united states
Environmental Protection
Agency
Office of Toxic Substances
Washington, DC 20460
EPA 560/13-79-009
July, 1979
Toxic Substances
oEPA
Acute Toxicity
Testing Criteria for
New Chemical Substances
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DISCLAIMER
This report has been reviewed by the Office of Toxic Substances, EPA,
and approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the
Environmental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
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EPA-560/13-79-009
July, 1979
ACUTE TOXICITY TESTING CRITERIA
FOR CHEMICAL SUBSTANCES
Contract No. W 2227-NA5X
Prepared for EPA by: Mary Janet Normandy
Robert Reynolds
EPA Project Officer: Daphne Kamely
ENVIRONMENTAL PROTECTION AGENCY
PREMANUFACTURING REVIEW DIVISION
OFFICE OF TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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ABSTRACT
This report addresses the rationale, considerations, and limitations of
acute toxicity testing. General procedures are described for acute tests
including lethality studies in oral, dermal, and inhalation toxicity, and
irritation studies in dermal and eye toxicity, phototoxicity and skin
sensitization. Recommendations are given for the acute toxicity tests
which may be used to evaluate the risks associated with the manufacture
and processing of chemical substances. Suggested minimum protocols are
offered including the choice of test animals, dosage levels, laboratory
practices, animal diets, and necropsy requirements. A method is sug-
gested for interpreting the results of these tests and extrapolating to
some guidelines for a safety factor in human exposure.
This report was submitted in fulfillment of contract W2227-NA5X by Enviro
Control, Inc. under the sponsorship of the U.S. Environmental Protection
Agency, and was prepared from April to July, 1979.
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FORWARD
This study was conducted for the Premanufacturing Review Division, Office
of Toxic Substances, Environmental Protection Agency, to collect data and
develop procedures for the assessment of toxicity as an integral part of
the risk assessment scheme for new chemical substances. This publica-
tion, which focuses on acute toxicity assessment, was directed primarily
toward the initial screening operation of chemical substances.
While studies on acute toxicity have appeared in the literature, this is
the first report useful for the evaluation of the risks posed by new
chemical substances to human health and the environment. Although this
study focuses on new chemical substances, it is important to note that
the procedures developed herein may have direct application to all areas
of environmental health analysis.
This document was published with the expert advice of Dr. Peter Voytek,
Office of Toxic Substances. Further information can be obtained from the
Premanufacturing Review Division; telephone: 202-426-2601; address:
Office of Toxic Substances (TS-794), U.S. Environmental Protection
Agency, 401 M Street, SW, Washington, D.C. 20460.
Daphne Kamely, Ph.D.
Premanufacturing Review Division
m
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CONTENTS
Page
Abstract ii
Foreward i i i
Section
1 Introduction 1
2 Types of Acute Toxicity Tests
Acute Oral Toxicity 6
Acute Dermal Toxicity 7
Acute Inhalation Toxicity 8
Acute Eye Irritation Toxicity 9
Dermal Irritation Toxicity 14
Dermal Sensitization Toxicity 17
Phototoxicity 19
Oral Irritation Toxicity and
Corrosive Effects 21
3 Acute Toxicity Testing Criteria
When Should Acute Toxicity Testing
Be Performed? 22
What Acute Toxicity Studies Should
Be Conducted? 23
What are the Minimal Protocols for
the Acute Studies? 24
How Should the Results be Interpreted
and Used to Estimate a Tolerable Level? 31
How Should the Data be Submitted For
Easy Interpretation by Reviewers? 34
References 35
iv
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Tables
1. Comparisons of Oral and Dermal L^QS within the
Same Animal Species 2
2. Grades for Ocular Lesions 12
3. Classification of Test Substances 13
4. Erythema and Edema Scoring 15
5. National Institute for Occupational Safety and Health
Interpretation of Skin Test Ratings 16
6. NIH-7 Open Formula Rat and Mouse Ration 27
7. Acute Study Price Test 29
8. Prediction of Human TLEs from Oral LD50s Derived From Animal
Studi es 33
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SECTION 1
INTRODUCTION
This report includes various suggestions for the collection and
utilization of acute toxicity data in the screening of chemical
substances.
Acute toxicity studies include lethality, irritation, sensitization
and corrosion testing. Acute lethality protocols usually involve expos-
ing an experimental animal to a single dose of a test substance for 24
hours or less. The animal is then observed for up to 14 days after
exposure (1). The results may be expressed as a simple mortality res-
ponse (death or no death) over a broad range of dose levels which are
reported as mg test substance per kg body weight of experimental animal.
These dose levels and associated mortality responses are then used to
extrapolate the LD50 (lethal dose for 50% of a group of test animals)
for the test substance. Specific toxic responses can also be measured
and used to determine another useful statisticthe TD5Q. This is the
dose at which 50% of all test subjects develop a given toxic response
such as tremors, erythema, convulsions, loss of consciousness, or
malfunction of a specific organ system.
Acute irritation, sensitization, and corrosion tests are used to as-
sess a chemical's potential for injury to specific tissue, usually the
eye or skin. Interpretation of test results is more complex than for
lethality testing and may include complex systems for grading toxic
responses. Because death is not expected to result from these studies,
LD^Q'S are not calculated.
It would be of obvious benefit to be able to use the results derived
from one type of acute test to predict the level of toxicity that would
be seen for that same compound using the same animal model in another
type of acute test. A major impediment to the feasibility of such corre-
lations is that the toxicity data most commonly available for any given
compound are usually collected for two different animal species - an oral
LD5Q in the rat and a dermal 1059 in the rabbit. However if both
oral and dermal LDcgs are available in the same animal species, even
when the data has been collected by two different laboratories, there are
some potentially useful generalizations to be made. Table 1 shows the
oral and dermal 10595 where both are available for the same animal
species for 51 compounds using 4 animal species in 57 tests (2). Dermal
LD5ps are higher than, or at least equal to, oral 10595 in about 88%
of the cases. In every case but one, hexabutyl-distannoxane tested in
the rat, the oral and dermal LD50s are within one order of magnitude of
each other. Data such as these suggest that substances which show
toxicity upon dermal application will probably be as toxic or more toxic
when administered orally. This is generally attributed to the more rapid
systemic absorption of ingested substances.
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TABLE 1
COMPARISONS OF ORAL AND DERMAL LD5QS
WITHIN THE SAME ANIMAL SPECIES
CHEMICAL
ACROLEIN
ACRYLONITRILE
AMMONIUM, (2-CHLOROETHYL)
TRIMETHYL-CHLORIDE
ANILINE
p-CHLORO-ANILINE
NITROBENZENE
BENZENETHIOL
4,4' - BIPYRIDINIUM, 1,V-
DIMETHYL-DICHLORIDE
2-ETHYLBUTANOL
t-BUTYLHYDROPEROXIDE
CARBAMIC ACID METHYL -,
1-NAPHTHYL ESTER
CARBON TETRACHLORIDE
m-CRESOL
o-CRESOL
p-CRESOL
4,6 - DINITRO-o-CRESOL
p-DIOXANE
HEXABUTYL - DISTANNOXANE
4-METHYL -6- PYRIMIDINYL
PHOSPHOROTHIOATE
1,2 - DIBROMO - ETHANE
2-BUTOXY-ETHANOL
2-(2-BUTOXY-ETHOXY) ETHANOL
ACETATE
2-CHLORO-ETHANOL
2-ETHOXY-ETHANOL
ORAL LD50 DERMAL LD5Q
SPECIES mg/kg mg/kg
RABBIT
RABBIT
RABBIT
RAT
RAT
RAT
RAT
RAT
RABBIT
RAT
RABBIT
RAT
RAT
RAT
RAT
RAT
RABBIT
RAT
RAT
RAT
RABBIT
GUINEA PIG
RABBIT
RAT
RABBIT
7
93
150
440
420
640
46
57
1200
406
710
2800
242
121
207
10
2000
87
2000
108
55
1200
2600
58
3100
562
250
232
1400
3200
2100
300
80
1260
790
2000
5070
620
1100
750
200
7600
11,700
8000
300
300
230
15,000
84
3500
DERMAL LDso
ORAL LD50 '
80.29
2.69
1.55
3.18
7.62
3.28
6.52
1.40
1.05
1.95
2.82
1.81
2.56
9.09
3.62
20.00
3.80
134.48
4.00
2.78
5.45
0.19
5.77
1.45
1.13
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TABLE 1 (CONTINUED)
CHEMICAL
SPECIES
ORAL LD50
mg/kg
DERMAL DERMAL
mg/kg ORAL LDso
2-(2-ETHOXYETHOXY) -
ETHANOL RAT 6500
Z'-HYDROXY - 2,4,4' -
TRICHLORODIPHENYL ETHER
N,N - DIMETHYL - FORMAMIDE
N '-(4-CHLORO-o-TOLYL)-N,N-
DIMETHYL-FORMAMIDINE
FORMIC ACID ETHYL ESTER
METHYL-HEPTANETHIOL
2-ETHYL-l,3-HEXANEDIOL
4-METHYL-3-PENTEN-2-ONE
PHENETHYL ALCOHOL
PHENOL
2-sec-BUTYL-4,6-
DINITROPHENOL RAT 25
4,4' - ISOPROPYLIDENEDI-
PHENOL
PENTACHLORO-PHENOL
PHOSPHONIC ACID (2,2,2-
TRICHLORO-1-HYDROXYETHYL)-
DIMETHYL ESTER
PHOSPHORIC ACID 1,2-DIBROMO-
2,2-DICHLORO-ETHYL DIMETHYL
ESTER RAT 250
S-((5-METHOXY-2-OXO-l,3,4-
THIADIAZOL-3 (2H)-YL) RAT 25
METHYL) 0,0-METHYL PHOS- RABBIT 63
PHORO-DITHIOATE
PHOSPHORODITHIOIC ACID, RAT 25
S-((2-ETHYLTHIO)ETHYL)
0,0-DIMETHYL ESTER
PHOSPHOROTHIOIC ACID, 0,0- RAT 76
DIETHYL 0-(2-ISOPROPYL-6-
METHYL-4-PYRIMIDINYL ESTER)
PHOSPHOROTHIOIC ACID, 0,0- RAT 1000
DIMETHYL ESTER, 0,0-DIESTER
WITH 4,4'-THIODIPHENOL
6000
0.93
RAT
RAT
RAT
MOUSE
RABBIT
RABBIT
RAT
RABBIT
RABBIT
GUINEA
RAT
3700
2800
170
160
625
2075
85
2600
1000
PIG 400
414
9300
5000
4000
225
640
20,000
1954
2000
5990
5000
669
2.51
1.79
23.53
1.41
1.02
9.64
22.99
0.77
5.99
12.5
1.62
80
800
20
375
179
455
1370
3.20
RABBIT
RAT
RAT
RABBIT
2230
50
450
1450
3000
105
2000
5000
1.35
2.10
4.44
3.45
3.20
0.80
5.95
7.16
5.99
1.37
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TABLE 1 (CONTINUED)
CHEMICAL
PHTHALIC ACID, BIS (2-ETHYL-
HEXYL) ESTER
POLYPROPYLENE GLYCOL MONO-
BUTYL ETHER
1,2-DIBROMO-3-CHLORO-PROPANE
1,2-EPOXY-PROPANE
TOXAPHENE
2-CHLORO-4-ETHYLAMINO-
6-ISOPROPYLAMINO-S-TRIAZINE
4-UNDECANOL, 7- ETHYL-2-
METHYL-,HYDROGEN SULFATE,
SODIUM SALT
2,6 - XYLENOL
SPECIES
RABBIT
ORAL LD5Q
mg/kg
DERMAL
mg/kg
34
RABBIT 23,900
RABBIT 180
GUINEA PIG 690
RAT 40
RABBIT
750
GUINEA PIG 650
MOUSE
RABBIT
980
700
25
21,000
1400
8640
600
7500
650
920
1000
DERMAL LD5Q/
ORALLDsn
0.74
0.88
7.78
12.52
15.00
10.00
1.00
0.94
1.43
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Acute toxicity testing is valuable in risk assessment in these ways:
as a screening procedure to help identify substances of such
low toxicity that extensive further acute testing is not
justified;
as a method to indicate specific toxic effects of a substance
which might be associated with either a single massive exposure
or with frequent use at a high level of exposure.
t as a range finder for studies by producing data concerning
lethal levels, signs of intoxication, and possible target organ
effects.
as an aid in the design of appropriate clinical management pro-
grams for individuals involved in the misuse of or an accident
with a chemical substance.
as a means to develop an index of the relative hazard of acute
exposure to various substances by comparing LDgQS and the
slopes of their dose-response curves.
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SECTION 2
TYPES OF ACUTE TOXICITY TESTS
ACUTE ORAL TOXICITY
Acute oral toxicity studies are designed to investigate the qualita-
tive and quantitative nature of the toxic effects from a single oral ex-
posure to a large dose of the chemical substance in question. The doses
are selected to provide data sufficient to estimate the LD50 and to
determine the slope of the dose-response curve. The numbers of test
animals per dosage level should be sufficient to make these statistical
analyses according to a particular method of evaluation. The test ani-
mals should be characterized as to species, strain, and physiological and
morphological characteristics. There is no standardized animal that is
suitable for all acute oral tests. It is extremely important that the
test animals be randomly selected for the dose level groups.
The test substance, if it is not a liquid, should be administered in
an appropriate carrier which facilitates absorption.
It is essential to note carefully all toxic signs including type,
time of onset, severity, and duration. The time at which deaths occur or
signs of toxicity appear is important, particularly if there is any
tendency for deaths to be delayed. Observations of the animal should
continue until signs of toxicity are absent in surviving animals. A
14-day observation period is sufficient for most compounds. At that time
survivors should be sacrificed and submitted to a gross pathologic
examination.
Ideally, to assess potential health hazards to humans, toxicity
studies should be conducted only in those species of animals whose meta-
bolism of the test substance is similar to that of humans. Since com-
parative metabolism is unknown for most new substances, and since studies
in humans are rarely feasible at this stage, rodents make good initial
test subjects. While extrapolation of the results to humans may not
always be valid, the correlation is reasonably good for single oral
doses. Acute toxicity testing can be conducted in several species if
necessary. A similar degree of toxicity in several species would suggest
that toxicity to humans might be comparable; marked variation in the res-
ponses of different species necessitates the assumption that humans are
at least as sensitive as the most sensitive species studied.
It is suggested that these tests be performed on two species of
animal-the albino rat, and the albino mouse. All female animals should
be nulliparous. The minimum sample number and dosage schedule should be:
- 5 male and 5 female animals of each test species at each of five
dose levels - preferably to include 2 levels above and 2 below the
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expected LD5n. A suggested dosage schedule is: 5000 mg/kg, 500
mg/kg, 50 mg/kg, and 5mg/kg.
ACUTE DERMAL TOXICITY
The ability of some chemicals to penetrate either intact or abraded
skin and produce systemic toxicity has been well established. A test for
acute dermal toxicity should evaluate the potential for systemic toxic
effects of chemicals expected to come in contact with the skin. This is
done by determining the LD5g of a single dermal exposure to the com-
pound in question by the animal test species. Since this LD50 will be
used in hazard evaluation, the test conditions should be related to the
anticipated human exposure, if at all possible.
The albino rabbit is the animal most frequently used in assessing
dermal toxicity and the most commonly used procedure is the method of
Draize.(S) It is also suggested that other animal species, such as mice,
rats, guinea pigs, or dogs, be used in addition to rabbits. The rabbit
appears to be exquisitely sensitive to dermal insult, and thus the eli-
cited reactions may not be valid for humans. The skin of guinea pigs has
permeability characteristics more like those of humans. The albino rat
is somewhat less reactive than the rabbit, and more reactive than the
guinea pig or humans. The rat is a preferred species for dermal
lethality testing because it is the model used most often for acute oral
studies and as well as for other types of toxicological studies.
Shortly before testing, fur from the trunks of healthy animals should
be clipped so that no less than 10% of the dorsal body surface area is
available for application of the substance. Care should be taken to
avoid abrading the skin, since this would alter its permeability. How-
ever, a dermal LDcQ for an animal with abraded skin may be desired
since the human skin which may eventually be exposed to the test sub-
stance will not necessarily be intact.
For some applications, it may be appropriate or necessary to use a
vehicle. If such is the case, any effect of the vehicle on the penetra-
tion of the test substance should be established. The test dose must
remain in contact with the skin throughout the exposure period, usually
24 hours.
At the end of the exposure period, any residual material is gently
removed with a gauze compress, the exposed area is examined, and any
lesions are noted. The animals should be examined at least once daily
for 14 days for signs of systemic toxicity and localized dermal reac-
tions. All animals that succumb or are moribund are necropsied. At the
end of the 14-day observation period, all survivors are subjected to a
thorough clinical examination, including examination of the exposed area
of skin. They are then sacrificed and necropsied. The degree of skin
irritation, signs of intoxication, changes in body weight, mortality, and
gross pathological findings as a function of dose and time are noted.
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The method of calculating the acute dermal LDgQ is the same as that
for the acute oral LD50. Both of these LDcg's are convenient for
estimating acute toxic hazard. Although there is always risk in extra-
polation from animals to humans, it is usually safe to presume that sub
stances with lower dermal LD^gs in animals will be potentially more
toxic to humans than those with higher
It is suggested that these tests be conducted on two species of
animal-the albino rabbit and the albino rat. Female animals should be
nulliparous. The following sample numbers and dosages are recommended:
- 2 male and 2 female animals of each test species at each of five
dose levels - preferably to include 2 levels above and 2 below the
expected LD50. A suggested dosage schedle is: 5000 mg/kg, 500
mg/kg, 50 mg/kg, 5mg/kg, and 0.5 mg/kg.
ACUTE INHALATION TOXICITY
The respiratory tract is particularly vulnerable to many substances
since it is generally less protected than most body systems. Moreover,
it can be subjected to injury not only when a toxicant enters the body
through the respiratory tissues, but also, in some instances, when a
toxicant leaves the body via the respiratory tract after having gained
entry by a different route. Consequently, injuries to the lung and other
body tissues resulting from inhaled toxic substances can have numerous
ramifications depending on the degree of toxicity of the substance, the
concentration and duration of exposure, and the existence of an immediate
or latent effect. The anatomy and physiology of the respiratory tract
can have great influence on the toxicity of inhaled vapors, gases, and
particularly, inhaled particles.
Single, high concentration inhalation exposures are used to deter-
mine the approximate toxicity level of a chemical substance or mixture
for comparative purposes. The nature of the toxic effect, if any, should
also be determined through this process; in this way the concentrations
to be used in subchronic inhalation exposure tests may be established.
These procedures are also applicable to brief and intermittent human
exposures.
The most informative and useful technique for determining the acute
toxic effects of inhalation exposure is the one used to determine the
LC5Q value for rats (that atmospheric concentration statistically esti-
mated to kill 50% of the exposed animals within a specified post-exposure
period, usually four hours.) Although death is the measured endpoint for
the LC5Q determination, observation of other toxic responses should
also be recorded. Test animals should be observed for at least two hours
for signs of irritation of eyes, nose, and lung tissue. Gross changes in
the respiratory rate, diaphragmatic breathing, gasping, and frothing and
bleeding from the nares are some signs of irritation of lung tissue.
Other evidence of discomfort may be pawing at the eyes or nose. In addi-
tion to these observations, records should include time of death and gross
-------
pathological changes noted at necropsy. An acceptable post-exposure
observation period is 14 days, with an optional extension to 21 days.
Exposure by inhalation is perhaps the most time-consuming and expen-
sive of all toxicological dosing procedures. If a test substance pro-
duces a systemic toxic effect by a route of administration other than
inhalation, one can assume that inhalation of a similar dosage would pro-
duce at least as great an effect (4). Thus, it may be expedient to post-
pone the inhalation tests or to perform them last, even though inhalation
might be the most likely route of exposure. It may be possible to form a
negative decision on a substance likely to be inhaled based on the re-
sults of exposure by routes other than inhalation.
The effects on the respiratory tract itself however, can be measured
only by inhalation studies. These effects may be transient such as tem-
porary inflammation, or irreversible such as death. A common direct ef-
fect is acute chemical irritation, which can affect any part of the in-
halation route or the gas-exchange surfaces of the lung. These acute
effects are often reversible except in cases where they produce pulmonary
edema or inflammation of such severity that the lung is no longer
functional (4).
It is recommended that these tests be conducted on two species of
animal-the albino rat and the albino mouse. Female animals should be
nulliparous. The following sample numbers and dosage schedule are
recommended:
- 5 male and 5 female or 10 male animals of each species at each of
two dose levels 2.0 mg/1 and 0.2 mg/1, for four hours of exposure.
ACUTE EYE IRRITATION TOXICITY
Test procedures to assess the surface toxicity of chemical substances
to ocular tissues of laboratory animals should show the potential for
substantial human eye injury. Albino rabbits have been most commonly
used in these test procedures because their eyes are large and have no
pigmentation. In addition, the tractable nature of this animal facili-
tates handling and examination. However, the rabbit eye differs in
several anatomical and physiological respects from the human eye. The
rabbit cornea is thinner, their blink reflex is not well developed, and
they have nictitating membranes and thick fur on their lids.
Limited comparative data from controlled exposures of humans and
rabbits show responses of the rabbit eye to be much more severe and long
lasting. Other nonprimate laboratory species such as rats, guinea pigs,
dogs, and cats are either less satisfactory than the rabbit or have not
been thoroughly evaluated. Among nonhuman primates, the rhesus monkey
has been used most frequently, but squirrel monkeys are also suitable.
The potential use of monkey species as human models seems obvious, as
their eyes are structurally and functionally similar to those of humans.
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However, the limited availability, cost, and potential hazards in the
handling of monkeys prevent their extensive use. Therefore, the albino
rabbit is the species of choice, with the rhesus monkey as the preferred
second species when confirmatory data are necessary.
In a given test only one eye of each animal should be used and the
animal should not be subjected to extraneous test procedures or stresses.
Most standard methods call for the instillation of a measured amount,
usually 0.1 gm or 0.1 ml, of the test substance directly to the cornea of
the test animal.
Epidemiological evidence suggests that most eye accident victims
rinse their eyes with water within one minute of exposure. Certainly
most physicians recommend prompt irrigation for accidental exposures to
chemical substances with the rationale that the chemical on the surface
is diluted and irrigated away. Some experimental animal studies indicate
that irrigation may decrease the amount of irritation caused by a chemi-
cal, but is not likely to change an apparent irritant to a nonirritant.
With some chemicals, such as 1% sodium hydroxide, irrigation markedly di-
minishes the toxic effects. With 5% sulfuric acid, irrigation exacer-
bates the reaction. The variability of irrigation techniques and the
arbitrary nature of any one regimen further complicate this test.
Consequently, irrigation is not a recommended requirement in a test of
the ocular irritancy of a substance.
Interlaboratory and temporal variability in rabbit eye testing makes
it difficult to determine the accuracy of any given result. Assuming
that the factors that cause variability consistently affect all observa-
tions in a single test, it should be possible to compensate for them.
This is done by testing control materials of established ocular irritancy
and by rating unknown substances with respect to them. If the human res-
ponse to the control material is known, animal response data can be
extrapolated to potential human response. In such cases, the more nearly
alike the test material and control are in irritancy, the more confidence
can be placed in the extrapolation.
If healing of the cornea and conjunctiva follow chemical injury, it
is usually completed within 14 days. Therefore, observation for 21 days
is essential in any test for toxicity. The recommended times for obser-
vations are 1 hour, 1, 3, 7, 14, and 21 days, though slight deviations
from this schedule will not seriously affect results.
Scoring the results of ocular irritancy tests is complex and several
scoring systems have been developed to assess the degree of damage. Ob-
servations of the cornea, iris, conjunctivae should be reported as well
as serious lesions such as pannus, phlyctena, and rupture of the eye-
ball. The grades of ocular reaction must be recorded at each examina-
tion. Evaluation of these reactions can be facilitated by use of a
binocular loupe or hand slit-lamp.
10
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At the 24 hour observation, the eyes of any or all test animals may
be further examined after applying fluorescein stain. For this optional
examination, one drop of fluorescein solution is dropped directly on the
cornea. After flushing out the excess fluorescein with tap water or
saline solution, the injured areas of the cornea will appear yellow in
ultraviolet light.
A record of the discharge from treated eyes in not required; however,
any exudate above normal can be recorded as additional information.
In grading these tests, an animal has exhibited a positive reaction
if the test substance has produced one or more of the following signs at
any observation:
a) Ulceration of the cornea beyond a fine stippling effect
b) Opacity of the cornea, other than a slight dulling
c) Inflammation of the iris, other than a slight deepening of the
rugae or a slight hyperemia of the circumcorneal blood vessels
d) Swelling in the conjunctivae (excluding the cornea and iris) with
partial eversion of the eyelids and a diffuse crimson color with
individual vessels not easily discernible.
In Table 2 a grading system for these parameters is presented. Fre-
quency counts are then made for each ocular parameter and Table 3 can be
used to aid in labelling the test substance.
The test should be considered positive if four or more animals ex-
hibit a positive reaction. If only one animal exhibits a positive reac-
tion, the test should be regarded as negative. If two or three animals
exhibit a positive reaction, the substance is considered to be an
irritant.
A clear distinction must be made between those substances that pro-
duce transient irritation and those that produce substantial injury.
Substances that produce conjunctivitis which clears within 2-3 days with
no further reactions generally require no further investigation. Materi-
als that produce serious cornea! injury or internal injury to the eye are
serious hazards and appropriate controls should be placed on their use.
It is also important to note any signs of systemic toxicity in the course
of these tests.
The recommended test species are the albino rabbit and albino rat.
The suggested sample numbers and dosage are:
6 animals of each test species at one dose of 0.1 gm (or O.lml)
in a single eye of each animal.
11
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TABLE 2
Grades for Ocular Lesions
CORNEA
No ulceration of opacity 0
Scattered or diffuse areas of opacity 1
Translucent areas, details of iris slightly obscured 2
Nacreous areas, no details of iris visible, size of pupil
barely discernible 3
Opaque cornea, iris not discernible 4
IRIS
Normal 0
Markedly deepened rugae, congestion, swelling or circumcorneal
hyperemia, but iris still reacting to light 1
Hemorrhage, gross destruction, or no reaction to light 2
CONJUNCTIVAE
Blood vessels normal 0
Some blood vessels hyperemic 1
Diffuse, crimson color, individual vessels not easily discernible 2
Diffuse, beefy red color 3
LIDS AND/OR NICITATING MEMBRANES
No swelling 0
Slight swelling 1
Moderate swelling with partial eversion of lids 2
Swelling with lids about half closed 3
Swelling with lids more than half closed 4
12
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TABLE 3
Classification of Test Substances
Classification
Non-irritant
Irritant
Severe Irritant
Ocular Reaction
No positive reaction in any
category in more than 1 out
of 6 test animals at 1-3
days and all eyes normal at
7th day.
Corneal opacity grades of
1.0 to 2.0 at any observa-
tion up to 7 days, but all
corneas cleared by 14th day.
Iritis grades of 1.0 at 1-7
days, but all iritis
cleared by 14th day.
Conjunctivitis grade of 2.0
or more at 1-7 days.
Lid or nictitating membrane
swelling grades of 2.0 or
more at 1-7 days.
A positive reaction in any
category which has not
cleared by 14 days.
13
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DERMAL IRRITATION TOXICITY
A reliable test for skin irritation should provide a means for dif-
ferentiating among substances that will produce different degrees of ir-
ritation or corrosion of the skin. In this context, irritation is the
local inflammatory response of normal living skin to direct injury by
single, repeated, or prolonged contact with a chemical agent without the
involvement of an immunologic mechanism. The macroscopic manifestations
are erythema and edema. Corrosion is direct chemical action on normal
living skin that results in its disintegration and irreversible altera-
tion at the site of contact. Its important manifestations are ulcera-
tion, necrosis, and with time, the formation of scar tissue. It is es-
pecially important to be able to distinguish between materials that will
produce minor or inconsequential degrees of skin irritation from materi-
als that can produce substantial irritation or corrosive injury as a re-
sult of either customary use or accidental exposure.
The voluminous literature on primary irritation test methods lacks
consensus on the animal model or procedure most likely to give accurate
and dependable results. Test procedures for human subjects are as nume-
rous as those for animals, suggesting that the problem does not lie sole-
ly in selection of the test species. The most standardized procedure is
a 24-hour, semiocclusive patch test of a full-strength substance on the
skin of albino rabbits. A common test procedure is to demarcate about
100 cm? of the skin into quadrants - two abraded and two intact. The
test substance can then be applied to one abraded and one intact quad-
rant, leaving another quadrant of each type to serve as controls. Most
of the conventional laboratory animals and some of the more exotic
species have been tried in skin irritancy testing. None provide perfect
models for human skin. The albino guinea pig and albino rabbit, though
commonly used, lack the broad spectrum of human responses to skin irri-
tants. They show only degrees of erythema and edema. Both species react
more strongly than humans to mild-to-moderate irritants. In fact, some
materials that appear unsafe when tested on rabbits may be nonirritating
to human skin. The response to guinea pig skin is more like that of
human skin over a wide range of materials; thus it is preferable to the
rabbit.
The chemical substance can be applied to intact and abraded test
sites on clipped animals for periods of exposure that range up to 24
hours. A statistically significant number of test sites (usually 2-4
sites on each of six animals) should be available for evaluating each
substance.
After removal of the test substance, 30-60 minutes should lapse be-
fore the patch sites are read to allow sufficient time for pressure and
hydration effects to subside. Additional readings should be made 24 and
72 hours after the patch application. Some persistent effects such as
corrosion are better determined at 7 days.
14
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Evaluation of skin effects involves using a scoring system for the
degree of redness and the degree of edema at the site of application of
the test substance. The scoring system usesd is commonly that which has
been published in the Federal Register in Section 191.11 of the Federal
Hazardous Substances Act of the United States. Their scoring system, as
seen in Table 4, involves assignment of numbers for the relative degree
of erythema and the degree of edema formation.
TABLE 4. ERYTHEMA AND EDEMA SCORING
Erythema
0 =
1 =
2 =
3 =
4 =
Edema
0 =
1 =
2 =
3 =
4 =
no erythema
slight, barely perceptible erythema
well defined erythema
moderate to severe erythema
severe, beet red erythema with injuries in depth
no edema
very slight, barely perceptible edema
slight edema with raised edges
moderate edema with surface raised approximately 1 mm
severe edema with the area raised more than 1 mm and
extending beyond the area of exposure
The scores obtained for both erythema and edema at each scoring
period and for both the abraded and intact skin are listed and the mean
for each group and for each type of effect is calculated. All 8 mean
values are then added together and divided by 4 since there are four mean
values for each effect (erythema and edema) thereby giving a final
numerical figure which is the primary irritation score.(5)
In some cases it may be useful to retain animals for 2 weeks after
application but because such delayed readings usually only confirm ef-
fects seen at 7 days, the value of further observation should be measured
against the cost of maintaining the animals.
Table 5 reflects the kinds of warnings which can be developed from
the numerical results of these tests.
The suggested test species are the albino rabbit and the albino rat.
Recommended sample numbers and dosage schedule are:
6 animals of each test species at 100%, 30%, 10%, 1%, and 0.3% of
the test compound. If the lowest of these produces positive re-
sults, a dose below 0.1% is used to find a no-effects level.
15
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TABLE 5
NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND HEALTH
INTERPRETATION OF SKIN TEST RATINGS (6)
Rating
Interpretation
Intact skin
Abraded skin
0-0.9
1-1.9
2-4
0-0.9
1-1.9
2-4
Nonirritant; probably safe for intact
human skin contact
Mild irritant; may be safe for use, but
appropriate protective measures are
recommended during contact
Too irritant for human skin contact;
avoid contact
Nontoxic to cellular components of
abraded skin; probably safe for human
skin contact
Mild cellular toxins; may be safe for
abraded skin contact provided protec-
tive measures are employed
Cellular toxins too irritant for
abraded skin contact; avoidance of con-
tact is advised
1-1.9
2-4
Mixed reactions
Intact
skin
0-0.9
Abraded
skin
0-0.9
1-1.9
Safe
Safe
for
for
human skin
contact
intact human
skin contact;
may
2-4
1-1.9
2-4
2-4
be safe for abraded skin contact when
protection is maintained
Safe for intact human skin; contact
with abraded skin should be avoided
May be safe for intact and abraded skin
contact when protection is maintained
May be safe for intact human skin con-
tact when protectio is maintained, but
contact with abraded skin is to be
avoided
Unsafe for intact and abraded human
skin contact; avoid contact
16
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DERMAL SENSITIZATION TOXICITY
Dermal contact sensitization refers to a delayed, immunologically-
mediated, allergic reaction to a chemical. With few exceptions, contact
sensitization develops as a result of one or more contacts with a chemi-
cal which initiate the sensitization process. The latent sensitized
condition generally develops no sooner than 1-2 weeks after the effective
exposure. Subsequent exposure of the skin of the sensitized individual
to a lower concentration of the sensitizer or related substance (cross-
sensitizer) can elicit a more intense response than to the initial expo-
sure to the chemical. This response may take hours or even days to
develop, hence it is "delayed." Responses may be characterized by pruri-
tis, erythema, edema, or induration, papules, vesicles, bullae, or combi-
nations of these. Reactions generally subside over a period of days if
there is no further contact with the sensitizer, but the state of sensi-
tization may be permanent.
Occasionally an individual who has been sensitized through the skin
will exhibit a systemic reaction, anaphylactic shock, whose symptoms in-
clude irritability, dyspnea, cyanosis, convulsions, unconsciousness and
death.
Dermal sensitization tests should detect materials that are capable
of inducing either a substantial incidence of or degree of sensitization
responses among individuals exposed during use or accidental misuse.
Laboratory animal species are generally much less responsive to con-
tact sensitizers than are humans. The most responsive is the guinea pig,
particularly albino varieties. Animals from 1-3 months of age are pre-
ferred as they are more easily sensitized than very young or older
animals. There is no appreciable difference in the proclivity of male
and female guinea pigs to develop sensitization, but pregnant females
should be avoided since pregnancy may alter the allergic response.
Topical application techniques for the determination of skin sensiti-
zation have been in common use for the last twenty years. A method which
involves intradermal injection of the test material in guinea pigs is
also used, although this technique does not represent true topical sensi-
tization.
This testing involves both an induction phase and a challenge phase.
The concentrations of test compound to be used in the induction phase as
suggested by Klecak (7) are 100%, 30%, 10%, 3%, 1% and 0.3%. On the
first day of testing, a O.lml solution or suspension of each of these
17
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concentrations of the test substance is applied to an area measuring 8
cm2 on the clipped flank skin of six to eight animals per concentration
group. Applications are repeated daily for 3 weeks, or five times weekly
for 4 weeks, always using the same skin site. The application site re-
mains uncovered and the reactions (if continuous daily applications are
performed) are read 24 hours after each application or at the end of each
week. When strong local irritation reactions are elicited, the applica-
tion site is changed. A control group of six to eight animals is not
induced.
The concentrations to be used in the challenge phase will have been
determined largely from the results of the dermal irritation studies.
Each test animal and each control will be challenged by each of four
concentrations of the test substance:
the minimal irritating concentration (the lowest dosage at which
25% of the test animals develop a mild erythema, an erythema
score of 1, but no edema.),
the maximum nonirritating concentration (the highest dosage which
elicits no skin reaction), and
two scaled doses below the maximum nonirritating concentration
On days 21 and 35, applications of 0.025 ml of each of these four
concentrations are applied to the contralateral flank of each animal on a
skin site measuring 2 cm2. Reactions are read at 24, 48, and 72 hours.
This procedure determination of the minimal sensitizing concentration
necessary for inducing allergic contact hypersensitivity and the minimal
eliciting concentration necessary to cause a positive reaction. A con-
centration is considered allergenic when at least one of the animals in
that concentration group concerned shows a positive reaction with non-
irritant concentrations.
Because cutaneous responses are visible, they can be readily evalu-
ated by a trained observer. The delayed reactions of contact sensitiza-
tion are best evaluated by making sequential observations of test sites
on the skin. The area is scored for the degree of erythema and edema as
described above in the dermal irritation section. Numerical scores are
then averaged for the animals in each group. The initial observation
should be made 48 hours after the first application of the test mixture
and these scores are used as an index of the irritant properties of the
substance. A second observation should be made 24 hours after the chal-
lenging injection in order to allow primary irritation to subside, but
subsequent scorings should be made 48 hours later after the challenging
dose. The scores that are taken following the challenging dose are com-
pared with the irritant scores. If the challenge scores are two to four
times the irritant scores the compound is considered to be a mild sensi-
tizer. Compounds that have high sensitizing activity will show sensitiz-
ing scores that are four to seven times the irritant scores. (8)
18
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Reactions to the test substance at challenge that are stronger than
reactions to negative controls or to those seen during induction should
be suspected as results of sensitization. Responses that are marginally
more intense than control response or that occur in very few animals
should be confirmed by a second challenge after 1 or 2 weeks. Rechal-
lenge after a longer delay can produce unreliable results, because
sensitization in guinea pigs is short-lived compared to that in humans.
Whether or not a rechallenge is performed, a judgement confirming the
presence or absence of sensitization should be recorded for each animal.
Mean scores or indices, which are customarily calculated for each experi-
mental group, are useful only for showing relative intensity of res-
ponse. Two or more unequivocally positive responses in a group of 10-20
animals should be considered significant. A negative, equivocal, or
single response probably assures that a substance is not a strong sensi-
tizer, though this is best confirmed by further testing with human
subjects.
The albino guinea pig and the albino rat are suggested as the test
species and the following sample numbers and dosage is recommended:
5 male and 5 female animals per concentration group each induced
with 0.1 ml of the test substance at 100%, 30%, 10%, 3%, 1%, 0.3%
or 0% (controls). Each animal is then challenged with 0.025 ml
applications of the test substance at four concentrations as
determined from the dermal irritation studies: 1) the minimum
irritating concentration, the maximum non-irritating concentra-
tion, 3 & 4) two scaled does below the maximum non-irritating
concentration.
PHOTOTOXICITY
A phototoxic response refers to irritation that is not immunologi-
cally-mediated and which depends on light exposure for its presence.
This response is not to be confused with photosensitization or the photo-
allergy response in which light energy results in the promotion of hapten
formation and the consequent appearance of antigen. In this case it is
the resultant antigen-antibody response that is the cause of cell
damage. At this point neither the need nor the exact protocol require-
ments have been established for photosensitization testing, thus it is
not presently recommended at this level of acute testing.
Responses to phototoxic chemicals are elicited by ultraviolet ligh-
wavelengths, those between 280 and 430 nm. The agent plus light energy
results in the excitation of the agent; its energy state is raised to a
higher level. In the process of returning to its original ground state
of energy, the compound can react with constituents of the cell.
Although the mechanism of action of phototoxicity is not clear, the
chemicals which elicit this reaction are thought to be free radical ini-
tiators. These compounds can react with molecular oxygen in the presence
of light energy to form peroxides that cause damage to cell membranes.(8)
19
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The chemical to be tested is administered in a solvent which will not
alter the test situation by reacting with the chemical or otherwise ab-
sorbing energy upon exposure to ultraviolet light. Dosing should be on a
microgram or milligram per square centimeter basis, simplifying the
extrapolation to dosing in humans. The skin site can be conveniently
demarcated with a marking pen and the chemical can be delivered to the
skin with a micropipette. Following application, the animals are exposed
to ultraviolet light from a high output source.
It is customary to administer one high dose of the test substance.
This may be up to 10% of the oral LD50. No situation has yet been
found in which a compound has been negative at a high dose and positive
at a low dose. If the high dose elicits a positive response, the least
effective dose is then determined. Each animal may be used as its own
control. Control sites include 1) negative (the vehicle), 2) positive (a
known phototoxic chemical), and 3) unirradiated, chemical-treated sites.
Groups of 4-10 animals, are sufficient for this testing.
The phototoxic response is usually elicited quickly. For maximum ef-
fect the site should be irradiated within 30 minutes to 2 hours after the
chemical application. Scoring as described for dermal irritation tests
is performed 12-24 hours later. The result most commonly found in photo-
toxic responses is a visible and palpable dermatitis consisting of ery-
thema, induration, and at times, frank necrosis. The phototoxic response
is dramatic; there are few tests easier to read. Certain phototoxic
chemicals and solvents may irritate the skin without irradiation. When
this occurs, attempts should be made to decrease the effective tissue
dose so that the irritancy is not seen at the site where the chemical has
been applied but has not been exposed to light.
Occasionally, extrapolation of results obtained from animal photo-
toxicity tests to humans may be questionable. In such cases, tests with
humans may be necessary if the basic systemic toxicololgic data are
available. The experimental procedure resembles that used with animals;
however, it is usually necessary to make human skin more permeable to the
test substance by removing most of the stratum corneum by repeated cel-
lophane tape stripping. A stripped skin site control is also used. The
dose should be administered in one small application.
20
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ORAL IRRITATION TOXICITY AND CORROSIVE EFFECTS
It is particularly important to be able to detect materials which, if
ingested, can produce corrosive injury to the mucosal surfaces of the
oral cavity, pharynx, esophagus, and stomach. Severe corrosive injury to
these tissues can be fatal or can result in strictures or other perman-
ently disabling injuries. Chemical properties are reliable predictors of
corrosive potential. Strong alkalis are likely to injure the esophagus
and strong acids to injure the stomach and duodenum. Either may injure
the tongue and pharynx.
There is no standardized procedure for predicting corrosive potential
to the alimentary tract, though several techniques have been described.
Materials have been admininstered by intraoral, intraesophageal, and in-
tragastric gavage and by timed application to specific tissues by solu-
tions or impregnated tampons. Rats, rabbits, cats, dogs, and swine have
been tested, but a preferred animal model has not been identified. The
experts agree that more research is needed before an animal model is se-
lected and a reliable procedure is established.
The need for a special test for esophageal corrosivity has been ques-
tioned on the grounds that information on the chemical and physical pro-
perties of a substance, as well as the results of dermal irritation
studies, can provide reasonable presumptive evidence of severe irritation
or corrosive hazard upon ingestion even in the absence of specific cor-
rosivity testing.
21
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SECTION 3
ACUTE TOXICITY TESTING CRITERIA
WHEN SHOULD ACUTE TOXICITY TESTING BE PERFORMED?
It is recommended that all chemicals substances be screened for acute
toxicity and their LDgQS be determined. It has been suggested that
certain substances should not be required to undergo testing for toxi-
cological effects including those whose physical and chemical properties
and structure-activity relationships indicate that no adverse health
effects are probable.
Examples of this situation include:
nonreactive insoluble polymers which pose no potential human ex-
posure through inhalation of particles in air
0 nonreactive insoluble polymers which contain no monomeric con-
taminants or have them at levels safely below those known to
cause human toxicity
new chemical substances which differ only slightly in structure
from known substances which have already been shown to be inno-
cuous by existing toxicity data.
These may be reasonable exemptions in theory but in practice, biolo-
gical nonreactivity should be demonstrable and not merely assumed. It is
difficult to assure "safe" levels of monomeric contaminants in polymers
without first measuring those levels and evaluating their toxicity. It
is difficult to define a "slight" modification in structure. This is a
very ambigious exemption and may ultimately be abused. Structural-func-
tional relationships may be useful predictors but it is also true that
small differences in chemical structures can significantly influence the
biological effects of chemicals.
A common example is found in the compounds benzene and toluene. Ben-
zene is associated with severe acute toxicity including irritation of the
mucous membranes, convulsions, and respiratory failure. Chronic effects
include bone marrow depression, aplasia, and occasionally leukemia.
Toluene, on the other hand, which differs structurally from benzene only
by the addition of a methyl group, is considerably less toxic than ben-
zene. Toluene at high concentrations may cause mild macrocytic anemia
but it is not associated with the broad spectrum of hematologic toxicity
which is seen with benzene exposure.
Another example is found in the drug amphetamine (racemic
-phenylisopropylamine) whose effects include central nervous system
stimulation and activation of receptors which are normally innervated by
the sympathetic nervous system. The d-isomer is three to four times more
22
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potent than the 1-isomer in its effects on the central nervous system
whereas the 1-isomer is twice as effective as the d-isomer in its effect
on the heart. An effect of valence number on the toxicity of arsenic can
also be seen. The trivalent arsenites are much more lethal than are the
pentavalent arsenates for lower plants and animals including protozoa,
bacteria, and yeast(8). In essence, most predictions about toxicity and
structural-functional relationships should be verifiable and not simply
left as assumptions.
Testing exemptions may be considered for minor alterations to sites
other than the reactive group on such compounds as epoxides, nitro-
samines, and nitrosoureas. But here too there must be some consensus as
to what changes would have no effect on the biological reactivity of the
molecule. In these cases it will be easier to implicate a compound as
toxic than to exonerate it as safe.
Another group of compounds that has been suggested as possible exemp-
tions from toxicological testing includes those in which adverse health
effects are anticipated but human exposure will be strictly controlled.
This includes:
substances which are extremely acidic (pH <2) or basic
(pH >13), which are not volatile, which are used solely for
industrial purposes, and for which human contact would be
strictly prevented by protective measures.
Compounds of extreme acidity or basicity in their industrial stages
may remain as trace residues in or on some resultant product intended for
human use. At this point corrosivity may no longer be a factor but other
toxic properties might possibly become evident. Exemptions to substances
in this category do not seem to be justified unless it can be shown un-
equivocally that detectable levels never remain in the products to which
humans may ultimately be exposed.
WHAT ACUTE TOXICITY STUDIES SHOULD BE CONDUCTED:
The first level of testing should provide enough information to be
suggestive of the need for further testing in any area of toxicity and
should also be as simple, fast, and inexpensive as possible. It is
apparent that complete testing for long-term, irreversible effects will
not be required for all substances, and thus should not be included in
the first level of tests. It seems prudent to require a complete battery
of acute toxicity tests in the initial testing stage in order to obtain
useful data and possibly give some indication of the need for subchronic
testing as a second series of tests.
Acute toxicity tests recommended are as follows:
acute oral toxicity - for all chemical substances except gases
23
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acute dermal toxicity - for all chemical substances except gases
acute inhalation toxicity tests - for those substances whose
physical nature suggests that they would be airborne and thus
where inhalation is expected to be a possible route of human
exposure including:
- all gases
- liquids with a vapor pressure higher than 1 torr
- solids with a particle size below 5 micra in diameter
Irritation and sensitization tests - for all chemical substances
except gases.
WHAT ARE THE MINIMAL PROTOCOLS FOR THE ACUTE STUDIES?
Several studies have examined the variability of acute toxicity test
results (specifically the LDcQ calculations) obtained on substances
examined in different laboratories. Even when the same testing procedure
was followed in each of eight laboratories, LDgg's differed by up to
5-fold; averaging a 2-3-fold difference between highest and lowest
values. This indicates that general guidelines for protocols would be
more appropriate than rigid procedures for acute toxicity studies.
Acute toxic effects in animals can range from no observed effect to
sedation, loss of motor function, loss of consciousness, respiratory
depression, and death. The intensity of effect is related to time, and
also to blood concentration. Thus, there are two major factors in acute
toxicity tests: the rate of absorption and the rate of elimination of
the test substance. These two rates are in turn influenced by several
factors, including:
physical/chemical properties of the substance
concentration
volume
route of administration
0 biotransformation
transport
excretion routes
24
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t species (age, sex)
t pre-existing pathology.
These various influencing factors will not be identical between
laboratories, or even within the same laboratory at different times.
In general, it would help to check on the testing laboratory's ac-
creditation as at least a general indication that the test was conducted
in accordance with good laboratory practices.
In toxicity testing a primary consideration is the purity of the sub-
stance to be tested. The purity of the test substance should be known
and any contaminating components should be identified and quantitated as
thoroughly as possible to account for 100% of the test substance. All
materials should be stored appropriately to maintain their purity and
chemical integrity. Whenever possible, all studies should be done using
the same lot of test sample.
It is suggested that for each type of acute test to be performed, two
species of test animal should be chosen, and one of these should be the
albino rat. This will allow comparison of the effects of a given chemi-
cal substance administered to the same animal model by all test routes.
Correlations can then be attempted between oral, dermal, and inhalation
LD5Q's. Rats are preferred also because there is abundant reference
toxicologic data available for this species.
The second species should be the albino rabbit in tests of dermal
toxicity, dermal irritation and eye irritation; the albino guinea pig
should be used in tests of dermal sensitization; the albino mouse would
probably suffice as the second species in tests of oral and inhalation
toxicity.
In order to standardize results within and among laboratories, all
protocol requirements should be as specific as possible. Therefore, if
the general test animal is to be the albino mouse, an example of the
specific requirement would be:
C57B1/C3H mice (F-l) from Caesarean-derived animals, maintained
free from Mycoplasmosis (CRD) Salmonella, external parasites,
internal parasites, and other disease are to be used. The sup-
plier should provide information about the type of diet the ani-
mals have been fed and their bedding. Arrange animal delivery in
time to be ready for assignment to treatments at about 42 days of
age.
25
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Dietary requirements should also be carefully delineated. Any diet
which meets the general nutritional requirements of the species in ques-
tion can be used as a standard diet in toxicity studies so long as the
exact formulation and processing of the diet are known. For standardiza-
tion among testing agencies, the NIH Interagency Toxicology Support Com-
mittee has recently recommended use of the NIH-7 Open Formula Rat and
Mouse Ration (Table 6) as a standard diet. This formula is available
from several manufacturers and is priced competitively with other commer-
cial feeds. It is suggested that this be the required diet in all
studies using rodents as the test animal.
Caging requirements and animal care procedures should also be very
specific since these features can influence not only reproducibility of
experimental results but can also affect worker safety. A suggested
requirement would be:
Test animals should be housed as a single animal per cage in
hanging stainless steel wire cages. The use of wire mesh bottoms
without bedding is preferred since this will prevent coprophagy
and induction of mixed function oxidases. However, closed hous-
ing with false wire cage bottoms may be used if precautions are
taken to prevent coprophagy.
It is recommended that animal care during these studies include the
following standard requirements:
Temperatures will be kept at 27-29°C with a 12 hour dark-12
hour light cycle.
Clean drinking water will be provided and changed twice weekly.
Food consumption and body weights will be recorded at weekly
intervals.
Cages and racks will be cleaned and sanitized once every two
weeks.
Floors will be sanitized daily.
Animals will be observed daily by trained animal care
personnel.
Sick or dead animals will be removed for complete necropsy,
after notification of the investigator or his representative.
Proper records will be kept daily.
Facilities and equipment will be monitored for sanitation on a
monthly schedule.
26
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TABLE 6, NIH-7 OPEN FORMULA RAT AND MOUSE RATIONl
Ingredient
Dried skim milk
Fish meal
Soybean meal
Alfalfa meal
Corn gulten meal
Ground No. 2 yellow shelled corn
Ground hard winter wheat
Wheat middlings
Brewers dried yeast
Dry molasses
Soybean oil
Sodium Chloride
Dicalcium phosphate
Ground limestone
Vitamin and Mineral Pre-mixes2
5.00
10.00
12.00
4.00
3.00
24.50
23.00
10.00
00
50
50
0.50
1.25
0.50
0.25
100.00
^Calculated approximate composition = crude protein, 23.5%; crude
fat, 5.0%; crude fiber, 4.5%; ash, 7.0%.
^Vitamin and mineral pre-mixes shall provide per kg diet: vitamin A
(stabilized), 6,050 IU; vitamin D^, 5,060 IU; vitamin K, 3.1 mg;
a-tocopheryl acetate, 22 IU; choline 0.6 g; folic acid, 2.4 mg; niacin,
33 mg; d-pantothenic acid, 20 mg; riboflavin, 3.7 mg; thiamin, 11 mg;
vitamin B-12, 4.4 yg; pyridoxine, 1.9 mg; biotin, 0.15 mg; cobalt, 0.44
mg; copper, 4.4 mg; iron, 132 mg; manganese, 66 mg; zinc 18 mg; iodine,
1.5 mg.
27
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Another important consideration is the gross pathological examina-
tions that are required. It is suggested that the following requirements
be considered for adoption:
Any animal which dies in the course of the study must be re-
frigerated at temperatures low enough to minimize autolysis if
necropsy cannot be performed immediately. Necropsy must be per-
formed on all animals who die or who are sacrificed within 24
hours and preferably within 16 hours of death. The gross dissec-
tion and evaluation shall be performed by or under the
supervision of the pathologist.
The necropsy is defined as an external examination, including
body orifices, and examination and fixation of the following
tissues:
Gross lesions Lungs & bronchi
Tissue masses or suspect tumors Heart
& regional lymph nodes Thyroids
Skin Parathyroids
Mandibular lymph node Esophagus
Mammary gland Stomach
Salivary gland Duodenum
Larynx Jejunum
Trachea Ileum
Cecum Spleen
Colon Kidneys
Rectum Adrenals
Mesenteric lymph node Bladder
Liver Seminal vesicles
Thigh muscle Prostate
Sciatic nerve Testes
Sternebrae, vertebrae, Ovaries
or femur (plus marrow) Uterus
Costochondral junction, Nasal cavity
rib Brain
Thymus Pituitary
Gallbladder Eyes
Pancreas Spinal cord
Specific requirements for gross necropsy procedures, and patho-
logy reports are described in NCI publication CG-TR-1 which might
be distributed to the test laboratories. Histopathological
examinations are considered to be too costly for this stage of
testing and are not expected to reveal significantly more infor-
mation than gross pathology reports.
Estimated costs for each test will vary with time and the location of
the laboratory. An example of the costs of various acute toxicity tests
is shown in Table 7.
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TABLE 7, ACUTE STUDY PRICE LIST
(July, 1979)
EYE IRRITATION $390.00
6 rabbits
72 hour evaluation
EYE IRRITATION $510.00
9 rabbits
6 unwashed
3 washed
1 week evaluation
PRIMARY SKIN IRRITATION $360.00
6 rabbits
14-day evaluation
ACUTE ORAL TOXICITY $900.00
Five levels of ten rats/level
72 hour evaluation
ACUTE ORAL TOXICITY $325.00
Six levels of two rats/level
72 hour evaluation
ACUTE DERMAL TOXICITY $850.00
Two levels of ten rabbits/level
14 day evaluation
ACUTE DERMAL TOXICITY $1050.00
Four levels of four rabbits/level
14 day evaluation
ACUTE DERMAL TOXICITY $3400.00
Five levels of eight rabbits/level
14 day evaluation
ACUTE ORAL TOXICITY $900.00
Five levels of ten mice/level
14 day evaluations
29
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Table 7 continued
SENSITIZATION $1200.00
One level of ten guinea pigs/level
Five week evaluation
SENSITIZATION $2300.00
Two levels of ten guinea pigs/level
Five week evaluation
30
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HOW SHOULD THE RESULTS BE INTERPRETED AND USED TO ESTIMATE A TOLERABLE
LEVEL?
In order to extrapolate the results of acute tests performed on
laboratory animals to "safe" levels of chronic human exposure, certain
obvious considerations are necessary. One such consideration is the
relationship of animal toxic responses to human toxic responses and the
other is the relationship of short-term high-dose toxicity to long-term
low-dose toxicity.
A convenient approach to the question of the animal-human sensitivity
differential is to use the hundredfold safety margin. This is based on a
convention which assumes that humans may be up to tenfold more sensitive
than the experimental animals used and that additonally there may be a
tenfold variation in sensitivity among individuals.
The second consideration, extrapolation of data from short-term high-
dose studies to predict the outcome of long-term low levels of exposure,
is a more complex problem. Several noted toxicologists have stated that
any acute toxic sign that will be produced at a given dose will occur
within 90 daysof the initial administration of the test substance.
(9-17) Also, the results from two major studies have indicated that
lifetime no-effect doses can be predicted from short-term studies.
The Weil and McCollister study (18) determined the 3 month and 2 year
no-effect doses of 33 compounds of diverse chemical structure, pharamco-
logic type, and toxicity in rats and dogs. These compounds included
pesticides, veterinary chemicals, food additives, thickeners, stabili-
zers, antimycotics, and water treatment substances.
The animals were observed for 36 criteria of toxicity including mor-
tality, food intake, weight, pathology, blood chemistry, hematology,
cholinesterase, fertility, and neoplasia. The most sensitive criteria
were body weight, ratios of liver and kidney weight to body weight, and
kidney pathology. It was the feeling of these authors that only these
sensitive parameters need to be followed.
There were various numerical relationships by which the long-term
no-effect dose for each compound could be derived from its short-term
no-effect doses. But in 97% of the compounds tested (or 32 out of 33) it
was seen that 1/10 of the 3 month no-effect dose could be given repeat-
edly throughout a lifetime without producing toxic effects; 100% of these
compounds were non-toxic given at 1/12 of the 3 month no-effect dose.
This approach is further supported by a recent literature survey by
McNamara (19) in which data was collected on the short-term and long-term
no-effect doses of 122 compounds of diverse chemical and pharmacological
31
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types. These studies were conducted in various laboratories using dif-
ferent methods and animal species. The resulting frequency distributions
of the 1050 levels and short term and long term no-effect doses demon-
strated three basic relationships:
any compound administered to an animal at one hundredth of its
LD^Q will produce no effects in a short-term (3 month) study
any compound administered daily at one tenth of its 3 month
no-effect dose will produce no effects in the lifetime of that
animal
any compound administered daily at one thousandth of its
LDtjQ will produce no effects in the lifetime of the test
animal.
Using the third observation, we can use the LDgg derived from short
term acute testing to predict the lifetime NOEL (no observable effects
level) within the same animal species.
The lifetime NOEL of the test animal may be further modified, as
described above, to accomodate the human-animal sensitivity differential.
The lifetime NOEL for the test animal divided by 100 becomes the proposed
TLE (tolerable level of exposure) for humans.
The working hypothesis therefore becomes:
The LDcQ derived from short term animal studies divided by
100,000 is the TLE of chronic human exposure.
This safety factor of 100,000 is very conservative and its use is
most justified when the results of acute testing produce a flat dose-
response curve or a delay in the onset of and recovery from toxic signs.
These data may be suggestive of a potential for cumulative toxicity and
thus the need for longer term testing (1). Physical/chemical properties
of the toxicant may also indicate the need to apply a greater safety
factor when determining a NOEL in humans.
In other situations, where a less conservative approach seems to be
justified, one might reduce the human-animal sensitivity differential to
10 and use the LD from animal studies divided by 10,000 to predict
the tolerable level of chronic human exposure.
An example of how one might use each of these factors is demonstrated in
Table 8 using some oral LD50S from the literature(21):
32
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TABLE 8. PREDICTIONS OF HUMAN TLEs FROM
ORAL LD50s DERVIED FROM ANIMAL STUDIES
Compound
Species
Conservative
Predicted Predicted
OralLD50 Human Human TLE&
(mg/kg) TLEa( q/kg day) ( g/kg/day)
Methyl Sal icy late
Thiabendazole
Propylene glycol
Pimaricin
dog
rat
rat
rat
2100
3100
27000
3700
210
310
2700
370
21
31
270
370
a. figures given are oral LD50s divided by 10,000.
b. figures given are oral LD50s divided by 100,000.
33
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HOW SHOULD THE DATA BE SUBMITTED FOR EASY INTERPRETATION BY REVIEWERS?
All dose and response data should be presented in tabular form.
Doses should be calculated in mg/kg body weight. Responses should be
listed as number of animals killed per number of animals exposed in each
dosage group. Other toxic effects should also be noted and recorded for
each dosage level group.
It is suggested that manufacturers perform their own calculations of
dose-response relationships, slopes, standard deviations, and LD50s,
the use of a second party to observe and monitor these calculations is
also recommended. Simple computer programs can be used to facilitate
operations and minimize errors.
Experimental protocols should also be fully described by each labora-
tory in order to check for any procedural errors which could influence
the accuracy of the data.
34
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References
1. Loomis, T.A. 1976. Essentials of Toxicology, Vol. 2, pp 180-184,
Philadelphia: Lea and Febiger.
2. Registry of Toxic Effects of Chemical Substances, NIOSH, July 1979.
3. Draize, J. H., G. Woodard, and H.O. Calvery. 1944. Methods of the
study of irritation and toxicity of substances applied topically to
the skin and mucous membranes. J. Pharmacol. Exp. Ther. 82:377-390.
4. Nelson, N., chairman. 1975. Acute and subchronic toxicity.
Principles for Evaluating Chemicals in the Environment, pp 97-114.
Washington:National Academy of Sciences Printing and Publishing
Office.
5. Loomis, T.A., 1976. pp 207-215.
6. Campbell, K. I., E. L. George, L. L. Hall and J. S. Stara. 1975.
Dermal irritancy of metal compounds. Arch. Environ. Health
30:168-170.
7. Klecak, G., 1977. Identification of contact allergens, in
Dermatology and Toxicology , Vol. 2, eds. Marzulli, F. N. and H. I.
Maibach, pp 321-328, New York: Halsted Press.
8. Loomis, T.A., 1976. pp 163-165.
9. Weil, C. S., M. D. Woodside, J. R. Bernard and C. P. Carpenter.
1969. Relationship between single-peroral one-week and ninety-day
rat feeding studies. Toxicol. Appl. fharmacol. 14:426-31.
10. Paget, G.E. 1963. Standards for the laboratory evaluation of the
toxicity of a drug - Viewpoint of the Expert Committee on Drug
Toxicity of the Association of British Pharmaceutical Industry.
Proc. Eur. Soc. Study Drug Toxic. 11: 7-12.
11. Peck, H. M. 1968. An appraisal of drug safety evaluation in animals
and the extrapolation of results to man. In Importance of
Fundamental Principles in Drug Evaluation, eds. D.H. Tedeschi and
R.E. Tedeschi, pp. 449-471. New York: Raven Press.
12. Barnes, J. M. and F. A. Denz. 1954. Experimental methods used in
determining chronic toxicity. Pharmacol Rev. 6:191-242.
13. McCollister, D. C. chairman. October 1974. Society of Toxicology Ad
Hoc Committee Comments on Health Research Group Petitions. Soc.
Toxicol. Newslett., p 2.
35
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14. Bein, H. J. 1963. Time to appearance of toxic signs in Ciba studies
of 46 compounds at Basel, Switzerland and Summit, New Jersey. Proc.
Eur. Soc. Study Drug Tox. 11:15-24.
15. Hayes, W., Jr. 1972. Essays on Toxicology, vol. 3, pp. 65-67. New
York, Academic Press.
16. Frawley, J. P. 1967- Scientific evidence and common sense as a
basis for food-packaging regulations. Food Cosmet. Toxicol.
5:293-309.
17. Kurlyandskiy, B. A. 1975. Premorbid conditions of chemical
etiology. U.S. Joint Publications Research Service (JPRS 64252).
(Translation of an article that appeared in Moscow Farmakologiya
Khimioterapevticheskiye Sredstva Toksikologiya Problemy Toksikologii,
1972, vol. 4, pp. 8-42.)
18. Weil, C. S., and D. D. McCollister. 1963. Safety evaluation of
chemicals. Relationship between short-and long-term feeding studies
in designing an oral toxicity test. J. Agri. Food Chem. 11:486-91.
19. McNamara, B. P. 1976. Concepts in Health Evaluation of Commercial
and Industrial Chemicals. In Advances in Modern Toxicology vol 1, pt
1: New Concepts in Safety Evaluation, ed. M. A. Mehlman, pp. 61-140.
New York, Halsted Press.
36
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50272-101
REPORT DOCUMENTATION
PAGE
1. REPORT NO.
EPA 560/13-79-009
3. Recipient's Accession No.
4. Title and Subtitle
Acute Toxicity Testing Criteria for New Chemical Substances
5. Report Date
July. 1979
6.
7. Author(s)
M.J. Normandy, R.6. Reynolds
8. Performing Organization Rept. No.
9. Performing Organization Name and Address
Enviro Control, Incorporated
11300 Rockville Pike
Rockville, Maryland '20852
10. Project/Task/Work Unit No.
EPA 560/13-79-009
11. Contract(C) or Grant(G) No.
(o W2227-NA5X
(G)
12. Sponsoring Organization Name and Address
Premanufacturing Review Division
Office of Toxic Substances
Environmental Protection Agency
Washington, D.C. **"AX
13. Type of Report & Period Covered
Technical Report
April-July, 1979
14.
15. Supplementary Notes
Project Officer - Dr. Daphne Kamely
Technical Assistant - Felix Santos
16. Abstract (Limit: 200 words)
This report addresses the rationale, considerations, and limitations of acute
toxicity testing. General procedures are described for acute tests including letha-
lity studies in oral, dermal, and inhalation toxicity, and irritation studies in dermal
and eye toxicity, phototoxicity and skin sensitization. Recommendations are given for
the acute toxicity tests which may be used to evaluate the risks associated with the
manufacture and processing of chemical substances. Suggested minimum protocols are
offered including the choice of test animals, dosage levels, laboratory practices,
animal diets, and necropsy requirements. A method is suggested for interpreting the
results of these tests and extrapolating to some guidelines for a safety factor in
human exposure.
17. Document Analysis a. Descriptors
Toxicity, Chemical Compounds, Test Methods, Laboratory Animals, Evaluation Criteria
b. Identifiers/Open-Ended Terms
c. COSATI Field/Group 06T,14B
18. Availability Statement
Release unlimited
19. Security Class (This Report)
Unclassified
20. Security Class (This Page)
Unclassified
21. No. of Pages
36
22. Price
(See ANSI-Z39.18)
See Instructions on Reverse
OPTIONAL FORM 272 (4-77)
(Formerly NTIS-35)
Department of Commerce
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