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A new method to determine the irritation of drugs after intramuscular injection in rabbits
TOXICOLOGY
AND
A New
APPLIED
PHARMACOLOGY
Method
to Determine
Intramuscular S. SHINTANI, M. YAMAZAKI, Takeda
11,
Chemical
293-301 ( 1967)
the
Irritation
Injection
of
Drugs
after
in Rabbits
M. NAKAMURA, AND I. NAKAYAMA Industries,
Ltd.,
Osaka, Japan
Received h-lay 12, 1967
Muscular damage caused by the intramuscular administration of drugs is an important criterion for the evaluation of irritation. Several procedures have been devised to study this property. Hanson ( 1961) observed local tissue changes resulting from the intramuscular injection of several broad spectrum antibiotics into the gluteal muscles of the rabbit. Hagan (1959) suggested that the irritating property of drugs might be studied by intramuscular injection using the M. sacrospinalis of the rabbit as the site for intramuscular injection. Benitz and Dambach (1966) have also used this muscle for their studies. In 1953, after having tried various injection sites in the rabbit with unsatisfactory results, we selected the M. vastus lateralis because of its topographical relationship to skin and other muscle entities. We were able to standardize the injection procedures and the dissection methods in such a manner that they could be used for routine work. Our experience over a period of thirteen years showed that this method was satisfactory with respect to the following characteristics in comparison with other methods: (1) the whole muscle is easily dissected from other tissues, (2) the site of injection is readily detected, and (3) injections can be repeated more precisely. It is the purpose of this report to describe our method for the irritation test, which has been performed with a number of drugs for more than ten years. METHODS Test solutions. The solutions for injections were prepared in normal saline except for the commercially available forms of antibiotics, which were diluted according to the manufacturers’ instructions. New experimental drugs submitted for testing were usually administered at the concentrations intended for clinical use. The relationship between irritation and concentration was determined using 2- to 5-fold dilutions of the original concentration and the same diluent. Animals. Male albino rabbits weighing 2.5-3.5 kg were used; they received a commercial solid diet and tap water ad libitum. The animal quarters were kept at a uniform temperature (25 + 1” ) and uniform humidity (4060% ). The experiments were carried out on two or more rabbits at each dose. The thigh muscle of one side was used as the injection site for the test solution, and the other side for the control solution such as normal saline or the diluent.
294
S.
SHINTANI,
M.
YAMAZAKI,
hi.
NAKAMURA,
AND
I.
NAKAYAMA
Znjection procedure. Intramuscular injection of the test solutions into the M. vastus lateralis of the rabbit was carried out as follows: the rabbit was fastened securely in a supine position without anesthesia. The hind legs were fixed together so that one was placed upon the other. This procedure made it easier to inject the test solutions into the center of the muscle. Fur was removed from the injection site with an electric clipper. A 22-gauge needle attached to a 2-ml syringe was inserted through the skin of the thigh at an angle of about 30 degrees to approximately the center of the muscle as indicated in Fig. 1-A. A volume of 1 ml of the test solution was then slowly injected.
FIG. 1. Illustration of the procedures for the injection of the test solution into the center of the M. vastus lateralis and the dissection of the muscle, (A ) Insertion of a Ugauge needle with its tip in the center of the muscle. ( B ) Removal of the M. tensor fasciae and a part of the M. rectus femoralis. (C) Separation of the M. vastus lateralis from the underlying muscles by blunt dissection. (D) Removal of the M. vastus lateralis.
During the injection the investigator should notice a slight resistance if the tip of the needle is correctly placed in the center of the muscle. The contralateral muscle was used for the injection of the control solution. If the intended clinical use of a drug formulation required repeated administration of the drug, the above-mentioned procedure was repeated daily for 3 or 5 days using the same injection site. Dissecting the injection site. After predetermined intervals when the local tissue irritation reaction was at its peak, the rabbit was sacrificed by intravenous injection of sodium pentobarbital. The animal was placed in a supine PO-
TEST
FOR
INTRAMUSCULAR
295
IRRITATION
sition and the skin over the injection site was removed. As shown in Fig. l-B, -C and -D, the M. tensor fasciae latae was then removed together with a part of the M. rectus femoris, thus exposing the M. vastus lateralis. This muscle originates with a short tendon on three parts of the collum femoris, trochanter major, and laterointermuscular septum and conjoins with the common tendon of the knee. Finally, the M. vastus lateralis was carefully cut off at both ends, that is, at the common tendon of the knee and the origin of the muscle. The dissected muscle was cut longitudinally along its midline until the scalpel fully exposed the injection site as indicated by grossly detectable injury (see Fig. 2-A). The first incision exposed the primary cut surface as shown in Figures 2-A and -B. A second incision was made along the largest diameter of the damaged area on both sides of the primary incision thus exposing the entire lesion ( see Fig. 2-B to -E ) . was grossly Scoring of the local tissue reaction. The local tissue irritation scored from 0 to 5 according to the following criteria (see Fig. 3): score O-no
1I I i I I I I
@ A
B
C
D
E
Fro. 2. Illustration of the incision process for gross examination on the cut surface of the M. vastus lateralis. The muscle is cut and opened in alphabetical order and the incisions follow the direction of the arrows and on the dotted lines. Upper: cross-section views. Lower: outside views. The regions showed by the oblique line represent the damaged area. (A) The dissected muscle is cut along its midline until the scalpel fully exposes the damaged area. (B ) The cut surface is exposed by bending back the muscle. ( C ) Secondary incisions are made at the damaged area on both sides of the initial incision. (D) Each incision is extended to both ends of the muscle. (E ) The cut surface of the damaged area is fully exposed by bending back the muscle. FIG. 3. Photograph of gross local reactions with six different scores. 0 = No discernible gross reaction; 1 = slight hyperemia and discoloration; 2 = moderate hyperemia and discoloration; 3 = distinct discoloration in comparison with the color of the surrounding area; 4 = brown degeneration with small necrosis; 5 z widespread necrosis with an appearance of “cooked meat” and occasionally an abscess involving the malor portion of the muscle.
Printed
in Japan
296
S. SHINTANI,
M.
YAMAZAKI,
M.
NAKAMURA,
AND
I.
NAKAYAMA
discernible gross reaction; score l-slight hyperemia and discoloration; score 2-moderate hyperemia and discoloration; score S-distinct discoloration in comparison with the color of the surrounding area; score 4-brown degeneration with small necrosis; score 5-widespread necrosis with an appearance of “cooked meat” and occasionally an abscess involving the major portion of the muscle. Evaluation of irritation. The final score for the local responses was indicated by the average irritation score from two or more rabbits at one dose level. The irritation of the test or control solutions was graded using the following cate“slight” for 0.5-1.4; “mild” for 1.5-2.4; “moderate” gories : “none” for 0.4 or less; for 2.5-3.4; “marked” for 354.4; and “severe” for 4.5 or more. The irritation score of a test solution in a rabbit that showed an irritation reaction corresponding to score 3 or more with the control solution was deleted from the calculation of the average. For a quantitative comparison of the irritation of compounds, the results were expressed in terms of the threshold irritation dose ( TID ), as defined by the dose in milligrams per injection site that produced no more than a moderate irritation. RESULTS
Maximum
Tissue
Response
with
Relation
to Time
Gross findings following a single injection of seven compounds were observed at different intervals and are summarized in Table 1. The time of maximum irritation varied from 5 to 120 hours after injection. The muscles that gave TABLE AVERAGE
IRRITATION INJECTION
SCORES OF \<~RIOUS
AND
MAXIMUM
Compounds Urethane Calcium chloride Polyoxethylene (20) sorbitan monolauratr” Polyoxyethylene (93) lauryl ethera Tetracyclinec Chloramphenicold Chromomycin Aae
Cont. Pi;;)
REACTION
COMPOUNDSINTO
Average
TIME
M.
THE
irritation Hours
1 VASTUS
score
after
injection
AFTER
A SINGLE
LATERALIS
Maximum reaction time (hours)
INTRAMUSCULAR
IN 3 RABBITS
Gross findings at maximum reaction time
5
24
48
7%
120
10.0 1.0
4.0
3.3
4.3
5
3.3
3.0
4.0 2 7
4.0
4.7
9.7
a4
Brown degeneration Discoloration
5.0
2.7
4.3
-
3.7
2.5
24
Brown
5.0
2.3
-
24
3.0
3.7 3.0
3.3 4.3 3.7
4.0
2.5
3.3
3.3
72 73
3.0
3.7
4.7
120
Brown degeneration Necrosis Brown degeneration Necrosis, swelling
“5. 0
1.7
4.3 3.7 4.7
0.005
0.7
2.3
u Tween W@, Atlas Chemical Industries, Inc. b Brij 358, Atlas Chemical Industries, Inc. c Achromycin@ for intramuscular use, Lederle (Japan), Ltd. d Kemicetine@ for intramuscular use, Fujisawa Pharmaceutical c Toyomycin@, Takeda Chemical Industries, Ltd.
Industries,
Ltd.
degeneration
TEST
FOR
INTRAMUSCULAR
IRRITATION
297
the maximum irritation score showed grossly either a marked necrosis or distinct discoloration with a surrounding hemorrhagic area as shown in Fig. 4. Injections of a 10% solution of urethane caused small necroses or brown degenerations surrounded by hemorrhages that corresponded to score 4 at 5 hours after injection. This response was about the same as that seen 48 hours after injection (see Fig. 4-A and -B). The local reactions for two nonionic surfactants, polyoxyethylene sorbitan monolaurate and polyoxyethylene lauryl ether, peaked 24 hours after injection. The maximum irritation scores of two antibiotics were
Brown a
The
white
deposit
of
the
degeneration
suspension.
FK. 4. Gross local reaction at the early and the peak stages following a single injection of various compounds indicated in Table 1. (A) 5 hours after 10% urethane solution (score 4). ( B ) 48 hours after the above solution (score 4). ( C ) 24 hours after 1% calcium chloride solution (score 3). ( D) 24 hours after 5% polyoxyethylene sorhitan monolaurate solution (score 4). ( E ) 24 h ours after 5% polyoxyethylene lauryl ether solution (score 4). (F) 5 hours after 0.005’% chromomycin A3 solution (score 1). (G) 120 hours after the above solution (score 5). ( H ) 72 h ours after 2.5% tetracycline solution for intramuscular (I ) 24 hours after 25% suspension of chloramphenicol for intramuscular (score (score Tj ). 3 ). ( 1) 24 hours after the above suspension ( score 4).
obtained at 72 hours. A peak of the local reaction for chromomycin A3 was observed on day 5 after injection. In the case of chloramphenicol, a white deposit of the suspension remained at the site of application for about 24-72 hours after injection and a hemorrhagic area was present in the surrounding tissue (see Fig. 4-I and -J). Effect of Repeated
Zntramuscular
Znjections
The effects of single daily injections oxyethylene nonionic surfactants were
for 5 days with several types of polycompared with the effects of a single
298
S.
SHINTANI,
M.
YAMAZAKI,
ht.
NAKAMURA,
AND
I.
NAKAYAhrA
injection. The local reaction was determined 24 hours after a single intramuscular injection and 24 hours after the last of 5 daily injections. A 5% solution was used to achieve the maximum irritation score. As shown in Table 2, the intensity of the irritation after repeated injections was greater than after one injection with the exception of hydrogenated castor oil ester, which showed no irritation. Efect
of pH
The effects of normal saline, at various pH values ranging from 2.1 to 12, upon the local tissue response after intramuscular injection were examined. As shown in Table 3, there was “moderate” irritation at the extreme pH’s of 2.1 TABLE COYPARISOX
JWTIOSS
2
OF IXTRAMIJSCTI,\H IRRIT~TIOY AFTER SINGLE ANJ REPIL~TED INTKAMUSCTLAR INTO THE M.V.AST~~Y LATERALIS OF THE RABBIT USING V,ARIOUS N~NI~~I~,~URF.~CTA~.TS OF POLYOXYETIIYLENE DERIVATIVES
Chemical composition of esters condensed with polyoxyethylenea Sorbitan laurateb Cetyl ether? Stearated Hydrogenated castor Sorbitan laurate” Cetyl etherc Stearated Hydrogenated castor
oiP
oile
Sumber of OXYethylene units
24 hours after single injection
24 hours after daily injections
_____ Average score from
5
Average score from 3 rabhits
Average irritation score
20 20
9 .7
Moderate Severe
4 3 -
Marked
“0 20 fro .50 -50 50
3.7 0. 0 0. 0 3 3 4.7 0. 0
Marked None None
1.7 0.3 1 3 5. 0 3 3 0. 3
Severe NOM Slight Severe Moderate None
* Injected as 5”/G solutions. b TL 20 and 508, Atlas Chemical Industries, c BC 20 and 50@, Atlas Chemical Industries, ’ AMYS 20 and 50@, Atlas Chemical Industries, e HCO 20 and 508, Atlas Chemical Industries,
5 0
Moderate
Moderate None
3 rahhith
Is-
Average irritation SCOW
Inc. Inc.
Inc. Inc.
and 12. The local irritating effect of the solutions ranging from pH 2.5 to 11 was not more than “mild” after a single injection. The peak response was found 24 hours after the injection and was no longer visible on day 3 after the injection in the case of saline at pH 2.1 while the peak response of saline at pH 12 still remained on day 3 and disappeared on day 7 after the injection. Relationship between Dosage and Irritation The standard procedure using the M. vastus lateralis has been limited to 1 ml of test solution because of the limited volume of the muscle; however, to determine the effects of volume of injection and drug concentration upon irritation, we used volumes ranging from 0.125 to 4 ml with various concentrations of solute as shown in Table 4. The average irritation scores were proportional to the drug concentration in milligrams per individual injection site and gen-
TEST
FOR
INTRAhIUSCULAR
IRRITATION
299
erally did not vary in proportion to the volume of injection except when 2 and 4 ml were used. The threshold irritation doses (TID’s) for tetracycline were constant at 12.5 mg per injection site with all the volumes ranging from 0.125 to 2.0 ml and at 25 mg per injection site in the large volume of 4.0 ml. The TID’s for chromomycin A, varied from 0.002 to 0.01 mg per injection site and did not give constant results with variations in the volume of injection. The TID’s for polyoxyethylene sorbitan monolaurate were constant at 12.5 mg per injection site through all the volumes from 0.125 to 1.0 ml and at 25.0 and 50.0 mg per injection site with volumes of 2 and 4 ml, respectively. TARLE
3
EFFECTOF pH ON INTRAMUSCULARIRRITATIONSP~ HOUHS ~FTERA SIKGLE INJECTION OF NORMAL SALINE INTO THE M.Vas~ms I,\TERALIS OF RABBITS
PH
Average score from 3 rabbits Acidic
2.1 2 5 3.4 6.3
Average irritation score side
Moderate Mild Slight NOIW
2.7 1.7 1.3 0.0 Alkaline
8.0 10.0
0.0 0.0
11.0 12.0
0 7 3.0
side
NOIW None Slight Moderate
DISCUSSION
The rabbit has been widely used for irritation studies, and any one of a number of muscles can be used as the injection site. The M. sacrospinalis of the rabbit has been recommended by Hagan (1959) for the reason that the injected depot will remain between the fibers of this muscle entity. Hanson (1961) has used the gluteal muscle of the rabbit to determine the irritation of antibiotics. However, it is difficult to locate the site of injection (the area of local tissue damage) in these muscles. Our choice of injection site, i.e., one of the distinct femoral muscles of the rabbit, has led to the standardization of the procedure. The injection site can be quickly and accurately located, this simpliiies the injection of drugs and the detection and examination of muscle damage. Gross examination and scoring of lesions can easily be made on the cut surface of the muscle, making our procedure advantageous. The evaluation of local reactions should always be made at the peak of the response indicating a maximum irritation. Usually, the peak of the response can be estimated by preliminary determinations at 24, 72, and 120 hours after the injection. Intramuscular administration of drugs in clinical use is frequently repeated
300
S. SHINTANI,
M.
YAMAZAKI,
M.
NAKAMURA,
AND
I.
NAKAYAMA
one or more times per day for a given period. One, therefore, can postulate that the local irritant properties of drugs should be studied not only after single injections, but also after repeated administration. In this connection, our procedure has the advantage that repeated injections can be placed more precisely in the M. vastus lateralis than in any other muscle of the rabbit. According to our experience with this procedure, drugs that cause an irritation grade of not more than “mild” offer practically no problems because the local reactions appear mostly within 24 hours and do not increase after repeated injecirritation in animals do not generally tions. The drugs that give “moderate” cause difficulties in the clinic, but repeated injections should be given cautiously because of the increased response, as shown in Table 2. There is a, possibility that the grades of local reaction may be underestimated when injection volumes of 0.25 ml or less are used. This can be explained by the relatively small damaged area that is usually caused by these volumes. However, the examples cited in Table 4 show that no correlation was found between the volume of injection and the intensity of irritation when the volume EFFECT SCORE
OF THE FROM
VOLUME
3 RABBITS)
OF INJECTIOV BY
Dosage (mg/injection site)
Antibiotics Polyoxyethylene (20) sorbitan monolauraten
6.25 19.5
Locar,
TISSUE
INJECTION R.*BBIT
The
0.125
RESPONSE
INTO
IRHITATION
VASTUS
LATEHALIS
IN
volume of the injection (ml/injection site)
0.0.5
0. 5
1.0
c? 0
4.0
2.3
2 0
2.3
1 0
0 7
2.7 3. 0
2.7 3 3
3 0 2.7
1.7 2.7
1.3 1.3
100.0
3.3 37
4.0 4.3
4.3 4 0
4.0 4.3
3.3 4.7
3.7 4.3
TID”
12.5
I?.5
6.25
0.0
1.7
” 0
“.3
1 3
0 3
” 7 -d
3 0 3.7
3.3 4 0
3.3 4 0
3. 0 3.7
1.7 4.3
5. 0
5.0
4.3
4.7
14.5 25.0
12.5
50.0
TID A.6
(AVER.*GE 31.
0.0
50.0
Chromomycin
THE
3.0 2.7
25.0
Tetracyclinec
ON THE
INTRAMUSC~-L.AR THE
0.001
12.5
12.5
15.5
12.5
12.5
“5.
0
1”. 5
50
-
0
25.0
O.OOcL 0. 005
1.7 2. 0 2.3
2. 0 2. 0 3.3
1.0 3. 0 4.3
1 0 2.7 4. 0
0 0 1 0 3 3
0 3 0.3 3.0
0.010
4.0
4.3
5.0
47
4. 0
4.7
0.005
0.00%
0.002
TID
0.010
a Tween %O@, Atlas Chemical Industries, Inc. b TID = Threshold irritation dose in mg per injection “moderate” irritation. c Achromycinm, IM, Lederle (Japan), Ltd. d Not determined because of poor solubility. D Toyomycin@, Takeda Chemical Industries, Ltd.
site
which
produced
0,005
no more
0.010
than
a
TEST
FOR
INTRAMUSCULAR
IRRITATION
301
was 1 ml or less. On the other hand, accurate evaluation with injections of 2 ml or more is difficult since the injected solution may easily leak out of the muscle and into the surrounding connective tissue because of the disproportion of muscle volume to the volume of injection. We have injected 2 ml or more of ink into the M. vastus lateralis of the rabbit and found that some of the ink escaped from a circumscribed area on the posterior side of the muscle. Injections of 0.5 to 1 ml did not cause this phenomenon. Mima et al. (1962) used our procedure and found that the local irritation of nonionic surfactants of polyoxyethylene derivatives became weaker as the degree of polymerization of ethylene oxide was increased or hydrophilic property was raised. Our procedure has also been used to obtain muscle samples for the determination of chromomycin A 3 remaining in the muscle after intramuscular injection. Also some estimates of muscle edema can be made by weighing the muscle. This is possible only because the whole muscle can be removed by simple surgical operation (Shintani et al., 1963). SUMMARY A new method for gross evaluation of the local tissue irritation of drugs by intramuscular injection in rabbits is presented. The preparations to be tested were injected into the M. vastus lateralis of rabllits which were then sacrificed at predetermined intervals. The intact muscle entity was dissected, and the entire lesion was exposed by various incisions, grossly examined, and scored using six numerical grades of damage. The final evaluation of irritation was based on these scores that led to a classification by an average irritation score ranging from “slight” to “severe.” Tests were usually made on two or more rabbits at each dose. One muscle was used as the injection site for the test solution and the contralateral muscle for the control, usually normal saline or the diluent used for the standard or the test solutions. The results of time sequence studies, multiple injections, variability of pH, and the volume of injection are presented. Preparations tested were urethane, calcium chloride, tetracycline, chloramphenicol, chromomycin AZ, and the following condensed with polyoxyethylene: sorbitan monolaurate, lauryl ether, cetyl ether, stearate, hydrogenated castor oil. ACKNOWLEDGMENT The authors wish to thank Drs. K. Kaziwara and Y. Aramaki, Biological Research Laboratories of Research and Development Division of Takeda Chemical Industries, Ltd. for their interest and helpful advice. We are also grateful to Messrs. K. Nakanishi, S. Minami, M. Maeda, S. Imanishi, and T. Tsukamoto for their technical assistance in various phases of this work. REFERENCES K.-F., and DAMBACH, G. (1966). Morphologic quantification of muscular lesions after injections of aqueous solutions. Arsneimittel-For&. 16, 658-661. HACAN. E. C. (1959). Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, p. 19. Association of Food and Drug Officials of the United States, Austin, Texas. HANSON, D. F. (1961). Local toxic effects of broad-spectrum antibiotics following injection. Antibiot. Chemotherapy 11, 396-464. MIMA, H., YASHIKI, T., NAKATANI, H., SHINTANI, S., and Usur, T. (1962). Studies on pharmaceutical preparations. XIII. Irritating action of aqueous injection of non-ionic surface active agents. J. PhaTm. Sot. Japan 82, 1171-1176. SHINTANI, S., KAWAJI, H., KOMEDA, T., YAMAZAKI, M., NAKAMUHA, M., and KAZIWARA, K. (1963). Observations on the local tissue toxicity of chromomycin An. Takeda Kenkyusho Nempo (Ann. Rept. Takeda Res. Laboratories) 22, 227-253. BENITZ,
AND
A New
APPLIED
PHARMACOLOGY
Method
to Determine
Intramuscular S. SHINTANI, M. YAMAZAKI, Takeda
11,
Chemical
293-301 ( 1967)
the
Irritation
Injection
of
Drugs
after
in Rabbits
M. NAKAMURA, AND I. NAKAYAMA Industries,
Ltd.,
Osaka, Japan
Received h-lay 12, 1967
Muscular damage caused by the intramuscular administration of drugs is an important criterion for the evaluation of irritation. Several procedures have been devised to study this property. Hanson ( 1961) observed local tissue changes resulting from the intramuscular injection of several broad spectrum antibiotics into the gluteal muscles of the rabbit. Hagan (1959) suggested that the irritating property of drugs might be studied by intramuscular injection using the M. sacrospinalis of the rabbit as the site for intramuscular injection. Benitz and Dambach (1966) have also used this muscle for their studies. In 1953, after having tried various injection sites in the rabbit with unsatisfactory results, we selected the M. vastus lateralis because of its topographical relationship to skin and other muscle entities. We were able to standardize the injection procedures and the dissection methods in such a manner that they could be used for routine work. Our experience over a period of thirteen years showed that this method was satisfactory with respect to the following characteristics in comparison with other methods: (1) the whole muscle is easily dissected from other tissues, (2) the site of injection is readily detected, and (3) injections can be repeated more precisely. It is the purpose of this report to describe our method for the irritation test, which has been performed with a number of drugs for more than ten years. METHODS Test solutions. The solutions for injections were prepared in normal saline except for the commercially available forms of antibiotics, which were diluted according to the manufacturers’ instructions. New experimental drugs submitted for testing were usually administered at the concentrations intended for clinical use. The relationship between irritation and concentration was determined using 2- to 5-fold dilutions of the original concentration and the same diluent. Animals. Male albino rabbits weighing 2.5-3.5 kg were used; they received a commercial solid diet and tap water ad libitum. The animal quarters were kept at a uniform temperature (25 + 1” ) and uniform humidity (4060% ). The experiments were carried out on two or more rabbits at each dose. The thigh muscle of one side was used as the injection site for the test solution, and the other side for the control solution such as normal saline or the diluent.
294
S.
SHINTANI,
M.
YAMAZAKI,
hi.
NAKAMURA,
AND
I.
NAKAYAMA
Znjection procedure. Intramuscular injection of the test solutions into the M. vastus lateralis of the rabbit was carried out as follows: the rabbit was fastened securely in a supine position without anesthesia. The hind legs were fixed together so that one was placed upon the other. This procedure made it easier to inject the test solutions into the center of the muscle. Fur was removed from the injection site with an electric clipper. A 22-gauge needle attached to a 2-ml syringe was inserted through the skin of the thigh at an angle of about 30 degrees to approximately the center of the muscle as indicated in Fig. 1-A. A volume of 1 ml of the test solution was then slowly injected.
FIG. 1. Illustration of the procedures for the injection of the test solution into the center of the M. vastus lateralis and the dissection of the muscle, (A ) Insertion of a Ugauge needle with its tip in the center of the muscle. ( B ) Removal of the M. tensor fasciae and a part of the M. rectus femoralis. (C) Separation of the M. vastus lateralis from the underlying muscles by blunt dissection. (D) Removal of the M. vastus lateralis.
During the injection the investigator should notice a slight resistance if the tip of the needle is correctly placed in the center of the muscle. The contralateral muscle was used for the injection of the control solution. If the intended clinical use of a drug formulation required repeated administration of the drug, the above-mentioned procedure was repeated daily for 3 or 5 days using the same injection site. Dissecting the injection site. After predetermined intervals when the local tissue irritation reaction was at its peak, the rabbit was sacrificed by intravenous injection of sodium pentobarbital. The animal was placed in a supine PO-
TEST
FOR
INTRAMUSCULAR
295
IRRITATION
sition and the skin over the injection site was removed. As shown in Fig. l-B, -C and -D, the M. tensor fasciae latae was then removed together with a part of the M. rectus femoris, thus exposing the M. vastus lateralis. This muscle originates with a short tendon on three parts of the collum femoris, trochanter major, and laterointermuscular septum and conjoins with the common tendon of the knee. Finally, the M. vastus lateralis was carefully cut off at both ends, that is, at the common tendon of the knee and the origin of the muscle. The dissected muscle was cut longitudinally along its midline until the scalpel fully exposed the injection site as indicated by grossly detectable injury (see Fig. 2-A). The first incision exposed the primary cut surface as shown in Figures 2-A and -B. A second incision was made along the largest diameter of the damaged area on both sides of the primary incision thus exposing the entire lesion ( see Fig. 2-B to -E ) . was grossly Scoring of the local tissue reaction. The local tissue irritation scored from 0 to 5 according to the following criteria (see Fig. 3): score O-no
1I I i I I I I
@ A
B
C
D
E
Fro. 2. Illustration of the incision process for gross examination on the cut surface of the M. vastus lateralis. The muscle is cut and opened in alphabetical order and the incisions follow the direction of the arrows and on the dotted lines. Upper: cross-section views. Lower: outside views. The regions showed by the oblique line represent the damaged area. (A) The dissected muscle is cut along its midline until the scalpel fully exposes the damaged area. (B ) The cut surface is exposed by bending back the muscle. ( C ) Secondary incisions are made at the damaged area on both sides of the initial incision. (D) Each incision is extended to both ends of the muscle. (E ) The cut surface of the damaged area is fully exposed by bending back the muscle. FIG. 3. Photograph of gross local reactions with six different scores. 0 = No discernible gross reaction; 1 = slight hyperemia and discoloration; 2 = moderate hyperemia and discoloration; 3 = distinct discoloration in comparison with the color of the surrounding area; 4 = brown degeneration with small necrosis; 5 z widespread necrosis with an appearance of “cooked meat” and occasionally an abscess involving the malor portion of the muscle.
Printed
in Japan
296
S. SHINTANI,
M.
YAMAZAKI,
M.
NAKAMURA,
AND
I.
NAKAYAMA
discernible gross reaction; score l-slight hyperemia and discoloration; score 2-moderate hyperemia and discoloration; score S-distinct discoloration in comparison with the color of the surrounding area; score 4-brown degeneration with small necrosis; score 5-widespread necrosis with an appearance of “cooked meat” and occasionally an abscess involving the major portion of the muscle. Evaluation of irritation. The final score for the local responses was indicated by the average irritation score from two or more rabbits at one dose level. The irritation of the test or control solutions was graded using the following cate“slight” for 0.5-1.4; “mild” for 1.5-2.4; “moderate” gories : “none” for 0.4 or less; for 2.5-3.4; “marked” for 354.4; and “severe” for 4.5 or more. The irritation score of a test solution in a rabbit that showed an irritation reaction corresponding to score 3 or more with the control solution was deleted from the calculation of the average. For a quantitative comparison of the irritation of compounds, the results were expressed in terms of the threshold irritation dose ( TID ), as defined by the dose in milligrams per injection site that produced no more than a moderate irritation. RESULTS
Maximum
Tissue
Response
with
Relation
to Time
Gross findings following a single injection of seven compounds were observed at different intervals and are summarized in Table 1. The time of maximum irritation varied from 5 to 120 hours after injection. The muscles that gave TABLE AVERAGE
IRRITATION INJECTION
SCORES OF \<~RIOUS
AND
MAXIMUM
Compounds Urethane Calcium chloride Polyoxethylene (20) sorbitan monolauratr” Polyoxyethylene (93) lauryl ethera Tetracyclinec Chloramphenicold Chromomycin Aae
Cont. Pi;;)
REACTION
COMPOUNDSINTO
Average
TIME
M.
THE
irritation Hours
1 VASTUS
score
after
injection
AFTER
A SINGLE
LATERALIS
Maximum reaction time (hours)
INTRAMUSCULAR
IN 3 RABBITS
Gross findings at maximum reaction time
5
24
48
7%
120
10.0 1.0
4.0
3.3
4.3
5
3.3
3.0
4.0 2 7
4.0
4.7
9.7
a4
Brown degeneration Discoloration
5.0
2.7
4.3
-
3.7
2.5
24
Brown
5.0
2.3
-
24
3.0
3.7 3.0
3.3 4.3 3.7
4.0
2.5
3.3
3.3
72 73
3.0
3.7
4.7
120
Brown degeneration Necrosis Brown degeneration Necrosis, swelling
“5. 0
1.7
4.3 3.7 4.7
0.005
0.7
2.3
u Tween W@, Atlas Chemical Industries, Inc. b Brij 358, Atlas Chemical Industries, Inc. c Achromycin@ for intramuscular use, Lederle (Japan), Ltd. d Kemicetine@ for intramuscular use, Fujisawa Pharmaceutical c Toyomycin@, Takeda Chemical Industries, Ltd.
Industries,
Ltd.
degeneration
TEST
FOR
INTRAMUSCULAR
IRRITATION
297
the maximum irritation score showed grossly either a marked necrosis or distinct discoloration with a surrounding hemorrhagic area as shown in Fig. 4. Injections of a 10% solution of urethane caused small necroses or brown degenerations surrounded by hemorrhages that corresponded to score 4 at 5 hours after injection. This response was about the same as that seen 48 hours after injection (see Fig. 4-A and -B). The local reactions for two nonionic surfactants, polyoxyethylene sorbitan monolaurate and polyoxyethylene lauryl ether, peaked 24 hours after injection. The maximum irritation scores of two antibiotics were
Brown a
The
white
deposit
of
the
degeneration
suspension.
FK. 4. Gross local reaction at the early and the peak stages following a single injection of various compounds indicated in Table 1. (A) 5 hours after 10% urethane solution (score 4). ( B ) 48 hours after the above solution (score 4). ( C ) 24 hours after 1% calcium chloride solution (score 3). ( D) 24 hours after 5% polyoxyethylene sorhitan monolaurate solution (score 4). ( E ) 24 h ours after 5% polyoxyethylene lauryl ether solution (score 4). (F) 5 hours after 0.005’% chromomycin A3 solution (score 1). (G) 120 hours after the above solution (score 5). ( H ) 72 h ours after 2.5% tetracycline solution for intramuscular (I ) 24 hours after 25% suspension of chloramphenicol for intramuscular (score (score Tj ). 3 ). ( 1) 24 hours after the above suspension ( score 4).
obtained at 72 hours. A peak of the local reaction for chromomycin A3 was observed on day 5 after injection. In the case of chloramphenicol, a white deposit of the suspension remained at the site of application for about 24-72 hours after injection and a hemorrhagic area was present in the surrounding tissue (see Fig. 4-I and -J). Effect of Repeated
Zntramuscular
Znjections
The effects of single daily injections oxyethylene nonionic surfactants were
for 5 days with several types of polycompared with the effects of a single
298
S.
SHINTANI,
M.
YAMAZAKI,
ht.
NAKAMURA,
AND
I.
NAKAYAhrA
injection. The local reaction was determined 24 hours after a single intramuscular injection and 24 hours after the last of 5 daily injections. A 5% solution was used to achieve the maximum irritation score. As shown in Table 2, the intensity of the irritation after repeated injections was greater than after one injection with the exception of hydrogenated castor oil ester, which showed no irritation. Efect
of pH
The effects of normal saline, at various pH values ranging from 2.1 to 12, upon the local tissue response after intramuscular injection were examined. As shown in Table 3, there was “moderate” irritation at the extreme pH’s of 2.1 TABLE COYPARISOX
JWTIOSS
2
OF IXTRAMIJSCTI,\H IRRIT~TIOY AFTER SINGLE ANJ REPIL~TED INTKAMUSCTLAR INTO THE M.V.AST~~Y LATERALIS OF THE RABBIT USING V,ARIOUS N~NI~~I~,~URF.~CTA~.TS OF POLYOXYETIIYLENE DERIVATIVES
Chemical composition of esters condensed with polyoxyethylenea Sorbitan laurateb Cetyl ether? Stearated Hydrogenated castor Sorbitan laurate” Cetyl etherc Stearated Hydrogenated castor
oiP
oile
Sumber of OXYethylene units
24 hours after single injection
24 hours after daily injections
_____ Average score from
5
Average score from 3 rabhits
Average irritation score
20 20
9 .7
Moderate Severe
4 3 -
Marked
“0 20 fro .50 -50 50
3.7 0. 0 0. 0 3 3 4.7 0. 0
Marked None None
1.7 0.3 1 3 5. 0 3 3 0. 3
Severe NOM Slight Severe Moderate None
* Injected as 5”/G solutions. b TL 20 and 508, Atlas Chemical Industries, c BC 20 and 50@, Atlas Chemical Industries, ’ AMYS 20 and 50@, Atlas Chemical Industries, e HCO 20 and 508, Atlas Chemical Industries,
5 0
Moderate
Moderate None
3 rahhith
Is-
Average irritation SCOW
Inc. Inc.
Inc. Inc.
and 12. The local irritating effect of the solutions ranging from pH 2.5 to 11 was not more than “mild” after a single injection. The peak response was found 24 hours after the injection and was no longer visible on day 3 after the injection in the case of saline at pH 2.1 while the peak response of saline at pH 12 still remained on day 3 and disappeared on day 7 after the injection. Relationship between Dosage and Irritation The standard procedure using the M. vastus lateralis has been limited to 1 ml of test solution because of the limited volume of the muscle; however, to determine the effects of volume of injection and drug concentration upon irritation, we used volumes ranging from 0.125 to 4 ml with various concentrations of solute as shown in Table 4. The average irritation scores were proportional to the drug concentration in milligrams per individual injection site and gen-
TEST
FOR
INTRAhIUSCULAR
IRRITATION
299
erally did not vary in proportion to the volume of injection except when 2 and 4 ml were used. The threshold irritation doses (TID’s) for tetracycline were constant at 12.5 mg per injection site with all the volumes ranging from 0.125 to 2.0 ml and at 25 mg per injection site in the large volume of 4.0 ml. The TID’s for chromomycin A, varied from 0.002 to 0.01 mg per injection site and did not give constant results with variations in the volume of injection. The TID’s for polyoxyethylene sorbitan monolaurate were constant at 12.5 mg per injection site through all the volumes from 0.125 to 1.0 ml and at 25.0 and 50.0 mg per injection site with volumes of 2 and 4 ml, respectively. TARLE
3
EFFECTOF pH ON INTRAMUSCULARIRRITATIONSP~ HOUHS ~FTERA SIKGLE INJECTION OF NORMAL SALINE INTO THE M.Vas~ms I,\TERALIS OF RABBITS
PH
Average score from 3 rabbits Acidic
2.1 2 5 3.4 6.3
Average irritation score side
Moderate Mild Slight NOIW
2.7 1.7 1.3 0.0 Alkaline
8.0 10.0
0.0 0.0
11.0 12.0
0 7 3.0
side
NOIW None Slight Moderate
DISCUSSION
The rabbit has been widely used for irritation studies, and any one of a number of muscles can be used as the injection site. The M. sacrospinalis of the rabbit has been recommended by Hagan (1959) for the reason that the injected depot will remain between the fibers of this muscle entity. Hanson (1961) has used the gluteal muscle of the rabbit to determine the irritation of antibiotics. However, it is difficult to locate the site of injection (the area of local tissue damage) in these muscles. Our choice of injection site, i.e., one of the distinct femoral muscles of the rabbit, has led to the standardization of the procedure. The injection site can be quickly and accurately located, this simpliiies the injection of drugs and the detection and examination of muscle damage. Gross examination and scoring of lesions can easily be made on the cut surface of the muscle, making our procedure advantageous. The evaluation of local reactions should always be made at the peak of the response indicating a maximum irritation. Usually, the peak of the response can be estimated by preliminary determinations at 24, 72, and 120 hours after the injection. Intramuscular administration of drugs in clinical use is frequently repeated
300
S. SHINTANI,
M.
YAMAZAKI,
M.
NAKAMURA,
AND
I.
NAKAYAMA
one or more times per day for a given period. One, therefore, can postulate that the local irritant properties of drugs should be studied not only after single injections, but also after repeated administration. In this connection, our procedure has the advantage that repeated injections can be placed more precisely in the M. vastus lateralis than in any other muscle of the rabbit. According to our experience with this procedure, drugs that cause an irritation grade of not more than “mild” offer practically no problems because the local reactions appear mostly within 24 hours and do not increase after repeated injecirritation in animals do not generally tions. The drugs that give “moderate” cause difficulties in the clinic, but repeated injections should be given cautiously because of the increased response, as shown in Table 2. There is a, possibility that the grades of local reaction may be underestimated when injection volumes of 0.25 ml or less are used. This can be explained by the relatively small damaged area that is usually caused by these volumes. However, the examples cited in Table 4 show that no correlation was found between the volume of injection and the intensity of irritation when the volume EFFECT SCORE
OF THE FROM
VOLUME
3 RABBITS)
OF INJECTIOV BY
Dosage (mg/injection site)
Antibiotics Polyoxyethylene (20) sorbitan monolauraten
6.25 19.5
Locar,
TISSUE
INJECTION R.*BBIT
The
0.125
RESPONSE
INTO
IRHITATION
VASTUS
LATEHALIS
IN
volume of the injection (ml/injection site)
0.0.5
0. 5
1.0
c? 0
4.0
2.3
2 0
2.3
1 0
0 7
2.7 3. 0
2.7 3 3
3 0 2.7
1.7 2.7
1.3 1.3
100.0
3.3 37
4.0 4.3
4.3 4 0
4.0 4.3
3.3 4.7
3.7 4.3
TID”
12.5
I?.5
6.25
0.0
1.7
” 0
“.3
1 3
0 3
” 7 -d
3 0 3.7
3.3 4 0
3.3 4 0
3. 0 3.7
1.7 4.3
5. 0
5.0
4.3
4.7
14.5 25.0
12.5
50.0
TID A.6
(AVER.*GE 31.
0.0
50.0
Chromomycin
THE
3.0 2.7
25.0
Tetracyclinec
ON THE
INTRAMUSC~-L.AR THE
0.001
12.5
12.5
15.5
12.5
12.5
“5.
0
1”. 5
50
-
0
25.0
O.OOcL 0. 005
1.7 2. 0 2.3
2. 0 2. 0 3.3
1.0 3. 0 4.3
1 0 2.7 4. 0
0 0 1 0 3 3
0 3 0.3 3.0
0.010
4.0
4.3
5.0
47
4. 0
4.7
0.005
0.00%
0.002
TID
0.010
a Tween %O@, Atlas Chemical Industries, Inc. b TID = Threshold irritation dose in mg per injection “moderate” irritation. c Achromycinm, IM, Lederle (Japan), Ltd. d Not determined because of poor solubility. D Toyomycin@, Takeda Chemical Industries, Ltd.
site
which
produced
0,005
no more
0.010
than
a
TEST
FOR
INTRAMUSCULAR
IRRITATION
301
was 1 ml or less. On the other hand, accurate evaluation with injections of 2 ml or more is difficult since the injected solution may easily leak out of the muscle and into the surrounding connective tissue because of the disproportion of muscle volume to the volume of injection. We have injected 2 ml or more of ink into the M. vastus lateralis of the rabbit and found that some of the ink escaped from a circumscribed area on the posterior side of the muscle. Injections of 0.5 to 1 ml did not cause this phenomenon. Mima et al. (1962) used our procedure and found that the local irritation of nonionic surfactants of polyoxyethylene derivatives became weaker as the degree of polymerization of ethylene oxide was increased or hydrophilic property was raised. Our procedure has also been used to obtain muscle samples for the determination of chromomycin A 3 remaining in the muscle after intramuscular injection. Also some estimates of muscle edema can be made by weighing the muscle. This is possible only because the whole muscle can be removed by simple surgical operation (Shintani et al., 1963). SUMMARY A new method for gross evaluation of the local tissue irritation of drugs by intramuscular injection in rabbits is presented. The preparations to be tested were injected into the M. vastus lateralis of rabllits which were then sacrificed at predetermined intervals. The intact muscle entity was dissected, and the entire lesion was exposed by various incisions, grossly examined, and scored using six numerical grades of damage. The final evaluation of irritation was based on these scores that led to a classification by an average irritation score ranging from “slight” to “severe.” Tests were usually made on two or more rabbits at each dose. One muscle was used as the injection site for the test solution and the contralateral muscle for the control, usually normal saline or the diluent used for the standard or the test solutions. The results of time sequence studies, multiple injections, variability of pH, and the volume of injection are presented. Preparations tested were urethane, calcium chloride, tetracycline, chloramphenicol, chromomycin AZ, and the following condensed with polyoxyethylene: sorbitan monolaurate, lauryl ether, cetyl ether, stearate, hydrogenated castor oil. ACKNOWLEDGMENT The authors wish to thank Drs. K. Kaziwara and Y. Aramaki, Biological Research Laboratories of Research and Development Division of Takeda Chemical Industries, Ltd. for their interest and helpful advice. We are also grateful to Messrs. K. Nakanishi, S. Minami, M. Maeda, S. Imanishi, and T. Tsukamoto for their technical assistance in various phases of this work. REFERENCES K.-F., and DAMBACH, G. (1966). Morphologic quantification of muscular lesions after injections of aqueous solutions. Arsneimittel-For&. 16, 658-661. HACAN. E. C. (1959). Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, p. 19. Association of Food and Drug Officials of the United States, Austin, Texas. HANSON, D. F. (1961). Local toxic effects of broad-spectrum antibiotics following injection. Antibiot. Chemotherapy 11, 396-464. MIMA, H., YASHIKI, T., NAKATANI, H., SHINTANI, S., and Usur, T. (1962). Studies on pharmaceutical preparations. XIII. Irritating action of aqueous injection of non-ionic surface active agents. J. PhaTm. Sot. Japan 82, 1171-1176. SHINTANI, S., KAWAJI, H., KOMEDA, T., YAMAZAKI, M., NAKAMUHA, M., and KAZIWARA, K. (1963). Observations on the local tissue toxicity of chromomycin An. Takeda Kenkyusho Nempo (Ann. Rept. Takeda Res. Laboratories) 22, 227-253. BENITZ,