National Toxicology Program

Mice: Male and/or female Hemizygous Tg.AC; 7-9 weeks of age (resting or telogen stage of the hair cycle and approximately 20 - 25 g body weight)

Husbandry: Males should be individually caged, females may be singly or group caged (4 -5/cage); when group caged, siblings held together since weaning should be caged together; cages may be shoebox with hardwood chip bedding or wire bottom cages; randomize cages to treatment groups; 12 hour light:dark cycle; tatoo or microchip identification; Ralston Purina mouse diet #20 is used at the NIEHS.

Route: Primarily by topical application; 2 - 5 times/wk in a volume up to 200 µL. Dorsal hair is clipped (electric clippers e.g., Oster Finisher, #59-03H) as needed between the suprascapular area and the base of the tail to expose a skin application site of approximately 2 x 4 cm.

Dose Selection: Doses are selected to approximate the ones used in the cancer bioassay. If no bioassay studies have been done, dose range finding studies must be conducted to establish the highest concentration that produces only minimal responses in the skin when applied topically, e.g., erythema, epidermal hyperplasia, etc. without inducing overt toxic effects such as erosion and ulceration. Doses between studies are usually calculated on g/Kg body weight basis for comparison. Depending on the chemical, dosing frequency may be adjusted for daily, 1, 2, 3, etc. times a week applications. Both positive and negative (vehicle) controls should be included in each study.

Duration of Treatment: Up to 26 weeks treatment followed by 6 weeks of observation.

Study Design: Typically,

^a 1.25-1.5 µg TPA per mouse per application

Measurements: Weekly Body Weights/Clinical Observations; mapping of papilloma sites optional; record time to first tumor (latency), # mice bearing papillomas and # of papillomas at site of application:

Necropsy/Pathology: Currently, in most Tg.AC studies only skin, site of application, is examined grossly and histopathologically. To begin to establish a historical pathology database, some studies will begin to follow the same procedures for necropsy and histopathology as used by the NTP in their subchronic studies. (NTP Statement of Work, revised 1994). In addition to a complete gross necropsy, select tissues, i.e., liver, thymus, right kidney, right testicle, heart, lung and spleen are weighed. A complete histopathologic evaluation inclusive of treatment-related gross lesions in all early death animals regardless of dose group, all control animals and all animals in the highest dose groups with at least 60% survivors at the time of sacrifice plus all animals in higher dose groups. Chemical-related lesions (target organs) are identified and these organs plus gross lesions are examined to a no-effect level.