A ten-day acute toxicity study was conducted using twenty male cynomolgous monkeys ranging in weight from 25 to 40 kg. These animals were divided into four groups of five monkeys each. Each of the four groups received a daily intravenous dose of Borazidine, delivered at a concentration of either 100, 250, 500, or 10,000 mg/kg/day.
Within the high-dose group (10,000 mg/kg/day) effects were immediate and catastrophic, resulting in death within 20 mins of dosing for all but one of the five animals. Animals 93445 and 93557, pre-death, exhibited vomiting and disorientation. These two animals almost immediately entered a catatonic state and were sacrificed moribund. Animals 93001 and 93458 exhibited vomiting, anxiety, disorientation, and digging at their abdomens. These animals also quickly entered a catatonic state and were sacrificed moribund.
Only one animal within this high-dose group, animal 93990, a diminutive 26 kg male, appeared unaffected.
All of the animals that had succumbed were removed from the enclosure and necropsied. Cause of death was seen, in all cases, to be renal failure.
No effects were seen on Day 1 in any of the three lower-dose groups (i.e., 100, 250, or 500 mg/kg/day).
On Day 2, after the second round of dosing, animals in the 500 mg/kg/day group began to exhibit vomiting, and, in some cases, aggressive behavior. This aggressive behavior most often consisted of a directed shrieking, with or without feigned biting. Some animals in the two lowest-dose groups (100 and 250 mg/kg/day) were observed to vomit, and one in the 250 mg/kg/day group (animal 93002) appeared to exhibit self-scratching behaviors similar to those seen earlier in the high-dose group (i.e., probing and scratching at abdomen, with limited writhing).
By the end of Day 3, three of five animals in the 500 mg/kg/day group had entered a catatonic state and the other two animals in this dose group were exhibiting extreme writhing punctuated with attempted biting and pinching of their fellows, often with shrieking. Some hair loss, ranging from slight to extreme, was observed, as was some "playing" with the resulting hair bundles. This "playing" behavior ranged from mild to quite energetic. This "playing" behavior was adjudged to be typical of the type of "play" such an animal might initiate with a smaller animal such as a rodent, i.e., out of a curiosity impulse, i.e., may have been indicative of hallucinogenic effects. Several animals were observed to repeatedly grimace at the hair bundles, as if trying to elicit a fear behavior from the hair bundles. Animal 93110 of the 500 mg/kg/day group was observed to sit in one corner of the cage gazing at its own vomit while an unaffected animal (93222) appeared to attempt to rouse the interest of 93110 via backpatting, followed by vigorous backpatting. Interestingly, the sole remaining high-dose animal (93990, the diminutive male), even after the second day's dosage, still showed no symptoms. Even though this animal was the smallest in weight within the highest-dose group, it showed no symptoms. It showed no vomiting, disinterest, self-scratching, anxiety, or aggression. Also no hair loss was observed. Although no hair bundles were present (because no hair loss occurred), this animal was not seen to "play" with inanimate objects present in the enclosure, such as its food bowl or stool or bits of rope, etc. This animal, rather, was seen only to stare fixedly at the handlers through the bars of the cage and/or to retreat rapidly when the handlers entered the enclosure with the long poking sticks to check under certain items (chairs, recreational tire) for hair bundles and or deposits of runny stool.
By the middle of Day 3, all of the animals in the 500 mg/kg/day group had succumbed. Pre-death, these showed, in addition to the effects noted above, symptoms ranging from whimpering to performing a rolling dementia-type motion on the cage floor, sometimes accompanied by shrieking or frothing. After succumbing, all five animals were removed from the enclosure and necropsied. Renal failure was seen to be the cause of death in all cases. Interestingly, these animals did not enter a catatonic state pre-death, but instead appeared to be quite alert, manifesting labored breathing and, in some cases, bursts of energetic rope-climbing. Coordination was adjudged to be adversely affected, based on the higher-than-normal frequency of falls from the rope. Post-fall reactions ranged from no reaction to frustration reactions, with or without self-punishment behaviors (i.e., self-hitting, self-hair-pulling, rapid shakes of head).
Toward the end of Day 3, all animals in the two lowest dose groups (250 and 100 mg/kg/day) were observed to be in some form of distress. Some of these had lapsed into a catatonic state; some refused to take food; many had runny brightly colored stools; some sat eating their stool while intermittently shrieking.
Animals 93852, 93881, and 93777, of the 250 mg/kg/day group, in the last hours before death, appeared to experience a brief period of invigoration and renewed activity, exhibiting symptoms of anxiety, as well as lurching, confusion, and scratching at the eyes with the fingers. These animals were seen to repeatedly walk or run into the cage bars, after which they would become agitated. Blindness or partial blindness was indicated. When brightly colored flags were waved in front of these animals, some failed to respond, while others responded by flinging stool at the handlers.
By noon on Day 4, all of the animals in the 250 mg/kg/day group had succumbed, been removed from the enclosure, and necropsied. In every case the cause of death was seen to be renal failure.
By the end of Day 4, only the five 100 mg/kg/day animals remained, along with the aforementioned very resilient diminutive male in the highest dose group (93990), who continued to manifest no symptoms whatsoever. This animal continued to show no vomiting, retching, nausea, disorientation, loss of motor skills, or any of the other symptoms described above. This animal continued to move about the enclosure normally and ingest normal amounts of food and water and in fact was seen to have experienced a slight weight gain and climbed the rope repeatedly with good authority.
On Day 5, animal 93444 of the 100 mg/kg/day group was observed to have entered the moribund state. Because of its greatly weakened condition, this animal was not redosed in the morning. Instead, it was removed from the enclosure, sacrificed moribund, and necropsied. Renal failure was seen to be the cause of death. Animal 93887 (100 mg/kg/day group) was seen to repeatedly keel over on one side while wincing. This animal succumbed at 1300 hrs on Day 5, was removed from the enclosure, and necropsied. Renal failure was seen to be the cause of death. Between 1500 hrs on Day 5 and 2000 hrs on Day 5, animals 93254 and 93006 of the 100 mg/kg/day dose group succumbed in rapid succession while huddled in the NW cornet of the large enclosure. Both animals exhibited wheezing and rapid clutching and release of the genitals. These two animals were removed from the enclosure and necropsied. In both cases the cause of death was seen to be renal failure.
This left only animal 93555 of the 100 mg/kg/day dose group and animal 93990, the diminutive male of the highest dose group. 93555 exhibited nearly all of the aforementioned symptoms, along with, toward the end of Day 5, several episodes during which it inflicted scratches and contusions on its own neck and face by attempting to spasmodically reach for something beyond the enclosure. This animal also manifested several episodes of quick spinning. Several of these quick-spinning episodes culminated in sudden hard falling. In two cases, the sudden hard fall was seen to result in tooth loss. In one of the cases of tooth loss, the animal was seen to exhibit the suite of aggressive behaviors earlier exhibited toward the hair bundles. In addition, in this case, the animal, after a prolonged period of snarling at its tooth, was observed to attack and ingest its own tooth. It was judged that, if these behaviors continued into Day 6, for humanitarian reasons, the animal would be sacrificed, but just after 2300 hrs, the animal discontinued these behaviors and only sat listlessly in its own stool with occasional writhing and therefore was not sacrificed due to this improvement in its condition.
By 1200 hrs of Day 5, the diminutive male 93990 still exhibited no symptoms. He was observed to be sitting in the SE corner of the enclosure, staring fixedly at the cage door. This condition was at first mistaken to be indicative of early catatonia but when a metal pole was inserted and a poke attempted, the animal responded by lurching away with shrieking, which was judged normal. It was also noted that 93990 occasionally seemed to be staring at and/or gesturing to the low-dose enclosure, i.e., the enclosure in which 93555 was still sitting listlessly in its own stool occasionally writhing. By the end of Day 5, 93990 still manifested no symptoms and in fact was observed to heartily eat the proffered food and weighing at midday Day 6 confirmed further weight gain. Also it climbed the rope. Also at times it seemed to implore. This imploring was judged to be, possibly, a mild hallucinogenic effect. This imploring resulted in involuntary laughter on the part of the handlers, which resulted in the animal discontinuing the imploring behavior and retreating to the NW corner where it sat for quite some time with its back to the handlers. It was decided that, in the future, handlers would refrain from laughing at the imploring, so as to be able to obtain a more objective idea of the duration of the (unimpeded) imploring.
Following dosing on the morning of Day 6, the last remaining low-dose animal (93555), the animal that earlier had attacked and ingested its own tooth, then sat for quite some time writhing in its own stool listlessly, succumbed, after an episode that included, in addition to many of the aforementioned symptoms, tearing at its own eyes and flesh and, finally, quiet heaving breathing while squatting. This animal, following a limited episode of eyes rolling back in its head, entered the moribund state, succumbed, and was necropsied. Cause of death was seen to be renal failure. As 93555 was removed from the enclosure, 93990 was seen to sit quietly, then retreat to the rear of the enclosure, that is, the portion of the enclosure farthest from the door, where it squatted on its haunches. Soon it was observed to rise and move toward its food bowl and eat heartily while continuing to look at the door.
Following dosing on Day 7, animal 93990, now the sole remaining animal, continued to show no symptoms and ate and drank vigorously.
Following dosing on Day 8, likewise, this animal continued to show no symptoms and ate and drank vigorously.
On Day 9, it was decided to test the effects of extremely high doses of Borazadine by doubling the dosage, to 20,000 mg/kg/day. This increased dosage was administered intravenously on the morning of Day 9. No acute effects were seen. The animal continued to move around its cage and ear and drink normally. It was observed to continue to stare at the door of the cage and occasionally at the other, now-empty, enclosures. Also the rope-climbing did not decrease. A brief episode of imploring was observed. No laughter on the part of the handlers occurred, and the unimpeded imploring was seen to continue for approximately 130 seconds. When, post-imploring, the stick was inserted to attempt a poke, the stick was yanked away by 93990. When a handler attempted to enter the cage to retrieve the poking stick, the handler was poked. Following this incident, the conclusion was reached to attempt no further retrievals of the poking stick, but rather to obtain a back-up poking stick available from Supply. As Supply did not at this time have a back-up poking stick, it was decided to attempt no further poking until the first poking stick could be retrieved. When it was determined that retrieving the first poking stick would be problematic, it was judged beneficial that the first poking stick was now in the possession of 93990, as observations could be made as to how 93990 was using and/or manipulating the poking stick, i.e., effect of Borazadine on motor skills.
On Day 10, on what was to have been the last day of the study, upon the observation that animal 93990 still exhibited no effects whatsoever, the decision was reached to increase the dosage to 100,000 mg/kg/day, a dosage 10 times greater than that which had proved almost immediately lethal to every other animal in the highest-dose group. This was adjudged to be scientifically defensible. This dosage was delivered at 0300 hrs on Day 10. Remarkably, no acute effects were seen other than those associated with injection (i.e., small, bright purple blisters at the injection site, coupled with elevated heart rate and extreme perspiration and limited panic gesturing) but these soon subsided and were judged to be related to the high rate of injection rather than to the Borazadine itself.
Throughout Day 10, animal 93990 continued to show no symptoms. It ate and drank normally. It moved energetically about the cage. It climbed the rope. By the end of the study period, i.e., midnight of Day 10, no symptoms whatsoever had been observed. Remarkably, the animal leapt about the cage. The animal wielded the poking stick with good dexterity, occasionally implored, shrieked energetically at the handlers. In summary, even at a dosage 10 times that which had proved almost immediately fatal to larger, heavier animals, 93990 showed no symptoms whatsoever. In all ways, even at this exceptionally high dosage, this animal appeared to be normal, healthy, unaffected, and thriving.
At approximately 0100 hrs of Day 11, 93990 was tranquilized via dart, removed from the enclosure, sacrificed, and necropsied.
No evidence of renal damage was observed. No negative effects of any kind were observed. A net weight gain of 3 kg since the beginning of the study was observed.
All carcasses were transported off-site by a certified medical waste hauler and disposed of via incineration.