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Arch Toxicot (1983) 54:275-287 TOXICOLOGY

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 46- 522 11.3
II 800-4,150 5.2
III 350-1,280 3.7
IV 805-5,420 6.7
V 70- 513 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-math-
ematical 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 Doses chosen for the second test

10 100 1,000
0/3* 0/3 0/3 1,600 2,900 5,000
0/3 0/3 1/3 600 1,000"* 1,600 2,900
0/3 0/3 2/3 200 400 800 1,600
0/3 0/3 3/3 140 225 370 600
0/3 1/3 3/3 50 100** 200 400
0/3 2/3 3/3 20 40 80 160
0/3 3/3 3/3 15 25 40 60
1/3 3/3 3/3 5 10"* 20 40
2/3 3/3 3/3 2 4 8 16
3/3 3/3 3/3 1 2 4 8
* Number of animals which died/number of animals used
** The result from the first test is taken over for these doses
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 .
A New Approach to Practical Acute Toxicity Testing 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 1st part of investigation 2nd part of investigation

Doses Mortality Doses Number of animals in each group

1 2 3 5 Total

1. KCN 10 3/3 2 0/1 0/2 0/3 0/5 0/11

100 3/3 4 0/1 0/2 1/3 1/5 2/11
1,000 3/3 9 1/1 1/2 3/3 5/5 10/11
14 1/1 2/2 3/3 5/5 11/11
LD5o 6 9 5 5 6
(Table continued)
280 D. Lorke

Table 3 (continued)

Substance 1st part of investigation 2nd part of investigation

Doses Mortality Doses Number of animals in each group

1 2 3 5 Total

2. HgC12 10 0/3 10 0/3 0/3 0/3 0/3 0/3

100 3/3 15 0/1 0/2 0/3 1/5 1/11
1,000 3/3 25 0/1 0/2 0/3 1/5 1/11
40 0/1 1/2 3/3 3/5 7/11
60 1/1 1/2 3/3 5/5 10/11
100 3/3 3/3 3/3 3/3 3/3
LDso 50 50 32 32 37

3. R 1513 10 0/3 14 0/1 0/2 0/3 0/5 0/11

100 3/3 22 0/1 0/2 0/3 0/5 0/11
1,000 3/3 37 0/1 0/2 1/3 0/5 1/11
60 1/1 2/2 3/3 4/5 i0/11
100 3/3 3/3 3/3 3/3 3/3
LDs0 50 50 40 60 50

4. Diphenyl- 10 0/3 14 0/1 0/2 0/3 0/5 0/11

thiourea 100 3/3 23 0/1 0/2 0/3 0/5 0/11
1,000 3/3 37 0/1 0/2 0/3 0/5 0/11
60 0/1 2/2 0/3 1/5 3/11
100 3/3 3/3 3/3 3/3 3/3
LDso 80 50 80 70 70

5. Oftanol C 10 0/3 50 0/1 0/2 0/3 0/5 0/11

100 1/3 100 1/3 1/3 1/3 1/3 1/3
1,000 3/3 200 1/1 2/2 3/3 5/5 11/11
400 1/l 2/2 3/3 5/5 11/11
LD5o 100 100 115 120 105

6. BAY h 1127 10 0/3 20 0/1 0/2 0/3 0/5 0/11

100 2/3 40 0/1 0/2 0/3 0/5 0/11
1,000 3/3 80 0/1 0/2 0/3 0/5 0/11
100 2/3 2/3 2/3 2/3 2/3
160 0/1 1/2 3/3 4/5 8/11
1,000 3/3 3/3 3/3 3/3 3/3
LDso 160 160 90 120 140

7. ARG 2032 10 0/3 100 0/3 0/3 0/3 0/3 0/3

100 0/3 140 1/1 2/2 2/3 2/5 7/11
1,000 3/3 225 1/1 2/2 3/3 5/5 11/11
370 1/1 2/2 3/3 5/5 11/11
600 1/1 2/2 3/3 5/5 11/11
LD5o 120 120 130 150 140
A New Approach to Practical Acute Toxicity Testing 281

Table 3 (continued)

Substance 1st part of investigation 2nd part of investigation

Doses Mortality Doses Number of animals in each group

1 2 3 5 Total

8.3-Bromo-l,4- 10 0/3 100 0/3 0/3 0/3 0/3 0/3

toluidine 100 0/3 140 0/1 0/2 1/3 0/5 1/11
1,000 3/3 225 0/1 1/2 1/3 3/5 5/11
370 1/1 2/2 3/3 5/5 11/11
600 1/1 2/2 3/3 5/5 11/11
LDs0 290 225 200 180 220

9. HOL 4578 10 0/3 140 0/1 1/2 0/3 0/5 1/11

100 0/3 225 0/1 0/2 2/3 1/5 3/11
1,000 3/3 370 0/1 2/2 1/3 5/5 7/11
600 1/1 2/2 3/3 4/5 10/11
1,000 3/3 3/3 3/3 3/3 3/3
LDs0 470 290 285 270 285

10. Cadmium 10 0/3 140 0/1 0/2 0/3 0/5 0/11

sulphate 100 0/3 225 0/1 0/2 0/3 1/5 1/11
1,000 3/3 370 0/1 2/2 2/3 3/5 7/11
600 1/1 2/2 3/3 5/5 11/11
1,000 3/3 3/3 3/3 3/3 3/3
LDs0 470 290 350 320 330

11. Levamisole 10 0/3 100 0/3 0/3 0/3 0/3 0/3

(HC1) 100 0/3 140 0/1 0/2 0/3 0/5 0/11
1,000 3/3 225 0/1 2/2 0/3 2/5 4/11
370 1/1 1/2 3/3 3/5 8/11
600 1/1 2/2 3/3 3/5 9/11
1,000 3/3 3/3 3/3 3/3 3/11
LDs0 290 180 290 350 310

12. Benzo- 10 0/3 140 0/1 0/2 0/3 0/5 0/11

thiazole 100 0/3 225 1/1 0/2 0/3 1/5 2/11
1,000 3/3 370 1/1 0/2 1/3 3/5 5/11
600 1/1 2/2 3/3 4/5 10/11
1,000 3/3 3/3 3/3 3/3 3/3
LDs0 180 470 370 375 375

13. Digitoxin 10 0/3 140 0/1 0/2 0/3 0/5 0/11

100 0/3 225 0/1 0/2 0/3 0/5 0/11
1,000 3/3 370 0/1 2/2 1/3 4/5 7/11
600 1/1 1/2 2/3 5/5 9/11
1,000 3/3 3/3 3/3 3/3 3/3
LDs0 470 290 460 350 450
282 D. Lorke

Table 3 (continued)

Substance 1st part of investigation 2nd part of investigation

Doses Mortality Doses Number of animals in each group

1 2 3 5 Total

14. Na barbitone 10 0/3 100 0/3 0/3 0/3 0/3 0/3

100 0/3 140 0/1 0/2 0/3 0/5 0/11
1,000 3/3 225 0/1 0/2 0/3 0/5 0/11
370 0/1 0/2 0/3 1/5 1/11
600 0/1 1/2 1/3 2/5 4/11
1,000 3/3 3/3 3/3 3/3 3/3
LDs0 770 600 650 600 650

15.4-Nitro-2- 10 0/3 140 0/1 0/2 0/3 0/5 0/11

aminophenol 100 0/3 225 0/1 0/2 0/3 0/5 0/11
1,000 3/3 370 0/1 0/2 0/3 0/5 0/11
600 0/1 0/2 0/3 2/5 2/11
1,000 3/3 3/3 3/3 3/3 3/3
LDs0 775 775 775 600 700

16. Folithion 50 EC 10 0/3 100 0/3 0/3 0/3 0/3 0/3

100 0/3 200 0/1 1/2 1/3 0/5 2/11
1,000 2/3 400 0/1 1/2 0/3 1/5 2/11
800 1/1 1/2 1/3 2/5 5/11
1,000 2/3 2/3 2/3 2/3 2/3
1,600 1/1 2/2 3/3 5/5 11/11
LDs0 570 400 900 700 700

17. Atropine 10 0/3 200 0/1 0/2 0/3 0/5 0/11

sulphate 100 0/3 400 0/1 0/2 0/3 0/5 0/11
1,000 2/3 800 0/1 0/2 1/3 1/5 2/11
1,000 2/3 2/3 2/3 2/3 2/3
1,600 1/1 2/2 3/3 5/5 11/!1
LDs0 950 950 900 950 900

18. MVK 0987 10 0/3 200 0/1 0/2 0/3 0/5 0/11
100 0/3 400 0/1 0/2 0/3 0/5 0/11
1,000 3/3 800 1/1 2/2 1/3 1/5 5/11
1,000 3/3 3/3 3/3 3/3 3/3
1,600 0/1 2/2 3/3 5/5 10/11
LDs0 570 570 680 830 900

19. Pentafluoro- 10 0/3 200 0/1 0/2 0/3 0/5 0/11

benzyl alcohol 100 0/3 400 0/1 0/2 0/3 0/5 0/11
1,000 2/3 800 0/1 0/2 1/3 1/5 2/11
1,000 2/3 2/3 2/3 2/3 2/3
1,600 1/1 2/2 3/3 5/5 11/11
LDs0 950 950 900 980 980
A New Approach to Practical Acute Toxicity Testing 283

Table 3 (continued)

Substance 1st part of investigation 2nd part of investigation

Doses Mortality Doses Number of animals in each group

1 2 3 5 Total

20, Na hexo- 10 0/3 100 0/3 0/3 0/3 0/3 0/3

barbitone 100 0/3 600 0/1 1/2 0/3 2/5 3/11
1,000 1/3 1,000 1/3 1/3 1/3 1/3 1/3
1,600 0/1 2/2 1/3 5/5 8/11
2,900 1/1 2/2 3/3 5/5 11/11
LDs0 2,150 1,000 1,600 1,000 1,000

21. DIC 3202 t0 0[3 1,000 1/3 1/3 1/3 1/3 1/3
100 0/3 1,600 1/1 2/2 3/3 5/5 11/11
1,000 1/3 2,900 1/1 2/2 3/3 5/5 lull
5,000 1/1 2/2 3/3 5/5 11/11
LDso 1,250 1,250 1,250 1,250 1,250

22. Dihydro- 10 0/3 1,000 0/3 0/3 0/3 0/3 0/3

methylindole 100 0/3 1,600 1/1 2/2 3/3 5/5 11/11
1,000 0/3 2,900 1/1 2/2 3/3 5/5 11/11
5,000 1/1 2/2 3/3 5/5 11/11
LDs0 1,250 1,250 1,250 1,250 1,250

23. Ethylcresidine 10 0[3 t ,000 0/3 0/3 0/3 0/3 0[3

(distilled) 100 0/3 1,600 1/1 2/2 3/3 5/5 11/11
1,000 0/3 2,900 1/1 2/2 3/3 4/5 10/11
5,000 0/1 2/2 3/3 5/5 11/11
LDs0 1,250 1,250 1,250 1,250 1,250

24. STJ 2900 t0 0/3 1,000 0/3 0/3 0/3 0/3 0/3
100 0/3 1,600 1/1 2/2 3/3 5/5 11/11
1,000 0/3 2,900 1/1 2/2 3/3 5/5 11/11
5,000 1/1 2/2 3/3 5/5 11/11
LD5o 1,250 1,250 1,250 1,250 1,250

25. KRA 3344b 10 0/3 1,000 0/3 0/3 0/3 0/3 013
100 0/3 1,600 1/1 1/2 3/3 3/5 7/11
1,000 0/3 2,900 1/1 2/2 2/3 5/5 10/11
5,000 1/1 2/2 3/3 5/5 11/11
LDso 1,250 1,600 1,250 1,250 1,500

26. BAY 1 8201 10 0/3 1,00f~ 0/3 0/3 0/3 0/3 013
100 0/3 1,600 1/1 1/2 1/3 2/5 5/11
1,000 0/3 2,900 1/1 2/2 3/3 5/5 11/11
5,000 1/1 2/2 3/3 5/5 lull
LD50 1,250 1,600 1,800 1,800 1,600
284 D. Lorke

Table 3 (continued)

Substance 1st part of investigation 2nd part of investigation

Doses Mortality Doses Number of animals in each group

1 2 3 5 Total

27. Surcopur EC 25 10 0/3 1,000 0/3 0/3 0/3 0/3 0/3

100 0/3 1,600 1/1 1/2 1/3 1/5 4/11
1,000 0/3 2,900 1/1 2/2 3/3 5/5 11/11
5,000 1/1 2/2 3/3 5/5 11/11
LD50 1,250 1,600 1,770 1,800 1,750

28. Hedonal MP-T 10 0/3 1,000 0/3 0/3 0/3 0/3 0/3
100 0/3 1,600 0/1 1/2 0/3 2/5 3/11
1,000 0/3 2,900 1/1 2/2 3/3 5/5 lull
5,000 1/1 2/2 3/3 5/5 11/11
LDso 2,150 1,600 2,150 2,000 1,900

29. FCR 1272 10 0/3 1,000 0/3 0/3 0/3 0/3 0/3
Y 10 WP 100 0/3 1,600 1/1 0/2 2/3 3/5 6/11
1,000 0/3 2,000 1/1 0/2 3/3 4/5 8/11
5,000 1/1 2/2 3/3 5/5 11/11
LDso 1,250 3,900 1,650 1,800 2,000

30. N,N-bis 10 0/3 1,600 0/1 0/2 0/3 0/5 0/11

(2-acetoxyethyl)- 100 0/3 2,900 0/1 2/2 3/3 5/5 10/11
aniline 1,000 0/3 5,000 1/1 2/2 3/3 5/5 11/11
LDso 3,800 2,150 2,150 2,150 2,150

31. p-Isooctyl- 10 0/3 1,000 0/3 0/3 0/3 0/3 0/3

phenol 100 0/3 1,600 0/1 0/2 1/3 1/5 2/11
1,000 0/3 2,900 1/1 1/2 2/3 3/5 7/11
5,000 1/1 2/2 3/3 5/5 lull
LDso 2,150 2,900 2,150 2,150 2,250

32. ZnSO 4 10 0/3 1,000 0/3 0/3 0/3 0/3 0/3

7H20 100 0/3 1,600 0/1 0/2 1/3 0/5 1/11
1,000 0/3 2,900 1/1 2/2 2/3 4/5 9/11
5,000 1/1 2/2 3/3 5/5 11/11
LDso 2,150 2,150 2,250 2,500 2,280
33. SLJ 0312 10 0/3 1,000 0/3 0/3 0/3 0/3 0/3
85VM 100 0/3 1,600 1/1 0/2 0/3 0/5 1/11
1,000 0/3 2,900 0/1 1/2 3/3 4/5 8/11
5,000 1/1 2/2 3/3 5/5 11/11
LDso 2,900 2,900 2,150 2,500 2,400

34. CCI 4 10 0/3 1,500 0/1 0/2 0/3 0/5 0/11

100 0/3 2,000 0/1 2/2 0/3 3/5 5/11
1,000 0/3 2,800 1/1 1/2 1/3 3/5 6/11
3,900 1/1 2/2 3/3 5/5 11/11
LD50 2,350 2,500 2,850 2,500 2,500
A New Approach to Practical Acute Toxicity Testing 285

Table 3 (continued)

Substance 1st part of investigation 2nd part of investigation

Doses Mortality Doses Number of animals in each group

1 2 3 5 Total

35. HST 1474 10 0/3 1,000 0/3 0/3 0/3 0/3 0/3
100 0/3 1,600 0/1 0/2 2/3 4/5 6/11
1,000 0/3 2,900 0/1 0/2 1/3 5/5 6/11
5,000 0/1 0/2 3/3 5/5 8/11
LDs0 >5,000>5,000 2,160 1,400 2,500

36. Dyrene 75 WP 10 0/3 1,600 0/1 0/2 0/3 0/5 0/11

100 0/3 2,900 1/1 2/2 2/3 4/5 9/11
1,000 0/3 5,000 1/1 1/2 3/3 5/5 10/11
LDs0 2,150 2,150 2,750 2,600 2,800

37. Preventol B2 10 0/3 1,600 0/1 0/2 0/3 0/5 0/11

100 0/3 2,900 1/1 0/2 2/3 3/5 6/11
1,000 0/3 5,000 1/1 2/2 3/3 4/5 10/11
LDso 2,150 3,800 2,600 2,750 2,800

38. Eulan SP 10 0/3 1,600 0/1 0/2 0/3 0/5 0/11

(816-143) 100 0/3 2,900 0/1 0/2 1/3 2/5 3/11
1,000 0/3 5,000 1/1 2/2 3/3 4/5 10/11
LDso 3,800 3,800 3,000 3,250 3,250

39. SMY 1500 10 0/3 600 0/1 0/2 0/3 0/5 0/11
100 0/3 1,000 1/3 1/3 1/3 1/3 1/3
1,000 1/3 1,600 0/1 0/2 0/3 0/5 0/11
2,900 0/1 0/2 2/3 1/5 3/11
LDs0 >2,900>2,900 2,000>2,900>2,900

40. NTN 19701 10 0/3 1,600 0/1 0/2 0/3 0/5 0/11
100 0/3 2,900 0/1 0/2 0/3 0/5 0/11
1,000 0/3 5,000 0/1 0/2 0/3 0/5 0/11
LDs0 >5,000>5,000>5,000>5,000>5,000

41. BAY a 1040 10 0/3 1,600 0/1 0/2 0/3 0/5 0/11
100 0/3 2,900 0/1 1/2 1/3 0/5 2/11
1,000 0/3 5,000 0/1 1/2 1/3 1/5 3/11
LDso >5,000 2,900>5,000>5,000>5,000

42. SSH 0860 10 0/3 1,600 0/1 0/2 0/3 0/5 0/11
100 0/3 2,900 0/1 0/2 0/3 1/5 1/11
1,000 0/3 5,000 1/1 i/2 1/3 3/5 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 1st part of investigation 2nd part of investigation

Doses Mortality Doses Number of animals in each group

1 2 3 5 Total

1. BAY k 5552 10 3/3 1 0/1 0/2 0/3 2/5 2/11

100 3/3 2 1/1 2/2 2/3 5/5 10/11
4 1/1 2/2 3/3 4/5 10/11
8 1/1 2/2 2/3 4/5 9/11
10 3/3 3/3 3/3 3/3 3/3
LDs0 1,4 1,4 2 2 2

2. BAY e 9736 10 2/3 2 0/1 0/2 0/3 0/5 0/11

100 3/3 4 0/1 0/2 1/3 2/5 3/11
8 1/1 1/2 2/3 5/5 9/11
10 2/3 2/3 2/3 2/3 2/3
16 1/1 2/2 3/3 5/5 11/11
100 3/3 3/3 3/3 3/3 3/3
LDs0 6 8 6 5 6

3, Strophanthin 10 3/3 1 0/1 0/2 0/3 0/5 0/11

100 3/3 2 0/1 0/2 0/3 0/5 0/11
4 0/1 0/2 0/3 0/5 0/11
8 0/1 0/2 2/3 3/5 5/11
10 3/3 3/3 3/3 3/3 3/3
LDs0 9 9 7 8 8

4. BAY a 1040 10 2/3 4 0/1 0/2 0/3 0/5 0/11

100 3/3 8 1/1 1/2 0/3 1/5 3/11
10 2/3 2/3 2/3 2/3 2/3
16 1/1 2/2 2/3 3/5 8/11
100 3/3 3/3 3/3 3/3 3/3
LDs0 6 9 10 12 10

5. Sisomycin 10 0/3 10 0/3 0/3 0/3 0/3 0/3

100 3/3 25 0/1 0/2 0/3 0/5 0/11
40 1/1 1/2 0/3 0/5 2/11
60 1/1 2/2 2/3 4/5 9/11
100 3/3 3/3 3/3 3/3 3/3
LDso 32 40 55 55 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
A New Approach to Practical Acute Toxicity Testing 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