# 407 Adopted: 27.07.95



## O E C D G UI D E L I N E F O R T H E T E ST I N G O F C H E M I C A L S

Adopted by the Council on 27 th 

### July 1995

R ep ea t ed Dose 28-d a y O r a l Toxi ci t y S t u d y i n R od en t s

I N T RO DUC T I O N
1. OECD Guidelines for the Testing of Chem icals are periodically reviewed in the light of scientific progress. The original g uideline 407 was adopted in 1981. In this revised version changes have been m ade with the objective of obtaining additional in form ation 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 W orking Group of Experts on Systemic Sh ort-term and (Delayed) Neurotoxicity, h eld 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 Mem ber coun tries.

I N I T I

A L C O NSI D E RA T I O NS
3. In the assessment and evaluation of the toxic characteristics of a chem ical, the determination of oral toxicity using repeated doses may be carried out after initial in form ation on tox icity has been obtained by acute testing. This study provides in form ation on the poss ible h ealth haz ards likely to arise from repeated exposure over a relatively limited period of time. The method comprises the basic repeated dose toxicity s tudy that may be used for chem icals on which a 90 day study is not warranted (e.g. when the production volume does not exceed certain limits) or as a prelimin ary to a long term study. The duration of expos ure 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 n eurolog ical effects as a specific en dpoint, and the need for careful clin ical obs ervations of the animals, so as to obtain as much in form ation as possible, is stressed. The method should iden tify chemicals with n eurotoxic potential, which may warrant further in-depth investigation of this as pect. In addition, the method may g ive an in dication of immunolog ical effects and reproductive organ toxicity.
5. Definitions used are g iven in the Annex.

P RI N C I P L E O F T H E T E S T 6. The test substance is orally administered daily in graduated doses to several groups of experimen tal animals, one dose level per group for a period of 28 days. During the period of administration the animals are observed clos ely, each day for signs of toxicity. Animals which die or are killed during the test are necropsied and at the conclusion of the test s urviving animals are killed and necropsied.

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D E SC RI P T I O N O F T H E M E T H O D

Select ion of a n im a l s p ecies 7. The preferred rodent s pecies is the rat, although other rodent s pecies may be used. Commonly used laboratory s trains of young healthy adult animals should be employed. The females should be nulliparous and non -pregnant. Dosing sh ould begin as soon as possible after weaning and, in any case, before the animals are nine w eeks old. At the commencement of the study the weight variation of animals used should be minimal and not ex ceed ± 20 % of the mean weight of each sex. Where a repeated dose oral study is conducted as a prelimin ary to a long term s tudy, preferably animals from the same s train and s ource should be used in both s tudies.

H ou s in g a n d feed in

g con d it ion s 8. The temperature in the ex perimen tal animal room sh ould be 22°C (± 3°C). Although the relative humidity should be at least 30% and preferably not to ex ceed 70% other than during room cleaning, the aim should be 50-60%. Lighting should be artificial, the sequence being 12 h ours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking w ater. The choice of diet may be in fluen ced by the need to ensure a s uitable 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.
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ion of a n im a ls 9. Healthy young adult animals are randomly assigned to the control and treatment g roups. Cages should be a rranged in such a way that poss ible effects due to cage placement are minimized. The animals are iden tified uniquely and kept in their cages for at least five days prior to the start of the study to allow for acclim atis ation to the laboratory con ditions.

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ion of d os es 10. The test compound is adminis tered by gavage or via the diet or drinking w ater. The method of oral adminis tration is dependent on the purpose of the study, and the physical/chem ical properties of the test material.
11. Where necess ary, the test substance is dissolved or suspen ded in a suitable vehicle. It is
recommen ded that, wherever poss ible, the use of an aqueous s olution/suspen sion be cons idered first, followed by consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible s olution in other vehicles. For veh icles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined.

P RO C E DURE

Nu m b er a n d s ex of a n im a ls
12. At least 10 animals (five female and five m ale) should be used at each dose level. If in terim kills are plann ed, the number should be increased by the number of animals scheduled to be killed before the completion of the s tudy. Consideration should be g iven to an additional sa tellite group of ten animals (five per sex) in the control and in the top dose group for obs ervation of reversibility, persistence, or delayed occurrence of toxic effects, for at least 14 days post treatment.

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Dos a ge 13. Generally, at least three test g roups 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 th ere are no suitable data available, a range finding study may be performed to aid the determination of the doses to be used. Ex cept for treatment with the test substance, animals in the control g roup should be handled in an identical manner to the test group subjects. If a veh icle is used in administering the test substance, the control g roup should receive the vehicle in the highest volume used. 14. Dose levels should be selected taking into account any existing toxicity and (toxico-) kin etic data available for the test com pound or related materials. The highest dose level should be chosen with the aim of inducing toxic effects but not death or s evere s uffering. T hereafter, 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 frequen tly optimal for s etting the descending dose levels and addition of a fourth test g roup is often preferable to using very large in tervals (e.g. more than a factor of 10) between dosages.

L im it t es

t 15. If a test at one dose level of at least 1000 mg/kg body weight/day or, for dietary or drinking water adminis tration, an equivalent percentage in the diet, or drinking w ater (based upon body weight determinations), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be ex pected based upon data from structurally related com pounds, then a full study using three dose levels may not be considered n ecess ary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.

A d m in is

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ion of d os es 16. The animals are dosed with the test substance daily s even days each w eek for a period of 28 days; use of a five-day per w eek dosing regime or a 14-day exposure period n eeds to be jus tified. When the test substance is administered by g avage, this should be done in a single dose to the animals using a stomach tube or a suitable in tubation cann ula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume sh ould not exceed 1ml/100g body weight, except in the case of aqueous s olutions wh ere 2m l/100g body weight may be used. Except for irritating or corrosive substances which w ill norm ally reveal exacerbated effects with higher concentrations, variability in test volume sh ould be minimised by adjusting the concen tration to ensure a constant volume at all dose levels.
17. For substances administered via the diet or drinking w ater it is im portant to ens ure that the quan tities of the test substance involved do not interfere with normal n utrition or w ater 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 altern ative used must be specified. For a substance administered by g avage, the dose sh ould be g iven at sim ilar times each day, and adjusted as n ecess ary to main tain a constant dose level in terms of animal body weight. Where a repeated dose study is used as a preliminary to a long term s tudy, a similar diet should be used in both studies.

O b s er v a t ion s 18. The observation period should be 28 days, unless the study duration is 14 days (see paragraph 3). Animals in a s atellite group scheduled for follow -up observations should be kept for at least a further 14 days with out treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

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19. General clin ical obs ervations should be m ade at least once a day, preferably at the same time(s) each day and cons idering the peak period of anticipated effects after dosing. The h ealth condition of the animals sh ould be recorded. At least twice daily, all animals are observed for morbidity and mortality. 20. Once before the first exposure (to allow for within-s ubject comparisons), and at least once a week th ereafter, detailed clin ical obs ervations should be m ade in all animals. These obs ervations should be made outs ide the home cage in a standard arena and preferably at the same time, each time. They should be carefully recorded, preferably using s coring systems, ex plicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test con ditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs n oted should include, but not be limited to, changes in skin, fur, eyes, mucous mem branes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual res piratory pattern). Changes in gait, posture and response to han dling as w ell as the presence of clonic or tonic movements, s tereotypies (e.g. excessive g rooming, repetitive circling) or biz arre behaviour (e.g. s elfmutilation, walking backw ards) should also be recorded (2). 21. In the fourth expos ure w eek sens ory reactivity to stimuli of different types (2) (e.g. auditory, visual and proprioceptive stimuli) (3)(4)(5), assessment of grip s trength (6) and motor activity assessment (7) should be conducted. Further details of the procedures that could be followed are g iven in the respective references. However, altern ative procedures than those referen ced could also be used.
22. Functional observations con ducted in the fourth expos ure week may be om itted when the study is conducted as a prelimin ary study to a subs equent 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 fun ctional observations from the repeated dose s tudy may enhance the ability to select dose levels for a subs equent subchronic s tudy. 23. Exceptionally, functional observations may also be om itted for groups that otherwise reveal signs of toxicity to an extent that would sign ifican tly interfere with the fun ctional test performance.

Bod y weigh t a n d food /wa t er con s u m p t ion 24. All animals should be weighed at least once a w eek. Meas urements of food consum ption should be m ade at least weekly. If the test substance is administered via the drinking w ater, water consumption sh ould also be measured at least weekly.

H a em a t

ology 25. The following haematolog ical examin ations sh ould be m ade at the end of the test period: haematocrit, haemog lobin concentration, eryth rocyte count, total and differen tial leucocyte count, platelet count and a meas ure of blood clotting time/poten tial. 26. Blood samples should be taken from a named s ite just prior to or as part of the procedure for killing the animals, and s tored under appropriate con ditions.

C lin ica l Bioch em is

t r y
27. Clin ical biochemistry determin ations to investig ate major toxic effects in tissues and, specifically, effects on kidney and liver, sh ould 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 m oribund

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and/or intercurren tly killed). Overnight fasting of the animals prior to blood sam pling is recommended (1) . Investigations of plasma or s erum sh all include s odium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanine aminotrans ferase, aspartate amin otrans ferase, alkaline phosphatase, gamma g lutamyl trans peptidase, and sorbitol deh ydrogenase). Meas urements of additional enzymes (of h epatic or other origin) and bile acids may provide useful in form ation under certain circumstances. 28. Optionally, the following urin alysis determinations could be performed during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, g lucose and blood/blood cells. 29. In addition, studies to investig ate s erum m arkers of general tissue damage should be considered. Other determinations that sh ould be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium, phosphate, fasting trig lycerides, specific hormones, methaemog lobin and cholines terase. These need to be iden tified for chemicals in certain classes or on a case-by-case basis. 30. Overall, there is a n eed for a flex ible approach, depending on the s pecies and the observed and/or expected effect with a g iven compound.
31. If historical bas eline data are inadequate, consideration should be g iven to determination of haematolog ical and clin ical biochemistry variables before dosing commences.

P a t h ology Gross necropsy 32. All animals in the study shall be subjected to a full, detailed gross necropsy which includes careful examin ation of the external s urface of the body, all orifices, and the cran ial, thoracic and abdominal cavities and their contents. The liver , kidn eys , adren als , tes tes , epididym ides , th ym us , s pleen , brain and h eartof all animals (apart from those found m oribund an d/or intercurren tly killed) should be trimmed of any adh erent tiss ue, as appropriate, and their wet weight taken as soon as possible after diss ection to avoid drying. 33. The following tissues sh ould be preserved in the most appropriate fix ation medium for both the type of tissue and the intended subs equent histopath olog ical examin ation: all g ros s les ion s , brain (representative regions in cluding cerebrum, cerebellum and pons), s pin al cord , s tom ach , s m all an d larg e in tes tin es (including Peyer's patches), liver , kidn eys , adren als , s pleen , h eart , th ym us , th yroid , trach ea and lun g s (preserved by in flation with fix ative and then immersion), g on ads , acces s ory s ex org an s (e.g. uterus, prostate), urin ary bladder , lym ph n odes (preferably one lymph n ode covering the route of adminis tration and another one distant from the route of adminis tration to cover systemic effects), periph eral n erve (sciatic or tibial) preferably in close proxim ity to the muscle, and a section of bon e m arrow (or, alternatively, a fresh moun ted bone m arrow as pirate). The clin ical 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 in creased 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 s tudies, may disturb the daily exposure to the test substance. If overnight fasting is adopted, clin ical biochemical determinations sh ould be performed after the conduct of fun ctional observations in week 4 of the s tudy.
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# 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 histopath ology sh ould be carried out on the preserved organs and tissues of all animals in the control and high dose groups. These examin ations sh ould be extended to animals of all other dosage groups, if treatmen t-related changes are observed in the high dose group. 35. All gross lesions shall be examined. 36. When a satellite group is used, his topathology sh ould be performed on tissues and organs iden tified as showing effects in the treated groups.

DA T A A N D RE P O RT I N G

D a t a 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 des cription of the signs of tox icity observed, in cluding time of onset, duration, and s everity 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.

T es t r ep or t 39. The test report must include the following in form ation:
Test substance: - physical nature, purity and phys icochem ical properties; - identification data.
Vehicle (if appropriate): - justification for ch oice of vehicle, if other than water.
Test animals: - species/s train used; - number, age and sex of animals; - source, housing con ditions, diet, etc.; - individual weights of animals at the start of the test.
Test conditions:
- ration ale for dose level selection;

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- details of test substance form ulation/diet preparation, ach ieved concen tration, stability and homogeneity of the preparation; - details of the adminis tration of the test substance; - conversion from diet/drinking w ater test substance concen tration (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 consum ption, and w ater consum ption, if applicable; - toxic response data by sex and dose level, in cluding signs of toxicity; - nature, severity and duration of clin ical obs ervations (whether revers ible or n ot); - sensory activity, grip s trength and m otor activity assessments; - haematolog ical tests with relevant base-line values; - clin ical biochemistry tests with relevant bas e-line values; - body weight at killing and organ weight data; - necropsy findings; - a detailed des cription of all his topatholog ical findings; - absorption data if available; - statistical treatment of res ults, where appropriate.
Discussion of res ults.
Conclusions.

L I T E RA T URE (1) OECD (Paris, 1992). Ch airman's R eport of the Meeting of the ad hoc Working Group of Experts on Systemic Sh ort-term and (Delayed) Neurotoxicity. (2) IPCS (1986). Prin ciples and Methods for the Assessment of Neurotoxicity Ass ociated with Exposure to Chemicals. En vironmen tal H ealth 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 Neuromus cular 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: Bas eline V alues and Effects of Am itraz. Tox icol. Appl. Pharmacol., 108 , 267-283. (6) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method for the R outine Assessment of Fore- and Hin dlimb Grip Strength of Rats and Mice. Neurobehav. Toxicol., 1 , 233-236. (7) Crofton K.M., How ard J.L., Moser V.C., Gill M.W., Reiter L.W., Tilson H.A., MacPh ail R.C. (1991). Interlaboratory Comparison of Motor Activity Ex periments: Implication for Neurotoxicolog ical Assessments. Neurotox icol. Teratol. 13 , 599-609.

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A NNE X

D E F I

N I T I O NS
Dos e 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 concen trations (ppm).
Dos ag e is a general term com prising of dose, its frequency and the duration of dosing.
Eviden t tox icity is a general term describing clear signs of toxicity following administration of test substance. These should be s ufficient for haz ard assessment and should be such that an increase in the dose administered can be expected to res ult 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 treatmen t-related findings are observed.

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