ANIMAL TOXICITY STUDIES

Preclinical Toxicological Information Concerning Antineoplastic Agents.
The objectives of the studies of toxicity of a substance in animals are basically two: 1) The prediction of the different types of toxic effects that can be caused in man and 2) the frequency with which they occur. Toxicity is understood to be the capacity of a substance to cause destructive changes in the organism.

During toxicity studies, effects are frequently reported that could be of interest arid usefulness for the clinics regarding the therapeutic effectiveness of the substance, since studies concerning toxicity frequently are intimately related to those of therapeutic efficacy.

Thanks to these studies, the probability of eliminating substances with great toxicity is very high. even with experiments as simple as the determination of LD₅₀ (dosage capable of causing death in 50% of the animals exposed) in mice and in general it can be said that the studies with animals have been successful if one considers how infrequently serious toxic reactions are found in humans and that they are not anticipated in accordance with the studies of animals.

Concerning the research protocol which should be folfowed and the species that should be studied. There is still important controversy, but in general it is accepted that in as much as there has not been found an animal that reacts to the medicaments exactly "like man," it is prudent to include in the studies rodents and non-rodents. Litchfield in his toxicological studies on rats and dogs, concluded that when these two species are used, one can predict the toxicity that a substance will have in humans with a certainty that exceeds 75%. Other authors often recommend mice, rats, rabbits and dogs. It is really exceptional that the toxicological data found about monkeys is more useful than data about dogs, mammals that are easy to keep in captivity and easy to acquire. On the other hand it is emphasized that the knowl edge gained about anatomy, physiology and histology of the dog is greater than that concerning the monkey.

Another method of dividing the different toxicity experiments with animals are the so-called (a) Ouantitative experiments: Toxicity with relationship to the magnitude of the dosage; and (b) Cualitative experiments: Toxicity with relationship to the different tolerance of tissues to the same dosage. It is ac cepted that it is more useful to use rodents for the quantitative toxicity experiments and non-rodent mam mals (dogs) for those of qualitative toxicity. In the former is determined, besides LD₅₀g (lethal dose 50). the MLD (minimum lethal dose) and LD₁₀₀ (lethal dose 100) for each one of the methods studied. In the experiments with dogs, it is generally recommended that histopathological studies of eight organs be performed (liver, kidney, lung, duodenum, heart, spleen, pancreas and brain) on the exposed dogs whether to a single dose or to several intermittent doses and to continuous infusion over various life spans, according to the substance being studied.

A very important aspect of the preclinical animal toxicity studies is that they provide the necessary information for the patients who will be exposed experimentally to an antineoplastic substance.

 

BIBLIOGRAPHICAL REFERENCES

Introduction
Studies concerning acute toxicity through intravenous amygdalin infusion on white mice reported by other laboratories, report LETHAL DOSAGE 50(LD₅₀) between 4,500 mg/kg of body weight (B.W.) and 14,600 mg/kg B.W.

This study was designed to establish the LD₅₀ of intravenous KEMDALIN.

Material and Methods
White mice were used, apparently healthy, males and females in equal proportion, weighing between 30 and 40g. and kept in common cages, with a balanced diet and purified water which was changed daily.

The symptoms, signs, appetite and behavior of the KEMDALIN exposed mice were monitored in comparison with mice not exposed to KEMDALIN. After 24 hours, the deaths were reported and the percentage of the same was calculated.

Initial l.V. dosage of KEMDALIN (AMYGDALIN) was 1,750 mg/kg B.W, increased by increments to double, according to toxicity and tolerance. Six mice for each dosage and six comparative mice were used.

Chart No. 1

·

Lethal Dose 50 by l.V. KEMDALIN in White Mice

Commentary

We consider the acute toxicity of KEMDALIN (AMYGDALIN) to be amazingly low by I.V., even in comparison to many foods, vegetable products and medicines.

According to findings in agreement with the present study, l.V. toxicity of KEMDALIN (AMYGDALIN) is nil with dosages up to 20 times greater than those usually prescribed in humans and LB₅₀in mice, corresponds to a dosage 66 times greater than those usually prescribed for humans.
 
 

Introduction
The previous toxicity studies of AMYGDALIN by intraperitoneal application In white mice have reported an LB₅₀of up to 14,600 mg/kg of body weight (B.W.). This study was designed to establish the intraperitoneal application in white mice.

Material and Methods
Apparently healthy white mice, males and females in equal proportions with an average weight of 30 to 40 g. were used. The mice were kept in communal cages during the study, and were fed a balanced and supplemented diet with purified water which was changed daily.

A solution of 300 mg/mI of KEMDALIN in distilled water, free from fever causing agents and of a proven purity of 99.5% was used. All of the solution used was from lot #004-80, manufactured on January 23, 1980, by KEMSA LABS.

KEMDALIN solution was administered in the usual form, taking the necessary precautions in order to avoid, as much as possible, puncturing the abdominal viscera during the injection.

The symptoms and signs that developed during the first 24 hours after administering the KEMDALIN were monitored. The percentage of the deaths was calculated and the LB₅₀was calculated.

The initial dosage of KEMDALIN was 833 mg/kg B.W., and doubled until signs of severe toxicity appeared. All other fractional doses necessary to obtain the LB₅₀ were included. In each dosage three male and three female mice were used.

Results
The results are summarized In Chart 2. The lethal minimum dosage was calculated to be 6,666 mg/kg B.W., even though there was one death at the initial dosage of 833 mg/kg B.W., believed to have been caused by the accidental puncturing of the intestine, with subsequent passage of beta-glucoside into the peritoneal cavity, the spreading of the AMYGDALIN and massive liberation of hydrocyanic acid.

Chart No. 1.

All other intermediate dosages between 10,000 and 26,666 mg/kg B.W. were 100% lethal.

.

Graph No. 1

Lethal Dose 50 of KEMDALIN by Peritoneal Application in White Mice

Br. Jose Ernesto Contreras Pulido, M.D. Medical Director, KEMSA LABS

Q.F.B. Martha Navarrete Del Rio

Chief of the Quality Control Dept., KEMSA LABS

Introduction
Studies on acute l.V. toxicity of AMYGDALIN in rabbits give an LB₅₀ of 3,200 mg/kg B.W. This study was designed to determine acute l.V. toxicity of KEMDALIN (100% pure Mexican AMYGDALIN) in white rabbits.

Material and Methods
Thirty-one New Zealand white rabbits were used, males and females in equal number, weighing between 2,300 and 3,300 g. Ampules with 300 mg/mI of KEMDALIN S in a sterile aqueous solution free from pyrogenic agents, from lot #004-80, manufactured January 23, 1980.

After placing the rabbits in special boxes and cleaning their ears with alcohol, the rabbits were injected using plastic, disposable 10 ml. syringes, pediatric type #25.

Temperature and any other sign or symptom that suggested toxicity were monitored. With each dosage, the day and hour of deaths was recorded.

The initial dosage was 300 mg/kg B.W. and was doubled until the lethal dosage 100 was reached. Intermediate dosages were then administered to find the LB₅₀.

The recorded symptoms from the minimum toxic dosage were adipsia, anorexia and apathy, which disappeared 48 hours after discontinuing KEMDALIN. To the lethal dosage, a typical tableau of cyanide poisoning was added, showing cyanosis, generalized convulsions, coma and respiratory arrest.

The LB₅₀ is illustrated in Graph 3.

Chart No. 1

Acute Toxicity of Intravenous KEMDALIN in Rabbits

Studies Sponsored by the McNaughton Foundation of California

Summary
Neither acute, sub-acute nor chronic toxicity of AMYGDALIN (D-MANDELONITRILE-BETA GLUCOSIDE-6-BETA-B.GLUCOSIBE) was found in oral, intramuscular, intraperitoneal and intravenous applications in rats, mice, rabbits and dogs, in dosages 100 times greater than those used in humans. Antigenicity was not observed in guinea pigs. Toxicity from oral application was 39 to 44 times greater than parenteral in the various species. Parenteral toxicity was considered to be comparable to that of glucose. Cytotoxicity in animals is duplicated with the previous administration of beta-glucosidase AMYGDALIN. That is the way the hydrolysis of AMYGDALIN in vivo' by the beta-glucosidase. In the animals that survived dosages of AMYGDALIN up to 3,000 mg/kg B.W. by oral, intraperitoneal, in tramuscular and intravenous applications, alterations of ingestion, corporal weight, chemistry, blood, urinalysis and histopathological study by autopsy were not found.

Subacute Toxicity by Intravenous Application in Dogs
In accordance with this study, "It was not possible to cause toxic manifestations' by AMYGDALIN in the dogs that received intravenous dosages of 150, 300, and from 300 to 1,000 mg/kg B.W., five to seven times per week, for eight weeks. Twenty-two dogs between seven and nine months of age and weighing between 7.3 and 12.7 kg were included in the study.

Antigenicity of AMYGBALIN in Guinea Pigs
They proceeded to sensitize guinea pigs with subcutaneous dosages of 100 mg/kg B.W., for a period of five consecutive days. The animals were allowed to rest for 21 days and afterward they were given an equal dosage of AMYGDALIN. There were no reactions of hypersensitivity observed in any of the animals, while in the control animals,, the experiment with egg albumin did indeed trigger a typical anaphylactic reaction.

Changes in the Toxicity of AMYGBALIN in Mice by the Previous Administration of Beta-Glucosidase
To mice with an LB₅₀for AMYGDALIN of 14,600 mg/kg R.W., was administered intravenously a non-toxic dosage of beta-glucosidase. Thirty minutes later, they received an intraperitoneal injection of AMYGDALIN and showed that the LB₅₀diminished to 7,700 mg/kg B.W. In the histopathological studies, it was also shown that tissue toxicity was practically duplicated by the administration of beta glucosidase (an enzyme that hydrolizes AMYGDALIN, causing the freeing of hydrocyanic acid). It was concluded that the cytotoxicity of AMYGDALIN at the cell and tissue level, is proportional to (a) the concentration of beta-glucosidase in cells or tissue and (b) the concentration of AMYGDALIN in the previously mentioned cells or tissue.

Teratogenicity of AMYGDALIN in Rats
AMYGDALIN was administered subcutaneously in dosages of 30 and 300 mg/kg B.W., and orally in dosages of five and 25 mg/kg B.W., from the first to nineteenth day of pregnancy. At birth, the viscera and skeleton (previous coloring with alizarin) of the progeny were studied in great detail. They found no abnormalities in the length, weight and mortality of the newly born. They showed no visceral or skeletal alterations that would indicate teratogenicity. One fetus from the control group had renal deformities. Also one fetus from the group that received five mg/kg B.W., orally and two of those that received five mg/kg B.W., orally had renal abnormalities. One fetus from the group that received five mg/kg B.W., orally, showed hydrocephalus. All these sporadic abnormalities were not considered to be related to the AMYGDALIN, because they are within the normal frequency rate in rats.

Chronic Toxicity of AMYGDALIN in Dogs
Three groups of dogs were included that received each one of the following dosages of AMYGDALIN: 15 mg/kg B.W. intravenously, five days per week; 15 mg/kg B.W. intravenously, two days per week; and 7.5 mg/kg B.W. orally, seven days per week. The study lasted six months. Mortality, vital signs, daily ingestion and weekly weight were monitored. At the conclusion of the study, hemograms, urnianlysis and blood tests were taken. The animals were killed, the viscera weighed and histopathological studies per formed. In none of the dogs could any changes attributable to AMYGDALIN toxicity be found.

Tolerance of AMYGDALIN and Acute Toxicity by Oral Application in Dogs
It was determined that the minimm lethal dosage (MLD) is from 2,000 to 2,500 mg/kg of AMYGDALIN per kilogram of corporal weight.

Determination of the LB₅₀ of AMYGDALIN in Mice

AMYGDALIN was administered intravenously in progressive dosages to mice with the purpose of calculating the LB₅₀ of AMYGDALIN in this specie. It was concluded that with 75% certainty, one can affirm that the LB₅₀ of AMYGDALIN given intravenously to mice is 4,500 mg/kg B.W. with margins between 4,300 and 4,600 mg/kg B.W.

ACUTE AND SUBACUTE TOXICITY OF KEMDALIN
(100% PURE, MEXICAN AMYGDALIN) GIVEN INTRAVENOUSLY IN DOGS
Br. Jose Erensto Contreras Pulido, M.D.
Medical Director of KEMSA LABS

M.V.Z. Luis Cota Alvarez
Medical Veterinarian and Zootechnologist of KEMSA LABS

A study sponsored and performed by KEMSA LABS, Playas de Tijuana, B.C.N., Mexico, 1980.

Introduction

The studies concerning acute and subacute toxicity in dogs reported by various authors, have shown that dosages even of 1,000 mg/kg B.W. (body weight), five to seven times per week for eight consecutive weeks, have not caused any toxicity intravenously, and that dosages of up to 3,000 mg/kg B.W. by rapid, intravenous infusion, only caused slight diminishing of arterial tension and heartbeat, for less than 30 minutes in one dog in which through monitoring the coronary flow and the cardiac contractility, found no changes. On the other hand, autopsies performed on dogs subjected to dosages of AMYGDALIN of up to 15 mg/kg B.W. given intravenously, five days per week, for six consecutive months, showed no visceral changes.

This study was designed with the purpose of accessing the intravenous toxicity of KEMDALIN (100% pure Mexican AMYGDALIN).

Material and Methods

Four healthy dogs, kept in separate cages, with no apparent pathology upon clinical examination, fed a balanced and supplemented commercial formula and purified water which was changed daily. The four dogs were allowed free, daily exercise and their vital signs were taken routinely as well as a clinical examination in search of signs and symptoms of toxicity.

All were started on progressive dosages of KEMBALIN (AMYGDALIN) in a sterile, aqueous solution of 300 mg/kg, free from pyrogenic agents, the initial dosage being 500 mg/kg B.W., which increased by increments in order to arrive at the MLB (minimum lethal dosage).

Results
The results are reported in Chart 1, in which it is seen that even dosages of 7,500 mg/kg B.W. failed to cause the death of the dog, so MLD was not achieved. On the other hand, the MTD (minimum toxic dosage) was 1,000 mg/kg B.W., with which symptoms such as adipsia and anorexia appeared, disappearing within 24 hours after discontinuing KEMDALIN.

Conclusion
It is concluded that the tolerance of IV. KEMDALIN in dogs is extraordinarily good. This coincides with previous reports by other authors. It was also proven that accumulated dosages of up to 39.37 g/kg B.W. did not produce subacute toxicity (315g. of IV. KEMDALIN administered-over a 10-day period), since all symptoms disappeared 24 hours after discontinuing the KEMDALIN.

Material and Methods
They used four native, adult dogs which were healthy, wormed, kept in captivity, fed a balanced and supplemented commercial formula and with water ad libitum, and which were allowed daily exercise in the gardens of the laboratory.
KEMDALIN in 500 mg tablets of AMYGDALIN was utilized, which were administered to the dogs orally with an initial dosage of 7,685 mg per kg B.W. (corporal weight), until arriving at a dosage of 14,318 mg/kg B.W.

The vital signs and the signs and symptoms that might suggest toxicity were monitored and the at tempt was made to arrive at the minimum lethal dosage.
 

Conclusions
Without being able to make a comparison of the acute toxicity between oral and intravenous applications, since the minimum lethal oral dosage was not achieved in our studies, we can say that MLD of KEMDALIN (AMYGDALIN) is very superior to that reported by other authors (14,318 mg/kg B.W. vs. 2,000 to 2,500 mg/kg B.W.) with other AMYGDALINS, and that the dosage normally used in humans of 30 mg/kg B.W., orally, per day, is found to be considerably below the Minimum Lethal Dosage found in this study.

SUBACUTE TOXICITY OF KEMDALIN (100% PURE MEXICAN AMYGDALIN)
APPLIED ORALLY IN DOGS

Dr. Jose Ernesto Contreras Pulido, M.D.
Medical Director of KEMSA LABS

M.V.Z.
Luis Cota Alvarez
Medical Veterinarian and Zootechnologist of KEMSA LABS

A study sponsored and performed at KEMSA LABS, Playas de Tijuana, B.C.N., Mexico, 1980

They used tablets of KEMDALIN, 500 mg each which were given orally in progressive dosages according to tolerance, starting with an initial dosage of 100 mg/kg B.VV.

The signs and symptoms that might suggest poisoning or toxicity were recorded and the attempt was made to arrive at a dosage of 1,000 mg/kg B.W.

Results
The first four days 100 mg/kg B.W. were administered orally, reporting on the fourth day only slight apathy. The dosage was raised to 150 mg/kg B.W. per day for two days and 200 mg/kg B.W. per day for another two days. The dosage was raised to 300 mg/kg B.W. per day for another two days and there were no symptoms whatsoever. The first day that 400 mg/kg B.W. were given, the dog experienced total anorexia, adipsia, nausea, persistent vomiting, hyperthemia, apathy and a heavy shedding of hair began. The dosage was repeated the second day and after three hours, there appeared nongeneralized tonic-clonic convulsions, persistent vomiting, slavering, apathy, complete anorexia, loss of weight, adipsia, diarrhea, dehydration and ocular hyperemia. The dosage was increased to 500 mg/kg B.W. the next day and outside of a little vomiting, the rest of the signs and symptoms diminished or disappeared. Dosages of 600 mg/kg B.W. per day were given for three consecutive days, and the dog continued with only ocular hyperemia and loss of hair, but completely recovered his appetite and thirst, along with his spirit and desire to play. The dosage was increased to 800 mg/kg B.W. per day for four consecutive days and he experienced no signs of toxicity besides ocular hyperemia. The loss of hair ceased. The dosage was increased to 1000 mg/kg B.W. per day for five consecutive days and no other symptoms were added to the ocular hyperemia. The dog increased his activity in exaggerated form which was interpreted as disorder in conduct. Food ingestion was normal and the ingestion of liquids adequate. On no day were the vital signs altered appreciably.

The ocular hyperemia and hyperactivity ceased 72 hours after suspending the KEMDALIN.

Conclusions
The Minimum Toxic Dosage (MTB) of KEMDALIN (AMYGDALIN) given orally was 100 mg/kg B.W. which is some 12 times greater than that commonly given to humans, and the minimum lethal dosage (MLD) was not achieved in dosages of up to 1000 mg/kg B.W. for five consecutive days, or a total dosage of 143,000 mg in a period of 25 days: 13,172 mg/kg B.W.

We believe that the dosage of 30 to 35 mg/kg B.W. of KEMDALIN in humans can be used with an adequate margin of safety, for toxicity studies (Phase I), and antineoplastic activity (Phase II and Ill).

NOTE: After completing the study, Dr. Cota stated that to the same dog, after two weeks of rest, he administered orally 12 g. of KEMDALIN per day for seven consecutive days and the dog only experienced ocular hyperemia, apathy and diminution in the ingestion of food and water. On the eighth day he gave the dog a dosage of 15 g with which appeared generalized convulsions, cyanosis, lethargy and coma within two hours which lasted for a period of approximately eight hours. In less than three days after discontinuing the AMYGDALIN (KEMDALIN) there was complete recovery of activity and ingestion and the disappearance of the ocular hyperemia. The dog was still alive two months after the study was finalized.