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RANDOMIZATION OF ANIMALS
Computer-generated randomization tables were used for assigning the dose and housing groups (copies of these tables were obtained and are attached as Exhibit #6). Each housing group consisted of 30 animals (12 controls, 6 low, 6 medium, and 6 high dose). Each animal was assigned a letter to designate the housing group (A through M), a cage number (1 through 30), a letter to indicate dose group (C,L,M & H), and a letter to indicate sex (M or F). For example, animal A30CM would be a control male, in housing group A, occupying cage number 30 (Exhibit #69).
Each rack (30 animals) contained a random distribution of control and treated animals. An example of a typical housing group is shown in the diagram attached as Exhibit #7.
The specific problems of feeding animals housed in the above manner were discussed in the report generated by the task force investigation of Aspartame in l975/l976. We will reiterate them here:
Housing experimental animals in this manner (controls, low, medium, & high dose animals randomly distributed on the same rack) would greatly increase the chance of administering the wrong diet to the animals. The chance of error was compounded by the method used to feed the animals which was as follows: At the specified intervals, the animals were weighed, and the empty food jars were removed, weighed and new food jars placed in the cages. The new (filled) food jars were placed on a mobile cart in rows corresponding to dose group (See Exhibit #8). The cart was wheeled to the Intec Unit and placed up against it with the rows of high dose jars farthest away from the operator. The operator started from the upper left corner of the housing rack, (See Exhibit #7), removed the mylar card from the cage and inserted it into the Intec Unit. This printed out the animal's identification number. A color coded card for dose level, also bearing the animal number, remained on the cage. The technician then opened the cage, removed the animal, placed it on the scale pan, pushed the button to register the weight and returned the animal to its cage. He then removed the empty food jar, placed it on the scale and pushed the button to record the empty feeder weight. The empty jar was placed on another mobile cart provided for that purpose. The new (filled) jar was selected from the appropriate row according to dose level (Exhibit #8), placed on the scale, weight recorded, and the jaw placed in the cage. The Card was then removed from the Unit and replaced on the cage.
This procedure was repeated for all cages proceeding from left to right. It is important to note that none of the food jars were identified in any manner, as to animal number or dose level. The position of the jar on the cart was the only means of identifying the proper dose level.
This procedure was used when concentrations were changed. At other times, the feeder jars were weighed; filled with the appropriate diet/dose, weighed and replaced in the cage. If a feed container was almost empty, or contained feces, it would be replaced with a new container.
IDENTIFICATION OF ANIMALS
No ear clips or other methods of uniquely identifying each animal were used. Animals were individually housed (one animal per cage) and a color coded card containing the cage number, compound number, project number (Path-Tox No.), and dose level was attached to each cage. When an animal died during the study, the color coded identification card was removed from the cage and accompanied the animal to the necropsy laboratory.
Also attached to each cage was a Mylar Card which identified the animals for the Computer System.
At the time of death or sacrifice the animals were assigned a pathology number which was used to identify the animal during tissue processing, and on all records pertaining to pathology. The pathology number was a five-digit sequential number assigned by the pathology department. An example of a pathology number is 94.893. The number was etched on all slides as a permanent identification.
All animals that died during the study were assigned an additional number called a "Master Number". The master numbers denote the chronological order in which the animals died during the study, by dose group. For example, CM38 would be the 38th Control Male to die during the study. Master numbers were noted only on the gross pathology sheets and not recorded for all animals. Four animals that died during the study had no master numbers. We were not able to locate any other records providing the missing numbers. Also, two of the master numbers (CM28 and CM29) were out of sequence. A chart was made by the FDA team, which shows the animal number (cage #), pathology number, master number, and the complete pathology history of each animal. The chart is organized by dose group, and is attached as Exhibit #35. |
