Toxicological Principles for the Safety Assessment of Food Ingredients
Return to Redbook 2000 table of contents
This guidance represents the Food and Drug Administration's (FDA's) current thinking on this topic. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. You can use an alternative approach if the approach satisfies the requirements of the applicable statutes and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance.
In the U. S., the Food and Drug Administration (FDA) is the agency responsible for ensuring that the are safe for all consumers. In order to determine the safety of these food ingredients for consumption, appropriate information and results from a series of tests must be made available to the agency. In 1982, in an effort to provide guidance to the food industry concerning the appropriate tests for the determination of safety, the FDA issued the Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Foods, commonly referred to as the Redbook.(23) In 1993, based on the expansion of technology and the use of food ingredients, as well as the refinement of the scientific criteria for establishing safety, the FDA updated its guidelines and issued the draft Redbook II.(24) Since the draft Redbook II was issued, additional refinements have been made in the procedures for the multigeneration reproduction study and for the assessment of effects on male reproduction. The latest proposed guidelines for multigeneration studies are provided here, in Redbook 2000.
During the past several decades, the technology of food processing has changed dramatically and the use and variety of food ingredients have increased. In the U. S., the Food and Drug Administration (FDA) is the agency responsible for ensuring that food ingredients are safe for all consumers. Safety, as it pertains to food ingredients , is defined in the Code of Federal Regulations as a "reasonable certainty ... that the substance is not harmful under the intended conditions of use".(25) In order to obtain a "reasonable certainty" of meeting the regulation, appropriate information and results from a series of tests must be made available to the agency. Just as the technology and use of food ingredients have expanded, so the scientific criteria for establishing safety have also been refined.
In an effort to provide guidance to industry concerning the appropriate tests for the determination of safety, the FDA issued the Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Foods.(23) The book is commonly referred to as the Redbook. In 1993, based on increased knowledge of toxicological processes and procedures as well as changes in the food industry, the FDA updated its guidelines and issued the draft Redbook II.(24)
In 1982, a three-generation reproduction study in rats was recommended, with a teratology phase as part of the battery of tests for substances in concern levels two and three. In 1993, a multigeneration reproduction study with a teratology phase for concern levels two and three was still required, but the multigeneration study was streamlined to two generations with a single litter per generation.
Concern levels, as determined by the agency, are "relative measures of the degree to which the use of an additive may present a hazard to human health".(24) The concern level is based on the extent of human exposure (dose) and the toxicological effects on biological systems. There are three broad bands of concern levels. Concern level three represents the highest probable risk to human health. Concern level one represents the lowest probable risk. Concern level two is intermediate between high and low risk.
Draft Redbook II also included general guidelines for assessing effects on male reproductive function and optional neurotoxicity and immunotoxicity screens. Since Draft Redbook II was released, additional refinements have been made in the procedures for the multigeneration reproduction study and for the assessment of effects on male reproduction. The latest proposed guidelines for multigeneration studies are provided here.
In a multigeneration reproduction study, the test substance is administered to parental (F0) males and females prior to and during mating, gestation, and through the weaning of F1 offspring. The test substance is then given to selected F1 generation offspring during their growth and development to adulthood, and through the mating period. Pregnant F1 generation females continue to receive the test substance throughout gestation and until the F2 generation offspring until the offspring are weaned.
III. Guideline for Reproduction Studies
The guideline for reproduction studies detailed below pertains to substances given orally to rodents. It is designed to evaluate the effects of a test substance on the reproductive systems of both males and females, the postnatal maturation and reproductive capacity of offspring, and possible cumulative effects through several generations. A study can provide information concerning the effects of a substance on gonadal function, estrous cycles, mating behavior, conception, parturition, neonatal morbidity, mortality, lactation, weaning, growth and development of the offspring, and target organs in the offspring. The study may also serve as a guide for subsequent tests. The end points evaluated and the indices calculated must provide sufficient information and statistical power to permit the FDA to determine whether the chemical is associated with changes in reproduction and fertility. Additional and historical information is found in Collins,(4) Francis and Kimmel,(6) and U.S. Environmental Protection Agency.(21)
The minimal reproduction study recommended consists of two generations, with one litter per generation (see Figure 1). If results of developmental and other toxicity tests indicate that a test substance may be associated with developmental toxicity, the minimal reproduction study should be expanded. This guideline contains optional procedures for inclusion of additional litters per generation, additional generations, a test for teratogenic and developmental toxicity effects, optional neurotoxicity screening, and optional immunotoxicity screening.
Figure 1. 2-Generation Reproduction and Teratology Study
A. General Recommendations
The following recommendations are applicable to all FDA toxicity studies:
- Studies should be conducted according to Good Laboratory Practice Regulations (GLPs).(22)
- Animals should be cared for, maintained, and housed according to the recommendations contained in the Guide for the Care and Use of Laboratory Animals.(10)
- Healthy animals that have not been subjected to previous experimental procedures should be used. Generally, it is not possible to treat animals for infection during the course of a study without the risk of interaction between the treatment drug and the test substance. The females should not be pregnant and should be nulliparous.
- Test animals should be characterized by reference to their species, strain, sex, and weight or age.
- Animals should be assigned to control and experimental groups in a stratified random manner to minimize bias and ensure compatibility across experimental and control groups for statistical purposes. Each animal must be assigned a unique number.
B. Dose Range-Finding Study
A dose range-finding study is recommended to determine the most appropriate doses, unless suitable pharmacokinetic and metabolic data concerning the test substance are available prior to the start of the study. The dose range-finding study should preferably, but not necessarily, be done in pregnant animals. Comparison of the results from a trial study in non-pregnant animals and a main study in pregnant animals should establish whether the test substance is more or less toxic in pregnant animals than in non-pregnant animals.
C. Main Study
1. Experimental Animals, Species and Strain Selection and Housing
Because of the expense and length of time needed for multigeneration studies, the species selected should be one that will yield the greatest amount of information per unit cost. Rodents such as rats and mice are usually selected for use in multigeneration studies. The rat is the preferred species because of the small size of the animals, ease of breeding in the laboratory, gestation length of approximately three weeks, high fertility rate, and spontaneous ovulation. The litters are large enough to allow for inter- and intra-litter comparisons, and the animals are less susceptible to stress effects than are mice. Strains with low fecundity should not be used. Single housing of the animals is recommended, except during mating. The animals' diet should meet all nutritional requirements to support pregnancy and lactation in the test species.
2. Number, Sex, and Age
Exposure to the test substance typically begins when the rats are five to nine weeks of age. All test and control animals should be acclimated to the study conditions before treatment begins. The acclimation period is usually one week except under unusual conditions. Each test and control group should consist of animals of uniform weight and age and should start with a number of animals sufficient to contain approximately 20 males and 20 pregnant females near term. In order to achieve this number, it is usually necessary to start with 30 animals per sex per group in the first parental group (F0) and 25 animals per sex per group (at least one male and one female from each litter, with a maximum of two of each sex per litter) in the parents of each consecutive generation.
3. Assignment to Dose Groups
Animals should be assigned to test and control groups in a stratified random manner to minimize intergroup weight differences. Each animal should be uniquely identified and the litter of origin for each F1 animal should be identified.
4. Dose Selection
A minimum of three doses of the test substance (high, intermediate, and low doses) should be used to facilitate the separation of dose-related responses from experimental variation. The high dose should produce some parental toxicity (such as reduced body weight or weight gain) but not more than 10% parental mortality. The dose should not exceed 5% of the diet for non-nutritive additives. In dietary studies for macronutrient additives, high doses should be based on nutritional effects rather than toxicological end points. The lowest dose should not induce observable adverse parental effects and should be set at a level which is expected to provide a minimal margin of safety. The intermediate dose(s) should be spaced to allow an arithmetic or geometric progression between the high and low doses. The addition of one or more extra groups is preferable to large intervals between doses.
5. Control Group(s)
A concurrent control group is required. Control animals should be fed and handled the same as dosed animals and should be caged in such a way as to preclude airborne or other contamination by the test substance. For dietary studies, the control group should be fed the basal diet. When a carrier vehicle for the test substance is used, the volume of vehicle given to control rats should be equal to the maximal amount of vehicle given to any dosed group. If there are insufficient data on the toxic properties of the vehicle used in administering the test substance, a sham control group could be included. An additional control group that is not exposed to the vehicle should be included in the study. If a test substance causes reduced dietary intake, a pair-fed control group should be considered.
6. Duration of Testing
Animals should be exposed to the test substance during the entire study. Males of the first parental group (F0) should be dosed for the duration of spermatogenesis and epididymal transit (at least ten weeks) before mating and throughout the mating period, in order to detect adverse effects on spermatogenesis by the test substance. The first parental females (F0) should be exposed before mating for the same length of time (at least ten weeks) as the males and then through mating and pregnancy, to the weaning of the F1a litter. Litters (usually F1a and F2a) should be exposed from the prenatal period throughout their entire postnatal lives. If a third generation is planned, these litters also should be exposed from the prenatal period and throughout their entire lives.
7. Substance Administration
The test substance may be administered to rodents in the diet, in drinking water, or by gavage (stomach tube intubation). The same route of administration should be used for all animals throughout the study. If the test substance is given by gavage it is best to adjust the volume daily or every three days based on the animal's body weight.
8. Mating Procedures
For each mating, a female should be placed with a single randomly selected male (one:one mating) from the same dose group until pregnancy occurs or two to three weeks have elapsed. Mating of one male with two female rats is permitted in the event that a male dies.
Animals should be separated as soon as possible after evidence of copulation has been observed. If mating has not occurred after two to three weeks, the animals should be separated without further opportunity for mating. Mating pairs should be clearly identified in the data. Each morning, all females should be examined for the presence of sperm in the vaginal lavage or the presence of a vaginal plug; if sperm and/or a vaginal plug are found, this is considered day zero of gestation. Near parturition, pregnant females should be caged separately in delivery or maternity cages and may be provided with nesting materials. Pregnant females in test and control groups should be allowed to litter normally.
9. Standardizing the Number of Pups per Litter
Standardization of the number of pups per litter through culling is optional. Litters may be standardized to ten (or eight) based on historical litter size for the strain. It is recommended that standardization be performed on postnatal day four by reducing all litters of more than ten (or eight) to ten (or eight) in a random manner. If possible, the retained litter-mates should consist of equal numbers of males and females. Random selection is important to guard against the human tendency to keep the most fit animals in the study.
10. Selection of Parental Animals for Next Generation
At least one male and one female should be randomly selected from each litter for mating with another pup of the same dose level but different litter to produce the next generation. If there are insufficient litters from which to make a selection, then no more than two males and two females per litter should be included in the group. As many litters as possible should be represented. The mating procedures for the F1 males and females should be carried out in the same manner as the F0 parental animals. Care should be taken that siblings are not mated on a study. F1 males and females not selected for mating should be terminated after weaning.
11. Optional Third Generation
If overt reproductive, morphologic, and/or toxic effects of a test substance are observed in offspring during the two-generation reproduction study, the study may be extended to a third generation to determine cumulative effects of the substance. Selection of animals for mating and mating for an additional generation should be carried out by the same procedures as for the first generation. Randomly mated animals from the F2a litter should be mated to produce the third generation. F3a animals should be weaned and either necropsied or used for a longer-term toxicity study.
12. Optional Second Mating
If production of a second litter is necessary, the dams should be mated again approximately one to two weeks after weaning of the F1a or F2a litter.
13. Optional Teratology Phase
The teratology phase should be incorporated into the multigeneration reproduction study unless justification can be provided for conducting a separate developmental study. In a reproduction study, either the F2b or the F3b litter can be used to determine fetotoxic effects of the test substance. If a teratology phase is to be performed, pregnancy should be timed by the presence of sperm in the vaginal lavage or by the presence of a vaginal plug, and this considered as day zero of gestation. Approximately one day before expected parturition, the dams should be euthanized and Cesarean sections performed. The uterus should be opened and examined for the presence of early and late deaths, and corpora lutea should be counted. Each live fetus should be removed from the uterus. The weight and sex of each live fetus should be determined. Each live fetus should be examined for gross malformations and then for skeletal or soft-tissue abnormalities. Additional, detailed procedures are found in the Food and Drug Administration Proposed Testing Guidelines for Developmental Toxicity Studies.(13)
14. Clinical Observations
Each animal should be observed at least twice each day. The first observation should be a thorough clinical examination. The second may involve the observation of the animals through their cages. Observation of the animals through their cages is satisfactory for pregnant animals near term and for animals nursing their litters. Observation times should be selected to permit detection of the onset and progression of all toxic and pharmacologic effects of the test substance and to minimize the loss of animals and organs/tissues. Relevant behavioral changes and all signs of toxicity, including mortality, should be recorded. Estrous cycle length and normality should be evaluated daily by vaginal smears for all F0 and F1 females during a minimum of three weeks prior to mating and during cohabitation. The duration of gestation should be calculated from day zero of pregnancy. Care should be taken to prevent the induction of pseudopregnancy.
Individual records should be maintained for all adult animals and offspring selected for the next generation. Toxicological and pharmacological symptoms and signs, including behavioral abnormalities, should be recorded daily; records should include the date of onset, duration, and intensity of symptoms and signs.
Animals should be weighed immediately before the test substance is first administered, once weekly thereafter, and at necropsy. Feed consumption also should be recorded weekly at a minimum. If the substance is given in the diet, weekly body weights are acceptable. If the substance is given by gavage it is best to adjust the volume daily or every three days on the basis of the animal's body weight. Water consumption should be measured if the test substance is administered in the water and may also be measured if it is thought that the substance might influence fluid consumption.
15. Growth of Offspring
Each litter should be examined as soon as possible after delivery for the number of pups, stillbirths, live births, and the presence of gross anomalies. Dead pups should be necropsied and observed for possible gross defects and the cause of death unless excessive autolysis renders specimens useless.
The neonates should be carefully observed, and their sex and weight should be noted on postnatal days zero (day of birth), four, seven, fourteen, and 21. Other appropriate days are acceptable to monitor postnatal growth and other developmental indices.
Anogenital distance should be measured at day zero for all F2 pups that show treatment-related effects in F1 sex ratio or sexual maturation. The age and weight of each animal on the day of vaginal opening or balano-preputial separation should be recorded for F1 weanlings selected for mating.
16. Optional Neurotoxicity Screening
Multigeneration reproduction studies provide an excellent vehicle to screen for potential developmental neurotoxicity. Periodic examination of the developing offspring provides information to help detect treatment-related changes in development, the appearance of neurological disorders, and other signs of nervous system toxicity. The examination of the offspring should be as brief as possible to minimize the period of separation from the dam. During the examination, any abnormalities in the animal's appearance or behavior should be noted as well as markers to gauge age-appropriate physical development (such as eye opening, genital development, and incisor eruption) and functional development (such as righting reflex, startle response, and motility). As an alternative option, satellite groups of litters (using suitable numbers for adequate statistical analysis) may be used to screen for developmental neurotoxicity. The inclusion of other endpoints should be encouraged, such as the assessment of cognitive function development. All data derived from the examination of the experimental offspring, including positive and negative findings, should be documented, statistically analyzed as appropriate, using the litter as the statistical unit, and reported. Additional information is available in Sobotka et al.(19)
17. Optional Immunotoxicity Screening
Multigeneration reproduction studies can screen the effects of a test substance on a developing immune system and evaluate the potential for immunotoxicity by the use of non-invasive (Type I) tests and invasive (Type II) tests.(24) Since the effects of in-utero exposure are not normally assessed in chronic, acute, and subchronic studies, Type I immunotoxicity testing should be conducted on exposed dams and F1 male and female offspring. By careful planning, animals used or produced in the reproduction study may be evaluated for Type I indicators, and possibly Type II. For example, when F0 parental males have completed the mating cycle, representative animals could be sacrificed or fed longer if data from chronic, acute, and subchronic studies are not available. After weaning, F0 parental females that are no longer needed are an excellent source of animals for evaluating potential immunotoxicity effects. For evaluating the effects of in-utero exposure, neonatal specimens from culled litters could be used for histologic evaluation of the neonatal lymphoid organs. Only a small number of weanlings of each sex are selected for further use in the reproduction study; the remaining animals could be sacrificed and evaluated at three weeks or allowed to mature to six or eight weeks of age. At this time, Type I testing and/or functional Type II testing could be performed. Similar opportunities exist in the F2 generation. Additional information is available in Hinton et al.(9)
18. Gross Necropsy and Microscopic Examination
All adult males and females should be terminated when they are no longer needed for the assessment of reproductive findings. Any dam that shows signs of imminent abortion, premature delivery, or moribund condition should be necropsied on the day such signs are observed. Dead pups (pups that die spontaneously during the postpartum phase) should be necropsied and observed for possible gross defects and the cause of death unless they are excessively autolyzed.
At the time of termination, all parental animals should be examined macroscopically for any structural abnormalities or structural changes. This should include examination of external surfaces, orifices, cranial cavity, carcass, and all organs. Special attention should be paid to the organs of the reproduction system. The uterus should be examined for the presence of implantation sites and resorptions. The uterus may be stained with sodium or ammonium sulfide (16) or other appropriate chemical to help visualize the implantation sites.
Necropsy of Weanlings
At the time of termination, at least two pups per sex per litter from unselected F1 and F2 weanlings should be examined macroscopically for any structural abnormalities or structural changes. Special attention should be paid to the organs of the reproduction system.
Brain, thymus, and spleen should be weighed from the F1 and F2 weanlings that are examined macroscopically for structural abnormalities or structural changes. At necropsy, grossly abnormal organs and tissues from pups from all dose groups should be preserved and then examined histopathologically.
Necropsy of Parental Animals
At necropsy, the following organs of all F0 and F1 control and treated parental animals should be observed and weighed: brain, pituitary, liver, kidneys, adrenal glands, spleen, known target organs, and reproductive organs. Uterus and ovaries of females should be weighed. For males, both testes, seminal vesicles with coagulating glands, and the prostate should be weighed. In addition, the total epididymal weight should be determined for one epididymis that will be fixed for histopathology, and both total epididymal and cauda epididymal weight should be determined for the epididymides that will be used for observing sperm morphology, numbers, and motility. Seminal vesicles and prostates should be weighed separately. The source of the prostate weight should be identified (e.g., as ventral and/or dorsal and/or dorsolateral prostate). At necropsy, the contralateral testis and epididymis (the non-fixed testis and epididymis) should be utilized for the determination of homogenization resistant spermatid numbers and cauda epididymal sperm reserves, respectively. Additionally, sperm from the cauda epididymis (or proximal vas deferens) should be collected for the evaluation of sperm motility and sperm morphology.
Since testis weight varies only slightly within a given species, a change in testis weight may indicate that a test substance has had an adverse effect on the testis. Seminal vesicles and prostates are androgen-dependent organs and changes in their weights may indicate a change in the endocrine status of the animal or the ability of the testis to produce androgen.
Organ weight should be reported as absolute weight and as a relative weight (e.g., organ-to-body or organ-to-brain weight).
Fixation of Tissues and Organs
At necropsy, the following organs and tissues, or representative samples thereof, from all parental animals, should be fixed and stored in a medium suitable for histopathological examination. For parental females, the vagina, uterus with cervix, ovaries with oviducts, adrenal and pituitary glands, target organs, and grossly abnormal tissue should be preserved. For parental males, one testis, one epididymis, seminal vesicles, coagulating glands, prostate, and adrenal and pituitary glands, target organs, and grossly abnormal tissue should be preserved. Testicular tissues should be fixed in Bouin's or a comparable fixative and stored in a suitable medium for histopathological examination. Several articles and books have been written recently to describe methods that can be used to preserve testicular tissue and evaluate histopathology.(8),(15) If an immunotoxicity screen is being included in the study, the appropriate procedures and organs mentioned in Redbook II(24) should be followed.
Full histopathological examination of the organs should be performed for ten randomly selected control and high-dose F0 and F1 animals per sex. If the high-dose group reveals a treatment-related effect, ten animals from each intermediate dose group should be randomly selected and examined. Tissues and organs preserved from the additional animals in each group may be examined to provide additional data.
In addition, a full histopathological examination should be performed on the reproductive organs of animals suspected of reduced fertility from intermediate dose groups. Signs of reduced fertility include failure to mate, conceive, sire, or deliver healthy offspring; effects on estrous cycle; reduced reproductive organ weight; and reduced testicular spermatid counts or cauda epididymal sperm counts.
Histopathology of Female Reproductive Organs
The post-lactational ovary should contain primordial and growing follicles as well as the large corpora lutea of lactation. Histopathological examination should detect qualitative depletion of the primordial follicle population. A quantitative evaluation of primordial follicles should also be conducted. If the high-dose animals reveal a treatment-related effect, all groups should be examined. The following evaluation technique may be used, but others may be used if the number of animals, ovarian section selection, and section sample size are statistically appropriate. Substance-induced depletion of primordial follicles can be identified by removing five sections from the inner third of each ovary. The sections should be at least 0.1 mm (100 µm) thick. Examination should include enumeration of the total number of primordial follicles from these ten sections for comparison with control ovaries. Examination should also confirm the presence or absence of growing follicles and corpora lutea in comparison with control ovaries. Additional information can be found in Bolon et al.,(1) Bucci et al.(2) and Heindel.(7)
Histopathology of Male Reproductive Organs
Histopathological assessment of the epididymis should include an evaluation of the corpus, cauda and caput epididymis. This can be accomplished by examining a longitudinal section through all three regions of the epididymis in order to identify such lesions as sperm granulomas, leukocyte infiltration, aberrant cell types within the lumen, or the absence of clear cells in the cauda epididymal epithelium.(5)
Careful histopathological examination of the testis is recognized as a sensitive method to identify effects on spermatogenesis. Testicular tissue should be examined with a knowledge of testis structure, the process of spermatogenesis, and the classification of spermatogenesis. If an effect is observed, it should be described in detail.(15) If testicular effects are quantitated, the methods used should be described in detail.
A thorough histological evaluation of the testis should include an examination of the interstitial compartment and the seminiferous tubule compartment. A histopathological evaluation of the intertubular cell compartment of the testis should include a general assessment of the Leydig cells, the blood vessels, and the cell types other than the Leydig cells typically found in the intratubular space. The general appearance of the seminiferous tubules should be noted This should be followed by an examination of the seminiferous tubule compartment to detect any disruption in the normal sequence of the events that occurs during the normal process of spermatogenesis. The seminiferous epithelium should then be carefully observed to detect any of the following: presence of multinucleated cells, missing germ cell layers, increased germ-cell degeneration, abnormal development in germ cells, sperm release delay or failure, presence of germ cells in the seminiferous tubule lumen, and any changes in the Sertoli cells (vacuolization, sloughing, or nuclear changes). The general condition of the boundary layer should be noted.
D. End Points of Female Reproductive Toxicity
End points of reproductive toxicity are usually expressed as indices that encompass the animals' responses to the test substance from conception to weaning. The following indices should be calculated for each reproduction study: female fertility, gestation, and live-born indices; weaning index or lactation index; sex ratio; and viability indices at postnatal days four, seven, fourteen, and 21.
1. Female Fertility Index
The female fertility index represents the percent of matings that result in pregnancies. It is calculated as follows: (number of pregnancies/number of matings) X 100. This index reflects the total number of dams that have achieved pregnancy, including those that deliver at term, abort, or have fully resorbed litters. This index depends on male libido and fertility as well as female cyclicity and receptiveness.
2. Gestation Index
The gestation index evaluates the efficiency of pregnancy that results in at least one live offspring. In this index, the litter with one live offspring is counted the same as one with more than one live offspring. The index is calculated as follows: (number of litters with live pups/number of pregnancies) X 100.
3. Live-born Index
Related to the gestation index, the live-born index (number of pups born alive/total number of pups born) X 100 is a measure of the total number of offspring lost, regardless of litter.
4. Weaning Index
The weaning index represents the ability of pups to survive from day four to day 21. It is calculated as follows: (number of pups alive at day 21/number of pups alive and kept on day four) X 100. This index corrects for the reduction of pups on day four. If the pups are not reduced, a related index, the lactation index, is calculated: (number of pups alive on day 21/number of pups alive on day four) X 100. Regardless of the etiology, a decrease in the weaning index indicates adverse reproductive effects.
5. Sex Ratio and Percentage by Sex
Determining the sex of pups at birth and verifying their sex at each weighing permits the relative fitness of each sex to be calculated as the offspring mature. The sex ratio is useful in detecting if the test substance is preferentially affecting one sex. This parameter is usually calculated as follows: (number of males/number of females). The related calculation (number of females or males/total number of animals) X 100 yields the percentage of total animals that are male or female.
6. Viability Indices
The viability indices are measures of the offsprings' ability to survive during specific brief intervals of their lives, from birth (day zero) to day four, day four to day seven, day seven to day fourteen, day fourteen to day 21, or they may reflect longer intervals, such as day zero to day seven, day zero to day 21, etc. For example, the day seven viability index is calculated as follows: (number of pups alive on day seven/number of pups alive and kept on day four) X 100. The pups' ability to survive may reflect the adequacy of postnatal nourishment, maternal neglect, and postnatal absorption of a toxic substance that is excreted in the mother's milk. Regardless of etiology, decreases in viability indices indicate adverse reproductive effects. Other appropriate days are acceptable to monitor postnatal growth and other developmental indices.
E. End Points of Male Reproductive Toxicity
The following end points of male reproductive toxicity should also be assessed if there is evidence of male-mediated effects on developing offspring. End points should be measured in all animals in each of the control and high dose groups. If treatment-related effects are observed, then animals from each intermediate dose group should be evaluated.
1. Evaluation of Testicular Spermatid Numbers
Testicular spermatid enumeration is a measure of sperm production from the stem cells and their survival through all phases of spermatogenesis. The enumeration of spermatid numbers should primarily be used in chronic studies where spermatid numbers have stabilized; in short-term studies treatment may not have impacted the late spermatid population. From the number of spermatids per testis, the efficiency of sperm production and daily sperm production rate can be calculated.(14)
The second testis (first testis was used for histopathology) from all F0 and F1 generation males used for mating should be collected and stored frozen until testicular spermatid numbers are enumerated. Homogenization-resistant spermatid numbers may be determined by enumerating elongated spermatid nuclei after the testis has been homogenized in a medium containing detergent.(14)
2. Sperm Evaluation for Motility, Morphology and Numbers
Motility is influenced by abstinence, the time between obtaining and evaluating the sample, pH of the medium, sample chamber depth, and temperature. Sperm samples obtained from the cauda epididymis (or proximal vas deferens) should be collected and evaluated for the percent of progressively motile sperm. (17) Care should be taken to avoid artifactual cell death during sample preparation so that the percentage of progressively motile sperm from control animals is consistently high (>70%).(11)
Sperm motility can be assessed by microscopic techniques or with a computer-assisted sperm analysis (CASA) system.(17) For microscopic evaluation, an acceptable counting chamber of sufficient depth is used to combine the assessment of motility with sperm numbers and sperm morphology. When the CASA system is utilized,(3),(18),(20) the derivation of progressive motility relies on user-defined thresholds for average path velocity and straightness or linear index. All samples should be videotaped or otherwise recorded. The video may be retained as raw data. In the event that sperm motility is not videotaped, then a sperm motility assessment from all animals in all dose levels should be performed.
Inasmuch as sperm morphology in rodents is generally stable, characteristic of the animal strain, and exhibits little variability, an increase in the number of morphologically abnormal sperm indicates that the test substance has gained access to the germ cells. This should be considered an adverse reproductive effect. Sperm should be collected from all F0 and F1 generation males selected for mating from the control and all dose levels for sperm morphology analysis.
Sperm (minimum 200 per sample) from the cauda epididymis or proximal vas deferens should be examined as a fixed wet preparation (12),(17) and classified as either normal (both head and midpiece appear normal) or abnormal (i.e., fusion, isolated heads, misshapen heads and/or tails).(26)
The total number of sperm in the cauda epididymis should be enumerated.(14) Cauda sperm reserves can be derived from the concentration and volume of sperm in the suspension used to complete the qualitative evaluations, and the number of sperm recovered by subsequent mixing and/or homogenizing the remaining cauda tissue. Sperm in the concentrated suspension can be frozen for subsequent evaluation of cauda epididymal sperm numbers. If sperm counts are reported in relation to the weight of the epididymis, the absolute counts should be reported in order to clarify declines in sperm number.
F. Analysis of Data
Values from control and test groups of animals should be compared statistically. The following techniques may be used, but others may be substituted if they are appropriate. The fertility and gestation indices may be analyzed by a one-tailed Fisher exact test. For the sex ratio index, a two-tailed Fisher exact test may be used. Data for the viability and weaning indices may be transformed by the Freeman-Tukey arc-sine transformation for binomial proportions. The transformed data may then be analyzed by an analysis of variance (ANOVA) followed by a one-tailed protected least significant difference (LSD) test to compare the control with the treated groups if the ANOVA p<0.10. The average litter size and the number of viable pups throughout the reproduction phase may be analyzed by ANOVA followed by a protected LSD test (one-tailed). For the growth (weight gain) and organ weight analyses, an analysis of covariance may be used followed by a protected LSD test (two-tailed) to compare the control and treated groups.
G. Reporting the Results of Reproduction Studies
Reports of all reproduction studies should contain the information required by the Good Laboratory Practice Regulations, including a copy of the study protocol and all amendments, absolute values for all parameters, complete data (individual pups) and tables of data summarized and analyzed by litter. Because the maternal animal and not the developing organism is the individual treated during gestation, data generally should be calculated as incidence per litter or as number and percent of litters with particular end points. All major indices and end points discussed in the previous section should be calculated. The dosage rate of test substance (doses) should be reported as mg/kg/day (milligrams of test substance per kilogram of body weight per day).
Problems commonly encountered in the review of multigeneration reproduction studies include: insufficient numbers of pregnant animals per control or treatment group, non-random selection procedures, and statistical analyses of data on a per-pup basis instead of a per-litter basis. Careful consideration of recommended guidelines and the submission of protocols for review by FDA before conducting the studies should help eliminate such problems.
In addition to the various indices in reproduction studies, data on the average number of pups that survived during a specific interval (e.g., average number of pups that survived from birth to day four, or the average number of pups that were weaned) should be examined. This analysis considers the total effect of the test substance at all stages to that point and is a more sensitive indicator than each index separately.
Relevant historical control data may be used to increase the understanding of the study results. When used, historical data should be compiled and presented with appropriate additional information, such as dates of study, strain of animals, vehicle, and route of administration.
Bolon, B., Bucci, T.J., Warbritton, A.R., Chen, J.J., Mattison, D.R., and Heindel, J.J. (1997). Differential Follicle Counts as a Screen for Chemically Induced Ovarian Toxicity in Mice: Results from Continuous Breeding Bioassays. Fundament. Appl. Toxicol. 39:1-10.
Chapin, R.E., Filler, R.S., Gulati, D., Heindel, J.J., Katz, D.F., Mebus, C., Obasaju, F., Perreault, S.D., Russell, S., Schrader, S., Slott, V., Sokol, R.Z. and Toth, G. (1992). Methods for Assessing Rat Sperm Motility. Repro. Toxicol. 6:267-273.
Heindel, J.J. (1998). Oocyte Quantitation and Ovarian Histology. In An Evaluation and Interpretation of Reproductive Endpoints for Human Health Risk Assessment. (G. Daston and C. Kimmel, Eds.), pp. 57-74. International Life Sciences Institute, Health and Environmental Sciences Institute, Developmental and Reproductive Toxicology Committee, Washington, D. C.
Klinefelter, G.R., Grey, L.E. and Suarez, J.D. (1991). The Method of Sperm Collection Significantly Influences Sperm Motion Parameters Following Ethane Dimethanesulfonate Administration in the Rat. Repro. Toxicol. 5:39-44.
Linder, R.E., Strader, L., Slott, V. and Suarez, J.D. (1989). Endpoints of Spermatotoxicity in the Rat After Short Duration Exposures to Fourteen Reproductive Toxicants. In Reproductive Toxicology. (A.W. Hayes, Ed.). Raven Press, New York, NY.
Revision Committee, FDA Guidelines for Developmental Toxicity and Reproduction, Food and Drug Administration. (1999). Food and Drug Administration proposed testing guidelines for developmental toxicity studies. Regulat. Toxicol. Pharmacol. 30:39-44.
Seed, J., Chapin, R.E., Clegg, E.D., Darney, S.P., Dostal, L., Foote, R.H., Hurtt, M.E., Klinefilter, G.R., Makris, S., Schrader, S., Seyler, D., Sprando, R.L., Treinen, K.A., and Veeranachaneni, R. (1996). Consensus report: Methods for Assessing Sperm Motility, Morphology, and Counts in the Rat, Rabbit and Dog. Repro. Toxicol. 10:237-244.
Sobotka, T.J., Ekelman, K.B., Slikker, W., Jr., Raffaele, K. and Hattan, D.G. (1996). Food and Drug Administration Proposed Guidelines for Neurotoxicological Testing of Food Chemicals. NeuroToxicol. 17:825-836.
Toth, G.P., Stober, J.A., Read, E.J., Zenick, H. and Smith, M.K. (1989). The Automated Analysis of Rat Sperm Motility Following Subchronic Epichlorohydrin Administration: Methodologic and Statistical Considerations. J. Androl. 10:401-415.
U.S. Food and Drug Administration. (1982). Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food. U.S. Food and Drug Administration, Bureau of Foods, Washington, DC.
U.S. Food and Drug Administration. (1993). Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food, Redbook II (Draft). U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Washington, D C.
- Zenick, H. and Clegg, E.D. (1986). Issues in Risk Assessment in Male Reproductive Toxicology. J. Amer. Coll. Toxicol. 5:249-259.
Submit comments on this guidance document electronically via docket ID: FDA-2013-S-0610 - Specific Electronic Submissions Intended For FDA's Dockets Management Staff (i.e., Citizen Petitions, Draft Proposed Guidance Documents, Variances, and other administrative record submissions)
If unable to submit comments online, please mail written comments to:
Food and Drug Administration
5630 Fishers Lane, Rm 1061
Rockville, MD 20852
All comments should be identified with the title of the guidance.