United States       Prevention, Pesticides     EPA712-C-96-201
          Environmental Protection    and Toxic Substances     June 1996
          Agency        (7101)
&EPA   Health Effects Test
          OPPTS 870.3200
          Repeated Dose Dermal
          Toxicity—21/28 Days
                "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 870.3200  Repeated dose dermal toxicity—21/28 days.
     (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 are  OPP 82-2 21-Day Dermal (Pesticide
Assessment Guidelines, Subdivision F—Hazard Evaluation; Human and
Domestic Animals) EPA report 540/09-82-025,  1982; and OECD  guide-
line 410 Repeated Dose Dermal Toxicity: 21-28-Day.

     (b) Purpose.  A 21/28-day repeated dose dermal study will provide
information on possible health hazards likely to arise from repeated dermal
exposure to a test substance for a period  of 21/28 days. This  study is
not capable of  determining those effects that have a long latency  period
for development (e.g.,  carcinogenicity and life shortening). Extrapolation
from the results of this study to humans is valid only to  a limited degree.
It can, however, provide useful information on the degree of percutaneous
absorption, target organs, the possibilities of accumulation, and can be of
use in selecting dose levels for  longer-term  studies and for establishing
safety criteria for human exposure.

     (c) Definitions. The definitions in section 3 of the Toxic Substance
Control Act (TSCA) and the definitions in 40 CFR Part  792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline.

     Cumulative toxicity is  the adverse effect of repeated doses occurring
as a result of prolonged action or increased concentration of the adminis-
tered test substance or its metabolites in susceptible tissues.

     Dose in  a 21/28-day  repeated dose dermal study is the amount of
test substance applied daily to the skin for 21/28 days. Dose is expressed
as weight of the test substance (grams, milligrams), or  as weight  of  the
test  substance  per  unit body weight of test animal (milligrams per kilo-
gram), or as weight of test substance per unit of surface area (milligrams
per square centimeter)

     No-observed-effect level (NOEL) is the maximum dose used in a study
which produces no adverse effects. The NOEL level is expressed in terms
of the weight of a test substance given daily per unit weight of test animal
(milligrams per kilogram per day).

     The term repeated dose dermal study is used to embrace  the toxic
effects associated  with repeated  doses of a chemical  over part of a  life
span of the test animal. Dosing periods lying between a single dose and
10 percent of life span are often referred to as subacute. The term subacute
is  semantically incorrect. To distinguish such a dosing period from  the

classical subchronic  period, it may be  described as short-term repeated
dose study. Study durations have been  14, 21, or 28 days.

     Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance.

     (d) Limit  test.  If a test at one  dose level of at least 1,000 mg/kg
body weight (expected human exposure may indicate the need for a higher
dose level), using the procedures  described for this guideline, produces
no observable toxic effects, or if toxic  effects would  not be expected based
upon data on structurally related compounds, a full study using three dose
levels might not be necessary.

     (e) Test procedures—(1) Animal selection—(i) Species and strain.
A mammalian species should be used for testing. The rat, rabbit, or guinea
pig may be used. Commonly used laboratory  strains should be employed.
If other mammalian  species are  used, the tester should provide justifica-
tion/reasoning for their selection. When a subchronic dermal study is con-
ducted as a preliminary to a chronic  study, the same  species and strain
should be used in both studies.

     (ii) Age. (A) Testing  should be  started with young healthy  animals
as soon as possible after weaning and acclimatization.

     (B) Dosing should generally begin no later than  8 weeks of age.

     (C) At the commencement of the study,  the weight variation of ani-
mals used  should not exceed + 20 percent of the mean weight for each

     (iii) Sex. Equal numbers  of animals of each sex with healthy skin
should be used  at each dose level.  The females should be nulliparous and
nonpregnant except for specially designed studies.

     (iv) Numbers. (A) At least 20 animals (10 animals per sex) should
be used at each  dose level.

     (B) If interim sacrifices are planned, the  number should be increased
by  the number of animals scheduled  to be sacrificed before completion
of the study.

     (C) To avoid bias, the use of adequate randomization procedures for
the  proper allocation of animals to test and control groups is required.

     (D) Each animal must be assigned a unique identification number.
Dead animals, their preserved  organs  and tissues, and microscopic slides
should be identified by reference to an  animal's unique number.

     (v) Husbandry.  (A) Animals should be housed in individual cages.

     (B) The temperature of the  experimental animal rooms should be at
22 + 3 °C for rodents and 20 + 3 °C for rabbits.

     (C) The relative humidity of the experimental animal rooms should
be 30 to 70 percent.

     (D) Where lighting is artificial, the sequence should be  12 h light/
12 h dark.

     (E) Control and test animals should be fed from the same batch and
lot. The feed should be analyzed  to assure adequacy of nutritional require-
ments  of the  species tested  and for impurities that might influence the
outcome of the test. Animals should be fed and watered ad libitum with
food replaced at least  weekly. For nonrodents feeding should be at least
daily and water ad libitum.

     (F) The study should not be  initiated until animals have been allowed
a period of acclimatization/quarantine to  environmental conditions, nor
should animals from outside sources be placed on test without an adequate
period of quarantine.

     (2) Control  and test  substances, (i) Where  necessary, the test sub-
stance is dissolved or suspended  in a suitable vehicle. If a vehicle or dilu-
ent is needed, the vehicle  should not elicit toxic effects or substantially
alter the chemical or toxicological properties of the test substance.

     (ii) One lot of the  test substance should be used if possible, through-
out the duration of the study, and the research sample should be stored
under conditions that maintain its purity and stability.  Prior to the initiation
of the study, there  should  be a characterization of the test substance, in-
cluding the  purity of the test compound, the amount of contaminants and
impurities, and their identity, if possible.

     (iii) If the test or control substance is to be incorporated into feed
or another  vehicle, the period during which the  test substance is  stable
in such a mixture should be determined prior to the initiation of the study.
Its homogeneity and concentration should be determined prior to the  initi-
ation of the study and periodically  during the study.  Statistically random-
ized samples of the mixture should  be analyzed to ensure that proper mix-
ing,  formulation,  and storage procedures are being followed, and that the
appropriate  concentration of the  test or control substance is contained in
the mixture.

     (3)  Control groups.  A  concurrent control group  is required.  This
group should be an untreated or sham-treated control group or, if a vehicle
is used in the application  of the test  substance,  a vehicle control group.
If the toxic  properties of the vehicle are not known or not available, both
untreated/sham-treated and vehicle control groups are required.

     (4) Satellite group. A satellite group of 20 animals (10 animals per
sex) may be treated with the high dose level for 21/28 days and observed
for reversibility, persistence, or delayed occurrence of toxic effects for a
post-treatment period of 14 days. In addition a control group of 20 animals
(10 animals of each sex) should be added to the satellite study.

     (5) Dose levels and  dose selection, (i) For the repeated dose study,
it  is  desirable to determine  a dose-response relationship  as  well as a
NOEL. Therefore, at least three dose levels plus a control and, where ap-
propriate,  a vehicle control (corresponding to the concentration of vehicle
at the highest dose level) group  should be used. Doses should be spaced
appropriately to produce  test groups with a range of toxic effects. The
data should be sufficient to produce a dose-response curve.

     (ii) The  highest  dose  level should result  in toxic  effects but not
produce severe skin irritation or an incidence of fatality which would pre-
vent a meaningful evaluation. If application of the  test substance produces
severe skin irritation, the concentration may be reduced, although this may
result in a  reduction in, or absence of, other toxic  effects at the high dose
level. If the skin has been badly damaged early in  the study, it may be
necessary to terminate the study and undertake a  new one  at lower con-

     (iii) The intermediate dose levels should be spaced to produce a gra-
dation of toxic effects.

     (iv) The lowest dose level should  not produce any evidence of toxic

     (6) Preparation of animal skin. Fur should be  clipped from not less
than 10 percent of the body surface area shortly before testing for applica-
tion of the test  substance. In order to  dose approximately  10  percent of
the body surface, the area starting at the  scapulae  (shoulders) to the wing
of the ileum (hipbone) and half way down the flank on each side of the
animal should be shaved. Shaving should be carried out approximately
24  h before the test. Repeated clipping  or shaving  is usually needed  at
approximately weekly intervals.  When clipping or shaving the fur, care
should be  taken to  avoid abrading the skin (which could alter its per-

     (7) Preparation of test substance, (i) Liquid  test substances are gen-
erally used undiluted, except as indicated in paragraph (e)(5)(ii) of this

     (ii) Solids should be pulverized when possible. The substance should
be moistened sufficiently with water or, when necessary, a suitable vehicle
to ensure good contact with the skin. When a vehicle is used, the influence
of the vehicle on toxicity of, and penetration of the skin by, the test sub-
stance should be taken into account.

     (iii) The volume of application should be kept constant, e.g. less than
300 (j,L for the rat; different concentrations  of test solution should be pre-
pared for different dose levels.

     (8) Administration of test  substance, (i)  The duration of exposure
should be at least for 21/28 days.

     (ii) The animals should be treated with test substance for at  least 6
h/day on a 7-day per week basis.  However, based on practical  consider-
ations, application on a 5-day per week basis is  acceptable. Dosing should
be conducted at approximately the same time each day.

     (iii) The test substance should be applied uniformly over the treatment

     (iv) The surface area covered may be less for highly toxic substances.
As much  of the area should be covered with as thin  and uniform a film
as possible.

     (v) During the exposure period,  the test substance should be held in
contact with the  skin with a porous  gauze  dressing. The  test site  should
be further covered with nonirritating tape to retain the gauze dressing and
the  test substance and to  ensure that the animals cannot  ingest the test
substance. Restrainers may be used  to prevent the ingestion of the test
substance, but complete immobilization is not recommended.

     (9) Observation period. The animals should be observed for a period
of 21/28 days. Animals in the satellite group, (if one  is used) scheduled
for follow-up observations should be kept for at  least 14 days further with-
out treatment to assess reversibility.

     (10)  Observation of animals, (i) Observations  should be made at
least  once each  day  for  morbidity  and mortality.  Appropriate actions
should be taken to minimize loss of animals to the study (e.g.,  necropsy
or refrigeration of those animals found dead and isolation or  sacrifice of
weak or moribund animals).

     (ii) A careful clinical examination should be made  at least once week-
ly. Observations  should  be detailed  and carefully recorded, preferably
using explicitly defined scales. Observations should include,  but  not be
limited to, evaluation of skin and fur, eyes and mucous membranes,  res-
piratory  and  circulatory  effects,  autonomic effects  such as salivation,
central nervous system effects, including tremors and convulsions, changes
in the level of motor activity,  gait and posture, reactivity  to handling or
sensory stimuli, grip  strength,  and stereotypies or bizarre  behavior (e.g.,
self-mutilation, walking backwards).

     (iii) Signs of toxicity should be recorded as they are observed includ-
ing the time of onset, degree, and duration.

     (iv) Individual weights of animals should be determined shortly be-
fore the test substance  is administered, weekly thereafter, and at death.

     (v) Food consumption should also be determined weekly if abnormal
body weight changes are observed.

     (vi) Moribund animals should be removed and sacrificed when no-
ticed. The time of death  should be recorded as precisely as possible.

     (vii) At termination, all survivors in  the treatment groups should be

     (11) Clinical pathology. Hematology and clinical chemistry examina-
tions should  be  made on all animals,  including controls, of each sex in
each group at the end of the study.

     (i) Hematology. The recommended parameters are: Hemoglobin and
hematocrit concentrations, red  blood cell count,  white blood cell count,
differential leukocyte count, platelet count, and a measure of clotting po-
tential such as prothrombin time or thromboplastin time.

     (ii)  Clinical chemistry.  Parameters which are considered  appropriate
to all studies are 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 and signs of clinical
toxicity. Suggested blood clinical chemistry determinations:

     (A) Electrolytes.

     (7) Calcium.

     (2) Chloride.

     (3) Magnesium.

     (4) Phosphorus.

     (5) Potassium.

     (6) Sodium.

     (B) Enzymes.

     (7) Alkaline phosphatase.

     (2) Alanine aminotransferase.

     (3) Aspartate aminotransferase.

     (4) Gamma glutamyl transferase.

     (5) Sorbitol dehydrogenase.

     (C) Other.

     (7) Albumin.

     (2) Blood creatinine.

     (3) Blood urea nitrogen.

     (4) Globulin.

     (5) Glucose (fasting) .

     (6) Total bilirubin.

     (7) Total cholesterol.

     (8) Total serum protein.

Other  determinations which  may  be  necessary  for  an  adequate  toxi-
cological evaluation include  analyses of lipids, hormones, acid/base bal-
ance, methemoglobin and cholinesterase activity. Additional clinical bio-
chemistry may be employed where necessary to extend the  investigation
of observed effects.

     (iii) Urinalysis is not recommended on a routine basis, but only  when
there is an indication based on expected  OR observed toxicity.

     (12) Optional immunotoxicity screen. In partial fulfillment of re-
quirements for an immunotoxicity screen, subpopulations of splenic or pe-
ripheral blood lymphocytes in the rodents should be enumerated and  quan-
tified. Total T-, Total B-, Total T-helper, T-suppressor/cytotoxic and Natu-
ral Killer (NK) cell populations should be determined on at least 10 ro-
dents of each sex in each group at the end of 21/28 days.

     (13)  Ophthalmological  examination.  Ophthalmological examina-
tions using an  ophthalmoscope  or an equivalent device should be  made
on all animals prior to the administration of  the test substance  and on
all high  dose and control  groups at termination. If changes  in the  eyes
are detected, all animals in the other dose groups  should be examined.

     (14) Gross necropsy, (i) All animals should 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

     (ii)  The liver,  lungs, brain,  kidneys,  spleen,  thymus,  and  gonads
should be trimmed and weighed wet, as soon as possible after dissection.

     (iii) The following organs and tissues, or representative samples there-
of,   should  be  preserved  in a suitable  medium  for  possible  future
histopathological examination:

(A) Digestive system.
(7) Salivary glands.
(2) Esophagus.
(3) Stomach.
(4) Duodenum.
(5) Jejunum.
(6) Ileum.
(7) Cecum.
(8) Colon.
(9} Rectum.
(10} Liver.
(11} Pancreas.
(12} Gall bladder (dogs).
(B) Nervous system.
(1} Brain (multiple sections).
(2) Pituitary.
(3} Peripheral nerve(s).
(4) Spinal cord (three levels).
(5) Eyes (retina,  optic nerve).
(C) Glandular system.
(1} Adrenals.
(2) Parathyroids.
(3} Thyroids.
(D) Respiratory system.
(1} Trachea.
(2} Lung.
(3} Pharynx.
(4) Larynx.

     (5) Nose.
     (E) Cardiovascular/Hematopoieitic system.
     (7) Aorta (thoracic).
     (2) Heart.
     (3) Bone marrow.
     (4) Lymph nodes.
     (5) Spleen.
     (6) Thymus.
     (F) Urogenital system.
     (7) Kidneys.
     (2) Urinary bladder.
     (3) Prostate.
     (4) Testes.
     (5) Epididymides.
     (6) Seminal vesicles.
     (7) Uterus.
     (8) Ovaries.
     (G) Others.
     (7) All gross lesions and masses.
     (2) Skin.
     (3) Sternum and/or femur
     (15) Histopathology. (i) The following histopathology should be per-
     (A) Full histopathology on the organs and tissues, listed under para-
graph (e)(14)(iii)  of this guideline, of all animals in the control and high
dose groups.
     (B) All gross lesions in all animals.
     (C) Target organs in all animals.
     (D) Lungs, liver and kidneys of all animals. Special attention to exam-
ination of the lungs of rodents  should be made for evidence of infection

as this provides a convenient assessment of the state of health of the ani-

     (ii) If excessive early deaths or other problems occur in the high dose
group, compromising the  significance  of the  data, the next dose level
should be examined for complete histopathology.

     (iii) An attempt should be made to correlate gross observations with
microscopic findings.

     (iv) Tissues and organs designed for microscopic examination  should
be fixed in  10 percent buffered  formalin or a recognized suitable fixative
as soon as necropsy is performed and no less than 48 hours prior to trim-
ming. Tissues should be trimmed to a maximum thickness  of 0.4 cm for

     (v)  When a satellite  group is used,  histopathology should be  per-
formed on tissues and organs identified as  showing toxic  effects  in the
treated groups.

     (f) Data and reporting—(1) Treatment of results, (i) Data  should
be summarized in tabular form,  showing for each test group—number of
animals at the  start  of the test,  the  number of animals  showing lesions,
the types of lesions and the percentage of animals displaying each  type
of lesion.

     (ii) All observed results, (quantitative and qualitative) should be eval-
uated by an appropriate statistical method. Any generally accepted statis-
tical method should be used; the statistical methods including  significance
criteria should be selected during the design of the study.

     (2)  Evaluation of study results.  The  findings including observed
toxic effects, necropsy, and histopathological changes should be evaluated.
The  evaluation  should include the  relationship between the dose  of the
test substance, the incidence and  severity of abnormalities including behav-
ioral and clinical  abnormalities, gross  lesions, identified target organs,
body weight changes, effect on mortality and any other general or specific
toxic effects. A properly conducted 21/28-day repeated dose dermal study
will  provide information on the effects of repeated application of  a  sub-
stance  and a satisfactory estimation  of  a NOEL. It also can indicate the
need for an additional longer-term study and provide information  on the
selection of dose levels.

     (3) Test report. In addition to reporting requirements specified under
EPA Good  Laboratory  Practice Standards at 40 CFR  part 792, subpart
J and  40 CFR part 160,  and the OECD  principles of  GLP  (ISBN 92-
64-12367-9), the following specific information should be reported:

     (i) Test substance characterization should include:.


     (A) Chemical identification.
     (B) Lot or batch numbers
     (C) Physical properties.
     (D) Purity/impurities.
     (E) Identification and composition of any vehicle if used.
     (ii) Test system should contain data on:
     (A)  Species and strain  of animals  used and rationale for selection
if other than that recommended.
     (B) Age including body weight data  and sex.
     (C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods.
     (D) Identification of animal diet.
     (iii) Test procedure should include the  following data:
     (A) Method of randomization used.
     (B) Full description of experimental  design and procedure.
     (C) Dose regime including levels, method, and volume.
     (iv) Test results should include:
     (A) Group animal data:  Tabulation of toxic response data by species,
strain, sex and exposure level for:
     (7) Number of animals exposed.
     (2) Number of animals showing signs of toxicity.
        Number of animals dying.
     (B)  Individual animal data. Data  should be presented  as  summary
(group mean) as well as for individual animals.
     (7) Date of death during the  study or whether animals survived to
     (2) Date of observation of each abnormal  sign and its subsequent
        Body weight data.
     (4) Feed consumption data, when collected.
     (5) Results of ophthalmological examination, when performed.

     (6} Results of the hematology tests performed.

     (7) Results of the clinical chemistry tests performed.

     (8) Results of the immunochemistry screen, when performed.

     (9) Necropsy findings, including absolute  and relative organ weight

     (10) Detailed description of all histopathological findings.

     (11} Statistical treatment of results, where appropriate.

     (g) Quality control. A system should be developed and maintained
to assure and document adequate  performance of laboratory  staff and
equipment. The study must be conducted in compliance with GLP regula-

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

     (1) Draize,  J.H.  Dermal toxicity, Appraisal of Chemicals in Food,
Drugs and Cosmetics. The Association of Food and Drug Officials of the
United States, 3rd printing 1975. pp. 46-59 (1959).

     (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)  Organization for  Economic  Co-Operation  and  Development.
Guidelines for Testing of Chemicals, Section 4—Health Effects, Part 410,
Repeated Dose Toxicity Study, Paris (1981).

     (4) World Health Organization. Part I. Environmental Health Criteria
6, Principles and Methods for Evaluating  the Toxicity of Chemicals.
(World Health Organization, Geneva) (1978).