The manufacturer
(2) Recovery group. The manufacturer shall include a group of 20 animals (10 animals per sex) in the test population, exposing them to the highest concentration level for the entire length of the study's exposure period. This group shall then be observed for reversibility, persistence, or delayed occurrence of toxic effects during a post-exposure period of not less than 28 days. (3) Inhalation exposure. (i) All data developed within this study shall be in accordance with good laboratory practice provisions under 79.60. (ii) The general conduct of this study shall be in accordance with the vehicle emissions inhalation exposure guideline in 79.61. (4) Observation of animals. (i) All toxicological (e.g., weight loss) and neurological signs (e.g., motor disturbance) shall be recorded frequently enough to observe any abnormality, and not less than weekly for all study animals. Animals shall be weighed weekly. (ii) The following is a minimal list of measures that shall be noted: (A) Body weight; (B) Subject's reactivity to general stimuli such as removal from the cage or handling; (C) Description, incidence, and severity of any convulsions, tremors, or abnormal motor movements in the home cage; (D) Descriptions and incidence of posture and gait abnormalities observed in the home cage; (E) Description and incidence of any unusual or abnormal behaviors, excessive or repetitive actions (stereotypies), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose, or mouth, and any other observations that may facilitate interpretation of the data. (iii) Any animal which dies during the test is necropsied as soon as possible after discovery. (5) Clinical examinations. (i) The following examinations shall be performed on the twenty animals designated as the 90-day study population, exclusive of pregnant dams and those study animals targeted for perfusion by gravity: (A) The following hematology determinations shall be carried out at least two times during the test period (after 30 days of exposure and just prior to terminal sacrifice at the end of the exposure period): hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as prothrombin time, thromboplastin time, or platelet count. (B) Clinical biochemistry determinations on blood shall be carried out at least two times during the test period, after 30 days of exposure and just prior to terminal sacrifice at the end of the exposure period, on all groups of animals including concurrent controls. Clinical biochemical testing shall include assessment of electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance. In the absence of more specific tests, the following determinations may be made: calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum alanine aminotransferase, serum aspartate aminotransferase, sorbitol dehydrogenase, gamma glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, methemoglobin, bile acids, total bilirubin, and total serum protein measurements. Additional clinical biochemistry shall be employed, where necessary, to extend the investigation of observed effects, e.g., analyses of lipids, hormones, acid/base balance, and cholinesterase activity. (ii) The following examinations shall initially be performed on the high concentration and control groups only: (A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to exposure to the test substance and at the termination of the study. If changes in the eyes are detected, all animals shall be examined. (B) Urinalysis is not required on a routine basis, but shall be done when there is an indication based on expected and/or observed toxicity. (iii) Preservation by whole-body perfusion of fixative into the anaesthetized animal for lung histology of ten animals from the 90-day study population for each experimental and control group. (6) Gross pathology. With the exception of the whole body perfusion-fixed test animals cited in paragraph (d)(1)(ii)(A) of this section, all rodents shall be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices and the cranial, thoracic, and abdominal cavities and their contents. Gross pathology shall be performed on the following organs and tissues: (i) The liver, kidneys, lungs, adrenals, brain, and gonads, including uterus, ovaries, testes, epididymides, seminal vesicles (with coagulating glands), and prostate, constitute the group of target organs for histology and shall be weighed as soon as possible after dissection to avoid drying. In addition, for other than rodent test species, the thyroid with parathyroids, when present, shall also be weighed as soon as possible after dissection to avoid drying. (ii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungswhich shall be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brainincluding sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; reproductive organs: uterus; cervix; ovaries; vagina; testes; epididymides; prostate; and, if present, seminal vesicles; aorta; (skin); gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph node; (mammary gland); (thigh musculature); peripheral nerve/tissue; (eyes); (femurincluding articular surface); (spinal cord at three levelscervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands). (7) Histopathology. Histopathology shall be performed on the following organs and tissues from all rodents: (i) All gross lesions. (ii) Respiratory tract and other organs and tissues, listed in paragraph (d)(6)(ii) of this section (except organs/tissues in parentheses), of all animals in the control and high dose groups. (iii) The tissues mentioned in parentheses, listed in paragraph (d)(6)(ii) of this section, if indicated by signs of toxicity or target organ involvement. (iv) Lungs of animals in the low and intermediate dose groups shall also be subjected to histopathological examination, primarily for evidence of infection since this provides a convenient assessment of the state of health of the animals. (v) Lungs and trachea of the whole-body perfusion-fixed test animals cited in paragraph (d)(1)(ii)(A) of this section are examined for inhaled particle distribution.