This document discusses various aspects of dermatotoxicity testing including skin structure and function, dermatotoxic reactions, OECD testing guidelines, and specific dermatotoxicity tests. It summarizes common dermatotoxicity tests including acute dermal toxicity tests, repeated dose dermal toxicity tests lasting 21-28 days, subchronic dermal toxicity tests lasting 90 days, and in vitro tests for skin corrosion. Testing procedures, observations, data collection, and reporting requirements are outlined for each test.

1. Presented by:
Chaudhari Sandip
M.Pharm[sem-II]
Department of Pharmacology
R. C. Patel Institute Of Pharmaceutical Education &
Research, Shirpur

2. 1. Skin
 Structure and Function
 Pathology and Toxicological Pathology
2. Dermatotoxic Reactions
3. OECD Guidelines for Dermatotoxicity Testing.
4. Dermatotoxicity Tests.
2

3. 1.The Epidermis
 Stratum basale- germinal layer, melanocytes + langerhans
cells epidermal cells (differentiated).
 Stratum spinosum – cells in process of keratinization.
 Stratum granulosum – granulated cells.
 Stratum lucidum – between keratinized layer and granular
layer.
 Stratum corneum – Horny layer, Flattened keratinized cells.
Skin

4.  The Dermis
 Connective tissue cells + collagen producing cells + Mast
cells + Blood vessels and other nerve endings.
 The Hypodermis
Fat cells + blood vessels + nerve fibres.

5. 5
 Impermeable to polar compounds.
 Transport via passive diffusion.
 Lipophilic compounds dissolve into lipid matrix of the skin.
 Specific barrier – A linoleic acid derivative involved.
SUBSTANCESUBSTANCE
EPIDERMAL
BARRIER
EPIDERMAL
BARRIER
DERMAL
LAYER
DERMAL
LAYER
SYSTEMIC
EFFECTS
Primary route of absorption
HAIR FOLLICLES &
SEBACIOUS GLANDS
HAIR FOLLICLES &
SEBACIOUS GLANDS
Secondary route of absorption

6. 6
1. Primary irritations or Irritant dermatitis.
Mild
Moderate
Severe
2. Allergic Reactions or Allergic dermatitis.
3. Phototoxic and Photoallergic skin disorders.

7. 4. Acne.
 Acne venenata
 Chloracne
 Acne vulgaris
5. Contact urticaria.
6. Pigment disorders
Hypopigmentation and Hyperpigmentation
7. Various forms of skin cancers

8. Number Title Most recently
updated
402 Acute dermal toxicity 24th
february 1987
410 Repeated dose dermal
toxicity:
21/28-day Study
12th
may 1981
411 Subchronic dermal toxicity:
90 days study
12th
may 1981
434 Acute dermal toxicity –
Fixed dose procedure
14th
may 2004(First version)
435 Invitro membrane barrier
method for skin corrosion
19th
july 2006
OECD Guidelines For Dermal Toxicity
8

9. Definations
Acute dermal toxicity
Chemical detection system
False negative rate
False positive rate
Performance standards
Reference chemicals
9

10. 10
-Developed by The Office of Prevention , Pesticides and Toxic substances US-EPA.
Acute Dermal Toxicity- “Adverse effects occurring within a short time of dermal
application of single dose or multiple doses of a substance within 24 hours period.”
Objectives –
Provides information on health hazards on short term dermal exposure.
Limit Test – When data on structurally related compounds is inadequate.
Administer a limit dose of at least 2000 mg/Kg to a single group of 5 males and 5
females.
Conventional Acute Toxicity Test –
Principle – “Dermal application of substance in graduated doses to each group.”

11. 11
Test procedure:
 Animal selection :
 Species, strain
 Age
 Weight
 Number and sex : 5 males (group I).
5 females (group II).
 Marking
 5 days acclimatization period before the study.
 Housing & feeding:
 Dose levels & dose selection:
 3 dose levels
 Vehicle – water or corn oil.
 Exposure time- 24 hrs.
 Skin preparation – Shaving 24 hrs prior.
 Application of test substance.

12. 12
 Observation period –
 Fixed observation period.
 14 days.
 Observation of animals -
 Once each day
 Daily in early periods
 Observations include - changes in fur, skin color, eyes n mucus membranes,
changes in reactivity to sensory stimuli, weight.
 Gross pathology –
 Sacrifice at the end of test for necropsy.
 Data & reporting –
a) Treatment of results:
No. of animals used.
Body weights
Time of death.
No. of animals showing other signs of toxicity.
Description of toxic effects.
Necropsy findings.
Method for LD50 determination (with reference).

13. 13
b) Evaluation of results :
 Relationship between exposure and toxic signs and other clinical
abnormalities.
 LD50
8.Test report -

14. 14
Principle :–
Test substance is applied daily to the skin in graduated
doses for 21/28 days.1 dose is given per group.
Test Procedure :
Preparation –
Animals are acclimatized for a period of 5 days before test.
They are randomly divided into treatment & control group.

15. Fur clipping/shaving is done 24 hours prior testing.
Liquid substances are used undiluted.
As in case of solids, they may be pulverized & moistened
with water or any other vehicle.
15

16. Animal selection :
Species, strain :- Rat, Rabbit, Guinea pig.
Weight
Number and sex : 5 males(group I). 5 females(group II).
A satellite group of another 10 animals is used to treat with
higher doses.
Housing & feeding condition :-
Test conditions :-
Dose levels - 3 dose levels.
16

17. Procedure :-
Animals are treated for at least 6 hours per day on a 7 days per
week basis (for 21-28 days).
Limit tests :-
It is carried out at the dose of 1000 mg/kg.
Observation :-
Once in a day.
The satellite group to be kept for 14 additional days without
treatment.
Application of test substance :- 17

18. Observations :-
Changes in fur, skin color, eyes n mucus membranes, changes in
reactivity to sensory stimuli, weight etc. are observed.
At the end of the test all animals from the non-satellite test group
are sacrificed.
Moribund and the weak animals are humanely killed.
Clinical examination:
Haematology
Clinical biochemistry
18

19. Data and Reporting :-
Treatment of results :-
Data summarized in the tabular form for each dose level group
including the number of animals at the start of the test, animals
showing lesions & the type of lesions, & the percentage of animals
displaying each type of lesions.
Pathology :–
Gross necropsy
Histopathology
19

20. Evaluation of results :-
It includes the relationship between dose of test substance &
the presence & absence, the incidence & sensitivity,
abnormalities, including behavioral & clinical abnormalities, gross
lesions, identified target organs, body weight changes, effect on
mortality & any other toxic effects.
20

21. 21
Introduction :-
This guideline was suggested by the British Toxicological
Society in 1984.
In this guideline death of animal as an endpoint concept is
removed &evident toxicity is considered as an endpoint.
Initial Considerations :-
All available information on the test substance should be
considered prior to conducting the study.

22. Principle :-
Only moderatory toxic doses are used & the dose levels that are
expected to be lethal or causing marked pain & distress should be
avoided.
Moribund animals are humanely killed & are considered in the
results.
Groups of animals of single sex are given fixed doses.
Test method :-
Selection of animal species
Housing & feeding condition
Preparation of animals
. 22

23. Administration of doses :-
Substance applied as thin, uniform layer, Porous gauze dressing
& non-irritant tape till 24 hour exposure period.
Moistening of test substance if in Solid form, with water or
suitable vehicle.
Liquids used undiluted.
Removal of test substance at the end of exposure period
23

24. Procedure –
1. Sighting study
2. Main study.
Sighting study
Objective : “Selection of an appropriate starting dose for the main study.”
-Completes when either a staring dose has been selected or a death is seen
at lowest fixed dose.
-Starting dose for the sighting study is selected from the fixed dose levels of 50,
200, 1000, 2000 mg/Kg.
-A period of at least 24 hours as test period for each animal.
-All animals are observed for 14 days.
24

25. 25
Main study
No. of animals and dose levels-
- Total of 5 animals to be used for each dose level to be tested.
(1 animal from the sighting study + 4 additional animals but all of one sex)
- The dose that caused death in the sighting study.
- Dosing interval decided by onset, duration & severity of toxic signs.
- Next dosing after 3-4 days in order to keep a check on delayed toxicity.
Higher dosing i.e. 5000 mg/Kg must only be done when concerned human
health benefit outweighs.
25

26. Limit Test :–
Only used when the experimenter has the information that the test
material is non-toxic.
Information can be gained from the test data of similar compounds.
If information is absent then directly main test is performed.
For the limit test the sighting study begins at 2000 mg/Kg followed
by subsequent dosing of 4 animals.
26

27. Observations :–
For signs of toxicity animals to be observed.
Immediately after dosing during first 30 minutes.
Periodically during first 24 hours.
Daily thereafter for a total of 14 days.
Other observations – changes in fur, skin color, eyes n mucus
membranes, respiratory system, ANS(salivation, diarrhea etc.),
CNS(tremors, convulsions etc.)
Body Weight & Pathology. 27

28. Data and Reporting :–
Individual data for each animal in tabular form showing:
No. used animals.
Animals showing toxicity.
Animals found dead during test or killed.
Time of death of individual animals.
Time course of Toxic effects.
Necropsy findings.
28

29. Test report –
a) Test substance
b) Vehicle
c) Test animals
d) Test conditions
29

30. e) Results –
Tabulation of response data and dose level of each animal.
Weight of animals.
Time course of onset of toxicity symptoms (Reversible or not).
Necropsy and histopathological findings
Discussion & Interpretation of results -
Conclusions -
30

31. 435 - In vitro Membrane Barrier Test method for skin
corrosion
Introduction : –
Skin corrosion :-
It refers to the production of irreversible damage to
the skin as visible necrosis through the epidermis & into the dermis,
following the application of a test material.
31

32. Advantages of this method.
In vitro test that utilizes an artificial membrane, so no living
animal is required & thuis reducting & refining the use of animal
testing.
It is an alternative for the standard in vivo rabbit skin procedure.
32

33. Disadvantages of this method
Validation studies are carried out to check reliability of test
results.
Many non corrosive chemicals & their mixtures & some
corrosive chemicals & their mixtures may not qualify.
Aqueous substances with a ph range of 4.5 to 8.5 often do not
qualify.
33

34. Initial considerations :– Identification and GHS classification
of corrosive agents.
34

35. Principle :–
The entire test system is composed of two main components:
a). Macromolecular bio-barrier.
b). Chemical detection system (CDS)
Membrane barrier damage after the application of corrosive substance
is detected.
Penetration of membrane barrier is measured by a no. of procedures,
including a change in the colour of a pH indicator dye.
35

36. Procedure :–
The following is a generic description of the components and
procedures of an artificial membrane barrier test method for
corrosivity assessment.
The membrane barrier and the compatibility/indicator and
categorisation solutions can be constructed, prepared or obtained
commercially, e.g., Corrositex®.
A sample test method protocol for the validated reference test
method can be obtained at. Testing should be performed at ambient
temperature (17-25ºC) and the components should comply with the
following.
36

37. Test substance categorization test.
It is a type of screening test to distinguish weak and strong
acids/bases.
Two different time scales used for GHS classification.
37
Test substance compatibility test.
Whether the compound is detectable by CDS or not.
The CDS & the exposure conditions used in this test should
reflect the exposure in MBT.

38. MB test method components :–
MB consists of 2 components:
a) Proteinaceous macromolecular aqueous (PMA)gel.
Composed of protein e.g. keratin, collagen or protein mixtures.
Acts as target for the test substance.
It should be of equal thickness & without air bubbles as it could
affect its functional integrity.
b) Permeable supporting membrane (SM).
It provides mechanical support.
38

39. The CDS :–
It is an indicator solution used to detect the passage of test
substance through the barrier.
Various pH indicator dyes & their combinations e.g. cresol red,
methyl orange are used.
The measurement system can be Electronic or visual.
39

40. Test performance :–
Assembly of the test method components:
MB positioned in vial or tube containing indicator solution.
Application of TS:-
500 µL of liquid or 500 mg solids (finely powdered).
Applied to upper surface of MB.
Replicates for each TS & its corresponding controls.
Time of application is noted.
40

41. Measurement of barrier penetration:-
Monitoring each vial.
Time of first change in indicator solution “Barrier Penetration”
is noted.
Time elapsed b/w Application and Penetration is determined.
41

42. Controls –
-Vehicle/solvent with the test substance- Compatible to MB.
42
Controls
+ve control
chemical
e.g. NaOH
pellets
With the TS
2nd
+ve control
Group
Other agent of
same class to
TS
Solvent control
group
-ve control
group
(non-corrosive)
e.g. 6%
propionic acid,
10% citric acid
Demonstrates
the reliability of
test method.
Demonstrates
The
compatibility of
solvent with
MB.
For evaluating
the corrosivity
potential of TS.
Demonstrates
The functional
integrity of
MB.
42
Solvent control
group
+ve control
chemical
e.g. NaOH
pellets
With the TS
2nd
+ve control
Group
Other agent of
same class to
TS
-ve control
group
(non-corrosive)
e.g. 6%
propionic acid,
10% citric acid
Demonstrates
the reliability of
test method.
For evaluating
the corrosivity
potential of TS.
Demonstrates
The functional
integrity of
MB.
Solvent control
group

43. Study acceptability criteria :-
Time elapsed is used to predict corrosivity of the test substance.
For study acceptability:-
a)+ve control = expected penetration response time.
b)-ve control = neighter be corrosive & not should alter the corrosive
potential of test substance.
c)12 proficiency chemicals = demonstrates the technical
proficiency/skill.
Interpretation of results & corrosivity classification of TS.
Time in minutes is used. 43

44. Study acceptability criteria –
- Time elapsed- in prediction of corrosivity.
For acceptability:
1) +ve control – expected penetration time.
2) -ve control – neither be corrosive nor should alter corrosive potential of TS.
3) 12 Proficiency chemicals – demonstrates the technical proficiency/skill.
Interpretation of results and corrosivity classification of TS –
-Time elapsed is used.
Data & Reporting –
Data :
-Data in tabular form.
-Means and standard deviation.
Test report :
-Detailed information of Test & control substances.
-Justification for use of this in vitro method.
-Test conditions
44

45. Data & reporting :-
All data should be reported in a tabular form
Means & standard deviations for each trial is also recorded.
Test report :-
Test & control substances.
Test condition. 45

46. Discussion of results –
Conclusions -
Results
1) Tabulation of data from individual test samples.
2) Description of :
a) Effects observed.
b) Evaluation and classification criteria.
46

47. 47
-Acute toxicity testing before this study.
Definition – “Adverse effects occurring as a result of repeated daily dermal application of a
chemical to experimental animals for a part of life span(not exceeding 10%).”
Principle – “Test drug applied in graduated doses to animal groups for 90 days period.”
Test Procedure – 1 dose per group
• Preparations -
• Experimental animals -
• No. & sex –
At least 20 animals(10 per sex)
+
satellite group (20 animals, high dose treatment, observed for extra 28 days )
• Housing and feeding conditions –

48. 48
Test conditions –
• Dose levels - 3 dose levels.
- If the skin gets damaged in the early study phase then terminate the study.
• Limit test - If a test at one dose level of at least 1000 mg/Kg produces no observable toxic
effects & if toxicity wouldn’t be expected based upon data from structurally related
compounds then full study
• Observation period-
Procedure :
• Dosing – Animals are treated for at least 6 hours per day on a 7 days per week basis (for 90
days).
-Satellite group 90 days high dose level + 28 days follow-up.
• Application of test substance –
• Observations --

49. 49
•Clinical examinations –
-Ophthalmological examinations.
-Hematology.
-Clinical biochemistry.
•Pathology –
a) Gross necropsy :
- External surfaces, cavities, orifices.
-Adrenals, kidneys, testes, liver are weighed while wet.
b) Histopathology :
-To be done on the high dose group, the control group & satellite group animals.
•Data and Reporting –
a)Treatment of results :
b)Evaluation of results :
•Test report & Interpretation of results –

50. Referances
1. OECD Guideline for Testing of Chemicals (1981).Repeated
Dose Dermal Toxicity:21/28-day Study. Page no. 1-8
2. OECD Guideline for Testing of Chemicals (1981).Subchronic
Dermal Toxicity:90-day Study.1-8
3. OECD Guideline for Testing of Chemicals (2004).Proposal
for a New Draft Guideline 434:Acute Dermal Toxicity-Fixed
Dose Procedure.1-13

51. 4. OECD Guideline for Testing of Chemicals (2004).Proposal for
a New Draft Guideline 435:In Vitro Membrane Test Method
for
Skin Corrosion.1-14
5. Health Effects Test Guidelines (1998). OPPTS 870.1200.
Acute
Dermal Toxicity. EPA 712–C–98–192:1-8
6. Robbins, Kumar,Cotrans, (2005).Basic Pathology. The Skin.
6th
edition, Elsevier, Page no. 697-712.
7.www.fda.com
8.www.wikipedia.com

52. Thankyou