[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR799.1560]
[Page 231-236]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE
TESTING REQUIREMENTS--Table of Contents
Subpart B_Specific Chemical Test Rules
Sec. 799.1560 Diethylene glycol butyl ether and diethylene glycol butyl
ether acetate.
(a) Identification of test substances. (1) Diethylene glycol butyl
ether (DGBE), CAS Number 112-34-5, and diethylene glycol butyl ether
acetate (DGBA), CAS Number 124-17-4, shall be tested in accordance with
this section.
(2) DGBE of at least 95 percent purity and DGBA of at least 95
percent purity shall be used as the test substances.
(b) Persons required to submit study plans, conduct tests, and
submit data. All persons who manufacture (including import) or process
or intend to manufacture or process DGBE and/or DGBA, other than as an
impurity, after April 11, 1988, to the end of the reimbursement period
shall submit letters of intent to conduct testing, submit study plans
and conduct tests, and submit data, or submit exemption applications as
specified in this section, subpart A of this part, and parts 790 and 792
of this chapter for single-phase rulemaking. Persons who manufacture or
process DGBE are subject to the requirements to test DGBE in this
section. Only persons who manufacture or process DGBA are subject to the
requirements to test DGBA in this section.
(c) Health effects testing--(1) Subchronic toxicity--(i) Required
testing. (A) A 90-day subchronic toxicity test of DGBE shall be
conducted in rats by dermal application in accordance with Sec.
798.2250 of this chapter except for the provisions in paragraphs
(e)(9)(iv), (10)(i)(A) and (ii)(B), (11) (ii) and (iii), and (12)(i) of
Sec. 798.2250.
(B) For the purpose of this section, the following provisions also
apply:
(1) A satellite group to evaluate fertility shall be established.
Control males shall be cohabited with control females, and males and
females administered the high dose shall be cohabited. Endpoints to be
evaluated shall include percent mated; percent pregnant; length of
gestation; litter size; viability at birth, on Day 4, and weaning, on
Day 21; sex of the offspring; and litter weights at birth and Days 4, 7,
14, and 21. Litters shall be standardized on day 4 in accordance with
the reproductive and fertility effects guideline, Sec.
798.4700(c)(6)(iv) of this chapter. Gross examinations shall be made at
least once each day and physical or behavioral anomalies in the dam or
offspring shall be recorded. At weaning, dams shall be sacrificed and
examined for resorption sites indicative of post-implantation loss. An
additional 20 males and 40 females will have to be added to the
subchronic study for this test. If the animals in the high dose group
exhibit marked toxicity (e.g. greater than 20 percent
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weight loss), then the fertility tests shall be conducted in the next
highest dose group.
(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
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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.
(ii) Reporting requirements. (A) The subchronic test shall be
completed and the final report submitted to EPA within 15 months of the
effective date of the final test rule.
(B) Progress reports shall be submitted to EPA every 6 months,
beginning 6 months from the effective date of the final rule until
submission of the final report to EPA.
(2) Neurotoxicity/behavioral effects--(i) Required testing--(A) (1)
Functional observational battery. A functional observational battery
shall be performed in the rat by dermal application of DGBE for a period
of 90 days according to Sec. 798.6050 of this chapter except for the
provisions in paragraphs (b)(1), (d)(4)(ii), (5), and (8)(ii)(E) of
Sec. 798.6050.
(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 Sec. 798.6050(f)(8) of this chapter have described a
neurologic exam for this purpose; these procedures are also discussed by
Deuel (1977), under Sec. 798.6050(f)(4) of this chapter. Irwin (1968)
under Sec. 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
Sec. 798.6050(f)(1); D'Amour and Smith (1941) under Sec.
798.6050(f)(3); and Evans (1971) under Sec. 798.6050(f)(6) of this
chapter.
(B)(1) Motor activity. A motor activity test shall be conducted in
the rat by dermal application of DGBE for a period of 90 days according
to Sec. 798.6200 of this chapter except for the provisions in
paragraphs (c), (d)(3)(ii), (4)(ii), (5), (8)(i), and (iii) of Sec.
798.6200.
(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
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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.
(C)(1) Neuropathology. A neuropathology test shall be conducted in
the rat by dermal application of DGBE for a period of 90 days according
to Sec. 798.6400 of this chapter except for the provisions in
paragraphs (d)(4)(ii), (5), (8)(iv)(C), and (E)(2) of Sec. 798.6400.
(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 Sec.
798.6400(f)(1), Ralis et al. (1973) under Sec. 798.6400(f)(5), and
Chang (1979) under Sec. 798.6400(f)(2) of this chapter. The nerve fiber
teasing technique is discussed in Spencer and Schaumberg (1980) under
Sec. 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.
(ii) Reporting requirements. (A) The neurotoxicity/behavioral tests
required under paragraph (c)(2) of this section shall be completed and
the final reports submitted to EPA within 17 months of the effective
date of the final rule.
(B) Interim progress reports shall be submitted to EPA at 6-month
intervals, beginning 6 months from the effective date of the final rule
until submission of the applicable final report to EPA.
(3) Developmental neurotoxicity--(i) Required testing. A
developmental neurotoxicity test of DGBE shall be
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conducted after a public program review of the Tier I data from the
functional observational battery, motor activity, and neuropathology
tests in paragraph (c)(2) of this section, and the reproductive tests in
paragraph (c)(1) of this section, and if EPA issues a Federal Register
notice or sends a certified letter to the test sponsor specifying that
the testing shall be initiated. The test shall be performed in rats in
accordance with Sec. 795.250 of this chapter.
(ii) Reporting requirements. (A) The developmental neurotoxicity
test shall be completed and the final report submitted to EPA within 15
months of EPA's notification of the test sponsor by certified letter or
Federal Register notice under paragraph (c)(3)(i) of this section that
the testing shall be initiated.
(B) Progress reports shall be submitted to EPA every 6 months,
beginning 6 months after the date of notification that the testing shall
be initiated, until submission of the final report to EPA.
(4) Pharmacokinetics--(i) Required testing. (A) Pharmacokinetics
testing of DGBE and DGBA will be conducted in rats by the dermal route
of administration in accordance with Sec. 795.225 of this chapter,
except for the provisions in paragraphs (b) (1)(ii) and (3)(i) of Sec.
795.225.
(B) For the purpose of this section, the following provisions also
apply:
(1) Animals. Adult male and female Sprague Dawley rats shall be
used. The rats shall be 7 to 8 weeks old and weigh 180 to 220 grams.
Prior to testing, the animals shall be selected at random for each
group. Animals showing signs of ill health shall not be used.
(2) Observation of animals--Urinary and fecal excretion. The
quantities of \14\C excreted in urine and feces by rats dosed as
specified in paragraph (b)(2)(iv) of Sec. 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.
(ii) Reporting requirements. (A) The pharmacokinetics tests shall be
completed and the final reports submitted to EPA within 8 months of the
effective date of the final amendment.
(B) A progress report shall be submitted to EPA 6 months from the
effective date of the final amendment.
(d) References. For additional background information the following
references should be consulted:
(1) Lamb, J.C. and Chapin, R.E. ``Experimental models of male
reproductive toxicology.'' In: ``Endocrine Toxicology.'' Thomas, J.A.,
Korach, K.S., and McLachlan, J.A., eds. New York, NY: Raven Press. pp.
85-115. (1985).
(2) Clermont, Y. and Perey, B. ``Quantitative study of the cell
population of the seminiferous tubules in immature rats.'' American
Journal of Anatomy. 100:241-267. (1957).
(3) Sadleir, R.M.F.S. ``Cycles and seasons.'' In: ``Reproduction in
Mammals: I. Germ Cells and Fertilization.'' Austin, C.R. and Short,
R.V., eds. New York, NY: Cambridge Press. Chapter 4. (1978).
(4) Mattison, D.R. and Thorgiersson, S.S. ``Ovarian aryl hydrocarbon
hydroxylase activity and primordial oocyte toxicity of polycyclic
aromatic hydrocarbons in mice.'' Cancer Research. 39:3471-3475. (1979).
(5) Pederson, T. and Peters, H. ``Proposal for classification of
oocytes and follicles in the mouse ovary. Journal of Reproduction and
Fertility. 17:555-557. (1968).
(6) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H.
``Neuropathological methods for the detection of neurotoxic disease.''
In: ``Experimental and Clinical Neurotoxicology.'' Spencer, P.S. and
Schaumburg, H.H., eds. Baltimore, MD: Williams & Wilkins, pp. 743-757.
(1980).
(7) Hafez, E.S., ed., ``Reproduction and Breeding Techniques for
Laboratory Animals.'' Chapter 10. Philadelphia: Lea & Febiger (1970).
(e) Effective date. (1) The effective date of the final rule is
April 11, 1988, except for paragraph (c)(2)(ii)(A) of this section. The
effective date for paragraph (c)(2)(ii)(A) of this section is March 1,
1990. The effective date for paragraphs (c)(4)(ii)(A) and (c)(4)(ii)(B)
of this section is November 27, 1989.
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(2) The guidelines and other test methods cited in this rule are
referenced as they exist on the effective date of the final rule.
[53 FR 5950, Feb. 26, 1988, as amended at 54 FR 27357, June 29, 1989; 54
FR 41835, Oct. 12, 1989; 55 FR 7326, Mar. 1, 1990; 58 FR 34205, June 23,
1993]