OECD GUIDELINE 406

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Skin sensitisation (OECD. 406)
Prepared by:
Momin Asran Zunnurain Khalid Ak
Guided by: Dr. Aman B. Upganalwar
M. PHARM FIRST YEAR
(department of pharmacology)
SNJB’s SSDJ College of Pharmacy, Chandwad

2. Skin Sensitisation(OECD GUIDELINE NO. 406)
INTRODUCTION:
• Skin sensitisation is defined as allergic response to a
substance after skin contact.
• Sensitisation is a immune system respose to chemical.
• Symptoms of sensitisation are often seen in skin.
• There are two type of animal use
1. Guinea Pig
2. Mouse

3. Guinea Pig
Two type of test:
1. Freuds complete adjuvants
(Guinea Pig Maximisation test)
2. Non adjuvants test
(Buehler test)
In the original guideline 406,
Four adjuvants test and three non adjuvants test

4. Mouse
Immune system of the mouse has been investigated more
extensive than that of the Guinea pig
Advantages – short duration and minimal animal treatment
Example:
1. Mouse ear swelling test
2. Local lymph node assay

5. Guinea pig maximisation test
• The guinea pig maximisation test (GPMT) is an in
vivo test to screen for substance that cause human
skin sensitisation (i.e. Allergens)
• It was first proposed by B. Magnussom and albert
kligman in 1669 and described in their 1970 book
contact dermatitis in the guinea pi

6. General principle of sensitisation test
• The test animal are initially exposed to the test substance by
intra dermal injection and epidermal application
• Following a rest period of 10 to 14 days during which an
immune response developed, the animal are exposed to a
challange dose
• The degree of skin reaction to the challange exposure in test
animal is compared with control group animals

7. Element common to sensitisation test in
Guinea Pig
01
Guinea pig
Animal
03 250-500g
Body weight
05 Temperature=20℃
HR=30-70%
12hrs light & 12hrs dark
Housing
02 Male
Female (non pregnant)
Sex
04 At least 5 days prior to
the test
Acclimatization
06
Feeding

8. Method
NUMBER OF ANIMAL
A minimum of 10 animal are used in the treatment group and at least 5
animals in the control group are used
(20 test and 10 control)
INDUCTION: INTRADERMAL INJECTION
Day 0 – treated group
1. Three pair of intradermal injection are given in the shoulder region
1/1 mixture of FCA/water or saline
2. Test substance in an appropriate vehicle at the selected concentration
3. Test substance in a 1/1mixture of FCA/water or saline

9. Day 0 – control group
Control groupe also receive three intradermal injection, but only vehicle
is used
Day 5-7: Treated and control group
24 hrs before topical application,
The test area after close clipping or saving painted with 0.5 ml of 10%
sod lauryl sulphate In Vaseline, in order to creat a local irritation

10. Day 6-8 : treated group
Shaving
Test substance applied to test area for 48 hrs
Day 6-8 : control group
Shaving
Only vehicle are applied in similar manner for 48 hrs

11. Day 20-22 : treated and control group
A patches loaded with test substance is applied to flank of animal
And
A patches loaded with vehicle is applied to other side of flank
For 24 hrs
Observation : treated and control group
Remove patch after 24 hrs
Clean area after 21 hrs of patch removal
Observe after 3 hrs (appropriate 48 hrs after challange)
Approximately 24 hrs after this observation A second observation 72 hrs

12. Scale for the evaluation of challenge patch
test reaction
3=Intense
erythema and
swelling
2=Moderate erythema
1=Discrete or patchy
erythema
0=No visible change

13. Rechallenge
• If it is necessary to clarify the results obtaine
d in the first challenge
• A second challenge
1. With a new control group / original group
2. Should be considered approximately one
week after the first one
3. Rechallenge may also be performed on the
original control group

14. Clinical observation
All skin reactions and any unusual findings,
including systematic reactions, result from
induction and challenge procedures should
be observed and recorded

15. Buehler test
• The bhehler test is an in vivo test to screen for
substance that cause human skin sensitisation
(i.e. Allergens)
• It was first proposed by Edwin Buehler in 1965
and further explained in 1980.

16. Number of animals :
• A minimum of 20 animals is used in the treatment group and at least 10
animals in the control group
Dose levels :
• The concentration for each induction exposure should be the highest to
cause (mild irritation)
• The concentration used for the challenge exposure should be the highe
st (non-irritating dose)
• The appropriate concentration can be determined from pilot study Using
two or three animals

17. Methode
Induction : Topical application
Day 0 – treated group
One flank is cleared of hair (closely clipped)
The test patch system should be fully loaded with test substance in a suitable
vehicle is applied to the test area and held in contact with the skin for 6 hours.
Day 0 - control group
One flank is cleared of hair (closely clipped)
The only vehicle applied on a similar manner of test application.

18. Days 6 – 8 and 13 – 15 treated and control group
The some application as on day 0 is carried out on the same test area of
same flank on day 6-8 and again on day 13-15.
Challenge
Day 27 – 29 treated and control groups
• The untreated flank of treated and control animals is cleared of hair.
• An occlusive patch or chamber containing the appropriate amount of
test substance is applied, at the maximum non irritant concentration, to
the posterior untreated flank of treated and control animals
• The patches or chambers are held in contact by a suitable dressing for
6 hours.

19. Observation – treated and control groups :
• Challenge area is cleared of hair 21 hours after removing the patch
• 3 hours later the skin reaction are observed and recorded
• Approximately 24 hours after the skin reaction observed and recorded
Rechallenge:
To clarify the results obtained in the first challenge

20. Clinical observations
• All skin reactions
• Any usual findings
1. Systemic reactions
• To clarify doubtful reactions
1. Histopathological examination
2. Measurement of skin fold thickness

21. Reference
OECD (1992) , Skin sensitisation. OECD Guideline for
the testing of Chemicals No. 406, Paris, France.

22. Thank you
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