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Toxicity studies on tetrasul III. Short-term comparative studies in rats with tetrasul and structurally related acaricides
Toxocology, 1 (1973) 113--123 © Elsevier / North-Holland, Amsterdam -- The Netherlands
TOXICITY STUDIES ON TETRASUL III. SHORT-TERM COMPARATIVE STUDIES IN RATS WITH TETRASUL AND STRUCTURALLY RELATED ACARICIDES
H.G. VERSCHUUREN, R. KROES and ENGELINA M. DEN TONKELAAR
Laboratory of Toxicology, National Institute of Public Health, Bilthoven (The Netherlands) (Received October 8th, 1972) SUMMARY
In a comparative study 0, 50, 200, 1000 and 3000 ppm {parts per million) of five acaricides, tetrasul ( 2 , 4 , 5 , 4 ' - t e t r a c h l o r o d i p h e n y l s u l p h i d e ) , V l l 0 (2,4,5,4'-tetrachlorodophenylsulphoxide), tetradifon, chlorfenson and kelthane were given to groups of 10 male and 10 female rats in separate 90-day toxicity experiments. Growth, food intake, mortality, haematology, blood and liver biochemistry, organ weight, histopathology, number of liver parenchymal cells per surface unit and the diameter of liver-cell nuclei were used as criteria. The acaricides investigated, with the exception of kelthane, induced both functionally and structurally the same liver alterations. SER (smooth endoplasmic reticulum) whorls were found in the higher dose-levels of all 5 compounds. In all compounds, except kelthane, enlarged thyroids were found accompanied by histological alterations. Testrasul was more toxic than V l l 0 , followed by tetradifon. Chlorfenson was equally as toxic as tetrasul. Kelthane can be regarded as the most toxic of this group of compounds. The 50-ppm dosage appeared to be a n o - t o x i c effect level only in the case of tetradifon. INTRODUCTION
Tetrasul is used as an acaricide in orchards and is effective against both eggs and larvae. As a result of the application of this c o m p o u n d residues may be found on fruit. Due to factors such as composition of the diet, difference in species or strains, climatic and seasonal influences, the results of the toxicity investigations of one c o m p o u n d recorded by different authors may vary 1 For an objective evaluation of the toxicity data of one c o m p o u n d it is very useful to have comparative toxicity data of other structurally related Abbreviations: Alk. Pase, alkaline phosphatase; GI-6-Pase, glucose-6-phosphatase; Hex. ox., hexobarbital oxidase; ppm, parts per million; SER, smooth endoplasmic reticulum; SGPT, serum glutamic pyruvic acid transaminase; tetrasul, 2,4,5,4'-tetrachlorodiphenylsulphide; V l l 0 , 2,4,5,4'-tetrachlorodiphenylsulphoxide.
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cL-C s-C c, CI
Tetrasul, 2,4, 5, 4'-tetrachlorophenylsulphide C[
chlorfenson,
4-chlorophenyl
4-chlorobenzenesulphonate
............
o,- / % -
o,
2,z,,5,4'-tetrachlorophenylsulphoxide
{ V 110)
kelthane, 11 his ( Z, - c h l o r o p h e n y l } - 2,2, 2 ~ trichtoroethanol
tetradifon, 2, ~, 5, 4'- t et rachlofodiphenylsulphon
Fig. 1. S t r u c t u r a l f o r m u l a of tetrasul a n d s t r u c t u r a l l y r e l a t e d acaricides.
and partly well-known compounds used for similar technological purposes. In this paper the toxicity of tetrasul is compared with that of V l l 0 , tetradifon, chlorfenson and kelthane *. Chemical names and structural formulas are given in Fig. 1. Another objective of these studies was the search for "SER whorls", previously noted with tetrasul 2, in experiments with the other compounds. Acute and long-term studies of tetrasul 3 and a comparative short-term study with tetrasul in 6 animal species have already been submitted for publication 4 EXPERIMENTAL
Material
The sample of tetrasul (also called Animert) was supplied by the manufacturer Philips Duphar Ltd., Weesp, The Netherlands. The technical product with a purity of 92% was a white crystalline powder. The molecular weight was 324. V l l 0 , with a purity of 80% {impurities 5--10% 2,4,5,2'-tetrachlorodiphenylsulphoxide, 2--5% tetrasul and 2--5% tetradifon) was obtained from Philips Duphar Ltd., as was tetradifon with a purity of more than 90%. Vondelingenplaat Ltd., Rotterdam, provided chlorfenson with a purity of > 99%, and kelthane with a purity of 74% was supplied by AAgrunol Ltd., Groningen. Kelthane contained as impurities 11% 1-(2-chlorophenyl)-l-(4chlorophenyl)-2,2,2-trichloroethanol, and 15% inactive compounds. A n i m a l s and diet
Conventional Wistar rats, from a strain bred in our laboratory for more than 20 years, were used. The diet {WMS) consisted of 66% wheat flour, 33% whole milk powder, 0.5% CaCO 3 and 0.5% NaC1. The iodine content of the diet was rather low, about 0.06 ppm. * Full d e t a i l e d r e p o r t s are available in t h e archives o f t h e N a t i o n a l I n s t i t u t e o f Public Health: Tetrasul report Tox 98/69, Vll0 report Tox 91/69, chlorfenson report Tox 88/69, tetradifon report Tox 87/69, kelthane report Tox 80/68.
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Methods Alk. Pase, SGPT, G1-6-Pase and Hex. ox. were det erm i ned according to t he m e t h o d s described earlier 4. T he ranking m e t h o d of Wilcoxon 5 was used for statistical evaluations.
Experimental design and conduct The design was identical for each separate experiment. Five groups o f 10 male and 10 female rats received 0, 50, 200, 1000 and 3000 ppm of one of the five acaricides in their diet for 12 weeks. F o o d c o n s u m p t i o n was recorded in weeks 1, 2, 5, 9 and 12. B o d y weight was r eco r d e d weekly. Blood cell counts and blood pictures were examined after 12 weeks. In a n u m b e r of animals from each group SGPT and Alk. Pase in serum and G1-6-Pase and Hex. ox. in the liver were det erm i ned at the end of the exper i m ent . In all species the weight of the following organs was determined: liver, kidneys, heart, spleen, adrenals, thyroid, pituitary, uterus, ovaries, testes and brain. These organs, and lungs, pancreas, thymus, duodenum, caecum, r e c t um , urinary bladder, musculus quadriceps and peripheral nerves (N. ischiadicus) were examined microscopically. In 2 to 4 animals per group the n u m b e r of h e p a t o c y t e s per unit {0.0543 m m 2) were c o u n t e d in liver slides. T he diameter of hepatocellular nuclei was measured 6 in the same slides. T he five experiments were not c o n d u c t e d at the same time for practical reasons. RESULTS
Grow th and mortality F r o m the figures on growth, the 12-week stage was selected and recorded in Table I. O t her stages were similar. T o compare m ore easily the different c o m p o u n d s t he mean weights per group are expressed as percentages of the b o d y weight of the appropriate cont r o l group. G r o w t h retardation of the males fed kelthane in their diet was not ed already at t he 50-ppm dose level. At the 1000-ppm dosage growth retardation was first n o t e d with V l l 0 and chlorfenson. For V l l 0 this was not dose-related in females and not significant in males. In the tetrasul experim e n t only the 3000-ppm group showed growth inhibition. This did not occur at any of the t et r a di f on dose-levels. Kelthane, in a dosage of 3000 p p m, was lethal within 5 weeks bot h for males and females.
Blood Significant differences in blood cell counts were only f o u n d on 5 occasions. This is far less than 5% of the calculations carried out. Because of the statistical m e t h o d 5 and a P-value of 0.05, 5% of the comparisons m ay wrongly indicate a significant difference. For this reason no furt her a t t e n t i o n will be given here to this criterion.
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TABLE I BODY WEIGHTS AFTER 12 WEEKS, GIVEN AS PERCENTAGES OF THE APPROPRIATE CONTROL GROUPS The original mean weights in g of the control groups are also given. Compound
Rats
0 ppm
50 ppm
200 ppm
1000 ppm
3000 ppm
Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
Females 10 10 10 10 10
197~100 (11) 192~100 219~100 200~100 202~100
96 101 104 98 104
98 97 100 92 96 (9)
96 92 a 98 92 b 79 b (11)
85 b 97 98 89 b c
Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
Males 10 10 10 10 10
286~100 (9) 287~100 343~100 325~100 330~100
115 99 95 100 91a
101 94 97 95 84b(11)
95 92 101 96 62b(9)
85 b 93 99 84 b c
a P~0.05. bp~0.01. c All animals already dead. Number of animals if different from the number in column 2 is given in brackets.
Liver function tests Alk. Pase a n d SGPT values o f t h e serum are given in Table II. D u e t o t h e small n u m b e r o f animals e x a m i n e d we can o n l y speak o f i n d i c a t i o n s f o r a l o w e r Alk. Pase value in the females o f the c h l o r f e n s o n groups, in t h e females o f t h e 1 0 0 0 - and 3 0 0 0 - p p m tetrasul g r o u p s and in the 3 0 0 0 - p p m V l l 0 group. SGPT values were all n o r m a l . I n d i c a t i o n s for a lower GI-6-Pase activit y (Table III) are o b t a i n e d for tetrasul in males, c h l o r f e n s o n in males and k e l t h a n e starting f r o m t h e 1 0 0 0 - p p m dose-levels. T h e same i n d i c a t i o n was f o u n d for the males o f t h e 3 0 0 0 - p p m V l l 0 dose level. T h e activity o f Hex. ox. r e p r e s e n t i n g the m i c r o s o m a l liver e n z y m e s was increased b y all test c o m p o u n d s starting f r o m the following dose levels: 50 p p m f o r k e l t h a n e a n d 200 p p m f o r the others. T h e increase in the t e t r a d i f o n g r o u p s was c o m p a r a tively low.
Organ weights O n l y figures f o r liver and t h y r o i d weight in relation t o b o d y w e i g h t are given in Table IV, because o n l y these organs p r o v e d t o be target organs. A n increased liver w e i g h t was f o u n d in t h e 2 0 0 - p p m dosages o f V l 1 0 , chlorf e n s o n and kelthane. In t h e 1 0 0 0 - a n d 3 0 0 0 - p p m g r o u p s all c o m p o u n d s i n d u c e d an increase in liver weight. T h y r o i d w e i g h t was increased at t h e 5 0 - p p m dose levels o f V l 1 0 a n d kelthane. F o r t h e o t h e r c o m p o u n d s this t h y r o i d w e i g h t increase started at 2 0 0 p p m . In all cases o f t e t r a d i f o n females
116
TABLE II Alk.Pase AND GPT ACTIVITY IN SERUM A F T E R 12 WEEKS Compound
Tetrasul Vll0 Tetradi~n Chlor~nson Kelthane Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
Tetrasul Vll0 Tetradifon Chlorfenson Kelthane Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
Rats
0 ppm
Females
Alk.PaseinB.L. units
3 3 3 3
5.0±0.9 6.6±2.8 6.9±0.9 5.4±0.4 .
Males 3 3 3 3 3
5.1 5.0 8.8 5.1 6.7
Females
SGPT in Karmen units
3 3 3 3
19 ± 22+ 27 + 21 + .
Males 3 3 3 3 3
± ± ± ± ±
50 ppm
0.6 0.8 1.7 1.4 0.8
4 1 2 6
18+3 23+3 32+4 22+2 28_+4
200 ppm
1000 ppm
3000 ppm
4.9±0.9 4.6±1.3 7.8±1.1 2.9±0.5 . .
3.8±0.9 4.0±1.1 7.6±1.9 3.5±1.8 .
3.4±0.3 4.0±0.6 6.5±0.8 3.1±0.4 .
3.5±0.4 3.6±1.0 5.7±0.8 3.220.5
5.2 5.1 7.2 6.1 8.9
6.0 5.3 8.9 4.3 4.2
3.8 3.7 6.8 5.2 8.4
4.3 3.3 7.8 6.3
_+ 1.0 + 0.3 ± 0.9 ± 2.0 + 1.8
15 + 4 27+3 27 + 5 16 + 1 . 26+13 24+ 5 33+ 7 25+ 4 24_+ 2
.
± + + + +
0.3 0.9 1.9 1.3 2.2
± 2 + 2 ±
0.8 0.7 0.8 2.4 1.6
± ± ± + a
0.5 0.8 1.3 2.2
18 + 4 25+2 25 + 2 16 _+ 8 .
13 + 1 26+3 21 + 2 16 _+ 3 .
14 + 4 24± 1 27 + 3 18 _+ 5
19+7 22+4 32±8 23_+1 38+9
17+ 17+ 32+ 22± 33 +
21+ 5 22+ 5 37 ± 1 0 18+ 4 a
1 6 10 6 1
a All animals already dead. _+, S.D. w e r e less s e n s i t i v e t h a n m a l e s . F o r t h e o t h e r c o m p o u n d s , to be more s~nsitive for this parameter.
too, males proved
Histopa thology T h e t y p e o f a l t e r a t i o n s in l i v e r a n d t h y r o i d a r e d e s c r i b e d e a r l i e r 2 - 4 . I n l o w e r d o s a g e s " V 1 0 1 - c e l l s " o c c u r , w h i c h c a n b e b r i e f l y d e s c r i b e d as f o l l o w s : e n l a r g e d l i v e r p a r e n c h y m a l c e ll s m o s t l y w i t h e n l a r g e d n u c l e i , - s o m e t i m e s hyperchromatic or with unbalanced chromatin distribution. A basophilic g r a n u l a t i o n w a s u s u a l l y s e e n in t h e p e r i p h e r y o f t h e e n l a r g e d cells. T h e remainder of the cytoplasm contained many fine granules and was eosinop h i l i c in c h a r a c t e r . S m a l l a n d l a r g e r l i p i d - c o n t a i n i n g v a c u o l e s w e r e o f t e n f o u n d in c e l l s l y i n g b e t w e e n n o r m a l a n d e n l a r g e d cells. T h e t h y r o i d a l t e r a t i o n s c a n b e d e s c r i b e d as h y p e r p l a s t i c c h a n g e s f o r m i n g n o d u l a r o r m i c r o f o l licular types of goitre. For the sake of brevity Table V only contains the frequency of the alterations "V101-cells", SER whorls and thyroid altera117
T A B L E III Gl-6-Pase A N D Hex.ox. A C T I V I T Y IN T H E L I V E R A F T E R 12 W E E K S Compound
Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
Rats
0 ppm
Females
Gl-6-Pase in u n i t s
2 2 2
123; 127 175; 131 138; 1 5 0 .
Males 2 2 2 2 3
156; 126 153; 150 158; 167 158; 150 148 _+ 9
50 p p m
99 (1) 154; 129 134; 143 .
.
119; 199 141; 118 217; 137 154; 129 245 _+ 31
Males
Hex.ox. a in %
2 4 2 4 2
35; 34 17 +_4 10; 17 15 _+ 2 35; 28
38; 44 21+_ 3 10; 12 23 + 9 60; 64
200 ppm
1000 ppm
3000 ppm
121; 134 160; 129 138; 136
126; 128 123; 156 115; 133
126; 124 151; 112 1 1 8 ; 112
.
c
135; 147 141; 137 188; 141 137; 137 147 + 12
114; 108 123; 136 164; 144 123; 134 118 + 10
100; 108; 181; 119; c
92 111 142 122
66; 74 39b+12 20; 26 27 b _+ 8 98; 97
90; 90 59b+8 22; 32 54 b + 16 99; 100
83; 96 76b+14 36; 36 61 b + 15 c
a Hex.ox. was d e t e r m i n e d in male rats only. b P~0.05. c All animals already dead. N u m b e r of a n i m a l s if d i f f e r e n t f r o m t h e n u m b e r in c o l u m n 2 is given in b r a c k e t s . +, S.D.
180[nuclei
150I
I00
50
tetrasuI V 110 tetradifon chlorfenson kelthane I~ 0 ppm 1-1 50 ppm I~! 200 PPm ~ t000 ppm I~ 3000 pprn Fig. 2. N u m b e r o f c e n t r i l o b u l a r liver cell n u c l e i p e r 0 . 0 5 4 3 m m 2 in r e l a t i o n t o increasing dosages o f s t r u c t u r a l l y r e l a t e d acaricides d u r i n g 12 weeks. F o r each c o l u m n t h e s t a n d a r d d e v i a t i o n is given. M e a s u r e m e n t s in 4 m a l e rats p e r group, in 5 l o c a t i o n p ~ liver.
118
TABLE IV LIVER AND THYROID WEIGHT A F T E R 12 WEEKS GIVEN AS PERCENTAGES OF THE APPROPRIATE CONTROL GROUPS The original mean weights of the control groups are given as organ weight/body weight ratio. Compound
Rats
0 ppm
50 ppm
200 ppm
1000 ppm
3000 ppm
Females
Liver
Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
10 10 10 10 10
3.74~100 (11) 3.79~100 3.76~100 3.64~100 3.80~100
103 102 99 107 105
110 99 (8) 104 133 b 116 a (9)
135 130 118 176 199
b c (9) b b a (11)
190 165 138 232 d
b c c b
Tetrasul Vl10 Tetradifon Chlor fenson Kelthane
Males 10 10 10 10 10
4.31~100 (8) 3.75~100 3.81~100 3.81~100 3.89~100
98 103 106 106 105
105 107 a 107 118 a 114(11)
166 138 121 147 156
b c(8) b b a(9)
233 165 144 202 d
b c c b
320 b 182 c 143 244 b d
Females
Thyroid
Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
10 10 10 10 10
0.015~100 0.016~100 0.014~100 0.016~100 0.012~100
107 129 a 86 106 108
127 a 142 107 137 a 117
200 194 121 194 275
b c
Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
Males 10 10 10 10 10
0.015~100 0.014~100 0.012~100 0.015~100 0.011~100
143 125 a 108 133 127 a
186 180 a 125 a 166 b 118
357 325 208 633 182
b c c b
b a
500 413 242 360 d
b c c b
a P(0.05. b P(0.01. c p ( 0.001. d All animals already dead. Number of animals if different from the number in column 2 is given in brackets. tions. " V 1 0 1 - c e l l s " are f o u n d already at t h e 5 0 - p p m dosages o f tetrasul, V l l 0 a n d c h l o r f e n s o n , a n d a t t h e 2 0 0 - p p m d o s a g e s o f t e t r a d i f o n a n d kelthane. SER whorls were found at the 1000-ppm dose levels of each test compound. Thyroid alterations are already found at the 50-ppm dosages of Vl10 and chlorfenson and at the 1000-ppm dose levels of tetrasul and tetradifon. Kelthane did not induce thyroid alterations.
Size of liver parenchymal cells and their nuclei The number of centrilobular liver parenchymal cell nuclei per surface unit is g i v e n in F i g . 2. T h e r e is a c l e a r r e l a t i o n s h i p b e t w e e n t h e n u m b e r o f n u c l e i 119
TABLE V FREQUENCY OF CERTAIN LIVER AND THYROID L I V E R A N D T H Y R O I D A F T E R 12 W E E K S " V 1 0 1 - c e l l s " are d e s c r i b e d in t h e R E S U L T S s e c t i o n . Compound
0 ppm
50 p p m
ALTERATIONS
FOUND
IN
200 p p m
1000 ppm
3000 ppm
4 18 7 16 7
19 19 20 20 18
20 20 20 20 a
0 0 0 0 0
8 14 13 16 9
16 20 19 19 a
" V 1 0 1 - c e l l s " in t h e liver Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
0 0 0 0 0
5 9 0 5 0
" S E R w h o r l s " in t h e liver Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
0 0 0 0 0
0 0 0 0 0
N o d u l a r a n d m i c r o f o l l i c u l a r goitre in t h e t h y r o i d Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
0 1 1 5 1
4 15 1 12 0
1 17 1 20 0
10 20 19 20 0
16 19 16 19(19) a
a All a n i m a l s a l r e a d y dead. b N u m b e r o f a n i m a l s if d i f f e r e n t f r o m 20 is given in b r a c k e t s .
diameter in N 9,0 8.5 8.0 7.5 7.0
~i~
6.5
tetrasul [] 0 ppm
V 110 ~ tetradifon chlorfenson kelthane [] 50 ppm [] 200ppm []1000 ppm Fi3000ppm
Fig. 3. D i a m e t e r o f c e n t r i l o b u l a r liver cell n u c l e i in r e l a t i o n t o i n c r e a s i n g d o s a g e s o f s t r u c t u r a l l y r e l a t e d acaricides d u r i n g 12 w e e k s . F o r e a c h c o l u m n t h e s t a n d a r d d e v i a t i o n is given. M e a s u r e m e n t s in 4 m a l e r a t s per g r o u p . In e a c h rat 50 liver cells w e r e m e a s u r e d .
120
as a measure of cell-size and the increasing dosage. The 50-ppm tetrasul and V l l 0 dosages are evidently not different from the controls. The five test compounds behave similarly at comparative dosages, but kelthane was less effective. Chlorfenson, tetrasul, V l l 0 and tetradifon induce larger nuclei at the 1000- and 3000-ppm dosages. Kelthane does not affect the size of the nuclei (Fig. 3). DISCUSSION The number of animals used for determining the biochemical parameters is very low. Only indications and no firm conclusions can be derived from the results. However, these indications are backed up by control values obtained in a larg~ number of other experiments. It is remarkable that each of the structurally related acaricides induced qualitatively the same alterations in the liver both functionally and structurally. Only kelthane forms a partial exception. Prolonged and strong stimulation of microsomal liver enzymes induces the formation of a large a m o u n t of smooth endoplasmic reticulum, laid down in the form of whorls. This is also observed after induction with phenobarbitone 7, chlordane 8 and others. In this series of experiments it was found for the first time that not only tetrasul but also V l l 0 , tetradifon, chlorfenson and kelthane can induce SER whorls at high dose levels. Liver parenchymal cells generally occur largely as tetraploids, and less frequently as diploids or octoploids. This has been shown by photometric determination of DNA content in cell nuclei of liver slides of rats 9. After subcutaneous administration of 100 mg phenobarbitone per kg body weight an increase in the diameter of liver cell nuclei was found, due to an increase of octoploidal and a decrease in diploidal nuclei 10. In this experiment no distinction was made between di-, tetra- and octoploidal nuclei. However, the optical density of larger nuclei was not different from those of smaller nuclei in slides of the same thickness. For this reason it is likely that in the present experiments the number of octoploidal cells is increased as a result of administration of these acaricides, with the exception of kelthane. The compounds with a close structural relationship, tetrasul, V l 1 0 and tetradifon, perform quantitatively in a fixed sequence for a number of criteria, like liver and thyroid weight and Hex. ox. activity. Tetrasul(sulphide) was most effective in comparative dosages, followed by V l l 0 (sulphoxide) and tetradifon (sulphone). Chlorfenson can be regarded equally toxic as tetrasul. Kelthane induces qualitatively other effects than tetrasul. The lack of histological thyroid alterations and the unchanged diameter of liver parenchymal cells may be an illustration of this. The mortality at the 3000-ppm dose level of kelthane and the growth reduction found at dosages of 50 ppm and above are another indication of this difference. Therefore kelthane might be regarded as the most toxic of the 5 acaricides. The 50ppm dose levels of tetrasul, V l l 0 , chlorfenson and kelthane still induce 121
t o x i c effects, b u t t h e 5 0 - p p m dosage o f t e t r a d i t i o n was a " n o - t o x i c e f f e c t level" in this e x p e r i m e n t . In t h e l i t e r a t u r e n o o t h e r a u t h o r s describe 9 0 - d a y t o x i c i t y investigations o f t e t r a s u l and V l l 0 . A c o m p a r i s o n o f results is t h e r e f o r e n o t possible. In a l o n g - t e r m e x p e r i m e n t w i t h t e t r a d i f o n in rats a t e n d e n c y t o v a c u o l i s a t i o n o f t h e a d r e n a l m e d u l l a was f o u n d at a d o s e level o f 100 p p m . A t t h e 30 a n d 1 0 0 - p p m dose level increased h a e m o s i d e r o s i s was f o u n d in t h e spleen 11 A l t h o u g h in o u r e x p e r i m e n t w i t h t e t r a d i f o n no h a e m o s i d e r o s i s was f o u n d in t h e spleen, t h e o t h e r results are n o t c o n t r a d i c t o r y . C h l o r f e n s o n was given t o rats at d o s e levels o f 300, 1 0 0 0 and 3 0 0 0 p p m . In t h e 1 0 0 0 a n d 3 0 0 0 p p m dosage levels liver w e i g h t was increased and slight d e g e n e r a t i o n was o b s e r v e d . T h e 3 0 0 - p p m g r o u p was r e g a r d e d as a n o - t o x i c e f f e c t level 1 2 In o u r e x p e r i m e n t t h e 2 0 0 - p p m d o s e level caused an increase in liver a n d t h y r o i d w e i g h t a n d even at t h e 5 0 - p p m d o s e level histological c h a n g e s w e r e o b s e r v e d in t h e s e organs (Tables IV a n d V). In a l a t e r l o n g - t e r m e x p e r i m e n t a n o n - t o x i c e f f e c t level o f 25 p p m c h l o r f e n s o n was f o u n d 13. This c o u l d be in line w i t h o u r findings. K e l t h a n e was given at d o s e levels o f 0, 20, 1 0 0 , 500, 1 2 5 0 a n d 2 5 0 0 p p m t o rats 14. T h e results o b t a i n e d are well in a c c o r d a n c e w i t h t h e p r e s e n t investigation. In 1 o u t o f 10 m a l e r a t s at t h e 2 0 - p p m d o s e level liver a l t e r a t i o n s such as focal swollen a c i d o p h i l i c c y t o p l a s m w i t h v a c u o l e s w e r e found. In o n e o f t h e r e f e r e n c e s t h y r o i d a l t e r a t i o n s are m e n t i o n e d . T h e r e a s o n m i g h t be t h a t t h e diets used c o n t a i n e d high a m o u n t s o f iodine. U n p u b l i s h e d w o r k o f t h e a u t h o r s s h o w e d a clear r e l a t i o n s h i p b e t w e e n t h e a m o u n t o f iodine in t h e diet a n d t h e e f f e c t o f t e t r a s u l o n t h e t h y r o i d . In t h e p r e s e n t e x p e r i m e n t a diet was given w h i c h was l a t e r p r o v e d t o be v e r y l o w in iodine.
ACKNOWLEDGEMENT T h e a u t h o r s a c k n o w l e d g e t h e c o o p e r a t i o n o f Dr. P. H e l l e m a n a n d his s t a f f in t h e h a e m a t o l o g i c a l w o r k a n d also of t h e s t a f f o f t h e A n i m a l H o u s e . REFERENCES 1 2 3 4 5 6 7 8 9 10 11
E. Schi~tz, Arzneimittel-Forsch., 18 (1968) 466. H.G. Verschuuren, Food Cosmet. Toxicol., 5 (1967)450. H.G. Verschuuren, R. Kroes and G.J. van Esch, Toxicology, 1 (1973) 63. H.G. Verschuuren,R. Kroes and E.M. den Tonkelaar, Toxicology, 1 (1973) 103. F. Wilcoxon, Biometr. Bull., 1 (1945) 80. H.G. Verschuuren, Toxicologisch Onderzoek van Tetrasul en Verwante Verbindingen, Diss. Utrecht, 1970, p. 20. A.H. Conney, C. Davison, R. Gastel and J.J. Burns, J. Pharmacol. Exptl. Ther., 130 (1960) 1. J.R. Fours and L.A. Rogers, J. Pharmacol. Exptl. Ther., 147 (1965) 112. M. Alfert and J.J. Geschwind, Exptl. Cell Res., 15 (1958) 230. W. St}iubli, R. Hess and E.R. Weibel, J. CeUBiol., 42 (1969) 92. Unpublished report 1957, Philips Duphar Ltd., Weesp, The Netherlands.
122
12 Technical Bulletin, Dow Chemical Company, Midland, Michigan, 1951. 13 C.S. Weil and D.D. McCollister, Jo Agr. Food Chem., 11 (1963) 486. 14 K.B. Smith, P.S. Larson, K.J. Finnegan, H.B. Haag, G.R. Hennigan and F. Coley, Toxicol. Appl. Pharmacol., 1 (1959) 119.
123
TOXICITY STUDIES ON TETRASUL III. SHORT-TERM COMPARATIVE STUDIES IN RATS WITH TETRASUL AND STRUCTURALLY RELATED ACARICIDES
H.G. VERSCHUUREN, R. KROES and ENGELINA M. DEN TONKELAAR
Laboratory of Toxicology, National Institute of Public Health, Bilthoven (The Netherlands) (Received October 8th, 1972) SUMMARY
In a comparative study 0, 50, 200, 1000 and 3000 ppm {parts per million) of five acaricides, tetrasul ( 2 , 4 , 5 , 4 ' - t e t r a c h l o r o d i p h e n y l s u l p h i d e ) , V l l 0 (2,4,5,4'-tetrachlorodophenylsulphoxide), tetradifon, chlorfenson and kelthane were given to groups of 10 male and 10 female rats in separate 90-day toxicity experiments. Growth, food intake, mortality, haematology, blood and liver biochemistry, organ weight, histopathology, number of liver parenchymal cells per surface unit and the diameter of liver-cell nuclei were used as criteria. The acaricides investigated, with the exception of kelthane, induced both functionally and structurally the same liver alterations. SER (smooth endoplasmic reticulum) whorls were found in the higher dose-levels of all 5 compounds. In all compounds, except kelthane, enlarged thyroids were found accompanied by histological alterations. Testrasul was more toxic than V l l 0 , followed by tetradifon. Chlorfenson was equally as toxic as tetrasul. Kelthane can be regarded as the most toxic of this group of compounds. The 50-ppm dosage appeared to be a n o - t o x i c effect level only in the case of tetradifon. INTRODUCTION
Tetrasul is used as an acaricide in orchards and is effective against both eggs and larvae. As a result of the application of this c o m p o u n d residues may be found on fruit. Due to factors such as composition of the diet, difference in species or strains, climatic and seasonal influences, the results of the toxicity investigations of one c o m p o u n d recorded by different authors may vary 1 For an objective evaluation of the toxicity data of one c o m p o u n d it is very useful to have comparative toxicity data of other structurally related Abbreviations: Alk. Pase, alkaline phosphatase; GI-6-Pase, glucose-6-phosphatase; Hex. ox., hexobarbital oxidase; ppm, parts per million; SER, smooth endoplasmic reticulum; SGPT, serum glutamic pyruvic acid transaminase; tetrasul, 2,4,5,4'-tetrachlorodiphenylsulphide; V l l 0 , 2,4,5,4'-tetrachlorodiphenylsulphoxide.
113
cL-C s-C c, CI
Tetrasul, 2,4, 5, 4'-tetrachlorophenylsulphide C[
chlorfenson,
4-chlorophenyl
4-chlorobenzenesulphonate
............
o,- / % -
o,
2,z,,5,4'-tetrachlorophenylsulphoxide
{ V 110)
kelthane, 11 his ( Z, - c h l o r o p h e n y l } - 2,2, 2 ~ trichtoroethanol
tetradifon, 2, ~, 5, 4'- t et rachlofodiphenylsulphon
Fig. 1. S t r u c t u r a l f o r m u l a of tetrasul a n d s t r u c t u r a l l y r e l a t e d acaricides.
and partly well-known compounds used for similar technological purposes. In this paper the toxicity of tetrasul is compared with that of V l l 0 , tetradifon, chlorfenson and kelthane *. Chemical names and structural formulas are given in Fig. 1. Another objective of these studies was the search for "SER whorls", previously noted with tetrasul 2, in experiments with the other compounds. Acute and long-term studies of tetrasul 3 and a comparative short-term study with tetrasul in 6 animal species have already been submitted for publication 4 EXPERIMENTAL
Material
The sample of tetrasul (also called Animert) was supplied by the manufacturer Philips Duphar Ltd., Weesp, The Netherlands. The technical product with a purity of 92% was a white crystalline powder. The molecular weight was 324. V l l 0 , with a purity of 80% {impurities 5--10% 2,4,5,2'-tetrachlorodiphenylsulphoxide, 2--5% tetrasul and 2--5% tetradifon) was obtained from Philips Duphar Ltd., as was tetradifon with a purity of more than 90%. Vondelingenplaat Ltd., Rotterdam, provided chlorfenson with a purity of > 99%, and kelthane with a purity of 74% was supplied by AAgrunol Ltd., Groningen. Kelthane contained as impurities 11% 1-(2-chlorophenyl)-l-(4chlorophenyl)-2,2,2-trichloroethanol, and 15% inactive compounds. A n i m a l s and diet
Conventional Wistar rats, from a strain bred in our laboratory for more than 20 years, were used. The diet {WMS) consisted of 66% wheat flour, 33% whole milk powder, 0.5% CaCO 3 and 0.5% NaC1. The iodine content of the diet was rather low, about 0.06 ppm. * Full d e t a i l e d r e p o r t s are available in t h e archives o f t h e N a t i o n a l I n s t i t u t e o f Public Health: Tetrasul report Tox 98/69, Vll0 report Tox 91/69, chlorfenson report Tox 88/69, tetradifon report Tox 87/69, kelthane report Tox 80/68.
114
Methods Alk. Pase, SGPT, G1-6-Pase and Hex. ox. were det erm i ned according to t he m e t h o d s described earlier 4. T he ranking m e t h o d of Wilcoxon 5 was used for statistical evaluations.
Experimental design and conduct The design was identical for each separate experiment. Five groups o f 10 male and 10 female rats received 0, 50, 200, 1000 and 3000 ppm of one of the five acaricides in their diet for 12 weeks. F o o d c o n s u m p t i o n was recorded in weeks 1, 2, 5, 9 and 12. B o d y weight was r eco r d e d weekly. Blood cell counts and blood pictures were examined after 12 weeks. In a n u m b e r of animals from each group SGPT and Alk. Pase in serum and G1-6-Pase and Hex. ox. in the liver were det erm i ned at the end of the exper i m ent . In all species the weight of the following organs was determined: liver, kidneys, heart, spleen, adrenals, thyroid, pituitary, uterus, ovaries, testes and brain. These organs, and lungs, pancreas, thymus, duodenum, caecum, r e c t um , urinary bladder, musculus quadriceps and peripheral nerves (N. ischiadicus) were examined microscopically. In 2 to 4 animals per group the n u m b e r of h e p a t o c y t e s per unit {0.0543 m m 2) were c o u n t e d in liver slides. T he diameter of hepatocellular nuclei was measured 6 in the same slides. T he five experiments were not c o n d u c t e d at the same time for practical reasons. RESULTS
Grow th and mortality F r o m the figures on growth, the 12-week stage was selected and recorded in Table I. O t her stages were similar. T o compare m ore easily the different c o m p o u n d s t he mean weights per group are expressed as percentages of the b o d y weight of the appropriate cont r o l group. G r o w t h retardation of the males fed kelthane in their diet was not ed already at t he 50-ppm dose level. At the 1000-ppm dosage growth retardation was first n o t e d with V l l 0 and chlorfenson. For V l l 0 this was not dose-related in females and not significant in males. In the tetrasul experim e n t only the 3000-ppm group showed growth inhibition. This did not occur at any of the t et r a di f on dose-levels. Kelthane, in a dosage of 3000 p p m, was lethal within 5 weeks bot h for males and females.
Blood Significant differences in blood cell counts were only f o u n d on 5 occasions. This is far less than 5% of the calculations carried out. Because of the statistical m e t h o d 5 and a P-value of 0.05, 5% of the comparisons m ay wrongly indicate a significant difference. For this reason no furt her a t t e n t i o n will be given here to this criterion.
115
TABLE I BODY WEIGHTS AFTER 12 WEEKS, GIVEN AS PERCENTAGES OF THE APPROPRIATE CONTROL GROUPS The original mean weights in g of the control groups are also given. Compound
Rats
0 ppm
50 ppm
200 ppm
1000 ppm
3000 ppm
Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
Females 10 10 10 10 10
197~100 (11) 192~100 219~100 200~100 202~100
96 101 104 98 104
98 97 100 92 96 (9)
96 92 a 98 92 b 79 b (11)
85 b 97 98 89 b c
Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
Males 10 10 10 10 10
286~100 (9) 287~100 343~100 325~100 330~100
115 99 95 100 91a
101 94 97 95 84b(11)
95 92 101 96 62b(9)
85 b 93 99 84 b c
a P~0.05. bp~0.01. c All animals already dead. Number of animals if different from the number in column 2 is given in brackets.
Liver function tests Alk. Pase a n d SGPT values o f t h e serum are given in Table II. D u e t o t h e small n u m b e r o f animals e x a m i n e d we can o n l y speak o f i n d i c a t i o n s f o r a l o w e r Alk. Pase value in the females o f the c h l o r f e n s o n groups, in t h e females o f t h e 1 0 0 0 - and 3 0 0 0 - p p m tetrasul g r o u p s and in the 3 0 0 0 - p p m V l l 0 group. SGPT values were all n o r m a l . I n d i c a t i o n s for a lower GI-6-Pase activit y (Table III) are o b t a i n e d for tetrasul in males, c h l o r f e n s o n in males and k e l t h a n e starting f r o m t h e 1 0 0 0 - p p m dose-levels. T h e same i n d i c a t i o n was f o u n d for the males o f t h e 3 0 0 0 - p p m V l l 0 dose level. T h e activity o f Hex. ox. r e p r e s e n t i n g the m i c r o s o m a l liver e n z y m e s was increased b y all test c o m p o u n d s starting f r o m the following dose levels: 50 p p m f o r k e l t h a n e a n d 200 p p m f o r the others. T h e increase in the t e t r a d i f o n g r o u p s was c o m p a r a tively low.
Organ weights O n l y figures f o r liver and t h y r o i d weight in relation t o b o d y w e i g h t are given in Table IV, because o n l y these organs p r o v e d t o be target organs. A n increased liver w e i g h t was f o u n d in t h e 2 0 0 - p p m dosages o f V l 1 0 , chlorf e n s o n and kelthane. In t h e 1 0 0 0 - a n d 3 0 0 0 - p p m g r o u p s all c o m p o u n d s i n d u c e d an increase in liver weight. T h y r o i d w e i g h t was increased at t h e 5 0 - p p m dose levels o f V l 1 0 a n d kelthane. F o r t h e o t h e r c o m p o u n d s this t h y r o i d w e i g h t increase started at 2 0 0 p p m . In all cases o f t e t r a d i f o n females
116
TABLE II Alk.Pase AND GPT ACTIVITY IN SERUM A F T E R 12 WEEKS Compound
Tetrasul Vll0 Tetradi~n Chlor~nson Kelthane Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
Tetrasul Vll0 Tetradifon Chlorfenson Kelthane Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
Rats
0 ppm
Females
Alk.PaseinB.L. units
3 3 3 3
5.0±0.9 6.6±2.8 6.9±0.9 5.4±0.4 .
Males 3 3 3 3 3
5.1 5.0 8.8 5.1 6.7
Females
SGPT in Karmen units
3 3 3 3
19 ± 22+ 27 + 21 + .
Males 3 3 3 3 3
± ± ± ± ±
50 ppm
0.6 0.8 1.7 1.4 0.8
4 1 2 6
18+3 23+3 32+4 22+2 28_+4
200 ppm
1000 ppm
3000 ppm
4.9±0.9 4.6±1.3 7.8±1.1 2.9±0.5 . .
3.8±0.9 4.0±1.1 7.6±1.9 3.5±1.8 .
3.4±0.3 4.0±0.6 6.5±0.8 3.1±0.4 .
3.5±0.4 3.6±1.0 5.7±0.8 3.220.5
5.2 5.1 7.2 6.1 8.9
6.0 5.3 8.9 4.3 4.2
3.8 3.7 6.8 5.2 8.4
4.3 3.3 7.8 6.3
_+ 1.0 + 0.3 ± 0.9 ± 2.0 + 1.8
15 + 4 27+3 27 + 5 16 + 1 . 26+13 24+ 5 33+ 7 25+ 4 24_+ 2
.
± + + + +
0.3 0.9 1.9 1.3 2.2
± 2 + 2 ±
0.8 0.7 0.8 2.4 1.6
± ± ± + a
0.5 0.8 1.3 2.2
18 + 4 25+2 25 + 2 16 _+ 8 .
13 + 1 26+3 21 + 2 16 _+ 3 .
14 + 4 24± 1 27 + 3 18 _+ 5
19+7 22+4 32±8 23_+1 38+9
17+ 17+ 32+ 22± 33 +
21+ 5 22+ 5 37 ± 1 0 18+ 4 a
1 6 10 6 1
a All animals already dead. _+, S.D. w e r e less s e n s i t i v e t h a n m a l e s . F o r t h e o t h e r c o m p o u n d s , to be more s~nsitive for this parameter.
too, males proved
Histopa thology T h e t y p e o f a l t e r a t i o n s in l i v e r a n d t h y r o i d a r e d e s c r i b e d e a r l i e r 2 - 4 . I n l o w e r d o s a g e s " V 1 0 1 - c e l l s " o c c u r , w h i c h c a n b e b r i e f l y d e s c r i b e d as f o l l o w s : e n l a r g e d l i v e r p a r e n c h y m a l c e ll s m o s t l y w i t h e n l a r g e d n u c l e i , - s o m e t i m e s hyperchromatic or with unbalanced chromatin distribution. A basophilic g r a n u l a t i o n w a s u s u a l l y s e e n in t h e p e r i p h e r y o f t h e e n l a r g e d cells. T h e remainder of the cytoplasm contained many fine granules and was eosinop h i l i c in c h a r a c t e r . S m a l l a n d l a r g e r l i p i d - c o n t a i n i n g v a c u o l e s w e r e o f t e n f o u n d in c e l l s l y i n g b e t w e e n n o r m a l a n d e n l a r g e d cells. T h e t h y r o i d a l t e r a t i o n s c a n b e d e s c r i b e d as h y p e r p l a s t i c c h a n g e s f o r m i n g n o d u l a r o r m i c r o f o l licular types of goitre. For the sake of brevity Table V only contains the frequency of the alterations "V101-cells", SER whorls and thyroid altera117
T A B L E III Gl-6-Pase A N D Hex.ox. A C T I V I T Y IN T H E L I V E R A F T E R 12 W E E K S Compound
Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
Rats
0 ppm
Females
Gl-6-Pase in u n i t s
2 2 2
123; 127 175; 131 138; 1 5 0 .
Males 2 2 2 2 3
156; 126 153; 150 158; 167 158; 150 148 _+ 9
50 p p m
99 (1) 154; 129 134; 143 .
.
119; 199 141; 118 217; 137 154; 129 245 _+ 31
Males
Hex.ox. a in %
2 4 2 4 2
35; 34 17 +_4 10; 17 15 _+ 2 35; 28
38; 44 21+_ 3 10; 12 23 + 9 60; 64
200 ppm
1000 ppm
3000 ppm
121; 134 160; 129 138; 136
126; 128 123; 156 115; 133
126; 124 151; 112 1 1 8 ; 112
.
c
135; 147 141; 137 188; 141 137; 137 147 + 12
114; 108 123; 136 164; 144 123; 134 118 + 10
100; 108; 181; 119; c
92 111 142 122
66; 74 39b+12 20; 26 27 b _+ 8 98; 97
90; 90 59b+8 22; 32 54 b + 16 99; 100
83; 96 76b+14 36; 36 61 b + 15 c
a Hex.ox. was d e t e r m i n e d in male rats only. b P~0.05. c All animals already dead. N u m b e r of a n i m a l s if d i f f e r e n t f r o m t h e n u m b e r in c o l u m n 2 is given in b r a c k e t s . +, S.D.
180[nuclei
150I
I00
50
tetrasuI V 110 tetradifon chlorfenson kelthane I~ 0 ppm 1-1 50 ppm I~! 200 PPm ~ t000 ppm I~ 3000 pprn Fig. 2. N u m b e r o f c e n t r i l o b u l a r liver cell n u c l e i p e r 0 . 0 5 4 3 m m 2 in r e l a t i o n t o increasing dosages o f s t r u c t u r a l l y r e l a t e d acaricides d u r i n g 12 weeks. F o r each c o l u m n t h e s t a n d a r d d e v i a t i o n is given. M e a s u r e m e n t s in 4 m a l e rats p e r group, in 5 l o c a t i o n p ~ liver.
118
TABLE IV LIVER AND THYROID WEIGHT A F T E R 12 WEEKS GIVEN AS PERCENTAGES OF THE APPROPRIATE CONTROL GROUPS The original mean weights of the control groups are given as organ weight/body weight ratio. Compound
Rats
0 ppm
50 ppm
200 ppm
1000 ppm
3000 ppm
Females
Liver
Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
10 10 10 10 10
3.74~100 (11) 3.79~100 3.76~100 3.64~100 3.80~100
103 102 99 107 105
110 99 (8) 104 133 b 116 a (9)
135 130 118 176 199
b c (9) b b a (11)
190 165 138 232 d
b c c b
Tetrasul Vl10 Tetradifon Chlor fenson Kelthane
Males 10 10 10 10 10
4.31~100 (8) 3.75~100 3.81~100 3.81~100 3.89~100
98 103 106 106 105
105 107 a 107 118 a 114(11)
166 138 121 147 156
b c(8) b b a(9)
233 165 144 202 d
b c c b
320 b 182 c 143 244 b d
Females
Thyroid
Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
10 10 10 10 10
0.015~100 0.016~100 0.014~100 0.016~100 0.012~100
107 129 a 86 106 108
127 a 142 107 137 a 117
200 194 121 194 275
b c
Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
Males 10 10 10 10 10
0.015~100 0.014~100 0.012~100 0.015~100 0.011~100
143 125 a 108 133 127 a
186 180 a 125 a 166 b 118
357 325 208 633 182
b c c b
b a
500 413 242 360 d
b c c b
a P(0.05. b P(0.01. c p ( 0.001. d All animals already dead. Number of animals if different from the number in column 2 is given in brackets. tions. " V 1 0 1 - c e l l s " are f o u n d already at t h e 5 0 - p p m dosages o f tetrasul, V l l 0 a n d c h l o r f e n s o n , a n d a t t h e 2 0 0 - p p m d o s a g e s o f t e t r a d i f o n a n d kelthane. SER whorls were found at the 1000-ppm dose levels of each test compound. Thyroid alterations are already found at the 50-ppm dosages of Vl10 and chlorfenson and at the 1000-ppm dose levels of tetrasul and tetradifon. Kelthane did not induce thyroid alterations.
Size of liver parenchymal cells and their nuclei The number of centrilobular liver parenchymal cell nuclei per surface unit is g i v e n in F i g . 2. T h e r e is a c l e a r r e l a t i o n s h i p b e t w e e n t h e n u m b e r o f n u c l e i 119
TABLE V FREQUENCY OF CERTAIN LIVER AND THYROID L I V E R A N D T H Y R O I D A F T E R 12 W E E K S " V 1 0 1 - c e l l s " are d e s c r i b e d in t h e R E S U L T S s e c t i o n . Compound
0 ppm
50 p p m
ALTERATIONS
FOUND
IN
200 p p m
1000 ppm
3000 ppm
4 18 7 16 7
19 19 20 20 18
20 20 20 20 a
0 0 0 0 0
8 14 13 16 9
16 20 19 19 a
" V 1 0 1 - c e l l s " in t h e liver Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
0 0 0 0 0
5 9 0 5 0
" S E R w h o r l s " in t h e liver Tetrasul Vll0 Tetradifon Chlorfenson Kelthane
0 0 0 0 0
0 0 0 0 0
N o d u l a r a n d m i c r o f o l l i c u l a r goitre in t h e t h y r o i d Tetrasul Vl10 Tetradifon Chlorfenson Kelthane
0 1 1 5 1
4 15 1 12 0
1 17 1 20 0
10 20 19 20 0
16 19 16 19(19) a
a All a n i m a l s a l r e a d y dead. b N u m b e r o f a n i m a l s if d i f f e r e n t f r o m 20 is given in b r a c k e t s .
diameter in N 9,0 8.5 8.0 7.5 7.0
~i~
6.5
tetrasul [] 0 ppm
V 110 ~ tetradifon chlorfenson kelthane [] 50 ppm [] 200ppm []1000 ppm Fi3000ppm
Fig. 3. D i a m e t e r o f c e n t r i l o b u l a r liver cell n u c l e i in r e l a t i o n t o i n c r e a s i n g d o s a g e s o f s t r u c t u r a l l y r e l a t e d acaricides d u r i n g 12 w e e k s . F o r e a c h c o l u m n t h e s t a n d a r d d e v i a t i o n is given. M e a s u r e m e n t s in 4 m a l e r a t s per g r o u p . In e a c h rat 50 liver cells w e r e m e a s u r e d .
120
as a measure of cell-size and the increasing dosage. The 50-ppm tetrasul and V l l 0 dosages are evidently not different from the controls. The five test compounds behave similarly at comparative dosages, but kelthane was less effective. Chlorfenson, tetrasul, V l l 0 and tetradifon induce larger nuclei at the 1000- and 3000-ppm dosages. Kelthane does not affect the size of the nuclei (Fig. 3). DISCUSSION The number of animals used for determining the biochemical parameters is very low. Only indications and no firm conclusions can be derived from the results. However, these indications are backed up by control values obtained in a larg~ number of other experiments. It is remarkable that each of the structurally related acaricides induced qualitatively the same alterations in the liver both functionally and structurally. Only kelthane forms a partial exception. Prolonged and strong stimulation of microsomal liver enzymes induces the formation of a large a m o u n t of smooth endoplasmic reticulum, laid down in the form of whorls. This is also observed after induction with phenobarbitone 7, chlordane 8 and others. In this series of experiments it was found for the first time that not only tetrasul but also V l l 0 , tetradifon, chlorfenson and kelthane can induce SER whorls at high dose levels. Liver parenchymal cells generally occur largely as tetraploids, and less frequently as diploids or octoploids. This has been shown by photometric determination of DNA content in cell nuclei of liver slides of rats 9. After subcutaneous administration of 100 mg phenobarbitone per kg body weight an increase in the diameter of liver cell nuclei was found, due to an increase of octoploidal and a decrease in diploidal nuclei 10. In this experiment no distinction was made between di-, tetra- and octoploidal nuclei. However, the optical density of larger nuclei was not different from those of smaller nuclei in slides of the same thickness. For this reason it is likely that in the present experiments the number of octoploidal cells is increased as a result of administration of these acaricides, with the exception of kelthane. The compounds with a close structural relationship, tetrasul, V l 1 0 and tetradifon, perform quantitatively in a fixed sequence for a number of criteria, like liver and thyroid weight and Hex. ox. activity. Tetrasul(sulphide) was most effective in comparative dosages, followed by V l l 0 (sulphoxide) and tetradifon (sulphone). Chlorfenson can be regarded equally toxic as tetrasul. Kelthane induces qualitatively other effects than tetrasul. The lack of histological thyroid alterations and the unchanged diameter of liver parenchymal cells may be an illustration of this. The mortality at the 3000-ppm dose level of kelthane and the growth reduction found at dosages of 50 ppm and above are another indication of this difference. Therefore kelthane might be regarded as the most toxic of the 5 acaricides. The 50ppm dose levels of tetrasul, V l l 0 , chlorfenson and kelthane still induce 121
t o x i c effects, b u t t h e 5 0 - p p m dosage o f t e t r a d i t i o n was a " n o - t o x i c e f f e c t level" in this e x p e r i m e n t . In t h e l i t e r a t u r e n o o t h e r a u t h o r s describe 9 0 - d a y t o x i c i t y investigations o f t e t r a s u l and V l l 0 . A c o m p a r i s o n o f results is t h e r e f o r e n o t possible. In a l o n g - t e r m e x p e r i m e n t w i t h t e t r a d i f o n in rats a t e n d e n c y t o v a c u o l i s a t i o n o f t h e a d r e n a l m e d u l l a was f o u n d at a d o s e level o f 100 p p m . A t t h e 30 a n d 1 0 0 - p p m dose level increased h a e m o s i d e r o s i s was f o u n d in t h e spleen 11 A l t h o u g h in o u r e x p e r i m e n t w i t h t e t r a d i f o n no h a e m o s i d e r o s i s was f o u n d in t h e spleen, t h e o t h e r results are n o t c o n t r a d i c t o r y . C h l o r f e n s o n was given t o rats at d o s e levels o f 300, 1 0 0 0 and 3 0 0 0 p p m . In t h e 1 0 0 0 a n d 3 0 0 0 p p m dosage levels liver w e i g h t was increased and slight d e g e n e r a t i o n was o b s e r v e d . T h e 3 0 0 - p p m g r o u p was r e g a r d e d as a n o - t o x i c e f f e c t level 1 2 In o u r e x p e r i m e n t t h e 2 0 0 - p p m d o s e level caused an increase in liver a n d t h y r o i d w e i g h t a n d even at t h e 5 0 - p p m d o s e level histological c h a n g e s w e r e o b s e r v e d in t h e s e organs (Tables IV a n d V). In a l a t e r l o n g - t e r m e x p e r i m e n t a n o n - t o x i c e f f e c t level o f 25 p p m c h l o r f e n s o n was f o u n d 13. This c o u l d be in line w i t h o u r findings. K e l t h a n e was given at d o s e levels o f 0, 20, 1 0 0 , 500, 1 2 5 0 a n d 2 5 0 0 p p m t o rats 14. T h e results o b t a i n e d are well in a c c o r d a n c e w i t h t h e p r e s e n t investigation. In 1 o u t o f 10 m a l e r a t s at t h e 2 0 - p p m d o s e level liver a l t e r a t i o n s such as focal swollen a c i d o p h i l i c c y t o p l a s m w i t h v a c u o l e s w e r e found. In o n e o f t h e r e f e r e n c e s t h y r o i d a l t e r a t i o n s are m e n t i o n e d . T h e r e a s o n m i g h t be t h a t t h e diets used c o n t a i n e d high a m o u n t s o f iodine. U n p u b l i s h e d w o r k o f t h e a u t h o r s s h o w e d a clear r e l a t i o n s h i p b e t w e e n t h e a m o u n t o f iodine in t h e diet a n d t h e e f f e c t o f t e t r a s u l o n t h e t h y r o i d . In t h e p r e s e n t e x p e r i m e n t a diet was given w h i c h was l a t e r p r o v e d t o be v e r y l o w in iodine.
ACKNOWLEDGEMENT T h e a u t h o r s a c k n o w l e d g e t h e c o o p e r a t i o n o f Dr. P. H e l l e m a n a n d his s t a f f in t h e h a e m a t o l o g i c a l w o r k a n d also of t h e s t a f f o f t h e A n i m a l H o u s e . REFERENCES 1 2 3 4 5 6 7 8 9 10 11
E. Schi~tz, Arzneimittel-Forsch., 18 (1968) 466. H.G. Verschuuren, Food Cosmet. Toxicol., 5 (1967)450. H.G. Verschuuren, R. Kroes and G.J. van Esch, Toxicology, 1 (1973) 63. H.G. Verschuuren,R. Kroes and E.M. den Tonkelaar, Toxicology, 1 (1973) 103. F. Wilcoxon, Biometr. Bull., 1 (1945) 80. H.G. Verschuuren, Toxicologisch Onderzoek van Tetrasul en Verwante Verbindingen, Diss. Utrecht, 1970, p. 20. A.H. Conney, C. Davison, R. Gastel and J.J. Burns, J. Pharmacol. Exptl. Ther., 130 (1960) 1. J.R. Fours and L.A. Rogers, J. Pharmacol. Exptl. Ther., 147 (1965) 112. M. Alfert and J.J. Geschwind, Exptl. Cell Res., 15 (1958) 230. W. St}iubli, R. Hess and E.R. Weibel, J. CeUBiol., 42 (1969) 92. Unpublished report 1957, Philips Duphar Ltd., Weesp, The Netherlands.
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12 Technical Bulletin, Dow Chemical Company, Midland, Michigan, 1951. 13 C.S. Weil and D.D. McCollister, Jo Agr. Food Chem., 11 (1963) 486. 14 K.B. Smith, P.S. Larson, K.J. Finnegan, H.B. Haag, G.R. Hennigan and F. Coley, Toxicol. Appl. Pharmacol., 1 (1959) 119.
123