United States Prevention, Pesticides EPA712-C-00-366
Environmental Protection and Toxic Substances July 2000
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.3050
Repeated Dose 28-Day
Oral Toxicity Study in
Rodents
-------
INTRODUCTION
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
(OECD).
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.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/OPPTS Harmonized) under the heading "Informa-
tion Resources/Test Methods/OPPTS Harmonized Test Guidelines."
-------
OPPTS 870.3050 Repeated dose 28-day oral toxicity study in ro-
dents.
(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, (i) The source material used in developing this har-
monized OPPTS test guideline is OECD guideline 407, Repeated Dose
28-Day Oral Toxicity Study in Rodents. While editing the OECD source
guideline has resulted in some minor changes in format and style, all es-
sential elements of this guideline and its OECD counterpart are equivalent.
(b) Purpose. (1) In the assessment and evaluation of the toxic charac-
teristics of a chemical, the determination of oral toxicity using repeated
doses may be carried out after initial information on toxicity has been
obtained by acute testing. This study provides information on the possible
health hazards likely to arise from repeated exposure over a relatively lim-
ited period of time. The method comprises the basic repeated dose toxicity
study that may be used for chemicals on which a 90-day study is not
warranted (e.g. when the production volume does not exceed certain limits)
or as a preliminary to a long term study. The duration of exposure should
normally be 28 days although a 14-day study may be appropriate in certain
circumstances; justification for use of a 14-day exposure period should
be provided.
(2) This guideline places emphasis on neurological effects as a spe-
cific endpoint, and the need for careful clinical observations of the animals,
so as to obtain as much information as possible, is stressed. The method
should identify chemicals with neurotoxic potential, which may warrant
further in-depth investigation of this aspect. In addition, the method may
give an indication of immunological effects and reproductive organ tox-
icity.
(c) Definitions. The definitions in section 3 of the 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.
Dosage is a general term comprising of dose, its frequency and the
duration of dosing.
Dose is the amount of test substance administered. Dose is expressed
as weight (g, mg) or as weight of test substance per unit weight of test
animal (e.g. mg/kg), or as constant dietary concentrations (parts per million
(ppm)).
No-observed-adverse-effect-level (NOAEL) is the maximum dose used
in a study which produces no adverse effects. The NOAEL is usually ex-
-------
pressed in terms of the weight of a test substance given daily per unit
weight of test animals (milligrams per kilograms per day).
(d) Principle of the test. The test substance is orally administered
daily in graduated doses to several groups of experimental animals, one
dose level per group for a period of 28 days. During the period of adminis-
tration the animals are observed closely, each day for signs of toxicity.
Animals which die or are killed during the test are necropsied and at the
conclusion of the test surviving animals are killed and necropsied.
(e) Description of the method—(1) Selection of animal species. The
preferred rodent species is the rat, although other rodent species may be
used. Commonly used laboratory strains of young healthy adult animals
should be employed. The females should be nulliparous and non-pregnant.
Dosing should begin as soon as possible after weaning and, in any case,
before the animals are 9 weeks old. At the commencement of the study
the weight variation of animals used should be minimal and not exceed
±20% of the mean weight of each sex. Where a repeated dose oral study
is conducted as a preliminary to a long term study, preferably animals
from the same strain and source should be used in both studies.
(2) Housing and feeding conditions. The temperature in the experi-
mental animal room should be 22°C (±3°C). Although the relative humid-
ity should be at least 30% and preferably not to exceed 70% other than
during room cleaning, the aim should be 50-60%. Lighting should be arti-
ficial, the sequence being 12 hours light, 12 hours dark. For feeding, con-
ventional laboratory diets may be used with an unlimited supply of drink-
ing water. The choice of diet may be influenced by the need to ensure
a suitable admixture of a test substance when administered by this method.
Animals may be housed individually, or be caged in small groups of the
same sex; for group caging, no more than five animals should be housed
per cage.
(3) Preparation of animals. Healthy young adult animals are ran-
domly assigned to the control and treatment groups. Cages should be ar-
ranged in such a way that possible effects due to cage placement are mini-
mized. The animals are identified uniquely and kept in their cages for
at least 5 days prior to the start of the study to allow for acclimatization
to the laboratory conditions.
(4) Preparation of doses, (i) The test compound is administered by
gavage or via the diet or drinking water. The method of oral administration
is dependent on the purpose of the study, and the physical/chemical prop-
erties of the test material.
(ii) Where necessary, the test substance is dissolved or suspended in
a suitable vehicle. It is recommended that, wherever possible, the use of
an aqueous solution/suspension be considered first, followed by consider-
ation of a solution/emulsion in oil (e.g. corn oil) and then by possible
-------
solution in other vehicles. For vehicles other than water the toxic charac-
teristics of the vehicle must be known. The stability of the test substance
in the vehicle should be determined.
(f) Procedure—(1) Number and sex of animals. At least 10 animals
(five female and five male) should be used at each dose level. If interim
kills are planned, the number should be increased by the number of ani-
mals scheduled to be killed before the completion of the study. Consider-
ation should be given to an additional satellite group of 10 animals (five
per sex) in the control and in the top dose group for observation of revers-
ibility, persistence, or delayed occurrence of toxic effects, for at least 14
days post treatment.
(2) Dosage, (i) Generally, at least three test groups and a control
group should be used, but if from assessment of other data, no effects
would be expected at a dose of 1000 mg/kg bodyweight/per day, a limit
test may be performed. If there are no suitable data available, a range
finding study may be performed to aid the determination of the doses to
be used. Except for treatment with the test substance, animals in the con-
trol group should be handled in an identical manner to the test group sub-
jects. If a vehicle is used in administering the test substance, the control
group should receive the vehicle in the highest volume used.
(ii) Dose levels should be selected taking into account any existing
toxicity and (toxico-) kinetic data available for the test compound or re-
lated materials. The highest dose level should be chosen with the aim of
inducing toxic effects but not death or severe suffering. Thereafter, a de-
scending sequence of dose levels should be selected with a view to dem-
onstrating any dosage related response and NOAEL at the lowest dose
level. Two to four fold intervals are frequently optimal for setting the de-
scending dose levels and addition of a fourth test group is often preferable
to using very large intervals (e.g. more than a factor of 10) between dos-
ages.
(3) Limit test. If a test at one dose level of at least 1000 mg/kg
body weight/day or, for dietary or drinking water administration, an equiv-
alent percentage in the diet, or drinking water (based upon body weight
determinations), using the procedures described for this study, produces
no observable toxic effects and if toxicity would not be expected based
upon data from structurally related compounds, then a full study using
three dose levels may not be considered necessary. The limit test applies
except when human exposure indicates the need for a higher dose level
to be used.
(4) Administration of doses, (i) The animals are dosed with the test
substance daily 7 days each week for a period of 28 days; use of a 5-
day per week dosing regime or a 14-day exposure period needs to be
justified. When the test substance is administered by gavage, this should
-------
be done in a single dose to the animals using a stomach tube or a suitable
intubation cannula. The maximum volume of liquid that can be adminis-
tered at one time depends on the size of the test animal. The volume should
not exceed Iml/lOOg body weight, except in the case of aqueous solutions
where 2ml/100g body weight may be used. Except for irritating or corro-
sive substances which will normally reveal exacerbated effects with higher
concentrations, variability in test volume should be minimized by adjusting
the concentration to ensure a constant volume at all dose levels.
(ii) For substances administered via the diet or drinking water it is
important to ensure that the quantities of the test substance involved do
not interfere with normal nutrition or water balance. When the test sub-
stance is administered in the diet either a constant dietary concentration
(ppm) or a constant dose level in terms of the animals' body weight may
be used; the alternative used must be specified. For a substance adminis-
tered by gavage, the dose should be given at similar times each day, and
adjusted as necessary to maintain a constant dose level in terms of animal
body weight. Where a repeated dose study is used as a preliminary to
a long term study, a similar diet should be used in both studies.
(5) Observations, (i) The observation period should be 28 days, un-
less the study duration is 14 days (see paragraph (b)(l) of this guideline).
Animals in a satellite group scheduled for follow-up observations should
be kept for at least a further 14 days without treatment to detect delayed
occurrence, or persistence of, or recovery from toxic effects.
(ii) General clinical observations should be made at least once a day,
preferably at the same time(s) each day and considering the peak period
of anticipated effects after dosing. The health condition of the animals
should be recorded. At least twice daily, all animals are observed for mor-
bidity and mortality.
(iii) Once before the first exposure (to allow for within-subject com-
parisons), and at least once a week thereafter, detailed clinical observations
should be made in all animals. These observations should be made outside
the home cage in a standard arena and preferably at the same time, each
time. They should be carefully recorded, preferably using scoring systems,
explicitly defined by the testing laboratory. Effort should be made to en-
sure that variations in the test conditions are minimal and that observations
are preferably conducted by observers unaware of the treatment. Signs
noted should include, but not be limited to, changes in skin, fur, eyes,
mucous membranes, occurrence of secretions and excretions and auto-
nomic activity (e.g. lacrimation, piloerection, pupil size, unusual res-
piratory pattern). Changes in gait, posture and response to handling as well
as the presence of clonic or tonic movements, stereotypies (e.g. excessive
grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation,
walking backwards) should also be recorded (see paragraph (h)(2) of this
guideline).
-------
(iv) In the fourth exposure week sensory reactivity to stimuli of dif-
ferent types (see paragraph (h)(2) of this guideline) (e.g. auditory, visual
and proprioceptive stimuli) (see paragraphs (h)(3), (h)(4), and (h)(5) of
this guideline), assessment of grip strength (see paragraph (h)(6) of this
guideline) and motor activity assessment (see paragraph (h)(7) of this
guideline) should be conducted. Further details of the procedures that
could be followed are given in the respective references. However, alter-
native procedures than those referenced could also be used.
(v) Functional observations conducted in the fourth exposure week
may be omitted when the study is conducted as a preliminary study to
a subsequent subchronic (90-day) study. In that case, the functional obser-
vations should be included in this follow-up study. On the other hand,
the availability of data on functional observations from the repeated dose
study may enhance the ability to select dose levels for a subsequent sub-
chronic study.
(vi) Exceptionally, functional observations may also be omitted for
groups that otherwise reveal signs of toxicity to an extent that would sig-
nificantly interfere with the functional test performance.
(6) Body weight and food/water consumption. All animals should
be weighed at least once a week. Measurements of food consumption
should be made at least weekly. If the test substance is administered via
the drinking water, water consumption should also be measured at least
weekly.
(7) Hematology. (i) The following hematological examinations
should be made at the end of the test period: hematocrit, haemoglobin
concentration, erythrocyte count, total and differential leukocyte count,
platelet count and a measure of blood clotting time/potential.
(ii) Blood samples should be taken from a named site just prior to
or as part of the procedure for killing the animals, and stored under appro-
priate conditions.
(8) Clinical Biochemistry, (i) Clinical biochemistry determinations
to investigate major toxic effects in tissues and, specifically, effects on
kidney and liver, should be performed on blood samples obtained of all
animals just prior to or as part of the procedure for killing the animals
(apart from those found moribund and/or intercurrently killed). Overnight
fasting of the animals prior to blood sampling is recommended.1 Investiga-
1 For a number of measurements in serum and plasma, most notably for glucose,
overnight fasting would be preferable. The major reason for this preference is that the
increased variability which would inevitably result from non-fasting, would tend to mask
more subtle effects and make interpretation difficult. On the other hand, however, over-
night fasting may interfere with the general metabolism of the animals and, particularly
in feeding studies, may disturb the daily exposure to the test substance. If overnight
Continued
-------
tions of plasma or serum shall include sodium, potassium, glucose, total
cholesterol, urea, creatinine, total protein and albumin, at least two en-
zymes indicative of hepatocellular effects (such as alanine
aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma
glutamyl transpeptidase, and sorbitol dehydrogenase). Measurements of ad-
ditional enzymes (of hepatic or other origin) and bile acids may provide
useful information under certain circumstances.
(ii) Optionally, the following urinalysis determinations could be per-
formed during the last week of the study using timed urine volume collec-
tion; appearance, volume, osmolality or specific gravity, pH, protein, glu-
cose and blood and blood cells.
(iii) In addition, studies to investigate serum markers of general tissue
damage should be considered. Other determinations that should be carried
out if the known properties of the test substance may, or are suspected
to, affect related metabolic profiles include calcium, phosphate, fasting
triglycerides, specific hormones, methemoglobin and cholinesterase. These
need to be identified for chemicals in certain classes or on a case-by-
case basis.
(iv) Overall, there is a need for a flexible approach, depending on
the species and the observed and/or expected effect with a given com-
pound.
(v) If historical baseline data are inadequate, consideration should be
given to determination of hematological and clinical biochemistry variables
before dosing commences.
(9) Pathology—(i) Gross necropsy. (A) All animals in the study
shall be subjected to a full, detailed gross necropsy which includes careful
examination of the external surface of the body, all orifices, and the cra-
nial, thoracic and abdominal cavities and their contents. The liver, kidneys,
adrenals, testes, epididymides, thymus, spleen, brain and heart of all ani-
mals (apart from those found moribund and/or intercurrently killed) should
be trimmed of any adherent tissue, as appropriate, and their wet weight
taken as soon as possible after dissection to avoid drying.
(B) The following tissues should be preserved in the most appropriate
fixation medium for both the type of tissue and the intended subsequent
histopathological examination: all gross lesions, brain (representative re-
gions including cerebrum, cerebellum and pons), spinal cord, stomach,
small and large intestines (including Peyer's patches), liver, kidneys,
adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by
inflation with fixative and then immersion), ovaries, uterus, testes,
epididymides, accessory sex organs (e.g., prostate, seminal vesicles), uri-
fasting is adopted, clinical biochemical determinations should be performed after the
conduct of functional observations in week 4 of the study.
-------
nary bladder, lymph nodes (preferably one lymph node covering the route
of administration and another one distant from the route of administration
to cover systemic effects), peripheral nerve (sciatic or tibial) preferably
in close proximity to the muscle, and a section of bone marrow (or, alter-
natively, a fresh mounted bone marrow aspirate). The clinical and other
findings may suggest the need to examine additional tissues. Also any or-
gans considered likely to be target organs based on the known properties
of the test substance should be preserved.
(ii) Histopathology. (A) Full histopathology should be carried out
on the preserved organs and tissues of all animals in the control and high
dose groups. These examinations should be extended to animals of all
other dosage groups, if treatment-related changes are observed in the high
dose group.
(B) All gross lesions shall be examined.
(C) When a satellite group is used, histopathology should be per-
formed on tissues and organs identified as showing effects in the treated
groups.
(g) Data and reporting—(1) Data, (i) Individual data should be pro-
vided. Additionally, all data should be summarized in tabular form show-
ing for each test group the number of animals at the start of the test,
the number of animals found dead during the test or killed for humane
reasons and the time of any death or humane kill, the number showing
signs of toxicity, a description of the signs of toxicity observed, including
time of onset, duration, and severity of any toxic effects, the number of
animals showing lesions, the type of lesions and the percentage of animals
displaying each type of lesion.
(ii) When possible, numerical results should be evaluated by an ap-
propriate and generally acceptable statistical method. The statistical meth-
ods should be selected during the design of the study.
(2) Test report. The test report must include the following informa-
tion:
(i) Test substance:
(A) Physical nature, purity and physicochemical properties.
(B) Identification data.
(ii) Vehicle (if appropriate): Justification for choice of vehicle, if
other than water.
(iii) Test animals:
(A) Species/strain used.
-------
(B) Number, age and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weights of animals at the start of the test.
(iv) Test conditions:
(A) Rationale for dose level selection.
(B) Details of test substance formulation/diet preparation, achieved
concentration, stability and homogeneity of the preparation.
(C) Details of the administration of the test substance.
(D) Conversion from diet/drinking water test substance concentration
(ppm) to the actual dose (mg/kg body weight/day), if applicable.
(E) Details of food and water quality.
(v) Results:
(A) Body weight/body weight changes.
(B) Food consumption, and water consumption, if applicable.
(C) Toxic response data by sex and dose level, including signs of
toxicity.
(D) Nature, severity and duration of clinical observations (whether
reversible or not).
(E) Sensory activity, grip strength and motor activity assessments.
(F) Hematological tests with relevant base-line values.
(G) Clinical biochemistry tests with relevant base-line values.
(H) Body weight at killing and organ weight data.
(I) Necropsy findings.
(J) A detailed description of all histopathological findings.
(K) Absorption data if available.
(L) Statistical treatment of results, where appropriate.
(vi) Discussion of results.
(vii) Conclusions.
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline.
8
-------
(1) OECD (Paris, 1992). Chairman's Report of the Meeting of the
ad hoc Working Group of Experts on Systemic Short-term and (Delayed)
Neurotoxicity.
(2) IPCS (1986). Principles and Methods for the Assessment of
Neurotoxicity Associated with Exposure to Chemicals. Environmental
Health Criteria Document No. 60.
(3) Tupper, D.E., Wallace, R.B. (1980). Utility of the Neurologic Ex-
amination in Rats. Acta Neurobiological Exposure, 40:999-1003.
(4) Gad, S.C. (1982). A Neuromuscular Screen for Use in Industrial
Toxicology. Journal of Toxicology and Environmental Health, 9:691-704.
(5) Moser, V.C., McDaniel, K.M., Phillips, P.M. (1991). Rat Strain
and Stock Comparisons Using a Functional Observational Battery: Base-
line Values and Effects of Amitraz. Toxicology and Applied Pharma-
cology, 108:267-283.
(6) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Meth-
od forthe Routine Assessment of Fore- and Hindlimb Grip Strength of
Rats and Mice. Neurobehavioral Toxicology, 1:233-236.
(7) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W.,
Tilson H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor
Activity Experiments: Implication for Neurotoxicological Assessments.
Neurotoxicology and Teratology, 13:599-609.
------- |