Toxicology, 7 ( 1977 ) 215--224 © Elsevier/North-Holland Scientific Publishers, Ltd.

SEMICHRONIC O R A L TOXICITY OF CADMIUM 1. STUDIES ON RATS

E. LOESER and D. LORKE

lnsititut fiir Toxikologie, Bayer A.G., Wuppertal (W. Germany) (Received April 7tb, 1976) (Revision received May 9th, 1976) (Accepted October 10th, 1976)

SUMMARY

Cadmium (in the form of CdC12) was fed to groups of 20 male and 20 female rats each over a period of 3 m o nt hs in concentrations o f 0, 1, 3, 10 and 30 ppm. Appearance, behaviour, f o o d consum pt i on, growth and mortality o f the treated rats of all groups were not affected during the 3-month period. The cadmium concentrations did n o t cause blood, liver or kidney damage. The systolic blood pressure of the treated animals was n o t increased. Autopsies and histopathological investigation of the animals showed no sign o f any alterations. Cadmium accumulated dose 0.05). During the 3-month period, three male rats died, one from the 1 ppm group, another from the 3 ppm group, the third from the 30 ppm group. No connection with the cadmium treatment was established.

Haematological investigations The haematological investigations showed that after 1 month's treatment, slightly diminished haemoglobin contents and reduced haematocrit values were found in the 10 ppm and 30 ppm groups. MCH and MCV values were correspondingly lower. This result was not confirmed later on after 3 m o n t h s ' treatment with cadmium (Table I). Differences between the treated groups and controls in reticulocyte, leucocyte and t h r o m b o c y t e counts and prothrombin time and differential blood counts did not reveal statistical significance at the time measured.

Liver function Enzyme activities (ALP, GOT, GPT and SDH) and amounts of bilirubin and total protein in the plasma do not indicate that cadmium administered for 1 and 3 months had any effect on the liver. There was no dose-dependent difference between the treated and control animals as regards the proportion of albumin/globulin and distribution of individual globulin fractions show~l by serum albumin electrophoresis.

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Fig. 1. Graphs of body weights of male and female rats which had been given cadmium with their feed for a period of 3 months.

Renal function T h e results o f t h e urine tests d o n e a f t e r 1 m o n t h ' s a n d 3 m o n t h s ' treatm e n t s h o w e d no significant d i f f e r e n c e s b e t w e e n t h e c o n t r o l s a n d t r e a t e d animals. T h e c o n c e n t r a t i o n s o f uric acid, u r e a and c r e a t i n i n e in p l a s m a w e r e within t h e n o r m a l range f o r t r e a t e d a n d u n t r e a t e d animals 1 a n d 3 m o n t h s a f t e r t h e s t a r t o f t r e a t m e n t (Table II). T h e e x c r e t i o n o f p r o t e i n via t h e urine c o r r e s p o n d e d to t h e physiological values in all groups. L A P a n d G O T activities in urine s h o w e d no d o s e < l e p e n d e n t changes. U r i n a r y p r o t e i n e l e c t r o phoresis revealed no d i f f e r e n c e b e t w e e n t r e a t e d g r o u p s and c o n t r o l s . Blood sugar and cholesterol B l o o d sugar and c h o l e s t e r o l s h o w e d no d o s e < l e p e n d e n t changes f o l l o w i n g 1- and 3 - m o n t h t r e a t m e n t w i t h c a d m i u m . Systolic blood pressure T h e systolic b l o o d pressure was m e a s u r e d o n a l l animals (Table I I I ) . T h e b l o o d pressure was n o t significantly or d o s e < l e p e n d e n t l y increased as a result o f t r e a t m e n t w i t h c a d m i u m . T h e r e was n o incidence o f h y p e r t e n s i o n .

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t~ t'~

0 1 3 10 30

Female rats

0 1 3 10 30

Male rats

Cadmium in f o o d ppm

15.4 15.8 15.6 15.8 15.0

15.7 15.7 16.4 15.6 15.4

Haemoglobin g / l O 0 ml

48 47 47 45 46

48 46 46 46 46

Haematocrit %

7.87 7.66 7.12 6.58 7.63

8.11 7.86 6.12 6.72 7.67

Erythrocytcs 106/pl

7.4 13.0 9.8 12.8 10.6

12.4 11.2 10.0 14.2 8.4

Reticulocytes %0

20 21 22 24 20

19 20 27 23 20

MCHC 77

61 61 66 68 60

59 59 75 68 60

MCV /2m3

9.8 10.9 9.9 11.3 10.1

8.1 7.6 8.8 8.9 9.6

Leucocytes 103//21

670 516 722 626 729

553 538 626 486 610

Thrombocytes lOa//11

13.5 13.6 13.4 12.1 13.1

12.4 13.6 14.3 14.0 14.5

Prothrombir time/sec

B L O O D I N V E S T I G A T I O N S ( H A E M A T O L O G Y ) O F R A T S G I V E N C A D M I U M WITH T H E I R F E E D F O R A P E R I O D O F 3 M O N T H S

TABLE I

TABLE II RENAL FUNCTION TESTS ON RATS GIVEN CADMIUM WITH THEIR FEED FOR 3 MONTHS Cadmium in food (ppm)

Uric acid

Urea

Creatinine

GOT

LAP

mg/100 ml

mU/ml

in plasma

in urine

Total albumin mg/100 ml

Male rats 0 1 3 10 30

1.3 1.5 1.8 1.8 2.0

28.4 32.4 31.6 31.6 33.3

1.02 1.03 1.05 1.01 1.06

7.1 5.7 5.2 5.7 7.0

11.7 12.5 13.0 15.2 13.3

24.6 29.7 32.7 30.7 33.2

1.2 1.4 1.6 1.5 2.3

33.2 33.9 32.3 31.9 36.4

1.02 1.04 1.05 1.11 1.14

4.7 3.3 4.1 3.0 4.9

4.4 4.8 3.5 5.2 5.5

26.2 27.7 28.1 28.8 30.7

Female rats 0 1 3 10 30

C a d m i u m in k i d n e y s , liver, urine and faeces B e f o r e t h e s t a r t o f f e e d i n g 1 0 m a l e a n d 1 0 f e m a l e rats w e r e k i l l e d a n d t h e c a d m i u m i n t h e i r k i d n e y s a n d livers was d e t e r m i n e d (all c a d m i u m c o n c e n t r a t i o n s are w i t h r e f e r e n c e t o w e t w e i g h t ) . O n average, t h e c a d m i u m c o n t e n t o f t h e liver in m a l e r a t s was 0 . 0 3 6 m g C d / k g w e t w e i g h t , in f e m a l e r a t s 0 . 0 1 9 mg Cd/kg wet weight, in the k i d n e y s 0.075 mg Cd/kg wet weight (male rats) a n d 0 . 0 3 2 m g C d / k g w e t w e i g h t ( f e m a l e r a t s ) . T h u s , t h e r e was a slight

TABLE III SYSTOLIC BLOOD PRESSURE OF RATS GIVEN CADMIUM IN THEIR FEED FOR 3 MONTHS Cadmium in food (ppm)

0 1 3 10 30

Systolic blood pressure measured on the tail vein of the rat mm Hg 2 months after the beginning of the test

3 months after the beginning of the test

Male rats

Female rats

Male rats

Female rats

116.9 121.9 111.7 124.3 121.9

100.7 104.3 108.4 101.3 106.9

121.4 123.9 121.4 125.8 121.1

106.2 103.8 103.1 1.01.1 104.2

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Fig. 2. C a d m i u m c o n c e n t r a t i o n s in t h e liver a n d k i d n e y s o r r a t s w h i c h h a d b e e n given cadm i u m w i t h t h e i r feed.

222

a m o u n t of cadmium in the kidneys and liver, the concentrations in the kidneys being about twice as high as in the liver. These amounts are attributed to the cadmium trace element naturally taken with the food. Cadmium content of the food administered was between 0.15 and 0.3 ppm. The course of cadmium concentrations in the liver and kidneys is shown graphically in Fig. 2. From this it can be seen that after 1 m o n t h ' s treatment there is a dose-dependently increased cadmium content in the kidneys and livers of all groups compared with the controls. The highest concentrations were found in the kidneys. The longer the treatment period the higher the cadmium content. In the 1 ppm and 3 ppm groups the increase of cadmium content in the kidneys and livers diminished as treatment continued. Minimal dose-independent amounts of cadmium only could be detected in the urine of animals of all groups; most of the excreted cadmium was found in the faeces and the a m o u n t was dose-dependent.

Autopsies, organ weights No pathological-anatomical changes attributable to treatment were determined either in the three animals dying during the course of treatment (one from the I ppm group, the second from the 3 ppm group and the third from 30 ppm group) or in those killed at the end of the investigation. There were no significant or dose-dependent differences between the treated animals and the controls as regards absolute and relative organ weights.

Histopathological investigations (We would like to thank C. Urwin and A.J. Newman, Huntingdon Research Centre, England and Dr. G Luckhaus, Inst. fi~r Toxikologie, Bayer AG for the histopathological investigations.) Chronic inflammatory phenomena occurred in the respiratory tract of animals in all groups. Slight variations in hepatocyte size and occasional mononuclear cell infiltration were determined. No morphological changes attributable to treatment were found. Apart from small changes in the kidneys which also occurred in the control animals and were not dose-dependent, no changes in the kidneys were f o u n d attributable to treatment with cadmium. Neither was there any sign of damage to the other organs (heart, brain, pituitary, gonads, urinary bladder, uterus, spleen, thyroid gland, adrenals, thymus, stomach, and intestines}. DISCUSSION

The above is a report on the results of a semichronic feeding test in rats with cadmium for a period of 3 months. During this treatment period, the animals were continuously given cadmium in concentrations of 1, 3, 10 and 30 plain in t h e i r feed. These concentrations were well tolerated. There was no influence on behaviour, body weight or food consumption. No systemic effects were observed after administration of cadmium up to a dosage of 30 ppm in the feed over a period of 3 months. In particular, the clinical-chem-

223

ical, p a t h o l o g i c a l - a n a t o m i c a l and h i s t o p a t h o l o g i c a l investigations s h o w e d n o sign o f changes t o t h e k i d n e y s caused b y t r e a t m e n t . C o n s t r i c t i o n o f smaller renal arteries, as r e c e n t l y d e s c r i b e d [14] c o u l d n o t be d e t e c t e d in a n y animal. N e i t h e r was t h e r e a n y sign o f p r o t e i n u r i a o r e l e v a t e d o c c u r r e n c e o f a n y l o w - m o l e c u l a r p r o t e i n in urine; s e r u m p r o t e i n f r a c t i o n s w e r e n o r m a l l y distrib u t e d . C o n c e n t r a t i o n s o f u p to 30 p p m c a d m i u m in t h e feed given o v e r a p e r i o d o f 3 m o n t h s caused n o t u b u l a r d y s f u n c t i o n with p r o t e i n u r i a as was d e s c r i b e d in o t h e r e x p e r i m e n t s using injections [1]. T h e oral c a d m i u m treatm e n t h a d n o e f f e c t on the organs investigated histologically. Also n o influe n c e on s p e r m a t o g e n e s i s c o u l d be d e t e c t e d . In this s t u d y t h e b l o o d pressure o f t h e rats o f all g r o u p s w e r e in t h e norm a l range a n d did n o t d i f f e r significantly f r o m t h o s e o f t h e c o n t r o l s . I n c r e a s e d b l o o d pressure has b e e n a t t r i b u t e d t o c a d m i u m in an earlier s t u d y w i t h c a d m i u m a d m i n i s t r a t i o n in the d r i n k i n g w a t e r a n d was f o u n d o n l y in s o m e old rats [ 1 5 ] . B u t this s t u d y d e m o n s t r a t e s n o d o s e - - e f f e c t r e l a t i o n s h i p a n d the high b l o o d pressure o c c u r r e d o n l y in females. A d d i t i o n a l l y old unt r e a t e d rats f r e q u e n t l y d e v e l o p s p o n t a n e o u s high b l o o d pressure. A c c u m u l a t i o n o f c a d m i u m in t h e k i d n e y s , liver a n d o t h e r organs d e p e n d e n t o n t h e c o n c e n t r a t ' o n a d m i n i s t e r e d and length of t r e a t m e n t , s h o w n also in o u r o w n feeding s t u d y , c a n n o t b y itself b e t a k e n t o o seriously. I t r e m a i n s t o be clarified a f t e r w h a t d u r a t i o n o f u p t a k e a n d c o n c e n t r a t i o n s in f o o d a critical level is r e a c h e d . Swedish a u t h o r s [1] e s t a b l i s h e d a c o n n e c t i o n b e t w e e n a c o n c e n t r a t i o n o f 2 0 0 m g C d / k g in t h e renal c o r t e x a n d t h e s t a r t o f renal d a m a g e . When 30 p p m was given t o rats in t h e i r f e e d o v e r a p e r i o d o f 3 m o n t h s , this level was n o t , h o w e v e r , r e a c h e d . T h e results p r e s e n t e d a b o v e s h o w t h a t f o r c a d m i u m t o o a " n o t o x i c e f f e c t l e v e l " m u s t be d e t e r m i n e d . In t h e case o f rats, it can be a s s u m e d t h a t the " n o t o x i c e f f e c t l e v e l " is at least 30 p p m f o r a f e e d i n g p e r i o d o f 3 m o n t h s . C h r o n i c tests f o r longer p e r i o d s o f a d m i n i s t r a t i o n m u s t be carried o u t t o d e t e r m i n e t o l e r a b l e levels. REFERENCES 1 L. Friberg, M. Piscator and G. Nordberg, in R.C. Weast (Ed.), CRC-Press, Cleveland, 1971. 2 Wld. Hlth. Org., Techn. Rep. Ser. 1972, No. 505, Sixteenth Report of the Joint FAO/WHO Expert Committee on Food Additives. 3 E~W. Schwartze and C.L. Alsberg, J. Pharm. Exp. Therap. 21 (1923) 1. 4 L. Schwarz, and A. Otto, Z. Hyg. Infektionskr., 104 (1925) 364. 5 L. Prodan, J. Indust. Hyg., 14 (1932) 132 and 174. 6 0 . G . Fitzhugh, and F.H. Meiller, J. Pharm. Exp. Therap., 72 (1941) 15. 7 L.E. Decker, R.U. Byerrum, C.F. Decker, C.A. Hoppert and R.F. Langham, Arch. Ind. Health, 18 (1958) 228. 8 C.W. Weber, and B.L. Reid, Toxicol. Appl. Pharmacol., 14 (1969) 420. 9 H.A. Schroeder, W.H. Vinton and J.J. Balassa, J. Nutr., 80 (1963) 48. 10 G,W. Powel|, W.J. Miller, J.D. Morton and C.M. Clifton, J. Nutr., 84 (1964) 205. 11 E. Loeser and D. Lorke, Arzneimittelforsch., 22 (1972) 1174. 12 H. Breuninger, Arzneimittelforsch., 6 (1956) 222. 13 F. Wilcoxon, Biometrics, 3 (1947) 119. 14 B.A. Fowler, S.H. Jones, H.W. Brown and J.K. Haseman, Toxicol. Appl. Pharmacol. 34 (1975) 233. 15 H.A. Schroeder, Am. J. Physiol., 207 (1964) 62. 224