Toxicology, 6 (1976) 97--106 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
TOXICITY OF M E T H Y L M E R C U R Y C H L O R I D E IN RATS II. R E P R O D U C T I O N STUDY H.G. VERSCHUUREN, R. KROES, ENGELINA M. DEN TONKELAAR, J.M. BERKVENS, P.W. HELLEMAN, A.G. RAUWS, P.L. SCHULLER and G.J. VAN ESCH National Institute of Public Health, Bilthoven (The Netherlands)
(Received November 25th, 1975) (Accepted February 10th, 1976)
SUMMARY A r e p r o d u c t i o n study over 3 generations of rats was carried out in which groups of 20 female and 10 male rats received in the diet 0, 0.1, 0.5 and 2.5 p p m MeHgC1. The parameters studied included growth, f o o d intake, haematology, serum and urinalysis, organ weights and reproductive performance. No effect was exerted on fertility index, lactation index or on the 21-day b o d y weights of pups but the viability index was impaired at 2.5 ppm in the F1 and F2 generations. Weight gain reductions observed at 12 weeks for the 2.5 p p m level were n o t accompanied by reductions in f o o d intake. At 6 months, Fla females on 2.5 ppm showed a reduced l eucocyt e c o u n t whilst P males on 0.5 and 2.5 ppm showed an increase in neutrophils and a decrease in l y m p h o c y t e s . The relative weights of the kidneys, heart, spleen, brain and t h y r o i d were increased at 2.5 ppm and in some cases the increases of kidney weights were inconsistently seen at the 0.1 and 0.5 ppm levels. No significant histological changes were seen at any level. In a special 7-week study involving the F3a generation, weanling rats obtained f r om the four different F2a groups, each comprising 20 females and 10 males, were all transferred to diets containing 25 p p m MeHgC1. T o x i c i t y signs were evident at 7 weeks. No evidence was obtained o f increased susceptibility to the t o x i c i t y of MeHgC1 in successive generations.
INTRODUCTION An i n t r o d u c t i o n to the t o x i c i t y of organic m ercury c o m p o u n d s , especially m e t h y l m e r c u r y , was presented in the first of the three papers in this series
[1]. Abbreviations: AH, aniline hydroxylase; Alk.Pase, alkaline phosphatase; APDM, aminopyrine demethylase; GPT, glutamic-pyruvie transaminase.
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In addition to studying the effect of MeHgC1 on breeding performance, the current r e p r o d u c t i o n study examined the possibility of increased susceptibility to MeHgC1 toxicity in successive generations. EXPERIMENTAL The material testea, animals and diets used, analytical methods and enz y me assays ana statistical analysis c o n a u c t e d are as described in the first paper in this series [1] except that 10% cellulose was aaued to the stanaard diet in order to reduce the nutritive value.
Experimental design and conduct Four groups, each of 20 female and 10 male weanling rats on 0, 0.1, 0.5 and 2.5 ppm MeHgC1 were taken from the long-term study [ 2 ] and used for the p r o d u c t i o n of two litters and then returned to complete the long-term test. Each male was allowed to mate with 2 females for one week to produce the first litter ( F l a ) and this was repeated after 6 weeks to p r o a u c e the second litter ( F l b ) . The same procedure was followed for each generation. The animals of F l a and F2a generations were used to produce two nests of the following generations F2a + F2b and F3a + F3b, respectively. The F3a generation was used in a special 7-week experiment. The F l b , F2b and F3b litters were killed at weaning. Offspring were count ed on days 1, 5 and 21 and the nest weights were recorded at days 5 and 21. All pups were observed for macroscopic changes. The F l a and F2a generations were observed for weight gain, food intake, behaviour and mortality. Haematological examination was carried out on 10 female and 10 male rats of each group after 6 months for haemoglobin concentration, packed cell volume, total e r y t h r o c y t e and total and differential leucocyte counts. After 6 months, serum levels of Alk.Pase, GPT and urea and hepatic levels of AH, APDM and glycogen were determined in 4--10 animals per group. Urinalysis was carried o u t in 10 female and 10 male animals per group for pH, the presence o f protein, glucose, ketone bodies, bilirubin and blood and for osmolality. The F l a and F2a generations were killed after 6--7 months and the heart, brain, liver, kidneys, spleen, adrenals, thyroid, pituitary, uterus, ovaries, testes and prostate were weighed. Histopathological examination was p er f o r m e d on brain, liver, kidneys, eye, spinal cord and Nervus ischiadicus of rats of all groups. In the special 7-week study with the F3a generation, weanling rats obtained from the f our different F2a groups, each comprising 20 females and 10 males on 0, 0.1, 0.5 and 2.5 ppm were all transferred to diets containing 25 ppm. A fifth group of 20 females and 10 males on 0 ppm remained on the basal diet. F o o d intake was recorded at weeks 1, 2 and 5 and body weights weekly. After 7 weeks total m e r cur y or MeHgC1 c o n c e n t r a t i o n was det erm i ned in blood, kidneys, brain and liver of 5 males per group. Urinalysis (Bililabstix ® )
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was carried o u t on 10 females and 10 males per group. The e x p e r i m e n t was t e r min ated after 7 weeks. Liver, kidneys and brain were weighed. These organs and the spinal cord, peripheral nerves and eyes were examined microscopically. RESULTS
Significant reductions in weight gain were seen in P females on 0.1 ppm at 6 and 12 weeks, increased weights were seen in F l a females on 0.1 and 2.5 p p m at 6 weeks and in F l a males on 0.1 p pm at 6 weeks, decreased weights in F2a females on 0.1, 0.5 and 2.5 ppm at 12 weeks and in F2a males on 2.5 p p m at 12 weeks (Table I). G r o w t h retardation was n o t accompanied by a corresponding r e duc t i on in f o o d intake and in several cases food intake was significantly increased. Alterations in the head profile and the regularity of the teeth as described in the long-term test were observed in t h e F l a generation (2 females and 1 male on 0 ppm, 1 male on 0.1 ppm and 3 females on 0.5 ppm) and in the
TABLE I W E I G H T G A I N (g) O V E R 6 A N D 12 W E E K S IN T H R E E G E N E R A T I O N S O F R A T S
Generation
Females P
Week
Number o f animals
Initial w e i g h t (g) a n d w e i g h t gain (g) 0 ppm
0.1 p p m
0.5 p p m
2.5 p p m
0 6 12
25 25 25
47 112 149
48 102 xx 141 xx
47 110 146
46 108 145
Fla
0 6 12
20 20 20
65 88 117
58 97 x 125
70 91 123
60 95 122
F2a
0 6 12
10 10 10
48 113 153
47 108 142 x
53 109 140 x
54 105 134
0 6 12
35 35 34
50 183 271
50 180 271
50 189 276
50 178 260
Fla
0 6 12
10 10 10
62 173 250
67 194 x 267
73 192 260
69 186 250
F2a
0 6 12
10 10 10
55 198 283
54 209 295
63 213 294
56 191 258
Males P
x, P ~ 0 . 0 5 ; xx, P ~
0.01.
99
F2a generation (2 females on 0 ppm, 1 female on 0.1 ppm and 1 male on 2.5 ppm). It is unlikely that this p h e n o n o m e n o n is caused by MeHgC1 as it also occurs in control animals. Haematological examination of the P, F l a and F2a generations after 6 months revealed a reduction in the leucocyte c o u n t in F l a females on 2.5 ppm and an increase in neutrophils and a decrease in l y m p h o c y t e s in P males on 0.5 and 2.5 ppm (Table II). The opposite effect, namely lowered neutrophil and raised l y m p h o c y t e c o u n t was seen in P females on 0.1 ppm and in F2a males on 0.5 ppm. Serum levels at 6 months of GPT, Alk.Pase and urea were normal in all groups of all generations, except for a decreased urea c o n t e n t in F2a females on 2.5 ppm. Urinalysis of the F l a and F2a generations gave normal values for all groups at 6 months. Liver microsomal enzym e assays showed the activity of AH to be decreased non-significantly in F l a males on 0.5 aria 2.5 ppm. Liver glycogen was decreased non-significantly in F l a males and females on 2.5 ppm. The most notable changes in relative organ weights (Table III) were the increases in the relative kidney weight at 2.5 ppm in the P, F l a and F2a generations, at 0.5 ppm in F2a males and females and at 0.1 ppm in F2a females and F l a males. Significant increases were also seen in the relative heart weight in F l a and F2a males on 2.5 ppm (F2a females on 2.5 ppm showed a decrease), in the relative brain weight of F2a females on 0.5 and 2.5 ppm, in the relative spleen weight in F l a males on 2.5 ppm and in the relative th y r o id weight of F2a females and males on 2.5 ppm and in F l a males on 2.5 ppm. The results obtained on reproductive performance showed no effect on the fertility index, lactation index and body weight of pups at 21 days. The viability index was decreased at 2.5 ppm in the F1 and F2 generations (Table IV). No significant macroscopic changes nor histological changes were seen in the F l a and F2a generations at any level, except for a slight degeneration of the granular cells in the brain of 1 male on 2.5 ppm. In the special study involving the F3a generation, all groups which received 25 p p m MeHgC1 showed a similar degree of growth retardation and decreased f o o d intake and no differences in relation to the original dose levels were observed (Table V). In some animals fractures in the Os nasale or loose incisors were observed. The animals recovered f r om this discomfort except in one case. From week 7 onwards rats receiving 25 ppm MeHgC1 started to show signs of paralysis and for this reason the e xpe r i m e nt was terminated. Although the first signs of clinical intoxication were observed somewhat earlier than in the short-term toxicity study, this was n o t regarded as significant. Urinalysis revealed higher protein contents in all groups given 25 ppm and also the presence of ke t one bodies in males (Table VI). In all rats which had received 25 ppm MeHgC1, the weights of the kidneys
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T A B L E II H A E M A T O L O G I C A L E X A M I N A T I O N IN 3 G E N E R A T I O N S A F T E R 6 M O N T H S
MeHgCl
Females
Males
(ppm) P
Fla 9.6 9.4 9.6 9.6
H a e m o g l o b i n (mmoles/1)
0 0.1 0.5 2.5
Packed cell v o l u m e (%)
0 0.1 0.5 2.5
Eryhthrocytes (106/ mm s )
0 0.1 0.5 2.5
7.5 7.2 7.4 7.3
8.1 8.1 8.1 8.0
7.7 8.0 8.0 8.4 x
Total l e u c o c y t e s (10s/ram s )
0 0.1 0.5 2.5
8.7 9.0 8.8 8.7
7.9 7.4 7.3 6.5 xx
9.9 9.2 8.9 8.6
47 44 46 46
10.3 9.8 x 9.7 9.9
F2a
51 48 xx 48 48 x
9.4 9.5 9.8 9.9 46 46 47 48
P
Fla 9.5 9.4 9.6 9.3
46 45 46 45
9.6 9.6 9.4 9.6 47 47 46 47
F2a 9.0 9.3 9.3 9.1 45 46 45 44
7.8 7.7 7.7 7.5
8.3 8.3 8.2 8.1
8.0 8.3 8.3 8.3
10.2 10.4 9.7 9.5
9.8 8.9 8.9 8.6
12.5 11.7 11.0 10.7
Differential leucocyte count (%) Basophils
0 0.1 0.5 2.5
0.4 0.4 0.8 0.5
0.3 0.1 0:4 0.2
0.4 0.4 0.1 x 0.2
0.2 0.5 0.3 0.5
0.2 0.4 0.5 0.2
0.3 0.5 0.3 0.2
Eosinophils
0 0.1 0.5 2.5
2.4 2.0 2.6 2.2
1.6 1.4 1.6 1.5
1.9 1.5 1.9 2.1
1.2 2.2 1.7 2.6
1.5 1.3 1.5 1.7
1.5 1.9 1.4 2.5
Neutrophils
0 0.1 0.5 2.5
15.1 9.9 x 17.8 13.3
12.0 14.2 16.6 14.4
10.6 11.5 15.5 13.1
11.1 13.3 17.3 x 16.4 xx
12.0 14.1 13.4 12.7
14.0 9.9 9.7 x 11.0
Lymphocytes
0 0.1 0.5 2.5
79.7 85.1 76.6 81.6
84.0 81.7 79.1 82.4
83.5 82.6 77.6 81.1
85.4 81.9 78.7 x 78.8 xx
84.1 81.4 82.6 83.2
79.8 83.6 85.5 x 82.4
Monocytes
0 0.1 0.5 2.5
2.5 2.7 2.4 2.5
2.2 2.8 2.5 1.6
3.6 4.0 4.9 3.5
2.2 2.3 2.1 1.8
2.4 2.8 2.1 2.2
4.4 4.1 3.1 3.9
A t y p i c a l cells
0 0.1 0.5 2.5
2.5 2.2 3.1 2.7
3.3 3.4 3.5 3.3
5.1 7.2 5.7 13.1 x
2.3 2.2 2.8 2.1
2.6 3.4 3.1 2.5
6.5 6.1 6.1 6.6
Values are f o r g r o u p s o f 10 males and 10 females. x, P ~ 0.05; xx, P ~ 0.01.
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T A B L E III R E L A T I V E O R G A N W E I G H T S (%) Females
Number of animals P-generation b Heart Brain Liver Kidneys Spleen Adrenals Thyroid Pituitary Uterus/prostate Ovaries/testes
Number of animals F l a-generation a Heart Brain Liver Kidneys Spleen Adrenals Thyroid Pituitary Ovaries/testes Uterus/prostate
Number of animals F2a-generation a Heart Brain Liver Kidneys Spleen Adrenals Thyroid Pituitary Ovaries/testes Uterus/prostate
Males
0 ppm
0.1 p p m 0.5 p p m 2.5 p p m 0 p p m
0.1 p p m 0.5 p p m 2 . 5 p p m
10
10
10
10
10
0.306 0.434 2.87 0.698 0.169 0.008
0.309 0.444 3.20 0.842 0.200 0.009
0.345 0.452 3.26 0 . 9 2 9 xx 0.198 0.010
0.003 0.667 0.110
0.003 0.647 0.103
0.003 0.679 0.102
10
10
10
9
0.404 0.417 0.667 0.656 3.31 3.09 0.797 0.780 0.203 0.182 0.019 0.017 . . . 0.005 0.005 0.225 0.188 0.023 0.026
0.396 0.650 3.36 0.866 0.186 0.018 . 0.005 0.224 0.028
0.461 0.318 0.668 0.442 3.55 3.06 1 . 0 8 6 xx 0 . 7 1 7 0.219 0.205 0.020 0.009 . . 0.006 0.003 0.209 0.720 0.026 0.101
10
10
10
10
10
10
0.365 0.767 4.44 0.919 0.196 0.022 0.006 0.006 0.189 0.036
0.342 0.726 4.14 0.841 0.167 0.018 0.006 0.005 0.188 0.029
0.352 0.817 3.91 x 1.003 0.190 0.019 0.006 0.006 0.231 0.031
0.264 0.482 4.14 0.716 0.134 0.010 0.004 0.003 0.817 0.094
0 . 2 9 3 xx 0 . 2 6 0 0.282 0.481 0.480 0.501 4.45 4.09 4.31 0 . 8 0 2 xx 0 . 7 7 0 0.856 0.140 0.135 0.172 0.011 0.010 0.009 0.005 0.004 0.005 0.003 0.003 0.003 0.825 0.704 x 0.783 0.077 0.083 0.087
13
17
8
8
7
0.438 0.736 4.06 0.834 0.174 0.021 0.004 0.006 0.175 0.035
0.356 0.364 0.760 0 . 7 8 6 xx 3.82 3.67 xx 0 . 9 2 7 xx 0 . 9 4 7 xx 0.179 0.179 0.020 0.021 0.005 x 0.005 0.006 0.005 0.178 0.191 0.036 0.035
x 0.328 x 0.749 2.77 xx 0.929 x 0.159 x x 0.019 0.005 0.005 0.168 x 0.032
20
x, P ~ 0.05; x x , P ~ 0 . 0 1 ; x x x , P ~ 0 . 0 0 1 . a A f t e r 6 m o n t h s o n test. b A f t e r 2 y e a r s o n test.
102
0.377 0.822 3.80 1.105 0.189 0.019 0.005 0.006 0.179 0.030
9
xx 0 . 2 7 0 x 0.511 4.04 xx 0 . 7 5 6 0.148 x 0.010 xx 0 . 0 0 4 0.003 0.867 0.081
.
.
10
x
xxx xxx xx
5
0.269 0.267 0.298 x 0.467 x 0.501 0.559 3.83 3.62 x 3.77 0.741 0.811 x 0.893xx 0.148 0.131 0.149 0.009 0.009 0.009 0.004 0.004 0.005 x 0.002 0.003 0.003 0 . 7 7 3 x 0 . 7 1 6 xx 0 . 7 3 4 0.070 0.093 0.075
T A B L E IV REPRODUCTION PERFORMANCE Generation
0 ppm
0.1 p p m
0.5 p p m
2.5 p p m
81 72 79
68 74 80
75 70 89
83 66 74
86 59 70
85 28 52
57 70 65
77 86 88
66 88 92
34 34 34
33 36 38
Fertility index a P F1 F2
78 75 70 Viability i n d e x b
P F1 F2
86 66 64 Lactation index c
P F1 F2
68 69 94
Mean b o d y w e i g h t at day 21 (g) P F1 F2
30 34 34
31 32 36
N u m b e r o f r a t s i n each g r o u p
P F1 F2
F
M
F
M
F
M
F
M
20 20 15
10 10 8
16 18 17
8 9 9
16 18 20
8 9 10
18 20 9
9 10 5
a N u m b e r o f p r e g n a n c i e s X 100 divided b y t h e n u m b e r o f m a t i n g s b N u m b e r o f p u p s alive at day 5 X 100 divided b y t h e n u m b e r o f p u p s b o r n . c N u m b e r o f p u p s alive at day 21 X 100 divided b y the n u m b e r o f p u p s alive at day 5.
TABLE V WEIGHT G A I N IN S P E C I A L F 3 a G E N E R A T I O N S T U D Y A T 6 W E E K S Parameter
0 ppm
Females Initial w e i g h t (g) Weight gain (g) at w e e k 6
87 75
Males Initial w e i g h t (g) Weight gain (g) at w e e k 6
109 159
0 + 25 ppm
89 56 xxx
105 137 x
0.1 + 25 ppm
0.5 + 25 ppm
2.5 + 25 ppm
93 60 xxx
87 55 xxx
76 59 xx
104 120 xxx
90 127 xx
i13 132 x
x , P < 0 . 0 5 ; x x , P < 0 . 0 1 ; x x x ; P < 0.001. Significant d i f f e r e n c e s are t h o s e b e t w e e n e x p e r i m e n t a l groups, as c o m p a r e d w i t h 0 p p m group. No significant d i f f e r e n c e exists b e t w e e n e x p e r i m e n t a l groups and 0 + 25 p p m group.
T A B L E VI U R I N A L Y S I S IN S P E C I A L F 3 a G E N E R A T I O N S T U D Y A T 6 W E E K S Parameter
0 ppm
0 + 25 ppm
0.1 + 25 ppm
0.5 + 25 ppm
2.5 + 25 ppm
Females Protein content
±a + ++ +++
1 8 1 0
0 1 7 2
0 2 7 1
0 2 7 1
0 1 7 2
+
10 0
10 0
10 0
9 1
10 0
+ ++ +++
2 8 0
1 7 2
0 8 2
0 8 2
0 3 7
1
1
3
2
3 5 1 0
4 3 2 0
1 6 0 0
3 3 1 1
Ketones
--
Males Protein content
Ketones
--
± + ++ +++
10
0 0 0 0
a __, n o n e ; -+, trace; +, s o m e ; ++, m o d e r a t e ; +++, m u c h . T h e p H was n e a r l y a l w a y s 6.0, in a few cases 6.5. G l u c o s e , b i l i r u b i n a n d b l o o d were n o t detected.
T A B L E VII RELATIVE 6 WEEKS
ORGAN
Organ
WEIGHTS
(%) IN S P E C I A L F 3 a G E N E R A T I O N
STUDY AT
0 ppm
0 + 25 ppm
0.1 + 25 ppm
0.5 + 25 ppm
2.5 + 25 ppm
Number of animals
20
19
20
201
17
Liver Kidneys Brain
3.92 0.915 0.998
4.39 1.319 1.142
4.21 1.385 1.098
4.38 1.458 x 1.217 x
4.50 1 . 4 3 8 xx 1 . 2 6 1 xx
Number of animals
10
9
8
7
6
Liver Kidneys Brain
4.51 0.860 0.647
4.29 1.309 0.752
4.57 1 . 4 9 0 xx 0.763
4.20 1.394 0.790
4.18 1.443 x 0.815
Females
Males
x , P < 0 . 0 5 ; x x , P < 0.01. Significance was c a l c u l a t e d in c o m p a r i s o n w i t h t h e 0 + 25 p p m g r o u p . T h e relative w e i g h t s o f k i d n e y s a n d brain o f t h e f o u r e x p e r i m e n t a l g r o u p s were s i g n i f i c a n t l y i n c r e a s e d in b o t h s e x e s in c o m p a r i s o n w i t h t h e 0 p p m g r o u p (P < 0.01 or 0 . 0 0 1 ) as did t h e relative liver w e i g h t in f e m a l e s .
TABLE VIII TISSUE LEVELS OF Hg A F T E R 7 WEEKS IN SPECIAL F3a GENERATION STUDY Tissue
0 ppm
0 + 25 ppm
0.1 + 25 ppm
Blood (total Hg) Kidneys (total Hg) Liver (MeHgCl) Brain (MeHgC1)
0.18 + 0.034 0.47 + 0.15 ---
146 86.8 67.9 10.6
227 92.8 84.8 12.9
Tissue
0.5 + 25 ppm
2.5 + 25 ppm
Blood (total Hg) Kidneys (total Hg) Liver (MeHgCl) Brain (MeHgC1)
227 79.2 103 11.0
151 81.3 87.6 11.2
+ + + +
37.0 xx 13.1 24 xx 1.3
+ + + +
+ + + +
18.1 8.75 2.2 0.9
+ 21.5 xx + 17.5 -+ 6.5 xx + 1.3 x
26.1 15.2 3.6 xx 1.5
Values are for group of 5 males. x, P ~ 0.05; xx, P ~ 0.01. Significance is calculated compared with the 0 + 25 ppm group.
and the brain were significantly increased when compared with the 0 ppm control group (Table VII). The relative liver weight of females was increased n o n - s i g n i f i c a n t l y in all 2 5 p p m g r o u p s . W h e n t h e i n d i v i d u a l g r o u p s w e r e c o m p a r e d t o t h e 0 + 2 5 p p m g r o u p , i n c r e a s e d r e l a t i v e w e i g h t s w e r e s e e n in the kidneys and brain of females on 0.5 + 25 ppm and on 2.5 + 25 ppm and in t h e k i d n e y s o f m a l e s o n 0 . 1 + 2 5 p p m a n d o n 2 . 5 + 2 5 p p m . The characteristic lesions of the nervous system and kidneys seen histol o g i c a l l y in t h e s h o r t - t e r m s t u d y w e r e f o u n d in all g r o u p s w h i c h r e c e i v e d 25 ppm. No differences between the treated groups could be observed. Lesions in t h e c e r e b e l l u m w e r e o n l y o b s e r v e d in t r e a t e d f e m a l e s . MeHgC1 a d m i n i s t r a tion to the parent generation did not profoundly affect tissue concentrat i o n s in t h e F 3 a g e n e r a t i o n a n d e x c e p t f o r b l o o d t h e c o n c e n t r a t i o n s o b tained at various dose levels were comparable (Table VIII). DISCUSSION F o r e a s e o f c o m p a r i s o n , t h e r e s u l t s o b t a i n e d in t h e r e p r o d u c t i o n s t u d y h a v e b e e n d i s c u s s e d in t h e l i g h t o f t h o s e o b t a i n e d in t h e s h o r t - a n d l o n g - t e r m s t u d i e s . T h e d i s c u s s i o n a p p e a r s in t h e r e p o r t o n t h e l o n g - t e r m s t u d y .
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REFERENCE 1 H.G. Verschuuren, R. Kroes, Rauws, P.L. Schuller and G.J. 2 H.G. Verschuuren, R. Kroes, Rauws, P.L. Schuller and G.J.
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E.M. den Tonkelaar, J.M. Berkvens, P.W. Helleman, A.G. van Esch, Toxicology, 6 (1976) 85. E.M. den Tonkelaar, J.M. Berkvens, P.W. Helleman, A.G. van Esch, Toxicology 16 (1976) 107.
TOXICITY OF M E T H Y L M E R C U R Y C H L O R I D E IN RATS II. R E P R O D U C T I O N STUDY H.G. VERSCHUUREN, R. KROES, ENGELINA M. DEN TONKELAAR, J.M. BERKVENS, P.W. HELLEMAN, A.G. RAUWS, P.L. SCHULLER and G.J. VAN ESCH National Institute of Public Health, Bilthoven (The Netherlands)
(Received November 25th, 1975) (Accepted February 10th, 1976)
SUMMARY A r e p r o d u c t i o n study over 3 generations of rats was carried out in which groups of 20 female and 10 male rats received in the diet 0, 0.1, 0.5 and 2.5 p p m MeHgC1. The parameters studied included growth, f o o d intake, haematology, serum and urinalysis, organ weights and reproductive performance. No effect was exerted on fertility index, lactation index or on the 21-day b o d y weights of pups but the viability index was impaired at 2.5 ppm in the F1 and F2 generations. Weight gain reductions observed at 12 weeks for the 2.5 p p m level were n o t accompanied by reductions in f o o d intake. At 6 months, Fla females on 2.5 ppm showed a reduced l eucocyt e c o u n t whilst P males on 0.5 and 2.5 ppm showed an increase in neutrophils and a decrease in l y m p h o c y t e s . The relative weights of the kidneys, heart, spleen, brain and t h y r o i d were increased at 2.5 ppm and in some cases the increases of kidney weights were inconsistently seen at the 0.1 and 0.5 ppm levels. No significant histological changes were seen at any level. In a special 7-week study involving the F3a generation, weanling rats obtained f r om the four different F2a groups, each comprising 20 females and 10 males, were all transferred to diets containing 25 p p m MeHgC1. T o x i c i t y signs were evident at 7 weeks. No evidence was obtained o f increased susceptibility to the t o x i c i t y of MeHgC1 in successive generations.
INTRODUCTION An i n t r o d u c t i o n to the t o x i c i t y of organic m ercury c o m p o u n d s , especially m e t h y l m e r c u r y , was presented in the first of the three papers in this series
[1]. Abbreviations: AH, aniline hydroxylase; Alk.Pase, alkaline phosphatase; APDM, aminopyrine demethylase; GPT, glutamic-pyruvie transaminase.
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In addition to studying the effect of MeHgC1 on breeding performance, the current r e p r o d u c t i o n study examined the possibility of increased susceptibility to MeHgC1 toxicity in successive generations. EXPERIMENTAL The material testea, animals and diets used, analytical methods and enz y me assays ana statistical analysis c o n a u c t e d are as described in the first paper in this series [1] except that 10% cellulose was aaued to the stanaard diet in order to reduce the nutritive value.
Experimental design and conduct Four groups, each of 20 female and 10 male weanling rats on 0, 0.1, 0.5 and 2.5 ppm MeHgC1 were taken from the long-term study [ 2 ] and used for the p r o d u c t i o n of two litters and then returned to complete the long-term test. Each male was allowed to mate with 2 females for one week to produce the first litter ( F l a ) and this was repeated after 6 weeks to p r o a u c e the second litter ( F l b ) . The same procedure was followed for each generation. The animals of F l a and F2a generations were used to produce two nests of the following generations F2a + F2b and F3a + F3b, respectively. The F3a generation was used in a special 7-week experiment. The F l b , F2b and F3b litters were killed at weaning. Offspring were count ed on days 1, 5 and 21 and the nest weights were recorded at days 5 and 21. All pups were observed for macroscopic changes. The F l a and F2a generations were observed for weight gain, food intake, behaviour and mortality. Haematological examination was carried out on 10 female and 10 male rats of each group after 6 months for haemoglobin concentration, packed cell volume, total e r y t h r o c y t e and total and differential leucocyte counts. After 6 months, serum levels of Alk.Pase, GPT and urea and hepatic levels of AH, APDM and glycogen were determined in 4--10 animals per group. Urinalysis was carried o u t in 10 female and 10 male animals per group for pH, the presence o f protein, glucose, ketone bodies, bilirubin and blood and for osmolality. The F l a and F2a generations were killed after 6--7 months and the heart, brain, liver, kidneys, spleen, adrenals, thyroid, pituitary, uterus, ovaries, testes and prostate were weighed. Histopathological examination was p er f o r m e d on brain, liver, kidneys, eye, spinal cord and Nervus ischiadicus of rats of all groups. In the special 7-week study with the F3a generation, weanling rats obtained from the f our different F2a groups, each comprising 20 females and 10 males on 0, 0.1, 0.5 and 2.5 ppm were all transferred to diets containing 25 ppm. A fifth group of 20 females and 10 males on 0 ppm remained on the basal diet. F o o d intake was recorded at weeks 1, 2 and 5 and body weights weekly. After 7 weeks total m e r cur y or MeHgC1 c o n c e n t r a t i o n was det erm i ned in blood, kidneys, brain and liver of 5 males per group. Urinalysis (Bililabstix ® )
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was carried o u t on 10 females and 10 males per group. The e x p e r i m e n t was t e r min ated after 7 weeks. Liver, kidneys and brain were weighed. These organs and the spinal cord, peripheral nerves and eyes were examined microscopically. RESULTS
Significant reductions in weight gain were seen in P females on 0.1 ppm at 6 and 12 weeks, increased weights were seen in F l a females on 0.1 and 2.5 p p m at 6 weeks and in F l a males on 0.1 p pm at 6 weeks, decreased weights in F2a females on 0.1, 0.5 and 2.5 ppm at 12 weeks and in F2a males on 2.5 p p m at 12 weeks (Table I). G r o w t h retardation was n o t accompanied by a corresponding r e duc t i on in f o o d intake and in several cases food intake was significantly increased. Alterations in the head profile and the regularity of the teeth as described in the long-term test were observed in t h e F l a generation (2 females and 1 male on 0 ppm, 1 male on 0.1 ppm and 3 females on 0.5 ppm) and in the
TABLE I W E I G H T G A I N (g) O V E R 6 A N D 12 W E E K S IN T H R E E G E N E R A T I O N S O F R A T S
Generation
Females P
Week
Number o f animals
Initial w e i g h t (g) a n d w e i g h t gain (g) 0 ppm
0.1 p p m
0.5 p p m
2.5 p p m
0 6 12
25 25 25
47 112 149
48 102 xx 141 xx
47 110 146
46 108 145
Fla
0 6 12
20 20 20
65 88 117
58 97 x 125
70 91 123
60 95 122
F2a
0 6 12
10 10 10
48 113 153
47 108 142 x
53 109 140 x
54 105 134
0 6 12
35 35 34
50 183 271
50 180 271
50 189 276
50 178 260
Fla
0 6 12
10 10 10
62 173 250
67 194 x 267
73 192 260
69 186 250
F2a
0 6 12
10 10 10
55 198 283
54 209 295
63 213 294
56 191 258
Males P
x, P ~ 0 . 0 5 ; xx, P ~
0.01.
99
F2a generation (2 females on 0 ppm, 1 female on 0.1 ppm and 1 male on 2.5 ppm). It is unlikely that this p h e n o n o m e n o n is caused by MeHgC1 as it also occurs in control animals. Haematological examination of the P, F l a and F2a generations after 6 months revealed a reduction in the leucocyte c o u n t in F l a females on 2.5 ppm and an increase in neutrophils and a decrease in l y m p h o c y t e s in P males on 0.5 and 2.5 ppm (Table II). The opposite effect, namely lowered neutrophil and raised l y m p h o c y t e c o u n t was seen in P females on 0.1 ppm and in F2a males on 0.5 ppm. Serum levels at 6 months of GPT, Alk.Pase and urea were normal in all groups of all generations, except for a decreased urea c o n t e n t in F2a females on 2.5 ppm. Urinalysis of the F l a and F2a generations gave normal values for all groups at 6 months. Liver microsomal enzym e assays showed the activity of AH to be decreased non-significantly in F l a males on 0.5 aria 2.5 ppm. Liver glycogen was decreased non-significantly in F l a males and females on 2.5 ppm. The most notable changes in relative organ weights (Table III) were the increases in the relative kidney weight at 2.5 ppm in the P, F l a and F2a generations, at 0.5 ppm in F2a males and females and at 0.1 ppm in F2a females and F l a males. Significant increases were also seen in the relative heart weight in F l a and F2a males on 2.5 ppm (F2a females on 2.5 ppm showed a decrease), in the relative brain weight of F2a females on 0.5 and 2.5 ppm, in the relative spleen weight in F l a males on 2.5 ppm and in the relative th y r o id weight of F2a females and males on 2.5 ppm and in F l a males on 2.5 ppm. The results obtained on reproductive performance showed no effect on the fertility index, lactation index and body weight of pups at 21 days. The viability index was decreased at 2.5 ppm in the F1 and F2 generations (Table IV). No significant macroscopic changes nor histological changes were seen in the F l a and F2a generations at any level, except for a slight degeneration of the granular cells in the brain of 1 male on 2.5 ppm. In the special study involving the F3a generation, all groups which received 25 p p m MeHgC1 showed a similar degree of growth retardation and decreased f o o d intake and no differences in relation to the original dose levels were observed (Table V). In some animals fractures in the Os nasale or loose incisors were observed. The animals recovered f r om this discomfort except in one case. From week 7 onwards rats receiving 25 ppm MeHgC1 started to show signs of paralysis and for this reason the e xpe r i m e nt was terminated. Although the first signs of clinical intoxication were observed somewhat earlier than in the short-term toxicity study, this was n o t regarded as significant. Urinalysis revealed higher protein contents in all groups given 25 ppm and also the presence of ke t one bodies in males (Table VI). In all rats which had received 25 ppm MeHgC1, the weights of the kidneys
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T A B L E II H A E M A T O L O G I C A L E X A M I N A T I O N IN 3 G E N E R A T I O N S A F T E R 6 M O N T H S
MeHgCl
Females
Males
(ppm) P
Fla 9.6 9.4 9.6 9.6
H a e m o g l o b i n (mmoles/1)
0 0.1 0.5 2.5
Packed cell v o l u m e (%)
0 0.1 0.5 2.5
Eryhthrocytes (106/ mm s )
0 0.1 0.5 2.5
7.5 7.2 7.4 7.3
8.1 8.1 8.1 8.0
7.7 8.0 8.0 8.4 x
Total l e u c o c y t e s (10s/ram s )
0 0.1 0.5 2.5
8.7 9.0 8.8 8.7
7.9 7.4 7.3 6.5 xx
9.9 9.2 8.9 8.6
47 44 46 46
10.3 9.8 x 9.7 9.9
F2a
51 48 xx 48 48 x
9.4 9.5 9.8 9.9 46 46 47 48
P
Fla 9.5 9.4 9.6 9.3
46 45 46 45
9.6 9.6 9.4 9.6 47 47 46 47
F2a 9.0 9.3 9.3 9.1 45 46 45 44
7.8 7.7 7.7 7.5
8.3 8.3 8.2 8.1
8.0 8.3 8.3 8.3
10.2 10.4 9.7 9.5
9.8 8.9 8.9 8.6
12.5 11.7 11.0 10.7
Differential leucocyte count (%) Basophils
0 0.1 0.5 2.5
0.4 0.4 0.8 0.5
0.3 0.1 0:4 0.2
0.4 0.4 0.1 x 0.2
0.2 0.5 0.3 0.5
0.2 0.4 0.5 0.2
0.3 0.5 0.3 0.2
Eosinophils
0 0.1 0.5 2.5
2.4 2.0 2.6 2.2
1.6 1.4 1.6 1.5
1.9 1.5 1.9 2.1
1.2 2.2 1.7 2.6
1.5 1.3 1.5 1.7
1.5 1.9 1.4 2.5
Neutrophils
0 0.1 0.5 2.5
15.1 9.9 x 17.8 13.3
12.0 14.2 16.6 14.4
10.6 11.5 15.5 13.1
11.1 13.3 17.3 x 16.4 xx
12.0 14.1 13.4 12.7
14.0 9.9 9.7 x 11.0
Lymphocytes
0 0.1 0.5 2.5
79.7 85.1 76.6 81.6
84.0 81.7 79.1 82.4
83.5 82.6 77.6 81.1
85.4 81.9 78.7 x 78.8 xx
84.1 81.4 82.6 83.2
79.8 83.6 85.5 x 82.4
Monocytes
0 0.1 0.5 2.5
2.5 2.7 2.4 2.5
2.2 2.8 2.5 1.6
3.6 4.0 4.9 3.5
2.2 2.3 2.1 1.8
2.4 2.8 2.1 2.2
4.4 4.1 3.1 3.9
A t y p i c a l cells
0 0.1 0.5 2.5
2.5 2.2 3.1 2.7
3.3 3.4 3.5 3.3
5.1 7.2 5.7 13.1 x
2.3 2.2 2.8 2.1
2.6 3.4 3.1 2.5
6.5 6.1 6.1 6.6
Values are f o r g r o u p s o f 10 males and 10 females. x, P ~ 0.05; xx, P ~ 0.01.
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T A B L E III R E L A T I V E O R G A N W E I G H T S (%) Females
Number of animals P-generation b Heart Brain Liver Kidneys Spleen Adrenals Thyroid Pituitary Uterus/prostate Ovaries/testes
Number of animals F l a-generation a Heart Brain Liver Kidneys Spleen Adrenals Thyroid Pituitary Ovaries/testes Uterus/prostate
Number of animals F2a-generation a Heart Brain Liver Kidneys Spleen Adrenals Thyroid Pituitary Ovaries/testes Uterus/prostate
Males
0 ppm
0.1 p p m 0.5 p p m 2.5 p p m 0 p p m
0.1 p p m 0.5 p p m 2 . 5 p p m
10
10
10
10
10
0.306 0.434 2.87 0.698 0.169 0.008
0.309 0.444 3.20 0.842 0.200 0.009
0.345 0.452 3.26 0 . 9 2 9 xx 0.198 0.010
0.003 0.667 0.110
0.003 0.647 0.103
0.003 0.679 0.102
10
10
10
9
0.404 0.417 0.667 0.656 3.31 3.09 0.797 0.780 0.203 0.182 0.019 0.017 . . . 0.005 0.005 0.225 0.188 0.023 0.026
0.396 0.650 3.36 0.866 0.186 0.018 . 0.005 0.224 0.028
0.461 0.318 0.668 0.442 3.55 3.06 1 . 0 8 6 xx 0 . 7 1 7 0.219 0.205 0.020 0.009 . . 0.006 0.003 0.209 0.720 0.026 0.101
10
10
10
10
10
10
0.365 0.767 4.44 0.919 0.196 0.022 0.006 0.006 0.189 0.036
0.342 0.726 4.14 0.841 0.167 0.018 0.006 0.005 0.188 0.029
0.352 0.817 3.91 x 1.003 0.190 0.019 0.006 0.006 0.231 0.031
0.264 0.482 4.14 0.716 0.134 0.010 0.004 0.003 0.817 0.094
0 . 2 9 3 xx 0 . 2 6 0 0.282 0.481 0.480 0.501 4.45 4.09 4.31 0 . 8 0 2 xx 0 . 7 7 0 0.856 0.140 0.135 0.172 0.011 0.010 0.009 0.005 0.004 0.005 0.003 0.003 0.003 0.825 0.704 x 0.783 0.077 0.083 0.087
13
17
8
8
7
0.438 0.736 4.06 0.834 0.174 0.021 0.004 0.006 0.175 0.035
0.356 0.364 0.760 0 . 7 8 6 xx 3.82 3.67 xx 0 . 9 2 7 xx 0 . 9 4 7 xx 0.179 0.179 0.020 0.021 0.005 x 0.005 0.006 0.005 0.178 0.191 0.036 0.035
x 0.328 x 0.749 2.77 xx 0.929 x 0.159 x x 0.019 0.005 0.005 0.168 x 0.032
20
x, P ~ 0.05; x x , P ~ 0 . 0 1 ; x x x , P ~ 0 . 0 0 1 . a A f t e r 6 m o n t h s o n test. b A f t e r 2 y e a r s o n test.
102
0.377 0.822 3.80 1.105 0.189 0.019 0.005 0.006 0.179 0.030
9
xx 0 . 2 7 0 x 0.511 4.04 xx 0 . 7 5 6 0.148 x 0.010 xx 0 . 0 0 4 0.003 0.867 0.081
.
.
10
x
xxx xxx xx
5
0.269 0.267 0.298 x 0.467 x 0.501 0.559 3.83 3.62 x 3.77 0.741 0.811 x 0.893xx 0.148 0.131 0.149 0.009 0.009 0.009 0.004 0.004 0.005 x 0.002 0.003 0.003 0 . 7 7 3 x 0 . 7 1 6 xx 0 . 7 3 4 0.070 0.093 0.075
T A B L E IV REPRODUCTION PERFORMANCE Generation
0 ppm
0.1 p p m
0.5 p p m
2.5 p p m
81 72 79
68 74 80
75 70 89
83 66 74
86 59 70
85 28 52
57 70 65
77 86 88
66 88 92
34 34 34
33 36 38
Fertility index a P F1 F2
78 75 70 Viability i n d e x b
P F1 F2
86 66 64 Lactation index c
P F1 F2
68 69 94
Mean b o d y w e i g h t at day 21 (g) P F1 F2
30 34 34
31 32 36
N u m b e r o f r a t s i n each g r o u p
P F1 F2
F
M
F
M
F
M
F
M
20 20 15
10 10 8
16 18 17
8 9 9
16 18 20
8 9 10
18 20 9
9 10 5
a N u m b e r o f p r e g n a n c i e s X 100 divided b y t h e n u m b e r o f m a t i n g s b N u m b e r o f p u p s alive at day 5 X 100 divided b y t h e n u m b e r o f p u p s b o r n . c N u m b e r o f p u p s alive at day 21 X 100 divided b y the n u m b e r o f p u p s alive at day 5.
TABLE V WEIGHT G A I N IN S P E C I A L F 3 a G E N E R A T I O N S T U D Y A T 6 W E E K S Parameter
0 ppm
Females Initial w e i g h t (g) Weight gain (g) at w e e k 6
87 75
Males Initial w e i g h t (g) Weight gain (g) at w e e k 6
109 159
0 + 25 ppm
89 56 xxx
105 137 x
0.1 + 25 ppm
0.5 + 25 ppm
2.5 + 25 ppm
93 60 xxx
87 55 xxx
76 59 xx
104 120 xxx
90 127 xx
i13 132 x
x , P < 0 . 0 5 ; x x , P < 0 . 0 1 ; x x x ; P < 0.001. Significant d i f f e r e n c e s are t h o s e b e t w e e n e x p e r i m e n t a l groups, as c o m p a r e d w i t h 0 p p m group. No significant d i f f e r e n c e exists b e t w e e n e x p e r i m e n t a l groups and 0 + 25 p p m group.
T A B L E VI U R I N A L Y S I S IN S P E C I A L F 3 a G E N E R A T I O N S T U D Y A T 6 W E E K S Parameter
0 ppm
0 + 25 ppm
0.1 + 25 ppm
0.5 + 25 ppm
2.5 + 25 ppm
Females Protein content
±a + ++ +++
1 8 1 0
0 1 7 2
0 2 7 1
0 2 7 1
0 1 7 2
+
10 0
10 0
10 0
9 1
10 0
+ ++ +++
2 8 0
1 7 2
0 8 2
0 8 2
0 3 7
1
1
3
2
3 5 1 0
4 3 2 0
1 6 0 0
3 3 1 1
Ketones
--
Males Protein content
Ketones
--
± + ++ +++
10
0 0 0 0
a __, n o n e ; -+, trace; +, s o m e ; ++, m o d e r a t e ; +++, m u c h . T h e p H was n e a r l y a l w a y s 6.0, in a few cases 6.5. G l u c o s e , b i l i r u b i n a n d b l o o d were n o t detected.
T A B L E VII RELATIVE 6 WEEKS
ORGAN
Organ
WEIGHTS
(%) IN S P E C I A L F 3 a G E N E R A T I O N
STUDY AT
0 ppm
0 + 25 ppm
0.1 + 25 ppm
0.5 + 25 ppm
2.5 + 25 ppm
Number of animals
20
19
20
201
17
Liver Kidneys Brain
3.92 0.915 0.998
4.39 1.319 1.142
4.21 1.385 1.098
4.38 1.458 x 1.217 x
4.50 1 . 4 3 8 xx 1 . 2 6 1 xx
Number of animals
10
9
8
7
6
Liver Kidneys Brain
4.51 0.860 0.647
4.29 1.309 0.752
4.57 1 . 4 9 0 xx 0.763
4.20 1.394 0.790
4.18 1.443 x 0.815
Females
Males
x , P < 0 . 0 5 ; x x , P < 0.01. Significance was c a l c u l a t e d in c o m p a r i s o n w i t h t h e 0 + 25 p p m g r o u p . T h e relative w e i g h t s o f k i d n e y s a n d brain o f t h e f o u r e x p e r i m e n t a l g r o u p s were s i g n i f i c a n t l y i n c r e a s e d in b o t h s e x e s in c o m p a r i s o n w i t h t h e 0 p p m g r o u p (P < 0.01 or 0 . 0 0 1 ) as did t h e relative liver w e i g h t in f e m a l e s .
TABLE VIII TISSUE LEVELS OF Hg A F T E R 7 WEEKS IN SPECIAL F3a GENERATION STUDY Tissue
0 ppm
0 + 25 ppm
0.1 + 25 ppm
Blood (total Hg) Kidneys (total Hg) Liver (MeHgCl) Brain (MeHgC1)
0.18 + 0.034 0.47 + 0.15 ---
146 86.8 67.9 10.6
227 92.8 84.8 12.9
Tissue
0.5 + 25 ppm
2.5 + 25 ppm
Blood (total Hg) Kidneys (total Hg) Liver (MeHgCl) Brain (MeHgC1)
227 79.2 103 11.0
151 81.3 87.6 11.2
+ + + +
37.0 xx 13.1 24 xx 1.3
+ + + +
+ + + +
18.1 8.75 2.2 0.9
+ 21.5 xx + 17.5 -+ 6.5 xx + 1.3 x
26.1 15.2 3.6 xx 1.5
Values are for group of 5 males. x, P ~ 0.05; xx, P ~ 0.01. Significance is calculated compared with the 0 + 25 ppm group.
and the brain were significantly increased when compared with the 0 ppm control group (Table VII). The relative liver weight of females was increased n o n - s i g n i f i c a n t l y in all 2 5 p p m g r o u p s . W h e n t h e i n d i v i d u a l g r o u p s w e r e c o m p a r e d t o t h e 0 + 2 5 p p m g r o u p , i n c r e a s e d r e l a t i v e w e i g h t s w e r e s e e n in the kidneys and brain of females on 0.5 + 25 ppm and on 2.5 + 25 ppm and in t h e k i d n e y s o f m a l e s o n 0 . 1 + 2 5 p p m a n d o n 2 . 5 + 2 5 p p m . The characteristic lesions of the nervous system and kidneys seen histol o g i c a l l y in t h e s h o r t - t e r m s t u d y w e r e f o u n d in all g r o u p s w h i c h r e c e i v e d 25 ppm. No differences between the treated groups could be observed. Lesions in t h e c e r e b e l l u m w e r e o n l y o b s e r v e d in t r e a t e d f e m a l e s . MeHgC1 a d m i n i s t r a tion to the parent generation did not profoundly affect tissue concentrat i o n s in t h e F 3 a g e n e r a t i o n a n d e x c e p t f o r b l o o d t h e c o n c e n t r a t i o n s o b tained at various dose levels were comparable (Table VIII). DISCUSSION F o r e a s e o f c o m p a r i s o n , t h e r e s u l t s o b t a i n e d in t h e r e p r o d u c t i o n s t u d y h a v e b e e n d i s c u s s e d in t h e l i g h t o f t h o s e o b t a i n e d in t h e s h o r t - a n d l o n g - t e r m s t u d i e s . T h e d i s c u s s i o n a p p e a r s in t h e r e p o r t o n t h e l o n g - t e r m s t u d y .
105
REFERENCE 1 H.G. Verschuuren, R. Kroes, Rauws, P.L. Schuller and G.J. 2 H.G. Verschuuren, R. Kroes, Rauws, P.L. Schuller and G.J.
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E.M. den Tonkelaar, J.M. Berkvens, P.W. Helleman, A.G. van Esch, Toxicology, 6 (1976) 85. E.M. den Tonkelaar, J.M. Berkvens, P.W. Helleman, A.G. van Esch, Toxicology 16 (1976) 107.
