ASTM E1073-91(1996)

NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: E 1073 – 91 (Reapproved 1996)
Standard Test Method for
Obtaining a Pharmacological Profile with Mice
This standard is issued under the fixed designation E 1073; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3.2 This test method is applicable to the study of most drugs
and chemicals, and will properly estimate both lethal and
1.1 This test method is designed as a simple and inexpen-
minimally effective dose levels. Although it is designed for the
sive initial screening procedure for new compounds with
study of single components, it can be used to study the
unknown pharmacological properties, or for the comparative
comparative toxicity of mixtures or formulations. The method
bioassay of new members of a chemical series with class
may not be applicable to oily substances which cause embo-
reference standards. The test method, which is applicable to
lism upon injection.
most pharmacologically active compounds including pesti-
3.3 This test method requires only small quantities of test
cides, will properly rank order both acute lethality and potency
materials (approximately 1 g), a fact that enhances its utility as
with a minimum expenditure of test material. It is intended as
the initial biological study for newly synthesized substances.
the first step in a multi-tiered development program.
3.4 It is equally economical in its requirements for equip-
1.2 This standard does not purport to address all of the
ment, space, personnel, and animals. Only a small laboratory,
safety concerns, if any, associated with its use. It is the
simple test equipment, and two technicians are needed to
responsibility of the user of this standard to establish appro-
conduct the experiments. Furthermore, an average of only
priate safety and health practices and determine the applica-
thirty mice are required to conduct the entire test method.
bility of regulatory limitations prior to use.
3.5 The procedure is applicable to a wide variety of mate-
2. Summary of Test Method rials. When results of this test were compared with those from
more detailed and specific animal tests, a high degree of
2.1 Mice are injected intravenously with a dose of the test
correlation was obtained. Further evidence of the utility of the
material and then observed for reaction signs, first those that
test was demonstrated by the fact that a high correlation of rank
can be detected by nonmanipulative tests, and then those
order potencies was found for a series of anticholinergics
sensed during a series of manipulative tests.
studied in both mice and men.
2.2 Two technicians are required to perform the experiment.
One injects the mice and records the data, while the other
4. Apparatus
conducts the experiment at regularly scheduled time intervals
4.1 All of the apparatus used for this test method is simple
and dictates the findings.
and inexpensive. It can be made in any well-equipped labora-
2.3 The results obtained using this procedure indicate the
tory workshop or, in some cases, obtained commercially.
approximate median lethal dose (LD50), the approximate
4.2 The major components of the apparatus are illustrated in
median level of nonlethal reactions (MED50), the reaction
Figs. 1-5.
signs elicited at each dose level tested, and the degree of
4.3 Syringes and Needles:
severity of those signs. In contrast to the more commonly used
4.3.1 One-quarter millilitre glass Tuberculin syringes, fitted
median effective dose (ED50), the MED50 value may have
with 0.75 in. (19 mm), 27-gage needles are recommended for
been generated by one or more responses at the same dose, and
injection of all solutions except undiluted polyethylene glycol
thus does not imply the 50 % level for a specific reaction sign.
(PEG) solutions. In the latter case, 24-gage needles must be
2.4 By judicious selection of acceptance criteria, the elimi-
used to compensate for the viscosity of the solvent, and 50-μL
nation rate for compounds tested using this procedure can be
syringes are required to measure accurately the small volumes
tailored to any desired level.
that are administered.
3. Significance and Use
4.3.2 Although disposable syringes could be used for most
of this work, calibrations on the glass syringes are more
3.1 This test method is designed as an initial screening
accurate. Furthermore, the possibility of a reaction between the
procedure for the selection of compounds worthy of more
plastic or rubber parts of disposable syringes with the test
detailed study.
solutions, especially those containing PEG, exists.
This test method is under the jurisdiction of ASTM Committee E-35 on
Pesticides and is the direct responsibility of Subcommittee E35.26 on Safety to Man. Hamilton Microliter syringes with stainless steel plunger using detachable
Current edition approved March 15, 1991. Published May 1991. Originally needles available from Hamilton Co., Reno, NV have been found suitable for this
published as E 1073 – 85. Last previous edition E 1073 – 86. purpose.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
E 1073
6.2 If mixtures or formulations are to be studied, up to 10-g
quantities may be needed depending on the expected dilutions
to be tested.
6.3 Test solutions are usually made in volumes of 10 mL.
Liquid test materials are prepared as volume/volume (v/v)
solutions, and solids are prepared as weight/volume (w/v)
solutions.
6.4 Stock concentrations of 10 mg/mL (10 mm /mL for
liquids) shall be prepared initially. Serial dilutions are prepared
from this stock solution as needed, keeping the number of
dilutions to the minimum number required to allow total
injection volumes of 0.05 to 0.25 mL for aqueous solutions or
suspensions. When undiluted PEG200 is required as the
solvent, injected volumes must be restricted to 0.01 to 0.05 mL
per mouse. Acetone, NaOH, and ethyl alcohol solutions can
only be injected at volumes up to 0.125 mL.
7. Test Animals
7.1 Male ICR Swiss mice, weighing 18 to 25 g, are used for
this test method. Although either sex may be used if desired,
experience has shown that results are more reproducible if only
males are employed.
7.2 The number of mice required for screening each com-
pound will depend on the potency of the material being tested
but, overall, approximately 30 mice are required.
7.3 Quarantine all animals for a period of at least one week
before use to ensure their good health and to acclimate them to
their quarters and diet. Allow animals continuous access to
feed and water.
1. Hinge with easily removed pin.
2. Permanent hinge.
7.4 In experiments where female mice are used, pregnant or
3. 24 by 24-in. plywood base with Lucite covering.
lactating animals shall be excluded.
4. 2 by 6 by 24 in. long.
5. 6 in. high, ⁄4 in. thick Lucite divider, interlocked in egg-crate manner.
8. Procedure
FIG. 1 Observation Platform
8.1 Conduct the experiment in two phases, one in which the
5. Solvents animals are carefully observed without being disturbed (see
9.4), and a second that requires handling (see 9.5).
5.1 Any of the following solvents can be used up to the
8.2 Because a complete evaluation of the treated mice may
volumes shown for this test method if necessary. Usually, one
take as long as 5 min, depending on the signs elicited, the
of the first four is used for testing solids. Methylcellulose
injection schedule is left flexible until a response pattern is
solutions are used to prepare suspensions, not true solutions.
established.
The test material is finely ground using an agate mortar and
8.3 Observe the injected mice at 3, 15, 30, and 60 min after
pestle before suspending it.
intravenous injection in a lateral tail vein and hourly thereafter
Maximum Volume to
Solvent Use throughout the workday.
Distilled water 10 mL/kg
8.4 Conduct a range-finding study first. For aqueous solu-
25 % aqueous solution PEG200 10 mL/kg
tions, inject the first animal with the stock concentration at 10
Undiluted PEG200 2 mL/kg
0.5 % aqueous methylcellulose 10 mL/kg mL/kg of body weight, with a resultant dose of 100 mg/kg.
0.1 N Hydrochloric acid 10 mL/kg
Frequently this dose will prove to be lethal, with death
0.1 N Sodium hydroxide 5 mL/kg
occurring in 1 to 2 min. Should that be the case, dose a second
10 % aqueous acetone 5 mL/kg
10 % aqueous ethyl alcohol 5 mL/kg mouse at 3.16 mL/kg body weight (31.6 mg/kg).
0.5 % aqueous acetic acid 10 mL/kg
8.5 Make a 1 + 9 dilution of the stock solution for the next
1 % aqueous lactic acid 10 mL/kg
two doses, which are spaced at 0.5-log intervals below the
6. Test Solutions preceding one. Repeat this procedure until a mouse survives
for 5 min.
6.1 One-gram quantities of test materials are sufficient for
8.6 At this point, test a second mouse at the apparent
both solubility testing and injection.
nonlethal dose. If both mice survive, test doses decreasing by
0.5-log intervals using 2 mice per dose until the no-effect level
Methocelt-4000 cps, Type F4M available from Dow Chemical Co., Midland,
is reached. Test each animal’s responses at the predetermined
MI has been found suitable for this purpose.
4 time intervals, so that the effect of intermediate doses is
PEG200 or Carbowax 200 available from Union Carbide Corp., Ethylene
Oxide/Glycol Div., Danbury, CT has been found suitable for this purpose. obtained in detail.
E 1073
1. Bodine speed reducer motor—Type NSE-11 RG, 21 r/min reducer output rating. Voltage to the motor is regulated using a variable voltage transformer (“Powerstat”).
2. Lucite divider.
3. 1-in. diameter wood dowel.
4. Laboratory time (“Gra-Lab”).
FIG. 2 Rota-rod Apparatus
1. Support rod.
2. Protractor.
3. End panels and base are 10 in. wide.
4. 12 by 9 ⁄2 by 5-in. elliptical sheet metal enclosure for air blast box. The enclosure is centered across the base of the narrow strip apparatus; the air blast box is centered
inside the elliptical enclosure.
FIG. 3 Narrow Strip Apparatus, Showing the Location of the Air-Blast Enclosure
8.7 These data are sufficient to allow a decision to be made Inject 2 or 3 additional mice at the apparent LD50 level, and 4
as to the desirability of further testing. at each of the three next three lower levels for the observations
8.8 If further testing is indicated, obtain a more accurate in Section 9. Use a similar design for the MED50, except that
estimate of the LD50 and MED50. Use the lethal and no-effect the successive 0.1 log doses are above the apparent no effect
levels observed in the range-finding study as the points for level.
additional dosage selection. 8.10 Include solvent injected control mice in these studies
8.9 Test a total of 4 mice at each of four doses spaced as needed. At least 2 control mice are required for comparison
0.1-log apart for both the LD50 and MED50 determinations. with test material treated mice. One should be injected first and
E 1073
1. Compressed air supply.
9 3
2. Rubber pressure tubing, ⁄16-in. outside diameter, ⁄16-in. inside diameter.
3. Brass T tube, ⁄32-in. inside diameter. T arms.
4. Ashcroft pressure gage (0 to 60 psi) Manning, Maxwell and Moore, Inc. Stratford, CT.
5. Brass hosecock with ⁄8-in. diameter bore.
6. Rotating shaft ( ⁄8-in. diameter hole) resting in steel bearing.
7. Steel bearing.
1 3
8. Side-armed steel tube, 2 ⁄2 in. long, 2-in. side arms, and ⁄16-in. diameter bore.
9. Thirteen-inch long pieces of rubber tubing, 1-cm outside diameter, 0.5-cm inside diameter.
3 1
10. Lucite nipple cemented at right angles at the bottom of each corner. Each nipple is 1 in. long, ⁄8-in. outside diameter, ⁄8-in. inside diameter Lucite tubing.
1 1 1 1
11. Air blast box with internal dimensions 4 ⁄2 in. (L) by 2 ⁄2 in. (W) by 1 ⁄2in. (H), made of clear ⁄4-in. Lucite. The hole through the wall of the box at the bottom of each
corner is ⁄8 in. diameter and is aimed at the top of the diagonally opposite corner.
FIG. 4 Air Blast Apparatus
the second 20 min later, since the animals tend to lose interest 9.2 At the highest doses, effects of the test material are
in their surroundings after that time and go to sleep. Additional usually immediately apparent, while delays of a minute or two
controls should be added at 20-min increments.
before the onset of effects is the norm at lower levels.
9.3 Observe the reactions that can be assessed without
9. Observations
touching the animal (nonmanipulative effects) first followed by
9.1 Place a fresh sheet of paper on the floor of the
those that require handling of the mice (manipulative effects).
observation platform (Fig. 1) before the experiment is begun.
9.4 Nonmanipulative Effects:
As each mouse is injected, place the animal in a numbered
compartment of the platform. 9.4.1 Make observations in a standard sequence as follows:
E 1073
1. ⁄4-in. mesh.
2. 3.5-mm cadmium plated frame.
3. 1-in. mesh.
4. Wooden block.
5. ⁄4-in. mesh.
6. Nail or staple.
7. Wooden block.
8. 2 by 4-in. open section (wire mesh removed) where mice are tested.
FIG. 5 Modified Test-Tube Rack (Horizontal Wire Apparatus)
tremors, convulsive reactions, prostration, and death; changes of the pencil. A repeatable vigorous head twitch is indicative of
in locomotor activity and rearing; changes in respiration; compound effect.
changes in posture and gait; changes in somatic responses; all 9.5.3 Observe the response upon being scooped up in the
other generalized changes, for example, bizarre reactions, palm of the hand or picked up by the tail. At this time, evaluate
cyanosis, piloerection; then localized changes, for example, the muscle tone as the animal moves about in the experiment-
phonation, exophthalmos, straub tail. er’s palm.
9.4.2 Periodically during the testing period, note the volume
9.5.4 Observe eye effects by placing the mouse on a wire
and appearance of both feces and urine excreted by each mesh and lightly restraining it by holding its tail. Illuminate the
mouse. Alterations in the normal excretory pattern may indi-
surface of the eye from above, allowing pupillary or other eye
cate important pharmacological effects. effects to be noted.
9.5 Manipulative Effects: 9.5.5 Following the eye examination, touch the cornea with
the tip of a horsehair bristle. Then touch the walls of the ear
9.5.1 Do not conduct the battery of manipulative tests when
either convulsions or a subconvulsive state (resulting in con- canal (not the eardrum)
...