OECD/OCDE 408

Adopted:
25 June 2018
│

OECD GUIDELINE FOR THE TESTING OF CHEMICALS

Repeated dose 90-day oral toxicity study in rodents

INTRODUCTION
1. OECD Guidelines for the Testing of Chemicals are periodically reviewed in
the light of scientific progress, changing regulatory needs, and animal welfare
considerations. The original guideline 408 was adopted in 1981. In 1998 a revised
version was adopted, to obtain additional information from the animals used in the
study, based on the outcome of an OECD Consultation Meeting of Experts on Sub-
chronic and Chronic Toxicity Testing held in Rome in 1995 (1).

2. This Test Guideline (TG) was updated in 2018 to add endocrine-sensitive

endpoints intended to improve detection of potential endocrine activity of test
chemicals and mirrors updates to TG 407 (Repeated Dose 28-Day Oral Toxicity Study
in Rodents).

INITIAL CONSIDERATIONS
3. In the assessment and evaluation of the toxic characteristics of a
chemical, the determination of sub-chronic oral toxicity using repeated doses may
be carried out after initial information on toxicity has been obtained from acute or
repeated dose 28-day toxicity tests. The 90- day study provides information on the
possible health hazards likely to arise from repeated exposure over a prolonged
period of time covering post-weaning maturation and growth into adulthood of the
test animals. The study will provide information on the major toxic effects, indicate
target organs and the possibility of accumulation of test chemical, and can provide
an estimate of a no-observed-adverse-effect level (NOAEL) of exposure which can
be used in selecting dose levels for chronic studies and for establishing safety
criteria for human exposure. Alternatively, this study yields dose related response data
that may be used to estimate point of departure for hazard assessment using appropriate
modelling methods (e.g., benchmark dose analysis).

© OECD 2018

You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.

In accordance with the Decision of the Council on a Delegation of Authority to amend Annex I of the Decision of the Council on the Mutual
Acceptance of Data in the Assessment of Chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meeting of the
Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.
2│ 408 OECD/OCDE
4. The revised Guideline places additional emphasis on endocrine endpoints to
combine with the existing sensitivity to neurological and immunological and
reproductive effects. The need for careful clinical observations of the animals, so as to
obtain as much information as possible, is also stressed. Required endpoints include
the measurement of thyroxine (T4), triiodothyronine (T3), thyroid stimulating hormone
(TSH) and thyroid gland weight, which are responsive to thyroid pathway
perturbation (2). In addition, serum total cholesterol, low-density lipoproteins (LDL)
and high-density lipoproteins (HDL) should also be determined as levels of these
parameters are directly controlled by thyroid hormone action and contribute (with other
thyroid endpoints) to evidence of thyroid effects. (3). Optional endpoints include other
hormone measurements, as well as assessments of sperm parameters. Required and
optional measures that may be altered by endocrine effects are listed in Annex B.
Assessment of the optional measures may be considered if existing information for the
test chemical or similar chemicals suggests potential to influence these or can be
triggered by observations from required measures collected as part of this guideline.
This study should allow for the identification of chemicals with the potential to cause
neurotoxic, endocrine, immunological or reproductive organ effects, which may
warrant further in-depth investigation.

5. The results obtained for the endocrine related parameters should be evaluated
in the context of the “OECD Conceptual Framework for Testing and Assessment of
Endocrine Disrupting Chemicals” (4). In this Conceptual Framework, TG 408 is
included in level 4 as an in vivo assay providing data on adverse effects on endocrine
relevant endpoints.

6. All animal-based procedures should conform to standards of animal care; the

descriptions of care and treatment set forth below are minimal performance standards,
and will be superseded by local regulations where more stringent. Further guidance of
the humane treatment of animals is provided in OECD Series on Testing and
Assessment 19 (19).
7. Definitions used in this test guideline are provided in Annex A.

PRINCIPLE OF THE TEST

8. The test chemical is orally administered daily in graduated doses to
several groups of experimental animals, one dose level per group for a period of
at least 90 days. During the period of administration, the animals are observed
closely for signs of toxicity as recommended by the OECD (19). Animals which die or
are humanely killed during the test are necropsied and at the conclusion of the test,
remaining animals are also humanely killed and necropsied after the full dosing period.

DESCRIPTION OF THE METHOD

Selection of animal species

9. The preferred species is the rat, although other rodent species (e.g., the mouse)
may be used. If the parameters specified within this TG 408 are investigated in another
rodent species, a detailed justification for the choice of species should be given,
including adaptations to the parameters measure. Although it is biologically plausible

© OECD 2018
 OECD/OCDE 408
Adopted:
25 June 2018
│
that other species should respond to toxicants in a similar manner to the rat, the use of
smaller species may result in increased variability in endpoint measurements due to
technical challenges of dissecting smaller organs. 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 the study is conducted preliminary to a long term
chronic toxicity study, animals from the same strain and source should be used in both
studies.

Housing and feeding conditions

10. All procedures should conform to local standards of laboratory animal care.
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 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 chemical
when administered by this method. Care should be taken to avoid diets or animal
bedding that may contain unacceptably high levels of hormonally active substances
prone or likely to interfere with the interpretation of the study results (e.g.,
phytoestrogens). High levels of phytooestrogens in laboratory diets have been known
to increase uterine weights in rodents. As a guide, dietary levels of phytooestrogens
should not exceed 350 μg of genistein equivalents/gram of rodent laboratory diet.

11. Animals should be housed in small groups of the same sex. Animals may be
housed individually if scientifically justified and the duration of single housing shall be
limited to the minimum period necessary. (5), (6), (7).

Preparation of animals
12. Healthy animals, which have been acclimated to laboratory conditions for at
least 5 days and have not been subjected to previous experimental procedures,
should be used. The test animals should be characterised as to species, strain,
source, sex, weight and/or age. Animals should be randomly assigned to the control
and treatment groups. Cages should be arranged in such a way that possible effects
due to cage placement are minimised. Each animal should be assigned a unique
identification number. The least invasive method of uniquely identifying animals must
be used. Appropriate methods include ringing, tagging, micro-chipping and biometric
identification.

© OECD 2018

You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.

In accordance with the Decision of the Council on a Delegation of Authority to amend Annex I of the Decision of the Council on the Mutual
Acceptance of Data in the Assessment of Chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meeting of the
Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.
4│ 408 OECD/OCDE
Preparation of doses
13. The test compound is to be administered by oral gavage, incorporated in the
diet or dissolved in drinking water. The method of oral administration is dependent on
the purpose of the study and the physical/chemical properties of the test material.

14. Where necessary, the test chemical 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 homogeneity and stability of the test chemical under the
conditions of administration should be determined.

PROCEDURE
Number and sex of animals
15. At least 20 animals (ten female and ten 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. Based on
previous knowledge of the chemical or a close analogue, consideration should be
given to including an additional satellite group of at least ten animals (five per sex) in
the control and in the top dose group for observation after the treatment period,
for the potential reversibility or persistence of any toxic effects. The duration of this
post-treatment period should be fixed appropriately with regard to the effects observed.

Dosage
16. At least three dose levels and a concurrent control shall be used, except where
a limit test is conducted (see paragraph 18). Dose levels may be based on the
results of repeated dose or range finding studies and should take into account any
existing toxicological and toxicokinetic data available for the test compound or
related materials. Unless limited by the physical-chemical nature or biological effects
of the test chemical, the highest dose level should be chosen with the aim to induce
toxicity but not death or severe suffering (see OECD Series on Testing and
Assessment No. 19 (19)). A descending sequence of dose levels should be selected
with a view to demonstrating any dosage related response and a 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 about 6-10) between dosages.

17. The control group shall be an untreated group or a vehicle-control group if a

vehicle is used for administering the test chemical. Except for treatment with the
test chemical, animals in the control group should be handled in an identical
manner to those in the test groups. If a vehicle is used, the control group shall
receive the vehicle in the highest volume used. If a test chemical is administered in
the diet, and causes reduced dietary intake, then a pair-fed control group may be
useful in distinguishing between reduced food intake due to palatability or
toxicological alterations in the test animals.

© OECD 2018
 OECD/OCDE 408
Adopted:
25 June 2018
│
18. Consideration should be given to the following characteristics of the
vehicle, as appropriate: effects on the absorption, distribution, metabolism, or
retention of the test chemical; effects on the chemical properties of the test chemical
which may alter its toxic characteristics; and effects on the food or water consumption
or the nutritional status of the animals.

Limit Test
19. Using the methods described for this study, if a test at one dose level
equivalent to at least 1000 mg/kg body weight/day produces no observed adverse
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
20. The animals are dosed with the test chemical daily seven days each week for at
least 90 days. Any other dosing regimen ( e.g., five days per week) needs to be
justified. When the test chemical 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 1 ml/100g body weight,
except in the case of aqueous solutions where 2 ml/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.

21. For chemicals administered via the diet or drinking water it is important to
ensure that palatability of the test chemical involved do not interfere with normal
nutrition or water balance. When the test chemical is administered in the diet, either
a constant dietary concentration (ppm) or adjusted as necessary to maintain a constant
dose level in terms of the animal’s body weight (e.g., mg/kg body weight/day) may
be used; the alternative used must be specified. For a chemical 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.

Observations
22. The observation period should be at least 90 days. If a satellite group is
included in the study, animals in the satellite recovery group scheduled for follow-up
observations should be kept for an appropriate period without treatment to detect
persistence of, or recovery from toxic effects.

© OECD 2018

You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.

In accordance with the Decision of the Council on a Delegation of Authority to amend Annex I of the Decision of the Council on the Mutual
Acceptance of Data in the Assessment of Chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meeting of the
Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.
6│ 408 OECD/OCDE
23. General clinical observations should be made at least once a day, preferably
at the same time(s) each day, taking into consideration the peak period of
anticipated effects after dosing. The clinical condition of the animals should be
recorded. At least twice daily, usually at the beginning and end of each day, all
animals are inspected for signs of morbidity and mortality (19).

24. At least once prior to the first exposure (to allow for within-subject
comparisons), and once a week thereafter, detailed clinical observations should be
made in all animals. These observations should be made outside the home cage,
preferably in a standard arena and at similar times on each occasion. 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 observation
conditions are minimal. 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, pilo-erection, pupil size, and unusual respiratory
pattern). Changes in gait, posture and response to handling as well as the presence of
clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling)
or bizarre behaviour (e.g., self-mutilation, walking backwards) should also be
recorded (8, 19).

25. Ophthalmological examination, using an ophthalmoscope or equivalent

suitable equipment, should be made prior to the administration of the test chemical
and at the termination of the study, preferably in all animals but at least in the high
dose and control groups. If changes in the eyes are detected all animals should be
examined.

26. Towards the end of the exposure period and not earlier than in week 11,
sensory reactivity to stimuli of different types (5) (e.g., auditory, visual and
proprioceptive stimuli) (9), (10), (11), assessment of grip strength (12) and motor
activity assessment (13) 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.

27. Functional observations conducted towards the end of the study may be
omitted when data on functional observations are available from other studies or when
daily clinical observations did not reveal any functional deficits.

28. 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

29. All animals should be weighed at least once a week. Measurements of food
consumption should be made at least weekly. If the test chemical is administered
via the drinking water, water consumption should also be measured at least weekly.
Measurement of water consumption may also be considered for dietary or gavage
studies.

© OECD 2018
 OECD/OCDE 408
Adopted:
25 June 2018
│
Haematology and Clinical Biochemistry
30. Blood samples should be taken from a designated site and stored, if
applicable, under appropriate conditions. At the end of the test period, samples are
collected just prior to or as part of the procedure for killing the animals.

31. The following haematological examinations should be made at the end of

the test period and when any interim blood samples have been collected:
haematocrit, haemoglobin concentration, erythrocyte count, reticulocyte count, total and
differential leukocyte count, platelet count and a measure of blood clotting
time/potential.

32. Clinical biochemistry determinations to investigate major toxic effects in

tissues and, specifically, effects on kidney and liver, should be performed on blood
samples obtained from each animal just prior to or as part of the procedure for
killing the animals (apart from those found moribund and/or intercurrently killed).
In a similar manner to haematological investigations, interim sampling for clinical
biochemical tests may be performed. Overnight fasting of the animals prior to blood
sampling is recommended1. Determinations in plasma or serum should include
sodium, potassium, glucose, total cholesterol, HDL, LDL, urea, blood urea nitrogen,
creatinine, total protein and albumin, and more than two enzymes indicative of
hepatocellular effects (e.g., alanine aminotransferase, aspartate aminotransferase,
alkaline phosphatase, gamma glutamyl transpeptidase, and sorbitol dehydrogenase).
Measurements of additional enzymes (of hepatic or other origin) and bile acids,
which may provide useful information under certain circumstances, and bilirubin may
also be included.

33. Clinical biochemistries should be evaluated as potential markers of general

tissue damage. Other determinations that should be carried out if the known
properties of the test chemical may, or are suspected to, affect related metabolic
profiles include calcium, phosphorus, fasting triglycerides, specific hormones,
methaemoglobin and cholinesterase. These determinations may be identified for
chemicals in certain classes or on a case-by-case basis.

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 chemical. If overnight fasting is adopted, clinical
biochemical determinations should be performed after the conduct of functional observations of
the study.
© OECD 2018

You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.

In accordance with the Decision of the Council on a Delegation of Authority to amend Annex I of the Decision of the Council on the Mutual
Acceptance of Data in the Assessment of Chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meeting of the
Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.
8│ 408 OECD/OCDE
34. Serum total T4, T3 and TSH should be measured on samples obtained from
each animal in the main group and in satellite and/or recovery groups at study
termination. Other hormones, e.g., testosterone, oestradiol, follicle stimulating
hormone (FSH), luteinizing hormone (LH) should be considered as on a case-by-case
basis. Serum may be stored frozen to allow time to determine the most informative
hormone analyses based on results observed for other endpoints (e.g., organ weight
and histology). Hormones may be measured in plasma if appropriate validation and
historical control data are available.

35. The following factors might influence the variability and absolute
concentration of the hormone determinations:

 time of humane killing because of diurnal variation of hormone concentration

 stage of the oestrus cycle
 method of humane killing to avoid undue stress to the animals that may affect
hormone concentrations
 test kits for hormone determinations that may differ by their standard curves.

36. Blood samples specifically intended for hormone determination should be

obtained at a comparable time of the day. The numerical values obtained when
analysing hormone concentrations differ with various commercial assay kits.
Consequently, it may not be possible to provide performance criteria based upon
uniform historical data. Control hormone levels (measured in the same lab, same
rodent strain, and using the same method) should be taken into account to differentiate
between incidental and treatment-related changes. Whenever possible, best practices
for blood sample collection, handling and analysis should be used (23). Laboratories
should strive, as far as possible, to keep control coefficients of variation below 25 for
T3 and T4 and below 35 for TSH. All concentrations are to be recorded in ng/ml.
Stability of T3, T4 and TSH under selected storage conditions should be tested as part
of the hormonal assay validation

37. Optionally, the following urinalysis determinations may 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.

38. If historical baseline data are inadequate, consideration should be given

as to whether haematological and clinical biochemistry variables need to be
determined before dosing commences; however, it is generally not recommended that
these data be generated before treatment (14).

Pathology
39. At termination, testis and epididymis weights are recorded for all males. At
least one epididymis from each male should be reserved for histopathological
examination. The remaining epididymis may be used for optional enumeration of
cauda epididymis sperm reserves, sperm morphology or sperm motility (15).

40. For the optional evaluation of sperm morphology, an epididymal (or vas
deferens) sperm sample should be examined as fixed or wet preparations and at least
200 spermatozoa per sample classified as either normal (both head and midpiece/tail

© OECD 2018
 OECD/OCDE 408
Adopted:
25 June 2018
│
appear normal) or abnormal. Examples of morphologic sperm abnormalities would
include fusion, isolated heads, and misshapen heads and/or tails. Misshapen or large
sperm heads may indicate defects in spermiation. Sperm motility can either be
evaluated immediately after humane killing or recorded for later analysis. The
percentage of progressively motile sperm may be determined either visually or by
computer-assisted motion analysis.

41. Analyses of sperm parameters may be restricted to control and high-dose

males. However, if treatment-related effects are observed, the lower dose groups
should also be evaluated.

42. At necropsy, the oestrus cycle of all females should be determined by taking
vaginal smears. These observations will provide information regarding the stage of
oestrus cycle at the time of humane killing and assist in histological evaluation of
oestrogen sensitive tissues (see OECD Guidance Document 106, part 3 (17)).

Gross necropsy
43. 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, prostate + seminal vesicles with coagulating
glands as a whole (alternatively, first weigh the entire prostate with seminal
vesicles/coagulation glands together, then dissect and weigh the prostate gland
separately), uterus, ovaries, thymus, spleen, brain, and heart of all animals (should
be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon
as possible after dissection to avoid drying. The pituitary gland may be weighted fresh,
immediately after dissection, or post-fixation. Care must be exercised when trimming
the prostate complex to avoid puncture of the fluid filled seminal vesicles.
Alternatively, seminal vesicles and prostate may be trimmed and weighed after
fixation.

44. Weighing of the thyroid gland must be performed with extreme care as this
tissue is easily damaged (for guidance, see reference 20). Tissue damage could
compromise histopathology analysis. Therefore, the thyroid trimming and weighing
should be done very carefully and should preferably be conducted after fixation in
order to avoid tissue damage.

45. The following tissues should be preserved in the most appropriate fixation
medium for both the type of tissue and the intended subsequent histopathological
examination (16, 17): all gross lesions, brain (representative regions including
cerebrum, cerebellum and medulla/pons), spinal cord (at three levels: cervical, mid-
thoracic and lumbar), pituitary, thyroid, parathyroid, thymus, oesophagus, salivary
glands, stomach, small and large intestines (including Peyer’s patches), liver,
© OECD 2018

You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.

In accordance with the Decision of the Council on a Delegation of Authority to amend Annex I of the Decision of the Council on the Mutual
Acceptance of Data in the Assessment of Chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meeting of the
Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.
10 │ 408 OECD/OCDE
pancreas, kidneys, adrenals, spleen, heart, trachea and lungs (preserved by inflation
with fixative and then immersion), aorta, ovaries, uterus, cervix, vagina, testes,
epididymides, prostate, seminal vesicles, coagulation glands, mammary gland (male
and female), urinary bladder, gall bladder (mouse), 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), skeletal muscle, and bone, with bone
marrow (section or alternatively, a fresh bone marrow aspirate), skin and eyes (if
changes were observed during ophthalmological examinations). It is recommended to
preserve testes by immersion in Bouin’s or modified Davidson’s fixative and
histopathological assessment should consider staging of seminiferous tubule cross
sections as described (16). See also OECD GD 106 (17) for fixation and histological
evaluation of endocrine organs. 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 chemical should be evaluated.

46. Histopathological assessment of the testes should be performed with attention

to stage specific changes as described (14). Detailed histopathological examination
should be conducted in order to identify treatment related effects such as retained
spermatids, missing germ cell layers or types, multinucleate giant cells or sloughing of
spermatogenic cells into the lumen (21). Also see OECD GD 106 (17) fixation and
histological evaluation of endocrine organs.

Histopathology
47. 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, for those tissues where treatment-
related changes are observed in the high dose group.

48. All gross lesions should be examined.

49. When a satellite group is used, histopathology should be performed on

tissues and organs identified as showing effects in the treated groups.

DATA AND REPORTING

Data
50. 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; number of animals found dead during the test or killed for humane
reasons and the time of any death or humane kill; number of animals showing signs
of toxicity, a description of the signs of toxicity observed, including time of onset,
duration, and severity of any toxic effects; and the number of animals showing
lesions, the type of lesions and the percentage of animals displaying each type of
lesion.

51. When applicable, numerical results should be evaluated by an appropriate

and generally acceptable statistical method. The statistical methods and the data to be
analysed should be selected during the design of the study.

© OECD 2018
 OECD/OCDE 408
Adopted:
25 June 2018
│
52. For quality control it is proposed that control data are compared to historical
control values originating from the same laboratory, species, strain, and collected
under similar conditions. In addition, coefficients of variation are calculated for the
continuous parameters in Annex B for endocrine activity. These data can be used for
comparison among studies. Differences between rat strains should be taken into
account when evaluating historical control data.

Test report
53. The test report must include the following information:

Test chemical:
 chemical identification, such as IUPAC or CAS name(s), CAS registry
number(s), SMILES or InChI code, structural formula, and/or other identifiers
 source, lot number, limit date for use (if available)
 stability of chemical, if known
 physical nature and, where relevant, physiochemical properties
 identification including CAS number if known/established; and
 purity.

Mono-constituent substance:
 Physical appearance, water solubility, and additional relevant physicochemical
properties.

Multi-constituent substance, UVBCs and mixtures:

 Characterised as far as possible by chemical identity (see above), quantitative
occurrence and relevant physicochemical properties of the constituents.

Vehicle if appropriate:
 Justification for choice of vehicle, if other than water.

Test animals:
 species and strain used
 number, age and sex of animals
 source, housing conditions, diet, etc.
 individual weights of animals at the start of the test; and
 justification for species if not rat.

Test conditions:
 rationale for dose level selection
© OECD 2018

You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.

In accordance with the Decision of the Council on a Delegation of Authority to amend Annex I of the Decision of the Council on the Mutual
Acceptance of Data in the Assessment of Chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meeting of the
Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.
12 │ 408 OECD/OCDE
 details of test chemical formulation/diet preparation, achieved concentration,
stability and homogeneity of the preparation
 details of the administration of the test chemical
 actual doses (mg/kg body weight/day), and conversion factor from
diet/drinking water test chemical concentration (ppm) to the actual dose, if
applicable; and
 details of food and water quality.

Results:
 body weight and 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)
 results of ophthalmological examination
 sensory activity, grip strength and motor activity assessments (when available)
 haematological tests with relevant baseline values
 clinical biochemistry tests with relevant baseline values
 circulating thyroid hormones (T4, T3, TSH; required)
 other hormone measures (optional)
 method of determining hormone values (assay type, supplier, protocol, etc.)
 terminal body weight, organ weights and organ/body weight ratios
 necropsy findings
 terminal vaginal cytology
 a detailed description of all histopathological findings
 total cauda epididymal sperm number, percent progressively motile sperm,
percent morphologically normal sperm, and percent of sperm with each
identified abnormality (optional)
 absorption (e.g., ADME or TK information) data if analysed
 statistical treatment of results, where appropriate
 for animals killed pre-terminally, the rationale behind the decision should be
reported; and
 for animals found dead during the study, the cause of death should, when
possible, be established..

Discussion of results.

Conclusions.

© OECD 2018
 OECD/OCDE 408
Adopted:
25 June 2018
│
LITERATURE

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You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.

In accordance with the Decision of the Council on a Delegation of Authority to amend Annex I of the Decision of the Council on the Mutual
Acceptance of Data in the Assessment of Chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meeting of the
Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.
14 │ 408 OECD/OCDE
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© OECD 2018
 OECD/OCDE 408 │ 15

ANNEX A

Definitions
Androgenicity is the capability of a chemical to act like a natural androgenic hormone
(e.g., testosterone) in a mammalian organism.
Antiandrogenicity is the capability of a chemical to suppress the action of a natural
androgenic hormone (e.g., testosterone) in a mammalian organism.
Antioestrogenicity is the capability of a chemical to suppress the action of a natural
oestrogenic hormone (e.g., oestradiol) in a mammalian organism.
Antithyroid activity is the capability of a chemical to suppress the action of a natural
thyroid hormone (e.g., T3) in a mammalian organism.
Dosage is a general term comprising of dose, its frequency and duration.
Dose is the amount of test chemical administered. Dose is expressed as weight (g, mg) or
as weight of test chemical per unit weight of test animal (e.g., mg/kg), or as constant
dietary concentrations (ppm).
Evident toxicity is a general term describing clear signs of toxicity following
administration of test chemical. 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.
HDL High density lipoprotein
LDL Low Density lipoprotein
NOAEL is the abbreviation for no-observed-adverse-effect level and is the highest dose
level where no adverse treatment-related findings are observed.
Oestrogenicity is the capability of a chemical to act like a natural oestrogenic hormone
(e.g., oestradiol) in a mammalian organism.
T3 Tri-iodothyronine – the active form of thyroid hormone
T4 Thyroxine – The main circulating thyroid gland product is converted to T3
Thyroid activity is the capability of a chemical to act like a natural thyroid hormone (e.g.,
T3) in a mammalian organism.
TSH Thyroid Stimulating Hormone – pituitary hormone that thyroid hormone production
and release from the thyroid gland
Validation is a scientific process designed to characterise the operational requirements
and limitations of a test method and to demonstrate its reliability and relevance for a
particular purpose.

© OECD 2018
16 │ 408 OECD/OCDE
ANNEX B.

Endpoints recommended for detection of endocrine activity

Required Measures Optional Measures

Organ Weights

Testes
Epididymides
Adrenal glands
Prostate + seminal vesicles with coagulating glands as a whole complex
Uterus
Ovaries
Pituitary gland
Thyroid gland
Histopathology
Thyroid and parathyroid glands Pancreatic islets
Adrenal glands
Pituitary gland2
Testis
Epididymides
Ventral and dorsolateral prostate
Seminal vesicles and coagulating glands
Ovaries2
Cervix2
Vagina2
Uterus2
Vaginal smear (collected at necropsy) to determine stage of oestrus
cycle2
Mammary glands (female and male)2
Serum/Plasma Biochemistry
Total cholesterol
HDL
LDL
Serum/Plasma Hormone Analyses
Thyroxine (T4) FSH
TSH LH
T3 Oestradiol
Testosterone
Sperm Measures
Cauda epididymis sperm reserves
Sperm motility
Sperm morphology
2
The condition of the oestrogen-sensitive organs in the female should be assessed with reference to the stage
of oestrus cycle at termination as endocrine active test agents may cause histological changes that, while not
overtly pathological, may differ from the condition anticipated based on the stage of ovarian cycle
Source: (OECD Guidance Document 106, parts 3, 4 (17)).

© OECD 2018
For Official Use