A new approach to practical acute toxicity testing - Springer Link

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TOXICOLOGY

Arch Toxicot (1983) 54:275-287

9 Springer-Verlag 1983

A New Approach to Practical Acute Toxicity Testing Dietrich Lorke Institut f~ir Toxikologie, Bayer AG, Friedrich-Ebert-Strasse 217, D-5600 Wuppertal, Federal Republic of Germany

Abstract. A method for the investigation of the acute toxicity of an unknown chemical substance, with an estimation on the LDs0, is described. Using this, it is possible to obtain with 13 experimental animals adequate information on the acute toxicity and on the LDs0. This method has no limitations and applies to drugs, agricultural and industrial chemicals. It can be used for every route of administration. Key words: Acute toxicity - LDs0 - Fewer animals Introductory Considerations Investigation of the acute toxicity is the first step in the toxicological investigations of an unknown substance. The index of the acute toxicity is the LDs0. However, the LDs0 should not be regarded as a biological constant, since differing results are obtained on repetition or when the determinations are carried out in different laboratories. This has been shown very clearly in a multicentre study carried out in the European community with five substances (Hunter et al. 1979; Lingk 1979). In this study the LDs0 values varied as follows in Table 1. Further examples, which show that LDs0 values can never be regarded as biological constants, have been surveyed by Zbinden and Flury-Roversi (1981).

Table 1 Compound

LDs0 mg/kg

Ratio of largest figure to smallest figure

I II III IV V

46- 522 800-4,150 350-1,280 805-5,420 70- 513

11.3 5.2 3.7 6.7 7.3

276

D. Lorke

The conclusion to be drawn from these results is that it is impossible in principle to specify an LDs0 for a substance which is generally valid and exact. If it is impossible in principle to determine a generally valid LDs0 , why are so many animals sacrificed for the sole purpose of determining this figure with a high precision? In order to answer this question the historical development of LDs0 test needs to be considered. In an effort to assess the acute toxicity of a substance, a simple means of grading acute poisonous effects was sought. It was desirable to have a test with which it was possible to determine whether a chemical substance was very toxic, toxic, less toxic, or whether the toxic effects were of no significance for dealing with the substance in practice. With this objective, a figure which expresses the toxic effects was sought. This figure was intended to indicate the amount of the substance which is injurious after a specified mode of intake. Since the term injuries always involves a high degree of subjectivity, an objective criterion was selected, namely death. Thus, a means of determining the likelihood of death as exactly as possible was sought. The LDs0 was recognised and justified of being the best parameter by Trevan (1927). Unfortunately, this has led to the determination of the LDs0 being equated with investigation into acute toxicity. A scientifically valid investigation into acute toxicity is necessary and from many points of view of great value (Lorke 1981). It is however not the intention to deal with this aspect again here. It is misleading to equate the determination of acute toxicity with that of the determination of the LDs0 although this often occurs.

Due to this misunderstanding various mathematical and statistical methods have been developed to estimate the LDs0 and its range of variation in order to report the acute toxicity as accurately as possible justified by statistical-mathematical methods. All these methods require a considerable number of animals in order to calculate an LDs0, and, implicit in their mathematical design is, that using more animals increases the accuracy of the figure reported. The question arises as to the value of very great accuracy for an individual case in view of the poor reproducibility of such biological determinations. Moreover, even if the LDs0 could be measured exactly and reproducibly, the knowledge of its precise numerical value would barely be of practical importance, because an extrapolation from the experimental animals to man is hardly possible. On the other hand, the knowledge of the signs of intoxication, the target organs of acute toxicity, reversibility of the lesions etc. are of great interest, and this information can be obtained from careful studies with small number of animals. Having criticised the prevailing evaluation of acute toxicity and the associated calculation of the LDs0, the question arises, how determinations of acute toxicity should be carried out using the smallest number of animals per group. It is now proposed that the acute toxicity should be tested in two steps:

A New Approach to Practical Acute Toxicity Testing

277

1. In the initial investigations the range of doses producing the toxic effects is established. 2. Based on these results, further specific doses are administered to calculate an LDs0. The m e t h o d described here is based on the assumption that the chemical substance to be investigated is completely unknown and that the investigation is to be carried out with a minimum n u m b e r of experimental animals. It is initially necessary to determine the approximate range of the acute toxicity. This is achieved by giving widely differing doses to the animals, e.g., 10, 100, and 1,000 mg/kg b.w. The results show whether a substance is very toxic, toxic, less toxic, or only slightly toxic. H o w m a n y animals in each group should be used for these investigations? We have examined the p r o b l e m using either a single animal or three animals per dose level. F r o m this emerged the effect that using a single animal in each group can lead to false assessment when, by chance, the animal in the non-toxic range dies or that in the toxic range survives. For this reason it is proposed that three animals in each group should be used to determine the toxic range. The result of this test is used as a basic for selecting the subsequent doses. The following assumptions are made with respect to the subsequent dosage schedules: 1. Substances m o r e toxic than 1 mg/kg are so highly toxic that it is not so important to calculate the LDs0 exactly. 2. LDs0 values greater than 5,000 mg/kg are of no practical interest. 3. A n approximate figure for the LDs0 is usually adequate to estimate the risk of acute intoxication. Based on these considerations and practical experience the doses listed in the table are based on the results of the first investigation. The new dosages are administered to the animals in the second test (Table 2). In the Table 3, some doses deviate from those given, since the doses finally r e c o m m e n d e d only emerged during the course of the investigations. Table 2

Doses in mg/kg body weight Result of the initial investigation 10

100

1,000

0/3* 0/3 0/3 0/3 0/3 0/3 0/3 1/3 2/3 3/3

0/3 0/3 0/3 0/3 1/3 2/3 3/3 3/3 3/3 3/3

0/3 1/3 2/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3

Doses chosen for the second test

600 200 140 50 20 15 5 2 1

1,600 1,000"* 400 225 100** 40 25 10"* 4 2

* Number of animals which died/number of animals used ** The result from the first test is taken over for these doses

2,900 1,600 800 370 200 80 40 20 8 4

5,000 2,900 1,600 600 400 160 60 40 16 8

278

D. Lorke

H o w m a n y a n i m a l s in e a c h g r o u p a r e n e c e s s a r y for t h e s e c o n d tests? I n o r d e r to e s t a b l i s h this, e a c h o f t h e p r o p o s e d d o s e s was given to g r o u p s consisting o f o n e a n i m a l , two a n i m a l s , t h r e e a n i m a l s a n d five a n i m a l s . T h e a l g e b r a i c s u m o f t h e s e results is p r e s e n t e d as i n c i d e n c e for a g r o u p c o n t a i n i n g e l e v e n animals.

Methods The investigations reported in the tables on pages 10-21 were carried out as follows. Animals were treated with substances after at least 5 days of adaptation. They were observed frequently on the day of treatment during normal working hours and the nature and time of all adverse effects were noted. Where any animals died the time of death was noted, during the 1st day in hours and thereafter in days. Dead animals were autopsied and examined macroscopically for any pathological changes. The surviving animals were weighed according the OECD guideline no. 401. If animals recovered and gained weight again, this was taken as a sign of having survived the acute intoxication. Observations and weighings were carried on for 14 days and the experiment was then terminated. If no recovery was noted during this period or if late deaths occurred, then the period of observation was extended intil the body weight of the surviving animals increased clearly. The experiment was then considered as completed. All surviving animals were sacrificed at the end of each test and then autopsied and examined macroscopically for any pathological changes.

Results T h e i n v e s t i g a t i o n s w e r e c a r r i e d o u t in f o u r d i f f e r e n t l a b o r a t o r i e s in o u r I n s t i t u t e o f T o x i c o l o g y . T h e results a r e c o m p i l e d in t h e T a b l e 3. T h e LDs0 was e s t i m a t e d in t h e f o l l o w i n g m a n n e r : G e o m e t r i c m e a n on t h e d o s e s for which 0/1 a n d 1/1 w e r e f o u n d . One animal group: E x a m p l e 1 : 2 , 9 0 0 m g / k g b o d y w e i g h t 0/1 5,000 m g / k g b o d y w e i g h t 1/1 LDs0 = 3,800 mg/kg. E x a m p l e 2: 370 m g / k g b o d y w e i g h t 0/1 600 m g / k g b o d y w e i g h t 1/1 LDs0 = 470 mg/kg. E x a m p l e 3: 200 m g / k g b o d y w e i g h t 1/1 400 m g / k g b o d y w e i g h t 0/1 600 m g / k g b o d y w e i g h t 1/1 LDs0 = 400 mg/kg. W h e n t h e results o f t h e first test give rise to p a r t i c u l a r situations ( e . g . , s u b s t a n c e s nos. 20 a n d 39) it is t h e n n e c e s s a r y to e s t i m a t e the results a p p r o p r i a t e l y , o r to r e p e a t t h e test. Other groups: If a d o s e w i t h 0 % l e t h a l i t y is f o l l o w e d b y o n e o f 100%, t h e n t h e LDs0 is e s t i m a t e d as for t h e o n e - a n i m a l g r o u p .

279


If a result > 0% and < 100% is found between the 0% and 100% lethality, then an estimation is carried out using the probit-log scale. If there is more than one result available between the 0% and 100% lethality, then the LDs0 is estimated by a procedure proposed by Rosiello et al. (1977). This examination is based on the maximum likelihood method of Bliss (1938). When comparing the LDs0 values obtained from investigations with different numbers of animals it was assumed that the figures obtained with 11 animals per group are the best estimates. The estimated LDs0s of the other groups were then assessed by their agreement with the figure from the ll-animal group. An agreement was assumed to exist when the difference did not exceed a factor of 2 (Bass et al. 1982). The substances were selected so that the entire range of toxicity from high acute toxicity to virtual non-toxicity, was tested. The substances not only included drugs and agricultural chemicals but also industrial chemicals. Among the 42 substances which were investigated using the male rat, the factor of 2 was exceeded twice in the one-animal group. These two compounds are HST 1474 and benzothiazole. Two out of 42 test means, only 7% of all tests exceeded the factor 2. The LDs0 for the substance HST 1474 was 2,500 mg/kg in the ll-animal group and > 5,000 mg/kg in the one-animal group, and for benzothiazole it was 375 mg/kg in the 11-animal group and 180 mg/kg in the one-animal group. In this latter instance, the factor is not remarcably larger than 2 (= 2.08). Thus even in these two cases, the results from the one-animal group are reasonable and of practical use. Only one LDs0 in the 2-animal group has a factor greater than 2 (HST 1774), and in the 3-animal and 5-animal group no results had a factor greater than 2. The results of oral administration agree with the five results from intravenous administration (Table 4), so that the method is not restricted to oral administration, but is valid, in principle for all routes of administration. Since the substances represented different classes of chemical structures the method can be used irrespective of whether the substance is a drug, an agricultural chemical or an industrial chemical.

Table 3. Investigations of the acute oral toxicity in male rats. All reported doses relate to mg/kg body weight Substance

1. KCN

1st part of investigation

2nd part of investigation

Doses

Doses

10 100 1,000

Mortality

3/3 3/3 3/3

2 4 9 14 LD5o

Number of animals in each group 1

2

3

5

Total

0/1 0/1 1/1 1/1

0/2 0/2 1/2 2/2

0/3 1/3 3/3 3/3

0/5 1/5 5/5 5/5

0/11 2/11 10/11 11/11

6

9

5

5

6

(Table continued)

280

D. Lorke

Table 3 (continued) Substance

2. HgC12



Doses

Doses

10 100

1,000

3. R 1513

4. Diphenylthiourea

5. Oftanol C

6. BAY h 1127

7. ARG 2032

10 100 1,000

10 100 1,000

10 100 1,000

10 100 1,000

10 100 1,000

Mortality

0/3 3/3 3/3

0/3 3/3 3/3

0/3 3/3 3/3

0/3 1/3 3/3

0/3 2/3 3/3

0/3 0/3 3/3


2

3

5

Total

10 15 25 40 60 100

0/3 0/1 0/1 0/1 1/1 3/3

0/3 0/2 0/2 1/2 1/2 3/3

0/3 0/3 0/3 3/3 3/3 3/3

0/3 1/5 1/5 3/5 5/5 3/3

0/3 1/11 1/11 7/11 10/11 3/3

LDso

50

50

32

32

37

14 22 37 60 100

0/1 0/1 0/1 1/1 3/3

0/2 0/2 0/2 2/2 3/3

0/3 0/3 1/3 3/3 3/3

0/5 0/5 0/5 4/5 3/3

0/11 0/11 1/11 i0/11 3/3

LDs0

50

50

40

60

50

14

0/1

23 37 60 100

0/1 0/1 0/1 3/3

0/2 0/2 0/2 2/2 3/3

0/3 0/3 0/3 0/3 3/3

0/5 0/5 0/5 1/5 3/3

LDso

80

50

80

70

70

50 100 200 400

0/1 1/3 1/1 1/l

0/2 1/3 2/2 2/2

0/3 1/3 3/3 3/3

0/5 1/3 5/5 5/5

0/11 1/3 11/11 11/11

LD5o

100

100

115

120

105

0/1 0/1 0/1 2/3 0/1 3/3

0/2 0/2 0/2 2/3 1/2 3/3

0/3 0/3 0/3 2/3 3/3 3/3

0/5 0/5 0/5 2/3 4/5 3/3

LDso

160

160

90

120

140

100 140 225 370 600

0/3 1/1 1/1 1/1 1/1

0/3 2/2 2/2 2/2 2/2

0/3 2/3 3/3 3/3 3/3

0/3 2/5 5/5 5/5 5/5

0/3 7/11 11/11 11/11 11/11

LD5o

120

120

130

150

140

20 40 80 100 160 1,000

0/11 0/11 0/11 3/11 3/3

0/11 0/11 0/11 2/3 8/11 3/3


281


8.3-Bromo-l,4toluidine



Doses

Doses

10 100

1,000

9. HOL 4578

10 100 1,000

Mortality

0/3 0/3 3/3

0/3 0/3 3/3

1

2

3

5

Total

100 140 225 370 600

0/3 0/1 0/1 1/1 1/1

0/3 0/2 1/2 2/2 2/2

0/3 1/3 1/3 3/3 3/3

0/3 0/5 3/5 5/5 5/5

0/3 1/11 5/11 11/11 11/11

LDs0

290

225

200

180

220

0/1 0/1 0/1 1/1 3/3

1/2 0/2 2/2 2/2 3/3

0/3 2/3 1/3 3/3 3/3

0/5 1/5 5/5 4/5 3/3

1/11 3/11 7/11 10/11 3/3

470

290

285

270

285

0/1 0/1 0/1 1/1 3/3

0/2 0/2 2/2 2/2 3/3

0/3 0/3 2/3 3/3 3/3

0/5 1/5 3/5 5/5 3/3

0/11 1/11 7/11 11/11 3/3

470

290

350

320

330

0/3 0/1 0/1 1/1 1/1 3/3

0/3 0/2 2/2 1/2 2/2 3/3

0/3 0/3 0/3 3/3 3/3 3/3

0/3 0/5 2/5 3/5 3/5 3/3

LDs0

290

180

290

350

310

140 225 370 600

0/1 1/1 1/1 1/1 3/3

0/2 0/2 0/2 2/2 3/3

0/3 0/3 1/3 3/3 3/3

0/5 1/5 3/5 4/5 3/3

0/11 2/11 5/11 10/11 3/3

LDs0

180

470

370

375

375

140 225 370 600

0/1 0/1 0/1 1/1 3/3

0/2 0/2 2/2 1/2 3/3

0/3 0/3 1/3 2/3 3/3

0/5 0/5 4/5 5/5 3/3

470

290

460

350

140 225 370 600 1,000 LDs0

10. Cadmium sulphate

10 100 1,000

0/3 0/3 3/3

140 225 370 600 1,000 LDs0

11. Levamisole (HC1)

12. Benzothiazole

10 100 1,000

10 100 1,000

0/3 0/3 3/3

0/3 0/3 3/3

100 140 225 370 600 1,000

1,000

13. Digitoxin

10 100 1,000

0/3 0/3 3/3

Number of animals in each group

1,000 LDs0

0/3 0/11 4/11 8/11 9/11 3/11

0/11 0/11 7/11 9/11 3/3 450

282

D. Lorke


14. Na barbitone



Doses

Doses

10 100 1,000

Mortality

0/3 0/3 3/3

1

2

3

5

Total

0/3 0/1 0/1 0/1 0/1 3/3

0/3 0/2 0/2 0/2 1/2 3/3

0/3 0/3 0/3 0/3 1/3 3/3

0/3 0/5 0/5 1/5 2/5 3/3

0/3 0/11 0/11 1/11 4/11 3/3

770

600

650

600

0/1 0/1 0/1 0/1 3/3

0/2 0/2 0/2 0/2 3/3

0/3 0/3 0/3 0/3 3/3

0/5 0/5 0/5 2/5 3/3

775

775

775

600

700

0/3 0/1 0/1 1/1 2/3 1/1

0/3 1/2 1/2 1/2 2/3 2/2

0/3 1/3 0/3 1/3 2/3 3/3

0/3 0/5 1/5 2/5 2/3 5/5

0/3 2/11 2/11 5/11 2/3 11/11

570

400

900

700

700

0/1 0/1 0/1 2/3 1/1

0/2 0/2 0/2 2/3 2/2

0/3 0/3 1/3 2/3 3/3

0/5 0/5 1/5 2/3 5/5

0/11 0/11 2/11 2/3 11/!1

950

950

900

950

900

0/1 0/1 1/1 3/3 0/1

0/2 0/2 2/2 3/3 2/2

0/3 0/3 1/3 3/3 3/3

0/5 0/5 1/5 3/3 5/5

0/11 0/11 5/11 3/3 10/11

LDs0

570

570

680

830

900

200 400 800

0/1 0/1 0/1 2/3 1/1

0/2 0/2 0/2 2/3 2/2

0/3 0/3 1/3 2/3 3/3

0/5 0/5 1/5 2/3 5/5

0/11 0/11 2/11 2/3 11/11

950

950

900

980

980

100 140 225 370 600 1,000 LDs0

15.4-Nitro-2aminophenol

10 100

1,000

0/3 0/3 3/3

140 225 370 600 1,000 LDs0

16. Folithion 50 EC

10 100 1,000

0/3 0/3 2/3

100 200 400 800 1,000 1,600 LDs0

17. Atropine sulphate

10 100

1,000

0/3 0/3 2/3

200 400 800 1,000 1,600 LDs0

18. MVK 0987

19. Pentafluorobenzyl alcohol

10 100 1,000

10 100 1,000

0/3 0/3 3/3

0/3 0/3 2/3


200 400 800 1,000 1,600

1,000 1,600 LDs0

650 0/11 0/11 0/11 2/11 3/3


283


20, Na hexobarbitone

21. DIC 3202

22. Dihydromethylindole

23. Ethylcresidine (distilled)

24. STJ 2900



Doses

Doses

10 100 1,000

t0 100 1,000

10 100 1,000

10 100 1,000

t0 100

1,000

25. KRA 3344b

26. BAY 1 8201

10 100 1,000

10 100 1,000

Mortality

0/3 0/3 1/3

0[3 0/3 1/3

0/3 0/3 0/3

0[3 0/3 0/3

0/3 0/3 0/3

0/3 0/3 0/3

0/3 0/3 0/3

100 600 1,000 1,600 2,900


2

3

5

Total

0/3 0/1 1/3 0/1 1/1

0/3 1/2 1/3 2/2 2/2

0/3 0/3 1/3 1/3 3/3

0/3 2/5 1/3 5/5 5/5

0/3 3/11 1/3 8/11 11/11

LDs0

2,150 1,000 1,600 1,000 1,000

1,000 1,600 2,900 5,000

1/3 1/1 1/1 1/1

1/3 2/2 2/2 2/2

1/3 3/3 3/3 3/3

1/3 5/5 5/5 5/5

LDso

1,250

1,250

1,250

1,250 1,250

1,000 1,600 2,900 5,000

0/3 1/1 1/1 1/1

0/3 2/2 2/2 2/2

0/3 3/3 3/3 3/3

0/3 5/5 5/5 5/5

LDs0

1,250 1,250 1,250 1,250 1,250

t ,000 1,600 2,900 5,000

0/3 1/1 1/1 0/1

LDs0

1,250 1,250 1,250 1,250 1,250

1,000 1,600 2,900 5,000

0/3 1/1 1/1 1/1

LD5o

1,250 1,250 1,250 1,250 1,250

1,000 1,600 2,900 5,000

0/3 1/1 1/1 1/1

LDso

1,250 1,600 1,250 1,250 1,500

1,00f~ 1,600 2,900 5,000

0/3 1/1 1/1 1/1

LD50

1,250 1,600 1,800 1,800 1,600

0/3 2/2 2/2 2/2

0/3 2/2 2/2 2/2

0/3 1/2 2/2

2/2

0/3 1/2 2/2 2/2

0/3 3/3 3/3 3/3

0/3 3/3 3/3 3/3

0/3 3/3 2/3 3/3

0/3 1/3 3/3 3/3

0/3 5/5 4/5 5/5

0/3 5/5 5/5 5/5

0/3 3/5 5/5 5/5

0/3 2/5 5/5 5/5

1/3 11/11 lull 11/11

0/3 11/11 11/11 11/11

0[3 11/11 10/11 11/11

0/3 11/11 11/11 11/11

013 7/11 10/11 11/11

013 5/11 11/11 lull

284

D. Lorke


27. Surcopur EC 25

28. Hedonal MP-T



Doses

Doses

10 100 1,000

10 100

1,000

29. FCR 1272 Y 10 WP

10 100 1,000

30. N,N-bis 10 (2-acetoxyethyl)- 100 aniline 1,000

31. p-Isooctylphenol

10 100

1,000

Mortality

0/3 0/3 0/3

0/3 0/3 0/3

0/3 0/3 0/3

0/3 0/3 0/3

0/3 0/3 0/3

32. ZnSO 4 7H20

10 100 1,000

0/3 0/3 0/3

33. SLJ 0312 85VM

10 100 1,000

0/3 0/3 0/3

34. CCI 4

10 100 1,000

0/3 0/3 0/3


2

3

5

Total

1,000 1,600 2,900 5,000

0/3 1/1 1/1 1/1

0/3 1/2 2/2 2/2

0/3 1/3 3/3 3/3

0/3 1/5 5/5 5/5

0/3 4/11 11/11 11/11

LD50

1,250 1,600 1,770 1,800 1,750

1,000 1,600 2,900 5,000

0/3 0/1 1/1 1/1

LDso

2,150 1,600 2,150 2,000 1,900

1,000 1,600 2,000 5,000

0/3 1/1 1/1 1/1

LDso

1,250 3,900 1,650 1,800 2,000

1,600 2,900 5,000

0/1 0/1 1/1

LDso

3,800 2,150 2,150 2,150 2,150

1,000 1,600 2,900 5,000

0/3 0/1 1/1 1/1

LDso

2,150 2,900 2,150 2,150 2,250

1,000 1,600 2,900 5,000

0/3 0/1 1/1 1/1

LDso

2,150 2,150 2,250 2,500 2,280

1,000 1,600 2,900 5,000

0/3 1/1 0/1 1/1

LDso

2,900 2,900 2,150 2,500 2,400

1,500 2,000 2,800 3,900

0/1 0/1 1/1 1/1

LD50

2,350 2,500 2,850 2,500 2,500

0/3 1/2 2/2 2/2

0/3 0/2 0/2 2/2

0/2 2/2 2/2

0/3 0/2 1/2 2/2

0/3 0/2 2/2 2/2

0/3 0/2 1/2 2/2

0/2 2/2 1/2 2/2

0/3 0/3 3/3 3/3

0/3 2/3 3/3 3/3

0/3 3/3 3/3

0/3 1/3 2/3 3/3

0/3 1/3 2/3 3/3

0/3 0/3 3/3 3/3

0/3 0/3 1/3 3/3

0/3 2/5 5/5 5/5

0/3 3/5 4/5 5/5

0/5 5/5 5/5

0/3 1/5 3/5 5/5

0/3 0/5 4/5 5/5

0/3 0/5 4/5 5/5

0/5 3/5 3/5 5/5

0/3 3/11 lull 11/11

0/3 6/11 8/11 11/11

0/11 10/11 11/11

0/3 2/11 7/11 lull

0/3 1/11 9/11 11/11

0/3 1/11 8/11 11/11

0/11 5/11 6/11 11/11


285


35. HST 1474



Doses

Doses

10 100 1,000

Mortality

0/3 0/3 0/3

1,000 1,600 2,900 5,000 LDs0

36. Dyrene 75 WP

37. Preventol B2

10 100 1,000

10 100

1,000

38. Eulan SP (816-143)

39. SMY 1500

10 100 1,000

10 100

1,000

0/3 0/3 0/3

0/3 0/3 0/3

0/3 0/3 0/3

0/3 0/3 1/3

1,600

10 100 1,000

0/3 0/3 0/3

10 100 1,000

0/3 0/3 0/3

10 100 1,000

0/3 0/3 0/3

3

5

0/3 0/1 0/1 0/1

0/3 0/2 0/2 0/2

0/3 2/3 1/3 3/3

0/3 4/5 5/5 5/5

>5,000>5,000

0/3 6/11 6/11 8/11

2,160 1,400 2,500

2,150 2,150 2,750 2,600 2,800

1,600 2,900 5,000

0/1 1/1 1/1

LDso

2,150 3,800 2,600 2,750 2,800

1,600 2,900 5,000

0/1 0/1 1/1

LDso

3,800 3,800 3,000 3,250 3,250

600

1,000 1,600 2,900

1,600 2,900 5,000

1,600 2,900 5,000

0/1 1/3 0/1 0/1

0/2 2/2 1/2

Total

LDs0

LDso 42. SSH 0860

2

0/1 1/1 1/1

LDs0 41. BAY a 1040

1

2,900 5,000

LDs0 40. NTN 19701


0/2 0/2 2/2

0/2 0/2 2/2

0/2 1/3 0/2 0/2

>2,900>2,900 0/1 0/1 0/1

0/2 0/2 0/2

0/3 2/3 3/3

0/3 2/3 3/3

0/3 1/3 3/3

0/3 1/3 0/3 2/3

0/5 4/5 5/5

0/5 3/5 4/5

0/5 2/5 4/5

0/5 1/3 0/5 1/5

0/11 9/11 10/11

0/11 6/11 10/11

0/11 3/11 10/11

0/11 1/3 0/11 3/11

2,000>2,900>2,900 0/3 0/3 0/3

0/5 0/5 0/5

0/11 0/11 0/11

>5,000>5,000>5,000>5,000>5,000 0/1 0/1 0/1

0/2 1/2 1/2

0/3 1/3 1/3

0/5 0/5 1/5

0/11 2/11 3/11

>5,000 2,900>5,000>5,000>5,000

1,600 2,900 5,000

0/1 0/1 1/1

0/2 0/2 i/2

0/3 0/3 1/3

0/5 1/5 3/5

0/11 1/11 6/11

LDs0

3,800 5,000 5,000 3,500 4,500

286

D. Lorke

Table 4. Investigations of the acute intravenous toxicity in male rats. All reported doses relate to mg/kg body weight Substance

1. BAY k 5552

2. BAY e 9736

3, Strophanthin

4. BAY a 1040

5. Sisomycin



Doses

Doses

10 100

10 100

10 100

10 100

10 100

Mortality

3/3 3/3

2/3 3/3

3/3 3/3

2/3 3/3

0/3 3/3


2

3

5

Total

1 2 4 8 10

0/1 1/1 1/1 1/1 3/3

0/2 2/2 2/2 2/2 3/3

0/3 2/3 3/3 2/3 3/3

2/5 5/5 4/5 4/5 3/3

2/11 10/11 10/11 9/11 3/3

LDs0

1,4

1,4

2

2

2

2 4 8 10 16 100

0/1 0/1 1/1 2/3 1/1 3/3

0/2 0/2 1/2 2/3 2/2 3/3

0/3 1/3 2/3 2/3 3/3 3/3

0/5 2/5 5/5 2/3 5/5 3/3

0/11 3/11 9/11 2/3 11/11 3/3

LDs0

6

8

6

5

6

0/1 0/1 0/1 0/1 3/3

0/2 0/2 0/2 0/2 3/3

0/3 0/3 0/3 2/3 3/3

0/5 0/5 0/5 3/5 3/3

LDs0

9

9

7

8

4 8 10 16 100

0/1 1/1 2/3 1/1 3/3

0/2 1/2 2/3 2/2 3/3

0/3 0/3 2/3 2/3 3/3

0/5 1/5 2/3 3/5 3/3

LDs0

6

9

10

12

10 25 40 60 100

0/3 0/1 1/1 1/1 3/3

0/3 0/2 1/2 2/2 3/3

0/3 0/3 0/3 2/3 3/3

0/3 0/5 0/5 4/5 3/3

LDso

32

40

55

55

1 2 4 8 10

0/11 0/11 0/11 5/11 3/3 8 0/11 3/11 2/3 8/11 3/3 10 0/3 0/11 2/11 9/11 3/3 55

Discussion The method of investigating acute toxicity which has been tested in practice s h o w s t h a t t h e r e is n o a d v a n t a g e i n u s i n g m o r e t h a n f i v e a n i m a l s p e r d o s e l e v e l . Indeed three animals per test group are entirely adequate. Nevertheless, three a n i m a l s p e r d o s e g r o u p is t h r e e t i m e s m o r e t h a n a s i n g l e a n i m a l p e r g r o u p . T h e


287

saving in animals by using one animal per group needs to be balanced against the unreliability in only 7% of the determinations of acute toxicity. It may be necessary to repeat approximately every 14th investigation. Balancing the advantages against the disadvantages, a one-animal group is recommended, since it provides adequate information about the acute toxicity. The saving in experimental animals using this method is outstanding. The determinations of acute toxicity described here by way of example show that, when the doses are appropriately selected, adequate information on the acute toxicity is generally obtained using 13 animals only. This is true in principle irrespective of the substance for all routes of administration and all dose ranges. Acknowledgement. I would like to thank my co-workers, Dr. Pauluhn, Dipl.-Biol. Mihail, Dr. Kr6tlinger and Mr. SchOngel for the technical conduct of the investigations. I also acknowledge the many valuable suggestions by Prof. Zbinden, Prof. Elias and Dr. Pauluhn.

References Bass R, Giinzel P, Henschler D, K6nig J, Lorke D, Neubert D, Schiitz E, Schuppan D, Zbinden G (1982) LDs0 Versus Acute Toxicity, Critical Assessment of the Methodology Currently in Use. Arch Toxicol 51:183-186 Bliss CI (1938) The determination of the dosage-mortality curve from small numbers. Q J Pharm Pharmacol 11 : 192-216 Hunter WJ, Lingk W, Recht R (1979) Intercomparison study on the determination of single administration toxicity in rats. J Assoc Off Anal Chem 62:864-873 Lingk W (1979) Eine Ringuntersuchung auf EG-Ebene zur Bestimmung der akuten oralen Toxizit~it an Ratten. Commission of the European Communities, Industrial Health and Safety: Quality assurance of toxicological data. Proceedings of the International Colloquium Luxembourg 1979. Report EUR 7270 EN p 89 Lorke D (1981) Zur Bedeutung von akuten Toxizit~itspriifungen. AMI-Bericht 1/1981. Inst Arzneimittel des BGA Rosiello AP, Essigmann JM, Wogan GN (1977) Rapid and accurate determination of the median lethal dose (LDs0) and its error with a small computer. J Toxicol Environm Health 3 : 797-809 Trevan JW (1927) The error of determination of toxicity. Proc R Soc (London) Ser. B. 101 : 483-514 Zbinden G, Flury-Roversi M (!981) Significance of the LDs0-Test for the toxicological evaluation of chemical substances. Arch Toxicol 47:77-99 Received May 5, 1983