[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: 40CFR798.4350]
[Page 173-177]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
Subpart E_Specific Organ/Tissue Toxicity
Sec. 798.4350 Inhalation developmental toxicity study.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics
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of an inhalable material such as a gas, volatile substance, or aerosol/
particulate, determination of the potential developmental toxicity is
important. The inhalation developmental toxicity study is designed to
provide information on the potential hazard to the unborn which may
arise from exposure of the mother during pregnancy.
(b) Definitions. (1) Developmental toxicity is the property of a
chemical that causes in utero death, structural or functional
abnormalities or growth retardation during the period of development.
(2) ``Aerodynamic diameter'' applies to the behavioral size of
particles of aerosols. It is the diameter of a sphere of unit density
which behaves aerodynamically like the particles of the test substance.
It is used to compare particles of different sizes, shapes, and
densities and to predict where in the respiratory tract such particles
may be deposited. This term is used in contrast to ``optical,''
``measured'' or ``geometric'' diameters which are representation of
actual diameters which in themselves cannot be related to deposition
within the respiratory tract.
(3) ``Geometric mean diameter'' or ``median diameter'' is the
calculated aerodynamic diameter which divides the particles of an
aerosol in half based on the weight of the particles. Fifty percent of
the particles by weight will be larger than the median diameter and 50
percent of the particles will be smaller than the median diameter. The
median diameter and its geometeric standard deviation are used to
statistically describe the particle size distribution of any aerosol
based on the weight and size of the particles.
(4) ``Inhalable diameter'' refers to that aerodynamic diameter of a
particle which is considered to be inhalable for the organism. It is
used to refer to particles which are capable of being inhaled and may be
deposited anywhere within the respiratory tract from the trachea to the
deep lung (the alveoli). For man, the inhalable diameter is considered
here as 15 micrometers or less.
(5) ``Concentration'' refers to an exposure level. Exposure is
expressed as weight or volume of test substance per volume of air (mg/
1), or as parts per million (ppm).
(6) ``No-observed-effect level'' is the maximum concentration in a
test which produces no observed adverse effects. A no-observed-effect
level is expressed in terms of weight or volume of test substance given
daily per unit volume of air.
(c) Principle of the test method. The test substance is administered
in graduated concentrations, for at least that part of the pregnancy
covering the major period of organogenesis, to several groups of
pregnant experimental animals, one exposure level being used per group.
Shortly before the expected date of delivery, the pregnant females are
sacrificed, the uteri removed, and the contents examined for embryonic
or fetal deaths, and live fetuses.
(d) Limit test. If a test at an exposure of 5 mg/1 (actual
concentration of respirable substances) or, where this is not possible
due to physical or chemical properties of the test substance, the
maximum attainable concentration, produces no observable developmental
toxicity, then a full study using three exposure levels might not be
necessary.
(e) Test procedures--(1) Animal selection--(i) Species and strain.
Testing shall be performed in at least two mamalian species. Commonly
used species include the rat, mouse, rabbit, and hamster. If other
mamalian species are used, the tester shall provide justification/
reasoning for their selection. Commonly used laboratory strains shall be
employed. The strain shall not have low fecundity and shall preferably
be characterized for its sensitivity to developmental toxins.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant female animals shall be used at each exposure
level.
(iv) Number of animals. At least 20 pregnant rats, mice, or hamsters
or 12 pregnant rabbits are required at each exposure level. The
objective is to ensure that sufficient pups are produced to permit
meaningful evaluation of the potential developmental toxicity of the
test substance.
(2) Control group. A concurrent control group shall be used. This
group shall be exposed to clean, filtered air
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under conditions identical to those used for the group exposed to the
substance of interest. In addition, a vehicle-exposed group may be
necessary when the substance under study requires a vehicle for
delivery. It is recommended that during preliminary range finding
studies, air vs. vehicle exposure be compared. If there is no
substantial difference, air exposure itself would be an appropriate
control. If vehicle and air exposure yield different results, both
vehicle and air exposed control groups are recommended.
(3) Concentration levels and concentration selection. (i) At least
three concentration levels with a control and, where appropriate, a
vehicle control, shall be used.
(ii) The vehicle shall neither be developmentally toxic nor have
effects on reproduction.
(iii) To select the appropriate concentration levels, a pilot or
trial study may be advisable. Since pregnant animals have an increased
minute ventilation as compared to non-pregnant animals, it is
recommended that the trial study be conducted in pregnant animals.
Similarly, since presumably the minute ventilation will vary with
progression of pregnancy, the animals should be exposed during the same
period of gestation as in the main study. In the trial study, the
concentration producing embryonic or fetal lethalities or maternal
toxicity should be determined.
(iv) Unless limited by the physical/chemical nature or biological
properties of the substance, the highest concentration level shall
induce some overt maternal toxicity such as reduced body weight or body
weight gain, but not more than 10 percent maternal deaths.
(v) The lowest concentration level should not produce any grossly
observable evidence of either maternal or developmental toxicity.
(vi) Ideally, the intermediate concentration level(s) shall produce
minimal observable toxic effects. If more than one intermediate
concentration is used, the concentration levels shall be spaced to
produce a gradation of toxic effects.
(4) Exposure duration. The duration of exposure shall be at least
six hours daily allowing appropriate additional time for chamber
equilibrium.
(5) Observation period. Day 0 in the test is the day on which a
vaginal plug and/or sperm are observed. The exposure period shall cover
the period of major organogenesis. This may be taken as days 6 to 15 for
rat and mouse, 6 to 14 for hamster, or 6 to 18 for rabbit.
(6) Inhalation exposure. (i)(A) The animals shall be tested in
inhalation equipment designed to sustain a minimum dynamic air flow of
12 to 15 air changes per hour and ensure an adequate oxygen content of
19 percent and an evenly distributed exposure atmosphere. Where a
chamber is used, its design should minimize crowding of the test animals
and maximize their exposure to the test substance. This is best
accomplished by individual caging. To ensure stability of a chamber
atmosphere, the total ``volume'' of the test animals shall not exceed 5
percent of the volume of the test chamber.
(B) Pregnant animals shall not be subjected to beyond the minimum
amount of stress. Since whole-body exposure appears to be the least
stressful mode of exposure, it is the method preferred. In general oro-
nasal or head-only exposure, which is sometimes used to avoid concurrent
exposure by the dermal or oral routes, is not recommended because of the
associated stress accompanying the restraining of the animals. However,
there may be specific instances where it may be more appropriate than
whole-body exposure. The tester shall provide justification/reasoning
for its selection.
(ii) A dynamic inhalation system with a suitable flow control system
shall be used. The rate of air flow shall be adjusted to ensure that
conditions throughout the exposure chamber are essentially the same.
Test material distribution should be established before animals are
committed to dosing. Maintenance of slight negative pressure inside the
chamber will prevent leakage of the test substance into the surrounding
areas.
(iii) The temperature at which the test is performed should be
maintained at 22 [deg]C (2[deg]) for rodents or 20
[deg]C (3[deg]) for rabbits. Ideally, the relative
humidity should be maintained between 40 to 60 percent, but in certain
instances
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(e.g., tests of aerosols, use of water vehicle) this may not be
practicable.
(7) Physical measurements. Measurements or monitoring should be made
of the following:
(i) The rate of airflow shall be monitored continuously but shall be
recorded at least every 30 minutes.
(ii) The actual concentration of the test substance shall be
measured in the breathing zone. During the exposure period the actual
concentrations of the test substance shall be held as constant as
practicable, monitored continously or intermittently depending on the
method of analysis and measured at least at the beginning, at an
intermediate time and at the end of the exposure period.
(iii) During the development of the generating system, particle size
analysis shall be performed to establish the stability of aerosol
concentrations with respect to particle size. During exposure, analysis
shall be conducted as often as necessary to determine the consistency of
particle size distribution.
(iv) Temperature and humidity shall be monitored continuously and be
recorded at least every 30 minutes.
(8) Food and water during exposure period. Food should be withheld
during exposure. Water may or may not be withheld. If it is not withheld
it should not come in direct contact with the test atmospheres.
(9) Observation of animals. (i) A gross examination shall be made at
least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of animals found dead and isolation or sacrifice of
weak or moribund animals).
(iii) Signs of toxicity shall be recorded as they are observed,
including the time of onset, the degree and duration.
(iv) Cage-side observations shall include, but not be limited to:
Changes in skin and fur, eye and mucous membranes, as well as
respiratory, autonomic and central nervous systems, somatomotor activity
and behavioral pattern. Particular attention should be directed to
observation of tremors, convulsions, salivation, diarrhea, lethargy,
sleep, and coma.
(v) Measurements should be made weekly of food consumption for all
animals in the study.
(vi) Animals shall be weighed at least weekly.
(vii) Females showing signs of abortion or premature delivery shall
be sacrificed and subjected to a thorough macroscopic examination.
(10) Gross necropsy. (i) At the time of sacrifice or death during
the study, the dam shall be examined macroscopically for any structural
abnormalities or pathological changes which may have influenced the
pregnancy.
(ii) Immediately after sacrifice or death, the uterus shall be
removed, weighed, and the contents examined for embryonic or fetal
deaths and the number of viable fetuses. Gravid uterine weights should
not be obtained from dead animals if autolysis or where decomposition
has occurred. The degree of resorption shall be described in order to
help estimate the relative time of death.
(iii) The number of corpora lutea shall be determined for all
species except mice.
(iv) The sex of the fetuses shall be determined and they shall be
weighed individually, the weights recorded, and the mean fetal weight
derived.
(v) Following removal, each fetus shall be examined externally.
(vi) For rats, mice and hamsters, one-third to one-half of each
litter shall be prepared and examined for skeletal anomalies, and the
remaining part of each litter shall be prepared and examined for soft
tissue anomalies using appropriate methods.
(vii) For rabbits, each fetus shall be examined by careful
dissection for visceral anomalies and then examined for skeletal
anomalies.
(f) Data and reporting--(1) Treatment of results. Data shall be
summarized in tabular form, showing for each test group: the number of
animals at the start of the test, the number of pregnant animals, the
number and percentages of live fetuses and the number of fetuses with
any soft tissue or skeletal abnormalities.
(2) Evaluation of results. The findings of a developmental toxicity
study shall
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be evaluated in terms of the observed effects and the exposure levels
producing effects. It is necessary to consider the historical
developmental toxicity data on the species/strain tested. A properly
conducted developmental toxicity study should provide a satisfactory
estimation of a no-effect level.
(3) Test report. In addition to the reporting requirements as
specified under 40 CFR part 792, subpart J, the following specific
information shall be reported:
(i) Test conditions. (A) Description of exposure apparatus including
design, type, dimensions, source of air, system for generating
particulates and aerosols, methods of conditioning air, and the method
of housing the animals in a test chamber when this apparatus is used.
(B) The equipment for measuring temperature, humidity, and
particulate aerosol concentrations and size shall be described.
(ii) Exposure data. These shall be tabulated and presented with mean
values and a measure of variability (e.g., standard deviation) and shall
include:
(A) Airflow rates through the inhalation equipment.
(B) Temperature of air.
(C) Nominal concentration--total amount of test substance fed into
the inhalation equipment divided by volume of air (no standard
deviation).
(D) Measured total concentrations (particulate and/or gaseous
phases) in test breathing zone.
(E) Particle size distribution (e.g., median aerodynamic diameter of
particles with geometric standard deviation) including estimates of the
percents of inhalable and non-inhalable portions for the test animals.
(iii) Animal data. (A) Toxic response data by concentration.
(B) Species and strain.
(C) Date of death during the study or whether animals survived to
termination.
(D) Date of onset and duration of each abnormal sign and its
subsequent course.
(E) Feed, body weight and uterine weight data.
(F) Pregnancy and litter data.
(G) Fetal data (live/dead, sex, soft tissue and sketetal defects,
resorptions).
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Department of Health and Welfare. The Testing of Chemicals for
Carcinogenicity, Mutagenicity and Teratogenicity. Minister of Health and
Welfare (Canada: Department of Health and Welfare, 1975).
(2) National Academy of Sciences. ``Principles and Procedures for
Evaluating the Toxicity of Household Substances.'' A report prepared by
the Committee for the Revision of NAS Publication 1138, under the
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(3) World Health Organization. Principles for the Testing of Drugs
for Teratogenicity. WHO Technical Report Series No. 364. (Geneva: World
Health Organization, 1967).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19076, May 20, 1987;
52 FR 26150, July 13, 1987; 54 FR 21064, May 16, 1989]