407

Adopted:

27.07.95

OECD GUID ELIN E FOR THE TESTIN G OF CHEMICALS

Adopted by the Council on 27th July 1995

Repeated Dose 28-day Oral Toxicity Study in Rodents

IN TRODUCTION

1. OECD Guidelines for the Testing of Chemicals are periodically reviewed in the light of

scientific progress. The original guideline 407 was adopted in 1981. In this revised version changes

have been made with the objective of obtaining additional information from the animals used in the

study.

2. This updated version of Guideline 407 is the outcome of a Consultation Meeting of an ad

hoc Working Group of Experts on Systemic Short-term and (Delayed) Neurotoxicity, held in Paris in

February 1992 (1). It is based on an earlier proposal by the United Kingdom, dated February 1991,

and the comments received on that proposal from Member countries.

IN ITIAL CONSID ERATIONS

3. 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.

4. The revised Guideline places more 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.

5. Definitions used are given in the Annex.

PRIN CIPLE OF THE TES T

6. 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.

1/8
407 OCD E/OECD

D ESCRIPTION OF THE METHOD

Selection of animal species

7. 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 nine 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.

Housing and feeding conditions

8. 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.

Preparation of animals

9. 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 five days prior to the start of

the study to allow for acclimatisation to the laboratory conditions.

Preparation of doses

10. 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.

11. 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 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.

PROCEDURE

Number and sex of animals

12. 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 

ten 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.

2/8
OCD E/OECD 407

Dosage

13. 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 1000mg/kg bw/d, 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.

14. 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 no-observed-adverse effects at the lowest dose level (NOAEL). 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.

Limit test

15. 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. 

Administration of doses

16. The animals are dosed with the test substance daily seven days each week for a period of

28 days; use of a five-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

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 minimised by adjusting the concentration

to ensure a constant volume at all dose levels.

17. 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.

Observations

18. The observation period should be 28 days, unless the study duration is 14 days (see

paragraph 3). 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.

3/8
407 OCD E/OECD

19. 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.

20. 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. selfmutilation, walking backwards) should also be recorded (2).

21. In the fourth exposure week sensory reactivity to stimuli of different types (2) (e.g. auditory,

visual and proprioceptive stimuli) (3)(4)(5), assessment of grip strength (6) and motor activity

assessment (7) 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.

22. 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.

23. 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.

Body weight and food/water consumption

24. 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. 

Haematology

25. The following haematological examinations should be made at the end of the test period:

haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count,

platelet count and a measure of blood clotting time/potential.

26. 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.

Clinical Biochemistry

27. 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

4/8
OCD E/OECD 407

and/or intercurrently killed). Overnight fasting of the animals prior to blood sampling is

recommended(1). Investigations 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.

28. 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/blood cells.

29. 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, methaemoglobin and cholinesterase. These need to be identified for

chemicals in certain classes or on a case-by-case basis.

30. Overall, there is a need for a flexible approach, depending on the species and the observed

and/or expected effect with a given compound.

31. If historical baseline data are inadequate, consideration should be given to determination of

haematological and clinical biochemistry variables before dosing commences.

Pathology

Gross necropsy

32. 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.

33. 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), gonads, accessory sex

organs (e.g. uterus, prostate), urinary 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

 

 (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 fasting is adopted, clinical

biochemical determinations should be performed after the conduct of functional observations

in week 4 of the study.

5/8
407 OCD E/OECD

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.

Histopathology

34. 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.

35. All gross lesions shall be examined.

36. When a satellite group is used, histopathology should be performed on tissues and organs

identified as showing effects in the treated groups.

DATA AN D REPORTIN G

D ata

37. Individual data should be provided. Additionally, all data should be summarised 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.

38. 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.

Test report

39. The test report must include the following information:

Test substance:

- physical nature, purity and physicochemical properties;

- identification data.

Vehicle (if appropriate):

- justification for choice of vehicle, if other than water.

Test animals:

- species/strain used;

- number, age and sex of animals;

- source, housing conditions, diet, etc.;

- individual weights of animals at the start of the test.

Test conditions:

- rationale for dose level selection;

6/8
OCD E/OECD 407

- details of test substance formulation/diet preparation, achieved concentration, stability

and homogeneity of the preparation;

- details of the administration of the test substance;

- conversion from diet/drinking water test substance concentration (ppm) to the actual

dose (mg/kg body weight/day), if applicable;

- details of food and water quality.

Results:

- body weight/body weight changes;

- food consumption, and water consumption, if applicable;

- toxic response data by sex and dose level, including signs of toxicity;

- nature, severity and duration of clinical observations (whether reversible or not);

- sensory activity, grip strength and motor activity assessments;

- haematological tests with relevant base-line values;

- clinical biochemistry tests with relevant base-line values;

- body weight at killing and organ weight data;

- necropsy findings;

- a detailed description of all histopathological findings;

- absorption data if available;

- statistical treatment of results, where appropriate.

Discussion of results.

Conclusions.

LITERATURE

(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

Neurobiol. Exp., 40, 999-1003.

(4) Gad, S.C. (1982). A Neuromuscular Screen for Use in Industrial Toxicology. J. Toxicol

Environ. 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. Toxicol.

Appl. Pharmacol., 108, 267-283.

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

Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice. Neurobehav. Toxicol.,

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. Neurotoxicol. Teratol. 13, 599-609.

7/8
407 OCD E/OECD

ANNEX

D EFIN ITIONS

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

(ppm).

Dosage is a general term comprising of dose, its frequency and the duration of dosing.

Evident toxicity is a general term describing clear signs of toxicity following administration of test

substance. These should be sufficient for hazard assessment and should be such that an increase in

the dose administered can be expected to result in the development of severe toxic signs and probable

mortality.

NOAEL is the abbreviation for no-observed-adverse-effect level and is the highest dose level where

no adverse treatment-related findings are observed.

8/8