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The guinea pig is the laboratory animal par except for spoken judgements of too frequent
failures.
not sensitize by injection: viz., tetraehlorosalicylanilide, monobenzyl ether of hydroquiHowever, sensitization to salts of mercury, cobalt and nickel was not obtained though these
sign is that of Landsteiner & Jacobs (3) in are well-known allergens in humans.
their renowned study of experimental contact
dermatitis. A series of ten intradermal injections is given on alternate days and the animals
challenged intraeutsneously two weeks after the
last injection. The literature does not contain
data which would enable one to appraise ac-
Gothenburg, Sweden, and the Department of picrio acid, and picryl chloride, the prospect
Dermatology, University of Pennsylvania School they hold forth of using the split adjuvant
of Medicine, Philadelphia, Pennsylvania 19104.
t Present address: Department of Dermatology, technique to identify contact allergens would
to indifferent results obtained with weaker sensitizers such as formaldehyde, penicillin, and
iodoform. However, the specifications of this
technique are not yet fixed and further modifications may well demonstrate an improved
269
grams are used. It is important to avoid older made with the allergen incorporated in
animals since they are appreciably less sensitiz- Frcund's adjuvant and also independently. It is
able. While susceptibility is not influenced by sex, simplest to purchase Preund's Complete Adjuwe prefer females because of their greater tract- vant; we have found that the Difco product'
ability. The combativeness of males often dam- gives results entirely comparable to the emulages the test sites. Pregnant animals are entirely
unsuitable because of decreased capacity to man- sion prepared according to Frcund's original deifest an inflammatory reaction.
scription (16).
The standard outbred Hartley strain should
Immediately before injection the emulsion is
be used unless the investigator has empirically prepared by blending the commercial adjuvant
verified the equivalent sensitizability of another
genotype. Although Chase (13, 14) and recently with an equal volume of water. The adjuvant
Polk ef al. (15) have clearly demonstrated the is placed in a container and the aqueous phase
possibility of selecting genotypes with either in- is added in several installments while homogecreased or decreased susceptibilities to specific nizing with a rotating stirrer. Water soluble
allergens, most breeds should be acceptable because the antigenic dose is extreme.
'Difco Laboratories, Detroit, Mich.
270
allergens are first dissolved in the water phase; is 5% by weight provided that the injection
oil soluble or insoluble chemicals are dissolved does not produce local necrosis or ulceration
or suspended in the adjuvant (a mixture of and is sufficiently free of systemic toxicity as
paraffin oil and an emulsifier with mycobac to not impair the health of the animal. Otherteria). The final concentration of the allergen wise, the concentration is adjusted to the
highest level that can be well tolerated locally
and generally; this will usually fall within the
15% range.
I
L
Fm. 1. induction. First stage. A row of three
mi ections are made on each side: (1) 0.1 ml of
B. Topical application. Solids are finely pulverized and incorporated in petrolatum at 25%
concentration by weight if not excessively irritating or deleterious to general health. Otherwise the concentration is the highest one which
produces a mild to moderate irritation.
Liquids are used at the highest concentration
which does not produce excessive inflammation,
undiluted if not irritating. Otherwise the concentration in petrolatum or water should be so
adjusted as to produce a mild to moderate irritation.
Induction Procedure
Fro. 2. Induction. First stage. An area of 4 X 6 cm over the shoulders is clipped short
with an electric clipper. Into this area three pairs of symmetrical intradermal injections are
given simultaneously as diagrammed in fig. 1.
Fm. 3. Induction. Second stage Preparation of the patch. A 2 X 4 cm filter paper patch is
loaded with the test substance, backed successively by the impermeable plastic tape and the
elastic bandage.
271
A. Intradermol injections. A row of 3 injections, six in all, are made on each side as follows: (1) 0.1 ml of the adjuvant without the
son).
With fluids, however, it is best to place the wetted patch directly on the skin and then
2
Model X 555 M, Sunbeam Electric Ltd., Ncrstone, East Kilbridge, Glasgow, Scotland.
3W. & R. Balston Ltd., Maidstone, England.
Paul, Minn.
272
Fio. 6. Challenge. The challenge test is performed on a 5 >< 5 cm chpped and shaved area
of the flank. The test agent is applied on a 2 H 2 cm piece of filter paper under a sealed
dressing as for induction.
FIG. 7. Challenge. The occluded patch is firmly secured by an encircling elastic adhesive
bandage for 24 hours.
petrolatum at 25% concentration and liquids soureesG are obtainable which simulate "dayare used as is. Otherwise a sub-irritating con- light".
centration is empirically found which will not
Redness constitutes the minimum criterion of
cause redness in any of ten unexposed animals.
It is essential to avoid toxic concentrations in
order to eliminate false positive readings.
The animals are challenged two weeks after
the topical induction. Hair is removed from a
5 x 5 cm area on the flank by clipping and tologic examination can usually distinguish beshaving as before.
wound around the trunk (Fig. 7). The im- redness, 2; intense redness and swelling, 3. The
portance of a secure dressing which affords important statistic in maximization testing
complete occlusion cannot be too strongly em- however, is the frequency of sensitization not
phasized.
intensity.
Reading of challenge reactions. The challenge
Rating of ollergenicity. Based upon the persite is evaluated 24 hours after removal of the centage of animals sensitized we assigned each
patch. Any irritation produced by the plastic substance to one of five grades of allergenic
tape will usually have subsided by then and the potency ranging from 0 to weak (I) to extreme
allergic reaction will generally be at its peak. (V) (Table I). We could thus judge whether
The sites arc again examined in an additional the results of maximization testing were similar
24 hours, mainly to detect weak, slowly de- in humans and guinea pigs.
veloping reactions.
RESULTS
genicity were assayed by both procedures conether. The readings are preferably made in
o
by Macbeth Corporation, P.O.
indoor daylight at noon. Artificial light BoxManufactured
950, Newburgh, New York.
273
TABLE I
Maximization grading
Sensitization
rate (%)
Grade
Classification
I
II
III
08
928
2964
6580
IV
V
81100
Weak
Mild
Moderate
Strong
Extreme
majority of animals usually becoming sensitized. Neither technique was successful with
lanolin and hexachlorophene, marginal sensitizers at best. The maximization procedure un-
Maximization Test
Induction
Substance
Intradermal
Concentration
in Adjuvant
Challenge
Topical
Concentration
in Petrolatum
Topical
Concentration
Sensitization
Rate
Sensitization
Rate
21/25
0/25
16/20
0/25
in Petrolatum
%
Acrylic monomer
Aluminum chloride
Apresoline
Atabrine
Benzocaine
Formalin
Hexachlorophene
Lanolin
Malathion
Marfanil
Mercaptobenzothiazole
Mercuric chloride
Monobenzyl ether of hydroquinone
Neomycin
Nickel sulfate
Penicillin G
Potassium dichromate
Sodium lauryl sulfate
Streptomycin
Sulfathiazole
Tetrachlorosalicylanilide
Turpentine
Tween 80
Vioform
* vehicle
t vehicle
H20.
ethanol 70%.
5
2
2
1
2
5
5
5
10
5
1
0.1
0.5
25
5
3
1
1
10
5
5
5
5
5
10'
25
5
25
25
5*
25
25
10
1
10
5
2*
1.5
15
20
20
25
15
25
25*
5*
0.1*
25
25*
0.5*
18/20
7/25
16/20
0/25
0/25
13/24
20/20
8/20
8/25
10/20
.18/25
11/20
20/20
18/24
10
0. 1*
0.5
0/25
10
25
1
25
25
25
0.5*
18/25
10
it
20
20
9/25
18/25
16/25
0/25
5/25
1/25
6/20
5/20
0/25
1/20
0/25
0/25
0/20
6/20
0/20
0/25
0/20
0/25
0/20
7/20
3/20
0/25
0/25
0/25
2/25
0/20
0/25
0/25
274
TABLE III
Grades of allergenic potency by the Landsteiner-Draize test, the maximization test in hu?nans
and the maximization test in guinea pigs
Substance
Acrylic monomer
Aluminum chloride
Apresoline
Atabrine
Benzocaine
Formalin
Hexachlorophene
Lanolin
Malathion
Marfanil
Mercaptobenzothiazole
Mercuric chloride
Monobenzyl ether of hydroquinone
Neomycin
Nickel sulfate
Penicillin G
Potassium dichromate
Sodium lauryl sulfate
Streptomycin
Sulfathiazole
Tetrachlorosalicylanilide
Landsteiner-Draize
Test
Guinea Pig
Maximization Test
% pos.
Grade
% pus.
4
0
30
I
I
III
II
I
I
I
84
0
80
90
28
80
0
0
54
100
40
32
50
72
55
100
75
0
25
0
5
0
0
0
30
0
0
0
0
0
35
15
Turpentine
Tween80
Vioform
0
0
0
8
0
0
0
I
III
I
I
I
I
III
II
I
I
I
I
I
I
72
Grade
Test'
% poe.
Grade
ND
0
IV
100
78
22
II
IV
72
IV
II
I
V
IV
I
I
III
0
0
I
I
100
ND
III
III
III
III
38
92
92
28
48
67
IV
100
IV
80
4
88
IV
72
IV
IV
III
I
36
III
72
64
IV
0
20
Human Maximization
III
I
II
0
0
V
V
II
III
IV
V
I
I
I
I
The L-D test rated 14 substances as weak al- in a way comparable to that of the human
lergens (Grade I) whereas 12 of these had maximization assay. The procedure has proved
grades of II or more by maximization testing. both specific and sensitive. In regard to sulMoreover, no substance was graded higher than fathiazole and Vioform the G.P. test was even
III by the L-D test whereas fully 10 achieved more sensitive. The latter was entirely missed
that status by the maximization test.
in humans but achieved grade II status in
As regards maximization testing in guinea guinea pigs. Grade I for sulfathiazole on human
pigs and humans, the results are remarkably testing doubtless underrates its allergenic pocongruent (human data from Kligman (12)).
tentiality; grade iii in guinea pigs seems more
Agents which sensitized humans invariably in accord with clinical experience.
did so in the guinea pig. The quantitative
In a total experience which is larger than
similarities are noteworthy. The ratings for the the results presented here, specificity of the
two tests were within a single grade level for test has been upheld. Guinea pigs do not be18 of the test substances; for the other four come sensitized to substances which do not
the discrepancy was two grades. Vioform induce contact allergy in humans.
sensitized guinea pigs but not man.
Although we are persuaded that the guinea
pig test can identify contact allergens as reliDISCUSSION
ably as the human, it is all too easy to make
The guinea pig maximization procedure ap- misjudgements. If unwarranted conclusions are
parently detects and rates allergenic substances
275
kind of decisions are permissible. Sound inter- finisher, an insecticide, etc. The end product,
pretation requires both judgement and experi- not the chemical itself, is assayed. If this results
ence. Our views have been presented previ- in little or no sensitization, exaggerated exposure testing in humans would be a likely next
ously (12).
The aim of the test clearly defines its limita- step. One might apply the product five times
tions. It simply establishes to what extent a daily instead of once, or perhaps under occlu-
particular substance has the potentiality for sion or in overly generous amounts to large
acting as a contact sensitizer. It reveals that a areas, or perhaps to skin deliberately damaged
chemical possesses immunogenic capabilities but by a chemical irritant. So varied are the apthe percentage of animals sensitized does not plications of substances to human skin that one
indicate the probable human incidence of sen- cannot lay down the conditions of further testsitization. The antigenic stimulus in the test ing in anything more than general terms.
Such exaggerated use or stress testing proprocedure is enormously greater than under
conceivable conditions of use; this magnifica- vides a safety factor in deciding to go ahead
tion is necessary in order not to miss weak al- with commercial exploitation even if one or
lergens. Whereas the L-D test seriously under- more ingredients are known to be potent alestimates the hazard by failing to identify fairly lergens.
potent sensitizers, the G.P. maximization test
Finally, the timid should be apprised that
may mislead the unforewarned into an over- certain substances known to be moderate to
estimation of risk.
strong sensitizers by maximization testing are
Actually there is one particular result which in fact in widespread use. Examples of these are
is predictive and enables a rather firm estimate neomyein, penicillin, streptomycin, Malaof safety in use; this is when none of the ani- thion, and p-phenylenediamine.
mals becomes sensitized. This indicates an alRes ipsa loquitur!
lergenic potential so low that no imaginable
SUMMARY
human exposure is likely to be attended by a
significant incidence of sensitization. We emA new procedure has been described, the
phasize that it does not mean that the sub- guinea pig maximization test, for identifying
stance will never sensitize anyone but rather contact sensitizers. Injections are given intrathat the probability of sensitization is very low. dermally with and without complete Freund's
Interpretation becomes more troublesome adjuvant and one week later the test agent is
when a high porportion of the animals becomes applied topically over the injection site. The
allergic. Let it be stated forthwith that this animals are challenged by patch test two weeks
outcome does not necessarily compel one to later.
abandon interest in the substance. This result
The sensitizing potentialities of about twenty
merely warns the toxicologist of the possibility allergens of differing potencies were determined
of harmfulness. Whether the agent should be concomitantly by the maximization and Landdiscarded or studied further requires careful steiner-Draize procedures. The sensitivity of
consideration of many factors. These include the latter was quite low, eleven substances
whether the material is to be nsed in high or failed to sensitize a single animal although
low concentration, whether for a short or long these were clearly allergenic by the maximizaperiod, xvhether it will be applied to normal or tion test.
The results of maximization testing in the
diseased skin, whether it is likely to be leached
out of the product, whether its effects are so guinea pig were quite comparable to humans.
unique and advantageous that even an appreci- Human allergens invariably sensitized the
able risk is justified.
guinea pig.
276
Geschlechtskronkheiten,
Erg.-Werk 11/1.
94
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein,
demonstration of lysosomes in the diseased
J. H.: Effect of ultraviolet light on RNA
skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci.,
105: 73, 1964.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362375, Lysasames Eds.,
de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.