ASTM E1073-01(2005)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: E1073 – 01 (Reapproved 2005)
Standard Test Method for
Obtaining a Pharmacological Profile with Mice
This standard is issued under the fixed designation E1073; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Significance and Use
1.1 This test method is designed as a simple and inexpen- 3.1 This test method is designed as an initial screening
sive initial screening procedure for new compounds with procedure for the selection of compounds worthy of more
unknown pharmacological properties, or for the comparative detailed study.
bioassay of new members of a chemical series with class 3.2 This test method is applicable to the study of most drugs
reference standards. The test method, which is applicable to and chemicals, and will properly estimate both lethal and
most pharmacologically active compounds including pesti- minimally effective dose levels.Although it is designed for the
cides, will properly rank order both acute lethality and potency study of single components, it can be used to study the
with a minimum expenditure of test material. It is intended as comparative toxicity of mixtures or formulations. The method
the first step in a multi-tiered development program. may not be applicable to oily substances which cause embo-
1.2 This standard does not purport to address all of the lism upon injection.
safety concerns, if any, associated with its use. It is the 3.3 This test method requires only small quantities of test
responsibility of the user of this standard to establish appro- materials (approximately 1 g), a fact that enhances its utility as
priate safety and health practices and determine the applica- the initial biological study for newly synthesized substances.
bility of regulatory limitations prior to use. 3.4 It is equally economical in its requirements for equip-
ment, space, personnel, and animals. Only a small laboratory,
2. Summary of Test Method
simple test equipment, and two technicians are needed to
2.1 Mice are injected intravenously with a dose of the test conduct the experiments. Furthermore, an average of only
material and then observed for reaction signs, first those that
thirty mice are required to conduct the entire test method.
can be detected by nonmanipulative tests, and then those 3.5 The procedure is applicable to a wide variety of mate-
sensed during a series of manipulative tests.
rials. When results of this test were compared with those from
2.2 Two technicians are required to perform the experiment.
more detailed and specific animal tests, a high degree of
One injects the mice and records the data, while the other correlation was obtained. Further evidence of the utility of the
conducts the experiment at regularly scheduled time intervals
testwasdemonstratedbythefactthatahighcorrelationofrank
and dictates the findings. order potencies was found for a series of anticholinergics
2.3 The results obtained using this procedure indicate the
studied in both mice and men.
approximate median lethal dose (LD50), the approximate
4. Apparatus
median level of nonlethal reactions (MED50), the reaction
signs elicited at each dose level tested, and the degree of 4.1 All of the apparatus used for this test method is simple
severity of those signs. In contrast to the more commonly used and inexpensive. It can be made in any well-equipped labora-
median effective dose (ED50), the MED50 value may have tory workshop or, in some cases, obtained commercially.
been generated by one or more responses at the same dose, and 4.2 The major components of the apparatus are illustrated in
thus does not imply the 50 % level for a specific reaction sign. Figs. 1-5.
2.4 By judicious selection of acceptance criteria, the elimi- 4.3 Syringes and Needles:
nation rate for compounds tested using this procedure can be 4.3.1 One-quarter millilitre glass Tuberculin syringes, fitted
tailored to any desired level. with 0.75 in. (19 mm), 27-gage needles are recommended for
injection of all solutions except undiluted polyethylene glycol
(PEG) solutions. In the latter case, 24-gage needles must be
This test method is under the jurisdiction of ASTM Committee E35 on
PesticidesandisthedirectresponsibilityofSubcommitteeE35.26onSafetytoMan.
used to compensate for the viscosity of the solvent, and 50-µL
Current edition approved April 1, 2005. Published May 2005. Originally
syringes are required to measure accurately the small volumes
approved in 1985. Last previous edition approved in 2001 as E1073 – 01. DOI:
that are administered.
10.1520/E1073-01R05.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1073 – 01 (2005)
6. Test Solutions
6.1 One-gram quantities of test materials are sufficient for
both solubility testing and injection.
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,
experiencehasshownthatresultsaremorereproducibleifonly
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.
1. Hinge with easily removed pin.
7.3 Quarantine all animals for a period of at least one week
2. Permanent hinge.
before use to ensure their good health and to acclimate them to
3. 24 by 24-in. plywood base with Lucite covering.
4. 2 by 6 by 24 in. long. their quarters and diet. Allow animals continuous access to
5. 6 in. high, ⁄4 in. thick Lucite divider, interlocked in egg-crate manner.
feed and water.
FIG. 1 Observation Platform
7.4 In experiments where female mice are used, pregnant or
lactating animals shall be excluded.
4.3.2 Although disposable syringes could be used for most
8. Procedure
of this work, calibrations on the glass syringes are more
accurate. Furthermore, the possibility of a reaction between the
8.1 Conduct the experiment in two phases, one in which the
plastic or rubber parts of disposable syringes with the test
animals are carefully observed without being disturbed (see
solutions, especially those containing PEG, exists.
9.4), and a second that requires handling (see 9.5).
8.2 Because a complete evaluation of the treated mice may
5. Solvents
take as long as 5 min, depending on the signs elicited, the
5.1 Any of the following solvents can be used up to the
injection schedule is left flexible until a response pattern is
volumes shown for this test method if necessary. Usually, one
established.
of the first four is used for testing solids. Methylcellulose
8.3 Observe the injected mice at 3, 15, 30, and 60 min after
solutions are used to prepare suspensions, not true solutions.
intravenous injection in a lateral tail vein and hourly thereafter
The test material is finely ground using an agate mortar and
throughout the workday.
pestle before suspending it.
8.4 Conduct a range-finding study first. For aqueous solu-
Maximum Volume to
tions, inject the first animal with the stock concentration at 10
Solvent Use
mL/kg of body weight, with a resultant dose of 100 mg/kg.
Distilled water 10 mL/kg
Frequently this dose will prove to be lethal, with death
25 % aqueous solution PEG200 10 mL/kg
Undiluted PEG200 2 mL/kg
0.5 % aqueous methylcellulose 10 mL/kg
0.1 N Hydrochloric acid 10 mL/kg
The sole source of supply of the apparatus known to the committee at this time
0.1 N Sodium hydroxide 5 mL/kg
is PEG200 or Carbowax 200 available from Union Carbide Corp., Ethylene
10 % aqueous acetone 5 mL/kg
Oxide/Glycol Div., 39 Old Ridgeberry Rd., Danbury, CT 06817-0001. If you are
10 % aqueous ethyl alcohol 5 mL/kg
aware of alternative suppliers, please provide this information to ASTM Interna-
0.5 % aqueous acetic acid 10 mL/kg
tional Headquarters.Your comments will receive careful consideration at a meeting
1 % aqueous lactic acid 10 mL/kg
of the responsible technical committee , which you may attend.
E1073 – 01 (2005)
1. Bodinespeedreducermotor—TypeNSE-11RG,21r/minreduceroutputrating.Voltagetothemotorisregulatedusingavariablevoltagetransformer(“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. 12by9 ⁄2by5-in.ellipticalsheetmetalenclosureforairblastbox.Theenclosureiscenteredacrossthebaseofthenarrowstripapparatus;theairblastboxiscentered
inside the elliptical enclosure.
FIG. 3 Narrow Strip Apparatus, Showing the Location of the Air-Blast Enclosure
occurring in 1 to 2 min. Should that be the case, dose a second 0.5-log intervals using 2 mice per dose until the no-effect level
mouse at 3.16 mL/kg body weight (31.6 mg/kg). is reached. Test each animal’s responses at the predetermined
8.5 Make a 1 + 9 dilution of the stock solution for the next time intervals, so that the effect of intermediate doses is
two doses, which are spaced at 0.5-log intervals below the obtained in detail.
preceding one. Repeat this procedure until a mouse survives 8.7 These data are sufficient to allow a decision to be made
for 5 min. as to the desirability of further testing.
8.6 At this point, test a second mouse at the apparent 8.8 If further testing is indicated, obtain a more accurate
nonlethal dose. If both mice survive, test doses decreasing by estimate of the LD50 and MED50. Use the lethal and no-effect
E1073 – 01 (2005)
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 ⁄2 in. (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
levels observed in the range-finding study as the points for in Section 9. Use a similar design for the MED50, except that
additional dosage selection. the successive 0.1 log doses are above the apparent no effect
8.9 Test a total of 4 mice at each of four doses spaced level.
0.1-log apart for both the LD50 and MED50 determinations. 8.10 Include solvent injected control mice in these studies
Inject 2 or 3 additional mice at the apparent LD50 level, and 4 as needed.At least 2 control mice are required for comparison
at each of the three next three lower levels for the observations with test material treated mice. One should be injected first and
E1073 – 01 (2005)
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)
the second 20 min later, since the animals tend to lose interest 9.4.2 Periodically during the testing period, note the volume
in their surroundings after that time and go to sleep.Additional and appearance of both feces and urine excreted by each
controls should be added at 20-min increments.
mouse. Alterations in the normal excretory pattern may indi-
cate important pharmacological effects.
9. Observations
9.5 Manipulative Effects:
9.1 Place a fresh sheet of paper on the floor of the
9.5.1 Do not conduct the battery of manipulative tests when
observation platform (Fig. 1) before the experiment is begun.
either convulsions or a subconvulsive state (resulting in con-
As each mouse is injected, place the animal in a numbered
vulsions when the animals are handled) exists. The additional
compartment of the platform.
stress produced by handling may be sufficiently severe to
9.2 At the highest doses, effects of the test material are
impede recovery, or even cause death in animals that would
usually immediately apparent, while delays of a minute or two
otherwise survive. Record the fact that manipulative tests were
before the onset of effects is the norm at lower levels.
not conducted.
9.3 Observe the reactions that can be assessed without
9.5.2 Observe the reaction of the mouse to repetitive light
touching the animal (nonmanipulative effects) first followed by
contact with the trunk using the tapered surface of a sharpened
those that require handling of the mice (manipulative effects).
wooden pencil. An exaggerated response indicates sensitivity
9.4 Nonmanipulative Effects:
to touch. Next touch lightly the back of the neck with the point
9.4.1 Make observations in a standard sequence as follows:
of the pencil.Arepeatable vigorous head twitch is indicative of
tremors, convulsive reactions, prostration, and death; changes
compound effect.
in locomotor activity and rearing; changes in respiration;
9.5.3 Observe the response upon being scooped up in the
changes in posture and gait; changes in somatic responses; all
other generalized changes, for example, bizarre reactions, palm of the hand or picked up by the tail.At th
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