Acute toxicity of corticoids in the mouse

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ACUTE TOXICITY OF CORTICOIDS IN THE MOUSE by George Tonelli Department of Endocrine Research Lederle Laboratories, American Cyanamid Company Pearl River, New York

Received July 20, 1966

ABSTRACT Median lethal doses following subcutaneous administration to mice of several corticoids were calculated after observation periods of 7, lh and 21 days. During the experimental period, lethality increased with time and was attributed to a supervening generalized septicemia, due to interference with the immune response mechanism of the animal. Such a conclusion was based on the observations that a) deaths were delayed and b) the majority of animals showed multiple small abscesses of the liver, kidneys and/or lungs.

Published information on the acute toxicity of corticoids is surprisingly limited.

Figdor et al.

I

have reported on the median

lethal doses in mice of intravenously administered deoxycorticosterone derivatives.

Recently, we determined 2 the median lethal doses of

several corticoids,

administered subcutaneously to the rat, and showed

that mortality was a function not only of dose but of time as well. During the 21-day experimental period corticoid lethality increased with time as a result of a supervening generalized septicemia, presumably due to interference with the in~nune response mechanism of the animal.

Such a conclusion was based on the observations that a) deaths

were delayed, the earliest deaths occurring 6 to 7 days after treatment; b) a majority of animals showed multiple small abscesses of the liver, kidneys and/or lungs; and, c) the addition of a broad-spectrum anti-

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biotic to the diet reduced the number of deaths and the number of animals showing abscessation of major organs.

It was apparent that#

unlike that of other therapeutic agents~ lethality following administration of single large doses of corticoids was the result not of direct but of secondary effects. In view of these findings, it was deemed of interest to determine whether administration of these same corticoids to mice would elicit a similar sequence of events. MATERIALS AND METHODS CF1 female mice (Carworth Farms), weighing 20-24 g, were randomly distributed with respect to treatment and cage assignment (five mice per cage). Rat pellets and tap water were allowed ad libitum. The relative humidity were 72 ± 2o F and 45 ± 5%, respectively. The doses of the various corticoids were administered subcutaneously in 2.0 ml of an aqueous vehicle consisting of 0.5% carboxymethylcellulose, 0.4% polysorbate 80 and 0.9¢0 sodium chloride. This volume was distributed to four distinct sites (0.5 ml per site) in an effort to facilitate absorption of the injected materials. Control animals received the vehicle alone. Deaths were recorded daily; the duration of the experiment was 21 days. Ten or 20 mice were used for each dose. The median lethal doses were calculated3 after observation periods of 7, 14 and 21 days. The following corticoids were employed: hydrocortisone: ll~,17G, 21-trihydroxypregna-4-ene-3,20-dione; triamcinolone: 9G-fluoro-ll~,l~, 17G,21-tetrahydroxypregna-l,4-diene-3,20-dione; dexamethasone: 9G fluoro-ll~,lT~,21-trihydroxy-16G-methylpregna-l-4-diene-3,20-dione; prednisolone: llS,17G,21-trihydroxypregna-l,4-diene-B,20-dione; triamcinolone acetonide: 9G-fluoro-ll~,21-dihydroxy-16~,17G-(isopropylidenedioxy)-pregna-l,4-diene-3,20-dione; 21-deoxytriamcinolone acetonide: 9G-fluoro-ll~-hydroxy-l~,lTG-(isopropylidenedioxy)-pregna-l,4-diene3,20-dione; 9G,11~-dichloro-21-hydroxy-16~,lTG-(isopropylidenedioxy)pregna-l,4-diene-3,20-dione; 9~,ll~-dichloro-16(~,17G-(isopropylidenedioxy)-pregna-l,4-diene-3,20-dione; and, 21-tertiary butylacetate-9~fluoro-ll~-hydroxy-16~,lTG-(isopropylidenedioxy)-pregna-l,h-diene-3, 20-dione. RESULTS AND DISCUSSION

Cumulative mortality after observation periods of 7, 14 and 21 days after single subcutaneous doses of corticoids to mice is pre-

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S T E R O ID

sented in Table I.

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In spite of the massive doses employed, no deaths

occurred among animals receiving either dexamethasone, 21-deoxytriamcinolone

acetonide or 9~,ll~-dichloro-16G,17~-(isopropyl-

idenedioxy)-pregna-l,4-diene-3,20-dione observation.

prednisolone,

during the first week of

Among the other corticoid-treated animals, deaths were

not observed until after the fourth day of observation.

Of 660

corticoid-treated mice, 32 or 4.8 per cent, died during the first week. Deaths for the second week numbered 191 of 628 mice, or 30.4 per cent. The corresponding values for the third week were 52 of 437 mice, or ll.9 per cent.

As was found in the rat 2, autopsy revealed that from

50 to 75 per cent of the mice had multiple,

small, abscess-like foci

of infection in the liver, kidneys and/or lungs.

Hence, death was

ascribed to a supervening generalized infection, resulting from suppression of the immune defense mechanism by the corticoids.

The

recent report 4 that the incidence of death in hydrocortisone treated mice was significantly less in germ-free animals than in conventionally reared animals would support this conclusion.

Furthermore,

several

publications 5-9 have appeared on the increased incidence of deaths in mice, rats or rabbits given corticoids and subsequently experimentally infected with pneumococci, hemolytic streptococci, tubercle bacilli or influenza virus. Median lethal doses were calculated or gross estimates made for each of the observation periods and are presented in Table II.

The

median lethal doses of these same corticoids in the rat, which were in part previously published 2 have been included for comparison. There is an increase in lethality with time for each of the administered corticoids in both the mouse and rat.

It will be noted that

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TABLE I. Cumulative mortality in mice following a single subcutaneous administration of corticoids Observation Period Dose 7 Days 14 Days 21 Days mg/~ D/T* D/T D/T CMC 0/20 o/~o 0/.20 Hydroc ort isone 8o00 i/io 5/1o 711o 4000 1/20 6/20 12/20 Triamcinolone

2000

2/20

8/20

100o

1/2o

~/2o

4o00 2000

o11o 2/20

9/lO 17/20

iooo 500

Dexamethasone

70o 35o 175 87.5 43..7

Prednisolone

35o0 175o 87.5 43.7 21.8

Triamcinolone Acetonide

21-Deoxytriamcinolone Acetonide

80o 400

200 i00 . . 50 8000 4000

2000 i000 500 9G, ll6-Dichloro-21hydroxy-16G,17~-(isopropylidenedioxy)-pregna1,4-diene-3,20-dlone 9~,ll~Dichloro-l~z, lV~(isopropylidenedioxy)pregna-l,h-diene-3,20dione 21- Tertiary butylacetate9(z-fluoro- i16-hydr oxy- 1Ex~, 170~(isopropylidenedioxy)pregna-l,4-diene-3,20dione

4000 2000 lO00

500

8/20 ,, 4/20

O/lO

6/io

O/lO O/lO

1/1o I/IO

O/lO

2/lO

0/i0

0/i0

o/to o/io

o/to o/1o

0/I0 0/i0 O/lO

0/i0 0/i0 O/lO

5/lO 3/20 4/20 0/20 0/2,0 0/i0

,,.

iO/lO 19/20

7/10

i0/i0 5/20

1/20 3/20 5/20

0/10

9/10

2/20

11/20

0•20 0/20

112o

8•20 4/20

112o lo/lo

iooo O/lO 5o0. . . . . . o/1o 8000 7/10 4000 0/20

111o

4000

2000 lOOO 500

o/zo

4/20 0•20 0/20 0/20

4/20 7/10 2/10 2/lO i/i0 O/i0 7/lO 2/10 i/i0 0/i0 0/I0 lO/lO 19/20

19/20 3/20

0/20 0/20 0/20

o/2o

19/2o 8/20

17/20 3/20 1/20

0•20 O/lO

250 8000

25o

* Dead/Treated

0/20 0/20

8/20

4/2o 10/10

611o

..... 111o lO/lO

h/20 8•20 5•20 7/20

0•20

1/20 512o 5/20

1/20 i0/I0

13/20 10/20

~/2o i/2o i0/i0

7/lO

3/lO 1/1o i0/iO

9/20 10/20

8120 8/20

0/20

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the rat is much more susceptible to the "toxic" effects of corticoids than the mouse.

With rare exceptions,

it was possible to calculate

7, l~ and 21-day median lethal doses in the rat for each corticoid. In contrast, mainly very gross estimates of the median lethal doses could be obtained for the mouse even though doses of these same corticoids were lO to 20 times greater than those employed for the rat. Undoubtedly,

the combination of absorption and biologic potency

of each corticoid has influenced these studies.

With respect to the

latter factor, the median lethal doses obtained in the rat for any one corticoid appear, in the overall, to reflect the respective thymolytic activities of hydrocortisone,

prednisolone,

triamcinolone, dexamethasone,

triamcinolone acetonide lO and of 21-deoxytriamcinolone

acetonide ll.

From unpublished thymolytic activity data from these laboratories, similar conclusions can be reached for the remaining three corticoids of Table II.

Unfortunately,

information on the thymolytic activities

of these compounds in the mouse is not available; hence, an assessment of the correlation between toxicity and biologic potencies cannot be made in this species. Another aspect meritorious of a brief discussion are the median lethal doses, as reported.

Conventionally,

"acute toxicity" relates

to the effects occurring during a short period of observation after the single administration of a compound.

However, if longer periods

of observation are employed, the results may yield several estimates of toxicity which may differ from each other, depending on the type of compound under investigation.

Where changes do occur, these can

be interpreted as manifestations of delayed toxicity.

Thus, the

changing median lethal doses obtained following corticoid treatment

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TABLE II. Median lethal doses of corticoids following a single subcutaneous administration to mice and rats calculated after 7, 14 or 21 day observation periods

Hydrocortisone

Observation

Median lethal dose (95% confidence limits)

perioddays

'MO'~se

7 14 21

Triamcinolone

7 14 21

Dexamethasone

Prednisolone

7

21-Deoxytriamcinolone Acetonide

9a, llO-Dichloro-21hydroxy-16~,17G-(isopropylidenedioxy)-pregnal~4-diene-B~20-dione 9a, ll~-Dichloro-16a,17~(isopropylidenedioxy)pregna-l,4-diene-3,20dione 21- Tertiary butylacetate9~- fluoro- ll8- hydroxy- 16G, 17~-(isopropylidenedioxy)pregna-1 4-diene-B~20-dione

1326(943-1711) 748(480-1044)

3073(1901-6646 ) >bOO0

566(391-799) 86h(396-1888)

i097(853-1447)

142(109-185)

954(769-119o) >700

,99(75-13~). .~'~120

14

648(376-3815)

25(14-43)

21

514(315-1673)

14(6-32)

7 14 21

Triamcinolone Acetonide

>8000 -~8000

Ra.t,*

7

>3500 >3500

2613(1821-5344) ~800

>240 223(161-337)

147(100-216) 94(60-3o5)

14

141(114-174)

35(25-50)

21

.132(107-162 ) >8000

32(24-43)

7 14

>2400

<8000

415(274-628)

21

~4000

194(156-242)

7 ].4

>%000

1672(I056-9192)

21 7

14 21 7 14 21

1404(1010-2198)

94(63-145)

1103(829-1528 !

52(39-71)

>8000

>13oo

2394(1447-3985) >13oo 1774(lO39-2994) ~8ooo

712(5o8-1o92) 419(272-18o6)

.~2ooo ~iooo

21(8.7-36) N.D.

* These data , with the exception of the last two corticoids, were obtained from Tonelli, G., Tox. Appl. Phann.8, 250 (1966). N.D.

Not determined.

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S T E R O I D S

(Table II) are to be ascribed to the delayed effects of the compounds. In summary, the single administration of a corticoid to either mice or rats has resulted in markedly different median lethal doses. These results appear to be related to the duration of the observation period. REFERENCES 1.

Figdor, S. K., Kodet, M. J., Bloom, B. M.~ Agnells, E. J., P'An, S. Y. and Laubach, G. D., J. Pharmacol. Exptl. Therap., ll9, 299 (1956).

2.

Tonelli, G., Tox. Appl. Pharm. 8, 250 (1966).

3.

Finney, D. J.~ Probit Analysis, Cambridge University Press, Cambridge. 1947, p. 60.

4.

Reed, N. D. and Jutila, J. W., Science, 150, 356 (1965).

5.

Magabgab 3 W. J., Thomas, L. and Floyd, J., J. Lab. Clin. Med., 39, 271 (1952).

6.

Denny, F. W. and Thomas, L., Proc. Soc. Exp. Biol. and Med.,

26o (1955). 7.

Morgan, T. E., Wanzer, S. H. and Smith, D. T., J. Bacteriol.,

257 (195 ). 8.

Kass, E. H., Lundgren, M. M. and Finland, M., Ann. N. Y. Acad.

sci., 56, 765 (1953). 9.

LeMaistre, C., Tompsett, R. and McDermott, W., Ann. N. Y. Acad. Sci., 56, 772 (1953).

lO.

Tonelli, G., Partridge, R. and Ringler, I., Proc. Soc. Exp. Biol. and Med., I19, 136 (1965).

ll.

Tonelli, G. and Ringler, I., Second International Congress on Hormonal Steroidsj Excerpta Medica Foundation, New York. 1966, p. 274.

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