OECD guidelines for toxicity testing Guinea Pig Maximization Test Mouse Ear Swelling Test Local Lymph Node Assay

1. SKIN SENSITISATION
By Prashant Shivgunde

2. Contents
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 Guinea Pig
 Mouse
 GPMT
 LLNA

3. Guinea Pig
 Two types of tests-
 Freunds Complete Adjuvant (FCA), and
 Non-adjuvant tests
 In the original guideline 406,
 Four adjuvant tests and three non-adjuvant tests were
considered to be acceptable
 In updated version (17 Jul 1992) preference given to-
 The Guinea Pig Maximisation Test (GPMT) of Magnusson
and Kligman which uses adjuvant and
 The non-adjuvant Buehler Test
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4. Mouse
 Immune system of the mouse has been investigated more
extensively than that of the guinea pig
 Advantages- short duration and minimal animal treatment.
 The mouse ear swelling test (MEST) and the local lymph
node assay (LLNA) appear to be promising
 Both assays
 Have undergone validation in several laboratories and
 Has been shown that they are able to detect reliably moderate
to strong sensitisers.
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5. Mouse
 Both Test-
 Can be used as a first stage in the assessment of skin
sensitisation potential.
 If a positive result is seen in either assay-
 A test substance may be designated as a potential
sensitiser, and
 It may not be necessary to conduct a further guinea pig test.
 However, if a negative result is seen-
 A guinea pig test should be referred
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6. GENERAL PRINCIPLE OF
SENSITISATION TESTS IN GUINEA PIGS
 The test animals are initially exposed to the test substance
by intradermal injection and/or epidermal application
(induction exposure).
 Following a rest period of 10 to 14 days (induction period),
during which an immune response may develop, the animals
are exposed to a challenge dose.
 The extent and degree of skin reaction to the challenge
exposure in the test animals is compared with that
demonstrated by control animals which undergo sham
treatment during induction and receive the challenge
exposure.
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7. ELEMENTS COMMON TO
SENSITISATION TESTS IN GUINEA
PIGS
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8. Sex of animals
 Male and/or female healthy young adult animals
can be used.
 If females are used they should be nulliparous and
non-pregnant.
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9. Housing and feeding conditions
 Temperature = 20oC (+ 3oC)
 Rh= 30-70 %
 lighting is artificial, (12 hours light, 12 hours dark)
 For feeding, conventional laboratory diets may be used with an
unlimited supply of drinking water.
 It is essential that guinea pigs receive an adequate amount of
 ascorbic acid.
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10. Preparation of the animals
 Acclimatisation
 Randomization
 Assignment to the treatment groups
 Clipping, shaving, or chemical depilation
 Weighing before the test commences
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11. Reliability check
 Assessed every six months by use of substances
which are known to have mild-to-moderate skin
sensitisation properties
 A response of at least
 30% in an adjuvant test and
 15% in a non-adjuvant test
should be expected for mild/moderate sensitisers
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12. Reliability check
 Preferred substances are
 hexyl cinnamic aldehyde (CAS No. 101-86-0),
 mercaptobenzothiazole (CAS No. 149-30-4) and
 benzocaine (CAS No. 94-09-7).
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13. Removal of the test substance
 Achieved using water or an appropriate solvent
without altering the existing response or the
integrity of the epidermis
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14. GUINEA-PIG
MAXIMISATION TEST
METHOD
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15. Number of animals
 Minimum No.
 10 animals is used in the treatment group and
 5 animals in the control group
 When not possible to conclude that the test
substance is a sensitiser-
 Testing in additional animals to give a total of at least
20 test and 10 control animals is strongly
recommended.
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16. Dose levels
 The concentration of test substance used for each induction exposure
should be
 Well-tolerated systemically and
 Highest to cause mild-to-moderate skin irritation.
 The concentration used for the challenge exposure should be the
 Highest non-irritant dose.
 The appropriate concentrations can be determined from a pilot
study using two or three animals.
 Consideration should be given to the use of FCA-treated animals for
this purpose.
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17. Induction: Intradermal Injections
 Day 0 - treated group
 Three pairs of intradermal injections of 0.1 ml
volume are given in the shoulder region which is
cleared of hair so that one of each pair lies on
each side of the midline.
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18. Injection
1
a 1:1 mixture (v/v) FCA/water or physiological saline
Injection
2
the test substance in an appropriate vehicle at the
selected concentration
Injection
3
the test substance at the selected concentration
formulated in a 1:1 mixture
(v/v) FCA/water or physiological saline.
Induction: Intradermal Injections
Day 0 - treated group
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19. Induction: Intradermal Injections
 In injection 3,
 Water soluble substances are dissolved in the aqueous phase
prior to mixing with FCA.
 Liposoluble or insoluble substances are suspended in FCA prior to
combining with the aqueous phase.
 The concentration of test substance shall be equal to that
used in injection 2
 Injections 1 and 2 are given close to each other and nearest
the head, while 3 is given towards the caudal part of the
test area.
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20. Induction: Intradermal Injections
 Day 0 - control group
 Three pairs of intradermal injections of 0.1 ml volume
are given in the same sites as in the treated animals.
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21. Induction: Intradermal Injections
 Day 0 - control group
Injection
1
a 1:1 mixture (v/v) FCA/water or physiological saline
Injection
2
the undiluted vehicle
Injection
3
a 50% w/v formulation of the vehicle in a 1:1 mixture
(v/v) FCA/water or
physiological saline.
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22. Induction: Topical Application
 Day 5-7 – treated and control groups-
 24 hours before the topical induction application,
 The test area, after close-clipping and/or shaving is
painted with 0.5 ml of 10% sodium lauryl sulphate in
vaseline, in order to create a local irritation (if the substance
is not a skin irritant).
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23. Induction: Topical Application
 Day 6-8 - treated group
 Shaving
 A filter paper (2 x 4 cm) is fully-loaded with test
substance applied to the test area and held in contact
for 48 hours.
 The choice of the vehicle should be justified
 Solids -pulverised and a suitable vehicle
 Liquids can be applied undiluted, if appropriate.
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24. Induction: Topical Application
 Day 6-8 - control group-
 Test area cleared of hair.
 The vehicle only is applied in a similar manner and held
in contact for 48 hours.
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25. Challenge: Topical Application
 Day 20-22 - treated and control groups-
 Flanks cleared of hair
 A patch loaded with the test substance is applied to one
flank of the animals and
 A patch or chamber loaded with the vehicle only may also
be applied to the other flank.
 The patches are held in contact by an occlusive dressing
for 24 hours.
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26. Observations - treated and control
groups
 Remove patch after 24 hrs
 Clean Area after 21 hrs of patch removal
 Shave
 Observe after 3 hrs (Apprx. 48 hrs after challenge)
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27. Observations - treated and control
groups
 Approximately 24 hours after this observation a
second observation (72 hours) is made and once
again recorded.
 Blind reading of test and control animals is
encouraged.
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28. SCALE FOR THE EVALUATION OF
CHALLENGE PATCH TEST REACTIONS
 0 = no visible change
 1 = discrete or patchy erythema
 2 = moderate and confluent erythema
 3 = intense erythema and swelling
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29. 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.
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30. Clinical observations
 All skin reactions and any unusual findings,
including systemic reactions, resulting from
induction and challenge procedures should be
observed and recorded.
 Other procedures, e.g. histopathological
examination, the measurement of skin fold
thickness, may be carried out to clarify doubtful
reactions.
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31. GPMT OECD 406
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32. Irritancy Development
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33. BUEHLER TESTMETHOD
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34. Number of animals
 A minimum of 20 animals is used in the treatment
group and at least 10 animals in the control group
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35. 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 highest -
 non-irritating dose
 The appropriate concentration can be determined from
a pilot study using two or three animals.
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36. 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.
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37. Induction: Topical application
 Day 0 - control group
 One flank is cleared of hair (closely-clipped).
 The vehicle only is applied in a similar manner to that
used for the treated group.
 The test patch system is held in contact with the skin for
6 hours.
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38. Induction: Topical application
 Days 6-8 and 13-15 - treated and control groups
 The same application as on day 0 is carried out on the
same test area (cleared of hair if necessary) of the
same flank on day 6-8, and again on day 13-15.
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39. Challenge
 Day 27-29 - treated and control groups
 The untreated flank of treated and control animals is
cleared of hair (closely-clipped).
 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
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40. Observations - treated and control
groups
 challenge area is cleared of hair 21 hours after removing
the patch the
 Three hours later (i.e. 30 hours after application of the
challenge patch) the skin reactions are observed and
recorded;
 Approximately 24 hours after the 30 hour observation (i.e.
54 hours after application of the challenge patch) skin
reactions are again observed and recorded
 Blind reading of test and control animals is encouraged.
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41. Rechallenge
 To clarify the results obtained in the first challenge
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42. Clinical observations
 All skin reactions
 Any unusual findings-
 Systemic reactions
 to clarify doubtful reactions-
 Histopathological examination,
 Measurement of skin fold thickness
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43. Reference
 OECD (1992), Skin Sensitisation. OECD Guideline
for the Testing of Chemicals No 406, Paris, France.
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44. Skin Sensitization:
Local Lymph Node Assay
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45. Salient features
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 LLNA provides advantages over TG 406 with regard to
animal welfare
 LLNA studies the induction phase of skin sensitization
and provides quantitative data suitable for dose-
response assessment
 A reduced LLNA (rLLNA) approach – use up to 40%
fewer animals –
 Used when there is a regulatory need to confirm a
negative prediction

46. LIMITATIONS
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 Alternative method for identifying potential skin
sensitizing test substances
 Assay is of equal merit and may be employed as
an alternative for TG 406
 All available information on the test substance is
required prior to conducting study

47. LIMITATIONS
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 in vivo method - will not eliminate the use of animals
 But have potential to reduce the number of animals
required for this purpose,
 Cause less pain and distress
 LLNA does not require that challenge-induced
dermal hypersensitivity reactions be elicited

48. LIMITATIONS
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 LLNA does not require the use of an adjuvant
 Despite the advantages there are certain limitations
that may necessitate the use of TG 406
 Limited validation database

49. PRINCIPLE OF THE TEST
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 Sensitizers induce proliferation of lymphocytes in the
lymph nodes draining the site of test substance
application
 Proliferation is proportional to the dose and to the
potency of the applied allergen and provides a simple
means of obtaining a quantitative measurement of
sensitization.
 Proliferation is measured by comparing the mean
proliferation

50. PRINCIPLE OF THE TEST
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 Stimulation Index (SI), should be ≥3 to classify test
substance as a potential skin sensitizer
 Measurement method based on the use of in vivo
radioactive labelling

51. DESCRIPTION OF THE ASSAY
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 Selection of animal species
 Mouse is the species of choice
 Young adult female mice of CBA/Ca or CBA/J strain, which
are nulliparous and non-pregnant
 between 8-12 weeks old

52. DESCRIPTION OF THE ASSAY
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 Housing and feeding conditions
 Group-housed
 The temperature = 22 ± 3ºC.
 Relative humidity should be aimed- 50-60%
 Lighting should be artificial
 For feeding, conventional laboratory diets may be used
with an unlimited supply of drinking water.

53. DESCRIPTION OF THE ASSAY
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 Preparation of animals
 Acclimatization
 Randomization with individual identification (but not by
any form of ear marking)
 Veterinary examination to ensure that test animal have
no observable skin lesions

54. DESCRIPTION OF THE ASSAY
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 Preparation of dosing solutions
 Solid
 Liquid
 Insoluble substances
 should be prepared daily unless stability data
demonstrate the acceptability of storage

55. DESCRIPTION OF THE ASSAY
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 Reliability check
 PC test substances are
 25% hexyl cinnamic aldehyde (Chemical Abstracts Service
[CAS] No 101-86-0) in acetone: olive oil (4:1, v/v) and
 5% mercaptobenzothiazole (CAS No 149-30-4) in N,N-
dimethylformamide
 PC dose should be chosen such that it does not cause
excessive skin irritation or systemic toxicity and the
induction is reproducible but not excessive (i.e. SI > 20).

56. DESCRIPTION OF THE ASSAY
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 Reliability check
 A concurrent PC group should always be included when
there is a procedural change to the LLNA e.g.
 Change in trained personnel,
 Change in test method materials and/or reagents,
 Change in test method equipment,
 Change in source of test animals

57. TEST PROCEDURE
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 Number of animals and dose levels
 A minimum of 4 animals is used per dose group,
 A minimum of three concentrations of the test substance,
 A NC group treated only with the vehicle for the test
substance, and
 A PC
 Consecutive doses are normally selected from an
appropriate concentration series such as 100%, 50%,
25%, 10%, 5%, 2.5%, 1%, 0.5%, etc

58. TEST PROCEDURE
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 Number of animals and dose levels
 Recommended vehicles are
 acetone: olive oil (4:1, v/v),
 N,N-dimethylformamide,
 methyl ethyl ketone,
 propylene glycol, and
 dimethyl sulphoxide

59. TEST PROCEDURE
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 Pre-screen test
 To determine the highest dose to be tested
 Where information on the concentration that induces
systemic toxicity and/or excessive local skin irritation is
not available
 Maximum dose level tested should be
 100% of the test substance for liquids or
 The maximum possible concentration for solids or
suspensions.

60. TEST PROCEDURE
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 Pre-screen test
 Conducted under conditions identical to the main LLNA
study,
 Except there is no assessment of lymph node proliferation
 Fewer animals per group can be used
 Observed daily for any clinical signs of systemic
toxicity or local irritation at the application site
 Study Termination on day 6,
 Both ears of each mouse are observed for erythema,
ear thickness measurement (on day 1, 3 and 6)

61. Table 1: Erythema Scores
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Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3

62. TEST PROCEDURE
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 Pre-screen test
 Excessive local skin irritation is indicated by an
erythema score ≥3 and/or an increase in ear thickness
of ≥25% on any day of measurement
 Highest dose selected for the main LLNA -
 Next lower dose in the pre-screen concentration series
that does not induce systemic toxicity and/or excessive
local skin irritation

63. Main study experimental schedule
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 Day 1:
 Individually identify and record the
weight of each animal and any clinical
observation
 Apply 25 μL of the appropriate dilution
of the test substance, the vehicle alone,
or the PC, to the dorsum of each ear.
 Days 2 and 3:
 Repeat the application procedure
carried out on Day 1.

64. Main study experimental schedule
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 Days 4 and 5:
 No treatment.

65. Main study experimental schedule
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 Day 6:
 Record the weight of each animal.
 Inject 250 μL of sterile PBS containing 20 μCi of (3H)-
methyl thymidine into all test and control mice via the
tail vein.
 Alternatively, inject 250 μL sterile PBS containing 2 μCi
of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine
into all mice via the tail vein.
 Five hours (5 h) later, humanely kill the animals.

66. Main study experimental schedule
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 Day 6:
 Individual animal approach
 Excise the draining auricular lymph nodes from each mouse ear and
process together in PBS for each animal;
 Pooled treatment group approach
 Excise and pool the lymph nodes from each ear in PBS for each
treatment group

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Lateral dissection

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Ventral
Dissection

69. Main study experimental schedule
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 Day 6:
 To further monitor the local skin response in the main study,
additional parameters such as –
 Scoring of Ear erythema or
 Ear thickness measurements (obtained either by using a thickness
gauge, or ear punch weight determinations at necropsy)
may be included in the study protocol.

70. Main study experimental schedule
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71. Main study experimental schedule
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 Preparation of cell suspensions
 A single-cell suspension of lymph node suspension
prepared using 200 micron-mesh stainless steel gauze
or another acceptable technique
 Washed twice with an excess of PBS
 DNA is precipitated with 5% trichloroacetic acid (TCA)
at 40C for 18h
 Pellets are either re-suspended in 1 mL TCA and
transferred to scintillation vials containing 10 mL of
scintillation fluid for 3H-counting, or transferred directly
to gamma counting tubes for 125I-counting

72. Main study experimental schedule
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 Determination of cellular proliferation (incorporated
radioactivity)
 Incorporation of 3H-methyl thymidine is measured by β-
scintillation counting as disintegrations per minute
(DPM).
 Incorporation of 125I-iododeoxyuridine is measured by
125I-counting and also is expressed as DPM.
 Depending on the approach used, the incorporation is
expressed as DPM/mouse (individual animal approach)
or DPM/treatment group (pooled treatment group
approach).

73. Main study experimental schedule
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74. Main study experimental schedule
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 Reduced LLNA
 When regulatory need to confirm a negative prediction
 Using fewer animals
 With strict adherence to all other LLNA protocol
specifications
 If a positive or equivocal result is obtained,
additional testing may be needed in order to
interpret or clarify the finding

75. OBSERVATIONS
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 Clinical observations
 Each mouse should be observed daily for any clinical
signs, either of local irritation at the application site or
of systemic toxicity.
 Promptly identify those mice exhibiting systemic toxicity,
excessive local skin irritation, or corrosion of skin for
euthanasia

76. CALCULATION OF RESULTS
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 Individual animal approach
 SI is derived by dividing the mean DPM/mouse within
each test substance group, and the PC group, by the
mean DPM/mouse for the solvent/VC group
 SI for the VCs = one

77. CALCULATION OF RESULTS
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 For pooled treatment group approach
 SI is obtained by dividing the pooled radioactive
incorporation for each treatment group by the
incorporation of the pooled VC group
 Test result as positive when SI ≥ 3
 Collecting radioactivity data at the level of the
individual mouse will enable a statistical analysis
for presence and degree of dose-response
relationship in the data.

78. REPORTING
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 Test substance and control test substances:
 – identification data (e.g. CAS number, if available;
source; purity; known impurities; lot number);
 – physical nature and physicochemical properties (e.g.
volatility, stability, solubility);
 – if formulation, composition and relative percentages
of components;

79. Test report
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 Solvent/vehicle:
 – identification data (purity; concentration, where
appropriate; volume used);
 – justification for choice of vehicle;

80. Test report
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 Test animals:
 – source of CBA mice;
 – microbiological status of the animals, when known;
 – number and age of animals;
 – source of animals, housing conditions, diet, etc;

81. Test report
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 Test conditions:
 – details of test substance preparation and application;
 – justification for dose selection (including results from pre-
screen test, if conducted); – vehicle and test substance
concentrations used, and total amount of test substance
applied;
 – details of food and water quality (including diet
type/source, water source);
 – details of treatment and sampling schedules;
 – methods for measurement of toxicity;
 – criteria for considering studies as positive or negative;
 – details of any protocol deviations and an explanation on
how the deviation affects the study design and results;

82. Test report
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 Reliability check:
 – summary of results of latest reliability check, including
information on test substance, concentration and vehicle
used;
 – concurrent and/or historical PC and concurrent NC
data for testing laboratory;
 – if a concurrent PC was not included, the date and
laboratory report for the most recent periodic PC and
a report detailing the historical PC data for the
laboratory justifying the basis for not conducting a
concurrent PC;

83. Test report
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 Results:
 – individual weights of mice at start of dosing and at
scheduled kill; as well as mean and associated error
term (e.g. SD, SEM) for each treatment group;
 – time course of onset and signs of toxicity, including
dermal irritation at site of administration, if any, for
each animal;
 – a table of individual mouse (individual animal
approach) or mean/median (pooled treatment group
approach) DPM values and SI values for each
treatment group;

84. Test report
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 – mean and associated error term (e.g. SD, SEM) for
DPM/mouse for each treatment group and the results of
outlier analysis for each treatment group when using the
individual animal approach;
 – calculated SI and an appropriate measure of
variability that takes into account the inter-animal
variability in both the test substance and control groups
when using the individual animal approach;
 – dose-response relationship;
 – statistical analyses, where appropriate;

85. Test report
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 Discussion of results:
 – a brief commentary on the results, the dose-response
analysis, and statistical analyses, where appropriate,
with a conclusion as to whether the test substance should
be considered a skin sensitizer.

86. Reference
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 OECD (2010), Skin Sensitisation: Local Lymph Node
Assay. OECD Guideline for the Testing of Chemicals
No 429, Paris, France

87. Mouse Ear Swelling Test (MEST)
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88. Mouse Ear Swelling Test (MEST)
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 The MEST comprises both the induction phase and
the elicitation phase of the immune response.
 Several weeks prior to and during the test period
mice are fed a diet enriched in vitamin A since this
has been shown to enhance contact sensitisation .
 Induction phase comprises clipping of the fur on the
belly region and removal of the outer layers of the
epidermis by tape stripping

89. MEST
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 Freund's Complete Adjuvant (FCA) is injected
intradermally before the test substance in vehicle (test
mice) or vehicle alone (control mice) is applied topically
 The skin is tape stripped each day during the following
four days and
 on days 1, 3 and 5 after FCA injection the same
amounts of test substance or vehicle alone are again
applied topically.

90. MEST
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 Give a rest period of five days
 Challenge phase begins on day 10
 Apply topical application of test substance at the maximum
non-irritating concentration to one ear and of vehicle alone
to the other ear of all (test and control) mice
 Measure ear thickness of test and control ears under
ether anaesthesia with a micrometer
 24 and 48 hours after application of test substance
 Ear swelling is expressed as the difference between test
and control ears in percent

91. Tape Stripping
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92. MEST
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 The MEST has been evaluated independently by
several laboratories and in inter-laboratory studies.
 It was concluded that the MEST is a useful model for
identifying strong contact sensitisers.
 The Mouse Ear Swelling Assay (MESA) is a variant
of the MEST with some modifications in the test
protocol. The use of the MESA is very limited.

93. Status of validation and/or
standardisation
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 No standardised test guideline is available for the
MEST.

94. References
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 Asherson and Ptak, 1968; Gad, 1986; 1994
Evaluation: Cornacoff et al., 1988; Descotes, 1988;
Dunn et al., 1990; Gad et al., 1987 (Cornacoff et
al., 1988; Hignet et al., 1989; Dunn et al., 1990)

95. Thank You!
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PPS