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Tna JOURNAL OF INVESTIGATIVE DERMATOLOGY

Copyright

Vol. 52, No. S

Printed in U.S.A.

1965 by The Williams & Wilkins Co.

THE IDENTIFICATION OF CONTACT ALLERGENS BY ANIMAL


ASSAY. THE GUINEA PIG MAXIMIZATION TEST5
BERTIL MAGNUSSON, MD.t AND ALBERT M. KLIGMAN, M.D., Pn.D.

The guinea pig is the laboratory animal par except for spoken judgements of too frequent
failures.

excellence for the establishment of allergic con-

tact sensitization. Basic knowledge of this

Divers efforts have been made to elevate the


form of delayed hypersensitivity has derived sensitivity of the guinea pig. Chase (4) estabalmost exclusively from studies of this animal lished an exquisite degree of sensitivity to
using potent allergens such as dinitrochloro- pieryl chloride by his "combination" method.
benzene and p-nitroso-dimethylaniline.
First pierylated erythrocyte stromata in adjuIt is the consensus however that the guinea vant was injected, followed by a series of
pig is less sensitizable than the human. The key weekly contact tests with pieryl chloride in
difficulty is that some, perhaps many, sub- which each exposure progressively intensified
stances which are known to be troublesome the degree of sensitivity. This procedure is
human sensitizers have failed to sensitize the suitable for chemicals which can couple with
guinea pig. These are generally weaker allergens red blood cells; it is probably too cumbersome
such as penicillin, neomyein, and heavy metals.
This deficiency seriously compromises the use-

for routine screening.

the guinea pig and the human.

evident with a number of substances which did

The Draize test (1, 2) recommended by the


U.S. Food & Drug Administration, is perhaps
the most widely used, especially by industries
producing new chemicals. Its fundamental de-

none, benzoeaine, thioglyeerol, and others.

Voss (5) improved the Draize test by using


fulness of this animal in screening new sub- the highest concentration of the test agent that
stances for their allergenic potentialities. On was non-irritating instead of a fixed concenthe other hand, there seem to be no instances tration of 0.1%. Even so, of 44 mercaptans
in which a substance sensitizing the guinea pig studied, eleven sensitized men but failed in
fails to do so in the human, It is obviously de- guinea pigs.
Still better results were secured by Buehler
sirable to use animals rather than humans in
preliminary screening but until false negative (6) who compared the Landsteiner intradermal
results can be eliminated, guinea pig testing test to repeated topical applications by closed
cannot be relied upon to identify contact al- patch. The animals were restrained for 6-hour
lergens. Investigators have been much con- periods during the topical exposures. The supecerned with closing the sensitivity gap between riority of occlusive topical exposure was very

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-

curately either its strengths or weaknesses,

Chase and Maguire (7) have elaborated the


"combination" method into a "split adjuvant"
technique. Typically 5 sites are injected intradermally with paraffin oil containing killed
tuberele bacilli. Each site is reinjected with the
allergen 24 hours later. A subsequent series of

patch tests boosts the sensitivity to a very


high
level. Although these workers demonReceived August 16, 1968; accepted for publi-

strated the possibility of inducing an exquisite


* From the Department of Dermatology, Uni- state of sensitization to dinitrochlorobenzene,
versity of Gothenburg, Sahlgrenska Sjukhuset,
cation August 29, 1968.

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

seem to be somewhat spoiled by their allusion

Almiinaa Sjukhuset, 214 01, Malmo, Sweden.


268

IDENTIFICATION OF CONTACT ALLEROENS

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

capacity to detect weak allergens. Hood's


method (8) has apparently proved satisfactory
in an industrial setting (du Pont). The design
calls for thrice weekly topical applications for
9 exposures using the highest concentrations
which do not excessively irritate. The applications are made to abraded skin in half the test

269

Though most of our basic studies have been


conducted on groups of 25 animals, it seems likely

that ten will generally suffice for preliminary


screening. If none become sensitized or, conversely, nearly all become allergic, one may confidently certify the chemical to be a weak at best
or strong allergen, respectively. A result between
these extremes may justify expanding the sample
to secure more accurate appraisal.
Test substonces. The agents included substances

not known to sensitize humans: aluminum chlo-

ride, sodium lauryl sulfate and Tween 80, as

well as those which could be clinically rated as


strong sensitizers, e. p. formalin and streptomycin.

Most of the test agents have mild to moderate

group while all animals receive a total of 4 allergenicity in man.


intradcrmal injections over a 3-week period.

LAND5TETNER-DEAIzE (u-n) TEST


We mounted an extensive research which
had the prime objective of enhancing the useThe same battery of allergens was tested by
fulness of the guinea pig in screening contact the L-D method in order to compare the efallergens. The evolution of this work over an ficiency of the two procedures. The procedure
8-year period has followed the tactics and was as follows: A 0.1% solution or suspension
principles utilized in developing the maximiza- of the test material in saline was injected intration test for identifying contact allergens in dermally into male albino guinea pigs of 300-.
humans (9, 10, 11, 12). The variables which 500 grams. Injections of 0.1 ml were made
control the induction and elicitation of contact every other day or three times a week for a
sensitization have been studied quantitatively; total of ten, keeping the injections within a
these results will he the subject of a forthcom- field 3 to 4 ems square. The site was read 24
ing monograph. The knowledge gained has been hours after each injection. Two weeks after the
combined into a test procedure which we now 10th injection, the animals were challenged by
consider highly reliable for detecting allergenic an intradermal injection of 0.05 ml into a fresh
chemicals.
skin area. The animal was judged to be senThe purposes of this paper arc: (1) to sitized if the reaction was clearly greater than
provide specifications for the performance of the average reaction of the inducing injections.
the guinea pig "maximization test", (2) to comSPECIFICATIONS OF THE GUINEA PIG
pare the sensitivity of this procedure to that of
(op.) MAxIMIzATION TEST
the Landstciner-Draize test and (3) to correlate the results of human and guinea pig maxiPreparation of Test Material
mization testing.
for Induction
MATERIALs AND METHODS

Animol.s. Albino guinea pigs weighing 300-500

A. Intradermal injections. Injections are

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

THE J OTJRNAL OF INVESTIGATIVE DERMATOLOGY

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.

The allergen which is to be injected without


adjuvant is dissolved or suspended in an appropriate vehicle, water if it is soluble in that
medium. Insoluble substances are incorporated
into either paraffin oil, peanut oil or propylene
glycol, whichever enables the best solution or
dispersion.

I
L
Fm. 1. induction. First stage. A row of three
mi ections are made on each side: (1) 0.1 ml of

adjuvant alone, (2) 0.1 ml of test substance

alone and (3) 0.1 ml of the test agent emulsified


in the adjuvant. The rectangle outlines the area
to which the test substance will be applied topically one week later.

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

Induction is a two-stage operation. First, 3


pairs of injections are made simultaneously.
See Figs. 1 and 2. Second, closed patch exposure is performed over the injection sites

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.

ITJRNTIFICATION OF CONTACT ALLERGENS

271

one week later. See Figs. 3, 4 and 5. The


shoulder region is the induction site. An area
4 x 6 em is clipped short with an electric
clipper.

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

test agent, (2) 0.1 ml of test agent without


adjuvant and (3) 0.1 ml of the test substance
emulsified in complete adjuvant (Fig. 1). It

should be noted that the injection sites are


just within the boundaries of the 2 x 4 cm
patch, which will be applied one week later.

The adjuvant injections should he made deep


into the dermis to minimize sloughing.

B. Topical application. One week after the


injections the same area is clipped and shaved
closely with an electric razor. We find the Sunbeam Shavemaster2 most suitable for the latter

operation. If the test agent is non-irritating,


the area is pretreated with 10% sodium lauryl
sulfate (SLS) in pctrolatum 24 hours before
the patch is applied. The SLS is massaged into
FIG. 4. Induction. The occlusive bandage unit
the skin with a glass rod without bandaging. with
tbe loaded patch is apphed over the sites inThis concentration of SLS enhances sensitiza- jected a week earlier.
tion by provoking a mild inflammatory reaction.
The test agent in petrolatum is spread over
a 2 x 4 em patch of Whatman No. 3MM filter

paper2 in a thick even layer or, if liquid, to


saturation. The patch is covered by an overlapping impermeable, plastic adhesive tape
(1" 3M Blenderm4). This in turn is firmly
secured by elastic adhesive bandage (Tcnsoplast5, 6.4 em in width), wound around the
torso of the animal. This dressing is left in
place for 48 hours (Figs. 3, 4 and 5).

It is expedient to prepare beforehand all the


occlusive bandage units required for one session. Lengths of elastic bandage, about 25 cm

secured by elastic adhesive bandage, wound around

the impermeable plastic tape, adhesive side up,

ship of the Swedish Medical Research Council


(Project No. l9X-1036-Ol) and was supported, in

plastic tape and loaded with the test substance.

son).

Fm. 5. lndu.ction. The occluded patch is firmly

shoulder region of the animal and left in


long, are cut and placed with the adhesive the
place for 48 hours.
This study was conducted under the sponsorsurface up on the worktable. A 6 cm strip of
is applied to one end of the elastic bandage. part, by funds from "Edvard Welanders stiftelse",
Finally, the patch is placed centrally on the and "Riksforbundet mot allergi" (to B. Magnus-

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.

Mining & Manufacturing Co., St.


P. J. Smith & Nephew Ltd., Hull & Welwyn

Paul, Minn.

Garden City, England.

apply the Blenderm-Tensoplast covering.


Challenge Procedure

Challenge is by topical application. Provided


there is no irritation, solids are incorporated in

272

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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

an allergic reaction. This presupposes of course

that identical tests on non-sensitized animals


cause na reaction. Uncertainty concerning the
validity of mild reactions may be reduced by
rechallenging within three to four days. His-

5 x 5 cm area on the flank by clipping and tologic examination can usually distinguish beshaving as before.

The test agent is apphed on a 2 x 2 em piece


of filter paper in the same fashion as for topical
induction. The patch is sealed to the flank for
24 hours under a 4 cm strip of 1" Blenderm
(Fig. 6). This in turn is secured by Tens oplast

tween allergic and irritant responses if doubt


still persists (17, 18). Strongly sensitized animals display a vivid redness, associated with
indurated swelling. If desired one can score the

reactions on a 4-point scale: no reaction, 0;


scattered mild redness, 1; moderate and diffuse

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.

Three hours prior to the first reading, the


test site is shaved with the electric razor and
the skin gently cleansed of excess chemical with

RESULTS

Twenty-four substances of differing aller-

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

IDENTIFICATION OF CONTACT ALLERGENS

TABLE I

chloride, monobenzyl ether of hydroquinone,


neomycin, nickel sulfate, streptomycin, sulfathiazole, turpentine, and Vioform failed to

Maximization grading
Sensitization
rate (%)

Grade

Classification

sensitize a single animal by the L.-D. test.


Maximization testing readily identified these, a

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-

equivocally identified every clinically significant


allergen. No reactions were obtained with noncomitantly. The results are given in Table II. allergens, viz, sodium lauryl sulfate, Tween 80
There was a startling disparity between t.he and aluminum chloride.

Table III compares the allergenicity grades


capabilities for identifying contact allergens.
Eleven known allergens (Benzocaine, Mal- achieved by the L-D test with those of maximercuric mization testing in guinea pigs and humans.
athion,
mercaptobenzothiazole,
TABLE II
Corn parizon of Landsteiner-Draize with maximization test
Landsteiner.

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

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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

1 Results from Kligman (1966d).


ND = not done.

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

to be avoided one must clearly understand what

JDENTIFICATION OF CONTACT ALLERGENS

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.

Guidelines are set forth for interpreting the


If a substance is found to be a potent allergen but has virtues which merit continuing in- results and obtaining further data to estimate
terest, we would propose the following guide- the hazard of clinical sensitization in use.
lines in estimating the hazard. Instead of the
REFERENCES
chemical itself, it can be tested in the form and
1.
Draize,
J.
H.,
Woodard, G. and Calvery, H.
concentration in which it will be actually used,
0.: Methods for the study of irritation and
viz, as a cosmetic, a topical drug, a fabric
toxicity of substances applied topically to

276

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

the skin and mucous membranes. J. Pharm.


Exp. Ther., 82: 377, 1944.

2. Draize, J. H.: Dermal toxicity, p 46, Appraisal


of the Safety of Chemicals in. Foods, Drugs
and Cosmetics. The Assoc. of Food & Drug

Officials of the United States, Texas State


Dept. of Health, Austin, Texas. 1959.

3. Landsteiner, K. and Jacobs, J.: Studies on the


sensitization of animals with simple chemi-

allergens by human assay. II. Factors in-

fluencing the induction and measurement of


allergic contact dermatitis. J. Invest. Derm.,
47: 375, 1966.

12. Kligman, A. M.: The identification of con-

tact allergens by human assay. III. The

maximization test: A procedure f or screening and rating contact sensitizers. J. Invest.


Derm., 47: 393, 1966.

13. Chase, M. W.: Inheritance in guinea pigs of


the susceptibility to skin sensitization with
4. Chase, M. W.: Experimental sensitization with
particular reference to p'icryl chloride. mt.
simple chemical compounds. J. Exp. Med.,
73: 711, 1941.
Arch. Allerg., 5:163, 1954.
5. Voss, J. G.: Skin sensitization by mercaptans 14. Chase, M. W.: The inheritance of susceptibilof low molecular weight. J. Invest. Derm.,
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