articulo lorke 17115

7/30/2019 Articulo Lorke 17115

1/13

Arch Toxicot (1983) 54:275-287 AfI~IvSST O X I C O L O G Y9 Springer-Verlag 1983

A New Approach to Pract i ca l Acute Toxic i ty Test ingD i e t r i c h L o r k eInstitut f~ir Toxikolog ie, Bayer A G ,Friedrich-Ebert-Strasse 217, D -5600 W uppertal, Federal R epublic of G ermany

A b s tr a c t . A m e t h o d f o r th e i n v e s t i g a ti o n o f t h e a c u t e t o x i c i ty o f a n u n k n o w nc h e m i c a l s u b s t a n c e , w i t h a n e s t i m a t i o n o n t h e L D s 0 , is d e s c r i b e d . U s i n g t h i s ,i t is p o s s ib l e t o o b t a i n w i t h 1 3 e x p e r i m e n t a l a n i m a l s a d e q u a t e i n f o r m a t i o n o nt h e a c u t e t o x i c it y a n d o n t h e L D s 0. T h i s m e t h o d h a s n o l i m i t at io n s a n da p p l i e s t o d r u g s , a g r i c u l t u r a l a n d i n d u s t r i a l c h e m i c a l s . I t c a n b e u s e d f o re v e r y r o u t e o f a d m i n i s t ra t i o n .K e y w o r d s : A c u t e t o x i c it y - L D s 0 - F e w e r a n i m a ls

I n tr o d u c to r y C o n s i d e r a t i o n sI n v e s t i g a t i o n o f t h e a c u t e t o x i c i t y is th e f ir s t st e p i n t h e t o x i c o l o g i c a li n v e s ti g a t io n s o f a n u n k n o w n s u b s t an c e . T h e i n d e x o f th e a c u t e t o x i c it y is t h eL D s 0. H o w e v e r , t h e L D s 0 s h o u l d n o t b e r e g a r d e d a s a b io l o g ic a l c o n s t a n t , s i n c ed i f fe r in g r e s u lt s a r e o b t a i n e d o n r e p e t i t i o n o r w h e n t h e d e t e r m i n a t i o n s a r ec a r r i e d o u t i n d i f fe r e n t l a b o r a t o ri e s . T h i s h a s b e e n s h o w n v e r y c l e a rl y i n am u l t i c e n t r e s tu d y c a r r i e d o u t i n t h e E u r o p e a n c o m m u n i t y w i t h f iv e s u b s t a n c e s( H u n t e r e t a l. 1 9 79 ; L i n g k 1 9 7 9 ). I n t h is s t u d y t h e L D s 0 v a l u e s v a r i e d a s f o l l o w si n T a b l e 1 .

F u r t h e r e x a m p l e s , w h i c h s h o w t h a t L D s 0 v a l u e s c a n n e v e r b e r e g a r d e d a sb i o lo g i c a l c o n s t a n t s, h a v e b e e n s u r v e y e d b y Z b i n d e n a n d F l u r y - R o v e r s i (1 9 8 1) .Table 1Com pound LDs0 mg/kg Ratio of largest figure

to smallest figureI 4 6 - 522 11.3II 800-4,150 5.2III 350-1,280 3.7IV 805-5,420 6.7V 70 - 513 7.3


2/13

276 D. Lorke

The conclusion to be drawn from these results is that it is impossible in principleto 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 ahigh precision?In order to answer this question the historical development of LDs0 testneeds to be considered.In an effort to assess the acute toxicity of a substance, a simple means ofgrading acute poisonous effects was sought. It was desirable to have a test withwhich 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 dealingwith the substance in practice. With this objective, a figure which expresses thetoxic effects was sought. This figure was intended to indicate the amount of thesubstance which is injurious after a specified mode of intake. Since the terminjuries always involves a high degree of subjectivity, an objective criterion wasselected, namely death. Thus, a means of determining the likelihood of death asexactly as possible was sought. The LDs0 was recognised and justified of beingthe best parameter by Trevan (1927). Unfortunately, this has led to thedetermination of the LDs0 being equated with investigation into acutetoxicity.A scientifically valid investigation into acute toxicity is necessary and f rommany points of view of great value (Lorke 1981). It is however not the intentionto deal with this aspect again here. It is misleading to equate the determinationof acute toxicity with that of the determination of the LDs0 although this ofteno c c u r s .Due to this misunderstanding various mathematical and statistical methodshave been developed to est imate the LDs0 and its range of variation in order toreport the acute toxicity as accurately as possible justified by statistical-math-ematical methods.

All these methods require a considerable number of animals in order tocalculate an LDs0, and, implicit in their mathematical design is, that using moreanimals increases the accuracy of the figure reported.The question arises as to the value of very great accuracy for an individualcase in view of the poor reproducibility of such biological determinations.

Moreover, even if the LDs0 could be measured exactly and reproducibly, theknowledge of its precise numerical value would barely be of practicalimportance, because an extrapolation from the experimental animals to man ishardly 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 greatinterest, and this information can be obtained from careful studies with smallnumber of animals.

Having criticised the prevailing evaluation of acute toxicity and theassociated calculation of the LDs0, the question arises, how determinations ofacute toxicity should be carried out using the smallest number of animals pergroup.It is now proposed that the acute toxicity should be tested in twosteps:


3/13

A New App roach to Practical Acute Toxicity Testing 2771 . I n t h e i n it ia l i n v e s t i g a t i o n s t h e r a n g e o f d o s e s p r o d u c i n g t h e t o x i c e f fe c t s ise s t a b l i s h e d .2 . B a s e d o n t h e s e r e s u l ts , f u r t h e r s p e c if i c d o s e s a r e a d m i n i s t e r e d t o c a lc u l a t e a nL D s 0 .T h e m e t h o d d e s c r ib e d h e r e is b a s e d o n t h e a s s u m p t i o n t h a t th e c h e m i c a ls u b s t a n c e t o b e i n v e s t i g a t e d is c o m p l e t e l y u n k n o w n a n d t h a t t h e i n v e s t i g a t io n i st o b e c a r r ie d o u t w i t h a m i n i m u m n u m b e r o f e x p e r i m e n t a l a n im a l s .

I t is i n it ia l ly n e c e s s a r y t o d e t e r m i n e t h e a p p r o x i m a t e r a n g e o f th e a c u t et o x i c it y . T h i s is a c h i e v e d b y g i v i n g w i d e l y d i f fe r i n g d o s e s t o t h e a n i m a l s , e . g . , 1 0 ,1 0 0, a n d 1 ,0 0 0 m g / k g b . w . T h e r e s u lt s s h o w w h e t h e r a s u b s t a n c e is v e r y t o x ic ,t o x i c , l e s s t o x i c , o r o n l y s l i g h t l y t o x i c .

H o w m a n y a n i m a l s i n e a c h g r o u p s h o u l d b e u s e d f o r t h e s e i n v e s t i g a t i o n s ?W e h a v e e x a m i n e d t h e p r o b l e m u s in g e i th e r a s in g le a n i m a l o r th r e e a n i m a l s p e rd o s e l e v e l . F r o m t h is e m e r g e d t h e e f f e c t t h a t u s i n g a s in g le a n i m a l in e a c h g r o u pc a n l e a d t o fa l se a s s e s s m e n t w h e n , b y c h a n c e , t h e a n i m a l in th e n o n - t o x i c r a n g ed i e s o r t h a t i n t h e t o x i c r a n g e s u r v i v e s . F o r t h i s r e a s o n i t i s p r o p o s e d t h a t t h r e ea n i m a l s in e a c h g r o u p s h o u l d b e u s e d t o d e t e r m i n e t h e t o x i c r a n g e . T h e r e s u lt o ft h i s t e s t i s u s e d a s a b a s i c f o r s e l e c t i n g t h e s u b s e q u e n t d o s e s .

T h e f o ll o w in g a s s u m p t i o n s a r e m a d e w i th r e s p e c t to t h e s u b s e q u e n t d o s a g es c h e d u l e s :1 . S u b s t a n c e s m o r e t o x i c t h a n 1 m g / k g a r e s o h i g h l y t o x i c t h a t i t is n o t s oi m p o r t a n t t o c a l c u l a te t h e L D s 0 e x a c t ly .2 . L D s 0 v a l u e s g r e a t e r t h a n 5 , 0 0 0 m g / k g a r e o f n o p r a c t i c a l i n t e re s t .3 . A n a p p r o x i m a t e f i g u r e f o r th e L D s 0 is u s u a l ly a d e q u a t e t o e s t i m a t e t h e r i sk o fa c u t e i n t o x i c a t i o n .

B a s e d o n t h e s e c o n s i d e r a t i o n s a n d p r a c t ic a l e x p e r i e n c e t h e d o s e s l i st e d i n t h et a b l e a r e b a s e d o n t h e r e s u l ts o f th e f i rs t i n v e s t i g a t i o n . T h e n e w d o s a g e s a r ea d m i n i s t e r e d t o t h e a n i m a l s i n t h e s e c o n d t e s t ( T a b l e 2 ) .

I n t h e T a b l e 3 , s o m e d o s e s d e v i a t e f r o m t h o s e g i v e n , s in c e th e d o s e s f in a l lyr e c o m m e n d e d o n l y e m e r g e d d u r in g t h e c o u r s e o f t h e i nv e s ti g at io n s .T a b l e 2Doses in m g/kg body weightResult of the initial investigation Doses cho sen for the seco nd test10 100 1,0000/3 * 0/3 0/3 1,600 2,900 5,0000/3 0/3 1/3 600 1,000"* 1,600 2,9000/3 0/3 2/3 200 400 800 1,6000/3 0/3 3/3 140 225 370 6000/3 1/3 3/3 50 100 ** 200 4000/3 2/3 3/3 20 40 80 1600/3 3/3 3/3 15 25 40 601/3 3/3 3/3 5 10"* 20 402/3 3/3 3/3 2 4 8 163/3 3/3 3/3 1 2 4 8

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


4/13

278 D. Lor keH 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 fo r t h e s e c o n d t e s ts ?I n o r d e r t o e s t a b l i s h t h i s , e a c h o f t h e p r o p o s e d d o s e s w a s g i v e n t o g r o u p s

c o n s i s t i n g o f o n e a n i m a l , t w o a n i m a l s , t h r e e a n i m a l s a n d f iv e a n im a l s . T h ea l g e b r a i c s u m o f t h e s e r e s u l t s is p r e s e n t e d a s i n c i d e n c e f o r a g r o u p c o n t a i n i n ge l e v e n a n i m a l s .

Me t h o d s

The invest igat ions reported in the tables on pages 10-21 were carried out as fol lows.Anim al s were t rea t ed wi th subs tances a f t e r a t l eas t 5 days of adap ta t ion . They w ere observedfrequent ly on the day of t rea tmen t dur ing normal working hours and the na ture and t ime of a l ladverse e ffec ts w ere noted . W here any an imal s d i ed the t ime of dea th w as noted , dur ing the 1s t dayin hours and ther eafte r in days. Dea d animals were autop sied and exam ined macroscopically for anypatho logical changes. The surviving animals were weighe d according the O EC D guidel ineno. 401.If animals rec over ed and gained weight aga in, this was taken as a sign of having survived theacute intoxicat ion. Observa t ions and weighings were carried on for 14 days and th e ex perim ent wasthen t e rm inated . I f no recovery was noted dur ing th is per iod or i f l a te dea ths occurred , t hen theper iod of obse rvat ion was exten ded int i l the bod y weight of the surviving animals increased clearly.Th e exp erim ent was then cons idered as comp leted. Al l surviving animals were sacri ficed at the endof each test and the n autop sied and exa min ed macroscopically for an y pathological changes.

Re su l t s

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 i n 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 eo f T o x i c o l o g y . T h e r e s u l t s a r e c o m p i l e d in th e T a b l e 3 . T h e L D s 0 w a s e s t i m a t e di n 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 o n t h e d o s e s f o r w h i c h 0 /1 a n d 1 /1 w e r e f o u n d .O n e a n i m a l g r o u p :E x a m p l e 1 : 2 , 9 0 0 m g / k g b o d y w e ig h t 0/1

5 , 00 0 m g / k g b o d y w e i g h t 1 /1L D s 0 = 3 , 8 0 0 m g / k g .

E x a m p l e 2 :

E x a m p l e 3 :

3 7 0 m g / k g b o d y w e i g h t 0 /16 0 0 m g / k g b o d y w e i g h t 1 /1L D s 0 = 4 7 0 m g / k g .2 00 m g / k g b o d y w e i g h t 1 /14 0 0 m g / k g b o d y w e i g h t 0 /16 0 0 m g / k g b o d y w e i g h t 1 /1L D s 0 = 4 0 0 m g / k g .

W h e n t h e r e s u l t s o f t h e f i r s t t e s t g iv e r is e to p a r t i c u l a r s i t u a t io n s ( e . g . , s u b s t a n c e sn o s . 2 0 a n d 3 9 ) i t i s t h e n n e c e s s a r y t o e s t i m a t e t h e r e s u l t s a p p r o p r i a t e l y , o r tor e p e a t t h e t e s t .

O t h e r g r o u p s :I f a d o s e w i t h 0 % l e t h a l i t y i s f o l l o w e d b y o n e o f 1 0 0 % , t h e n t h e L D s 0 is

e s t i m a t e d a s f o r t h e o n e - a n i m a l g r o u p .


5/13

A New Approach to Practical Acute Toxicity Testing 279If 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 proc edure 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 differentnumbers of animals it was assumed that the figures obtained with 11 animals pergroup are the best estimates. The est imated LDs0s of the ot her groups were thenassessed by their agreement with the figure from the ll-animal group.

An agreement was assumed to exist when the difference did not exceed afactor of 2 (Bass et al. 1982). The substances were selected so that the entirerange of toxicity from high acute toxicity to virtual non-toxicity, was tested. Thesubstances not only included drugs and agricultural chemicals but also industrialchemicals.

Among the 42 substances which were investigated using the male rat, thefactor of 2 was exceeded twice in the one-animal group. These two compoundsare HST 1474 and benzothiazole. Two out of 42 test means, only 7% of all testsexceeded the factor 2. The LDs0 for the substance H ST 1474 was 2,500 mg/kg inthe ll-animal group and > 5,000 mg/kg in the one-animal group, and forbenzothiazole it was 375 mg/kg in the 11-animal group and 180 mg/kg in theone-animal group. In this latter instance, the factor is not remarcably larger than2 (= 2.08). Thus even in these two cases, the results from the one-animal groupare 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 fromintravenous administration (Table 4), so that the method is not restricted to oraladministration, but is valid, in principle for all routes of administration.Since the substances r epresente d different classes of chemical structures themethod can be used irrespective of whether the substance is a drug, anagricultural chemical or an industrial chemical.Table 3. Investigations of the acute oral toxicity in male rats. Al l repor ted doses relate to mg/kg bodyweightSubstance 1st part of investigation

Doses Mortality2nd part of investigationDoses Number of animals in each group

1 2 3 5 Total1. KCN 10 3/3100 3/31,000 3/3

2 0/1 0/2 0/3 0/5 0/114 0/1 0/2 1/3 1/5 2/119 1/1 1/2 3/3 5/5 10/1114 1/1 2/2 3/3 5/5 11/11LD5o 6 9 5 5 6

(Table continued)


6/13


7/13

A New Approach to Practical Acute Toxicity TestingTable 3 (continued)

281

Substance 1st part of investigationDoses Mortality

2nd part of investigationDoses Number of animals in each group

1 2 3 5 Total8.3-Bromo-l,4-toluidine

9. HOL 4578

10. Cadmiumsulphate

11. Levamisole(HC1)

10 0/3100 0/31,000 3/3

10 0/3100 0/31,000 3/3

10 0/3100 0/31,000 3/3

10 0/3100 0/31,000 3/3

100 0/3 0/3 0/3 0/3 0/3140 0/1 0/2 1/3 0/5 1/11225 0/1 1/2 1/3 3/5 5/11370 1/1 2/2 3/3 5/5 11 /11600 1/1 2/2 3/3 5/5 11/11LDs0 290 225 200 180 220

140 0/1 1/2 0/3 0/5 1/11225 0/1 0/2 2/3 1/5 3/11370 0/1 2/2 1/3 5/5 7/11600 1/1 2/2 3/3 4/5 10/111,000 3/3 3/3 3/3 3/3 3/3LDs0 470 290 285 270 285

140 0/1 0/2 0/3 0/5 0/11225 0/1 0/2 0/3 1/5 1/11370 0/1 2/2 2/3 3/5 7/11600 1/1 2/2 3/3 5/5 11/11

1,000 3/3 3/3 3/3 3/3 3/3LDs0 470 290 350 320 330

100 0/3 0/3 0/3 0/3 0/3140 0/1 0/2 0/3 0/5 0/11225 0/1 2/2 0/3 2/5 4/11370 1/1 1/2 3/3 3/5 8/11600 1/1 2/2 3/3 3/5 9/11

1,000 3/3 3/3 3/3 3/3 3/11LDs0 290 180 290 350 310

12. Benzo-thiazole

13. Digitoxin

10 0/3100 0/31,000 3/3

10 0/3100 0/31,000 3/3

140 0/1 0/2 0/3 0/5 0/11225 1/1 0/2 0/3 1/5 2/11370 1/1 0/2 1/3 3/5 5/11600 1/1 2/2 3/3 4/5 10 /111,000 3/3 3/3 3/3 3/3 3/3LDs0 180 470 370 375 375

140 0/1 0/2 0/3 0/5 0/11225 0/1 0/2 0/3 0/5 0/11370 0/1 2/2 1/3 4/5 7/11600 1/1 1/2 2/3 5/5 9/111,000 3/3 3/3 3/3 3/3 3/3LDs0 470 290 460 350 450


8/13

282Table 3 (continued)

D. Lorke

Substance 1st part of investigationDoses Mortality

2nd part of investigationDoses Number of animals in each group

1 2 3 5 Total14. Na barbitone

15.4-Nitro-2-aminophenol

16. Folithion 50 EC

17. Atropinesulphate

18. MVK 0987

10 0/3100 0/31,000 3/3

10 0/3100 0/31,000 3/3

10 0/3100 0/31,000 2/3

10 0/3100 0/31,000 2/3

10 0/3100 0/31,000 3/3

19. Pentafluoro- 10 0/3benzyl alcohol 100 0/31,000 2/3

100 0/3 0/3 0/3 0/3 0/3140 0/1 0/2 0/3 0/5 0/11225 0/1 0/2 0/3 0/5 0/11370 0/1 0/2 0/3 1/5 1/11600 0/1 1/2 1/3 2/5 4/11

1,000 3/3 3/3 3/3 3/3 3/3LDs0 770 600 650 600 650

140 0/1 0/2 0/3 0/5 0/11225 0/1 0/2 0/3 0/5 0/11370 0/1 0/2 0/3 0/5 0/11600 0/1 0/2 0/3 2/5 2/11

1,000 3/3 3/3 3/3 3/3 3/3LDs0 775 775 775 600 700

100 0/3 0/3 0/3 0/3 0/3200 0/1 1/2 1/3 0/5 2/11400 0/1 1/2 0/3 1/5 2/11800 1/1 1/2 1/3 2/5 5/111,000 2/3 2/3 2/3 2/3 2/3

1,600 1/1 2/2 3/3 5/5 11 /11LDs0 570 400 900 700 700

200 0/1 0/2 0/3 0/5 0/11400 0/1 0/2 0/3 0/5 0/11800 0/1 0/2 1/3 1/5 2/111,000 2/3 2/3 2/3 2/3 2/31,600 1/1 2/2 3/3 5/5 11/!1LDs0 950 950 900 950 900

200 0/1 0/2 0/3 0/5 0/11400 0/1 0/2 0/3 0/5 0/11800 1/1 2/2 1/3 1/5 5/111,000 3/3 3/3 3/3 3/3 3/31,600 0/1 2/2 3/3 5/5 10 /11LDs0 570 570 680 830 900

200 0/1 0/2 0/3 0/5 0/11400 0/1 0/2 0/3 0/5 0/11800 0/1 0/2 1/3 1/5 2/111,000 2/3 2/3 2/3 2/3 2/31,600 1/1 2/2 3/3 5/5 11/11LDs0 950 950 900 980 980


9/13

A N e w A p p r o a c h t o P r a c t i c a l A c u t e T o x i c i t y T e s t i n gT a b l e 3 ( c o n t i n u e d )

283

S u b s t a n c e 1s t pa r t o f i nve s t i ga t i onD o s e s M o r t a l i t y

2 n d p a r t o f i n v e s t i g a t i o nD o s e s N u m b e r o f a n i m a ls i n e a c h g r o u p

1 2 3 5 To t a l2 0 , N a h e x o -b a r b i t o n e

2 1 . D I C 3 2 02

10 0/3100 0/3

1,000 1 /3

t 0 0 [3100 0/3

1,000 1 /3

2 2 . D i h y d r o - 1 0 0 / 3m e t h y l i n d o l e 1 00 0 /3

1,000 0 /3

23 . E t hy l c re s i d i n e 10 0 [3(dis t i l l ed ) 100 0 /3

1,000 0 /3

24 . S TJ 2900

2 5 . K R A 3 3 4 4b

2 6 . B A Y 1 8 20 1

t 0100

1,000

10100

1,000

101001,000

0 /30 /30 /3

0 /30 /30 /3

0 /30 /30 /3

100 0/3 0 /3 0 /3 0 /3 0 /3600 0/1 1/2 0/3 2/5 3/11

1,000 1/3 1/3 1/3 1/3 1/31,60 0 0/1 2/2 1/3 5/5 8/112,900 1 /1 2 /2 3 /3 5 /5 11/11

LDs0 2 ,150 1 ,000 1 ,600 1 ,000 1 ,000

1,000 1/3 1/3 1/3 1/3 1/31,600 1/1 2/2 3/3 5/5 11/112 ,900 1 /1 2 / 2 3 / 3 5 / 5 l u l l5 ,000 1 /1 2 /2 3 /3 5 /5 11/11LDso 1,250 1,250 1,250 1,250 1,250

1,000 0 /3 0 /3 0 /3 0 /3 0 /31,600 1/1 2/2 3/3 5/5 11/112,900 1 /1 2 /2 3 /3 5 /5 11/115,000 1 /1 2 /2 3 /3 5 /5 11/11LDs0 1 ,250 1 ,250 1 ,250 1 ,250 1 ,250

t ,000 0/3 0 /3 0 /3 0 /3 0[31,600 1/1 2/2 3/3 5/5 11/112,900 1 /1 2 /2 3 /3 4 /5 10/115,000 0 /1 2 /2 3 /3 5 /5 11/11LDs0 1 ,250 1 ,250 1 ,250 1 ,250 1 ,250

1,000 0 /3 0 /3 0 /3 0 /3 0 /31,600 1/1 2/2 3/3 5/5 11/112,90 0 1/1 2/2 3/3 5/5 11/115,00 0 1/1 2/2 3/3 5/5 11/11LD5o 1 ,250 1 ,250 1 ,250 1 ,250 1 ,250

1,000 0 /3 0 /3 0 /3 0 / 3 0 1 31,600 1/1 1/2 3/3 3/5 7/112,900 1 /1 2/2 2/3 5/5 10/115,00 0 1/1 2/2 3/3 5/5 11/11LDso 1 ,250 1 ,600 1 ,250 1 ,250 1 ,500

1,00f~ 0/3 0/3 0/3 0/3 0131,60 0 1/1 1/2 1/3 2/5 5/112,900 1 /1 2 /2 3 /3 5 /5 11/115 ,000 1 /1 2 / 2 3 / 3 5 / 5 l u l lLD50 1 ,250 1 ,600 1 ,800 1 ,800 1 ,600


10/13

2 8 4 D . L o r k eTable 3 ( c o n t i n u e d )S u b s t a n c e 1s t pa r t o f i nve s t i ga t i on

D o s e s M o r t a l i t y2 n d p a r t o f i n v e s t i g a t i o nD o s e s N u m b e r o f a n i m a l s i n e a c h g r o u p

1 2 3 5 To t a l2 7 . S u r c o p u r E C 2 5 1 0 0 / 3

100 0/31,000 0/3

2 8 . H e d o n a l M P - T 1 0 0 /3100 0/31,000 0 /3

29 . F C R 1272 10 0 / 3Y 10 W P 100 0 / 3

1,000 0 /3

30 . N ,N -b i s 10 0 / 3(2 -a c e t ox ye t hy l ) - 100 0 / 3a n i l i ne 1 ,000 0 / 3

31. p-Iso oc ty l - 10 0 /3p h e n o l 1 00 0 / 3

1,000 0 /3

3 2 . Z n S O 4 1 0 0 / 37 H 2 0 1 00 0 / 31,000 0 /3

33. SLJ 0312 10 0 /38 5 V M 1 00 0 /31,000 0 /3

1,000 0 /3 0 /3 0 /3 0 /3 0 /31,600 1/1 1/2 1/3 1/5 4/112,90 0 1/1 2/2 3/3 5/5 11/115,00 0 1/1 2/2 3/3 5/5 11/11LD50 1 ,250 1 ,600 1 ,770 1 ,800 1 ,7501,000 0 /3 0 /3 0 /3 0 /3 0 /31,600 0/1 1/2 0/3 2/5 3/112 ,900 1 /1 2 / 2 3 / 3 5 /5 l u l l5,00 0 1/1 2/2 3/3 5/5 11/11LDso 2 ,150 1 ,600 2 ,150 2 ,000 1 ,9001,000 0/3 0/3 0/3 0/3 0/31,600 1/1 0/2 2/3 3/5 6/112,00 0 1/1 0/2 3/3 4/5 8/115,00 0 1/1 2/2 3/3 5/5 11/11LDso 1 ,250 3 ,900 1 ,650 1 ,800 2 ,0001,600 0/1 0/2 0/3 0/5 0/112,90 0 0/1 2/2 3/3 5/5 10/115,00 0 1/1 2/2 3/3 5/5 11/11LDso 3 ,800 2 ,150 2 ,150 2 ,150 2 ,1501,000 0 /3 0 /3 0 /3 0 /3 0 /31,600 0/1 0/2 1/3 1/5 2/112,90 0 1/1 1/2 2/3 3/5 7/115,000 1 /1 2 /2 3 /3 5 /5 l u l lLDso 2 ,150 2 ,900 2 ,150 2 ,150 2 ,2501,000 0 /3 0 /3 0 /3 0 /3 0 /31,600 0/1 0/2 1/3 0/5 1/112,90 0 1/1 2/2 2/3 4/5 9/115,00 0 1/1 2/2 3/3 5/5 11/11LDso 2 ,150 2 ,150 2 ,250 2 ,500 2 ,2801,000 0/3 0/3 0/3 0/3 0/31,600 1/1 0/2 0/3 0/5 1/112,90 0 0/1 1/2 3/3 4/5 8/115,00 0 1/1 2/2 3/3 5/5 11/11LDso 2 ,900 2 ,900 2 ,150 2 ,500 2 ,400

34. CC I 4 101001,000

0 /30 /30 /3

1,500 0/1 0/2 0/3 0/5 0/112,00 0 0/1 2/2 0/3 3/5 5/112,80 0 1/1 1/2 1/3 3/5 6/113,90 0 1/1 2/2 3/3 5/5 11/11LD50 2 ,350 2 ,500 2 ,850 2 ,500 2 ,500


11/13

A N e w A p p r o a c h t o P r a c t i c a l A c u t e T o x i c i t y T e s t i n gT a b l e 3 ( c o n t i n u e d )

285

S ub s t a nc e 1 st pa r t o f i nve s t i ga t i onD o s e s M o r t a l i t y

2 n d p a r t o f i n v e s t i g a t i o nD o s e s N u m b e r o f a n i m a ls i n e a c h g r o u p

1 2 3 5 To t a l3 5 . H S T 1 4 74

3 6 . D y r e n e 7 5 W P

3 7 . P r e v e n t o l B 2

3 8 . E u l a n S P( 8 1 6 - 1 4 3 )

3 9 . S M Y 1 5 0 0

40 . NT N 19701

4 1 . B A Y a 1 0 4 0

42 . S S H 0860

10 0/3100 0/3

1,000 0 /3

10 0/3100 0/31,000 0 /3

10 0/3100 0/31,000 0 /3

10 0/3100 0/3

1,000 0 /3

10 0/3100 0/31,000 1/3

10 0/3100 0/3

1,000 0 /3

10 0/3100 0/3

1,000 0 /3

10 0/3100 0/3

1,000 0 /3

1,000 0/3 0 /3 0 /3 0 /3 0 /31 ,600 0 /1 0 /2 2 /3 4 /5 6 /112,90 0 0/1 0/2 1/3 5/5 6/115,000 0 /1 0 /2 3 /3 5 /5 8 /11L D s0 > 5 , 0 0 0 > 5 , 0 0 0 2 , 1 6 0 1 , 40 0 2 , 5 0 0

1,600 0/1 0/2 0/3 0/5 0/112,900 1 /1 2 /2 2 /3 4 /5 9 /115,00 0 1/1 1/2 3/3 5/5 10/11LDs0 2 ,150 2 ,150 2 ,750 2 ,600 2 ,800

1,600 0/1 0/2 0/3 0/5 0/112,900 1 /1 0 /2 2 /3 3 /5 6 /115,000 1 /1 2 /2 3 /3 4 /5 10/11LDso 2 ,150 3 ,800 2 ,600 2 ,750 2 ,800

1,600 0/1 0/2 0/3 0/5 0/112,90 0 0/1 0/2 1/3 2/5 3/115,00 0 1/1 2/2 3/3 4/5 10/11LDso 3 ,800 3 ,800 3 ,000 3 ,250 3 ,250

600 0/1 0 /2 0 /3 0 /5 0 /111,000 1/3 1/3 1/3 1/3 1/31 ,600 0 /1 0 /2 0 /3 0 /5 0 /112,90 0 0/1 0/2 2/3 1/5 3/11L D s0 > 2 , 9 0 0 > 2 , 9 0 0 2 , 0 0 0 > 2 , 9 0 0 > 2 , 9 0 01,600 0 /1 0 /2 0 /3 0 /5 0 /112,90 0 0/1 0/2 0/3 0/5 0/115,00 0 0/1 0/2 0/3 0/5 0/11L D s 0 > 5 , 0 0 0 > 5 , 0 0 0 > 5 , 0 0 0 > 5 , 0 0 0 > 5 , 0 0 0

1,600 0 /1 0 /2 0 /3 0 /5 0 /112,90 0 0/1 0/2 0/3 1/5 1/115,00 0 1/1 i /2 1/3 3/5 6/11LDs0 3 ,800 5 ,000 5 ,000 3 ,500 4 ,500

1,600 0/1 0/2 0/3 0/5 0/112,90 0 0/1 1/2 1/3 0/5 2/115,00 0 0/1 1/2 1/3 1/5 3/11L D so > 5 , 0 0 0 2 , 9 0 0 > 5 , 0 0 0 > 5 , 0 0 0 > 5 , 0 0 0


12/13

2 8 6 D . L o r k eT a b l e 4 . I n v e s t i g a t i o n s o f t h e a c u t e i n t r a v e n o u s t o x i ci t y i n m a l e r a t s . A l l r e p o r t e d d o s e s r e l a t e t om g / k g b o d y w e i g h tS u b s t a n c e 1 s t p a r t o f i n v e s t i g a t i o n

D o s e s M o r t a l i t y2 n d p a r t o f i n v e s t i g a t i o nD o s e s N u m b e r o f a n i m a l s i n e a c h g r o u p

1 2 3 5 To t a l1 . B A Y 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 10/114 1/1 2/2 3/3 4/5 10/118 1/1 2/2 2/3 4/5 9/11

10 3/3 3/3 3/3 3/3 3/3LDs0 1 ,4 1 ,4 2 2 2

2 . B A Y e 9736 10 2 /3 2 0 /1 0 /2 0 /3 0 /5 0 /11100 3/3 4 0/1 0/2 1/3 2/5 3/11

8 1/1 1/2 2/3 5/5 9/1110 2/3 2 /3 2 /3 2 /3 2 /316 1/1 2/2 3/3 5/5 11/11

100 3/3 3/3 3/3 3/3 3/3LDs0 6 8 6 5 6

3 , S t ro ph a n t h i n 10 3 / 3 1 0 /1 0 / 2 0 / 3 0 / 5 0 /11100 3/3 2 0/1 0/2 0/3 0/5 0/11

4 0/1 0/2 0/3 0/5 0/118 0/1 0/2 2/3 3/5 5/11

10 3/3 3/3 3/3 3/3 3/3LDs0 9 9 7 8 8

4 . B A Y a 1040 10 2 /3 4 0 /1 0 /2 0 /3 0 /5 0 /11100 3/3 8 1/1 1/2 0/3 1/5 3/11

10 2/3 2/3 2/3 2/3 2/316 1/1 2/2 2/3 3/5 8/11

100 3/3 3/3 3/3 3/3 3/3LDs0 6 9 10 12 10

5. S isom ycin 10 0 /3 10 0 /3 0 /3 0 /3 0 /3 0 /3100 3/3 25 0/1 0/2 0/3 0/5 0/11

40 1/1 1/2 0/3 0/5 2/1160 1/1 2/2 2/3 4/5 9/11

100 3/3 3/3 3/3 3/3 3/3LDso 32 40 55 55 55

Di s cu s s i on

T h e m e t h o d o f i n v e s t i g a t i n g a c u t e t o x i c i t y w h i c h h a s b e e n t e s t e d i n p r a c t i c es h o w s t h a t t h e r e i s 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 .I n d e e d t h r e e a n i m a l s p e r t e s t g r o u p a r e e n t i r e l y a d e q u a t e . N e v e r t h e l e s s , t h r e ea n i m a l s p e r d o s e g r o u p i s 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


13/13

A N e w A p p r o a c h t o P r ac tic al A c u t e T o x i c i t y T e s t i n g 2 8 7s a v i n g in a n i m a l s b y u s i n g o n e a n i m a l p e r g r o u p n e e d s t o b e b a l a n c e d a g a i n s t t h eu n r e l i a b i li t y in o n l y 7 % o f t h e d e t e r m i n a t i o n s o f a c u t e to x i c i t y . I t m a y b en e c e s s a r y t o r e p e a t a p p r o x i m a t e l y e v e r y 1 4 t h i n v e s t i g a t i o n .

B a l a n c i n g t h e a d v a n t a g e s a g a i n s t t h e d i s a d v a n t a g e s , a o n e - a n i m a l g r o u p i sr e c o m m e n d e d , s i n c e it p r o v i d e s a d e q u a t e i n f o r m a t i o n a b o u t t h e a cu t etox ic i ty .

T h e s a v i n g i n e x p e r i m e n t a l a n i m a l s u s i n g t h i s m e t h o d i s o u t s t a n d i n g .T h e d e t e r m i n a t i o n s o f a c u te t o x i c it y d e sc r i b ed h e r e b y w a y o f e x a m p l e s h o w

t h a t , w h e n t h e d o s e s a r e a p p r o p r i a t e l y s e l e c t e d , a d e q u a t e i n f o r m a t i o n o n t h eacute tox ic i ty i s gen eral ly ob ta ine d us ing 13 an im als on ly . Th is is t rue in pr inc ip lei r r e s p e c t i v e o f t h e s u b s t a n c e f o r a l l r o u t e s o f a d m i n i s t r a t i o n a n d a l l d o s er a n g e s .Acknowledgement. I w o u l d l i k e t o t h a n k m y c o - w o r k e r s , D r . P a u l u h n , D i p l . - B i o l . M i h a i l , D r .K r 6 t l in g e r a n d M r . S c h O n g e l f o r t h e t e c h n i c a l c o n d u c t o f t h e i n v e s t i g a t io n s . I a l s o a c k n o w l e d g e t h em a n y v a l u a b l e s u g g e st i o n s b y P r o f. Z b i n d e n , P r o f . E l i a s a n d D r . P a u l u h n .

R e f e r e n c e sB a s s R , G i i n z e l P , H e n s c h l e r D , K 6 n i g J , L o r k e D , N e u b e r t D , S c h i it z E , S c h u p p a n D , Z b i n d e n G(1982) LDs 0 Ver s us Acu te Tox ic i ty , Cr i t i ca l As s es s men t o f t h e M e t h o d o l o g y C u r r e n t l y i n U s e .A r c h T o x ic o l 5 1 : 1 8 3 - 1 8 6B l i s s CI (1938) T h e d e t e r m i n a t i o n o f t h e d o s a g e - m o r t a l i t y c u r v e fr o m s m a l l n u m b e r s . Q J P h a r mP h a r m a c o l 1 1 : 1 9 2 - 2 1 6H u n t e r W J , L i n g k W , R e c h t R ( 1 9 7 9 ) I n t e r c o m p a r i s o n s t u d y o n t h e d e t e r m i n a t i o n o f s i n g l e

a d m i n i s t r a t i o n t o x i c i t y i n r a t s . J A s so c O f f A n a l C h e m 6 2 : 8 6 4 - 8 7 3L i n g k W ( 19 79 ) E i n e R i n g u n t e r s u c h u n g a u f E G - E b e n e z u r B e s t i m m u n g d e r a k u t e n o r a l e n T o xiz it ~i ta n R a t t e n . C o m m i s si o n o f t h e E u r o p e a n C o m m u n i ti e s , I n d u s t r i a l H e a l t h a n d S a f e t y : Q u a l i t ya s s u r a n c e o f t o x i c o l o g i c a l d a t a . P r o c e e d i n g s o f t h e I n te r n a t i o n a l C o l l o q u i u m L u x e m b o u r g 1 9 7 9.R e p o r t E U R 7 27 0 E N p 8 9Lo rke D (1981) Z ur B edeu tung v on aku ten Tox iz it~i ts p ri ifungen . AM I-B er ich t 1 /1981 . In s tA r z n e i m i t t e l d e s B G AR o s i e ll o A P , E s s ig m a n n J M , W o g a n G N (1 97 7) R a p i d a n d a cc ur at e d e t e r m i n a t i o n o f t h e m e d i a nl e t h a l d o s e ( L D s 0 ) a n d i t s e r r o r w i t h a s m a l l c o m p u t e r . J T o x i c o l E n v i r o n m H e a l t h3 : 7 9 7 - 8 0 9T r e v a n J W ( 19 27 ) T h e e r r o r o f d e t e r m i n a t i o n o f t ox i c it y . P r o c R S o c ( L o n d o n ) S e r . B .101 : 48 3-5 14

Z b i n d e n G, F lu ry -Ro ver s i M ( !981) S ign i f i cance o f the LDs 0-Test fo r the tox ico log ica l eva lu a t ion o fc h e m i c a l s u b s t a n c e s . A r c h T o x ic o l 4 7 : 7 7 - 9 9

R e c e i v e d M a y 5, 1983

articulo lorke 17115

Documents