roxIcoLom

AND

Oral

APPLIED

PHARMACOLOCY

A9-Tetrahydrocannabinol for Periods

32, 399-417

(1975)

Toxicity in Rats up to Six Months1

Treated

HARRIS ROSENKRANTZ,* ROSA A. SPRAGUE,~ ROBERT W. FLEISCHMAN~ AND MONIQUE C. BRAUDE~ Mason Research Institute, Worcester, Massachusetts 01608 Received August 12,1974; accepted November 27,1974

Oral d9-Tetrahydrocannabinol Toxicity in Rats Treated for Periods up to Six Months ROSENKRANTZ, H., SPRAGUE, R. A., FLEISCHMAN, R. W. AND BRAUDE, M. C. (1975). Toxicol. Appl. Pharmacol. 32, 399-417. The chronic toxicity of A9-tetrahydrocannabinol (A9-THC) given orally 28, 90, and 180days to Fischerrats at dosesof 2,10, and 50 mg/kg wasinvestigated. Some 180-day treated animals were monitored after a 30-day recovery interval. The lowerdosesusedcorrespondedto the d9-THC content of marihuanaor hashish.In the first 10 daysCNS-depression,incoordination, ataxia andpassivity, poikilothermia, andhypopneaoccurredto which tolerance developed. During days 10-20, irritability, hypersensitivity, hyperactivity, and aggressionpredominated. Fighting occurred between days 20-100. Tremors and clonic convulsionsappearedafter day 70 in 50% of the animalsat 50 mg/kg and 12% at 10mg/kg. Tolerancedeveloped to CNS-stimulation, fighting and neurotoxicity and lethal cumulative toxicity wasseenalthough the causeof death wasnot established.Growth rate of both sexeswasinhibited despitean elevation in food consumption after a transient anorexia No morphologicalchangescould be ascribedto d9-THC. Except for a rise of 2845% in SGOT and 4669% in SGPT in malesat higher dosesand a belatedhyperglycemiain both sexes,clinical chemistry, hematological,and urinalysis parameterswere within normal ranges.The greatestchangeswereseenin ratios of organ/FBW at 50mg/kg: 10-20% increasein brain, lungs,kidneys, heart, and liver; 45% increasein adrenals; 96‘A increasein male pancreasand 13-25‘A increasein testis and prostate. The present investigation implicated reasonabledosesof d9-THC in undesirablebehavioral changeshighlightedby fighting aggression, convulsive activity, and lethal cumulative toxicity. The absenceof morphological changesdespitechangesin growth rate and organ weights indicateda functional impairmentthat wasnot an immediatethreat to the life of the organism becauseof initiation of as yet unknown protective mechanisms. Initial toxicological evaluations of the most pharmacologically active cannabinoid, A9-tetrahydrocannabinol (d9-THC), employed massive doses of A9-THC in order to establish lethal and safe dosesfor man (Thompson et al., 1973a, 1974a). As would be expected, larger dosesof A9-THC required the oral route of administration particularly ’ Supportedby ContractHSM 42-71-79. 2MasonResearch Institute,Worcester,Massachusetts 01608. 3Presentaddress:BiomedicalLiterary Services, Auburn, Massachusetts 01501. 4 NationalInstituteon Drug Abuse,Rockville,Maryland 20852. Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

399

400

ROSENKRANTZ

ET AL.

in chronic studies (Thompson et al., 1973b, 1974b). In all the previous prolonged chronic investigations (approx. 28-100 days of treatment), four major observations were recorded: (1) a biphasic behavioral response consisting of CNS-depression for several days followed by CNS-stimulation for days to weeks; (2) the development of tolerance at varying rates to different parameters of inhibition and excitation; (3) cumulative toxicity exhibited as lethality without morphologic changes at nonlethal acute doses; and (4) neurotoxicity expressed as spontaneous fighting aggression and tremors terminating in clonic-tonic convulsions. Other investigations have seen the biphasic response and the development of tolerance in a variety of species (Nahas, 1973; Singer, 1971). Lethal cumulative toxicity, spontaneous aggression, and neurotoxicity have not been a common finding presumably due to variation in species used, route of administration, dosage formulation and duration of treatment (Marihuana and Health, 1972 and 1974; Rosenkrantz et al., 1972). However, neurochemical changes in brain biopolymers and enzymes have occurred (Luthra and Rosenkrantz, 1974). Recently, spontaneous fighting aggression and the biphasic behavioral response have been confirmed by the inhalation route in rats at dg-THC doses similar to those used by man (Rosenkrantz et af., 1974 and Rosenkrantz and Braude, 1974). In light of the findings of aggression and neurotoxicity with large doses of cannabinoids given over extended periods of treatment, it seemed of considerable importance to investigate dg-THC toxicity at reasonable doses. The present investigation fulfilled this requirement since rats were orally administered dg-THC doses between 2-50 mg/kg for a period of 6 mo. Pharmacologic, physiologic, and toxicologic parameters were assessed at four time intervals. METHODS

Fischer rats,5 weighing approximately 100 g, were housed three to a 7 x 9.5 x 7 in. wire suspension cage. A 12-hr circadian light (8.00 AM to 8.00 PM) and 12-hr dark cycle was maintained. Commercial rat chow and water were available ad libitum. Ambient room temperature was adjusted to 23 + 2°C. Four treatment groups were arranged to have 48 male and 48 female rats of which approximately 6-l 5 of each sex were sacrificed after 28,90, and 180 days of treatment and after 180 days of treatment plus a 30-day recovery interval. Individual groups received orally, either vehicle (sesame oil) or a dg-THC dose of 2, 10, or 50 mg/kg in a gavage volume of 1 ml/100 g of body wt. Synthetic 96 % pure dg-THC6 was used and treatments were performed each morning. The dg-THC doses were selected on the basis of doses consumed by man (Kiplinger and Manno, 1971). Using an average of 0.1 mg/kg as a mean inhalation dose for the human, an equivalent oral human dose is 0.3 mg/kg. Correcting for surface area (Freireich et al., 1966), a corresponding marihuana rat oral dose was 2 mg/kg while 10 mg/kg, simulated the dg-THC content of hashish (Lerner and Zeffert, 1968). The 50 mg/kg dose served as a reference point to the earlier studies with large doses of A g-THC. Daily behavioral signs of CNS-depression or stimulation were recorded. Clinical 5 Charles River Breeding Laboratory, Inc., Wilmington, Massachusetts. 6 Supplied under contract by NIDA, Rockville, Maryland.

L~~-TETRAHYDROCANN.~IUNOL

SIX MONTH TOXICITY

401

measurements of respiration rate, rectal temperature, activity, body weight, and food and water consumption were carried out once a week. Rectal temperatures were determined by a thermistor7 and exploratory activity was measured by an activity meter.*, 9 One day prior to necropsy, all three animals from the same cage were transferred to a metabolism cage for collection of a urine specimen pool. Urinalysis included specific gravity by refractometer, pH, sugar, protein, ketone bodies, and occult blood by Lab-Stix reactions,‘O microscopic estimation of RBC, WBC, casts and crystals, and estimation of color and volume (Davidsohn et al., 1969). Animals were sacrificed by guillotine and blood specimens were submitted for measurements of hematocrit (Hct), hemoglobin (Hglb), RBC, WBC, differential white blood cell count, reticulocyte count, and prothrombin consumption time (Davidsohn et al., 1969). Blood glucose was determined by the hexokinase reaction, BUN by the urease procedure, alkaline phosphatase by hydrolysis of p-nitrophenylphosphate, SGOT, and SGPT by formation of keto acid dinitrophenylhydrazones, and Na+ and K+ by flame photometry (Tietz, 1970). All animals were inspected for gross pathological changes. Brain, lung, kidney, liver, heart, spleen, testis, ovary, pituitary, thymus, pancreas, adrenals, prostate, and uterus weights were recorded. Tissues were fixed in 10 % neutral buffered formalin, paraffin blocks were prepared by standard techniques and 6 pm sections were stained with hematoxylin and eosin (H & E). Histopathological evaluation was primarily performed on tissues from rats treated with vehicle or d9-THC at 50 mg/kg. Significance of differences between treated groups and the placebo group was evaluated by Student’s t test. RESULTS Behavioral and clinical data were obtained on all animals including those scheduled for sacrifice at specific time intervals. Hemochemical, urinalysis, organ weights, histopathology, and brain neurochemical data were obtained at the time of necropsy. Behavioral and neurochemical changes are reported elsewhere (Luthra et al., 1975). The number of rats of each sex studied for each time interval was : 192 through 28 days, 120 through 90 days, 72 through 180 days, and approximately 24 during the recovery period. Briefly, chronic oral treatment induced a dose-related CNS-depression within the first 10 days associated with incoordination, ataxia, passivity, poikilothermia, and hypopnea. With the onset of tolerance, hypersensitivity and hyperactivity predominated during days 10-20. Hyperactivity culminated in fighting aggression at 10 mg/kg during days 14-40 and at 50 mg/kg during days 30-90. Fighting activity was evident in the 2 mg/kg group between days 130-140. Neurotoxicity as manifested by tremors and clonic convulsions appeared between days 80-90 at 10 mg/kg and between days SO--l 50 at 50 mg/kg. The animals after a 30-day recovery period displayed no abnormal behavior. 7 Tele-Thermometer, Yellow Springs Instrument Co., Yellow Springs, Ohio. ’ Photoelectric Activity Cage, Woodard Research Co., Herndon, Virginia. 9 Selective Activity Meter, Model S, Columbus Instrument Co., Columbus, Ohio. lo Ames Co., Elkhart, Indiana.

402

ROSENKRANTZ

Physiological

ET AL.

Findings

The large number of rats studied generated a massive quantity of physiological, hematological, blood chemistry, and urinalysis data. In order to present this data in a reasonably brief manner, trends of changes are indicated in Tables 1 and 2 based on the mean of all observations performed during a particular treatment period. Actually, 5-6 individual measurements on 2448 rats of each sex in each group were made for body weight, food and water consumption, rectal temperature, and respiration during the 28-day treatment period; 9 individual recordings on 28-33 rats per sex per group were made between days 30-90; 11-13 individual determinations on 15-18 rats per group were performed between days 90-180; and 3-5 individual measurements on 5-6 rats per sex per group were obtained during the recovery period. TABLE SUMMARY

OF

PHYSIOLOGIC ORALLY FOR

DATA

28-180

ON DAYS

1

FISCHER RATS GIVEN dg-TETRAHYDROCANNABINOL AND A XI-DAY RECOVERY PERIOD”

dg-THC mg/kg x days

Sex

FBW/IBWb

Food mean + SD (g/3 days)

Vehicle x 28

M

1.97 f0.18 1.53 fO.ll

47.6 f2.7 32.5 +2.0

41.7 f2.1 29.2 L-1.1

37.2 kO.6 37.3 kO.6

37.3 f4.0 39.4 f3.8

1.93 f0.24 1.55 kO.11

45.2 +2.5 31.0 k1.6

37.9” f2.0 26.4’ k1.6

37.4 f0.6 37.6 kO.6

35.5 f3.0 36.3d +2.5

1.84 rtO.25 1 .3gd f0.04

42.2’ +3.5 29.3” +2.2

34.0” +1.0 22.9’ f1.9

37.3 f0.6 37.4 f0.7

27.9” f3.0 29.2’ f3.5

1.74d fO.ll 1.35d f0.07

46.1 f5.8 35.6 +3.7

32.8’ f2.4 23.2’ F1.8

37.3 kO.6 37.3 kO.7

26.3” rt4.8 28.7” 54.9

2.64 +0.17 1.84 f0.04

40.8 f1.8 27.8 k1.2

39.5 +2.7 29.1 +2.6

36.8 +0.5 36.8 +0.4

34.6 f1.8 36.9 k3.5

2.58 +0.17 1.89 f0.04

40.2 +2.8 27.1 +2.3

37.7 +2.8 28.4 52.2

36.9 50.5 36.8 50.4

34.5 i2.5 33.8 f3.8

2.44d +0.18 1 .72d +0.08

41.1 i3.2 28.4 k3.1

38.7 k4.5 26.8 +2.2

37.0 f0.5 36.6 +0.5

31.6’ +1.9 31.3” t-2.6

F 2 x 28

M F

10 x 28

M F

50 x 28

M F

Vehicle x 90

M F

2 x 90

M F

10 x 90

M F

Water Rectal temp mean + SD mean _+ SD (ml/2 days) (“Cl

Respiration mean f SD (rate/l 5 set)

d9-TET~~HYDROCANNABINOL

SIX MONTH

TABLE

&‘-THC mg/kg x days

Sex

x 90 -continued 50 x 90

FBW/IBWb

403

TOXICITY

l-conrimed

Food mean f SD (g/3 days)

Water Rectal temp mean ctrSD mean k SD (ml/2 days) (“0 -

Respiration mean + SD (rateil5sec)

Vehicle

M F

Vehicle x 180

M F

2x 180

M F

10 x 180

M F

50 x 180

M F

Recovery after 180 days treat. Vehicle x 30

M F

2 x 30

M F

10 x 30

M F

50 x 30

M F

2.24d +0.04 1 .67d f0.05 3.34 +0.42 2.09 kO.01 3.04 +0.19 2.13 +o.os 2.84d +0.23 1.92d f0.03 2.77’ zbo.04 1.93d f0.03

3.26 f0.31 2.05 +0.13 3.09 f0.17 2.13 f0.06 2.92 kO.27 2.07 kO.09 2.95 kO.29 2.07 kO.05

47.4” k4.5 37.2” +4.8 35.5 +3.4 28.0 +2.9 37.9 +3.2 27.0 k2.2 40.4” +4.6 30.8’ ,3.7 48.0’ f5.4 42.9’ f5.1

50.6 +1.1 34.1 +3.6 48.8d a1.2 38.1 f3.6 53.9 +4.0 42.0’ f3.1 65.7’ f1.9 52.7” f0.9

39.8 k4.4 32.3 k3.8 43.8 k1.4 31.1 +1.2 40.2” k1.9 32.4” +1.3 42.8 a3.1 31.6 k2.5 50.4’ +4.1 41.6” k4.1

44.4 +1.5 33.6 k1.3 44.2 i-1.6 36.3’ +0.8 49.2d k3.4 37.6’ k1.2 50.4” +3.3 43.2’ k1.5

36.9 +0.5 36.9 +0.4 36.4 +3.0 37.4 kO.2 37.2 kO.1 37.3 kO.3 37.4 +0.3 37.1’ +0.1 37.2 +0.2 37.5 50.4

37.0 fO.0 37.0 +0.1 37.0 +0.1 37.3d kO.2 37.0 kO.0 37.0 +0.1 37.1 kO.1 37.2’ kO.0

31.1d +3.8 31.3‘ k2.0 40.5 +3.5 43.7 +5.3 41.1 +3.5 44.1 +6.1 37.1 f5.9 33.4d k11.1 30.7’ k3.2 33.5’ k-3.4

37.4 +1.9 39.0 +0.9 39.3 +0.8 37.3 +1.4 36.3 +2.2 38.3 +1.0 37.9 +2.2 38.0 +2.0

n N = 48 rats/group of each sex for 28 days, 30 for 90 days, 18 for 180 days, and 6 for the recovery interval. b Final body weight: initial body weight. c p < 0.01 as compared to vehicle control. d p < 0.05 as compared to vehicle control. 14

D0S.Z

50x90

10x90

2x90

Vehicle

50X28

10x28

x 90

2 x 28

F

M

F

M

F

M

F

M

F

M

F

h

F

M

F

M

Vehicle

x 28

S.2.X

mg/kg Y days

A(-THC

SUMMARY

4S.Sc f2.0 42.7’ +1.9

42.7 f1.6 41.2 11.2

42.2 zk3.4 40.3 ztl.8

42.7 12.4 39.0 f2.4

47.3 *3.0 46.6 f1.3

48.0 ztl.6 45.8 zt1.9 47.3 *2.5 46.8 f1.5 43.2 58.2 42.9 1t4.6

Hct %

OF MEAN

15.8 f0.9 15.1 f0.6

15.1 f0.7

f0.9

15.0

fl.O

14.8 50.5 13.9

8.00~ zko.51 6.88” to.40

7.41 Yk0.47 6.7@+ f0.31

7.08 Ito. 6.18 to.36

7.15 zto.20 6.14 zkO.36

15.5

10.6 14.2 *1.1

7.74 hO.49 6.78 f0.31

7.58 f0.27 7.03 f0.53 7.43 -LO.66 7.16 f0.33 6.77 f1.46 6.00 *1.19

RBC 106imm3

5.22 f1.29 6X& +1.02

5.12 11.10 5.34b 10.73

4.68 zk1.22 4.12’ *to.47

11.94 3.46 *0.41

6.49

7.18 zt1.2-1 7.30 i3.09

8.27 k1.80 7.88 f2.33 7.03 12.81 8.82 f2.40 9.16 +3.52 6.57 11.11

WBC 103 mm3

23.3 f6.3 13.3 14.1

19.0 zt7.8 15.3 f3.1

21.6 f7.5 29.8’ f 10.6

25.8 zk7.6 17.8 f3.6

11.8 f1.2 14.0 fS.5

24.0 f3.6 11.0 fS.6 11.7 13.5 9.8 A-2.2 13.0 14.1 16.0 zt4.0

POIYS %

RATS GIVEN

(mean+SD)

ON FISCHER

Hematology

DATA

16.2 f0.8 15.8 f0.4

15.8 f0.5 15.0 zkO.8 15.0 f0.9 15.7 zko.7 14.4 f3.3 14.3 Zk1.S

Hglb g/l00 ml

HEMOCHEMISTRY

TABLE

2

75.5 f6.9 85.0 fS.0

80.2 zt8.2 82.8 f2.5

76.8 *7.3 68.8= ztlO.1

72.3 17.5 80.8 f4.2

85.0 -c2.4 84.4 fS.9

83.8 *3.9 86.0 rt4.5 83.2 fS.6 81.0 k-5.6

72.8 f4.7 86.5 17.2

Lymphs %

1.93= +1.20 2.37 A-o.87

2.27’ *to.64 1.88 zkO.60

3.20 10.66 2.27 f0.48

3.52 50.79 2.22 fO.52

1.47 f0.42 2.00 f1.04

1.94 f1.40 2.87 52.07

1.90 zko.37 2.33 *0.42

1.46 f0.46 1.50 +1.14

Reties ‘A RBC

As-TETRAHYDROCANNA~INOL

109b

1146 *s 108 *4

116* *4

f5

97 *4 104 f4 99 f4 105 f3

177 CtlO 210 127

178 *17 166 *14 190 5x23 175 +11 197 *40 172 116

GlUCOSe mg/ 100 ml

ORALLY

--

14.2’ f0.2 14.7 zto.3

14.6 f0.5 14.2 10.2

14.6 f0.4 14.7 f0.5

14.8 10.2 14.6 f0.4

16.9 f2.2 19.5 zk1.9

18.7 12.4 17.1 52.0 16.0 xt2.3 16.2 11.2

17.5 f1.9 18.3 rt1.9

BUN g/LOO ml

FOR 28-180

18.1 f15.3 8.3 11.8

10.7 zkO.6 7.4 *1.7

10.5 10.9 8.5 rt2.2

11.6 f3.2 7.5 zt1.1

26.9 f3.7 26.3 zk2.6

25.7 11.8 23.8 ztl.9 32.4 -16.7 26.6 k2.4

28.1 f3.1 22.0 zk1.9

Alk-aS.e KAU

Blood

A

199 f22 215 ItO

196 f14 206 *0

153 f3 174 +6

156 rt7 173 zts

151 *3 173 +14 d

f9

*

d

so 196

f22

163 +22 183

160 f7 176 114

192 131 214 126 172 It7 199 121

Na+ mEq/liter

150 *2 d

35.6 i 12.3 so.7 139.3

37.3 14.0 35.6 f 10.3 44.4 zt8.6 29.7 zkO.6

49.0 f14.8 32.5 f0.7

SGPT U/ml

PERIOD’

d

189

182 153 203 122

198 f40 163 zk6 165 f35 170 f43 188 *13 184 fll

SGOT U/ml

k SD)

RECOVERY

(mean

30-DAY

Chemistry

DAYS AND

7.3’ 10.5 7.3 zto.3

7.1 $ 0.8 7.3 zko.4

10.7 7.5 to.6

6.9

1,o.s

6.2

6.3 zk2.1 5.8 10.1

5.1 10.2 6.2 2.0

7.0 %I.7 8.1 ~‘~0.6

7.8 LkO.5 8.2 f1.3

K+ r&q/liter

180

50x

30

46.8 11.8 46.7’ el.6

47.8 l 1.3 48.0’ f3.0

M

M

F

F

F

a ,V= 6 of each sex. b n co.01 as CornDared to V&i& c ~~0.05 as corn&wed to vehicle d Insufficient blood specimens.

50*30

44.8C 11~1.5 45.3 12.2

M

F

ContrOl. control.

8.73 1kO.25 7.84 LO.16

8.89 i 0.26 8.20 zto.30

17.1 f0.7 16.9 It-o.5

17.1 -co.4 17.1 10.7

8.45 ho.27 7.88 *to.21

8.77 zko.29 7.75 kO.46

8.42 ho.38 7.43h 1~0.06

7.78 zkO.26 l.SSb ho.29

8.14 *to.57 6.12 ho.28

8.27 il.19 6.95 f0.25

16.5 f0.6 16.6 rtO.8

16.8 f0.4 16.1 f0.8

15.6 &OS IS.3 10.4

43.0 I!: 1.4 42.0b 10.8

46.8 rt1.3 43.7 i2.0

15.2 to.9 15.4 +0.7

15.8 hO.7 14.7 f0.3

15.6 to.6 14.6 10.4

41.3 k2.0 42.2b 12.3

43.0 12.1 39.8C kl.2

k1.0

41.6 k1.8 38.0

M

F

M

F

M

F

M

rats/group

x 30

10 .. 30

2,

Vehicle

Received after 1 SOdays treatment

I80

1oi

21.180

F

M

8.13 ~1.10 8.18 11.55

9.97 Lko.73 6.78 +1.2s

7.19= It 1.66 7.08 rkl.99

10.20 k-2.20 4.99 11.77

7.12 f0.80 6.38 I 0.87

9.72 13.13 .5.07= h1.20

9.19 zk2.83 7.12 11.43

8.19 yt2.60 6.89 10.92

29.0 f7.5 20.3 h4.0

31.8* f5.4 21.5 zks.7

26.6 +4.9 25.3 zk6.7

22.7 12.9 21.5 rt4.7

25.8 112.1 19.8 A4.1

30.3 k8.2 19.2 f5.2

24.0 si5.2 20.0 18.0

24.0 19.6 20.6 k11.4

70.8 17.9 79.7 A4.0

67.2b is.1 78.3 15.6

72.8 d-4.6 74.5 1-6.3

77.0 Yt3.1 78.0 14.8

72.8 fl2.0 78.3 f4.5

68.8 f8.1 78.3 zk4.8

73.8 15.3 78.5 f7.3

73.6 :I- 10.3 77.8 i 12%

1.2sb f0.50 2.23= f0.38

2.96 f0.64 2.57= -to.98

2.66 $1.02 2.176 ztO.68

3.17 zko.43 3.98 zko.94

d

d

d

d

d

d

d

d

&8

1936 +21 1576 rfr.11

137= +12 112* 19 l49b 121 1 42b

114 114 97 16

146b :I: 7 144 1k6 l46= i5 145 :I.10 --_

1536 i-8 143 15

129 ? 10 147 *9

lS.3C il.1 15.0 k1.0

lS.lC *1.2 14.8 10.6

14.0b il.5 15.0 10.5

18.0 f2.0 14.5 10.8

15.5 h-1.0 18.2 rto.9

18.2 f2.3 17.9 Al.4

15.5 12.1 18.1 -t1.7

18.4 t3.7 20.8 L4.5

24.9’ zk4.1 20.5 rt3.s

22.5’ 12.8 22.0 i5.4

28.0b *4.9 16.7 13.1

18.7 12.1 19.1 f5.3

19.0 11.9 17.5 k1.8

22.2 &8.3 17.3 Al.5

17.8 il.4 18.4 zk3.4

18.2 L1.7 17.9 zk2.8

147 tt38 207 10

153 *F-13 137 118

149 Yi.25 150

148 It. 33 123

257’ :A:12 187 *ss

+32

228 f27 247

228 3.28 217 124

178 z-60 218 19

44.3 i lj.0

30.0’ +s.o 33.0

41.8 + 162.6

52.6 if.8

43.Sb 16.2 38.0 .l:O.O

38.2 f 12.8 26.5 zt7.6

38.2’ ltlO.5 31.5 AI 10.8

26.4 .I 4.4 32.0 i4.6

129 +19 179 10 132 f8 179 ItO

123 115 204

12s j-9

146 .t3 149 +7

144 *3 146 13

14s _t2 146 k3

12

144 il 149

*::i

7.0* io.5

6.8 f1.4 8.3 10.0

6.3 40.6 9.2

5.8 qJ.4

1.5 ztO.7 7.3 -10.6

7.3 *0.7 7.1 zko.3

7.2 lro.5 7.0 to.7

6.8 10.5 7.0 *0.7

406

ROSENKRANTZ

ET AL.

28 Days of treatment. The data in Table 1 reveal a dose-related 8-12 % decline in rate of body growth of both sexes. Food consumption significantly decreased (about 10 %) only at 10 mg/kg but water intake diminished 10-20x in a dose-related manner. Respiration rate fell approximately 27 % at the two higher doses while rectal temperature remained unchanged. The weekly temporal pattern indicated a decreased body weight gain in 10 and 50 mg/kg groups in the frrst week. The difference between treated and vehicle groups slightly but progressively widened each of the next 3 wk. Generally, decreased water intake paralleled the temporal pattern of growth rate while food consumption was decreased in the first 2 wk and tended to increase in the second 2 wk of treatment. Hypopnea was most evident between days l-10 and returned to normal thereafter. 90 Days of treatment. Body growth declined at a similar rate as that seen during 28 days of treatment. Food ingestion increased 16-33 % for both sexes at 50 mg/kg. The increased food consumption was apparent at each weekly measurement throughout this interval. While higher-dosed groups exhibited a 13 % decrement in respiration rate, both rectal temperature and water consumption were unaltered. The diminished respiration rate was observed during days 35-70 but dyspnea was sporadic thereafter. 180 Days of treatment. Although the decrease in the rate of growth continued, it was only 9 ‘A for females and approximately 17 o/o for males. A dose-related increase in food consumption was 10-53 % for females and 13-25 % for males. Water consumption was similarly elevated but only at the higher dose. At the latter dose, both sexesexhibited a 25 % drop in respiration rate. Rectal temperature was generally normal for all groups. Temporally, food and water intake in dg-THC-treated rats were greater and respiration rate lower at each weekly measurement during days 98-l 75. 30 Days of recovery. Rate of body growth was restored to normal for all treated groups. A nearly dose-related rise in food and water consumption were observed: food, 6-29 % for males and 1 l-54 % for females; water, 10-l 3 % for males and 8-28 % for females. Generally, rectal temperature and respiration rate were unchanged. Hemochemical and Urinalysis Findings At each sacrifice interval, individual blood specimens from six animals of each sex from each group were subjected to hematological and blood chemical analyses within the limitations of blood volume. Differential white cell counts were conducted, but since no significant changes in number or morphology of basophils, eosinophils, monocytes, or bands were seen, their quantitation is not given in Table 2. Urine specimens were collected and analyzed on six males and six females per group before necropsy. No urinalysis data are provided because no drug-related changes were found at any time interval. 28 Days of treatment. Lymphocytes increased and polymorphonuclear cells decreased in treated males while reticulocytes rose in treated females, but all the changes were of borderline significance. In addition, an apparent hypokalemia at higher doses for both sexes was not statistically significant. 90 Days of treatment. At the 50 mg/kg dose, both sexes displayed an 8% rise in hematocrit and approximately 11% elevation in RBC. A dose-related 19-74 % increase in female WBC was found but the WBC value for vehicle female was unusually low. Other changes were inconsistent.

LI~-TET~H~DR~CANN,~BINOL

SIX MONTH

TOXICITY

407

280 Days of freatment. At the higher doses, hematocrit and RBC in the females rose 11 ‘A and 7 %, respectively. A nondose-related 14 “/, increase in blood glucose was seen. A trend toward increased SGOT and SGPT activity (44 and 65 %, respectively, at 50 mg/kg) occurred among males. Prothrombin consumption time (no data shown) was within normal limits for all groups. 30 Days of recovery. Borderline increases in hematocrit for higher-dosed females occurred. In addition, reticulocytes were elevated at higher doses for females. Blood glucose concentrations increased approximately 15-60x for both sexes in a doserelated fashion. Alkaline phosphatase activity was generally increased in the males treated with dg-THC. Other changes were inconsistent or insufficient analyses were available. Organ Weights

The wet weight of major organs and endocrine organs were recorded at four sacrifice intervals: six to nine rats of each sex per group were studied at 28 and 90 days, six per sex per group at 180 days and after the recovery period. In addition, some endocrine organs were weighed in nine rats of each sex at 14 days. The data have been summarized in Table 3 and are expressed as percent of final body weight. 28 Days of treatment. No significant alteration of weight was observed for major organs. Increases in adrenal weight for both sexes were dose-related and as large as 30 7:. The weight of the pancreas rose 10 7: and the prostate weight decreased 30% in male animals receiving the high dose. A slight increase in testicular and ovarian weight occurred. 90 Days of treatment. A dose-related increase of 3-25?: for brain and 7-18x for kidney took place for both sexes. Adrenal weights remained elevated for both sexes. Prostate weight was down 6-21x while testes weight increased. Pancreas in the males tended to be higher. 180 Days of treatment. At the two higher doses, brain weight increased 16x, lung IO-23 %, kidney 1 l-20 %, and heart 12 % for both sexes. Liver weight rose 10 “/; at 50 mg/kg in rats of both sexes. At 50 mg/kg, thymus of the males decreased 17x, pancreas of males increased 96x, adrenals of both male and female were elevated 34-56 %, prostate increased 13 % and testes 25 %, while uterus decreased 14 “/. Many of the changes were dose-related but of borderline significance. 30 Days ofrecouery. Heart weights in animals of both sexes (50 mg/kg) were elevated less than 10 % pancreas of the males was up 30 % and adrenals of both sexes 20 “/;. Histopathological

Findings

Microscopic examination of approximately 14 organs was performed generally on vehicle controls and animals treated with dg-THC at 50 mg/kg. Since approximately ten rats per group were sacrificed at 14 days and their tissues showed similar changes to those treated for 28 days, their histopathological findings have been included with the latter group. The primary findings on sacrificed animals are outlined in Table 4 and on deceased animals in Table 5. 28 Days of treatment. All lesions seen were considered incidental. Similar morphological changes in lungs and thymus were seen in vehicle and dg-THC-treated animals. The presence of fat globules within macrophages seen in areas of pneumonitis in vehicle and d9-THC-treated rats suggested aspiration of compound into the pulmonary tree

3

50 x 28

10 x 28

2x28

M

Vehicle x 28

F

M

F

M

F

M

F

Sex

Dose dg-THC mg/kg x days

0.642 kO.043 0.958 kO.041 0.680 kO.046 1.029 +0.051 0.772 f0.078 1.097 &0.081 0.764 kO.066 1.079 +0.080

Brain

0.401 f0.028 0.528 +0.008 0.438 kO.051 0.504 kO.053 0.451 kO.046 0.613 f0.036 0.491 kO.062 0.582 kO.103

Lungs

0.696 kO.039 0.707 f0.056 0.692 kO.015 0.709 f0.038 0.716 kO.028 0.808 kO.043 0.743 kO.039 0.793 kO.049

Kidneys 4.490 kO.333 3.938 +0.286 4.096 kO.256 3.710 kO.258 3.660 kO.280 3.974 kO.144 4.087 f0.229 4.270 Ifr0.224

Liver 0.274 kO.018 0.316 kO.021 0.290 +0.019 0.310 -to.006 0.328 kO.024 0.340 kO.030 0.301 kO.012 0.357 kO.023

Heart 0.181 +0.009 0.225 kO.021 0.181 +0.010 0.228 kO.043 0.236 kO.066 0.227 f0.032 0.182 f0.022 0.207 kO.012

Spleen 0.003 f0.000 0.007 +o.ooo 0.004 +o.ooo 0.007 +o.ooo 0.003 +0.000 0.006 +0.003 0.003 ~0.000 0.007 +_0.003

Pituitary 0.124 kO.013 0.162 kO.014 0.129 20.013 0.157 kO.014 0.125 f0.009 0.169 kO.021 0.118 t-o.01 1 0.156 f0.012

Thymus 0.215 L-0.039 0.272 kO.030 0.216 kO.033 0.246 kO.027 0.241 50.029 0.226 kO.053 0.272b f0.043 0.278 kO.053

Pancreas

0.016 kO.003 0.029 to.000 0.017 &O.OOO 0.030 to.003 0.023” +0.003 0.040” +O.OlO 0.023b -to.003 0.0436 kO.004

Adrenals

0.074 +0.008 0.158 kO.022 0.069 -to.010 0.142 +0.03 1 0.070 f0.008 0.189 50.063 0.05gb kO.012 0.179 kO.045

Prostate or uterus

1.166 kO.094 0.051 +0.004 1.182 kO.062 0.050 f0.003 1.265” kO.086 0.058” kO.004 1.290b kO.051 0.057’ -to.008

or ovaries

ORALLY FOR 28-180 DAYS AND A 30-DAY RECOVERY

Organ weights as percent of final body weight (mean + SD)

SUMMARY OF MEAN ORGAN WEIGHTS ON FISCHER RATS GIVEN ~~~~~~~~~~~~~~~~~~~~~~ PERIOD”

TABLE

b

q

ti k-4 3 $ 3 N

50 x 180

10 x 180

2x 180

Vehicle x 180

50 x 90

10x90

2 x 90

Vehicle x 90

F

M

F

M

F

M

F

M

F

M

F

M

F

M

F

M

0.554 kO.074 0.928 k-O.058 0.554 20.071 0.924 kO.083 0.634’ kO.020 0.955 kO.024 0.687* kO.029 1.044 k-0.077 0.488 t-o.003 0.762 t-O.042 0.489 kO.045 0.814 t-O.038 0.569h kO.041 0.879’ kO.094 0.566’ to.053 0.833’ kO.056

0.362 L-o.059 0.456 kO.038 0.389 kO.084 0.527 *0.004 0.387 kO.032 0.514 k0.129 0.375 kO.021 0.507 kO.053 0.395 kO.076 0.429 kO.022 0.377 to.048 0.495 kO.065 0.452” kO.045 0.512b 20.046 0.439 kO.156 0.527h LO.034

0.560 kO.031 0.576 rfIo.030 0.570 kO.028 0.570 kO.040 0.601’ kO.018 0.617” kO.020 0.657b kO.033 0.670 kO.050 0.582 20.032 0.587 kO.057 0.592 LO.014 0.635 t-O.006 0.648’ LO.041 0.706* kO.023 0.658’ rto.045 0.712b kO.023 2.071 kO.044 2.043 to.123 2.106 kO.036 2.057 kO.130 2.118 kO.067 2.097 kO.099 2.146’ kO.060 2.299 kO.174 2.877 to.114 2.639 kO.254 2.871 kO.176 2.778 kO.202 2.910 kO.106 2.768 kO.152 3.147 to.253 2.974’ kO.133

0.267 -to.020 0.320 kO.051 0.275 kO.014 0.308 rtro.021 0.273 20.035 0.307 kO.008 0.303b kO.019 0.313 kO.005 0.240 kO.029 0.272 kO.021 0.246 -to.01 4 0.283 kO.023 0.278’ kO.019 0.323* kO.020 0.265 kO.007 0.310’ kO.027

0.150 20.024 0.170 20.012 0.147 20.007 0.189 L-o.01 1 0.147 kO.014 0.187 1-0.010 0.138 20.009 0.190 t-o.01 1 0.138 +0.010 0.174 kO.014 0.138 +0.013 0.180 -to.01 1 0.155 20.041 0.181 kO.014 0.142 kO.007 0.184 rto.004

0.003 +o.ooo 0.007 +o.ooo 0.003 ~0.000 0.007 +o.ooo 0.003 fO.OOO 0.007 f0.000 0.004 _+o.ooo 0.005 +0.003 0.002 -to.ooo 0.004 +0.001 0.002 +o.ooo 0.005 Lko.001 0.002 +o.ooo 0.006b +0.001 0.002 +o.ooo 0.006* +0.001 0.029 L-o.004 0.055 kO.008 0.031 ~0.004 0.05 I -to.010 0.022’ h0.005 0.061 20.005 0.024 -to.010 0.057 kO.009

0.222 kO.046 0.274 20.048 0.209 kO.044 0.246 kO.072 0.271 kO.073 0.283 kO.051 0.288’ kO.066 0.275 kO.067 0.163 kO.050 0.335 kO.119 0.177 rtO.048 0.322 to.120 0.229 kO.056 0.342 kO.077 0.320’ io.120 0.348 kO.084 0.014 +0.003 0.025 kO.003 0.015 kO.003 0.029” +0.003 0.015 kO.003 0.031b *0.005 0.021b +_0.003 0.035b *0.005 0.007 0.002 0.023 +0.004 0.012” kO.001 0.022 +0.001 0.012’ kO.002 0.026 kO.003 0.014h -to.001 0.032b Iko.003

0.087 kO.019 0.232 kO.088 0.069” +0.009 0.284 +0.118 0.082 kO.014 0.209 kO.077 0.076 -to.020 0.246 kO.117 0.063 +0.008 0.210 kO.027 0.070 kO.014 0.196 t-o.050 0.072 -to.012 0.215 kO.032 0.071 fO.O1O 0.181 to.053

0.978 ~0.040 0.044 kO.012 0.966 kO.053 0.050 *0.010 1.021 kO.052 0.050 kO.006 1 .210b kO.172 0.052 +0.008 0.802 kO.035 0.031 +0.006 0.848 kO.035 0.010 +0.006 0.919’ kO.104 0.035 kO.008 0.996’ kO.077 0.036 +0.004 g w

5 2 iz e $3 5 2 4

K

$

?E

Pi $

n ,a 2 2 & a”

M

50 x 30

0.368 kO.056 0.431

f0.055 0.362 f0.057 0.465 f0.004

fO.058 0.524 10.048 0.840 kO.042

0.306 kO.047 0.443 f0.055 0.342 f0.038 0.411 f0.038

Lungs

0.538 50.107 0.829

0.473 -to.030 0.785 kO.096 0.496 kO.036 0.834 f0.048

Brain

f0.030 0.575 kO.039 0.661 +0.041

0.591 kO.120 0.633

0.591 kO.089 0.593 kO.042 0.597 kO.059 0.630 +0.046

Kidneys

y.g f0:104

to.124 2.026

2.068 kO.327 2.001

1.925 kO.124 2.016 +O.lOO 2.134* kO.072 2.075 kO.196

Liver

‘o”.;;; c *0:019

50.015 0.264’

0.269 f0.032 0.302

0.235 kO.021 0.291 +0.015 0.266’ f0.022 0.296 f0.023

Heart

kO.021 0.139” +0.007 0.186 +0.008

0.138 kO.032 0.179

0.116 +0.017 0.213 kO.105 0.132 kO.011 0.176 kO.018

Spleen

0.003 +O.OOl 0.006 ~0.001 0.003 f0.001 0.006 +0.002

0.002 +o.ooo 0.006 _+O.OOl 0.003 +O.OOO 0.006 f0.001

Pituitary

0.028 +_O.OlO 0.052 +O.OOS 0.034 +O.OlO 0.055 +0.008 0.037 f0.009 0.053 +0.011 0.036 +0.007 0.055 f0.009

Thymus

a N= 6-9 rats/group of each sex for 28 and 90 days and 6 rats/group of each sex for 180 days and recovery. *p c 0.01 as compared to vehicle control. ‘p < 0.05 as compared to vehicle control.

F

M F

F

M

F

M

Sex

10x30

2x30

Vehicle x 30

Recovery after 180 days treatment

Dose Ag-THC mg/kg x days

3-continued

0.193 kO.031 0.289 kO.106 0.209 f0.067 0.296 f0.074 0.234 +0.004 0.300 kO.082 0.252’ kO.030 0.335 f0.097

Pancreas

Organ weights as percent of final body weight (mean f SD)

TABLE

+O.OOl 0.013* +0.001 0.024’ kO.002

0.010 +_oo.OO1 0.020 &0.002 0.011 +o.OOl 0.020 +0.002 0.012” *0.002 0.024*

Adrenals

to:047 0.056 50.013 0.228 kO.031

0.059 +_0.007 0.236 kO.053 0.074 kO.024 0.252 kO.038 0.069 yo;

Prostate or uterus

0.774 10.054 0.045 kO.008 0.825 kO.028 0.039 +0.008 0.898 f0.135 0.040 kO.017 0.864 kO.076 0.042 -to.01 2

Testes or ovaries

q ,$ ’

g # E $! $

22 0

LI~-T~HYDI~~~ANNABIN~L

six MONTH Toxrcm

F II m

6 -

-

II

67 -

-

II

3

5

+

cc+)) +

(+)

+++ ++

(+I (+) (+)

very severe.

++

++

+ ++++

+ ++

+

+++ +

+ ++ +

+ ++

-t + + +

+

120 228 171 131 Dead

168 203 168 128 Morib.

+++

+

+

98 155 181 142 Morib.

F 16-1

F 16-5

M 16-6

M 16-5 102 190 185 145 Dead

141

2811

145

107 189 171 131 Dead

305 170 132 Good

2834

131

128

117 246 145 105 Fair

104

208 170 132 Good

128

2780

d9-THC 50 mg/kg

2742

F 15-2

M

F 3-l

M 3-l

131

2779

d9-THC ORALLY'

24-1

105

132

2781

132

Sesameoil

277%

’ Grading legend: No entry = no lesion; (+) slight; + moderate; ++ marked+ +++ severe; ++++

Number of treatments Sex Rat number Body weight (g) Initial Final As (days) Day of death or sacrifice Condition Incidental pathology Brain, vacuolar change Adrenal, fine discretevacuolar changein cortex Lymphoid tissue,atrophy Thymus, focal hemorrhages Heart, focal subacutemyocarditis Lungs,focal hemorrhages Lungs,edema Lungs,focal acute bronchitis pneumonia Lungs,focal subacute: perivasculitis peribronchiolitis peribronchitis Lungs,congestion Lungs,blood vessels,focal proliferative vasculitis Kidney, right, infarct Focal eosinophiliccasts Skin, subcutis,axilla, abscess Bonemarrow, myeloid hyperplasia Mediastinum, acute mediastinitis Organs,liver, kidney, congestion

Treatment

TABLE MORIBUND AND DECEASED FIXZHER RATSTREATEDWITH

Histology number 72- 2777

SUMMARYOF HISTOPATHOLOGYIN P

b

3

3

E

TI i4 z

i-z

dg-TETRAHYDROCANNABINOL

SIX MONTH

TOXICITY

413

or faulty gavage technique. However, when other routes of administration are used, there is an affinity of dg-THC for lung tissue: therefore, a systemic contribution of compound to lungs in dg-THC-treated groups cannot be eliminated. Most of the pulmonary changes and thymic hemorrhages were interpreted to be agonal and related to the method of sacrifice which was decapitation. No dose-related changes were observed in sections of heart, kidneys, ovaries, spleen, liver, testis, prostate, adrenals, pituitary, and pancreas. 90 Days of treatnzent. Pulmonary changes at this sacrifice interval were also not drug related and incidental or *induced by the procedure of decapitation. Interstitial myocarditis was found in 3/17 rats treated with low and high doses of dg-THC. 180 Days of treatment. Among eight males and eight females in the 50 mg/kg group, two of each sex had exhibited multiple seizures before sacrifice but no morphological lesions were evident. Incidental histopathological changes were observed in some rats : one control and one treated male animal, subacute myocarditis; one treated male. renal tubular degeneration ; one low-dosed male, focal degeneration in left testicle: two high-dosed females, acute endometritis; one high-dosed male, focal cyst in the pituitary. Gross incidental pathological changes included: mottled lungs in one low-dosed female and an ovarian bursal cyst in another; at 10 mg/kg, one female had pulmonary bronchiectasis and one male had an atrophied adrenal gland; and among control rats, one male had testicular atrophy. Because of increased adrenal weight, the distribution of adrenal fat was investigated histochemically. Oil red 0 stained frozen sections of adrenal glands showed no difference in fat distribution between dg-THC treated (2 and 10 mg/kg) and vehicle control animals of both sexes. Since earlier work with massive doses of dg-THC had induced changes in total nucleated cells (TNC) in bone marrow after 90 days (Thompson et al., 1973b), TNC counts were performed on rats treated for 180 days in the present study. Among male rats, the mean + SD TNC was 16.9 of:1.2, 13.4 + 5.5, 14.2 i 7.4, and 23.1 & 8.4 for vehicle, 2, 10, and 50 mg/kg, respectively (no significant change). Among female rats, the corresponding values were 3.7 + 1.4,9.5 + 2.7, 13.4 i- 6.7, and 20.9 + 7.9 : p values were between 0.01-0.05. Vehicle-treated female rats exhibited TNC counts which were somewhat low and additional control TNC counts should be made before any interpretation of the findings is contemplated. Several rats died during days 90-180 and three control animals were sacrificed on day 132 for comparative purposes (Table 5). Animal 15-l F (50 mg/kg) died over a weekend (approx. day 96) and was examined in rigor mortis (tissue was unsuitable for histopathology). Lung hemorrhages and oil droplets in the trachea suggested that the cause of death was aspiration pneumonia; however, this rat displayed convulsions a few days before death. Animal 15-3M (50 mg/kg) died immediately after a convulsive episode on day 121. Lungs were dark red and an oily foam was present in the bronchial tree; fine oil globules were seen in the edema fluid. Probable cause of death was acute pulmonary hemorrhages and edema related to compound aspiration into lungs. Animal 16-5M (50 mg/kg) was sacrificed in a moribund condition on day 128 after a 41 g loss in body weight during the previous week. The animal could not maintain an

414

ROSENKRANTZ

ET AL.

upright posture and had rhinitis. Muscle tone was poor and respiration erratic. Gross pathological assessment revealed a normal tracheal tree but lungs were mottled, atelectatic, and rubbery. The absence of oily material in the pulmonary tree indicated death was not due to faulty gavage. The alimentary tract was normal. Whereas the left kidney was normal, the right kidney had an abnormal pale tan color and was internally mottled. Toxic state was associated with infarction of the right kidney and rhinitis. This animal convulsed on two occasions prior to death. Animal 16-6M (50 mg/kg) died on day 131 after an 89 g loss in body weight over a 2-wk interval. The emaciated rat had little subcutaneous fit and gastric distention due to gas. The lungs had marked edema and the bronchial tree contained a thick, tenacious, mucoid material. Cerebral blood vessels were congested and this rat had exhibited convulsive seizures. A necropsy was performed in animal 24-l M (sesame oil control) on day 106 because of a large subcutaneous mass in the right axila; the 3 cm (diameter) firm necrotic mass contained a light green inspissated pus. Except for this abcess and marked myeloid hyperplasia, all major organs appeared normal. Animal 15-2F (50 mg/kg) was found dead on day 131. Marked congestion of the lungs and brain occurred and the animal convulsed prior to death. Animal 16-1F (50 mg/kg) was sacrificed on day 141 while moribund and having convulsions involving the hind legs. The forelegs were paretic. Necropsy revealed focal areas (5 mm diam.) of degeneration in the liver. A focal 6 mm nodule was seen between the right and left medial lobes. The adrenals were enlarged but all other organs appeared normal. Animal 16-5F (50 mg/kg) was found dead on day 145 and had no history of convulsions. There were severe pulmonary hemorrhages but no clear evidence implicating compound aspiration. All major organs were grossly normal. Animal 16-2F (50 mg/kg) died on a weekend on approximately day 173. This was not a convulsive rat and acute pulmonary hemorrhages and edema may have been associated with faulty gavage although compound was not discerned in the bronchial tree. Postmortem changes prevented evaluation of histopathology. Animal 36-3M (50 mg/kg) expired on approximately day 164. Severe postmortem changes and cannibalism prevented a proper pathological evaluation. The animal did display convulsive activity during the week preceding death. Animal 28-2111 (2 mg/kg) was found dead on day 177 and had no history of convulsions. Intense pulmonary hemorrhages and edema were discerned. Compound formulation was not seen in the tracheal-bronchial passages. Other organs were normal and the cause of death was probably hypoxia. A summary of histopathological findings on moribund and deceased rats is provided in Table 5. Data on vehicle control animals are also shown. Despite aberrations in heart, lungs, and kidneys (16-5M) in some of the chronically treated rats, the changes were not considered to be drug related. 30 Days of recovery. No drug-related lesions were observed. Incidental pathological findings (Table 4) were limited to a vacuolar change in the adrenal cortex observed in both vehicle control and treated male rats, focal pulmonary hemorrhages related to the method of sacrifice, occasional focal aggregates of alveolar macrophages, and focal subacute interstitial myocarditis.

d9-TETRAHYDROCANNABIN~L

SIX MONTH

TOXICITY

415

DISCUSSION

Alterations in physiological parameters and lethality have now been demonstrated in rats chronically treated with reasonable doses of d9-THC. Despite lack of definitive morphological evidence to explain the changes and mortality, the changes were of importance because some existed through the whole period of treatment while others persisted into the recovery period. Rectal temperature variations were minimal although larger doses of d9-THC are known to evoke a dose-related hypothermia to which tolerance developed (Thompson et al., 1973b). A poikilothermic effect of cannabinoids has been shown to be doserelated (Sofia, 1972) and both the effect of room temperature (Haavik and Hardman, 1973) and time of day of treatment (Abel, 1973) affect the response of body temperature to d9-THC and other drugs. d9-THC at 10 and 50 mg/kg inhibited the rate of growth of both sexes and despite an elevation in food consumption after a transient anorexia, growth rate remained depressed. It seems possible that increased food consumption was utilized to stimulate general metabolism to provide energy for adaptive and detoxification mechanisms. In subchronic studies, others have reported tolerance to anorexia and bodyweight loss (Pirch et al., 1972; Rating et al., 1972) or continued inhibition of growth rate (Sofia and Barry, 1972; Sjijden et al., 1973; Manning et al., 1971). Hematology, clinical chemistry, and urinalysis revealed little except an increase in SGOT and SGPT for males. There were no drug-related morphological alterations in liver or heart to explain the changes in transaminase activities. Hyperglycemia first became evident for both sexes at 180 days of treatment and apparently intensified and persisted during the recovery phase. Increased blood sugar concentrations were consistent with the need to restore depleted liver glycogen stores (Sprague et al., 1973) and the synthesis of increased quantities of glucose-6-phosphate would provide substrate for phosphatase generation of blood glucose. Because of similar organ weight measurements at four time intervals, the validity of weight changes can be explored. For example, the elevation of pancreas weight in the males took place at 14, 28, 90, and 180 days of necropsy. A similar observation was apparent for adrenal weights of animals of both sexes. Testis weight increased at 28, 90, and 180 days. Thymus weight in the males seemed depressed at all time intervals studied, whereas, prostate weight appeared depressed at earlier time periods but showed a borderline increment at 180 days. In the instance of the female pituitary gland, lowered weights were characteristic at 14,28, and 90 days but an increased weight was obtained on day 180. Ovarian and uterine weights were too variable to detect a trend in weight alterations. Organ/FBW ratios were affected by changes in growth rates. The changes in weights of endocrine glands suggested possible endocrinopathies despite lack of histopathological findings. If there were endocrine imbalances, they would appear to be functional and not morphological. Despite the normal appearance of recovered animals and lack of neurotoxicity, biochemical derangements may persist during a 30-day recovery period. The loss of tolerance is known to vary from more than a month in dogs (McMillan et al., 1972) and monkeys (Stadnicki et al., 1974) to approximately 2 wk in rats.‘l I1 Rose&rank,

H., unpublished data.

416

ROSENKRANTZ

ET AL.

It may be concluded that marked aggression, other behavioral changes, and neurotoxicity can be induced by levels of d9-THC used by man. Absence of deleterious changes in blood and tissue suggest the initiation of protective mechanisms which sustain the organism in the face of chronic administration of d9-THC. ACKNOWLEDGMENTS The authors express their appreciation to Paul Bunnell, Robert Lezinski, and Bruce Calkins for competent technical assistance. REFERENCES ABEL, E. L. (1973).Chronopharmacologyof dg-tetrahydrocannabinolhypothermia in mice. Experientia 15, 1528-l 529. DAVIDSOHN, I. AND HENRY, J. B. (1969). Todd-Sanford Clinical Diagnosis by Laboratory Methods, 14ed. Saunders,Philadelphia,Pennsylvania. FREIREICH, E. J., GEHAN, E. A., RALL, D. P., SCHMIDT, L. H. AND SKIPPER, H. E. (1966).

Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey and man. Cancer Chemother. Rep. 50,219-244. ~AVIK, C. 0. AND ~RDMAN, H. F. (1973).Evaluation of the hypothermic action of tetrahydrocannabinolsin mice and squirrel monkeys. J. Pharmacol. Exp. Ther. 187, 568-574. KWLINGER, G. F. AND MANNO, J. E. (1971). Dose-responserelationshipsto Cannabisin humansubjects.Pharmacol. Rev. 23,339-347. LERNER, M. AND ZEFFERT, J. T. (1968). Determination of tetrahydrocannabinol isomersin marihuanaand hashish.Bull. Narcotics 20, 53-54. LCJTHRA, Y. K. AND ROSENKRANTZ, H. (1974). Cannabinoids: neurochemicalaspectsafter oral chronic administrationto rats. Toxicol. Appl. Pharmacol. 27, 158-168. LUTHRA, Y. K., ROSENKRANTZ, H., HEYMAN, I. A. AND BRAUDE, M. C. (1975). Differential neurochemistryand temporal pattern in rats treated orally with dg-tetrahydrocannabinol for periodsup to six months. Toxicol. Appl. Pharmacol. 32,418-431. MANNING, F. J., MCDONOUGH, J. H., JR., ELSMORE, T. F., SALLER, C. AND SODETZ, F. J. (1971). Inhibition of normal growth by chronic administration of Ag-tetrahydrocannabinol. Science 174,424-426. MARIHUANA AND HEALTH, SECOND ANNUAL REPORT TO CUNGRESS (1972). Department of Health, Education and Welfare Publ. No. (HSM) 72-9113,pp. l-l 50. MARIHUANA AND HEALTH, THIRD ANNUAL REPORT TO CONGRESS (1974). Department of Health, Education and Welfare Pzzbl. No. (ADM) 74-50,pp. 1-163. MCMILLAN, D. E., DEWEY, W. L. AND HARRIS, L. S. (1972). Characteristicsof tetrahydrocannabinoltolerance.Ann. N. Y. Acad. Sci. 191, 83-99. NAHAS, G. (1973).Marihuana-Deceptive Weed, pp. l-334. Raven Press,New York. F’IRCH, J. H., COHN, R. A. AND OSTERHOLM, K. C. (1972).Toleranceto the effectsof marihuana on behavior and body weightsof rats. Fzyth Znt. Cong. Pharmacol., p. 8. RATING, D., BROERMANN, I., HONECKER, H., KLUWE, S. AND &PER, H. (1972). Effect of

subchronic treatment with (-)A*-trans-tetrahydrocannabinol (AS-THC) on food intake, body temperature, hexobarbital sleeping time, and hexobarbital elimination in rats. Psychopharmacologia 27, 349-357. ROSENKRANTZ, H. AND BRAUDE, M. C. (1974).Acute, subacuteand 23-day chronic marihuana inhalation toxicities in Fischerrat. Toxicol. Appl. Pharmacol. 28,428-441. ROSENKRANTZ, H., HEYMAN, I. A. AND BRAUDE, M. C. (1974).Inhalation, parenteraland oral LD50 values of Ag-tetrahydrocannabinol in Fischer rats. Toxicol. Appl. Pharmacol. 28,

18-27. ROSENKRANTZ, H., THOMPSON, G. R. AND BRAUDE, M. C. (1972).Oral and parenteralformulations of marihuanaconstituents.J. Pharm. Sci. 61, 1106-l112. SINGER, A. J. (1971). Marijuana: chemistry, pharmacology and patternsof socialuse.Ann. N. Y. Acad. Sci. 191, l-269.

L~~-TETRAHYDROC.~NNABINOL six

MONTH

TOXICITY

417

SJ~DEN,P-O, JARBE,T. U. C. AND HENDRIKSEN, B. G. (1973).Influence of tetrahydrocannabinols (As-THC and A9-THC) on body weight, food and water intake in rats. Phurmacol. Biochem. Behav. 1,395-399.

SOFIA,R. D. (1972).Paradoxical effect for A’-tetrahydrocannabinol on rectal temperaturein rats. Res. Commun. Chem. Pathol. Pharmacol. 4,281-288. SOFIA,R. D. ANDBARRY,H., III (1972). Food intake by rats following acute and chronic administrationof A’-tetrahydrocannabinol. Fifth Znt. Gong. Pharmacol., p. 218. SPRAGUE, R. A., ROSENKRANTZ, H. AND BRAUDE, M. C. (1973).Cannabinoideffectson liver glycogenstores.Life Sci. l&409-416. STADNICKI, S. W., SCHAEPPI, U., ROSENKRANTZ, H. AND BRAUDE, M. C. (1974).Crudemarihuanaextract: EEG and behavioraleffectsof chronic oral administrationin rhesusmonkeys. Psychopharmacologia

37,225-233.

G. R., FLEISCHMAN, R. W., ROSENKRANTZ, H. AND BRAUDE, M. C. (1974a).Chronic oral toxicity of cannabinoidsin monkeys. Toxicol. Appl. Pharmacol. 28,7 (Abstr.). THOMPSON, G. R., FLEISCHMAN, R. W., ROSENKRANTZ, H. AND BRAUDE, M. C. (1974b).Oral and intravenous toxicity of A9-tetrahydrocannabinol in rhesusmonkeys. Toxicol. Appl. THOMPSON,

Pharmacol.

27,648-665.

G. R., MASON, M. M., ROSENKRANTZ, H. AND BRAUDE, M. C. (1973b).Chronic oral toxicity of cannabinoidsin rats. Toxicol. Appl. Pharmacol. 25, 373-390. THOMPSON, G. R., ROSENKRANTZ, H., SCHAEPPI, U. H. AND BRAUDE, M. C. (1973a).Comparison of acute oral toxicity of cannabinoidsin rats, dogsand monkeys. Toxicol. Appl. THOMPSON,

Pharmacol. 25,353-372. N. W. (1970). Fundamentals

TIETZ,

of Clinical Chemistry. Saunders,Philadelphia,Pennsylvania.