[Code of Federal Regulations]
[Title 40, Volume 28]
[Revised as of July 1, 2002]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR799.9365]

[Page 355-363]
 
                   TITLE 40--PROTECTION OF ENVIRONMENT
 
         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
 
PART 799--IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE TESTING REQUIREMENTS--Table of Contents
 
                Subpart H--Health Effects Test Guidelines
 
Sec. 799.9365  TSCA combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

    (a) Scope--(1) Applicability. This section is intended to meet 
testing requirements of the Toxic Substances Control Act (TSCA) (15 
U.S.C. 2601).
    (2) Source. The source material used in developing this TSCA test 
guideline is the Office of Prevention, Pesticides and Toxic Substances 
(OPPTS) harmonized test guideline 870.3650 (July 2000, final 
guidelines). This source is available at the address in paragraph (h) of 
this section.
    (b) Purpose. (1) This screening test provides limited information on 
systemic toxicity, neurotoxicity, and/or immunotoxicity following 
repeated exposure over a limited time period. In addition, it can be 
used to provide initial information on possible effects on male and 
female reproductive performance such as gonadal function, mating 
behavior, conception, development of the conceptus, and parturition. It 
is not an alternative to, nor does it replace, the existing test 
guidelines in Secs. 799.9370, 799.9380, 799.9620, and 799.9780 of this 
part.
    (2) This test does not provide complete information on all aspects 
of reproduction and development. In particular, it offers only limited 
means of detecting postnatal manifestations of prenatal exposure, or 
effects that may be induced during postnatal exposure. Due (amongst 
other reasons) to the selectivity of the end points, and the short 
duration of the study, this method will not provide evidence for 
definite claims of no reproduction/developmental effects.
    (3) This test can be used to provide initial information either at 
an early stage of assessing the toxicological properties of chemicals, 
or chemicals of high concern. It can also be used as part of a set of 
initial screening tests for existing chemicals for which little or no 
toxicological information is available or when otherwise considered 
relevant. It also can serve as an alternative to conducting two separate 
screening tests for repeated dose toxicity as described in Sec. 799.9305 
of this part and reproductive/developmental toxicity as described in 
Sec. 799.9355 of this part.
    (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR 
Part 792--Good Laboratory Practice Standards apply to this section. The 
following definitions also apply to this section.
    Dosage is a general term comprising dose, its frequency and the 
duration of dosing.
    Dose is the amount of test substance administered. Dose is expressed 
as weight (g, gm) or as weight of test substance per unit weight of test 
animal (e.g., mg/kg), or as constant dietary concentration (parts per 
million (ppm)).
    No-observed-effects level (NOEL) is the maximum dose used in a study 
which produces no adverse effects. The NOEL is expressed in terms of the 
weight of a test substance given daily per unit weight of test animal 
(milligrams per kilogram per day).
    (d) Principle of the test. (1) The test substance must be 
administered in graduated doses to several groups of males and females. 
Males should be dosed for a minimum of 4 weeks, up to and including the 
day before scheduled sacrifice (this includes a minimum of 2 weeks prior 
to mating, during the mating period and, approximately, 2 weeks post 
mating). In view of the limited pre-mating dosing period in males, 
fertility may not be a particularly sensitive indicator of testicular 
toxicity. Therefore, a detailed histological examination of the testes 
is essential. The combination of a pre-mating dosing period of 2 weeks 
and subsequent mating/fertility observations with an overall dosing 
period of at least 4 weeks, followed by detailed histopathology of the 
male gonads, is considered sufficient to enable detection of the 
majority of effects on male fertility and spermatogenesis.
    (2) Females should be dosed throughout the study. This includes 2 
weeks prior to mating (with the objective of covering at least two 
complete oestrous cycles), the variable time to conception, the duration 
of pregnancy and at least 4 days after delivery, up to and including the 
day before scheduled sacrifice.

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    (3) Duration of study, following acclimatization, is dependent on 
the female performance and is approximately 54 days, (at least 14 days 
pre-mating, (up to) 14 days mating, 22 days gestation, 4 days 
lactation).
    (4) During the period of administration, the animals are observed 
closely each day for signs of toxicity. Animals which die or are 
sacrificed during the test are necropsied and, at the conclusion of the 
test, surviving animals are sacrificed and necropsied.
    (e) Description of the method--(1) Selection of animal species. This 
test guideline is designed for use with the rat. If other species are 
used, appropriate modifications will be necessary. Strains with low 
fecundity or well-known high incidence of developmental defects should 
not be used. Healthy virgin animals, not subjected to previous 
experimental procedures, should be used. The test animals should be 
characterised as to species, strain, sex, weight and/or age. At the 
commencement of the study the weight variation of animals used should be 
minimal and not exceed [plusmn] 20% of the mean weight of each sex. 
Where the study is conducted as a preliminary study to a long-term or a 
full-generation study, preferably animals from the same strain and 
source should be used in both studies.
    (2) Housing and feeding conditions. (i) The temperature in the 
experimental animal room should be 22 [deg]C ([plusmn] 3[deg]). The 
relative humidity should be at least 30% and preferably not exceed 70% 
other than during room cleaning. 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.
    (ii) 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. Mating procedures should be carried out in cages 
suitable for the purpose. Pregnant females should be caged individually 
and provided with nesting materials.
    (3) Preparation of the animals. Healthy young adult animals must be 
randomised and assigned to the treatment groups and cages. Cages should 
be arranged in such a way that possible effects due to cage placements 
are minimized. The animals must be uniquely identified and kept in their 
cages for at least 5 days prior to the start of the study to allow for 
acclimatisation to the laboratory conditions.
    (4) Preparation of doses. (i) It is recommended that the test 
substance be administered orally unless other routes of administration 
are considered more appropriate. When the oral route is selected, the 
test compound is usually administered by gavage; however, alternatively, 
test compounds may also be administered via the diet or drinking water.
    (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 solution in other vehicles. For non-aqueous vehicles 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. It is recommended that 
each group be started with at least 10 animals of each sex. Except in 
the case of marked toxic effects, it is expected that this will provide 
at least eight pregnant females per group which normally is the minimum 
acceptable number of pregnant females per group. The objective is to 
produce enough pregnancies and offspring to assure a meaningful 
evaluation of the potential of the substance to affect fertility, 
pregnancy, maternal and suckling behaviour, and growth and development 
of the F1 offspring from conception to day 4 post-partum. If 
interim sacrifices are planned, the number should be increased by the 
number of animals scheduled to be sacrificed before the completion of 
the study. Consideration should be given to an additional satellite 
group of five animals per sex in the control and the top dose group for

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observation of reversibility, persistence or delayed occurrence of 
systemic toxic effects, for at least 14 days post treatment. Animals of 
the satellite groups must not be mated and, consequently, must not used 
for the assessment of reproduction/developmental toxicity.
    (2) Dosage. (i) Generally, at least three test groups and a control 
group should be used. If there are no suitable general toxicity 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. It should also be taken into account that there may 
be differences in sensitivity between pregnant and non-pregnant animals. 
The highest dose level should be chosen with the aim of inducing toxic 
effects but not death nor obvious suffering. Thereafter, a descending 
sequence of dose levels should be selected with a view to demonstrating 
any dosage related response and no adverse effects at the lowest dose 
level. Two- to four-fold intervals are frequently optimum 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 an oral study at 1-dose level of at least 1000 
mg/kg body weight/day or, for dietary 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 
several 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. For other types of administration, such as inhalation 
or dermal application, the physical chemical properties of the test 
substance often may dictate the maximum attainable exposure.
    (4) Administration of doses. (i) The animals are dosed with the test 
substance daily for 7 days a week. 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 1 ml/100 g 
body weight, except in the case of aqueous solutions where 2 ml/100 g 
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 (parts per million (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 at least weekly to 
maintain a constant dose level in terms of animal body weight.
    (5) Experimental schedule. (i) Dosing of both sexes should begin 2 
weeks prior to mating, after they have been acclimatized for at least 5 
days. The study should be scheduled in such a way that mating begins 
soon after the animals have attained full sexual maturity. This may vary 
slightly for different strains of rats in different laboratories, e.g., 
Sprague Dawley rats 10 weeks of age, Wistar rats about 12 weeks of age. 
Dams with offspring should be sacrificed on day 4 post-partum, or 
shortly thereafter. In order to allow for overnight fasting of dams 
prior to blood collection (if this option is preferred),

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dams and their offspring need not necessarily be sacrificed on the same 
day. The day of birth (viz. when parturition is complete) is defined as 
day 0 post-partum. Females showing no-evidence of copulation are 
sacrificed 24-26 days after the last day of the mating period. Dosing is 
continued in both sexes during the mating period. Males should further 
be dosed after the mating period at least until the minimum total dosing 
period of 28 days has been completed. They are then sacrificed, or, 
alternatively, are retained and continued to be dosed for the possible 
conduction of a second mating if considered appropriate.
    (ii) Daily dosing of the parental females should continue throughout 
pregnancy and at least up to, and including, day 3 post-partum or the 
day before sacrifice. For studies where the test substance is 
administered by inhalation or by the dermal route, dosing should be 
continued at least up to, and including, day 19 of gestation.
    (iii) Animals in a satellite group scheduled for follow-up 
observations, if included, must not mated. They should be kept at least 
for a further 14 days after the first scheduled sacrifice of dams, 
without treatment to detect delayed occurrence, or persistence of, or 
recovery from toxic effects.
    (iv) The experimental schedule is given in the following figure 1.
    [GRAPHIC] [TIFF OMITTED] TR15DE00.065
    
    (6) Mating procedure. Normally, 1:1 (one male to one female) matings 
should be used in this study. Exceptions can arise in the case of 
occasional deaths of males. The female should be placed with the same 
male until pregnancy occurs or 2 weeks have elapsed. Each morning the 
females should be examined for the presence of sperm or a vaginal plug. 
Day 0 of pregnancy is defined as the day a vaginal plug or sperm is 
found. In case pairing was unsuccessful, re-mating of females with 
proven males of the same group could be considered.
    (7) Observations. (i) 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 must be observed for morbidity and mortality.
    (ii) Once before the first exposure (to allow for within-subject 
comparisons),

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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 
day. 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), 
difficult or prolonged parturition or bizarre behaviour (e.g., self-
mutilation, walking backwards) should also be recorded.
    (iii) At one time during the study, sensory reactivity to stimuli of 
different modalities (e.g., auditory, visual and proprioceptive stimuli) 
assessment of grip strength and motor activity assessment should be 
conducted in five males and five females, randomly selected from each 
group. 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. In males, these functional 
observations should be made towards the end of their dosing period, 
shortly before scheduled sacrifice but before blood sampling for 
hematology or clinical chemistry. Females should be in a physiologically 
similar state during these functional tests and should preferably be 
tested during lactation, shortly before scheduled sacrifice. In order to 
avoid hypothermia of pups, dams should be removed from the pups for not 
more than 30 to 40 minutes. Examples of procedures for observation are 
described in the references in paragraphs (h)(3), (h)(4), (h)(5), 
(h)(6), and (h)(7) of this section.
    (iv) Functional observations made once towards the end of the study 
may be omitted when the study is conducted as a preliminary study to a 
subsequent subchronic (90-day) or long-term 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 
this repeated dose study may enhance the ability to select dose levels 
for a subsequent subchronic or long-term study.
    (v) 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.
    (vi) The duration of gestation should be recorded and is calculated 
from day 0 of pregnancy. Each litter should be examined as soon as 
possible after delivery to establish the number and sex of pups, 
stillbirths, live births, runts (pups that are significantly smaller 
than corresponding control pups), and the presence of gross 
abnormalities.
    (vii) Live pups should be counted and sexed and litters weighed 
within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 
post-partum. In addition to the observations on parental animals, 
described by paragraphs (f)(7)(ii) and (f)(7)(iii) of this section, any 
abnormal behaviour of the offspring should be recorded.
    (8) Body weight and food/water consumption. (i) Males and females 
should be weighed on the first day of dosing, at least weekly 
thereafter, and at termination. During pregnancy, females should be 
weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 
0 or 1 post-partum), and day 4 post-partum. These observations should be 
reported individually for each adult animal.
    (ii) During pre-mating, pregnancy and lactation, food consumption 
should be measured at least weekly. The measurement of food consumption 
during mating is optional. Water consumption during these periods should 
also be measured, when the test substance is administered by that 
medium.
    (9) Hematology. (i) Once during the study, the following 
hematological examinations should be made in five males and five females 
randomly selected from each group: hematocrit, hemoglobin concentration, 
erythrocyte

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count, total and differential leucocyte count, platelet count and a 
measure of blood clotting time/potential.
    (ii) Blood samples should be taken from a named site. Females should 
be in a physiologically similar state during sampling. In order to avoid 
practical difficulties related to the variability in the onset of 
gestation, blood collection in females may be done at the end of the 
pre-mating period as an alternative to sampling just prior to, or as 
part of, the procedure for sacrificing the animals. Blood samples of 
males should preferably be taken just prior to, or as part of, the 
procedure for sacrificing the animals. Alternatively, blood collection 
in males may also be done at the end of the pre-mating period when this 
time point was preferred for females.
    (iii) Blood samples should be stored under appropriate conditions.
    (10) 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 from 
the selected five males and five females of each group. Overnight 
fasting of the animals prior to blood sampling is recommended\1\. 
Investigations of plasma or serum must 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 and sorbitol 
dehydrogenase) and bile acids. Measurements of additional enzymes (of 
hepatic or other origin) may provide useful information under certain 
circumstances.
---------------------------------------------------------------------------

    \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 (pregnant) animals, 
disturbs lactation and nursing behaviour, 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.
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    (ii) Optionally, the following urinalysis determinations could be 
performed in five randomly selected males of each group during the last 
week of the study using timed urine volume collection; appearance, 
volume, osmolality or specific gravity, pH, protein, glucose and blood 
or 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 and fasting glucose, specific hormones, 
methemoglobin and cholinesterase. These need to be identified on a case-
by-case basis.
    (iv) Overall, there is a need for a flexible approach, depending on 
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.
    (11) Pathology--(i) Gross necropsy. (A) All adult animals in the 
study must 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. Special attention should be paid to the organs of the 
reproductive system. The number of implantation sites should be 
recorded. Corpora lutea should be counted.
    (B) The testes and epididymides of all adult males should be weighed 
and the ovaries, testes, epididymides, accessory sex organs, and all 
organs showing macroscopic lesions of all adult animals, should be 
preserved.
    (C) In addition, for five adult males and females, randomly selected 
from each group, the liver, kidneys, adrenals, thymus, spleen, brain and 
heart should be trimmed of any adherent tissue, as appropriate and their 
wet weight taken as soon as possible after dissection to avoid drying. 
Of the selected males and females, the following

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tissues should also 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), uterus, urinary bladder, 
lymph nodes (preferably 1 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 marrow aspirate).
    (D) Formalin fixation is not recommended for routine examination of 
testes and epididymides. An acceptable method is the use of Bouin's 
fixative for these tissues. 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.
    (E) Dead pups and pups sacrificed at day 4 post-partum, or shortly 
thereafter, should, at least, be carefully examined externally for gross 
abnormalities.
    (ii) Histopathology. (A) Full histopathology should be conducted on 
the preserved organs and tissues of the selected animals in the control 
and high dose groups and all gross lesions. These examinations should be 
extended to animals of other dosage groups if treatment-related changes 
are observed in the high dose group.
    (B) Detailed testicular histopathological examination (e.g., using 
Bouin's fixative, paraffin embedding and transverse sections of 4-5 
[plusmn]m thickness) should be conducted with special emphasis on stages 
of spermatogenesis and histopathology interstitial testicular cell 
structure. The evaluation should identify treatment-related effects such 
as retained spermatids, missing germ cell layers or types, 
multinucleated giant cells or sloughing of spermatogenic cells into the 
lumen (the specifications for the evaluation are discussed in paragraph 
(g)(2) of this section). Examination of the intact epididymis should 
include the caput, corpus, and cauda, which can be accomplished by 
evaluation of a longitudinal section. The epididymis should be evaluated 
for leukocyte infiltration, change in prevalence of cell types, aberrant 
cell types, and phagocytosis of sperm. Periodic acid-Schiff (PAS) and 
hematoxylin staining may be used for examination of the male 
reproductive organs. Histopathological examination of the ovary should 
detect qualitative depletion of the primordial follicle population.
    (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. Individual animal 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 sacrificed for 
humane reasons, the time of any death or humane sacrifice, the number of 
fertile animals, the number of pregnant females, the 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, the types of histopathological changes, and all relevant litter 
data.
    (2) Evaluation of results. (i) The findings of this toxicity study 
should be evaluated in terms of the observed effects, necropsy and 
microscopic findings. The evaluation will include the relationship 
between the dose of the test substance and the presence or absence, 
incidence and severity of abnormalities, including gross lesions, 
identified target organs, infertility, clinical abnormalities, affected 
reproductive and litter performance, body weight changes, effects on 
mortality and any other toxic effects.
    (ii) Because of the short period of treatment of the male, the 
histopathology of the testes and epididymides must be considered along 
with the fertility data, when assessing male reproduction effects. The 
use of

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historic control data on reproduction/development (e.g. for litter size) 
where available may also be useful as an aid to the interpretation of 
the study.
    (iii) When possible, numerical results should be evaluated by an 
appropriate and general acceptable statistical method. The statistical 
methods should be selected during the design of the study. Due to the 
limited dimensions of the study, statistical analysis in the form of 
tests for ``significance'' are of limited value for many endpoints, 
especially reproductive endpoints. Some of the most widely used methods, 
especially parametric tests for measures of central tendency, are 
inappropriate. If statistical analyses are used then the method chosen 
should be appropriate for the distribution of the variable examined and 
be selected prior to the start of the study.
    (3) Test report. The test report must include the following 
information:
    (i) Test substance:
    (A) Physical nature and, where relevant, physicochemical properties.
    (B) Identification data.
    (ii) Vehicle (if appropriate): Justification for choice of vehicle, 
if other than water.
    (iii) Test animals:
    (A) Species/strain used.
    (B) Number, age and sex of animals.
    (C) Source, housing conditions, diet, etc.
    (D) Individual weights of animals at the start of the test.
    (iv) Test conditions:
    (A) Rationale for dose level selection.
    (B) Details of test substance formulation/diet preparation, achieved 
concentration, stability and homogeneity of the preparation.
    (C) Details of the administration of the test substance.
    (D) Conversion from diet/drinking water test substance concentration 
(parts per mission (ppm)) to the actual dose (mg/kg body weight/day), if 
applicable.
    (E) Details of food and water quality.
    (v) Results (toxic response data by sex and dose):
    (A) Time of death during the study or whether animals survived to 
termination.
    (B) Nature, severity and duration of clinical observations (whether 
reversible or not).
    (C) Body weight/body weight change data.
    (D) Food consumption and water consumption, if applicable.
    (E) Sensory activity, grip strength and motor activity assessments.
    (F) Hematological tests with relevant baseline values,
    (G) Clinical biochemistry tests with relevant baseline values.
    (H) Effects on reproduction, including information on mating/
precoital interval, fertility, fecundity and gestation duration.
    (I) Effects on offspring, including number of pups born (live and 
dead), sex ratio, postnatal growth (pup weights) and survival (litter 
size), gross abnormalities and clinical observations during lactation.
    (J) Body weight at termination and organ weight data for the 
parental animals.
    (K) Necropsy data, including number of implantations and number of 
corpora lutea.
    (L) Calculations of pre- and postimplantation loss.
    (M) Detailed description of histopathological findings.
    (N) Statistical treatment of results, where appropriate.
    (vi) Discussion of results.
    (vii) Conclusions.
    (h) References. For additional background information on this test 
guideline, the following references should be consulted. These 
references are available for inspection at the TSCA Nonconfidential 
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M 
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, 
except legal holidays.
    (1) Mitsumori, K., Kodama, Y., Uchida, O., Takada, K., Saito, M. 
Naito, K., Tanaka, S., Kurokawa, Y., Usami, M., Kawashima, K., Yasuhara, 
K., Toyoda, K., Onodera, H., Furukawa, F., Takahashi, M. and Hayashi, 
Y., (1994). Confirmation Study, Using Nitro-Benzene, of the Combined 
Repeat Dose and Reproductive/ Developmental Toxicity Test Protocol 
Proposed by the Organization for Economic Cooperation and Development 
(OECD). Journal of Toxicology and Science, 19:141-149.
    (2) Tanaka, S., Kawashima, K., Naito, K., Usami, M., Nakadate, M., 
Imaida, K.,

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Takahashi, M., Hayashi, Y., Kurokawa, Y. and Tobe, M. (1992). Combined 
Repeat Dose and Reproductive/Developmental Toxicity Screening Test 
(OECD): Familiarization Using Cyclophosphamide. Fundamental and Applied 
Toxicology, 18:89-95.
    (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 
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Neurotoxicology and Teratology 13:599-609.

[65 FR 78793, Dec. 15, 2000]