Monique C. Braude

Chronic oral toxicity of cannabinoids in rats

Abstract The preclinical toxicity of Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-THC and a crude marihuana extract (CME) was evaluated in Fischer rats. The compounds were dissolved in sesame oil and administered po for 119 consecutive days at doses of 50, 250, 400 or 500 mg/kg/day for Δ9- and Δ8-THC, or 150, 750, 1200 or 1500 mg/kg/day for CME. These high dose levels were 30–300 times the usual oral dose in man and were chosen specifically to induce toxicity in rats. A biphasic toxicity pattern was characterized by generalized depression to which tolerance developed during the first week. Thereafter, hyperactivity, irritability and aggressiveness progressed and convulsions occurred by day 50. Cumulative toxicity occurred during the first few days of depression as indicated by increased chronic vs acute mortality. The singledose LD1 and LD4 for Δ9-THC produced 23 and 70% mortality in male rats which were treated chronically. A consistently higher incidence of mortality occurred in females than in males, but essentially all deaths from all three compounds occurred during the interval 36 to 72 hr after initial treatment. Dose-related hypothermia and bradypnea were prevalent and coincided with the interval for mortality. Growth in all treated groups was persistently decreased in a dose-related manner (30–130%) despite an increased consumption of food and water after approximately day 50; abdominal fat stores were depleted for extended periods. Organ weights decreased for uterus, prostate, ovary and spleen, and increased for adrenals, but pathologic changes in these organs were limited to splenic hypocellularity and vacuolization of the adrenal. Hematologic and biochemical parameters were generally normal except for mild leukopenia, increased SGOT and decreased coagulation time. Toxicity was similar for the three compounds and suggested a prominent effect on endocrinologic systems.

Comparison of acute oral toxicity of cannabinoids in rats, dogs and monkeys

Abstract For preclinical toxicologic evaluation, Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-THC, and Cannabis extract were administered po to rats, dogs and monkeys as solutions in either absolute ethanol, sesame oil, or sesame oil with 2.5–9.0% ethanol. All three compounds were significantly more potent in female than in male Wistar-Lewis and Fischer rats. However, within the dosage range of 225–3600 mg/kg, Δ9-THC and Δ8-THC produced the same lethality, while both isomers were approximately twice as potent as the Cannabis extract. Death due to all three compounds consistently occurred between 36 and 72 hr after treatment regardless of the dose level or sex of the rats. Mortality in rats apparently resulted from severe hypothermia and other central effects. Toxicity was characterized by severe hypothermia, bradypnea, rapid weight loss, inactivity, wide stance, ataxia, muscle tremors, and prostration. Rats treated with equimolar amounts of tetrahydrocannabinol from the three compounds exhibited equivalent diversities and severities of clinical signs. In dogs and monkeys, single oral doses of Δ9-THC and Δ8-THC between 3000 and 9000/mg/kg were nonlethal. Predominant toxic signs in dogs included drowsiness, ataxia, prostration, anesthesia, tremors, mild hypothermia, salivation, emesis, and anorexia. Toxic signs in monkeys included hyperreactivity to stimuli, lethargy, drowsiness, characteristic huddled posture, slow movements, abnormal eating procedures and sedation. Histopathologic alterations did not occur in either dogs or monkeys.

Acute, subacute and 23-day chronic marihuana inhalation toxicities in the rat

The acute, 5-day and 23-day toxicities of Δ9-THC-impregnated (9% Δ9-THC) and nonimpregnated marihuana (2.2% Δ9-THC) cigarettes were determined in Fischer rats. Human smoking conditions were simulated by providing a 50-ml puff of 2-sec duration with a 30-sec exposure interval followed by a 30-sec purge period, each min, through use of an automatic smoking machine. The calculated inhalation doses ranged from 7 to 60 mg/kg acutely from 3.4 to 18.1 mg/kg subacutely, and from 0.7 to 4.2 mg/kg after chronic administration. Acute toxicity was manifested by dose-related hypothermia, hypopnea, loss of coordination, ataxia and prostration. Upon removal from the inhalator, high-dosed animals were depressed and low-dosed animals displayed increased activity rapidly followed by depression. Recovery was nearly complete by 24 hr in the acute trial and by day 6 in the subacute study. Body weights were normal; histopathologic examinations did not reveal any abnormalities. In the 5-day subacute toxicity study rats displayed transitory incoordination, hypothermia, hypopnea and initial increased activity at the lower doses followed by a persistent depression. Rate of body growth was normal, but there was an increase in the weight of reproductive organs. A transient hyperkalemia and hyperproteinemia were seen, but histopathologic examinations gave essentially normal results. Food intake and liver glycogen were increased. When lethality occurred, it was due to respiratory arrest. In the chronic study, there was a dose-related CNS depression and vocalization in the first week of exposure. Low- and middle-dosed animals were hyperactive in the first 10 min post-exposure, but all rats were depressed within 1–2 hr and generally recovered by 6 hr. In the second week of exposure, dose-related ataxia, prostration, “popcorn” response and vocalization were observed; cumulative toxicity occurred in 2 of the high-dosed males. Tolerance to prostration, ataxia, vocalization and exploratory activity developed after 8–12 exposures. After 3 weeks of exposure, hyperactivity and hypersensitivity were seen in low- and middle-dosed groups while high-dosed females exhibited fighting aggression and high-dosed males were depressed or tranquil. During the fourth and last week of exposure, tolerance developed to CNS stimulation in all groups, and animals were either normal or tranquil. Clinical signs were primarily affected in the first week of exposure: a 30–75% dose-related decrease in exploratory activity, hyperthermia at lower doses and hypothermia at the high dose, and a 15–30% decrease in respiration rate at mid and high doses. On day 14, hemochemistry, urinalysis and histopathology were normal but a 10% increase in testes (0.7 mg/kg), and a 15–40% increase in adrenal weights for both sexes was observed, although growth rate was normal. During prolonged exposure, there was a 10% decline in growth rate for males receiving the high dose, but food intake increased 14–29%; in females, body weight was maintained while food consumption fell 5–12%. Generally, continued exposure did not alter rectal temperature, respiration rate, urinalysis or hemochemistry, but a slight decline in hematocrit value was observed at the high-dose level. Histopathologic findings were essentially negative. The inhalation studies reveal that doses of Δ9-THC, similar to those used by man, rapidly initiate behavioral and physiological changes. Tolerance developed to most of these changes during chronic treatment. Some cumulative toxicity and fighting episodes can occur in the rat after inhalation of marihuana smoke, despite the lack of morphologic changes.

Oral Δ9-tetrahydrocannabinol toxicity in rats treated for periods up to six months

Abstract The chronic toxicity of Δ 9 -tetrahydrocannabinol ( Δ 9 -THC) given orally 28, 90, and 180 days to Fischer rats at doses of 2, 10, and 50 mg/kg was investigated. Some 180-day treated animals were monitored after a 30-day recovery interval. The lower doses used corresponded to the Δ 9 -THC content of marihuana or hashish. In the first 10 days CNS-depression, incoordination, ataxia and passivity, poikilothermia, and hypopnea occurred to which tolerance developed. During days 10–20, irritability, hypersensitivity, hyperactivity, and aggression predominated. Fighting occurred between days 20–100. Tremors and clonic convulsions appeared after day 70 in 50% of the animals at 50 mg/kg and 12% at 10 mg/kg. Tolerance developed to CNS-stimulation, fighting and neurotoxicity and lethal cumulative toxicity was seen although the cause of death was not established. Growth rate of both sexes was inhibited despite an elevation in food consumption after a transient anorexia No morphological changes could be ascribed to Δ 9 -THC. Except for a rise of 28–45% in SGOT and 46–69% in SGPT in males at higher doses and a belated hyperglycemia in both sexes, clinical chemistry, hematological, and urinalysis parameters were within normal ranges. The greatest changes were seen in ratios of organ/FBW at 50 mg/kg: 10–20% increase in brain, lungs, kidneys, heart, and liver; 45% increase in adrenals; 96% increase in male pancreas and 13–25% increase in testis and prostate. The present investigation implicated reasonable doses of Δ 9 -THC in undesirable behavioral changes highlighted by fighting aggression, convulsive activity, and lethal cumulative toxicity. The absence of morphological changes despite changes in growth rate and organ weights indicated a functional impairment that was not an immediate threat to the life of the organism because of initiation of as yet unknown protective mechanisms.

Oral and intravenous toxicity of Δ9-tetrahydrocannabinol in rhesus monkeys☆☆☆

The toxicity of Δ9-THC was evaluated in 35 rhesus monkeys treated acutely po or iv; in 28 monkeys treated po for 28 days with 0, 50, 250 or 500 mg/kg/day; or in 16 monkeys treated iv for 28 days with 0, 5, 15 or 45 mg/kg/day. The high subacute doses selected from acute toxicity studies were chosen to establish toxicity, not to simulate human dosages. No deaths occurred in monkeys treated acutely po with up to 9000 mg/kg, but all monkeys treated acutely iv with 128 mg/kg or more died from respiratory arrest and cardiac failure. In the subacute oral study 2 of 8 monkeys treated with 500 mg/kg/day became moribund on days 10 and 14, and 1 of 6 monkeys treated with 50 mg/kg/day became moribund on day 16. Primary pathologic changes in these 3 moribund monkeys included atrophy of the pancreas, hemorrhagic ulcerative colitis, myeloid hyperplasia of the bone marrow, vacuolar nephrosis and severe serum electrolyte imbalance. All other monkeys were normal at necropsy. In the subacute iv trials, 2 of 4 monkeys treated with 45 mg/kg/day died on days 8 and 19 as a result of acute hemorrhagic pneumonia, but injection site edema, necrosis, ulceration and fibrosis also occurred. Behavioral and physiologic changes were similar in both studies and included lethargy, huddled posture, bradypnea, hypothermia, bradycardia, weight loss, anorexia and constipation. Tolerance and cumulative toxicity were also similar for the 2 studies, but monkeys treated iv showed dose-related anemia and increased BSP retention while no blood changes occurred in monkeys treated po. Pathologic changes in deceased monkeys were associated with the route of administration.

Inhalation, parenteral and oral LD50 values of Δ9-tetrahydrocannabinol in Fischer rats

Abstract In order to resolve the differences in reported LD50 values for Δ 9 -tetrahydrocannabinol ( Δ 9 -THC) obtained with various vehicles and rat strains, the oral LD50 values of Δ 9 -THC in a natural vegetable oil vehicle and in an aqueous emulsion were determined in the same rat strain. In addition an iv LD50 value obtained with the emulsion formulation was compared with an inhalation LD50. The natural vegetable oil was pure sesame oil and the aqueous emulsion used orally was comprised of 13% sesame oil, 1% polysorbate 80 and isotonic saline. A similar emulsion (7% sesame oil and 0.5% polysorbate 80) was used to establish the iv and ip LD50 values. The young adult Fischer rat was used in all experiments and the polysorbate 80 concentration in emulsion formulations was maintained below toxic levels for the rodent. Marihuana cuttings were impregnated with Δ 9 -THC and formed into cigarettes which were smoked under controlled conditions of puff volume and duration in an automatic smoking machine to obtain an inhalation LD50. It was demonstrated that behavioral and physiological responses to Δ 9 -THC occurred sooner with the oral emulsion formulation than with the vegetable oil. The intragastric LD50 with the emulsion was 800 mg/kg and with the sesame oil formulation, 1270 mg/kg. The iv LD50 was 36–40 mg/kg, similar to the inhalation dose when the latter was corrected for Δ 9 -THC losses in the rodent nasal passages. The results from this study affirmed that the LD50 values obtained were reliable and that the vehicle did not contribute to the toxicity.

Chronic marihuana inhalation toxicity in rats

Abstract Exposure of rats to marihuana or placebo smoke for periods of up to 87 days was performed with an automatic inhalator. Δ9-Tetrahydrocannabinol (Δ9-THC) concentrations in the marihuana smoke were similar to those inhaled by man and were presented to rats in a 50-ml puff volume of 2-sec duration and a 30-sec exposure interval followed by a 30-sec period of fresh air each minute. By varying the number of puffs from three simultaneously smoked marihuana cigarettes (2.1% Δ9-THC), 8–10 Fischer rats simultaneously received a single daily Δ9-THC dose of 0.7, 2, or 4 mg/kg, 6 consecutive days per week for 27, 57, or 87 days. Lethal cumulative toxicity in male rats began in the first week, which eventually resulted in 60% deaths, at two peak intervals, in weeks 3 and 8. During the first week, some dose-related CNS inhibition, hypothermia, and hypopnea occurred to which tolerance developed at different rates. In the second and third weeks, CNS stimulation was prevalent at lower doses. Neurotoxicity (“popcorn” reaction) occurred in 70% of both sexes on the high dose and reached a peak in weeks 3 and 8. Tolerance to these manifestations developed in subsequent weeks. Generally, growth rates and food intake decreased in males treated with the high dose but, in the other treated groups, food and water consumption were evaluated throughout the study. Increased organ to body weight ratios for the brain, lungs, and heart of males reflected decreases in final body weight. No convulsive episodes were noted. In weight-decreased rats, congestion in major organs and pulmonary edema suggested circulatory failure. In the treated animals, which were sacrificed, a dose-related moderate focal pneumonitis, characterized by the accumulation of aggregates of yellow-brown, sudanophilic alveolar macrophages, polymorphonuclear leukocytes, and lymphocytes, was observed. Female rats evoked as yet unknown protective mechanisms more efficiently than males in the face of neurotoxicity and morphological changes.

Crude marihuana extract: EEG and behavioral effects of chronic oral administration in Rhesus monkeys

Three Rhesus monkeys were treated daily po with crude marihuana extract (CME) containing δ9-THC 22–25%, cannabidiol 2–3% and cannabinol 2–3%, but no δ8-THC. CME with the equivalent of δ9-THC 12.5 mg/kg or more caused sedation, ptosis, ataxia, huddled posture, spontaneous jerky body movements and increased EEG synchrony without an initial phase of increased motor activity. Thereafter, δ9-THC 12.5 mg/kg in 2/3 monkeys produced specific EEG changes including the appearance of protracted trains of 20–25 cps rhythmic activity in thalamus and cerebellar nuclei. δ9-THC 37.5 mg/kg or more, in all 3 monkeys tested, caused 1.5–2 cps slow waves in hippocampus, amygdala and septum. Spikes or other epileptiform EEG activity were not observed. EEG manifestations after oral treatment were therefore different from those previously observed following i.v. injection or smoke inhalation. Behavioral effects consistently preceded and outlasted EEG changes, and tolerance developed more rapidly to specific EEG changes than to behavioral effects. The two monkeys that became tolerant after 50 daily treatments with δ9-THC 37.5 mg/kg responded to termination of treatment with withdrawal signs manifested by increased aggressiveness. One of the two exhibited “hallucinations” and increased periods of EEG desynchronization. Withdrawal signs were, therefore, more severe for behavioral than for EEG effects.

Cerebral and cerebellar neurochemical changes and behavioral manifestations in rats chronically exposed to marijuana smoke

Abstract Groups of male and female Fischer rats were exposed to marijuana cigarette smoke via an automatic smoking machine. Inhaled Δ 9 -tetrahydrocannabinol doses of 0.7, 2, and 4 mg/kg were relevant to man and were given for 12, 18, 27, 57, and 87 days. Another group of rats treated for 87 days was studied after a recovery of 20 days. Control animals inhaled smoke produced by placebo cigarettes. In the first week of exposure, 20% of lower-dosed rats were hyperactive and 50% at the high dose were prostrate or ataxic upon removal from the inhalator. Behavioral aberrations ameliorated within a few hours except for the depression exhibited by males at the high dose. Tolerance to CNS inhibition developed in 1–2 weeks. CNS stimulation, as manifested by hypersensitivity and hyperactivity, progressively involved more animals, primarily females, in all groups during Days 27–57. Tolerance to CNS stimulation developed thereafter. Fighting was displayed by 90% of females and 50% of males at 4 mg/kg by weeks 6–7. Neurotoxicity was expressed by involuntary vertical jumping, predominantly among high-dosed males in weeks 3 and 8. Normal behavior was displayed after cessation of treatment. At necropsy, homogenates of cerebrum and cerebellum were prepared and were analyzed for protein, RNA and acetylcholinesterase (AChE) activity. Cerebral AChE activity in females increased 33–71% after 12 exposures, decreased 10–23% after 57 exposures and rose 12% after 87 exposures. Cerebellar enzyme activity initially increased 15–35% in animals of both sexes during the subchronic phase but declined in females after 27 exposures. The extent of change in enzyme activity was generally reduced with continued treatment. Cerebellar RNA increased approximately 20% in rats of both sexes, but at different time intervals during the subchronic phase, and remained elevated in females at 87 days. Neurochemical changes were sex related and coincided with behavioral manifestations, and some changes extended into the recovery period. Inhalation findings were similar to those obtained earlier by the oral route; however, females demonstrated a greater facility to adapt to the cumulative toxic effects of marijuana smoke.

Δ9-tetrahydrocannabinol: Subcortical spike bursts and motor manifestations in a fischer rat treated orally for 109 days☆

Abstract A total of 12 Fischer rats was prepared surgically for chronic EEG recording from cortical and subcortical sites. Most rats, within 2 to 9 weeks after electrode implantation, developed polyspike activity in cortical and subcortical recordings that were without motor manifestations. Six of these rats, chronically treated po with Δ 9 -tetrahydrocannabinol (Δ 9 -THC) 10 mg/kg exhibited acute EEG changes with more frequent occurrence of EEG desynchronization and polyspike activity. On day 109 one of 6 rats displayed consulsive activity, with jerky movements of the head and paws, characteristics of Δ 9 -THC neurotoxicity. EEG alterations concomitant with motor signs included bursts of spikes of approximately 0.2 sec that occurred in subcortical, but not in cortical, recordings. It is concluded that in the Fischer rat acute and chronic treatment with Δ 9 -THC facilitated the occurrence of surgically-induced “polyspike” activity while chronic treatment caused occasional transient subcortical spike bursts with concomitant motor manifestations.