ArchToxicol (1992) 66:455-470
Archives of
Toxicology 9 Springer-Verlag 1992
Review
A national validation study of the acute-toxic-class m e t h o d an alternative to the LD50 test E. Schlede, U. Mischke, R. Roll, and D. Kayser AbteilungChemikalienbewertung, Max yon Pettenkofer-Insdtut, Bundesgesundheitsamt, Thielallee 88-92, W-1000 Berlin 33, Federal Republic of Germany Received 1 May 1992/Accepted 19 May 1992
Abstract. In a national colloborative study an alternative to the classical LDs0 test - the acute-toxic-class method was validated. With this testing procedure mortality ranges are determined between defined dose levels that are used for classification and labelling in the European Community. The results were compared with LDs0 data obtained from the literature which were categorized according to the defined dose levels. The results of this collaborative study have shown that the acute-toxic-class method allows allocation to the toxicity classes of very toxic, toxic, harmful and unclassified in the same manner as on the basis of the classical LDs0 tests. The acute-toxic-class method uses fewer animals and subjects fewer animals to pain and distress than the LDs0 test and yields the same information on toxic signs in the treated animals. Identical classifications were obtained by the six participating laboratories in 86% of the tests. This demonstrates that the acute-toxic-class method results in excellent reproducibility in comparison to the classical LDs0 test and that this new method is a reliable alternative to the LDs0 test. Key words: Acute-toxic-class method - Alternative LDs0 test - Animal welfare - Classification - Risk assessment
Introduction The determination of the mean lethal dose (LDso) for the evaluation of the acute oral toxicity of a chemical or any other test material is usually the first step in a series of other follow-up toxicological studies. An LDso value also serves for risk assessment purposes for the classification of chemicals that in turn leads to specific labelling, packaging and use criteria. With the LDso test, groups of experimental animals are treated with graduated doses of a test substance
Correspondence to: E. Schlede
with the aim of obtaining a 50% or even higher mortality at the highest doses. In addition, this test can give information on the dose response of toxic signs and to some extent on pathological findings. Internationally accepted guidelines recommend the use of at least three doses with ten animals (five males and five females) for each dose (OECD 1981; Commission of the European Communities 1984) or with five animals for each dose level (three groups of one sex and one group of the other sex; OECD 1987). A single dose of 2000 mg/kg body weight with the use of ten animals (five males and five females) is recommended for substances where no death is anticipated, the so-called limit test (OECD 1987). The scientific significance of the classical LDs0 test has been questioned on the basis of the relatively broad variability of the test results and for animal welfare reasons (Zbinden and Flury-Roversi 1981; Bass et al. 1982; Tattersall 1982). For the evaluation of the acute oral toxicity of substances it is not necessary to derive this information with an LDs0 test using large numbers of animals but instead with the use of only few animals that are carefully monitored for signs of toxicity and signs of recovery or mortality. Until now no validated in vitro method is available to investigate the acute oral toxicity and for the time being this information has to be gained from animal studies. Numerous alternatives to the LD50 test have been published, with the aim of reducing the number of animals and gaining detailed information on toxic signs and mortality ranges or mortality (Deichmann and LeBlanc 1943; Lorke 1983; Bruce 1985; Kennedy et al. 1986; Yamanaka et al. 1990). None of these methods are accepted as official guidelines by national regulatory authorities or by international organizations like the OECD. Recently, another alternative test procedure - the fixed dose method - has gained acceptance from the OECD and the European Community (van den Heuvel et al. 1990). In principle this method relies on signs of toxicity rather than on mortality for the evaluation of the toxicity of a test material and it uses 50% fewer animals when compared to the LDs0 test (OECD 1981).
456 TaMe 1. List of participants in the national validation study
Schering AG, Berlin Institute of Experimental Toxicology (P. GiJnzel, Chr. Sehrbel)
known. In general all available information, including that on structure activity relationships (SAR), is used for the selection of the starting dose. For the selection of the vehicle it was recommended to consider the use of water (including saline) first, followed by considerations of a solution in arachis oil, then of an aqueous suspension using Tween, carboxylmethylcellulose or polyethyeleneglycol as a formulating agent. All participants used the Wistar rat for the tests because this rat strain was being used for other toxicological tests in their laboratories. The experiments were started in April 1989 and all tests were completed by November 1990. The substances were supplied from the Bundesgesundheitsamt to the participants. They were at least of analytical grade and each substance was part of one lot. One laboratory was unable to perform the testing with phenobarbital. Therefore althogether only 179 tests instead of 180 tests were carried out.
Bundesgesundheitsamt, Berlin Abteilung Chemikalienbewertung, Max yon Pettenkofer Institut (U. Mischke, R. Hagen)
Method
BASF AG, Ludwigshafen Department of Toxicology (P. Kirsch) Bayer AG, Wuppertal Institute of Industrial Toxicology (J. A. Ivens-Kohl, R. Jaeger) Boehringer Ingelheim, Ingelheim Department of Experimental Pathology and Toxicology (A. Schuster) Henkel KGaA, Dtisseldorf Abteilung Forschung and Entwicklung (M. Potokar)
A t the B u n d e s g e s u n d h e i t s a m t ( B G A ) a n o t h e r a l t e r n a t i v e test has b e e n d e v e l o p e d - the a c u t e - t o x i c - c l a s s m e t h od. W i t h this m e t h o d e v e n f e w e r a n i m a l s are u s e d , at the s a m e t i m e p r e s e r v i n g the a s p e c t s o f h u m a n h e a l t h p r o t e c tion. T h i s test p r o c e d u r e has b e e n v a l i d a t e d in a n a t i o n a l c o l l o b o r a t i v e s t u d y a n d the results o f this s t u d y are rep o r t e d here.
Study design The testing procedure of the acute-toxic-class method was evaluated on a mathematical basis with the use of the probit analysis (Finney 1971) before the start of the validation study. Based on the mathematical results it could be demonstrated that the probability of the accurate classification is equal or even better for hazard assessment with this method than with the classical LDso method (Roll et al. 1989). The testing procedure for the acute-toxic-class method is also applicable for dosages that are used in other countries or organisations for classification purposes (Siccha et al. 1992). Five laboratories from the German chemical industry and one laboratory from the Bundesgesundheitsamt participated in this study (Table 1). The Bundesgesundheitsamt was responsible for the coordination of the study. Thirty substances were selected on the basis of their physico-chemical properties and of their toxicity profile, including also four substances (cadmium chloride, aniline, sodium salicylate and acetanilide) that have been already tested in two previous LDs0 validation studies (Hunter et al. 1979; Lingk 1982). The results of the acute-toxic-class tests were taken in relation to the LDso data derived from the literature. According to the German Act on Animal Welfare, it is prohibited to repeat animal studies for which data are already available in the literature. Therefore a thorough literature search was carried out in order to find as many oral LDs0 values for rats as possible for each substance (Table 2). For one substance only one reference but otherwise at least two and up to ten references were documented in the literature. As expected, the values varied widely for a substance from one reference to the next but also within one publication. The substances are listed according to their LDs0 values, aldicarb being the most toxic and ethylene glycol being the least toxic substance. The allocation of the substances to the different toxicity classes followed the classification criteria of the European Community (Commission of the European Communities 1983, Table 3). In the literature signs of toxicity and autopsy findings were inconsistently described or not recorded at all. Therefore they were not included in Table 2. The national validation study was not conducted "blind". The participants argued that they preferred to perform the tests under conditions of practice. In the majority of the laboratories before testing the chemical structure and the physico-chemical properties of a new substance are
The acute-toxic-class method is described in detail in Appendix 1. It includes three graphic charts of the test procedure with the options to start with 25, or 200 or 2000 mg/kg body weight. Also included are the criteria after which no further testing is necessary for classification purposes in the European Community (Appendix 2). The format follows the OECD Guidelines for toxicity testing. In the collaborative study a slightly different testing procedure as outlined in Appendix 1 was used for doses of 2000 mg/kg body weight. No further testing was necessary when either in step 1 and/or in step 2 none or one animal had died. For future testing with this dose the procedure was changed in order to meet international classification requirements: no further testing is now necessary when none of the animals tested either in step 1 and/or step 2 had died. As already indicated, the dose levels of 25,200 and 2000 mg/kg body weight follow the classification criteria of the European Community. In principle the testing is a stepwise procedure with the use of only three rats per step. The choice of the sex is optional at the starting dose but it is recommended to use the more sensitive sex if there is any information available. The time interval between treatments is determined by the duration of toxic signs. The animals had to be free of toxic signs for at least 2 days before the next treatment (if necessary) could be started with the use of additional three rats. The participants decided to use mainly mortality as the endpoint in this study. The reason for this decision was that definitions such as "extreme distress" or "evident toxicity" are not clearly defined in the literature. The participants argued that from their experience rats showing severe toxic signs may be able to survive. Nevertheless, they were free to kill the animals for animal welfare reasons if they considered it necessary. Great importance was attached to the observations of toxic signs following administration. Observations had to be made at least 6 times during the day of treatment followed by daily observations until the end of the study (or until death). The signs of toxicity that had to be observed are shown in Table 4.
Results T h e test r e p o r t s w e r e e v a l u a t e d a c c o r d i n g to t h e following criteria: 1) c l a s s i f i c a t i o n o f the s u b s t a n c e s to any of the f o u r t o x i c i t y c l a s s e s in c o m p a r i s o n to the LD50 data and their a l l o c a t e d t o x i c i t y class; 2) t a b u l a t i o n o f signs o f toxicity; 3) a u t o p s y f i n d i n g s a n d 4) n u m b e r o f a n i m a l s used and n u m b e r o f d e a d a n i m a l s . B a s e d o n t h e p u b l i s h e d LD50 d a t a a l o n e ( T a b l e 2) the a l l o c a t i o n to o n e o f t h e f o u r t o x i c i t y c l a s s e s w a s possible f o r the f o l l o w i n g 17 s u b s t a n c e s : Aldicarb, parathion, di-isopropylfluorophosphate, t h i o s e m i c a r b a z i d e , i n d o m e t h a c i n a n d p h e n y l t h i o u r e a are very toxic. S o d i u m a r s e n i t e , a l d r i n a n d a l l y l a l c o h o l are toxic.
457 Table 2. LDso values cited in the literature and EC classification Nr.
Substance
CAS no.
LDso (mg/kg body weight) (95% confidence limits or ___ se in brackets)
Literature
Classification according to EC criteria
1
Aldicarb
116-06-3
f ca. 1.0 m 0.8 (0.6-1.0); f0.65 (0.50-0.85) rn/f 3.2-5.0
Weiden et al. 1965 Gaines 1969 van den Heuvel et al. 1990
very toxic
2
Parathion
56-38-2
m 15; f 6 m 5; f 1.75 m 1.8 (1.26-2.57) m 30 (+3.6); f 3 (+0.25) (m/f) 6.5 m5-15 f3.2 (2.7-3.8) m 13 (10-17); f 3.6 (3.2-4) m8.5;f10 fca. 2,8
Du Bois et al. 1949 Hazleton and Holland 1950 Weiss and Orzel 1967 Frawley et al. 1952 Wirth 1954 Klimmer and Pfaff 1955 Edson and Noakes 1960 Gaines 1960 Nishizawa et al. 1961 Casida and Sanderson 1963
very toxic
3
Di-isopropylfluorophosphate
55-91-4
(m/f) 6.0 (m/f) 5.0- 10.0 m 13.5 (--+0.35); f7.7 (-+0.64)
Boyland and McDonald 1944 Coon 1946 Frawley et al. 1952
very toxic
4
Thiosemiearbazide
79-19-6
(m/f) 11.0 (-I-2); (m/f) 18 (-+2) (m/f) 9.2
Dieke 1949 Marhold 1977
very toxic
5
Indomethacin
53-86-1
(m/f) 12 (10-14) (m/f) 14
Goldenthal 1971 Niemegeers et al. 1975
very toxic
6
Phenylthiourea
103-85-5
(m/f) 8.6 ( -+0.6)
Dieke et al. 1947
very toxic
7
Mercury (II) oxide
21908-53-2
m 18 (7-44); (m/f) 46 ( + 5 )
Vernot et al. 1977 Trakhtenberg et al. 1981
very toxic/ toxic
8
Sodium arsenite
7784-46-5
(m/f) 41 m 36 ( 2 7 - 52), f 4 8 (37-76)
Smyth et al. 1969 van den Heuvel et al. 1990
toxic
9
Aldrin
309-00-2
(m/f) 67 (m/f) 38; (m/f) 49 m 59.6 (-+ 1.86); f52.3 (-+5.75) m 49; f45.9 (35.8-54.2) m 39 (34-47); f 6 0 (50-72) (m/f) 80.0
Lehman 1951 Bergmann et al. 1952 Ball et al. 1953 Treon and Cleveland 1955 Gaines 1960 Deichmann and Keplinger 1965
toxic
10
Allylalcohol
107-18-6
(m/f) 64 (56-74) (m/f) 99 (75-130)
Smyth et al. 1951 Dunlop et al. 1958
toxic
11
Bis (tributyltin) oxide
56-35-9
m 148 - 194 (m/f) 112 (m/f) 234 (m/f) 180 (-+48) (m/f) 87
Elsea and Paynter 1958 Klimmer 1969 Sheldon 1975 Truhaut et al. 1976 Marhold 1986
toxic/harmful
12
Acrylamide
79-06-1
f 150-180 f203 (160-249)
toxic/harmful
(m/f) 124
McCollister et al. 1964 Fullerton and Barnes 1966 Marhold 1972 Paulet and Vidal 1975
(m/f) 170 13
Cadmiumchloride
10108-64-2
(m/f) 88 m 70-513; f 140-500 m 103-482; f 8 4 - 4 8 2
Lehman 1951 Hunter et al. 1979 Lingk 1982
toxic/harmful
14
Methyl Chloroformate
79-22-1
(m/f) 60 (31.5-118) m 90 (100 -280); f 110 (70-160) rrdf 313 (329-427)
Gurova et al. 1977 Vernot et al. 1977 Gelbke and Jiickh 1981
toxic/harmful
15
Phenobarbital
50-06-6
(m/f) 220; (m/f) 162 -+ 14; (m/f) 318___23
Goldenthal 1971
toxic/harmful
16
Caffeine
58-08-2
(m/f) 233 -+ 14 (m/f) 192 ( _ 18) m 355 ( 3 1 2 - 403); f 247 (220- 277) m 483
Scott and Chen 1944 Boyd 1965 Palm et al. 1978 Kennedy et al, 1986
toxic/harmful
458 Table 2. Continue Nr..
Substance
CAS no.
LDs0 (mg/kg body weight) (95 % confidence limits or ___ se in brackets)
Literature
Classification according to EC criteria
17
Bariumcarbonate
513-77-9
(m/f) 630- 750
harmful
(m/l) 800
Farm Chemicals Handbook 1980 Izmerow 1982 Merck Index 1989
18
Amline
62-53-3
m 440 (340- 570) m 450 m 350-1280; f 378-1062 m 479-1169; f 4 7 9 - 1 8 5 0
Jacobson 1972 Czajkowska et al. 1977 Hunter et al. 1979 Lingk 1982
harmful
19
Fe~ocene
102-54-5
(m/l) 1320
harmful
(m/l) 418
m >2000; f 1260-2000
Shell Chemical Comp. 1961 Documentation 1986 van den Heuvel et al. 1990
20
m-Dichlorobencene
541-73-1
f 2300 (2052- 2591) m 580 (400-825)
Leist and Weigand 1979 Bayer 1980
harmful/ unclassified
21
Sodium salicylate
54-21-7
(m/l) 1600 (m/f) 1190, 1200 m 800-4150; f 958-4028 m 930-2329; f 8 8 3 - 2 5 5 5
Hart 1947 Drebinger 1950 Hunter et al. 1979 Lingk 1982
harmful
22
Acetanilide
103-84-4
(m/f) 800 m 804-5420; f 915-4350 m 723-3060; f 594-3250 m 2033 (1368-2858); f 1893 (1218-2459)
Smith and Hambourger 1935 Hunter et al. 1979 Lingk 1982 van den Heuvel et al. 1990
harmful
23
Sodiumlaurylsulphate
151-21-3
m/f 1288 (1216-1376) (m/f) 2640
Gale and Scott 1953 Walker et al. 1967
harmful/ unclassified
24
Acetonitrile
75-05-8
(m/l) 3800
Smyth and Carpenter 1948 Pozzani et al. 1959 Kimura et al. 1971 Purchase et al. 1987 van den Heuvel et al. 1990
harmful/ unclassified
(m/l) 1000
m 1312-3507; f 1730-6662 m 3046 (m/f) 1170-6650 m 1453 (1123-1879); f>2000 25
Benzyl benzoate
120-51-4
(m/f) 2800 (m/f) 1700
Graham and Kuizenga 1945 Draize et al. 1948
harmful/ unclassified
26
o-Phenylphenol
90-43-7
(m/l) 3000 m 2700 (2400- 3100) (m/l) 2000 - 4000
Macintosh 1945 Hodge et al. 1952 Kaneda et al. 1978
harmful/ unclassified
27
Butylated hydroxyanisole
25013-16-5
(m/f) 4100 (m/f) 2200 (m/f) 2900 (m/f) 2200 m 2950 (m/f) 2000 (m/f) 4100-5000
Wilder and Kraybill 1949 Lehman 1951 Kaprylik 1959 Dacre 1960 Daniyalov 1966 Hiraga et al. 1970 Furia and Belanca 1977
harmful/ unclassified
28
N,N- Dimethyl formamide
68-12-2
(m/f) 70oo (m/f) 3000
unclassified
(m/f) 2800 m/f 7205 - 8100 m/f >2000
Smyth et at. 1951 Thiersch 1967 Druckrey et al. 1967 Bartsch et al. 1976 van den Heuvel et al. 1990
29
Quercetin dihydrate
6151-25-3
m/f >2000
van den Heuvel et at. 1990
unclassified
30
Ethylene Glycol
107-21-1
(m/f) 8540
Smyth et al. 1950 Filatova et al. 1982
unclassified
(m/l) 4700 m = male, f = female; m/f = male and female groups combined; (m/l) = sex not specified. Values on slopes were only available for four substances: parathion m 1.50 (1.00-2.25), (Weiss and Orzel, 1950); sodium arsenite m 7.6 ( + 2.3), (van den Heuvel et al., 1990); caffeine m 5.1, f 7.7
(Palm et al. 1978); acetanilide m 7.0 (+2.2), (van den Heuvel et al, 1990). The LDso value of phenylthiourea (m/f 126-400 mg/kg body weight) was not included because of the difficulties in the preparation of the test solution (van den Heuvel et al. 1990)
459 Table3. Toxicityclasses of the EuropeanCommunity LDs0range
Toxicityclass
LD 50 < 25 25
Archives of
Toxicology 9 Springer-Verlag 1992
Review
A national validation study of the acute-toxic-class m e t h o d an alternative to the LD50 test E. Schlede, U. Mischke, R. Roll, and D. Kayser AbteilungChemikalienbewertung, Max yon Pettenkofer-Insdtut, Bundesgesundheitsamt, Thielallee 88-92, W-1000 Berlin 33, Federal Republic of Germany Received 1 May 1992/Accepted 19 May 1992
Abstract. In a national colloborative study an alternative to the classical LDs0 test - the acute-toxic-class method was validated. With this testing procedure mortality ranges are determined between defined dose levels that are used for classification and labelling in the European Community. The results were compared with LDs0 data obtained from the literature which were categorized according to the defined dose levels. The results of this collaborative study have shown that the acute-toxic-class method allows allocation to the toxicity classes of very toxic, toxic, harmful and unclassified in the same manner as on the basis of the classical LDs0 tests. The acute-toxic-class method uses fewer animals and subjects fewer animals to pain and distress than the LDs0 test and yields the same information on toxic signs in the treated animals. Identical classifications were obtained by the six participating laboratories in 86% of the tests. This demonstrates that the acute-toxic-class method results in excellent reproducibility in comparison to the classical LDs0 test and that this new method is a reliable alternative to the LDs0 test. Key words: Acute-toxic-class method - Alternative LDs0 test - Animal welfare - Classification - Risk assessment
Introduction The determination of the mean lethal dose (LDso) for the evaluation of the acute oral toxicity of a chemical or any other test material is usually the first step in a series of other follow-up toxicological studies. An LDso value also serves for risk assessment purposes for the classification of chemicals that in turn leads to specific labelling, packaging and use criteria. With the LDso test, groups of experimental animals are treated with graduated doses of a test substance
Correspondence to: E. Schlede
with the aim of obtaining a 50% or even higher mortality at the highest doses. In addition, this test can give information on the dose response of toxic signs and to some extent on pathological findings. Internationally accepted guidelines recommend the use of at least three doses with ten animals (five males and five females) for each dose (OECD 1981; Commission of the European Communities 1984) or with five animals for each dose level (three groups of one sex and one group of the other sex; OECD 1987). A single dose of 2000 mg/kg body weight with the use of ten animals (five males and five females) is recommended for substances where no death is anticipated, the so-called limit test (OECD 1987). The scientific significance of the classical LDs0 test has been questioned on the basis of the relatively broad variability of the test results and for animal welfare reasons (Zbinden and Flury-Roversi 1981; Bass et al. 1982; Tattersall 1982). For the evaluation of the acute oral toxicity of substances it is not necessary to derive this information with an LDs0 test using large numbers of animals but instead with the use of only few animals that are carefully monitored for signs of toxicity and signs of recovery or mortality. Until now no validated in vitro method is available to investigate the acute oral toxicity and for the time being this information has to be gained from animal studies. Numerous alternatives to the LD50 test have been published, with the aim of reducing the number of animals and gaining detailed information on toxic signs and mortality ranges or mortality (Deichmann and LeBlanc 1943; Lorke 1983; Bruce 1985; Kennedy et al. 1986; Yamanaka et al. 1990). None of these methods are accepted as official guidelines by national regulatory authorities or by international organizations like the OECD. Recently, another alternative test procedure - the fixed dose method - has gained acceptance from the OECD and the European Community (van den Heuvel et al. 1990). In principle this method relies on signs of toxicity rather than on mortality for the evaluation of the toxicity of a test material and it uses 50% fewer animals when compared to the LDs0 test (OECD 1981).
456 TaMe 1. List of participants in the national validation study
Schering AG, Berlin Institute of Experimental Toxicology (P. GiJnzel, Chr. Sehrbel)
known. In general all available information, including that on structure activity relationships (SAR), is used for the selection of the starting dose. For the selection of the vehicle it was recommended to consider the use of water (including saline) first, followed by considerations of a solution in arachis oil, then of an aqueous suspension using Tween, carboxylmethylcellulose or polyethyeleneglycol as a formulating agent. All participants used the Wistar rat for the tests because this rat strain was being used for other toxicological tests in their laboratories. The experiments were started in April 1989 and all tests were completed by November 1990. The substances were supplied from the Bundesgesundheitsamt to the participants. They were at least of analytical grade and each substance was part of one lot. One laboratory was unable to perform the testing with phenobarbital. Therefore althogether only 179 tests instead of 180 tests were carried out.
Bundesgesundheitsamt, Berlin Abteilung Chemikalienbewertung, Max yon Pettenkofer Institut (U. Mischke, R. Hagen)
Method
BASF AG, Ludwigshafen Department of Toxicology (P. Kirsch) Bayer AG, Wuppertal Institute of Industrial Toxicology (J. A. Ivens-Kohl, R. Jaeger) Boehringer Ingelheim, Ingelheim Department of Experimental Pathology and Toxicology (A. Schuster) Henkel KGaA, Dtisseldorf Abteilung Forschung and Entwicklung (M. Potokar)
A t the B u n d e s g e s u n d h e i t s a m t ( B G A ) a n o t h e r a l t e r n a t i v e test has b e e n d e v e l o p e d - the a c u t e - t o x i c - c l a s s m e t h od. W i t h this m e t h o d e v e n f e w e r a n i m a l s are u s e d , at the s a m e t i m e p r e s e r v i n g the a s p e c t s o f h u m a n h e a l t h p r o t e c tion. T h i s test p r o c e d u r e has b e e n v a l i d a t e d in a n a t i o n a l c o l l o b o r a t i v e s t u d y a n d the results o f this s t u d y are rep o r t e d here.
Study design The testing procedure of the acute-toxic-class method was evaluated on a mathematical basis with the use of the probit analysis (Finney 1971) before the start of the validation study. Based on the mathematical results it could be demonstrated that the probability of the accurate classification is equal or even better for hazard assessment with this method than with the classical LDso method (Roll et al. 1989). The testing procedure for the acute-toxic-class method is also applicable for dosages that are used in other countries or organisations for classification purposes (Siccha et al. 1992). Five laboratories from the German chemical industry and one laboratory from the Bundesgesundheitsamt participated in this study (Table 1). The Bundesgesundheitsamt was responsible for the coordination of the study. Thirty substances were selected on the basis of their physico-chemical properties and of their toxicity profile, including also four substances (cadmium chloride, aniline, sodium salicylate and acetanilide) that have been already tested in two previous LDs0 validation studies (Hunter et al. 1979; Lingk 1982). The results of the acute-toxic-class tests were taken in relation to the LDso data derived from the literature. According to the German Act on Animal Welfare, it is prohibited to repeat animal studies for which data are already available in the literature. Therefore a thorough literature search was carried out in order to find as many oral LDs0 values for rats as possible for each substance (Table 2). For one substance only one reference but otherwise at least two and up to ten references were documented in the literature. As expected, the values varied widely for a substance from one reference to the next but also within one publication. The substances are listed according to their LDs0 values, aldicarb being the most toxic and ethylene glycol being the least toxic substance. The allocation of the substances to the different toxicity classes followed the classification criteria of the European Community (Commission of the European Communities 1983, Table 3). In the literature signs of toxicity and autopsy findings were inconsistently described or not recorded at all. Therefore they were not included in Table 2. The national validation study was not conducted "blind". The participants argued that they preferred to perform the tests under conditions of practice. In the majority of the laboratories before testing the chemical structure and the physico-chemical properties of a new substance are
The acute-toxic-class method is described in detail in Appendix 1. It includes three graphic charts of the test procedure with the options to start with 25, or 200 or 2000 mg/kg body weight. Also included are the criteria after which no further testing is necessary for classification purposes in the European Community (Appendix 2). The format follows the OECD Guidelines for toxicity testing. In the collaborative study a slightly different testing procedure as outlined in Appendix 1 was used for doses of 2000 mg/kg body weight. No further testing was necessary when either in step 1 and/or in step 2 none or one animal had died. For future testing with this dose the procedure was changed in order to meet international classification requirements: no further testing is now necessary when none of the animals tested either in step 1 and/or step 2 had died. As already indicated, the dose levels of 25,200 and 2000 mg/kg body weight follow the classification criteria of the European Community. In principle the testing is a stepwise procedure with the use of only three rats per step. The choice of the sex is optional at the starting dose but it is recommended to use the more sensitive sex if there is any information available. The time interval between treatments is determined by the duration of toxic signs. The animals had to be free of toxic signs for at least 2 days before the next treatment (if necessary) could be started with the use of additional three rats. The participants decided to use mainly mortality as the endpoint in this study. The reason for this decision was that definitions such as "extreme distress" or "evident toxicity" are not clearly defined in the literature. The participants argued that from their experience rats showing severe toxic signs may be able to survive. Nevertheless, they were free to kill the animals for animal welfare reasons if they considered it necessary. Great importance was attached to the observations of toxic signs following administration. Observations had to be made at least 6 times during the day of treatment followed by daily observations until the end of the study (or until death). The signs of toxicity that had to be observed are shown in Table 4.
Results T h e test r e p o r t s w e r e e v a l u a t e d a c c o r d i n g to t h e following criteria: 1) c l a s s i f i c a t i o n o f the s u b s t a n c e s to any of the f o u r t o x i c i t y c l a s s e s in c o m p a r i s o n to the LD50 data and their a l l o c a t e d t o x i c i t y class; 2) t a b u l a t i o n o f signs o f toxicity; 3) a u t o p s y f i n d i n g s a n d 4) n u m b e r o f a n i m a l s used and n u m b e r o f d e a d a n i m a l s . B a s e d o n t h e p u b l i s h e d LD50 d a t a a l o n e ( T a b l e 2) the a l l o c a t i o n to o n e o f t h e f o u r t o x i c i t y c l a s s e s w a s possible f o r the f o l l o w i n g 17 s u b s t a n c e s : Aldicarb, parathion, di-isopropylfluorophosphate, t h i o s e m i c a r b a z i d e , i n d o m e t h a c i n a n d p h e n y l t h i o u r e a are very toxic. S o d i u m a r s e n i t e , a l d r i n a n d a l l y l a l c o h o l are toxic.
457 Table 2. LDso values cited in the literature and EC classification Nr.
Substance
CAS no.
LDso (mg/kg body weight) (95% confidence limits or ___ se in brackets)
Literature
Classification according to EC criteria
1
Aldicarb
116-06-3
f ca. 1.0 m 0.8 (0.6-1.0); f0.65 (0.50-0.85) rn/f 3.2-5.0
Weiden et al. 1965 Gaines 1969 van den Heuvel et al. 1990
very toxic
2
Parathion
56-38-2
m 15; f 6 m 5; f 1.75 m 1.8 (1.26-2.57) m 30 (+3.6); f 3 (+0.25) (m/f) 6.5 m5-15 f3.2 (2.7-3.8) m 13 (10-17); f 3.6 (3.2-4) m8.5;f10 fca. 2,8
Du Bois et al. 1949 Hazleton and Holland 1950 Weiss and Orzel 1967 Frawley et al. 1952 Wirth 1954 Klimmer and Pfaff 1955 Edson and Noakes 1960 Gaines 1960 Nishizawa et al. 1961 Casida and Sanderson 1963
very toxic
3
Di-isopropylfluorophosphate
55-91-4
(m/f) 6.0 (m/f) 5.0- 10.0 m 13.5 (--+0.35); f7.7 (-+0.64)
Boyland and McDonald 1944 Coon 1946 Frawley et al. 1952
very toxic
4
Thiosemiearbazide
79-19-6
(m/f) 11.0 (-I-2); (m/f) 18 (-+2) (m/f) 9.2
Dieke 1949 Marhold 1977
very toxic
5
Indomethacin
53-86-1
(m/f) 12 (10-14) (m/f) 14
Goldenthal 1971 Niemegeers et al. 1975
very toxic
6
Phenylthiourea
103-85-5
(m/f) 8.6 ( -+0.6)
Dieke et al. 1947
very toxic
7
Mercury (II) oxide
21908-53-2
m 18 (7-44); (m/f) 46 ( + 5 )
Vernot et al. 1977 Trakhtenberg et al. 1981
very toxic/ toxic
8
Sodium arsenite
7784-46-5
(m/f) 41 m 36 ( 2 7 - 52), f 4 8 (37-76)
Smyth et al. 1969 van den Heuvel et al. 1990
toxic
9
Aldrin
309-00-2
(m/f) 67 (m/f) 38; (m/f) 49 m 59.6 (-+ 1.86); f52.3 (-+5.75) m 49; f45.9 (35.8-54.2) m 39 (34-47); f 6 0 (50-72) (m/f) 80.0
Lehman 1951 Bergmann et al. 1952 Ball et al. 1953 Treon and Cleveland 1955 Gaines 1960 Deichmann and Keplinger 1965
toxic
10
Allylalcohol
107-18-6
(m/f) 64 (56-74) (m/f) 99 (75-130)
Smyth et al. 1951 Dunlop et al. 1958
toxic
11
Bis (tributyltin) oxide
56-35-9
m 148 - 194 (m/f) 112 (m/f) 234 (m/f) 180 (-+48) (m/f) 87
Elsea and Paynter 1958 Klimmer 1969 Sheldon 1975 Truhaut et al. 1976 Marhold 1986
toxic/harmful
12
Acrylamide
79-06-1
f 150-180 f203 (160-249)
toxic/harmful
(m/f) 124
McCollister et al. 1964 Fullerton and Barnes 1966 Marhold 1972 Paulet and Vidal 1975
(m/f) 170 13
Cadmiumchloride
10108-64-2
(m/f) 88 m 70-513; f 140-500 m 103-482; f 8 4 - 4 8 2
Lehman 1951 Hunter et al. 1979 Lingk 1982
toxic/harmful
14
Methyl Chloroformate
79-22-1
(m/f) 60 (31.5-118) m 90 (100 -280); f 110 (70-160) rrdf 313 (329-427)
Gurova et al. 1977 Vernot et al. 1977 Gelbke and Jiickh 1981
toxic/harmful
15
Phenobarbital
50-06-6
(m/f) 220; (m/f) 162 -+ 14; (m/f) 318___23
Goldenthal 1971
toxic/harmful
16
Caffeine
58-08-2
(m/f) 233 -+ 14 (m/f) 192 ( _ 18) m 355 ( 3 1 2 - 403); f 247 (220- 277) m 483
Scott and Chen 1944 Boyd 1965 Palm et al. 1978 Kennedy et al, 1986
toxic/harmful
458 Table 2. Continue Nr..
Substance
CAS no.
LDs0 (mg/kg body weight) (95 % confidence limits or ___ se in brackets)
Literature
Classification according to EC criteria
17
Bariumcarbonate
513-77-9
(m/f) 630- 750
harmful
(m/l) 800
Farm Chemicals Handbook 1980 Izmerow 1982 Merck Index 1989
18
Amline
62-53-3
m 440 (340- 570) m 450 m 350-1280; f 378-1062 m 479-1169; f 4 7 9 - 1 8 5 0
Jacobson 1972 Czajkowska et al. 1977 Hunter et al. 1979 Lingk 1982
harmful
19
Fe~ocene
102-54-5
(m/l) 1320
harmful
(m/l) 418
m >2000; f 1260-2000
Shell Chemical Comp. 1961 Documentation 1986 van den Heuvel et al. 1990
20
m-Dichlorobencene
541-73-1
f 2300 (2052- 2591) m 580 (400-825)
Leist and Weigand 1979 Bayer 1980
harmful/ unclassified
21
Sodium salicylate
54-21-7
(m/l) 1600 (m/f) 1190, 1200 m 800-4150; f 958-4028 m 930-2329; f 8 8 3 - 2 5 5 5
Hart 1947 Drebinger 1950 Hunter et al. 1979 Lingk 1982
harmful
22
Acetanilide
103-84-4
(m/f) 800 m 804-5420; f 915-4350 m 723-3060; f 594-3250 m 2033 (1368-2858); f 1893 (1218-2459)
Smith and Hambourger 1935 Hunter et al. 1979 Lingk 1982 van den Heuvel et al. 1990
harmful
23
Sodiumlaurylsulphate
151-21-3
m/f 1288 (1216-1376) (m/f) 2640
Gale and Scott 1953 Walker et al. 1967
harmful/ unclassified
24
Acetonitrile
75-05-8
(m/l) 3800
Smyth and Carpenter 1948 Pozzani et al. 1959 Kimura et al. 1971 Purchase et al. 1987 van den Heuvel et al. 1990
harmful/ unclassified
(m/l) 1000
m 1312-3507; f 1730-6662 m 3046 (m/f) 1170-6650 m 1453 (1123-1879); f>2000 25
Benzyl benzoate
120-51-4
(m/f) 2800 (m/f) 1700
Graham and Kuizenga 1945 Draize et al. 1948
harmful/ unclassified
26
o-Phenylphenol
90-43-7
(m/l) 3000 m 2700 (2400- 3100) (m/l) 2000 - 4000
Macintosh 1945 Hodge et al. 1952 Kaneda et al. 1978
harmful/ unclassified
27
Butylated hydroxyanisole
25013-16-5
(m/f) 4100 (m/f) 2200 (m/f) 2900 (m/f) 2200 m 2950 (m/f) 2000 (m/f) 4100-5000
Wilder and Kraybill 1949 Lehman 1951 Kaprylik 1959 Dacre 1960 Daniyalov 1966 Hiraga et al. 1970 Furia and Belanca 1977
harmful/ unclassified
28
N,N- Dimethyl formamide
68-12-2
(m/f) 70oo (m/f) 3000
unclassified
(m/f) 2800 m/f 7205 - 8100 m/f >2000
Smyth et at. 1951 Thiersch 1967 Druckrey et al. 1967 Bartsch et al. 1976 van den Heuvel et al. 1990
29
Quercetin dihydrate
6151-25-3
m/f >2000
van den Heuvel et at. 1990
unclassified
30
Ethylene Glycol
107-21-1
(m/f) 8540
Smyth et al. 1950 Filatova et al. 1982
unclassified
(m/l) 4700 m = male, f = female; m/f = male and female groups combined; (m/l) = sex not specified. Values on slopes were only available for four substances: parathion m 1.50 (1.00-2.25), (Weiss and Orzel, 1950); sodium arsenite m 7.6 ( + 2.3), (van den Heuvel et al., 1990); caffeine m 5.1, f 7.7
(Palm et al. 1978); acetanilide m 7.0 (+2.2), (van den Heuvel et al, 1990). The LDso value of phenylthiourea (m/f 126-400 mg/kg body weight) was not included because of the difficulties in the preparation of the test solution (van den Heuvel et al. 1990)
459 Table3. Toxicityclasses of the EuropeanCommunity LDs0range
Toxicityclass
LD 50 < 25 25
