SCREENING METHODS FOR SKIN

SENSITIZATION, SKIN IRRITATION AND DERMAL

TOXICITY STUDIES

Presented by

P. Raga Amrutha
Y17MPHPY452
Department Of Pharmacology
Chalapathi Institute Of Pharmaceutical Sciences, Lam,
Guntur.
CONTENTS
o SKIN SENSITIZATION
- INTRODUCTION
- SCREENING METHODS

o SKIN IRRITATION
- INTRODUCTION
- SCREENING METHOD

o DERMAL TOXICITY
- INTRODUCTION
SCREENING METHOD

o REFERENCES
 Skin Sensitization

o Skin sensitisation is defined as allergic response to a

substance after skin contact.
o Substances are classified as skin sensitizers if there is
evidence in humans that the substance can lead to
sensitisation by skin contact or if there are positive results
from an appropriate animal test.
o Sensitization test estimate the potential for contact
sensitization to medical devices or materials.
o Symptom of sensitization are often seen in skin.
o Sensitization is a immune system response to chemicals.
o The sensitization tests are used to determine the allergic or
sensitizing capacity to the repeated or prolonged exposure
of a test material.
o Sensitization is characterized by the fact that reactions are
delayed, not localized, and independent of dose.
o Skin sensitization is a delayed type humoral immune
response mediated by the T- cell.
o Sensitized T cells migrate to the site and, on contacting the
allergen, liberate cytokines.
o These cytokines attract leukocytes to the site and appear to
raise the temperature of the area.
 A variety of methods are available for the prospective
identification of skin sensitizing chemicals. The methods
that have been used are the :

 Traditional Guinea Pig models for skin sensitization

• Guinea pig maximization test(GPMT)
• Buehler test

 New alternative: Local lymph node assay(LLNA)

Guinea pig maximization test:
• Animals: Guinea pig
• Sex: Male(females, should be nulliparous & non-pregnant).
• Body weight: 250-500g.
• Acclimatization: at least 5 days prior to the test.
• Dose: An average, a guinea pig requires 10-30 mg/kg daily.
Preparation of the animals:
• Before the test, animals are randomised & assigned to the
treatment groups.
• Removal of hair is by clipping , shaving or possibly by
chemical depilation , depending on the test method used.
• Care should be taken to avoid abrading the skin.
• The animals are weighed before the test and at the end of
the test.
It is an adjuvant sensitization test requiring intradermal
injections of the test substance, in combination with
freund’s complete adjuvant which stimulates non-
specifically the immune system of treated animals,
enhancing their ability to respond to sensitizing chemicals.
PROCEDURE:
A minimum of 10 animals used in the treatment group and at
least 5 animals in the control group are used (or 20 test and
10 control animals).

Induction is done initially on skin of shoulder region with

three intradermal injections on day 0:
–1/1 mixture of Freund’s complete adjuvant (FCA)/water
or saline
– Test substance (appropriate concentrations may be
determined from a pilot study using two or three animals)
– Test substance in a 1/1 mixture of FCA/water or saline.
Control animals also receive three intradermal injections, but only
vehicle is used instead of the test substance.

5–7 days later skin is painted with 0.5 mL of 10% sodium lauryl
sulfate in vaseline, to create a local irritation,

24 h later test substance is applied under occlusion for 48 h.

Challenge is done on days 20–22 with reapplication of patches for

24 h and results are assessed at 48 and 72 h after challenge.
Magnusson and Kligman grading scale is used for
evaluation:
0=no visible change,
1=discrete or patchy erythema,
2=moderate and confluent erythema,
3=intense erythema and swelling).

Rechallenge: If it is necessary to clarify the results

obtained in the first challenge,
A second challenge
with a new control group/ original control group
should be considered approximately one week after the
first one.
Irritancy development:
Observations of skin appearance after Guinea pig
maximization test:
• Buehler Test Method
• A minimum of 20 animals in the treatment group and 10
animals in the control groups is used.
• Test material is applied on skin of flank under occlusion for
6 h vehicle is applied to the control group hair on the
application sites are closely clipped.
• Same process is repeated total of three times within 2
weeks.
• Followed by 2 weeks rest and then challenge. To challenge
patches are again applied for 6 h under occlusion and then
evaluated at 24 and 56 h after application.
• If necessary rechallenge is done by repeating the same
procedure 1 week later.
• Due to multiple limitations of these assays including
subjective assessment of the frequency of responses, it is
not possible to assess sensitizing potencies of a chemical
using this assay.
 Buehler Test Method

A minimum of 20 animals in the treatment group and 10

animals in the control groups is used.

Test material is applied on skin of flank under occlusion for

6 h vehicle is applied to the control group hair on the
application sites are closely clipped.

Same process is repeated total of three times within 2 weeks.

Followed by 2 weeks rest and then challenge.

To challenge patches are again applied for 6 h under
occlusion and then evaluated at 24 and 56 h after
application.

If necessary rechallenge is done by repeating the same

procedure 1 week later.

Due to multiple limitations of these assays including

subjective assessment of the frequency of responses,

It is not possible to assess sensitizing potencies of a chemical

using this assay.
 Local Lymph Node Assays

The assay is based on the fact that sensitizers induce a

primary proliferation of lymphocytes in the lymph nodes
draining the site of application.
This proliferation is proportional to the dose applied and
provides objective data on of sensitization potentials.
Radioactive labeling with (3H) thymidine is done to
measure cell proliferation.
Procedure:
A minimum of four animals is used per dose group
(groups of four CBA/Ca female mice; 7–12 weeks of age)
with a minimum of three concentrations of the test
substance, plus a concurrent negative control group treated
only with the vehicle for the test substance, and a positive
control (concurrent or recent).

Animals are treated topically on the dorsum of both ears

with 25 μL of test material, or with an equal volume of
the vehicle; once a day for 3 days.
Five days after the initial exposure, all mice will be injected
with 20 μCi (7.4×105 Bq) of tritiated (3H)-methyl
thymidine via the tail vein.

Five hours later, all animals are humanely killed; draining

auricular lymph nodes are excised and processed.

Cellular proliferation is determined by measuring

incorporated radioactivity.
LLNA: DA; Cellular proliferation is determined by
measurement of the adenosine triphosphate (ATP) content
by bioluminescence as an indicator of this proliferation.

Results are calculated based on stimulation index (SI).

Result is regarded positive when SI≥3.
They can be classified as skin sensitizers.

Dose – response curves are plotted in order to provide

information on the relative potencies of skin sensitizers.
SKIN IRRITATION:
o Topical exposure to chemicals can lead to adverse skin
effects. According to the severity and reversibility of effects
one distinguishes skin corrosion (=skin burns) from skin
irritation.
o Corrosive substances irreversibly damage the skin beyond
repair, while irritant substances lead to a reversible local
inflammatory reaction caused by the innate (non-specific)
immune system of the affected tissue

o While some chemicals will only trigger an irritant response

after repeated exposure of the same skin area (=cumulative
irritants), other chemicals will even after a one-time
exposure cause irritation (=acute irritants).
 PRINCIPLE:
o The test chemical to be tested is applied in a single dose to
the skin of an experimental animal, untreated skin areas of
the test animal serve as the control.
o The degree of irritation/corrosion is read & scored at
specified intervals.
o Animals showing continuing signs of severe distress and/or
pain at any stage of the test should be humanely killed , and
the test chemical assessed accordingly.
Requirements:
o Animals should be individually housed.
o Rabbit at least 12 weeks old and body weight 1.5kg- 3.0kg.
o The temperature should be 200C for rabbit.
Selection of animal species:
o The albino rabbit is the preferable laboratory animal &
healthy young adult rabbits are used.
o A rationale for using other species should be provided.
Preparation of the animals:
o Approximately 24 h before the test, fur should be removed
by closely clipping the dorsal area of the trunk of the
animals.
o Care should be taken to avoid abrading the skin, and only
animals with healthy, intact skin should be used.
o Some strains of rabbit have dense patches of hair that are
more prominent at certain times of the year.
o Such areas of dense hair growth should not be used as test
sites.
TEST PROCEDURE:
Application of the test procedure:

o The test chemical should be applied to a dorsal/flank region

(approximately 6 cm2) of skin and covered with a gauze patch.
o In cases in which direct application is not possible (eg: liquids or
some pastes),the test chemical should first be applied to the gauze
patch , which is then applied to the skin.
o The patch should be loosely held in contact with the skin by means of
a suitable semi- occlusive dressing for the duration of the exposure
period.
o If the test chemical is applied to the patch , it should be attached to the
skin in such a manner that there is good contact & uniform
distribution of the test chemical on the skin.
o Liquid test chemicals are generally used undiluted.
o When testing solids , the test chemical should be diluted
with the smallest amount of water (or another suitable
vehicle) sufficient to ensure good skin contact.
o At the end of the exposure period , which is normally 4
hours, residual test chemical should be removed.

Dose level:
o A dose of 0.5mL of liquid or 0.5g of solid or paste is applied
to the test site.
Initial test(using one animal):
o When a test chemical has been judged to be corrosive or
irritant is determined on the basis of a weight of
evidence analysis or in vivo testing.

o The test is performed initially using and applying the

following approach.

o Up to three test patches are applied sequentially to the

animal.

o The first patch is removed after 3 minutes.

o If no serious skin reaction is observed, a second
patch is applied at a different site and removed after
one hour.

o If the observations at this stage is humanely be

allowed to extend to 4 hours, a third patch is applied
& removed after 4 hours, and the response is graded.

o If a corrosive effect is observed after the 3

sequential exposures, the test is immediately
terminated.
o If a corrosive effect is not observed after the last patch is
removed , the animal is observed for 14 days, unless
corrosion develops at an earlier time point.

o In those cases in which the test chemical is not expected to

produce corrosion but may be irritating, a single patch
should be applied to one animal for 4 hours.
Confirmatory test(with additional animals):

o If a corrosive effect is not observed in the initial test, the

irritant response should be confirmed using up to 2
additional animals, each with one patch, for an exposure
period of 4 hours.

o If an irritant effect is observed in the initial test, the

confirmatory test may be conducted by exposing 2
additional animals simultaneously.
o In the exceptional case , in which the initial test is not
conducted , 2 or 3 animals may be treated with a single
patch, which is removed after 4 hours.

o When 2 animals are used, if both exhibit same response, no

further testing is needed. Otherwise, the third animal is also
used.

o Equivocal responses may need to be evaluated using

additional animals.
Clinical observations & Grading of skin reactions:
o All animals should be examined for signs of erythema and
oedema, and the responses scored at 60 minutes , and then
24,48 & 72 hours after patch removal.

o For the initial test in one animal , the test site is also
examined immediately after the patch has been removed.

o Dermal reactions are graded and recorded according to the

grades in table below.

o Histopathological examination should be considered to

clarify equivocal responses.
Grading of skin reactions:
 DERMAL TOXICITY:
o Dermal toxicity involves the assessment and evaluation of
the toxic characteristics of a substance.
o It is useful where the exposure to dermal route is likely.
o Data from an acute dermal toxicity study may serve as a
basis for classification and labelling.
PRINCIPLE:
o The test substance is applied to the skin in graduated doses
to several groups of experimental animals.
o Animals which die during the test are necropsied and
surviving animals are also done the same at the conclusion..
o Animals showing severe and enduring signs of distress and
pain may need to be humanely killed.
Requirements:
o Selection of species: Rat, other species.
o Body weight: Rat- 200 to 300g
Guinea pig- 350 to 450g
Rabbit-1.5 to 3.0kg
o Age: Rat-8 to 12 weeks old
Guinea pig- 5 to 6 weeks old
Rabbit- at least 12 weeks old
o Sex: male / female (nulliparous & non- pregnant)
o Acclimatization: Minimum 5 days
o Dose: 200mg/kg
Preparation of test item:
Solids-Moistened with distilled water/vehicle.
Liquids-applied directly with out dilution.

Preparation of test animals:

o One day before application of test item, 10% of the total
body surface area of the animals will be clipped with
electric clipper/shaved with razor blade/depilated with
VEET cream.

o The position of clipping will be the area starting at the

scapulae(shoulders) to the wing of the ilium(hipbone) and
half way down the flank on each side.
Test procedure:
o Test material should be applied on shaved skin covering not
less than 10% of the total body surface area.

o Porous gauze dressing should be used to hold liquid

material in place.
• Formulations with different concentrations ( at least 3) of
test substance, several fold higher than the clinical dosage
form should be used.

• Period of application may vary from 7 to 90 days depending

on the clinical duration of use.

• Where skin irritation is grossly visible in the initial studies,

a recovery group should be included in the subsequent
repeated study.

• Local signs ( erythema, oedema and Escher formation ) as

well as Histopathological examination of sites of application
should be used for evaluation of results.
After the test animals look like:
REFERENCES
• Magnusson B. and Kligman A.M. (1969). The identification of contact allergens by
animal assay. The guinea pig maximisation test. J. Invest. Dermatol., 52, 268.
• Magnusson B. and Kligman A.M. (1970). Allergic Contact Dermatitis in the Guinea
Pig. Charles G. Thomas; Springfield, Illinois.
• Magnusson B. (1980). Identification of contact sensitizers by animal assay. Cont.
Derm., 6, 46.
• Magnusson B., Fregert S. and Wahlberg J. (1979). Determination of skin
sensitization potential of chemicals. Predictive testing in guinea pigs. Arbete och
Hälsa, 26(E).
• Buehler E.V. (1965). Delayed contact hypersensitivity in the guinea pig. Arch.
Dermatol., 91, 171.
• Ritz H.L. and Buehler E.V. (1980). Procedure for conducting the guinea pig assay.
Current Concepts in Dermatology, Drill V.A. and Lazar P. (eds), Academic Press,
New York, N.Y., 25-40.
• UN (2009), United Nations Globally Harmonized System of Classification and
Labelling of Chemicals (GHS), Third revised edition, UN New York and Geneva.
Available [http://www.unece.org/trans/danger/publi/ghs/ghs_rev03/03files_e.html]
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Descotes, 1988; Dunn et al., 1990; Gad et al., 1987 (Cornacoff et al., 1988; Hignet
et al., 1989; Dunn et al., 1990)
• WHO Publication: Environmental Health Criteria 6, Principles and Methods for
Evaluating the Toxicity of Chemicals. Part I, Geneva, 1978.
• National Academy of Sciences, Committee for the Revision of NAS Publication
1138, Principles and Procedures for Evaluating the Toxicity of Household S
Substances, Washington, 1977.
• Litchfield, J.T. and Wilcoxon, F., J. Pharmacol., Exp. T her., 96, 99-113, 1949.
• Bliss, C.I., Quart. J. Pharm. Pharmacol., 11, 192-216, 1938.
• Finney, D.G., Probit Analysis. (3rd Ed.) London, Cambridge University Press, 1971.
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• Thompson, W., B act. Rev., 11, 115-141, 1947.
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