
56. Kajs T. M. ,Gartstein V Review of the instrumental
assessment of skin: Effects of cleansing products J.
Soc.Cosmet. Chem. 1991,42, 249-271
57. Khan G, Frum Y, Sarheed O, Eccleston GM, Meidan
VM. Assessment of drug permeability distribution in
two different model skins. Int J Pharm. 2005;303:81–7.
58. Kishore A S, Surekha P A, Sekhar P V R et al.
Chorioallantoic Membrane Bioassay: An In Vitro
93
Alternative to Draize Eye Irritation Test for Pesticide
Screening. International Journal of Toxicology 2008;
27: 449-453.
59. Kobayashi, Y.; Iwai,L.; Akutsu, N.; et al., Increased
carbonyl protein levels in the stratum corneum of the
face during winter, Int. J. Cosmetic. Sci., 2008, 30, 3540.
60. Kogan, A.; Garti,N., Microemulsions as transdermal
drug delivery vehicles. Adv. Colloid. Interfac., 2006,
123-126, 369-385.
61. Kónya, M.; Sorrenti, M.; Ferrari F.; et al., Study of the
microstructure of o/w creams with thermal and
rheological methods, Journal of Thermal Analysis and
Calorimetry 2003, 73, 623-632.
62. Michaels A, Chandrasekaran SK, Francis W, Meredith
M. Drug permeation through human skin: theory and in
vitro experimental measurement. A I Ch E J.
1975;21:985–96.
63. Morell, J.L.P.; Claramonte, M.D.C.; Vialard, A.P.
Validation of a release diffusion cell for topical dosage
forms. Int. J. Pharm. 1996, 137, 49–55.
64. Oatley, C.W., 1982. The early history of the scanning
electron microscope. J. Appl. Phys. 53 (2).
94
65. *** OECD, 2008. Guideline for the Testing of
Chemicals, Draft Proposal for a New Guideline: In
vitro Skin Irritation: Human Skin Model Test.
Available at: http://www.oecd.org/ searchResult /
0,3400,en-2649-201185-1-1-1-1-1,00.html.
66. Otto, A.; Plessis, J.; Wiechers, J.W., Formulation
effects of topical emulsions on transdermal and dermal
delivery. International Journal of Cosmetic Science,
2009, 31, 1–19.
67. Park J.-H., Allen M.G., Prausnitz M.R., 2005, Journal
of Controlled Release, 104, 51-66;
68. Pellett, M.A.; Castellano, S.; Hadgraft, J.; Davis, A.F.
The
penetration
of
supersaturated
solutions
of
piroxicam across silicone membranes and human skin
in vitro. J. Control. Release 1997, 46, 205–214.
69. Prausnitz M.R., Mitragotri S., Langer R., 2004, Nat.
Rev. Drug. Discov., 3, 115-124;
70. Sanchez L, Mitjans M, Infante MR et al. Determination
of interleukin-1_ in human NCTC 2544 keratinocyte
cells as a predictor of skin irritation from lysine-based
surfactants. Toxicol Lett 2006; 167: 40–46.
71. Santerre J.P., Woodhouse K., Laroche G., Labow R.S.,
2005, Biomaterials, 26, 7457-7470;
95
72. Santoyo, S.; Arrelano, A.; Ygartua, P.; Martin, C. In
vitro percutaneous absorption of piroxicam through
synthetic membranes and abdominal rat skin. Pharm.
Acta. Helv. 1996, 71, 141–146.
73. Savic, S.; Weber, C.,; Savic, M.M.; Muller-Goymann,
C., Natural surfactant-based topical vehicles for two
model drugs: Influence of different lipophilic excipients
on in vitro/in vivo skin performance. International
Journal of Pharmaceutics 2009, 38, 220–230.
74. Sivak, W.N., Zhang J., Petoud S., Beckman E.J., 2010,
Acta Biomaterialia, 6, 144-153;
75. Schaefer H., Redelmeier T.E., 1996, „Skin Barrier:
Principles of Percutaneous Absorption”, Karger, Basel,
12-18;
76. Shah, V.P.; Elkins, J.S.; Lam, S.Y.; Skelly, J.P.
Determination
of
in
vitro
drug
release
from
hydrocortisone creams. Int. J. Pharm. 1989, 53, 53–39.
77. Shah, V.; Elkins, J.; Hanus, J.; Noorizadeh, C.; Skelly,
J. In vitro release of Hydrocortisone from topical
preparations and automated procedure. Pharm. Res.
1991, 8, 55–59.
78. Tatai L., Moore T.G., Adhikari R., Malherbe F.,
Jayasekara R., Griffiths I., Gunatillake A., 2007,
Biomaterials, 28, 5407-5417;
96
79. Teichmann,
A.;Heuschkel,S.;Jacobi,U.;
et
al.,
Comparison of stratum corneum penetration and
localization of a lipophilic model drug applied in an
O/W microemulsion and an amphiphilic cream, Eur. J.
Pharm. Biopharm., 2007, 67, 699-706.
80. Thakker, K.D.; Chern, H. Development and validation
of in vitro release tests for semisolid dosage forms case study. Disso. Tech. 2003, 10, 10–15.
81. Twist, J.; Zatz, J. Membrane - solvent - solute
interaction in a model permeation system. J. Pharm.
Sci. 1988, 77, 538–540.
82. Twist, J.N.; Zatz, J.L. Influence of solvents on paraben
permeation through idealized skin model membranes. J.
Soc. Cosmet. Chem. 1986, 37, 429–444.
83. Uchiyama T., Watanabe J., Ishihara K., 2002, Journal
of Membrane Science, 208, 39;
84. Ueda, C.T.; Shah, V.P.; Derdzinski, K.; Ewing, G.;
Flynn, G.; Maibach, H.; Marques, M.; Rytting, H.;
Shaw, S.; Thakker, K.; Yacobi, A. Topical and
Transdermal Drug Products. Pharmacopeial Forum.
2009, 35, 750–764.
85. Urşica L, C.Dehelean, C. Peev, V.Vlaia, G.ConeacFormulation and quality evaluation of some topical
semisolid preparations containing dried extract of birch
97
tree, Recent Developments in Pharmaceutical Analysis,
Sept.2005 pp.207.
86. Van Ruissen, F.; Carroll, Le, M.; Van der Valk, J.M.;
Schalkwijk, J., Differential effects of detergents on
keratinocyte gene expression. J. Invest. Dermatol.
1998, 110, 358–363.
87. Vinardell MP, Garcıa L. The quantitive chlorioallantoic
membrane test using trypan blue stain to predict the eye
irritancy of liquid scintillation cocktails. Toxicol In
Vitro 2000; 14: 551–555.
88. Vinardell MP., Mitjans M. Alternative Methods for Eye
and Skin Irritation Tests: An Overview. J Pharm Sci
2008; 97:46–59.
89. Vinardell MP, Rimbau V, Mitjans M. Potential eye
irritation of some „„biodegradable‟‟ liquid scintillation
cocktails determined in vitro. Food Chem Toxicol 2004;
42:1287–1290.
90. Vinardell
MP,
Mitjans
M.
The
chorioallantoic
membrane test as a model to predict the potential
human eye irritation induced by commonly used
laboratory solvents. Toxicol In Vitro 2006; 20: 1066–
1070.
91. Welin-Berger, K.; Neelissen, J.A.M.; Bergenstahl, B.
The effect of rheological behaviour of a topical
98
anaesthetic formulations on the release and permeation
rates of the active compound. Eur. J. Pharm. Sci. 2001,
13, 309–318.
92. Wester
R,
Maibach
H.
Review:
percutaneous
absorption of drugs. Clin Pharmacokinet. 1992;23:253–
66.
93. Williams, A.C.; Barry, B.B., Penetration enhancers,
Adv. Drug Deliv. Rev. 2004,56, 603–618
94. Wu, S.T.; Shiu, G.K.; Simmons, J.E.; Bronaugh, R.L.;
Skelly, J.P. In vitro release of nitroglycerin from topical
products by use of artificial membranes. J. Pharm. Sci.
1992, 81, 1153–1156.
95. Zorin, S.; Kuylenstierna, F.; Thulin, H. In vitro test of
Nicotine's permeability through human skin. Risk
evaluation and safety aspects. Am. Occup. Hyg. 1999,
43, 405–413.
99
100