§799.1560. Diethylene glycol butyl ether and diethylene glycol butyl ether acetate.

(2) Cage-side observations shall include, but not be limited to, changes in skin and fur; eyes and mucous membranes; respiratory, circulatory autonomic, and central nervous systems; somatomotor activity; and behavior pattern. In addition a daily examination for hematuria shall be done.

(3) Certain hematology determinations shall be carried out at least three times during the test period: Just prior to initiation of dosing (baseline data), after approximately 30 days on test, and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leucocyte count, mean corpuscular volume, and a platelet count.

(4) Urinalyses shall be done at least three times during the test period: Just prior to initiation of dosing (baseline data), after approximately 30 days into the test, and just prior to terminal sacrifice at the end of the test period. The animals shall be kept in metabolism cages, and the urine shall be examined microscopically for the presence of erythrocytes and renal tubular cells, in addition to measurement of urine volume, specific gravity, glucose, protein/albumin, and blood.

(5) The liver, kidney, adrenals, brain, gonads, prostate gland, epididymides, seminal vesicles, and pituitary gland shall be weighed wet, as soon as possible after dissection, to avoid drying.

(6) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs—which should 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; brain—including 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; gonads; uterus; oviducts; vagina; vas deferens; accessory genital organs (epididymis, 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; (eyes); (femur—including articular surface); (spinal cord at three levels—cervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands).

(7) (i) Full histopathology on normal and treated skin and on organs and tissues listed in paragraph (c)(1)(i)(B)(6) of this section, as well as the accessory genital organs (epididymides, prostate, seminal vesicles) and the vagina, of all animals in the control and high dose groups.

(ii) The integrity of the various cell stages of spermatogenesis shall be determined, with particular attention directed toward achieving optimal quality in the fixation and embedding; preparations of testicular and associated reproductive organ samples for histology should follow the recommendations of Lamb and Chapin (1985) under paragraph (d)(1) of this section, or an equivalent procedure. Histological analyses shall include evaluations of the spermatogenic cycle, i.e., the presence and integrity of the 14 cell stages. These evaluations should follow the guidance provided by Clermont and Perey (1957) under paragraph (d)(2) of this section. Information shall also be provided regarding the nature and level of lesions observed in control animals for comparative purposes.

(iii) Data on female cyclicity shall be obtained by performing vaginal cytology over the last 2 weeks of dosing; the cell staging technique of Sadleir (1978) and the vaginal smear method in Hafez (1970) under paragraphs (d) (3) and (7) of this section or equivalent methods should be used. Data should be provided on whether the animal is cycling and the cycle length.

(iv) The ovary shall be serially sectioned with a sufficient number of sections examined to adequately detail oocyte and follicular morphology. The methods of Mattison and Thorgiersson (1979) and Pederson and Peters (1968) under paragraphs (d) (4) and (5) of this section may provide guidance. The strategy for sectioning and evaluation is left to the discretion of the investigator, but shall be described in detail in the study plan and final report. The nature and background level of lesions in control tissue shall also be noted.

(2) For the purpose of this section, the following provisions also apply:

(i) Definition. Neurotoxicity is any adverse acute and/or lasting effect on the structure or function of the central and/or peripheral nervous system related to exposure to a chemical substance.

(ii) Lower doses. The data from the lower doses shall show either graded dose-dependent effects in at least two of all the doses tested including the highest dose, or no neurotoxic (behavioral) effects at any dose tested.

(iii) Duration and frequency of exposure. Animals shall be exposed for 6 hours/day, 5 days/week for a 90-day period.

(iv) Sensory function. A simple assessment of sensory function (vision, audition, pain perception) shall be made. Marshall et al. (1971) in § 798.6050(f)(8) of this chapter have described a neurologic exam for this purpose; these procedures are also discussed by Deuel (1977), under § 798.6050(f)(4) of this chapter. Irwin (1968) under § 798.6050(f)(7) of this chapter described a number of reflex tests intended to detect gross sensory deficits. Many procedures have been developed for assessing pain perception (e.g., Ankier (1974) under § 798.6050(f)(1); D'Amour and Smith (1941) under § 798.6050(f)(3); and Evans (1971) under § 798.6050(f)(6) of this chapter.

(2) For the purpose of this section, the following provisions also apply:

(i) Principle of the test method. The test substance is administered to several groups of experimental animals, one dose being used per group. Measurements of motor activity are made. Where possible, the exposure levels at which significant changes in motor activity are produced are compared to those levels which produce toxic effects not originating in the central and/or peripheral nervous system.

(ii) Positive control data. Positive control data are required to document the sensitivity of the activity measuring device and testing procedure. These data should demonstrate the ability to detect increases or decreases in activity and to generate a dose-effect curve or its equivalent using three values of the dose or equivalent independent variable. A single administration of the dose (or equivalent) is sufficient. It is recommended that chemical exposure be used to collect positive control data. Positive control data shall be collected at the time of the test study unless the laboratory can demonstrate the adequacy of historical data for this purpose.

(iii) Lower doses. The data from the lower doses shall show either graded dose-dependent effects in at least two of all the doses tested including the highest dose, or no neurotoxic (behavioral) effects at any dose tested.

(iv) Duration and frequency of exposure. Animals shall be exposed for 6 hours/day, 5 days/week for a 90-day period.

(v) General. Motor activity shall be monitored by an automated activity recording apparatus. The device used shall be capable of detecting both increases and decreases in activity, i.e. baseline activity as measured by the device shall not be so low as to preclude decreases nor so high as to preclude increases. Each device shall be tested by a standard procedure to ensure, to the extent possible, reliability of operation across devices and across days for any one device. In addition, treatment groups shall be balanced across devices. Each animal shall be tested individually. The test session shall be long enough for motor activity to approach asymptotic levels by the last 20 percent of the session for most treatments and for the session control animals. All sessions should be of the same duration. Treatment groups shall be counter-balanced across test times. Effort should be made to ensure that variations in the test conditions are minimal and are not systematically related to treatment. Among the variables which can affect motor activity are sound level, size and shape of the test cage, temperature, relative humidity, lighting conditions, odors, use of home cage or novel test cage, and environmental distractions. Tests shall be executed by an appropriately trained individual.

(vi) Subchronic. All animals shall be tested prior to initiation of exposure and at 30 ±4, 60 ±4, and 90 ±4 days during the exposure period. Testing shall occur prior to the daily exposure. Animals shall be weighed on each test day and at least once weekly during the exposure period.

(2) For the purpose of this section, the following provisions also apply:

(i) Lower doses. The data from the lower doses shall show either graded dose-dependent effects in at least two of all the doses tested including the highest dose, or no neurotoxic (behavioral) effects at any dose tested.

(ii) Duration and frequency of exposure. Animals shall be exposed for 6 hours/day, 5 days/week for a 90-day period.

(iii) Clearing and embedding. After dehydration, tissue specimens shall be cleared with xylene and embedded in paraffin or paraplast except for the sural nerve which should be embedded in plastic. Multiple tissue specimens (e.g. brain, cord, ganglia) may be embedded together in one single block for sectioning. All tissue blocks shall be labeled to provide unequivocal identification. A method for plastic embedding is described by Spencer et al. in paragraph (d)(6) of this section.

(iv) Special stains. Based on the results of the general staining, selected sites and cellular components shall be further evaluated by the use of specific techniques. If hematoxylin and eosin screening does not provide such information, a battery of stains shall be used to assess the following components in all appropriate required samples: Neuronal body (e.g., Einarson's gallocyanin), axon (e.g., Bodian), myelin sheath (e.g., Kluver's Luxol Fast Blue), and neurofibrils (e.g., Bielchosky). In addition, peripheral nerve fiber teasing may be used. Detailed staining methodology is available in standard histotechnological manuals such as Armed Forces Institute of Pathology (AFIP) (1968) under § 798.6400(f)(1), Ralis et al. (1973) under § 798.6400(f)(5), and Chang (1979) under § 798.6400(f)(2) of this chapter. The nerve fiber teasing technique is discussed in Spencer and Schaumberg (1980) under § 798.6400(f)(6) of this chapter. A section of normal tissue shall be included in each staining to assure that adequate staining has occurred. Any changes shall be noted and representative photographs shall be taken. If a lesion(s) is observed, the special techniques shall be repeated in the next lower treatment group until no further lesion is detectable.

(2) Observation of animals—Urinary and fecal excretion. The quantities of 14C excreted in urine and feces by rats dosed as specified in paragraph (b)(2)(iv) of § 795.225 shall be determined at 8, 24, 48, 72, and 96 hours after dosing, and if necessary, daily thereafter until at least 90 percent of the dose has been excreted or until 7 days after dosing (whichever occurs first). Four animals per sex per dose group shall be used for this purpose.