United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–00–366

July 2000

Health Effects Test

Guidelines

OPPTS 870.3050

Repeated Dose 28–Day

Oral Toxicity Study in

Rodents
i

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

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 ‘‘Information Resources/Test Methods/OPPTS Harmonized Test Guidelines.’’
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OPPTS 870.3050 Repeated dose 28–day oral toxicity study in rodents.

(a) Scope—(1) Applicability. This guideline is intended to meet testing 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 harmonized 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 essential elements of this guideline and its OECD counterpart are equivalent.

(b) Purpose. (1) In the assessment and evaluation of the toxic characteristics 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 limited 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 specific 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 toxicity.

(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-
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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 administration 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 experimental animal room should be 22°C (±3°C). Although the relative humidity 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 artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking 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 randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimized. 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 properties 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 consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible
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solution in other vehicles. For vehicles other than water the toxic characteristics 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 animals scheduled to be killed before the completion of the study. Consideration 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 reversibility, 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 control group should be handled in an identical manner to the test group subjects. 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 related materials. The highest dose level should be chosen with the aim of

inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and NOAEL at the lowest dose

level. Two to four fold intervals are frequently optimal for setting the descending 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 dosages.

(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 equivalent 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
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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 administered at one time depends on the size of the test animal. The volume should

not exceed 1ml/100g body weight, except in the case of aqueous solutions

where 2ml/100g body weight may be used. Except for irritating or corrosive 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 substance 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 administered 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, unless the study duration is 14 days (see paragraph (b)(1) 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 morbidity and mortality.

(iii) Once before the first exposure (to allow for within-subject comparisons), 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 ensure 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 autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory 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).
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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, overnight 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

(iv) In the fourth exposure week sensory reactivity to stimuli of different 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, alternative 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 observations 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 subchronic study.

(vi) Exceptionally, functional observations may also be omitted for

groups that otherwise reveal signs of toxicity to an extent that would significantly 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 appropriate 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-
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fasting is adopted, clinical biochemical determinations should be performed after the

conduct of functional observations in week 4 of the study.

tions of plasma or serum shall include sodium, potassium, glucose, total

cholesterol, urea, creatinine, total protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanine

aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma

glutamyl transpeptidase, and sorbitol dehydrogenase). Measurements of additional enzymes (of hepatic or other origin) and bile acids may provide

useful information under certain circumstances.

(ii) Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose 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-bycase 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 compound.

(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 cranial, thoracic and abdominal cavities and their contents. The liver, kidneys,

adrenals, testes, epididymides, thymus, spleen, brain and heart of all animals (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 regions 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-
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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, alternatively, a fresh mounted bone marrow aspirate). The clinical and other

findings may suggest the need to examine additional tissues. Also any organs 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 performed on tissues and organs identified as showing effects in the treated

groups.

(g) Data and reporting—(1) Data. (i) Individual data should be provided. Additionally, all 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 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 appropriate and generally acceptable statistical method. The statistical methods should be selected during the design of the study.

(2) Test report. The test report must include the following information:

(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.
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(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 additional background material on this test guideline.
9

(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 Examination 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: Baseline Values and Effects of Amitraz. Toxicology and Applied Pharmacology, 108:267–283.

(6) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method 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.