United States       Prevention, Pesticides      EPA712-C-96-206
          Environmental Protection    and Toxic Substances      June 1996
          Agency         (7101)
&EPA    Health Effects Test
           OPPTS 870.3600
           Developmental Toxicity
                 'Public Draft"

     This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.

     The Office of Prevention,  Pesticides and Toxic Substances (OPPTS)
has  developed this guideline through  a  process of harmonization that
blended the testing  guidance and requirements that existed in the Office
of Pollution Prevention and Toxics  (OPPT) and appeared in Title 40,
Chapter I,  Subchapter R of the Code of Federal Regulations  (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical  Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development

     The purpose of harmonizing these guidelines into a single set of
OPPTS  guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic  Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide,  Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).

     Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that  need to  be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access  Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public    Docket    at    (703)    305-5805    or     by    e-mail:

     To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency,  401  M  St.  SW.,  Washington, DC 20460. In  person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted  electronically by  sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.

     Final  Guideline Release: This guideline is available  from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin  Board.   By  modem   dial   202-512-1387,   telnet   and  ftp:
fedbbs.access.gpo.gov (IP,  or  call 202-512-0132 for disks
or paper copies.  This  guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access  Gopher
(gopher.epa.gov) under the heading  "Environmental Test Methods and

OPPTS 870.3600   Inhalation developmental toxicity study.
     (a) Scope—(1) Applicability. This guideline is intended to meet test-
ing  requirements   of both  the  Federal  Insecticide,  Fungicide,  and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).

     (2) Background. The source material used in developing this har-
monized OPPTS test guideline is  40  CFR 798.4350 Inhalation Devel-
opmental Toxicity Study.

     (b) Purpose. In the assessment and evaluation of the toxic character-
istics of an inhalable material such as a gas, volatile substance, or aerosol/
particulate, determination of the potential developmental toxicity is impor-
tant. 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.

     (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP)  apply to this  test
guideline. The following definitions also apply to this test guideline.

     Aerodynamic diameter applies to  the behavioral size of particles of
aerosols. It is the diameter of a sphere of unit density which behaves aero-
dynamically 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 relat-
ed to deposition within the respiratory tract.

     Concentration  refers to an exposure  level.  Exposure is expressed as
weight or volume of test substance per volume of air (milligrams per liter),
or as parts per million (ppm).

     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.

     Geometric mean diameter or median diameter is the calculated aero-
dynamic 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 par-

     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 any-
where within the respiratory tract from the trachea to the  deep lung  (the
alveoli). For man, the inhalable diameter is considered here as  15 (im or

    No-observed-effect level (NOEL) is the maximum concentration  in a
test which produces no observed adverse  effects. A NOEL is  expressed
in terms of weight or volume of test substance given daily per unit volume
of air.

    (d) 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 experi-
mental 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.

    (e) 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 con-
centration, produces  no observable  developmental  toxicity, then  a  full
study using three exposure levels might not be necessary.

    (f)  Test  procedures—(1) Animal selection—(i) Species and strain.
Testing  should 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 should provide justification/reasoning for their
selection.  Commonly used laboratory strains  should be  employed.  The
strain should not have low fecundity and should preferably be  character-
ized for its sensitivity to developmental toxins.

    (ii) Age.  Young adult animals (nulliparous females) should be used.

    (iii) Sex. Pregnant female animals should be used at each exposure

    (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 evalua-
tion of the potential developmental toxicity of the test substance.

    (2) Control group. A concurrent control group should be  used. This
group should be exposed to clean, filtered air  under conditions identical
to those used for the group exposed to  the substance of interest. In addi-
tion, a vehicle-exposed group may be necessary when the substance under
study requires a vehicle for delivery. It is  recommended that during  pre-
liminary range finding studies, air vs. vehicle  exposure be compared. If
there is no substantial difference, air exposure itself would be  an appro-

priate control. If vehicle and air exposure yield different results, both vehi-
cle 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, should be used.

     (ii) The  vehicle should neither be developmentally toxic nor have ef-
fects 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 presum-
ably 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 prop-
erties of the substance, the highest concentration level should 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)  should produce
minimal observable toxic effects. If more than one intermediate concentra-
tion is used, the concentration levels should be spaced to produce a  grada-
tion of toxic effects.

     (4) Exposure duration. The duration of exposure should be at least
six hours  daily allowing  appropriate additional time  for chamber equi-

     (5) Observation period. Day 0 in the test is the day on which a
vaginal plug  and/or sperm are observed. The exposure period should 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 should be tested in inha-
lation 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 maxi-
mize 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 should not exceed 5  percent of the volume
of the test chamber.

     (B) Pregnant animals should not be subjected to beyond the minimum
amount of stress. Since whole-body exposure appears to be the least stress-
ful 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 should provide justification/reasoning for its selection.

     (ii) A  dynamic inhalation system with a suitable flow  control system
should be used. The  rate of air flow should 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  main-
tained at 22+2 °C for rodents  or 20+3 °C for  rabbits. Ideally, the relative
humidity should be maintained  between 40 to 60 percent, but in certain
instances (e.g. tests  of aerosols, use of water vehicle) this may not  be

     (7) Physical measurements.  Measurements or monitoring should be
made of the following:

     (i) The rate of airflow should be monitored continuously but should
be recorded at least every 30 minutes.

     (ii) The actual concentration of the test substance should be measured
in the breathing zone. During the exposure period the actual concentrations
of the test substance should 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 should be performed to establish the stability of aerosol concentra-
tions  with  respect to particle  size. During  exposure, analysis should  be
conducted as often  as necessary to determine the  consistency of particle
size distribution.

     (iv) Temperature and humidity should be  monitored continuously and
be recorded at least every 30 minutes.

     (8) Food and water during exposure period. Food should be with-
held 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 should 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 should be recorded as they are observed, includ-
ing the time of onset, the degree and duration.

     (iv) Cage-side observations  should include, but not be  limited to:
Changes in skin and fur, eye  and mucous membranes, as well as res-
piratory, 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 should be weighed at least weekly.

     (vii) Females  showing signs of abortion or premature delivery should
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  should be examined macroscopically for any structural ab-
normalities or pathological changes which may have influenced  the preg-

     (ii) Immediately after  sacrifice or  death,  the uterus  should  be re-
moved,  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 oc-
curred. The degree  of resorption should  be described in order to  help esti-
mate the relative time of death.

     (iii) The number of corpora lutea should be determined for all species
except mice.

     (iv) The sex  of the fetuses  should be determined and they  should
be weighed individually, the weights recorded, and the mean fetal  weight

     (v) Following removal,  each fetus should be examined externally.

     (vi) For rats,  mice and hamsters, one-third to  one-half of each  litter
should be prepared and examined for skeletal anomalies, and the remaining
part of each litter should be  prepared and examined for soft tissue anoma-
lies using appropriate methods.

     (vii) For rabbits, each fetus should be examined by careful dissection
for visceral anomalies and then examined for skeletal anomalies.

     (g) Data and reporting—(1) Treatment of results. Data should 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 should be evaluated in terms of the observed effects and the expo-
sure levels producing effects. It is necessary to consider the historical de-
velopmental  toxicity data on the species/strain tested. A properly con-
ducted developmental  toxicity  study should provide a satisfactory esti-
mation 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
should 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 particu-
late aerosol concentrations and size should be described.

     (ii) Exposure  data. These should  be tabulated and  presented with
mean values and a measure of variability  (e.g.  standard  deviation) and
should 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 per-
cents 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

     (D) Date of onset and duration of each abnormal sign and its subse-
quent 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,

     (h) References. The following references should be consulted for ad-
ditional background information on this test guideline.

     (1) Department of Health and Welfare. The Testing of Chemicals for
Carcinogenicity, Mutagenicity and Teratogenicity. Minister of Health and
Welfare. Department of Health and Welfare, Canada (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 aus-
pices of the Committee  on Toxicology, National Research Council, Na-
tional Academy of Sciences, Washington, DC (1977).

     (3) World Health Organization.  Principles for  the Testing of Drugs
for Teratogenicity. WHO Technical Report Series No. 364. World Health
Organization, Geneva (1967).