The toxicologic and pharmacologic effects of hafnium salts

TOXICOLOGY

AXD

The

THOMAS

APPLIED

PHARMACOLOGY

Toxicologic

J. HALEY,

Ii.

4, 238-246 (1962)

and

Pharmacologic

Hafnium

Salt&”

Effects

of

N. KOMESU, AND H. C. UPHAM

RAYMOND,

Laboratory of Nuclear Medicine and Radiation Biology of the Department of Biophysics and Nuclear Medicine, School of Medicine, Univerisity of Californiaat Los Angeles Received

July

31, 1961

Modern technological advances have resulted in the industrial application of the rarer elements as alloying agents (Hale and Carmichael, 1961; Spink, 1961). However, our knowledge of the biological effects of these elements has not kept pace, and information on such aspects is scarce. Van Niekerk (1937) studied the pharmacology of hafnium and found that it had a depressanteffect on most isolated tissuesand caused death by hypotension and respiratory paralysis. It is difficult to be certain whether such effects were produced by hafnium or by some impurity, becausethe exact purity of the salts used is unknown. Therefore, we have reinvestigated the toxicology and pharmacology of hafnium using hafnium chloride of 98.5% purity. EXPERIMENTAL

METHODS

The intraperitoneal LDbO was determined on 86 male CF-1 mice. The chronic toxicity of hafnium chloride was determined by including 0.01, 0.1, and 1% of the compound in the diet and feeding it over a period of 12 weeks to three groups of CFN rats. Each group contained 6 males and 6 females. Observations were made of the following: total erythrocytes, total leucocytes, differential cell count, hemoglobin, hematocrit, and body weight. At the conclusion of the period, the following tissues were subjected to histologic examination: heart, lung, liver, kidney, pan1 A preliminary report was presented at a meeting of the American Society of Pharmacology and Experimental Therapeutics at Chicago, April 14-18, 1960 [Federation Proc. 19, 389 (1960) 1. 2 These studies were supported by Contract AT(04-l)GEN-12 between the Atomic Energy Commission and the University of California. 238

TOXICOLOGY

OF HAFNIUM

SALTS

239

cress, spleen, adrenal, and small intestine. The method of Draize et al. (1944) was used to study ocular and skin irritation in rabbits and intradermal irritation in guinea pigs. Six rabbits were used in the ocular studies, each rabbit had one eye exposed to 0.1 ml of 1:lOOO hafnyl chloride while the other eye received 0.1 ml of a control saline solution adjusted to the same pH value. Three rabbits had 1 mg of hafnium chloride placed in one eye for further study of ocular irritation. Rabbit skin irritation studies employed 6 animals according to the design of Draize et al. (1944). Five guinea pigs were used in the intradermal studies, and the concentrations of hafnyl chloride were 1:lO to 1: 10”. Effects of hafnyl chloride on isolated guinea pig ileal strips bathed in Locke-Ringer’s solution were studied in a thermostatically regulated 25ml bath using the Trendelenburg method (1917). Studies were also made on the isolated rabbit ileum in the presenceof 0.5 ng of nicotine or 2.5 ug of acetylcholine. Eighteen cats of both sexes,weighing 3.54.25 kg were anesthetized with 0.5 ml/kg of Dial-urethane intraperitoneally. A six-channel Offner Dynagraph with Statham transducers was used to record carotid arterial pressure, respiration, nictitating membrane contraction, ECG Lead II, femoral arterial pressure, and femoral arterial flow. The latter was obtained with a 25ml Shipley-Wilson flowmeter (1951). Preganglionic stimulation of the cervical sympathetic fibers and the contralateral vagus fibers was accomplished with a Grass model S-4 stimulator at 8v/lO sec. Two hours were allowed to elapse prior to beginning drug administration. Intravenous dosesof the drugs used were: hafnyl chloride 0.5-10 mg/kg; hafnyl chloride citrate complex OSacetylcholine 5 pg/kg; histamine 75 m&kg ; epinephrine 5 pg/kg; 0.5 pg/kg; atropine 2 mg/kg; and sodium citrate 36.8-441.6 mg/kg. The hafnyl compounds were injected at a constant volume of 2 ml per dose. Inasmuch as the hafnyl chloride solution had a low pH, control solutions containing HCI at pH 2.33 to 0.8 were also studied. Where appropriate, the results were analyzed statistically by the LitchfieldWilcoxon method (1949) or standard errors were calculated. RESULTS

Chemical

Considerations

When hafnium chloride (HfC14) contacts moisture or is dissolved in water there is an immediate liberation of HCl and the formation of hafnyl chloride (HfOCla). This reaction, whether it occurs in the test tube or in the gastrointestinal tract, results in a marked lowering of the

240

THOMAS

J.

HALEY

ET

AL.

pH. Thus the studies reported herein are in essence based upon the responses to hafnyl chloride rather than the original compound, hafnium chloride. Acute Toxicity The symptoms of acute toxicity were immediate urination and lethargy. The first deaths occurred within 24 hours, but the peak was not reached until 48 hours. A few animals did not die until the sixth day of the study and all animals were unthrifty during this interval. The intraperitoneal

360

0

CONTROL

0

1.0%

no.l%

t

0

0

0.01%

2

4

6

8

IO

12

WEEKS

FIG. 1. of hafnium

Response chloride

of male and female CFN rats to the feeding of various in the diet for 90 days. Bars signify the standard errors.

levels

TOXICOLOGY

OF

HAFNIUM

LD50 for 7 days for hafnyl chloride was 112 (93.3-134.4) slope of 1.44 (0.97-2.13). Chronic

241

SALTS

mg/kg

with a

Toxicity

Throughout the chronic feeding period of 12 weeks, the animals receiving the hafnium chloride did not appear to differ from the controls. The growth curves shown in Fig. 1 indicate that, at the dietary levels of hafnium chloride studied, there was no significant effect on growth. Furthermore, the chemical produced no significant effect on the hematology of the rats (see Table 1 for a comparison between groups and sexes). The increases in the cells and hemoglobin between the beginning and the end of the experiment were probably related to the normal growth pattern of the animals and not to any influence of the chemical, because the control group did not differ significantly from the medicated groups. It should also be noted that all the values in Table 1 were within the ranges given for the rat by Gardner (1947). The data on the differential counts are not given in Table 1, because there was no significant difference between the medicated and control groups. Also the data were equivalent to the normal distribution given by Gardner (1947). At autopsy the internal organs of all groups of rats appeared normal and there were no outward signs of damage from ingestion of hafnium chloride for 12 weeks. Histopathologic examination revealed that, of all the tissues examined, only the liver showed consistent damage. This consisted of perinuclear vacuolization of the parenchymal cells and coarse granularity of the cytoplasm. Both sexes showed this damage in S/6 animals at the 1% feeding level whereas only 2/6 males and l/6 females showed such liver changes at the 0.1% and 0.01% levels. Ocular Irritation Direct application of 0.1 of 1:lOOO hafnyl chloride pH 2.1 to the eyes of six rabbits produced no evidence of damage to the cornea or iris or irritation of the conjunctiva at the l- and 24-hour observation periods. A similar application of diluted HCl of the same pH also did not affect the eyes. The placement of 1 mg of hafnium chloride in the eyes of rabbits resulted in an immediate increase in the rate of blinking and a redness of the palpebral conjunctiva within 1 hour. At 24 hours there was no evidence of cornea1 damage, iris damage, conjuctival irritation, chemosis, or increased lacrimal discharge. Apparently, the natural buffering capacity of the lacrimal fluid was sufficient to counteract the

u Values

1 .O%

Hafnium

os7c

Hafnium

0.01%

Hafnium

are

M

Control

&

means

F

M

-c .31

plus

or minus

6.01 Ir, .21 5.36-6.88

.30

standard

error

9.89 k .39 7.92-10.01

9.13-12.14

-c .44

10.82

4.24-6.20

21 .28

5.56

k

7.99-10.23

9.19

8.99-11.30

-c .40

-c 1.19

and

ranges

14.85 ?I 1.42 11.20-19.65

6.40-14.35

10.60

1.05 1.80

& 2.49

k

7.60-12.25

10.05

5.60-I

8.80

-c 1.25

10.32

10.40

11.10 -c- 1.75 7.05-18.00 5.90-15.40

-+- .48

.29

13.20 lr 7.00-18.60

.S8

-c .47

103) 12

2

2.02

? k ?

3.19

1.24

3.96

0 and

9.6s32.00

16.70

11.20-18.65

14.95

7.05-24.25

17.90

17.60 -r- 2.07 11.45-24.20

15.50 zk 2.76 9.45-28.45

12.05 c 1.47 7.6&16.00

10.6-30.80

18.20

at time

x

Leucocytes

1 OF HAFNIUM

11.60 * 1.39 7.25-16.05

TABLE EFFECTS

(-3

10.2-19.45

15.00

0

7.89-10.95

9.27

10.79 k 9.86-12.03

8.99 zk .38 7.67-11.00

9.59-11.29

10.38

12

4.96-6.80

.29

5.81

F

+

5.57 r?r: .24 4.56-6.00

7.75 2 .40 6.50-9.15

F

M

6.67 f .23 5.82-7.45

5.08-8.35

6.51

.32

-c .25

5.4s6.95

6.20

0

M

F

Sex

Treatment

Erythrocytes (mm3 X 10”)

HLMATOL~CIC

at

(vol.

Hematocrit

1.49

1.78

1.07

12

.82

1.30

weeks.

44-50

48 2

42-50

46 f

47 21 .89 44-50

47 k 1.70 42-52

44-53

49 k

39-50

44 ?

43-54

47 2

47 2 1.79 4&58

0

CHLORIDES

%)

.80

1.02

1.02

.91

.71

49 k .80 47-52

52-58

54 k

47-52

50 k

53 k 1.05 50-56

48-54

so f

54 c 1.14 52-59

47-54

50 ?

47-53

52 k

12 k ?

.25

.41

(.LT - %)

? 2

.36

.39

13.4 -I- .26 12.5-14.2

11.6-14.0

13.0

11.5-14.3

12.8

12.9 f .58 11.6-15.0

13.0 & .30 12.3-13.9

11.9 2 .56 10.5-14.3

11.3-13.6

12.6

11.6-14.3

12.4

0

Hemoglobin

f ?

.24

.25

f

c 14.1-16.0

15.3

.28

.2s

-I- .30 14.6-16.5

15.8

13.6-15.4

14.1

15.4 k .39 13.6-16.1

15.9 k .33 15.3-17.2

16.6 iI .I4 16.0-17.0

12.5-15.2

13.9

14.4-15.9

15.2

12

TOXICOLOGY

nascent hydrochloric the lacrimal fluids.

OF

HAFNIUM

SALTS

243

acid released when the hafnium chloride dissolved in

Skin Irritation The direct application of hafnium chloride crystals to unabraded rabbit skin produced an irritation index of 2 within 24 hours for both edema and erythema. These reactions terminated within 72 hours. The reaction on abraded skin was very severe, resulting in the maximum irritation index of 8 within 24 hours. No change was observed at 72 hours, and within 7 days perforating ulcers 25-30 mm in diameter developed with penetration through the skin to the underlying muscle layers. Inasmuch as healing did not occur, the animals were sacrificed at 14 days. The differences in response between intact and abraded skin were probably related to the liberation of nascent HCl by tissue fluids. Intradermal injection of hafnyl chloride in guinea pigs at concentrations of 1 X lo4 to 1 X lo6 produced 24-hour erythema indexes of 1 and complete remission to normal with no scar formation at 7 days. Erythema, edema, and partial necrosis were observed 2 hours after injection of the 1: 10 concentration of the chemical. The 24-hour irritation indexes, erythema plus edema formation, for concentrations of l:lO, 1: 100, and 1: 1000 were, respectively, 8, 6, and 3. Only the two highest concentrations produced eschars and the diameters were 8 and 6 mm. Scar formation with epilation of the area occurred at 7 days with the 1: 1000 concentration and after 14 days with the 1: 10 and 1: 100 concentrations. In these latter cases, complete healing occurred only after 4 weeks. These results again point out the irritating nature of hafnpl chloride and are probably related to the acidic nature of the compound. Efi,ects on the Isolated Intestine Within the dosage range of lo-200 mg, hafnyl chloride produced an increasing depression of intestinal tonus and contractions ending in complete paralysis of the rabbit ileum. Repeated washing of the five ileal strips did not restore the contractility. This depressant effect counteracted the spasmogenic effects of both acetylcholine and nicotine. The antispasmodic EDBO’s were 128 (83.7-195.8) mg and 99 (79.8122.8) mg, respectively. A similar depression was seen with the Trendelenburg guinea pig ileal preparation where the EDSO’s for blocking the circular and longitudinal muscular contractions by hafnyl chloride were 11.8 (5.8-23.8) mg and 20 (8-50) mg, respectively. Inasmuch

244

THOMAS

J.

HALEY

ET

AL.

as the contractions seen with this preparation are induced by pressure stimulation of the intestinal ganglia, it would appear that hafnyl chloride may exert its intestinal depression by ganglionic blockade. However, experiments with the superior cervical ganglion preparation of the cat indicated that this was unlikely because this latter preparation never showed any evidence of blockade and was functional even after the animals had expired. Pharmacologic

Effects

Hafnyl chloride at doses of 0.5-2 mg/kg produced no observable pharmacologic effects in the cat. A transient hypotension of 15-25 mm Hg occurred in both carotid and femoral blood pressure coupled with a decreased femoral blood flow after administration of 5 mg/kg. ,4t this dose the electrocardiogram showed an increase in the height of the P wave and a notching of the T wave. Complete cardiovascular collapse occurred at 10 mg/kg and was followed by respiratory paralysis. Prior to exitus, the respiratory rate was not affected. The terminal electrocardiographic changes included a transient increase in the height of the P wave until it equaled the QRS complex, decreased height of the P wave coupled with an increased T wave, absence of the P wave, a high take-off of the T wave, and finally ventricular fibrillation. Within the dosage range studied, hafnyl chloride did not affect the physiologic responses to acetylcholine, epinephrine, histamine, or vagal stimulation. Furthermore, it had no effect on transmission in the superior cervical ganglion or on contraction of the nictitating membrane. The above effects of hafnyl chloride could not be modified by atropine, and the cardiovascular collapse could not be counteracted by epinephrine. When complexed with citrate, hafnyl chloride produced pharmacologic effects that appeared to be identical with those observed with the uncomplexed chemical, and cardiovascular collapse occurred at a dose of 60 mg/kg. However, evaluation of the action of citrate alone in doses equivalent to those used in complexing the hafnyl chloride indicated that the effects observed were due to citrate, not to hafnium. pH was not a factor in the results obtained with hafnyl chloride, because injection of the same volume, 2 ml/dose, of diluted HCl within the pH range 2.33-0.8 produced no pharmacologic effects and was not lethal to the animals.

TOXICOLOGY

OF HAFNIUM

SALTS

245

DISCUSSION

The results herein presented confirm and extend the observations of van Niekerk (1937) concerning the pharmacologic effects of hafnyl chloride. The toxic effects of the compound are, in general, greatest on abraded skin where nonhealing ulcers are formed. However, all local external effects, major or minor, can be prevented by proper application of good industrial hygiene practices, e.g., protective clothing and washing to remove skin or ocular deposits. Acute toxicity from hafnium chloride was a delayed type of reaction, a fact indicating the possibility that incorporation into a vital cell constituent or enzyme system may have been involved. The fact that chronic feeding of the compound caused liver damage without affecting either the growth rate or the hemogram indicates that the liver damage was not extensive enough to impair any vital function essential to survival. However, such liver changes might not be conducive to survival if hafnium chloride exposure is prolonged for periods greater than the go-day interval used in the present study. SUMMARY The toxicology and pharmacology of hafnium salts have been investigated. Acute intraperitoneal toxicity of hafnyl chloride is of the delayed type with an LD,,, per 7 days of 112 (93.3-134.4) mg/kg. Chronic feeding for 90 days at levels of 1.0, 0.1, and 0.01% of the diet produced liver changes at the 1.0% level. No changes were observed in the growth rate or the hemogram. Ocular application produced a transient conjunctivitis. Topical application to intact rabbit skin produced a transient irritation, whereas similar studies on abraded skin resulted in nonhealing ulcers. Intradermal injections also produced localized irritation and scar formation. Hafnyl chloride had a depressant effect upon the isolated rabbit and guinea pig ileum. In the intact cat, the compound produced hypotension and death by cardiovascular collapse and respiratory paralysis. The chemical had no effect on the responses to acetylcholine, epinephrine, or histamine or those elicited by stimulation of the cervical sympathetic or vagus nerves. REFERENCES DRAIZE, J. H., WOODAFCD, G., and CALVERY, H. 0. (1944). Method for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Phatvnucol. Exptl. Therap. 82, 377-390. GARDNER, M. V. (1947). The blood picture of normal laboratory animals. A review of the literature 19361946.1. Fmnklin Inst. 243, 77-86. HALE, R. W., and CARMICHAEL, R. L. (1961). Less common elements. Economics. Znd. Eng. Chem. 63, 108-111. LITCHFIELD, J. T., JR., and WILCOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pkarmacol. Exptl. Therap. 96, 99-113.

246

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HALEY

ET

AL.

R. E., and WILSON, C. (1951). An improved recording rotameter. Proc. Exptl. Biol. Med. 78, 724-728. SPINK, D. R. (1961). Less common metals. Reactive metals, zirconium, hafnium and titanium. Ind. Eng. Cbm. 69, 97-104. TT~ENDELENBURG, P. (1917). Physiologische und pharmakoiogische Versuche tiber die Diinndarmperistaltik. Arch. exptl. Pathol. Pharmakol. Naunyn-Schmiedeberg’s 81, 55-129. VAN NIEKERK, J. (1937). Einige pharmakologische Untersuchungen mit Salzen von reinum Zirkonium und reinem Hafnium. Arch. exptl. Pathol. Pkar&akol. NawynSchmiedeberg’s 184, 686-693. SHIPLEY,

Sot.